DIVISION OF AUTOTROPHIC MICROORGANISMS

DIVISION OF AUTOTROPHIC MICROORGANISMS
Head
Ondřej Prášil, Assoc. Prof., PhD.
The division is located in Třeboň, a small town about 150 km south of Prague. The research is focused on autotrophic microorganisms – algae, cyanobacteria and other photosynthetic bacteria. The division is formed by three research laboratories: Laboratory of Photosynthesis, Laboratory of Algal Biotechnology and Laboratory of Algal Cell Cycles. Research
in the Laboratory of Photosynthesis is oriented towards biochemical and molecular biological
studies of biogenesis of pigment-protein complexes in thylakoid membranes and on the ecophysiology of photosynthesis in phytoplankton and photoheterotrophic bacteria. Important
part of the research also involves development on new biophysical instrumentation for field
studies of phytoplankton photosynthesis. Laboratory of Algal Cell Cycles is involved in the
studies of the mechanisms of regulation of cell cycles in algae and the role of various environmental factors on regulatory proteins during the cell cycle. Laboratory of Algal Biotechnology develops and tests new techniques of microalgal production and isolates and identifies
new valuable compounds from algae and cyanobacteria.
The Division closely collaborates on everyday basis with the University of South Bohemia – mostly with the Faculty of Science in České Budějovice and with the Institute of Physical Biology in Nové Hrady. The collaboration involves standard courses given for the
students at the University, supervision of research projects for students at all levels, shared
use and maintenance of expensive instruments and participation in international summer
courses “Schola ludus“ for both high school students and undergraduate students.
The research facilities in Třeboň are undergoing major reconstruction with the aim of
upgrading them to suit current needs.
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Laboratory 131
PHOTOSYNTHESIS
Head
Ondřej Prášil, Assoc. Prof., PhD.
Scientific staff
Radek Kaňa, PhD.
Michal Koblížek, PhD.
Josef Komenda, Prof., PhD.
Part-time scientists
Eva Kotabová, PhD.
Martin Tichý, PhD.
Roman Sobotka, PhD.
Ondřej Komárek, PhD.
Michal Mašín, PhD.
Ivan Šetlík, PhD.
Eva Šetlíková, PhD.
Technical staff
Barbora Hošková
Jana Hofhanzlová
Jana Knoppová
Vendula Krynická
Eva Prachová
Doctoral students
Zuzana Čuperová, MSc., Marika Dobáková, MSc., Jana Kopečná, MSc.,
Petra Krausová, MSc., Stanislava Kuviková, MSc., Hana Medová, MSc.,
Jarmila Mlčoušková, MSc., Jiří Patera, MSc., Kamooltip Promnares, MSc.,
Kateřina Rottnerová, MSc., Eva Žišková, MSc.
Undergraduate students
Kristína Felcmanová, Markéta Foldýnová, Eva Hojerová, Jitka Kručínská,
Markéta Muroňová
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Research field and principal results
The main research topics include: (i) assembly and degradation of the thylakoid membranes, with emphasis on Photosystem II (ii) regulation of the chlorophyll synthesis,
(iii) regulation of photosynthesis in diazotrophic cyanobacteria, (iv) ecology and physiology
of aerobic anoxygenic photosynthetic bacteria, and (v) development and application of novel
instrumentation to study photosynthesis in microorganisms.
Localization of small chlorophyll-binding proteins
Cyanobacterium Synechocystis PCC 6803 contains four small proteins designated
ScpB-ScpD that code small one-helix proteins with significant sequence similarity to plant
chlorophyll a/b-binding proteins. The tagged ScpD protein together with other homologues
ScpC and ScB were identified in Photosystem II (PSII). In contrast, ScpE did not co-isolate
with any major protein complexes in thylakoids. On the basis of obtained data we suggested
that members of the SCP family can associate with damaged PSII and serve as a temporary
pigment reservoir during PSII repair (Yao et al. 2007).
Mechanism of the FtsH-mediated degradation of the D1 protein in cyanobacteria
Genome of Synechocystis PCC 6803 contains genes encoding homologues of the protease FtsH. Previously we confirmed that the FtsH homologue plays a crucial role in the degradation of the D1 protein and regulates the level of PSII assembly. Since the bacterial FtsH is
known to start degradation of substrates from N-terminus, we tested the role of the N-terminal
region of D1. Removal of 5 or 10 residues blocked D1 synthesis whereas removal of 20 residues restored the ability to assemble functional PSII but inhibited D1 degradation. Our results
identify an important physiological role for the N-terminal tail of D1 in selective D1 degradation (Fig. 23).
Structural role of the PsbI protein in the cyanobacterial Photosystem II complex
The PsbI protein belongs to small protein subunits of PSII with unknown function.
Analysis of PSII assembly in a number of mutants of Synechocystis PCC 6803 showed that
PsbI is an early assembly partner for D1 and increases its stability in the unassembled state.
PsbI is not required for formation of PSII reaction center complexes but its absence leads to
a dramatic destabilization of CP43 binding within PSII core complexes. Despite the close
structural relationship between D1 and PsbI in the PSII complex, PsbI turns over much slower
than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI.
