Abstracts – Poster Presentations

Transkript

28
th
Symposium on Biotechnology
for Fuels and Chemicals
Hosted by the Oak Ridge National Laboratory
Welcome from the Organizing Committee
We are living in exciting times and times of change. Many countries are focusing significant efforts toward new, improved,
or affordable products from renewable resources. We have designed the program for the 28th Symposium on Biotechnology for Fuels and Chemicals to deliver the latest research breakthroughs and results in biotechnology that stimulate such
research activities. This year, we have an exciting program with six Oral Presentation Sessions, two Special Topics, and
two Poster Presentation Sessions. Whether you represent the industrial, academic, or government sector, we invite you to
join us and participate in this exciting exchange of information and ideas. You will find valuable opportunities for productive
interactions with your colleagues, both from a national and international perspective.
This year the Symposium is being managed by the Society for Industrial Microbiology (SIM) to assist the organizing
committee with non-technical operations. The Symposium continues in the tradition of providing an informal, congenial
atmosphere that our participants find conducive to discussing technical program topics. All this is made possible through
the generosity of our sponsors, and we urge you to join us in thanking them. This year’s sponsors include:
Abengoa Bioenergy Corporation
Battelle Nanotechnology Innovation Alliance
BRI Energy
Cargill
DOE Office of the Biomass Program
Genencor International
Idaho National Laboratory
Iogen
Katzen International
National Renewable Energy Laboratory
Natural Resources Canada
The Samuel Roberts Noble Foundation
Novozymes
Oak Ridge National Laboratory
P&G
Southeastern SunGrant Center
Tate & Lyle
USDA Agricultural Research Service
Each year at this Symposium we recognize an individual, or individuals, who have distinguished themselves by their contributions to the field of applied biotechnology for renewables as a whole, and this Symposium, in particular. This award
is named in honor of Dr. Charles D. Scott, founder of the Symposium on Biotechnology for Fuels and Chemicals and its
chair for the first ten years. At Oak Ridge National Laboratory, Chuck performed research and development on many
novel bioprocessing systems. This year’s award(s) go to two very deserving individuals, Drs. Brian Davison, Oak Ridge
National Laboratory and Mark Finkelstein, National Renewable Energy Laboratory. Both are being recognized for their
excellent technical leadership in the area of biotechnology and bioprocessing, and for their outstanding support of this
Symposium and the Proceedings for many years.
We also hope you will join us for the 29th Symposium in Denver, Colorado, at the Denver Adam’s Mark Hotel, April 29May 2, 2007. The Web link to the 29th Symposium is www.simhq.org/html/meetings.html.
28th Symposium on Biotechnology for Fuels and Chemicals
Charles D. Scott Award
Established in 1995, the Charles D. Scott Award has since been presented to individuals who have made distinguished
contributions to the field of biotechnology for fuels and chemicals. Named in honor of Dr. Charles D. Scott, founder of
the Symposium on Biotechnology for Fuels and Chemicals and its chair for the first ten years, this award acknowledges
contributions to the field as a whole or to this Symposium, particularly innovation in fundamental and applied biotechnology, insight into bioprocessing fundamentals, or commitment to facilitate commercialization of products from renewable
resources. In his years of work at ORNL, Chuck performed research and development on many novel bioprocessing
systems including high production bioreactors, immobilized microbes, enzymes in organic media, and coal bioprocessing.
Brian Davison
Oak Ridge National Laboratory
Brian H. Davison is the Chief Scientist for Systems Biology and
Biotechnology at Oak Ridge National Laboratory. He recently served
two years as the Director of Life Sciences Division. He was previously
a Distinguished Researcher & BioChemical Engineering Research
Group leader. In his twenty years at ORNL he has performed biotechnology research in a variety of areas including bioconversion of
renewable resources (ethanol, organic acids, solvents), non-aqueous
biocatalysis, systems analysis of microbes (cultivation and proteomics),
biofiltration of vocs, mixed cultures, immobilization of microbes and
enzymes, metal biosorption, and extractive fermentations.
If there is a theme connecting his work, it is at the interface of solid,
liquid, and gas phases between biocatalysts and their environments.
This has resulted in over 90 publications and six patents. He led a multilab team which received an R&D100 Award for “Production of Chemicals from Biologically Derived Succinic Acid,” in 1997.
He received his Ph.D. in Chemical Engineering from the California
Institute of Technology, and his B.S.E. in Chemical Engineering, from
the University of Rochester. He co-chaired the 15th to 26th Symposia
on Biotechnology for Fuels and Chemicals and served as editor of
Proceedings in Appl. Biochem. Biotechnol., (1994 – 2005). The Symposium grew from 150 to over 400 attendees
during these twelve years (ten with Mark Finkelstein).
He is the Chair of the ORNL Institutional Biosafety Committee from 2001 to present. He was just named a
Fellow in the American Institute for Medical and Biological Engineering (AIMBE), 2006. He is also an Adjunct Professor of Chemical Engineering at the University of Tennessee-Knoxville where he supervises one Ph.D. student
and four Masters students.
Mark Finkelstein
Luca Technologies
A keen interest in microbes and their utility has remained at the core of
Mark Finkelstein’s academic and professional career. After obtaining B.S.,
M.S. and Ph.D. degrees in biology (Rochester Institute of Technology),
microbiology (Miami University), and molecular biology (SUNY at Buffalo),
respectively, Mark secured a research fellowship at University of Wisconsin to continue his postdoctoral studies.
Mark began his career in biotechnology in the pharmaceutical industry
at Schering Plough and worked his way up to senior principal scientist,
and section head of microbial biochemistry. Mark’s team was responsible
for building Schering’s bacterial expression system for interferon that remains in production today. In 1988 Mark moved to Colorado to join Coors
Biotech as group leader of molecular genetics, where he had oversight of
the metabolic and genetic manipulation of microbes for the production of
a variety of commercially useful products.
Mark joined NREL in 1992 as the Branch Manager of Applied Biological
Sciences. Mark began to introduce industrial approaches to problem solving and helped establish numerous successful business relationships during his tenure at NREL. He helped initiate work on Zymomonas within the
Biofuels Program that resulted in numerous patents, publications, NREL’s
first team Staff Award, and an R&D 100 award in 1995. Mark soon became Director of the Biotechnology Division
in the National Bioenergy Center. In 2004 Mark joined Luca Technologies, a small biotechnology company using
microbes to create in-situ hydrogen/methane from buried hydrocarbons, which has huge sustainable and commercial ramifications.
He has served in a variety of capacities in support of the Symposium on Biotechnology for Fuels and Chemicals before becoming co-chair from 1996 to 2004.
Past Awardees of the Charles D. Scott Award
1995—17th Symposium—Donald L. Johnson
1996—18th Symposium—Bruce Dale
1997—19th Symposium—Raphael Katzen
1998—20th Symposium—Jack Saddler
1999—21st Symposium—Charles E. Wyman
2000—22nd Symposium—Karel Grohman
2001—23rd Symposium—Patrick Foody
2002—24th Symposium—Sharon Shoemaker
2003—25th Symposium—Thomas W. Jeffries
2004—26th Symposium—Guido Zacchi
2005—27th Symposium—Lee Lynd
General information
All meeting sessions will be held at the Nashville Airport
Marriott Hotel.
Registration
On-site registration and distribution of meeting packets to
preregistrants will take place in the Nashville Ballroom
Foyer, Main floor, during the following hours:
Sunday, April 30
Monday, May 1
Tuesday, May 2
Wednesday, May 3
8:30 am – 5:00 pm
8:15 am – 5:00 pm
8:15 am – 12:00 pm
8:15 am – 5:00 pm
Programs will be distributed at the meeting to all attendees. Extra copies of the meeting program at the meeting
site are $50. Name badges must be worn for admittance
to the scientific sessions, exhibits and special functions.
Smoking is not permitted in the hotel.
Information and message center
Messages and announcements will be posted on the
bulletin board in the Registration Area and updated daily. In
case of emergency, registrants may be contacted through
the Nashville Airport Marriott Hotel at 615-889-9300.
Speaker ready room
Speakers may review their presentations in the Knoxville
Room during the following hours:
Sunday
8:15 am – 8:00 pm
Monday–Wednesday 8:00 am – 5:00 pm
Prior to their session, speakers should coordinate
delivery of their presentations to their chairs.
Membership table
The SIM Membership Committee would like to welcome
new members, first-time attendees and students to the
Tabletop exhibits
New this year! Company representatives will be available during the Symposium to discuss their company’s products
and/or services. Please visit the exhibitors during the show hours listed in the program.
ATR (appropriate Technical Resources)
laurel, MD
ATR is the US partner of Infors. With 40 years of experience, INFORS provides innovative fermenter and control systems for research and process engineering. The Multitron environmental stackable shakers; provide cooling, lighting,
humidity and CO2 control. Terrafors solid state fermenter is a unique instrument for the study of bioremediation and
biodegradation.
Knoxville, TN
The SunGrant Initiative is a concept to solve America’s energy needs and revitalize rural communities with landgrant university-based research, education, and extension programs on renewable energy and biobased, non-food
industries. The University of Tennessee Agricultural Experiment Station is one of five regional centers across the
nation and leads the Southeastern Regional Center.
U.S. Department of Energy Genomics:GTL Program
Oak Ridge, TN
Learn about the Department of Energy’s Genomics: GTL program, sponsoring systems environmental microbiology
research. Armed with next generation biology facilities that will serve as engines of discovery for new solutions to
energy and environmental challenges, Genomics: GTL will provide the impetus for dramatic growth in industrial and
environmental biotechnology.
Symposium. Please visit the membership table to learn
more about SIM and its membership benefits. The membership table will be located in the Registration area.
Airport shuttle
The Nashville Airport Marriott provides complimentary
daily shuttle service to and from the Nashville Airport.
Shuttle buses leave every half hour on the hour from the
lobby; and on the quarter hour from the airport.
Shuttle service to downtown Nashville/
Opry Mills
For those interested in visiting downtown Nashville or Opry
Mills, the hotel is offering complimentary shuttle service to
attendees on Tuesday afternoon. Details will be available
at the registration desk.
Editor’s desk
The editor’s desk will be open Sunday, April 30 from 10:00
am to 5:00 pm and at convenient hours throughout the
Symposium. Please turn manuscripts in immediately upon
arrival at the Symposium. We need to distribute many of
them to reviewers who are attending.
Internet cafe
An Internet Cafe is available to all attendees and their
guests in the Jackson Room during Symposium hours.
27th Symposium Proceedings
If you attended the 27th Symposium and are to receive a
copy of the proceedings book, please pick up your copy at
the Editor’s Desk during registration hours.
Special needs
The Organizing Committee and SIM want to ensure your
comfort and convenience at the Symposium. If you have
any special needs, please let us know at the registration
desk.
Organizing committee
Jonathan Mielenz, Conference Chairman
Oak Ridge National Laboratory, Oak Ridge, TN
Thomas Klasson, Conference Co-Chairman
USDA-Agricultural Research Service, New Orleans, LA
Jim McMillan, Conference Co-Chairman
National Renewable Energy Laboratory, Golden, CO
Bill Adney
Doug Cameron
Cargill, Inc., Minneapolis, MN
Brian Davison
Oak Ridge National Laboratory, Oak Ridge, TN
Jim Duffield
Don Erbach
USDA-Agricultural Research Service, Beltsville, MD
Thomas Jeffries
USDA-Forest Service, Madison, WI
Lee Lynd
Dartmouth College, Hnover, NJ
Amy Miranda
USDOE Office of the Biomass Program, Washington, DC
Dale Monceaux
Katzen International, Inc., Cincinnati, OH
Lisbeth Olsson
Technical University of Denmark, Kgs Lyngby, Denmark
Jack Saddler
University of British Columbia, Vancouver, BC, Canada
Jin-Ho Seo,
Seoul National University, Seoul, Korea
Sharon Shoemaker
University of California, Davis, CA
David Short
DuPont, Inc., Newark, DE
Dave Thompson
Idaho National Laboratory, Idaho Falls, ID
Jeff Tolan
Iogen Corporation, Ottawa, ON, Canada
Charles Wyman
University of California, Riverside
Gisella Zanin
State University of Maringa, Maringa, Parana, Brazil
Local information
Heralded as Music City, USA, and the country-music
capital of the world, Tennessee’s fast-growing capital city
also shines as a leading center of higher education, appropriately known as the Athens of the South. Nashville
has prospered from both labels, emerging as one of the
South’s most vibrant cities in the process. The Gaylor Entertainment Center (formerly Nashville Arena), a 20,000seat facility spanning three blocks at 5th and Broadway,
opened in 1996. Connected to the city’s convention center
by a tunnel, the Arena hosted the U.S. Figure Skating
Championship in 1997. A successful drive to land both a
National Football League and a National Hockey League
franchise, coupled with a population gain that has pushed
Nashville ahead of Memphis, has put Nashville into the
major leagues of American cities.
Nashville’s radio program, which began as station
WSM’s in 1925 and thrived throughout the Great Depression right into today’s MTV years, established the town
as a music center. The Opry now performs in a sleek $15
million Opry House. An infusion of new talent is attracting
another generation of fans. The Opry is still as gleeful and
down-home informal as it was when ticket holders used to
jam into the old Ryman Auditorium.
Bolstering Nashville’s reputation as a music town are
dozens of clubs, performance stages (including the revitalized Ryman), and television tapings open to the public,
as well as memorials to many country-music stars. The
District, the downtown area along 2nd Avenue and historic
Broadway, has emerged as another destination for tourists
and locals alike, with restaurants, specialty shopping, and
entertainment options. And, of course, legendary Music
Row continues to beckon aspiring singers, musicians, and
songwriters with stars in their eyes and lyrics tucked in
their back pockets.
Much of Nashville’s role as a cultural leader, enhanced
by the presence of the Tennessee Performing Arts Center,
is derived from the presence of 16 colleges and universities, two medical schools, two law schools, and six
graduate business schools. Several, including Vanderbilt
University, have national or international reputations, and
many have private art galleries. As ancient Athens was the
“School of Hellas,” so Nashville, where a full-size replica
of the Parthenon graces Centennial Park, fills this role in
the contemporary South. The historic sites throughout the
city -- such as the Hermitage, Belle Meade Plantation, and
Travellers’ Rest -- add another dimension.
Belle Meade Plantation
Known as the “queen of the Tennessee plantations,” this
stunning Greek Revival house is recognizable by the Civil
War bullet holes that riddle its columns. Guides in period costumes lead you through the mansion, which is furnished antebellum style. The mansion is the centerpiece of a 5,300-acre
estate that was one of the nation’s first and finest thoroughbred breeding farms. It’s also the site of the famous Iroquois,
the oldest amateur steeplechase in America, a society event
now run each May in nearby Percy Warner Park.
Belmont Mansion
This 1850s Italian-style villa was the home of Adelicia Acklen, Nashville’s answer to Scarlett O’Hara, who married
“once for money, once for love, and once for the hell of it.”
On Belmont College’s campus, it’s a gem right down to
its sweeping staircase designed for grand entrances and
cast-iron gazebos perfect for romance.
Carl Van Vechten Gallery
Alfred Stieglitz rewarded Fisk University’s progressive arts
program with a bequest from his collection of 20th-century
paintings and his own superb photographs. Stieglitz’s wife,
Georgia O’Keeffe, helped install the collection, which holds
some of her own paintings as well as works by Picasso and
Renoir. The gallery also exhibits African sculpture.
Cheekwood Botanical Garden and Museum
of Art
Thirty acres of gardens at the Cheekwood Botanical
Garden and Museum of Art showcase annuals, perennials,
and seasonal wildflowers. The museum is in a carefully
restored neo-Georgian mansion. The permanent exhibition
shows American art to 1945, while the “Temporary Contemporary” gallery presents local and national artists.
Country Music Hall of Fame and Museum
This tribute to country-music’s finest, among them Hank Williams, Loretta Lynn, Patsy Cline, and Johnny Cash, reopened
in 2001 in a new facility across from the Gaylord Entertainment Center. A block long, the museum contains plaques
honoring country greats, a two-story wall with every gold and
platinum country record, a theater that screens a digital film
on the industry, and daily live entertainment. Not to mention
Elvis Presley’s solid-gold 1960 Cadillac limo. A digital film
presentation surveys country music around the world.
Fort Nashborough
museum. Jackson built the mansion on 600 acres for his
wife, Rachel, for whose honor he fought and won a duel.
Both are buried in the family graveyard. The Andrew Jackson Center, a 28,000-square-ft museum, visitor center,
and education center, contains many Jackson artifacts
never before exhibited.
High on limestone bluffs along the river, a crude log fort,
whose original was built in 1779 to protect and shelter early
settlers, overlooks Nashville. This painstaking re-creation,
built in 1962, serves as a monument to courageous city
founders. In five log cabins, costumed interpreters evoke
the indomitable spirit of the American age of settlement.
Opry Mills
Frist Center for Visual Arts
Parthenon
This art gallery boasts 20,000 ft of exhibit space and offers
an ambitious showcase of paintings, sculpture, and other
visual art. The historic building, with its original Art Deco
exterior, was formerly Nashville’s downtown post office.
In its current incarnation as the Frist Center, the building
houses four art galleries, a children’s discovery gallery, a
250-seat auditorium, gift shop, café, art workshops, and
an art resource center.
Gibson Guitar Factory
Gibson’s largest Nashville factory: the Electric Guitar
Division. This is the heart of Gibson’s operation, and the
facility dwarfs the other two. Guitars come in as raw wood,
and every scrap gets either used or recycled. (According
to Gibson’s Fred Greene, General Manager of the Electric
Guitar division, the sawdust is sold to Jack Daniels and
used to fire the stills. Funny, we thought it was the Jack
that fuels the music, not the other way around.) It takes up
to six weeks for Gibson to fashion those slabs of wood into
instruments; in an era of CNC machines and automated
production, Gibson still does much of its work by hand.
Grand Ole Opry
This enormously popular radio show, performed in the
Grand Ole Opry House, has been broadcasting country
music for more than 70 years. You can see superstars,
legends, and up-and-coming stars on this stage. The Opry
seats about 4,400 people and is broadcast live on WSM
AM 650 every Friday and Saturday night at 6:30 and 9:30.
Hermitage
The life and times of Andrew Jackson, known as Old
Hickory, are reflected with great care at this house and
Opry Mills features the best names in manufacturers’ and
retail outlets including Nike, OFF 5th Saks Fifth Avenue,
Gap Outlet and Old Navy; high quality entertainment
venues and themed restaurants including Regal Cinemas,
Dave & Buster’s and The Aquarium.
An exact copy of the Athenian original, Nashville’s Parthenon was constructed to commemorate Tennessee’s 1897
centennial. Across the street from Vanderbilt University’s
campus, in Centennial Park, it’s a magnificent sight,
perched on a gentle green slope beside a duck pond.
Inside are the Cowan Collection, with 63 works of art by
American artists, traveling exhibits, and such exquisite
statuary as the 42-ft the tallest indoor sculpture in the
Western world.
Ryman Auditorium and Museum
A country-music shrine, the Ryman Auditorium and
Museum was home to the Grand Ole Opry from 1943 to
1974. The auditorium seats 2,000 for live performances of
classical, jazz, pop, gospel, and, of course, country. The
museum displays photographs and memorabilia of past
Ryman Auditorium performances that provide a history of
both the facility and country music.
State Capitol
The state capitol was designed by noted Philadelphia
architect William Strickland (1788-1854), who was so impressed with his Greek Revival creation that he requested
-- and received -- entombment behind one of the building’s
walls. On the grounds you’ll also find the graves of the 11th
U.S. president, James K. Polk, and his wife.
Tennessee State Museum
More than 6,000 artifacts and rotating art and history
exhibits trace the state’s history from the days of Native
American settlement through the Civil War and into the
20th century.
Nashville Airport Marriott
Hotel and meeting rooms
floor plan, Lobby level
Program at a glance
Sunday, April 30, 2006
8:30 am - 5:00 pm
10:00 am - 5:00 pm
1:00 pm - 5:30 pm
5:30 pm - 9:00 pm
6:00 pm - 9:00 pm
Registration/Editor’s Desk
Nashville Foyer
Poster set up
Cumberland Ballroom
Opening remarks
Nashville Ballroom
Session 1A (Concurrent)
Nashville ABCD
Session 1B (Concurrent)
Nashville EFGH
Welcoming reception/Exhibits open
Nashville Foyer
Poster Session I (Sections 1A, 1B, 3A, 3B, 4) Cumberland Ballroom
Monday, May 1, 2006
7:15 am - 5:00 pm
7:15 am - 8:00 am
7:15 am - 8:00 am
8:00 am - 12 noon
12 noon - 1:30 pm
1:30 pm - 5:30 pm
6:00 pm - 9:00 pm
Continental Breakfast
Speakers Breakfast
Session 2
Lunch on own
Session 3A (Concurrent)
Session 3B (Concurrent)
Poster Session II (Sections 2, 5, 6)
Nashville Foyer
Nashville Foyer
Memphis Room
Nashville Ballroom
Nashville ABCD
Nashville EFGH
Cumberland Ballroom
Tuesday, May 2, 2006
7:15 am - 12:00 noon
7:15 am - 8:00 am
7:15 am - 8:00 am
7:00 am - 10:00 am
8:00 am - 12 noon
12:15 pm - 1:45 pm
7:00 pm - 9:30 pm Registration/Editor’s Desk
Speakers Breakfast
Poster removal
Session 4
Organizing committee lunch Free afternoon
Special Topic A (Concurrent)
Special Topic B (Concurrent)
Nashville Foyer
Nashville Foyer
Memphis Room
Cumberland Ballroom
Nashville Ballroom
Memphis Room Nashville Ballroom ABCD
Nashville Ballroom EFGH
Wednesday, May 3, 2006
7:15 am - 5:00 pm
7:15 am - 8:00 am
7:15 am - 8:00 am
8:00 am-12 noon
1:30 pm-5:00 pm
5:00 pm
6:00 pm
7:00 pm
Speakers Breakfast
Session 5
Session 6
Closing remarks
Reception
Banquet
Nashville Foyer
Nashville Foyer
Memphis Room
Nashville Ballroom
Nashville Ballroom
Nashville Ballroom
Nashville Foyer
Cumberland Ballroom
28
th
Technical Program
Sunday, April 30
Registration
Location: Nashville Ballroom Foyer
Time: 8:30 am – 5:00 pm
3:10 pm
Coffee break
3:40 pm
1A-05: Modeling the disruption of crystalline cellulose by the carbohydrate binding
module from cellobiohydrolase I
M.R. Nimlos,* W.S. Adney, M.E. Himmel, NREL,
Golden, CO; X. Qian, Rx-Innovation, Inc., Ft.
Collins, CO; M.F. Crowley, Scripps Research
Inst., La Jolla, CA; G. Chukkapalli, M. Cleary,
San Diego Super Computer Center, La Jolla,
CA; J.F. Mathews, J. Brady, Cornell University,
Ithaca, NY
Poster set-up
Location: Cumberland Ballroom
Time: 10:30 am – 5:00 pm
Opening remarks
4:05 pm
Location: Nashville Ballroom
Time: 1:00 – 1:15 pm
Session 1A: Enzyme catalysis and
engineering
Chairs: E. Vlasenko, Novozymes, Davis, CA and M. Himmel, NREL, Golden, CO
Location: Nashville Ballroom ABCD
1:30 pm 1A-01: Grass lignocellulose: Strategies to
overcome recalcitrance
D.E. Akin, USDA-ARS, Athens, GA
4:30 pm
4:55 pm
1:55 pm
2:20 pm
2:45 pm
12
1A-02: Plant cell wall microfibril structure
and changes during biomass conversion
processes
S-Y. Ding,* D.K. Johnson, J.O. Baker, X. Qian,
M.E. Himmel, NREL, Golden, CO
1A-03: Thermophilic enzyme systems for
efficient total hydrolysis of lignocellulosic
materials
L. Viikari,* M. Siika-aho, VTT Biotechnology,
Espoo, Finland; J. Vehmaanper, ROAL Oy, Rajamaki, Finland; G. Zacchi, Lund University, Lund,
Sweden; K. Reczey, Budapest University of
Technology and Economics, Budapest, Hungary
1A-04: The role of xylanases in the enzymatic hydrolysis of lignocellulose
A. Berlin,* N. Gilkes, J. Saddler, University of
British Columbia, Vancouver, BC, Canada
1A-06: Adsorption and desorption of cellulase, beta-glucosidase and BSA protein on
pretreated corn stover, cellulose and lignin
D. Willies,* B. Yang, Dartmouth College, Hanover, NH; C.E. Wyman, University of California, Riverside, CA
1A-07: Enzyme optimization for cell free
ethanol production
E.J. Allain,* A. Madison, D. Dardugno, R.A. Salinas, E. Marland, Appalachian State University,
Boone, NC
1A-08: Enzymatic hydrolysis of lignocellulose at very high solids concentrations
H. Jorgensen,* C. Felby, The Royal Veterinary
and Agricultural University, Taastrup, Denmark;
J. Larsen, M.O. Petersen, Elsam Engineering
A/S, Fredericia, Denmark
Have you picked up
your copy of the
27th Symposium
Proceedings Book?
now available at Editor’s desk
Sunday, April 30
Session 1B: Plant biotechnology
and genomics
Chairs: M. Sticklen, Michigan State University, E. Lansing,
MI and G. Tuskan, ORNL, Oak Ridge, TN
Location: Nashville Ballroom EFGH
1:30 pm 1B-01: Application of plant genomics to the
improvement of a dedicated energy crop
S. Thomas,* R. Flavell, K. Feldmann and the
Ceres Genomics Teams, Ceres, Thousand
Oaks, CA
2:00 pm
2:30 pm
1B-02: Regulation of cell wall polysaccharide composition
C. Somerville,* A. Paredez, D. Ehrhardt, S.
Persson, S. Bauer, S. Chen, Carnegie Institution, Stanford, CA
1B-03: Energy crops for fuel ethanol production
B.W. Ferguson,* D.A. Lee, Edenspace Systems
Corp., Dulles, VA; and M. Sticklen, Michigan
State University, E. Lansing, MI
Tabletop exhibits open
Time: 5:30 - 9:00 pm
Companies participating include:
ATR (Appropriate Technical Resources)
U.S. Dept of Energy Genomics:GTL Program
Representatives will be available to discuss their company’s products and/or services.
Poster session 1
Sections: 1A, 1B, 3A, 3B, 4
Chair: J. Duffield, NREL, Golden, CO
Sponsored by the USDA Agricultural Research Service
1A-09
3:00 pm
Coffee Break
3:30 pm
1B-04: Development of brown midrib sweet
sorghum as a biomass crop
A. Saballos,* G. Ejeta, W. Vermerris, Purdue
University, West Lafayette, IN
1B-05: Conversion of cellulose to glucose
using endo-1, 4-b-glucanase (E1) enzyme
produced in transgenic rice plants
H. Oraby,* R. Ahmad, C. Ransom, M. Sticklen,
B. Venkatesh, B. Dale, Michigan State University, East Lansing, MI
1A-10
4:00 pm
1A-11
Molecular cloning of a gene encoding the
sucrose phosphorylase from Leuconostoc
mesenteroides B-1149 and its use for transglucosylation
J-S. Ahn,* X-J. Jin, D-H. Park, H-K. Kang, D.
Kim, J-H. Lee, Chonnam National University,
Gwangju, Korea; G.J. Kim, Korean Minjok
Leadership Academy, Gwangwon-do, Korea
Enzymatic micro reactors for the determination of ethanol by an automatic sequential
injection analysis system
E.M. Alhadeff,* A.M. Salgado, N. Pereira, Jr.,
B. Valdman, Univ. Federal do Rio de Janeiro,
Rio de Janeiro, Brazil; F. Valero, O. Cos, Univ.
Autonoma de Barcelona, Barcelona, Spain
Purification, characterization and substrate
specificity analysis of the glycosyl hydrolase family 1 (Cel1b) enzyme of Trichoderma
reesei
M.K. Ali,* J.O. Baker, E. Knoshaug, T. Jeoh,
M.E. Himmel, W.S. Adney, NREL, Golden, CO
13
Sunday, April 30
1A-12
Optimization of cyclodextrin glucanotransferase production from Bacillus clausii
strain E16 in submerged fermentation using
response surface methodology
H.F. Alves-Prado,* D.A. Bocchini, E. Gomes, R.
Da Silva, L.C. Baida, IBILCE/UNESP, San Jose
do Rio Preto, Brazil; I.C. Roberto, Faenquil,
Lorena, Brazil
1A-19
1A-20
1A-13
1A-14
1A-15
Purification and characterisation of a cyclodextrin glucanotransferase (CGTase) from
Paenibacillus campinasensis H69-3
H.F. Alves-Prado,* E. Gomes, R. da Silva;
IBILCE-UNESP, San Jose do Rio Preto, Brazil
1A-21
Electrochemical regeneration of cofactor for
continuous bioreactor product formation
M.B. Arora,* Y.J. Lin, S.W. Snyder, E.J. St.
Martin, Argonne National Laboratory, Argonne,
IL; L. de la Garza, Valdosta State University,
Valdosta, GA
Cellulolytic enzymes from streptomycetes
isolated from Brazilian soil
A.L.G. de Lima, R.P. do Nascimento, A.S.A. da
Silva, E.P.S. Bon,* R.R.R. Coelho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
K. Willquist, Lund University, Lund, Sweden
1A-22
1A-23
1A-16
1A-17
1A-18
14
Effect of poultry by-product on protease
production by Bacillus subtilis in solid state
fermentation
V.F. Soares, E.P.S. Bon,* D.M.G. Freire, L.A.I.
de Azeredo, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil
Screening of cellulolytic fungal strains from
Brazilian biodiversity and culture collections
L.T.C. Pereira, L.T.C. Pereira, M. Lobao, A.S.A.
da Silva, E.P.S. Bon,* Federal University of Rio
de Janeiro, Rio de Janeiro, Brazil; K. Willquist,
Lund University, Lund, Sweden
The effect of nitrogen regulation on cellulolytic enzymes production by a wild type and
a nit-2 mutant strain of Neurospora crassa
A.S.A da Silva, F. Knopp, E.P.S. Bon,* Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil
1A-24
Further improvements in performance of
salt-activated enzymes via chemical modification and use of surfactants
A.P. Borole,* B.H. Davison, Oak Ridge National
Laboratory, Oak Ridge, TN
Novel endoglucanase of Clostridium thermocellum is specific for beta-glucans
P.J. Brumm,* D.A. Mead, Lucigen, Inc., Middleton, WI
Effect of different carbon sources on xylanase production by Bacillus circulans D1:
Maltose is the best inductor of xylanase
production
D.A. Bocchini, E. Gomes, R. Da Silva,* IBILCEUNESP, San Jose do Rio Preto and Araraquara,
Brazil
Enzymatic activity of peroxidase and polyphenoloxidase from mate tea leaves (Ilex
paraguariensis St. Hill.) in compressed CO2
M.S. Primo, D. Oliveira, J.V. Oliveira, M.L.
Corazza, C. Dariva,* Univ. Reg. Integrada to
Alto Uruguai e das Missoes Campus de Erechim, Erechim, Brazil; O.A.C. Antunes, IQ/
UFRJ, Rio de Janeiro, Brazil
Screening of microorganisms towards the
conversion of carveol to carvone by PCR
technique
L. Lerin, I. Rottava, D. de Oliveira,* C. Dariva,
R.L. Cansian, F. Padilha, Univ. Reg. Integrada
do Alto Uruguai e das Missoes Campus de
Erechim, Erechim, Brazil; G. Toniazzo, O.A.C.
Antunes, IQ/UFRJ, Rio de Janeiro, Brazil
Characterization of inulinase obtained by
solid state fermentation and submerged
fermentation: A comparative study
M.A. Mazzutti, M. Di Luccio,* H. Treichel, Univ.
Reg. Integrada do Alto Uruguai e das Missoes
Campus de Erechim, Erechim, Brazil; M.I.
Rodrigues, Cidade Universitaria Zefereino Vaz,
Caixa, Brazil
Sunday, April 30
1A-25
Evaluation of lipase activity from seeds of
different varieties of Ricinus communis L
E.D.C. Cavalcanti, D.M.G. Freire,* Universidade
Federal do Rio de Janeiro, Rio de Janeiro,
Brazil; P. Villeneuve, CIRAD/AMIS, Montpellier,
France; R. Lago, Embrapa Food Technology,
Rio de Janeiro, Brazil
1A-33
1A-34
1A-26
1A-27
1A-28
1A-29
Immobilization of Candida antarctica lipase
B by covalent attachment to green coconut
fiber
A.I.S. Brigida, A.D.T. Pinheiro, A.L.O. Ferreira,
L.R.B. Goncalves*, Universidade Federal do
Ceara, Fortaleza, Brazil
Use of agricultural and agro-industrial solid
wastes for depolymerizing enzymes in solidstate fermentation
E.B.N. Graminha,* A.Z.L. Goncalves, R. Da
Silva, E. Gomes, UNESP, San Jose do Rio
Preto, Brazil; M.A.A, Balsalobre, Bellman Nutricao Animal, Mirassol, Brazil
Pectinase production by solid-state fermentation using agro-industrial residues using a
thermophilic fungi strain
M.A.U. Guez, N. Martin, E.B.N. Graminha,* J.C.
Thomeo, R. da Silva, E. Gomes, UNESP, San
Jose do Rio Preto, Brazil
1A-35
1A-36
1A-37
Activity determination of cellulase components using non-crystalline cellulose
R. Gupta,* Y.Y. Lee, Auburn University, Auburn, AL
1A-38
1A-30
1A-31
1A-32
Poly(L-lactic acid) synthesis by uncatalyzed
polycondensation and enzymatic ring opening polymerization methods
D. Omay, Y. Guvenilir,* Istanbul Technical University, Istanbul, Turkey
Protease immobilization by covalent and
ionic binding on various carriers
C. Tasdelen, Y. Guvenilir,* Istanbul Technical
University, Istanbul, Turkey
Enantioselective reduction of ketones by
carbonyl reductases
L. Hua,* D. Zhu, K. Yang, Southern Methodist
University, Dallas, TX
1A-39
A study of layer-by-layer nano self-assembly
techniques for enzyme immobilization in
microchannels
J. Wen, B. Elmore, Louisiana Tech University,
Ruston, LA; F. Jones*, University of Tennessee,
Chattanooga, TN
b-D-xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43
D.B. Jordan,* X.-L. Li, C.A. Dunlap, T.R. Whitehead, M.A. Cotta, USDA-ARS-NCAUR, Peoria,
IL
Cellobiose production using cellulase inhibition
M. Kim*, Lousiana State University, Baton
Rouge, LA; C-H. Chung, D.F. Day, Audubon
Sugar Institute, St. Gabriel, LA
Investigation of factors limiting hydrolysis at
high solids concentrations
J.B. Kristensen*, C. Felby, H. Jorgensen, The
Royal Veterinary and Agricultural University,
Taastrup, Denmark; and J. Larsen, Elsam Engineering A/S, Fredericia, Denmark
Solvent-free biolubricant synthesis using
lipase
P.T.C. Dias, M.A.P. Langone,* Univ. do Estado
do Rio de Janeiro, Rio de Janeiro, Brazil; M.A.P.
da Silva, Univ. Federal do Rio de Janeiro, Rio
de Janeiro, Brazil
Production of biodiesel from vegetable oil
using immobilized lipase
O.L. Bernardes, M.A.P. Langone,* Univ. do
Estado do Rio de Janeiro, Rio de Janeiro,
Brazil; J.V. Bevilaqua, Centro de Pesquisa &
Desenvolvimento da Petrobras, Cenpes, Brazil;
D.M.G. Freire, Univ. Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
Biodiesel synthesis via enzymatic esterification
A.P. de Alencar Vieira, M.A.P. da Silva, Univ.
Federal do Rio de Janeiro, Rio de Janeiro, Brazil; M.A.P. Langone,* Univ. do Estado do Rio de
15
Sunday, April 30
1A-40
1A-41
1A-42
1A-43
1A-44
1A-45
1A-46
Solvent-free ethyl oleate synthesis by noncommercial lipases
M.A.P. Langone,* Univ. do Estado do Rio de
Janeiro, Rio de Janeiro, Brazil; J.V. Bevilaqua,
Centro de Pesquisa da Petrobras, Brazil; A.G.
Cunha, M.M.R. Leal, D.M.G. Freire, Univ. Fed.
do Rio de Janeiro, Rio de Janeiro, Brazil
1A-47
Cloning and characterization of cold-active
xylanase enzymes
C.C. Lee,* R.E. Kibblewhite-Accinelli, K. Wagschal, G.H. Robertson, D.W.S. Wong, USDAARS-WRRC, Albany, CA
1A-48
Stability and performance of glucose isomerase for the production of high fructose
corn syrup
L.H. Lim,* B.A. Saville, University of Toronto,
Toronto, ON, Canada
Life cycle analysis of the AFEX process in consideration of recent process improvements
E. Newton,* H. Alizadeh, B. Dale, S. Kim, M.
Laser, L. Lynd, Michigan State University, E.
Lansing, MI
Removal of phenolic solubles from hydrothermally pretreated wheat straw
N.P.K. Nielsen*, H. Jorgensen, C. Felby, The
Royal Veterinary and Agricultural University,
Taastrup, Denmark
Improved analytical methods and quantifying mass deposition of urease immobilized
using layer-by-layer nano self-assembly
S. Reddy, J. Elmore, J. Cook, L.D. Snow, J.D.
Palmer,* Lousiana Tech University, Ruston, LA;
B.B. Elmore, Mississippi State University, Mississippi State, MS
Filter paper-based assays for low activity
enzyme preparations
T.S. Nordmark, M.H. Penner,* Oregon State
University, Corvallis, OR
1A-49
Enzymatic hydrolysis optimization of ethanol production by simultaneous saccharification and fermentation
M.P. Vasquez, J.N.C. da Silva, M.B. de Souza,
Jr., N. Pereira, Jr.,* Federal University do Rio de
Characterization of cellulase by non-crystalline cellulose and cello-oligosaccharides
S. Peri*, Y.Y. Lee, Auburn University, Auburn, AL
1A-50
Ethanolysis of corn oil using crude papain
E.B. Saad, N. Krieger, L.P. Ramos,* Federal
University of Parana, Curitiba, Brazil
1A-51
Chemical characterization of pulp components in unbleached softwood kraft fibers
that were recycled with the assistance of a
laccase/HBT system
T.A. da Silva, L.P. Ramos,* Federal Univ. of
Parana, Curitiba, Brazil; P. Mocchiutti, M.A. Zanuttini, Universidad Nacional del Litoral, Santa
Fe, Argentina
1A-52
1A-53
1A-54
16
Production and characterization of cellulases obtained from a sugarcane bagasse
cellulignin by Aspergillus niger and Trichoderma harzianum
A.M. de Castro, D.F.Carvalho, K.C. Nascimento,
R. Pedro, S.G.F. Leite, N. Pereira, Jr.,* Univ.
Federal do Rio de Janiero, Rio de Janeiro, Brazil
Elucidation of the factors responsible for celulose fragmentation activity of Trichoderma
reesei
A.A.N. Saqib,* P.J. Whitney, University of Surrey, Guildford, UK
The effect of feruloyl esterase on the enzymatic saccharification of pretreated corn stover
S.R. Decker, E. Knoshaug, W.S. Adney, M.J.
Selig,* NREL, Golden, CO
A tray bioreactor for hyper production of the
industrially significant xylanase by a mutant
Aspergillus niger NKUCn-3.40
N. Kapur, D. Dutt, R.P. Singh,* Indian Institute
of Technology, Roorkee, India
Sunday, April 30
1A-55
2-hydroxychromene-2-carboxylate isomerase: a Kappa class GST involved in naphthalene catabolism from Pseudomonas putida
L. Thompson,* R. Armstrong, Vanderbilt University, Nashville, TN; J. Ladner, G. Gilliland,
Center for Advanced Research in Biotechnology, Rockville, MD
1A-63
1B-06
1A-56
1A-57
1A-58
1A-59
Spontaneous formation of the polykaryons
of Trichoderma reesei by long-term treatment with colchicine
H. Toyama,* M. Yano, T. Hotta, N. Toyama,
Minamikyushu University, Miyazaki, Japan
Use of glucose oxidase in a membrane reactor for the gluconic acid production
L.C. Matins das Neves, M. Vitolo,* University of
Sao Paulo, Sao Paulo, Brazil
Production of high fructose syrup using immobilized invertase in a membrane reactor
E. Junko Tomotani, M. Vitolo,* University of Sao
Paulo, Sao Paulo, Brazil
Anion exchange resin as support for enzymes
M. Vitolo,* University of Sao Paulo, Sao Paulo,
Brazil; R.R. Ribeiro, Presbyterian Mackenzie
University, Sao Paulo, Brazil
1B-07
1B-08
1B-09
1A-60
1A-61
1A-62
Membrane bioreactor used for the conversion of sucrose in fructose and gluconic
acid
L.C. Martins das Neves, M. Vitolo,* University of
Sao Paulo, Sao Paulo, Brazil
Enzyme production by industrially relevant
fungi growing on feed co-product from dry
mill ethanol plants
E.A. Ximenes,* M.R. Ladisch, N. Mosier, Purdue University, West Lafayette, IN; B.S. Dien,
M.A. Cotta and X-L. Li, USDA/ARS, Peoria, IL
Changes in the enzymatic hydrolysis rate of
avicel cellulose with conversion
B. Yang,* D.M. Willies, Dartmouth College,
Hanover, NH; C.E. Wyman, Dartmouth College,
Hanover, NH/University of California, Riverside, CA
3A-07
3A-08
Assays and methods to engineer enzymes
for biomass conversion
M.M. Yang,* S.J. Robles, W.J. Coleman, KAIROS Scientific, Inc., San Diego, CA
Controlling cellulose production through
gene expression
J. Milne,* Stanford University, Stanford, CA; N.
Kitrov, Carnegie Institution, Stanford, CA; and
C. Somerville, Stanford University and Carnegie
Institution, Stanford, CA
Breeding, selection, testing and commercialization of high-yielding varieties of shrub
willow for bioenergy, biofuels and bioproducts
L.B. Smart,* K.D. Cameron, T.A. Volk, L.P.
Abrahamson, SUNY College of Environmental
Science and Forestry, Syracuse, NY
Enhanced bio-processing of corn stover
with modified cell wall composition
W. Vermerris,* J. Campos, B. Phillips, M.R.
Ladisch, N.S. Mosier, Purdue University, West
Lafayette, IN
Glycosylation modification of Trichoderma
reesei cellobiohydrolases I expressed in
Pichia pastoris
L. Wei, G. Wu, G. Zhuang,* Y. Qu, Shandong
University, Shandong Province, China
Liquid-liquid extraction process for separating acrylic acid produced from biomass
M.E.T. Alvarez,* A.B. Machado, A.P.A. Pinho,
E.B. Moraes, M.R. Wolf-Maciel, State University
of Campinas, Campinas, Brazil
Further characterization of the bio-burden in
United States aviation fuel: A comparison of
FTA paper, direct PCR and traditional culture
methods for obtaining genetic material from
aviation fuel microbes
S.K. Chelgren, E.M. Strobel, Air Force Research Laboratory, PRTG, Dayton, OH; L.M.
Balster,* M.D. Vangness, L.L. Bowen, University
of Dayton Research Institute, Dayton, OH
17
Sunday, April 30
3A-09
Xylitol production from wheat straw hemicellulose in a 15-L STR
L. Canilha,* W. Carvalho, J.B. Almeida de Silva,
College of Lorena, Lorena, Sao Paulo, Brazil
Brazil; F.P. de Franca,* E.F.C. Servulo, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil
3A-16
3A-10
3A-11
3A-12
3A-13
3A-14
3A-15
18
Stability of a bioreactor for the production of
ethanol in sequential batch operation from
molasses-stillage medium using a selected
yeast strain immobilized in calcium alginate
S.M.D.S. Carvalho,* S.A. Filho, Federal University of Amazon, Amazon, Brazil; L. Pantoja, R.N.
Maeda, National Research Institute of Amazon,
Amazon, Brazil; N. Pereira, Jr., Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Microbiological production of fatty acids:
Performances of a new synthesis path of
molecules to energetic or chemical use
J. Cescut,* L. Fillaudeua, S. Alfenore, C.
Bideaux, X. Cameleyre, N. Goret, S. Guillouet,
J. Lesage, C. Mollina-Jouve, J.L. Uribelarrea,
INSA, Toulouse, France
Aqueous-phase reforming of biomass-derived compounds to hydrogen and alkanes
R.D. Cortright, Virent Energy Systems, Inc.,
Madison, WI
Analysis of economics for food waste conversion to ethanol
R. Davis,* N. Mosier and M. Ladisch, Purdue
University, West Lafayette, IN; and J. Warner,
Defense Life Sciences Institute, McLean, VA
Optimization of parameters for lactic acid
fermentation by Lactobacillus delbrueckii
EQ-2
F.P. de Franca,* A.C. da Silva, F.J.S. Oliveira,
Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil; A.C.A. da Costa, Unversidade
do Estado do Rio de Janeiro, Rio de Janeiro,
Brazil
Enhancement of rhamnolipid production in
soybean residual oil by an isolated strain of
Pseudomonas aeruginosa
C.J.B. de Lima, R.B. Veira, V.L. Cardoso, Universidade Federal de Uberlandia, Uberlandia,
3A-17
3A-18
3A-19
3A-20
3A-21
3A-22
Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis
isolated strain
R.R. Fonseca, F.P. de Franca,* E.F.C. Servulo,
A.J.R. da Silva, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; V.L. Cardoso,
Universidade Federal de Uberlandia, Uberlandia, Brazil
A new procedure for the acrylic acid synthesis by fermentative process
B.H. Lunelli, R.M. Filho,* State University of
Campinas, Campinas, Brazil
Obtainment of ethanol/water pulps from
sugarcane straw and the pulps biobleaching
with xylanase by Bacillus pumilus
R.Y. Moriya, A.R. Goncalves,* FAENQUIL,
Lorena, Brazil
Biopulping of sugarcane straw and biobleaching using xylanases
L.R.M. Oliveira, M.B.W. Saad, A.R. Goncalves,*
FAENQUIL, Lorena, Brazil
Biological hydrogen production from wastewater via immobilized bacteria
B. Hu,* Z. Chi, S. Chen, Washington State University, Pullman, WA Nisin production utilizing skimmed milk aiming process cost reduction
A.F. Jozala,* M.S. de Andrade, L.J. de Arauz,
A. Pessoa, Jr., T.C.V. Penna, University of Sao
Paulo, San Paulo, Brazil; and O. Cholewa, Molecular Probes, Inc., Eugene, OR
Production of bacterial cellulose by Gluconacetobacter sp. RKY5 in a rotary biofilm
contactor
Y-J. Kim, J-N. Kim*, Y-J. Wee, H-W. Ryu, Chonnam National University, Gwangju, Korea
Sunday, April 30
3A-23
3A-24
L(+)-lactic acid production from commercial
starches and corn steep liquor by Enterococcus faecalis RKY1
Y-J. Wee, D-H. Park, H-W. Ryu, J-N. Kim,*
Chonnam National University, Gwangju, Korea;
J-S. Yun, BioHelix, Jeonnam, Korea
3A-29
Characterization of novel epigallocatechin
gallate glycosides using glucansucrase
from Leuconostoc mesenteroides B-1299CB
Y-H. Moon,* H-J. Chung, S-H. Nam, D. Kim,
Chonnam National University, Gwangju, Korea;
G.J. Kim, N-Y. Woo, J-B. Pyo, Korean Minjok
Leadership Academy, Gwangwon-do, Korea;
D.F. Day, Audubon Sugar Institute, LSU, Gabriel, LA
3A-30
WITHDRAWN
3A-31
Biopulping of sugarcane straw: Improvement of the organosolv process
M.B.W. Saad, A.R. Goncalves,* FAENQUIL,
Lorena, Brazil
3A-32
3A-25
Biosurfactants production by Bacillus atrophaeus ATCC9372 and Bacillus subtilis W
in TSB (tryptone soya broth) culture media
enriched with glucose, soy flour and casein
digested
L.C.M. das Neves,* K.O. da Silva, M.J. Kobayashi, T.C.V. Penna, University of Sao Paulo,
Sao Paulo, Brazil; O. Cholewa, Molecular
Probes, Inc., Eugene, OR
3B-08
3B-09
3A-26
Evaluation of the resistance parameters for
the recombinant green fluorescent protein
(GFPuv) exposed to sodium chloride solutions at different concentrations, pH values
and temperature conditions
T.C.V. Penna,* M. Ishii, J.S. Kunimura, University of Sao Paulo, Sao Paulo, Brazil; O. Cholewa,
Molecular Probes, Inc., Eugene, OR
3B-10
3A-27
3A-28
Utilization of green fluorescent protein as
biosensor of drugs stability in physiological
solutions
C.A. dos Santos,* P.G. Mazzola, T.C.V. Penna,*
University of Sao Paulo, Sao Paulo, Brazil
Mechanical properties of polyurethane
foams prepared from liquefied corn stover
T. Wang, Z. Mao, L. Liang, D. Li, Y. Yu, L.
Zhang, China Agricultural University, Beijing,
China; Y. Li, A. Shahbazi,* North Carolina A&T
State University, Greensboro, NC
3B-11
Genetic modification of a gluconic acid
producing Aspergillus niger AR-12 strain
through combined mutagenesis for enhanced metal tolerance
A. Sharma, O.V. Singh, R.P. Singh,* Indian
Institute of Technology Roorkee, Roorkee, India
Recovery of the enzyme xylanase in the
biobleaching of bagasse pulps
L.R.M. Oliveira, A.R. Goncalves,* FAENQUIL,
Lorena, Brazil
Microalgae for biofuels and biochemicals
production
D.E. Brune,* Clemson University, Clemson,
SC; G. Schwartz, M. Massingill, Kent Sea Tech
Corp., San Diego, CA; J. Benemann, Walnut
Creek, CA
Logistic aspects related to the production of
biofuels from lignocellulosic residues and
dedicated crops: The case of some Italian
districts
I. DeBari,* A. Enzo, M. Vincenzo, G. Braccio,
ENEA CR TRISAIA, Policoro, Italy
The logistical implications of optimizing
anaerobic digestion of manure and waste
E. Ghafoori,* P.C. Flynn, University of Alberta,
Edmonton, AB, Canada
The upgrade of agro-food industries byproducts and wastes in European mediterranean countries: The Portuguese current
situation and future prospects
L.C. Duarte, M.P. Esteves, F. Carvalheiro, F.M.
Girio,* INETI, Lisboa, Portugal
19
Sunday, April 30
3B-12
The real value of selective harvest
K.L. Kenney,* C.T. Wright, C.W. Radtke, J.R.
Hess, R.L. Hoskinson, P.A. Pryfogle, Idaho
National Laboratory, Idaho Falls, ID
3B-13
Effect of acid and alkaline pretreatment
severity on the enzymatic hydrolysis of corn
stover fractions
M.D. Montross,* K.B. Duguid, S.A. Shearer,
C.L. Crofcheck, University of Kentucky, Lexington, KY; R.L. Hoskinson, C.W. Radtke, Idaho
3B-19
The relative cost of biomass energy transport
E. Searcy,* P. Flynn, A. Kumar, University of
Alberta, Edmonton, AB, Canada
3B-20
Agronomic management of switchgrass:
Soil, seeding rate, fertility, cultivar and weed
control evaluations
D. Tyler,* B.C. English, R. Roberts, M. Walsh, L.
Steckel, University of Tennessee, Jackson, TN
3B-21
3B-14
New insights on the value of mechanical
preprocessing
C.T. Wright,* P.A. Pryfogle, J.R. Hess, N.A. Stevens, K.L. Kenney, C.W. Radtke, R.L. Hoskinson, Idaho National Laboratory, Idaho Falls, ID
Effect of acid and alkaline pretreatment severity on the enzymatic hydrolysis of wheat
stover fractions
M.D. Montross,* K.B. Duguid, S.A. Shearer,
C.L. Crofcheck, University of Kentucky, Lexington, KY; R.L. Hoskinson, C.W. Radtke, Idaho
4-08
3B-15
WITHDRAWN
4-09
3B-16
Application of imaging techniques for optimizing biomass fractionation processes
P.A. Pryfogle,* C.T. Wright, J.R. Hess, N.A. Stevens, K.L. Kenney, C.W. Radtke, R.L. Hoskinson, Idaho National Laboratory, Idaho Falls, ID
Xylose-fermenting Saccharomyces cerevisiae expressing a fungal xylose isomerase:
Further steps toward industrial implementation
E. Bellissimi,* J.P. van Dijken, J.T. Pronk, TU
Delft, Delft, The Netherlands; A.A. Winkler, J.P.
van Dijken, Bird Engineeering BV, Rotterdam,
The Netherlands; W.T.A.M. de Laat, Royal
Nedalco, Bergen op Zoom, The Netherlands
4-10
Withdrawn
4-11
Construction and evaluation of Clostridium
thermocellum 27405 partial-genome
oligonucleotide microarray
S.D. Brown,* S. Kale, Z. He, J.R. Mielenz, Oak
Ridge National Laboratory, Oak Ridge, TN; C.K.
McKeown, Xavier University of Louisiana, New
Orleans, LA
3B-17
3B-18
20
Quality changes during bunker-ensiled storage of corn stover for the biorefining industry
C.W. Radtke,* C.T. Wright, K.L. Kenney, P.A.
Pryfogle, R.L. Hoskinson, H.G. Silverman, D.B.
Blackwelder, N.A. Yancey, D.F. Bruhn, C.R.
Breckenridge, J.R. Hess, Idaho National Laboratory, Idaho Falls, ID; E. Bonner, W.E. Bond, L.T.
McNear, Livingstone College, Salisbury, NC; T.M.
Schechinger, Iron Horse Farms, Harlan, IA
Quality changes during dry storage of cereal
straw for the biorefining industry
Blackwelder, NA. Yancey, D.F. Bruhn, C.R.
Breckenridge, J.R. Hess, Idaho National Laboratory, Idaho Falls, ID; E. Bonner, W.E. Bond,
L.T. McNear, Livingstone College, Salisbury, NC
4-12
Effect of initial cell concentration on glucose
and xylose fermentation using Pichia stipitis
F.K. Agbogbo,* M. Torry-Smith, G. CowardKelly, K. Wenger, Novozymes, Franklinton, NC
Microbial modification of corn stover to
increase ethanol yield
N.K. Budhavaram,* A. Gidh, C.W. Williford, A.
Mikell, University of Mississippi, University, MS
Sunday, April 30
4-13
4-14
4-15
Bioethanol production from biomass hydrolyzates mediated by enzymatic isomerization
of xylose to xylulose
I. De Bari,* V. De Matteo, ENEA CR TRISAIA,
Italy
4-21
Investigation of latex micro-photobioreactor
for hydrogen production using non-growing
Rhodopseudomonas palustris
J.L. Gosse, M.C. Flickinger,* University of Minnesota, St. Paul, MN
4-22
Tanase production by solid state fermentation of cashew apple bagasse
T.H.S. Rodrigues, M.A.A. Dantas, L.R.B.
Goncalves,* Universidade Federal do Ceara,
Fortaleza, Brazil; G.A.S. Pinto, Embrapa Agroindustria Tropical, Fortaleza, Brazil
4-23
4-24
4-16
4-17
4-18
4-19
4-20
A novel microbial platform for the anaerobic
synthesis of oxidized chemicals: Anaerobic
production of acetic acid in E. coli
K. Smith, Y. Dharmadi, R. Gonzalez,* Rice University, Houston, TX
The fermentation inhibitor furfural causes cellular damage to Saccharomyces cerevisiae
S.W. Gorsich,* P.J. Slininger, USDA-ARSNCAUR, Peoria, IL; J.M. McCaffery, Johns
Hopkins University, Baltimore, MD
Comparison of multiple gene assembly
methods for metabolic engineering
C. Lu,* K. Mansoorabadi, University of Wisconsin, Madison, WI; T.W. Jeffries, University
of Wisconsin, Madison/USDA Forest Products
Laboratory, Madison, WI
Production of succinic acid by metabolically
engineered Escherichia coli
J.L. Buday, S. Lu,* M.A. Eiteman, J.R. Kastner,
E. Altman, University of Georgia, Athens, GA
Loop configuration in the chromosome
region containing [Fe]-hydrogenase gene of
Chlamydomonas reinhardtii
S.A. Markov,* DePauw University, Greencastle,
IN; E.R. Eivazova, B. Durr, Vanderbilt University, Nashville, TN
4-25
Withdrawn
4-26
5-hydroxymethyl furfural reduction in lignocelulose hydrolysates with Saccharomyces
cerevisiae strains and its connection to
tolerance
T. Modig,* A. Petersson, G. Liden, J.R.M. Almeida,
K. Karhumaa, B. Hahn-Hagerdal, M.F. GorwaGrauslund, Lund University, Lund, Sweden
Developing desulfurization biocatalysts for
removing organic sulfur from fossil fuels at
higher temperatures
Y. Bai, M. Sedlak, N.W.Y. Ho,* Purdue University, West Lafayette, IN
Improvement of e-caprolactone production
in recombinant Escherichia coli by optimization of substrate feeding and cofactor
regeneration
W-H. Lee,* K-J. Lee, M-D. Kim, J-H. Seo, Seoul
National University, Seoul, Korea
A new approach of pellet formation of a
filmamentous fungi-Rhizopus oryzae
W. Liao,* Y. Liu, S. Chen, Washington State
University, Pullman, WA
Study of pellet formation of filamentous
fungi Rhizopus oryzae using a multiple logistic regression model
Y. Liu,* W. Liao, S. Chen, Washington State
University, Pullman, WA
4-27
4-28
Modified expression contexts of phosphoglucose isomerase (PGI1) and glucose
6-phosphate dehydrogenase (ZWF1) genes
to increase xylitol production in recombinant Saccharomyces cerevisiae
Y-J. Oh,* D-H. Kwon, T-H. Lee, W-K. Min, M-D.
Kim, J-B. Park, J-H. Seo, Seoul National University, Seoul, Korea
Succinic acid production by genetically
modified Corynebacterium glutamicum under oxygen-deprivation
S. Okino,* A.A. Vertest, M. Inui, H. Yukawa,
RITE, Kyoto, Japan
21
Sunday, April 30
4-29
Microbial community of biofilm from a thermopohilic trickling biofilter used for continuous biohydrogen production
Y. Ahn, E-J. Kim, KAIST, Korea; Y-K. Oh, S.
Park,* Pusan National University, Korea; G.
Webster, A.J. Weightman, University of Cardiff,
Cardiff, UK
4-37
4-38
4-30
Ethanol and co-products from sugar beets
J.B. Doran Peterson,* K. Patel, T. Rogers,
University of Georgia, Athens, GA; A. Hotchkiss, M. Fishman, K. Hicks, USDA-ARS-ERRC,
Wyndmoor, PA; W. Widmer, USDA-ARS, Winter
Haven, FL
4-39
4-31
Optimization of gallic acid production by
Aspergillus niger CNPAT 001 in submerged
fermentation
A.C. de Freitas, G.A.S. Pinto,* EMBRAPA, Fortaleza, Brazil
4-40
4-32
PDU-scale fed-batch cultivation of yeast on
spruce hydrolysates
A. Rudolf,* M. Galbe, G. Liden, Lund University,
Lund, Sweden
4-41
4-33
4-34
4-35
4-36
22
Co-fermentation of glucose, xylose and cellobiose by genetically modified Saccharomyces yeast
M. Sedlak,* N.W.Y. Ho, Purdue University, West
Lafayette, IN
Effect of furfural and HMF on the cofermentation of glucose and xylose from pretreated
lignocellulosic biomass by recombinant
yeast
R. Warner,* M. Sedlak, N. Ho, N.S. Mosier,
Purdue University, West Lafayette, IN
Effect of loading rate on lipid production
from microalgae grown on dairy manure effluent
W. Mulbry, USDA-ARS, Beltsville, MD; Z. Wen,*
Virginia Polytechnic Institute and State University, Blacksburg, VA
Genetic engineering and characterisation
of a yeast strain fermenting both pentose
sugars, L-arabinose and D-xylose
B. Wiedemann,* M. Keller, E. Boles, Universitaet Frankfurt/Main, Frankfurt, Germany
Production of lactic acid by metabolically
engineered Escherichia coli
Y. Zhu,* M.A. Eiteman, E. Altman, University of
Georgia, Athens, GA
Production of lactic acid from aqueous ammonia-treated corn stover via simultaneous
saccharification and co-fermentation
Y. Zhu,* Y.Y. Lee, Auburn University, AL
Recombinant Saccharomyces cerevisiae as
source of glucose 6-phosphate dehydrogenase
L.C.M. das Neves, A. Pessoa, Jr., M. Vitolo,*
University of Sao Paulo, Sao Paulo, Brazil
Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate
dehydrogenase production
A.S.M. Miguel, M. Vitolo,* A. Pessoa, Jr., University of Sao Paulo, Sao Paulo, Brazil
Transposome-mediated directed evolution
for improving rhamnolipids production in
Pseudomonas aeruginosa
Q. Wang,* X. Fang, X. Liang, P.J. Shuler, Y.
Tang, W.A. Goddard III, California Institute of
Technology, Covina and Pasadena, CA
Join SIM!
www.simhq.org
Monday, May 1
Registration
Time: 7:15 am – 5:00 pm
Time: 7:15 am – 8:15 am
Speaker breakfast
Location: Memphis Room
Time: 7:15 am – 8:00 am
Tabletop exhibits
8:25 am 2-02: Recent process improvements for the
ammonia fiber explosion (AFEX) process
and resulting reductions in projected ethanol prices
B.E. Dale,* E. Newton, Michigan State University, E. Lansing, MI and L. Lynd, M. Laser,
Dartmouth College, Hanover, NH
8:50 am 2-03: Research challenges and opportunities
for cellulose conversion technology in a dry
mill pathway
M. Ladisch,* W. Tyner, N. Mosier, Purdue University, West Lafayette, IN; M. Cotta, B. Dien,
USDA-ARS-NCAUR, Peoria, IL; H. Blaschek,
University of Illinois, Champaign-Urbana, IL; B.
Dale, Michigan State University, E. Lansing, MI;
B. Shanks, Iowa State University, Ames, IA; G.
Petersen, DOE, Golden, CO
Time: 7:00 am - 10:30 am
9:15 am 2-04: Identification of quantitation of water
extractives in corn stover
S-F. Chen, R.A. Mowery, C.K. Chambliss,* G.P.
van Walsum, Baylor University, Waco, TX;
U.S. Dept of Energy Genomics: GTL Program
9:40 am Coffee break (Sponsored by the Southeastern
SunGrant Center)
Representatives will be available to discuss their company’s
products and/or services.
Session 2: Biomass fractionation
and hydrolysis
Chairs: A. Miranda, DOE OBP, Washington, DC and C.
Wyman, University of California Riverside, Riverside, CA
8:00 am 2-01: A novel concentrated phosphoric acid/
acetone lignocellulose fractionation featuring modest reaction conditions and reagent
recycling
Y-H.P. Zhang,* Virginia Polytechnic Institute and
State University, Blacksburg, VA; L.R. Lynd,
10:10 am 2-05: Comparative sugar recovery data from
application of leading pretreatment technologies to corn stover and poplar
C.E. Wyman,* Dartmouth College, Hanover, NH;
B.E. Dale, Michigan State University, East Lansing, MI; R.T. Elander, NREL, Golden, CO; N.
Gilkes, University of British Columbia, Vancouver, BC, Canada; M.T. Holtzapple, Texas A&M
University, College Station, TX; M.R. Ladisch,
Purdue University, West Lafayette, IN; Y.Y. Lee,
Auburn University, Auburn, AL; M. Moniruzzaman, Genencor International, Beloit, WI
10:35 am 2-06: Corn stover to ethanol - moving the
focus toward process integration
G. Coward-Kelly,* M. Torry-Smith, D. Milam,
Novozymes North America, Franklinton, NC
10:50 am 2-07: Design, installation and start up of
biomass fractionation plants
A. Richard,* M. Burke, J. Kangudie, SunOpta
BioProcess Group, Norval, ON, Canada
23
Monday, May 1
Lunch on own
Time: 12:00 noon – 1:30 pm
A buffet lunch is available for purchase in the hotel restaurant, as well as in the Capitol Ballroom for attendees.
Session 3A: New and developing industrial bioproducts
Chairs: M. Moniruzzaman, Genencor International, Beloit,
WI and C. Stevens, Ghent University, Gent, Belgium
1:30 pm 3A-01: BioBased chemicals from ethanolThe ethanol platform
R.W. Glass,* National Corn Growers Assn.,
Chesterfield, MO; R. Cascone, J. Plotkin, Nexant, White Plains, NY; N. Danielson, BioCognito, Collegeville, PA
2:00 pm 3A-02: Improving the value of corn proteins
via reforming with anaerobic bacteria
T. Eggeman,* D. Verser, S. Hunter, ZeaChem,
Inc., Lakewood, CO
2:30 pm 3A-03: DuPont introduces bio-based high
performance polyetherglycols
R.W. Miller,* H. Ng, H. Sunkara, E.I. DuPont,
Wilmington, DE
3:00 pm Coffee break (Sponsored by the Southeastern
SunGrant Center)
3:30 pm 3A-04: BioAmber-The first large scale commercial production of bio-based succinic
acid
K.A. Berglund,* BioAmber, Pomacle, France,
and Lulea Univ. of Technology, Lulea, Sweden;
P. Jacobson, Diversified Natural Products, Inc.,
New York, NY; Y. Le Henaff, Agro-Industrie
Rech. et Dev., Pomacle, France; U. Rova, Lulea
Univ. of Technology, Lulea, Sweden
24
4:00 pm 3A-05: Techno-economic analysis of biocatalytic processes for production of alkene
epoxides
A.P. Borole,* B.H. Davison, ORNL, Oak Ridge,
TN
4:30 pm 3A-06: Enzyme mediated production of
microcrystalline cellulose from agricultural
residues
F.A. Agblevor,* Virginia Polytech Inst. and State
Univ., Blacksburg, VA; M. Ibrahim, W.K. El-Zawawy, National Research Center, Cairo, Egypt
Session 3B: Feedstock supply and
logistics
Chairs: R.D. Perlack, ORNL, Golden, CO and J.R. Hess,
Idaho National Laboratory, Idaho Falls, ID
1:30 pm 3B-01: Biomass resource feedstock supply
for replacing 30% of U.S. gasoline consumption by 2030
R.D. Perlack,* ORNL, Golden, CO; J.R. Hess,
INL, Idaho Falls, ID
2:00 pm 3B-02: Biomass energy: Is there enough
land?
A.O. Converse, Dartmouth College, Hanover,
NH
2:30 pm 3B-03: Switchgrass for biomass: Commercial-scale production costs in the Northern
Great Plains
R.K. Perrin,* University of Nebraska, Lincoln,
NE; K.P. Vogel, R. Mitchell, M.R. Schmer,
USDA-ARS, Lincoln, NE
3:00 pm Coffee break (Sponsored by the Southeastern
SunGrant Center)
3:30 pm 3B-04: Switchgrass for biomass: Farm scale
production practices affect feedstock costs
and quantities in the Northern Great Plains
K.P. Vogel,* R. Mitchell, M.R. Schmer, USDAARS, Lincoln, NE; R.K. Perrin, University of
Nebraska, Lincoln, NE
Monday, May 1
4:00 pm 3B-05: Comparison of single-pass harvesting of corn grain and stover with conventional harvesting schemes
K.J. Shinners,* G.C. Boettcher, J.T. Munk, M.F.
Digman, G.S. Adsit, University of Wisconsin,
Madison, WI; R.E. Muck, P.J. Wiemer, USDAARS, Madison, WI
Poster Session II
Sections: 2, 5, 6
Chair: M. Downing, ORNL, Oak Ridge, TN
Time: 6:00 - 9:00 pm
Sponsored by the USDA Agricultural Research Service
4:30 pm 3B-06: Preparation and characterisation of
pyrolytic biomass slurries: A new feedstock
for gasification
K. Raffelt,* E. Henrich, C. Kornmayer, C. Renck,
R. Stahl, J. Steinhardt, F. Weirich, Institut fur
Technische Chemie, Eggenstein-Leopoldshafen, Germany
5:00 pm 3B-07: Preprocessing effects on lignocellulosic feedstock cost and quality for ethanol
conversion
Blackwelder, N.A. Yancey, D.F. Bruhn, C.R.
Breckenridge, J.R. Hess, Idaho National Laboratory, Idaho Falls, ID
2-08
2-09
2-10
Tabletop exhibits
Time: 6:00 pm - 9:00 pm
2-11
2-12
2-13
Investigation of the impact of enzyme characteristics on corn stover preservation and
pretreatment
H. Ren,* T.L. Richard, Pennsylvania State
University, University Park, PA; K.J. Moore, P.
Patrick, Iowa State University, Ames, IA
The effect of flow rate, temperature and acid
concentration on protein extraction from
alfalfa in a flowthrough system
C.E. Wyman,* T. Abifarin, Dartmouth College,
Hanover, NH and University of California, Riverside, CA
Optimizing AFEX pretreatment and enzymatic hydrolysis of cassava
N.S. Achampong,* B. Venkatesh. B.E. Dale,
Michigan State University, East Lansing, MI
Fractionation of Cynara cardunculus (cardoon) biomass by dilute-acid pretreatment
M. Ballesteros,* M.J. Negro, P. Manzanares, I.
Ballesteros, F. Saez, J.M. Oliva, CIEMAT, Madrid, Spain
Extracting proteins from switchgrass within
an integrated biorefinery
B. Bals,* B. Dale,* V. Balan, Michigan State University, East Lansing, MI
Heat-extraction of corn fiber hemicellulose
Z. Benko,* Z. Szengyel, M. Gaspar, K. Reczey,
Budapest University of Technology and Economics, Budapest, Hungary; A. Andersson, H.
Stalbrand, Lund University, Lund, Sweden
25
Monday, May 1
2-14
2-15
2-16
2-17
2-18
Fortifying spent sulphite pulping liquor
using levoglucosan derived derived from
pyrolysis oil to improve ethanol yields
N.M. Bennett,* S.S. Helle, S.J.B. Duff, University of British Columbia, Vancouver, BC, Canada
Evaluation of British Columbian beetle-killed
hybrid spruce for bioethanol production
A. Berlin,* N. Gilkes, S. Alamuti, J. Saddler,
University of British Columbua, Vancouver, BC,
Canada; C. Munoz, J. Baeza, University of Concepcion, Concepcion, Chile
2-22
2-23
Optimization of SO2-catalysed steam explosion for hybrid poplar
R. Bura,* A. Berlin, N. Gilkes, J. Saddler,
University of British Columbia, Vancouver, BC,
Canada
2-24
Maximizing overall sugar recovery of hydrolysable and fermentable sugars during SO2catalysed steam explosion of corn stover
Canada
2-25
Robustness of SO2-catalysed steam explosion pretreatment process for bioconversion of agricultural, hardwood and softwood
residues
Canada
2-26
2-19
WITHDRAWN 2-20
Analysis of modes of detoxification of dilute
acid pretreatment hydrolysate by ion exchange and overliming treatments
K. O’Bric, S-F. Chen,* C.K. Chambliss, G.P. van
Walsum, Baylor University, Waco, TX
2-27
2-21
26
The development of a microplate technique
for screening enzyme mixtures and synergistic additives for hydrolysis of AFEX
treated lignocellulosic biomass
S.P.S. Chundawat,* B. Venkatesh, B.E. Dale,
Solid state fermentation of agricultural
wastes for cellulases and hemicellulases
production
R.S.R. Leite, E. da Silva Martis, E. Gomes, R.
da Silva*, Universidade Estadual Paulista, Sao
Jose do Rio Preto, Brazil
Development and pilot scale performance
trials of a steep delignification process
M.C. Dale,* B. Billings, Bio-Process Innovation, W. Lafayette, IN; D. Musgrove, Universal
Entech, Phoenix, AZ
The effect of particle size on hydrolysis
reaction rates and rheological properties in
cellulosic slurries
R.K. Dasari,* E. Berson, University of Louisville,
Louisville, KY
Oxidative fractionation of sugarcane bagasse using a combination of hypochlorite
and peroxide
Y-J. Lee, Louisiana State University, Baton
Rouge, LA; C-H. Chung, D.F. Day,* Audubon
Sugar Institute, Baton Rouge, LA
Enzyme hydrolysis and phenols recovery
post alkaline and organosolv treatment of
sugarcane bagasse
C-H. Chung, D.F. Day, G.A. DeQueiroz,* Louisiana State University, St. Gabriel, LA
Effect of dissolved carbon dioxide on accumulation of organic acids in liquid hot water
pretreated biomass hydrolysates
G.P. van Walsum, M. Garcia-Gill,* S-F. Chen,
C.K. Chambliss, Baylor University, Waco, TX
2-28
Enzymatic hydrolysis of tissue paper
O. Garcia-Kirchner,* A.S. Suazo, G.M. Segura,
UPIBI-IPN, Mexico DF, Mexico
2-29
Separation of glucose and pentose sugars
by selective enzyme hydrolysis of AFEXtreated corn fiber
R.J. Hanchar,* F. Temouri, C.D. Nielson, D. McCalla, M.D. Stowers, MBI International, Lansing,
MI
Monday, May 1
2-30
2-31
Pilot scale measurement of viscosity for a
biomass slurry composed of 15-20% corn
fiber in light stillage
R. Hendrickson,* Y. Kim, Y. Lu, N. Mosier, M.
Ladisch, Purdue University, West Lafayette IN;
R. Dreschel, G. Welch, Aventine Renewable
Energy Company, Pekin, IL
2-37
Investigating factors affecting enzymatic hydrolysis of dilute acid pretreated corn stover
slurries
D.B. Hodge,* D.J. Schell, J.D. McMillan, NREL,
Golden, CO
2-38
2-39
2-32
2-33
2-34
2-35
2-36
The potential in bioethanol production from
fiber sludges in pulp mill-based biorefineries
A. Sjode, Karlstad Univ., Karlstad, Sweden/
STFI,-Packforst, Stockholm, Sweden; B. Alriksson, L.J. Jonsson,* Karlstad Univ., Karlstad,
Sweden; N-O. Nilvebrant, STFI-Packforsk,
Stockholm, Sweden
Ethanolic fermentation and enzymatic convertibility of dilute sulfuric acid-pretreated
agricultural and agro-industrial residues
C. Martin, Karlstad Univ., Karlstad, Sweden/
Univ. of Matanzas, Matanzas, Cuba; B. Alriksson, Karlstad Univ., Karlstad, Sweden; A. Sjode,
Karlstad Univ/Swedish Pulp and Paper Res.
