Enhanced biodegradation and detoxification of disperse

Enhanced biodegradation and detoxification of disperse

International Biodeterioration & Biodegradation 72 (2012) 94e107
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International Biodeterioration & Biodegradation
journal homepage: www.elsevier.com/locate/ibiod
Enhanced biodegradation and detoxification of disperse azo dye Rubine GFL
and textile industry effluent by defined fungal-bacterial consortium
Harshad S. Lade a, Tatoba R. Waghmode a, Avinash A. Kadam b, Sanjay P. Govindwar a, *
a
b
Department of Biochemistry, Shivaji University, Vidyanagar, Kolhapur, Maharashtra 416004, India
Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur, Maharashtra 416004, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 April 2012
Received in revised form
28 May 2012
Accepted 1 June 2012
Available online
In this study, a defined consortium-AP of Aspergillus ochraceus NCIM-1146 fungi and Pseudomonas sp.
SUK1 bacterium was studied to assess its potential for enhanced decolorization and detoxification of azo
dye Rubine GFL and textile effluent. Developed consortium-AP showed enhanced decolorization of dye
(95% in 30 h) and effluent (98% ADMI removal in 35 h) without formation of aromatic amines under
microaerophilic conditions. Individual A. ochraceus NCIM-1146 showed only 46% and 5% decolorization of
the dye and effluent. However, Pseudomonas sp. SUK1 showed 63% and 44% decolorization of the dye and
effluent respectively with the production of aromatic amines. Induction of laccase, veratryl alcohol
oxidase, azo reductase and NADH-DCIP reductase in the consortium-AP suggests synergetic reactions of
fungal and bacterial cultures for enhanced decolorization process. Differential fate of metabolism of
Rubine GFL by an individual and consortium-AP cultures were proposed on the basis of enzymatic status,
FTIR and GC-MS analysis. Furthermore, consortium-AP also achieved a significant reduction in COD
(96%), BOD (82%) and TOC (48%) of textile effluent. The results of toxicity studies suggest that this
consortium may effectively be used for complete detoxification of dye and effluent and has potential
environmental implication in cleaning up azo dyes containing effluents.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Rubine GFL
Consortium-AP
Decolorization
Biodegradation
Veratryl alcohol oxidase
Detoxification
1. Introduction
Among the many different groups of synthetic dyes, azo (containing one or more azo group, R1eN NeR2) dyes are extensively
used as raw material in textile processing industry. Azo dyes are
resistant to degradation and remains persistent for long time due to
its fused aromatic structure (Xu et al., 2006). The treatment of
wastewater coming from dying and textile industries becomes
most difficult due to its high chemical oxygen demand (COD) and
excess content of suspended solids such as surfactants, detergents
and dyestuff. This results in severe ecological damages when
released into the water resources such as rivers and lakes, which
alters its pH, increases COD and gives intense coloration. It is quite
undesirable to discharge azo dyes wastewater into the environment due to its high toxicity and toxic intermediates produced
(Levine, 1991). The toxicity of most of the azo dyes is one of the
serious environmental concerns (Dong et al., 2003; Wang et al.,
2009) as the effluents coming from dye processing and
manufacturing industries are known to be carcinogenic as well as
* Corresponding author. Tel.: þ91 231 2609152; fax: þ91 231 2691533.
E-mail address: [email protected] (S.P. Govindwar).
0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ibiod.2012.06.001
mutagenic to various organisms (Mathur et al., 2005; Chen, 2006;
Novotny’ et al., 2006; Mathur and Bhatnagar, 2007). This increasing
toxicity of discharged wastewater affects the human beings in
a number of ways making dye contamination both, an environmental as well as public health issues.
A number of conventional physico-chemical wastewater treatment processes such as electrocoagulation, adsorption on activated
carbon, ion exchange, flocculation, froth flotation, ozonation,
membrane filtration and reverse osmosis have been suggested for
decolorization of textile effluent. However, most of the dyes form
textile effluents escape from such conventional treatment
processes and persist in the environment for long time as a result of
their high stability against light, temperature and oxidizing agents.
These conventional physico-chemical processes cannot be used
widely due to their high cost, secondary pollution generated by the
excessive use of chemicals and inapplicability to a wide variety of
dyes. Compared with physical and chemical processes, bio-friendly
approaches have been the main focus for remediation of dyecontaminated wastewater since they require lower costs, are ecofriendly and produce fewer toxic metabolites (Kobayashi and
Rittmann, 1982; Stolz, 2001).
A lot of research on the treatment of textile dyestuff and effluent
has been carried out using individual bacterial and fungal cultures.
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
Several microorganisms belonging to different taxonomic groups of
fungi (Parshetti et al., 2007), bacteria (Kalyani et al., 2008) and yeast
(Waghmode et al., 2011a) proved their ability to decolorize dyes by
bioadsorption, biotransformation or degradation. Numerous
bacterial species have been studied for degradation of azo dyes by
virtue of their rapid growth and faster degradation rates, although
many of them produce colorless carcinogenic and mutagenic
aromatic amines (Levine, 1991; Joshi et al., 2008). On the other
hand, some bacterial cultures are able to reduce azo compounds
aerobically with the help of oxygen catalyzed azo reductase but also
produce aromatic amines (Lin et al., 2010). Besides bacterial
cultures, diverse fungal species have been investigated for
biodegradation of textile dyestuff due to their excellence in large
biomass production, hostile growth, spacious hyphal reach and
high surface to cell ratio. White-rot fungi Pyricularia oryzae is
known to degrade phenolic azo dyes without the formation of
aromatic amines (Chivukula and Renganathan, 1995). However, the
time consuming growth, long hydraulic retention time for
complete decolorization and low decolorization efficiency limits
the use of fungi for bioremediation of textile effluent (Banat et al.,
1996; Chang et al., 2004).
Despite their great promise, both bacteria and fungi have
suffered certain limitations with respect to their individual abilities
to completely degrade and detoxify azo dyes. A synergistic action of
fungal-bacterial consortium leads to the enhanced degradation and
detoxification of azo dyes and, thus provides an alternate way for
efficient removal of contaminants (Khelifi et al., 2009; Su et al.,
2009; Qu et al., 2010). Moreover the high rates of dye decolorization by fungal-bacterial synergism suggests an appropriate
powerful tool for the efficient degradation and detoxification of azo
dyes as well as textile effluent (Khelifi et al., 2009; Su et al., 2009;
Qu et al., 2010). Eco-friendly, efficient and short degradation times
are some of the highlights of fungal-bacterial synergism over
individual cultures. Such synergisms are more effective due to the
concerted metabolic activities, which might attack dye molecules at
various positions or utilize intermediate degradation metabolites
for further mineralization into non-toxic form (Keck et al., 2002;
Chen and Chang, 2007). It is known that, addition of intermediate
metabolites of dye decolorizing culture into another culture could
enhance the azo dye decolorization rates (Chang et al., 2004).
Microorganisms can decolorize the dyes with different enzyme
systems. Fungal enzymes are non-specific towards different structures of dyes and thus oxidize a wide range of them (Aust, 1990).