Critical role of the C-terminal extension of ferrochelatase for functioning of the
tetrapyrrole pathway in Synechocystis PCC 6803
Heme and chlorophyll share a common biosynthetic pathway up to the branch point
where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for chlorophyll biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in
cyanobacteria is their C-terminal extension, which contains a putative chlorophyll-binding
motif. We analyzed the Δh324 strain of Synechocystis 6803, which contains a truncated FeCH
enzyme lacking this C-terminal domain. Our measurements revealed that the Δh324 mutation
dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis, implying a direct role for heme in the regulation of flux through the
pathway. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the
C-terminal extension is critical for activity of the FeCH and is strictly required for oligomerization of this enzyme.
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Fig. 23. Proposed model for selective D1 replacement during PSII repair in Synechocystis. A: Intact PSII core complex with the functional and correctly folded D1 protein.
B: High-light induced inactivation of PSII is followed by release of CP43 and extrinsic
proteins. In the resulting core complex lacking CP43 (RC47), the structure of damaged
D1 protein (D1 dam) is destabilized, the protein is recognized by FtsH and its released
N-terminus is caught by the protease. C: The damaged D1 subunit is degraded (D1 deg)
by FtsH processively from N- and C-temrinus releasing short oligopeptides but no distinct larger fragments. D: Insertion of the new D1 molecule and re-assembly of the active
dimeric PSII core complex (RCII).
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Physiology and ecology of aerobic anoxygenic phototrophic bacteria
We have developed a new method, which uses bacteriochlorophyll a as natural in situ
tracer of aerobic anoxygenic phototrophs (AAPs). On measuring its turnover we have found
that AAP bacteria in the Atlantic oligotrophic gyres grow at rates of about one division per
day. In more productive marine regions, such as equatorial upwelling or North Atlantic AAPs
grew at rates up to three divisions per day. This is much faster than current growth estimates
for the total bacterial community. Thus, in spite of the fact that AAP bacteria represent only
2-4% of total prokaryotes, they appear to be a very dynamic part of marine microbial community and contribute significantly to organic carbon recycling in the upper ocean.
We demonstrated that AAP bacteria are present also in many freshwater environments.
We have found these organisms in many oligotrophic and mesotrophic lakes in the Czech
Republic. In some specific habitats AAP bacteria even dominated local microbial comunities.
Regulation of photosynthesis in diazotrophic cyanobacterium Trichodesmium
Anthropogenic increases in atmospheric pCO2 are accompanied by higher concentrations of CO2 in seawater. This can influence cyanobacterial nitrogen-fixers via the coupling
between photosynthesis and nitrogen-fixation. We showed that elevated pCO2 levels will
enhance nitrogen fixation and photosynthesis in the bloom-forming, diazotrophic, cyanobacterium Trichodesmium spp. that contributes greatly to primary production in the oligotrophic
subtropical and tropical oceans. We suggest that in the high pCO2 cultures nitrogen-fixation
and growth are enhanced by shifting energy and reductants from a Ci-uptake system, operating in the low and ambient pCO2. This enables higher nitrogen fixation and growth. Decreasing dependence on CCM and active Ci-uptake in the high CO2 oceans may also be important
in other diazotrophs – thereby enhancing inputs of new N and increasing primary productivity
in the oceans.
Low temperature emission spectroscopy as a tool in phytoplankton ecophysiology
We have developed and tested new sensitive portable emission spectrometer that can
detect emission spectra of natural phytoplankton samples that are cooled to low temperature
(77K). This instrument was developed in collaboration with the company PSI s.r.o. and has
been successfully used on several scientific cruises and during field measurements. The low
temperature emission spectra provide information about the photophysiology of phototrophic
microorganisms present in the sample. Each group of photosynthetic microorganisms has
unique light harvesting complex that provides specific signature in the emission spectrum.
The instrument is controlled by the newly developed software can detect not only the sole
presence and relative quantity of various phototrophs (oxygenic phytoplankton – algae and
cyanobacteria, anoxygenic prokaryotic phototrophs – purple sulphur and aerobic bacteria,
green bacteria) but also changes in their photophysiology. This opens new possibilities for the
ecophysiological research of phytoplankton.
Publications
Andersen E., Lohscheider J., Šetlíková E., Adamska I., Šimek M., Kupper H.: Acclimation of Trichodesmium
erythraeum ISM 101 to high and low irradiance analysed in the physiological, biophysical and biochemical level. New Phytologist, in press.
Boehm M., Nield J., Zhang P., Aro,E.M, Komenda J., Nixon P.J.: Structural and mutational analysis of band 7
proteins in the cyanobacterium Synechocystis sp. PCC 6803. J. Bacteriol. doi:10.1128/JB.00644-09 (in
press).
Bogos B., Uchy B., Domonkos I., Laczkó-Dobos H., Komenda J., Abasova L., Cser K., Vass I., Sallai, Wada
H., Gombos Z.: Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC
7942 cells. Photosynth. Res., in press.
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Bonnet, S., Guieu, C., Bruyant, F., Prášil, O., Van Wambeke, F., Raimbault, P., Moutin, T., Grob, C., Gorbunov,
M.Y., Zehr, J.P., Masquelier, S.M., Garczarek, L., Claustre, H.: Nutrient limitation of primary productivity in the Southeast Pacific (BIOSOPE cruise). Biogeosciences 5, 215-225 (2008).
Červený J., Šetlík I., Trtílek M., Nedbal L.: Photobioreactor for cultivation and real-time, in-situ measurement of
O2 exchange rates, growth dynamics, and of chlorophyll fluorescence emission of photoautotrophic microorganisms. Eng. Life. Sci. 9, 247-253(2009).