Inst., Stockholm, Sweden; L.J. Jonsson,* Karlstad University, Karlstad, Sweden
Optimization of dilute-acid prehydrolysis of
rice straw using small-scale bomb reactors
J.S. Park, H-j. Lee, K.H. Kim*, Korea University,
Seoul, Korea
Cellobiose production using b-glucosidase
inhibition
M. Kim*, Louisiana State University, Baton
Rouge, LA; C-H. Chung, D.F. Day, Audubon
Sugar Institute, St. Gabriel, LA
The impact of dilute sulfuric acid on the
selectivity of xylooligomer depolymerization
to monomers
R. Kumar,* Dartmouth College, Hanover, NH and
C.E. Wyman, University of California, Riverside, CA
2-40
2-41
2-42
2-43
2-44
Studying effects of dilute acid treatment on
components of dairy manure
W. Liao,* Y. Liu, Z. Wen, S. Chen, Washington
State University, Pullman, WA
Simultaneous saccharification and fermentation of steam-pretreated barley straw at
low enzyme and yeast concentrations
M. Linde,* M. Galbe, G. Zacchi, Lund University, Lund, Sweden
Enrichment of gamma-linolenic acid from
borage oil via lipase-catalyzed hydrolysis
L.V. Fregolente, P. Bogalhos, L. Fregolente,
C.B. Batistella, M.R.W. Maciel,* State University
of Campinas, Campinas-SP, Brazil
Use of different enzymatic mixtures in hydrolysis from barley straw
M.P. Garcia-Aparicio, M. Ballesteros, P. Manzanares,* I. Ballesteros, A. Gonzales, M.J.
Negro, CIEMAT, Madrid, Spain
Improving hydrolysis of poorly pretreated biomass by rough mixing scaled to the laboratory
K.C. McFarland,* J. Cherry, Novozymes, Inc., Davis, CA
Application of statistical experimental
design for optimization of the conditions
for eucalyptus hemicellulosic hydrolysate
detoxification
G.B.M. de Carvalho, S.I. Mussatto,* J.B. de Almeida e Silva, Rodovia Itajuba-Lorena, Lorena,
Brazil
Phenolic acids release by alkaline hydrolysis of brewer’s spent grain
S.I. Mussatto,* I.C. Roberto, Rodovia ItajubaLorena, Lorena, Brazil
High throughput production of pretreatment
residues for use in advanced characterization methods to understand biomass recalcitrance
N. Nagle,* F. Michel, Jr., N. Weiss, M. Davis, R.
Elander, NREL, Golden, CO
27
Monday, May 1
2-45
Bioconversion of hybrid poplar to bioethanol using organosolv ethanol pretreatment:
Effect of processing variables on substrate
features and enzymatic digestibility
X. Pan,* N. Gilkes, D. Xie, J.N. Saddler, University of British Columbia, Vancouver, BC,
Canada
2-53
2-54
2-46
2-47
2-48
2-49
2-50
The effect of reaction conditions upon lipase
catalysed hydrolysis of canola and soybean
oil
R.C.Pinheiro,* F.F. de Moraes, G.M. Zanin, Universidade Estadual de Maringa, Maringa, Brazil;
C.M.F. Soares, H.F. de Castro, Lorena, Brazil
Advanced confocal imaging of corn fiber
using starch- and cellulose-specific fluorescent probes
S.E. Porter,* Q. Xu, S-. Ding, M.E. Himmel,*
NREL, Golden, CO
Liquid hot water pretreatment of olive tree
pruning
C. Cara, I. Romero,* E. Castro, University of
Jaen, Jaen, Spain
2-55
2-56
2-52
28
Production of cellulolytic and xylanolytic
enzymes by Aspergillus niger H2 grown on
Pennisetum amcrieanum x Pennisetum purpureum in solid state fermentation
D. Su, J. Sun,* China Agricultural University,
Beijing, China; B. Yang, Dartmouth College, Hanover, NH; D. Su, Henan University of Technology, Zhengzhou, China
Effect of xylan and lignin removal by batch
pretreatment on the enzymatical digestibility
of corn leaf and corn stalk
S. Donghai, J. Sun,* China Agriculture University, Beijing, China; B. Yang, Dartmouth College,
Hanover, NH
Fraction insoluble solids (FIS): Procedure
and effect on high solids saccharification
yield calculations
D.W. Templeton,* R.O. Ruiz, M.P. Tucker,
B.R.Hames, NREL, Golden, CO
Enzymatic hydrolysis of pretreated olive tree
pruning at different biomass concentrations
C. Cara, M. Moya, E. Ruiz,* University of Jaen,
Jaen, Spain
2-57
Withdrawn
Pre-treatment of sugarcane leaves and
bagasse pith with lime-impregnation and
steam explosion for conversion to fermentable sugars
M. Saska,* M. Gray, Louisiana State University,
St. Gabriel, LA
2-58
Trials on the Danish pilot-scale pre-treatment plant (IBUS): Identification of inhibitors, detoxification and SSF
M.H. Thomsen,* A. Thygesen, A.B. Thomsen,
Risoe National Laboratory, Roskilde, Denmark
2-59
2-51
Technical-economical analysis for a new concentrated acid hydrolysis process of sugar
cane bagasse for bio-ethanol production
B.F. Sarrouh, S.S. da Silva,* R.D.F. Branco,
D.T. Dos Santos, FAENQUIL, Lorena, Brazil;
and J. Jover, Santa Clara, Cuba
Techno-economic evaluation of a bioethanol
process for three different lignocellulosic
materials
P. Sassner,* M. Galbe, G. Zacchi, Lund University, Lund, Sweden
Wet oxidation of softwood and hardwood
resulting in convertible cellulose and hemicellulose
W. Varga, M.H. Thomsen,* A.B. Thomsen, Risoe National Laboratory, Roskilde, Denmark; R.
Reczey, Budapest University of Technology and
Economics, Budapest, Hungary
Improved total solids measurement of acid
pretreated biomass by Karl Fischer titration
C. Scarlata,* NREL, Golden, CO
Monday, May 1
2-60
2-61
2-62
2-63
2-64
Quantitation of organic degradation products in response to severity of dilute acid
pretreatment of corn stover
S-F. Chen, R.A. Mowery, C.K. Chambliss, G.P.
van Walsum,* Baylor University, Waco, TX
5-10
Enhancement of cellulose saccharification
kinetics using an ionic liquid pretreatment
strategy
A.P. Dadi, C.A. Schall, S. Varanasi,* University
of Toledo, Toledo, OH
5-11
Process options in AFEX pretreatment and
fermentation of rice straw
B. Venkatesh,* C. Shishir, B.E. Dale, Michigan
State University, East Lansing, MI
5-12
Characterization of changes in viscosity and
undissolved solids content of corn stover
slurries during enzymatic hydrolysis
E.D. Wyatt*, R.E. Berson, University of Louisville, Louisville, KY
Evaluation of different biomass materials as
feedstock for fermentable sugar production
R. Zhang,* Y. Zheng, Z. Pan, J. Labavitch, B.M.
Jenkins, University of California, Davis, CA; D.
Wang, Kansas State University, Manhattan, KS
5-13
5-14
5-07
Pretreatment and bioconversion of chipped
yard waste to carboxylic acids using a percolation column apparatus
R. Adapala,* G.P. van Walsum, Baylor University, Waco, TX
5-15
5-08
5-09
Influence of temperature on glucose and
fructose adsorption process by activated
carbon
R.M.R.G. Almeida,* P.S. Oliveira, G.T.S. Gonzaga, M.M.S. Cabral, Federal University of
Alagoas, Maceio, Brazil
Continuous esterification of bio-based organic acids: Formation of triethyl citrate
N. Asthana,* A. Kolah, D. Vu, C. Lira, D. Miller,
5-16
5-17
Performance of a thermophilic anaerobic digester at different feed loading frequencies
J. Bombardiere,* M. Domaschko, M. Chatfield,
West Virginia State University, Institute, WV; T.
Espinosa-Solares, Chapingo, Mexico
A comparison of cellulases activity restoration using starch or dextrin following foam
fractionation
E. Booth,* I. Snyder,* V. Burapatana, R.D. Tanner, Vanderbilt University, Nashville, TN
Removal of hydrogen sulfide by immobilized
Thiobacilli on polyvinylpyridine carrier
J-M. Cha,* I-J. Song, Bio & Environmental Tech.
Co. Ltd., Jeonnam, Korea; G-T. Jeong, K-M.
Lee, W-T. Lee, D-H. Park, Chonnam National
University, Gwang-ju, Korea; G-Y. Lee, Dong-A
College, Jeonnam, Korea
Study on the production of biodiesel by
magnetic cell catalyst based on lipase-producing B. subtilis
M. Ying, G. Chen,* Tianjin University, Tianjin, China
Production of omega-3 polyunsaturated
fatty acids from cull potato with the algae
culture process
Z. Chi,* S. Chen, Washington State University,
Pullman, WA
Hybrid neural model of an industrial ethanol
fermentation process considering the effect
of temperature
I.C.C. Mantovanelli, A.C. da Costa,* R.M. Filho,
UNICAMP, Campinas, Brazil
A software sensor for the ethanol fermentation process
E.A.C. Rivera, R.R. Andrade, D.P. Atala, F.M.
Filho, R.M. Filho, A.C. da Costa,* UNICAMP,
Campinas, Brazil
Process intensification for ethyl acetate
production from bioethanol
A.F. Custodio,* M.R. Wolf, R.M. Filho, State
University of Campinas, Campinas, Brazil
29
Monday, May 1
5-18
Characterization of thermostructural damages observed in a seaweed used for biosorption of cadmium: Effects on the kinetics and
uptake
A.C.A. da Costa,* A.S. Luna, R. Pafume, Universidade do Estado do Rio de Janeiro, Rio de
Janeiro, Brazil
5-25
5-26
5-19
Effect of physiochemical properties of catalysts on the conversion of glycerol to valueadded liquid chemicals
K. Pathak, D. Ferdous, A.K. Dalai,* N.N. Bakhshi, University of Saskatchewan, Saskatoon,
SK, Canada
5-20
Withdrawn
5-21
Bio-PSA: A tool for aid in the bioprocesses
design and operation
V.L.R. de Gouveia,* R.M. Filho, State University
5-23
5-24
30
Fermentation inhibitors from pretreated lignocellulosic materials: Problems and solutions
W. de Laat,* F. Aboka, S.F. Maltha, Royal
Nedalco B.V.; Z. Kadar, K. Reczey, Budapest
University of Technology and Economics, Budapest, Hungary
Modeling complex experimental behavior in
the production of ethanol by Zymomonas
mobilis: Comparison between phenomenological and hybrid-neural approaches
M.B. de Souza, Jr.,* A.C.R. Camelo, T.L.M. Alves,
Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil; I.O. Pinheiro, C.E. Lopes, Federal
University of Pernambuco, Pernambuco, Brazil
Comparison of methanogenic and acidogenic digestion of cattle manure for production of fuels and reduction of phosphorus
bioavailability
E. Doyle,* G.P. van Walsum, Baylor University,
Waco, TX
A non-linear control strategy of a fixed bed
catalytic reactor for bio-ethanol oxidation
R.M. Filho,* E.R. Duarte, State University of
Campinas, Campinas, Brazil; L. Enger, Regional University of Blumenau, Blumenau, Brazil
5-27
New silicone based antifoams for fermentation
A. Etoc,* J-P. Lecomte, Dow Corning, sa.,
Seneffe, Belgium; F. Delvigne, P. Thonart, Unversitaire des Sciences Agronomiques, Gembloux, Belgium
5-28
A structure kinetic model for bioethanol
production by fermentation
B.H. Lunelli, E.A.C. Rivera, D.P. Stremel, E. Coselli, V. de Toledo, R.M. Filho,* State University
5-22
Control of an extractive fermentation process to bio-ethanol production
R.M. Filho,* D.I.P Atala, E.R. Duarte, State
University of Campinas, Campinas, Brazil; L.
Ender, Regional University of Blumenau, Blumenau, Brazil
5-29
5-30
5-31
On-site lime pretreatment and acidogenic
digestion of dairy manure employing counter-current fermentation with minimal solids
handling
M. Flatt,* G.P. van Walsum, Baylor University,
Waco, TX
The effect of age and inoculum concentration on the production of biosurfactants and
protein-related virulence factors by Pseudomonas aeurginosa PA1
L.F.D. Tavares, V.F. Soares, D.M.G. Freire,*
Janeiro, Brazil
Investigation of new inoculum strategies for
solid-state fermentation
M.E.L. Gutarra, D.M.G. Freire,* L.R. Castilho,
Janeiro, Brazil
Monday, May 1
5-32
5-33
Separation of solid residues from liquids
produced during ethanol production from
lignocellulosic materials
M. Galbe,* C.F. Roslander, Lund University,
Lund, Sweden
5-40
Continuous production of ethanol from
sugar cane molasses using yeast immobilized in pectin gel
R.L.C. Giordano,* R.C. Giordano, Universidade
Federal de Sao Carlos, Sao Carlos, Brazil; W.S.
Netto, Universidade Federal de Santa Catarina,
Santa Catarina, Brazil
5-41
5-42
5-34
5-35
5-36
5-37
Development of inorganic membranes for
improved efficiency of ethanol extraction
R.J. Higgins,* R.A. Foti, CeraMem Corp.,
Waltham, MA
Improved ethanol-water separation using
fatty acids
T.M. Boudreau. G.A. Hill,* University of Saskatchewan, Saskatoon, SK, Canada
Purification of green fluorescent protein
(GFPuv) from E. coli cell lysate
M. Ishii,* M. Minaguti, P.G. Mazzola, T.C.V.
Penna, University of Sao Paulo, Brazil; O. Cholewa, Molecular Probes, Inc., Eugene, OR
The effects of engineering design on heterogeneous biocatalysis in microchannels
F. Jones,* J. Hiestand, University of Tennessee,
Chattanooga, TN; R. Bailey, Loyola College in
Maryland, Baltimore, MD
5-43
5-44
5-45
5-38
A study on characteristics of reverse micellar systems by measuring viscosity and
conductivity
C-H. Kang,* J-S. Shin, Chonnam National University, Kwangju, Korea
5-46
5-39
Bioconversion of barley hull into ethanol using
a soaking in aqueous ammonia pretreatment
T.H. Kim,* F. Taylor, K.B. Hicks, USDA-ARSERRC, Wyndmoor, PA
Continuous simultaneous saccharification
and fermentation of cellulose to ethanol
C. Liu,* J. Bardsley, Dartmouth College, Hanover, NH; C.E. Wyman, University of California, Riverside, CA
Value-added products from condensed distillers solubles
C. Liu,* University of North Dakota, Grand
Forks, ND; B. Hu, S. Chen, Washington State
University, Pullman, WA; R.W. Glass, National
Corn Growers Assn., Chesterfield, MO
Simulation of the recovery of acrylic acid
from sugar cane bagasse using chromatographic adsorption process
A.P.A Pinho, M.E.T. Alvarez, A.B. Machado,
M.R. Wolf Maciel, State University of Campinas,
Campinas, Brazil; A.P. Scheer, University of
Parana, Curitiba, Brazil
Optimization of tocopherol concentration
process from SODD using response surface
methodology
V.M. Ito, C.B. Batistella, M.R. Wolf Maciel,*
State University of Campinas, Campinas, Brazil
Effects of solids concentration on simultaneous saccarification and fermentation
of steam exploded wheat straw for ethanol
production
J. Miguel Oliva, I. Ballesteros, M. J. Nebro, P.
Manzanares,* F. Saez, M. Ballesteros, CIEMAT,
Madrid, Spain
Green fluorescent protein extraction from
E. coli cell lysate using two-phase aqueous
micellar systems
P.G. Mazzola,* A. Pessoa, Jr., T.C.V. Penna,
University of Sao Paolo, Sao Paolo, Brazil
Influence of pH in the production of biopolymer of Sphingomonas capsulata ATCC
14666 and rheological characterization
L.T. Vanzo, A. Babicz; L. Rigo, F.F. Padilha,*
URI, Erechim, Brazil; A.R.P. Scamparini, UICAMP, Campilnas, Brazil
31
Monday, May 1
5-47
5-48
Production and characterization of the
xanthan gum produced from Xanthomonas
campestris pv magiferaendicae 1230 using
conventional and industrial media
I. Rottava, E. Mayeski, F. Rauber, F.P Padilha,*
URI, Brechim, Brazil; Y. Rosato, A.R.P. Scamparini, UNICAMP, Campinas, Brazil
Improved sampling for biotechnological
systems
C.J. Pedersen,* K.H. Esbensen, Aalborg Universitet Esbjerg, Esbjerg, Denmark
5-54
5-55
5-56
5-49
Performance of continuous detoxification of
dilute-acid hydrolyzates by Ca(OH)2
R. Purwadi, University of Boras, Boras,
Sweden;Chalmers Univ. of Technology, Goteborg, Sweden; M.J. Taherzadeh, University of
Boras, Boras, Sweden
5-57
5-50
5-51
5-52
Semi-continuous production of lactic acid
from cheese whey using integrated membrane reactor
Y. Li, A. Shabazi,* M.M. Mims, North Carolina
A&T State University, Greensboro, NC
Using cheese whey to produce penicillin G
acylase by Bacillus megaterium ATCC 14945
V.R. Souza,* R.L.C. Giordano, Federal University de Sao Carlos, Sao Carlos, SP, Brazil
Microbial community changes in a mixed microbial consortia producing PHA from waste
carbon sources
E.R. Coats, University of Idaho, Moscow, ID;
W.A. Smith, D.N. Thompson,* Idaho National
Laboratory, Idaho Falls, ID; F.J. Loge, University
of California, Davis, CA; M.P. Wolcott, Washington State University, Pullman, WA
5-58
6-06
6-07
5-53
32
Xanthan gum production by Xanthomonas
campestris pv manihotis 1182 using cheese
whey
M.F. Silva, R.G. Fornari, F.F. Padilha, H.
Treichel,* Univ. Regional Integrada do Alto
Uruguai e das Missoes, Erechim, Brazil; A.P.
Scamparini, UNICAMP, Campinas, Brazil
6-08
The effect of temperature on ethanol production from concentrated solka floc in a
three-liter bench scale bioreactor
B-H. Um,* T.R. Hanley, Auburn University, Auburn, AL
Effects of saccharification and particle size
on the rheology of biomass slurries at high
solids concentrations
S. Viamajala,* D.J. Schell, J.D. McMillan, R.T.
Elander, NREL, Golden, CO
Separating a mixture of egg yolk from egg
white using foam fractionation
T.M. Ward,* R.D. Tanner, Vanderbilt University,
Nashville, TN
Conditioning and glucose/xylose cofermentation of pretreated lignocellulosic biomass
R. Warner,* M. Sedlak, N. Ho, N.S. Mosier,
Purdue University, West Lafayette, IN
Molecular imprinting in silica matrices in
presence or absence of ß-cyclodextrin
F.F. de Moraes, G.M. Zanin,* Universidade
Estadual de Maringa, Maringa, Brazil; F.M.F.
Soares, H.F. de Castro, Lorena, Brazil
An environmentally friendly biorefinery for
the production of fuel-grade ethanol and
value-added co-products from the Uruguayan rice industry
A. Berlin,* V. Meximenko, N. Gilkes, J. Keating,
W. Mabee, R. Chedgy, A. Alvarez, P. Acosta, R.
Horta, R. Ortiz, J. Saddler, University of British
Columbia, Vancouver, BC Canada
Potential for integrated bioethanol and biogas
production from high dry matter olive pulp
T.I. Georgieva,* B.K. Ahring, Technical University of Denmark, Lyngby, Denmark
Testing performance of an integrated corn
stover to ethanol process
E.W. Jennings,* D.J. Schell, NREL, Golden, CO
Monday, May 1
6-09
Impact of recycle water on fermentation of a
dilute acid pretreated corn stover hydrolysate
A. Mohagheghi,* D.J. Schell, NREL, Golden, CO
6-12
6-10
6-11
Plant design and economical evaluation of
an ethanol production process from sugarcane bagasse: A Brazilian conception
L.A.F.S. Schlittler,* C.A.G. Perlingeiro, University of Rio de Janeiro, Rio de Janeiro, Brazil
Physical and chemical characteristics of
liquids from microwave pyrolysis of corn
stover
F. Yu,* P. Chen, University of Minnesota, St.
Paul, MN; X. Lin, Y. Liu, R. Ruan, University of
Minnesota, St. Paul, MN; Nanchang University,
Nanchang, China
Microwave pyrolysis of corn stover
F. Yu,* P. Chen, University of Minnesota, St.
Paul, MN; A.I.M. Tunheim, Univ. of Minnesota,
St. Paul, MN/The Norwegian Univ. of Life
Sciences, Norway; Y. Lin, Y. Liu, Nanchang
University, Nanchang, China; R. Ruan, Univ. of
Minnesota, St. Paul, MN/Nanchang University,
Nanchang, China
33
Tuesday, May 2
Registration
Session 4: Microbial catalysis and
metabolic engineering
Time: 7:15 am – 12:00 noon
Time: 7:15 am – 8:15 am
Speaker breakfast
Time: 7:15 am – 8:00 am
Chairs: S. Bower, Tate & Lyle, Decatur, IL and M. Eiteman,
University of Georgia, Athens, GA
8:00 am 4-01: Construction of cellulolytic Saccharomyces cerevisiae strains for consolidated
bioprocessing
R. Den Haan,* R. van Rooyen, S.H. Rose, W.H.
Van Zyl, University of Stellenbosch, Stellenbosch, South Africa; J.E. McBride, L.R. Lynd,
8:30 am 4-02: Development of Saccharomyces cerevisiae strains with increased HMF reduction
capacity
J.R. Moreira de Almeida,* K. Karhumaa, B.
Hahn-Hagerdal, M.F. Gorwa-Grauslund, A.
Petersson, T. Modig, G. Liden, Lund University,
Lund, Sweden
Tabletop exhibits
Time: 7:00 am - 12:00 noon
9:00 am 4-03: The growth-independence bioprocess
for ethanol production using Corynebacterium glutamicum
M. Inui, A.A. Vertes, S. Okino, H. Yukawa,*
RITE, Kyoto, Japan; T. Watanabe, Kyoto University, Kyoto, Japan
9:30 am Coffee break
Poster Tear down
Time: 7:00 am - 12:00 noon
Posters not removed during this time will be discarded.
10:00 am 4-04: Metabolic engineering of the xylose
utilizing thermophile Thermoanaerobacterium saccharolyticum JW/SL-YS485 for
ethanol production
A.J. Shaw, M. Tyurin, K. Podkaminer, L.R.
Lynd,* Dartmouth College, Hanover, NH
10:30 am 4-05: The engineering of the solvent-tolerant
Pseudomonas putida S12 for the production
of phenol and other aromatic compounds
J.A.M. de Bont,* N.J.P Wierckx, K. Nijkamp,
J. Wery, TNO Quality of Life, Apeldoorn, The
Netherlands
34
Tuesday, May 2
11:00 am 4-06: Development of succinic acid producing Zymomonas mobilis strain
J.Y. Kim, J-H. Kim, H. Chong,* Macrogen,
Seoul, South Korea
11:30 am 4-07: Production of hydrogen and heterologous hydrogenase in metabolically engineered Escherichia coli strains
J. Mathews*, Q. Li, R. Boyer, G. Wang, University of Hawaii, Honolulu, HI
Organizing committee lunch
Time: 12:15 pm – 1:45 pm
Free afternoon
The hotel is offering to take attendees downtown or to
Orpy Mills in their complimentary shuttle buses. Buses will
be leaving every half on the hour. Space is limited, your
patience is appreciated. Details available at the registration desk.
Special topic A: Life cycle
analysis/sustainability
Chairs: B. Dale, Michigan State University, E. Lansing, MI
and M. Wang, Argonne National Laboratory, Argonne, IL
7:00 pm STA-01: GIS-based fuelshed analysis: Net
energy yields and economics of cellulosic
and grain biomass production
M.P. Russelle,* USDA-ARS, St. Paul, MN; A.S.
Birr, Minnesota Dept. of Agriculture, St. Paul,
MN; D.G. Tiffany, Univ. of Minnesota, St. Paul,
MN
7:40 pm STA-03: Reducing the environmental footprint of NatureWorks® polylactide (PLA)
polymers
E.T.H. Vink,* NatureWorks, BV, Naarden, The
Netherlands; D.A. Glassner, J. Kolstad, R.
Wooley, NatureWorks LLC, Minnetonka, MN
8:00 pm STA-04: Using life cycle assessment to develop sustainable bio-refineries
R. Jenkins,* C. Alles, J. Ginn, D. Culver, B.
Vrana, R. Sylvester, J. Friend, M. Emptage, S.
Hennessey, Dupont, Wilmington, DE
8:20 pm STA-05: Life cycle evaluation of four lignocellulosic ethanol pretreatment technologies
S. Spatari,* H. MacLean, University of Toronto,
Toronto, ON, Canada
8:40 pm STA-06: Energy and greenhouse gas emission impacts of fuel ethanol in the U.S.
M. Wang, Argonne National Laboratory, Argonne, IL
Special topic B: International
Biomass/Biofuels Update
Chairs: J.-H. Seo, Seoul University, Seoul, Korea and B.
Hahn-Hagerdal, Lund University, Lund, Sweden
7:00 pm STB-01: Current aspects and future prospects of bioenergy in Korea
J.-S. Lee,* S-C. Park, Korea Institute of Energy
Resarch, Daejeon, Korea
7:20 pm STB-02: Recovery of acrylic acid produced
from sugar cane using simulated moving
bed adsorption
A.B. Machado, M.E.T. Alvarez, A.P.A. Pinho,
A.P. Scheer, M.R. Wolf Maciel,* University of
Campinas, Campinas, Brazil
7:20 pm STA-02: More than just carbon dioxide: Nitrous oxide is the dominant greenhouse gas
in corn based biofuels and bioproducts
S. Kim, B.E. Dale,* Michigan State University,
East Lansing, MI
35
Tuesday, May 2
7:40 pm STB-03: Bioethanol and lactic acid production from lignocellulose; R&D progress in
The Netherlands
H. Reith, H. den Uil, A. Boersma, J. Kuijvenhoven, Energy Research Centre of The Netherlands, Petten, The Netherlands; J. de Bont,* J.
van Groenestijn, H. van Buijsen, D. Binnema,
G. Bos, J. Zeevalkink,TNO, Apeldoorn, The
Netherlands; M. Kabel, H. Schols, R. Beeftink, Wageningen University, Wageningen,
The Netherlands; R. Bakker, R. Weusthuis, R.
Maas, E. de Jong, Agrotechnology and Food
Innovations, Wageningen, The Netherlands;
W. de Laat, Royal Nedalco, Bergen op Zoom,
The Netherlands; D. Visser, Purac biochem,
Gorinchem, The Netherlands; H. Haan, Shell
Global Solutions International, Amsterdam, The
Netherlands
8:20 pm STB-05: Enzymatic modification of spruce
hemicellulose, O-acetyl galactoglucomannan
L. Anderson, A. Andersson, R. Peterson, H.
Stalbrand,* G. Zacchi, Lund University, Lund,
Sweden; J. Le Nours, L. Lo Leggio, University
of Copenhagen, Copenhagen, Denmark; A.
Larsson, J. Stahlberg, Uppsala University, Uppsala, Sweden
8:40 pm STB-06: New improvements for lignocellulosic ethanol (NILE), an integrated project in
FP6 of the European Commission
F. Monot, Institut Francais du Petrole, RueilMalmaison, France
8:00 pm STB-04: The potential for cellulosic ethanol
production in China
B. Yang,* Y. Lu, Dartmouth College, Hanover,
NH; J. Sun, S. Donghai, China Agriculture University, Beijing, China
Surf the
SIM
Wave!
Learn more about SIM . . .
• Membership benefits
• Online member directory
• Publications
• SIM Corporate members
• Meetings
• Career information
Visit us online www.simhq.org
36
Wednesday, May 3
Registration
Time: 7:15 am – 5:00 pm
Time: 7:15 am – 8:15 am
Speaker breakfast
Time: 7:15 am – 8:00 am
Session 5: Bioprocessing and separations R&D
Chairs: L. Zullo, Cargill, Minneapolis, MN and S. Snyder,
Argonne National Laboratory, Argonne, IL
8:00 am 5-01: Influence of the scale-up process on a
Saccharomyces cerevisiae fed-batch process: Modeling and experimental investigations
A. Lejeune, F. Delvigne,* P. Thonart, Universitaire des Sciences Agronomiques, Gembloux,
Belgium
8:30 am 5-02: An innovative membrane bioreactor for
very high ethanol performances
F.B. Chaabane,* A.S. Aldiguier, S. Alfenore, C.
Bideaux, X. Cameleyre, S. Guillouet, C. MolinaJouve, G. Roux, INSA, Toulouse, France
9:00 am 5-03: Economical evaluation of extraction of
hemicelluloses from process streams from
thermo-mechanical pulping of spruce
T. Persson*, A-S. Jonsson, G. Zacchi, Lund
University, Lund, Sweden
9:30 am Coffee break
10:00 am 5-04: Scale-up of stirred tank reactors using
CFD simulations
J. Hannon,* C.E. Wyman, Dartmouth College,
Hanover, NH
10:30 am 5-05: Procedure for development of a robust
mathematical model for alcoholic fermentation process
R.R. Andrade, A.C. Costa,* R. Maciel, D.P.
Atala, F. Maugeri, State University of Campinas,
Campinas, Brazil
11:00 am 5-06: Pervaporation membrane systems for
volatile fermentation product recovery and
dehydration
L.M. Vane, U.S. Environmental Protection
Agency, Cincinnati, OH
Lunch on own
Time: 12:00 noon – 1:30 pm
A buffet lunch is available for purchase in the hotel restaurant, as well as in the Capitol Ballroom for attendees.
Session 6: Bio/Thermo-chemical integrated biorefinery
Chairs: A. Ragauskas, Georgia Institute of Technology,
Atlanta, GA and T. Foust, NREL, Golden, CO
1:30 pm 6-01: Enhancing the successful development of North American biorefineries with
integrated regional bioproduct innovation
alliances
A. Kawczak, Battelle Memorial Institute, Columbus, OH
2:00 pm 6-02: Biorefining challenges: Choosing technologies in establishing a lignocellulosicbased biorefinery
W. Mabee,* J. Saddler, University of British
Columbia, Vancouver, BC, Canada
37
2:30 pm 6-03: Hybrid thermochemical/biological processing: Putting the cart before the horse?
R.C. Brown, Iowa State University, Ames, IA
Closing remarks
3:00 pm Coffee break
3:30 pm 6-04: Putting advanced biorefineries in
context: Integration with mature feedstock
supply systems, processing options and
nitrogen recycle
M. Laser,* H. Jin, L.R. Lynd, Dartmouth College, Hanover, NH; S. Sokhansanj, Oak Ridge
National Laboratory, Oak Ridge, TN; D. Bransby, Auburn University, Auburn, AL; E. Larson,
Princeton University, Princeton, NJ
4:00 pm 6-05: Integration of a biorefinery working
at a high dry matter content with a power
plant: Concepts and feasibilities
J. Larsen,* F. Iversen, Elsam Engineering A/S,
Fredericia, Denmark; B.H. Christensen, Sicco
K/S, Aalsgaarde, Denmark; M.H. Thomsen, A.B.
Thomsen, Riso National Laboratory, Roskilde,
Denmark; H. Jorgensen, C. Felby, The Royal
Veterinary and Agricultural University, Taastrup,
Denmark
29
th
Reception
Time: 6:00 – 7:00 pm
Sponsored by Novozymes, Inc.
Banquet
Time: 7:00 pm – 10:00 pm
7:00 pm Dinner
7:45 pm Charles D. Scott Award presentations
8:00 pm Entertainment: Butch Robins Trio
Sponsored by Novozymes, Inc.
See you
next time!
April 29–May 3, 2007
Adam’s Mark Hotel
Denver, CO
38
28
th
oral Presentation
abstracts
Abstracts – Oral Presentations
Oral presentation 1A-01
Grass Lignocellulose: Strategies to Overcome Recalcitrance
Danny E. Akin*
Russell Research Center, ARS-USDA, Athens, Georgia
Grass lignocelluloses are limited in bioconversion by aromatic constituents of various types and include both lignin and
phenolic acids esters. Histochemistry, ultraviolet absorption microspectroscopy, and response to microorganisms and
specific enzymes have been used to determine the significance of aromatics toward recalcitrance. Coniferyl-type lignin
appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues; cell walls with
syringyl-type lignin, e.g., sclerenchyma, are less recalcitrant. Phenolic acid esters often constitute the major chemical
limitation to cell wall biodegradation in grasses, especially for warm season species. Methods to improve biodegradability
include: plant breeding that mitigates the recalcitrance within specific cell walls, use of lignin-degrading white rot fungi,
and addition of esterases. Plant breeding for new cultivars has been especially effective for nutritionally improved forages,
e.g., bermudagrasses. In laboratory studies, selective white rot fungi, which lack cellulases, delignified the lignocellulosic
materials and improved bioconversion. Phenolic acid esterases released p-coumaric and ferulic acids for potential coproducts and improved the available sugars for fermentation. The separation and removal of the aromatic components for
co-products, while enhancing the availability of sugars for bioconversion, would improve the economics of bioconversion.
Address for correspondence:
PO Box 5677, Russell Research Center, ARS-USDA, Athens, Georgia 30604 USA
Ph: 706-546-3482 FAX: 706-546-3607 [email protected]
Oral presentation 1A-02
Plant Cell Wall Microfibril Structure
And Changes During Biomass Conversion Processes
Shi-You Ding*, David K. Johnson, John O. Baker, Xianghong Qian,
and Michael E. Himmel
National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401
Phone: 303-384-7758, Fax: 303-384-7752, Email: [email protected]
The major technical barrier to the realization of an advanced biorefinery economy is the low conversion efficiency of lignocellulosic polymers to fermentable sugars. In nature, these polysaccharides form a microfibril network in plant cell walls.
Each microfibril is approximately 3-5 nm in diameter, and chemically and structurally heterogeneous. In order to understand the deconstruction processes, it is therefore critical to characterize the microfibril at the nanometer scale. Atomic
force microscopy (AFM) offers the unique capability of imaging biomaterials at the atomic scale without extensive sample
preparation that may significantly alter the original structure. In this study, maize cell wall materials from coleoptiles, living
and mature parenchyma cells, and corn stover treated with dilute sulfuric acid and cellulases have been directly visualized using AFM in air and under aqueous conditions. Preliminary results have shown more accurate measurement of
native microfibrils in mature parenchyma cells, their synthesizing and assembling in meristematic cells, and disordering
and aggregating during chemical and enzymatic hydrolysis in corn stover. These observations have led to a new model of
molecular structure of the microfibril, and provided a deep insight into the cell wall deconstruction processes. Computer
simulation has also been used to delineate the interactions between cell wall polysaccharides.
40
oral presentation 1A-03
Thermophilic enzyme systems for efficient total hydrolysis of lignocellulosic materials
Liisa Viikari, Matti Siika-aho
VTT Biotechnology, P.O.Box 1500, FIN-02044, Espoo, Finland
Jari Vehmaanperä
ROAL Oy, P.O.Box 57 FIN-05200 Rajamäki, Finland
Guido Zacchi
Lund University, Dept.of Chemical Engineering, P.O.Box 124 SE-22100 Lund, Sweden
Kati Réczey
Budapest University of Technology and Economics, Dept. Agricultural Chemical Technology, Szent Gellért tér 4 HU-1521
Budapest Hungary
Thermophilic enzymes are expected to improve the overall efficiency of enzymatic hydrolysis of lignocellulosic materials,
due to potentially higher specific activities and increased hydrolysis rates. Moreover, they would provide flexibility in terms
of process configurations and parameters. One of the objectives of the European project TIME was to develop efficient
mixtures of new thermophilic enzymes and to evaluate new process concepts by using these enzymes in hydrolysis of
different raw materials. The role of enzymatic removal of residual hemicelluloses in pre-treated materials for improving the
overall hydrolysis was also investigated.
New thermophilic enzymes hydrolysing cellulose and hemicellulose were purified, characterised and produced in T.
reesei. The new enzymes were evaluated and mixtures of them were assembled and studied in hydrolysis experiments.
The presence of additional hemicellulolytic activities was shown to be essential for an efficient hydrolysis of corn stover
or hardwood. As a result, efficient multi-enzyme mixtures of thermostable enzymes were obtained for total hydrolysis.
Several of the new thermophilic cellulases performed comparably or better than the T. reesei enzymes. The optimised
mixtures could be succesfully used in the total hydrolysis of steam pre-treated materials at elevated temperatures.
The Role of Xylanases in the Enzymatic Hydrolysis of Lignocellulose
Alex Berlin*, Neil Gilkes, Jack Saddler
Lignocellulosic residues from forestry and agriculture contain large amounts of potentially fermentable sugars in the form
of cellulose and hemicelluloses and are attractive feedstocks for production of bioethanol and other chemicals. Complete
hydrolysis of the polymeric carbohydrates into monosaccharides for subsequent fermentation requires a complex mixture
of hydrolytic enzymes with broad substrate specificity; these include endo- and exocellulases, endo- and exoxylanases,
β-glucosidases and β-xylosidases. We have recently reported data suggesting that the hydrolytic performance of several commercial and experimental enzyme preparations on pretreated lignocellulose can be significantly improved by
supplementation with “accessory” enzymes. Here we present data on the effects of supplementing a commercial cellulase preparation with various xylanase activities. Enzyme preparations were evaluated using a panel of lignocellulosic
feedstocks from forestry and agricultural that had been pretreated by steam-explosion, organosolv pulping or dilute-acid
pretreatment. These data provide an empirical approach to enzyme improvement and as well as fundamental insights into
the mechanism of lignocellulose hydrolysis by complex enzyme mixtures.
41
Modeling the Disruption of Crystalline Cellulose by the Carbohydrate Binding Module from Cellobiohydrolase I
Mark R. Nimlos*, James F. Mathews4, Xianghong Qian1, Michael F. Crowley2, Giridhar Chukkapalli3,
John Brady4, William S. Adney, Michael Cleary3, and Michael E. Himmel
1
Rx-Innovation, Inc., Fort Collins, CO 80526
2
Scripps Research Institute, La Jolla, CA 92037
3
San Diego Super Computer Center, La Jolla, CA 92093-0505
4
Department of Food Science, Cornell University , Ithaca, NY 14853-7201
Trichoderma reesei cellobiohydrolase I is thought to “process” from the reducing end of a cellulose chain in crystalline
cellulose producing cellobiose as its product. Endoglucanases hydrolyze glycosyl linkages on the exposed surfaces of
cellulose, producing new reducing chain ends. It is clear that the CBH I cellulose binding modules (CBM) absorb to the
substrate to increase the local concentration of reducing chain ends for the enzyme. However, it is not certain what role
the binding module plays in decrystallizing and feeding the strand to the catalytic domain. In this study, computational
Molecular Dynamics (MD) simulations have been used to investigate the interaction of a Type A CBM with crystalline
cellulose. The objective is to understand the role that the CBM plays in disrupting the inter-chain hydrogen bonding of
cellulose and extraction of a chain from the crystal. These simulations on the (1,0,0) surface contained a model crystal of
cellulose Iβ with a hydrolyzed glycosyl linkage and a T. reesei CBH I CBM enclosed in a box of water molecules. Several
simulations were started with the CBM in different orientations relative to the hydrolyzed bond and were allowed to run for
up to 10 ns of simulation time on TeraGrid at SDSC. The results of this study show that in some orientations, the amino
acids on the distal end of the CBM form hydrogen bonds with the O6 hydroxyl group of the reducing end glucose of the
broken chain. This allows the terminal glucose to twist out of the crystal, which breaks the cellulose inter-chain hydrogen
bond. These results suggest that this CBM aids in the decrystallization of cellulose and may be instrumental in feeding a
strand to the catalytic domain.
*National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401
Phone: 303-384-7704, Fax: 303-384-6363, Email: [email protected]
42
Adsorption and Desorption of Cellulase, Beta-Glucosidase, and BSA Protein on
Pretreated Corn Stover, Cellulose, and Lignin
Deidre Willies, Bin Yang
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755
Charles E. Wyman*
Center for Environmental Research and Technology (CE-CERT), University of California, Riverside, California 92521
Addition of non catalytic proteins (e.g., BSA) and certain surfactants has been shown to enhance cellulose hydrolysis by
enzymes or reduce the amount of enzyme required to realize a particular conversion. One common explanation is that
the additives can reduce the nonproductive adsorption of enzyme on lignin. However, the interactions among substrate
features, enzymes, and proteins are not clear, and experiments were conducted to better understand the mechanism by
which these additives influence hydrolysis. Adsorption and desorption profiles were measured to better clarify the interaction of BSA, cellulase, and beta-glucosidase with cellulose, pretreated corn stover, and lignin prepared in different ways.
Adsorption of cellulase and beta-glucosidase on BSA-treated corn stover and lignin was also observed. This data indicated that both corn stover and lignin adsorb large amounts of all these proteins although in different relative amounts while
cellulose only adsorbs cellulase significantly. Adsorption and desorption patterns of other compounds that could be viable
hydrolysis enhancers will also be presented.
Enzyme Optimization for Cell Free Ethanol Production
Eric J. Allain*, Andrew Madison, Diana Dardugno, Rene A. Salinas, and Eric Marland
Appalachian State University, Boone, NC 28608-2036
Economic improvements in the production of fuel ethanol are largely dependent upon increasing the rate or yield of the
ethanol production process. Ultimately, both of these are limited by the microorganism being used to make the ethanol.
While strain improvements are possible, the constraints of maintaining a viable organism will eventually limit success in
this area. These limitations may be avoided by getting rid of the microorganism altogether and using only the enzymes
involved in the ethanol production pathway. This ‘cell free’ approach to ethanol production has a number of advantages
over the microbial process including greater process flexibility (i.e. higher temperature, or higher ethanol concentration),
more freedom to manipulate enzymes (i.e. removing feedback inhibition mechanisms), and the ability to easily optimize
the production process by altering enzyme levels.
The feasibility of cell free ethanol production was investigated by utilizing a metabolic model comprising of the twelve enzymatic reactions involved in the anaerobic glycolysis pathway of Saccharomyces cerevisiae. Metabolic control analysis
was used to optimize enzyme levels needed to result in a substantial increase in ethanol production rate compared to the
in vivo process. These results were used to evaluate the potential economics of cell free ethanol production and suggest
areas of future research.
43
Enzymatic hydrolysis of lignocellulose at very high solids concentrations
Henning Jørgensen*, Claus Felby
Department of Forestry and Forest Products, Danish Center for Forest, Landscape and Planning
The Royal Veterinary and Agricultural University, Højbakkegård Allé 1, DK-2630 Taastrup, Denmark
Phone: +45 35281704 Email: [email protected]
Jan Larsen, Mai Østergaard Petersen
Elsam Engineering A/S, Kraftværksvej 53, DK-7000 Fredericia, Denmark
The ability to process lignocellulosic materials at high solids concentrations in a bioethanol production is beneficial for
process economics. Operating at higher solids concentrations means less water consumption, less material to be handled
during heating and cooling and in general a reduced size of equipment. Previously, performing enzymatic hydrolysis and
fermentation of lignocellulosic materials with initial solids concentrations above 15 to 20 % DM has been regarded impossible due to the problems of mixing the material. This paper describes a method that enables enzymatic hydrolysis of
lignocellulosic material with up to initially 40 % DM.
Enzymatic hydrolysis of pretreated wheat straw was performed at solids concentrations ranging from 2 to 40 % DM. The
resulting material was readily fermentable by Saccharomyces cerevisiae without any addition of nutrients. The method
was also tested for simultaneous hydrolysis and fermentation of pretreated wheat straw over the same range of solids
concentrations.
Simultaneous hydrolysis and fermentation of whole crop wheat was investigated using pretreated wheat straw mixed with
dry milled wheat grain (1:1 ratio on a dry basis). A mixture of cellulases and low temperature amylases (cold mash) was
used for hydrolysis and saccharification. The process could be operated with up to 40 % initial DM.
oral presentation 1B-01
Application of plant genomics to the improvement of a dedicated energy crop
Steven Thomas, Richard Flavell, Kenneth Feldmann and the Ceres genomics teams
Ceres, Thousand Oaks, CA 91320
The economics of production of ethanol from a lignocellulosic feedstock depends on many factors, one of which is the
yield of digestible polysaccharides per unit of land. This is directly coupled to the biomass yield of the crop. Crop yield is
a combination of the environment and inputs provided and the genetic potential of the germplasm. The latter is increased
traditionally by plant breeding. This process involves establishing large numbers of new combinations of genes via genetic
crossing and selecting new gene combinations that enhance the yield potential in given environments. This process is
labor intensive and time-consuming. Transgenes offer faster ways of improving germplasm performance in a shorter time
but it is essential to have relevant transgenes available for insertion. Current progress to find transgenes to enhance biomass produced by biomass crop species by high throughput genomics using selection in both Arabidopsis and rice will be
described. Genes obtained to date address needs for making improvements in crop architecture, stress tolerance, growth
rate and germination efficiency. Examples will be discussed.
44
Regulation of Cell Wall Polysaccharide Composition
Chris Somerville, Alex Paredez, David Ehrhardt, Staffan Persson, Stefan Bauer, Shaolin Chen
Carnegie Institution, Stanford CA 94305
To facilitate an investigation of how cellulose synthesis is regulated we have developed transgenic plants in which subunits of cellulose synthase have been tagged with fluorescent proteins. The tagged proteins are fully functional based on
their ability to genetically complement mutations in the corresponding genes. Using spinning-disc confocal microscopy we
have visualized individual cellulose synthase complexes making cellulose in live plant cells that also contain microtubules
that are labeled with a second fluorophore. These studies have clarified the role of microtubules in orienting cellulose
deposition and have allowed measurements of the rate of cellulose synthesis and the lifetime of cellulose synthase complexes. The materials and methods developed for this study will be useful in understanding the role of other proteins that
have been implicated in the overall process of cellulose synthesis by genetic and genomic methods.
We have recently used genomic methods to identify a number of proteins that are relevant to secondary wall synthesis. In
particular, we have identified genes that appear to participate in xylan and xyloglucan synthesis.
Energy Crops for Fuel Ethanol Production
Bruce W. Ferguson,* David A. Lee
Edenspace Systems Corporation, Dulles, VA 20151
Mariam Sticklen,
Michigan State University, East Lansing, MI 48824
While cellulosic biomass from corn stover, grasses and other cultivated plants represents a tremendous amount of stored
energy, current techniques for converting this biomass to renewable fuels such as ethanol are too costly for widespread
commercial use. Lower costs can be achieved by tailoring the plants with traits such as cellulase expression and higher
biomass that reduce operating costs or increase production efficiencies. With Michigan State University and collaborators at the National Renewable Energy Laboratory and U.S. Department of Agriculture, Edenspace is developing a suite
of such traits in transgenic plants that include greater biomass, production of enzymes to aid post-harvest hydrolysis of
plant biomass to simple sugars, and bioconfinement features to reduce migration of the transgenes. Results to date with
tobacco and corn transformed with Acidothermus cellulolyticus E1 endoglucanase gene indicate that such tailoring of
plant traits appears to offer substantial potential for reducing cellulosic fuel ethanol production costs.
*Corresponding author: Bruce W. Ferguson, [email protected] Edenspace Systems Corporation, 3810 Concorde Parkway, Suite 100,
Dulles, VA 20151. Tel. 703-961-8700, Fax 703-961-8939.
45
oral presentation 1b-04
Development of brown midrib Sweet Sorghum as a biomass crop
Ana Saballos1, Gebisa Ejeta1, and Wilfred Vermerris1,2,3*
Department of Agronomy, 2Department of Agriculture & Biological Engineering,
3
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN, USA
1
Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. Traits
affecting biomass utility are lignin content and composition, soluble carbohydrate content and overall biomass yield.
Sorghum offers inherent advantages that make it an attractive biomass crop. It is tolerant to drought, performs well on
marginal lands, and possesses a deep root system that helps prevent the loss of soil organic matter. We are working
towards improving the quality of sorghum as a feedstock by combining traits from useful germplasm sources of brown
midrib (bmr) lignin mutants, sweet sorghums, and high biomass producers. Unfortunately, the genetic basis of these
characteristics is poorly understood. In order to elucidate the genetic basis and enhance the efficiency of combining these
traits, we have developed a population of recombinant inbred lines from crosses between a sweet sorghum and a bmr
mutant. Characterization of this population will allow identification of loci associated with biomass yield, cell wall composition and soluble sugar content in the phloem. Through a combination of genetic and chemical approaches we have also
established the number of independent bmr loci associated with our collection of bmr mutants. A candidate-gene approach, based on the chemical composition of the mutants’ cell wall and mapping data, is being used to clone the genes
underlying some of these mutations.
Conversion of Cellulose to Glucose using endo-1, 4-β-glucanase (E1) Enzyme
Produced in Transgenic Rice Plants
Hesham Oraby, Rashid Ahmad, Callista Ransom, Mariam Sticklen*
Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824
Balan Venkatesh, Bruce Dale
Department of Chemical Engineering and Material Sciences, Michigan State University, East Lansing, MI 48824
A great deal of efforts has been exerted towards improving ethanol yield and reducing its production costs. The cheap
availability of lignocellulosic biomass, the less greenhouse effect and the large quantity of ethanol that could be produced
from crop biomass compared to maize seeds might have the potential to make the cost of ethanol competitive to gasoline.
While rice seed is the useful portion of this crop, its remaining biomass has limited use and its burning has raised health
concerns. Therefore, rice could be recommended for use as a viable bio-based energy crop. The catalytic domain of
Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1, 4-β-glucanase enzyme or E1) was
constitutively expressed in rice. Molecular analyses of T1 plants confirmed presence and expression of the transgene.
The amount of E1 enzyme extracted from T1 plants accounted for up to 4.9% of the plant total soluble proteins. Approximately 22 and 30% of the cellulose in the AFEX-pretreated rice and maize biomass respectively was converted into
glucose using rice E1 heterologous enzyme. These results may suggest an economically successful strategy for producing biologically active hydrolysis enzymes in rice straw for alcohol fuel while substituting the wasteful practice of rice straw
burning with an environmentally conscious technology.
46
oral presentation 2-01
A Novel Concentrated Phosphoric Acid/Acetone Lignocellulose Fractionation
Featuring Modest Reaction Conditions and Reagent Recycling
1
Y.-H. Percival Zhang *1, Lee R. Lynd 2
Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
24060, USA; 2 Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755, USA
*Corresponding Author, Y.-H. Percival Zhang ([email protected]),
Tel: (540) 231-7414; Fax: (540) 231-3199
Overcoming the recalcitrance of lignocellulosic materials is one of the largest technical obstacles for emerging biobased
products and bioenergy industries. Here we demonstrate a novel lignocellulose pretreatment using concentrated phosphoric acid/acetone, based on the principle of significantly different chemical-physical properties of the individual lignocellulose components in concentrated phosphoric acid, organic solvent, and water. This lignocellulose pretreatment has four
distinctive features: modest reaction conditions, fractionation of lignocellulose into amorphous cellulose, hemicellulose
sugars, lignin, and acetic acid, generation of highly reactive amorphous cellulose, and cost-effective reagent recycling. Images of scanning electron microscope showed that the original supramolecular structures of corn stover did not exit after
the pretreatment. Hydrolysis results of pretreated corn stover showed that ca. 97% cellulose was digested after 24 hours
of hydrolysis at an enzyme loading of 15 filter paper unit/gram cellulose. This technology would offer several potentially
economic benefits: 1) an increase in total revenue, 2) a decrease in processing costs, 3) a low capital investment for a
small biorefinery, 4) lower transportation costs for feedstock, and 5) low investment risks due to the integration of several
mature technologies
Recent Process Improvements for the Ammonia Fiber Explosion (AFEX) Process
and Resulting Reductions in Projected Ethanol Prices
Bruce E. Dale* and Elizabeth Newton
Michigan State University, East Lansing, MI 48824-1226
Lee Lynd and Mark Laser
Thayer School, Dartmouth College, Hanover, NH 03755
Biomass processing technology, particularly for lignocellulose conversion, is not mature. We can expect considerable
improvements in the technical and economic performance of related processes as these are further developed. The
ammonia fiber explosion (AFEX) process is one such biomass pretreatment that is still being improved. Recent process
modeling work by the Consortium for Applied Fundamentals and Innovation (CAFI) team highlighted the importance of
factors such as ammonia concentration, ammonia to biomass ratio and ammonia recovery as cost sensitive areas in the
AFEX process.
Subsequent research work at Michigan State and collaborative process design efforts between Michigan State and
Dartmouth College have led to very significant cost reductions in the AFEX process. When these AFEX improvements
are combined with consolidated bioprocessing (CBP) as a means of converting pretreated biomass directly to ethanol,
projected minimum ethanol selling prices of around $0.80 per gallon are achieved. We describe the improvements made
in AFEX and how AFEX might be integrated with CBP in an advanced bioprocessing system.
47
Research Challenges and Opportunities for Cellulose Conversion Technology in a Dry Mill Pathway
Michael Ladisch*, Wally Tyner, Nathan Mosier
Purdue University, West Lafayette, IN 47907
Mike Cotta, Bruce Dien, USDA-ARS-NCAUR-FBT, Peoria, IL 61604
Hans Blaschek, University of Illinois, Champaign-Urbana, IL 61801
Bruce Dale, Michigan State University, E. Lansing, MI 48824
Brent Shanks, Iowa State University, Ames, IA 50011-2230
Gene Petersen, Golden Field Office, DOE, Golden, CO 80401
Most of the current expansion in ethanol production capacity is attributable to newly constructed dry grind facilities that
convert corn into fuel ethanol. These facilities process the corn into ethanol, with the remaining solid material, known as
distillers’ grains (DG) or distillers dried grains with solubles (DDGS), being marketed as a value-added animal feed. For
each bushel of corn fermented to ethanol, approximately 17 lb of DDGS is produced. Thus, DDGS production is expanding as dry grind ethanol production expands. As a consequence, the Midwest Consortium is examining methods of
adding value to the distillers’ grains by processing them further to produce ethanol from the cellulose and hemicellulose
which also results in higher protein residual. An overview of The Midwest Consortium and its work, as well as technical
challenges in applying pretreatment, enzyme, and fermentation advances to processing of DG into both 5- and 6-carbon
sugars, ethanol, and other value-added products, as well as the fit of these new processes into existing dry mills currently
under construction, will be discussed. An economic framework for assessing the impact of changes in a dry grind operation due to enhanced DG utilization will also be summarized in this presentation.
Identification of Quantitation of Water Extractives in Corn Stover
Shou-Feng Chen, Richard A. Mowery, C. Kevin Chambliss*
Department of Chemistry & Biochemistry, Baylor University, Waco, TX 76798-7348
G. Peter van Walsum, Department of Environmental Studies, Baylor University
Previous work has demonstrated that a significant fraction of corn stover feedstock is composed of water-soluble materials. These components have typically been classified only as ‘extractives’ or ‘total water-soluble material’ in compositional
analyses, and little effort has been extended to explore quantitative assessments of individual components. However,
there is increasing interest in this fraction of feedstocks due to the possibility for leaching of potentially valuable components from materials stored outdoors during prolonged rain events.
A developing suite of analytical methodologies based on initial class fractionation of water extracts followed by chromatographic separation and analysis of individual components has been used to investigate the composition of six different
corn stover feedstocks. To date, quantitative protocols have resulted in greater than 80% mass closure for water-soluble
materials. It is important to point out that up to 50% of the mass balance is represented by sugars, with approximately
35% of total sugar present in monomeric form. The remaining 30% is comprised of various organic acids, inorganic ions,
and higher-molecular-weight materials. These data suggest that as much as 6-12% of the dry weight a feedstock is composed of sugars that could potentially be lost upon prolonged contact with water.
48
Comparative Sugar Recovery Data from Application of
Leading Pretreatment Technologies to Corn Stover and Poplar
Charles E. Wyman*,
Dartmouth College, Hanover, New Hampshire 03755
(now at University of California, Riverside, California)
Bruce E. Dale, Michigan State University, East Lansing, Michigan 48824
Richard T. Elander, National Renewable Energy Laboratory, Golden, Colorado 80401
Neil Gilkes, University of British Columbia, Vancouver, British Columbia V6T 1Z4
Mark T. Holtzapple, Texas A&M University, College Station, Texas 77843
Michael R. Ladisch, Purdue University, West Lafayette, Indiana 47907
YY Lee, Auburn University, Auburn, Alabama 36849
Mohammed Moniruzzaman, Genencor International, Beloit, Wisconsin 53511
Pretreatment is essential to high yields and low costs for biological processing of cellulosic biomass to fuels and chemicals. A team experienced in biomass hydrolysis research formed a Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) and developed the first comparative data for the promising pretreatment options of ammonia
explosion (AFEX), aqueous ammonia recycle (ARP), controlled pH, dilute acid, flowthrough, and lime. A single source of
corn stover was initially employed, and material balances were developed based on use of shared analytical methods. In
addition, comparative data were developed on the digestibility of the pretreated solids using a controlled source of cellulase, and initial estimates were made of the costs. All pretreatments were effective in making cellulose accessible to enzymes with high yields, with trends slightly better for high pH technologies. Xylose recovery yields were high for all these
pretreatments, although hemicellulase activity in the enzyme formulation was vital to recover the substantial amounts of
xylan left in the residual solids for pretreatments at high pH. Initial applications of the same technologies to poplar wood
suggest that lignin impacts the digestibility of cellulose substantially and that different feedstocks may benefit from different pretreatments.
49
Corn Stover to ethanol - Moving the focus toward process integration
Guillermo Coward-Kelly, Mads Torry-Smith, David Milam
Novozymes North America, 77 Perry Chapel Church Road, Franklinton, NC 27525
The Biomass Program seeks to cost-effectively produce ethanol, other fuels and chemicals from biomass resources. The
economical viability of converting biomass into ethanol is affected by numerous factors, some of them include: substrate
digestibility, enzyme loading, and final potential ethanol concentration. Economical viability through improved process
performance can be achieved by tackling each of these factors separately or through an integrated process development.
At Novozymes, our primary goal is to reduce the enzyme related cost of converting biomass into ethanol. This can be accomplished by two different approaches: 1. Increased specific activity and enzyme yield in the cellulase complex, and 2.
Increased process performance. This talk will focus on process optimization.
Several pretreatments technologies exist for increasing substrate digestibility. Dilute-acid-hydrolysis followed by steam explosion has the advantage of solubilizing the hemicellulose fraction into free monomers and leaving the cellulose fraction
accessible to enzymatic attack. This pretreatment technology was used as focus for the optimization work.
Generating high final ethanol concentrations requires high solid; however, high solid loadings (>13%) generate mixtures
with poor mixing quality and traditionally requires higher enzyme loads. Nevertheless, enzymatic hydrolysis of the cellulose fraction in biomass reduces the viscosity of the solid mixture significantly and a fed-batch approach can be used to
step-wise increase the solid content, hence generating a higher ethanol concentration.
Oral presentation 2-07
Design, Installation and Start Up of Biomass Fractionation Plants
Andrew Richard*, Murray Burke, Joelle Kangudie
SunOpta BioProcess Group
2838 Bovaird Drive West, Norval, Ontario, Canada L7A 0H2
Phone: 905-455-2528
Fax: 905-455-5744
Email: [email protected]
The SunOpta BioProcess Group (formerly Stake Technology) has been designing, engineering and building biomass
plants since 1978. End products include xylitol, butanol, ethanol, dietary fibre, cellulose derivatives, cattle feed and unbleached pulp. Raw materials include wheat straw, ryegrass straw, sugarcane bagasse, oat hulls, wood chips and wood
fines and SunOpta’s focus has been on raw material preparation, steam explosion pretreatment and downstream solid-liquid separation. This paper will present SunOpta’s experiences and knowledge gained in the scale up, design, installation
and start up of these facilities. In particular, overcoming the challenges inherent to processing biomass on a continuous
basis will be presented with emphasis on issues encountered and design solutions implemented.
50
BioBased Chemicals from Ethanol – The Ethanol Platform
Richard W. Glass*, National Corn Growers Association, Chesterfield, MO 63005
Ron Cascone, Jeff Plotkin, Nexant, White Plains, NY 10601
Nathan Danielson, BioCognito, Collegeville, PA 19426
Anticipated passage of a Renewable Fuels Standard and the potential increase in rural ethanol production from corn, led
the National Corn Growers Association (NCGA) several years ago, with the financial support of the Office of the Biomass Program of the US DOE Office of Energy Efficiency and Renewable Energy, to initiate a study similar to the “Top
Value Added Chemicals from Biomass” that could identify biobased chemicals produced from corn ethanol. This study is
unique in that it extends the concept of the sugar platform specifically to ethanol and drives towards the “ethanol platform.”
The study considered parameters such as market size, cost of production, available technology, and geographic conditions to identify candidates, but mandated ethanol as a starting raw material. A primary objective was to identify opportunities for rural development that are compatible with small dry grind ethanol facilities. The conclusions of this study
establish that there are several classes of compounds made from ethanol that may replace the same product made by
conventional petrochemical route(s), other products of similar functionality, and entirely new products or applications that
can compete on price as well as benefiting from the natural and ‘green’ concept.
Improving the Value of Corn Proteins Via Reforming with Anaerobic Bacteria
Tim Eggeman*, Dan Verser, Shirley Hunter, Scott Walmsley
ZeaChem Inc., Lakewood, CO 80228
The projected expansion of the corn processing industry over the next decade will likely result in severe oversupply conditions for related animal feed co-product markets. Today, the proteins of processed corn are sold either into ruminant feed
markets that place little value on amino acid profiles, or into poultry markets where the pigment content, rather than the
amino acid profile, is the key factor for diet formulation.
This work examines a novel concept for reforming the amino acids in corn by using non-gas forming anaerobic bacteria
in a fermentation step that co-produces fuels and chemicals. Our approach potentially maximizes the value obtained
from the whole kernel since we derive improved value from both the protein and carbohydrate fractions. The cell mass
produced by the fermentation is processed into a high protein concentrate. Reforming the native proteins of corn in this
manner results in a more valuable feed co-product slate suitable for use in a broad range of feed formulations. While cell
mass yields for anaerobic fermentations are low when compared to aerobic processes, use of anaerobic bacteria has the
advantage that starch and other complex carbohydrates are simultaneously converted into desirable biobased fuels and
chemicals rather than CO2 and water.
51
DuPont Introduces Bio-Based High Performance Polyetherglycols
Ray W. Miller* , Howard Ng, and Hari Sunkara, E. I. DuPont de Nemours and Co, Inc.
DuPont Bio-Based Materials, Chestnut Run Plaza 728/1419, Wilmington, DE 19805
DuPont has developed and is commercializing a bio-based process for producing 1,3 propanediol (PDO) from fermentation of dextrose in a joint venture with Tate & Lyle, Ltd. at Loudon, Tennessee. Separately, DuPont has developed and
patented a process which polymerizes PDO directly into a proprietary new family of polyetherglycols. These long-chain
ether-linked diols have exciting properties which make them exceptionally attractive as soft segments in a variety of high
performance elastomeric polymers. This paper will overview the bio-based polyetherglycol process, highlight the benefits
of this new class of high-performance ether-linked diols, and provide some examples of the unique performance enhancements they bring to today’s demanding elastomeric fibers and molded parts markets.
BioAmber- The First Large Scale Commercial Production of Bio-based Succinic Acid
Kris Arvid Berglund*1,2, Managing Director
BioAmber, Route de Bazancourt, F-51110 Pomacle, FRANCE
Paul Jacobson, CEO
Diversified Natural Products, Inc., 145 West 57th Street, New York, NY 10019 USA
1
Yvon Le Hénaff, Managing Director
Agro-Industrie Recherches et Developpements, Route de Bazancourt, F-51110 Pomacle, FRANCE
2
Ulrika Rova, Head
Div. of Biochem. & Chem. Process Engg., Luleå University of Technology, SE 971 87 Luleå SWEDEN
BioAmber is a joint venture between Diversified Natural Products, Inc (DNP). from the USA and Agro-Industrie Recherches et Developpements (ARD) from France formed for the commercial production and marketing of bio-based succinic
acid. This venture has resulted in the first successful large scale production of bio-based succinic acid. BioAmber’s technology platform has been developed by a number of participants over the past decade. A full description of the product
portfolio of the company including derivatives of succinic acid will be presented.
The presentation will detail the successful commercialization of technology that was initially developed at the USDOE and
further refined at Michigan State University, Luleå University of Technology, and DNP. Discussion of the role of public-private partnerships will be included. The international elements of the effort will also be described.
52
Techno-economic analysis of biocatalytic processes for production of alkene epoxides
Abhijeet P. Borole*, Brian H. Davison
Oak Ridge National Laboratory, Oak Ridge, TN 37831‑6226
A techno-economic analysis of two different bioprocesses, one for conversion of propylene to propylene oxide and other
for conversion of styrene to styrene epoxide have been performed. The first process was based on a lipase-mediated
chemo-enzymatic reaction, while the second one was based on a one-step enzymatic process using chloroperoxidase
(CPO). The propylene oxide produced via the chemo-enzymatic process is a racemic product, while the latter process
(based on CPO) produces an enantio-pure product. The former process thus falls under the category of low-volume high
value product, while the latter is a high-volume commodity chemical.
A simulation of the process was conducted using the bioprocess engineering software, Superpro Designer to determine
the potential energy savings and economics of the process. The purpose of the exercise was to compare biocatalyst
based processes, (that use biocatalyst formulations such as salt-activated biocatalysts, surfactant-activated biocatalysts
or immobilized biocatalysts) with existing chemical processes for production of alkene expoxides. The results show where
improvements are needed to make bioprocesses competitive with chemical processes.
Enzyme mediated production of microcrystalline cellulose from agricultural residues
Foster A Agblevor*,
Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Maha Ibrahim and Waleed K. El-Zawawy
Cellulose and Paper Department, National Research Center, El-Tahrir Street, Cairo, Egypt
Microcrystalline cellulose has a wide application in food, pharmaceuticals, and other industries. Most microcrystalline cellulose is produced from dissolving pulps using concentrated acids. We investigated steam explosion and partial enzyme
hydrolysis of corn cobs and cotton gin waste for the production of microcrystalline cellulose. Steam explosion conditions
were varied from severities of 2.8 to 5.0. The corn cob was converted into a fine powder after steam explosion and the
hemicellulose fraction was easily extracted water. The lignolytic fraction was extracted with sodium hydroxide solution and
the residual cellulose was bleached with hydrogen peroxide and converted into microcrystalline cellulose using hydrochloric acid, sulfuric acid and cellulase enzyme preparation. The resulting microcrystalline cellulose samples had properties
that were comparable to commercial microcrystalline cellulose.
Similarly, cotton gin residue was steam exploded and converted into microcrystalline cellulose. However, the steam
exploded cotton gin residue was more difficult to extract the crude cellulose product was very difficult to bleach using
hydrogen peroxide. Whereas the microcrystalline cellulose from the cotton gin residue appeared like fiber strands, those
from the corn cob were particles when analyzed with scanning electron microscope. The shape of the particles could be
controlled depending on the enzyme treatment.
53
Biomass Resource Feedstock Supply for Replacing 30% of U.S. Gasoline consumption by 2030
Bob Perlack1 and J. Richard Hess2
Oak Ridge National Laboratory, Oak Ridge, TN 37831‑6422
2
Idaho National Laboratory, Idaho Falls, ID 83415-2210
1
This presentation will first summarize the results of a joint USDOE/USDA study to determine whether the land resources
of the United States are capable of producing a sustainable supply of biomass sufficient to support a biorefinery industry large enough to have a significant impact on energy supply and oil imports. Study results show a potential resource
exceeding 1.3 billion dry tons with agricultural crop residues, perennial energy crops, and forest residues being the largest
unexploited sources of biomass feedstocks. The second part of the presentation will report on an initiative to displace
30% of the country’s present light-duty vehicle gasoline demand with biofuels by 2030. This initiative will require the
annual production of about 60 billion gallons of ethanol and the development and use of a large fraction of the potential
resources identified in the joint DOE/USDA resource assessment. This part of the presentation will discuss alternative
biomass supply scenarios including supply limitations and cost reductions required in feedstock production, collection and
harvesting, preprocessing, storage, and transportation and handling.
Biomass Energy, Is there Enough Land?
Alvin O. Converse
The recent literature on the land requirements of biomass energy is reviewed. In particular the global analysis of the
potential conflict with land needed for food, recently published by S. Nonhebel (2005), is examined and extended to the
situation in the U S. The effect of possible improvements in agricultural production and the combined production of food
and energy are considered. Various values of the solar energy efficiency of biomass formation are compared, and the
variation in the estimates of the net energy to produce liquid fuels from biomass is discussed.
Nonhebel, S., Renewable energy and food supply: will there be enough land?, Renewable and Sustainable Energy Rev. 9 (2005) 191-201
54
Switchgrass for Biomass: Commercial-Scale Production Costs in the Northern Great Plains1
Richard K. Perrin*2, K.P. Vogel3, Rob Mitchell3, and Marty R. Schmer4
Switchgrass is a potential commercial biomass feedstock but field scale economic data has been limited. Here we report
initial results on switchgrass cost of production for five years by ten farmer-cooperators on 15-24 acre fields in the Northern Great Plains. Four fields in Nebraska were grown during 1999 to 2004, six fields in North and South Dakota were
grown during 2000 to 2005. A total of 173 acres were planted, 10 acres of which were abandoned because of poor establishment due to drought. Total five-year expenditures on these 173 acres were $63,0005, plus land rent totaling $48,416.
Total production was 2,240 US tons of air-dry switchgrass, for an average yield over the four production years of 3.4 US
tons per acre per year, and resulting in an average cost of $33/ton plus $17/ton for land rent. If establishment costs are
pro-rated over nine production years, cost falls an additional $6/t. Yields through the five years averaged 0.4, 2.5, 3.2, 4.2
and 3.2 t/acre. Year 1 was the establishment year. Total costs for individual farms ranged from 26 to 66 $/ton. Reducing
establishment costs and increasing establishment and post-establishment year yields could reduce total farm gate cost of
switchgrass biomass.
*Corresponding author: 208 Filley Hall, UNL East Campus, Lincoln, NE 68583-0922, 402-472-9818; fax 402-472-3460; [email protected].
1
Paper submitted for oral presentation in Session 3B (Feedstock Supply and Logistics) of the 28th Symposium on biotechnology for Fuels and Chemicals, Nashville, April 30-May3, 2006
2
Professor, Department of Agricultural Economics, U. of Nebraska
3
Research Geneticist and Rangeland Scientist, respectively, USDA-ARS, Lincoln, NE
4
Biological Research Technologist, USDA-ARS, Lincoln, NE.
5
Numbers presented here are correct as of November, 2005, but are subject to revision as final data from 2005 are fully incorporated.
55
Switchgrass for Biomass: Farm Scale Production Practices Affect Feedstock Costs
and Quantities in the Northern Great Plains1
K.P. Vogel*2, Richard K. Perrin3, Rob Mitchell2, and Marty R. Schmer4
Switchgrass was managed as a biomass energy crop for five years on 10 farms in Nebraska and South and North Dakota
during the period 2000 to 2005. Primary production factors that affected feedstock costs were stand establishment which
affected first and second year yields, fertilization and harvest management which affected post-establishment yields, and
available precipitation which affected both establishment and yields. All factors except for precipitation can be significantly
improved by management. Farmers that had the lowest production costs used recommended planting guidelines, best
available herbicides, followed fertilizer guidelines, and harvested at the recommend cutting height and stage of maturity.
Improvements in yield and reduction in per ton costs can be achieved by improving seed quality and seed quality testing,
pre-establishment year cropping sequences, seedbed preparation and herbicide applications, improved fertilizer recommendations based on production and soil tests, improved cultivars, and improved harvesting and storage technologies.
Case examples will be provided to illustrate the effect of production practices on feedstock costs. Biorefineries will need
to have agronomists on their staffs to provide technical production information to their producers.
*Corresponding author: USDA-ARS, 344 Keim Hall, P.O. Box 830937, Univ. of Nebraska, Lincoln, NE 68507. Phone 402-472-1564, fax 402-4724020, [email protected].
1
Paper submitted for oral presentation in Session 3B (Feedstock Supply and Logistics) of the 28th Symposium on biotechnology for Fuels and Chemicals, Nashville, April 30-May3, 2006
2
Research Geneticist and Rangeland Scientist, respectively, USDA-ARS, Lincoln, NE
3
Professor, Department of Agricultural Economics, U. of Nebraska
4
Biological Research Technologist, USDA-ARS, Lincoln, NE
Comparison of Single-Pass Harvesting of Corn Grain and Stover with Conventional Harvesting Schemes
Kevin J. Shinners*, Garritt C. Boettcher, Jesse T. Munk, Matthew F. Digman, Graham S. Adsit
Department of Biological Systems Engineering, University of Wisconsin – Madison, Madison, WI 53706
Richard E. Muck, Paul J. Wiemer
US Dairy Forage Research Center, USDA-ARS, Madison, WI 53706
To minimize cost of corn stover as a bio-mass feedstock, a single pass, split-stream combine harvester was investigated
to harvest clean grain and stover simultaneously. Two innovative single-pass harvester configurations have been developed and performance data collected. This data will allow comparisons of this harvesting scheme with conventional
baled or chopped systems. Comparison of the performance of these three harvesting schemes will be made based on
equipment needs; number of passes in the field; stover and grain harvesting rates; fractional stover DM and water capture
rates; soil contamination; stover moisture content and particle-size; stover transport density; post harvest ground cover;
storage losses; and stover physical condition after storage. Chemical analysis of the harvested stover fractions using Stover9 NIR calibration from NREL will allow prediction of specific ethanol production from the three harvesting schemes.