Fungi have been extensively studied to degrade textile dyes due to
their extracellular oxidoreductive, nonspecific and nonstereoselective enzyme system, including lignin peroxidase, laccase, manganese peroxidase and tyrosinase (Hofrichter, 2002;
Kaushika and Malik, 2009). The bacterial biodegradation is associated with its intracellular and extracellular oxidoreductive
enzyme system such as azo reductase, DCIP-reductase and laccase
(Chen et al., 2003; Kalyani et al., 2008; Telke et al., 2009a). The
selected pure cultures of Aspergillus ochraceus NCIM-1146 and
Pseudomonas sp. SUK1 are well known for the degradation of
different dyes due to induction in the activities of oxidoreductive
enzymes under certain environmental conditions (Parshetti et al.,
2007; Kalyani et al., 2009). In recent years, different consortial
approaches have been studied due to their enhancing degradation
abilities. Consortial systems can provide advantages over individual
cultures as they involve the combined and inductive effects of
various enzymes which can work synergistically. Few cases have
been reported that demonstrated the potential of fungal-bacterial
consortium for enhanced degradation of textile dyestuff (Kadam
et al., 2011). There is, however a great need for further research
to set up eco-friendly remediation technologies without the
formation of toxicants by virtue of fungal-bacterial synergism.
95
Though most of the research works on dye decolorization have
been carried out using individual fungal and bacterial cultures but
the work pertained to fungal-bacterial synergism for biodegradation and detoxification of azo dyes is missing. Keeping this view as
well as to overcome the problems of partial degradation, long
reaction time and formation of toxic metabolites, a developed
consortium-AP of fungal-bacterial synergism was investigated for
biodegradation of model azo dye Rubine GFL and textile effluent
without the formation of toxic aromatic amines.
2. Material and methods
2.1. Chemicals and dye stuff used
Catechol, L-ascorbic acid, o-tolidine, veratryl alcohol, methyl red,
nutrient medium (NM) and potato dextrose broth (PDB) were
obtained from Hi Media Laboratories Pvt. Ltd., Mumbai, India.
Chloranil, Dimethylformamide (DMF) and Aniline-2-sulfonic acid
were procured form SigmaeAldrich, USA. Remaining chemicals were
purchased from Sisco Research Laboratories (SRL), India. All chemicals used were of highest purity available and of an analytical grade.
2.2. Dye stuff and effluent collection
Disperse azo textile dye Rubine GFL (98% purity) (C.I. Disperse
red 78) was generously gifted by Mahesh Textile Processors,
Ichalkaranji, India. The highly colored effluent of the same textile
processing industry utilizing various dyes v.z. azoic, sulphonic,
reactive and disperse dyes as raw materials was collected in airtight
plastic can and tightly stoppered. The collected effluent was
transported to laboratory and filtered through Whatman grade no.
1 filter paper to remove large suspended particles. The pH of the
filtered effluent was maintained at 7.0 and stored at 4 1 C
temperature until processing to prevent contamination by nonindigenous microbes.
2.3. Determination of aromatic amines
Samples were taken after decolorization, frozen and freeze-dried
in Upright Freeze Dryer Model: FDU5003/8603 (Operon Co. Ltd.,
Korea) and the aromatic amines formed were determined spectrophotometrically as per the method of Elisangela et al. (2009). A
calibration curve of aniline-2-sulfonic acid as a model amine
product of azo dyes reduction was prepared and the concentration
of sample amine was calculated in mM l1. The pre-grown cultures
without addition of dye and effluent were used as control.
2.4. Microorganism and culture conditions
A. ochraceus NCIM-1146 culture was obtained from National
Center for Industrial Microorganisms, National Chemical Laboratories (Pune, India). The stock culture was maintained on potato
dextrose agar slants at 4 C. Pseudomonas sp. SUK1 culture previously isolated from textile dye contaminated site was used (Kalyani
et al., 2008). The stock culture was maintained on nutrient medium
agar slants at 4 C. The composition of PDB used for decolorization
studies was (g l1); potatoes infusion 200.0, dextrose 20.0 and yeast
extract 5.0. The composition of NM used for decolorization studies
was (g l1); sodium chloride 5.0, beef extract 1.5, yeast extract 4.0
and peptic digest of animal tissue 5.0.
2.5. Development of consortium-AP for decolorization study
Two A. ochraceus NCIM-1146 discs (8 mm diameter) of 96 h old
culture were inoculated into 250 ml Erlenmeyer flasks containing
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H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
100 ml of PDB and incubated for 96 h at 30 C under microaerophilic (no agitation) as well as aerobic conditions (shaking at
120 rpm). One loopful of 24 h old Pseudomonas sp. SUK1 culture
was inoculated into 100 ml of NM and incubated for 24 h at 30 C
under microaerophilic and aerobic conditions. Consortium-AP was
prepared by aseptically transferring the 50 ml of 96 h grown A.
ochraceus NCIM-1146 culture into a 250 ml Erlenmeyer flasks
containing 50 ml of 24 h grown Pseudomonas sp. SUK1 culture. The
pre-grown individual cultures and its developed consortium were
then used as inoculums for further degradation studies.
2.6. Optimization of media composition
Optimization of media composition for enhanced decolorization
of dye and effluent by using A. ochraceus NCIM-1146 was carried out
in PDB (pH 5.8) supplemented with 2.5, 5.0 and 10.0 g ll of yeast
extract and peptone as additional nitrogen source and 2.5, 5.0 and
10.0 g ll of dextrose and lactose as additional carbon source. For
Pseudomonas sp. SUK1, factorial experiments were designed in NM
(pH 7.0) supplemented with same concentration of additional
nitrogen and carbon sources.
2.7. Decolorization experiment and physicochemical parameters
Decolorization of Rubine GFL was carried out under microaerophilic conditions with 100 ml culture of A. ochraceus NCIM1146 and Pseudomonas sp. SUK1 pre-grown in PDB and NB supplemented with 5.0 and 2.5 g ll of yeast extract respectively.
Consortium-AP was prepared as per the method described in
Section 2.5 and used for decolorization of dye. 100 mg l1 of dye
was added into each 250 ml Erlenmeyer flask containing 100 ml of
individual pre-grown cultures as well as its developed consortium
and further incubated until decolorization was observed. Aliquots
of the culture supernatant were withdrawn at regular time intervals during the process of decolorization. Suspended particles from
the culture supernatant were removed by adding equal volume of
methanol followed by centrifugation at 7500 rpm for 15 min. The
decolorization was monitored by measuring the change in absorbance maxima of the dye (lmax of Rubine GFL 530 nm) using
a UVevis spectrophotometer (Hitachi U-2800; Japan). All decolorization experiments were performed in triplicate and the % decolorization was calculated as follows:
Decolorizationð%Þ ¼
Initial absorbance Observed absorbance
Initial absorbance
100
The above mentioned protocol was followed while studying the
decolorization of textile azo dye Rubine GFL by using individual
cultures as well as its consortium at wide range of pH (3e11),
temperature (20, 30, 37, 40 and 50 C) and increasing dye
concentrations (50 mg ll to 250 mg ll). The dissolved oxygen (DO)
level in the individual and consortium culture was measured with
Hanna HI 9146 dissolved oxygen meter (Hanna Instruments, USA).
Decolorization experiments were carried out in triplicate and the
abiotic (without microorganism) controls were always included to
measure the photodecolorization or abiotic loss of dye.