Dobáková M., Tichý M., Komenda J.: Role of the PsbI protein in photosystem II assembly and repair in the
cyanobacterium Synechocystis sp. PCC 6803. Plant Physiology 145, 1681–1691 (2007).
Dobáková M., Sobotka R., Tichý M., Komenda J.: Psb28 protein is involved in the biogenesis of the photosystem II inner antenna CP47 (PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiology
149, 1076-1086 (2009).
Gasol J.M., Pinhassi J., Alonso–Sáez L., Ducklow H., Herndl G.J., Koblížek M., Labrenz M., Luo Y., Morán
X.A.G., Reinthaler T., Simon M.: Towards a better understanding of microbial carbon flux in the sea.
Aquatic Microbial Ecology 53, 21–38 (2008).
Herbstová M., Litvín R., Gardin Z., Komenda J., Vácha F.: Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica. BBA Bionenergetics; doi:10.1016/j.bbabio.2009.09.002
(in press).
Kaňa R., Kotabová E., Prášil O.: Acceleration of plastoquinone pool reduction by alternative pathways precedens a decrease in photosynthetic CO2 assimilation in preheated barely leaves. Physiol. Plantarum 133,
794–806 (2008).
Kaňa R., Vass I.: Thermoimaging as a tool for studying light–induced heating of leaves Correlation of heat
dissipation with the efficiency of photosystem II photochemistry and non–photochemical quenching. Environ. Exp. Botany 64, 90–96 (2008).
Kaňa R., Prášil O., Komárek O., Papageorgiou G.C., Govindjee: Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942). BBA Bioenergetics 1787, 1170-1178
(2009).
Kaňa R., Prášil O., Mullineaux C.W.: Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffucion in the lumen is very restricted. FEBS Lett. 583, 670-674 (2009).
Koblížek M., Mašín M., Ras J., Poulton A.J., Prášil O.: Rapid growth rates of aerobic anoxygenic phototrophs in
the ocean. Environ. Microbiol. 9(10), 2401–2406 (2007).
Komenda J., Kuviková S., Granvogl B., Eichacker L.A., Diner B.A., Nixon P.J.: Cleavage after residue Ala352
in the C–terminal extension is an early step in the maturation of the D1 subunit of photosystem II in
Synechocystis PCC 6803. Biochim. Biophys.Acta Bioenergetics 1767, 829–867 (2007a).
Komenda J., Nickelsen J., Tichý M., Prášil O., Eichacker L.A., Nixon P.J.: The cyanobacterila homologue of
HCF136/YCF48 is a component of an early photosystem II assebly complex and is importatn for both the
efficient assembly and repair of photosystem II in synechocystis sp. PCC6803. J. Biol. Chem. 283 (33),
22390–22399 (2008).
Komenda J., Tichý M., Prášil O., Knoppová J., Kuviková S., de Vries R., Nixon P.J.: The exposed N-terminal
tail of the D1 subunit is required for rapid D1 degradation during Photosystem II reapir in Synechocystis
sp. PCC 6803. Plant Cell 19, 2839–2854 (2007b).
Kotabová E., Kaňa R., Kyseláková H., Lípová L. Novák O., Ilík, P.: A pronounced light-induced zeaxanthin
formation accompanied by an unusually slight increase in non-photochemical quenching: A study with
barley leaves treated with methyl viologen at moderate light. Journal of Plant Physiology 165, 1563–
1571 (2008).
Küpper H., Parameswasran A., Leitenmaier B., Trtílek M., Šetlík I.: Cadmium–induced inhibition of photosynthesis and long–term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens. New
Phytologist 175, 655–674 (2007).
Küpper H., Šetlík I., Seifert S., Prášil O., Šetlíková E., Strittmatter M., Levitan O., Lohscheider J., Adamska I.,
Berman–Brank I.: Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into
regulation of photosynthesis and nitrogen fixation. New Phytologist 179, 784–798 (2008).
Küpper H., Andersen E., Wiegert S., Šimek M., Leitenmaier B., Šetlík I.: Reversible coupling of individual
phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements. Biochim. Biophys. Acta 1787, 155-167 (2009).
Lacskó–Dobos H., Ughy B., Tóth S.Z., Komenda J., Zsiros O., Domonkos I., Párducz Á., Bogos B., Komura
M., Itoh S., Gombos Z.: Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsAinactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks
phycobilisomes. Biochim. Biophys. Acta 1777, 1184–1194 (2008).
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Lami R., Čuperová Z., Ras J., Lebaron P., Koblížek M.: Distribution of free living and particle attached aerobic
anoxygenic phototrophic bacteria in marine environments. Aquatic Microbial Ecol. 55, 31-38 (2009).
Levitan O., Rosenberg G., Šetlík I., Šetlíková E., Grígel J., Klepetář J., Prášil O., Berman-Frank I.: Elevated
CO2 enhances nitrogen fixation and growth in the marine cyanobacterium Trichodesmium. Global
Changes Biol. 13, 1–8 (2007).
Lípová L., Krchňák P., Komenda J., Ilík P.: Heat-induced disassembly and degradation of chlorophyllcontaining protein complexes in vivo. A study with linearly heated barley leaves. BBA-Bioenergetics;
doi:10.1016/j.bbabio.2009.08.001 (in press).
Mašín M., Nedoma J., Pechar L., Koblížek M.: Distribution of aerobic anoxygenic phototrophs in temperate
freshwater systems. Environ. Microbiol. 10 (8), 1988–1996 (2008).
