Bacterial-yeast consortium as an effective biocatalyst for biodegradation of sulphonated azo dye Reactive Red 198

Article abstract of DOI:10.1039/c4ra15834b

A novel bacterial-yeast consortium (Brevibacillus laterosporus and Galactomyces geotrichum) acts as a proficient biocatalyst. It decolorized 92% of sulphonated azo dye Reactive Red 198 (RR 198) within 18 h at a dye concentration of 50 mg L^-1 as compared to 58 and 42% decolorization using Brevibacillus laterosporus and Galactomyces geotrichum alone, respectively, in the same experimental conditions (pH 7, 40 °C, in static condition). The cumulative action of enzymes such as veratryl alcohol oxidase, laccase, NADH-DCIP reductase and azoreductase in the consortium culture was responsible for dye degradation. Fourier transform infrared spectroscopy and high performance thin layer chromatography analysis of the dye and its extracted metabolites suggested the biotransformation of RR 198 into simple metabolites; whereas the biotransformation of the same by individual microorganisms was different than by consortial biodegradation. According to gas chromatography-mass spectroscopy studies, RR 198 was biotransformed into much simpler compounds such as (ethylsulfonyl)benzene and 1,3,5-triazine by the bacterial-yeast consortium. This metabolic fate of the dye was entirely different in consortium than when compared to individual microbial treatment. Single microbial species could lead to only partial mineralization of the intact dye molecule; whereas, nearly complete degradation of the dye molecule was achieved using the consortium culture. This study clearly suggests that the consortium has an enormous strength to catalyze RR 198 within a short period as compared to individual microbial cultures. This journal is

Full text of DOI:10.1039/c4ra15834b

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: M. B. Kurade, T.
Waghmode, M. U. Jadhav, B. Jeon and S. P. Govindwar, RSC Adv., 2015, DOI: 10.1039/C4RA15834B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Bacterial-yeast consortium as an effective biocatalyst for
biodegradation of sulphonated azo dye Reactive red 198
Mayur B. Kurade,ab, Tatoba R. Waghmodecd, Mital U Jadhave, ByongHun Jeonb, Sanjay P.
Govindwar*c
aDepartment of Biotechnology, Shivaji University Kolhapurꢀ416004, India.
bDepartment of Natural Resources and Environmental Engineering, Hanyang University,
Seoul, South Korea.
cDepartment of Biochemistry, Shivaji University Kolhapurꢀ416004, India.
dInstitute of Agriculture and Life Sciences, Gyeongsang National University, Jinju 660ꢀ701,
South Korea
eDepartment of Microbioloy, Shivaji University Kolhapurꢀ416004, India.
Address for correspondence:
Prof. S. P. Govindwar
Professor and Head,
Department of Biochemistry,
Shivaji University, Kolhapurꢀ 416004, India.
Email: govindwarsp@gmail.com
Phone: +91ꢀ231ꢀ2609152
Fax: +91ꢀ231ꢀ2691533.
1

RSC Advances
Page 2 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Abstract
A novel developed bacterialꢀyeast consortium (Brevibacillus laterosporus and
Galactomyces geotrichum) has been acted as a proficient biocatalyst. It decolorized 92% of
sulphonated azo dyeꢀReactive red 198 (RR 198) within 18h at a dye concentration of 50 mg
Lꢀ1 as compared to 58 and 42% decolorization using Brevibacillus laterosporus and
Galactomyces geotrichum alone respectively in the same experimental conditions (pHꢀ7, 40
°C, in static condition). The cumulative action of enzymes such as veratryl alcohol oxidase,
laccase, NADHꢀDCIP reductase and azoreductase in the culture was responsible for dye
degradation. Fourier transform infrared spectroscopy and High performance thin layer
chromatography analysis of dye and its extracted metabolites suggested the biotransformation
of RR 198 into simple metabolites; whereas the biotransformation of the same by individual
microorganism was different than consortial biodegradation. According to Gas
chromatographyꢀMass spectroscopy studies, RR 198 was biotransformed into much simpler
compounds such as (ethylsulfonyl)benzene and 1,3,5ꢀtriazine by the bacterialꢀyeast
consortium. This metabolic fate of dye was entirely different in consortium when compared
to individual microbial treatment. Single microbial species could only lead to partial
mineralization of intact dye molecule; whereas, nearly complete degradation of dye molecule
was achieved using consortium culture. This study clearly suggests that the consortium has an
enormous strength to catalyze RR 198 within a short period as compared to individual
microbial cultures.
Keywords: Biocatalysis; Biodegradation, Biotransformation; Consortium, Decolorization;
High performance thin layer chromatography; Phytotoxicity
2

Page 3 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
1.0. Introduction
Among 12 classes of chromogenic groups of dyes, the azo dyes including sulphonated
and reactive dyes make up to 70% of all textile dyestuff produced. This leads to effluent
streams containing intense color due to the presence of azo dyes. Normally colors are
noticeable at a dye concentration of more than 1 mg Lꢀ1 and an average concentration of 300
mg Lꢀ1 has been reported in effluents from textile manufacturing processes.1 Azo dyes are of
concern in wastewater treatment because of their intense coloration, bioꢀrecalcitrance and
potential toxicity to animals and humans as it contains mutagenic and carcinogenic
properties.2,3 Several physicochemical techniques have been used for treating wastewater
containing dyes but the methodologies appear to face several technical and economic
limitations and lead to the generation of toxic byꢀproducts.4ꢀ7
On the other hand, a number of biotechnological approaches have been suggested by
recent research as of potential interest in combating this pollution source in an ecoꢀefficient
manner.8,9 Lower cost of treatment and amenability to scale up easily are the merits of
biological methods. Numerous studies have been carried out with isolated, selected, mutated
and genetically engineered microorganisms for effective bioremediation to achieve enhanced
rates of degradation of recalcitrant target compounds.10ꢀ14 However, the isolated/single
cultures are often specific to type of dye under consideration. Moreover, pure cultures cannot
be scaled up easily and maintained in largeꢀscale operations typical of effluent treatment
systems.15 Instead, microbial consortium could be an attractive tool to improve the rate of
decolorization process with better suitability in largeꢀscale applications.
Recent studies have suggested that mixed culture may be more appropriate for
decolorization of azo dyes.16ꢀ18 Different strains may attack dye molecule at different
positions or may use decomposition products produced by another strain for further
decomposition.19,20 In recent years, mixed microbial consortia are believed to have more
3