2.8. Decolorization of textile effluent
Decolorization of real textile effluent was carried out in the
250 ml Erlenmeyer flask containing 100 ml of pre-grown individual
cultures as well as its developed consortium-AP. 100 ml of sterilized
textile effluent (121 C for 20 min) was added into each flask
containing 100 ml of pre-grown individual cultures as well as its
consortium and further incubated under microaerophilic as well as
aerobic conditions. Aliquots of the culture supernatant were withdrawn at regular time intervals; suspended particles were removed
by adding equal volume of methanol followed by centrifugation at
7500 rpm for 15 min. The obtained clear supernatant was used to
determine the decolorization of effluent. Decolorization was monitored using the American Dye Manufacturer’s Institute (ADMI 3WL)
tristimulus filter method reported earlier (Chen et al., 2003). The
transmittance of the sample at three different wavelengths (590, 540
and 438 nm) were recorded and the ADMI value was calculated using
the ‘AdamseNickerson chromatic value formula’ (APHA, 1998). The
ADMI value provides a true measurement of water color, independent of hue and thus gives deep insight into the more précising
definition of effluent. Decolorization was expressed in terms of ADMI
removal ratio and calculated using the following formula:
ADMI removal ratioð%Þ ¼
Initial ADMIð0 hÞ Observed ADMIðtÞ
Initial ADMIð0 hÞ
100
Where, ADMI(0 h) and ADMI(t) are the initial ADMI values at (0 h)
and the ADMI value after a particular reaction time (t), respectively.
All decolorization experiments were carried out in three sets.
Control set (without effluent or inoculums) was also run under
identical conditions.
2.9. Characterization of textile effluent
The textile effluent was characterized for reduction in chemical
oxygen demand (COD) and biological oxygen demand (BOD) before
and after the biodegradation (APHA, 1998). The COD of the textile
effluent was measured by using automated COD analyzer (Spectralab
CT 15, India). The total organic carbon (TOC) was measured using
Hach DR 2700 spectrophotometer (Hach Co., USA) (Waghmode et al.,
2011b). The TOC removal ratio was calculated as follows:
TOC removal ratioð%Þ ¼
Initial TOC ð0 hÞ Observed TOC ðtÞ
Initial TOC ð0 hÞ
100
Where, TOC(0 h) and TOC(t) are the initial TOC value at (0 h) and the
TOC value after particular reaction time (t), respectively.
2.10. Metabolites analysis
After decolorization of Rubine GFL and textile effluent, the
fungal mycelium was removed by filtration; bacterial cells were
removed by centrifugation at 10,000 rpm for 20 min while the
consortium-AP biomass was removed by filtration followed by
centrifugation at 10,000 for 20 min. The supernatant obtained was
used to extract metabolites with an equal volume of ethyl acetate;
dried over anhydrous Na2SO4, dissolved in HPLC grade methanol
and used for further analytical studies like HPTLC, HPLC, FTIR and
GC-MS analysis.
Biodegradation of dye was confirmed by analyzing the obtained
metabolites with HPTLC system (CAMAG, Switzerland) as reported
earlier (Kurade et al., 2011). 15 ml of control dye and obtained
metabolites were applied on the pre-coated silica gel plates (HPTLC
Lichrospher silica gel 60 F254S, Merk, Germany) by micro syringe
using spray gas nitrogen sample applicator (Linomat V, CAMAG,
Switzerland). The dosage parameters for plate were set as 6 mm
bands, 10 mm apart from Y-axis, 10 mm from the lower edge of the
plate, first application position 20 mm from left edge. The
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
composition of developing solvent system used as mobile phase
was toluene: ethyl acetate: methanol (7:2:1 v/v). The twin trough
chamber was pre-equilibrated with developing solvent for a period
of 20 min prior to plate development. TLC plate was developed by
placing in the trough chamber containing pre-conditioning solvent
until the desired running distance is reached and then oven dried at
120 C for 20 min. After development, densitometric evaluation of
spots was carried out at 254 and 530 nm wavelength using
deuterium and tungsten lamp respectively with slit dimension of
5 0.45 mm using CAMAG TLC Scanner-3 (CAMAG, Switzerland).
The chromatograms were integrated using HPTLC WinCATS evaluation software (Version 1.4.4.6337).
HPLC analysis was carried out using Waters 2690 instrument
(Waters Corporation, UK) on C18 column (Symmetry,
4.6 250 mm) by isocratic method using the gradient of methanol
with a flow rate of 0.50 ml min1 for 10 min and UV detector at
280 nm (Kadam et al., 2011). 10 ml of filtered sample was manually
injected into the injector port.
FTIR analysis was performed in order to investigate the changes
in surface functional groups of the extracted metabolites, before
and after microbial decolorization. FTIR analysis was done on Shimadzu 8400S spectrophotometer (Shimadzu Corporation, Japan) in
the mid IR region of 400e4000 cm1 with 16 scan speed
(Waghmode et al., 2011b). The samples were prepared using
spectroscopic pure KBr (5:95), pellets were fixed in the sample
holder and analyzed.
The identification of metabolites formed after decolorization
was carried using GC-MS QP2010 (Shimadzu Corporation, Japan) by
modifying the procedure reported earlier (Kalyani et al., 2009). The
ionization voltage was 70 eV. Gas chromatography was conducted
in the temperature programming mode with a Restek column
(0.25 mm id, 60 m long, nonpolar; XTI-5). The initial column
temperature was 80 C for 2 min, then increased linearly at
10 C min1 to 280 C, and held for 7 min. The temperature of the
injection port was 280 C and the GC-MS interface was maintained
at 290 C. Helium was used as carrier gas with a flow rate of
1.0 ml min1. Degradation products were identified by comparison
of retention time and fragmentation pattern, as well as with mass
spectra in the NIST spectral library support stored in the GC-MS
solution software (version 1.10 beta, Shimadzu).
2.11. Enzyme extraction
The individual cultures were grown in their respective optimized medium while the consortium-AP was prepared as method
described in Section 2.5. A. ochraceus NCIM-1146 fungal mycelium
was collected by filtration, while Pseudomonas sp. SUK1 bacterial
cells were collected by centrifugation at 7500 rpm for 15 min and
the resulted fungal culture filtrate and bacterial supernatant was
used as extracellular enzyme source. The collected biomass of
individual cultures and consortium was separately suspended in
50 mM potassium phosphate buffer (pH 7.4), homogenized and
sonicated (Sonics-vibracell ultrasonic processor, 7 strokes of 30 s
each for 2 min interval based on 50 amplitude output) at 4 C. The
sonicated cells were centrifuged in cold condition (4 C, at
7500 rpm for 15 min) and resulting supernatant was used as the
source of intracellular enzymes. Similar procedure was carried out
to quantify enzyme activities after Rubine GFL decolorization by
individual cultures as well as its consortium.
2.12. Enzyme activities
2.12.1. Oxidative enzymes during decolorization
Activities of oxidative dye degrading enzymes such as laccase,
veratryl alcohol oxidase and tyrosinase were assayed
97
spectrophotometrically in the cell free extract (intracellular) as well
as culture supernatant (extracellular). Laccase activity was determined in a reaction mixture of 2.0 ml containing 1.7 ml sodium
acetate buffer (20 mM, pH 4.8) and 0.1 ml 50 mM o-tolidine. The
reaction was started by adding 0.2 ml of enzyme solution and an
absorbance increase due to oxidation of o-tolidine was monitored
at 366 nm (Telke et al., 2009a). Veratryl alcohol oxidase activity was
determined in a reaction mixture of 2.0 ml containing 4 mM
veratryl alcohol as substrate in citrate phosphate buffer (50 mM, pH
3.0). The reaction was started by adding 0.2 ml of enzyme solution
and an absorbance increase due to the formation of veratraldehyde
was monitored at 310 nm (Jadhav et al., 2009). Tyrosinase activity
was determined by modifying the earlier reported method
(Kandaswami and Vaidyanathan, 1973). The 3.0 ml reaction
mixture contained 0.1 ml 50 mM catechol and 0.1 ml 2.1 mM Lascorbic acid in potassium phosphate buffer (50 mM, pH 7.4)
equilibrated at 25 C. The DA265 nm was monitored until constant,
and then reaction was started by adding 0.1 ml of enzyme solution.