56
Preparation and Characterisation of Pyrolytic Biomass Slurries: A New Feedstock for Gasification
Klaus Raffelt, Edmund Henrich, Christoph Kornmayer,
Christian Renck, Ralph Stahl, Joachim Steinhardt, Friedhelm Weirich
Forschungszentrum Karlsruhe, Institut für Technische Chemie (ITC-CPV), Hermann-von-Helmholtz-Platz 1, D-76344
Eggenstein-Leopoldshafen, Germany; e-mail: [email protected], fax +49(0)7247/82-2244
The “BiomassToLiquid2” (BTL2) concept of Forschungszentrum Karlsruhe will be introduced: Dry biomass with a high ash
content is flash-pyrolysed in a regional plant (first step) and gasified in a central plant (second step). In the regional plant, a
slurry of pyrolysis products is mixed. The high costs of straw transport were one of the main arguments for investigating this
new approach of biomass utilisation, whereas the transport of pumpable and dense slurry is cost- and energy-efficient.
In previous and present experiments of flash-pyrolysis by means of the twin-screw LR-mixing reactor pyrolysis liquids and char of
softwood, beech, wheat straw and rice straw were produced. The condensation is carried out at two temperature levels and two
different and stable fractions are recieved: one with high water content and low heating value, the other with low water content
and high heating value. The latter is inmiscible with water and rich in pyrolytic lignin. Slurries of this pyrolysis oil and of char with
particle sizes of <20 micrometers are free of sedimentation over many weeks and even after a longer period homogenisation prior
to gasification is simple. Attention is paid on the particle size range of the char. If milling is required, the slurry viscosity is reduced
drastically by the simultaneous destruction of inner pores that would otherwise absorb a considerable amount of liquid. Apart from
pores also the void volume of agglomerates becomes soaked with liquid and may result in a non-flowable slurry. This is prevented
by a continuous colloid mixing process with high shear forces. For gasification in the 3 MW pressurised entrained flow gasifier of
Future Energy, Freiberg, Germany, ~45 t of wood pyrolysis products were mixed and successfully converted into synthesis gas.
Preprocessing Effects on Lignocellulosic Feedstock Cost and Quality for Ethanol Conversion
Corey W. Radtke*, Christopher T. Wright, Kevin L. Kenney, Peter A. Pryfogle, Reed L. Hoskinson, Heather G. Silverman,
D. Brad Blackwelder, Neal A. Yancey, Debby F. Bruhn, Cindy R. Breckenridge, and J. Richard Hess
The objective of this research was to evaluate the impact of several types of preprocessing on feedstock quality including both chemical composition and an estimation of in-refinery conversion efficiency. The quality and cost of biomass
feedstocks is impacted by feedstock assembly (preprocessing) operations. These quality and cost changes need to be
understood to minimize the negative impacts and to harness biomass upgrades due to preprocessing.
The preprocessing operations investigated were selective harvest and fractional milling, collectively termed mechanical
preprocessing, and in-storage preprocessing. Selective harvest involved the mechanical separation of stover anatomical components into two functionally similar streams during grain harvest. Fractional milling involved size reduction and
mechanical separation of biomass through full scale grinding. In-storage preprocessing considered both positive and
negative changes to biomass material while in wet and dry storage. Preprocessing costs and biomass-quality results will
be presented as pertaining to the ethanol-biorefining industry.
57
Construction of cellulolytic Saccharomyces cerevisiae strains for consolidated bioprocessing
Riaan Den Haan, Ronél van Rooyen, Shaunita H Rose, and WH (Emile) Van Zyl*
Department of Microbiology, University of Stellenbosch, Stellenbosch, 7600, South Africa
JE McBride, LR Lynd, Thayer School of Engineering, Dartmouth College, Hanover, NH 03755
Cellulase production, hydrolysis of biomass and fermentation of resulting sugars to desired products in a single process
step via a cellulolytic microorganism(s) is a promising approach to low-cost conversion of lignocellulose to fuels and
chemicals. One strategy for developing a microorganism capable of such consolidated bioprocessing involves engineering the non-cellulolytic yeast Saccharomyces cerevisiae so that it expresses a heterologous cellulase system enabling
cellulose utilization. We have successfully demonstrated expression of the three major cellulase activities (b‑glucosidase,
endo-glucanase and cellobiohydrolase) in S. cerevisiae. We have enabled this yeast to grow aerobically and anaerobically on cellobiose as sole carbon source at levels comparable to growth on glucose. Furthermore, strains co-producing
a b‑glucosidase and an endo-1,4-b-glucanase were constructed, that could grow aerobically (0.13 h-1) and anaerobically (0.03 h-1) on amorphous phosphoric acid swollen cellulose. To our knowledge, this is the first report of recombinant
strains of S. cerevisiae growing on cellulose. To enable growth on more crystalline substrates, successful co-expression
of cellobiohydrolase (CBH) activity is needed. Although functional expression of several CBHs in S. cerevisiae has been
achieved, activity levels reported to date are quite low. Strategies to improve CBH production will be discussed.
Development of Saccharomyces cerevisiae strains with increased HMF reduction capacity
João R.M. Almeida, Kaisa Karhumaa, Bärbel Hahn-Hägerdal, Marie F. Gorwa-Grauslund*
Department of Applied Microbiology, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
Anneli Petersson, Tobias Modig, Gunnar Lidén
Dept. of Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
In the process of bioethanol production from lignocellulosic hydrolysate, pre-treatment of lignocelluloses is necessary to
convert the polymeric carbohydrates to simple sugars available for yeast fermentation. Several types of inhibitory compounds, such as the furans 5-hydroxymethylfurfural (HMF) and furfural, are formed during acid pre-treatment. Furans
are known to inhibit yeast growth and viability and reduce ethanol productivity. Yeast strains with endogenous capacity to
reduce HMF and furfural to less toxic compounds have been isolated, however the identification of gene(s) responsible
for the conversion is lacking.
We used a genomic library and microarray analyses of two strains with different abilities to convert HMF in order to
identify HMF-reducing enzymes in S. cerevisiae. Genes were selected from microarray results and further analyzed using
corresponding clones from the yeast ExClone overexpression collection where in vitro and in vivo assays were used to
characterize HMF reduction capability. Physiological studies with new constructed strains were carried out under aerobic
and anaerobic conditions in order to analyze HMF uptake and strain tolerance. Both increased HMF reduction ability and
increase ethanol productivity were achieved.
58
The growth-independence bioprocess for ethanol production using Corynebacterium glutamicum
Masayuki Inui1, Alain A. Vertès1, Shohei Okino1, Takashi Watanabe2 and Hideaki Yukawa1
1
Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho,
Soraku-gun, Kyoto 619-0292 Japan. Phone: 81-774-75-2308; Fax 81-774-75-2321; E-mail: [email protected]
2
Research Institute for Sustainable Humanosphare, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
The depletion of fossil-fuel-based energy sources has fueled research on alternative renewable energy sources. Studies
on fuel ethanol production from various types of biomass using yeast, Zymomonas mobilis and genetically modified
Escherichia coli have been reported.
We previously developed a new bioprocess for ethanol production from cellulosic and hemicellulosic sugars using genetically
modified Corynebacterium glutamicum. The bioprocess is based on unique features that enable aerobic C. glutamicum to
maintain its main metabolic capabilities under oxygen deprivation even though its their proliferation is arrested. These features
allow for the use of a reactor filled to a high density with cells derived from aerobic culture, leading to a bioprocess with high
volumetric productivity. Recently, we found that this bioprocess is not significantly affected by inhibitors formed during saccharification of lignocellulosic biomass. Moreover, the amount of heat generation in our process was less than in other bioprocess for
ethanol production. These may be attributed to the growth-independence of ethanol the production process.
Metabolic Engineering of the Xylose Utilizing Thermophile
Thermoanaerobacterium saccharolyticum JW/SL-YS485 for Ethanol Production
A. Joe Shaw, Michael Tyurin, Kara Podkaminer, and Lee R. Lynd
Thermoanaerobacterium saccharolyticum JW/SL-YS485 is a gram positive, thermophilic, obligate anaerobe that grows
in a temperature range of 30°C - 66°C and a pH range of 3.9 – 6.5. This organism can consume a variety of hydrolysis
sugars found in lignocellulosic biomass as well as xylan and starch, but not cellulose, and produces ethanol, acetic acid,
and lactic acid as organic fermentation products.
Metabolic engineering of end-product metabolism in T. saccharolyticum is described with the goal of increasing the yield of
ethanol in lieu of organic acids. Using targeted gene knockout in conjunction with a high-frequency gene transfer system, a
strain (ALK1) has been created that produces ethanol as the only detectable fermentation product. Elimination of acetate
production is of particular note in that this involves significant changes in carbon and electron flux, ATP yield, and because
stoichiometric production of ethanol was achieved in the absence of pyruvate decarboxylase. Notwithstanding the substantial metabolic changes accompanying elimination of organic acid production, strain ALK1 grows at a rate similar to the
wild-type with slightly reduced cell yield. Results will be presented from experiments aimed at thermophilic simultaneous
saccharification and fermentation (SSF) and evaluating the ability of the engineered strain to produce ethanol at high titer
T. saccharolyticum strain ALK1 is attractive for use in an SSF process configuration, since the elevated fermentation temperature of this organism lowers the requirement for added cellulase. In addition, the ability of this strain to utilize xylose and
other five carbon sugars makes it attractive for use in consolidated bioprocessing in conjunction with a cellulolytic thermophile such as Clostridium thermocellum.
59
The Engineering of the Solvent-tolerant Pseudomonas putida S12 for the Production of
Phenol and Other Aromatic Compounds
Jan A. M. de Bont*, Nick J. P. Wierckx, Karin Nijkamp and Jan Wery
TNO Quality of Life, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands
We develop bioprocesses for the production from sugar of aromatic model compounds such as phenol (1) and cinnamic
acid (2) and of other aromatics that are of industrial importance. A major drawback in terms of the economics of biobased
processes is the susceptibility of host microorganisms to the toxicity of aromatic compounds. We use the solvent-tolerant
organism Pseudomonas putida S12 as a production platform because it has evolved several mechanisms to deal with toxic solutes, including modifications of the inner and outer membrane and active extrusion of a broad range of compounds.
Phenol is one of the most economically important hydroxylated aromatic compounds. With an annual production of more
than 7 million tons, it is utilized mostly for the production of biphenol A and phenolic resins. Currently, the main production
method for phenol is the chemical oxidation of cumene, which is produced from benzene. This process is very energy
intensive and produces toxic waste, and an hazardous explosive intermediate is involved. Furthermore, a sharp increase
in demand and a dramatic rise in oil prices have prompted us to seriously address the possibility of a “green” production
method for phenol. As for the near future, other aromatics of higher added value are economically more attractive.
The conversion of de novo-synthesized tyrosine into phenol in the solvent-tolerant host P. putida S12 was achieved by introduction of the tpl gene from Pantoea agglomerans. This gene encodes the pyridoxal 5-phosphate-dependent enzyme tyrosine phenol
lyase (TPL) (EC 4.1.99.2), which catalyzes the formation of phenol, pyruvate, and ammonia from tyrosine. We optimized the production host by a combination of targeted genetic alteration, random mutagenesis, antimetabolite selection, and high-throughput
screening. A further increase in phenol production was achieved by fed-batch cultivation in a biphasic medium-octanol system.
Current optimization work as based on comparative genomics (transcriptomics, proteomics) and reverse metabolic engineering will be reported for the production from glucose of compounds such as phenol and cinnamic acid.
(1) Wierckx et al (2005) Engineering the solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose. Appl. Environ. Microbiol.
71, 8221-8227.
(2) Nijkamp et al (2005) The solvemt-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose. Appl. Microbiol.
Biotechnol.; In Press.
Development of succinic acid producing Zymomonas mobilis strain
Jae Young Kim, Jeong-Hyun Kim and Hyonyong Chong*
Macrogen, 10F, World Meridian Venture Center, Kumchun-ku, Gasan-dong 60-24, Seoul 153-081, South Korea
Succinic acid overproducing Zymomonas mobilis strains have been developed by disruption of two genes for pyruvate
decarboxylase (pdc) and lactate dehydrogenase (ldh). It shows high yields of succnic acid production at close to the estimated
theoretical maximum with relatively low biomass (1.73 mole/mole glucose). The succinic acid production yield is more than 30%
increase comparing those of other succinic acid producing bacteria such as Actinobacillus succinogenes, and Mannheimia
succiniciproducens (about 1.34 mole/mole glucose). Recombinant Z. mobilis also shows higher specific rates of sugar uptake
and succinic acid production (1.62 g/l/h) than those of other succinic acid producing bacteria (1.35 g/l/h) under Na-bicarbonate
supplemented conditions. These characteristic higher yields and production rates of recombinant Z. mobilis strains make the mass
production of succinic acid from biomass possible which is much economic comparing chemical synthesis of succinic acid.
60
Production of hydrogen and heterologous hydrogenase in metabolically engineered Escherichia coli strains
Juanita Mathews, Quanzi Li, Richard Boyer and Guangyi Wang*
University of Hawaii at Manoa, Honolulu, HI 96822
Microbes have diverse biosynthetic pathways to produce molecular hydrogen and potentially hold the key to the viable
macroscale utilization and production of hydrogen from the renewable source. However, low production yield has been a
major limiting factor for large-scale biohydrogen production because of various metabolic bottlenecks. Using metabolic
engineering approach, we have engineered several E. coli strains for production of hydrogen and a functional hydrogenase. Hydrogenase 1, 2, 3, 4, and the hyc repressor of E. coli were sequentially deleted from the genome. Deletion
of hydrogenase 1 and 2 in conjunction with an over-expression of hydrogenase 3 produced significantly more hydrogen
than wild-type E. coli in an anaerobic batch fermenter. Also, the creation of a hydrogenase null E. coli strain allowed the
functional expression of the Ni-Fe hydrogenase (RH) from Ralstonia eutropha. This presentation will go over the results of
our study and discuss the possible applications of this knowledge towards developing a commercially efficient hydrogenproducing strain of E. coli.
oral presentation STA-01
GIS-based Fuelshed Analysis: Net Energy Yields and Economics of Cellulosic and Grain Biomass Production
Michael P. Russelle*
USDA-Agricultural Research Service, Saint Paul, MN 55108
Adam S. Birr, Minnesota Department of Agriculture, Saint Paul, MN 55155-2538
Douglas G. Tiffany, University of Minnesota, Saint Paul, MN 55108
Comprehensive planning for a biomass energy facility requires knowledge of fuel supply and physiographic characteristics of the fuelshed. This is especially true for herbaceous materials, which entail higher transportation costs. Perennial
forage production and corn stover harvest increase net energy yields, while perennials offer additional income streams
from environmental benefits. Crops differ, however, in the energy requirements and economics of production. We used a
GIS-based approach to evaluate the yield of harvested energy (i.e., before processing) of four biomass crops, both in the
field and when delivered to a central facility in a proposed fuelshed. Economic evaluations were conducted on competing
crop rotations.
The model fuelshed was centered on Madelia, Minnesota, site of several biomass-based initiatives. Maps of energy yield
were based on expected crop yields from the Soil Survey Geographic (SSURGO) database. Gross energy in harvested
soybeans was one-half that of corn grain, corn stover, or alfalfa hay, which yielded between 10 and 50 Gcal/ha. With
removal of all grain and 50% of corn stover, the net energy gain from a corn-soybean rotation would be only one-half that
of a rotation of four years of alfalfa, two years of corn, and one year of soybean.
61
More than Just Carbon Dioxide: Nitrous Oxide is the Dominant
Greenhouse Gas in Corn Based Biofuels and Bioproducts
Seungdo Kim and Bruce E. Dale*
Michigan State University, Department of Chemical Engineering & Materials Science
East Lansing, MI 48824-1226
Nitrous oxide (N2O) emissions from soil (via microbial nitrification and denitrification pathways) play a dominant and hitherto underappreciated role in overall greenhouse gas emissions associated with corn cultivation for biobased fuels and
chemicals. It may be correct to say that corn derived fuels and chemicals are carbon neutral, but that observation may
be nearly irrelevant if nitrous oxide emissions are significant. Nitrous oxide emissions from soil depend heavily on physical properties of soil, climate, application rate of nitrogen fertilizer, quantity of crop residues incorporated with soil, and
so forth. Thus, this study investigates nitrous oxide emissions and soil organic carbon level at corn farming sites in thirty
different counties in eight states (i.e., Indiana, Illinois, Iowa, Michigan, Minnesota, Missouri, Nebraska and South Dakota).
Each county is adjacent to an ethanol plant (either a dry or wet mill).
Soil organic carbon and soil nitrogen dynamics are estimated by the DAYCENT model. We estimate nitrous oxide emissions from soil when corn stover is removed and when winter cover crops are planted after harvesting corn grain. These
two practices would significantly reduce nitrous oxides from soil, eventually lowering greenhouse gas emissions associated with biomass production. Furthermore, we also estimate effects of these two agricultural practices on the greenhouse
gas profile of the overall ethanol production system.
The functional unit is defined as one hectare of cornfield. The system boundary includes biomass production, transportation of biomass to a biorefinery, biorefinery operations, transportation of ethanol to users, and ethanol fueled vehicle
operation. The allocation procedures for external functions are done by the system expansion approach. Thus, the system
boundary is expanded to include the avoided product systems (e.g., gasoline fueled vehicle operation, etc.). Ethanol is
used as a liquid fuel in a midsize passenger vehicle in the form of E10 (a mixture of 10 % ethanol and 90 % gasoline by
volume).
62
Reducing the environmental footprint of NatureWorks® Polylactide (PLA) Polymers
a
Erwin T.H. Vinka,*, David A. Glassnerb, Jeff Kolstadb, Robert Wooleyb
NatureWorks B.V., Gooimeer 6-10, 1411DD Naarden, The Netherlands, [email protected]
b
NatureWorks LLC, Minnetonka Boulevard, Minnetonka, Minnesota 55345, USA
NatureWorks® polylactide (PLA) is a versatile polymer made entirely from annually renewable resources. NatureWorks®
PLA applications include rigid packaging, films, disposable service ware and bottles. IngeoTM PLA fibers are used in
fiberfill applications and apparel. NatureWorks LLC currently has a manufacturing capacity of 140,000 metric tons of PLA
annually in Blair, Nebraska.
NatureWorks objectives are to eliminate the use of non renewable energy and the emissions of greenhouse gasses,
minimize non valuable co-products and reduce water use. As of January 1, 2006 all of the fossil-fuel-based electricity
used in NatureWorks facilities was replaced by wind power via the purchase of renewable energy certificates (REC). This
significantly reduced the use of non-renewable energy as well as greenhouse gas emissions. With the purchase of additional certificates to offset the remaining cradle-to-gate greenhouse gasses NatureWorks® PLA became the first and only
greenhouse gas neutral polymer world wide. The paper will explain the improved LCA for PLA along with the principle of
RECs and give the results for various impact categories.
In the near-future further improvement of the process technology will be combined with the utilization of renewable energy
for electricity requirements. Finally, the long term opportunity to improve the sustainability profile of PLA is to improve the
sustainability of carbohydrate feedstocks. Collecting and utilizing residues left in the field today, in a sustainable way, will
significantly improve the sustainability profile. NatureWorks® PLA also offers new possibilities to use polymer waste disposal/recycling options such as chemical recycling, industrial composting and anaerobic digestion. The paper will present
our projections for these improvements.
®
: Registered Trademark of NatureWorks LLC
: Trademark of NatureWorks LLC
TM
63
Using Life Cycle Assessment to Develop Sustainable Bio-Refineries
Robin Jenkins*, Carina Alles, Jamie Ginn, David Culver, Bruce Vrana, Robert Sylvester
(DuPont Engineering Research & Technology, Wilmington DE)
Julie Friend, Mark Emptage, Susan Hennessey
(DuPont Central Research and Development, Wilmington DE)
DuPont is leading the way towards demonstrating the feasibility of an Integrated Corn Bio-Refinery (ICBR) concept. In
the ICBR, a fermentation process is used to transform renewable resources such as corn biomass into ethanol and valueadded chemicals. DuPont received matching funds from the U.S. Department of Energy (DOE) for this program and
is partnering with Diversa Corporation, the National Renewable Energy Laboratory (NREL), Michigan State University
(MSU), and Deere & Company.
A sustainable ICBR will be both economically and environmentally favorable. In collaboration with MSU, DuPont is guiding ICBR project research to a more environmentally friendly design through the use of Life Cycle Analysis (LCA).
LCA is a holistic tool used to evaluate a product or process along the entire value chain. With an environmental perspective, LCA is integrating with ICBR process development by evaluating alternatives, identifying improvement opportunities,
and focusing on the high impact areas.
This presentation will reveal how LCA methodology is incorporated into the overall ICBR process development efforts.
Life Cycle Evaluation of Four Lignocellulosic Ethanol Pretreatment Technologies
Sabrina Spatari, Heather MacLean
Department of Civil Engineering, University of Toronto, Toronto, ON M5S 1A4
A life cycle inventory model is developed comparing four pretreatment and hydrolysis/fermentation options proposed for
producing lignocellulosic ethanol at commercial scales in North America. From the analysis, a set of metrics is identified
to compare the environmental and energy aspects of the four technologies for their near term development. We consider three feedstocks: switchgrass, a herbaceous crop, and corn stover, an agricultural residue, crops that are native
to North America, and likely candidates for this emerging technology; and Douglas fir, a softwood residue abundant in
Western Canada. Four pretreatment technologies combined with a hydrolysis/co-fermentation (SSCF) process are to be
compared for the near-term analysis. A more technologically advanced pretreatment combined with an advanced hydrolysis/ fermentation method, known as consolidated bioprocessing (CBP), which may develop in the mid-to-longer term
(between 2020-2030), is then compared to the near-term technologies in order to estimate the potential for reducing total
energy input to production and environmental emissions to the environment.
64
Energy and Greenhouse Gas Emission Impacts of Fuel Ethanol in the U.S.
Michael Wang
Center for Transportation Research, Argonne National Laboratory
9700 South Cass Avenue, Argonne National Laboratory, ESD362/E337, Argonne, IL 60439
(630) 252-2819; [email protected]
In 2005, the U.S. used 4.2 billion gallons of fuel ethanol for blending with gasoline. Virtually all fuel ethanol in the U.S.
is now produced from corn. Use of corn-based ethanol has been questioned for its energy and environmental effects.
Some past studies maintained that corn-based ethanol has a negative energy balance value (which means that more fossil energy is required to produce ethanol than the amount of energy in produced ethanol available for use) and results in
increases in greenhouse gas (GHG) emissions relative to petroleum gasoline.
Since 1996, the Center for Transportation Research of Argonne National Laboratory has been evaluating fuel ethanol’s
energy and GHG emission impacts as part of its overall efforts of evaluating well-to-wheels energy and emission effects
of various advanced vehicle technologies and transportation fuels. Argonne’s analysis, in agreement with many other
recently completed studies, concludes that corn-based ethanol achieves energy and GHG emission reduction benefits,
relative to conventional gasoline. This is mainly because of 1) improved corn productivity in U.S. corn farms in the past 30
years; 2) reduced energy use in ethanol plants built since mid-1990s; and 3) appropriately addressing ethanol co-products. Furthermore, cellulosic ethanol that can be produced from feedstocks such as woody or herbaceous biomass has
much larger energy and GHG emission reduction benefits.
This presentation will show updated results regarding ethanol’s energy and emission effects. In addition, the presentation will discuss ethanol’s role in a broad energy and emission perspective and the differences in ethanol’s results among
completed studies.
oral presentation STB-01
Current Aspects and Future Prospects of Bioenergy in Korea
Jin-Suk Lee and Soon-Chul Park
Biomass Center, Korea Institute of Energy Research, 71-2 Jang-dong Yuseong-ku Daejeon, 305-343 Korea.
Tel. 82-42-860-3553, Fax. 82-42-860-3739, E-mail. [email protected]
Due to the high oil price and global warming problems, bioenergy recently emerges in Korea as a promising option to
overcome the problems. Active works have been carried out to develop the bioenergy technologies which are good for the
situation in Korea. Unlike other countries, few biomass resources are available in Korea for bioenergy. Therefore, Korean
R&D activities have been focused on the development of bioenergy technologies which were mostly needed in Korea.
Some commercial technologies have been developed and currently on implementation. One of the commercialized technologies is the anaerobic digestion of food wastes. The other is biodiesel. Detailed information will be given to describe
the Korean R&D activities on those technologies. Other technologies, which are either close to the commercialization or
still under development, will be also mentioned in the presentation.
65
Recovery of acrylic acid produced from sugar cane using simulated moving bed adsorption
Machado, A.B*.;2Alvarez, M.E.T.; 3Pinho, A. P.A.; 4Scheer, A P; 5 Wolf Maciel, M.R.;
Laboratory of Separation Processes Development, School of Chemical Engineering, State University of Campinas,
Campinas– Brazil, [email protected]
1
Acrylic acid might become an important target for fermentative production from sugar cane, as an alternative carbon
sources to its current production from petrochemical.
The adsorption process is in the context of ecologically correct processes, comparing with other separation processes
like distillation, especially due to its low energy consumption and to its usefulness for thermally sensitive materials.
These simulations were carried out with the developed rigorous simulator, named ADMOV. The procedure to model
simulated moving bed adsorption column is to take series of fixed bed columns, with constant feeding and in pulse. The
answers and the analyses of the behavior can be obtained through the concentration profiles of throughout the column,
in fixed positions and time. The typical rupture curves are generated at the column exit which enables the analysis of
bed saturation and behavior of the adsorbate. The results depend on adsorption equilibrium constant, on axial dispersion
coefficient and on the mass transfer coefficient. Peaks of elution of the binary and multi component mixtures, applying
different numerical techniques, are also shown, as well as the effect of the variation of mixture feed composition, of the
column length and of the superficial speed.
66
Bioethanol and lactic acid production from lignocellulose; R&D progress in The Netherlands
Hans Reith, Herman den Uil, Arjen Boersma, Jarno Kuijvenhoven
Energy Research Centre of the Netherlands (ECN), P.O. Box 1, 1755 ZG Petten, The Netherlands.
Jan de Bont*, Johan van Groenestijn, Hugo van Buijsen, Doede Binnema, Gijsbert Bos, Jan Zeevalkink,
TNO, P.O. Box 342, 7300AH Apeldoorn, The Netherlands
Mirjam Kabel, Henk Schols, Rik Beeftink,
Wageningen University, P. O. Box 8129, 6700 EV Wageningen, The Netherlands
Robert Bakker, Ruud Weusthuis, Ronald Maas, Ed de Jong,
Agrotechnology and Food Innovations, P. O. Box 17, 6700 AA Wageningen, The Netherlands
Wim de Laat, Royal Nedalco, P. O. Box 6, 4600 AA Bergen op Zoom, The Netherlands
Diana Visser, Purac biochem, P. O. Box 21, 4200 AA Gorinchem, The Netherlands
Hans Haan, Shell Global Solutions International, P. O Box 38000, 1030 BN Amsterdam, The Netherlands
In the Netherlands a 4-year R&D project (2002-2006) has been performed with the objective to develop and evaluate
technologies for the use of lignocellulose as feedstock for bioethanol and lactic acid production. Wheat straw was selected as the model feedstock. The preparation of fermentable sugars from lignocellulose is a major challenge for both
bioethanol and lactic acid production. This requires integral optimization of the trajectory from feedstock through fermentation, as addressed in this project. Major R&D themes in the project are:
- Physical/chemical pre-treatment for mobilization of (hemi)cellulose from the lignocellulose matrix; either via mild
acid pressurized hot water or mild alkaline pre-treatment
- Optimization of enzymatic cellulose hydrolysis with commercially available enzymes
- Use of lignocellulose hydrolysates for ethanol and lactic acid fermentation
- Power and heat production from non-fermentable biomass fractions, including evaluation of the use of biomass
ashes as secondary building material or fertilizers
- System evaluation and integral process plant design including economic and ecological evaluation
- Application of bioethanol in blends with fossil fuels.
The optimization steps in the project have led to a concept for the production of ethanol from straw. In pilot scale trials,
ethanol was produced from straw at the scale of several liters.
67
The Potential for Cellulosic Ethanol Production in China
Bin Yang1*, Yanpin Lu1, Junshe Sun2 and Su Donghai2
2
Department of Bioengineering, China Agriculture University, Beijing, China 100083
1
China now faces very serious energy shortages and environmental pollution problems. Thus, the Chinese government
is encouraging ethanol use as an alternative transportation fuel by introducing fuel ethanol production and distribution in
several provinces. Although the current emphasis is on ethanol production from corn and other grains, China has huge
quantities of low cost cellulosic biomass such as corn stover, wheat straw, and rice hulls that could significantly expand
ethanol production volume and reduce feedstock costs. However, a number of important issues mush be addressed
including: Is there actually enough biomass to make a significant impact on energy supplies in China? How could cellulosic ethanol benefit China’s energy security? Is production of cellulosic ethanol technically and economically feasible in
China? Are appropriate technologies available for China? If not, what are the economic impacts if we use the best potential US technologies apply to Chinese market? This presentation will analyze the situation with regard to these questions
to provide a perspective for government, industry, and academics of the benefits that cellulosic ethanol could potentially
offer now and in the future.
68
Enzymatic Modification of Spruce Hemicellulose, O-Acetyl Galactoglucomannan
Anderson L.a, Andersson A.a, Peterson Ra Zacchi G.b, Le Nours J.c, Lo Leggio L.c,
Larsson Ad, Ståhlberg Jd & Stålbrand H.a
a, b
Departments of Biochemistrya, and Chemical Engineeringb, Lund University, Sweden
c
Center for Chrystallographic studies, University of Copenhagen, Denmark
d
Department of Molecular Biology, SLU and Uppsala university, Sweden
Hemicellulose is the second most abundant group of plant polysaccharides. Although, minor quantities of hemicellulose
is present in many paper products, it has no broad application on its own merits. However, the interest of using hemicellulose as a renewable resource for new polymer products is currently increasing. Knowledge of the polymers structure
and properties is therefore an important contribution to this development. We are studying the major softwood hemicellulose: O-acetyl galactoglucomannan (GGM). It is a complex branched heteropolysaccharide containing an O-acetylated
β-(1→4) linked glucomannan backbone with α-(1→6)-D-galactosyl side groups attached to some of the mannosyl units.
We have developed a method for the water extraction of GGM from spruce chips by microwave heat treatment and
further fractionation using size exclusion chromatography (1). The time, temperature and pH conditions during the extraction of GGM influence the molecular weight, substitution and yield of the extracted material (2). We are also studying
the enzymes (glycoside hydrolases) which catalyse hydrolysis of the GGM backbone (β-mannanase, β-mannosidase)
and galactosyl side-groups (β-galactosidases) (3,4). These enzymes are useful tools in the characterisation of GGM
and promising tools in the structural-functional modification of GGM for novel applications. Structural information about
the enzymes is important for knowledge about how the hydrolysis occurs. Two recently solved 3D structures of family 5
(MeMan5A) and family 26 β-mannanases (CfMan26A) will be discussed with a focus on implications on enzyme-substrate interactions, important for specificity and recognition of the different substrate monomers (5). These enzymes
displays a double-displacement mechanism, and they thus theoretically can catalyse transglycosylation reactions which
potentially are useful for the biosynthesis of new hemicellulose-based glyco-conjugates. However, only MeMan5A was
shown in practise transglycosylate and CfMan26A did not. Fine structural differences in the subsite organistation may
be the cause for this observation. Furthermore, the β-galactosidase B (AglB) from Aspergillus niger was used for partial
side group hydrolysis of water-soluble GGM. The effect on polymer properties such as rheology and solubility will be
discussed.
References:
1. Lundqvist, J., Teleman, A., Junel, L., Zacchi, G., Dahlman, O., & Stålbrand H. (2002) Carbohydrate polymers, 48, 29-39
2. Lundqvist J., Jacobs A., Palm M., Zacchi G., Dahlman O., & Stålbrand H. (2003). Carbohydrate Polymers, 51, 203-211.
3. Ademark P., Larsson M., Tjerneld F. & Stålbrand H. (2001). Enzyme and microbial Technology, 29, 441-448.
4. Ademark P. de Vries R. P. Hägglund P. Stålbrand H. & Visser J. (2001) European Journal of Biochemistry, 268, 2982-2990
5. Le Nours J., Anderson L., Stoll D., Stålbrand H. & Lo Leggio L.(2005) Biochemistry, 38, 12700-12708.
69
New Improvements for Lignocellulosic Ethanol (NILE), an integrated project in FP6 of the European Commission
Frédéric Monot*, IFP, Institut français du pétrole, 92852 Rueil-Malmaison, France
NILE is a 4-year integrated project with the overall objective of developing cost effective production of clean bioethanol
from lignocellulosic biomass (LCB), to enable its use as a transport biofuel. NILE will develop, investigate and evaluate
new technologies for efficient conversion of lignocellulose to bioethanol. The activities of NILE aim at overcoming critical
hurdles in process development. Key issues are:
− decreasing the cost of enzymatic hydrolysis of lignocellulose to fermentable sugars using new engineered enzyme
systems;
− removing current intrinsic limitations in the conversion of fermentable sugars to ethanol by i) constructing inhibitor-tolerant pentose-fermenting industrial yeast strains, and ii) combining fermentation and saccharification;
− validating the engineered enzyme systems and yeast strains in a fully integrated pilot plant using softwood and wheat
straw as model feedstock. Validation includes all process steps and recycling of process streams;
− analysing socio-economic and global environmental impacts of the production and use of bioethanol from LCB based
on the new data obtained;
− dissemination and training of target groups.
The project mobilizes the critical mass of expertise necessary to improve the whole bioethanol from LCB chain, involving
21 partners from 11 countries, including industrial companies, research Centres and Universities.
70
Influence of the scale-up process on a Saccharomyces cerevisiae fed-batch process:
Modeling and experimental investigations
Lejeune A., Delvigne F.*, Thonart P.
Faculté Universitaire des Sciences Agronomiques, Unité de bio-industries, CWBI,
Passage des Déportés, 2, 5030 Gembloux, Belgium
Bioprocesses scaling-up procedures are not a simple task owing to the interactions between the microorganisms and the
extracellular environment generated by the bioreactor mixing performances. From a descriptive point of view, there are
three components to take into account: the concentration gradient field developed by the bioreactor mixing operation, the
movements of the microorganisms induced by the circulation flow developed by the impeller system, and the physiological
states modification of the microorganisms in response to the environmental perturbations experienced in the bioreactor.
The first two components (i.e., the mixing and the circulation processes developed by the impeller system of the bioreactor) are mainly influenced by the bioreactor scale and operating conditions and are thus directly affected by the scaling-up
procedure. A stochastic hydrodynamic model has been developed and permits to obtain the environmental fluctuations
encountered by the microorganisms by superposing the concentration gradient field obtained by mixing simulation and
the circulation process. The third component (i.e., the physiological responses of the microorganisms in front of the environmental perturbation experienced) is more difficult to modelise and experimental data are needed. These experimental
data have been obtained by performing some experiments in scale-down reactor (SDR). The SDR is a reactor especially
designed to reproduce at small scale the hydrodynamics encountered in industrial scale reactor and comprises a stirred
vessel (20 litters) connected to a pipe section, this last section representing the bad mixed zones. As a case study, the
fed-batch culture of Saccharomyces cerevisiae has been chosen. Biomass yield drop from 0.5 to 0.35 can be observed
by comparing S. cerevisiae cultures in small-scale stirred bioreactor and in SDR. The stochastic simulations of the concentration encountered by the yeast cells during their sojourn in the SDR show that higher environmental perturbations
are perceived by the cells when the circulation and the mixing performances of the whole system drop.
In front of these SDR tests, perspectives about the elaboration of a general model taking into account the three components (i.e., the mixing, the circulation and the physiological components) are discussed.
71
An innovative membrane bioreactor for Very High Ethanol Performances
F. Ben Chaabane, A.S. Aldiguier, S.Alfenore, C. Bideaux, X. Cameleyre, S. Guillouet, C. Molina-Jouve
LBB, UMR CNRS 5504, UR INRA 792, INSA, 135 avenue de Rangueil, 31077 Toulouse, France
G. Roux, LAAS-CNRS, 7 avenue du colonel Roche, 31077 Toulouse, France
e_mail: [email protected], tel: +33 5 61559447 fax:+33 5 61559400
To achieve the european goals, the production of ethanol as biofuel must be increased from 0.5 Million tons in 2003 to 7.5
Million tons in 2010. Technological breakthroughs are necessary to define innovative bioprocess in order to optimize the
ethanol productivity, the ethanol concentration and/or the conversion yields.
An innovative bioreactor configuration with membrane cell recycling was developed in the Biotechnology and Bioprocess
Laboratory for continuous ethanol production (Figure 1) It was deduced taking into account the management of growth,
ethanol production and cell viability. The first stage was dedicated to the cell growth under non oxygen limitation. The
ethanol concentration targeted in this stage had to be low enough to limit the ethanol inhibition to the growth and to favour
the vitamin assimilation. The second stage was dedicated to the ethanol production, in a microaerated reactor, coupled to
an ultrafiltration module to reach very high cell density and ethanol concentration. The innovation was related to the recycle loop from the second stage to the first one : the deal was to manage the cell activity by the biomass transfer from the
second stage where very stressful conditions took place (high biomass and ethanol concentration) to the first stage where
the ethanol concentration was maintained low in order to limit growth and production inhibition by ethanol.
Vitamin
Feed
Qv
Qm
Medium
Feed
Qw
Water
Feed
Sfmax Q
s
Substrate
Feed
gas
gas
Sf Q f
Q21
Xt2, Xv2
Xt1, Xv1
S1
S2
P1
P2
G1
G2
Ac1
Ac2
Suc1
Suc2
Q
P
Permeate
Q12
air
Reactor 1 (R1)
Q pg
Bleed
Ultrafiltration
Reactor 2 (R2)
Figure 1. Schematic diagram of the two-stage reactor for the continuous ethanol production.
Saccharomyces cerevisiae CBS 8066 cultures were performed on mineral medium, at 30°C and pH 4. In continuous steady
state, an ethanol productivity and concentration above 40 g.L-1.h-1 and 64 g.L-1 were achieved with an ethanol yield on glucose
near 0.44 g.g-1 and a residual concentration in substrate equal to zero. Compared to the industrial process productivity,
these results are 15 to 20 fold higher. We developed a model to predict the dynamic behavior of the yeast during cultures in
this two-stage bioreactor and to quantify the influence of the operating parameters on its performances.
72
Economical evaluation of extraction of hemicelluloses from process streams
from thermo-mechanical pulping of spruce
Tobias Persson, Ann-Sofi Jönsson, Guido Zacchi*
Department of Chemical Engineering, Lund university, Sweden
More research and development is needed on the utilization of biomass to decrease the consumption of fossil raw materials in the future both for fuels, chemicals and materials. Around 80% of the biomass on earth is lignocellulosic materials
i.e. cellulose, lignin and hemicelluloses.
In this study the possibility to use an industrial separation process that extracts hemicelluloses from process streams from
thermo-mechanical pulping of spruce has been developed. The process consists of three steps, removal of high molecular species by filtration, preconcentration of hemicelluloses by ultrafiltration and reduction of low molecular species by diafiltration. Pretreatment by adjusting the pH and ionic strength prior to filtration to increase the recovery will be evaluated.
Several ultrafiltration membranes will be compared regarding flux, fouling and retention and their influence on the process
economy. Different operating conditions in the three process steps will be studied from a process-economic perspective.
Cost estimations will be presented both regarding investment and operational costs for the entire process. The advantages and disadvantages by using different methods and materials will also be discussed.
Scale-Up of Stirred Tank Reactors using CFD Simulations
John Hannon and Charles E. Wyman*
Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755-8000
Phone: 617-513-7092, Email: [email protected]
Scaling up biochemical processes from laboratory scale (1-10 L) to pilot scale (100-1000 L) to industrial scale (10,000–
1,000,000+ L) presents a significant engineering challenge because nearly every mixing factor changes. Typical scaleup techniques include maintaining one parameter constant such as 1) impeller tip speed, 2) Reynolds number, 3) mixing
time, or 4) the ratio of power number to volume. However, all other factors change, raising questions about changes in
performance with scale. Because impeller tip speed is often kept constant to avoid potential shear sensitivity effects on
fermentative organisms, a computational fluid dynamic (CFD) model was applied to estimate the effect of this strategy
on mixing performance, flow path, and power number differences during scale up. Laboratory, pilot, and industrial scale
vessel configurations equipped with four, 90 degree baffles and one 45 degree pitched blade turbine (PBT) impeller were
analyzed and compared to flow path data using laser doppler anemometry (LDA) and particle image velocimetry (PIV) at
various Reynolds numbers where available in the literature. A CFD simulation will also be presented of an actual industrial
scale fermentor described in the literature.
*Charles E. Wyman, PhD, Ford Motor Company Professor of Chemical and Environmental Engineering and Adjunct Professor of Engineering at
Dartmouth College, Bourns College of Engineering, Center for Environmental Research and Technology (CE-CERT), University of California, Riverside,
California 92521
73
Procedure for Development of a Robust Mathematical Model for Alcoholic Fermentation Process
a
Andrade, R.R a ; Costa, A.C* a ; Maciel, R a ; Atala, D.P b; Maugeri. F b
Department of Chemical Processes, School of Chemical Engineering, State University of Campinas, Campinas - Brazil
b
Department of Food Engineering, School of Foof Engineering, State University of Campinas, Campinas - Brazil
e-mail: [email protected]
Among the main current problems related to industrial ethanol production is the lack of robustness of the fermentation in
presence of fluctuations in the quality of raw material, which leads to changes in the kinetic behavior with impact on yield,
productivity and conversion. This lack of robustness can be taken into account through operational and control adjustments, which can be aided by the use of an accurate mathematical model.
In this work, a procedure to develop a robust mathematical model, valid in a large range of operational conditions, was
established. This model considers the effect of temperature on the kinetics. Experiments were performed in batch mode
and conditions for fermentation were determined through a factorial design 22 + axial points and four replications at the
center points. The microorganism used was Sacharomyces cerevisae and the culture media, sugar-cane molasses.
After the development of the model, it was evaluated to describe a fed batch experiment. This experiment was performed
in different conditions, with raw material cropped in another period of time to prepare the culture media and other lineage
of microorganisms. The objective is to assess the difficulty in updating the kinetic parameters when there are changes in
fermentation conditions.
Pervaporation Membrane Systems for Volatile Fermentation Product Recovery and Dehydration
Leland M. Vane, Ph.D.
Chemical Engineer, U.S. Environmental Protection Agency, 26 W. Martin Luther King Dr., M/S443, Cincinnati, OH 45268
Ph: 1-513-569-7799, Fax: 1-513-569-7677, e-mail: [email protected]
The economics of fermentative production of fuels and commodity chemicals can be a strong function of the efficiency
with which the fermentation products are removed from the biological media. Due to growth inhibition by some fermentation products, including ethanol, concentrations of these products in fermentors are generally on the order of 1 to 10
percent by weight (10 to 100 grams per liter). These low concentrations greatly increase product recovery costs using
traditional separation processes, such as distillation. Furthermore, the removal of trace water from the fermentation
products to meet commercial specifications may require relatively large amounts of energy. Membrane-based pervaporation technology has been proposed as an energy saving alternative to traditional technologies, especially for smaller
systems. In pervaporation, compounds permeate through a nonporous or molecularly porous membrane, and evaporate
into a vapor permeate stream. The properties of the membrane dictate the separation. For example, pervaporation can be
used for the dehydration of an organic stream, through the selection and use of a hydrophilic membrane. Alternatively, if a
hydrophobic membrane is used, organic compounds will preferentially pass through the membrane. Options for integrating pervaporation systems with fermentation processes and the state-of-the-art of pervaporation for this application will
be discussed.
*This is an abstract of a proposed presentation and does not necessarily reflect USEPA policy.
74
Enhancing the Successful Development of North American BioRefineries with
Integrated Regional BioProduct Innovation Alliances
Alex Kawczak- Vice President, Battelle Memorial Institute, Columbus, Ohio
The successful growth of existing and new North American “Biorefineries” that produce both biofuels and bio-derived
industrial products will become increasingly important to the global competitiveness of the agricultural and chemical
industries. The emergence of regional innovation alliances in North America will pave the path for the improved national
integration of research and commercialization alliances to create breakthroughs in next generation biopower, biofuels,
lower cost and higher quality commodity chemicals and advanced specialty chemicals and polymer systems. Primary
examples of regional research and development alliances include the Ohio BioProducts Innovation Center (OSU, Battelle,
OSC and industry) and the Washington State University/ Pacific Northwest National Laboratory Bioproducts Sciences and
Engineering Laboratory (BSEL) in Richland, Washington. These alliances derive benefits from collaborations with the National BioEnergy Center, headquartered at the National Renewable Energy Laboratory (NREL). The NBC was established
to support the science and technology mission of the U.S. Department of Energy (DOE) Biomass Program by integrating
key elements of research at the Oak Ridge National Laboratory, Idaho National Laboratory, Argonne National Laboratory,
Pacific Northwest National Laboratory and NREL.
This presentation proposes a vision of the “biorefinery of the future” which includes the strategic integration of sugar-platforms with plant-oil platforms to create novel “hybrid carbohydrate-oil platforms” that will be basis of globally competitive
“Bio-Verbunds” and “Biorefineries” in North America. The successful development and commercialization of these new
platforms will require the financially catalyzed integration of the regional bioproduct innovation centers with the USDA
research centers, DOE national laboratories, universities, and industrial corporate R&D programs. The proposed alliance
catalyst includes the collaborative development of a “National Biorefinery Technology Initiative” (biopower, biofuels and
bioproducts) that should be funded by industry and the federal government.
Biorefining challenges: Choosing technologies in establishing a lignocellulosic-based biorefinery
Warren Mabee, Jack Saddler, University of British Columbia, Vancouver BC, Canada
Recent discussions around the lignocellulosic-based biorefinery suggest two technological platforms as alternatives: the
biological platform and the thermochemical platform. However, most of the literature devoted to these platforms is highly
specialized and not suitable for general conclusions. Therefore, a trade-off analysis has been developed using two 2ndgeneration biofuels associated with these platforms, lignocellulosic-based ethanol and biosyn diesel. In this analysis, we
developed scenarios of 2nd-generation biofuel use, highlighting their ability to respond to major economic, environmental,
and social issues, and relating these fuels to the estimated available feedstocks. These scenarios are designed to examine the potential of these fuels, and the trade-offs between alternatives in terms of capital and processing costs, rural
development and employment, environmental returns, and biofuel availability. The study provides perspective on the ability of different technological platforms to meet policy goals within certain timeframes, and may be utilized to help financial
institutions, companies, and governments decide which platform is appropriate for meeting the needs of their respective
constituents.
75
Hybrid Thermochemical/Biological Processing: Putting the Cart before the Horse?
Robert C. Brown
Iowa State University, Center for Sustainable Environmental Technologies,
286 Metals Development Building, Ames, IA 50011
Tel: 515-294-7934, Fax: 515-294-3091, E-mail: [email protected]
The Biomass Research and Development Technical Advisory Committee of the U.S. Departments of Energy and Agriculture defines a biorefinery as:
“A processing and conversion facility that (1) efficiently separates its biomass raw material into individual components and (2)
converts these components into marketplace products, including biofuels, biopower, and conventional and new bioproducts.”
Implicit in this definition is the assumption that grains will be fractionated into starch, oils, proteins, and fiber and lignocellulosic crops will be fractionated into cellulose, hemicellulose, lignin, and terpenes before these components are converted into market products. Certainly, this is the approach of modern wet corn milling plants and wood pulp and paper mills.
Occasionally, these plants include a thermochemical step at the end of the process to convert recalcitrant plant components or mixed waste streams into heat to meet thermal energy demands elsewhere in the facility.
However, another possibility for converting high fiber plant materials is to start by thermochemically processing it into a
uniform intermediate product that can be biologically converted into a biobased product. This alternative route to biobased
products is known as hybrid thermochemical/biological processing. There are two distinct approaches to hybrid processing: (1) gasification followed by fermentation of the resulting gaseous mixture of carbon monoxide (CO), hydrogen (H )
2
and carbon dioxide (CO ), and (2) fast pyrolysis followed by hydrolysis and/or fermentation of the anhydrosugars found in
2
the resulting bio-oil. This paper explores this “cart before the horse” approach to a biorefinery.
Putting Advanced Biorefineries in Context: Integration with Mature Feedstock Supply Systems,
Processing Options, and Nitrogen Recycle
Mark Laser, Haiming Jin, Lee R. Lynd*, Thayer School of Engineering, Dartmouth College, Hanover, NH 03755
Shahab Sokhansanj, Oak Ridge National Laboratory, Oak Ridge, TN 37831
David Bransby, Department of Agronomy and Soils, Auburn University, Auburn, AL 36849
Eric Larson, Princeton Environmental Institute, Princeton University, Princeton, NJ 08544
When considering mature biorefining scenarios, it is instructive to analyze multiple links in the biomass-to-fuel supply
chain. Large scale feedstock supply systems (encompassing production, collection, storage, and transport ) will be defined and analyzed for production of switchgrass, a model cellulosic feedstock. Results will emphasize projecting the cost
and energy inputs for alternative mature feedstock supply systems. An extensive analysis of mature conversion technology options will be summarized, with options including stand-alone production of electricity, Fischer-Tropsch (FT) fuels,
dimethyl ether (DME), and hydrogen, as well as co-production scenarios involving ethanol-power, ethanol-power-FT fuels, ethanol-hydrogen, ethanol-FT fuels-natural gas, and several of these products in combination with feed protein. Most
of these options are compatible with recovering nitrogen in the form of ammonia, which can be returned to the field. Such
nitrogen recycle will be examined in terms of process technology, economics, and thermodynamics. Integrated scenarios
consisting of feedstock supply, conversion, and nitrogen recycle options will be assessed from an aggregated perspective
encompassing cost, efficiency, and environmental effects.
76
Integration of a Biorefinery Working at a High Dry Matter Content with a Power Plant.
Concepts and Feasibilities.
Jan Larsen*, Frank Iversen
Elsam Engineering A/S, Kraftværksvej 53, 7000 Fredericia, Denmark
Børge Holm Christensen
Sicco K/S, Odinshoejvej 116, 3140 Aalsgaarde, Denmark
Mette Hedegaard Thomsen, Anne Belinda Thomsen
Risø National Laboratory, Frederiksborgvej 399, 4000 Roskilde, Denmark
Henning Jørgensen, Claus Felby
The Royal Veterinary and Agricultural University (RVAU), Hoejbakkegaard Allé 1, 2630 Taastrup, Denmark
Elsam, the main power producer in Denmark, has in collaboration with Sicco, Risø National Laboratory and RVAU been
working on the EU-project “Integrated Biomass Utilization System”, referred to as IBUS, since 2002. The IBUS project
has developed technical, energy efficient and economical new solutions in pilot scale for production of ethanol from lignocellulose. This presentation will include an overview of the developed processes. It will emphasize the technical solutions
for a unique way of converting biomass into bioethanol at a high dry matter content of 20-40% in all steps from hydrothermal pre-treatment of large particles over enzymatic liquefaction and hydrolysis to fermentation. Process possibilities and
feasibility studies hereof.
A key element in the IBUS project is the integration of the current combined heat and power (CHP) plants with production
of liquid bio-fuel. The majority of the cellulose and hemicellulose in the lignocelluloses can be used for ethanol production,
and the remaining lignin can be used as a high-quality solid fuel in the CHP-plant to produce heat and power. Low-quality steam from the CHP-plant that cannot be used in the power production can be used in the ethanol production plant,
thereby improving the overall energy efficiency of the whole plant.
77
28
th
Poster abstracts
Poster 1A-09
Molecular cloning of a gene encoding the sucrose phosphorylase from
Leuconostoc mesenteroides B-1149 and its use for transglucosylation
Jun-Seob Ahn, Xing-ji Jin, Don-Hee Park, Hee-Kyung Kang, Doman Kim, Jin-Ha Lee*
Chonnam National University, Gwangju 500-110, Korea
Ghahyun J. Kim
Korean Minjok Leadership Academy, Gwangwon-do 225-823, Korea
Sucrose phosphorylase (EC 2.4.1.7) (SPase) belongs to family 13 of the glycosylhydrolases. It catalyzes the synthesis of
∂-D-glucose-1-phosphate (Glc-1-P) and D-fructose from sucrose and inorganic phosphate. Acceptor products by SPase
also have various potential applications as anticarigenic materials, probiotics, antibiotic substitutes and food ingredients.
There was a unique nucleotide sequence at the C-terminal ends of 1149SPase and lower similarity compared to other
Leuconostoc SPase genes.
Leuconostoc mesenteroides NRRL B-1149 SPase gene, 1149sp, was isolated and characterized. It is composed of 1,479
bp nucleotides and encodes a 1149SPase of 492 amino acid residues with a calculated molecular mass of 56.1 kDa. It
has unique C-terminal amino acid sequence (439DVETPSDTTIKITRKDKSGENVAVLVANAADKTFTITANGEE
ILANTEADKQQL492). 1149sp was expressed in Escherichia coli and the purified 1149SPase specific activity was 1.49
U/mg for sucrose. The optimum temperature and pH for SPase activities were ranged broad between 20 and 50ºC, between pH 6.0 and 7.5, respectively. The optimum temperature and pH were 37ºC at pH 6.7 and it showed Km of 6.3 mM
and kcat of 1.59/s for sucrose. It had a broad range of acceptor specificity and transferred the glucosyl moiety of sucrose or
glucose-1-phosphate to various acceptors.
Poster 1A-10
Enzymatic micro reactors for the determination of ethanol by an automatic sequential injection analysis system
Eliana Mossé Alhadeff1 *, Andrea Medeiros Salgado1, Oriol Cós2, Nei Pereira Jr.1, Belkis Valdman1, Francisco Valero2
1
Escola de Química, Centro de Tecnología, Universidade Federal do Rio de Janeiro, Ilha do Fundão,
Cidade Universitaria, CEP.: 21.949-900, Rio de Janeiro, Brasil; e-mail: [email protected]
2
Depto. de Enginyeria Química, Escola Técnica Superior d´Enginyeria, Universitat Autònoma de Barcelona,
08193 Bellaterra, Barcelona, Spain
An Automatic Sequential Injection Analysis (SIA) system was used to quantify ethanol based on the alcohol oxidase and
horseradish peroxidase enzymatic reactions with phenol and 4-aminophenazone. The coloured product could be detected with a colorimeter at 470 nm. Both enzymes were separately immobilized on glass aminopropyl beads, and packed
in two micro reactors, with 0.91 mL of void volume each, working in-line with the SIA system. For a detection range of 0 0.08 g ethanol/L the system showed good reproducibility of the response signal and a maximum mean absorbance value
of 0.112 ± 0.003. Two operational conditions were evaluated: continuous flow at 1.7, 3.3 and 7.4 mL/min and stop-flow
of 120 s at the alcohol oxidase immobilized micro reactor. The stop flow technique was selected, and a linear detection
range of 0 - 0.04 g ethanol/L with a relative standard deviation of 0.6 % and a sampling frequency of 7 analyses per hour
was obtained. The system was applied to measure ethanol concentrations in samples of distilled and non-distilled alcoholic beverages and for monitor an alcoholic fermentation. The results were compared with other analytical procedures
(GG and HPLC).
80
Poster 1A-11
Purification, Characterization, and Substrate Specificity Analysis of the Glycosyl Hydrolase Family 1 (Cel1b)
Enzyme of Trichoderma reesei
Mursheda K. Ali*, John O. Baker, Eric Knoshaug, Tina Jeoh, Michael E. Himmel and William S. Adney
National Renewable Energy Laboratory, Golden, CO 80401
The filamentous fungus, Trichoderma reesei, has been demonstrated to produce a diverse mixture of enzymes important
in the degradation of biomass. These enzymes include a total of seven confirmed or putative β-D-glucosidases. The
precise function and specificities of all of the reported T. reesei β-D-glucosidases has not been clearly established, nor
has their role in biomass conversion yet been clearly defined. In general, β-glucosidases are known to catalyze the hydrolysis of a variety of alkyl- and aryl-glucosides, as well as diglucosides and oligosaccharides. In this study, cel1b, a glycosyl hydrolase family 1 gene (GenBankTM accession no. AAP57758) was cloned from T. reesei using genomic DNA. The
gene was expressed in an Aspergillus awamori expression host under the control of the A. niger glucoamylase promoter.
Fusion of the Cel1b protein with a glucoamylase N-terminal signal peptide facilitated its extracellular secretion. The
rCel1b enzyme purified from the culture supernatant was found to have a molecular weight of approximately 55 kDa using
SDS-PAGE and an optimum temperature and pH of 50˚C and 3.0, respectively. The enzyme showed significant levels of
α-galactosidase and β-galactosidase activity.
Poster 1A-12
Optimization of cyclodextrin glucanotransferase production from Bacillus clausii strain E16
in submerged fermentation using response surface methodology
Heloiza F. Alves-Prado, Daniela A. Bocchini, Eleni Gomes, Roberto Da Silva
Biochemistry and Applied Microbiology Laboratory – IBILCE/UNESP, São José do Rio Preto, Brazil, CEP 15054-000
Luis C. Baida
Computation Sciences and Statistic Department – IBILCE/UNESP, São José do Rio Preto, Brazil
Inês C. Roberto
Faculty of Chemical Engineering of Lorena – Faenquil, Lorena, Brazil
Cyclodextrin glucanotransferase (CGTase) production from Bacillus clausii E16, a new bacterial isolated from Brazilian
soil sample was optimized in shake-flask cultures. A 24 full-factorial central composite design was performed to optimize
the culture conditions, using a response surface methodology. It was investigated the combined effect among the soluble
starch concentration, the peptone concentration, the yeast extract concentration and the initial pH value of the culture
medium. The optimum concentrations of the components, determined by a 24 full-factorial central composite design, were
13.4 g/L soluble starch, 4.9 g/L peptone, 5.9 g/L yeast extract and initial pH 10.10. Under these optimized conditions, the
maximum CGTase activity was 5.9 U/mL after a 48 hour fermentation. This yield was 65.4% higher than that obtained
when the microorganism was cultivated in basal culture medium.
81
poster 1A-13
Purification and characterisation of a cyclodextrin glucanotransferase (CGTase) from
Paenibacillus campinasensis H69-3
Heloiza Ferreira Alves-Prado, Eleni Gomes and Roberto da Silva
Biochemistry and Applied Microbiology Laboratory – IBILCE/UNESP, São José do Rio Preto, Brazil, CEP 15054-000.
A cyclodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) from a newly isolated alkalophilic moderate thermophilic
Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. CGTase
was produced in submerged fermentation at 45°C and purified by gel filtration on Sephadex G50, ion exchange using a
Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70
kDa by SDS-PAGE and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range
6.0 to 11.5. The optimum temperature was 65°C at pH 6.5, and it was thermally stable up to 60°C without substrate during
one hour in the presence of 10 mM CaCl2. The enzyme activity increased by the presence of Co2+, Ba2+, and Mn2+. Using
maltodextrin as substrate, the Km and Kcat were 1.65 mg ml-1 and 347.9 µmolmg-1min-1, respectively.
poster 1A-14
Electrochemical Regeneration of Cofactor For Continuous Bioreactor Product Formation
Michelle B. Arora*, Yupo J. Lin, Linda de la Garza†, Seth W. Snyder, Edward J. St.Martin
Argonne National Laboratory, Argonne, IL 60439
†
Current address: Valdosta State University, Valdosta, GA 31698
Within the active site of an enzyme, the cofactor shuttles electrons and protons to/from a substrate for conversion to a
chemical product. One cofactor is used for every product made. In a bioreactor, cofactors become exhausted, preventing
continuous enzyme activity and halting product formation. A method allowing cofactor regeneration would enable continuous product formation and maximize bioreactor production.
One method of cofactor regeneration involves electrochemical regeneration of the cofactor by controlling the transfer of
electrons and protons to/from the cofactor using electrodes in an electrochemical cell. A critical barrier to electrochemical
cofactor regeneration is the internal resistance of electron and proton transport caused by the physical gap between the
cofactor and the electrode. Most work considers electron transfer only, but true cofactor regeneration requires the transport of both protons and electrons. At Argonne, we have engineered a mixed electron/ion conductive matrix based on our
porous ion-exchange resin wafer (U.S. Patent #6,495,014). The novel matrix formed an electron/proton network that not
only physically bridged the gap between the cofactor and electrode but also allowed effective simultaneous transport of
both electrons and protons for cofactor regeneration. The novel matrix will provide a technology platform for continuous
product formation in cofactor-utilizing enzyme-based reactors.
82
poster 1A-15
Cellulolytic enzymes from streptomycetes isolated from Brazilian soil
André Luiz Grigorevski de Lima*1, Karin Willquist3, Rodrigo Pires do Nascimento1, Ayla Sant’Ana da Silva2, Elba P. S. Bon2
and Rosalie Reed Rodrigues Coelho1
1
Microbiology Institute and 2 Chemistry Institute of the Federal University of Rio de Janeiro,
3
Applied Microbiology – University of Lund
*[email protected]
The identification of more stable biocatalysts is needed for widening its industrial uses. The Brazilian tropical soils show a
rich biodiversity being promising for the search of actinomycetes with biotechnological applications such as the ability to
excrete enzymes with desirable industrial features. This work reports on cellulolytic enzymes produced by three streptomycete strains isolated from a soil under Cerrado vegetation, and a tropical forest soil. CMCase activity was evaluated
concerning the effect of pH and temperature on its activity and stability. Culture media were prepared using different
carbon and nitrogen sources, including some agro-industrial residues. Native PAGE and SDS PAGE zymograms were
prepared showing the characteristic bands for CMCase activity and the corresponding molecular mass. The cellulolytic
enzymes from the streptomycetes strains that were evaluated in this study displayed higher activity levels in the temperature range 50ºC to 60ºC and were able to perform in a quite wide pH range, noticeably in alkaline pH values. They were
also quite stable upon incubation for one hour at 50ºC at selected pH values.
Acknowledgements: This work was supported by the Brazilian Research Council – CNPq and by the “Swedish Foundation for International Cooperation in Research and Higher Education” – STINT.
poster 1A-16
Effect of poultry by-product on protease production by Bacillus subtilis in solid state fermentation
Valéria F. Soares, Elba P. S. Bon, Denise M. G. Freire*
Instituto de Química, Bioquímica, Universidade Federal do Rio de Janeiro, Brasil
Luciana A. I. de Azeredo
COPPE, Programa de Engenharia Química, Universidade Federal do Rio de Janeiro, Brasil
Feather, generated in large quantities as a by-product of commercial poultry processing, carries potent polluting implications. Keratin is degradable by some species of fungi, actinomycetes and some Bacillus strains. The aim of this study was
to investigate the effect of distinct poultry by-products on protease production by B. subtilis in solid state fermentation
(SSF). A strain of B. subtilis was cultivated in a casitone-molasses medium for 7 h at 30 °C under agitation for inoculum
preparation. One gram of feather meal, supplemented or not with poultry offal meal (9:1 and 1:1, w/w), moisturized with
water or corn steep liquor (CSL) mineral solution (70%, w/v), was seeded with inoculum suspension (3.5 mg g-1). SSF
was carried out for 160h at 30 °C.
The highest proteolytic activity (338 U g-1) were obtained for feather meal-offal meal media (1:1, w/w), moisturized with
water after 68h. Protease production was not significantly different in feather meal medium moisturized with CSL mineral
solution (203 U g-1) or with water (210 U g-1). These results indicate that poultry by-products can be used as low cost substrates for protease production by Bacillus subtilis in SSF. This strain shows excellent biotechnological potential for SSF.
83
poster 1A-17
Screening of Cellulolytic Fungal Strains from Brazilian Biodiversity and Culture Collections
Lucas T.C. Pereira1, Leonardo T.C. Pereira1, Mallú Lobão1, Ayla Sant’Ana da Silva1, Karin Willquist2 and Elba P.S.Bon1*
1
Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, CT, Bloco A, Ilha do Fundão,
CEP 21949-900, Rio de Janeiro, RJ, Brazil and 2 Applied Microbiology – University of Lund
* [email protected]
Effective and low cost cellulolytic enzyme blends are necessary for biomass hydrolysis. This study compared twenty cellulolytic
fungal strains from the genera Aspergillus and Trichoderma, both wild type and mutants, including the Trichoderma reseei Rut
C30 strain, regarding their extracellular endoglucanase and beta glucosidase activity levels and their corresponding zymogram
patterns. Robust, stable, sporulating microorganisms were selected from Brazilian and International culture collections and isolated from the Amazon rain forest. Enzymes production was performed in shake flask experiments at 30ºC and 200 rpm. Growth
medium presented 3,0 % (p/v) of either wheat bran, distillers grain or lactose as carbon sources and, as nitrogen sources, either
0,7% (p/v) yeast extract, 0,3% (v/v) corn steep liquor or 0,6% (p/v) (NH4)2SO4, plus salts. Supernatants from the fourth fermentation day (peak enzyme concentration), using optimized growth medium were analyzed for enzymes concentration (IU/L) and pH
and using SDS PAGE zymograms. The endoglucanase activity assay, based on Ghose, 1987, was optimized, concerning the
CMC substrate concentration and sampling procedure allowing enzyme activity measurements under initial rate conditions and by
extension a more comprehensive assessment of the strains ability to excrete cellulolytic enzymes. Results concerning fermentations time course, enzyme levels, zymogram patterns and the ability of the fungi strains to metabolize lactose will be presented.
Acknowledgements: This work has received financial support from the Brazilian Research Council / CNPq and from The Swedish Foundation for
International Cooperation in Research and Higher Education / STINT.
poster 1A-18
The effect of nitrogen regulation on cellulolytic enzymes production by a wild type and
a nit-2 mutant strain of Neurospora crassa
Ayla Sant’Ana da Silva, Felipe Knopp and Elba P.S.Bon*
Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, CT, Bloco A, Ilha do Fundão,
CEP 21949-900, Rio de Janeiro, RJ, Brazil
(*) [email protected]
Nitrogen regulation represses the expression of genes when cells are grown in preferred nitrogen sources such as glutamine or ammonia. It has been shown, however, that the nitrogen regulatory mechanisms have a wide domain of function.
Its action is integrated with a number of regulatory systems, affecting the expression of genes that are not directly related to
nitrogen metabolic pathways, being also connected to carbon regulation. In Aspergillus nidulans, nitrogen regulation involves
over a hundred genes, though not all are directly involved in nitrogen sources utilization. Cellulolytic enzymes are produced
by several species of fungi including the fungus Neurospora crassa, that has been extensively used for fungi genetic studies. In this work this microorganism was used to evaluate the effect of nitrogen regulation on cellulolytic enzymes production
using a wild type and a nit-2 mutant strain. The mutant lacks a regulatory protein that activates gene transcription in conditions of nitrogen depletion or in the presence of poor nitrogen sources such as proline, urea or nitrate. The wild type and the
mutant strain were cultivated in medium containing, as nitrogen sources, urea, proline, nitrate or ammonium and as carbon
sources either wheat bran or Avicel. Endoglucanase activity assay, based on Ghose, 1987, was carried out under initial rate
conditions. SDS-PAGE zymograms were performed using culture supernatants showing peak enzyme activity.
Acknowledgements: This work has received financial support from the Brazilian Research Council / CNPq and from The Swedish Foundation for
International Cooperation in Research and Higher Education / STINT. We are also grateful to the Fungal Genetic Stock Center (University Missouri,
Kansas City) for providing us with the wild type and the nit-2 mutant strains of Neurospora crassa.
84
poster 1A-19
Further improvements in performance of salt-activated enzymes via chemical modification
and use of surfactants
Abhijeet P. Borole*, Brian H. Davison, Oak Ridge National Laboratory, Oak Ridge, TN 37831‑6226
Salt-activated enzymes have been found to result in several orders of magnitude improvement in activity in organic media
compared to native enzymes. The current poster reports on further activation of these enzymes via chemical modification
and use of surfactants. Initial work was targeted to identify ways to achieve higher activity of enzymes in organic media
via chemical modification. This work focused on chemical modification of esterase enzymes with transesterification as
model reaction. Production of amino acid esters by subtilisin Carlsberg (SC) and other proteases/lipases were studied.
Two different chemical modifications were studied: Covalent attachment of (1) polyethylene glycol (PEG) and (2) alkyl
chains with 2-14 carbons. Chemical modification has been shown to enable solubilization of enzymes in organic media.
Potential to solubilize salt-immobilized enzymes was studied and will be presented. Use of surfactants (without covalent
attachment) was also studied and found to enhance activity for certain enzyme-solvent systems and will be presented.
The two approaches demonstrated in this study can be applied to enzyme catalysts to make them more effective as
catalysts in production of fine chemicals, pharmaceuticals as well as in developing improved methodologies to screen
enzymes in high throughput solid-phase synthesis.
poster 1A-20
Novel Endoglucanase of Clostridium thermocellum is Specific for beta-Glucans
Phillip J. Brumm*, David A. Mead, Lucigen, Inc., Middleton, WI 53562
During screening of a Clostridium thermocellum DNA library for cellulytic activity, a previously undescribed endoglucanase was discovered. The enzyme shows strong activity on an insoluble beta-glucan substrate (AZCL-barley-betaglucan, Megazyme). The enzyme is adsorbed by crystalline cellulose, but does not hydrolyze dye-linked cellulose
substrates or 4-methylumbelliferyl-beta-D-cellobioside, a soluble cellulase substrate. Unlike many endoglucanases, this
enzyme shows no activity toward a soluble xylanase substrate (4-methylumbelliferyl-beta-D-xyloside or an insoluble
xylan substrate (AZCL-xylan, Megazyme).
Sequencing of the ends of the DNA insert allowed identification of the gene product within the Cl. thermocellum genome.
The gene encodes a putative 59K protein; the protein was identified as a cellulase by annotation, but has not been characterized. Homology searches show that the protein is not homologous to other Cl. thermocellum endoglucanases or
xylanases.
85
poster 1A-21
Effect of different carbon sources on xylanase production by Bacillus circulans D1:
Maltose is the best inductor of xylanase production
a
Bocchini, D.A.a,b, Gomes, E.c, Da Silva, R.a,b*
Department of Chemistry and Environmental Sciences, IBILCE – UNESP, São José do Rio Preto, SP, Brazil
b
Chemistry Institute, UNESP, Araraquara, SP, Brazil
c
Department of Biology, IBILCE – UNESP, São José do Rio Preto, SP, Brazil
Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbons
sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that D-maltose is
the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48h), while D-glucose and D-arabinose lead to the
production of basal levels of xylanase. The crude enzyme solution is cellulase-free, even when the microorganism was
cultivated in medium with D-cellobiose. When oat spelt xylan was supplemented with D-glucose, the repressive effect of
this sugar on xylanase production was observed at 24 hours, only when used at 5.0 g/L, leading to a reduction of 60% on
the enzyme production. On the other hand, when the xylan medium was supplemented with D-xylose at 3.0 or 5.0 g/L,
this effect was more evident with 80 and 90% of reduction on the enzyme production, respectively. Unlike observed in
xylan medium, glucose repressed xylanase production in maltose medium, leading to a reduction of 55% on the enzyme
production at 24 hours of cultivation. Xylose, at 1.0 g/L, induced xylanase production on maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on xylan medium.
poster 1A-22
Enzymatic activity of peroxidase and polyphenoloxidase from mate tea leaves (Ilex paraguariensis St Hill.)
in compressed CO2
Maristela S. Primo, Débora Oliveira, J. Vladimir Oliveira, Marcos L. Corazza, Cláudio Dariva*
Department of Food Engineering, Av. Sete de Setembro 1621, Erechim, RS, 99700-000, Brazil
Octávio A.C. Antunes
Depto. de Bioquímica - IQ/UFRJ, Centro de Tecnologia, Bl. A, Sala 641, 21945-900, Rio de Janeiro, RJ, Brazil
The main objective of this work was to evaluate the activity of peroxidase (EC 1.11.1.7) and polyphenoloxidase (EC
1.14.18.1) obtained from mate tea leaves (Ilex paraguariensis St Hill.) treated in carbon dioxide at high pressures. The
effects of temperature (30-50oC), pressure (70-250bar), depressurization rate (10-200 Kg/m3/min), and exposure time
(1-6h) on the specific activity of both enzymes (U/mg) were assessed by a semi-factorial experimental design. Results
showed that in general the activity of peroxidase was increased after high-pressure treatment, while for polyphenoloxidase final activities were observed to be lower than the initial ones. The experimental condition (30ºC, 70bar, 10Kg/m3/min,
1h) that promoted the best results was employed to evaluate the effects of pressurization/depressurization cycles on
enzyme activity, and the results showed that polyphenoloxidase was more sensitive to the compression/decompression
cycles than peroxidase. The stability of the processed extracts with carbon dioxide were investigated by a period of 100
days, leading to a loss of around 20% for peroxidase and an enhancement of about 30% for polyphenoloxidase.
86
poster 1a-23
Screening of Microorganisms Towards the Conversion of Carveol to Carvone by PCR technique
Lindomar Lerin, Ieda Rottava, Débora de Oliveira*, Cláudio Dariva, Rogério L. Cansian, Francine Padilha
Geciane Toniazzo, Octávio A. C. Antunes
Depto. de Bioquímica - IQ/UFRJ, Centro de Tecnologia, Bl. A, Sala 641, 0Rio de Janeiro, 21945-900, RJ, Brazil
The essential oils comprise an abundant source of terpenes, substances plenty available in nature. Some monoterpenes
such as limonene can be obtained in large amounts from essential oils, with low price, to be used as a raw material in the
synthesis of high value added flavors and fine chemicals. Several routes for the microbial degradation of limonene making
use of the cytochrome P450-dependent monooxygenases have been postulated so far, but most of them lack of appropriate biochemical studies. The best studied microbial degradation pathway for limonene involves the conversion of carveol
to carvone by DCPIP – dependent carveol dehydrogenase (CDH), identified as gene limC. This work is focused on the
screening of microorganisms that presented the gene limC, responsible for the conversion of carveol to carvone, using
the PCR (Polymerase Chain Reaction) technique. The PCR technique selected fragments with size between 700 to 900
bp, whereas the size of fragment expected is 834 bp. These fragments were obtained in the strains of Aspergillus niger
ATCC 16404, Aspergillus niger ATCC 9642, Aspergillus oryzae ATCC 1003, Penicillium notatum ATCC 9478, Penicillium
camembertii (CT) ATCC 4845 and Paecilomyces variotii ATCC 22319.
poster 1A-24
Characterization of inulinase obtained by solid state fermentation and submerged fermentation:
A comparative study
Marcio A. Mazutti*, Marco Di Luccio, Helen Treichel
Maria Isabel Rodrigues
Department of Food Engineering/FEA/UNICAMP, Cidade Universitária Zeferino Vaz – Caixa Postal 16121, SP, 13083862, Brazil
Inulinase is an important enzyme in food processes, since it can be used to produce high fructose syrups by enzymatic
hydrolysis of inulin and fructooligosaccharides, which are used as a new functional food. The characteristics of inulinases
depends on many factors as: substrate, microorganism, mode of operation of the fermentative process, control of process
variables, among others. This worked aimed to characterize an inulinase obtained by solid state fermentation (SSF) and
submerged fermentation (SmF), using agroindustrial residues as substrates. The enzyme showed an optimal pH and temperature for hydrolytic activity in SSF of 5.0 and 55.0°C, respectively and optimal pH and temperature in SmF of 4.5 and
55°C, respectively. The higher thermostability for both enzymes was at 50 °C, with a D-value of 123.1 h and 230.4 h for
SSF and SmF, respectively. The enzyme obtained by SSF showed good stability on pH 4.5 to 4.8, maintained a residual
activity of 23% after 55 hours. The inulinase produced by SmF showed highest stability at pH 4.4 to 4.8, maintained a
residual activity of 49.9% after 82h of incubation. The results showed that inulinase obtained by SmF has higher thermostability than the enzyme obtained by SSF.
