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International Biodeterioration & Biodegradation 65 (2011) 1024e1034

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

A sequential aerobic/microaerophilic decolorization of sulfonatedmono azo dye Golden Yellow HER by microbial consortium GG-BL

Tatoba R. Waghmode a, Mayur B. Kurade b, Rahul V. Khandare b, Sanjay P. Govindwar a,*aDepartment of Biochemistry, Shivaji University, Kolhapur 416004, IndiabDepartment of Biotechnology, Shivaji University, Kolhapur 416004, India

a r t i c l e i n f o

Article history:Received 17 June 2011Received in revised form2 August 2011Accepted 2 August 2011Available online 30 August 2011

Keywords:Consortium GG-BLGYHERDecolorizationLaccaseHPTLCPAGE

* Corresponding author. Department of Biochemisnagar, Kolhapur 416004, Maharashtra, India. Tel.: þ92691533.

E-mail address: spg_biochem@unishivaji.ac.in (S.P

0964-8305/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.ibiod.2011.08.002

a b s t r a c t

This study is a part of efforts to develop new batch method with the help of prepared consortium GG-BLusing two microbial cultures viz. Galactomyces geotrichum MTCC 1360 and Brevibacillus laterosporusNCIM 2298, varying oxidation conditions for the bio-treatment processes to produce reusable water bydecolorization of Golden Yellow HER (GYHER) to less toxic metabolites. Consortium was found to bemuch faster for decolorization and degradation of GYHER as compared to the individual strains. Theintensive metabolic activity of these strains led to 100% decolorization of GYHER (50 mg l�1) within 24 hwith significant reduction in chemical oxygen demand (84%) and total organic carbon (63%). The pres-ence of veratryl alcohol oxidase, NADH-DCIP reductase and induction in laccase, tyrosinase, azo reduc-tase and riboflavin reductase during decolorization suggests their role in decolorization process.Substrate staining of nondenaturing polyacrylamide electrophoresis gel (PAGE) also confirms inductionof oxidative enzymes during GYHER degradation. The degradation of the GYHER into different metab-olites by individual organism and in consortium was confirmed using High Performance Thin LayerChromatography (HPTLC), High Performance Liquid Chromatography (HPLC), Fourier Transform Infra RedSpectroscopy (FTIR), Gas Chromatography Mass Spectroscopy (GCeMS) analysis. Phytotoxicity studiesrevealed nontoxic nature of the metabolites of GYHER.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Azo dyes are synthetic organic compoundswith one ormore azobonds (eN]Ne) and having criterions that are necessary for theirvarious applications in textile dyeing and many other industries. Asthey are designed to be stable and long-lasting colorants, they areusually recalcitrant in natural environment. The release of thesedyes into the environment without proper treatment may lead topotential risk of bioaccumulation that may eventually incorporateinto food chain and affect human health. Annual consumption ofdyes is around 7 � 104 tones throughout the world due to theirsuperior fastness to the applied fabric, high photolytic stability, andresistance to microbial degradation (Patil et al., 2010). Approxi-mately the 10e20% of azo dye released in environment duringmanufacturing and textile dying due to their low levels of fixationwith the fiber (Junnarkar et al., 2006). The release of these effluent

try, Shivaji University, Vidya-1 231 2609152; fax: þ91 231

. Govindwar).

All rights reserved.

from dying/textile industries into the environment (water bodies)causes change in pH, increases in biological oxygen demand,chemical oxygen demand, and total organic carbon as well asreduced light penetration and gas solubility in water bodies whichleads to adverse effects on living system including plant, animalsand humans (Phugare et al., 2010; Waghmode et al., 2011). Thetreatment of these dye containing textile effluent is becomesnecessary prior to their final discharge to the environment.