The formation of dehydro-ascorbic acid and o-benzoquinone and
decrease in optical density was measured at 265 nm. One unit of
tyrosinase activity was equal to a DA265 nm of 0.001 per min at pH
7.4 at 25 C in a 3.0 ml reaction mixture containing catechol and Lascorbic acid.
2.12.2. Reductase enzymes during decolorization
Activities of reductive dye degrading enzymes such as azo
reductase and NADH-DCIP reductase were determined spectrophotometrically in cell free extracts using the procedure reported
earlier. Azo reductase assay was performed in a reaction mixture of
2.0 ml containing 25 mM of methyl red and 0.2 ml of enzyme
solution in potassium phosphate buffer (50 mM, pH 7.4). The
reaction mixture was pre-incubated for 4 min at room temperature
followed by the addition of 1000 mM NADH. The decrease in color
absorbance due to enzymatic cleavage of azo dye methyl red (2-[4(dimethylamino)phenylazo] benzoic acid) into N,N-dimethyl-pphenylenediamine and 2-aminobenzoic acid was monitored at
430 nm. Methyl red reduction was calculated by using its molar
extinction coefficient of 0.023 mM1 cm1 (Chen et al., 2005).
Activity of NADH-DCIP reductase was determined by modifying the
procedure reported earlier (Salokhe and Govindwar, 1999). Briefly,
5.0 ml reaction mixture contained 25 mM DCIP (2,6-dichlorophenol
indophenol) and 0.1 ml enzyme solution in potassium phosphate
buffer (50 mM, pH 7.4). From this, 2.0 ml reaction mixture was
assayed at 590 nm by adding 250 mM NADH. The DCIP reduction
was calculated using the extinction coefficient of 0.019 mM1 cm1.
One unit of reductase enzyme activity was defined as amount of
enzyme required to reduce 1 mM of substrate min1 mg of
protein1.
All enzyme assays were carried at room temperature where
reference blank run along the test. All enzyme assays were run in
triplicate, average rates were calculated and one unit of enzyme
activity was defined as a change in absorbance unit min1 mg of
protein1. The protein content was determined by using the
method of Lowry et al. (1951) with bovine serum albumin as the
standard.
2.13. Toxicity studies
It is known that, the colouration of water due to presence of
textile dyes, even in small concentration may have inhibitory effect
on the process of photosynthesis and thus affects its growth. In
order to assess the toxicity of dye Rubine GFL, textile effluent and
it’s produced metabolites after decolorization by consortium-AP;
phytotoxicity tests were carried out on two kinds of common
Indian agricultural crops: Sorghum vulgare (monocot) and Phaseolus
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mungo (dicot) as described earlier (Telke et al., 2010). The 1000 ppm
solution of dye Rubine GFL and ethyl acetate extracted degradation
metabolites of dye and effluent were prepared in distilled water
and applied for toxicity testing. The filtered real textile effluent was
directly used to assess it toxicity. Ten healthy seeds of each crop
were separately sowed into the plastic pot containing 15.0 g of
washed and oven dried sand. The toxicity study was carried out at
room temperature i.e. 27 3 C by daily watering 5 ml of Rubine
GFL (1000 ppm), real textile effluent and its degradation metabolites (1000 ppm). Control set was carried out at the same time by
watering the seeds with distilled water (daily 5 ml). Germination
(%) and the length of plumle (shoot) and radicle (root) was recorded
after 13 days. Germination % was calculated as follows:
Germinationð%Þ ¼
No: of seeds germinated
100
No: of seeds sowed
2.14. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA)
with TukeyeKramer multiple comparison test (Hsu, 1996).
3. Results and discussion
3.1. Optimization of media composition and culture conditions
In view of optimizing the media composition for enhanced
decolorization of dye Rubine GFL and textile effluent by using
A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1; effect of
additional nitrogen and carbon sources was studied at microaerophilic conditions as no considerable decolorization performance was observed in aerobic conditions. A total of 46%
decolorization of Rubine GFL (100 mg l1) in 30 h and 5% ADMI
removal of textile effluent color in 35 h was observed using
A. ochraceus NCIM-1146 grown in PDB supplemented with
5.0 g l1 yeast extract as additional nitrogen source, while less
decolorization with other supplements of nitrogen source
(peptone) was observed (Data not shown). NM containing 2.5 g l1
yeast extract was found to be better additional nitrogen source for
enhanced decolorization of Rubine GFL (63% in 30 h) and textile
effluent (44% ADMI removal) using Pseudomonas sp. SUK1, while
less decolorization with peptone as nitrogen source was observed
(Data not shown). There are some evidences to suggest that the
azo dye decolorization by pure as well as mixed culture requires
additional complex organic sources. For example nitrogen sources
such as yeast extract or peptone could enhance the decolorization
efficiency of Aeromonas hydrophila for dye Red RBN (Chen et al.,
2003). Moreover, Pseudomonas aeruginosa NBAR 12 was able to
decolorize diazo dye Reactive blue rapidly when supplied with
additional yeast extract in the medium (Bhatt et al., 2005). The
organic nitrogen sources can regenerate NADH, which acts as an
electron donor for the reduction of azo dyes which ultimately
enhances decolorization (Hu, 1994). Furthermore, addition of
dextrose and lactose as additional carbon source decreases the
decolorization rate of dye Rubine GFL as well as textile effluent.
The negative effect of carbon sources like glucose on microaerophilic decolorization has been ascribed either due to decrease
in pH by acid formation or to catabolic repression (Chen et al.,
2003). The accelerating effect of consortium-AP of A. ochraceus
NCIM-1146 and Pseudomonas sp. SUK1 pre-grown in respective
medium with additional nitrogen sources showed great
improvement in the dye Rubine GFL (95% in 30 h) and textile
effluent (98% ADMI removal in 35 h) decolorization efficiency in
microaerophilic condition (Table 1).
Better growth of A. ochraceus NCIM-1146 (Dry weight 6.41 g l1
in 96 h at 30 C) and Pseudomonas sp. SUK1 (Dry weight 1.35 g l1 in
24 h at 30 C) was observed under aerobic condition when
compared with microaerophilic condition (Dry weight 4.41 g l1
and 0.55 g l1 respectively). The aerobic grown A. ochraceus NCIM1146 and Pseudomonas sp. SUK1 showed 46% and 34% decolorization of dye Rubine GFL in 30 h and 5% and 35% ADMI removal of
textile effluent within 35 h in microaerophilic conditions respectively (Table 1). On the other hand, individual cultures pre-grown at
microaerophilic conditions showed 43% and 63% decolorization of
dye and 4% and 44% ADMI removal of textile effluent when incubated at microaerophilic conditions (Table 1). The DO levels under
microaerophilic condition were found to be 0.04, 0.02 and
0.01 mg l1 for A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 and
consortium-AP
respectively.
Decolorization
efficiency
of
consortium-AP for dye (95% in 30 h) and effluent (98% ADMI
removal in 35 h) of aerobic grown A. ochraceus NCIM-1146 and
microaerophilic grown Pseudomonas sp. SUK1 was appreciably
improved in microaerophilic conditions suggest the involvement of
oxygen sensitive reductases in the process of decolorization
(Table 1). This is similar to previous report where nearly zero DO
level was observed in static decolorization of azo dye with Escherichia coli NO3 (Chang and Kuo, 2000). In contrast, only 5% decolorization of dye and 2% ADMI removal of effluent was observed
under aerobic condition by using the same consortium-AP (data not
shown). The presence of oxygen may inhibit the enzymatic
reduction of azo bond (eN]Ne), since aerobic condition may rule
over the utilization of NADH, thus preventing the electron transfer
from NADH to azo bonds (Stolz, 2001). The results obtained in the
present study are in agreement with the reports recorded during
decolorization of Reactive red 120 and Direct red 81 by A. niger as
well as decolorization of Reactive red 2 by Pseudomonas sp. SUK1,
where microaerophilic conditions were used in the best possible
Table 1
Determination of aromatic amines produced and % decolorization of Rubine GFL and textile effluent by using A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 and its
consortium-AP under microaerophilic conditions.