Prášil O., Sugget D.J., Cullen J.J., Babin M., Govindjee: Aquafluo 2007: chlorophyll fluorescence in aquatic
sciences, an international conference held in Nové Hrady. Photosynthesis Res. 95, 111–115 (2008).
Prášil O., Bína D., Medová H., Řeháková K., Zapomělová E., Veselá J., Oren A.: Emission spectroscopy and
kinetic fluorometry studies of phototrophic microbial communities along a salinity gradient in solar saltern evaporation ponds of Eilat, Israel. Aquatic Microbial Ecol. 56, 227-239 (2009).
Prosecka J., Orlov A., Fantin Y., Zinchenko V., Babykin M., Tichy M.: A novel ATP-binding cassette transporter is responsible for resistance to viologen herbicides in the cyanobacterium Synechocystis sp. PCC
6803. FEBS Journal 276, 4001-4011 (2009).
Řeháková K., Zapomělová E., Prášil O., Veselá J., Medová H., Oren A.: Composition changes of phototrophic
microbial communities along the salinity gradient in the solar saltern evaporation ponds of Eilat, Israel. –
Hydrobiologia 2009, in press.
Rontani J.–F., Koblížek M.: Regiospecific enzymatic oxygenation of cis–accenic acid in the marine phototrophic
bacterium Erythrobacter sp. strain MG3. Lipids 43, 1065–1074 (2008).
Salka I., Moulisová V., Koblížek M., Jost G., Jürgens K., Labrens M.: Abundance, depth distribution, and
composition of aerobic bacteriochlorophyll a-producing bacteria in four basins of the Central Baltic Sea.
Appl. Environ. Microbiol. 74 (14), 4398–4404 (2008).
Sobotka R., DühringU., Komenda J., Peter E., Gardian Z., Tichý M., Grimm B., Wilde A.: Improtance of the
cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes.
J. Biol. Chem. 283 (38), 25794–25802 (2008a).
Sobotka R., McLean S., Zuberová M., Hunter C.N., Tichý M.: The C–Terminal extension of ferrochelatase is
critical for enzyme aktivity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC
6803. J. Bacteriol. 190 (6), 2086–2095 (2008b).
Sugget D., Szambler N., Prášil O., Kolber Z., Quigg A., Vázquez-Dominguez E., Zohary T., Berman T., Iluz D.,
Levitan O., Lawson T., Meeder E., Lazar B., Baz-Zeev E., Medová H., Berman-Frank I.: Nitrogen and
phosphorus limitation of oceanic microbial growth during spring in the Gulf of Aqaba. Aquatic Microbial
Ecol. 56, 227-239 (2009).
Van Mooy B.A.S., Fredericks H.F., Pedler B.E., Dyhrman S.T., Karl D.M., Koblížek M., Lomas M.W., Mincer
T.J., Moore L.R., Moutin T., Rappé M.S., Webb E.A.: Phytoplankton in the ocean use non-phosphorus
lipids in response to phosphorus scarcity. Nature 458, 69-72 (2009).
Vredenberg W., Durchan M., Prášil O.: On the chlorophyll a fluorescence yield in chloroplasts upon excitation
with twin turnover flashes (TTF) and high frequency flash trains. Photosynthesis Res. 93, 183–192
(2007).
Vredenberg W., Durchan M., Prášil O.: Photochemical and photoelectrochemical quenching of chlorophyll
fluorescence in photosystem II. BBA Bioenergetics 1787, 1468-1478 (2009).
Yao D., Kiselbach R., Komenda J., Promnares K., Prieto M.A.H., Tichý M., Vermaas W., Funk C.: Localization
of the small CAB–like proteins in Photosystem II. J. Biol. Chem. 282, 267–276 (2007).
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Laboratory 132
ALGAL BIOTECHNOLOGY
Head
Jiří Masojídek,
Assoc. Prof., PhD.
Scientific staff
Jiří Kopecký, PhD.
Part-time scientists
Pavel Hrouzek, MSc.
Jiří Doucha, PhD.
Karel Lívanský, PhD.
Technical staff
Jan Hinterholzinger
Petr Novotný, MSc.
Soňa Pekařová
Lada Samcová
Pavel Souček, MSc.
Jaroslav Záruba
Doctoral students
Petra Briestenská, PhD., Jan Frolík, MSc., Lenka Hanzlíková, MSc., Jan Král, MSc.,
Lucie Marková, MSc., Jana Tomšíčková, MSc., Petr Zelík, PhD.
Undergraduate students
Eliška Hořejší, MSc., Petr Tomek, Daniel Hisem, MSc., Kateřina Skácelová
Research fields and principal results
The Laboratory of Algal Biotechnology has dealt with several research topics in applied
research of microalgae (i.e. prokaryotic cyanobacteria and eukaryotic algae): (i) design of
microalgae photobioreactors and optimization of cultivation regime; (ii) cultivation of microalgae biomass with high nutritional value and (iii) isolation and characterization of bioactive
compounds isolated from microalgae.