RSC Advances
Page 4 of 29
View Article Online
DOI: 10.1039/C4RA15834B
superiority over pure isolates in some biorefractory wastewater treatment. Recently, a
bacterial consortium (Enterobacter dissolvens and Pseudomonas aeruginosa) was used for
the treatment of diꢀazo dye Acid Maroon V, which showed 2.15 fold increase in
decolorization efficiency compared to individual microorganisms.21 Fungal species
Aspergillus terreus and Rhizopus oryzae in consortium enhanced the removal of metals and
dyes.22 Yeast species including Galactomyces geotrichum, Candida tropicalis and
Saccharomyces cerevisiae were effectively used for dye degradation.14,23ꢀ25; however, as far
as our knowledge is concerned, there is still no report on yeast consortium for dye
decolorization.
Although studies carried out in the past have used undefined microbial consortium1,26
or pure cultures for dye decolorization, there is very limited research to unravel the
application of bacterialꢀyeast/fungi consortium for the rehabilitation of our natural resources.
In the present study, a bacterialꢀyeast consortium has been developed using a yeastꢀ
Galactomyces geotrichum with another potential dye degrading microbial speciesꢀ
Brevibacillus laterosporus to decolorize a sulphonated azo dyeꢀ Reactive red 198 (RR 198).
Brevibacillus laterosporus is an aerobic bacterium and is a potent dye degrading species. Our
previous reports provide strong evidence on abilities of Brevibacillus laterosporus in
decolorization of several dyes, such as, Remazol red, Rubine GFL, Disperse brown 118,
Golden Yellow HER and mixture of dyes.27ꢀ30 In the yeast species, Galactomyces geotrichum
can be a strong highlight, as it has been used to eliminate different variety of textile dyes
which include Reactive yellow 84A, Rubine GFL and Methyl red.14,23 Presence of wide range
of biocatalytic enzyme system make these species strong candidates for enhanced dye
degradation. High efficiency and biocatalytic performance was expected using the
combination of these microbial species. RR 198 is a sulphonated monoazo dye which is toxic
and recalcitrant in nature due to the presence of azo and sulphonic groups. It gives intense
4

Page 5 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
coloration to water at very low concentration, hence effluent containing this dye cause
deterioration of water bodies and lead to harmful effects on aquatic flora and fauna. It is one
of the most consumable azo dye in the textile industries in Ichalkaranji, India; hence this dye
was used as a model azo dye for the decolorization experiments in the present study. This
study put forward the detailed mechanism of how this bacterialꢀyeast consortium is superior
over an individual microbial culture in eliminating the xenobiotic and toxic azo dye RR 198
with the evidence of enzymatic status, characterization of products extracted after dye
decolorization using Fourier transform infrared spectroscopy (FTIR) and High performance
thin layer chromatography (HPTLC) along with metabolic fate of RR 198 using Gas
chromatography–Mass spectroscopy (GCꢀMS) data.
2.0. Experimental
2.1. Microorganisms and culture conditions
Brevibacillus laterosporus MTCC 2298 was obtained from Microbial Type Culture
°
Collection, Chandigarh, India. The pure culture was maintained at 4 C on the Nutrient agar
slant containing (g Lꢀ1): NaCl, 5.0; bacteriological peptone, 5.0; yeast extract, 2.0; beef
°
extract, 1.0 and agar powder 15.0 at 4 C. Galactomyces geotrichum MTCC 1360 was
obtained from Microbial Type Culture Collection, Chandigarh, India. Pure culture was
°
maintained on malt yeast agar slants at 4 C, the composition of malt extract medium was (g
Lꢀ1): malt extract 3, yeast extract 3, peptone 5, and glucose 10.
2.2. Dyes and Chemicals
All required chemicals were obtained from Sigma Aldrich, USA, Hiꢀmedia
Laboratories Pvt. Ltd., Mumbai, India and Sisco Research Laboratory (SRLs), India. All
chemicals used were of the highest purity available and of the analytical grade. Textile dye
5