Cultures
Enrichment conditions
Rubine GFL
% Decolorization
A. ochraceus NCIM-1146
Pseudomonas sp. SUK1
Consortium-AP
A. ochraceus NCIM-1146
Pseudomonas sp. SUK1
A. ochraceus NCIM-1146
Pseudomonas sp. SUK1
0.58
1.00
1.53
0.58
Effluent
Amines (mM)
% ADMI removal
ND
ND
0.14 0.01
0.06 0.01
4
5
44
35
43
46
63
34
Aerobic
Microaerophilic
Microaerophilic
Microaerophilic
95 1.00
ND
98 1.00
ND
78 1.53
ND
82 1.53
ND
ND ¼ Not detected. Values are mean of three experiments, standard deviation (SD).
1.00
1.53
1.00
1.53
Amines (mM)
Microaerophilic
Aerobic
Microaerophilic
Aerobic
ND
ND
0.18 0.01
0.07 0.01
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
manner than aerobic (Husseiny, 2008; Kalyani et al., 2009). Thus,
further decolorization study of dye and effluent was carried out in
microaerophilic conditions only.
3.2. Decolorization experiment and physicochemical parameters
Microbial decolorization of model azo dye Rubine GFL, which is
suspected to be recalcitrant, was investigated at different physicochemical conditions by using pure cultures as well as its
consortium-AP. It is important to study the effect of pH on decolorization process, as transport of dye molecule into the cell is pH
dependent and thought to be rate limiting step for decolorization of
dyes (Lourenco et al., 2000). Both the individual cultures were able
to decolorize the dye at broad range of pH, however optimum pH
Decolorization (%)
a
100
80
60
40
20
0
3
4
5
6
7
8
9
10
11
12
pH
Decolorization (%)
b
100
80
60
40
20
0
20
30
37
40
50
Temperature (o C)
for dye decolorization was found to be 8.5 for consortium-AP and
8.0 for Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 (Fig. 1a).
Decrease in % decolorization was observed at lower pH (5e7) as
well as higher pH (9e12) for both the cultures. It is thought that
metabolites formed during the process of decolorization by individual and consortium cultures may significantly increase the pH of
culture medium towards alkaline. An incubation temperature of
37 C was found to be optimum for enhanced degradation of dye
Rubine GFL by using consortium-AP (Fig. 1b). Further increase in
the temperature decreased the extent of degradation for both
consortium-AP and individual cultures. Decolorization performance at increasing dye concentrations suggest its potential for
complete decolorization of 100 mg l1 of dye Rubine GFL (95% in
30 h), whereas individual A. ochraceus NCIM-1146 (46% in 30 h) and
Pseudomonas sp. SUK1 (63% in 30 h) showed less decolorization for
the same concentration of dye Rubine GFL (Fig. 1c). Waghmode
et al. (2012) reported the enhanced decolorization and degradation of azo dye Rubine GFL (50 mg l1 within 30 h) using defined
consortium GG-BL of Galactomyces geotrichum MTCC 1360 yeast
and Brevibacillus laterosporus MTCC 2298 bacterium, whereas
individual cultures fails to completely decolorize the dye.
We have made a comparison of the UVevis spectral analysis
(400e800) of control dye Rubine GFL and its decolorization by
individual cultures as well as its consortium-AP. The spectrophotometric analysis of culture supernatant after decolorization by
consortium-AP showed significant reduction in absorbance than
both of the individual cultures (Fig. 2). As expected, the rate of
decolorization of consortium-AP was significantly higher than that
of individual cultures. The increased decolorization rate might be
due to the synergistic enzymes actions of both the organisms in the
consortium. As previously reported, the degradation of intermediates metabolites by bacteria could decline the fungal inhibition
and thus enhances the decolorization efficiency of consortium (Gou
et al., 2009). It is also known that the degradation products of one
culture in the consortium may act as inducer for another co-culture,
which results in the further mineralization of dye and metabolites
(Chang et al., 2004; Forgacs et al., 2004). Similar finding were
reported by Kadam et al. (2011), who observed higher decolorization rate of azo dye Navy blue HE2R in solid state fermentation by
developed consortium-PA of A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1. However, such studies are limited up to the
decolorization of water soluble dyes as dye must adsorb on solid
substrate. Hence, to overcome the problem of adsorption, the
submerged cultures of same organisms were used to study the
decolorization of disperse azo dye Rubine GFL.
100
0.3
80
0.25
60
Absorbance
Decolorization (%)
c
99
40
20
0
0.2
0.15
0.1
0.05
50
100
150
200
250
Dye concentration (mg l-1 )
Fig. 1. Effect of pH [a], temperature [b] and initial dye concentration [c] on decolorization of Rubine GFL by using consortium-AP (-), A. ochraceus NCIM-1146 (:) and
Pseudomonas sp. SUK1 (). Data points represents the mean of three independent
replicates, standard error of mean (SEM) is indicated by error bars. Decolorization (%)
was measured after 30 h of incubation.
0
400
450
500
550
600
650
700
750
800
Wavelength (nm)
Fig. 2. UVevis spectra of Rubine GFL decolorization after 30 h at optimized conditions:
Control dye (C), consortium-AP (-), A. ochraceus NCIM-1146 (:) and Pseudomonas
sp. SUK1 ().
100
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
Table 2
Environmental parameters of untreated and treated textile industry effluent by using consortium-AP, A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1.
Environmental parameters
Untreated effluent
BOD (mg l1)
COD (mg l1)
TOC (mg l1)
Color (% ADMI removal)
260
3920
4175
100
Treated effluenta
A. ochraceus NCIM-1146
4.0
20.0
22.0
0.0
221
3489
3966
5
Pseudomonas sp. SUK1
2.5
19.0
20.0
1.5
63
391
2631
44
Consortium-AP
5.0
6.0
16.0
2.0
47
157
2171
98
3.5
5.0
19.0
1.5
Values are mean of three experiments, SD.
a
Treated effluent samples were analyzed after 35 h of incubation.
Table 3
Enzyme status during decolorization of Rubine GFL by A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 and consortium-AP.
Enzymes
Laccasea
Veratryl alcohol oxidasea
Tyrosinasea
Intracellular
Extracellular
Azoreductaseb
NADH-DCIP reductasec
Enzyme activity
A. ochraceus NCIM-1146
Pseudomonas sp. SUK1
Consortium-AP
Control
Test
Control
Test
Control
2.11 0.2
ND
566 5.0
692 6.0
ND
23 2.0
1.74 0.3
0.28 0.3***
708*** 7.0
775 7.0***
ND
27 3.0*
1.68 0.3
1.41 0.7
ND
ND
1.32 0.3
227 6.0
1.89 0.4**
0.36 0.3
ND
ND
1.80 0.4**
147 4.0
0.80
0.60
1038
657
1.95
52
Test
0.2
0.4
8.0
5.0
0.3
2.0
0.91
0.81
768
543
3.20
156
0.3**
0.6***
7.0
4.0
0.5***
4.0***
Control ¼ Enzyme extracted from culture medium without dye after 30 h; Test ¼ Enzyme extracted from dye decolorized culture medium after 30 h; ND ¼ Not detected.