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Design of microalgae photobioreactors and optimization of cultivation regime
Outdoor thin-layer cascade units used in Třeboň represent unique and highly productive
system for microalgae cultivation due to increased average cell irradiance. Its high productivity
is achieved due to the elevated ratio of irradiated surface-to-total volume (S/V). In fully-controlled experimental unit (24 m2; Fig. 24) the S/V ratio was adjusted between 80 and 135 m-1. At
the maximum value we found the seasonal productivity increase of 20–30% as compared with
large production units (224 m2, S/V ~100 m-1). We also clarified that high productivity is
achieved in the thin-layer turbulent culture due to fast light/dark cycles which match the turnover of the photosynthetic apparatus (frequency 0.1–1 kHz).
Fig. 24. The fully-controlled experimental unit of 24 m2 has been used to test automatic control and
regulation of cultivation parameters (pH, dissolved oxygen concentration, temperature, irradiance, circulation, surface-to-volume ratio) which are implemented in the production units.
The experimental unit was also used to test automatic control and regulation of cultivation
variables, which are to be implemented in the production units. Except for the irradiance, another important variable represents carbon dioxide supply which can be efficiently regulated by
pH measurement in microalgae suspension. Kinetics of photobiochemical activity measured by
chlorophyll fluorescence in-situ in the pH range of 6–9.5 proved that the value up to 8 does not
significantly limit photosynthesis of the culture. In this way CO2 supply can be substantially
lower to decrease its losses in open cultivation units.
Novel devices, the chlorophyll fluorometers AquaPen AP100 (produced P.S.I. Ltd. Brno)
and PAM 2500 (H.Walz, Germany) were tested to monitor photobiochemical activities of microalgal cultures. Selected fluorescence variables – Vj, Vi, NPQ, ΔF/Fm’ and Fv/Fm characterizing the function of photosynthetic electron transport were calculated by operating software from
polyphasic induction kinetics and saturating-pulse quenching analysis. These variables proved
to be good indicators of photosynthetic performance of microalgae cultures which influence
culture productivity.
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Two types of closed microalgae photobioreactors – annular-column and flat-panel were
tested for cultivation of shear-stress sensitive strains, e.g. Nostoc and Nannochloropsis.
Cultivation of microalgae biomass with high nutritional value
The biomass of some microalgae strains has high nutritional value due to the content of
carotenoids and polyunsaturated fatty acids (PUFA), which represent important supplement to
human and animal diet. We tested several microalgae strains – Haematococcus, Nannochloropsis and Chlorococcum. Changes in photobiochemical activities were studied during
the cell cycle of the microalga Haematococcus to induce the synthesis of astaxanthin under
various degrees of nitrogen limitation in combination with high irradiance in closed laboratory photobioreactors. The green alga Chlorococcum sp. was cultivated in open outdoor cascades under nitrogen limitation which increased carotenoid to chlorophyll ratio by about 40%.
During the visit to the National Center for Mariculture in Eilat (Israel), we studied environmental limitation of culture growth in the marine microalga Nannochloropsis sp.
The biomass of microalgae Chlorella, Haematococcus and Nannochloropsis was used
early stages of freshwater fish and crayfish aquaculture as feed additive (a source of carotenoids and polyunsaturated fatty acids) in joint experiments with Fisheries Nové Hrady s.r.o.
and the Research Institute of Fish Culture and Hydrobiology in Vodňany.
Isolation and characterization of bioactive compounds from microalgae
Molecular targets relevant for drug discovery evolved for millennia have only been isolated and characterized recently. We used novel test systems to identify drug candidates isolated from microalgae, with particular focus on two biological activities. The first is the
screening of anti-inflammatory, anti-tumoral, anti-metastatic and wound healing activities
using new cell-based technologies (e.g. Fig. 25). The second approach represents the screening for antioxidant activity. Over 100 fractions isolated from microalgae were tested of which
10 % anti-inflammatory, 6 % wound healing properties and 12 % antioxidant and 18 % displayed cytotoxic activity (see example in Fig. 25).
Fig. 25. HeLa human cancer cell line treated with a novel, unidentified cytotoxic compound (30 μg/mL)
isolated from the soil cyanobacterium Nostoc muscorum (left) as compared with untreated control cells
(right). Nuclei and mitochondria were visualized using DAPI (blue) and MitoTracker® (red) to observe
the condensation of chromatin and ‘puzzling’ mitochondria circularization. The results suggest a diversity of effects of natural compounds as there is still much to be explored in the nature’s pool of bioactive metabolites.
One promising cyanobacterium Cylindrospermum sp. C24 exhibited remarkable cytotoxic and anti-inflammatory activity. The mechanism of action was tested with two cell lines,
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human lung microvascular endothelial cells and lymphatic endothelial cells. We found that
the compound disrupts microfilament organization through inhibition of actin polymerization,
and causes in-vitro de-polymerization or fragmentation of the F-actin.
We also developed new rapid-resolution method to determine antioxidant activity combining HPLC/DAD/MS separation with SPE-SFE extraction, which was optimized to identify
antioxidative metabolites in submicrolitre (femtomol) sample volumes. The phenolic compounds and isoflavones were found in cell extracts of several cyanobacteria strains (Arthrospira platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). The analysis showed that these strains usually contain phenolic acids or aldehydes in microgram amounts per
gram of biomass. The proposed SPE/SFE extraction method is suitable for analysis of different plant species containing trace amounts of polar phenolics.
Publications
Caisová L., Kopecký J.: Relation of Pleurocapsa cuprea Hansgirg to the geneus Hildenbrandia (Rhodophyta).
Phycologia 47, 404–415 (2008).
Drápalová P., Štys D., Lukešová A., Kopecký J.: Genus Nostoc - a source of novel trypsin inhibitors. Arch.