RSC Advances
Page 6 of 29
View Article Online
DOI: 10.1039/C4RA15834B
RR 198 was a munificent gift from Manpasand textile processing industry, Ichalkaranji,
India.
2.3. Development of bacterial-yeast consortium
The consortium was prepared by the earlier reported protocol.31 The surface culture
biomass of G. geotrichum (1 cm pieces of mycelium cut from agar slants were used to
°
inoculate 100 mL malt extract medium in 250 mL Erlenmeyer flasks and grown at 30 C for
24 h) was aseptically transferred to the 24 h old culture of B. laterosporus (grown at 30 °C in
250 mL Erlenmeyer flask containing 100 mL of nutrient medium) to obtain the bacterialꢀ
yeast consortium.
2.4. Decolorization experiment
All decolorization experiments were performed in the 250 mL Erlenmeyer flasks.
Textile dye RR 198 was added at a concentration of 50 mg Lꢀ1 in the flask containing
developed consortium with 100 mL nutrient broth. Aliquot was centrifuged (4000 rpm for 20
min) to separate cell mass and clear supernatant was used to determine the decolorization.
Decolorization was determined by measuring absorbance maxima of the dye (λmax of RR 198ꢀ
530 nm) using UV visible spectrophotometer (Hitachi Uꢀ2800) by using following formula.
Initial absorbance – Final absorbance
Decolorization (%) =
× 100
Initial absorbance
To investigate the effect of different dye concentration on decolorization, the
concentrations of the RR 198 was varied from 50 to 400 mg Lꢀ1 concentration (50, 100, 200,
300 and 400 mg Lꢀ1) to determine the amount of dye that could be decolorized by consortium
and decolorization was determined by the formula mentioned above.
6

Page 7 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
All the media used in this study were preꢀsterilized before experiments by autoclaving
°
it at 121 C for 20 min. All decolorization experiments were carried out in triplicate. Abiotic
controls (without microorganisms) were always included.
2.5. Preparation of cell free extract
The consortium was prepared as mentioned above and individual organisms were
grown in their respective medium for 24 h at 30 °C and centrifuged at 10,000 rpm for 20 min.
This biomass of consortium and individual organisms were separately suspended in 50 mM
potassium phosphate buffer (pH 7.4), gently homogenized and used for sonication (sonicsꢀ
vibracell ultrasonic processor) keeping sonifier output at 60 amplitude maintaining
°
temperature below 4 C and giving 12 strokes each of 30 sec with one min interval. These
resulting extracts were then used as enzyme source. The culture supernatant obtained after
centrifugation during the harvesting of cell biomass was directly used as a source of
extracellular enzymes. Same procedure was carried out for samples obtained after
degradation of the dye.
2.6. Determination of enzyme activities
All enzyme activities were assayed spectrophotometrically (Hitachi Uꢀ2800) in the
°
cell free extract as well as culture media at room temperature (25 C). Veratryl alcohol
oxidase activity was determined by using the procedure reported earlier by Jadhav et al.32
Laccase activity was monitored according to the procedure reported earlier.33 2 mL reaction
mixture contained 5 mM 3’, 3’ꢀdiaminobenzidine tetrahydrate (DAB) in 0.1M acetate buffer
(pH 4.8) and increase in optical density was measured at 410 nm. Tyrosinase activity was
determined by the procedure as follows; 3 mL reaction mixture contained 50 mM of catechol
and 2.1 mM of ascorbic acid in 50 mM potassium phosphate buffer (pH 6.5) equilibrated at
25°C. ∆A265 nm was monitored until constant, and then 0.1 mL of the supernatant from the
7

RSC Advances
Page 8 of 29
View Article Online
DOI: 10.1039/C4RA15834B
reaction mixture was added. The formation of oꢀbenzoquinone and dehydroꢀascorbic acid and
decrease in optical density was measured at 265 nm. ∆A265 nm was obtained using the
maximum linear rate for both the test and control. One unit of tyrosinase activity was equal to
a ∆A265 nm of 0.001 per min at pH 6.5 at 25oC in a 3.0 mL reaction mixture containing Lꢀ
catechol and Lꢀascorbic acid.
The NADHꢀDCIP reductase and Riboflavin reductase activity were assayed by
modifying earlier reported methods by Waghmode et al.33 DCIP reduction was monitored at
590 nm and calculated using an extinction coefficient 0.019 µMꢀ1 cmꢀ1. The reaction mixture
(5.0 mL) prepared contained 25 µM substrate (DCIP) in the 50 mM potassium phosphate
buffer (pH 7.4) and 0.1 mL enzyme. From this, 2.0 mL reaction mixture was assayed at 590
nm by addition of 250 µM NADH. Riboflavin reductase NAD(P)H:flavin oxidoreductase
was measured by monitoring the decrease in absorbance at 340 nm. Cell free extract was
added to a solution (final volume 2 mL) containing 100 mM of TrisꢀHCl (pH 7.5), 25 µM of
NADPH and 10 µM of riboflavin. Reaction rates were calculated by using a molar extinction
coefficient of 0.0063 µMꢀ1 cmꢀ1. Azo reductase activity was assayed by earlier reported
method.33 The assay mixture (2 mL) contains 4.45 µM of methyl red (MR), 50 µM NADH,
1.2 mL of potassium phosphate buffer (20 mM, pH 7.5). The reaction mixture was preꢀ
incubated for 4 min followed by the addition of NADH and monitored for the decrease in
color absorbance (430 nm) at room temperature. The reaction was initiated by addition of 0.2
mL of the enzyme solution. Methyl red reduction was calculated by using its molar extinction
coefficient of 0.023 µMꢀ1 cmꢀ1. One unit of enzyme activity was defined as amount of
enzyme required to reduce 1 ꢁM of substrate min−1 mg of proteinꢀ1. All the enzyme assays
were run in triplicates.
2.7. Extraction and analysis of products obtained after dye decolorization
8