Values are mean of three experiments standard error mean (SEM), significantly different from control cells at *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way analysis of
variance (ANOVA) with Tukey Kramer comparison test.
a
Enzyme unit’s min1 mg protein1.
b
mM of methyl red reduced min1 mg protein1.
c
mg of DCIP reduced min1 mg protein1.
3.3. Biodegradation of textile effluent
Various azo dyes with fused aromatic structures are commonly
used in the textile processing industry and thus their waste stream
has marked variation in its composition. The physicochemical
status of an untreated textile effluent showed considerably high
values of BOD (260 mg l1), COD (3920 mg l1), TOC (4175 mg l1)
and color above the prescribed fresh water limits (Table 2).
However, a considerable decline in almost all studied parameters
such as BOD (82%), COD (96%), TOC (48%) and ADMI color removal
(98%) was observed after treatment with consortium-AP under
microaerophilic conditions within 35 h (Table 2). The higher
decolorization performance of consortium-AP at alkaline pH (8.5)
suggests the sign of its suitability for degradation of most of the
textile effluents as it have alkaline pH. In contrast, using individual
cultures of A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1
a lower reduction in COD (11% and 90%), BOD (15% and 76%), TOC
(5% and 37%) and ADMI color removal ratio (5% and 44%) was
achieved within same time (Table 2). Since the fungi and bacteria
alone cannot completely decolorize this textile effluent, it is suspected that the fungal-bacterial consortium could cooperatively
decolorize the effluent. This is consistent with the observation that
consortium-AP of fungal-bacterial synergism used in this study
showed considerably better decolorization performance than any
Fig. 3. a. HPTLC profile of control dye Rubine GFL [a] and its metabolites obtained after decolorization by using A. ochraceus NCIM-1146 [b], Pseudomonas sp. SUK1 [c] and
consortium-AP [d] after 30 h of incubation. b. HPTLC 3-D chromatogram of control dye Rubine GFL [a] and its metabolites obtained after decolorization by using A. ochraceus NCIM1146 [b], Pseudomonas sp. SUK1 [c] and consortium-AP [d].
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
individual culture. These results are better than a previous report
which showed that individual Pseudomonas sp. SU-EBT decolorized
90% effluent within 60 h with 50% and 45% reduction in COD and
BOD respectively (Telke et al., 2010). Our results suggest that
fungal-bacterial synergisms could be used as a better alternative for
bioremediation of textile effluent than individual cultures.
3.4. Aromatic amine determination
The focus on azo dyes degradation by fungal-bacterial synergism in recent years has attributed due to its higher ability to
complete mineralizes the dye without the formation of toxic
aromatic amines. Our studies have demonstrated that partial
decolorization of dye and effluent by individual Pseudomonas sp.
SUK1 culture produce aromatic amines, while the samples treated
with consortium-AP achieved complete removal of amines under
microaerophilic conditions. The microaerophilic pre-grown Pseudomonas sp. SUK1 showed amine concentration of 0.14 and
101
0.18 mM for Rubine GFL and textile effluent under microaerophilic
degradation conditions respectively. At the same time aerobic pregrown Pseudomonas sp. SUK1 culture showed presence of amines
for both the samples dye (0.06 mM) and effluent (0.07 mM) under
microaerophilic conditions (Table 1). Bacterial azo reductases are
known to be key enzymes responsible for reductive azo dyes
degradation and are capable of transforming them into aromatic
amines. This is consistent with a number of previous reports that
suggest the reductive cleavage of azo dye by bacterial cultures in
microaerophilic conditions which leads to the formation of
aromatic amines (Joshi et al., 2008). On the other hand, under same
conditions no presence of amines were detected in the dye and
effluent samples treated with aerobic and microaerophilic pregrown A. ochraceus NCIM-1146 fungal culture (Table 1). This is
probably due to the absence of reductase enzyme systems such as
azo reductase in the A. ochraceus NCIM-1146 fungal culture. Further,
the fungal-bacterial consortium used in our study suggests
increased dye and effluent degradation rates without the formation
of toxic aromatic amines.
3.5. Enzyme activities
Several microorganisms including bacteria and fungi have been
reported to decolorize azo dyes with its highly versatile enzyme
systems. In the present study, significant induction in the activity of
veratryl alcohol oxidase by 35% and 28% was observed in
consortium-AP and A. ochraceus NCIM-1146 cells respectively after
decolorization as compared to control (cultures without dye);
however there was no activity in Pseudomonas sp. SUK1 cells for the
same enzyme. In addition to this, laccase was also induced by 14% in
consortium-AP and 12% in Pseudomonas sp. SUK1 cells (after
decolorization) as compared to control, but it was reduced in
A. ochraceus NCIM-1146 cells. Intracellular and extracellular tyrosinase activity was induced in A. ochraceus NCIM-1146 cells by 25%
and 12% respectively after decolorization, but the same activity was
absent in Pseudomonas sp. SUK1. Reduced tyrosinase activity was
observed in consortium-AP after decolorization as compared to
control (Table 3). The higher induction of oxidoreductive enzymes
during decolorization of dye by consortium-AP might be due to
Fig. 4. HPLC chromatogram of control dye Rubine GFL [a] and its metabolites obtained
after decolorization by using consortium-AP [b], A. ochraceus NCIM-1146 [c] and
Pseudomonas sp. SUK1 [d] after 30 h of incubation.
Fig. 5. HPLC chromatogram of textile effluent [a] and its metabolites obtained after
decolorization by using consortium-AP [b].
102
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
synergistic effect of both cultures which supports their vigorous
role in the consortium. The role of oxidoreductive enzymes in the
decolorization of azo dye Reactive red 2 have been previously
characterized in Pseudomonas sp. SUK1 (Kalyani et al., 2009).
Available literature on degradation of dyes shows that reductive
cleavage of azo bond is the initial step in bacterial metabolism of
azo dyes under microaerophilic conditions. In our study, significant
induction of azo reductase (64%) and NADH-DCIP reductase (200%)
activities in consortium-AP suggests their involvement in decolorization of dye molecule. No consequential change was seen in
NADH-DCIP reductase activity of A. ochraceus NCIM-1146 culture
cells after decolorization, while it was reduced to 65% in Pseudomonas sp. SUK1 cells. Moreover, induction in azo reductase activity
up to 36% was observed in individual Pseudomonas sp. SUK1 cells
after decolorization, whereas it was absent in A. ochraceus NCIM1146 cells (Table 3). In the same contest, the inductive pattern of
reductase was reported during the decolorization of azo dye Navy
blue HE2R by developed consortium-PA of A. ochraceus NCIM-1146
Fig. 6. FTIR spectrum of control dye Rubine GFL [a] and its metabolites obtained after
decolorization by using consortium-AP [b], A. ochraceus NCIM-1146 [c] and Pseudomonas sp. SUK1 [d] after 30 h of incubation.
fungi and Pseudomonas sp. SUK1 bacterium (Kadam et al., 2011).
The reason why individual cultures alone cannot completely
degrade the dye molecule is not clear, but in the consortium it may
be due to the synergetic actions of oxidoreductases (Gou et al.,
2009; Telke et al., 2009b).