87
poster 1A-25
Evaluation of Lipase Activity from Seeds of Different Varieties of Ricinus communis L
Elisa D. C. Cavalcanti, Denise M. G. Freire*
Instituto de Química, Universidade Federal do Rio de Janeiro, CT, Bl A, Lab 549-2, Brasil
Pierre Villeneuve, CIRAD/AMIS, Montpellier, France
Regina Lago, Embrapa Food Technology, Rio de Janeiro, Brazil
Lipases are biocatalysts with large potential for technological application as they catalyze not only hydrolysis reaction
of esters but also reverse reactions such as esterification, transesterification and interesterification. Ricinus communis
seeds (castor seeds) are a low-priced source of lipase, with the advantage of not requesting germination of the seed
for enzyme production. This work refers to the attempt to characterize kinetically the activity of the lipase present in the
acetone powder of castor dormant seeds, variety BRS. Experiments focusing sunflower oil hydrolysis by the acetone powder and the seed oil hydrolysis by the endogenous lipase showed lipase activity only under an acid pH (pH 4.0). Lipase
activity of 538.9 U/g was observed when 5% (w/v) tributyrin was used as substrate. The Km and Vm determined with this
triglyceride were 134 g/L and 171.2 µmol/min, respectively. The results pointed out that the lipase originated from castor
oil dormant seeds presents an acid character and a hydrolytic preference for short chain triglycerides.
poster 1A-26
Immobilization of Candida antarctica Lipase B by Covalent Attachment to Green Coconut Fiber
Ana Iraidy Santa Brígida, Álvaro Daniel T. Pinheiro, Andréa L. O. Ferreira, Luciana Rocha B. Gonçalves*
Universidade Federal do Ceará – Depto. de Engenharia Química – Campus do Pici, Bloco 709 – 60.455-760
Fortaleza – CE, Brasil
Lipases constitute a group of enzymes that catalyze the hydrolysis of lipids in biological systems. In organic media, they
can be used to the synthesis of different lipids, which leads to several industrial applications in food and flavor making,
pharmaceuticals, cosmetics, among others. The use of immobilized enzymes offers some advantages over suspended
enzymes, such as reutilization, avoiding lost and reducing the cost of recuperation. Moreover, it is also possible to vary
the nature of the immobilized derivative in order to improve enzyme activity and stability. However, cost of support and immobilization reagents may be an economical drawback. In order to reduce those costs, an agro industrial residue, green
coconut fiber, was used to immobilize Candida Antarctica type B lipase by covalent coupling method. For this purpose,
prior to enzyme immobilization, silanization of green coconut fiber was performed 3-glycidoxypropyltrimethoxysilane. In
order to improve recovered activity, thermal and operational stabilities, some immobilization conditions were investigated,
using a factorial design: immobilization pH (7.0 or 10.0), enzyme concentration and use of polyethyleneglycol 6000 or
butyric acid during the enzyme attachment. Results indicated that the biocatalyst was active for butyl butyrate synthesis
and maintained 40% of its initial activity after 14 cycles of reaction.
88
poster 1A-27
Use of agricultural and agro-industrial solid wastes for depolymerizing enzymes in solid-state fermentation
Graminha, E. B. N.1*, Gonçalves, A. Z. L.1, Balsalobre, M. A. A.2, Da Silva, R.1, Gomes, E.1
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP), São José do Rio Preto – SP. Cristóvão Colombo, 2265. CEP: 15054-000, Brasil.
2
Bellman Nutrição Animal – Rodovia Washington Luiz, km 453, Mirassol - SP. CEP: 15130-000, Brasil.
1
There is an estimate that, in Brazil, the agricultural industries produce approximately 1000 ton/year of wastes. Only a
small part of this waste is used for animal nutrition. In this work enzyme production by solid state fermentation (SSF)
was evaluated using eight agricultural wastes (wheat bran, peanut bran, cotton bran, orange bagasse, soyhull, corn cob,
rice straw and grass) and five fungi strains (two of Thermomyces lanuginosus, Rhizomucor pussillus, Chaetomium sp.
and Rhizomucor sp.). The production of the enzymes (pectinase, Mn peroxidase - MnP, lignin peroxidase - LiP, lacase,
xilanase, amylase, avicelase e CMCase) in SSF was carried out using Erlenmeyer flasks at 45°C for 360 h. The highest
enzyme productions were: 66.2 U/g of pectinase on soy bean in 24 h by the Rhizomucor sp.; 19,555 U/g of MnP on grass
in 216 h by Chaetomium sp.; 4,686 U/g of LiP on peanut bran in 312 h by T. lanuginosus (TO 03); 2,986 U/g of xilanase
on corn cob in 360 h by T. lanuginosus (TO 03); 4,341 U/g on wheat bran in 216 h by T. lanuginosus (TO 04). There was
no lacase production and production of avicelase and CMCase were low in all the media.
*
Corresponding author – Fax: +55-017-3221 2390. Phone: +55-017-3221 2393. E-mail address: [email protected].
poster 1A-28
Pectinase production by solid-state fermentation using agro-industrial residues using a
thermophilic fungi strain
Marcelo Andrés Umsza Guez, Natália Martin, Érika Barbosa Neves Graminha, João Cláudio Thomeo, Roberto da Silva,
Eleni Gomes*
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Biociências, Letras e Ciências Exatas (IBILCE).
Universidade Estadual Paulista (UNESP), São José do Rio Preto – SP
Pectinases are one of the upcoming enzymes of fruit and textile industries. In this work a mixture of wheat bran, orange
bagasse and sugar cane bagasse (40, 40 and 20% respectively) with 70%moisture were used as substrate for production
of pectinases in solid-state fermentation (SSF) carried out in Erlenmeyer flasks at 45°C for 360 h, using a thermophilic
fungus Rhizomucor sp. N31. The substrate supported the growth of the microorganism, and afforded a exo-polygalacturonase production of 86,4 U/g in 48 h. The optimal conditions for the activity of the enzyme were pH 4,5 and temperature
60°C. 1374 U/g of endo-polygalacturonase was obtained after 144 h of fermentation. Other depolymerizing enzymes were
produced, such as celulase 12,3 U/g (144 h), xilanase 15,7 U/g (144 h), amylase 357 U/g (96 h), Mn peroxidase 4539,3
U/g (168 h) and lignin peroxidase 4077,4 U/g (216 h). The use of these agricultural wastes for enzyme synthesis could be
of great commercial significance and the fungal strain shows a good performance for the polygalacturonase production.
*
Corresponding author – Fax: +55-017-3224 8692. Phone: +55-017-3221 2393. E-mail address: [email protected].
89
poster 1A-29
Activity Determination of Cellulase Components Using Non-Crystalline Cellulose
Rajesh Gupta and Y.Y.Lee*, Department of Chemical Engineering, Auburn University, Auburn, AL 36849
Enzymatic hydrolysis of cellulose is a result of synergetic action of three different enzymes in cellulase system (endoglucanase, exo-glucansase and β-glucosidase). The hydrolytic reaction also depends upon the physical and chemical
properties of cellulosic substrate. The composition of the cellulase components is one of the key characteristics of the
cellulase system. The relative activities of each cellulase fraction ultimately control the overall reactivity. The data on individual enzymes are necessary for better understanding of cellulase reaction against lignocellulosic substrates.
In this study, a method to determine the relative activities of cellulase components in commercial enzymes (Spezyme CP
and GC-220 of Genencor International) was investigated. For this purpose, non-crystalline cellulose (NCC) was used as
the standard substrate. The NCC was prepared in our laboratory by acid treatment of pure natural cellulose. Examination
by X-ray crystallography has confirmed that NCC has non-crystalline structure presumably by breakage of the hydrogenbonds in the natural cellulose. Because of the amorphous nature and open structure of NCC, cello-oligosaccharides are
formed along with cellobiose and glucose as hydrolysis products. The time-course behavior of these three observables
changes sensitively with different level of enzyme and substrate loading. These data were used individually and collectively to estimate the activities of the individual enzymes. Exo-glucanase activity was estimated from initial hydrolysis rate
data obtained from experiments using extremely low enzyme loading. The β-glucosidase activity was calculated by cellobiose depletion data in the later phase obtained from high enzyme loading runs. Endo-glucanase activity was estimated
by cello-oligosaccharide and cellobiose data obtained during the initial phase of hydrolysis carried out with high enzyme
loading. The relationship between the individual activities and the overall activities (FPU) measured against filter paper
and Avicel is discussed.
Corresponding Author: Y.Y.Lee, E-mail: [email protected], Department of Chemical Engineering, Auburn University, Auburn, AL 36849-5127.
Phone: 334-844-2019/2034; Fax: 334-844-2063
90
poster 1A-30
Poly(L-Lactic Acid) Synthesis by Uncatalyzed Polycondensation and Enzymatic Ring Opening Polymerization Methods
Didem Omay, Yuksel Guvenilir*
Istanbul Technical University, Chemical Engineering Department, Maslak, Istanbul, Turkey. Email: [email protected]
Polylactic acid is a biodegradable polymer derived from lactic acid. It is a highly versatile material and is made from 100%
renewable resources like corn, sugar beets, wheat and other starch rich products. Polylactic acid exhibits many properties
that are equivalent to or better than many petroleum-based plastics, which makes it suitable for a variety of applications
[1]. As an important product from biological material, polylactic acid has excellent properties including biodegradability,
biocompatibility and biological resorbability, which made it, have extensive applications in many fields.
Polylactic acid is interesting as a biodegradable and bioabsorbable material, and is produced from lactic acid, either by
direct polycondensation of lactic acid or via ring opening polymerization of lactide. Conventional chemical polymerizations
of lactic acid or lactide require extremely pure monomers and anhydrous conditions as well as metallic catalysts which
must be completely removed before use, particularly for medical applications. To avoid these difficult restrictions for the
polymerizations of lactic acid or lactide by chemical methods, enzymatic polymerization may be one of the feasible methods to obtain polyesters [2].
In the present work, we present our experiences with the synthesis of poly(L- lactic acid) homopolymers with different
molecular weights. Poly(L-lactic acid) with low molecular weights was synthesized by direct polycondensation of lactic
acid under uncatalyzed reaction conditions. Also the goal of the work was to develop a methodology to synthesize high
molecular weights poly(L-lactic acid) by using enzymatic ring opening polymerization method. In this report, the preparation of polylactide by lipase catalyzed ring opening polymerization was studied with respect to the reaction conditions.
Poly(L-lactic acid) with low molecular weights were produced by direct polycondensation of the lactic acid monomer.
Poly(L-lactic acid) with different molecular weights in the range of 365-2685, were synthesized by changing polymerization time and temperature. Also it was found that the monomer conversion of lactide increased with time and showing
similar tendencies at 80oC and 100oC by lipase catalyzed ring opening polymerization.
References
1. R. Datta, S. Tsai, P. Bonsignore, S. Moon, J. Frank, 1995, ‘Technological and economic potential of poly(lactic acid) and lactic acid derivatives’
FEMS Microbiology Reviews, 16, 221-231.
2. Matsumura, S., Mabuchi, K., Toshima, K., 1997, ‘Lipase Catalyzed Ring Opening Polymerization of Lactide’ Macromol. Rapid Commun. 18, 477482.
91
poster 1A-31
Protease Immobilization by Covalent & Ionic Binding on Various Carriers
Cigdem Tasdelen, Yuksel Guvenilir*
Istanbul Technical University, Chemical Engineering Department, Maslak, Istanbul, Turkey
[email protected]
Different immobilization methods containing covalent binding, ionic binding and physical adsorption were used for the
immobilization of protease enzyme on various carriers. The usage of chitin, which supplies the enzyme to bind covalently,
as the carrier shifted the optimal temperature of the enzyme from 60oC to 70oC while chitosan didn’t make any change.
Covalently immobilized enzymes on chitosan and chitin improved the thermal stability and increased the optimum pH
towards the alkaline side by one unit. The pH stability, storage stability and re-usability of the immobilized enzyme that
was attached onto carriers of chitin and chitosan were investigated. Protease was immobilized on vermiculite by covalent
binding and optimum pH, pH stability, optimum temperature, thermal stability, storage stability and re-usability of the
immobilized enzymes were studied. The attachment capacity of protease enzyme was studied using cation (Dowex 50WX8, Amberlite IR-120) and anion (Dowex 1X8 Cl, Ecteola-23 Cellulose and DEAE Cellulose) carriers by ionic binding.
Physical adsorption studies were experienced with raw chitin and tannin-chitosan. The re-usage of the enzyme which
was immobilized on the tannin-chitosan made a decrease in the acitivity.
poster 1A-32
Enantioselective Reduction of Ketones by Carbonyl Reductases
Ling Hua,* Dunming Zhu, Kathy Yang
Department of Chemistry, Southern Methodist University, Dallas, TX 75275
Enzymatic asymmetric reduction of ketones has been developed as one of most attractive strategies for producing
homochiral alcohols as important fine chemicals and drug building blocks. Carbonyl reductases are widely distributed
in mammalian, plant and microorganism. Most carbonyl reductases are limited to reduce small ketones such as methyl
ketones and to give Prelog product alcohols. We are interested in seeking carbonyl reductases with capability of reducing
bulky ketones and/or affording anti-Prelog alcohols products. In this endeavor, several carbonyl reductases have been
cloned from microorganism and expressed in E. coli. Among them, a carbonyl reductase from Sporobolomyces salmonicolor is active toward bulky ketones such as 2,2-dimethylpropiophenone with high enantioselectivity. Another carbonyl
reductase from Candida magnoliae reduces ketones to anti-Prelog alcohols with greater than 99% enantioselectivity in
most cases. These enzymes were also engineered by rational mutagenesis based on their 3-D structures to improve their
properties. The impact of the mutation on activity and enantioselectivity will be discussed.
92
poster 1A-33
A Study of Layer-by-Layer Nano Self-Assembly Techniques for Enzyme Immobilization in Microchannels
Jie Wen and Bill Elmore, Louisiana Tech University, Ruston, LA 71272
Frank Jones*, University of Tennessee at Chattanooga, Chattanooga, TN 37403
Enzyme architectures are created as systems of polyion layers attached to surfaces where enzymes are immobilized
between polyion layers. In this study, urease is immobilized using polyethyleneimine (PEI) and polystyrenesulfonate
(PSS), which are used as polycations and polyanions respectively. The electrostatic charges on the polyions cause them
to adhere to one another in single, alternate layers. The urease has a negative charge (at an assembly pH of 7.5) causing
it to also adhere as a layer between two positively charged polymer layers.
The continuous flow microreactor consisted of parallel channels with cross sections of 125 x 500 micrometers. It is found
that increasing polyion layers increases both the reactivity and stability of the enzyme. Experiments show that increasing the number of enzyme layers increases conversion for up to 5 layers. However, useful reactor lifetime continued to
increase for up to 7 enzyme layers. Fifty percent of maximum reactivity was maintained for up to 2 months during continuous flow experiments in the microchannels.
poster 1A-34
β- d-Xylosidase from Selenomonas ruminantium of Glycoside Hydrolase Family 43
Douglas B. Jordan*1, Xin-Liang Li1, Christopher A. Dunlap2, Terence R. Whitehead1, and Michael A. Cotta1
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, U.S. Department of
Agriculture, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604
1
2
Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture,
Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604
Certain strains of the ruminal anaerobic bacterium, Selenomonas ruminantium, have been shown to enhance utilization of
xylooligosaccharides under fermentation conditions. Preparations of S. ruminantium β-d -xylosidase were shown to act on
4‑nitrophenyl-β-d -xylopyranoside and 4-nitrophenyl-α-l-arabinofuranoside with a 10-fold preference of the former substrate over the latter. Moreover, natural oligosaccharides, produced from reacting oatspelt xylan and wheat arabinoxylan
with a xylanase, were accepted as substrates by the enzyme. Recently, X-ray structures of β-d -xylosidase belonging to
glycoside hydrolase family 43 have been reported for the enzyme from different species. The amino acid sequence of the
xylosidase from Clostridium acetobutylicum has 72% identity with that of the S. ruminantium enzyme, and its X-ray structure was used to model the active site of the S. ruminantium enzyme. β-xylosidase from S. ruminantium, heterologously
expressed in Escherichia coli, was purified to homogeneity for structure and function studies. Steady-state kinetic studies
determined that the xylosidase is highly active on natural and artificial substrates. Substrate specificities, reaction stereochemistry, and effects of pH, temperature, and site-directed mutations were determined for the enzyme.
Correspondence: Douglas B. Jordan, USDA, ARS, NCAUR, FBT, 1815 N. University Street, Peoria, IL 61604
E-mail: [email protected], Phone: (309) 681-6472, Fax: (309) 681-6427
93
poster 1a-35
Cellobiose Production using Cellulase Inhibition
Misook Kim,
Department of Food Science, Louisiana State University, Baton Rouge, LA 70803
Chang-Ho Chung, Donal F. Day*
Audubon Sugar Institute, St. Gabriel, La. 70776
Typically, enzyme hydrolysis of ligno-cellulose produces primarily glucose and a small amount of cellobiose. Cellobiose in
its own right is a potentially valuable product as a non-nutritive sugar. Co-production of a β-glucosidase inhibitor, gluconic
acid, during enzymatic hydrolysis of cellulose altered the amounts of glucose and cellobiose produced.
Glucose oxidase produces gluconic acid from glucose via a glucolactone intermediate. Gluconic acid is an inhibitor of
β-glucosidase. Addition of either of these compounds or the enzyme to the cellulase (Trichoderma viride) and β-glucosidase (Aspergillus niger) cocktail used to hydrolyze ligno-cellulose altered the ratio of cellobiose to glucose produced from
the cellulose. Addition of gluconolactone, glucose oxidase, or gluconic acid significantly increased the amount cellobiose
with a corresponding decrease in amount of glucose produced during cellulose hydrolysis. The rate of cellulose hydrolysis
decreased on addition of gluconolactone but was not affected on addition of glucose oxidase. With suitable concentrations of glucose oxidase it was possible to convert over 60% of the cellulose to cellobiose.
poster 1A-36
Investigation of factors limiting hydrolysis at high solids concentrations
Jan B. Kristensen*, Claus Felby, Henning Jørgensen
Department of Forestry and Forest Products, Danish Centre for Forest, Landscape and Planning
The Royal Veterinary and Agricultural University, Højbakkegård Allé 1, DK-2630 Taastrup, Denmark
Phone: +45 35281687 Email: [email protected]
Jan Larsen, Elsam Engineering A/S, Kraftværksvej 53, DK-7000 Fredericia, Denmark
Wheat straw is a promising substrate for bioethanol production due to its availability and recent success with hydrothermal pretreatment improving the processability of the substrate.
We have developed a method that enables enzymatic hydrolysis and fermentation of lignocelluloses materials with an
initial dry matter (DM) content of up to 40%. Working at solids concentrations around 25 to 30% DM is important for the
feasibility of bioethanol production as it lowers costs associated with heating, cooling and bulk of substrate.
Initial experiments with steam-pretreated wheat straw at 5 to 40% DM, have revealed a negative linear relationship between
the DM content and the efficiency of the hydrolyzing enzymes despite a constant substrate-enzyme ratio. This was also
observed in simultaneous saccharification and fermentation by yeast where product inhibition by glucose was alleviated.
In order to improve hydrolysis yields at high DM we have, using a pure cellulose based system as model, investigated
potential causes for the reduced enzyme performance at increasing DM such as hemicellulose, salt and lignin content
and mass transfer.
94
poster 1A-37
Solvent-free biolubricant synthesis using lipase
Pollyanna Talida Coelho Dias, Marta Antunes Pereira Langone*
Instituto de Química, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier, 524, PHLC, IQ,
Rio de Janeiro, RJ, Brazil, CEP: 20559-900
Mônica Antunes Pereira da Silva
Escola de Química, Universidade Federal do Rio de Janeiro, RJ, Brazil
Because of their outstanding lubricating properties and biodegradability, vegetable oils and their derivatives are seeing
increased interest as base stocks for lubricants and functional fluids. A biolubricant should provide maximum wear protection without pollution effects to the environment. Oleic acid is one of the most important fatty acids in nature, present in
many varieties of plants. Oleic esters produced by enzyme catalysis can be applied as biolubricant. The purpose of this
work was to study the enzymatic synthesis of butyl oleate by direct esterification of oleic acid and butanol in a solvent-free
media. Reactions were carried out in an open batch reactor with constant stirring, using a commercial immobilized lipase
(Lipozyme RM-IM, Novo Nordisk). The influence of temperature, enzyme concentration and butanol/ oleic acid molar ratio
has been studied using a 2-level-3-factor central composite experimental design. The best conditions were obtained with
butanol/oleic acid molar ratio of 1.84, temperature of 50oC and enzyme concentration of 3.0% (w/w). Under these conditions, palmitic acid conversion was about 97% after 1 hour of reaction.
poster 1A-38
Production of biodiesel from vegetable oil using immobilized lipase
Otávio Luiz Bernardes, Marta Antunes Pereira Langone*
Juliana Vaz Bevilaqua, Centro de Pesquisa e Desenvolvimento da Petrobras - Cenpes
Denise Maria Guimarães Freire, Instituto de Química, Universidade Federal do Rio de Janeiro, RJ, Brazil
Biodiesel, an alternative diesel fuel, is defined as monoalkyl fatty acid ester (preferentially methyl and ethyl esters). Conventionally, biodiesel was produced by transesterification of triglycerides and alcohols in the presence of an alkaline catalyst. However, the chemical process has several drawbacks. As an alternative, lipases have been used as biocatalysts for
the synthesis of biodiesel from natural oils. In this work, enzymatic alcoholysis of soybean oil with methanol and ethanol
was investigated. The reactions were carried out in a closed batch reactor with constant stirring and coupled with condenser by transesterification of soybean oil by alcohol, using a commercial immobilized lipase (Lipozyme RM-IM, Novo
Nordisk). The influence of alcohol (methanol or ethanol), enzyme amount, molar ratio of alcohol to soybean oil, solvent (nhexane) and temperature on biodiesel production was determined. The ethanolysis of soybean oil by successive additions
of ethanol was also investigated. The best conditions were obtained in a solvent-free system with ethanol/oil molar ratio
of 3.0, temperature of 50oC and enzyme concentration of 7.0% (w/w). Three-step batch ethanolysis was most effective for
the production of biodiesel. In these conditions, ethyl esters yield was about 55% after 2 hours of reaction.
95
poster 1A-39
Biodiesel synthesis via enzymatic esterification
Ana Paula de Alencar Vieira, Mônica Antunes Pereira da Silva
Escola de Química, Universidade Federal do Rio de Janeiro, RJ, Brazil
Marta Antunes Pereira Langone*
Because of crude oil limited resources, high prices and increasingly environmental concerns there has been a renewed
focus on alternative processes to produce biodiesel. The main objective of this work was to study the synthesis of ethyl
hexadecanoate, one of the main products obtained in biodiesel synthesis. Direct esterification between palmitic acid and
alcohol in a solvent-free system was carried out in a closed batch reaction coupled with condenser under stirring, using
a commercial immobilized lipase (Lipozyme RM-IM, Novo Nordisk). The effects of palmitic acid/ethanol molar ratio (0.16
to 1.84), temperature (65 to 75ºC) and enzyme concentration (0.48 to 5.52% w/w) on the initial reaction rate were determined using an experimental design (central composite design 23 with six central points and six star points). The best
conditions were obtained with palmitic acid/ethanol molar ratio of 0.50, temperature of 67oC and enzyme concentration of
4.50% (w/w). Under these conditions, palmitic acid conversion was about 50% after 30 minutes of reaction. The effects
of carbon chain-length of alcohol with different molar ratio of reactants on the process were also investigated. Overall the
results show that enzymatic esterification of palmitic acid with alcohol could be a good alternative for the classic process
using strong acids.
poster 1A-40
Solvent-free ethyl oleate synthesis by non-commercial lipases
Marta Antunes Pereira Langone
Instituto de Química, Universidade do Estado do Rio de Janeiro, PHLC, IQ, Lab. 427,Brasil
Juliana Vaz Bevilaqua, Centro de Pesquisa da Petrobras, Brasil
Aline Gomes Cunha, Márcia M. Ribeiro Leal, Denise Maria Guimarães Freire*
Ethyl oleate is a fatty ester which finds wide application as biological additive, PVC plasticizer, water resisting agent, in the
production of tailored triglycerides or in diesel fuel additives. Among the high number of lipases described in the literature,
only the enzymes belonging to a few species have been demonstrated to have adequate stability and biosynthetic capabilities to allow routine use in organic reactions. Moreover, the commercial lipases currently available on the market are
imported and are themselves very costly. In the present work, the synthesis of ethyl oleate was investigated by different
non-commercial biocatalysts obtained by submerged and solid state fermentation using low cost agroindustrial waste. Lipases isolated from the cell-free culture medium of Penicillum restrictum, Penicillium simplcissimum, Aspergilllus sulfureos and Aspergillus parasiticus were immobilized on hydrophobic support. These non-commercial biocatalysts maintained
90% of their activity during 30 days’ storage at room temperature (25 oC). The syntheses of ethyl oleate were carried out
in an open batch reactor with constant stirring at 40oC. No solvent has been added to reaction medium consisting only
of oleic acid, ethanol (1:1) and immobilized lipase. In all experiments performed, lipases from Aspergillus parasiticus and
Penicillium simplissicinum showed promising results, with high esterification activities and conversions.
96
poster 1A-41
Cloning and characterization of cold-active xylanase enzymes
Charles C. Lee*, Rena E. Kibblewhite-Accinelli, Kurt Wagschal, George H. Robertson, Dominic W.S. Wong
USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA 94710
There is great interest in lignocellulolytic enzymes due to the wide variety of industrial applications for these reagents.
At the USDA Agricultural Research Service, we are screening DNA libraries to discover genes encoding new hemicellulolytic enzymes. The libraries were generated both from pure cultures as well as from mixed environmental populations
(metagenome). Among the new enzymes isolated are cold-active xylanase enzymes that are categorized as glycosyl
hydrolase family 8 and family 10 members. These enzymes have potential applications in processes where the products
are heat-sensitive. We describe the cloning, purification, and biochemical characterization of these enzymes.
poster 1A-42
Stability and Performance of Glucose Isomerase for the Production of High Fructose Corn Syrup
Leng Hong (Calvin) Lim, Bradley A. Saville
Department of Chemical Engineering and Applied Chemistry, University of Toronto
Glucose isomerase (GI) is a key enzyme used to produce high fructose corn syrup (HFCS) from glucose. Because GI is an
expensive intracellular enzyme, the ability to make the enzyme more stable could greatly reduce HFCS production costs. A
thorough understanding of the thermoinactivation mechanism of GI is the first step in making GI with higher stability.
In this study, the thermoinactivation of Streptomyces rubiginosus GI, in both soluble (SGI) and immobilized (IGI) forms,
was investigated from 40 to 90 oC both in batch and continuous reactors. The key issues addressed were the contributions of cysteine residue oxidation and the Maillard reaction between the enzyme and the sugars in the reaction medium
to the deactivation of GI. Cysteine residue oxidation and Maillard reaction contributed to SGI deactivation at 60 and 80 oC.
For IGI, the effects were less pronounced and seemed to diminish as temperature increased to 80 oC. This implies that
immobilization has altered the thermoinactivation mechanism of GI. A mathematical model was developed to describe the
reversible reaction as well as the enzyme deactivation as a function of temperature.
poster 1A-43
Life Cycle Analysis of the AFEX Process in Consideration of Recent Process Improvements
Elizabeth Newton*, Hasan Alizadeh, Bruce Dale, Seungdo Kim, Mark Laser, Lee Lynd
The Ammonia Fiber Explosion (AFEX) process has been shown to be an effective pretreatment for lignocellulosic biomass, but
little analysis has been done to determine the environmental impacts of this pretreatment in the context of a biorefinery. Recent
research has shown reduced overall ammonia requirements, and reduction in the required ammonia recycle concentrations.
Based on these new research results it is shown that the system environmental performance is enhanced. The AFEX process
and the overall biorefining system producing fuel ethanol from cellulosic biomass show many positive environmental metrics including increased soil organic matter, reduced greenhouse gas formation and, depending on cultivation practices, reduced nitrous
oxide emissions and nitrate leaching compared with corn ethanol and gasoline. Eutrophication and acidification potential may be
increased but appropriate system design can likely prevent serious consequences. Life cycle analysis (LCA) used in conjunction
with classical design for economic profitability can illuminate pathways that produce wealth and enhance the environment.
* Michigan State University, 2527 Engineering Building, East Lansing, MI 48823. E: [email protected]
97
poster 1A-44
Removal of Phenolic Solubles from Hydrothermally Pretreated Wheat Straw
Nielsen, N.P.K.*, Jørgensen, H., Felby, C.
Department of Forestry and Forest Products, Danish Centre for Forest & Landscape, KVL
The Royal Veterinary and Agricultural University, Højbakkegaard Allé 1, 2630 Taastrup, Denmark
Hydrothermally pretreated wheat straw is used as substrate for bio-ethanol production in the EU-project: Co-production
of biofuels (the IBUS project). The pretreated straw and the water from the pretreatment process contains a substantial
amount of phenolic solubles. These phenolic compounds are the result of lignin degradation during the pretreatment process. The purpose of the work presented here is to identify these low molecular weight phenolic solubles, and to examine
the effect of laccase catalyzed oxidation of these.
The laccase treatment was carried out as a separate step prior to hydrolysis and fermentation of the pretreated straw. An
easy method for separation of phenolic compounds from the aqueous phase was developed, and the phenolic compounds
with a molecular weight below 1200 Da was identified using GC-MS. The treatment was very efficient, as a drastic decrease
in the concentration of a number of the soluble phenolics was observed. Relatively small laccase dosages was used, and
the effect of laccase may find application for removal of potential inhibitors for enzymes and microorganisms.
poster 1A-45
Improved Analytical Methods and Quantifying Mass Deposition of Urease Immobilized using Layer-by-Layer
Nano Self-assembly
Shaila Reddy, John Elmore, Joseph Cook, L. Dale Snow, James D. Palmer*
Louisiana Tech University, Chemical Engineering Program, Ruston, LA 71272
Bill B. Elmore
Mississippi State University, Chemical Engineering Department, Mississippi State, MS 39762
Previous studies have been reported applying Urease in microchannels using layer-by-layer nano self-assembly. In this
technique, charged polymers are used to “encase” enzymes in a series of alternating layers. The polycations used were
polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were
polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The product concentration of the prior experiments was measured using pH sensitive dyes in the reactant. The Berthelot procedure has recently been applied to directly measure the
ammonium ion concentration allowing higher sensitivity to conversion. Studies have been conducted to determine the
degree of interference of the polyelectrolytes with the Lowry protein assay. Polyethylenimine (PEI) was found to interfere
with the Lowry assay but a sample preparation described in the article successfully addresses this issue. The amount of
enzyme immobilized using various polyelectrolyte combinations has been quantified with both direct protein assay and
Quartz Crystal Microbalance (QCM) experiments. The immobilization efficiency will be compared with observed catalytic
activity for various polyelectrolyte combinations.
98
poster 1A-46
Filter Paper-Based Assays for Low Activity Enzyme Preparations
Tor Soren Nordmark and Michael H. Penner*
Food Science & Technology, Oregon State University, Corvallis, OR 97331-6602
The traditional IUPAC-endorsed filter paper assay was adapted for use with enzyme preparations having relatively low
cellulase activities. The adaptation makes use of the principle that the extent of substrate conversion in an enzyme catalyzed reaction is directly dependent on the product of enzyme concentration and time, provided the enzyme is stable over
the course of the assay. Thus, activity may be determined from the amount of time required for a test enzyme preparation to catalyze the conversion of a fixed percentage of substrate, filter paper, to soluble saccharides. The adapted assay
targets the same extent of substrate conversion as does the traditional IUPAC assay. However, reaction times in the
adapted assay may be extended well beyond that allowed in the traditional assay due to the inclusion of common protein
stabilizers. The adapted assay thus differs from the traditional assay in that it is not limited to a one hour reaction period.
The assay may thus be used to obtain relative filter paper activities for enzyme preparations well below that required for
application of the IUPAC-endorsed filter paper assay.
poster 1A-47
Production and characterization of cellulases obtained from a sugarcane bagasse cellulignin
by Aspergillus niger and Trichoderma harzianum
Aline Machado de Castro, Daniele Fernandes Carvalho, Kelly Cristina Nascimento Rodrigues Pedro,
Selma Gomes Ferreira Leite and Nei Pereira Jr.*
Escola de Química, Universidade Federal do Rio de Janeiro, P.O. Box 68542, Zip code 21945-970, Rio de Janeiro, RJ,
Brazil, Phone: 55 21 25627639, Fax: 55 21 25627567, e-mail: [email protected]
Cellulases are enzymes that act sinergically to convert the cellulosic fiber into glucose. This monossacharide can be
fermented to several compounds, both commodities (e.g. ethanol) and high value-added (e.g. xylitol) by biological processes that are much more attractives when low cost biomasses are used. In Brazil, one of the most abundant residues
is sugarcane bagasse. Its production in 2004 was about 125 million tons. Therefore, this work aims to evaluate cellulase
production by Aspergillus niger ATCC 16404 and Trichoderma harzianum IOC 4038 on a sugarcane bagasse cellulignin
and characterize the final extracts. A. niger produced higher ß-glucosidase (1627 IU/L) but lower FPase (36 IU/L) and
endoglucanase (386 IU/L) activities comparatively to T. harzianum, that achieved 745 IU/L, 100 IU/L and 558 IU/L for
these activities, repectively. In the final extracts, cellobiohydrolase production by A. niger (88 IU/L) and T. harzianum (135
IU/L) and unexpressive protease and endoxylanase activities were observed. Assays on carboxymethylcellulose salts
showed, for both strains, that the lower their viscosity, the higher the activity. The study of the effect of nine metals in the
activities indicated Mn2+ the best of them. A binomial analysis revealed that the optimal pH values for enzymatic catalysis
ranged from 4.8 to 5.5 and the optimal temperatures were 50ºC and 60ºC, depending on the activity studied.
Financial support: CNPq.
99
poster 1A-48
Enzymatic Hydrolysis Optimization of Ethanol Production by Simultaneous Saccharification and Fermentation
Mariana Peñuela Vásquez; Juliana Nascimento C. da Silva; Maurício Bezerra de Souza Jr.; Nei Pereira Jr.*
School of Chemistry - Federal University of do Rio de Janeiro
Bloco E - Rio de Janeiro - R.J. - Brazil - CEP 21.949-900
Phone: (+5521) 2562 7565. - Fax: (+5521) 2562 7616 - e-mail: [email protected].
There is an enormous interest to use agro-industrial wastes and to explore them as starting materials for the production of
different metabolites, especially in order to obtain ethanol as fuel, employing the biorefinery concept.
The aim of this work was the optimization of the sugar enzymatic extraction from cellulignin and the study of the conversion to ethanol using a fermentation process by the yeast Saccharomyces cerevisiae. The cellulignin is the solid resulting
from the acid hydrolysis of the sugarcane bagasse.
Such optimization was accomplished by response surface methodology using the pH, enzymatic concentration, solid
percentage and temperature as the main factors. Glucose conversion, calculated from the initial dried weight of cellulignin, and glucose concentration were chosen as output dependent variables. Desirability function approach allowed the
simultaneous maximization of both output variables, being the following conditions found: 44.5oC, 10%, 25.3 FPU/g of
celulignina. The effect of the variable pH was not statistically significant.
Those conditions were evaluated to obtain ethanol by simultaneous saccharification and fermentation (SSF) process,
studying the yeast performance in the optimum conditions of the enzymatic hydrolysis.
poster 1A-49
Characterization of Cellulase by Non-Crystalline Cellulose and Cello-Oligosaccharides
Suma Peri and Y.Y.Lee*, Department of Chemical Engineering, Auburn University, Auburn, AL 36849
Hydrolysis of pure cellulosic substrates is affected by the crystalline structure of the substrate, the accessible surface
area/active sites, adsorption of the enzyme and the product inhibition. The purpose of this study is to identify the properties of the three different components in cellulase using non-crystalline cellulose (NCC) and cello-oligosaccharides
(COS). NCC and COS are the products of our laboratory. Because of the amorphous structure of NCC, use of this material as the substrate of cellulase may reveal the intrinsic nature of the enzymatic reaction unaffected by the crystalline
structural barrier. In this study, enzymatic hydrolysis experiments were conducted using NCC and COS independently and
in combinations. This paper reports on behavior of cellulase reaction yet to be found from previous work on the enzymatic
hydrolysis of natural crystalline cellulose.
Major findings of our experiments include:
• In hydrolysis of NCC by cellulase, cello-oligosaccharides as well as cellobiose were formed in significant quantities.
• Endo and exo-glucanases function only on solid substrate. They do not hydrolyze COS.
• Beta-glucosidase hydrolyzes cellobiose and COS. It does not function on solid glucan.
• COS inhibits endo and exo-glucanases.
100
poster 1A-50
Ethanolysis of corn oil using crude papain
Emir Bolzani Saad, Nadia Krieger, Luiz Pereira Ramos*
Research Center in Applied Chemistry (CEPESQ), Department of Chemistry,
Federal University of Paraná, Curitiba, PR 81531-990, Brazil
The ethanolysis of winterized corn oil was evaluated in aqueous-restricted media using crude papain as the biocatalyst of
choice. Crude papain was obtained directly from unripe fruits of Carica papaya and immediately freeze-dried at -45°C and
0.07 mBar for further use. The freeze-dried material contained 10% (w/w) moisture and a total lipolytic activity against
p-nitrophenyl palmitate of 40U/g of dry matter. Ethanolysis of corn oil was carried out for 120h in 15mL of n-hexane according to a 24 factorial design, employing temperatures of 37 and 55°C, two different types of ethanol (anhydrous and
95% proof), ethanol:oil molar ratios (MR) of 3:1 (210mmol/L:70mmol/L) and 6:1 (420mmol/L:70mmol/L) and total lipopytic
activities of 4.4 and 8.8U/g of corn oil. The best conditions for the ethanolysis of corn oil involved the use of 37°C, ethanol
95%, ethanol:oil MR of 3:1 and a lipopytic activity of 8.8U/g. HPLC analysis of the corn oil ethyl esters revealed a total
ester content of 95,9% (w/w) for an actual reaction yield of 91,52%, with the amount of unreacted glycerides restricted to
approximately 0.8% (w/w). Polar compounds such as glycerol, monoacylglycerides and ethanol were not detectable in the
ester product because of their low solubility in n-hexane.
Supported by CNPq, CAPES, Fundação Araucária
poster 1A-51
Chemical Characterization of Pulp Components in Unbleached Softwood Kraft Fibers That Were Recycled With
The Assistance of a Laccase/HBT System
Thiago A. da Silva, Luiz Pereira Ramos*
Department of Chemistry, Federal University of Paraná, Curitiba, PR 81531-990, Brazil
Paulina Mocchiutti, Miguel A. Zanuttini
Instituto de Tecnologia Celulósica, Universidad Nacional Del Litoral, Santa Fé, SF 53000AOJ Argentina
The application of laccases from Trametes hirsuta in the recycling of unbleached kraft fibers (kappa 91) has been demonstrated in the presence of oxygen and HBT as the enzyme mediator. The laccase treatment caused a partial reversion
of the detrimental effects of hornification without interfering in the carbohydrate moiety of the pulp fibers. A considerable
increase in the amount of carbonyl groups was observed as a result of the enzyme treatment and this helped explaining
the improved fiber-to-fiber bonding of laccase-treated recycled fibers. The amount of extractables in ethanol:toluene also
increased after laccase treatment and the kraft lignin underwent a noticeable decrease in its apparent molecular mass.
This latter effect was readily attributed to the hydrolysis of aryl-ether bonds that survived the severity employed in the
pulping process. These observations were collectively useful to explain why laccase-treated recycled fibers produced
handsheets with better properties, mainly characterized by their improved resistance to tensile index (c.a. 10%) in relation
to the control.
Supported by CNPq, CAPES, SECyT
101
poster 1A-52
Elucidation of the factors responsible for cellulose fragmentation activity of Trichoderma reesei
Abdul A.N. Saqib*, Philip J. Whitney
School of Biomedical & Molecular Sciences, University of Surrey, Guildford, GU2 7XH, UK.
Hydrolysis of cellulose to fermentable sugars is an important step in the utilization of biomass. To reduce the cost of
enzymes or increasing their efficiency involves understanding their mechanism of action. Ample work has been done on
endo-glucanses, exo-glucanases and β-glucosidases. However cellulose maceration or fragmentation has been largely
over-looked. If understood, this could be used to enhance the cellulolytic power of fungi, like Myrothecium verrucaria,
which produce full set of the conventional enzymes but fail to fragment native cellulose.
Our work on the cellulolytic fungus, T. reesei, has furthered our insight into this feature. Light microscopy, SEM imaging,
particle size analysis and filter paper maceration showed that the culture filtrate of T. reesei can macerate cellulose powder and filter paper. The culture filtrate of T. reesei was fractionated by gel filtration and ion-exchange chromatography.
SDS-PAGE was used to monitor the homogeneity of the fractions. None of the fractions alone could macerate cellulose
but a mixture of selected fractions could. This shows that the activity was the result of interaction of different components
of the culture filtrate rather than the effect of a previously undetected component.
poster 1A-53
The Effect of Feruloyl Esterase on the Enzymatic Saccharification of Pretreated Corn Stover
Stephen R. Decker*, Eric Knoshaug, William S. Adney, and Michael J. Selig*, National Bioenergy Center
National Bioenergy Center, National Renewable Energy Laboratory, Golden, 1617 Cole Boulevard, Golden, CO 80401,
E-mail: [email protected]; [email protected]
Ferulic acid, a common cell wall component in most energy crops, is bound by ester linkages to both hemicellulose and
lignin. Feruloyl esterase (ferulic acid esterase – FAE) actively cleaves these linkages, directly increasing hemicellulose
and lignin accessibility while indirectly providing better cellulose accessibility. In the lignocellulosic-to-ethanol industry,
FAE’s and other hemicellulose accessory enzymes have the potential to reduce the severity of thermochemical pretreatment necessary for economic enzymatic conversion of biomass to sugars. For this study we have cloned a single
component FAE from Penicillium funiculosum and expressed it in Aspergillus awamori. The enzyme effectively releases
ferulic acid when assayed with methyl ferulate as a substrate. Progressively pretreated (low to high severity) corn stover
samples were prepared by a number of methods, including dilute acid, ammonia fiber explosion (AFEX) and aqueous ammonia recycle percolation (AARP). The susceptibility of the substrates to enzymatic degradation with a commercial cellulase preparation (Spezyme CP, Genencor Intl.) and a component cellulase mix is assessed with and without the presence
of the FAE. In addition, we explore possible synergistic effects of the FAE in combination with additional cell wall degrading enzymes such as XynA from Thermomyces lanuginosis, Axe1 and Abf2 from Trichoderma reesei.
102
poster 1A-54
A Tray Bioreactor for Hyper production of the Industrially Significant Xylanase by a Mutant Aspergillus niger
NKUCN-3.40
Nidhi Kapura, Dharm Duttb and Rajesh P. Singha*
Department of Biotechnology and Department of Paper Technologyb, Indian Institute of Technology Roorkee,
Roorkee-247 667, India
E: [email protected], F: 91-1332-273560, T: 91-1332-285792
a
Xylanases have been denoted as a major group of enzymes, having significant application in the paper and pulp industry.
Xylanases are also employed in the baking and textile industry, fruit and vegetable processing, brewing, wine production, starch-gluten separation, bioconversion and bioremediation of agro-residues. A total of 42 fungal strains that were
isolated from a variety of natural sites, 22 isolates were capable of producing xylanases. The level of xylanase production
from A. niger NK-23 was found to be higher as compared to the other strains, this strain also produced notable levels of
β-xylosidase and had lower levels of cellulase production. An attempt to improve the xylanase production ability of the
selected strain was undertaken by physical, chemical and mixed mutagenesis of the strain. The survival rate and distribution profile i.e. positive, negative and corresponding mutants were determined after every stage of mutagenesis. Analysis of the resultant mutants had indicated that mutant strain A. niger NKUCN-3.40 had notably higher ability of xylanase
production. The multiple sub culturing of this strain had denoted the stable levels of the enzyme production. Scanning
electron microscopic studies revealed notable changes in the morphological features as the mycelia observed of the
mutant A. niger NKUCN-3.40 strain were compact and dirty white as compared to the mycelia of the wild type A. niger
NK-23 that was detected to be loose and brownish yellow in appearance. An attempt was made to further economize
the process of production by evaluating the solid-state fermentation using agro-industrial residues as the solid support.
Among ten different solid supports used, cotton hull was found to be the most effective for production (1705.67 IU g-1).
Under solid-state fermentation, maximum xylanase production was achieved on 4th day of incubation. Of the various additives evaluated, mustard oil cake at 5% (w/v) had further enhanced the xylanase production ability of A. niger NKUCN3.40 to 1783 IU g-1.
The semi-continuous production of xylanase was attempted by designing a tray bioreactor and the production was evaluated under the conditions as derived earlier. In the bioreactor, biomass was recycled for different cycles of the fermentation. Significant levels (1513-1645 IU g-1) of enzyme were produced up to 3 cycles of fermentation, the level of production
declined thereafter. SDS-PAGE and zymogram analysis revealed that the molecular weight of the enzyme produced
during the fermentation was 29 kD protein, which was stable up to 65oC and over a pH range of 7.0-9.0.
Xylanase obtained was critically evaluated for the biobleaching of the mixed wood pulp as well as wheat straw pulp.
Enzymatic treatment of mixed wood and wheat straw pulp resulted into 11.65 and 20% decrease in chlorine consumption
respectively due to reduction in the kappa number. This had further improved the properties of the pulp and paper.
103
poster 1A-55
2-Hydroxychromene-2-carboxylate Isomerase: a Kappa class GST involved in naphthalene catabolism from
Pseudomonas putida
Lawrence Thompson* and Richard Armstrong, Dept. of Biochemistry, Vanderbilt University, Nashville, TN 37232
Jane Ladner and Gary Gilliland, Center for Advanced Research in Biotechnology, Rockville, MD 20850
We have solved the structure and developed a hypothesis for the enzymatic mechanism of 2-Hydroxychromene-2-carboxylate (HCCA) Isomerase. Its three dimensional fold is reminiscent of the Kappa class GST (figure 1). The thioredoxin
fold is interrupted by the insertion of an alpha helical domain. The enzyme’s product is not a glutathione (GSH) conjugate
but we have demonstrated that GSH is directly involved in the enzyme’s chemistry. This data in conjunction with the tight
binding of GSH to the enzyme suggests that GSH is a cofactor in the enzymatic mechanism. Crystallographic and kinetic
data with native substrate/product and analogs of each suggest that the enzymatic mechanism follows two steps. First,
is the cleavage of the acetal bond of HCCA to form cis-o-Hydroxybenzylidene pyruvate. Next, the GSH sulfur attacks
C7 (in a 1,4 Michael addition) altering hybridzation and thereby assisting isomerization. Elimination of the enzyme/GSH
complex yields product.
Figure 1. Kappa GST (left) and HCCA Isomerase (right): ribbon diagram of a single monomer showning the alpha helical domain insertion (blue) and the thioredoxin fold in red (the N-terminal βαβ motif) and green (the C-terminal ββα motif).
104
poster 1A-56
Spontaneous formation of the polykaryons of Trichoderma reesei by long-term treatment with colchicine.
Hideo Toyama*, Makiko Yano, Takeshi Hotta, and Nobuo Toyama
Minamikyushu University, Kirishima 5-1-2, Miyazaki 880-0032, Japan
We already reported that the polykaryons of a cellulolytic fungus, Trichoderma reesei, could be produced using a mitotic
arrester, colchicine, and an haploidizing reagent, benomyl. But, it appeared that the polykaryons of the fungus could be
constructed by long-term treatment of colchicine with supplying fresh colchicine solution.
Trichoderma reesei RUT C-30 was used as a model strain. Green mature conidia of this fungus were treated with colchicine solution and observed by a microscope using nuclear staining. As colchicine treatment was prolonged, a nuclei in
the conidium became larger and an autopolyploid nucleus was produced at first but multiple smaller nuclei were generated from the autopolyploid nucleus afterward. Moreover, the diameter of such smaller nuclei in the conidium also enlarged and multiple larger nuclei existed in the conidium. Cellulose hydrolyzing ability was also investigated in the strains
derived from such conidia.
poster 1A-57
Use of glucose oxidase in a membrane reactor for the gluconic acid production
Luiz Carlos Martins das Neves and Michele Vitolo*
University of São Paulo, School of Pharmaceutical Sciences, Department of Biochemical and Pharmaceutical Technology, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, São Paulo, SP, Brasil. Phone:55-11-30912382, Fax: 55-11-38156386,
E-mail: [email protected].
Glucose oxidase (GO) (EC.1.1.3.4) was used as catalyst for oxidizing glucose (G) into gluconic acid (GA) utilizing a 10mLBioengineering Enzyme Membrane Reactor ® coupled with a Millipore UF-membrane (cutoff of 100 kDa) and operated for
24h under agitation of 100 rpm, pH 5.5, and 30oC. The effect of initial glucose concentration (2.5-40mM), GO concentration (2.5-20 UGO/mL), dissolved oxygen (DO) (8.0 and 16.0 mg/L), feeding rate (D) (0.5-6.0 h-1) and glucose oxidase/catalase activity ratio (UGO/UCAT)(1:0, 1:10, 1:20 and 1:30) on the G/GA conversion were studied. A conversion yield of 80%
and specific reaction rate of 40x10 -4 mmol/h.UGO were attained when the process was carried out under the following
conditions: D = 3.0 h-1, DO = 16.0 mg/L, [G] = 40mM and (UGO/UCAT) = 1:20.
105
poster 1A-58
Production of High Fructose Syrup Using Immobilized Invertase in a Membrane Reactor
Ester Junko Tomotani and Michele Vitolo*
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São
Paulo, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, Saõ Paulo, SP, Brazil. Phone: 0055-11-30912382, E-mail:
[email protected]
The aim of this work was to evaluate the performance of immobilized invertase (EC. 3.2.1.26; KM = 17.0 mM, Vmax =
0.0240 U/mL, optimal activity at 55oC and pH 5.0) in a continuous process using a membrane bioreactor (MB) at a dilution rate of 1.6 h-1 and agitation of 100 rpm. Commercial invertase (BIOINVERT®) was adsorbed on polystyrene beads of
DOWEX-anionic resin (1X4-200) and used for sucrose hydrolysis in the MB coupled with a UF (cut off 100 kDa) or a MF
(a pore diameter of 5 µm) membrane. Considering the reactor in a 20h-steady-state continuous operation, the yields attained were 84% (mean activity of 0.88 mmol/h.mgenzyme) and 99% (mean activity of 1.02 mmol/h.mgenzyme) when a UF and
a MF membrane were employed, respectively. No leakage of enzyme from the support was detected. The inverted sugar
attained was submitted to a chromatographic separation through a column packed with DOWEX-50W8-100 resulting in a
high fructose syrup with a concentration of 70%.
poster 1A-59
Anion Exchange Resin as Support for Enzymes
Michele Vitolo*
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences of University of São Paulo,
Av. Prof. Lineu Prestes, 580, B.16, 05508-900, São Paulo, SP, Brazil. Phone: 005511-30913682 email: [email protected]
Roberto Rodrigues Ribeiro
Presbyterian Mackenzie University, Faculty of Biological, Exact and Experimental Sciences, Rua da Consolação, 930,
01302-907, São Paulo, SP, Brazil. Phone: 3236-8150, email: [email protected]
The invertase (EC.3.2.1.26) from Saccharomyces cerevisiae was employed as model enzyme for evaluating the adsorption capability of the DOWEX-1X8-50 ®, a basic anion exchange resin, for using as support in the immobilization technique. Mixing 100 mg of resin with 27 mg of invertase (pI = 4.0) an adsorption of 93% was achieved, when the procedure
was carried out at pH 4.6, agitation of 100 rpm and 25oC. The activity (1U = mg reducing sugars formed/min) for soluble
and insoluble invertase was 0.086 U/mgE and 0.075 U/mgE, respectively, which led to an immobilization coefficient of
90%. The thermal stability of the immobilized invertase was higher than of the soluble form. The highest activity was
achieved at pH 4.5 for both forms of the enzyme, whereas the stability intervals regarding pH for soluble and insoluble
invertase were 3.5-5.0 and 4.5-5.5, respectively. The kinetic constants for soluble invertase were KM = 18.3 mM and Vmax
= 0.084 U/mgE, whereas for the insoluble form were KM = 29.1 mM and Vmax = 0.075 U/mgE. The resin tested had a good
adsorbing capability, provided that the enzyme molecule had a net negative charge, i.e., the immobilization and reaction
procedures must be carried out at pH>pI.
106
poster 1A-60
Membrane Bioreactor Used for the Conversion of Sucrose in Fructose and Gluconic Acid
Luiz Carlos Martins das Neves and Michele Vitolo*
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo.
Av. Prof. Lineu Prestes, 580, B.16, 05508-900, Saõ Paulo, SP, Brazil. Phone: 0055-11-30912382 email: [email protected]
The sucrose conversion in fructose and gluconic acid was studied through a two-step process using a stirred UF-cell-type
membrane bioreactor (MB). In all tests, a 100 kDa cut off membrane was adapted to the bottom of the 10mL capacity
stirred UF-cell. The first and second steps consisted, respectively, of the sucrose hydrolysis by invertase (EC.3.2.1.26)
and the glucose oxidation by glucose oxidase (EC.1.1.3.4) (GO). The performance of both steps was followed for 48h in
steady-state continuous process. In the first step were set the invertase concentration (3.5 U/mL), pH (5.5) and agitation
(100 rpm), whereas the sucrose concentration [S], the feeding specific rate (D) and the temperature were varied in the intervals of 5.0-20mM, 1.0-3.0 h-1 and 30-50oC, respectively. A sucrose conversion of 100% was achieved at [S] = 20 mM,
D= 2.0 h-1 and 35oC. In the second step were set the catalase concentration (EC.1.11.1.6) (200 U/mL), glucose concentration (20mM), pH (5.5) and agitation (100 rpm), whereas the GO concentration, D and the temperature were varied in the
intervals of 5.0-40 U/mL, 1.0–0.3 h-1 and 30-50oC, respectively. An average glucose conversion of 89% was attained at
[GO] = 10 U/mL, D = 2.0 h-1 and 30oC.
Poster 1A-61
Enzyme Production by Industrially Relevant Fungi Growing on Feed Co-Product from Dry Mill Ethanol Plants
Eduardo A. Ximenes2, Bruce S. Dien1, Michael R. Ladisch2, Nathan Mosier 2, Michael A. Cotta1, and Xin-Liang Li1
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA/ARS, 1815 N.
University Street, Peoria, IL 61604
2
Purdue University, LORRE, 500 Central Drive, West Lafayette, IN 47907-2022
1
The United States corn ethanol market has grown to 3.4 billion gal/yr (2004) and is targeted to reach 7.5 billion gal/yr
by 2012. Expanding ethanol production has raised concerns about finding a sufficient market for Distillers Dried Grain
with Solubles (DDGS), the major co-product produced at a dry grind facility. Currently, DDGS is sold primarily as a
ruminant animal feed. In this study, DDGS was investigated as carbon source for extracellular hydrolytic enzyme production. Three microorganisms, noted for their high cellulolytic and hemicellulolytic enzyme titers, were grown on DDGS:
Trichoderma reesei, Aspergillus niger, and Aureobasidium pullulans. DDGS was either used as delivered from the plant
(untreated) or after being pretreated with hot-water (HW-DDGS, 20 min and 160°C). All three microorganisms secreted
a broad range of enzymes when grown on DDGS. T. reesei and A. niger produced higher xylanase titers when cultured
on HW-DDGS compared to growth on untreated DDGS, while the converse was observed for A. pullulans. Maximum xylanase titers were produced in 3 days for A. niger and A. pullulans and 8 days for T. reesei cultures. Initial cultures were
cultivated in flasks. Larger amounts of enzymes were produced in bioreactors (5 L) either equipped with rushton (for T.
reesei) or updraft marine impellers (A. niger). Production titers were lower for bioreactor vs. flask cultures, especially for
T. reesei cultures. Attempts to improve enzyme titers will be addressed using fed batch feeding schemes.
107
poster 1A-62
Changes in the Enzymatic Hydrolysis Rate of Avicel Cellulose with Conversion
Bin Yang1, Deidre M. Willies1 and Charles E. Wyman1,2
2
Bourns College of Engineering, University of California, Riverside, California 92521
1
The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the
substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of
this phenomenon, the enzymatic reaction of Avicel cellulose was interrupted over the course of nearly complete hydrolysis. Our initial results showed that the rate of cellulose hydrolysis, expressed as the percentage of remaining substrate
at the testing time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was
approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the
drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate
reactivity, suggesting that other effects such as enzymes getting “stuck” or otherwise slowing down may be responsible.
A new mechanism for enzymatic hydrolysis of cellulose will be described.
poster 1A-63
Assays and Methods to Engineer Enzymes for Biomass Conversion
Mary M. Yang*, Steven J. Robles, and William J. Coleman, KAIROS Scientific Inc., San Diego, CA 92121 USA
Engineered enzymes offer a potentially rich source of catalysts for the fuels and chemical industries. Using directed evolution and high-throughput screening, one can engineer enzymes for industrial processes that are more efficient, involve
unusual substrates, or have activity under harsh reaction conditions.
KAIROS has created a system of assays and methods, known as Kcat Technology, for screening very large numbers of
enzyme variants expressed in microcolonies. Spectral and/or kinetic information on tens of thousands of individual enzyme variants can be simultaneously acquired using chromogenic or fluorogenic solid-phase assays. Only 10-50 nanoliters of substrate volume per microcolony is needed, while at the same time providing a throughput of one million variants
per instrument per day. The solid-phase format is ideally suited for insoluble or high molecular weight substrates such as
polysaccharides and polymers. Due to assay design flexibility, multiple properties can be evolved simultaneously in the
same variant. Almost no pipetting is necessary, enabling individual researchers to efficiently screen for enzymes meeting
their own performance criteria. Kcat Technology can also be used to re-design metabolic pathways or to facilitate host
strain improvement. Enzyme engineering data, including that from an NSF funded project to design cellulases for biomass conversion will be presented.
108
poster 1B-06
Controlling Cellulose Production Through Gene Expression
Jennifer Milne2, Natalia Kitrov1, and Chris Somerville1,2
Carnegie Institution, Dept. of Plant Biology1 and Stanford University2, Dept of Biological Science, Stanford, CA 94305
Plant cell walls are the primary source of terrestrial biomass. Any attempt to develop and use biomass-based sources of
energy must involve optimized production and utilization of cell walls. One of the principle components of plant cell walls
is cellulose, a polymer composed of β-1-4 linked glucose. The amount of cellulose varies from one cell type to another
within the various tissue types in a plant. Two main types of cell wall, primary and secondary, are known to exist in plants.
Secondary walls by definition are those laid down after cell division and expansion have ceased. These cells can have
thickenings of cellulose and other polymers that increase their width by more than 10 times compared to primary walls.
The Cellulose Synthase that makes the cellulose in these secondary wall thickenings is known to require three different protein subunits to make a functional complex. I propose to test the concept that the production of cellulose can be
increased by increasing the expression of the genes encoding the components of Cellulose Synthase.
poster 1B-07
Breeding, selection, testing, and commercialization of high-yielding varieties of shrub willow for bioenergy,
biofuels, and bioproducts
Lawrence B. Smart*, Kimberly D. Cameron, Timothy A. Volk, and Lawrence P. Abrahamson
SUNY College of Environmental Science and Forestry, Syracuse, NY 13210
High-yielding perennial energy crops must be bred, tested, and deployed for the U.S. to meet a national goal of producing
1 B tons of biomass annually for renewable energy and products. Analysis by Perlack and colleagues (2005) suggests
that this goal is attainable by cultivating 55 M acres of perennial energy crops yielding 8 dry tons ac-1 yr-1. Fast-growing
shrub willow is ideally suited as a dedicated energy crop for the Midwest and Northeast U.S. We have established and
maintain the largest willow breeding program in North America, which includes a genetically diverse collection of >700
varieties. We have completed more than 450 controlled crosses since 1998, incorporating over 20 species and species
hybrids and producing in excess of 4000 new varieties. >From among >2000 individuals produced by breeding in 1999,
82 were selected based on stem area and height after two seasons of growth. These were propagated and planted in
a selection trial with four current production varieties in 2002, which was harvested in 2005 after two growing seasons.
Eighteen varieties produced mean biomass yield that exceeded that of the best current production variety, ‘SV1’. The
highest mean yield in these small plots was 40% higher than that of ‘SV1’, which typically yields 4-5 dry tons ac-1 yr-1. A
number of these high-yielding varieties have been scaled-up and are being tested in regional yield trials, two of which
were established in 2005. They are also being transferred to a commercial nursery for production and sale of whips as
planting stock for expanded bioenergy plantings in New York.
109
poster 1B-08
Enhanced Bio-Processing Of Corn Stover With Modified Cell Wall Composition
Wilfred Vermerris1,2,3*, Javier Campos1, Brittany Phillips2, Michael R. Ladisch2,3, and Nathan S. Mosier 2,3
Department of Agronomy, 2Department of Agricultural & Biological Engineering and 3Laboratory of Renewable
Resources Engineering, Purdue University, West Lafayette, IN, USA
1
The modification of cell wall composition in planta offers great opportunities to improve the efficiency of the stoverto-ethanol process. We have performed a multi-year analysis of maize brown midrib mutants in near-isogenic lines to
investigate the role of cell wall composition on the yield of fermentable sugars obtained from enzymatic hydrolysis. In
addition, we have evaluated the agronomic performance of these lines, as well as the impact on pretreatment. We are
able to show a clear relationship between cell wall composition – predominantly the ratio of guaiacyl-to-syringyl residues
in the lignin – and the yield of fermentable sugars. The agronomic performance, especially pest resistance, is negatively
affected in some of the lines. These data will be helpful for the development of maize lines intended for whole-plant bioprocessing, whereby both the grain and the stover are harvested and used for ethanol production. We have developed
several new inbred lines with excellent agronomic properties and high yields of fermentable sugars that may be further
improved through metabolic engineering.
poster 1b-09
Glycosylation Modification of Trichoderma reesei Cellobiohydrolases I Expressed in Pichia pastoris
Lianguo Wei, Guochao Wu, Guoqiang Zhuang* and Yinbo Qu
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Shandong Province, China.
Email: [email protected]
The filamentous fungus T. reesei, one of the most potent cellulose-degrading organisms, produces a complex cellulase
system. CBH I is the principal component of the T. reesei cellulase system, representing 60% of the total cellulase protein
produced by the fungus on a mass basis. To fulfill the need of this enzyme on enzyme studies and enzyme engineering
studies, it is necessary to establish a better expression system since it is hard work and also time consuming to obtain the
component from complex cellulase system.
Pichia pastoris was thought to be a good expression system for CBH I, which could secrete very high level single protein
into almost protein-free medium. However, the expressed CBH I in P. pastoris was found to be hyperglycosylated, and the
activity was significantly lower than that of T. reesei CBH I.
In this study, asparagines of the four N-glycosylation sites-Asn45, Asn64, Asn270 and 384 of CBH I were replaced by
serines using site-directed mutagenesis. These Four mutants and T. reesei cbh1 gene were transformed into P. pastoris,
the recombinant enzymes were then purified, and analyzed kinetically. Data showed that the specific activity of mutant
Asn64 enzyme was higher than original P. pastoris expressed enzyme. This indicates the Asn64 glycosylation has a great
effect on activity of the CBH I enzyme expressed in P. pastoris. The glycosylation modified CBH I enzyme could have a
better expression in P. pastoris, making this yeast a much more appropriate host for CBH I expression.
110
Poster 2-08
Investigation of the impact of enzyme characteristics on corn stover preservation and pretreatment
a
Haiyu Rena, Tom L. Richarda*, Kenneth J. Mooreb, Patricia Patrickb
Department of Agricultural and Biological Engineering, Pennsylvania State University
b
Department of Agronomy, Iowa State University
An anaerobic fermentation process similar to ensilage has been proposed as a preservation and pretreatment method
for lignicellulosic biomass prior to industrial bioprocessing. To initiate this process, cell wall degrading enzymes may be
required to supplement the low sugar content in biomass materials harvested during senescence,. This study investigate
the impacts on corn stover preservation and pretreatment of 7 commercial enzyme mixtures. These enzyme products
were derived from three microbial sources: Aspergillus niger, Trichoderma reesei, and Trichoderma longibrachiatum..
Treatments included three size grades of corn stover (< 10 cm, < 1 cm, < 0.5 cm), two enzyme levels (1.67 IU/g DM and
5 IU/g DM based on hemicellulase), and various ratios of cellulase to hemicellulase (C:H) in products derived from each
microbial source. Higher lactic acid content and lower pH were obtained with increasing C:H ratios, especially with Trichoderma reesei enzymes. The highest C:H ratio tested, 2.38, resulted in the most effective fermentation, with lactic acid
the dominant product. Significant cellulose and hemicellulose degradation was observed in these high C:H ratio enzyme
mixtures derived from Trichoderma reesei, indicating the additive rates could be reduced if preservation is the primary
goal. More intensive enzymatic pretreatment during storage may complement industrial pretreatment strategies, creating
synergies that could reduce total bioconversion costs.
poster 2-09
The Effect of Flow Rate, Temperature, and Acid Concentration on Protein Extraction from Alfalfa in a
Flowthrough System
Charles E. Wyman
Bourns College of Engineering Center for Environmental Research and Technology, University of California, Riverside,
CA 92521
Toyin Abifarin*, Thayer School of Engineering, Dartmouth College, Hanover, NH 03755
Extraction of protein from cellulosics presents an opportunity to produce both animal feed and fuels and chemicals on
the same land, thereby enhancing the potential impact of biomass derived products. In addition, energy and other costs
for biomass production and cultivation are distributed over more products. However, the majority of current research on
biomass-based energy production is focused on increasing yields of fermentable sugars from biomass for subsequent
fermentation into ethanol and other products, with limited attention given to protein fate or utilization.
In our research, a flowthrough reactor system is being applied to clarify the effect of temperature and pH on protein
release patterns during processing of cellulosic materials and their relationship to solubilization of hemicellulose, lignin, and other constituents. In this study, just water and very low concentrations of sulfuric acid were passed through a
packed bed of alfalfa at specific temperatures, and the composition of the residual solids was analyzed at regular intervals
to track the removal of total mass, protein, glucan and xylan to determine favorable conditions for protein release. An
analysis is then presented of the trade-off between conditions that favor protein extraction compared to fermentable sugar
recovery.
111
poster 2-10
Optimizing AFEX Pretreatment and Enzymatic Hydrolysis of Cassava
Nana Siriboe Achampong*, Balan Venkatesh, Bruce E. Dale
Biomass Conversion Research Laboratory (BCRL), Department of Chemical Engineering & Material Science, Michigan
State University, East Lansing, MI – 48824.
The quest for a viable alternative source of fuel has generated interest in producing fuels and chemicals from various
sources of biomass. Our laboratory applies the ammonia fiber explosion (AFEX) process to increase the hydrolysis of
lignocellulosic biomass. With promising results in the AFEX pretreatment and bioconversion of biomass raw materials
like poplar, switch grass, corn stover and dried distillers grain (DDG), other biomass sources are being explored for AFEX
pretreatment. One such source is cassava. Cassava (Manihot esculenta) is a tuber plant grown mostly in tropical areas.
The major component of cassava is starch with very little protein and cellulose. Most of cassava cellulose is present in
the tuber’s skin.
There has been work done on liquefied cassava starch to produce ethanol using co-immobilized cells of Zymomonas
mobilis and Saccharomyces diastaticus. We report here the effectiveness of the ammonia fiber explosion (AFEX) pretreatment on cassava skin and its dry tuber part followed by enzymatic hydrolysis using different combination of enzymes
such as amylase, cellulase and hemicellulase.
poster 2-11
Fractionation Of Cynara Cardunculus (Cardoon) Biomass By Dilute-Acid Pretreatment
Mercedes Ballesteros*, M. José Negro, Paloma Manzanares, Ignacio Ballesteros, Felicia Saez, J. Miguel Oliva
Renewable Energies Department-CIEMAT, Avda. Complutense, 22 28040-MADRID SPAIN
Phone: +34 91 346 62 61 E-mail: [email protected]
Cardoon (Cynara cardunculus L.) is a perennial herb originally from the Mediterranean area that can be considered as
a potential lignocellulosic feedstock for biofuels production in Spain. Field studies have shown great adaptability to the
environmental conditions of the country and high biomass productivities. One of the energy uses that could be envisaged
for aboveground biomass, particularly steams and leaves fraction, is its biological conversion to ethanol. In an enzymebased biomass to ethanol process, pretreatment has been identified as a key step to increase substrate accessibility.
Among the different technologies that have been proved to fractionate biomass, pretreatment with dilute sulphuric acid at
high temperatures has been reported as an effective method for dissolving hemicelluloses and increasing the enzymatic
digestibility of cellulose in a great variety of lignocellulosic substrates.
In this work, cardoon biomass (steams and leaves) was pretreated with dilute-sulphuric-acid at high temperature in a Parr
batch reactor. The effect of temperature (160-200ºC), acid loading (0-0.2 %, w/w) and solid/liquid ratio (5-10 %, w/v) on
sugar recovery yields in the liquid and solid fractions and enzymatic hydrolysis was studied using a response surface
method according to a Box-Behnken experimental design. Detailed results of this study will be presented.
112
poster 2-12
Extracting Proteins from Switchgrass within an Integrated Biorefinery
Bryan Bals, Bruce Dale, Venkatesh Balan
Department of Chemical Engineering and Material Science, Michigan State University College of Engineering,
East Lansing, MI 48824‑1226
Proteins can be an important co-product in the production of biofuels, significantly reducing the cost of ethanol production. Switchgrass (Panicum virgatum), considered a potential source of biofuel, contains up to 15% protein if harvested
in the spring. Integrating a protein extraction process with ethanol production may be advantageous for both products. In
particular, the ammonia fiber explosion (AFEX) pretreatment process has been shown to improve both sugar and protein
yields.
This research focuses on recovering these proteins by solid-liquid extraction using aqueous ammonia and integrating this
process with ethanol production. Optimal conditions for protein extraction are determined, as well as optimal placement of
the extraction within the ethanol process. A proposed process flow diagram for biomass processing giving optimal ethanol
and protein yields will be presented with corresponding mass balances for carbohydrates, protein, and total biomass. The
presence of other components, such as lignin and ash, within each of the streams are also determined.
poster 2-13
Heat-extraction of corn fiber hemicellulose
Zsuzsa Benko, Zsolt Szengyel, Melinda Gáspár, Kati Réczey*
Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, Szent Gellért tér 4,
H-1521 Budapest, Hungary
Alexandra Andersson, Henrik Stålbrand
Department of Biochemistry, Center of Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100
Lund, Sweden
Water-soluble hemicellulose (mainly arabinoxylan) was extracted from corn fiber with microwave-assisted heat treatment. The effects of treatment temperature and the initial pH of the aqueous extraction media were investigated regarding
hemicellulose recovery and molecular weight of the extracted polysaccharides The apparent molecular weight distribution
of the water soluble polysaccharides was analyzed using size-exclusion chromatography. Water extraction at 180oC and
neutral pH gave a yield of 20% and an average molecular weight of 1.4x105. At higher temperatures the yield was higher,
but the molecular weight decreased. Lowering the temperature or using alkaline extraction conditions resulted in an
increased molecular weight but compromised yield. Acidic extraction conditions gave all together the lowest yield and the
lowest molecular weight of the extracted hemicellulose. The monomeric sugar composition of the hemicellulose extracted
at 180oC suggests that the major polysaccharide to be extracted is arabinoxylan.
113
poster 2-14
Fortifying Spent Sulphite Pulping Liquor using Levoglucosan Derived from Pyrolysis Oil to Improve Ethanol Yields
Nicole M. Bennett, Steve S. Helle and Sheldon J.B. Duff *
UBC Pulp and Paper Centre and Department of Chemical and Biological Engineering,
The University of British Columbia, 2360 East Mall, Vancouver, BC, V6T 1Z3
Lignocellulosic materials exhibit great potential as a renewable feedstock for the production of transportation fuel. Commercial ethanol production from lignocellulosic hydrolysates such as sulphite pulping liquor (SSL) are limited by the low
concentration of fermentable sugars. One potentially viable source of additional fermentable sugars is pyrolysis oil. Depending on the substrate and the operating conditions, pyrolysis oils contain variable amounts of 1,6-anhydro-β-D-glucopyranoses (levoglucosan or LG), an anhydrosugar that can be hydrolyzed to glucose. By using the levoglucosan fraction
of pyrolysis oil to increase the concentration of fermentable sugars in SSL (or any hydrolysate) prior to fermentation, it
may be possible to significantly increase ethanol yield and reduce distillation costs. The goal of this study was to evaluate
this potential.