Page 9 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
°
Biomass was removed after decolorization by centrifugation (10, 000 rpm at 4 C for
20 min) and supernatant was processed for extraction of degradation metabolites with equal
volume of ethyl acetate. The extracted residue were dried over anhydrous Na2SO4 and
evaporated to dryness in a rotary evaporator. The crystals obtained were dissolved in small
volume of High Pressure Liquid Chromatography (HPLC) grade methanol and used for High
Performance Thin Layer Chromatography (HPTLC), Fourier Transform Infrared
spectroscopy (FTIR) and Gas Chromatography ꢀ Mass Spectroscopy (GCꢀMS) analysis. The
FTIR analysis of extracted metabolites was carried out using protocol reported earlier.31 This
FTIR analysis of metabolites was carried out on Perkin–Elmer, spectrum one instrument and
compared with control dye in the mid IR region of 400ꢀ4000 cmꢀ1 with 8 scan speed. The
samples were mixed with spectroscopically pure KBr in the ratio 5:95, pellets were fixed in
sample holder, and the analysis was carried out. The GC–MS analysis of metabolites were
carried out using a Shimadzu 2010 MS Engine, equipped with integrated gas chromatograph
with a HP1 column (60 m long, 0.25 mm id, nonpolar). Helium was used as carrier gas at a
°
flow rate of 1 mL min−1. The injector temperature was maintained at 280 C with oven
°
°
°
conditions as: 80 C kept constant for 2 min and increased up to 200 C with 10 C minꢀ1 –
°
°
raised up to 280 C with 20 C min−1 rate. The interphase temperature of MS engine was
°
maintained at 280 C and ionization voltage to 70 eV. The minimum and maximum limit of
m/z was kept to 40 and 950 respectively. The compounds were identified on the basis of mass
spectra using the NIST library. To confirm the biodegradation of dyes, the same metabolites
were analyzed by HPTLC using silica gel plates (HPTLC Lichrospher Silica gel 60 F254S,
Merck). 15 µL of sample was applied on the plate by micro syringe using sample applicator
(Linomat V, Camag, Switzerland). The solvent system used for RR 198 was propanol: ethyl
acetate: methanol: toluene (3: 6: 0.5: 0.5). The chromatogram was analyzed using scanner
(Camag, Switzerland).
9

RSC Advances
Page 10 of 29
View Article Online
DOI: 10.1039/C4RA15834B
2.8. Phytotoxicity study
This test was performed to assess the toxicity of the untreated and treated effluent
with respect to two kinds of seeds commonly used in Indian agriculture; Sorghum vulgare
and Phaseolus mungo using earlier reported method.33 Ten seeds of each plant were sowed
into a plastic sand pot. The sand pot was prepared by adding 15 g of washed sand into the
plastic pot. Metabolites were extracted using procedure described in section 2.7 and these
crystals obtained after ethyl acetate extraction were dissolved in distilled water. Toxicity
study was carried out by watering the seeds of each plant with untreated textile effluent and
extracted metabolites of effluent (5 mL per day). Control was kept by watering the seeds in
distilled water (5 mL per day). Germination (%), lengths of the shoots and roots were
recorded after 7 days. The experiment was carried out at room temperature.
2.9. Statistical Analysis
Data were analyzed by oneꢀway analysis of variance using Tukey–Kramer multiple
comparison test. Values were the mean of three independent experiments. Values were
considered significant when P <0.05.
3.0. Results and discussion
3.1. Decolorization experiment and effect of physico-chemical parameters
The decolorization of dyes is directly dependent upon the catalytic potential of a
microorganism. Decolorization efficiencies of individual microorganism and the developed
consortium are presented in Fig 1 in terms of percentage decolorization and time required for
decolorization. This developed consortium is nothing but a combination of a bacterium (B.
laterosporus) which is known to be a proficient decolorizer27ꢀ29 with another yeast species (G.
geotrichum). Consortium exhibited 92% decolorization of RR 198 within 18 h with dye
°
concentration of 50 mg Lꢀ1 when optimized conditions (pHꢀ7, 40 C and static condition) for
10

Page 11 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
decolorization were provided to the consortial culture. In contrast, B. laterosporus and G.
geotrichum alone exhibited 58 and 42% decolorization respectively in same experimental
conditions. Recently, we have reported the higher decolorization of textile dye Scarlet RR
and textile industry effluent using the same consortium of yeast and bacteria.31,33
Decolorization of most of the dyes by microbial cultures is generally carried out in neutral to
slightly alkaline pH range. Consortium of Bacillus sp. and G. geotrichum also showed
effective decolorization of Brilliant blue G at neutral pH34 while natural microbial consortium
°
required the same temperature range (40 C) for maximum decolorization of mixture of dyes
and also with faster rate.35 The loss of cell viability or the negligible oxidoꢀreductive enzyme
activities might be the cause of reduced decolorization in culture broth which was incubated
beyond the optimum temperature range.
3.2. Effect of dye concentration on decolorization by consortium
An investigation of effect of dye concentration on decolorization ability of microbial
system is very important as it explores the limits of a biological system. To assess this,
culture of consortium was provided with higher doses of dye from 50 to 400 mg Lꢀ1. It has
been found that, higher concentration of dye decreased the decolorization performance of
consortium. However, the difference in decolorization was insignificant (P > 0.05) as
decolorization in flask with dye concentration of 400 mg Lꢀ1 was 59% as compared to 78%
decolorization in flask containing the dye concentration of 50 mg Lꢀ1 within first 12 h (Fig
2A). Interestingly, the decolorization of RR 198 using consortium was found to be much
higher with all dye concentration used with minor cessation in decolorization with increase in
dye concentrations as compared to the decolorization by individual microorganisms. The
decolorization of RR 198 by B. laterosporus was marginally reduced from 40% (at dye
concentration of 50 mg Lꢀ1) to 7% (at dye concentration of 400 mg Lꢀ1) within first 12h;
11