3.6. Biodegradation analysis
HPTLC analysis of metabolites obtained after biodegradation of
dye Rubine GFL was carried out to provide an additional insight to
the biotransformation of dye molecule. The HPTLC chromatogram
showed the absence of control dye band in the consortium-AP
metabolites lane, which indicates its complete mineralization,
whereas it was present in A. ochraceus NCIM-1146 and Pseudomonas sp. SULK1 metabolites lanes indicates its partial degradation
(Fig. 3a). Furthermore, the intensity of derivatized bands of individual cultures metabolites was found to be decreased in
consortium-AP metabolites suggesting its further biotransformation. With respect to Rf values, control dye Rubine GFL showed two
peaks (0.84, 0.94), where as individual A. ochraceus NCIM-1146
showed six peaks (0.13, 0.16, 0.38, 0.64, 0.84, 0.94), Pseudomonas
sp. SULK1 showed seven peaks (0.14, 0.42, 0.51, 0.55, 0.65, 0.84,
0.94) and its consortium-AP showed seven distinct peaks (0.13,
0.30, 0.42, 0.47, 0.56, 0.66, 0.93) indicates the differential degradation pattern of dye by individual cultures and its consortium-AP
(Fig. 3b).
HPLC analysis of the control dye Rubine GFL showed single peak
at retention time of 2.971 min (Fig. 4a), while formed metabolite
after decolorization by consortium-AP showed the disappearance
of the major peak as seen in case of control dye Rubine GFL and the
formation of two major peaks at retention time of 3.047 and
3.317 min and three minor peaks at retention times of, 2.265, 4.123
and 4.663 min (Fig. 4b), which were not seen in the control dye. The
appearance of five new peaks and disappearance of the single peak
in the metabolites formed after decolorization by consortium-AP
support the more mineralization of parent dye Rubine GFL into
different metabolites. In case of individual cultures, decolorized
product of Rubine GFL by A. ochraceus NCIM-1146 showed two
major peaks at retention times, 1.484 and 1.572 min (Fig. 4c), while
Fig. 7. FTIR spectrum of textile effluent [a] and its metabolites obtained after decolorization by using consortium-AP [b] after 35 h of incubation.
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
Pseudomonas sp. SUK1 showed two major and two minor peaks at
the retention time of 2.106, 2.462, 2.858 and 3.001 min (Fig. 4d).
This suggested the conversion of parent dye into various metabolites by individual cultures.
It is well known that textile industry consume large volume of
water for various dyeing processes and thus releases large volumes
of wastewater with numerous pollutants are discharged. Since the
effluent is a complex mixture of dyes, it showed different peaks
when characterized by HPLC. The HPLC chromatogram of the real
textile effluent showed the presence of four major peaks at retention times of 3.199, 3.325, 4.122 and 5.098 min and four minor
peaks at retention times of 3.758, 4.706, 4.516 and 7.895 min
(Fig. 5a). The degraded products of textile effluent by consortiumAP after 35 h of incubation showed the disappearance of several
Table 4
GC-mass spectral data of metabolites obtained after degradation of Rubine GFL by A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1.
Retention time (min)
103
m/z
Mol. weight
Name of metabolite
19.356
244
241
1-(2-methyl-4-nitrophenyl)2-phenyl diazene [I]
13.029
166
165
(2-methyl-4-nitrophenyl)
diazene [II]
15.339
256
257
4-[(2-methyl-4-nitrophenyl)
diazenyl] phenol [I]
14.132
154
152
2-methyl-4-nitroaniline [II]
I] A. ochraceus NCIM-1146
II] Pseudomonas sp. SUK1
Mass spectrum
104
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
peaks as seen in case of real textile effluent and the formation of
three major peaks at retention times of 3.461, 3.553 and 3.774 min,
while four new minor peaks at retention time of 3.223, 3.328, 4.125
and 4.644 min (Fig. 5b). The difference in the retention times of real
textile effluent and metabolites formed after degradation by
consortium-AP confirms the biodegradation of effluent into
different metabolites.
FTIR spectra obtained from control dye Rubine GFL showed
specific peaks at 779.766e910.90 cm1 and 1173.17 cm1 for CeH
deformation, 1202.15 cm1 for CeN vibrations, 1341.18 cm1 for
NO2 stretching of aromatic nitro compound, 1520.82 cm1 for N]O
stretching of aromatic nitro compound, 1599.45 cm1 for N]N
stretching in azo group, 2248.70 cm1 for C^N stretching in
saturated nitriles and 2926.58 cm1 for CeH stretching in alkanes
(Fig. 6a). After the consortium decolorization, a significant reduction in IR peaks was observed in the 2845.20 cm1 to 2322.06 cm1
regions of metabolites suggests absence of charged amines in the
produced metabolites. A significant peak at 1659.73 cm1 for NHþ
3
deformation suggest the possible alkenes conjugation with C]O.
1
1
Moreover, peaks at 992.89 cm and 1151.77 cm for CeH deformation suggests cleavage of dye molecule. The absence of peak at
1599.75 cm1 for N]N stretching vibrations indicates the reductive cleavage of azo bond (Fig. 6b). Vanishing of major peaks and
formation of new peaks in the IR spectrum of consortium-AP
metabolites suggests the biotransformation of dye into distinct
metabolites.
Metabolites obtained after partial decolorization of Rubine GFL
by A. ochraceus NCIM-1146 showed peaks at 756.51 cm1 to
942.51 cm1 and 1151.94 cm1 for CeH deformations, 1384.28 cm1
for alkanes CH3 deformation, 1456.49 cm1 for alkanes CeH
deformation, 1531.93 cm1 for N]O stretching and peaks at
2872.33, 2926.58 and 2958.63 cm1 for alkanes CeH stretching
(Fig. 6c). Metabolites obtained after the partial decolorization of
Rubine GFL by Pseudomonas sp. SUK1 showed peak at 810.76 cm1
for CeH deformation and 1333.90 cm1 for formation of primary
aromatic amine which has also been additionally confirmed by GCMS analysis. The peak at 1450.16 cm1 represents alkanes CeH
deformation while that at 2849.08, 2917.59 and 2961.46 cm1
represents alkanes CeH stretching (Fig. 6d).
Analysis of FTIR results of control textile effluent showed
specific peaks at 2925.65 cm1 for alkanes CeH stretching,
2862.31 cm1 for alkanes CeH stretching, 1637.41 cm1 for urea C]
N stretching, 1458.04 cm1 for alkanes CeH deformation,
1400.72 cm1 for phenols OeH deformation, 1261.09 cm1 for
nitrates OeNO2 vibration, 1097.15 cm1 for aliphatic ethers
stretching, 805.60 cm1 for benzene ring containing two adjacent H
atoms eCeH deformation and 601.68 cm1 for alkynes CeH
deformation (Fig. 7a). Metabolites obtained after complete decolorization of effluent by consortium-AP showed disappearance of
major peaks and formation of new peak at 2922.08 cm1 for
alkanes CeH stretching, 1650.88 cm1 for acyclic C]N stretching,
1461.65 cm1 for alkanes CeH deformation, 1400.54 cm1 for
ketones CeH deformation and 1109.02 cm1 for secondary alcohols
CeOH stretching (Fig. 7b). Considerable difference between the
FTIR spectrum of control textile effluent and the metabolites
obtained after complete decolorization by consortium-AP
confirmed the biodegradation of effluent into different metabolites.
GC-MS analyses of the metabolites raised from the degradation
of dye Rubine GFL by A. ochraceus NCIM-1146 demonstrated the
asymmetric cleavage of dye Rubine GFL mediated by veratryl
alcohol enzyme to yields two metabolites, one of them is identified
as 1-(2-methyl-4-nitrophenyl)-2-phenyl diazene (m/z ¼ 244).