Preliminary data demonstrates that LG can be efficiently extracted using minimum levels of an aqueous solution (water or
SSL); however significant concentrations of organic acids are also extracted. Complete hydrolysis of LG can be obtained
and parameter effects between temperature, time and acid concentration have been established. Fermentation production rates, inhibited by the organic acid fraction, can be improved using micro-aerophilic conditions and large yeast concentrations. Under such conditions, growth medium fortified up to 20% with the hydrolysate exhibits long growth cycles
(2x control) with proportionally higher final ethanol concentrations.
poster 2-15
Evaluation of British Columbian Beetle-Killed Hybrid Spruce for Bioethanol Production
Alex Berlin*,1, Neil Gilkes1, Sepideh Alamouti1, Claudio Muñoz2, Jaime Baeza2, Juanita Freer 2, Jack Saddler1
Forest Products Biotechnology, Faculty of Forestry, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada
2
Laboratory Renewable Resources, Biotechnology Center, University of Concepción, Concepción, Chile
1
The development of resources and conversion technologies for production of fuels, chemicals and power from renewable
resources is currently a high priority for developed nations such as the USA, Canada and the EU as a way to improve
national energy security and reduce greenhouse gas emissions. The widespread implementation of such technologies will
require a constant and secure supply of biomass from forestry and agriculture.
Forests are a major source of feedstocks for biofuels production in Canada. Woody biomass includes residues from
logging and forest thinning, and from wood processing and pulp production. More recently, damaged wood resulting
from beetle attack has become available on a large scale in Western Canada . This study evaluates beetle-killed British
Columbian hybrid spruce (Picea glauca x Picea engelmannii) as a feedstock for the production of bioethanol. In the past
30 years, attack by the beetle Dendroctonus rufipennis and associated fungi has resulted in estimated losses of >3 billion board feet in British Columbia alone. Here we describe the chemical and physical characteristics of both healthy and
beetle-killed British Columbian hybrid spruce and evaluate the technical feasibility of using these feedstocks as a source
of biomass for bioethanol production.
114
poster 2-16
Optimization of SO2-catalysed steam explosion for hybrid poplar
Renata Bura*, Alex Berlin, Neil Gilkes, Jack Saddler
University of British Columbia, Forest Products Biotechnology Group, Vancouver, BC V6T 1Z4
As a renewable resource that is potentially available in large quantities, hybrid poplar could provide a low cost feedstock
for manufacture of fuel-grade alcohol. In this study, we have examined acid-catalyzed steam explosion as a pretreatment
technology for the bioconversion for hybrid poplar. Pretreatment temperature (190oC-215oC), residence time-5min, and
SO2 concentration-3% (w/w dry matter) were varied to give pretreatments of low, medium and high severities. Subsequently, enzymatic hydrolysis of pretreated and water washed poplar was investigated at 2% (w/v) solids concentration.
Maximum soluble sugar yields after enzymatic hydrolysis were recovered from poplar that had been pretreated at 200oC,
5 min and 3% SO2. Sequential SO2-catalysed steam explosion and enzymatic hydrolysis resulted in very high conversion
of all polysaccharides in hybrid poplar to monomeric sugars.
poster 2-17
Maximizing overall sugar recovery of hydrolysable and fermentable sugars during SO2-catalysed steam
explosion of corn stover
A commercially-viable biomass-to-ethanol conversion process should provide maximum recovery of sugar in a form that
can be easily hydrolyzed and fermented. In an effort to increase overall sugar recovery, we have explored the possibility
of using lower pretreatment severity during SO2-catalysed steam explosion of corn stover, followed by enzymatic hydrolysis with a mixture of cellulases, β-glucosidases and xylanases.
In this study, we compared corn stover pretreated by SO2-catalysed steam explosion at low pretreatment severity (170oC,
9 min and 3% SO2) with material pretreated at medium severity (190oC, 5 min and 0/3% SO2). By supplementation of the
enzyme cocktail with additional xylanase, we showed that is feasible to use a low pretreatment severity, or a medium pretreatment severity without an SO2-catalyst, to obtain good sugar recovery in hydrolysable form and low levels of fermentation inhibitors.
115
poster 2-18
Robustness of SO2-catalysed steam explosion pretreatment process forbioconversion of agricultural, hardwood
and softwood residues
Pretreatment is the most critical sub-process for enzyme-based biomass-to-ethanol conversion, affecting later hydrolysis
and fermentation steps. Since no single lignocellulosic feedstock is likely to satisfy future demands for fuel ethanol production, future biomass-to-ethanol schemes require a robust pretreatment process able to accommodate a diverse range
of feedstocks. This study demonstrates a SO2-catalysed steam explosion pretreatment process for agricultural, hardwood
and softwood residues. Pretreatment robustness is evaluated in terms of overall recovery of hydrolysable and fermentable
sugars, and general process complexity. The versatility of SO2-catalysed steam explosion is demonstrated using mixed
feedstocks (corn fibre + corn stover + poplar and poplar + lodgepole pine).
poster 2-19
WITHDRAWN
116
poster 2-20
Analysis of modes of detoxification of dilute acid pretreatment hydrolysate by ion exchange and overliming treatments
Kerry O’Bric, Shou-Feng Chen, C. Kevin Chambliss, Department of Chemistry and Biochemistry, Baylor University
G. Peter van Walsum*, Department of Environmental Studies, Baylor University, Waco, TX 76798-7266
Overliming and ion-exchange are generally recognized as efficient methods for improving fermentability of dilute acid pretreated hydrolysates. This study aims at furthering the understanding of the detoxification characteristics of commercially
available ion exchangers and overliming.
Two strong-base anion exchangers (R4N+X−, X− = Cl− or OH−) and one weak-base anion exchanger (R3N) were investigated in a side-by-side comparison. Hydrolysate was generated by pretreatment of milled corn stover with dilute sulfuric acid
at high severity. Aliquots of the hydrolysate were treated by overliming and/or ion exchange. Fermentability by Saccharomyces cerevisiae was used to evaluate the detoxification strategies, and HPLC was used to identify the array of organic
acids and phenolics originating from the degradation.
Fermentation experiments demonstrated that the OH form strong-base anion-exchange resin was the most efficient
detoxifier of the three resins and that overliming used in combination with anion-exchange was a highly effective. Interestingly, HPLC analysis indicated that removal of organic acids and phenolics was essentially independent of the type of
anion-exchanger. Experiments combining overliming and ion exchange also indicated detoxification results independent
of resin type. These results indicate that the primary mode of extraction by the resins may have been adsorption instead
of anion-exchange. Overliming alone had a dramatic effect on hydrolysate fermentability, though it did not appear to appreciably reduce the concentrations of presumed fermentation inhibitors (organic acids, phenolics, etc.). This suggests a
potential dependence of fermentability on the ionic strength of the fermentation medium.
poster 2-21
The Development of a Microplate Technique for Screening Enzyme Mixtures & Synergistic Additives for
Hydrolysis of AFEX treated Lignocellulosic Biomass
Shishir P. S. Chundawat*, Balan Venkatesh, Bruce E. Dale
Biomass Conversion Research Lab (BCRL), Department of Chemical Engineering & Materials Science, Michigan State
University, East Lansing, MI 48824. Phone: 517-353-9948 email: [email protected]
Past biomass pretreatment research has often emphasized acidic pretreatments which hydrolyzed most hemicellulose
and some cellulose prior to further enzymatic hydrolysis. However, increasing attention is now being paid to alkaline or
neutral pretreatments (e.g, Liquid Hot Water or Ammonia Fiber Explosion (AFEX)) and we must identify multi-activity
enzyme mixtures to completely hydrolyze residual glucan and xylan. Screening of these multi-enzyme systems might be
rapidly performed using a microplate technique currently under development in our laboratory. The microplate technique
allows screening of cellulolytic enzymes (cellulases, hemicellulases and beta glucosidases) over a larger range of input
variables, in a fast and reproducible manner comparable to the conventional NREL LAP 009 hydrolysis protocol. Other
accessory enzymes, such as beta-xylosidases and other side chain hydrolyzing hemicellulases, and synergistic additives,
such as surfactants & proteins, were also screened using the microplate technique for various lignocellulosics. The goal
of the present project was to develop economic process options effective in reducing the net enzyme loading by determining a suitable concoction of hemicellulases, cellulases and additives tailored for several AFEX pretreated biomass
substrates.
117
poster 2-22
Solid state fermentation of agricultural wastes for cellulases and Hemicellulases production
Rodrigo Simoes Ribeiro Leite, Eduardo da Silva Martins, Eleni Gomes, Roberto da Silva*
Laboratorio de Bioquimica e Microbiologia Aplicada, Instituto de Biociencias,
Letras e Ciencias Exatas (IBILCE), Universidade Estadual Paulista (UNESP), Sao Jose do Rio Preto-SP.
Cristovao Colombo, 2265. CEP: 15054-000, Brasil.
Corresponding author: Fax: +55-017-3221-2390; phone: 55-017-3221-2453, email: [email protected]
Cellulase and hemicellulase enzymes have application in the paper and cellulose, food and chemical industries. In
this work a selected strain of the yeast-like AureobasidiumpPullulans was investigated for production of cellulases and
hemicellulases in solid state fermentation (SSF). The substrates used were wheat bran, soy bran, soy peel and corn
cob. Higher enzymatic production was obtained on wheat bran cultivation which was: 1.05 U/ml of endoglucanase (96
hours), 1.3 U/ml of ß-glucosidase (120 Hours) and 5.0 U/ml of xylanase (96 hours). It was not observed the production
of avicelase by the microorganism. The production of xylanase and endoglucanase obtained in the present work, when
compared with some other Aureobasidium species, was very expressive. Moreover, regarding the scarcity of works on
production of these enzymes by the microorganism Aureobasidium pullulans on SSF, the result stands out in its importance, extending the source of these enzymes for future works.
Support: FAPESP
poster 2-23
Development and Pilot Scale Performance Trials of a Steep Delignification Process
M. Clark Dale, Brian Billings, Bio-Process Innovation, 226 N 500W, W. Lafayette, IN 47906-8505
Daniel Musgrove, Universal Entech, 5501 N. 7th Ave, Phoenix, AZ 85013
A new ‘Steep Delignification’ (SD) process is under development by Bio-Process Innovation. The SD process is characterized by 1) mild temperatures (30 to 60 C) and pressures (atmospheric), and 2) low cost/ recyclable chemicals. The SD
process solubilizes lignin while leaving cellulose and hemicellulose intact. During the current trials, process performance
was evaluated based on key processing variables including temperature, stirring/shear, ozone addition rates, particle size
and pH. It was determined that rates of delignification could be measured and monitored via base addition/utilization rates
metered in by the unit’s pH controller.
With the assistance of a Recycling R&D Contract from the Arizona Department of Environmental Quality, two types of
urban-derived biomass were tested for effectiveness of the SD process; 1) ‘Urban Wood Waste’(UWW) sawdust consisting of milled pallets and commercial wood waste, and 2) ‘Urban Green Waste’ (UGW) consisting of milled yard trimming
debris collected from commercial landscape contractors. The green waste ‘UGW’ delignified much more quickly than the
sawdust (UWW), with particle size having a major impact on speed of delignification. Fermentation efficiencies via SSF of
the sawdust (UWW) were directly related to the degree of delignification. Effects of the various operating variables on the
UWW will be presented.
118
poster 2-24
The Effect of Particle Size on Hydrolysis Reaction Rates and Rheological Properties in Cellulosic Slurries
Rajesh K. Dasari and R. Eric Berson*
University of Louisville, Department of Chemical Engineering, Louisville, KY 40292
The enzymatic hydrolysis of agricultural residues in slurries consisting of varying initial particle sizes is investigated in
shaker flasks. Slurries with particle sizes ranging from 5 mm to less than 75 microns, are subjected to enzymic hydrolysis
using an enzyme dosage of 15 FPU/ g cellulose at 500C and 250 RPM. As the particle size decreases, reaction rates for
the conversion of cellulose to glucose increase. The effect of the initial particle size on viscosity over a range of shear
is also investigated. For equivalent initial solids concentration, smaller particle sizes result in lower viscosities. Results
indicate particle size reduction may provide a means for reducing the long residence time required for the enzymatic hydrolysis step in the conversion of biomass to ethanol. Furthermore, the corresponding reduction in viscosity may allow for
higher solids loading to help minimize reactor size during large-scale processing.
poster 2-25
Oxidative Fractionation of Sugarcane Bagasse using a Combination of Hypochlorite and Peroxide
Yong-Jae Lee, Louisiana State University, Food Science in Baton Rouge, LA, Chang-Ho Chung, Donal F. Day*, Audubon
Sugar Institute in Baton Rouge, LA
Ox-B (US Patent 6,866,870), a solution of sodium hypochlorite and hydrogen peroxide, was effective in fractionating
bagasse. Reactive Oxygen Species (ROS); singlet oxygen (1O2), superoxide (O2-), hydroxyl radicals (OH·), and hypochlorite ion (OCl-) produced by Ox-B removed both hemicellulose and lignin from sugarcane bagasse. Ox-B treated
cellulose was readily separated by filtration from lignin. After a 20:1 treatment (v/w) with an Ox-B solution the bagasse
lost 38.6% of its weight. The residual solids, contained 45% cellulose, 22% hemicellulose and 11.4% lignin. Untreated
bagasse contains 34.5% cellulose, 24.5% hemicellulose and 21.8% lignin. The treated solids were 97.4% digestible by
cellulases in a standard NREL 72 hrs digestion. Treatment temperature did not affect weight loss or cellulose digestibility.
Concentrations of Ox-B greater than 2% (v/w) removed hemicellulose as well as lignin. Sequential treatments doubled the
digestibility for the same concentration of Ox-B. A caustic wash of the treated solids with 0.6% NaOH produced a 20%
improvement in lignin removal above that produced by Ox-B.
119
poster 2-26
Enzyme Hydrolysis and Phenols Recovery Post Alkaline and Organosolv Treatment of Sugarcane Bagasse
Chang-Ho Chung, Donal F. Day, Giovanna A. DeQueiroz,
Audubon Sugar Institute, Louisiana State University Agricultural Center, St. Gabriel, LA 70776
Utilization of the lignocellulosic residues from sugarcane production (bagasse and field trash) for fuel, energy and
chemicals has become a vision for the future for the sugarcane industry. In 2004, approximately 2,209,691 tons of
bagasse was available as biomass in Louisiana and Florida, with conversion of the cellulose in a ton of sugarcane
bagasse can yield 80 L of ethanol which it is equivalent of 1.2 barrels of oil. An organosolv process using ethanol, in
the presence of an acid or a base, will delignify bagasse. Enzyme conversion of sugarcane bagasse to glucose and the
release of mono phenols from lignin was evaluated after treatment with 50% (w/w) ethanol and sodium hydroxide (5%
chemical on dry bagasse) and following sequential treatments with sodium hydroxide (80-100˚C at atmospheric pressure)
. The treated solids were greater than 80% digestible by cellulases in a standard NREL 72 hrs digestion test. Ethanol
with base hydrolysis produced high concentrations of mono phenolic compounds, ranging from 30 mg to 100 mg of
vanillin equivalent g-1 dry bagasse. The compounds produced by the various treatments were mostly substituted phenols,
benzofurans, and vanillin as characterized by GC/MS.
poster 2-27
Effect of Dissolved Carbon Dioxide on Accumulation of Organic Acids in Liquid Hot Water Pretreated
Biomass Hydrolysates
G. Peter van Walsum*, Maurilio Garcia-Gill
Department of Environmental Studies, Baylor University, Waco TX, 76798-7266
Shou-Feng Chen, C. Kevin Chambliss
Department of Chemistry and Biochemistry, Baylor University
Liquid Hot Water Pretreatment has been proposed as a possible means of improving rates of enzymatic hydrolysis of
biomass while maintaining low levels of inhibitory compounds. Supplementation of liquid hot water pretreatment with dissolved carbon dioxide, yielding carbonic acid, has been shown to improve hydrolysis of some biomass substrates compared to the use of water alone. Previous studies on the application of carbonic acid to biomass pretreatment have noted
a higher pH of hydrolysates treated with carbonic acid as compared to samples prepared with water alone.
This study has applied recently developed analytical methods to quantify the concentration of organic acids in Liquid Hot
Water pretreated hydrolysates prepared with and without the addition of carbonic acid. Results from these comparison
studies are presented.
120
poster 2-28
Enzymatic Hydrolysis of tissue paper
Oscar García-Kirchner , Samuel Suazo A.*, Marcela Segura G.*
Departamentos de Química y Bioprocesos* de la UPIBI-IPN, Av. Acueducto S/N. Barrio La Laguna Ticomán, México.
07340, D.F., MEXICO.
Our laboratory team has developed a novel enzymatic hydrolysis using a commercial cellulase to produce soluble sugars
together aluminum foils obtained from cigarettes boxes. During 12 hours of enzymatic hydrolysis it was possible obtain
75% of reducing sugars and 90% of aluminum foil separation after to stop the reaction. This system is now being further
developed for optimization of hydrolysis conditions. The results presentation will include the research development for
enzymatic hydrolysis of paper tissue used for cigarettes package using cellulase Sigma. It will emphasize the technical
feasibility for aluminum foils recuperation, the reducing sugar production obtained by cellulose hydrolysis reaction and the
enzymatic hydrolysis conditions.
Now we are studying the effect of different environmental conditions on the hydrolysis reaction and the separation of
aluminum foils released after 12 hours of enzymatic hydrolysis using a simple filtration procedure for the scale up the process to a major level. Preliminary economic estimates show this process to be another competitive possibility for recycle
of a waste obtained in high quantities.
poster 2-29
Separation of Glucose and Pentose Sugars by Selective Enzyme Hydrolysis of AFEX-treated Corn Fiber
Robert J. Hanchar, Farzaneh Teymouri, Chandra D. Nielson, Darold McCalla and Mark D. Stowers*
MBI International, 3900 Collins Road, Lansing, MI 48910, USA
A process was developed for separation of cellulose and hemicellulose of Ammonia Fiber Explosion (AFEX) treated
corn fiber by selective hydrolysis of cellulose by cellulase, followed by ethanol precipitation of solubilized hemicellulose.
Monomeric sugars were produced by either acid or enzyme hydrolysis of the generated hemicellulose fraction. Using this
method, 57% of xylose and 54% of available arabinose were recovered in a pentose-rich stream, and 83% of available
glucose was recovered in a glucose-rich stream. The experimental detail, process flow diagram (PFD) and the carbohydrate composition of both glucose and pentose rich streams as well as the overall mass balance will be presented.
121
poster 2-30
Pilot Scale Measurement of Viscosity for a Biomass Slurry Composed of 15-20% Corn Fiber in Light Stillage
Richard Hendrickson, Youngmi Kim, Yulin Lu, Nathan Mosier, Michael Ladisch*
Purdue University, West Lafayette, IN 47907
Richard Dreschel, Gary Welch
Aventine Renewable Energy Company, Pekin, IL 61555
The aqueous pretreatment of corn fiber at a pH of 4 to 7 while being pumped through a hold coil is effective in increasing
the rate of enzyme hydrolysis of the cellulose. However, scale-up of the pretreatment process depends on physical properties of the material to be pumped through the system. High concentrations of fermentable sugars require that aqueous
biomass streams from which these sugars are derived have a high solids content. Since the corn fiber solids at high
loading have characteristics that resemble a shear-thinning fluid, measurement of viscosity in the laboratory is difficult,
particularly at temperatures above ambient. Consequently, we carried out measurements in a plant setting. Corn fiber at
150 to 200 g/L were pumped at rates of 1 to 10 gal/minute through sections of jacketed tubing having diameters ranging
from 1 to 1.5 inches and a length of 17.25 feet. The temperatures and pressure drops were measured at the inlet and
outlet of the tubes and recorded through a LabVIEW programmed data acquisition system. The pressure drop and flow
rate enabled calculation of viscosity and determination of correlations that will be useful for scale-up.
poster 2-31
Investigating Factors Affecting Enzymatic Hydrolysis of Dilute Acid Pretreated Corn Stover Slurries
David B. Hodge*, Daniel J. Schell, and James D. McMillan
Enzymatic hydrolysis of cellulose is an important step in deploymerizing lignocellulosic biomass into its component
sugars. It is desirable to perform this step at high solids loadings to produce concentrated sugar streams and thereby
reduce production cost. But operating at high solids loadings can also concentrate soluble components produced during
pretreatment, some of which can be potent inhibitors of enzymatic hydrolysis and/or microbial fermentation. This work
used analysis of variance (ANOVA) and response surface modeling (RSM) to characterize the effects of (enzyme loading,
solids concentration, phenolics, furans, sugars, and acetic acid) on cellulose conversion. We found that the presence of
lignin-derived phenolic compounds and sugar-derived furans did not significantly affect reaction kinetics over the ranges
investigated. The major inhibitors to enzymatic hydrolysis were sugars (up to 130 g/L total sugars) produced during pretreatment, while acetic acid (15 g/L) was only slightly inhibitory. A significant finding was that enzymatic reaction kinetics
observed for high-solids hydrolyzate slurries could be duplicated using washed pretreated cellulosic solids with added
inhibitors. Thus, we are confident that this work has identified the major inhibitors affecting enzymatic hydrolysis of high
solid, dilute acid pretreated corn stover slurries.
122
poster 2-32
The potential in bioethanol production from fiber sludges in pulp mill-based biorefineries
Anders Sjöde1,2, Björn Alriksson1, Leif J. Jönsson1*, and Nils-Olof Nilvebrant 2
1
Biochemistry, Division for Chemistry, Karlstad University, SE-651 88 Karlstad, Sweden
2
STFI-Packforsk, P.O. Box 5604, SE-114 86 Stockholm, Sweden
Industrial production of bioethanol from fibers that are unusable for pulp production in pulp mills offers a novel approach
to product diversification and more efficient on-site exploitation of the raw material. In an attempt to utilize fibers flowing
to the biological waste treatment, three fiber sludges from different pulp mills were collected and tested for bioethanol
production. Another aim was to acquire a solid residue with a higher dry content than the original fiber sludge to achieve a
more effective final combustion. The chemical composition of the fiber sludges was determined.
Large amounts of cellulose were found, ranging from 32 to 65% of the dry matter. The samples were also characterized
with respect to lignin (1 to 25%) and wood extractives (0.2 to 5.8%). The carbohydrates were converted to fermentable
monosaccharides using two approaches: hydrolysis with cellulolytic enzymes and dilute-acid (H2SO4) hydrolysis. The
heat values of the solid residues were determined and compared to those of untreated fiber sludges. The glucose concentrations and glucose yields of the enzymatic hydrolysates ranged from 8 to 102 g/L and from 14 to 93%, respectively.
The hydrolysates were fermented to ethanol and the yields and productivities were determined.
poster 2-33
Ethanolic fermentation and enzymatic convertibility of dilute sulfuric acid-pretreated agricultural
and agro-industrial residues
Carlos Martín1,2, Björn Alriksson1, Anders Sjöde1,3, Nils-Olof Nilvebrant3, Leif J. Jönsson1*
1Biochemistry, Division for Chemistry, Karlstad University, SE-651 88 Karlstad, Sweden
2Department of Chemistry and Chemical Engineering, University of Matanzas, Matanzas 44740, Cuba
3Swedish Pulp and Paper Research Institute (STFI-Packforsk), P.O. Box 5604, SE-114 86 Stockholm, Sweden
Four raw materials, sugarcane bagasse, rice hulls, peanut shells and cassava stalks, were pretreated using 2% H2SO4
and a solid-to-liquid ratio of 1:10 at 122oC during 20, 40 or 60 min. The chemical composition of the raw materials and
the hydrolysates and the effects on enzymatic hydrolysis and fermentability were investigated. The formation of sugars
increased with increasing reaction time. Xylose, glucose, arabinose and galactose were detected in all of the prehydrolysates, whereas mannose was detected only in the prehydrolysates of peanut shells and cassava stalks. The sugarcane
bagasse gave high xylose (19.1 g/L) and arabinose concentrations (2.2 g/L). The high glucose concentration (26.0-33.5
g/L) in the prehydrolysates of rice hulls could partially be due to hydrolysis of starch of grain remaining in the hulls. The
glucose was generally consumed efficiently during fermentation of the prehydrolysates with Saccharomyces cerevisiae.
The length of the pretreatment did not affect the fermentability of the prehydrolysates using Saccharomyces cerevisiae,
except for cassava stalks, where the ethanol yield and productivity decreased with increasing pretreatment time. The enzymatic hydrolysis of the pretreated solid residues was evaluated with a commercial cellulase preparation. The dilute-acid
prehydrolysis led to a 2.7-3.7-fold increase of the enzymatic convertibility of bagasse but was less efficient with regard to
peanut shells, cassava stalks and rice hulls.
123
poster 2-34
Optimization of Dilute-Acid Prehydrolysis of Rice Straw Using Small-Scale Bomb Reactors
Jin Seop Park, Ho-joung Lee, and Kyoung Heon Kim*
College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea
Cheon-Seok Park
Department of Food Science and Biotechnology, Kyung Hee University, Yongin 449-701, Korea
Lignocellulosic materials can be used as a potential renewable biomass feedstock for the production of ethanol as an
alternative biofuel. However, cellulose in these materials, which is the major source for fermentable sugars, is protected by a cooperative network of lignin and hemicellulose. Therefore, without removing this protecting shield through
such as the dilute-sulfuric-acid pretreatment, it is not possible to make the cellulose susceptible to enzymatic digestion. In this study, a lignocellulosic feedstock available in South Korea, rice straw was pretreated with dilute sulfuric acid
at lower than 1.2 (%, w/w) at relatively high temperatures (140-180°C) in custom-made small-scale Hastelloy bomb
reactors which were designed to have relatively small diameters compared to their length to reduce the time taken to
reach the target temperature. The bomb reactors with controlling their temperatures in oil baths were effective for the
purpose of optimizing prehydrolysis conditions such time, temperature, and acid concentration on a laboratory scale.
The prehydrolysis results were kinetically modeled by using a series of first-order reactions. The enzyme digestibility of
unsolubilized solids obtained in the prehydrolysis were tested by cellulase.
poster 2-35
Cellobiose Production using ß-Glucosidase Inhibition
Misook Kim, Department of Food Science, Louisiana State University, Baton Rouge, LA 70803
Chang-Ho Chung, Donal F. Day*, Audubon Sugar Institute, St. Gabriel, La. 70776
Typically, enzyme hydrolysis of ligno-cellulose produces primarily glucose and a small amount of cellobiose. Cellobiose in
its own right is a potentially valuable product as a non-nutritive sugar. Co-production of a ß-glucosidase inhibitor, gluconic
acid, during enzymatic hydrolysis of cellulose altered the amounts of glucose and cellobiose produced.
Glucose oxidase produces gluconic acid from glucose via a glucolactone intermediate. Gluconic acid is an inhibitor of
ß-glucosidase. Addition of either of these compounds or the enzyme to the cellulase (Trichoderma viride) and ß-glucosidase (Aspergillus niger) cocktail used to hydrolyze ligno-cellulose altered the ratio of cellobiose to glucose produced from
the cellulose. Addition of gluconolactone, glucose oxidase, or gluconic acid significantly increased the amount cellobiose
with a corresponding decrease in amount of glucose produced during cellulose hydrolysis. The rate of cellulose hydrolysis
decreased on addition of gluconolactone but was not affected on addition of glucose oxidase. With suitable concentrations of glucose oxidase it was possible to convert over 60% of the cellulose to cellobiose.
124
poster 2-36
The Impact of Dilute Sulfuric Acid on the Selectivity of Xylooligomer Depolymerization to Monomers
Rajeev Kumar*, Charles E. Wyman1
Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, New Hampshire 03755
(603) 646-3193 phone; (603) 646-2277 fax; [email protected]
1
Now at University of California, Riverside, CA
Dilute acid hydrolysis of the hemicellulose in biomass produces high yields of dissolved xylose monomers and highly
digestible cellulose in the residual solids. It would be desirable to eliminate acid use to save money for the acid itself and
the base needed to neutralize it as well as significant costs for materials of construction and hydrolyzate conditioning.
However, performance suffers, although the effect of acid on xylose release is still not entirely clear. Thus, this research
was directed toward understanding the effect of acid concentration on the hydrolysis of soluble xylooliogmers. Previous
research by our group showed that the selectivity of xylose monomer formation from oligomers over a range of degrees
of polymerization from 1 to 5 was low without acid use. Furthermore, addition of acid for xylobiose hydrolysis increased
the ratio of the depolymerization rate constant to the total disappearance rate constant to close to one. In this study, the
effect of acid addition on xylobiose, xylotriose, xylotetrose, and xylopentose hydrolysis was followed at 160 oC over a pH
range from near neutral to 1.45, and kinetic models were developed to clarify how acid impacts the selectivity and yields
of xylose formation.
poster 2-37
Studying Effects of Dilute Acid Treatment on Components of Dairy Manure
Wei Liao*, Yan Liu, Zhiyou Wen, Shulin Chen
Department of Biological Systems Engineering, and Center for Multiphase Environmental Research Washington State
University, Pullman, WA 99163
Effects of dilute acid treatment on the main components of dairy manure were studied in terms of better understanding
the behavior of each component during the treatment as well as further optimizing accumulation of cellulose, which could
be utilized by enzyme to produce glucose. A 23 full factorial design was adopted to investigate the effects of the reaction
conditions of time, temperature and acid concentration on each individual component, further followed by a 3-factor central composite design which was used to obtain the optimal conditions for cellulose accumulation. The results indicated
that acid was the most important factor for changes of all the components. The results also presented that other two
individual factors of reaction time and temperature, and the interactions among three factors had significant influences on
the changes. In addition, the optimal conditions for cellulose accumulation were 2.8 hours reaction time, 140°C reaction
temperature and 1.0% acid concentration. Under these conditions cellulose reached 32%, and hemicellulose, lignin and
nitrogen were 3.15%, 20.8% and 2.35%, respectively.
125
poster 2-38
Simultaneous Saccharification and Fermentation of Steam-Pretreated Barley Straw at
Low Enzyme and Yeast Concentrations
Marie Linde*, Mats Galbe and Guido Zacchi
Department of Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
In the biomass-to-bioethanol-production process cellulose-rich raw materials are pretreated at high pressure and temperature to facilitate enzymatic degradation of the cellulose fraction to fermentable sugars. Barley straw is a cellulose-rich
agricultural residue and thus an interesting potential source of lignocellulose for ethanol production.
In previous studies it has been concluded that the concentration of enzymes and yeast in the simultaneous saccharification and fermentation (SSF) must be decreased and the concentration of water insoluble solids (WIS) increased in order
to reduce the ethanol production cost. In this study SSF was conducted on pretreated barley straw. The straw was prior
to SSF steam-pretreated at conditions found optimal in a previous study: 210ºC for 5 minutes after impregnation with 1%
H2SO4. The influence of the following parameters on ethanol productivity and yield in SSF were investigated; 1) decreased dosage of an enzyme mixture of Celluclast 1.5L and Novozyme 188 from 20 to 5 FPU/g cellulose; 2) decreased
concentration of baker´s yeast from 5 to 2 g/L; 3) increased concentration of water insoluble material from 5 to 10 wt-%.
The study also included a comparison of baker’s yeast cultivated on a glucose solution and yeast cultivated on hydrolyzate produced by steam-pretreatment of barley straw. Results from this study will be presented.
poster 2-39
Enrichment of Gamma-Linolenic Acid from Borage Oil via Lipase-Catalized Hydrolysis
Leonardo V. Fregolente*, Patricia Bogalhos L. Fregolente, César B. Batistella, Maria Regina W. Maciel
Separation Process Development Laboratory (LDPS), School of Chemical Engineering, State University of Campinas, CP
6066,13081-970, Campinas-SP, Brazil
Gamma-linolenic acid (GLA) is one of the essential fatty acids of interest of food and pharmaceutical industries, because
it has been reported to have beneficial effects in treating certain diseases. Therefore, in the last decades, researchers
have been developing different processes to enrich oils in GLA, such as urea fractionation, solvent winterization, lipase
mediated reactions, etc.
In this work, lipase-catalyzed selective hydrolysis was studied to enrich borrage oil in GLA. The commercial lipase Lipolase 100L Type EX was used, which is a food-grade liquid preparation of a microbial lipase from Thermomyces lanuginosus produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism. The conversion of
triglycerides into partial glycerides and free fatty acids was accompanied by high-performance size exclusion chromatography and the fatty acid composition was determined by gas-liquid chromatography. As results, the lipase used enriched
the unhydrolyzed acylglycerols in GLA. Starting from a GLA content of 21.9%, it was possible to obtain a 36.6% GLAcontaining oil, carrying out a hyldrolysis of borage oil at 60ºC for 20h in a solvent-free system, consisting of 70% (w/w) of
water and 30% of borrage oil.
126
poster 2-40
Use Of Different Enzymatic Mixtures In Hydrolysis From Barley Straw
María P.García-Aparicio, Mercedes Ballesteros, Paloma Manzanares, Ignacio Ballesteros, Alberto González, M. José Negro*
Renewable Energies Department-CIEMAT, Avda. Complutense, 22, 28040-MADRID, SPAIN.
E-mail: [email protected]
Current research and development on bioethanol production are being directed at the substitution of the higher-cost
sugars and starch feedstocks for low-cost lignocellulosic biomass as a way of reducing its final cost. Barley straw, an
important residue from grain industry in Spain, seems to be a promising substrate for microbial fermentation to ethanol to
be used as a fuel extender.
For an efficient production of ethanol from lignocellulose biomass by enzymatic hydrolysis and fermentation, the lignocellulosic substrate needs to be pretreated to make the cellulose more accessible to enzymes. Steam explosion has been proposed as an efficient pretreatment of lignocellulosic materials, and has the advantage to be developed at commercial scale.
In this study steam-exploded barley straw pretreated under different conditions (200-210ºC, 5-10 minutes) was used
as substrate in the enzymatic hydrolysis step. In order to maximize enzymatic hydrolysis yield, different combinations of
enzymes, i.e., cellulose complex, β-glucosidase and xylanase have been tested. The optimum ratio between enzyme
mixtures greatly depends on the composition and distribution of various fractions (cellulose, hemicellulose and lignin) in
the biomass hydrolysate. Results showed that supplementation with xylanase can have a significant effect on cellulase
performance, by improving cellulose accessibility. Results from hydrolysis studies using various enzymes preparations will
be presented.
poster 2-41
Improving Hydrolysis of Poorly Pretreated Biomass by Rough Mixing Scaled to the Laboratory
K.C. McFarland*, Joel R. Cherry, Novozymes, Inc., Davis, CA 95616
The processes necessary for conversion of biomass to sugars or ethanol are currently being scaled from laboratory to
pilot plant, with mixed results. Some mild pretreatments are insufficient for efficient enzymatic hydrolysis under current
laboratory methods. We present a laboratory scale model for improved mixing that demonstrates benefit for high solids
content and poorly pretreated samples.
Standard testing of enzyme hydrolysis of biomass is most often performed in shake flasks with orbital mixing (NREL LAP009). Mixing of high solids content or poorly treated biomass represents a challenge for orbital mixing. This is sometimes
addressed by impellers more suitable for low viscosity liquids or by lift-and-drop mechanisms (e.g. cement mixers) not
scaled for the laboratory. We built a small and inexpensive tumbler that is analogous to efficient lift-and-drop systems for
testing enzyme hydrolysis simultaneously on multiple samples, and demonstrated that poorly treated biomass is more efficiently hydrolyzed by rough mixing than by the standard laboratory process.
127
poster 2-42
Application of statistical experimental design for optimization of the conditions for eucalyptus hemicellulosic
hydrolysate detoxification
Giovani B.M. de Carvalho, Solange I. Mussatto*, João B. de Almeida e Silva
Departamento de Biotecnologia, Faculdade de Engenharia Química de Lorena
Rodovia Itajubá-Lorena Km 74,5 – CEP: 12600-970 - Lorena – SP, Brazil.
Phone: 0055 12 31595027; Fax: 0055 12 31533165; *E-mail: [email protected]
Eucalyptus hemicellulosic hydrolysate contains, besides sugars, several compounds that are toxic to the microorganisms
and negatively affect the hydrolysate fermentation efficiency. For this reason, the present work aimed to establish the best
condition for toxic compounds removal from eucalyptus hemicellulosic hydrolysate, so that it can be used in fermentative
processes.
The hydrolysate was produced by dilute acid hydrolysis, concentrated up to 68 g/l xylose and pH adjusted to 3.5, 5.25 or
7.0 by addition of CaO. Subsequently it was subjected to a sequence of two adsorption processes, the first based on the
use of activated charcoal or diatomaceous earths (2.4% w/v), and the second one using ion-exchange resin (Purolite A860 S - anionic) or adsorbent resin (Purolite MN-150). The assays were performed according to a 23 factorial design with
eight additional assays at the center point of pH. Using the statistical tool, it was concluded that the pH adjustment to 3.5
followed by activated charcoal adsorption and use of the adsorbent resin was the best condition for eucalyptus hydrolysate detoxification, since it promoted the highest removal rates (>82.5%) for the toxic compounds (acetic acid, furfural,
hydroxymethylfurfural and phenolics). Acknowledgements: CAPES, CNPq and FAPESP.
poster 2-43
Phenolic acids release by alkaline hydrolysis of brewer’s spent grain
Solange I. Mussatto*, Inês C. Roberto
Departamento de Biotecnologia, Faculdade de Engenharia Química de Lorena
Rodovia Itajubá-Lorena Km 74,5 – CEP: 12600-970 - Lorena – SP, Brazil.
Phone: 0055 12 31595027; Fax: 0055 12 31533165; *E-mail: [email protected]
Brewer’s spent grain (BSG) is an agro-industrial by-product proceeding from breweries, mainly composed by the barley malt residue obtained after the wort manufacture. In the present work, BSG was submitted to an alkaline hydrolysis
process aiming to break its structure and release the phenolic acids present, which are compounds of great interest for
the food and cosmetic industries. The used process consisted firstly in a pretreatment of the BSG with dilute sulfuric acid.
Subsequently, the resultant solid material was submitted to alkaline hydrolysis, and the effects of the three main operational variables affecting this process (NaOH concentration, 1.0%-2.0%; temperature, 80°C-120°C; and reaction time, 30
min-90 min) were explored through a 23 full factorial design.
The phenolic acids: ferulic, p-coumaric, p-hydroxybenzoic, syringic and vanillic were found in all the produced alkaline
liquors, ferulic and p-coumaric being the most abundant. NaOH concentration, temperature and reaction time presented a
significant (p<0.05) and positive influence on the phenolic acids release from BSG. The best results were attained when
the alkaline hydrolysis was performed with 2.0% NaOH concentration, at 120°C for 90 min, conditions that promoted
90.2% solubilization of the total lignin present in the pretreated BSG. Acknowledgements: CAPES, CNPq and FAPESP.
128
poster 2-44
High throughput Production of Pretreatment Residues for use in Advanced Characterization Methods
to Understand Biomass Recalcitrance
Nick Nagle*, Frederick Michel, Jr., Noah Weiss, Mark Davis and Richard Elander
National Renewable Energy Laboratory , National Bioenergy Center, 1617 Cole Blvd., Golden, CO 80401
Achieving a 30% displacement of current U.S petroleum consumption will require over one billion tons of sustainable biomass. Sources for the biomass will come from both agricultural and forest sectors, in a wide variety of feedstock type,
chemical composition and structural features. Meeting this aggressive target will depend not only on the production and
collection of biomass, but efficient conversion of biomass into fuels and chemicals. The challenge in biomass conversion
is the biomass recalcitrance, resulting from the interaction of chemical composition and structural features. New tools in
biomass imaging, spectrophotometry, such as NMR and MBMS methods, and advanced enzyme systems will facilitate
a better understanding of the root cause of biomass recalcitrance. These advanced methods of analysis require process
significant residues from a wide array of feedstocks, varieties and pretreated materials to understand biomass recalcitrance.
We report on the development of a high throughput screening method producing bench scale pretreated residues over conditions of varying pH, temperature and residence time using switchgrass as a model feedstock. The methodology consists
of pretreating biomass using a multiwell batch reactor, and using either sulfuric acid, sodium hydroxide or with water alone
as the catalyst across a robust and systematic set of reaction conditions. Following pretreatment, the solids are enzymatically hydrolyzed, using a combination of cellulase and hemicellulase enzymes. The combined yield of glucose and xylose in
the pretreatment and enzymatic hydrolysis steps provides a map of the feedstock’s reactivity under systematic and robust
pretreatment conditions. Producing process significant residues for advanced studies with known feedstock reactivity can
provide stakeholders with valuable information to understand biomass recalcitrance and feedstock performance.
129
poster 2-45
Bioconversion of hybrid poplar to bioethanol using organosolv ethanol pretreatment:
Effect of processing variables on substrate features and enzymatic digestibility
Xuejun Pan, Neil Gilkes, Dan Xie and Jack N. Saddler*
Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada, V6T 1Z4
*email: [email protected]
An organosolv process involving ethanol extraction is the basis of a biorefining strategy for conversion of lignocellulose
into fuels, chemicals and materials. Woody substrates pretreated using the organosolv process, including those with high
residual lignin content, have better enzymatic digestibility than those pretreated by alternative processes,. The organosolv
process also allows recovery of valuable chemicals such as hemicellulosic sugars, furfural, hydroxymethylfurfural, organic
acids, and low molecular weight polyphenols from lignin degradation. The process generates a particularly high-quality
lignin fraction with potential in many industrial applications.
In this presentation, hybrid poplar (Populus nigra x P. maximowiczii) chips were pretreated using an experimental matrix
of organosolv process conditions designed by Response Surface Methodology (RSM) within the ranges of 155-205°C,
26-94 min and 0.83-1.67% (w/w) H2SO4 catalyst and 25-75% (v/v) ethanol. The effects of pretreating conditions on various substrate characteristics (e.g., residual lignin and hemicellulose, fiber size, crystallinity and degree of polymerization
of cellulose) were examined. The enzymatic digestibilities of the various substrates were compared and correlated to
processing conditions and substrate features. Regression models were developed to enable quantitative prediction of
substrate properties digestibility based on the pretreatment conditions.
poster 2-46
The effect of reaction conditions upon lipase catalysed hydrolysis of canola and soybean oil
Rubiane C. Pinheiro, Flávio F.de Moraes, Gisella M. Zanin*
Chemical of Chemical Engineering, Universidade Estadual de Maringá, Maringá-PR, Brazil, TN +55 (44)32614745
Cleide M. F. Soares, Heizir F. de Castro,
Chemical of Chemical Engineering, Faculdade de Engenharia Química, Lorena-SP, Brazil, TN +55 (12)31595116
The hydrolysis of triglyceride esters to yield free fatty acids and glycerol represents an important group of chemical
reactions relevant to the industrial processing of natural oils and fats. Hydrolysis is the most important reaction for production of free fatty acids that may then be inter-esterified, trans-esterified, or converted into high-value fatty alcohols.
The mainstream current technologies for hydrolysis are based on high-temperature, high-pressure contacting processes
with steam or superheated liquid water. This study is concerned with the hydrolysis of two types of oils (canola and oil of
soybean) in the presence of lipase from the yeast Candida rugosa (CRL). Two 23 full factorial design with repetitions at the
center point were employed to evaluate the immobilization yield as a function of pH, temperature and substrate concentration. Similar pH optimum (7.0) and optimum temperature was found between 40°C for both oils. Optimum substrate
concentration was 50% also for oils. It was concluded that the oils type do not influence the hydrolytic activity of CRL
lipase in the range of the parameters studied.
130
poster 2-47
Advanced Confocal Imaging of Corn Fiber Using Starch- and Cellulose-Specific Fluorescent Probes
Stephanie E. Porter, Qi Xu, Shi-You Ding, Michael E. Himmel*, National Bioenergy Center, NREL, Golden, CO 80125
Kyle Beery, Charles Abbas, Archer Daniels Midland Co., Decatur, IL 62525
Ethanol is the primary renewable product currently being produced for transportation fuel in the U.S. Corn grain, the
primary feedstock for ethanol production, is typically processed in wet mills yielding such products as gluten feed, gluten
meal, starch, and germ. Starch extracted from the grain is used to produce fuel ethanol in saccharification and fermentation steps, but the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples
using a laser confocal microscope. These probes are based on the binding specificities of two types of carbohydrate
binding modules (CBMs), which are substrate-specific binding peptides derived from carbohydrate degrading enzymes.
CBMs are fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to a red fluorescent protein
called dsRed (RFP). Control experiments show that CBM3-RFP, which is specific for cellulose, binds only cellulose and
not starch, whereas CBM20-GFP, which is specific for starch, binds only starch and not cellulose. We are using these
probes to examine the in situ distribution of starch and cellulose in actual corn fiber samples from a commercial wet-milling process.
poster 2-48
Liquid hot water pretreatment of olive tree pruning
Cristóbal Cara, Inmaculada Romero, Eulogio Castro*
Department of Chemical, Environmental and Materials Engineering, University of Jaén,
Campus Las Lagunillas, 23071 Jaén (Spain)
Jose Miguel Oliva, Felicia Sáez
DER-CIEMAT, Avda. Complutense 22, 28040 Madrid (Spain)
Over eight million hectares of olive trees are cultivated worldwide, especially in Mediterranean countries. Olive tree pruning, an essential operation in this culture, eliminates old branches and prepares trees for the next crop, generating one of
the most abundant, renewable lignocellulose residue. Although olive tree wood obtained by pruning has a limited application as domestic firewood, thin branches and leaves are usually burnt on fields to prevent propagation of vegetal diseases, causing economic costs and environmental concerns. As an alternative, this work is aimed to study the use of these
cheap residues as a raw material to produce fuel ethanol.
Pretreatment of olive tree pruning by liquid hot water (LHW) was carried out at seven temperature levels in the range 170230ºC for 10 and 60 minutes. Sugar recoveries in both solid and liquid fractions resulting from pretreatment as well as
enzymatic hydrolysis yield of the solid were used to evaluate pretreatment performance.
The enzyme accessibility of cellulose in the pretreated solid fraction increased with pretreatment time and temperature
although sugar degradation in the liquid fraction was concomitantly higher. As a conclusion, LHW pretreatment at 210ºC
for 10 minutes are the best conditions for taking advantage of glucose content from olive tree pruning.
131
poster 2-49
Enzymatic hydrolysis of pretreated olive tree pruning at different biomass concentrations
Cristóbal Cara, Manuel Moya, Encarnación Ruiz*
Department of Chemical, Environmental and Materials Engineering, University of Jaén,
Campus Las Lagunillas, 23071 Jaén (Spain)
Ignacio Ballesteros, Mª José Negro, Alberto González
DER-CIEMAT, Avda. Complutense 22, 28040 Madrid (Spain)
Ethanol production from lignocellulose residues requires, as a first step, a residue pretreatment in order to obtain high
enzymatic hydrolysis yields by improving cellulose accessibility to enzymes. Besides that, high glucose concentrations
are also desirable to keep ethanol distillation costs as low as possible.
Olive tree pruning, a largely available renewable agricultural residue with no industrial applications, may be used as a raw
material for ethanol production. In this work, the enzymatic hydrolysis of olive tree pruning, pretreated by liquid hot water
under selected conditions, is studied. The pretreated material was submitted to a further pretreatment step by alkaline
delignification, the objective being to improve hydrolysis yields as well as increasing cellulose content in the pretreated
material.
The enzymatic hydrolysis of pretreated residues was performed using a commercial cellulolytic complex supplemented
with β-glucosidase, using a substrate concentration range from 2 to 30% (w/v). The influence of both glucose inhibition
and physical factors on enzymatic saccharification yields is evaluated. Comparative results with and without a delignification step are presented.
poster 2-50
Pre-treatment of sugarcane leaves and bagasse pith with lime-impregnation and steam explosion for
conversion to fermentable sugars
Michael Saska* and Matthew Gray
Audubon Sugar Institute, Louisiana State University Agricultural Center, 3845 Hwy 75, St. Gabriel, LA 70776
Based on the review of literature related to pretreatment of sugarcane biomass and previous work at Audubon Sugar Institute, ambient temperature impregnation with dilute milk-of-lime followed by low-severity steam explosion was chosen for
testing and scale up to a pilot scale. Impregnation or presoaking of the biomass with milk-of-lime at ambient temperature is
expected to be a low capital, low operating cost alternative to other pretreatment processes. In addition, industrial experience with large-scale wet storage of bagasse, a proven process at a number of bagasse pulping operations around the
world will be helpful in designing the industrial lime pretreatment process. Up to date, the preferred conditions for the impregnation are: the moisture content 90%, hydrated lime dose 20% on biomass dry matter, 50˚C and a residence time of about
5 to 10 days. The following short-duration steam treatment at about 165˚C and steam explosion sterilizes the biomass and
enhances the enzymatic conversion without any generation of the fermentation toxins, known products of the acid-catalyzed or un-catalyzed steam explosion. As the new process is envisioned for integration within the existing sugarcane milling
operations and if the use of a supplemental fossil fuel is to be avoided, some undigested fiber and all residual lignin are
required for generating steam and electric power for the sugar and ethanol parts of the integrated process.
132
poster 2-51
Techno-Economic Evaluation of a Bioethanol Process for Three Different Lignocellulosic Materials
Per Sassner*, Mats Galbe and Guido Zacchi
Department of Chemical Engineering, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden
Telephone: +46 46 222 82 93; Fax: +46 46 222 45 26; E-mail: [email protected]
The lignocellulosic materials spruce (softwood), Salix (hardwood), and corn stover (agricultural residue) are all potential
feedstock for ethanol production. In this study the utilization of these materials for bioethanol production were compared
in terms of production cost and energy demand using a process concept based on SO2-catalyzed steam pretreatment followed by simultaneous saccharification and fermentation (SSF).
A model including all major process steps was used, and process data were based on results recently obtained in the experimental work carried out at our department in lab-scale or in a process development unit. Mass and energy balances
were solved using the commercial flowsheeting program Aspen Plus and capital costs were estimated with either Icarus
Process Evaluator or from vendor quotations.
A model description will be presented together with key results from the study. These include the effects of pentose
fermentation and overall process steam requirement on the economic outcome of the ethanol production process. The
steam demand affects the amount of solid residue that may generate an income as a solid fuel co-product. Furthermore,
sensitivity analyses were performed on important process parameters, such as overall ethanol yield and substrate concentration in SSF, to give information on the potential for cost reduction for each raw material.
poster 2-52
Improved Total Solids Measurement of Acid Pretreated Biomass by Karl Fischer Titration
Christopher Scarlata*
National Renewable Energy Laboratory, Golden, CO 80401
Karl Fischer (KF) titration measures water selectively and specifically and can be considered a direct method for determining total solids (TS) content of biomass samples. Traditional oven drying methods for determining TS measure the
solid material in a sample after drying to a constant weight. These methods are accurate for feedstocks but are problematic when applied to chemically pretreated biomass samples.
Oven methods can underestimate biomass TS content because chemical processing can generate volatile materials,
other than water, which evaporate at temperatures at or below 105 °C (e.g., acetic acid). The volatile compounds would
then be falsely counted as water. This study compared the TS content of acid pretreated corn stover as measured by KF
titration and three standard oven methods; gravity convection, vacuum, and infrared.
Karl Fischer titration was shown to reduce and/or eliminate errors inherent in the oven methods. A further benefit of KF titration is it can significantly reduce the time required to measure biomass TS from over 24 hours (for the convection oven
case) to under 5 minutes per sample independent of moisture content.
133
poster 2-53
Technical-Economical Analysis for a New Concentrated Acid Hydrolysis Process of Sugar Cane Bagasse for
Bio-Ethanol Production
Boutros Fouad Sarrouh1; Josefina Jover2; Silvio Silvério da Silva*; Ricardo de Freitas Branco1; Diego Tresinari Dos Santos1
*Departament of Biotechnology, Faculty of Chemical Engineering, Lorena-SP.
E-mail: [email protected]. Tel: 00551231595146.
1
Departament of Biotechnology, Faculty of Chemical Engineering, Lorena-SP.
2
Department of Chemical Engineering, Faculty of Chemistry-Pharmacy, Santa Clara, Cuba.
Bio-ethanol is considered as the most promising and useful product from agricultural waste. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low
yield and high cost of the hydrolysis process.
This work studies, the acid hydrolysis conditions of sugar cane bagasse using concentrated acid in a single and a modified single step as well as their technical-economical analysis in a supposed pilot plant with a production capacity of 800
hl of alcohol/day was evaluated. The maximum obtained conversion in the single step hydrolysis was 87.65% of the total
fermentative sugars. Starting from these results the modified single step was carried out and a maximum conversion of
97.5%. A technical-economical study at industry level was achieved for both variants of this process in order to study their
economical feasibility. For the concentrated acid hydrolysis in a single step the bio-ethanol unitary production cost 18 $/hl
and a Payback Period (PP) of 3 years, while as for the new modified single step the bio-ethanol total unitary production
cost 19.6 $/hl, and a Payback Period (PP) of 7 years.
According to these results, the economical feasibility of the single step showed very promising results and thus we recommend its application in our proposed pilot-plant with a production capacity of 800hl of alcohol/day.
poster 2-54
Production of cellulolytic and xylanolytic enzymes by Aspergillus niger H2 grown on Pennisetum amcrieanum×
Pennisetum purpureum in solid state fermentation
Su Donghai, Sun Junshe*
China Agricultural University, Beijing 100083, China
Pennisetum amcrieanum× Pennisetum purpureum (PAPP) is a perfect kind of grass with excellent soil and water conservation property. The production of cellullolytic and xylanolytic enzymes with PAPP as substrate was studied. Effect of
liquid-solid ratio, initial pH, temperature, and concentration of nitrogen on FPase, β-glucosidase, xylanase, β-xylosidase
and CMCase production by solid-state fermentation was optimized. The liquid-solid ration the substrate was the influencing factor during the whole process most greatly. Nevertheless, The effect of initial pH were not significant. It is suggested
that water content, initial pH, the temperature and concentration of (NH4)2SO4 in the substrate are 75%, 4.02, 27 and 1%
respectively.
134
poster 2-55
Effect of Xylan and Lignin removal by Batch Pretreatment on the Enzymatical Digestibility of
Corn leaf and Corn Stalk
1
Su Donghai1, Bin Yang2 and Junshe Sun1*
Department of Bioengineering, China Agriculture University, Beijing, China 100083
2
Corn leaf and corn stalk was pretreated with liquid hot water using the same batch reactor, respectively. Solids concentration was 5%. Reaction temperature and time ranged from 160 to 220°C and 2 to 20 min, respectively. This study compare
liquid hot water pretreatment of corn leaf and corn stalk by measuring pH of liquid hydrolyzates, Xylose, oligomer yields,
Total mass recovery and Klason lignin removal. In uncatalyzed batch reactor runs, the maximum total xylose yield of corn
leaf over the range of conditions run was 9.2, 15.6, 45.4 and 56.8% at 160, 180, 200, and 220°C, respectively. The maximum total xylose yield of corn stalk over the range of conditions run was 25.2, 37.2, 80.3 and 86.1% at 160, 180, 200, and
220°C, respectively. The composition and digestibility data indicate that the xylose and lignin content in the biomass are
the major factors controlling the enzymatic digestibility. Under the same pretreatment condition, The enzymatic digestibility rate of corn stalk was higher than that of corn leaf.
poster 2-56
Fraction Insoluble Solids (FIS): Procedure and Effect on High Solids Saccharification Yield Calculations
David W. Templeton*, Raymond O. Ruiz, Melvin P. Tucker, and Bonnie R. Hames
National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401-3393
Phone: 303-384-7764; FAX: 303-384-7752; Email: [email protected]
The fraction insoluble solids (FIS) is an important parameter that describes the effectiveness of high solids saccharifications. The FIS is defined as the dry weigh of insoluble solids (washed clean of water-soluble carbohydrates and salts) per
dry weight of saccharification slurry. As saccharification starting solids increases, significant amounts of water are consumed by hydrolysis reactions. This reduces the final volume of liquor and increased the final carbohydrate concentrations. Carbohydrate yield calculations based on starting volume and final concentrations become less accurate at higher
solids loadings. It becomes necessary to experimentally determine the FIS and liquor density to accurately determine
saccharification yields. We will present model saccharification data showing the effects and magnitude of FIS on yield
calculations, critical starting solids concentrations, and procedures for FIS determination.
135
poster 2-57
Withdrawn
poster 2-58
Trials on the Danish Pilot Scale Pre-treatment Plant (IBUS).
Identification of inhibitors, detoxification and SSF.
Mette Hedegaard Thomsen, Anders Thygesen, Anne Belinda Thomsen
Biosystems Department, Risoe National laboratory,
POB 49, DK-4000 Roskilde, Denmark
In Denmark, a pilot scale reactor for hydrothermal treatment (with and without catalysts) of lignocellulosic materials has
been constructed as a part of the EU-project: Co-production of Biofuels. During the last year this plant has been expanded from a two-step pre-treatment reactor system with a maximum capacity of 100 kg/h to a complete plant with automatic
feeding and chopping of straw and a three reactor system working with increasing temperatures. (Reactor 1 max. 100°C,
reactor 2 max. 200°C, reactor 3 max. 230°C). The capacity is around 1000 kg straw/h.
In experiments performed with the two-step pre-treatment reactor a straw flow of 50 kg/h was used. In these trials, a high
cellulose convertibility was obtained (90%), but at temperatures above 195°C a significant part of the hemicellulose was
degraded (up to 50% at 200°C), resulting in formation of fermentation inhibitors. Analysis of the liquid fraction was performed in order to identify the inhibitors.
The first three-step pre-treatment trails were performed with a straw flow of 150 kg/h. The trials were performed at 8085°C for 20 min. in the first step, 170 and 180°C for 15 min. in the second step followed by 195°C for 3 minutes in the third
step. The enzymatic convertibility of the pre-treated straw as well as extraction and recovery of hemicellulose sugars in
the liquid fraction was compared to results obtained in the two-step process, including identification and comparison of
fermentation inhibitors. Detoxification of the most toxic liquid fractions has been performed with subsequent SSF.
136
poster 2-59
Wet Oxidation of Softwood and Hardwood Resulting in Convertible Cellulose and Hemicellulose
Enik Varga*, Risoe National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark,
Present address: Budapest University of Technology and Economics, H-1111 Budapest, Hungary
Mette H. Thomsen, Anne Belinda Thomsen, Risoe National Laboratory P.O. Box 49, DK-4000 Roskilde, Denmark
Kati Réczey, Budapest University of Technology and Economics, H-1111 Budapest, Hungary
In the production of bioethanol from lignocellulosic substrates, the subsequent enzymatic hydrolysis and fermentation processes are known to be affected by the pre-treatment technology applied. Wet oxidation has been successfully used to
different herbaceous biomasses such as corn stover or wheat straw, resulted in easily degradable cellulose rich biomass
for ethanol production. However, wet oxidation has only seldom investigated on woody materials.
In this study, hardwood (willow, Salix viminalis) and softwood (Norway spruce, Picae abies) were wet oxidized using different catalyst (Na2CO3, NH3 or H2SO4) in order to make the cellulose more accessible to the enzymes in the enzymatic
saccharification and to hydrolyze the hemicellulose. Since the optimal pretreatment conditions for hemicellulose and cellulose sugars are usually rather different, one- and two-stage wet oxidation experiments have also been performed at a
temperature ranging from 180°C to 220°C for 5-20 minutes.
In general, mild pretreatment conditions in the first stage extracted about two-third of the original hemicellulose content
both from willow and spruce. The harsher circumstances in the second stage helped to open up the structure of remained
cellulose and gave high overall glucose yield both for willow and spruce (80% and 73% respectively). Result from this
study will be presented.
poster 2-60
Quantitation of Organic Degradation Products in Response to Severity of Dilute Acid Pretreatment of Corn Stover
Shou-Feng Chen, Richard A. Mowery, C. Kevin Chambliss, Department of Chemistry and Biochemistry, Baylor University
G. Peter van Walsum*, Department of Environmental Studies, Baylor University, Waco, TX 76798-7266
A variety of degradation products are produced upon pretreatment of biomass with dilute acid. The complexity of these
samples has significantly limited the scope of efforts to perform summative analyses of degradation products. Qualitative
and quantitative interrogation of hydrolysates is also paramount to identifying potential correlations between pretreatment
chemistry and microbial inhibition in downstream bioconversion processes.
A developing suite of analytical methodologies based on chromatographic separation of analytes with UV-visible and/or
mass spectrometry detection modes has been applied to perform quantitative assessments of twenty degradation products present in hydrolysates produced by dilute acid pretreatment of corn stover. Accumulations of degradation components were tracked as a function of reaction severity, incorporating pretreatment time, temperature, and pH as well as
initial solids concentration. Correlations of product concentrations to the pretreatment severity function indicate differing
responses of various compounds to the kinetic influences of temperature and reaction time. Activation energies calculated
for several compounds indicate higher activation energies than are inherently assumed in the biomass pretreatment severity function. Accumulation trends in response to initial solids concentration indicate mass transfer limitations for some
but not all quantified compounds.
137
poster 2-61
Enhancement of Cellulose Saccharification Kinetics Using an Ionic Liquid Pretreatment strategy
Anantharam P. Dadi, Constance A. Schall, Sasidhar Varanasi*
Department of Chemical and Environmental Engineering, The University of Toledo, Toledo, OH 43606, USA
Biomass has the potential to be converted into value added products through a sugar platform. Sugar feedstock can also
be converted to ethanol for fuel production. Techno-economic analysis conducted by NREL indicates that efficiency of
the biomass saccharification step – hydrolysis of cellulose to glucose - is critically important in making the process costcompetitive. Cellulose hydrolysis in aqueous media suffers from slow reaction rates and low yields because cellulose
is a water-insoluble crystalline biopolymer. To accomplish its hydrolysis, the hydrolyzing enzymes (cellulases) and water
have to penetrate the crystalline fibrils. The tight packing arrangement of the fibrils not only excludes the enzymes but
also water. Pretreatment methods which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. We developed a novel technique wherein the cellulose
is dissolved in an ionic liquid (IL) and is subsequently regenerated as an amorphous precipitate by rapidly quenching the
solution with an anti-solvent. Hydrolysis kinetics of the regenerated cellulose were significantly enhanced as measured by
initial rate of glucose formation With an appropriate selection of enzymes, initial rates were approximately an order of
magnitude greater than those of untreated cellulose.
Because of their extremely low volatility ionic liquids are expected to have minimal environmental impact. A unique solvency characteristic of ILs which makes them ideal our work is that they are able to instantly reject all the dissolved cellulose in presence of anti-solvents such as water, methanol and ethanol. Indeed, by mere addition of about 1 to 5 wt % of
these anti-solvents, one is able to completely regenerate all the dissolved cellulose from the ILs as an amorphous precipitate. Once the cellulose is precipitated, the anti-solvent used for displacement can easily be stripped off the non-volatile
IL via flash distillation and the IL recovered for subsequent reuse.
poster 2-62
Process Options in AFEX Pretreatment and Fermentation of Rice Straw
Balan, Venkatesh.; Shishir, Chundawat.; Bruce E. Dale
Biomass Conversion Research Laboratory, Department of Chemical Engineering and Materials Science,
Michigan State University, East Lansing, MI – 48824
Rice straw is an important lignocellulosic biomass with nearly 800 million dry tons produced annually worldwide. Rice
straw therefore has great potential for making renewable fuels. However, rice straw appears to be a more recalcitrant
material than some others we have studied. For example, about two thirds of ammonia fiber explosion (AFEX) pretreated
rice straw glucan is hydrolyzed to glucose while we see almost 100 % glucan conversion for AFEX treated corn stover
under similar hydrolysis conditions.
Solid State Fermentation (SSF) of the rice straw prior to the AFEX pretreatment may help improve its overall conversion
to useful products. In this work, we report solid-state fermentation of rice straw and its effect on AFEX pretreatment and
enzymatic hydrolysis conditions, hydrolysis using different combinations of cellulase and hemicellulase, fermentation of
the hydrolyzate to ethanol and recovery of other SSF byproducts, such as mushrooms, organic acids, etc.
138
poster 2-63
Characterization of Changes in Viscosity and Undissolved Solids Content of Corn Stover Slurries
During Enzymatic Hydrolysis
Erin D. Wyatt and R. Eric Berson*
University of Louisville, Department of Chemical Engineering, Louisville, KY 40292
Enzymatic hydrolysis may be used to convert cellulose to glucose in pretreated corn stover slurries. As the saccharification reaction proceeds, insoluble particles are degraded into soluble sugars resulting in reduced mass of undissolved
solids. The reduction in undissolved solids corresponds to a significant reduction of viscosity. Viscosity and the mass of
undissolved material are measured over time for slurries with initial solids concentrations between 10% and 30%, which
provides parameters necessary for large-scale substrate loading strategies and processing equipment design. For each
initial solids concentration, the changes in viscosity and solids content over time are correlated to cellulose conversion as
determined by glucose concentration.
poster 2-64
Evaluation of Different Biomass Materials as Feedstock for Fermentable Sugar Production
Ruihong Zhanga, Yi Zhenga, Zhongli Pana, John Labavitchb, Bryan M. Jenkinsa
a
Biological and Agricultural Engineering Department
b
Plant Science Department
University of California - Davis, One Shields Avenue, CA 95616, USA
Donghai Wang, Agricultural and Biological Engineering Department, Kansas State University
Saline crops and pretreated municipal organic solid wastes are evaluated for their potential for use as feedstock for fermentable sugar production via dilute acid and enzymatic hydrolysis. The saline crops included two woods, Athel (Tamarix
aphylla L) and Eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose Tall Wheatgrass (Agropyron elongatum)
(JTW) and Creeping Wild Rye (Leymus triticoides) (CWR), which were produced by using high salt subsurface irrigation
water in California. The pretreated municipal organic solid wastes include autoclaved organic waste and source-separated
food waste. Each of the materials was first treated with dilute acid at selected temperatures and times and then treated
with cellulases. The original chemical composition (cellulose, hemicellulose and lignin) and the yield of total and different
types of sugars were determined. The results showed that among the saline crops evaluated, Creeping Wild Rye resulted
in the highest sugar yield (0.67g/g dry matter). Autoclaved municipal organic solid wastes also showed reasonable sugar
yields.
139
poster 3A-07
Liquid-Liquid Extraction Process for Separating Acrylic Acid Produced from Biomass
Alvarez, M.E.T. *; Machado, A.B.; Pinho, A.P.A.; Moraes, E.B. and Wolf-Maciel, M.R.
Separation Process Development Laboratory (LDPS). Chemical Engineering School. State University of Campinas,
(Unicamp), CP 6066, ZIP CODE 13081-970, Campinas-SP, Brazil.
[email protected], [email protected]
Renewable materials, such as sugar cane, are interesting alternative carbon sources for the production of chemicals,
especially considering the power of modern biotechnological methods, and one of the possible routes for obtaining acrylic
acid is through dehydration of lactic acid, which can be gotten via fermentation of biomass.
The liquid-liquid extration process is an alternative for the separation of acrylic acid. In order to represent the non-idealities present in the liquid–liquid equilibria (LLE), some thermodynamic models and methods are available, such as the
most used models NRTL and UNIQUAC, and the group contribution methods UNIFAC and their modifications. In this
work, these models were used to generate liquid–liquid equilibrium diagrams for systems composed by acrylic acid, lactic
acid and water. The results were compared with available LLE experimental data. Aspen Plus software was used for the
calculations. Among the solvents studied, the best ones, in terms of distribution coefficients, for carrying out the extraction process simulations were chosen. Accuracy in the phase equilibrium representation is one of the most critical condition to be evaluated. On the other hand, group-contribution methods can be applied to predict the LLE model parameters,
if no experimental data are available.
Poster 3A-08
Further Characterization of the Bio-burden in United States Aviation Fuel: A Comparison of FTA Paper, Direct
PCR, and Traditional Culture Methods for Obtaining Genetic Material from Aviation Fuel Microbes
Sarah K. Chelgren, Ellen M. Strobel, Air Force Research Laboratory/PRTG, Dayton, OH 45433
Lori M. Balster, Marlin D. Vangsness, Loryn L. Bowen, University of Dayton Research Institute, Dayton, OH 45469-0116
The natural presence of microorganisms in jet fuel can cause gradual degradation of aircraft and fuel handling systems,
a fact which has been well-documented for over forty years. The problem of microbial contamination continues today,
and with the expected increase in usage of biodiesels and other alternative fuels, the problems associated with microbial
contamination are expected to increase as well. In order to understand and improve mitigation of these problems, it is
necessary to characterize to the greatest extent possible the microbial consortia affecting aircraft systems today. This paper presents results from an ongoing study to describe the bio-burden in current aviation fuel systems, including a broad
range of aircraft, fuel storage tanks, and fuel delivery systems located across the continental U.S.
Traditional microbiology methods have been used successfully in the past to isolate microorganisms from aviation fuel
samples. Unfortunately, these methods do not take into account unculturable microorganisms and numerous other factors
introduced with the drastic environmental change from the microorganism’s fuel environment to a standard microbiological media plate. In this study, microorganisms were isolated from each fuel sample using FTA paper, direct polymerase
chain reaction (direct PCR), and traditional microbiological culture methods. The isolated microorganisms were further
identified using 16S Ribosomal RNA Sequencing. Through the refinement and comparison of these analytical techniques,
the microorganisms present in aviation fuel systems will be better characterized to achieve a more accurate picture of the
bio-burden, including its composition, location, and activity.
140
Poster 3A-09
Xylitol production from wheat straw hemicellulose in a 15-L STR
Larissa Canilha*, Walter Carvalho and João Batista Almeida e Silva
Chemical Engineering College of Lorena, Lorena, São Paulo, Brazil, 12600-970
Wheat straw represents a renewable and widely available source of carbohydrates that could be used as substrates in a
variety of bioconversion processes. Dilute-acid hydrolysis of its hemicellulose results in a hydrolysate rich in fermentable
sugars, mainly xylose, which can be used to produce xylitol. Xylitol is a sugar-alcohol with anticariogenic properties and
clinical applications that arouses interest in pharmaceutical and food industries. In the present study, the best conditions
to promote the xylose-to-xylitol bioconversion in wheat straw hemicellulosic hydrolysate, previously defined in Erlenmeyer flasks with the yeast Candida guilliermondii FTI 20037, were used to produce xylitol in a 2-L stirred tank reactor
(STR). By using an agitation speed of 300 rpm and an air flowrate of 0.4 vvm, which resulted in an initial KLa of 15 h-1, a
xylitol production of 28.6 g/L was observed after 70 h of fermentation, resulting in a bioconversion yield of 0.6 g/g and in
a productivity of 0.4 g/L h. The maintenance of the initial KLa was used as a criterion to produce xylitol in a 15-L STR. In
this semi-pilot reactor, the use of 300 rpm agitation speed and of 0.2 vvm air flowrate ensured the necessary conditions to
achieve an initial KLa of 15 h-1. Under these conditions, a xylitol production of 30.7 g/L was observed after 82 h of fermentation, resulting in a bioconversion yield of 0.7 g/g and in a productivity of 0.4 g/L h.
Acknowledgements: CNPq, Fapesp/Brazil
poster 3a-10
Stability of a Bioreactor for the Production of Ethanol in Sequential Batch Operation from Molasses-Stillage
Medium using a Selected Yeast Strain Immobilized in Calcium Alginate
Sônia M. da S. Carvalho*; Spartaco Astolfi Filho
Federal University of Amazon, Amazon, Brazil, CEP: 69.077-000
Lílian Pantoja; Roberto N. Maeda
National Research Institute of Amazon, Amazon, Brazil, CEP: 69060-001
Nei Pereira Jr.
Bioprocess Development Laboratory, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, CEP: 21949-900
The objective of this work was to evaluate the stability of a fixed bed bioreactor for 30 days, with a selected strain of Saccharomyces cerevisiae, immobilized in calcium alginate. The strain, isolated from a Brazilian Distillery, was selected by its
thermo and ethanol tolerance and fermentative activity. The bioreactor, operated in sequential batch with 6h-cycles, was
packed with roughly 16,000 beds, containing 300 x 108cells/bead, and fed with molasses-stillage medium in a total sugar
concentration of 100g.L-1. The medium was fortified with KH2PO4 1,0g/L, MgS04.7H20 1,0g/L, CaCl2 1,0g/L, YE 0,5g/L,
and tetracycline was added in a concentration of 10mg/L. The bioprocess monitoring was carried out by measurements
of sugar and ethanol concentrations, as well as pH and cell detachment. The system showed to be sufficiently stable to
withstand variations in the substrate feed rate, resulting in average values for ethanol yield on substrate consumed and
volumetric productivity of 0.43 g.g-1 and 7.5 g.L-1.h-1, respectively.
141
poster 3A-11
Microbiological production of fatty acids:
Performances of a new synthesis path of molecules to energetic or chemical use
J. Cescut*, L. Fillaudeau, S. Alfenore, C. Bideaux, X. Cameleyre, N. Goret, S. Guillouet, J. Lesage, C. Molina-Jouve et
J.L. Uribelarrea
Biotechnology and Bioprocesses Laboratory, UMR-CNRS 5504,UR-INRA 792,
Institut National des Sciences Appliquées, DGBA – 135, avenue de Rangueil 31077 Toulouse Cedex 4, France
In a context of petroleum resources dwindling, exploitation of renewable carbon for the production of biomolecules, substitute for oil in energetic or chemical road, represents important stake. Fatty acids are precursory molecules of chemical
or enzymatic reactions for producing compounds with valuable function depending on their number of carbon and their
insaturation degree. Microbiological production of fatty acid on carbohydrate constitutes an option compared to the usual
way (vegetal oil). This way has high advantages: a better control of the profile of the synthesized molecules and a diversification of the agricultural origin of the substrates: starch-based molecules, derived from hydrolysis, glycerol.
The aim of our research work consists in the optimization of the microbial production of fatty acids by the yeast Rhodotorula glutinis according to yield and content criteria compatible with a realistic industrial transposition.
Fed-batch cultures on synthetic medium and glucose, with two levels of temperatures allowed us to identify a relevant
strategy to obtain 164 g.L-1 of dry biomass, 59% (w/w) of lipid cell content with a conversion yield value of 0.20 gLip.gGlu-1.