Azo dyes are recalcitrant xenobiotic compound and theconventional wastewater treatment processes cannot efficientlydecolorize azo dyes (Kumar et al., 2006). The removal of textileeffluent with existing physical and chemical methods are verydifficult because of its high BOD, COD, TOC, color, pH and presenceof metals as well as methods having expensive nature, canproduce large amount of sludge and toxic substances (Senan andAbraham, 2004).

The general approach of bioremediation is to improve thenatural biodegradation capacity of the native organisms byproviding suitable environmental condition for degradation.Several microbial sources have been reported for dye decoloriza-tion including, fungal and bacterial cultures (Chander et al., 2004;Vitor and Corso, 2008; Dawkar et al., 2010). However the trend is

T.R. Waghmode et al. / International Biodeterioration & Biodegradation 65 (2011) 1024e1034 1025

shifting toward use of mixed microbial culture for degradation ascompared to individual strain. Several microbial consortia havebeen reported for efficient dye removal (Chen and Chang, 2007;Patil et al., 2010; Phugare et al., 2011). On the contrary azo dyesare xenobiotic and under anaerobic condition can produce aromaticamines which are carcinogenic andmutagenic in nature (Senan andAbraham, 2004). At aerobic condition, most of the sulfonated azodyes are harder to degrade (Kalme et al., 2007a). Existing studyreported that complete mineralization of dyes is possible onlywhen the anaerobic reduction is followed by aerobic oxidation ofthe aromatic amines formed in the reductive steps (Rajaguru et al.,2000; Kalme et al., 2007b). In the present study we have focusedour attention to develop new batch process for the degradation ofreactive mono azo dye Golden Yellow HER. The constructedconsortium applied via aerobic/microaerophilic batch process forcomplete mineralization of azo dye. Aerobic oxidation of GYHER isfollowed bymicroaerophilic reduction of metabolite formed duringaerobic degradation.

2. Materials and methods

2.1. Materials

Golden Yellow HER was obtained from the Manpasand TextileProcessing Industry, Ichalkaranji, India. L-catechol, Methyl Red,veratryl alcohol, peptone, yeast extract, and malt extract werepurchased from Hi-Media Laboratories Pvt. Ltd, Mumbai, India.Remaining chemicals were purchased from Sisco Research labora-tory (SRL), India. All chemicals used were of the highest purityavailable and of analytical grade.

2.2. Microorganism and culture condition

Galactomyces geotrichum MTCC 1360 and Brevibacillus later-osporus NCIM 2298 were obtained from Microbial Type CultureCollection, Chandigarh, and National Collection of IndustrialMicroorganism, Pune, India, respectively. The pure culture ofG. geotrichum and B. laterosporus were maintained on malt extractagar and nutrient agar slants at 4 �C respectively. The compositionof malt extract medium used for decolorization studies was (g l�1):malt extract 3.0, yeast extract 3.0, peptone 5.0 and glucose 10.0. Thecomposition of nutrient medium used for decolorization studieswas (g l�1): NaCl 5.0, bacteriological peptone 10.0, yeast extract 2.0,beef extract 1.0.

2.3. Development of consortium GG-BL for decolorization of GYHER

Consortium GG-BL was prepared by aseptically transferring themycelial biomass of 24 h grown culture of G. geotrichum at 30 �C in250 ml Erlenmeyer flasks containing 100 ml malt extract mediumto the flask containing 24 h grown B. laterosporus (grown at 30 �C in250 ml Erlenmeyer flasks containing 100 ml of nutrient medium).