RSC Advances
Page 12 of 29
View Article Online
DOI: 10.1039/C4RA15834B
while at the same time, 29 and 12% decolorization was achieved using G. geotrichum at dye
concentration of 50 and 400 mg Lꢀ1 respectively (Fig 2B and 2C). These results suggest that
consortial culture can tolerate higher dye concentration with effective decolorization of dyes
as compared to individual strains. It has been found that the higher dye concentration do not
alter the decolorization when bacterial coꢀculture was used instead of pure culture, and this
might be due to the synergistic effect of both microorganisms.31,36 The gradual decrease in
the decolorization rate at higher dye concentration in case of individual microbial cultures
was probably due to the toxic effect of dyes with regard to the individual bacteria and/or
inadequate biomass concentration or improper cell to dye ratio.34 Based on previous reports,37
the concentration of dye in the textile industry effluent is generally around 16ꢀ32 mg Lꢀ1 and
as consortium is unaffected with higher dye concentration, this can be used as an effective
tool in continuous wastewater treatment systems.
3.3. Enzyme analysis
A major mechanism of biodegradation by the microbial cells is controlled by the
biotransformation enzymes produced by the cell itself. To prove the role of enzymes involved
in biodegradation reaction, various biotransformation enzymes were monitored during the
decolorization of RR 198 (18 h) in consortium and also in the individual microorganism i.e.
B. laterosporus and G. geotrichum which are summarized in Table 1. In this set of
experiment, significant induction of laccase (115%) in consortium was observed as compared
to the activity in the cells of G. geotrichum. NADHꢀDCIP reductase activity was induced by
43 and 14% in consortium cells when compared to activity in the cells of G. geotrichum and
B. laterosporus respectively. In addition to that, 22% induction in azo reductase activity was
detected in consortium culture as compared to the activity detected in the cells of B.
laterosporus. It should be noted that, there were absence of few biotransformation enzymes
12

Page 13 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
in the individual microbial species during decolorization. In contrast, the same enzymes were
in positive action in consortium system during decolorization of RR 198. This collective
action of all the oxidoꢀreductive enzymes in consortium might be the key factor of rapid
decolorization. The communal enzyme activities of mixed microbial cultures are far better for
dye decolorization as compared to individual strains.31,36,37 These enzymes have been found
in an active role in the biodegradation of various textile dyes such as Acid Violet 19,
Amaranth,13,32 Green HE4B,38 Malachite Green39 and mixture of dyes.35
3.4. Product characterization
Chromatographic technique can be used to detect the biotransformation process of a
substrate molecule. In the present study, the biotransformation of RR 198 was monitored by
FTIR, HPTLC, and GCꢀMS. Fig 3A elucidated the FTIR spectrum of RR 198. The FTIR
spectrum of the RR 198 dye showed the peaks at 2889 cm−1 for CꢀH stretching vibrations in
alkanes, 1646 and 1490 cm−1 for C=N stretching vibrations in cyclic α, β unsaturated
compounds, 1553 cm−1 C=N stretching vibrations in pyridines and NꢀH deformation in
acyclic compounds, 1400 cm−1 for OꢀH deformation in phenols, 1209 cm−1 for CꢀO stretching
in phenols and also for NꢀH deformation in bonded NꢀH, 1137 cm−1 for S=O asymmetric
stretching in sulphones and CꢀH deformation in substituted benzenes, 1055 cm−1 for CꢀO
stretching in phenols, 740 cm−1 for CꢀCl stretching in halogenated compounds and benzene
ring with five adjacent free H atoms, 621 cm−1 for CꢀN stretching and NꢀH deformation in
acyclic compounds. On the other side, significant disappearance of major peaks and
formation of new peaks in the FTIR spectrum of metabolites obtained after dye
decolorization suggested the biotransformation and effective mineralization of RR 198 by
consortium than individual microorganisms (Fig 3BꢀD).
13

RSC Advances
Page 14 of 29
View Article Online
DOI: 10.1039/C4RA15834B
HPTLC analysis of control dye and metabolites obtained after decolorization clearly
suggest the biotransformation of RR 198 into simple compounds as multiple bands were
obtained in the metabolites lane of consortium and also in metabolites produced by individual
microorganisms (Fig 4). However, the number of separated compounds and their Rf values
were different than control dye (data not shown). In the lane of metabolites obtained via
consortial degradation, eleven bands were detected while nine and six bands were obtained in
the lane of metabolites extracted after degradation of RR 198 by B. laterosporus and G.
geotrichum respectively. These observations suggested the catalytic superiority of the
consortium in dye degradation. The change of Rf value of RR 198 (0.90) and presence of
multiple products in metabolites lane provided the supplementary substantiation for
biodegradation of dyes as recorded by FTIR.
In order to get an insight of difference between the metabolic fate of RR 198 by
consortium and pure cultures during degradation of dye, the degradative pathways was
proposed using GCꢀMS data of metabolites extracted after the degradation of RR 198, which
would help us to get the answer, how actually the consortium works? Fig 5 illustrates the
proposed pathway of RR 198, degraded by consortium and individual organisms on the basis
of GCꢀMS analysis of products formed within 18 h and Table 3 represents mass spectra of the
metabolites formed. In all three possibilities, biodegradation of RR 198 was initially followed
by reduction of azo bond. Azo dyes can be cleaved symmetrically and asymmetrically, with
an active site available for an enzyme to excite the molecule.27 Accordingly, the first step in
degradation of RR 198 by consortium was asymmetric cleavage of azo bond via azo
reductase which lead to formation of intermediate [A] 2ꢀ(phenylsulfonyl)ethanesulfonate
detected at retention time in GC, Rtꢀ24.325, having molecular weight of MWꢀ249 based on
the obtained mass to charge ratio of m/zꢀ247(+2) Fig 5(c). Further desulphonation of this
intermediate lead to the product [B] (ethylsulfonyl)benzene detected at retention time in GC,
14