Further asymmetric cleavage of intermediate metabolite [I] by
fungal laccase gave (2-methyl-4-nitrophenyl) diazene (m/z ¼ 166)
[II] (Table 4; Fig. 8a). In Pseudomonas sp. SUK1 individual culture,
the appearance of intermediate metabolite 4-[(2-methyl-4nitrophenyl) diazenyl] phenol (m/z ¼ 256) [I] indicates the initial
oxidative cleavage of parent dye Rubine GFL by bacterial laccase,
Fig. 8. Proposed pathways for the degradation of Rubine GFL by A. ochraceus NCIM-1146 [a] Pseudomonas sp. SUK1 [b] and consortium-AP [c].
Table 5
GC-MS spectral data of metabolites obtained after degradation of Rubine GFL by consortium-AP.
Retention time (min)
m/z
Mol. weight
Name of metabolite
18.964
284
284
N-ethyl-4-[(2-methyl-4-nitrophenyl)
diazenyl] aniline [I]
17.500
256
257
4-[(2-methyl-4-nitrophenyl)
diazenyl] phenol [II]
19.354
244
241
1-(2-methyl-4-nitrophenyl)-2-phenyl
diazene [III]
13.030
165
165
(2-methyl-4-nitrophenyl) diazene [IV]
14.137
152
152
2-methyl-4-nitrophenol [V]
Mass spectrum
106
H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107
Table 6
Phytotoxicity of Rubine GFL, textile effluent and its metabolites formed after degradation by consortium-AP for the S. vulgare and P. mungo.
Parameters
Germination
(%)
Plumule
(cm)
Radicle
(cm)
S. vulgare
P. mungo
Distilled
water
Rubine GFL
Rubine GFL
metabolites
Textile
effluent
Effluent
metabolites
Distilled
water
Rubine GFL
Rubine GFL
metabolites
Textile
effluent
Effluent
metabolites
100
50
100
40
100
100
60
100
50
100
4.99 0.77
1.95* 0.29
4.45$ 0.55
1.60* 0.32
4.15 0.38
7.88 0.54
4.55* 0.16
6.80$ 0.55
4.10* 0.13
6.65$ 0.42
2.29 0.39
0.86** 0.07
2.25$ 0.28
0.63* 0.09
1.65$ 0.28
1.52 0.26
0.95* 0.09
1.40$ 0.08
0.70* 0.07
1.30$$ 0.06
Values are mean of three experiments, SEM (). Seeds germinated in Rubine GFL and textile effluent are significantly different from the seeds germinated in distilled water at
*P < 0.05, **P < 0.001 and the seeds germinated in metabolites are significantly different from the seeds germinated in Rubine GFL and textile effluent at $P < 0.05, $$P < 0.001
by one-way analysis of variance (ANOVA) with TukeyeKramer comparison test.
which was further cleaved at azo position by azo reductase to gave
2-methyl-4-nitroaniline (m/z ¼ 154) [II] as identified aromatic
amine (Table 4; Fig. 8b). This is in agreement with a previous report
which supports the involvement of bacterial reductases in the
reductive cleavage of azo dyes to yield aromatic amines (Levine,
1991). In addition, with the cleavage of azo bonds by bacterial azo
reductase, most azo dyes get reduced microaerophilically to the
corresponding amines (Zimmerman et al., 1982). Pseudomonas sp.
SUK1 laccase is known for oxidative as well as asymmetric cleavage
of dye molecules, where as reductase is known for reductive
cleavage of azo dyes (Kalyani et al., 2009; Kadam et al., 2011).
In case of consortium-AP, enzymes from both bacteria and fungi
facilitates dye metabolism, as there was significant induction in
veratryl alcohol activity which results in asymmetric cleavage of
parent dye molecule to form an intermediate N-ethyl-4-[(2methyl-4-nitrophenyl) diazenyl] aniline (m/z ¼ 284) [I] (Table 5).
It is reported that veratryl alcohol oxidase brings about the asymmetric cleavage of azo dyes (Jadhav et al., 2009). Further oxidative
cleavage of intermediate [I] by laccase gives 4-[(2-methyl-4nitrophenyl) diazenyl] phenol (m/z ¼ 256) [II], which undergoes
dehydroxylation to form 1-(2-methyl-4-nitrophenyl)-2-phenyl
diazene (m/z ¼ 244) [III]. Furthermore, asymmetric cleavage of
intermediate [III] by veratryl alcohol enzyme leads to the formation
of (2-methyl-4-nitrophenyl) diazene (m/z ¼ 165) [IV], which
undergoes azo bond cleavage by azo reductase to form 2-methyl 4nitroaniline as unidentified aromatic amine. The earlier report
confirms the role of azo reductase in direct cleaves of azo bond
(Chen et al., 2003). This aromatic amine further get deaminated and
oxidised by laccase to gave 2-methyl-4-nitrophenol (m/z ¼ 152) [V]
as final metabolite (Table 5; Fig. 8c). The ability of consortium-AP to
completely decolorize the dye without forming aromatic amines
suggested its applicability over individual cultures. The intermediates not detected by GC-MS but rationalized as necessary intermediates during the degradation process were labeled
alphabetically.
S. vulgare and P. mungo was 60 and 50% respectively (Table 6). On
the other hand, complete germination (100%) as well as significant
growth in the plumule and radical was observed for both the plants
grown in consortium-AP metabolites as compared to that of dye
and effluent (Table 6). In addition to this, the length of plumule and
radicle was found to be lower in seeds germinated with dye and
effluent samples than those germinated in distilled water as well as
dye and effluent metabolites. This study suggest that the dye and
effluent was toxic to these plants, while the metabolites formed
after consortium degradation was less toxic, which signifies the
detoxification of dye and effluent by consortium-AP. These results
underline the importance of fungal-bacterium synergism for
bioremediation of textile effluent in terms of both decolorization
and detoxification.
4. Conclusions
A new biodegradation approach with fungal-bacterial synergism was first applied for degradation of disperse azo dye Rubine
GFL and textile effluent in submerged conditions. Overall studies
revealed that the combined metabolic activities of A. ochraceus
NCIM-1146 and Pseudomonas sp. SUK1 in the consortium led to
complete decolorization and detoxification of dye and effluent. In
contrast, individual cultures showed lesser decolorization rate with
the formation of toxicants. The enhanced decolorization efficiency
of consortium-AP could be due to the induced synergetic reactions
of oxidoreductases viz. laccase, veratryl alcohol oxidase, azo
reductase and NADH-DCIP reductase. Deep insight into the
different aspects presented here strongly supports its applicability
for enhanced biodegradation and detoxification of azo dyes which
are recalcitrant to degradation by individual cultures. With a better
understanding, this fungal-bacterium synergism would be further
exploited to develop a continuous treatment process for degradation and detoxification of textile effluent containing wide range of
azo dyes.
3.7. Toxicity studies
Acknowledgements
The assessment of toxicity of dyes, effluents and its degraded
products is often great concern as most of them exert toxic effect on
plants and animals when released in stream water. Use of bioassays
such as phytotoxicity for monitoring the toxic effect of dyes as well
as its metabolites on plants was suggested by many researchers
(Valerio et al., 2007; Jadhav et al., 2011). Plant bioassays have been
used to establish the toxicity levels of dye, effluent and its degraded
products on common agricultural crops. In this case, the phytotoxicity study revealed that there is an inhibition of germination in
solutions containing 1000 ppm of the dye Rubine GFL for both
S. vulgare and P. mungo by 50 and 40% respectively (Table 6).
Moreover the inhibition of germination in real textile effluent for
The author Dr. Harshad S. Lade would like to acknowledge
University Grant Commission, New Delhi, India for providing Dr.
D.S. Kothari Postdoctoral Fellowship.
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