The dynamical behavior of the yeast was quantified in order to understand the biological phenomena involved in lipid accumulation. The fatty acid profile obtained will be presented with his modulation according to the temperature of culture.
Poster 3A-12
Aqueous-Phase Reforming of Biomass-Derived Compounds to Hydrogen and Alkanes
Randy D. Cortright *, Virent Energy Systems, Inc., 3571 Anderson Street, Madison, WI 53704
Virent Energy Systems, Inc. is commercializing the Aqueous Phase Reforming (APR) process that allows the generation of
hydrogen and alkanes from biomass-derived compounds such as glycerol, sugars, and sugar alcohols. The APR process is a
unique method that generates hydrogen from aqueous solutions of these oxygenated compounds in a single step reactor process
compared to the three or more reaction steps required for hydrogen generation via conventional processes that utilize non-renewable fossil fuels. The key breakthrough of the APR process is that the reforming of these aqueous solutions is done in the liquid
phase. The patented APR process occurs at temperatures (150 oC to 270 oC) where the water-gas shift reaction is favorable,
making it possible to generate hydrogen with low amounts of CO in a single chemical reactor. Furthermore, the APR process
occurs at pressures (typically 15 to 50 bar) where the hydrogen-rich effluent can be effectively purified using either membrane
technology or pressure swing adsorption technology. The utilization of biomass-based compounds allows the APR process to be
a carbon neutral method to generate hydrogen. If carbon sequestration is added, the process would be carbon negative. In the
near term, the feed-stock of interest is waste glycerol that is being generated in large quantities as a byproduct in the production
of biodiesel. Virent has developed the APR system for on-demand generation of a hydrogen/alkane mixture from either glycerol
or sorbitol (the sugar alcohol formed by hydrogenation of glucose) to fuel a stationary internal combustion engine driven generator
(5 kW). Virent is also developing the APR process for on-demand generation of hydrogen for small PEM or SOFC fuel cells (less
than 500 watts). Under this development project, Virent has already built a small prototype system that combines the APR reactor system with a palladium-based hydrogen purifier to generate over 300 sccm of high purity hydrogen. Under a USDOE funded
project, Virent is currently developing the APR process to generate high yields of hydrogen from corn-derived glucose. This project
objective is to achieve the DOE 2010 cost target for distributed production from renewable liquid fuels of $3.60/gge (gasoline gallon
equivalent) delivered.
142
poster 3A-13
Analysis of Economics for Food Waste Conversion to Ethanol
Rebecca Davis, Nathan Mosier, Michael Ladisch*, Purdue University, West Lafayette, IN 47907
Jerry Warner, Defense Life Sciences Institute, McLean, VA 22101
The increase in both waste disposal and energy costs has provided an incentive to convert waste streams into fuel. For
example, dining halls and restaurants discard carbohydrate-rich foods that require tipping fees for removal. These wastes
undergo energy and time intensive processing and are usually composted. An effective use of food waste may be the
enzymatic hydrolysis of the food waste to glucose and fermentation of the sugars to ethanol. Since these wastes have
a complex composition, (lipids, proteins, particulates, wrapper materials, vegetable matter) that could be inhibitory to a
fermentation, experiments were carried out to test fermentability. A high carbohydrate mixture of food materials was prepared for this purpose. When enzymes were added hydrolysis was complete after 5 hours at 50°C. The sugars formed
readily fermented to 60 g/L ethanol within 15 hours when S. cerevisiae was used at 37°C. Simultaneous saccharification
and fermentation (SSF) using glucoamylase and cellulase enzymes and S. cerevisiae results in ethanol production in
one step at 37°C. These tests showed that fermentation is readily achieved. The experimental results were fitted with a
Monod-type model. The model enables estimation of bioreactor volumes, fermentation efficiencies, and costs of replacing landfill disposal with ethanol fermentation.
poster 3A-14
Optimization of Parameters for Lactic Acid Fermentation by Lactobacillus delbrueckii EQ-2
Francisca Pessôa de França1*, Antonio C. A. da Costa2, da Silva A. C.1 and Fernando J. S. Oliveira1
1 Universidade Federal do Rio de Janeiro, Escola de Química, Departamento de Engenharia Bioquímica, Centro de Tecnologia, Bl E, Ilha do Fundão, Rio de Janeiro, RJ, Brasil, 21949-900
2 Departamento de Tecnologia de Processos Bioquímicos, Universidade do Estado do Rio de Janeiro
Experiments were done to determine the best conditions to obtain lactic acid from molasses, using Lactobacillus delbrueckii EQ-2. Several experiments were done to select optimum conditions, including agitation, age of culture, volume of
inoculum/volume of broth ratio and bioreactor height/diameter ratio (H/D). Agitation was tested from 100 to 300 rpm, age
of the culture ranged from 24 to 72 h, H/D ratio ranged from 0.8 to 2.0. Fermentation tests were performed in 4L reactors,
equipped with pH, temperature and rotation velocity automatic controls.
Homolactic fermentation was verified by using high performance liquid chromatography. The highest fermentation yield
obtained was equal to 96.1, a result confirmed in several fermentations. This result was obtained under optimized conditions: H/D equal to 1.5, inoculum aged 24 hours, 5% of volume of inoculum/volume of broth ratio (biomass 0.35 g/L) and
agitation at 200 rpm. At same time the kinetic of bioprocess was established, from specific rates of lactic acid production
and cellular growth.
Authors thank to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and FAPERJ for financial support.
143
poster 3a-15
Enhancement of rhamnolipid production in soybean residual oil by
an isolated strain of Pseudomonas aeruginosa
Cristian Jacques Bolner de Lima1, Rafael Bruno Veira1, Francisca Pessoa de França2, Eliana Flávia Camporese Sérvulo2
and Vicelma Luiz Cardoso1
1
Universidade Federal de Uberlândia, FEQ, Campus Santa Mônica, Bloco 1K, Caixa Postal 593, CEP: 38400-902,
Uberlândia – MG, Brazil.
E-mail:[email protected]
2
Universidade Federal do Rio de Janeiro, Escola de Química, Rio de Janeiro – RJ, Brazil.
Biosurfactants are microbiological substances presenting high surface and interfacial activities. These products can be
produced from renewable raw materials and, in comparison to conventional surfactants, they present very low environmental impact. In the present work, the production of rhamnolipid from soybean residual oil from food frying facilities has
been studied employing a strain of Pseudomonas aeruginosa PATC, isolated from a hidrocarbon contaminated soil. The
optimization of soybean residual oil, ammonium nitrate and brewery residual yeast concentrations has been accomplished
by a central compound planning (CCP) and surface response analysis. The experiments were performed in 500 mL
Erlenmeyer flasks containing 50 mL of mineral medium, at 170 rpm and 30±1ºC, for 48-hour fermentation period. Rhamnolipid production has been monitored by measurements of surface tension, rhamninose cocncentration and emulsifying
activity. The best planned results, located on the central point, have corresponded to 22 g/L of soybean residual oil, 5.625
g/L of NH4NO3 and 11.5 g/L of brewery yeast. At the maximum point the values for rhamnose and emulsifying index were
2.2 g/L and 100%, respectively
poster 3a-16
Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis isolated strain.
Rayane Rodrigues Fonseca1, Francisca Pessoa de França1, Antonio Jorge Ribeiro da Silva3, Vicelma Luiz Cardoso2
and Eliana Flávia Camporese Sérvulo1
Universidade Federal do Rio de Janeiro, Escola de Química, Centro de Tecnologia Bloco E, Ilha do Fundão, CEP
21949-900, Rio de Janeiro – RJ, Brazil. E-mail: [email protected]
2
Universidade Federal de Uberlândia, FEQ, Uberlândia – MG, Brazil.
3
Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, Rio de Janeiro – RJ, Brazil.
1
A Bacillus subtilis isolated strain from a refinery-contaminated soil from has been screened for biosurfactant production in
soluble (commercial sugar) and insoluble (soybean oil) carbon sources, with or without the addition of trace elements and
yeast extract. The best result was achieved with a 48-hour fermentation of commercial sugar supplemented with trace elements. Although in a longer period of time, soybean oil has also stimulated biosurfactant synthesis. Concerning the commercial sugar mineral medium, ammonium nitrate among different nitrogen sources (NaNO3, (NH4)2SO4, urea and brewery
residual yeast) showed the highest reduction in surface tension. Optimization of carbon/ nitrogen (3, 9 and 15) and agitation (50, 150 and 250 rpm) rates for biosurfactant production was carried out using complete factorial design and response
surface analysis. The condition of C/N 3 and 250 rpm allowed the maximum increase in surface-activity of biosurfactant. A
suitable model has been developed, having presented great accordance with experimental data. Preliminary characterization of the bioproduct suggested it to be a lipopeptide with some isomers differing from those of a commercial surfactin.
144
poster 3A-17
A New Procedure for the Acrylic Acid Synthesis by Fermentative Process
Betânia H. Lunelli and Rubens Maciel Filho
Laboratory of Otimization, Project and Advanced Control – LOPCA, Departament of Chemical Process – School of
Chemical Engineering, State University of Campinas – UNICAMP
With the synthesis of chemical products through biotechnological processes is possible to discovery and to explore innumerable routes that can be used to obtain products of high added value. These routes form parallel and alternative ways
for the production of the desired product, what makes easier the preservation of the environment. A possible feedstock is
the sugar cane.
In fact, from the industrial point of view, the acrylic acid production by fermentative process is presented as an innovative
process of great importance, due to possibility of low cost for its production and a renewable raw material. It consists on
a biotechnological route, which has lower aggression to the environment, when compared to the conventional chemical
process. Bearing this in mind, the purpose of this work is the development of a deterministic model of the biotechnological process of the acrylic acid synthesis. It aims to propose a new methodology for its production. The proposed process
make possible to obtain Acrylic acid continuously from the sugar cane fermentation. The reactor is CSTR type (continuous stirred tank reactor) so that it is possible to define operating strategy and conditions to achieve the product with the
desired specifications. A detailed deterministic model based on the concepts of structured representation is developed to
take into account the main phenomena taking place in the system.
145
poster 3A-18
Obtainment of Ethanol/Water Pulps from Sugarcane Straw and the Pulps Biobleaching with Xylanase By
Bacillus pumilus
Regina Y. Moriya and Adilson R. Gonçalves
Departamento de Biotecnologia, FAENQUIL, CP 116, CEP 12600-970 Lorena-SP, Brazil
E-mail: [email protected], URL: http://www.debiq.faenquil.br/adilson
In this work the ethanol/water pulping was studied. A factorial design 22 was used to examine the influence of independent variables (viz. processing temperature and time) of the ethanol pulping of sugarcane straw. The experimental results
obtained in the determinations of the independent variables of the pulping carried out in the 200-mL vessel are shown in
the table 1.
Table 1. Conditions used in the ethanol pulping of sugarcane straw and experimental results for the yield, properties
kappa number, viscosity and the chemical composition of the pulps obtained.
XT
+1
+1
-1
-1
0
0
Xt
+1
-1
+1
-1
0
0
YI, %
55.6
53.7
47.6
45.2
51.7
52.8
KN
61.4
64.4
54.5
59.1
61.0
64.2
VI, cP
4.4
5.4
10.6
12.2
6.9
7.5
GL,%
60.3
59.6
58.6
57.5
62.2
63.6
PE,%
2.3
2.5
11.9
14.2
6.0
6.5
LI,%
33.0
33.3
26.1
25.6
26.8
25.7
X T=Temperature (+1=215°C, 0=200°C, -1=185°C); Xt=Time (+1=2.5 h, 0=2.0 h, -1=1.5 h);YI=Yield; NK=Kappa number;
VI=Viscosity; GL= Glucan; PE= Pentosan; LI= Total lignin.
The ethanol/water pulps to be used as dissolving pulps must have a maximum of 10% pentosan, thus the better time and
temperature of pulping achieved were 2.0 h and 200°C, respectively. Sugarcane straw ethanol/water pulping was carried
out in 40 L vessel with 1.5 kg sugarcane straw, the ratio straw/solvent was 1:10 (w/v), 15 L ethanol/water (1:1) at 200°C,
for 2.0 h. The pulp obtained was bleached with the XE sequence. The X step was conducted using different xylanase
dosage: 5, 10, 20, 50 UI/ g oven-dry pulp for 2 h at 50°C and 150 UI/ g oven-dry pulp for 20 h, pH 8.5 at a concentration
3%. The pulps treated with xylanase were bleached with NaOH 3.5 % (w/v) at a 5% concentration for 1 h at 65 °C. The
pulps treated with different xylanase dosage presented viscosity similar the control pulp (3 cP) and the pulps treated with
xylanase followed by alkaline extraction presented low increased in the viscosity compared with the pulps treated only
with xylanase. The kappa number of the pulps treated with diffrent enzyme dose was 60 and the pulps treated with enzyme followed by alkaline extraction presented kappa number 25. Using Principal Component Analysis (PCA) it was possible to differenciate the pulps treated with different enzyme dosage. The influence of infrared bands on PC scores can
be evaluated and the plot of PC is influenced by 1000cm-1 C-O bonds, characteristic of esters. The pulp obtained in this
work presented low pentosan quantity and high lignin, so the differences in the pulps treated enzymatically only can be
noted in the Principal Component Analysis. [Acknowledgements due to FAPESP, CAPES and CNPq – Brazilian agencies]
146
poster 3A-19
Biopulping of Sugarcane Straw and Biobleaching using Xylanases
Luís R. M. Oliveira, Marcelo Brant W. Saad, Adilson R. Gonçalves*
Vegetal Biomass Conversion and Mathematical Modeling Group
Biotechnology Department – Chemical Engineering School of Lorena - FAENQUIL
FAENQUIL, CP 116, 12600-970 – Lorena-SP, Brazil
In Brazil, the sugarcane is one of the largest agricultural monocultures, supplying an enormous amount of residues and
these can be used in the pulp production. Thus, this work considers the use of the straw in the pulp production. Firstly
a biological treatment of the raw material with fungi Ceriporiopsis subvermispora was performed, degrading selectively
lignin, and then, the pulping was performed using an acetic acid solution. A study for the optimization of the pulping of
the straw was performed through a factorial design 2^3, having as factors of the study the acetic acid concentration, the
consistency and the type of straw (treated and in natura). With this study one verified that the excellent conditions had
been 93% (w/w) for the acetic acid concentration 1/16 (w/w) for the consistency and treated straw. The alternative way for
the biobleaching was the use of the enzyme xylanase, in the case the Pulpzyme. As the cost presented for the enzyme in
the process is relatively high, this work considers the recovery of xylanase in the enzymatic daily pretreatment of the pulp.
The conditions used in this pretreatment was 36 UI of enzyme for gram of dry pulp, at the temperature of 50°C for 2h.
To verify the action of the enzyme, an alkaline extraction of pulps dealt with enzyme was carried through and had been
measured the residual lignin of the pulp (kappa number) and its viscosity. The accompaniment to the enzymatic recovery
activity in the beginning and the ending of the pretreatment was made measuring this activity. [Acknowledgments due to
FAPESP and CNPq – Brazilian agencies]
poster 3A-20
Biological Hydrogen Production from Wastewater via Immobilized Bacteria
Bo Hu*, Zhanyou Chi, Shulin Chen
Department of Biological System Engineering, Washington State University, Pullman WA 99164-6120
This paper explores the possibility of using immobilized hydrogen producing bacteria for treating wastewater with high
carbohydrate concentration to produce hydrogen gas while removing the BOD. Sewage sludge was pretreated by acid to
serve as the bacteria source for hydrogen production. Experiment results showed that the hydrogen producing bacteria
worked well within a wide range of pH from 5 to 8, and tolerated different nitrogen levels. The hydrogen producing bacteria were immobilized into alginate beads and then used to anaerobically treat different carbohydrate containing wastewaters, including: domestic wastewater, food processing wastewater, animal waste and other agricultural wastewaters.
During the tested anaerobic process, all these wastewaters produced carbon dioxide, hydrogen, and volatile fatty acids
such as acetate and butyrate; the BOD in these wastewaters was partly removed simultaneously.
147
poster 3A-21
Nisin Production Utilizing Skimmed Milk Aiming Process Cost Reduction
Angela Faustino Jozala, Maura Sayuri de Andrade, Luciana Juncioni de Arauz , Adalberto Pessoa Jr., Thereza Christina
Vessoni Penna*
School of Pharmaceutical Sciences, University of São Paulo, São Paulo – SP – Brazil
Olivia Cholewa
Molecular Probes, Inc. Eugene, Or, USA. 97402.
Nisin is widely used as a natural additive for conservation of food, pharmaceutical and dental products, and it can be used
as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth a variety of Gram-positive bacteria and Gramnegative bacteria (with the outer membrane first destabilized by EDTA). This study was performed to optimize large scale
nisin production in skimmed milk and derivatives aiming process cost reduction and stimulating its utilization. As nisin
expression is related to the growth conditions of Lactococcus lactis, the effects of growth parameters, media components
and incubation time, were also studied. L. lactis ATCC 11454 was developed (30º/36horas/100rpm) in MRS and skimmed
milk, and in both media diluted in two different concentrations (25% and 12.5%). The dilution of skimmed milk to 12.5%
improved the nisin expression by L. lactis by a factor of 5 (79 AU.L-1), in comparison to 17 AU.L–1 showed in MRS 12.5%.
Skimmed milk 12.5% was assayed in a New Brunswick fermenter (30oC/24h/200rpm) final volume 1.5 L, air flow of 1.5
mL.min-1, without pH control. Nisin activity was analyzed by agar diffusion using Lactobacillus sake ATCC 15521 and a
recombinant Escherichia coli DH5α expressing the recombinant green fluorescent protein (GFPuv), as the nisin-sensitive
test organisms. The titers of nisin expressed and released in culture media were expressed in arbitrary units (AU.mL-1 medium) and converted to standard nisin concentration (Nisaplin, 25 mg of pure nisin with an activity of 1.0 x 106 AU.mL-1).
The minimum concentration of nutrients was enough to improve nisin expression and its release into the media. Casein
and lactose impact nisin production was detected when proteins and sugars concentration decreased because of metabolic reactions. This work shows that the utilization of a low cost culture media (milk derivatives) to antimicrobial production can be exploited. In Brazil 50% of milk whey is disposed with no treatment in rivers and due to high organic matter
concentrations is considered an important pollutant. In this particular case an optimized production of an antimicrobial
would be lined up with industrial disposal recycling.
148
Poster 3A-22
Production of Bacterial Cellulose by Gluconacetobacter sp. RKY5 in a Rotary Biofilm Contactor
Yong-Jun Kim, Jin-Nam Kim
Department of Material Chemical and Biochemical Engineering, Chonnam National University, Gwangju 500-757, Korea
Young-Jung Wee, Hwa-Won Ryu*
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea
Cellulose is one of the most abundant biological macromolecules, which is a linear insoluble biopolymer that is composed
of the repeated unit of â-1,4 glycosidic bonds. A rotary biofilm contactor (RBC) has become a popular method for the
treatment of industrial wastewater during the last decades. RBC is capable of retaining considerable amounts of attached
biomass, which could provide successful performance.
The objective of this study is to optimize the fermentation conditions for the improvement of bacterial cellulose production
in RBC. We investigated the operational conditions of the RBC with Gluconacetobacter sp. RKY5 isolated from persimmon vinegar. The optimal number of discs for bacterial cellulose production was eight, and the optimal aeration rate and
rotating speed were found to be 1.25 vvm and 15 rpm, respectively. The operation of RBC without pH control during
fermentation showed higher production of bacterial cellulose than the operation with pH control. The optimal conditions
for the production of bacterial cellulose in RBC were established, and the amount of bacterial cellulose produced and cell
concentration was 6.17 g/L and 5.58 g/L, respectively, under the optimized conditions.
Poster 3A-23
l(+)-Lactic
Acid Production from Commercial Starches and Corn Steep Liquor by Enterococcus faecalis RKY1
Young-Jung Wee, Don-Hee Park, Hwa-Won Ryu*
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea
Jin-Nam Kim
Department of Material Chemical and Biochemical Engineering, Chonnam National University, Gwangju 500-757, Korea
Jong-Sun Yun
BioHelix, Noan-myeon, Naju, Jeonnam 520-811, Korea
Lactic acid is considered to be one of the most useful chemicals, used in the food, textile, pharmaceutical, and chemical
industries. Recently, lactic acid consumption has increased considerably because of its role as a monomer in the production of biodegradable poly(lactic acid), which is well-known as a sustainable bioplastic material. However, it is still necessary to screen the inexpensive raw materials for the economical production of lactic acid.
The commercial starches and corn steep liquor (CSL) was used as main nutrients for the production of l(+)-lactic acid.
Corn, potato, tapioca, and soluble starches were used as a carbohydrate source, and CSL obtained from a local corn
product company was used as a nitrogen source. For all experiments, after 2 or 4 h of saccharification with commercial
α-amylase and glucoamylase enzymes, Enterococcus faecalis RKY1 was inoculated. Lactic acid yields based on the
theoretical glucose concentration, were greater than 0.9 g/g in all cases experimented. The productivities of l(+)-lactic
acid were ranged between 2.9 and 3.5 g/L·h, when the starch concentrations were between 50 and 100 g/L. The commercial starches and CSL were good raw materials for the production of lactic acid by E. faecalis RKY1, with reasonable
productivity and yield.
149
Poster 3A-24
Characterization of Novel Epigallocatechin Gallate Glycosides Using Glucansucrase from
Leuconostoc mesenteroides B-1299CB
Young-Hwan Moon, Hyun-Ju Chung, Seung-Hee Nam, Doman Kim*
Chonnam National University, Gwangju 500-110, Korea
Ghahyun J. Kim, Na-Young Woo, Jun-Bum Pyo
Korean Minjok Leadership Academy, Gwangwon-do 225-823, Korea
Donal F. Day
Audubon Sugar Institute, LSU-Ag Center, LA 70776
EGCG (epigallocatechin gallate), which are main polyphenol compound in green tea, have strong antioxidative activities, anticancer, antimutagenic, antibacterial effects, prevention of dental caries, regulation of plasma cholesterol levels.
However, EGCG are poorly soluble in water, and degraded easily by light irradiation in water resulting in rapid browning.
To solve these problems, the transglucosylation of novel EGCG analogues was studied using glucansucrase from Leuconostoc mesenteroides B 1299CB.
EGCG analogues were synthesized by the reaction of EGCG with sucrose and glucansucrases from L. mesenteroides
B-1299CB. Their structures were assigned as epigallocatechin gallate 7-O-α-D-glucopyranoside (EGCG-G1), epigallocatechin gallate 4′-O-α-D-glucopyranoside (EGCG-G1′), and epigallocatechin gallate 7,4′-O-α-D-glucopyranoside
(EGCG-G2) after 1H, 13C, HSQC, H-H COSY, HMBC analyses. These EGCG analogues showed a different antioxidant
effects according to their structures (EGCG ≥ EGCG-G1 > EGCG-G1′ > EGCG-G2). Furthermore, EGCG analogues
showed the strong stability in a browning resistance than EGCG, even for 24 h. The solubility of the EGCG analogues
was 52, 76 and 140 times higher than that of EGCG, respectively.
150
Poster 3A-25
Biosurfactants Production by Bacillus Atrophaeus ATCC9372 and Bacillus Subtilis W in TSB (Tryptone Soya
Broth) Culture Media Enriched With Glucose, Soy Flour and Casein Digested
Luiz Carlos Martins das Neves, Kátia Oliveira da Silva, Márcio Junji Kobayashi, Thereza Christina Vessoni Penna *
School of Pharmaceutical Sciences, University of São Paulo, São Paulo – SP – Brazil
Olivia Cholewa
Molecular Probes, Inc. Eugene, Or, USA. 97402.
Biosurfactants are proteins which present detergent, emulsifier and antimicrobial actions. They present potential application in the environment protection, organic substances treatments and oil recoveries. Bacillus subtilis strains are
a non-pathogenic and suitable biosurfactants source, among others, surfactin. The aim of this work was established a
culture media composition that favored biosurfactants production by B. atrophaeus ATCC 9372 (standard strain) and B.
subtilis W expressing a recombinant green fluorescence protein (GFP). In preliminary tests in TSB (original composition),
B. subtilis W exhibited a maximum cell concentration 44% less than obtained for the standard strain B. atrophaeus. On
the other hand, the recombinant strain exhibited average values of the yields of extracellular surfactant on biomass and
total proteins about 9.6% and 61.2% higher than the reference one, which demonstrated the higher ability of the former
to overproduce the surfactant even at lower cell concentration. Tests were realized utilizing TSB (Tryptone Soy Broth)
enriched with glucose, casein and soy flour, at 150 rpm and 35°C for 24 h. Results show the addition of glucose until
30g/L in the culture media improved 17 times the biosurfactants production by B. atrophaeus ATCC9372. The presence
of casein (17 g/L) and soy flour (3.0 g/L) added in the media, simultaneously, with glucose (20 g/L and 30 g/L) resulted in
a diauxie effect during cell growth. Diauxie is characterized as an alternated utilization of a new nutrient as energy source
in a minimum concentration of glucose into the culture media, drawing out the cell growth. Nevertheless, after starting
diauxie effect, biosurfactants production was 50% less than observed before this phenomenon. The capability observed
by B. subtilis and B. atrophaeus strains to adapt and use several nutrients as an energy source favored its application in
bioremediation process.
151
Poster 3A-26
Evaluation of The Resistance Paramenters for the Recombinant Green Fluorescent Protein (GFPuv) Exposed to
Sodium Chloride Solutions at Different Concentrations, pH Values and Temperature Conditions
Thereza Christina Vessoni Penna*, Marina Ishii, Juliana Sayuri Kunimura
Department of Biochemical and Pharmaceutical Technology,
School of Pharmaceutical Science, University of São Paulo, SP, Br.
Olivia Cholewa
Molecular Probes, Inc., Eugene, Or, USA. 97402.
The thermal resistance of recombinant green fluorescent protein (GFPuv) in sodium chloride (NaCl) solutions at different concentrations, pH values and set temperatures were evaluated by assaying the loss of fluorescence intensity as a
measure of denaturation. GFPuv, extracted from E. coli cells by the three-phase partitioning (TPP) method and purified
through a butyl-HIC column, was diluted in WFI (pH 6-7) and in 10 mM buffer solutions (acetate, pH 5; phosphate, pH 6-7;
Tris-EDTA, pH 8) with 0.9%, 5%, 10%, 15%, 20%, 25% and 30% sodium chloride and exposed to 80oC, 85oC, 90oC, and
95oC. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (Dvalue); the interval of time required to reduce 90% or one decimal logarithm of the initial fluorescence intensity of GFPuv.
In acetate (pH 5), D-values for 90% reduction in the fluorescence intensity of GFPuv ranged from 0.99 min to 1.99 min,
independend of NaCl concentration and temperature. For glucophysiologic solution (0.9% NaCl added with 5% glucose),
D-value increased 3-4 min, for the same set conditions. The resistance of GFPuv diluted in WFI decreased linearly with
the increment of NaCl concentration; and D- values (at 80oC) ranged from 8.26 min (5% NaCl) to 3.44 min (30% NaCl).
However, the resistance of GFPuv in Tris-EDTA (pH 8) was directly dependent upon the NaCl concentration and 5-10
times higher than D-values for GFPuv in WFI at 80 oC. The addition of 5% glucose in 0.9% NaCl Tris-EDTA solution
did not interfere in the GFPuv resistance. GFPuv can be easily monitored by the convenient measure of fluorescence
intensity and be potencially used as an indicator to report the extent of denaturation rates of other proteins exposed to the
same conditions.
152
Poster 3A-27
Utilization of Green Fluorescent Protein as Biosensor of Drugs Stability in Physiological Solutions
Carolina Alves dos Santos, Priscila Gava Mazzola, Thereza Christina Vessoni Penna* School of Pharmaceutical Sciences, University of São Paulo, São Paulo – SP – Brazil
The physiological solutions (PS) are used as vehicles to administrate drugs into the organism. The development of new
techniques will enable the detection of incompatibilities between drugs and physiological solutions. This is necessary to
guarantee the correct association and rational utilization of drugs. The Green Fluorescent Protein (GFPuv) is sensitive
to pH variations and chemical compounds in solutions. GFPuv changes can indicate the potential use of this protein as
biosensor to detect incompatibilities among drugs added to physiological solutions. Therefore, this work aims to evaluate the association of drugs added to the physiological solution in the presence of GFPuv. The fluorescence intensity of
GFPuv (Ex /Emmax=394/509 nm) was analyzed with and without the presence of dopamine and ranitidine separated and
associated. The following assays were evaluated in the fluorimeter with both commercially and laboratory prepared physiological solutions: (i) 4.9mL solution of dopamine and + 0.1mL of GFPuv, (ii) 4.9mL solution of ranitidine and + 0.1mL of
GFPuv. The value of pH in physiological solution containing ranitidine was pH=6.85 ± 0.02, GFPuv fluorescence intensity
was similar to the fluorescence intensity observed for GFP in PS. Dopamine addition reduced the pH value is PS to 3.0 ±
0.2 and the GFP fluorescence intensity in about 100 times. Presence of the acidic molecule (dopamine) in solution with
GFPuv caused abruptly fluorescence intensity decrease. In presence of ranitidine (neutral molecule), the fluorescence
intensity emission was similar. Green fluorescent protein has presented better stability and pH values when added in solutions with neutral to basic character as described in previous studies. This study is important to develop new techniques
to detect drugs incompatibilities in physiological solutions and to promote the rational use of drugs in hospital patients.
GFPuv was sensitive to the presence of drugs in PS, and it is indicated as a potential biosensor for drugs stability in solution. Rational drug administration is mandatory to reduce costs to hospitals and also to reduce its disposal in the environmental.
Poster 3A-28
Mechanical Properties of Polyurethane Foams Prepared from Liquefied Corn Stover
Tipeng Wang, Zhihuai Mao*, Lingyun Liang, Dong Li, Yaoxian Yu, Lianhui Zhang China Agricultural University, Beijing
100083, China
Yebo Li, Abolghasem Shahbazi
North Carolina A&T State University, 1601 East Market Street, Greensboro, NC 27411
Corn stover was liquefied using ethylene carbonate as liquefying solvent and 98% sulfur acid as catalyst at 170°C for 90
min. The liquefied corn stover comprised up to 37% corn stover. The hydroxyl number and viscosity (25°C) of the liquefied corn stover was measured to be 191-40mg KOH/g and 0.5-2.2Pa.s, respectively. Corn stover-polyurethane foams
were prepared by the one-shot method, where liquefied corn stover and polymeric MDI, water, silicone, and triethanol
amineas were used as co-reactants, blowing agent, surfactant and catalyst, respectively. The above materials were
mixed at approximately 1500rpm for 10s. The effects of [NCO]/[OH] ratio, catalyst content and corn stover content on the
tensile properties and Young’s modulus of corn stover-polyurethane foam were studied. With the increase of corn stover
content, the Young’s modulus of corn stover-polyurethane foam was significantly increased and the maximum elongation
was markedly decreased. The rigid mechanical properties vary according to the increase of dissolved corn stover fragments.
153
Poster 3A-29
Genetic Modification of a Gluconic acid Producing Aspergillus niger AR-12 strain Through Combined
Mutagenesis for Enhanced Metal Tolerance
A. Sharma, Om V. Singha and Rajesh P. Singh*
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, India
E: [email protected], F: 91-1332-273560, T: 91-1332-285792
Gluconic acid (penta-hydroxycaproic acid) has wide applications in textile, cement, food, leather and pharmaceutical
industries. Large scale production of gluconic acid is normally accomplished by using glucose as the carbon source. Cost
of the gluconic acid production can be substantially reduced by using molasses as the substrate. Sugarcane molasses is
a cheaper carbohydrate source that is available in plenty as a byproduct of the sugarcane industries. Higher levels of the
heavy metal content of the molasses make it an unfavorable substrate for bioconversion process and result into remarkably decreased yield of the product for industrial applications. Strain AR-12 identified as Aspergillus niger ITCC 5483 was
selected from a number of isolates from the wastes of regional sugarcane industries. The isolated strains were evaluated
for their gluconic acid production ability using glucose as the carbon source. Among the isolated strains, Aspergillus niger
AR-12 (ITCC 5483) was observed having the maximum ability of gluconic acid production with glucose as the carbon
source. However, the level of production was considerably lower when molasses was used as the substrate. Since the
cost of molasses is manifold lower than the glucose, efforts were made to utilize the sugar cane molasses directly as the
carbohydrate substrate by developing a potent mutant strain having improved ability of heavy metal tolerance and simultaneously with higher ability for gluconic acid production. In the mutagenesis strategy, strain AR-12 was chemically mutagenized with Colchicine (0.01-2 µg ml-1) and with N-methyl-n-nitro-N-nitrosoguanidine (NTG, 100 µg ml-1). Mutagenized
spores were harvested and cultured on potato dextrose agar (PDA) plates containing different concentrations (0.05-1.20
mM) of manganese ions. Resulting colonies that had shown higher AU values and the metal tolerance were further analyzed for gluconic acid production. Mutant ARN-30 obtained by NTG treatment that showed higher gluconic acid production and metal resistance was selected for further mutagenesis by UV irradiation (2.5 J -2s-1, distance 0.43m) for different
time periods. Mutagenized spores were further analyzed on PDA plates containing 0.05-1.20 mM of Mn+2 as the heavy
metal content. Out of the mutants obtained, mutant ARNU-4 had maximal gluconic production ability in the bioconversion
reaction containing Mn+2 at the concentrations of 0.23mM respectively. Strain ARNU-4 was evaluated for gluconic acid
production under surface, submerged and solid-state fermentation conditions with molasses as the substrate. Solid-state
fermentation was found to be most favorable that had resulted into 81.5g/l of gluconic acid production.
a
Present address: Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA
154
Poster 3A-30
WITHDRAWN
Poster 3A-31
Biopulping of Sugarcane Straw: Improvement of the Organosolv Process
Marcelo Brant W. Saad, Adilson R. Gonçalves*
Vegetal Biomass Conversion and Mathematical Modeling Group, Biotechnology Department – Chemical Engineering
School of Lorena, FAENQUIL, CP 116, 12600-970 – Lorena-SP, Brazil. E-mail: [email protected]
In Brazil, the sugarcane is one of the largest agricultural monocultures, supplying an enormous amount of residues and
these can be used in the pulp production. The introduction of biodegradation process into the pulp and paper industry has
great economic potential and contributes to environmental preservation. The purpose of the present study was to determine the ability of Ceriporiopsis subvermispora CS1 to delignify sugarcane straw and evaluate the acetosolv pulping of
this raw material. First, the influence of the time and the load of inoculum in the biological treatment was studied. The load
of inoculum of 250 mg/kg and the time of 15 days of culture had approximately shown to be the best conditions for daily
pretreatment causing 23 % of loss of total lignin and only 8 % of loss of cellulose and 12 % of hemicelluloses. The pulping of straw was carried through in a reactor of 0.5 L at 115°C. With 2 h of pulping biopulp and pulp control had presented
8 and 12.5 % of total lignin and kappa number of 25 and 32, respectively. Through a screening design 2^3 it was verified
that the use of the biotreated straw is more important factor that the acetic acid concentration and the consistency of
pulping in the delignification and that the type of straw significantly does not influence the result and the viscosity of pulps
in the explored intervals. Of this form, it is possible to diminish the demand of reagent and the time in the pulping using
sugarcane straw daily pretreated and it can be decreased the acid concentration of and/or the consistency without significantly modifying viscosity and the yield, but still thus diminishing the kappa number. [Acknowledgements due to CNPq
and FAPESP, Brazilian agencies].
155
Poster 3A-32
Recovery of the enzyme xylanase in the biobleaching of bagasse pulps
Luís R. M. Oliveira, Adilson R. Gonçalves*
Vegetal Biomass Conversion and Mathematical Modeling Group, Biotechnology Department – FAENQUIL, Lorena-SP,
Brazil, CP 116, CEP 12.600-970
The pulp and paper industry is implementing changes in the bleaching process to minimize the use of chlorine in order
to satisfy regulatory and market demands. Biotechnology has a potentially important role to play in providing alternatives
to conventional chlorine bleaching of chemical pulps, principally with use of xylanases. The enzyme cost in pulp bleaching is still an economic barrier for its application. Two commercial xylanases were used in the acetosolv pulps treatment:
Cartazyme HS and Pulpzyme HC. These enzymes were recovered and reused for several times in the enzymatic treatment of pulps. At each new treatment, just the recovered enzyme was used, and there was no addition enzyme charge.
The accompaniment of the enzymatic activity was carried out through measurement at initial and finish of each enzymatic
treatment. Alkaline extraction of untreated and xylanase-treated pulps were carried out. The untreated-, xylanase-treated
and alkaline-extracted pulps were analyzed with regard to kappa number, viscosity and infrared associated to principal
component analyses (FTIR-PCA). The Cartazyme HS achieved 20% recovery for both solvents utilized (water and buffer),
however the Pulpzyme HC presente just 2% of recovery in aqueous medium and 84% in buffered medium. FTIR-PCA
showed differences among untreated, xylanase-treated and alkaline-extracted pulps. [Financial supporte from CNPq and
FAPESP, Brazilian agencies].
156
poster 3B-08
Microalgae for biofuels and biochemicals production
D.E. Brune, Dept of Biological and Agricultural Engineering, Clemson University, Clemson SC., 29634
G. Schwartz and M. Massingill, Kent SeaTech Corp, 11125 Flintkote Avenue, San Diego, CA 92121
J. Benemann, Consultant, Walnut Creek, CA., 94595
Microalgal production offers many advantages over conventional lignocellulosic biomass production technologies: 1) Four
to five-fold higher rate of biomass production requiring less land surface area, 2) Production of biomass on non-productive hardpan, desert, saline lands, 3) Short biomass generation time providing continuous production with full utilization
of harvest systems and reduced processing and storage requirements, 4) Hydraulic cultivation allowing for low cost fluid
transport of harvested algal concentrate, 5) Capability of recovery and reuse of nutrients from agricultural wastewaters
and municipal discharges and, 6) Short algal cell generation times allowing for rapid crop modification and improvement
The Partitioned Aquaculture System was originally developed at Clemson University, to integrate microalgae biofixation of CO2
with fish aquaculture promoting a high rate of harvestable algae and aquatic animal production. A modification of the PAS, the
Controlled Eutrophication Process is currently being evaluated by Kent SeaTech Corp at the Salton Sea in Southern California.
Methods for conversion of algal biomass to biofuels include anaerobic fermentation to directly convert the biomass into methane and hydrogen and an indirect biophotolysis process in which the harvested algae is converted into hydrogen. This presentation will review development of Clemson and Kent SeaTech algal production, harvest and conversion systems.
157
poster 3B-09
Logistic aspects related to the production of biofuels from lignocellulosic residues and dedicated crops:
The case of some Italian districts
De Bari Isabella*, Alois Enzo, Motola Vincenzo, and G. Braccio
ENEA CR TRISAIA, SS 106 Jonica, km 419+500, 75025 Policoro (MT), Italy
In Italy, the present biomass utilization accounts only for 2.5 % of the national energetic demand. This percentage is modest if
compared with the effective Italian potential that is much higher (21-23 Mtep). More than 92% of the used feedstock is still destined
to the thermal heating sector while scarcely 1% is used for biofuels production. These figures are destined to grow in the next few
years following the application of the European directive requiring the addition of increasing amounts of biofuels (up to 5.75% by
2010) to the fossil fuels for transportation. Specifically, this implies that roughly 2Mt biofuels should be produced by 2010. One big
distillery, Caviro, is going to integrate the existing process with the production of bioethanol from dedicated crops such as sugar
beet. Additionally, another big cereals producer, Grandi Molini Italiani, is building an industrial facility of 2.2 t/d of cereals for bioethanol production. These initiatives let foresee a market development for bioethanol. However, considering the Italian landscape
irregularity and the increasing desertification, the production of biofuels from dedicated crops needs be integrated with the production from lignocellulosic residues. Almost 66 mlt/y lignocellulosic residues have been estimated. However, part of this material has
already a non-energetic market utilization. Additional obstacles are due to the variability of the residues chemico-physical properties and degradability, to the dispersion through the territory, and to the seasonal production. Thus a wider use of the lignocellulosic residues in Italy is still hindered by two barriers: the identification of the most suitable biomass for the specific process and the
relevant supply; the optimization of the logistics from the production area to the processing plant.
This paper focuses on the analysis and optimization of the logistic aspects concerning the use of lignocellulosic residues
and/or dedicated crops for biofuels production in some Italian districts.
The GIS methodology has been applied to the assessment of the agricultural residues, dedicated and agro-industrial
crops either in case of describing the present situation either in case of a different management of the forest and marginal areas. A simulation software (SIMILE) has been run to optimize the process costs under different production scenarios
and transportation modalities.
poster 3B-10
The Logistical Implications of Optimizing Anaerobic Digestion of Manure and Waste
Emad Ghafoori and Peter C. Flynn, Department of Mechanical Engineering, University of Alberta
Anaerobic digestion of manure and other selected waste streams can deal with two problems: it can reduce some of
the impacts of manure spreading from confined feeding operations, and it can produce carbon neutral power. Anaerobic
digestion has most frequently applied on a farm by farm basis, but applications in Denmark have focused on centralized
digesters to achieve an economy of scale.
All plants process remote sources of biomass face a tradeoff: unit capital costs decrease with increasing plant size (the
economy of scale) while feedstock transportation cost increases as the area from which biomass is sourced increases with
plant size. We explore the tradeoff between these two competing cost factors and calculate the optimum digester size for a
range of biomass feedstock availability, i.e. the amount of digester feedstock per unit of gross area around the digester. We
use a range of values for biomass availability ranging from intense confined feeding operations to dispersed farms. In all the
cases we develop centralized digesters are more economic than farm or feedlot based units. We explore the logistical implications of optimizing digester size, including transportation of feedstock to the plant and removal of digestate from the plant.
158
poster 3B-11
The upgrade of Agro-food industries by-products and wastes in European Mediterranean countries:
The Portuguese current situation and future prospects
Luís C. Duarte, M. P. Esteves, Florbela Carvalheiro, Francisco M. Gírio*
INETI, Dep. Biotecnologia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal
Lignocellulosic biomass by-products and residues from agro-food processing plants are usually considered to be widespread, cheap and readily available resources. They may even constitute an environmental problem leading to an
economic burden for these industries. Nevertheless, the economic upgrade feasibility of such materials is extremely
dependent on many variables, e.g. quantities available, associated costs, transportation economics, current applications
and, also geographical, regional and political constrains.
To develop R&D–based upgrading programs to increase the value chain of the agro-food sector it is necessary a clear
characterization of such by-products and residues. With this purpose, it was developed a mail survey that reached about
1300 Portuguese companies selected from a comprehensive database constructed from official data. The obtained
response rate was 17%. The sample has a fairly good geographical and sub-sector coverage that adequately represents
the target agro-food sector.
This survey enabled us to characterize the agro-food industrial sector, namely its composition and regional distribution.
Furthermore, the characterization of the different by-products and residues produced by each sub-sector, respective
quantities, production periods, current applications and economic value were also possible. Opportunities and constrains
for the biotechnological upgrade of these materials will be presented and discussed based on the preliminary data for
their proximal composition.
poster 3B-12
The Real Value of Selective Harvest
Kevin L. Kenney*, Christopher T. Wright, Corey W. Radtke, J. Richard Hess, Reed L. Hoskinson, and Peter.A. Pryfogle
Idaho National Laboratory, Idaho Falls, Idaho 83415-2210
Composition and pretreatment studies of corn stover and wheat stover anatomical fractions clearly show that some corn
and wheat stover anatomical fractions are of higher value than others. This premise, along with soil sustainability and erosion control concerns, provides the basis of the selective harvest concept for separating and collecting the higher value
residue fractions in a combine during grain harvest. The value of selective harvest is determined by the impact on feedstock quality including both chemical composition and in-refinery conversion efficiency. Recognizing that residue separation in a grain combine is significant in affecting quality through selective harvest, this research focused on achieving
high-fidelity residue separation. The fidelity of residue separation obtained using a commercial grain combine was insufficient to effect significant quality differences in the straw and chaff residue streams. Using a computational engineering
approach, involving modeling, analysis and simulation, a residue separator was designed for accomplishing high-fidelity
separation of the residue streams. A selective harvest test combine was retrofit with the engineered residue separator
and tested to evaluate its effectiveness in achieving a high-fidelity residue separation. Subsequent compositional and
techno-economic analysis of the residue fractions was performed to assess the selective harvest fractionation impact on
feedstock quality.
159
poster 3B-13
Effect of Acid and Alkaline Pretreatment Severity on the Enzymatic Hydrolysis of Corn Stover Fractions
Michael D. Montross*, K. Blair Duguid, Scott A. Shearer, Czarena L. Crofcheck
Biosystems and Agricultural Engineering, University of Kentucky, Lexington, KY 40546-0276
Reed L. Hoskinson, Corey W. Radtke, Idaho National Laboratory, Idaho Falls, ID 83415
Due to concerns with biomass collection systems and soil sustainability there are opportunities to investigate the optimal plant
fraction to collect. An ideal feedstock would require less pretreatment and release a maximum amount of sugar after enzymatic
hydrolysis. Corn stover fractions were separated by hand and concentrated acid hydrolysis performed to determine the amount
of monomeric glucose, monomeric xylose, acid soluble lignin, acid insoluble lignin, and ash. Fractions were pretreated with DI
water, 0.4, and 0.8% NaOH for 2 hours at room temperature. The samples were filtered, washed, autoclaved, and 0.75 g of
enzyme per 3 g of biomass added (2500 CMCU/g biomass or 46 FPU/g biomass, 37,500 XU/g). After 65 hours, the glucose
and xylose was measured and the overall conversion efficiency calculated. There were considerable differences in the quantity
of glucose and xylose released as a function of pretreatment level and fraction. Corn husks released 0.39 g glucose/g biomass
and corn stalks below the ear released 0.18 g glucose/g biomass when pretreated with 0.8% NaOH. Over 90% of the available
glucose was recovered from corn husks when pretreated with 0.8% NaOH, while only 45% of the glucose was recovered from
corn stalks below the ear. Acid pretreatment followed by simultaneous enzymatic saccharification and fermentation produced
similar results. The bottom stalk responded poorly to pretreatment and only approximately 30% of the glucan was converted
to ethanol compared to 90% of the glucan from corn stover obtained in a pilot scale pretreatment run. The top stalk, leaves,
husks, and cobs had a considerably higher conversion to ethanol. Optimization of biomass collection systems to remove a sustainable amount of material and the needs of a biorefinery could be performed to minimize the overall production cost.
poster 3B-14
Effect of Acid and Alkaline Pretreatment Severity on the Enzymatic Hydrolysis of Wheat Stover Fractions
Michael D. Montross*, K. Blair Duguid, Scott A. Shearer, Czarena L. Crofcheck
Biosystems and Agricultural Engineering, University of Kentucky, Lexington, KY 40546-0276
Reed L. Hoskinson, Corey W. Radtke, Idaho National Laboratory, Idaho Falls, ID 83415
Due to concerns with biomass collection systems and soil sustainability there are opportunities to investigate the optimal
plant fraction to collect. An ideal feedstock would require less pretreatment and release a maximum amount of sugar after
enzymatic hydrolysis. Wheat stover fractions were separated by hand and concentrated acid hydrolysis performed to determine the amount of monomeric glucose, monomeric xylose, acid soluble lignin, acid insoluble lignin, and ash. Fractions
were pretreated with DI water, 0.4, and 0.8% NaOH for 2 hours at room temperature. The samples were filtered, washed,
autoclaved, and 0.75 g of enzyme per 3 g of biomass added (2500 CMCU/g biomass or 46 FPU/g biomass, 37,500
XU/g). After 65 hours, the glucose and xylose was measured and the overall conversion efficiency calculated. There were
considerable differences in the quantity of glucose and xylose released as a function of pretreatment level and fraction.
Internodes released 0.33 g glucose/g biomass and nodes released 0.28 g glucose/g biomass when pretreated with 0.8%
NaOH. Over 80% of the available glucose was recovered from leaves and chaff, while less than 80% was recovered from
nodes and internodes. Acid pretreatment followed by simultaneous enzymatic saccharification and fermentation produced
similar results. Nodes and internodes seemed to require a higher intensity of pretreatment, as relatively low conversion
to ethanol was observed with or without the dilute acid pretreatment. In contrast, the conversion of the wheat leaves
suggests that pretreatment was not necessary due to the high conversion to ethanol. Optimization of biomass collection
systems to remove a sustainable amount of material and the needs of a biorefinery could be performed to minimize the
overall production cost.
160
poster 3B-15
WITHDRAWN
poster 3B-16
Application of Imaging Techniques for Optimizing Biomass Fractionation Processes
Peter A. Pryfogle*, Christopher T. Wright, J. Richard Hess, Nathan A. Stevens, Kevin L. Kenney, Corey W. Radtke, and
Reed L. Hoskinson
The mechanical and chemical processing of biomass to prepare it for use in biofuels or other bioproducts can be very
labor and energy intensive. A significant reduction in production costs may be realized as the separations processes become more efficient. We have developed and are applying imaging techniques to investigate biomass fractionation as a
function of material characteristics, i.e. particle size, mechanical strength, chemical content, and the required energy input
into various production processes.
The imaging techniques developed provide metrics for structural analysis before, during, and after material stress failure.
Multiple tests can be run, incorporating a range of load factors, and materials with different load histories can be examined at varying magnifications to characterize mode of failure. Low magnification images are collected to observe whole
plant materials, while higher magnifications are necessary to observe tissues and specific structures. Several systems
are used to collect images at the magnifications necessary to document testing activities and failure analyses. Stereozoom dissecting scopes, light microscopes equipped with differential contrast, polarization, epifluorescence and confocal
capabilities, and video units are used separately or together to optimize analyses. A discussion and representation of
these methods are provided in this report.
161
poster 3B-17
Quality Changes During Bunker-Ensiled Storage of Corn Stover for the Biorefining Industry
Earic Bonner, William E. Bond, LaKenya T. McNear
Livingstone College, Salisbury, NC 28144
Tom M. Schechinger
Iron Horse Farms, Harlan, IA 51537
In many areas of higher rainfall, and particularly when using an agricultural residue, biomass may only be available at
moisture concentrations above 25% at the time of grain harvest. In these conditions, wet storage may be economically
advantageous over dry storage because of the large costs associated with commercial drying, and the unreliability of
drying the biomass in the field. However, another possible advantage of wet stored systems is that a percentage of the inbiorefinery pretreatment may be accomplished during the wet storage, defraying the overall impact of the wet system on
the minimum ethanol selling price (MESP).
The purpose of this research was to estimate the baseline compositional and relative ethanol conversion efficiencies of
control and wet stored corn stover on projected biorefinery operations, and to identify areas which will most likely result in
an increased payoff when further developed. Further, the costs of the wet stored system was quantified.
poster 3B-18
Quality Changes During Dry Storage of Cereal Straw for the Biorefining Industry
Earic Bonner, William E. Bond, LaKenya T. McNear
Livingstone College, Salisbury, NC 28144
Dry storage systems will likely support the first biorefineries. However, the baseline case of bulk losses and quality
changes of biomass through dry storage systems is not well understood, and particularly not sufficiently investigated from
the standpoint of specific biorefinery decision support. Large supply questions of feedstock availability, losses in storage,
and quality changes are unanswered. To help answer these questions, we investigated cereal straw dry storage using
three systems, 4x4x8 ft bales, loaves, and a chopped pile.
After outside storage for one year, compositional and refinery-specific chemical qualities were estimated. Two major
types of biomass damage were observed from wheat straw; one from regions in the storage units that became transiently
wet and those that remained continually wet throughout the storage year. The sugar compositions with each type of damaged area changed by about 10% of the total mass, and the damaged areas were further found to convert less efficiently
to ethanol, measured using a dilute acid pretreatment and SSF screening method.
162
poster 3B-19
The relative cost of biomass energy transport
Erin Searcy, Peter Flynn* and Amit Kumar
The University of Alberta
Logistics cost, the cost of moving feedstock or products, is a key component of the overall cost of recovering energy
from biomass. In practice, projects have the option of moving either feedstock (raw biomass) or products such as power,
ethanol or biodiesel. Both options are under consideration, for example movement of bulk biomass to Europe for processing and remote location of power plants using biomass. In this study we calculate the relative cost of transportation for
every known form of transportation, including truck, rail and pipeline for feedstock and transmission line, pipeline, truck
and rail for products. Costs include fixed and distance variable operating costs as well as product losses (as occurs with
power transmission). Costs are normalized to a common basis of a GJ of biomass input energy. The relative cost of moving products vs. feedstock is an approximate measure of the incentive for location of biomass processing at the source of
biomass rather than at the point of ultimate consumption of produced energy.
poster 3B-20
Agronomic Management of Switchgrass: Soil, Seeding Rate, Fertility, Cultivar and Weed Control Evaluations
Donald Tyler*, Department of Biosystems Engineering and Soil Science, The University of Tennessee,
605 Airways Blvd., Jackson, TN 38301
Burton C. English, Roland Roberts, and Marie Walsh, Bio-based Energy Analysis Group,
Department of Agricultural Economics, The University of Tennessee, Knoxville, TN 37996-4518
Larry Steckel, Department of Plant Sciences, The University of Tennessee, 605 Airways Blvd., Jackson, TN 38301
Second year switchgrass production has been evaluated on four common soil and landscape combinations at The
University of Tennessee’s Milan Experiment Station. These were: 1.) a level floodplain that was well to moderately well
drained, 2.) a level floodplain that was poorly to somewhat poorly drained, 3.) and upland sloping hillside that had a naturally occurring hardpan, and 4.) a level well drained upland with no hardpan. These combinations have resulted in large
differences in switchgrass productivity.
All four areas have a seeding rate and nitrogen interaction study imposed. This consists of four seeding rates split with
five nitrogen rates. Yields and interactions will be reported. Each area also has a cultivar comparison of the common
cultivar grown to three new synthetic cultivars from the Georgia and Oklahoma breeding programs. Yields will be reported
for these for both seasons.
Research on weed control in first and second year switchgrass is also underway. Grass species were especially detrimental on some areas in the establishment year. Certain weed control strategies look very promising for controlling both
grass and broadleaf species with no harm to the switchgrass.
163
poster 3B-21
New Insights on the Value of Mechanical Preprocessing
Christopher T. Wright*, Peter A. Pryfogle, J.Richard Hess, Nathan A. Stevens, Kevin L. Kenney, Corey W. Radtke, and
Reed L. Hoskinson
Idaho National Laboratory, Idaho Falls, Idaho 83415-2210
The purpose of mechanical preprocessing is to chop, grind, or otherwise format biomass into a suitable feedstock for use
in the biorefinery conversion process. Variables such as equipment cost and efficiency, and feedstock moisture content,
particle size, bulk density, compressibility, and flowability affect the location and implementation of this operation in the
assembly system. Initial designs locate this operation at the front-end of the biorefinery. However, data are presented
that show mechanical preprocessing at the field-side or in a fixed preprocessing facility (depot) can provide significant
cost benefits by producing a higher value feedstock with improved handling, transporting, and merchandising potential.
By exploiting the composite biological structure of the material through mechanical preprocessing, both a bulk flowable
and fractionated material can be produced that could potentially improve costs and efficiencies of the assembly system
and biorefinery unit operations. We used a hammermill grinder fitted with various screen sizes to evaluate the relationship between moisture content, particle size, bulk density, compressibility, and flowability of different feedstock fractions
with the respective cost and efficiency benefits to the assembly system and biorefinery.
164
poster 4-08
Effect of Initial Cell Concentration on Glucose and Xylose Fermentation Using Pichia stipitis
Frank K. Agbogbo*, Mads Torry-Smith, Guillermo Coward-Kelly, Kevin Wenger
77 Perry Chapel Church Road, P. O. Box 576, Franklinton, NC 27525-0576
E-mail: [email protected]; Phone: 919-494-3159
Pichia stipitis has an advantage over Saccharomyces cerevisiae in its ability to ferment xylose and cellobiose. Xylose is
the major component of the hemicellulose fraction (20-35%) of biomass. Efficient utilization of the hemicellulose component offers an opportunity to reduce the cost of ethanol production from biomass by about 25%. However P. stipitis
has a lower ethanol production rate, ethanol tolerance, inhibitor tolerance, and cell growth compared to Saccharomyces
cerevisiae. The purpose of this study is to determine the effect of initial cell concentration on the ethanol production rate,
ethanol concentration, and yield. This study was performed using synthetic media of glucose and xylose.
Different initial cell concentrations were used to inoculate synthetic media of glucose and xylose. Fermentations were performed with P. stipitis in 125 ml erlenmeyer flasks in an air-shaker incubator at 30ºC and 100 rev/min. The media consists
of xylose or glucose (containing yeast nitrogen base, peptone and urea). Samples were taken at regular time intervals
and the concentration of glucose, xylose, and ethanol were determined. The average ethanol production rate, substrate
consumption rate, and yield were determined.
poster 4-09
Xylose-fermenting Saccharomyces cerevisiae expressing a fungal xylose isomerase: further steps towards
industrial implementation
Eleonora Bellissimi, Johannes P. van Dijken and Jack T. Pronk*
Department of Biotechnology, Delft University of Technology & Kluyver Centre for Genomics of Industrial Fermentation,
Julianalaan 67, 2628 BC Delft, The Netherlands
Aaron A. Winkler, Johannes P. van Dijken, Bird Engineering B.V., Vlaardingweg 62, 3044 CK Rotterdam, The Netherlands
Wim T.A.M. de Laat, Royal Nedalco, PO Box 6, 4600 AA Bergen op Zoom, The Netherlands
After extensive genetic modification and strain selection via evolutionary engineering, we have obtained Saccharomyces
cerevisiae strains that rapidly and efficiently ferment glucose and xylose in anaerobic cultures. High-level functional expression of a fungal xylose isomerase played a key role in these successful experiments. Until recently, our efforts were
focused on ‘proof of principle’ experiments in synthetic laboratory media. A clear and urgent objective is to evaluate and
improve performance in the presence of inhibitors present in industrial media and, ultimately, in real-life plant biomass
hydrolysates. In this presentation, we will provide an update on the current status of our activities in this area. Special attention will be devoted to the improvement of acetic acid tolerance and to the fermentation of wheat-straw hydrolysates.
poster 4-10
WIthdrawn
165
poster 4-11
Construction and Evaluation of Clostridium thermocellum 27405 Partial-Genome Oligonucleotide Microarray
1
1
Steven D. Brown , Shubha P. Kale2, Zhili He , Catherine K. McKeown2, Jonathan R.Mielenz2
*
1
Microbial Ecology and Functional Genomics Group
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6038
2
Department of Biology, Xavier University of Louisiana, New Orleans, LA 70125
Bioconversion Science and Technology
Life Sciences Division, Oak Ridge National Laboratory, Post Office Box 2008, Building 4505, Room 27, MS-6226, Oak
Ridge, Tennessee 37831-6226. Telephone: 865-576-8522; Fax: 865-574-6442; E: [email protected]
3
Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into
ethanol. The Joint Genome Institute has determined the sequence for the 3.7 Mb genome of strain ATCC 27405, which
is predicted to encode 3,163 candidate protein-encoding genes. The application of DNA microarray analysis to study
gene expression and regulatory networks in C. thermocellum offers the potential to gain important insights into the basic
metabolism of this organism and to improve ethanol production. We have designed unique 70-mer oligonucleotide probes
that represent approximately 94% of the predicted coding sequences in the genome and in this study present the construction and evaluation of a partial-genome microarray containing 70 C. thermocellum specific probes. We detected a
signal to noise ratio of 3 with as little as 1 ng of genomic DNA and only low signals from negative control probes, indicating the array was sensitive and specific. In order to further test the specificity of the array we amplified and hybridized
PCR products that represented 10 C. thermocellum genes and found gene specific hybridization in each case. These
results suggest our oligonucleotide probe set can be used for sensitive and specific C. thermocellum microarray studies.
poster 4-12
Microbial Modification of Corn Stover to Increase Ethanol yield
Naresh K Budhavaram, Aarti Gidh, Clint W Williford*, Alfred Mikell
The University of Mississippi, MS – 38677
With higher petroleum prices, ethanol production from biomass is gaining importance. Our aim is to improve the ethanol
yield from corn stover using biological agents. We are isolating microbial agents from wood-eating insects, along with
other sources. Screening studies then assess activity toward pretreated and unpretreated corn stover. Beetles were
dissected and the microorganisms from the fore gut, mid gut and hind gut were isolated. Fourteen bacterial and fungal
isolates were observed, and eleven of them were grown successfully. Weight loss experiments were conducted using the
isolates. A 5ml slurry of pretreated corn stover was used as substrate, and 500μL of the isolate was added to it. Incubations were carried out for 120hrs. Experiments with isolates from the mid gut showed weight loss. The experiments were
repeated with untreated corn stover as substrate. No significant weight loss was seen.
Ongoing SSF experiments are being carried out using an NREL protocol for this purpose. The isolates that have shown
weight loss will be used in the SSF experiments to see their effect on the ethanol yield. The glucose, acetic acid, cellobiose and ethanol are being monitored using HPLC with Refractive Index detector. In addition, pH and temperature are
varied and the effect of isolates on the corn stover observed. Biolog experiments and RNA and DNA analyses have been
conducted to give us more detailed insight into the bacterial characteristics.
166
poster 4-13
Bioethanol Production from Biomass Hydrolyzates Mediated by Enzymatic Isomerization of Xylose to Xylulose
Isabella De Bari, Vincenzo Di Matteo
ENEA CR TRISAIA, SS 106 Jonica, km 419+500, 75026 Rotondella (MT), Italy
Phone: (+39) 835 974313; Fax: (+39) 835 974210, e-mail: isabella.debari @trisaia.enea.it.
Lignocellulosics conversion into bioethanol is destined to come into the European market in the next years. Some big
firms producing cellulolytic enzymes already announced the launch of new mixtures yielding an efficient hydrolysis of
cellulose at costs more competitive than they were in the past. On the other side, the microbial fermentation of all the
biomass sugars does not seem to have reached the same commercial maturity. In fact, the major progresses in this field
concern the development of new engineered microrganisms able to coferment C-6 and C-5 sugars. However, despite the
noticeable scientific achievements, most of these microrganisms are still not included in the strains collections accessible
through some important European organizations such as DSMZ. This underlines the necessity that the research activities
in this field further proceed toward a better definition of the fermentation strategies.
Besides the use of superior microrganisms (both yeasts and bacteria) able to coferment different carbon sources even
in harsh conditions (i.e. in presence of inhibiting molecules from thermal degradation and ethanol), another viable option
is converting the C-5 sugars, mainly xylose, into the corresponding ketose ready to be metabolized by Saccharomyces
Cerevisiae. This conversion can be made outside the cellular machine by a glucose isomerase enzyme (EC5.3.1.1.5; EC
5.3.1.1.8) able to convert xylose into xylulose.
In the present paper, two commercial isomerase enzymes, the one soluble (Gensweet) and the other immobilized (Sweetzyme), were tested. Both enzymes were isolated from Streptomyces species. High concentrations (10-70 g/L) of lyophilized
S. cerevisiae were used to ferment both isomerized hemicellulose rich-streams and mixed sugar syrups containing glucose
and xylose. The effect of increasing both the enzyme and the yeast dosages was assessed. Since the isomerase enzymes
working curves versus pH and temperature have maxima at 7-7.5 and 60°C respectively, some modulations of pH and
temperature trough the process were investigated. Concerning the process strategies, the simultaneous isomerization and
fermentation (SIF) and the semi-SIF processes were compared in terms of final bio-ethanol yield and process productivity.
In the semi-SIF approach the yeast was inoculated in the shaken flasks after a pre-isomerization time at optimized pH and
temperature. The process was called semi-SIF because the yeast was added when the isomerization had still not reached
the equilibrium concentration. This process configuration had the advantage of right-shifting the xylose isomerization equilibrium. The obtained results were discussed in terms of the isomerase activity and the yeast metabolism.
167
poster 4-14
Investigation of Latex Micro-photobioreactor for Hydrogen Production using
Non-Growing Rhodopseudomonas palustris
Jimmy L. Gosse, Michael C. Flickinger*
BioTechnology Institute, Dept. of Biochemistry, Molecular Biology, and Biophysics
University of Minnesota, St. Paul, MN 55108
Rhodopseudomonas palustris CGA009 is an excellent organism for optimization of nitrogenase meditated photosynthetic
hydrogen production from organic substrates. Rps. palustris contains three functional nitrogenase enzymes, a nonfunctional uptake hydrogenase, and has the capacity to utilize waste carbohydrates, organic acids, and aromatic compounds,
which could be derived from lignin. Many of the obstacles to large-scale biohydrogen production may be overcome by
using this organism under non-growth conditions in thin, nano-porous, multilayer translucent latex coating photobioreactors. The multilayer coating approach along with antenna pigment and light harvesting complex mutants in different layers
could increase photosynthetic efficiency by overcoming light saturation. Hydrogen production rates have been measured
for mono-layers of latex entrapped Rps. palustris CGA009 as a function of light intensity, substrate composition and thickness. In addition we have demonstrated the thin, translucent, nanoporous coatings remain active after hydrated storage
for over three months in the dark or frozen at -80oC.
poster 4-15
Tanase Production by Solid State Fermentation of Cashew Apple Bagasse
Tigressa H. S. Rodrigues; Maria Alcilene A. Dantas; Luciana R. B. Gonçalves*.
Universidade Federal do Ceará – Depto. de Engenharia Química – Campus do Pici, Bloco 709 – 60.455-760,
Fortaleza – CE, Brazil
Gustavo A. S. Pinto
Embrapa Agroindústria Tropical, Rua Dra Sara Mesquita, 2270, Planalto do Pici, CEP 60511-110, Fortaleza – CE, Brazil
Cashew apple (Anacardia naturalis) is a pseudofruit, representing 90% of the fruit. In the north coast of Brazil, especially
in the state of Ceará, the cashew agroindustry has an outstanding role in the local economy. In spite of the internal and
external market consumption of cashew nut, only 12% of the total pseudofruit was processed and the majority of its production spoils in the soil. Those facts, added to rich composition, turns it inexpensive and safe for culture medium. Therefore, the aim of this study is to evaluate the potentialities of tannase production using cashew apple bagasse as substrate
for Aspergillus oryzae during solid state fermentation. Tannase catalyses the hydrolysis of the ester and depside bonds in
hydrolysable tannins such as tannic acid to release glucose and gallic acid. The production of this enzyme has been carried out by different approaches, but several studies have indicated solid state fermentation to be advantageous in terms
of tannase productivity and tannin conversion, among others. In this work, to improve enzyme production, some culture
conditions were investigated, such as: water activity, tannic acid and ammonium sulfate concentration. Preliminary results
show this process is competitive when compared with other proposed for enzyme production.
168
poster 4-16
A novel microbial platform for the anaerobic synthesis of oxidized chemicals:
Anaerobic production of acetic acid in E. coli
K. Smith, Y. Dharmadi, and Ramon Gonzalez*
Assistant Professor in the Department of Chemical and Biomolecular Engineering, Rice University
Current aerobic methods used to produce oxidized fuels and chemicals do not compete with petrochemical routes due to
their high capital and operating costs. Fermentative production of acetate, for example, result in a loss of ~50% of sugar as
CO2 and can not use sugars obtained from lignocellulosic biomass, which constitute a low-cost (~0.05 $/lb), sustainable
alternative for producing oxidized materials. We are developing a novel microbial platform for the anaerobic production of
oxidized chemicals and fuels from plant biomass sugars. The proposed E. coli-based platform uses nitrate respiration to
convert plant biomass sugars into acetate. This process has lower capital and operating costs than existing aerobic processes. Furthermore, the reduction of nitrite obtained from nitrate respiration will generate ammonium thus reducing the use
of ammonium salts in the fermentation and purification processes. The work to be presented at the meeting includes the
engineering of E. coli for the efficient use of nitrate as electron acceptor and nitrogen source under anaerobic conditions and
the construction of strains for the anaerobic production of acetate acid as the main fermentation product.
*Department of Chemical and Biomolecular Engineering, MS-362, P.O. Box 1892, Houston, Texas 77251-1892
Phone: (713) 348 4893, Fax: (713) 348 5478; email: [email protected]
poster 4-17
The fermentation inhibitor furfural causes cellular damage to Saccharomyces cerevisiae.
Steven W. Gorsich and Patricia J. Slininger
National Center for Agricultural Utilization Research-ARS-USDA, Peoria, IL 61604.
J. Michael McCaffery
Integrated Imaging Center, Biology Department, Johns Hopkins University, Baltimore, MD 21218
The use of Bio-fuel ethanol is increasingly important for multiple environmental and economical reasons. To reach the
United States ethanol goals it will be essential to take advantage of various biomass substrates for ethanol production.
The release of fermentable sugars from lignocellulose biomass for ethanol fermentation is often facilitated by a weak acid
hydrolysis step. As a result, inhibitors such as furfural and 5-hydroxymethylfurfural are formed as degradation products
of xylose and glucose, respectively. These, and other, inhibitors present an environment which elicits the expression
of stress-related genes in Saccharomyces cerevisiae. Recently, 62 S. cerevisiae genes were identified as important in
furfural stress tolerance due to the inability of mutants lacking these genes to grow in furfural’s presence. These include
some pentose phosphate pathway genes (ZWF1, GND1, RPE1, and TKL1). When some of these genes are overexpressed we observe an increase in furfural tolerance. Using various fluorescent indicators and transmission electron
microscopy techniques we determined that furfural causes an increase in reactive oxygen species accumulation, cellular membrane damage (vacuole and mitochondrial membranes), chromatin damage, and actin damage in wild-type S.
cerevisiae. Whether or not overexpressing any of the previously identified genes will reduce oxidative damage is being
investigated.
169
poster 4-18
Developing Desulfurization Biocatalysts for Removing Organic Sulfur from Fossil Fuels at Higher Temperatures
Yinglin Bai, Miroslav Sedlak and Nancy W. Y. Ho*
Laboratory of Renewable Resources Engineering (LORRE), Purdue University, West Lafayette, IN 47907
Coal and petroleum will remain as major sources of energy for years to come. However, there are serious environmental concerns related to the use of these resources. One such concern is that coal and petroleum contain substantial
amounts of organic sulfur, which is the major cause of acid rain. Thus, the development of innovative, low-cost technologies for the removal of organic sulfur from these resources is greatly desired. Microbial desulfurization has long been
recognized to be a low-cost technology for the removal of organic sulfur from coal and petroleum. With unprecedented
advances in genetic manipulation of microorganism, the development of ideal microbial systems for desulfurization as
well as for the removal of other toxic contaminates from these resources is within reach. On key factor to successfully
developing a safe reliable microbial system for large-scale industrial operation is to make the microbial system function
effectively under conditions that contaminating microorganisms cannot survive, particularly those that are lethal to human
health or well being of other living species. In this paper, we demonstrate that the major genes responsible for removing
organic sulfur in petroleum or coal can be engineered for expression at higher temperatures.
poster 4-19
Improvement of ε-caprolactone production in recombinant Escherichia coli by optimization of substrate feeding
and cofactor regeneration
Won-Heong Lee, Kun-Jae Lee
Department of Agricultural Biotechnology, Seoul National University Seoul 151-742, Korea
Myoung-Dong Kim
Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, Korea
Jin-Ho Seo*
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials,
Seoul National University, Seoul, 151-742, Korea
A recombinant Escherichia coli strain harboring the NADPH-dependent cyclohexanone monooxygenase (CHMO) gene
was developed to produce ε-caprolactone from cyclohexanone through Baeyer-Villiger (BV) oxidation. A series of fedbatch fermentation were carried out in order to optimize a cyclohexanone feeding strategy. The cyclohexanone level
maintained less than 5 g/L showed the best results of 9.37 g/L ε-caprolactone concentration with 0.94 g/L·hr productivity
and 93% yield based on cyclohexanone. Coexpression of glucose-6-phosphate dehydrogenase (zwf1) gene enhanced εcaprolactone concentration to 14.22 g/L with 1.39 g/L-hr productivity. Such an improvement in ε-caprolactone production
seemed to be due to the optimized substrate level for microbial BV-oxidation and enhancement of NADPH regeneration.
170
poster 4-20
A New Approach of Pellet Formation of a Filamentous Fungus – Rhizopus oryzae
Wei Liao*, Yan Liu, Shulin Chen
Department of Biological Systems Engineering and Center for Multiphase Environmental Research, Washington State
University, Pullman, WA 99163
The effects of inoculum and medium compositions such as potato dextrose broth as carbon source, soybean peptone,
calcium carbonate, and metal ions on pellet formation of Rhizopus oryzae ATCC 20344 have been studied. It has been
found that metal ions had significantly negative effects on pellet formation while soybean peptone had positive effects. In
addition potato dextrose broth and calcium carbonate were beneficial to Rhizopus oryzae for growing small smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5´ 109 spores/L had no significant influence on pellet formation although it had large impacts on pellet growth. Thus, a new approach to form pellets
has been developed using only potato dextrose broth, soybean peptone, and calcium carbonate. And the pellet size can
be controlled by adjusting inoculum size and the concentrations of potato dextrose broth, soybean peptone, and calcium
carbonate in the medium.
poster 4-21
Study of Pellet Formation of Filamentous Fungi Rhizopus oryzae Using a Multiple Logistic Regression Model
Yan Liu,Wei Liao, Shulin Chen*
Department of Biological Systems Engineering and Center for Multiphase Environmental Research, and, Washington
State University, Pullman, WA 99163
Fungal pellet formation is an important topic of fermentation research. It has been reported that many factors such as
agitation, medium nutrients, pH, polymer additives, and inoculum size influence the formation of fungal pellets. However,
a comprehensive investigation on the effects of all of these factors on fungal pellet formation has not been reported. This
paper used a completely randomized design (CRD) on a filamentous fungus, Rhizopus oryzae NRRL 395, in order to
discover the effects of the above factors on fungal pellet formation. In addition, other factors, such as addition of biodegradable polymers and spore storage time that have never been reportedly studied were examined and their effects on
pellet formation were investigated. A multiple logistic regression model was established to predict the probability of pellet
formation using the above factors and their interactions as predictor variables. Model building and diagnostics was obtained using the Statistical Analysis System (SAS 8.0) program. The model developed in this study can be used to predict
the pellet formation of other R. oryzae strains as well.