Page 15 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Rtꢀ22.417 having molecular weight of MWꢀ170 and the mass to charge ratio of m/zꢀ170 by
the action of veratryl alcohol oxidase. On the other side, laccase was responsible for
dechlorination, desulphonation and deamination of postulated intermediate [1] which yielded
product [C] naphthalenꢀ1ꢀol [(Rtꢀ20.167, MWꢀ144) based on the obtained mass to charge
ratio of m/zꢀ146(ꢀ2)], and a reactive intermediate [D] 1,3,5ꢀtriazinꢀ2ꢀamine [(Rtꢀ21.508,
MWꢀ96) based on the mass to charge ratio of m/zꢀ97(ꢀ1)] and an unidentified product [2].
The formation of final product [E] 1,3,5ꢀtriazin [(Rtꢀ19.500, MWꢀ81) according to the mass
to charge ratio obtained at m/zꢀ83(ꢀ2)] indicates deamination of reactive intermediate [D] by
veratryl alcohol oxidase.
In case of degradation of RR 198 by individual organism, different metabolites were
obtained. In case of biodegradation of dye by B. laterosporus; formation of products [A] 2ꢀ
(phenylsulfonyl)ethanesulfonate [detected at retention time of Rtꢀ21.467 having molecular
weight of MWꢀ249 according to the mass to charge ratio of m/zꢀ248(+1)], [B]
(ethylsulfonyl)benzene [Rtꢀ20.625, MWꢀ170 based on the mass to charge ratio achieved at
m/zꢀ170] and [C] 6ꢀchloroꢀNꢀphenylꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiamine [detected at Rtꢀ17.350, MWꢀ
221 based on the mass to charge ratio of m/zꢀ223(ꢀ2)] occurred due to the positive role of
azoreductase and veratryl alcohol oxidase [Fig 5(a)]. Whereas, only one product i.e. [A]
(ethylsulfonyl)benzene [Rtꢀ19.892, MWꢀ170, according to the mass to charge ratio of m/zꢀ
170] was obtained in a single step via azo bond cleavage and desulphonation by azoreductase
and veratryl alcohol oxidase respectively from G. geotrichum [Fig 5(b)]. These results
suggested individual organisms partially mineralized the parent dye molecule; whereas,
consortium totally mineralized the RR 198 into simple forms. Recently, in a different
approach, 96% decolorization of RR 198 was attained within 60h using a plant system,40 but
totally different metabolites were obtained which were not completely mineralized as
15

RSC Advances
Page 16 of 29
View Article Online
DOI: 10.1039/C4RA15834B
consortium did in the present study. These observations suggest that, each species has its own
way to act upon a substrate molecule with different fate of metabolism.
3.5. Phytotoxicity
The untreated textile effluent released to the environment influence their toxicity,
when used directly for the agriculture purpose. Hence it is of prime intention to study the
toxic effect of treated as well as untreated dye samples on commonly used agricultural plants.
Table 2 represents the toxicity analysis of the RR 198 and its metabolites obtained after
decolorization. In the dye samples, seed germination was inhibited by 50 and 30 % in
Sorghum vulgare and Phaseolus mungo respectively. But after the treatment with consortium,
germination of Sorghum vulgare and Phaseolus mungo was enhanced by 100 and 30%
respectively when compared to untreated dye. In addition, the metabolites produced after
degradation of RR 198 might have played a nutritive role in plant growth regulation as both
the plants shown better growth in metabolites solution as compared to plants grown in plain
distilled water. Reduction in shoot and root length of both the plants clearly suggested the
toxicity of untreated azo dye. While, reduction in toxicity of dye after treatment proved the
effectiveness of consortium.
Conclusions
This study clearly suggests that the bacterialꢀyeast consortium has an enormous
strength to catalyze the azo dyeꢀRR 198 within short period rather than individual microbial
species. FTIR, HPTLC and GCꢀMS analysis indicated better mineralization of dye by
consortium with significant reduction in its toxicity. The tolerance capacity of this developed
consortium with high dye concentrations also recommends application of this consortium into
effluent treatment plant.
16