Hydrobiol. Suppl. Algol. Stud 127, 232–241 (2008).
Klejdus B., Kopecký J., Benešová L., Vacek J.: Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species. J. Chromat.
A, 1216, 763–771 (2009).
Klejdus B., Vacek J., Benešová L., Kopecký J., Lapčík O., Kubáň V.: Rapid-resolution HPLC with spectrophotometric detection for determination and identification of isoflavones in soy preparations and plant extracts. Anal. Bioanal. Chem. 389, 2277–2285 (2007).
Kromkamp J.C., Beardall J., Sukenik A., Kopecký J., Masojídek J., van Bergeijk S., Gabai S., Shaham E.,
Yamshon A.: Short term variation in photosynthetic parameters of Nannochloropsis grown in twodifferent types of outdoor mass cultures. Aquat. Microb. Ecol. Aquatic Microbial Ecology, 56, 309-322 (2009).
Madlener S., Svačinová J., Kitner M., Kopecký J., Eytner R., Lackner A., Phuong Nha Vo T., Frisch R., Grusch
M., De Martin R., Doležal K., Strnad M., Krupitza G.: In vitro anti-inflammatory and anticancer activities of extracts of Acalypha alopecuroidea (Euphorbiaceae). Int. J. Oncol., (in press) (2009).
Masojídek J., Vonshak A., Torzillo G.: Chlorophyll fluorescence applications in microalgal mass cultures. In:
Suggett D.J., Prášil O., Borowitzka M.A. (Eds. ) Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications (2009) (in press).
Masojídek J., Sergejevová M., Rottnerová K., Jirka V., Korečko J., Kopecký J., Zaťková I., Torzillo G., Štys D.:
A Two-stage Solar Photobioreactor for Cultivation of Microalgae based on Solar Concentrators. J. Appl
Phycol. 21, 55-63 (2009).
Masojídek J., Torzillo G.: Mass Cultivation of Freshwater Microalgae. pp. 2226–2235 In Jørgensen S.E., Fath
B.D. (Eds): Ecological Engineering, vol. 3, Encyclopedia of Ecology, Elsevier, Oxford, (2008).
Onofrejová L., Vašíčková J., Klejdus B., Stratil P., Mišurcová L., Kráčmar S., Kopecký J., Vacek J.: Bioactive
phenols in algae: The application of pressurized-liquid and solid-phase extraction techniques. J. Pharmac. Biomed. Anal., (in press) (2009).
Papaefthimiou D., Hrouzek P., Mugnai M.A., Rasmussen U., Lukesova A., Turicchia S., Ventura S.: Differential patterns of evolution and distribution of the symbiotic behaviour in ostocacean cyanobacteria. Int. J.
Syst. Evol. Micr. 58, 553–564 (2008).
Sukenik A., Beardall J., Kromkamp J.C., Kopecký J., Masojídek J., van Bergeijk S., Gabai S., Shaham E.,
Yamshon A.: Photosynthetic performance of outdoor Nannochloropsis mass cultures to extreme environmental conditions – assessment by chlorophyll fluorescence techniques. Aquat. Microb. Ecol. 56, 297308 (2009).
Voloshko L.N., Kopecky J., Titova N.N., Pljusch A.V., Safronova T.V., Morosova A.A., Drabkova V.G.,
Kapustina L.L., Pinevich A.V.: A variety of toxins produced by cyanobacteria in Lake Ladoga. Est. J.
Ecol. 57, 1–11 (2008).
Vondrák J., Šoun J., Hrouzek P., Říha P., Kubásek J., Palice Z., Søchting U.: Caloplaca subalpina and C. thracopontica, two saxicolous species from the Caloplaca cerina group (Teloschistales). Lichenologist 40,
375–386 (2008).
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Wiesner C., Kopecky J., Pflüger M., Hundsberger H., Entler B., Kleber C., Atzler J., Hrouzek P., Štys D.,
Lukešová A., Schütt W., Lucas R.: Novel cell-based methods for the detection of cyanobacterial antiinflammatory and wound-healing promoting metabolites. Drug Metab.Lett. 1, 254-260 (2007).
Wiesner C., Pflüger M., Kopecky J., Štys D., Hundsberger H., Schütt W.: Innovative test systems for screening
and identification of potential therapeutic drugs. Int. J. Artif. Organs 31, 658-659 (2008).
Zapomělová E. , Hisem D., Řeháková K., Hrouzek P., Jezberová J., Komárková J., Korelusová J., Znachor P.:
Experimental comparison and phenotypical plasticity and growth demands of two strains from the Anabaena circinalis / A. crassa complex (cyanobacteria). J. Plankton Res. 30, 1257–1269 (2008).
Zapomělová E., Hrouzek P., Řeháková K., Šabacká M., Stribal M., Caisová L., Komárková J., Lukešová A.:
Morphological variability in selected heterocystous cyanobacterial strains as a response to varied temperature, light intensity and medium compositon. Folia Microbiol. 53, 333-341 (2008).
Zelík P., Lukešová A., Čejka J., Buděšínský M., Havlíček V., Čegan A., Kopecký J.: Nostotrebin 6, a bis(cyclopentenedione) with cholinesterase inhibitory activity isolated from Nostoc sp. str. Lukešová 27/97. J. Enz.
Inh. Med. Ch., (2009) (in press).