2.4. Decolorization experiment and physicochemical parameters

Biodegradation of GYHER (50 mg l�1) by using consortium GG-BL was carried out at different experimental condition in 250 mlErlenmeyer flask containing 100 ml nutrient medium. Decoloriza-tion potential of individual organisms was also studied in theirrespective growth medium at the same experimental conditionsused for the consortium GG-BL. Aliquots (4 ml) of the culturesupernatant were withdrawn after regular time intervals duringdecolorization process. Suspended particles were removed fromthe culture medium by centrifugation at 4105 � g for 20 min anddecolorization was monitored by measuring absorbance by using

UVevis spectrophotometer (Hitachi U 2800, Tokyo, Japan) at420 nm. All decolorization experiments were performed in tripli-cate and the decolorization activity was expressed in terms ofpercent decolorization (Phugare et al., 2011). Abiotic controls(without microorganism) were always included. The above saidprotocol was followed while studying the potential of consortiumGG-BL to decolorize GYHER at wide pH (3e11) and temperaturerange (10e50 �C). The potential of consortium GG-BL to toleratehigher concentration of GYHER and repeated addition of GYHER(50 mg l�1) in a fed batch manner was also checked. Reduction inchemical oxygen demand (COD) (APHA, 1998) and total organiccarbon (TOC) were also measured using Hach DR 2700 spectro-photometer (Hach Co., USA) (Waghmode et al., 2011).

2.5. Effect of various carbon, nitrogen source and agriculturalwaste on decolorization

Bushnell Haas medium composed of (BHM) (g l�1) (MgSO4 0.2,K2HPO4 1.0, KH2PO4 1.0, CaCl2 0.02, FeCl3 0.05, NH4NO3 1.0) sup-plemented with yeast extract (0.5) was used to study the effect ofcarbon and nitrogen sources at the concentration of 5.0 g l�1 on thedecolorization of GYHER (50 mg l�1). In addition glucose (5.0 g l�1)and yeast extract (5.0 g l�1), effect of different agricultural wasteextract were seen on the decolorization of GYHER in BHM medium(5 ml extract of 10 g l�1 boiled agricultural residue).

2.6. Preparation of cell free extract

The consortium GG-BL was prepared as method described inSection 2.3 and individual organisms were grown in their respec-tive medium for 24 h at 30 �C and centrifuged at 9237 � g for25 min. The biomass of consortium and individual organisms wasseparately suspended in 50 mM potassium phosphate buffer (pH7.4) and sonicated (sonics-vibracell ultrasonic processor, 12 strokesof 30 S each for 1 min interval based on 60 amplitude output) at4 �C. The sonicated cells were centrifuged in cold condition (4 �C, at9237 � g for 25 min) and supernatant used as the source of intra-cellular enzymes. Similar procedure was used to determine theenzyme activities after GYHER decolorization.

2.7. Enzymatic assay

2.7.1. Oxidative enzymes during decolorizationActivities of laccase, veratryl alcohol oxidase and tyrosinasewere

assayed spectrophotometrically in cell free extract and culturesupernatant at room temperature (25 �C). Laccase and veratrylalcohol oxidase activitywere determined according to the procedurereported earlier (Tamboli et al., 2010a). The 2 ml reaction mixturecontained 5mM30, 30-diaminobenzidine tetrahydrate (DAB) in 0.1Macetate buffer (pH 4.8) and increase in optical density wasmeasuredat 410 nm. Veratryl alcohol oxidase activitywas determined by usingveratryl alcohol as a substrate. The reactionmixture contained 1mMveratryl alcohol, in 0.1 M citrate phosphate buffer, pH 3.0, and 0.2mlenzyme. Total volume of 2 ml was used for the determination ofoxidase activity. Oxidation of the substrate at room temperaturewasmonitored by an absorbance increase at 310 nmdue to the formationof veratraldehyde (Tamboli et al., 2010a). Tyrosinase activity wasdeterminedbymodifyingearlier reportedmethod (KandaswamiandVaidyanathan,1973). The 3ml reactionmixture contained 50mMofcatechol and 2.1mMof ascorbic acid in 50mMpotassiumphosphatebuffer (pH 6.5) equilibrated at 25 �C. The DA265 nm was monitoreduntil constant, and then 0.1 ml of the supernatant from the reactionmixture was added. The formation of o-benzoquinone and dehydro-ascorbic acid and decrease in optical density was measured at265 nm. One unit of tyrosinase activity was equal to a DA265 nm of

T.R. Waghmode et al. / International Biodeterioration & Biodegradation 65 (2011) 1024e10341026

0.001 per min at pH 6.5 at 25 �C in a 3.0 ml reaction mixture con-taining L-catechol and L-ascorbic acid.