Page 17 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Acknowledgement
M. B. Kurade and T. R. Waghmode wish to thank Department of Biotechnology, New
Delhi, India for providing funds for this study (DBT Grant Number ꢀ BT/PRꢀ
10094/BCE/08/599/2007).
References
1 B. D. Tony, D. Goyal, and S. Khanna, Int. Biodeterior. Biodegrad., 2009, 63, 462ꢀ469.
2 E. S. Yoo, J. Libra, and U. Wiesmann, Water Sci. Technol., 2000, 1, 15ꢀ22.
3 J. Lin, X. Zhang, Z. Li, and L. Lei, Bioresour. Technol., 2010, 101, 34ꢀ40.
4 K. Selvam, K. Swaminathan, and K.ꢀS. Chae, World J. Microbiol. Biotechnol., 2003, 19,
591ꢀ593.
5 O. J. Hao, H. Kim, and P.ꢀC. Chiang, Crit. Rev. Environ. Sci. Technol., 2000, 30, 449ꢀ505.
6 S. Mohanty, N. N. Rao, P. Khare, and S. N. Kaul, Water Res., 2005, 39, 5064ꢀ5070.
7 N. Rao, G. Bose, P. Khare, and S. Kaul, J. Environ. Eng., 2006, 132, 367ꢀ376.
8 G. McMullan, C. Meehan, A. Conneely, N. Kirby, T. Robinson, P. Nigam, I. M. Banat, R.
Marchant, and W. F. Smyth, Appl. Microbiol. Biotechnol., 2001, 56, 81ꢀ87.
9 K. Murugesan, Y. M. Kim, J. R. Jeon, and Y. S. Chang, J Hazard Mater. 2009, 168, 523ꢀ
529.
10 R. Renner, Environ. Sci. Technol., 1997, 31, 188Aꢀ189A.
11 D. H. Pieper, and W. Reineke, Curr. Opin. Biotechnol., 2000, 11, 262ꢀ270.
12 P. S. Patil, U. U. Shedbalkar, D. C. Kalyani, and J. P. Jadhav, J. Ind. Microbiol.
Biotechnol., 2008, 35, 1181ꢀ1190.
13 S. B. Jadhav, N. S. Patil, A. D. Watharkar, O. A. Apine, and J. P. Jadhav, Environ. Sci.
Pollut. Res. Int., 2013, 20, 2854ꢀ2866.
14 S. P. Govindwar, M. B. Kurade, D. P. Tamboli, A. N. Kabra, P. J. Kim, and T. R.
17

RSC Advances
Page 18 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Waghmode, Chemosphere, 2014, 109, 234ꢀ238.
15 N. Dafale, N. N. Rao, S. U. Meshram, and S. R. Wate, Bioresour. Technol., 2008, 99,
2552ꢀ2558.
16 S. S.. Phugare, D. C. Kalyani, S. N. Surwase, J. P. Jadhav, Ecotox. Environ. Safe., 2011,
74, 1288ꢀ1296.
17 M. F. Coughlin, B. K. Kinkle, and P. L. Bishop, Water Res., 2003, 37, 2757ꢀ2763.
18 L. Ayed, E. Khelifi, H. B. Jannet, H. Miladi, A. Cheref, S. Achour, A. Bakhrouf,
Chemical Engineering Journal, 2010, 165, 200ꢀ208.
19 N. Dafale, S. Wate, S. Meshram, T. Nandy, J. Hazard, Mater., 2008, 159, 319ꢀ328.
20 E. Forgacs, T. Cserhati, and G. Oros, Environ. Int., 2004, 30, 953ꢀ971.
21 Y. Patel, C. Mehta, and A. Gupte, Int. Biodeter. Biodegrad., 2012, 75, 187ꢀ193
22 A. Mishra, and A. Malik, Bioresour. Technol., 2014, 171, 217ꢀ226.
23 T. R. Waghmode, M. B. Kurade, A. N. Kabra, and S. P. Govindwar, Microbiology, 2012,
158, 2344ꢀ2352.
24 L.Tan, S. Ning, X. Zhang, and S. Shi, Bioresour. Technol., 2013, 138, 307ꢀ313.
25 M.S. Mahmoud, HBRC Journal, 2014, Article in press, doi:10.1016/j.hbrcj.2014.07.005
26 F. He, W. Hu, and Y. Li, Chemosphere, 2004, 57, 293ꢀ301.
27 M. B. Kurade, T. R. Waghmode, D. P. Tamboli, and S. P. Govindwar, J. Basic Microbiol.,
2013, 53, 136ꢀ146.
28 M. B. Kurade, T. R. Waghmode, A. N. Kabra, and S. P. Govindwar, Biotechnol. Lett.,
2013, 35, 1593ꢀ1598.
29 S. S. Gomare, D. P. Tamboli, A. N. Kagalkar, S. P. Govindwar, Int.. Biodeter.
Biodegrad., 2009, 63, 582ꢀ586.
30 M.B. Kurade, T.R. Waghmode, S.P. Govindwar, J. Hazard. Mater., 2011, 192, 1746ꢀ
1755.
18

Page 19 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
31 M. B. Kurade, T. R. Waghmode, A. N. Kagalkar, and S. P. Govindwar, Chem. Eng. J.,
2012, 184, 33ꢀ41.
32 S. B. Jadhav, S. M. Yedurkar, S. S. Phugare, and J. P. Jadhav, CLEAN – Soil, Air, Water,
2012, 40, 551ꢀ558.
33 T. R. Waghmode, M. B. Kurade, A. N. Kagalkar, and S. P. Govindwar, J. Environ. Sci.
(China), 2012, 24, 1295ꢀ1304.
34 S. U. Jadhav, M. U. Jadhav, A. N. Kagalkar, and S. P. Govindwar, J. Chi. Inst. Chem.
Eng., 2008, 39, 563ꢀ570.
35 S. M. Joshi, S. A. Inamdar, A. A. Telke, D. P. Tamboli, and S. P. Govindwar, Int.
Biodeterior. Biodegrad., 2010, 64, 622ꢀ628.
36 S. S. Phugare, D. C. Kalyani, A. V. Patil, and J. P. Jadhav, J. Hazard. Mater., 2011, 186,
713ꢀ723.
37 R. G. Saratale, G. D. Saratale, D. C. Kalyani, J. S. Chang, and S. P. Govindwar,
Bioresour. Technol., 2009, 100, 2493ꢀ2500.
38 A. N. Kabra, R. V. Khandare, M. B. Kurade, and S. P. Govindwar, Environ. Sci. Pollut.
Res. Int., 2011, 18, 1360ꢀ1373.
39 P. Sathishkumar, T. Palvannan, K. Murugesan, and S. KamalaꢀKannan, Environ. Technol.,
2013, 34, 139ꢀ147.
40 R. V. Khandare, A. N. Kabra, D. P. Tamboli, and S. P. Govindwar, Chemosphere, 2011,
82, 1147ꢀ1154.
19