Zelík P., Lukešová A., Voloshko L., Štys D., Kopecký J.: Screening for acetylcholinesterase inhibitory activity
in cyanobacteria of the genus Nostoc. J. Enz. Inh. Med. Ch. 23, 1-6 (2008).
Župčanová A., Arellano J.B., Bína D., Kopecký J., Psenčík J., Vácha F.: The length of esterifying alcohol
affects the aggregation properties of chlorosomal bacteriochlorophylls. Photochem. Photobiol. 84, 11871194 (2008).
Patent
Čejka J., Kopecký J., Lukešová A., Štys D., Zelík P.: Cyanobacterial strain Nostoc sp. Lukešová 27/97 and isolation of acetylcholin esterase inhibitor. Patent 299567, Czech Industrial Property Authority (2008).
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AUTOTROPHIC MICROORGANISMS
Laboratory 134
CELL CYCLES OF ALGAE
Head
Vilém Zachleder, PhD.
Scientific staff
Kateřina Bišová, PhD.
Mária Čížková, PhD.
Technical staff
Milada Vítová, PhD.
Jiří Doucha, PhD.
Helena Vondrková, Vlasta Grillová
Doctoral students
Irena Doušková, MSc., Monika Hlavová, MSc., Dáša Umysová, MSc.
Research field and principal results
The program of the laboratory includes both a basic and applied research.The topics investigated include: (i) the role of environmental factors like light intensity and spectral composition of light or different compounds (heavy metals, selenium) on the cell cycle progression; (ii) study of the molecular mechanisms responsible for regulation of algal cell cycle,
progression into mitosis and response to DNA damage; (iii) development of processes allowing usage of waste CO2 for algal biomass production, with controlled synthesis of large
amounts of starch and lipids for a bioethanol and biofuels production.
B-type cyclin dependent kinase in alga Chlamydomonas reinhardtii
Cyclin dependent kinases (CDKs) are key regulators of the cell cycle. In higher plants,
canonical A-type CDK is constitutively expressed and regulates progression through the cell
cycle. A plant-specific B-type CDK has a strictly regulated expression and coordinates mitosis and endoreduplication. C. reinhardtii is a model green alga, its genome encodes a single
CDKB gene, CrCDKB1. Similarly to higher plant homologs, CrCDKB1 expression is strictly
regulated, being high at commitment point and in the S/M phase. We have shown that contrary to higher plants, CrCDKB1 is a major cell cycle regulating kinase. CrCDKB1 is phos-
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phorylated on tyrosine (a feature typical for higher plant CDKA) and its kinase activity is
higher than that of CrCDKA1. Moreover, CrCDKB1 is, in response to DNA damage, tyrosine
phosphorylated and hence inhibited by WEE1 kinase. This implies that CDKBs were originally major mitotic kinases and later they evolved to fulfill other roles outside the cell cycle
regulation.
WEE1 kinase of Chlamydomonas reinhardtii
WEE1 kinase is a major regulator of mitosis. During S and G2 phase it phosphorylates
and inactivates cyclin dependent kinase (CDK), the key activator of cell cycle progression. In
mammals and higher plants is WEE1 a part of DNA damage checkpoint that ensures maintenance of cell viability in cells which have suffered genomic damage. We have produced
recombinant strains containing GFP tagged CrWEE1 cDNA under inducible promoter. The
overexpressing strains display a clear cell size phenotype. The mother cells overexpressing
CrWEE1 divide into less bigger daughter cells if compared to un-induced cells. Moreover, the
overexpressing strains are hyposensitive to DNA damage. This implied a role for CrWEE1 in
both cell cycle/cell size regulation and in response to DNA damage. More detailed analysis
showed that CrWEE1 is transcriptionally induced upon DNA damage and post-transcriptionally regulated in ataxia telangiectasia mutatated (ATM) and ATM and Rad3 related
(ATR) kinase dependent manner. Transcriptional activation of CrWEE1 lead to a cell cycle
arrest prior to S/M phase with high tyrosine phosphorylated inactive CDKB. DNA damage
response in C. reinhardtii includes a plant/alga specific component, CrCDKB1, and an evolutionary conserved component, ATM/ATR dependent regulation of WEE1 kinase.
Bioaccumulation and toxicity of selenium compounds
in the green alga Scenedesmus quadricauda
We selected three strains of S. quadricauda specifically resistant to the presence of high
concentrations of inorganic selenium added as selenite – strain SeIV, selenate – strain SeVI or
both - strain SeIV+VI. The total amount of Se and selenomethionine in biomass increased
with increasing concentration of Se in the culturing media. The selenomethionine made up
30-40% of the total Se in biomass. In both the wild type and Se-resistant strains, the activity
of thioredoxin reductase, increased rapidly in the presence of the form of selenium for which
the given algal strain was not resistant.
With sulfur deficiency, the selenium toxicity increases indicating interference of Se
with sulfur metabolism. The amount of selenium and SeMet in algal biomass was dependent
on both on the type of the compound and dose. The activity of thioredoxin reductase was
affected by selenium treatment in dose-dependent and toxic-dependent manner.