2.7.2. Reductive enzymes during decolorizationThe NADH-DCIP reductase and riboflavin reductase activities

were assayed by modifying earlier reported methods (Waghmodeet al., 2011). DCIP reduction was monitored at 590 nm and enzymeactivity was calculated using an extinction coefficient 0.019mM�1 cm�1. The 5.0 ml reaction mixture contained 25 mM substrate(DCIP) in the 50mMpotassium phosphate buffer (pH 7.4) and 0.1 mlenzyme. From this, 2.0ml reactionmixturewas assayed at 590nmbyaddition of 250 mM NADH. Riboflavin reductase NAD(P)H:flavinoxidoreductase was measured by monitoring the decrease inabsorbance at 340 nm. Cell free extract was added to a solution (finalvolume 2 ml) containing 100 mM of TriseHCl (pH 7.4), 25 mM ofNADPH and 10 mM of riboflavin. Reaction rates were calculated byusing a molar extinction coefficient of 0.0063 mM�1 cm�1. Azoreductase activitywas assayedbymodifying earlier reportedmethod(Telke et al., 2010). The 2 ml reaction mixture contained 25 mM ofMethyl Red (MR), 50 mMNADH,1.2ml of potassiumphosphate buffer(50 mM, pH 7.4). The reaction mixture was pre-incubated for 4 minfollowed by the addition of NADH andmonitored for the decrease incolor absorbance (430 nm) at room temperature. The reaction wasinitiated by addition of 0.2ml of the enzyme solution. The reductionof Methyl Red was calculated using extinction coefficient of0.023 mM�1 cm�1. One unit of enzyme activity was defined asamount of enzyme required to reduce 1 mMof substratemin�1mg ofprotein�1. All the enzyme assays were run in triplicates.

2.8. Nondenaturing polyacrylamide gel electrophoresis analysis

The cell free extract was used for protein profiling study as perthemethod described in Section 2.6. The clear supernatant obtainedafter centrifugation was directly used for nondenaturing poly-acrylamide gel electrophoresis. Polyacrylamide gel electrophoresisof cell free extract obtained after decolorization of GYHER byG. geotrichum, B. laterosporus and consortium GG-BL was carriedusing vertical slab gel electrophoresis unit with 10% separating geland 4% stacking gel (GeNei, Bangalore, India). The 10% separating gelcomprised of 33.33ml stock of acrylamide solution (30% acrylamide,0.8% bisacrylamide), 25 ml TriseHCl (pH 8.8) and 39.87% of distilledwater. Composition of 4% stacking gel was 5.36ml stock acrylamidesolution, 10 ml TriseHCl (pH 6.8) and 24 ml distilled water. Afterdegassing, 0.75 and 0.2 ml (10%) ammonium persulfate and 50 and40 ml TEMED was added for separating gel and resolving gelrespectively. The sample buffer was prepared by mixing 2.0 ml ofTriseHCl buffer (pH 6.8),1.6ml of glycerol, 0.4ml bromophenol blue(1% w/v) together and used further for sample loading. Proteinbands were stained using Coomassie Brilliant Blue R-250 for 4 h atroom temperature followed by distaining using methanol: aceticacid: water (v/v) (4:1:5). The protein concentration used for loading(in each well) was approximately 150 mg. Activity staining wascarried out by incubating the gel after nondenaturing PAGE at roomtemperature with 1 mM L-DOPA for 2 h.