RSC Advances
Page 20 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Figure captions
Figure 1 Decolorization of RR 198 by consortium (
♦
), B. laterosporus ( ■ ) and G.
°
geotrichum ( ▲ ) at pH 7, 40 C and in static condition with dye concentration of
50 mg lꢀ1.
Figure 2 Effect of different concentration of dye concentration [50 mg lꢀ1 (
♦
), 100 mg lꢀ1 (
■
), 200 mg lꢀ1 ( ▲ ), 300 mg lꢀ1 (
●
), 400 mg lꢀ1 ( × ) on decolorization by
°
consortium (A), B. latersporus (B) and G. geotrichum (C) at pH 7, 40 C and in
static condition.
Figure 3 FTIR spectrum of control dye RR 198 (a) and its metabolites extracted after
°
decolorization (18 h) at pH 7, 40 C in static condition by consortium (b), B.
laterosporus (c) and G. geotrichum (d).
Figure 4 HPTLC chromatogram (I) and HPTLC plate (II) of control dye RR 198 (a) and
metabolites produced after its degradation (18 h) at pH 7, 40 °C in static condition by
consortium (b), G. geotrichum (c) and B. laterosporus (d).
Figure 5 Proposed metabolic pathway of RR 198 by B. laterosporus (a), G. geotrichum (b)
and consortium (c).
20

Page 21 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Table 1 Enzyme activities of cells obtained after decolorization (18 h) of RR 198.
G. geotrichum B. laterosporus Consortium
Veratryl alcohol oxidasea
Laccasea
NA
1.846 ± 0.154
NA
1.051 ± 0.106*$
1.084 ± 0.087*$
NA
0.942 ± 0.046
Intracellular 1408 ± 151
Extracellular 1217 ± 111
1115 ± 18.57
253.4 ± 14.72
Tyrosinaseb
NA
NADHꢀDCIP reductasec
Riboflavin reductased
Azoreductasee
183.96 ±11.22 230.94 ± 34.36 263.03 ± 23.79*$
14.39 ± 0.296
5.546 ± 0.481
NA
4.80 ± 0.126*$
4.738 ± 0.227*$
3.890 ± 0.157
a Activity in units/min/mg protein.
b Activity in IU.
c µg of DCIP reduced/min/mg protein.
d µg of Riboflavin reduced /min/mg protein.
e µM of MR reduced /min/mg protein.
NAꢀ No activity.
Values are mean of three experiments ± SEM. Enzyme activities of consortium are significantly different from
$
the G. geotrichum at *P < 0.001 and significantly different from the B. laterosporus at P < 0.001 and by oneꢀ
way ANOVA with TukeyꢀKramer Multiple Comparisons Test.
21

RSC Advances
Page 22 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Table 2 Phytotoxicity study of RR 198 and the metabolites obtained after its decolorization
(18 h) by consortium.
Sorghum vulgare
Phaseolus mungo
Observations
I
II
III
I
II
III
Germination (%)
Plumule length (cm)
100
50
100
4.83
100
12.52
70
100
14.28
3.47
± 0.180
5.11
± 0.133
2.32
10.31
± 0.182* ± 0.217$ ± 0.271
± 0.469* ± 0.196$
1.38
4.91
5.72
2.35
4.76
Radicle length (cm)
± 0.22*
± 0.271$ ± 0.116
± 0.120* ± 0.211$
I: Seeds germinated in distilled water.
II: Seeds germinated in RR 198.
III: Seeds germinated in metabolites obtained after decolorization of RR 198.
Data was analyzed by one way analysis of variance (ANOVA) with TurkeyꢀKramer multiple comparison test
using mean values of germinated seeds of three experiments. Seeds germinated in RR 198 are significantly
different from the seeds germinated in plain Distilled water at *P<0.001 and the seeds germinated in
degradation products are significantly different from the seeds germinated in RR 198 at $P<0.001.
22

Page 23 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Table 3 The GCꢀMS analysis of metabolites obtained at 18 h decolorization of RR 198.
Retention
time (min)
Metabolite
m/z
Mass spectra
1. Consortium
[A] 2ꢀ(phenylsulfonyl)ethanesulfonate
24.325
249
[B] (ethylsulfonyl)benzene
22.417
20.167
21.508
170
146
97
[C] naphthalenꢀ1ꢀol
[D] 1,3,5ꢀtriazinꢀ2ꢀamine
[E] 1,3,5ꢀtriazin
19.500
83
2. B. laterosporus
23

RSC Advances
Page 24 of 29
View Article Online
DOI: 10.1039/C4RA15834B
[A] 2ꢀ(phenylsulfonyl)ethanesulfonate
21.467
249
[B] (ethylsulfonyl)benzene
20.625
17.350
170
223
6ꢀchloroꢀNꢀphenylꢀ1,3,5ꢀtriazineꢀ
[C]
2,4ꢀdiamine
3. G. geotrichum
[A] (ethylsulfonyl)benzene
19.892
170
24

Page 25 of 29
RSC Advances
View Article Online
DOI: 10.1039/C4RA15834B
Fig. 1
25

RSC Advances
Page 26 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Fig. 2
26

Page 27 of 29
RSC Advances
Fig. 3
27

RSC Advances
Page 28 of 29
View Article Online
DOI: 10.1039/C4RA15834B
Fig. 4
28

Page 29 of 29
RSC Advances
Fig. 5
29