171
poster 4-22
Comparison of multiple gene assembly methods for metabolic engineering
1
Chenfeng Lu 1, Karen Mansoorabadi 2 and Thomas W. Jeffries 1,2,3*
Department of Food Science; 2 Department of Bacteriology, University of Wisconsin, Madison, WI 53706;
and 3 USDA Forest Service, Forest Products Laboratory, Madison, WI 53726
Optimization of metabolic pathways requires the introduction and altered expression of multiple genes to enhance product
formation. This process can be facilitated by the simultaneous expression of several genes each with different promoters. High-throughput screening or strain competition can then identify which combination of genes and promoters is most
effective. A universal multiple gene assembling method resulting in efficient multi-gene plasmid construction, is important for biological research and industrial fermentation. Three different methods to achieve this goal were developed and
tested, including Uracil-DNA Glycosylase (UDG), joint PCR and SfiI method. Among these, the SfiI method proved to be
the most reliable and the most efficient method.
The SfiI method consists of three steps. The first was to construct a SfiI linker, the multiple cloning site (MCS) of which
was flanked by two three-base linkers matching the neighboring SfiI linkers upon SfiI digestion; in the second step the
linkers were attached to desired genes by cloning it into SfiI linker vectors; and in the final step, genes flanked with two
three-base linkers were released by SfiI digestion. These genes were then joined in a simple one-step ligation. In this
study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of TDH3p were successfully ligated together using the above method.
poster 4-23
Production of Succinic Acid by Metabolically Engineered Escherichia coli
Jessica L. Buday, Shiying Lu, Mark A. Eiteman*, James R. Kastner, Elliot Altman
Center for Molecular BioEngineering, Driftmier Engineering, University of Georgia, Athens, GA 30602 USA
(706) 542-0833 tel; (706) 542-8806 fax; *contact: [email protected]
Succinic acid (succinate) and its derivatives have a wide range of applications in the food and chemical industries.
Although several microorganisms can produce succinate by fermentation of glucose and other renewable resources,
Escherichia coli has many advantages including rapid growth, simple nutritional requirements and the ease of genetic
manipulation. One unique feature of succinate production by this organism is that the process consumes as much as 1
mole of carbon dioxide per mole of succinic acid generated, and thus it is a means to sequester CO2 while generating a
chemical product.
We report our on-going efforts on succinate production in a “two-phase” fermentation using chemically defined media.
The two-phase process involves an aerobic growth phase followed by an anaerobic production phase. We present
results comparing several strains for succinate production. These strains have perturbations in glucose uptake and in
central metabolism. The base strain, AFP111, contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase
(ldhA) and the phosphotransferase system (ptsG). Additional strains studied include AFP111/pTrc99A-pyc (containing
pyruvate carboxylase), AFP111 poxB (pyruvate oxidase mutation), AFP111 adhE (alcohol dehydrogenase mutation),
AFP111/pTrc99A-glk (overexpressed glucokinase). We also present results on the effect of controlling aerobic phase
growth rate, its relationship with overflow metabolism, and its effect on succinate production.
172
poster 4-24
Loop configuration in the chromosome region containing [Fe]-hydrogenase gene of Chlamydomonas reinhardtii
S. A. Markov*1, E.R. Eivazova2 and B. Durr
Department of Biology, DePauw University, Greencastle, IN, USA
2
Molecular Physiology & Biophysics,Vanderbilt University, Nashville, TN, USA
Phone: 765-658-5362, Fax: 765-658-4766, E-mail: [email protected]
1
Green algae utilize the enzyme hydrogenase, which catalyzes hydrogen production, but only under anaerobic conditions, because the enzyme is very sensitive to oxygen. In addition, the hydrogenase gene is not expressed in the presence of oxygen.
Molecular mechanisms that regulate the hydrogenase gene expression under anaerobic conditions are poorly understood.
Experimental evidence was provided recently that expression of a gene or gene family can be regulated through looping
and bending of chromosome. Our study tested the hypothesis that the hydrogenase gene region adopts a specific conformation required for gene expression in green alga Chlamydomonas reinhardtii. Conformation in the gene region was
assessed by a novel technique, chromosome conformation capture (3C), which is designed to estimate the interactions
between genomic regions in vivo and model a 3-D chromosome organization in a gene (Dekker et al., 2002).
We analyzed long range interactions in the 29 kilobase (kb) region containing [Fe]-hydrogenase gene (hydA1) of C. reinhardtii. Our results showed presence of a loop organization in the [Fe]-hydrogenase gene region, which changed upon
transition of cells from aerobic to anaerobic conditions. In such loop conformation, several regions in the chromosome were
in close spatial proximity to each other, thus creating possibility for hydrogenase gene regulation. Thus, our results described
a hypothetical spatial model for the hydrogenase gene region organization under different physiological conditions.
poster 4-25
Withdrawn
173
poster 4-26
5-hydroxymethyl furfural reduction in lignocellulose hydrolysates with Saccharomyces cerevisiae strains
and its connection to tolerance
Tobias Modig*, Anneli Petersson, Gunnar Lidén
Dept. of Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
João R.M. Almeida, Kaisa Karhumaa, Bärbel Hahn-Hägerdal, Marie F. Gorwa-Grauslund
Dept. of Applied Microbiology, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
In lignocellulose hydrolysates many inhibitory compounds are found that originate from the process of releasing the fermentable sugars in the lignocellulose material. Therefore, efficient conversion of lignocellulose hydrolysates requires high inhibitor tolerance of the yeast strain applied. The furan 5-hydroxymethyl furfural (HMF) is one compound that has been shown
to significantly decrease fermentability of lignocellulose hydrolysates. One - among many - desired features of a production
strain is therefore a high HMF conversion capacity, allowing effective in situ detoxification of HMF in the hydrolysate.
In the present study, strains of Saccharomyces cerevisiae were cultivated in a medium containing dilute-acid spruce
hydrolysate and investigated for their tolerance to the hydrolysate. Fermentations were performed in controlled reactors in
either batch or fed-batch mode. Ethanol productivities, in vitro and in vivo HMF reduction capacity, as well as the overall
metabolic response in terms of carbon dioxide evolution rate were measured. Results showed that strains possessing
high HMF reduction capacity, intrinsic or obtained by genetic modifications, performed better in fermentations of diluteacid lignocellulose hydrolysates.
poster 4-27
Modified expression contexts of phosphoglucose isomerase (PGI1) and glucose 6-phosphate dehydrogenase
(ZWF1) genes to increase xylitol production in recombinant Saccharomyces cerevisiae
Yong-Joo Oh, Do-Hyun Kwon, Tae-Hee Lee, Won-Ki Min,
Department of Agricultural Biotechnology, Seoul National University Seoul 151-921, Korea
Myoung-Dong Kim
Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea
Jun-Bock Park
Interdisciplinary Program for Biochemical Engineering and Biotechnology,
Seoul National Unversity, Seoul, 151-921, Korea
Jin-Ho Seo*
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials,
Seoul National University, Seoul, 151-921, Korea
Two independent approaches to increase the metabolic flux toward the pentose phosphate pathway (PPP) for enhanced
NADPH regeneration were made in attempt to increase xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis. Enhanced regeneration of NADPH was achieved by manipulating expression contexts of the ZWF1 and PGI1 genes encoding glucose 6-phosphate dehydrogenase and phosphoglucose isomerase,
respectively. An 8 fold increase in the glucose 6-phosphate dehydrogenase level improved xylitol productivity by 1.2~1.3 fold.
While down regulation of the phosphoglucose isomerase activity alone accomplished by replacing the inherent promoter with
the ADH1 promoter resulted in no appreciable influence on xylitol productivity, simultaneous manipulation of both enzyme
levels synergistically increased xylitol productivity by a 1.4~1.6 fold in the glucose-limited fed-batch fermentation.
174
poster 4-28
Succinic Acid Production by genetically modified Corynebacterium glutamicum under Oxygen-Deprivation
Shohei Okino, Alain A. Vertès, Masayuki Inui and Hideaki Yukawa
Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho, Soraku-gun,
Kyoto 619-0292 Japan
Phone: 81-774-75-2308; Fax 81-774-75-2321; E-mail: [email protected]
Succinic acid has a long history of use as a raw material in the food, chemical and pharmaceutical industries. However,
the market for succinic acid polymers of as alternative raw materials for biodegradable plastics has been dramatically
growing in the past few years.
The aerobe C. glutamicum has been used for the industrial production of various amino acids and nucleic acids.
Previously, we found that C. glutamicum produced organic acids, such as lactic acid and succinic acid, from glucose under oxygen deprivation even when its cell growth is arrested. These features allow for, under oxygen deprivation, the use
of a reactor filled to high density with cells derived from an aerobic culture, leading to a bioprocess with high volumetric
productivity. We developed a C. glutamicum mutant strain that lacks the lactate dehydrogenase gene and overexpresses
pyruvate carboxylase gene. Using this strain, we achieved production of succinic acid at high yield and high volumetric
productivity.
poster 4-29
Microbial community of biofilm from a thermophilic trickling biofilter used for
continuous biohydrogen production
Yeonghee Ahn 1, Eun-Jung Kim 1, You-Kwan Oh 2, Sunghoon Park 2,3,*, Gordon Webster 4, and Andrew J. Weightman 4
1
Department of Chemical and Biomolecular Engineering, KAIST, Korea
2
Department of Chemical and Biochemical Engineering, Pusan National University, Korea
3
Institute for Environmental Technology and Industry, Pusan National University, Korea
4
Cardiff School of Biosciences, University of Cardiff, Main Building, United Kingdom
Trickling biofilter reactors (TBR) employ biofilm formed on supporting matrices packed in the reactors. Biofilm in TBR
can degrade organic compounds in wastewaters that are trickled over the biofilm. Thermophilic (45-65oC) TBR can take
advantage of characteristics of biofilm and thermophilic bacteria to achieve high and stable production of hydrogen. Since
H2 is less soluble at high temperature, thermophilic TBR can reduce partial pressure of H2 and alleviate inhibition of H2
production. This study investigated microbial community in biofilms obtained from a thermophilic TBR that was longterm operated to produce H2. Biomass in the TBR gradually decreased as bed height increased from the bottom of the
reactor bed. Despite the biomass difference, bottom and middle heights of the TBR bed revealed similar microbial profiles
as determined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified partial 16S rDNA. Most bands of
the DGGE were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes. Nucleotide sequences of the
distinctively major bands were most closely related to Thermoanaerobacterium thermosaccharolyticum.
Corresponding Author: S. Park, Professor. Department of Chemical and Biochemical Engineering, Pusan National University, Pusan 609-735, Korea.
Tel, 82-510-2395/ Fax, 82-510-512-8563/ Email, [email protected]
*
175
poster 4-30
Ethanol and Co-Products from Sugar Beets
Joy B. Doran Peterson, Krishna Patel and Theresa Rogers
University of Georgia, Athens, GA 30605
Arland Hotchkiss, Marshall Fishman, and Kevin Hicks
USDA-ARS, ERRC, 600 E. Mermaid Lane, Wyndmoor, PA 19038-8598
Wilbur Widmer
Research Chemist, Citrus and Subtropical Products Lab, USDA / ARS, 600 Ave. S NW, Winter Haven, FL 33881
Approximately 400 million tons of sugar beets are produced annually, with U.S. production at 20-30 million wet tons. After
sucrose extraction, over 1.6 x 106 tons of sugar beet pulp remains. Production of livestock feed from beet pulp can be an
economically marginal part of processing due to low feed value and high drying costs. Energy required to pelletize pulp
can reach upwards of 30-40% of the overall energy cost of sugar production. Ethanol from whole sugar beets may be
self-limiting due to competing value as food (similar to corn-based fermentations), however, processing plants could retain
flexibility of fermenting sucrose and molasses streams to ethanol as well.
The project goal was to develop methods for separation of cellulose, hemicellulose, and pectin from sugar beets and
to determine ethanol yields for conversion of each fraction. Fermentation of all components in the beet pulp from one
midsized US processor could generate approximately six million gallons of fuel ethanol from 60,000 tons of pulp with our
present process using engineered bacteria originally developed in the laboratory of Dr. L.O. Ingram (University of Florida).
Co-product contribution will enhance the economic feasibility of producing ethanol from sugar beets and are the subject
of future studies.
poster 4-31
Optimization of gallic acid production by Aspergillus niger CNPAT 001 in submerged fermentation
Adriana Crispim de Freitas; Gustavo Adolfo Saavedra Pinto*
Brazilian Agricultural Research Corporation/EMBRAPA, National Research Center of Tropical Agroindustry - Fortaleza/
CE, Brazil - Email: [email protected]
Gallic acid (3,4,5-trihydroxybenzoic acid) is applied in various fields, mainly for production of trimethoprim and propylgallate. For microbial production of gallic acid a medium containing tannic acid, which is considered a thermolabel molecule,
is utilized. The aim of this work was the optimization gallic acid production in submerged fermentation by strain Aspergillus niger CNPAT 001. Initially, the medium containing was (in g.L-1): tannic acid, 20.0; (NH4)2SO4, 3.0; KH2PO4, 1.0; KCl,
0.5 and MgSO4, 0.5. The fermentation. All experiments were incubated at 30oC in orbital shaker at 150rpm for 96 hours.
For optimization were evaluated: two sterilization procedures: filtration (Millipore 0,25µm membrane) and autoclavation
(121oC/15min and 121oC/30min); amount of inoculum; effect of initial pH and of initial concentrations of tannic acid and
(NH4)2SO4. In contrast with expected, the sterilization by autoclavation were better for gallic acid accumulation in medium
than membrane procedure. In medium with 55g/L of tannic acid and 1.0g/L of (NH4)2SO4 at initial pH 5,0 and innoculation of1x106 spores/mL were most adequate conditions for production. In this condition after 72 hours of fermentation was
obtained 32.6g/L of gallic acid, which represented a yield of 60% and represents an increase of 280%, if compared with
the initial conditions.
176
poster 4-32
PDU-scale fed-batch cultivation of yeast on spruce hydrolysates
Andreas Rudolf*, Mats Galbe and Gunnar Lidén
Dep. of Chemical Engineering, Lund University, Box 124, SE-221 00 Lund
Previous work has shown that the inhibitor tolerance of the yeast can be significantly improved if the yeast is produced on
hydrolyzate. This is particularly important when fermenting undetoxified hydrolysate. In the present work Saccharomyces
cerevisiae TMB 3000 (Applied Microbiology, Lund University, Sweden) was cultivated aerobically on hydrolysate in a 20
L-reactor. A fed-batch process with a closed-loop control of the feed rate was used. The control strategy applied was to
maintain a low and constant ethanol concentration (0.5 – 1 g/l) in the broth during cultivation, and the ethanol concentration was obtained from on-line measurements of the ethanol mole fraction in the exhaust gas. Two different hydrolysates
were evaluated; a dilute acid hydrolysate from spruce and liquid obtained from steam-pretreatment of spruce. The two
hydrolysates used were rich in glucose and mannose, however the pentose concentration was rather low. Productivities,
biomass yields as well as by-product formation in the aerobic fed-batch cultivations were evaluated. The produced yeast
was, furthermore, evaluated in shake-flask fermentations in order to determine if it indeed had better fermentation performance in hydrolysate than yeast cultivated on pure glucose.
poster 4-33
Co-fermentation of glucose, xylose and cellobiose by genetically modified Saccharomyces yeast
Miroslav Sedlak and Nancy W. Y. Ho*
Laboratory of Renewable Resources Engineering (LORRE), Purdue University, West Lafayette, IN 47907
Cellulosic biomass is considered to be an ideal inexpensive feedstock for large-scale production of fuel ethanol and other
chemicals by microbial processes. However, cellulosic biomass contains large amounts of xylose in addition to glucose.
The naturally occurring Saccharomyces yeasts used for large-scale ethanol production from starch (glucose) cannot
metabolize xylose. We have succeeded in genetically engineering the Saccharomyces yeasts to effectively co-metabolize
glucose and xylose both aerobically and anaerobically. This was accomplished by cloning and overexpressing three major
xylose-metabolizing genes - xylose reductase, xylitol dehydrogenase, and xylulokinase genes (KDR) and by modifying
the control mechanisms present in microbial cells. The resulting genetically engineered yeast can efficiently use xylose
for growth as well as effectively co-ferment both glucose and xylose simultaneously to ethanol. In this paper we report
that we have further modified our glucose/xylose metabolizing Saccharomyces yeast by overexpressing the β-glucosidase gene from Saccharomycopsis fibuligera. The resulting yeast is able to effectively grow on cellobiose as well as efficiently ferment cellobiose, a mixture of xylose/cellobiose or glucose/cellobiose to ethanol.
Furthermore the new recombinant yeast is able to ferment cellulosic biomass hydrolysate containing a mixture of glucose, xylose and cellobiose to ethanol.
177
poster 4-34
Recombinant Saccharomyces cerevisiae as Source of Glucose 6-Phosphate Dehydrogenase
Luiz Carlos Martins das Neves, Adalberto Pessoa Jr., Michele Vitolo*
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São
Paulo. Av. Prof. Lineu Prestes, 580, B.16, 05508-900, São Paulo, SP, Brazil. Phone: 0055-11-30912382 email:
[email protected].
The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC
181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in
the formation of glucose 6-phosphate dehydrogenase (G6PD) (EC.1.1.1.49). Two liters of culture medium (10 g/L glucose,
3.7 g/L YNB, 0.02 g/L L-tryptophan, 0.02 g/L L-histidine, 0.02 g/L uracil and 0.02 g/L adenine) were inoculated with 1.0
g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30oC and agitation of 400 rpm. After
glucose concentration in the medium was lower than 1 g/L, the cell culture was fed with a solution of glucose (10 g/L) or
micronutrients (L-tryptophan, L-histidine, uracil and adenine each one at a concentration of 0.02 g/L) following the constant, linear or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2L up to 3L
during 5h. The highest G6PD specific activity (250 U/mg of protein; 1 U = 1 µmol of NADP/min), which is comparable with
those presented by commercial preparations (150-500 U/mg protein), occurred when the glucose solution was fed into
the fermenter through the decreasing linear mode.
poster 4-35
Fed-Batch Culture of Recombinant Saccharomyces cerevisiae for Glucose 6-Phosphate
Dehydrogenase Production
Ângelo Samir Melim Miguel, Michele Vitolo*, Adalberto Pessoa Jr
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences of University of
São Paulo, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, São Paulo, SP, Brazil. Phone: 005511-30913682 email:
[email protected].
The aim of this work was to study the glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation,
using a recombinant strain of Sacharomyces cerevisiae W303-181 overexpressing the enzyme. The cultivations were carried out in a 3L fermentor at pH 5.7, 30oC, aeration of 2.0 vvm (oxygen mass transfer coefficient of 42 h-1), agitation of 200
rpm and inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process was varied from
1.3L up to 2.0L by the addition of glucose 15.0 g/L at a filling up time of 5h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time
constant), namely: 0.2, 0.5 and 0.8 h-1. The G6PD activity unit (U) was defined as 1 µmol of NADP consumed per min.
The best enzyme production (847 U/L) was observed with exponentially increasing feeding rate corresponded to K = 0.2
h-1, and with a medium composed of glucose 15.0 g/L, YNB 1.85 g/L, adenine 67 mg/L, uracil 47 mg/L, histidine 66 mg/L
and tryptophan 80 mg/L. The preliminary results attained also showed that this process is promising for producing G6PD.
178
poster 4-36
Transposome-mediated directed evolution for improving rhamnolipids production in Pseudomonas aeruginosa.
Qinhong Wang1,2 Xiangdong Fang 1*, Xiaoling Liang 1, Patrick J. Shuler 1, Yongcun Tang 1, William A. Goddard III 1,2
1) Power, Environmental & Energy Research Center, California Institute of Technology, Covina, CA 91722
2) Chemistry and Chemical Engineering Division, California Institute of Technology, Pasadena, CA 91125
Phone: 626-858-5077 *Email: [email protected]
Rhamnolipids, the potent biosurfactant which could be produced by Pseudomonas aeruginosa, have a great wide of
potential biotechnological applications such as enhancing oil recovery and bioremediation. Rhamnolipids were composed of rhamnose sugar molecules and ß-hydroxyalkanoic acids. The carbon chain length of ß-hydroxyalkanoic acids in
rhamnolipds would significantly affect the properties of biosurfactant. Rhamnosyltransferase complex (RhlAB) was the
key enzyme which would determine substrate specificity and diversity of carbon chain length of product during biosynthesis of rhamnolipids in P. aeruginosa. Here, we applied transposome-mediated directed evolution to mutate RhlAB, and
then improve the production of rhamnolipids. First RhlAB gene was mutated by error-prone PCR; then mutated RhlAB
genes were constructed into the Tn5-derived transposon. The chimeric transposomes which constructed by supplementing transposase to transposon were randomly inserted into the chromosome of P. aeruginosa RhlA- strain in which RhlA
gene was disrupted and rhamnolipids were not produced to construct the library of mutant strains which could produce
rhamnolipids. Through several rounds of screening, the mutants which could produce the improved rhmanolipids with
lower interfacial tension (IFT) were screened. IFT of some mutants was about 50 fold lower than that of the P. aeruginosa
RhlA- strain that was inserted normal RhlAB gene by chimeric transposome. Preliminary LC-MS analysis of engineered
rhamnolipids revealed that low IFT resulted from the increase of the ratio of longer carbon chain of rhamnolipids. From
this study, we also confirmed the novel tool for directed metabolic engineering---transposome-mediated mutagenesis and
its combination with directed evolution.
poster 4-37
Effect of Furfural and HMF on the Cofermentation of Glucose and Xylose from Pretreated Lignocellulosic
Biomass by Recombinant Yeast
Ryan Warner1,2, Miroslav Sedlak1, Nancy Ho1, and Nathan S. Mosier1,2*
1 Laboratory of Renewable Resources Engineering, Purdue University
2 Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907
Pretreatment of lignocellulosic biomass, while improving enzymatic digestibility, can also produce fermentation inhibitors
such as furfural and HMF. Both furfural and HMF can decrease the fermentability and the ethanol yields from sugars
derived from lignocellulose. This paper reports a systematic study of the effect of furfural and HMF on the fermentation
of both glucose and xylose to ethanol by the recombinant yeast S. cerevisiae 424A(LNH-ST). Fermentations were run
with furfural, HMF, or both in a control solution of YEP with glucose and xylose as co-substrates. Inhibitor concentrations
were varied and range from 0 to 40 g/L. Further experiments varied inhibitor concentrations in the presence of a single
substrate, either glucose or xylose. Batch fermentations were carried out for 48 hours in 300 mL sidearm flasks at 30°C
and 200 rpm with periodic sampling for analysis by HPLC. Our results show that concentrations of either furfural or HMF
below about 5 g/L cause negligible inhibition for yeast cells in early stationary phase. We confirm that furfural is more
inhibitory than HMF. Lastly, xylose fermentation to ethanol is more sensitive to these inhibitors than glucose for fermentation to ethanol.
179
poster 4-38
Effect of loading rate on lipid production from microalgae grown on dairy manure effluent
Walter Mulbry1 and Zhiyou Wen2*
Environmental Management & Byproduct Utilization Lab, USDA/ARS, Beltsville, MD 20705
2
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Phone: 540-231 9356; Fax: 540-231 3199; Email: [email protected]
1
Microalgae are a potential source for renewable oil production. Although this research area has been the focus of numerous studies over nearly 50 years, microalgal bioenergy production has been limited largely because of the difficulty of
harvesting and drying biomass from suspended microalgal culture systems. However, as the quest for renewable energy
sources intensifies, there is renewed interest in microalgal production for bioenergy, especially in regard to oil production
from microalgal systems designed to treat agricultural wastewater or animal manure. Our research has focused on the
use of filamentous freshwater algae for manure treatment because the resulting biomass is relatively easy to harvest and
dewater. The objective of this study was to determine the effect of manure loading rates on the lipid content of freshwater
filamentous algae grown in algal turf scrubbers (ATS) with and without carbon dioxide supplementation. Algal biomass
was harvested weekly from laboratory scale ATS units using three loading rates of anaerobically digested dairy manure
effluent corresponding to daily loading rates of 0.37-1.8 g total N m-2 day-1. The harvested algal biomass was air-dried,
ground, and stored at 20 C prior to lipid analysis. The total lipid content of the dried biomass from ATS grown with CO2
supplementation was roughly 7% of dry weight and did not change with increased loading rate. In contrast, the total lipid
content of dried biomass from ATS grown without CO2 supplementation increased with loading rate from 7 to 11% of dry
weight. Further experiments will focus on manipulation of algal growth conditions and algal inocula to enrich for species
with increased lipid content.
poster 4-39
Genetic engineering and characterisation of a yeast strain fermenting both pentose sugars,
L-arabinose and D-xylose
Beate Wiedemann, Marco Keller, Eckhard Boles*
Institut fuer Molekulare Biowissenschaften, J. W. Goethe-Universitaet Frankfurt/Main, D-60439 Frankfurt, Germany
Bioethanol produced from plant biomass is a promising alternative to fossil transportation fuels. The yeast Saccharomyces cerevisiae is commonly used for ethanol production in industrial processes. However, to increase ecological and
economical revenue it is necessary to expand the substrate range and increase ethanol yields in this organism. Although
S. cerevisiae is able to produce high ethanol yields from hexose sugars it lacks the ability to ferment pentose sugars.
Therefore, the development of a yeast strain capable to ferment pentose sugars is of great industrial interest. We have
constructed a S. cerevisiae strain able to utilise both pentoses, L-arabinose and D-xylose, which are the most abundand
pentose sugars in our biosphere. To this end, we integrated a plasmid expressing the genes for a D-xylose-utilising
pathway into the genome of a yeast strain already engineered for L-arabinose fermentation. We will present physiological
analyses of a recombinant S. cerevisiae strain able to co-ferment both pentose sugars L-arabinose and D-xylose, together with glucose, into ethanol.
180
poster 4-40
Production of Lactic Acid by Metabolically Engineered Escherichia coli
Yihui Zhu, Mark A. Eiteman*, Elliot Altman
Center for Molecular BioEngineering, Driftmier Engineering, University of Georgia, Athens, GA 30602 USA
(706) 542-0833 tel; (706) 542-8806 fax; *contact: [email protected]
Lactic acid is a three-carbon compounds which each occur as biochemical intermediates in the central metabolism of
bacteria. Although several microorganisms produce this compound by fermentation of glucose and other renewable
resources, Escherichia coli has many advantages including rapid growth, simple nutritional requirements and the ease of
genetic manipulation.
This presentation reports on-going efforts at understanding central metabolism in order to design processes for the accumulation of lactic acid. Two-phase fed-batch fermentations (an aerobic growth phase followed by an anaerobic production phase) in a defined medium containing glucose were conducted by using metabolically engineered E. coli having the
genotype pflB aceEF poxB pps. The effects of several parameters such as pH and temperature were studied, with the
ultimate process generating about 100 g/L lactate in 36 hours. During the production phase of this two-phase fed-batch
fermentation, the volumetric lactate productivity was between 4-8 g/Lh. We furthermore report the effects of mutations in
the frdABCD and ppc genes on lactate production.
poster 4-41
Production of Lactic Acid from Aqueous Ammonia-Treated Corn Stover
via Simultaneous Saccharification and Co-Fermentation
Yongming Zhu, Y. Y. Lee*
Department of Chemical Engineering, Auburn University, AL 36849
Corn stover was pretreated by aqueous ammonia and further processed by simultaneous saccharification and co-fermentation (SSCF) for production of lactic acid. The enzyme and lactic acid bacteria used in the SSCF were: Spezyme
CP (Genencor International) and Lactobacillus pentosus ATCC 8041 (CECT-4023). In the SSCF of the pretreated corn
stover, the carbohydrates in cellulose as well as in hemicellulose (glucan, xylan, and arabinan) were effectively converted
to lactic acid and acetic acid. The lactic acid yield reached 92% of the theoretical on the basis of total fermentable sugar
(glucose, xylose, and arabinose). Small amount of acetic acid was also produced in the process. The impacts of four
major process variables of the SSCF on lactic acid production were studied by way of statistical experimental design.
The process variables investigated were: enzyme loading, inocula size, yeast extract concentration, and clarified corn
steep liquor [cCSL] concentration. The statistical analysis of the results showed that enzyme and yeast extract were the
most important factors affecting the lactic acid yield. In contrast, the impact of inocula size was found to be insignificant.
The response surface analysis indicated that cCSL can be used as a nitrogen source replacing yeast extract without
adversely affecting the lactic acid yield. The product concentration was increased by fed-batch operation the SSCF. The
maximum lactic acid concentration attainable by fed-batch operation was 74.8 g/L. Further improvement of the lactic acid
concentration was difficult due to severe product inhibition.
181
poster 5-07
Pretreatment and Bioconversion of Chipped Yard Waste to Carboxylic Acids using a Percolation
Column Apparatus
Reddy Adapala, G. Peter van Walsum*
Baylor University, Waco TX, 76798-7266
The MixAlco process is a method for converting biomass wastes such as solid wastes, manure and other waste organic
materials into marketable chemicals such as ethanol, acetone, and acetic acid. Processing steps are as follows: (1) oxidative lime pretreatment, using air and lime at ambient pressures, (2) non-sterile fermentation to carboxylate salts using a
mixed culture of acid-forming microorganisms, (3) dewatering, (4) thermal conversion of carboxylate salts to ketones, and
(5) hydrogenation of ketones to alcohols. To date, research on the MixAlco process has been applied primarily to slurried
substrates.
Chipped yard waste contributes a considerable waste stream in municipal landfills. Attempts to recycle yard waste as
compost or mulch has met with some success, but typically the quantities available quickly saturate local markets. This
project seeks to investigate the application of acidogenic digestion to the conversion of chipped yard waste using a percolation system to simulate in-situ solid state conversion of particulate biomass. This study presents results from oxidative
lime pretreatment and acidogenic fermentation carried out in 6’ tall percolation reactor column apparatus. The reactor is
capable of performing both the pretreatment and fermentation steps. Results on mixing effectiveness, percolation behavior and conversion to mixed acids are presented.
poster 5-08
Influence of temperature on Glucose and Fructose Adsorption Process by Activated Carbon
Renata M. R. G. Almeida*, Pollyanna S. Oliveira, Gustavo T. S. Gonzaga, Mirelle M. S. Cabral
Chemical Engineering Department, Federal University of Alagoas,
Rod Br 101, Km 97, 57070-970, Maceió, AL, Brazil
Fructose is an important natural sugar, sweeter than glucose and sucrose. One of the most important sources of fructose
is sucrose, that can be converted into glucose and fructose. To produce a high fructose syrup from sucrose, it is necessary to separate the glucose formed. Generally, the separation of sugar, like glucose and fructose, is difficult, because
these sugars are isomers. Adsorption is the process utilized in these cases.
In this work batch runs in stirred tank were carried out to study the adsorption of fructose and glucose by activated carbon
and ion exchange resin. The batch tests were carried out at different temperatures in order to verify influence of temperature in adsorption capacity. The glucose and fructose concentrations were determined by Somogyi & Nelson method and
enzymatic method. It was observed that the carbon size did not influence in the adsorption process and the treatment
of the carbon either. The temperature influenced fructose adsorption but not glucose. Analyzing the adsorption kinetic
curves, it was observed that activated carbon adsorption capacity is bigger with glucose than with fructose. Although,
with ion exchange resin the inverse occurred. Batch tests were carried out with a mixture of glucose and fructose to analyze the adsorption process.
182
poster 5-09
Continuous Esterification of Bio-based Organic Acids: Formation of Triethyl Citrate
Navinchandra Asthana*, Aspi Kolah, Dung Vu, Carl Lira & Dennis Miller*
Department of Chemical Engineering and Material Science, Michigan State University,
East Lansing, Michigan 48824, USA. Phone: 517-3555130, Fax: 517-4321105
Email: [email protected], [email protected]
Depletion of fossil fuel resources, coupled with heightened awareness of environmental and health concerns is providing
impetus to search for alternative materials that are biocompatible, biodegradable, and based on renewable resources.
Organic acids, typically produced by fermentation of carbohydrate feedstocks, constitute an important class of biorenewable platform chemicals that can be further converted to useful products. Tri-ethyl citrate, produced from citric acid and
ethanol, is currently gaining a lot of attention because it is a non-toxic, biocompatible plasticizer that can be used in place
of petroleum-based phthalate compounds. Phthalates have potential carcinogeneity and overall health concerns to the
extent that they have been banned in Europe from use in children’s toys and human contact applications.
The potential application of tri-ethyl citrate as a plasticizer is currently limited by the lack of large-scale, efficient, and
economic production facilities. Generally, there is a dearth of published literature on synthesis of tri-ethyl citrate from citric
acid and ethanol, and relatively little information pertaining to the kinetics and thermodynamics of its formation is available
in the open literature.
We present here a continuous process for the formation of triethyl citrate using cationic exchange resins as catalysts in
a reactive distillation column. In developing the process concept, we have studied the kinetics and equilibrium of tri-ethyl
citrate formation using citric acid and ethanol as reactants in batch and continuous reactors. Vapor-liquid equilibrium studies for important binary mixtures have also been conducted. In a standard batch reaction, equilibrium was achieved after
16 hours; 99% of the citric acid was converted to a mixture of monoethyl, diethyl, and triethyl citrate, with a batch triethyl
citrate selectivity of 64%. Kinetic parameters for an nth order, reversible reaction, activity-based model that includes
both ion-exhcanged catalyzed reactions and self-catalyzed reactions were generated by non-linear regression of batch
experimental data in MATLAB. Chemical and physical parameters thus obtained were inserted into AspenPlus process
simulation software to model citric acid esterification in our semi-pilot reactive distillation column (5 cm diameter x 4 m
height). Column performance parameters (liquid holdup, catalyst efficiency) determined by matching pilot-scale column
performance with the model were then used to design a commercial-scale triethyl citrate esterification process using
AspenPlus.
Production of triethyl citrate via reactive distillation will lead to expanded markets for bio-based citric acid and to substantially lower costs than the current price of $2.50 to $2.80/lb.
*Authors to whom correspondence can be addressed.
183
poster 5-10
Performance of a Thermophilic Anaerobic Digester at Different Feed Loading Frequencies
John Bombardiere, Teodoro Espinosa-Solares*, Max Domaschko, Mark Chatfield
Division of Agricultural, Consumer, Environmental, and Outreach Programs
West Virginia State University, Institute WV 25112-1000
* Departamento de Ingeniería Agroindustrial. Universidad Autónoma Chapingo
Chapingo, 56230, Edo. de Méx., México.
Feed input may be one of the most important events in process control of an anaerobic digester. Feed input volume and
concentration must be monitored carefully to ensure stable and efficient operating conditions. More frequent feeding
events give operators the flexibility to modify feed input and maintain a stable process. Therefore, the effect of feed loading frequency on digester performance was studied on a 40 m3 thermophilic anaerobic digester. The digester was fed 1
m3 of chicken litter slurry per day, containing 45 kg of volatile solids. The treatments were loading frequencies of 1, 2, 6
and 12 times per day. There was no difference between the treatments when average daily pH and average daily methane percent of the biogas were compared. However, total biogas production was 20-30% greater during periods of twice
daily and six times per day loading frequencies when compared to once per day and twelve times per day. The hourly pH,
biogas production, and methane percent of the biogas were less stable at lower feed frequencies. When methane production and process stability are considered, the feed loading frequency of six times per day provided the best performance
of the treatments tested in this experiment.
poster 5-11
A Comparison of Cellulase Activity Restoration Using Starch Or Dextrin Following Foam Fractionation
Elizabeth Booth, Ian Snyder, Vorakan Burapatana, and Robert D. Tanner *
Chemical Engineering Department, Vanderbilt University, Nashville, TN 37235
Foam Fractionation is an inexpensive adsorptive bubble separation technique that is an alternative to current protein
concentration and separation methods. However, foam fractionation has a few drawbacks that include: (1) Not all proteins
will create a foam layer at low concentrations when aerated and (2) If a foam layer forms many proteins will be denatured
in the air-liquid interface. Cellulase is a protein that suffers from both of these drawbacks. In order to deal with these
problems, cellulase is foamed with the surfactant Cetyltrimethylammonium bromide (CTAB) and refolded with a dextrin or
a starch. 100mg/L of (CTAB) added to a 200mg/L cellulase solution is used to create enough foam to collect the concentrated but denatured cellulase in the foam layer. Various starch-like materials are then added to the foamed protein to try
and recover some of the lost activity. Corn dextrin, potato starch, kuzu (kudzu) starch, “soluble” starch, and Stevioside
(Steviosideo-Inga, Stevia) were individually added to the collected foamate at varying concentrations to determine which
substance and at what concentration causes the most renaturation of the protein. The refolding process slightly dilutes
the collected foamate but increases the specific activity of the cellulase protein. Cellulase foamed with CTAB has previously been refolded with β-cyclodextrin to achieve a tripling of its activity. Corn dextrin and starches were considered because they are possible alternatives that are significantly cheaper than β-cyclodextrin. Refolding with the various starches
and Stevia showed very little increase in specific activity. Refolding with corn dextrin, however, greatly increases the
specific activity of the cellulase solution. Refolding with corn dextrin resulted in an increase in activity that is up to three
times greater than the activity when refolded with β-cyclodextrin for an absolute activity increase of ten-fold.
184
poster 5-12
Removal of Hydrogen Sulfide by Immobilized Thiobacilli on Polyvinylpyridine Carrier
Jin-Myeong Cha1, In-Ji Song1
Bio & Environmental Tech. Co. Ltd., Jeonnam 519-831, Korea
1
Gwi-Taek Jeong2, Kyoung-Min Lee2, Woo-Tae Lee4, Don-Hee Park 2,3,4*
School of Biological Sciences and Technology, 3Institute of Bioindustrial Technology,
4
Faculty of Applied Chemical Engineering, Chonnam National University, Gwang-ju 500-757, Korea
2
5
Gwang-Yeon Lee5
Dept. Ophthalmic Optics, Dong-A College, Jeonnam, 526-872, Korea
Hydrogen sulfide is an extremely odorous, toxic, and corrosive air pollutant. A number of microbial processes for H2S
removal have been proposed that are based on oxidation by Thiobacilli and other sulfur-oxidizing microbes. In this study,
Thiobacillus sp. IW, T. denitrificans, and T. thiooxidans, can capable of degrading sulfur-containing odor, was immobilized
on polyvinylpyridine (PVP) carrier to remove hydrogen sulfide using a bubble column bioreactor. T. sp. IW shows a fast
growth compared with other sulfur-oxidizing bacteria of T. denitrificans and T. thiooxidans. Three kinds of Thiobacilli could
oxidize thiosulfate to sulfate as the final product. Sulfate concentrations in medium accompanied by the increase of cell
growth and thiosulfate concentration. The removal efficiency of H2S was 6-18 times faster than that of T. denitrificans and
T. thiooxidans. It shows that the high cell growth is present high H2S removal. The removal efficiencies of 99% were observed in the range of inlet H2S concentration from 200 to 2,200 ppm at a constant gas flow rate 19.2L/min. Immobilized
T. sp. IW on PVP carrier showed better removal performance. Regenerated immobilized T. sp. IW on PVP carrier did
not shown marked pH change during the period of experiments compared to fresh immobilized T. sp. IW on PVP carrier.
Therefore, it is indicated that immobilized PVP carriers, which lost activity, can be reused.
poster 5-13
Study on the Production of Biodiesel by Magnetic Cell Catalyst based on Lipase-producing B. subtilis
Ming Ying, Guanyi Chen*,
Section of Bioenergy and Environment, Faculty of Environmental Science and Engineering,
Tianjin University, Weijin Road 92, 300072, Tianjin, China
Production of bio-diesel from waster cooking oils by Magnetic Cell biocatalyst immobilized in hydrophobic magnetic polymicrosphere is studied innovatively. The cells of lipase-producing B. subtilis was encapsulated within the net of hydrophobic carrier with magnetic particles (Fe3O4), and the secreted lipase can be conjugated with carboxyl at the magnetic
polymicrosphere surface. Environmental scanning electron microscope, transmission electron microscope and vibrating
magnetometer etc. were employed to characterize the Magnetic cell biocatalyst (MCB). The MCB was proved to be superparamagnetic; and could be repeatedly used more than 300 hours after recovered by magnetic separation; moreover
it could regenerate by 72 hours of cultivation. When methanolysis is carried out using MCB with waste cooking oils under
stepwise additions of methanol, the methyl esters (MEs) in the reaction mixture reaches more than 90% in solvent-free
system; and the by-product glycerol decreases obviously than alkali or acid-catalyzed process for cell metabolism. The
process presented here is environmentally friendly, and simple without purification and immobilized process required by
the current lipase-catalyzed process. The process is therefore very promising for development of biodiesel fuel industry.
1*
Corresponding author. Fax: 0086-22-27404833. E-mail address: [email protected]
185
poster 5-14
Production of Omega-3 Polyunsaturated Fatty Acids from Cull Potato with the algae culture process
Zhanyou Chi, Shulin Chen
Department of Biological Systems Engineering, Washington State University , WA 99163
This presentation reports the research results on developing a cost-effective algal cultivation process converting cull potato to ω-3 polyunsaturated fatty acids (ω-3 PUFA), i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA),
that have beneficial effects in treating and preventing human heart and immune diseases. Using under-utilized cull potato
to replace glucose as a carbon source can significantly reduced the medium cost and ultimately the price of the products.
The producers used for DHA and EPA are the algae strain Schizochytrium limacnum SR21 and Nitzschia laevis, respectively. The cull potato hydrolyzed broth (PHB) is used as the carbon and nitrogen source in the culture medium. A optimum ratio of the PHB to the whole medium needs was determined, due to the fact that the high nitrogen concentration in
the PHB inhibited the algae’s growth. A fed batch culture will be conducted to obtain a high cell density culture. The DHA
and EPA enriched algal biomass could be used as feed additives for dairy to enhance the nutritional value of the milk.
poster 5-15
Hybrid Neural Model of an Industrial Ethanol Fermentation Process Considering the Effect of Temperature
Ivana C. C. Mantovanelli1, Aline C. da Costa2 e Rubens Maciel Filho2
Department of Biotechnological Processes - School of Chemical Engineering - UNICAMP
2
Department of Chemical Processes - School of Chemical Engineering – UNICAMP
Cidade Universitária “Zeferino Vaz” - Caixa Postal 6066 - CEP 13083-970 - Campinas - SP – Brasil
1
Among the problems in industrial ethanol fermentations is the lack of robustness of the fermentation in the presence of
fluctuations in operational conditions, which leads to changes in the kinetic behavior, with impact on yield, productivity and
conversion. Another difficulty is associated with the influence of temperature in the kinetics of the process. It is difficult to
support a constant temperature during large-scale fermentation and variations in temperature affects productivity through
changes in kinetics and in microorganism lifetime.
Due to the difficulties described, the main difficulty in model-based techniques for definition of operational strategies for
the ethanol production process is the problem of obtaining an accurate model. In this work a procedure for the development of a robust mathematical model for an industrial alcoholic fermentation process is evaluated. The proposed model
is a hybrid neural model, which combines mass and energy balance equations with Functional Link Networks to describe
the kinetics. These networks have been shown to have a good non linear approximation capability, although the estimation of its weights is linear. The proposed model considers the effect of temperature on the kinetics and has the neural
network weights reestimated always that a change in operational conditions occurs.
186
poster 5-16
A Software Sensor for the Ethanol Fermentation Process
Elmer A. Ccopa Rivera1, Rafael R. Andrade1, Daniel I.P. Atala2, Francisco Maugeri Filho2, Rubens Maciel Filho1 e Aline C.
da Costa1
1
Department of Chemical Processes - School of Chemical Engineering – UNICAMP
Cidade Universitária “Zeferino Vaz” - Caixa Postal 6066, CEP 13083-970, Campinas/SP – Brasil
2
Department of Food Engineering - School of Food Engineering – UNICAMP
Cidade Universitária “Zeferino Vaz” - Caixa Postal 6121, CEP 13083-862, Campinas/SP – Brasil
Among the problems in industrial ethanol fermentations is the lack of robustness of the fermentation in the presence of
fluctuations in operational conditions, which leads to changes in kinetic behavior, with impact on yield, productivity and
conversion. These changes are common in industrial plants; they occur not only due to variations in the quality of raw
material but also due to variations of dominant yeasts in the process.
One of the sources of fluctuation in the quality of raw material is the operation of the sugar factories integrated with
ethanol plants, which changes the proportion of molasses and sugar-cane juice in the composition of the fermentors feed
media. Besides, sugar-cane molasses undergo composition variations in different harvests. Changes in dominant yeast
population are common due to the high microbial load in raw materials.
This problem can be solved by operational and control conditions adjustments, which requires an efficient monitoring
with reliable sensors. In this work we developed a software sensor to infere the process key variables (concentrations of
biomass, ethanol and substrate) from secondary measurements, such as pH, turbidity and CO2 flow rate. The objective is
to have a robust sensor, which describes the process even on operational conditions changes.
187
poster 5-17
Process Intensification for Ethyl Acetate Production from Bioethanol
A.F.Custódio, *M.R.Wolf, *R.Maciel Filho
Laboratory of Separation Process Development. Faculty of Chemical Engineering – State University of Campinas
(UNICAMP) P.O. BOX 6066, ZIP 13081-970, FAX +551937883965, Campinas, SP, Brazil. ¹ [email protected],
*[email protected]
Reactive distillation technology combines chemical synthesis steps with separations by distillation. This combination can
lead to intensified, high-efficiency process systems with significant green engineering attributes. Many of these potential
advantages are intimately linked to the principles and challenges for green engineering.
This work presents a study of a complete reactive distillation system for production of ethyl acetate via esterification of
acetic acid with bioethanol using ASPEN PLUS. Ethyl acetate is normally produced via reversible reaction of acetic acid
with petrochemical ethanol. There are only few papers in the literature on the subject of the production of ethyl acetate via
reactive distillation.
A suitable NRTL (nonrandom two-liquid) model parameter set for calculating liquid activity coefficients has been established with excellent prediction of the compositions and temperatures for the four azeotropes in the system, including
three homogeneous azeotropes and also one heterogeneous azeotrope of ethyl acetate/water.
The proposed process intensification of ethyl acetate production by reactive distillation was composed by two columns,
including the reactive distillation column and a second column, one decanter, and two recycle streams. The optimum
operating conditions of the overall system is also investigated in order to minimize the total costs while meeting product
specifications.
poster 5-18
Characterization of Thermostructural Damages Observed in a Seaweed Used for Biosorption of Cadmium:
Effects on the Kinetics and Uptake
Antonio Carlos Augusto da Costa*, Aderval Severino Luna and Robson Pafumé
Universidade do Estado do Rio de Janeiro, Programa de Pós-Graduação em Engenharia Química,
R. S. Francisco Xavier 524, Sala 427, Maracanã, Rio de Janeiro, RJ, Brasil, 20550-013
The use of biomass for biosorption of heavy metals is well documented in the literature. Several types of microbial species and seaweeds were already tested. Most of these works are conducted with dry biomass in order to prevent any
undesirable metabolic response. Several literature reports recommend drying the biomass under mild conditions, such as
sun-drying; on the other hand, several reports recommend the range from 40 to 80oC to ensure complete inactivation of
the biomass. The effect of sun-drying the biomass at 20oC and oven-drying at 60oC is here reported. Evaluation of results
was based on the kinetics and uptake capacity of cadmium by the seaweed Sargassum filipendula.
Results indicated that the maximum metal uptake was reduced by 30% when oven-dried biomass was used, for cadmium
concentrations ranging from 10.0 to 500.0 mgL-1. Kinetic tests performed indicated a better performance of the sun-dried
biomass, both for a dilute (10mgL-1) and a concentrated solution (100mgL-1). In both cases the effect of drying in an oven,
at 60oC, was reflected in the uptake. Results were best represented by the Langmuir model in comparison to the Freundlich model. Kinetics of the process indicated a second-order kinetics. Thermogravimetric analysis and infrared spectra
confirmed the structural damages produced after drying, markedly affecting cadmium uptake capacity.
Authors thank Faperj (Prociência Program) for financial support to conduct this work.
188
poster 5-19
Effect of Physiochemical Properties of Catalysts on the Conversion of Glycerol to Value-Added
Liquid Chemicals
K. Pathak, D. Ferdous, A.K. Dalai*, N.N. Bakhshi
Catalysis and Chemical Reaction Engineering Laboratories, Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9 Canada
Glycerol is one of the by-products from biodiesel production. Increased production of biodiesel would lead to glut of glycerol
in Canadian market. The value-added products from glycerol may improve the economics of biodiesel production. On the
other hand new processing methods can improve glycerol market. In this work, a systematic study has been conducted to
investigate the effect of physiochemical properties of catalysts on glycerol conversion and liquid product yield. The catalysts
studied were HZSM-5, HY, silica-alumina and γ-alumina. A statistical approach was used for design the experiments. The
experiments were conducted in a fixed bed down flow reactor at the temperature, carrier gas flow rate and WHSV of 350500 oC, 20-50 mL/min and 5.4- 21.6 h-1, respectively. Numerical optimization was performed using variable simplex method
to find out the optimum operating conditions for glycerol conversion and liquid product yield. With increase in catalysts pore
size from 0.54 to 0.74 nm the optimum liquid product yield increased from 59 to 83.5 g/100 g feed. HZSM-5 has the highest
total acidity out of the selected catalysts; however the optimum glycerol conversion and liquid product yield was least in this
case. No straight forward trend was observed between total acidity and liquid component yields.
poster 5-20
Withdrawn
189
poster 5-21
Bio-PSA: A tool for aid in the bioprocesses design and operation
Vera Lúcia Reis de Gouvêia* , Rubens Maciel Filho
School of Chemical Engineering (State University of Campinas)
Laboratory of Optimization, Design and Advanced Process Control (LOPCA)
P.O. Box 6066 – ZIP Code: 13081-970, Campinas - SP, Brazil, E-mail: [email protected]
The increasing raise of the bioprocess importance and the largest difficulty in its control and simulation it resulted in the
search and use of Artificial Intelligence (AI) tools. Artificial Intelligence is a part of the computer science related to the
project of computational intelligent systems. In the last years there was a substantial increase of the use of tools of AI
such as Artificial Neural Networks (A.N.N), Expert Systems and Fuzzy Logic in the bioprocess control and simulation.
This work had as objective the development of software that acts as an assistant, aiding in the bioprocess simulation. The
Bio-PSA (Bioprocess Simulation Assistant) has four modules:
- Module 1: Bioprocess simulation, including applications for simulation of ethanol and dextrana production, both
with normal and hybrid modeling.
- Module 2: Simulation of proteins purification processes, including processes modeling based in the CARE
(Continuous Affinity Recycle Extraction) model.
- Module 3: Expert System that aids the user in the bioprocesses design.
- Module 4: Software for A.N.N training.
The basic structure of the software was developed so that its new versions can add new processes and new rules for the
Expert System.
poster 5-22
Fermentation Inhibitors from Pretreated Lignocellulosic Materials: Problems and solutions
Wim de Laat*, Fredrick Aboka, San Feng Maltha
Royal Nedalco B. V., P.O. Box 6, 4600 AA, Bergen op Zoom, The Netherlands
Zsófia Kádár, Kati Réczey
Budapest University of Technology and Economics, Szent Gellért ter 4., 1111-Budapest, Hungary
This work has been carried out in a European project TIME (ENK6-CT-2002-00604), which was initiated at the end of
2002, and funded by the European Union. The project involved seven research institutions, companies and universities throughout Europe and focused on improving the European security of energy supply for reduction of environmental
impacts in the transport sector. The technological objective was to develop cost effective and sustainable production
methods for ethanol, based on lignocelluloses waste.
Our research work aimed to test whether at least 5(v/v)% final ethanol production could be achieved in the presence of
inhibitors (lignin and sugar degradation products), which are generated during pretreatment of lignocellulosic materials.
Ethanol was produced from steam pretreated spruce, willow and corn stover by a previously selected inhibitor resistant
Saccharomyces cerevisiae strain. To prevent bacterial contamination batch-wise alcoholic fermentation was carried at low
pH (pH4) with continuously monitored CO2 evolution.
This presentation will emphasize the effect of inhibitors on ethanol fermentation and will also include our latest results on
improving the fermentation ability of the selected yeast strain by adaptation to toxic components present in the pretreated
lignocellulosic materials on spruce matrix.
190
poster 5-23
Modeling Complex Experimental Behavior in the Production of Ethanol by Zymomonas mobilis:
Comparison between Phenomenological and Hybrid-Neural Approaches
Maurício B. de Souza Jr.*
School of Chemistry - Federal University of Rio de Janeiro, Bloco E - Rio de Janeiro - R.J. - Brazil - CEP 21.949-900
Anna Carolina R. Camêlo, Tito Lívio M. Alves
Chemical Engineering Program of COPPE, Federal University of Rio de Janeiro, Rio de Janeiro - R.J. – Brazil
Irapuan O. Pinheiro, Carlos E. Lopes
Antibiotics Department, Federal University of Pernambuco
Complex experimental behavior has been observed in continuous ethanol fermentations using different strains of Z. mobilis (Lee et al., 1979; Ghommidh et al. 1989). In the present work, sustained oscillations and steady state (SS) solutions
are reported for the strain ATCC 29129.
The development of a model is mandatory for the efficient control of this process. A model of the literature (Li et al., 1999)
was initially used to develop bifurcation diagrams. However, this model was developed for the strain 29129 and does not
use a unique set of parameters. So, two approaches were attempted in this paper: re-estimation of the parameters of Li’s
traditional model (1999) and development of an innovative hybrid-neural model (HNM). The HNM replaces the growth
rate by a neural network model. The present contribution uses statistical parameters to compare the models. It is shown
that the HNM has a much better prediction capability, using a unique set of parameters. The developed HNM model plays
an extremely helpful role as it predicts the initial conditions that lead to complex behavior.
poster 5-24
Comparison of methanogenic and acidogenic digestion of cattle manure for production of
fuels and reduction of phosphorous bioavailability
Erin Doyle, G. Peter van Walsum*
Acidogenic and methanogenic digestion are proposed to address dairy-derived phosphate water pollution in Central Texas. Biomass such as cattle manure can be converted to methane or to mixed-alcohol fuels or chemicals via the MixAlco
Process, which includes: alkaline pretreatment, acidogenic digestion, product concentration, thermal conversion and hydrogenation. Because it is non-sterile, digestion can process problem waste streams such as manure and biosludges and
utilizes low-cost materials for construction. It is hypothesized in this study that dairy farm-derived phosphate contamination of surface waters could be diminished by the application of the MixAlco conversion process to dairy waste streams.
Soluble reactive phosphorous concentrations were measured in samples taken from dairy manure before, during, and
after lime pretreatment and digestion steps. It was found that soluble reactive phosphate is present in each step, but at
different concentrations. Lime pretreatment reduced the amount of available phosphorus in dairy cattle manure, and the
fermentation step further precipitated out calcium phosphate salts with the addition of calcium carbonate. Comparing
acidogenic and methanogenic fermentation of manure demonstrated increased phosphate removal for the methanogenic
digestion, which was unexpected since it used less CaCO3 for neutralization.
191
poster 5-25
Control of an Extractive Fermentation Process to Bio-Ethanol Production
Rubens Maciel Filho, Daniel. I. P. Atala, Elis R. Duarte*
State University of Campinas (UNICAMP), 13.083-970, Campinas, SP, Brazil
Laércio Ender
Regional University of Blumenau,Campus II, 89010-971, Blumenau , SC, Brazil
An alternative procedure based on extractive alcoholic fermentation process is considered in this work. There is a great
interest in the optimisation of the ethanol process, especially due the many advantages of using ethanol as fuel. In order
to do that, it is necessary an efficient and robust control strategy because the control of biotechnological process is very
difficult due to the complex nature of the microbial metabolism, as well as no linearity of its kinetics.
Nowadays days with the globalisation, market competitively and environmental concern, there is a real need of products
with high quality, low cost and low emission of pollutants. The process control is the main form to reach such objectives.
That is the reason for the great advance on researches in the investigation of new methodologies and conceptions about
process development and process control in all areas.
As a case study an extractive alcoholic fermentation process is considered, it that consists on four interlinked units: fermentor (ethanol production unit), centrifuge (cell separation unit), cell treatment unit, and vacuum vessel (ethanol –water
separation unit), represented a non-linear process. The obtained results showed the potential of the proposed learning
strategy for non-linear process.
poster 5-26
A Non-Linear Control Strategy of a Fixed Bed Catalytic Reactor for Bio-Ethanol Oxidation
Rubens Maciel Filho, Elis R. Duarte*
State University of Campinas (UNICAMP), 13.083-970, Campinas, SP, Brazil
Laércio Ender
Regional University of Blumenau,Campus II, 89010-971, Blumenau , SC, Brazil
A fixed bed catalytic reactor for production of acetaldehyde by ethyl alcohol oxidation over Fe-Mo catalyst is considered
in this work as an alternative way to obtain chemical products. The use of bio-ethanol as feedstate for chemical product
production is nowadays attractive since it is renewable and without contaminants. However it is necessary to develop
advanced control strategies to be able to run the process in such was that high performance maybe achieved. This is a
important requirement especially due to economic reason and process competitive.The reaction is very exothermic, and
besides being an alternative procedure to obtain acetaldehyde, the process has a very complex dynamical behavior since
inverse response and temperature runaway may occur.
The effective control of these reactors is fundamental to obtain safe operations, especially when high performance is
desired. This work presented a control strategy based on neural networks that are on-line trained through an optimization
routine. The obtained results showed the potential of the proposed learning strategy for non-linear process. Such systems
present a complex dynamic behavior and even so the performance of the proposed control strategy has shown to be very
good in a large range of operating conditions.
192
poster 5-27
New silicone based antifoams for fermentation
# :
Ariane Etoc+, Frank Delvigne#, Jean-Paul Lecomte+*, Philippe Thonart #
Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, B-5030;
+:
Dow Corning sa, Seneffe, B‑7180
Fermentation is often accompanied by foam formation because of the high foaming tendency of solutions containing
biomaterials such as proteins. It is of interest to develop new material which can be used to control foam in fermentation
processes. In this perspective, the choice of a antifoaming material will be directed not only by its antifoam power but also
by some secondary properties in order to avoid problems during the fermentation process (e.g., oxygen transfer drop,
biomass yield drop,…) and the subsequent downstream processing operations (e.g., membrane fouling,…).
New silicone based antifoaming agents were developed for the fermentation and found efficient to control the foam during
sugar cane fermentation, especially when used as “foam conditioner”, as potential replacement of polyglycols.
The aim of this work was to better understand the effectiveness of these new antifoams, to assess their influence on various fermentation parameters such as the oxygen transfer rate and potential ultrafiltration membrane fouling, and try to
correlate surface tension measurements and antifoam efficiency.
Production of Lipase by Yarrowia lipolytica was also used as model fermentation system, as we experienced it to be especially foamy. It enabled us to extrapolate the efficiency of these new materials to other fermentation systems.
poster 5-28
A Structure Kinetic Model for Bioethanol Production by Fermentation
Betânia H. Lunelli, Elmer A. Ccopa Rivera, Dile Pontarolo Stremel, Eduardo Coselli Vasco de Toledo and
Rubens Maciel Filho
Laboratory of Otimization, Project and Advanced Control – LOPCA
Departament of Chemical Process – School of Chemical Engineering
State University of Campinas – UNICAMP
Bioethanol is nowadays the largest fermentation product obtained from the sugar cane. From ethanol many chemical
products may also be produced, making the sugar cane very interesting for the environmental point of view and economic
attractive raw material to obtain chemicals. From ethanol is possible to achieve high quality acetaldehyde, acetic acid,
ethyl acetate, ethylene and from them a huge amount of chemicals, including polymers. Either sugar cane or bagasse
may be used as feedstock, and several bio-chemical pathways are shown. Technical feasibility, however, is not sufficient:
economic potential is the driving force.
Compared with growing studies of microorganism populations, few improvements on the development and application
of structured models for product formation have appeared. This work presents an alternative dynamic structured model,
adapted from a structured growth model and modified with the objective to simulate the intrinsic reactions taking place
in the fermentation process to obtain bioethanol. A tower bioreactor type is considered and the original model of Sacharomyces cerevisae growth was adapted for ethanol production. The results show how the model may be used to find out
optimal operating conditions so that even cheaper bioethanol may be produced.
193
poster 5-29
On-site Lime Pretreatment and Acidogenic Digestion of Dairy Manure Employing Counter-Current
Fermentation with Minimal Solids Handling
Michael Flatt, G. Peter van Walsum*
The MixAlco process is an anaerobic fermentation system that converts biomass into organic chemicals and mix-alcohol
fuels via alkaline pretreatment, non-sterile, acidogenic digestion, product concentration, thermal conversion and hydrogenation. Because they have low capital costs and relatively simple operation, it is proposed that the pretreatment and
fermentation steps of the MixAlco process may be suitable to be carried out on location at confined animal feeding operations. This project focuses on converting lime-pretreated dairy manure into carboxylic acids using a three-stage countercurrent fermentation system installed on-site at a dairy farm.
The pretreatment and fermentation system was operated for several months to determine the effectiveness of on-site
conversion. The system consists of four plastic barrels that serve as both pretreatment and fermentation reactors. The
setup was configured to minimize solids handling by decanting fermentation vessels and transporting only liquids to enable the counter-current fermentation system. Residence times of one week for pretreatment and three weeks for counter
current fermentation were employed, with both functions carried out at 40°C. The temperature and pH of the fermentation
system was logged automatically to ensure steady state was achieved. Conversion results and comparisons with laboratory studies are presented.
poster 5-30
The effect of age and inoculum concentration on the production of biosurfactants and protein-related virulence
factors by Pseudomonas Aeruginosa PA1
Luiz Fernando D. Tavares, Valéria F. Soares, Denise M.G. Freire*
Lídia Maria de Melo Santanna
Brazilian Oil Research Center, Petrobrás, Brasil
Biosurfactants are amphipathic molecules produced by microorganisms, whose broad spectrum of activity makes them
suitable for a variety of environmental applications. One group of these are rhamnolipids, which are produced mainly by
strains of Pseudomonas. The aim of this work was to study the effect of the physiological age and inoculum concentration
on the production of rhaminolipids and the protein-related virulence factors by Pseudomonas aeruginosa PA1 previously
isolated from the water of oil production in Sergipe, Brazil. Cell growth, rhamnolipid production, substrate consumption
(glycerol and nitrate), the production of nonspecific proteases, total proteins and elastases were all investigated for different inoculum concentrations (0.3g/L, 0.6 g/L, 1.1 g/L and 2.2 g/L and physiological ages (20 and 40 hours). Greater
inoculum concentrations were also evaluated (4.5 g/L and 9.0 g/L) for the 40 h physiological age. It was found that the
inocula with a greater physiological age tended to yield better results than the younger ones. Under all conditions, nitrogen was the limiting substrate, and a fall in the virulence factors was noted when inocula with a greater physiological age
were used. For higher inoculum concentrations (4.5 and 9 g/L) the kinetic profiles indicating cell death and rhaminolipids
consumption were observed, probably associated to a lack of nutrients in the culture.
194
poster 5-31
Investigation of new inoculum strategies for solid-state fermentation
Melissa E. L. Gutarra, Denise M. G. Freire*
Federal University of Rio de Janeiro, IQ, Department of Biochemistry, CT, Lab. A-549-2, Ilha do Fundao, 21949-900
Rio de Janeiro/RJ, Brazil
Leda R. Castilho
Federal University of Rio de Janeiro, IQ, Department of Biochemistry, CT, Lab. A-549-2, Ilha do Fundao, 21949-900
Rio de Janeiro/RJ, Brazil
Lipases are industrially interesting enzymes and can be produced by either submerged or solid-state fermentation (SSF).
The latter allows the use of low-cost agroindustrial residues as raw materials, but limitations regarding inoculum development are found in large-scale SSF applications.
The aim of the present work was to study the effect of diferent types of inoculum on lipase production by Penicillium simplicissimum using SSF with babassu cake. Conventional inoculation with spores at concentrations of 107 and 108/g gave
lipase activities and productivities of 20 U/g and 0.4 U/g/h. Lower spore concentrations resulted in lower lipase production. Inoculum propagated in liquid medium delayed enzyme production in SSF, giving lipase activities of 11 U/g after
96h. Contrastingly, the use of inocula consisting of fermented solids (previously prepared by carrying out a small-scale
SSF inoculated with spores) allowed an anticipation of the lipase peak and, after optimizing inoculum age and concentration through experimental design, lipase activities and productivities of 30 U/g and 0.63 U/g/h were obtained. This
was achieved for an inoculum previously propagated for 36h, added at a concentration of 10% (m/m). Thus, the present
results indicate that the use of fermented solids is an interesting alternative for inoculum development in SSF.
poster 5-32
Separation of Solid Residues from Liquids Produced during Ethanol Production from Lignocellulosic Materials
Mats Galbe and Christian Roslander
Dept. of Chemical Engineering, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden
Ethanol can be produced not only from starchy materials, but also from various lignocellulosic materials, e.g. wood, agricultural residues or other materials. It is necessary to break down the polymeric chains to individual sugars, which can
then be fermented. In this study steam-pretreatment, followed by either enzymatic hydrolysis or simultaneous saccharification and fermentation (SSF) was used to produce fuel ethanol.
One of the major technical obstacles in an ethanol-from-cellulose process is actually to separate the lignin-containing
residues from the hydrolysate or the SSF-slurry. The residues form extremely compact filter cakes and tend to be very
difficult to filter and therefore demand long filtering times. One of the major ranges of uses for the lignin residue is to use
it for heating purposes, either in the plant, or in a pelletized form for external heating purposes. In any case it is important
to get as dry a filter cake as possible to minimize energy requirements in the dryer. This has a huge impact on the ethanol
production cost. Results from a filtering study on hydrolysis residues and SSF residues utilizing a filter press from will be
presented.
195
poster 5-33
Continuous Production of Ethanol from Sugar Cane Molasses using Yeast Immobilized in Pectin Gel
Raquel. L. C. Giordano1*; Roberto C. Giordano1 , Willibaldo Schmidell Netto2
Departamento de Engenharia Química, Universidade Federal de São Carlos,
13.565-905, C.P. 676, São Carlos, SP, Brazil (e-mail: [email protected])
2
Universidade Federal de Santa Catarina, SC, Brazil
1
A continuous process to produce ethanol from sugar cane molasses using Saccharomyces cerevisiae immobilized in
pectin gel is presented. The bio-catalyst was packed in a three fixed-bed reactors system, with two intermediate purges
for CO2. This work aimed at studying the stability of this system for high ethanol concentrations and the influence of the
bed density on the process performance. Initial substrate concentration in the feed ranged from 182.5 g/L to 234.0 g/L of
sucrose. Continuous operation was tested during 16 days. Bed porosity ranged from 0.83 to 0.53. Stable operation was
achieved for ethanol concentrations up to 86.6 g/L, with ethanol productivity of 21.7 g /L/h. The best results were obtained
using initial yeast load of 0.05 g wet yeast/mL in each reactor, corresponding to an initial bed porosity of 0.53. A mathematical model is used to assess reactor performance and mass transfer effects.
poster 5-34
Development of Inorganic Membranes for Improved Efficiency of Ethanol Extraction
Richard J. Higgins,* Robyn A. Foti
CeraMem Corporation, Waltham, MA 02453
Hydrophobic zeolite (silicalite) membranes are known to provide highly effective separation of ethanol from aqueous
media such as fermentation broths. CeraMem Corporation has fabricated silicalite membranes that can extract a product liquid of >80% ethanol by weight from a fermentation broth (“beer”) in a single stage. In concept, these membranes
have excellent potential for augmenting/displacing distillation in the ethanol manufacturing process train. Modeling of
conceptual “hybrid processes” employing ethanol-extracting membranes in combination with smaller distillation columns
has demonstrated that energy input to produce ethanol could be substantially decreased, resulting in ethanol production
cost savings. In addition, the relative advantages of membranes versus distillation are magnified for smaller ethanol plant
sizes that may be more typical of a next-generation lignocelluslosic-based ethanol industry.
However, there are significant obstacles to commercialization of hydrophobic zeolite membranes in the ethanol industry.
First, the ability of these membranes to produce ethanol-rich product streams at economically attractive throughputs over
extended periods of operation, when utilizing actual fermentation products as feed streams, has not yet been thoroughly
demonstrated. Second, in order that the capital costs of the membrane plant also be economically attractive, the ability to
scale up manufacturing to large-area, compact membrane modules must be demonstrated.
This presentation will describe the on-going activities of CeraMem Corporation’s product and process development
program aimed ultimately at industrial deployment of ethanol-extracting inorganic membranes. These activities include
device scale-up, lab process testing using feed from the beer well of an ethanol plant, and process/economic modeling.
196
poster 5-35
Improved Ethanol-Water Separation Using Fatty Acids
Tracy M Boudreau, Gordon A. Hill*
Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK, S7N 5A9, Canada
As a means to reduce the processing costs for separating ethanol from fermentation broth, a range of fatty acids have
been tested as liquid solvents for extracting ethanol from water. Valeric acid, the lowest molecular weight fatty acid used
in this study, extracted the most ethanol but also extracted a significant amount of water and was quite soluble in water.
Oleic acid, the highest molecular weight fatty acid used in this study, did not absorb water but extracted low amounts of
ethanol. Nonanoic acid was found to be the best extracting solvent, providing a reasonable ethanol distribution coefficient
(0.354) and a 69.5% vapour rich ethanol stream using flash separation. Using a commercial process simulator, nonanoic
acid extraction combined with the flash process is shown to require 38% less thermal energy to separate an equivalent
amount of ethanol from fermentation broth compared to the traditional distillation process.
poster 5-36
Purification of Green Fluorescent Protein (GFPuv) from E. coli Cell Lysate
Marina Ishii , Mariane Minaguti, Priscila Gava Mazzola, Thereza Christina Vessoni Penna*
Department of Biochemical and Pharmaceutical Technology,
School of Pharmaceutical Science, University of São Paulo, SP, Br.
Olivia Cholewa
Molecular Probes, Inc., Eugene, Or, USA. 97402.
The recombinant green fluorescent protein (GFPuv), intracellular expressed by E. coli, has been used as an interesting
molecular biology tool due to its fluorescence property and can be used to control process parameters in a fast and accurate way. The major challenge for GFPuv application in large scale is the adjustment of the extraction/purification methods. The recovering of GFPuv from E. coli lysate was evaluated from sonicated cells purified throughout anion exchange
chromatography (Q Sepharose FF) that was compared to the three-phase partitioning (TPP) extraction of GFPuv from E.
coli cells followed by a butyl- hydrophobic interaction chromatography column purification step. The best elution of GFPuv
from the Q Sepharose FF column was obtained with 0.1 M NaCl salt concentration among the range studied up to 0.6 M
NaCl. The collected fractions were analyzed by policrilamida gel SDS-PAGE and the eluted fraction with 0.1M NaCl was
represented by a single band of GFPuv. The one step anion exchange chromatography method provided larger amounts
(five times) of purified GFPuv from the E. coli lysate in a shorter period of time. The improvement of GFPuv purification
method increases the protein economic value; enlarges the industrial application and reduces costs by decreasing time
and assuring the level of purity of the protein.
197
poster 5-37
The Effects of Engineering Design on Heterogeneous Biocatalysis in Microchannels
Frank Jones*, and James Hiestand
University of Tennessee at Chattanooga, Chattanooga, TN 37403
Robert Bailey
Loyola College in Maryland, Baltimore, MD 21210
The results of a numerical study of the fundamental interactions of engineering design and micromixing on conversion in
packed microchannels are presented. Previously, channel-based microreactors made of molded silicon plastic were designed, fabricated, and experimentally tested. These reactors have enzymes immobilized on the channel walls by various
methods including layer-by-layer nano self-assembly techniques. They also contain molded packing particles to add reactive surface area and to redistribute the fluid. An arbitrary but intuitively sensible packing arrangement was initially chosen
and used in experimental studies.
A computer simulation study has been conducted in order to understand how changes in packing size, shape, density,
and position improve the conversion efficiency. The experimental reactors has been simulated using CFD-ACE+ multiphysics software. The focus of this study is to optimize the packing in order to meet conversion goals, taking into account
micro fabrication and operational constraints. Many micromixing fundamentals are also explored due to variations in design features, Reynolds number (Re), and Peclet number (Pe). The micro scale dimensions of the channel cross section
(125 by 500 micrometers) cause all flows to be laminar. Behavior in the range 0.1 < Re < 100 is examined.
poster 5-38
A Study on Characteristics of Reverse Micellar Systems by Measuring Viscosity and Conductivity
Choon-Hyoung Kang*, Jae-Soon Shin
Department of Chemical Engineering , Chonnam National University
Kwangju, 500-757, KOREA
The extraction method using the reverse micellar systems is one of promising techniques for separation/purification of
biologically active materials such as proteins, cell debris, amino acid, and enzymes. In particular, the characteristic properties of the micelle itself bear an immense influence on the feasibility and performance of the process. This work presents two properties such as the hydrodynamic radius and the percolation threshold of the reverse micellar solutions which
were determined by measuring the viscosity and the conductivity, respectively. The reverse micellar solution in this study
was comprised of an anionic surfactant sodium di(2-ethyl hexy) sulfosuccinate(AOT) and isooctane as an organic solvent.
From the conductivity measurement, it was noticed that incorporation of the protein(Bovine Serum Albumin) led to a
retarded occurrence of the percolation. In other words, the interaction between the micelles is reduced upon encapsulation of a charged solute in the core. The diameter determined from the viscosity as well as number of surfactant
molecules(aggregation number) increased and then leveled off with the water content. It implies that achievable micelle
size depends on the system factors such as the type and concentration of the surfactant and the solvent, and water content restricts the size of micelles.
*current address: Department of Chemical Engineering, Auburn University, Auburn, AL 36830
198
poster 5-39
Bioconversion of Barley Hull Into Ethanol Using A Soaking in Aqueous Ammonia Pretreatment
Tae Hyun Kim, Frank Taylor and Kevin B. Hicks*
ERRC-ARS-USDA, Wyndmoor, PA 19038
A method for the efficient conversion of cellulosic polysaccharides in barley hull to ethanol was studied. The Soaking in
Aqueous Ammonia (SAA) process was used as a pretreatment method, which has been show to achieve high enzymatic
digestibility of cellulose as well as high retention of xylan in the solid residue.