Utilization of waste sources of CO2 for cultivation of algae
CO2 represents a considerable part of the running costs when producing the microalgal
biomass in a large scale. In order to reduce the expenditures, flue gas originating from a municipal waste incinerator runned by TERMIZO Comp. in Liberec (Czech Republic) was found
to be suitable source of CO2 for cultivation of Chlorella vulgaris. Toxicological analysis of
the biomass produced using untreated flue gas showed the algal biomass composition compliant with all the foodstuff legislation requirements. Considering also non-nutritional utilization
of the biomass there is a great potential of its exploitation in the field of biofuels. We focussed especially on the production of starch (and consequently ethanol) in microalgae. Affecting
the cell cycle regulation can influence its content in the biomass. Several approaches making
possible to increase enormously (up to 80 % of dry mass) the starch content in cells (Fig. 26)
were successfully tested and applications for patents were filed.
AUTOTROPHIC MICROORGANISMS
103
Fig. 26. The electron microscopy of the cells Chlorella with markedly increased amount of starch.
Publications
Cepák V., Přibyl P., Vítová M.: The effect of light color on the nucleocytoplasmic and chloroplast cycle of the
green chlorococcal alga Scenedesmus obliquus. Folia Microbiol. 51, 342-346 (2006).
Cepák V., Přibyl P., Vítová M. Zachleder, V.: The nucleocytosolic and chloroplast cycle in the green alga
Scenedesmus obliquus (Chlorophyceae, Chlorococcales) grown under various temperatures. Phycologia
46, 263-269 (2007).
Čížková M., Pichová A., Vítová M., Hlavová M., Hendrychová J., Umysová D., Gálová E., Ševčovičová A.,
Umen J., Zachleder V., Bišová, K.: CDKA and CDKB kinases from Chlamydomonas reinhardtii are able
to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae. Protoplasma 232,
183-191 (2008).
Doucha J., Lívanský , K.: Outdoor open thin-layer microalgal photobioreactor: potential productivity. J. Appl.
Phycol. 21, 111-117 (2009).
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Doucha J., Lívanský K., Kotrbáček V., Zachleder V.: Production of Chlorella biomass enriched by selenium and
its use in animal nutrition: a review. Appl. Microbiol. Biotechnol. 83, 1001-1008 (2009).
Doucha J., Lívanský K.: Influence of processing parameters on disintegration of Chlorella in various types of
homogenizers. Appl. Microbiol. Biotech. 81, 431-440 (2008).
Douskova I., Doucha J., Livansky K., Machat J., Novak P., Umysova D., Zachleder V., Vitova M.: Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs. Appl. Microbiol. Biotechnol. 82, 179–185 (2009).
Merchant S.S., et al.:The Chlamydomonas genome reveals the evolution of key animal and plant functions.
Science 318, 245-250 (2007).
Oldenhof H., Zachleder V., van den Ende H.: The cell cycle of Chlamydomonas reinhardtii: the role of the
commitment point. Folia Microbiol. 52, 53-60 (2007).
Pribyl P., Cepák V., Zachleder, V.: Cytoskeletal alterations in interphase cells of the green alga Spirogyra
decimina in response to heavy metals exposure: II. The effect of aluminium, nickel and copper. Toxicol.
In Vitro 22, 1160-1168 (2008).
Trávníček J., Písek L., Herzig I., Doucha J., Kvíčala J., Kroupová V., Rodinová H.: Selenium content in blood
serum and urine of ewes receiving selenium-enriched unicellular alga Chlorella. Veter. Med. 52, 42-45
(2007).
Trávníček J., Racek J., Trefil L., Rodinová H., Kroupová V., Illek J., Doucha J., Písek L.: Activity of glutathione peroxidase (GSH-Px) in the blood of ewes and their lambs receiving the selenium-enriched unicellular alga Chlorella. Czech. J. Anim. Sci. 53, 292-298 (2008).
Umysová D., Vítová M., Doušková I., Bišová K., Hlavová M., Čížková M., Doucha J., Machát J., Zachleder V.:
Bioaccumulation and toxicity of selenium compounds in the green alga Scenedesmus quadricauda. BMC
Plant Biol. 9, 58, 1-16 (2009).
Vítová M., Hendrychová J., Čížková M., Cepák V., Umen J.G., Zachleder V., Bišová K. Accumulation, activity
and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga
Scenedesmus quadricauda under inhibition of nuclear DNA replication. Plant Cell Physiol. 49, 18051817 (2008).
Chapters in books
Bišová K.: Cell growth control in an algal model. – In: Bögre, L., Beemster, G. (eds.) Plant Growth Signaling,
pp. 351-373, Springer-Verlag, 2008.
Šetlík I., Doucha J.: Cyanobacteria and Microalgae. In:Quality of Plant Products at the Beginning of the 21st
Century. (in Czech), ed. F. Prugar, pp. 298-304, 2008.
Šetlík I., Doucha J., Prugar J., Smotlacha M.: Fungi. In:Quality of Plant Products at the Beginning of the 21st
Century. (in Czech), ed. F. Prugar, pp. 296-297, 2008.
Patents
Doucha J., Lívanský K.: Production strain of microalgae Chlorella vulgaris BEIJ. Strain Doucha et Lívanský
1996/H14. (in Czech). Cz.Patent 299352-2008.
Doucha J., Lívanský K., Zachleder V.: Production strain P13/1998 of unicellular alga Chlorella vulgaris BEIJ.
(in Czech). PV 2009-214 (Z6452), 2009.
Doušková I., Hlavová M., Umysová D., Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda
SeIV of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent 3008612009.
Doušková I.; Hlavová M.; Umysová D.; Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda
SeVI of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent 3008092009.
Doušková I., Hlavová M., Umysová D., Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda
SeIV+VI of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent
300808-2009.