SAA was conducted at a range of low temperatures (room temperature to 80oC). This process has many advantages as
compared to conventional pretreatment methods in terms of minimal loss of carbohydrates and low energy requirements.
In this study, the effects of various reaction and operational parameters of the SAA, such as solid-liquid ratio and reaction
time, were investigated as it applies to pretreatment of barley hull. In general, delignification and cellulase “digestibility” increased with the soaking time, ammonia concentration and liquid-to-solid ratio. The effects of mechanical treatment (size
reduction) were also compared to the effects of chemical treatment. The fermentabilities of the SAA-treated barley hulls
were also determined and optimized yields of >80 percent conversion of cellulose to ethanol was achieved.
poster 5-40
Continuous Simultaneous Saccharification and Fermentation of Cellulose to Ethanol
Chaogang Liu*1, John Bardsley1, and Charles E. Wyman2
1 Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755
Telephone: (603) 646-6505; Fax: (603) 646-2277; E-mail: [email protected]
2 Bourn College of Engineering, University of California, Riverside, CA 92521
Bioconversion of cellulosic biomass to ethanol for use as a transportation fuel has substantial environmental, economic,
and energy security benefits. However, high processing costs present an important barrier to commercialization of biomass-to-ethanol processes, and the biological steps of enzymatic hydrolysis and fermentation account for major fraction
of these costs. Combining both hydrolysis and fermentation in one vessel via the simultaneous saccharification and fermentation (SSF) process can significantly reduce capital investment and operating costs and realize high ethanol yields
and concentrations at lower enzyme loadings. Continuous ethanol production can further reduce costs through increased
biomass conversion, ethanol concentrations, ethanol productivity, and enzyme effectiveness, but little data or insight has
been developed on such systems. Therefore, an automatically controlled two stage continuous fermentation system was
applied initially to investigate continuous SSF of Avicel cellulose. Special attention was given to evaluating the effects of
dilution rate, enzyme loadings, and cellulose concentrations on yeast density, cellulose conversion, ethanol concentration
and yield, glucose accumulation, and cellobiose concentration in each fermentor. A mathematical model is also being developed based on this data to predict the performance of continuous SSF and enhance our understanding of fundamental
issues in continuous conversion of insoluble substrates.
199
poster 5-41
Value-added Products from Condensed Distillers Solubles
Chuanbin Liu*, Energy & Environmental Research Center, University of North Dakota, Gand Forks, ND, 58203
Bo Hu, Shulin Chen, Washington State University, Pullman, WA, 99164-6120
Richard W. Glass, National Corn Growers Association, Chesterfield, MO, 63005
The major challenge associated with the rapid growth of ethanol industry is the usage of the co-products, i.e., Condensed
Distillers Solubles (CDS) and Distillers Dried Grains (DDG), which are currently sold as animal feed supplements. As the
growth of the livestock industries remains flat, alternative usage of these co-products is urgently needed. CDS is obtained
after the removal of ethanol by distillation from the yeast fermentation of a grain or a grain mixture by condensing the thin
stillage fraction to a semi-solid. In this work, the strategies and related technologies to produce value-added products
from CDS were studied.
CDS was converted into two value-added products, in particular, renewable heavy metal adsorbent and nutrient supplement for the fermentation industry. The CDS-based adsorbent has the potential application to condense heavy metals,
since the adsorption capacity of Ni ions of the CDS-based adsorbent was almost equal to the commercially available synthesized resins. The nutrient supplement derived from CDS could be applied for the production of nisin by Lactococcus
lactis subsp. lactis (ATCC 11454).
poster 5-42
Simulation of the recovery of acrylic acid from sugar cane bagasse using chromatographic adsorption process
Pinho, A. P. A.*; Alvarez, M. E. T.; Machado, A.B.; Wolf Maciel, M. R.
Laboratory of Separation Process Development, School of Chemical Engineering, State University of Campinas,
Campinas - Brazil, [email protected]
Scheer A.P
Department of Chemical Engineering, Federal, University of Parana, Curitiba - Brazil
Acrylic acid and chemicals associated with acrylics have a world market volume of almost 3x10 6 tons per year. Although
its importance, relatively few attempts have been made to produce them with microorganisms. Currently 100% of acrylic
acid is produced from fossil fuels.
Acrylic acid might become an important target for fermentative production from sugar cane, as an alternative carbon
sources to its current production from petrochemical. The production from renewable resources is propagated via lactic
acid fermentation and subsequent chemical conversion to acrylic acid.
This work is concerned with the simulation of the recovery and purification of acrylic acid from the mixture produced from
sugar cane bagasse through chromatographic adsorption. Chromatographic adsorption is a natural choice to follow a bio
production process due to its low energy needs and potentiality for separating high added value product. These simulations were carried out with the developed rigorous simulator, named ADCRO.
The main results were the obtention of elution peaks of binary and multicomponent mixtures, simulated applying different
numerical methods. This allows exploring many separation possibilities that might come up from slight variations of production process. These simulations take into account feed composition change effect, height of the column, and surface
velocity.
200
poster 5-43
Optimization of tocopherol concentration process from SODD using response surface methodology
Vanessa M. Ito*, César Benedito Batistella, Maria Regina Wolf Maciel
State University of Campinas, Faculty of Chemical Engineering, Campinas, Brazil, PO Box 6066, Zip code 13084-970.
The recovery of the unsaponifiable fraction of the residues from the refining of vegetable oils is of great commercial interest, due to the fact that, in many cases, the “valuable products” have vitamin activities such as tocopherols (vitamin E);
as well as, anticarcinogenic properties such as sterols. The soybean oil deodorized distillate (SODD) is a product derived
from the refine of edible soybean oil.
Molecular distillation has large potential to be used in order to concentrate tocopherols, since it uses very low levels of
temperatures due to the high vacuum and short operating time for separation and, also, it does not use solvents. Then it
can be used to separate and purificate termosensitive material as well as vitamins.
In this work, the molecular distillation process was applied for tocopherol concentration, and response surface methodology (RSM) was used to optimize free fatty acids (FFA) elimination and tocopherol concentration in the residue and the
distillate streams, both of which are product of the molecular distiller. The independent variables studied were feed flow
rate (Q) and evaporator temperature (T). The experimental range was 4 to 12 ml/min for Q and 130 to 200 °C for T.
It can be noticed that the Q and T are important operating variables in the FFA elimination. To decreasing the FFA loss,
in the residue stream, the operational range should be dislocated, increasing the evaporator temperature and decreasing the feed flow rate. D/F ratio increases with the increasing T and decreasing Q. It was obtained high concentration of
tocopherols at low values of feed flow rate and high evaporator temperature.
poster 5-44
Effect Of Solids Concentration On Simultaneous Saccharification And Fermentation Of Steam Exploded
Wheat Straw For Ethanol Production
J. Miguel Oliva, Ignacio Ballesteros, M. José Negro, Paloma Manzanares*, Felicia Sáez, Mercedes Ballesteros
Renewable Energies Department-CIEMAT, Avda. Complutense, 22, 28040-MADRID SPAIN
Phone 34 91346 67 37 E-mail: [email protected]
In a biomass to ethanol process that uses steam-explosion technique as pretretament, the utilization of whole pretreated
material (both soluble and insoluble water solids) is an interesting way to avoid expensive equipment for solid/liquid
separation, reduce the amount of wastewater produced and attain higher carbohydrate concentrations in the fermentation
media. However, it involves high toxic compounds loading in the fermentation media.
In this work the whole slurry obtained from steam explosion of wheat straw at previously selected optima pretreatment
condition (220ºC and 2.5 minutes) was used as substrate in a simultaneous saccharification and fermentation (SSF)
process using commercial cellulases and baker’s yeast Saccharomyces cerevisiae.
Enzymatic hydrolysis (EH) and SSF experiments were performed at pH 4.8 on slurry from wheat straw pretreatment
containing total solids (TS) concentrations from 16 to 2% (w/v). Results show significant EH yields in all conditions tested.
However, SSF was inhibited when using slurry with TS higher than 10% (w/v), indicating elevated fermentation inhibition
at these conditions of high toxic compounds loading and low inoculum size (0.3 g/l).
In order to optimize SSF, further experiments were performed in slurries with 8-12% TS, at different pH and enzyme loadings using higher inoculum loading of 1 g/l. Fed-batch process configuration was also assessed as a way to increase final
solid content in SSF. The results from this study will be presented
201
poster 5-45
Green Fluorescent Protein Extraction from E. coli Cell Lysate Using Two-Phase Aqueous Micellar Systems
Priscila Gava Mazzola *, Adalberto Pessoa Jr., Thereza Christina Vessoni Penna
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science,
University of São Paulo, SP, Br.
Henry Lam, Daniel I. C. Wang, and Daniel Blankschtein
Department of Chemical Engineering, Massachusetts Institute of Technology; Cambridge, Massachusetts, EUA.
Mojgan Kavoosi, Charles A. Haynes
Biotechnology Laboratory and The Department of Chemical and Biological Engineering,
The University of British Columbia, Vancouver, British Columbia, Canada.
It has recently been proposed that Green Fluorescent Protein (GFP) can be utilized as a biological indicator to verify the
effectiveness of sterilization processes in various clinic applications. The aim of this study was to investigate a potentially
scalable and effective new method to purify recombinant GFP from Escherichia coli culture media by extraction using
two-phase aqueous micellar systems. Bioseparations have been conducted in different types of two-phase aqueous
complex-fluid systems. In particular, two-phase aqueous micellar systems exploit the fact that some aqueous micellar solutions, under appropriate conditions, can spontaneously separate into two water-based liquid phases between
which proteins and other biomolecules can distribute unevenly. A novel affinity tag family 9 carbohydrate-binding module
(CBM9) was fused to GFPuv by molecular engineering, and the resulting fusion protein was affinity-extracted in a decyl
β-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this simple system, the surfactant C10G1 acts simultaneously as the phase-forming surfactant and as the affinity surfactant. Buffered solutions, each with a total volume of
3 mL, were prepared in graduated 10-mL test tubes. In the system the surfactant final concentration was 0.05 g C10G1/g
total (5% of surfactant) and the cell homogenate was added, accordingly. The solutions were well mixed and equilibrated
at 4oC for each solution to exhibit a clear and homogeneous single phase. Subsequently, the solutions were placed in a
thermo-regulated device, previously set at the temperature of 29oC. Solutions were maintained at that temperature for at
least 6 h to attain partitioning equilibrium. After partitioning equilibrium was attained, the two coexisting micellar phases
formed were withdrawn separately with great care, using syringe and needle sets, and the protein concentration in each
phase was determined. The determination of CBM9-GFP concentration in aqueous surfactant solutions was carried out
by fluorimetry in a spectrofluorometer (Ex=395nm and Em=508nm). Total protein concentration (other than CBM9-GFP)
was analyzed using the bicinchoninic acid (BCA) method. GFP from the cell lysate is extracted preferentially into the
micelle-rich phase by virtue of affinity interactions. The GFP partition coefficient was found to be unaffected by the presence of the impurities, and to be equal to the partition coefficient obtained using purified GFP. Measurements of the total
protein concentration in the micelle-poor phase indicates that the majority of the contaminating proteins can be removed
in just one partitioning step.
202
poster 5-46
Influence of pH in the Production of Biopolymer of Sphingomonas capsulata ATCC 14666 and
Rheological Characterization
Larissa Tonial Vanzo, Aniassa Babicz; Lisandra Rigo; Francine Ferreira Padilha*
Department of Food Engineering, URI – Campus de Erechim, Av. Sete de Setembro 1621, Erechim, RS, 99700-000,
Brazil
Adilma Regina Pippa Scamparini
Department of Food Science, UNICAMP, CP 6121, Campinas, SP, 13083-970, Brazil
Sphingomonas sp, produced diutan gum is a more efficient viscosifier than xanthan and welan gums at ambient temperature in most low concentration salt systems. The main objective of this work was available the influence of the pH in the
fermentation and relationship with the production and rheological characterization of biopolymer by Sphingomonas capsulata ATCC 14666 using conventional medium in rotary shaker at 28ºC and 72h, at 200 rpm. The production of biopolymer in conventional medium was carried out using full 22 factorial design with 3 replicates at the center point to establish
the optimum conditions of each independent variable (pH and fermentation time). The rheology of the fermentation broth
was analyzed by apparent viscosity and the polymer was recovered with ethanol (1:3, v/v). After its recovery, the productivity evaluation was performed. The productivity increased in pH 11.0. The viscosity analysis was performed for aqueous
solutions 3%, at 25, 45 and 60ºC. The polysaccharides synthesized in different pH 9 and 11 largely indistinguishable from
native xanthan gum (pH 7.0). The rheology (apparent viscosity) presented similar values compared to those of the literature for other biopolymers in pH 7.0.
poster 5-47
Production and Characterization of the Xanthan Gum Produced from Xanthomonas campestris pv
magiferaendicae 1230 using Conventional and Industrial Media
Ieda Rottava; Edicléa Mayeski; Fernanda Rauber; Francine Ferreira Padilha*
Department of Food Engineering, URI – Campus de Erechim, Av. Sete de Setembro 1621, Erechim, RS, 99700-000,
Brazil,
Yoko Rosato; Adilma Regina Pippa Scamparini
Department of Food Science, UNICAMP, CP 6121, Campinas, SP, 13083-970, Brazil
The main objective of this work was available the influence of the fermentation media in the production and relationship
with the chemical characterization of the biopolymer synthesized by Xanthomonas campestris pv magiferaendicae 1230.
The fermentation was realized in rotary shaker at 28ºC and 96h, at 200 rpm, using conventional and industrial media. The
chromatographic characterization of the polymers was realized in HPLC with refractive index detector, the eluent was acetonitrile:water (75/25 p:v) at a flow-rate of 0.5 mL/min, 80ºC for monosacharides. For acid analysis was used diode array
detector, the eluent was 0.6 mL/min of aqueous perchloric acid solution (pH 1.9) at 50ºC. The best productivities was obtained using molasses 6% (wt/v) (0.13 g/Lh), molasses 4% (wt/v) (0.11g/Lh), conventional (0,105067 g/Lh) and molasses
2% (wt/v) (0.06 g/Lh), respectively. The chromatographic analysis showed the different composition of monosaccharides
for the polymers synthesized by strain 1230 and commercial xanthan: glucose, mannose, ramnose and glucuronic acid
in both media. The rheology presented similar values compared to those of the literature for other biopolymers; the best
apparent viscosity was obtained using gum synthesized with molasses 4% at 25ºC, 1373 cP (centipoise).
203
poster 5-48
Improved Sampling for Biotechnological Systems
Carina J. Pedersen* & Kim H. Esbensen
ACABS, Aalborg Universitet Esbjerg, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark
Phone: +45 79 127702, email: [email protected]
Process monitoring of fermentation processes is very often based on inadequate, non-representative sampling techniques, which do not comply the fundamental principle in the Theory of Sampling (TOS), creating large, unavoidable
– indeed uncontrollable errors; sampling errors can easily reach 50-100 x the analytical error. Even so, measurements
obtained by such methods are used for process control and important decision makings. Especially in systems containing
significantly heterogeneous materials like bio-refineries, these errors may be completely dominating, making the process
less cost effective and - competitive in the market
TOS analyses sampling scenarios and indicates solutions for all complicated systems and materials, and has a.o. proved
that the only way to obtain representative samples is to sample in vertically flowing streams. Based on this a new innovative sampling approach, applying recurrent looping, has been constructed. This approach also allows optimal application
of PAT (Process Analytical Technologies).
Results from extensive laboratory scale experiments on both model and real-world bio-technological systems are presented, documenting highly significant improvements in both mixing efficiency and sampling representativeness.
poster 5-49
Performance of Continuous Detoxification of Dilute-acid Hydrolyzates by Ca(OH)2
1
Ronny Purwadi*1,2, Mohammad J. Taherzadeh1
School of Engineering, University of Borås, 501 90 Borås, Sweden
2
Dept. of Chemical Engineering and Environmental Science,
Chalmers University of Technology, 412 96 Göteborg – Sweden
Continuous detoxification of dilute-acid hydrolyzates using Ca(OH)2 (overliming) were examined. The examination involved adjusting the pH of detoxification to 9, 10, 11 and 12 at different dilution rates of 0.216, 0.432 and 0.648 h-1. The
detoxified hydrolyzates was evaluated by batch fermentability test as well as cultivated continuously by immobilized
Saccharomyces cerevisiae in alginate beads. In addition, the concentration of total phenolic compounds and detoxification precipitate were also examined. Mathematical model of furfural, HMF and sugar degradations were developed and it
seems that the decomposition of furfural and HMF are much favorable than fermentable sugars regarding the detoxification pH. However, accumulation of furfural and HMF occurred by the dilution rate amplify the inhibitory effect of the hydrolyzates. Moreover, the concentration of phenolic compounds is increased at high detoxification pH. The fermentability of
detoxified hydrolyzates, in large extent, are depended by the amount of furans, while phenolic compound contributes less
toxic effect due to its concentration. The developed model can be used in designing an optimum detoxification condition
to suppress the toxicity of hydrolyzates as well as to reduce Ca(OH)2 usage.
204
poster 5-50
Semi-Continuous Production of Lactic Acid from Cheese Whey Using Integrated Membrane Reactor*
Yebo L1*i, Abloghasem Shahbazi1, Michele M. Mims1
1. Bioenvironmental Engineering Program Department of Natural Resources and Environmental Design, North Carolina
A&T State University, 1601 East Market Street, Greensboro, NC 27411
E-mail: [email protected] Phone: 336-334-7787; fax: 336-334-7270
The major accomplishment from our previous research is that lactic acid could be produced at high yield using free cells
of Bifidobacterium longum (B. longum) from cheese whey without any nutrient supplement, which could substantially reduce the production cost of lactic acid. An ultrafiltration and nanofiltration membrane unit was also successfully developed
and tested to separate the cells, proteins, and lactose from lactic acid. In this current study, semi continuous production of
lactic acid from cheese whey using membrane integrated bioreactor toward commercial production was studied.
An integrated membrane reactor was developed and tested for semi continuous production of lactic acid from cheese
whey. The lactic acid is harvested every 24 hour using a nanofiltration membrane unit. The cells and unutilized lactose
are kept in the reactor and converted together with new added cheese whey in the subsequent run. There was no negative effect on the viability of cells was observed as a result of applying this membrane separation. The fermentation
can be run semi continuously for five days without any significant affect on performance. Compared to the reactor only
system, the membrane integrated bioreactor has much higher lactic acid productivity. The outcome of this research will
enhance the utilization of cheese whey as an industrial material, which will create a significant impact on the local economy and environment.
poster 5-51
Using Cheese Whey to Produce Pencillin G Acylase by Bacillus megaterium ATCC 14945
Vanessa R. Souza e Raquel L. C. Giordano*
Departamento de Engenharia Química, Universidade Federal de São Carlos,
13.565-905, C.P. 676, São Carlos, SP, Brazil (e-mail: [email protected])
Penicillin G Acylase is an important industrial enzyme used in the β-lactam antibiotics production. Cheese whey, a byproduct of the dairy industry, contains a great amount of nutrients, among them proteins (7 g/L). The presence of this
by-product in the medium is very important for the production of penicillin G acylase by Bacillus megaterium. However,
the high amount of proteins in this nutrient makes more difficult the downstream process. In this work, it was studied
how these proteins could be eliminated or modified without losing the good properties of cheese whey. Bacillus megaterium was cultivated in flasks runs, at 300 rpm and 30ºC, in a medium containing: free amino acids (10,0 g/L), potassium
phenylacetate (3,5 g/L), salts (0,22 g/L) and cheese whey – powder and “in natura” after different pre-treatments: (1)
ultrafiltration through a 10 KDa cut-off membrane; (2) thermal coagulation, 90ºC, and (3) hydrolysis using immobilized
chymotrypsin, two different hydrolyses degrees (HD). The best results were found using hydrolyzed cheese whey (14%
HD). The use of this hydrolysate will facilitate the separation of the produced PGA. The mixture of amino acids and small
peptides resulting from the hydrolysis will be an important carbon and nitrogen source for enzyme production.
205
poster 5-52
Microbial Community Changes in a Mixed Microbial Consortia Producing PHA from Waste Carbon Sources
Erik R. Coats1, William A. Smith2, Frank J. Loge3, Michael P. Wolcott4, and David N. Thompson2*
1
Department of Civil Engineering, University of Idaho, Moscow, Idaho 83844-1022
2
Department of Biological Sciences, Idaho National Laboratory, Idaho Falls, Idaho 83415-2203
3
Department of Civil & Environmental Engineering, University of California, Davis, CA 95616
4
Department of Civil and Environmental Engineering, Washington State University, Pullman, WA 99164-2910
* Corresponding author: phone: (208) 526-3977, fax: (208) 526-0828, email: [email protected]
Polyhydroxyalkanoates (PHAs), naturally-occurring biological polyesters that are produced from a myriad of carbon
sources, can be utilized as biodegradable substitutes for petroleum-derived thermoplastics. Current PHA commercialization schemes are limited by high feedstock costs, the requirement for aseptic reactors, and high separation and purification costs. PHA-producing bacteria indigenous to municipal waste streams can accumulate large quantities of PHA under
environmentally controlled conditions; hence, a potentially more cost-effective method of PHA production would utilize
these consortia to produce PHAs from inexpensive waste carbon sources. However, few reports on PHA production,
characterization and optimization in mixed microbial consortia exist. In this study, PHA production was accomplished in
sequencing batch bioreactors utilizing mixed microbial consortia from municipal activated sludge as inoculum and forest
products and municipal wastewaters as feedstocks. PHA production averaged 85 and 53% of the dry cell weight from
methanol-enriched pulp-and-paper mill foul condensate and from fermented municipal primary solids, respectively. The
PHA-producing microbial consortia were examined to explore the microbial community changes that occurred during
reactor operations, employing denaturing gradient gel electrophoresis (DGGE) of 16S-rDNA from PCR-amplified DNA extracts. Distinctly different communities were seen after enrichment, with total numbers and dominant members depending on both feedstock and hydraulic residence time.
poster 5-53
Xanthan gum production by Xanthomonas campestris pv manihotis 1182 using Cheese Whey
Marceli Fernandes Silva, Rejane Gollo Fornari, Francine Ferreira Padilha, Helen Treichel
Department of Food Engineering , Av. Sete de Setembro 1621, Erechim, RS, 99700-000, Brazil
Adilma Pippa Scamparini
Department of Food Science/FEA/UNICAMP
Cidade Universitária Zeferino Vaz – Caixa Postal 16121, SP, 13083-862, Brazil
Cheese whey contains many proteins with high biological value. However it is treated as residue and inappropriately
discarded, what may cause damages to environment, and may be considered as an unacceptable waste of a valuable raw material, due to its nutritional properties. Xanthan gum is a polysaccharide produced by bacteria of the genus
Xanthomonas and is of great commercial importance. They find application in food, pharmaceutical and petrochemical
industry for their property to yield viscous solutions at a low concentration (0.05 – 1.0%) and to present high stability at a
broad pH and temperature range. These polymers have been used in food industry around the world. Their application is
approved by FDA (Food and Drug Administration). The aim of this work was to produce xanthan gum using cheese whey
as substrate. Experimental design methodology were performed and the maximum productivity obtained was 0.331 g/L.h.
The rheology (apparent viscosity) presented similar values compared to those of the literature for other biopolymers.
206
poster 5-54
The Effect of Temperature on Ethanol Production from Concentrated Solka Floc in a Three-Liter Bench Scale
Bioreactor
Byung-Hwan Um and Thomas R. Hanley*
Department of Chemical Engineering, Auburn University, Auburn, Alabama 36849
Phone: 334-703-0594; Fax: 502-228-0161; Email: [email protected]
To obtain the least five percent (v/v) ethanol production needed for an economically viable industrial-scale ethanol distillation, high carbonate concentration is required. High carbonate concentration can only be achieved with high initial
cellulose concentration combined with a favorable conversion yield of cellulose into soluble sugars. Many researchers
have reported repeatedly that solid concentrations above 10 percent resulted in poor ethanol yield due to inefficient mass
transfer and to the different operating temperatures required for enzymatic hydrolysis and fermentation.
To develop data for a full scale design, enzymatic hydrolysis of concentrated solka floc is evaluated at three operating
temperatures (30oC, 35oC and 40oC) in a three-liter bioreactor. The effects of mixing were evaluated using computational
fluid dynamics (CFD) simulations of the three-liter reactor.
poster 5-55
Effects of saccharification and particle size on the rheology of biomass slurries at high solids concentrations1
Sridhar Viamajala*, Daniel J. Schell, James D. McMillan, Richard T. Elander
National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401, E-mail:
[email protected], Ph: 303-384-7717
In this study, we report on the rheological characterization of dilute acid pretreated corn stover (PCS) slurries at high solid
concentrations. Using plate-plate type measurements under a continuous shear, we characterized the rheology of PCS
slurries as a function of insoluble solids concentration (up to 40%), extent of pretreatment (up to 75% removal of xylan)
and particle size (-20 and -80 mesh). Results show that PCS slurries are yield stress materials that exhibit shear thinning
characteristics which can be described using a Casson model. Further, results demonstrate that the apparent viscosity
and yield stress increase with increasing solids concentration (which corresponds to a decrease in free water) and that
dilute acid pretreatment, as well as the presence of smaller sized particles, improve rheological properties. Taken together, these results are consistent with the hypothesis that the availability of free water in the slurry plays a significant role in
determining its rheological behavior. In other words, as the solids concentration increases, lack of free water in the slurry
likely causes formation of a network-like structure among biomass particles that increases the apparent viscosity and
yield stress of the slurry. As shear rate increases, disruption of this network likely results in shear thinning behavior.
This work has been authored by an employee of the Midwest Research Institute under Contract No. DE-AC3699GO10337 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting
the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable,
worldwide license to publish or reproduce the published form of this work, or allow others to do so, for United States Government purposes.
1
207
poster 5-56
Separating a Mixture of Egg Yolk from Egg White Using Foam Fractionation
Tiffany M. Ward and Robert D. Tanner, *
Chemical Engineering Department, Vanderbilt University , Nashville, TN 37235
The product created by mixing an egg yolk with an egg white from the same egg can serve as a binary system for testing to see how well foam fractionation can be used to separate two different groups of proteins naturally found together.
This mixture is particularly attractive for such a study because the two groups can be visualized distinctively when in their
separated states. It has been shown that air alone and, with little or no water added, can effect visually clean separations
of egg yolk from egg white . The white precedes the yolk in the process which takes less than 10 minutes at a laboratory
scale. No additives other than air were needed to accomplish this separation making this a “Green” separation process.
poster 5-57
Conditioning and Glucose/Xylose Cofermentation of Pretreated Lignocellulosic Biomass
Ryan Warner1,2, Miroslav Sedlak1, Nancy Ho1, and Nathan S. Mosier1,2*
1 Laboratory of Renewable Resources Engineering, Purdue University
2 Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907
Pretreatment of lignocellulosic biomass, while improving enzymatic digestibility, can also produce fermentation inhibitors.
Two important inhibitors, furfural and HMF, are formed from the degradation of carbohydrates from lignocellulose. Both
furfural and HMF can decrease the fermentability and the ethanol yields for sugars derived from lignocellulose. Thus,
pretreated material may require conditioning to either remove or otherwise detoxify these inhibitors. This paper explores
some conditioning methods on hydrolysates obtained from corn stover and poplar pretreated by dilute acid, controlled
pH liquid hot water, SO2 steam explosion, and others. The effects of these conditioning methods on the subsequent
fermentation of both glucose and xylose by the recombinant yeast S. cerevisiae 424A(LNH-ST) is presented. Overliming
the pretreated corn stover to pH 9 or higher removes 100% of the HMF and furfural present in corn stover hydrolysates.
However, the fermentation is negatively affected, producing only 53% of theoretical ethanol yield as opposed to 82% yield
from the unconditioned material. Hydrophobic resins (Amberlite XAD2, XAD4, and XAD7) were also examined for their
ability to remove HMF and furfural. The resins were able to remove 100% of furfural and approximately 60% or more
HMF. The yield from fermentation was 87%; slightly better than the unconditioned corn stover hydrolysate.
208
poster 5-58
Molecular Imprinting in Silica Matrices in presence or absence of β-cyclodextrin
Flávio F.de Moraes, Gisella M. Zanin*
Chemical of Chemical Engineering, Universidade Estadual de Maringá, Maringá-PR, Brazil, TN+55 (44)32614745
Cleide M. F. Soares, Heizir F. de Castro,
Chemical of Chemical Engineering, Faculdade de Engenharia Química, Lorena-SP, Brazil,
TN +55 (12)31595116
Inorganic gels matrices can be prepared from silicon derived alkoxides through sol-gel polycondesation in the presence or absence of β-cyclodextrin (β-CD). In this case, the alkoxides function as crosslinkers and give rise to the matrix
network through sol-gel polycondesation. Cholesterol removal was studied in this work with three different hydrophobic
matrices: molecular imprinting polymer/β-CD-cholesterol (MIP/β-CD-cholesterol), MIP/cholesterol and Pure matrix.
MIP/β-CD-cholesterol as produced by the inclusion of target molecule (cholesterol) in host molecule (β-CD), followed by
molecular imprinting of the assembled complex in the hydrophobic matrix. MIP/cholesterol is made by direct molecular
imprinting of in the hydrophobic matrix in the absence of β-CD. The efficiency for cholesterol removal from a solution was
analyzed by adsorption tests at different concentrations (1 to 10 mg/mL) for the two hydrophobic matrices. Cholesterol
was removed from solution at 25°C using 1% (w/v) solid/liquid suspensions during 24h. The experimental data on cholesterol adsorption was well fitted by the Langmuir isotherm model with a maximum adsorption capacity of cholesterol 0.528,
0.515 and 0.0408g cholesterol/g-adsorbent, for MIP/β-CD-cholesterol, MIP/cholesterol and Pure matrix, respectively.
The enhanced adsorption capacity observed for the MIP/β-CD-cholesterol matrix is attributed to the presence of the
β-CD moieties in the network, wich allows the formation of an inclusion complex between β-CD and the host molecules.
Molecular imprinting is a new technology in solid-phase extraction and the use of imprinted hydrophobic matrices has application on the removal of cholesterol from lacteous products.
209
poster 6-06
An Environmentally-Friendly Biorefinery for the Production of Fuel-Grade Ethanol and Value-Added
Co-Products from the Uruguayan Rice Industry
Alex Berlin*, Vera Maximenko, Neil Gilkes, Jeff Keating, Warren Mabee,
Russell Chedgy, Álvaro Álvarez, Patricia Acosta, Roberto Horta, Ricardo Ortiz, Jack Saddler
Concerns about diminishing fossil-fuel resources, national energy security, and excessive production of greenhouse
gases continue to motivate the search for alternatives to petroleum. Lignocellulosic biomass contains large amounts of
polymeric carbohydrates that represent an attractive source of sugars to produce alternative fuels and other chemical
commodities. Potential feedstocks include agricultural residues such as corn stover, rice and wheat straw, “purposegrown” energy crops such as hybrid poplar, and hardwood or softwood wastes from the forest industry. Bioconversion is a
particularly attractive option for small countries like Uruguay that have economies based on agriculture and cattle breeding and are fully dependent on foreign oil.
Uruguay is the largest rice producer in Latin-American (FAO, 2004) and has an emerging forest industry. Uruguay has
about 200,000 ha of rice under cultivation which generate ~1.750.000 tons of rice straw and ~200.000 tons of rice hulls
annually. Currently, these residues are waste-products and the rice hulls are causing significant environmental problems.
However, rice straw and rice hulls are potential feedstocks for the production of fuel ethanol and other valuable commodities. This presentation describes technical, economical and environmental aspects of an integrated biorefinery strategy
that uses Uruguayan rice residues for the production of energy, fuels and added-value co-products. The study is presented in the context of parallel investigations into the use of other cereal straws for bioconversion.
poster 6-07
Potential for Integrated Bioethanol and Biogas Production from High Dry Matter Olive Pulp
Tania I. Georgieva*, Birgitte K. Ahring
Environmental Microbiology & Biotechnology Group, BioCentrum-DTU, Technical University of Denmark, Building 227,
DK-2800 Lyngby, Denmark
The olive oil industry is the largest agrindustrial sector in Southern Europe (i.e. Greece, Italy, Spain and Portugal) which is
the main olive production region, about 75% of global annual production. One major environmental issue in this region is
the generation of highly polluting olive mill wastewater from traditional, three-phase industrial olive processing. Recently,
employment of two-step-centrifugation process for extraction of olive oil has resulted in generation of a semi residue
called olive pulp. Olive pulp (OP) contains a high amount of carbohydrates (cellulose and hemicellulose) and lignin, and
thus it could be used as a renewable energy source for production of bioethanol, biogas (methane) and electricity.
The presentation will focus on utilization of olive pulp for production of bioenergy (bioethanol and biogas). Enzymatic
pre-treatment of high dry matter concentrations (20-31% DM) of olive pulp with different enzyme loadings shows that
enzymatic hydrolysis is independent of the DM concentration of the OP. Results from simultaneous saccharification and
fermentation (SSF) using baker’s yeast Saccharomyces cerevisiae as well as a glucose/xylose co-fermentation with the
thermophilic anaerobic bacterial strain HY10 and subsequent methane production from the effluent will be included in the
presentation. Finally, economic evaluation of the whole process will be presented.
210
poster 6-08
Testing Performance of an Integrated Corn Stover to Ethanol Process
Edward W. Jennings*, Daniel J. Schell
National Bioenergy Center, National Renewable Energy Laboratory
1617 Cole Blvd., Golden, CO 80401
The objective of this study was to generate performance data for an integrated corn stover to ethanol process based on
dilute acid pretreatment and enzymatic cellulose hydrolysis. Pretreated corn stover hydrolysate was produced in a pilotscale pretreatment reactor. Liquor was extracted from the pretreated slurry and conditioned by an “overliming” process.
The liquor was then recombined with the pretreated cellulosic solids and the resulting slurry was subjected to enzymatic
cellulosic hydrolysis followed by fermentation with a glucose/xylose fermenting Zymomonas mobilis. Shake flash studies
investigated the effect of enzymatic saccharification time and initial cell density on ethanol yield and rigorous performance
data backed up by mass balances were obtained in 1 L vessels. Good cellulose conversion (95%) was achieved but ethanol yields were low (50%) because of poor xylose utilization by the microorganism. Ethanol yields were improved 25% to
50% by inoculating the fermentation at higher initial cell densities.
poster 6-09
Impact of Recycle Water on Fermentation of a Dilute Acid Pretreated Corn Stover Hydrolysate
Ali Mohagheghi* and Daniel J. Schell
National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401
The current biorefining industry (corn starch conversion) uses recycle water (thin stillage) to reduce fresh water usage
thereby reducing cost. However, high recycle water use can negatively impact fermentation performance due to build up
of inhibitors. The objective of this study was to assess the impact of recycle water use on performance of a corn stover
to ethanol conversion process. A glucose/xylose fermenting Zymomonas mobilis was used to ferment the liquid portion
of a dilute acid pretreated corn stover hydrolysate conditioned by overliming. Stillage was recycled three times to achieve
steady state concentration of inert components and performance was evaluated as a function of the effective total solids
concentration of the hydrolysate liquor and fraction of the stillage recycled. Fermentation performance was unaffected
by recycle water at low solids concentration (15%), but high recycle water use (25% of the stillage recycled) significantly
affected performance at the highest solids concentrations (25%). Fermentation of pure sugar control containing the same
level of acetic acid as in the hydrolysate liquor revealed that other components besides acetic acid are inhibiting the
microorganism. Results shows that recycle of 25% or higher levels of stillage will be problematic without development of
more robust fermentation microbes and/or more effective hydrolyzate detoxification methods.
211
poster 6-10
Plant Design and Economical Evaluation of an Ethanol Production Process from Sugar Cane Bagasse:
A Brazilian Conception
Luiz A. F. S. Schlittler*, Carlos A. G. Perlingeiro.
Chemical School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, CEP: 21949-900
Nei Pereira Jr.
Bioprocess Development Laboratory, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, CEP: 21949-900
A group of Brazilian researchers has developed a technology to produce ethanol from sugar cane bagasse. Based upon
this development an industrial plant was designed to perform such process as well as to evaluate its feasibility. This
process is now being further studied for scaling up to a pilot plant by the Brazilian oil company. It emphasizes Brazilian
distilleries structure, the sugar cane agriculture and its residue generation, raw material logistic, process engineering,
economical analysis of the process and its potential of implementation. (83)
In this work, the two stream model, that consists of processing separately the cellulosic and hemicellulosic fractions, was
used. Hemicellulose is first separated by thermal hydrolysis, followed by fermentation with Picchia stipitis. The remaining solid residue, so called cellulignin, is treated to remove the lignin, and the cellulose is processed by SSF, starting with
commercial enzymes and fermented by Saccharomyces cerevisiae. The adoption of a very common concept in Brazilian
sugar and alcohol industries, called attached distillery, reduced drastically the capital, operating and mainly raw material
transportation costs, since both process have some common operations. This work shows an evolution in terms of costs,
comparing to what has been shown on the literature, and will be useful to locate the process development stage and help
to guide future efforts to the consolidation of this technology.
poster 6-11
Microwave Pyrolysis of Corn Stover
Fei Yu1, Alf Inge MyhreTunheim1,2, Xiangyang Lin3, Yuhuan Liu3, Paul Chen1, and Roger Ruan1,3,*
1 Center for Biorefining, Department of Biosystems and Agricultural Engineering, University of Minnesota, 1390 Eckles
Ave., St. Paul, MN 55108, USA
2 Dept. of Environmental Engineering, The Norwegian University of Life Sciences, Norway
3 MOE Key Laboratory of Food Science, Nanchang University, Nanchang, China
This study is to develop a novel pyrolytic process for the production of gas and liquid biofuels from agricultural wastes. The new
process is an environmental friendly technique in which energy transfer to the waste occurs via microwave radiation. Microwave
pyrolysis has several advantages over conventional pyrolytic processes and represents a new opportunity to use pyrolysis to
process biomass and municipal solid wastes as it overcomes the major difficulties that arise from other alternative methods.
The microwave pyrolysis process can be optimized to produce gas or liquid fuels, e.g. about 45% gas, 30% liquid, and 25%
char residue. The organic pyrolytic volatile will be condensed to produce liquids and gas hydrocarbon fractions that can be
used in power generation. The yields and chemical properties of individual phases will be determined. Effect of microwave
absorbers, such as the carbon residue produced in the pyrolysis itself or activated charcoal, as catalysts will be evaluated.
212
poster 6-12
Physical and Chemical Characteristics of Liquids from Microwave Pyrolysis of Corn Stover
Fei Yu1, Xiangyang Lin1,2, Yuhuan Liu1,2, Paul Chen1 , and Roger Ruan1,2,*
1 Center for Biorefining, Department of Biosystems and Agricultural Engineering, University of Minnesota, 1390 Eckles
Ave., St. Paul, MN 55108, USA
2 MOE Key Laboratory of Food Science, Nanchang University, Nanchang, China
Biomass pyrolytic liquid fuels differ from petroleum based fuels in both chemical composition and physical properties.
Pyrolytic liquid fuels contain more water, have low heating values, are acidic, and usually unstable in air. Characterization
of the physical and chemical properties of pyrolytic liquid fuels are critical to the development of processes to upgrade the
pyrolysis liquid fuels.
In this research corn stover will be pyrolyzed using a microwave reactor. The objective of this study is to test the applicability of the standard testing methods developed for petroleum-based fuels to the microwave pyrolytic liquid fuels. The
water content, pH, ash, minerals, CHNOS ratio, viscosity, and heating value of the pyrolytic liquid fuels will be evaluated.
The stability of the pyrolytic liquid fuels will also be assessed in this study.
213
Author
A
Abifarin, T.
Aboka, F.
Abrahamson, L.P.
Achampong, N.S.
Acosta, P.
Adapala, R.
Adney, W.S.
Adsit, G.S.
Agblevor, F.A.
Agbogbo, F.K.
Ahmad, R.
Ahn, J.-S.
Ahn, Y.
Ahring, B.K.
Akin, D.E.
Alamuti, S.
Aldiguier, A.S.
Alfenore, S.
Alhadeff, E.M.
Ali, M.K.
Alizadeh, H.
Allain, E.J.
Alles, C.
Almeida de Silva, J.B.
Almeida, J.R.M.
Almeida, R.M.R.G.
Alriksson, B.
Altman, E.
Alvarez, A.
Alvarez, M.E.T.
Alves, T.L.M.
Alves-Prado, H.F.
Anderson, L.
Andersson, A.
Andrade, R.R.
Antunes, O.A.C.
Armstrong, R.
Arora, M.B.
Asthana, N.
Atala, D.I.P.
Atala, D.P.
B
Babicz, A.
Baeza, J.
Bai, Y.
214
Abstract(s)
2-09
5-22
1B-07
2-10
6-06
5-07
1A-05, 1A-11, 1A-53
3B-05
3A-06
4-08
1B-05
1A-09
4-29
6-07
1A-01
2-15
5-02
3A-11, 5-02
1A-10
1A-11
1A-43
1A-07
STA-04
3A-09
4-26
5-08
2-32, 2-33
4-23, 4-40
6-06
3A-07, 5-42, STB-02
5-23
1A-12, 1A-13
STB-05
2-13, STB-05
5-05, 5-16
1A-22, 1A-23
1A-55
1A-14
5-09
5-25
5-05, 5-16
5-46
2-15
4-18
Author
Baida, L.C.
Bailey, R.
Baker, J.O.
Bakhshi, N.N.
Bakker, R.
Balan, V.
Ballesteros, I.
Ballesteros, M.
Bals, B.
Balsalobre, M.A.A.
Balster, L.M.
Bardsley, J.
Batistella, C.B.
Bauer, S.
Beeftink, R.
Bellissimi, E.
Benemann, J.
Benko, Z.
Bennett, N.M.
Berglund, K.A.
Berlin, A.
Bernardes, O.L.
Berson, E.
Berson, R.E.
Bevilaqua, J.V.
Bideaux, C.
Billings, B.
Binnema, D.
Birr, A.S.
Blackwelder, D.B.
Blaschek, H.
Bocchini, D.A.
Boersma, A.
Boettcher, G.C.
Bogalos, P.
Boles, E.
Bombardiere, J.
Bon, E.P.S.
Bond, W.E.
Bonner, E.
Booth, E.
Borole, A.P.
Bos, G.
Boudreau, T.M.
Bowen, L.L.
Bower, S.
Boyer, R.
Abstract(s)
1A-12
5-37
1A-02, 1A-11
5-19
STB-03
2-12
2-11, 2-40, 5-44
2-11, 2-40, 5-44
2-12
1A-27
3A-08
5-40
2-39, 5-43
1B-02
STB-03
4-09
3B-08
2-15
2-14
3A-04
1A-04, 2-15, 2-16, 2-17, 2-18, 6-06
1A-38
2-24
2-63
1A-38, 1A-40
3A-11, 5-02
2-23
STB-03
STA-01
3B-07, 3B-17, 3B-18
2-03
1A-12, 1A-21
STB-03
3B-05
2-39
4-39
5-10
1A-15, 1A-16, 1A-17, 1A-18
3B-17, 3B-18
3B-17, 3B-18
5-11
1A-19, 3A-05
STB-03
5-35
3A-08
4C
4-07
Author
Braccio, G.
Brady, J.
Branco, R.D.F.
Bransby, D.
Breckenridge, C.R.
Brigida, A.I.S.
Brown, R.C.
Brown, S.D.
Bruhn, D.F.
Brumm, P.J.
Brune, D.E.
Buday, J.L.
Budhavaram, N.K.
Bura, R.
Burapatana, V.
Burke, M.
C
Cabral, M.M.S.
Cameleyre, X.
Camelo, A.C.R.
Cameron, K.D.
Campos, J.
Canilha, L.
Cansian, R.L.
Cara, C.
Cardoso, V.L.
Carvalheiro, F.
Carvalho, D.F.
Carvalho, S.M.D.S.
Carvalho, W.
Cascone, R.
Castilho, L.R.
Castro, E.
Cavalcanti, E.D.C.
Cescut, J.
Cha, J-M.
Chaabane, F.B.
Chambliss, C.K.
Chatfield, M.
Chedgy, R.
Chelgren, S.K.
Chen, P.
Chen, S.
Chen, S-F.
Cheng, G.
Cheng, P.
Abstract(s)
Author
3B-09
1A-05
2-53
6-04
3B-07, 3B-17, 3B-18
1A-26
6-03
4-11
3B-07, 3B-17, 3B-18
1A-20
3B-08
4-23
4-12
2-16, 2-17, 2-18
5-11
2-07
Cheng, S.
Cherry, J.
Chi, Z.
Cholewa, O.
Chong, H.
Christensen, B.H.
Chukkapalli, G.
Chundawat, S.P.S.
Chung, C-H.
Chung, D-H.
Chung, H-J.
Cleary, M.
Coats, E.R.
Coelho, R.R.R.
Coleman, W.J.
Converse, A.O.
Cook, J.
Corazza, M.L.
Cortwright, R.D.
Cos, O.
Coseli, E.
Costa, A.C.
Cotta, M.A.
Coward-Kelly, G.
Crofcheck, C.L.
Crowley, M.F.
Culver, D.
Cunha, A.G.
Custodio, A.F.
5-08
3A-11, 5-02
5-23
1B-07
1B-08
3A-09
1A-23
2-48, 2-49
3A-15, 3A-16
3B-11
1A-47
3A-10
3A-09
3A-01
5-31
2-48
1A-25
3A-11
5-12
5-02
2-04, 2-20, 2-27, 2-60
5-10
6-06
3A-08
6-11
1B-02, 2-37, 3A-20, 4-20, 4-21, 5-14
2-04, 2-20, 2-27, 2-60
5-13
6-12
D
Abstract(s)
5-41
2-41
3A-20, 5-14
3A-21, 3A-25, 3A-26, 5-36
4-06
6-05
1A-05
2-21
1A-35, 2-25, 2-35
2-26
3A-24
1A-05
5-52
1A-15
1A-63
3B-02
1A-45
1A-22
3A-12
1A-10
5-28
5-05
1A-34, 1A-61, 2-03
2-06, 4-08
3B-13, 3B-14
1A-05
STA-04
1A-40
5-17
da Costa, A.C.
da Costa, A.C.A.
da Silva Martins, E.
da Silva, A.C.
da Silva, A.J.R.
da Silva, A.S.A.
da Silva, J.N.C.
da Silva, K.O.
da Silva, M.A.P.
da Silva, R.
da Silva, S.S.
da Silva, T.A.
Dadi, A.P.
Dalai, A.K.
5-15, 5-16
3A-14, 5-18
2-22
3A-14
3A-16
1A-15, 1A-17, 1A-18
1A-48
3A-25
1A-37, 1A-39
1A-12, 1A-13, 1A-21, 1A-27, 1A-28, 2-22
2-53
1A-51
2-61
5-19
215
Author
Dale, B.E.
Dale, M.C.
Danielson, N.
Dantas, M.A.A.
Dardugno, D.
Dariva, C.
das Neves, L.C.M.
Dasari, R.K.
Davis, M.
Davis, R.
Davison, B.H.
Day, D.F.
de Alencar Vieira, A.P.
de Almeida e. Silva, J.B.
De Andrade, M.S.
De Arauz, L.J.
de Azeredo, L.A.I.
De Bari, I.
De Bont, J.A.M.
de Carvalho, G.B.M.
de Castro, A.M.
de Castro, H.F. de Franca, F.P.
De Freitas, A.C.
de Gouveia, V.L.R.
de Jong, E.
de la Garza, L.
de Laat, W.T.A.M.
de Lima, A.L.G.
de Lima, C.J.B.
De Matteo, V.
de Moraes, F.F.
de Oliveira, D.
De Queiroz, G.A.
de Souza, M.B., Jr.
de Toledo, V.
DeBari, I.
Decker, S.R.
Delvigne, F.
Den Haan, R.
Den Uil, H.
Dharmadi, Y.
Di Luccio, M.
Dias, P.T.C.
Dien, B.S.
216
Abstract(s)
Author
1A-43, 1B-05, 2-02, 2-03, 2-05, 2-10, 2-12, 2-21, 2-62, STA-02, STAC
2-23
3A-01
4-15
1A-07
1A-22, 1A-23
3A-25, 4-34
2-24
2-44
3A-13
1A-19, 3A-05
1A-35, 2-25, 2-26, 2-35, 3A-24
1A-39
2-42
3A-21
3A-21
1A-16
4-13
4-05, STB-03
2-42
1A-47
5-58, 2-46
3A-14, 3A-15, 3A-16
4-31
5-21
STB-03
1A-14
4-09, 5-22, STB-03
1A-15
3A-15
4-13
2-46, 5-58
1A-23
2-26
5-23, 1A-48
5-28
3B-09
1A-53
5-01, 5-27
4-01
STB-03
4-16
1A-24
1A-37
1A-61, 2-03
Digman, M.F.
Ding, S-Y.
do Nascimento, R.P.
Domaschko, M.
Donghai, S.
Doran Peterson, J.B.
dos Santos, C.A.
Dos Santos, D.T.
Downing, M.
Doyle, E.
Dreschel, R.
Duarte, E.R.
Duarte, L.E.
Duff, S.J.B.
Duffield, J.
Duguid, K.B.
Dunlap, C.A.
Durr, B.
Dutt, D.
E
Eggeman, T.
Ehrhardt, D.
Eiteman, M.
Eivazova, E.R.
Ejeta, G.
Elander, R.T.
Ellmore, B.B.
Elmore, J.
El-Zawawy, W.K.
Emptage, M.
Ender, L.
English, B.C.
Enzo, A.
Esbensen, K.H.
Espinosa-Solares, T.
Esteves, M.P.
Etoc, A.
F
Fang, X.
Felby, C.
Feldmann, K.
Ferdous, D.
Ferguson, B.W.
Ferreira, A.L.O.
Filho, F.M.
Abstract(s)
3B-05
1A-02, 2-47
1A-15
5-10
2-55, STB-04
4-30
3A-27
2-53
Poster C
5-24
2-29
5-25, 5-26
3B-11
2-14
Poster Convener
3B-13, 3B-14
1A-34
4-24
1A-54
3A-02
1B-02
4C, 4-23, 4-40
4-24
1B-04
2-05, 2-44, 5-55
1A-33, 1A-45
1A-45
3A-06
STA-04
5-25, 5-26
3B-20
3B-09
5-48
5-10
3B-11
5-27
4-36
1A-08, 1A-36, 1A-44, 6-05
1B-01
5-19
1B-03
1A-26
5-16
Author
Filho, R.M.
Filho, S.A.
Fillaudeua, L.
Fishman, M.
Flatt, M.
Flavell, R.
Flickinger, M.C.
Flynn, P.C.
Fonseca, R.R.
Fornari, R.G.
Foti, R.A.
Foust, T.
Fregolente, L.
Fregolente, L.V.
Freire, D.M.G.
Friend, J.
Abstract(s)
Author
3A-17, 5-15, 5-16, 5-17, 5-21, 5-25, 5-26, 5-28
3A-10
3A-11
4-30
5-29
1B-01
4-14
3B-19, 3B-10
3A-16
5-53
5-34
6C
2-39
2-39
1A-16, 1A-25, 1A-38, 1A-40, 5-30, 5-31
STA-04
Gorsich, S.W.
Gorwa-Grauslund, M.F.
Gosse, J.L.
Graminha, E.B.N.
Gray, M.
Guez, M.A.U.
Guillouet, S.
Gupta, R.
Gutarra, M.E.L.
Guvenilir, Y.
H
Abstract(s)
4-17
4-02, 4-26
4-14
1A-27, 1A-28
2-50
1A-28
3A-11, 5-02
1A-29
5-31
1A-30, 1A-31
Haan, H.
Hahn-Hagerdal, B.
Hames, B.R.
Hanchar, R.J.
Hanley, T.R.
Hannon, J.
He, Z.
Helle, S.S.
Hendrickson, R.
Hennessey, S.
Henrich, E.
Hess, J.R.
Hicks, K.
Hicks, K.B.
Hiestand, J.
Higgins, R.J.
Hill, G.A.
Himmel, M.E.
Ho, N.
Ho, N.W.Y.
Hodge, D.B.
Holtzapple, M.T.
Horta, R.
Hoskinson, R.L.
Hotchkiss, A.
Hotta, T.
Hu, B.
Hua, L.
Hunter, S.
STB-03
4-02, 4-26, STBC
2-56
2-29
5-54
5-04
4-11
2-14
2-29
STA-04
3B-06
3B-01, 3B-07, 3B-12, 3B-16, 3B-17, 3B-18, 3B-21, 3BC
4-30
5-39
5-37
5-34
5-35
1A-02, 1A-05, 1A-11, 1AC, 2-47
4-37, 5-57
4-18, 4-33
2-31
2-05
6-06
3B-07, 3B-12, 3B-13, 3B-14,
3B-16, 3B-17, 3B-18, 3B-21
4-30
1A-56
3A-20, 5-41
1A-32
3A-02
217
G
Galbe, M.
Garcia-Aparicio, M.P.
Garcia-Gill, M.
Garcia-Kirchner, O.
Gaspar, M.
Georgieva, T.I.
Ghafoori, E.
Gidh, A.
Gilkes, N.
Gilliland, G.
Ginn, J.
Giordano, R.C.
Giordano, R.L.C.
Girio, F.M.
Glabe, M.
Glass, R.W.
Glassner, D.A.
Goddard, W.A., III
Gomes, E.
Goncalves, A.R.
Goncalves, A.Z.L.
Goncalves, L.R.B.
Gonzaga, G.T.S.
Gonzales, A.
Gonzalez, R.
Goret, N.
2-38, 2-51, 5-32
2-40
2-27
2-28
2-13
6-07
3B-10
4-12
1A-04, 2-05, 2-15, 2-16, 2-17, 2-18, 2-45, 6-06
1A-55
STA-04
5-33
5-33, 5-51
3B-11
4-32
3A-01, 5-41
STA-03
4-36
1A-12, 1A-13, 1A-21, 1A-27, 1A-28, 2-22
3A-18, 3A-19, 3A-31, 3A-32
1A-27
1A-26, 4-15
5-08
2-40
4-16
3A-11
Author
I
Ibrahim, M.
Inui, M.
Ishii, M.
Ito, V.M.
Iversen, F.
J
Jacobson, P.
Jeffries, T.W.
Jenkins, B.M.
Jenkins, R.
Jennings, E.W.
Jeoh, T.
Jeong, G-T.
Jin, H.
Jin, X-J.
Johnson, D.K.
Jones, F.
Jonsson, A-S.
Jonsson, L.J.
Jordan, D.B.
Jorgensen, H.
Jover, J.
Jozzala, A.F.
K
Kabel, M.
Kabel, M.
Kadar, Z.
Kale, S.
Kang, C- H.
Kang, H-K.
Kangudie, J.
Kapur, N.
Karhumaa, K.
Kastner, J.R.
Kawczak, A.
Keating, J.
Keller, M.
Kenney, K.L.
Kibblewhite-Accinelli, R.E.
Kim, D.
Kim, E-J.
Kim, G.J.
Kim, J.-H.
218
Abstract(s)
3A-06
4-03, 4-28
3A-26, 5-36
5-43
6-05
3A-04
4-22
2-64
STA-04
6-08
1A-11
5-12
6-04
1A-09
1A-02
1A-33, 5-37
5-03
2-32, 2-33
1A-34
1A-08, 1A-36, 1A-44, 6-05
2-53
3A-21
STB-03
STB-03
5-22
4-11
5-38
1A-09
2-07
1A-54
4-02, 4-26
4-23
6-01
6-06
4-39
3B-07, 3B-12, 3B-16, 3B-17, 3B-18, 3B-21
1A-41
1A-09, 3A-24
4-29
1A-09, 3A-24
4-06
Author
Kim, J.Y.
Kim, J-N.
Kim, K.H.
Kim, M.
Kim, M-D.
Kim, S.
Kim, T.H.
Kim, Y.
Kim, Y-J.
Kitrov, N.
Knopp, F.
Knoshaug, E.
Kobayashi, M.J.
Kolah, A.
Kolstad, J.
Kornmayer, C.
Krieger, N.
Kristensen, J.B.
Kuijvenhoven, J.
Kumar, A.
Kumar, R.
Kunimura, J.S.
Kwon, D-H.
L
Labavitch, J.
Ladisch, M.
Ladisch, M.R.
Ladner, J.
Lago, R.
Langone, M.A.P.
Larsen, J.
Larson, E.
Larsson, A.
Laser, M.
Le Henaff, Y.
Le Nours, J.
Leal, M.M.R.
Lecomte, J-P.
Lee, C.C.
Lee, D.A.
Lee, G-Y.
Lee, H-J.
Lee, J.-S.
Lee, J-H.
Lee, K-M.
Lee, S-J.
Lee, T-H.
Abstract(s)
4-06
3A-22, 3A-23
2-34
1A-35, 2-35
4-19, 4-27
1A-43, STA-02
5-39
2-29
3A-22
1B-06
1A-18
1A-11, 1A-53
3A-25
5-09
STA-03
3B-06
1A-50
1A-36
STB-03
3B-19
2-36
3A-26
4-27
2-64
2-03, 2-29, 3A-13
1A-61, 1B-08, 2-05
1A-55
1A-25
1A-37, 1A-38, 1A-39, 1A-40
1A-08, 1A-36, 6-05
6-04
STB-05
1A-43, 2-02, 6-04
3A-04
STB-05
1A-40
5-27
1A-41
1B-03
5-12
2-34
STB-01
1A-09
5-12
4-19
4-27
Author
Lee, W.-T.
Lee, W-H.
Lee, Y.Y.
Lee, Y-J.
Leite, R.S.R.
Leite, S.G.F.
LeJeune, A.
Lerin, L.
Lesage, J.
Li, D.
Li, Q.
Li, X-L.
Li, Y.
Liang, L. Liang, X.
Liao, W.
Liden, G.
Lim, L.H.
Lin, X.
Lin, Y.
Lin, Y.J.
Linde, M.
Lira, C.
Liu, C.
Liu, Y.
Lo Leggio, L.
Lobao, M.
Loge, F.J.
Lopes, C.E.
Lu, C.
Lu, S.
Lu, Y.
Luna, A.S.
Lunelli, B.H.
Lynd, L.R.
Lyng, L.
M
Maas, R.
Mabee, W.
Machado, A.B.
MacLean, H.
Madison, A.
Maeda, R.N.
Maltha, S.F.
Mansoorabadi, K.
Mantovanelli, I.C.C.
Manzanares, P.
Abstract(s)
Author
5-12
4-19
1A-29, 1A-49, 2-05, 4-41
2-25
2-22
1A-47
5-01
1A-23
3A-11
3A-28
4-07
1A-34, 1A-61
3A-28, 5-50
3A-28
4-36
2-37, 4-20, 4-21
4-02, 4-26, 4-32
1A-42
6-12
6-11
1A-14
2-38
5-09
5-40, 5-41
2-37, 4-20, 4-21, 6-11, 6-12
STB-05
1A-17
5-52
5-23
4-22
4-23
2-29, STB-04
5-18
3A-17, 5-28
2-01, 2-02, 4-01, 4-04, 6-04
1A-43
Mao, Z.
Markov, S.A.
Marland, E.
Martin, C.
Martin, N.
Martins das Neves, L.C.
Massingill, M.
Mathews, J.
Mathews, J.F.
Maugeri, F.
Mayeski, E.
Mazzola, P.G.
Mazzutti, M.A.
McBride, J.E.
McCaffery, J.M.
McCalla, D.
McFarland, K.C.
McKeown, C.K.
McMillan, J.D.
McNear, L.T.
Mead, D.A.
Meximenko, V.
Michel, F., Jr.
Mielenz, J.R.
Miguel, A.S.M.
Mikell, A.
Milam, D.
Miller, D.
Miller, R.W.
Milne, J.
Mims, M.M.
Min, W-K.
Minaguti, M.
Miranda, A.
Mitchell, R.
Mocchiutti, P.
Modig, T.
Mohagheghi, A.
Molina-Jouve, C.
Mollina-Jouve, C.
Moniruzzaman, M.
Monot, F.
Montross, M.D.
Moon,Y-H.
Moore, K.J.
Moraes, E.B.
Moreira de Almeida, J.R.
Moriya, R.Y.
STB-03
6-02, 6-06
3A-07, 5-42, STB-02
STA-05
1A-07
3A-10
5-22
4-22
5-15
2-11, 2-40, 5-44
Abstract(s)
3A-28
4-24
1A-07
2-33
1A-28
1A-57, 1A-60
3B-08
4-07
1A-05
5-05
5-47
3A-27, 5-36, 5-45
1A-24
4-01
4-17
2-29
2-41
4-11
2-31, 5-55
3B-17, 3B-18
1A-20
6-06
2-44
4-11
4-35
4-12
2-06
5-09
3A-03
1B-06
5-50
4-27
5-36
2C
3B-03, 3B-04
1A-51
4-02, 4-26
6-09
5-02
3A-11
2-05, 3AC
STB-06
3B-13, 3B-14
3A-24
2-08
3A-07
4-02
3A-18
219
Author
Mosier, N.S.
Mowery, R.A.
Moya, M.
Muck, R.E.
Mulbry, W.
Munk, J.T.
Munoz, C.
Musgrove, D.
Mussatto, S.I.
N
Nagle, N.
Nam, S-H.
Nascimento, K.C.
Negro, M.J.
Netto, W.S.
Newton, E.
Ng, H.
Nielsen, N.P.K.
Nielson, C.D.
Nijkamp, K.
Nilvebrant, N-O.
Nimlos, M.R.
Nordmark, T.S.
O
O’Bric, K.
Oh, Y-J.
Oh, Y-K. Okino, S.
Oliva, J.M.
Oliveira, D.
Oliveira, F.J.S.
Oliveira, J.V.
Oliveira, L.R.M.
Oliveira, P.S.
Omay, D.
Oraby, H.
Ortiz, R.
P
Padilha, F.
Padilha, F.F. Padilha, F.P.
Pafume, R.
Palmer, J.D.
Pan, X.
220
Abstract(s)
Author
1A-61,1B-08 2-03, 2-29, 3A-13, 4-37, 5-57,
2-04, 2-60
2-49
3B-05
4-38
3B-05
2-15
2-23
2-42, 2-43
Pan, Z.
Pantoja, L.
Paradez, A.
Park, D-H.
Park, J.S.
Park, J-B.
Park, S.
Park, S-C.
Patel, K.
Pathak, K.
Patrick, P.
Pedersen, C.J.
Pedro, R.
Penna, T.C.V.
Penner, M.H.
Pereira, L.T.C.
Pereira, N., Jr.
Peri, S.
Perlack, R.D.
Perlingeiro, C.A.G.
Perrin, R.K.
Persson, S.
Persson, T.
Pessoa, A., Jr.
Petersen, G.
Petersen, M.O.
Peterson, R.
Petersson, A.
Phillips, B.
Pinheiro, A.D.T.
Pinheiro, I.O.
Pinheiro, R.C.
Pinho, A.P.A.
Pinto, G.A.S.
Plotkin, J.
Podkaminer, K.
Porter, S.E.
Primo, M.S.
Pronk, J.T.
Pryfogle, P.A.
Purwadi, R.
Pyo, J-B.
2-44
3A-24
1A-47
2-11, 2-40, 5-44
5-33
1A-43, 2-02
3A-03
1A-44
2-29
4-05
2-32
1A-05
1A-46
2-20
4-27
4-29
4-03, 4-28
2-11, 5-44
1A-22
3A-14
1A-22
3A-19, 3A-32
5-08
1A-30
1B-05
6-06
1A-23
5-53, 5-46
5-47
5-18
1A-45
2-45
Abstract(s)
2-64
3A-10
1B-02
1A-09, 3A-23, 5-12
2-34
4-27
4-29
STB-01
4-30
5-19
2-08
5-48
1A-47
3A-21, 3A-25, 3A-26, 3A-27, 5-36, 5-45
1A-46
1A-17, 1A-17
1A-10, 1A-47, 1A-48, 3A-10
1A-49
3BC, 3B-01
6-10
3B-03, 3B-04
1B-02
5-03
3A-21, 4-34, 4-35, 5-45
2-03
1A-08
STB-05
4-02, 4-26
1B-08
1A-26
5-23
2-46
3A-07, 5-42, STB-02
4-15, 4-31
3A-01
4-04
2-47
1A-22
4-09
3B-07, 3B-12, 3B-16, 3B-17, 3B-18, 3B-21
5-49
3A-24
Author
Q
Qian, X.
Qu, Y.
R
Radkte, C.W.
Raffelt, K.
Ragauskas, A.
Ramos, L.P.
Ransom, C.
Rauber, F.
Reczey, K.
Reczey, R.
Reddy, S.
Reith, H.
Ren, H.
Renck, C.
Ribeiro, R.R.
Richard, A.
Richard, T.L.
Rigo, L.
Rivera, E.A.C.
Roberto, I.C.
Roberts, R.
Robertson, G.H.
Robles, S.J.
Rodrigues, M.I.
Rodrigues, T.H.S.
Rogers, T.
Romero, I.
Rosato, Y.
Rose, S.H.
Roslander, C.F.
Rottava, I.
Roux, G.
Rova, U.
Ruan, R.
Rudolf, A.
Ruiz, E.
Ruiz, R.O.
Russelle, M.P.
Ryu, H-W.
S
Saad, E.B.
Saad, M.B.W.
Abstract(s)
1A-02, 1A-05
1B-09
3B-07, 3B-12, 3B-13, 3B-14,
3B-16, 3B-17, 3B-18, 3B-21
3B-06
6C
1A-50, 1A-51
1B-05
5-47
1A-03, 2-13, 5-22
2-59
1A-45
STB-03
2-08
3B-06
1A-59
2-07
2-08
5-46
5-16, 5-28
1A-12, 2-43
3B-20
1A-41
1A-63
1A-24
4-15
4-30
2-48
5-47
4-01
5-32
1A-23, 5-47
5-02
3A-04
6-11, 6-12
4-32
2-49
2-56
STA-01
3A-22, 3A-23
1A-50
3A-19, 3A-31
Author
Saballos, A.
Saddler, J.N.
Saez, F.
Salgado, A.M.
Salinas, R.A.
Saqib, A.A.N.
Sarrouh, B.F.
Saska, M.
Sassner, P.
Saville, B.A.
Scamparini, A.R.P.
Scarlata, C.
Schall, C.A.
Schechinger, T.M.
Scheer, A.P.
Schell, D.J.
Schlittler, L.A.F.S.
Schmer, M.R.
Schols, H.
Schwartz, G.
Searcy, E.
Sedlak, M.
Seguara, G.M.
Selig, M.J.
Seo, J-H.
Servulo, E.F.C.
Shahbazi, A.
Shanks, B.
Sharma, A.
Shaw, A.J.
Shearer, S.A.
Shearer, S.S.
Shell D.J.
Shin, J-S.
Shinners, K.J.
Shishir, C.
Shuler, P.J.
Siika-aho, M.
Silva, M.F.
Silverman, H.G.
Singh, O.V.
Singh, R.P.
Sjode, A.
Slininger, P.J.
Smart, L.B.
Smith, K.
Smith, W.A.
Abstract(s)
1B-04
1A-04, 2-15, 2-16, 2-17, 2-18, 2-45, 6-02, 6-06
2-11, 5-44
1A-10
1A-07
1A-52
2-53
2-50
2-51
1A-42
5-46, 5-47, 5-53
2-52
2-61
3B-17
5-42, STB-02
2-31, 6-08, 6-09
6-10
3B-03, 3B-04
STB-03
3B-08
3B-19
4-18, 4-33, 4-37, 5-57
2-28
1A-53
4-19, 4-27, STBC
3A-15, 3A-16
3A-28, 5-50
2-03
3A-29
4-04
3B-13
3B-14
5-55
5-38
3B-05
2-62
4-36
1A-03
5-53
3B-07, 3B-17, 3B-18
3A-29
1A-54, 3A-29
2-32, 2-33
4-17
1B-07
4-16
5-52
221
Author
Snow, L.D.
Snyder, I.
Snyder, S.
Snyder, S.W.
Soares, C.M.F.
Soares, V.F.
Sokhansanj, S.
Somerville, C.
Song, I-J.
Souza, V.R. Spatari, S.
St. Martin, E.J.
Stahl, R.
Stahlberg, J.
Stalbrand, H.
Steckel, L.
Steinhardt, J.
Stevens, C.
Stevens, N.A.
Sticklen, M.
Stowers, M.D.
Stremel, D.P.
Strobel, E.M.
Su, D.
Suazo, A.S.
Sun, J.
Sunkara, H.
Sylgester, R.
Szengyel, Z.
T
Taherzadeh, M.J.
Tang, Y.
Tanner, R.D. Tasdelen, C.
Tavares, L.F.D.
Taylor, F.
Temouri, F.
Templeton, D.W.
Thomas, S.
Thomeo, J.C.
Thompson, D.N.
Thompson, L.
Thomsen, A.B.
Thomsen, M.H.
Thonart, P.
Thygesen, A.
Tiffany, D.G.
222
Abstract(s)
Author
1A-45
5-11
5C
1A-14
2-46, 5-58
1A-16, 5-30
5-04
1B-02, 1B-06
5-12
5-51
STA-05
1A-14
3B-06
STB-05
2-13, STB-05
3B-20
3B-06
3AC
3B-16, 3B-21
1B-03, 1B-05, 1BC
2-29
5-28
3A-08
2-54, 2-54
2-28
2-54, 2-55, STB-04
3A-03
STA-04
2-35
Tomotani, E.Junko
Toniazzo, G.
Torry-Smith, M.
Toyama, H.
Toyama, N. Treichel, H.
Tucker, M.P.
Tunheim, A.I.M.
Tuskan, G.
Tyler, D.
Tyner, W.
Tyurin, M.
5-49
4-36
5-56, 5-11
1A-31
5-30
5-39
2-29
2-56
1B-01
1A-28
5-52
1A-55
2-58, 2-59, 6-05
2-58, 2-59, 6-05
5-01, 5-27
2-58
STA-01
U
Um, B-H.
Uribelarrea, J.L.
V
Valdman, B.
Valero, F.
van Buijsen, H.
van Dijken, J.P.
van Groenestijn, J.
van Rooyen, R.
van Walsum, G.P.
Van Zyl, W.H.
Vane, L.M.
Vangness, M.D.
Vanzo, L.T.
Varanasi, S.
Varga, W.
Vasquez, M.P.
Vehmaanper, J.
Veira, R.B.
Venkatesh, B.
Vermerris, W.
Verser, D.
Vertes, A.A.
Viamajala, S.
Viikari, L.
Villeneuve, P.
Vincenzo, M.
Vink, E.T.H.
Visser, D.
Vitolo, M.
Vlasenko, E.
Abstract(s)
1A-58
1A-23
2-06, 4-08
1A-56
1A-56
1A-24, 5-53
2-56
6-11
1BC
3B-20
2-03
4-04
5-54
3A-11
1A-10
1A-10
STB-03
4-09
STB-03
4-01
2-04, 2-20, 2-27, 2-60,
5-07, 5-24, 5-29
4-01
5-06
3A-08
5-46
2-61
2-59
1A-48
1A-03
3A-15
1B-05, 2-10, 2-21, 2-62
1B-04, 1B-08
3A-02
4-03, 4-28
5-55
1A-03
1A-25
3B-09
STA-03
STB-03
1A-57, 1A-58, 1A-59, 1A-60, 4-34, 4-35
1AC
Author
Vogel, K.P.
Volk, T.A.
Vrana, B.
Vu, D.
W
Wagschal, K.
Walsh, M.
Wang, D.
Wang, G.
Wang, M.
Wang, Q.
Wang, T.
Ward, T.M.
Warner, J.
Warner, R.
Watanabe, T.
Webster, G.
Wee, Y-J.
Wei, L.
Weightman, A.J.
Weirich, F.
Weiss, N.
Welch, G.
Wen, J.
Wen, Z.
Wenger, K.
Wery, J.
Weusthuis, R.
Whitehead, T.R.
Whitney, P.J.
Widmer, W.
Wiedemann, B.
Wiemer, P.J.
Wierckx, N.J.P.
Willies, D.
Willies, D.M.
Williford, C.W.
Willquist, K.
Winkler, A.A.
Wolcott, M.P.
Wolf Maciel, M.R.
Wong, D.W.S.
Woo, N-Y.
Wooley, R.
Wright, C.T.
Abstract(s)
Author
3B-03, 3B-04
1B-07
STA-04
5-09
Wu, G.
Wyatt, E.D.
Wyman, C.E.
1A-41
3B-20
2-64
4-07
STA-06, STAC
4-36
3A-28
5-56
3A-13
4-37, 5-57
4-03
4-29
3A-22, 3A-23
1B-09
4-29
3B-06
2-44
2-29
1A-33
2-37, 4-38
4-08
4-05
STB-03
1A-34
1A-52
4-30
4-39
3B-05
4-05
1A-06
1A-62
4-12
1A-15, 1A-17
4-09
5-52
STB-02, 5-17, 3A-07,
2-39, 5-42, 5-43, 5-05
1A-41
3A-24
STA-03
3B-07, 3B-12, 3B-16, 3B-17, 3B-18, 3B-21
X
Xie, D.
Ximenes, E.A.
Xu, Q.
Y
Yancey, N.A.
Yang, B.
Yang, K.
Yang, M.M.
Yano, M.
Ying, M.
Yu, F.
Yu, Y.
Yukawa, H.
Yun, J-S.
Z
Zaachi, G.
Zanin, G.M.
Zanuttini, M.A.
Zeevalkink, J.
Zhang, L.
Zhang, R.
Zhang, Y-H.P.
Zheng, Y.
Zhu, D.
Zhu, Y.
Zhu, Y.
Zhuang, G.
Zullo, L.
Abstract(s)
1B-09
2-63
1A-06, 1A-62, 2C, 2-05, 2-09, 2-36, 5-04, 5-40
2-45
1A-61
2-47
3B-07, 3B-17, 3B-18
1A-06, 1A-62, 2-54, 2-55, STB-04
1A-32
1A-63
1A-56
5-13
6-11, 6-12
3A-28
4-03, 4-28
3A-23
1A-03, 2-38, 2-51, 5-03, STB-05
2-46, 5-58
1A-51
STB-03
3A-28
2-64
2-01
2-64
1A-32
4-40
4-41
1B-09
5C
223
Notes
224

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