2.9. Metabolites analysis

The decolorization of GYHER by G. geotrichum, B. laterosporus andconsortium GG-BL were monitored by using UVevis spectropho-tometer (Hitachi U 2800, Tokyo, Japan). The metabolites producedduring degradation, were extracted with equal volumes of ethylacetate; dried over anhydrous Na2SO4 and dissolved in small volumeofHPLCgrademethanol andused for further analysis. HPTLC analysiswas performed by using HPTLC system (CAMAG, Switzerland)(Kurade et al., 2011). 10 ml of control and extracted metabolites after

degradation were loaded on pre-coated HPTLC plates (Lichrosphersilica gel plate, Merck, Germany), by spray gas nitrogen and TLCsample loading instrument (CAMAG LINOMAT V). The compositionof mobile phase was methanol: ethyl acetate (6:4 v/v). The controldye andmetabolites obtained after its degradationwere visualized inUVchamber and scanned at 280nmwith slit dimension 5� 0.45mmby using TLC scanner (CAMAG) and the results were analyzed usingHPTLC software WinCATS 1.4.4.6337. HPLC analysis was carried out(Waters model no. 2690) on C18 column (symmetry, 4.6 mm �250 mm) by isocratic method with 10 min run time (Phugare et al.,2010). The mobile phase was methanol with flow rate of1 ml min�1 and UV detector was kept at 420 and 280 nm. Thesampleswere filteredwith a 0.2 mmmembrane filter and about 10 mlof sample was manually injected into the injector port.

FTIR (Shimadzu 8400S spectrophotometer) was used for inves-tigating the changes in surface functional groups of the samples,before and after microbial decolorization. FTIR analysis was done inthe mid IR region of 400e4000 cm�1 with 16 scan speed (Sarataleet al., 2009a). The pellets were prepared using spectroscopic pureKBr (5:95) and fixed in the sample holder for the analysis. Theidentification of metabolites formed after degradation was carriedusing a QP2010 gas chromatography coupled with mass spectros-copy (Shimadzu) (Kalyani et al., 2009). The ionization voltage was70 eV. Gas chromatography was conducted in the temperatureprogramming mode with a Restek column (0.25 mm, 60 m; XTI-5).The initial column temperature was 80 �C for 2 min, thenincreased linearly at 10 �C min�1 to 280 �C, and held for 7 min. Thetemperature of the injection port was 280 �C and the GCeMSinterface was maintained at 290 �C. The helium carrier gas flow ratewas 1.0 ml min�1. Degradation products were identified bycomparison of retention time and fragmentation pattern, as well aswithmass spectra in the NIST spectral library stored in the computersoftware (version 1.10 beta, Shimadzu) of the GCeMS.

2.10. Phytotoxicity analysis

Phytotoxicity of GYHER was performed in order to assess thetoxicity of textile industry effluent to common agricultural crop(Kalyani et al., 2009; Telke et al., 2010). The obtained product wasdissolved in water to form a final concentration of 1000 ppm. Tenseeds of Phaseolus mungo and Sorghum vulgare plants were sowedinto a plastic sand pot with daily watering of (5 ml) GYHER(1000 ppm) and its degradation metabolites (1000 ppm) obtainedafter degradation by Consortium GG-BL. Control set was carried outusing distilled water (daily 5 ml watering) at the same time.Germination (%) and length of shoot and root was recorded after 7days. The study was carried out at room temperature. Germination% was calculated by following formula as:

Germinationð%Þ ¼ No: of seeds germinatedNo: of seeds sowed

� 100

2.11. Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA)with TukeyeKramer multiple comparisons test.

3. Results

3.1. Decolorization experiment and physicochemical parameters

Dyes of different structures are often used in the textile pro-cessing industry, so the time and condition required for thedecolorization depends upon the structure of dye. Decolorization of

Fig. 1. Effect of pH (A) and Temperature (-) on the decolorization of GYHER by usingconsortium GG-BL.

T.R. Waghmode et al. / International Biodeterioration & Biodegradation 65 (2011) 1024e1034 1027

GYHER at different experimental conditionwas carried out by usingG. geotrichum, B. laterosporus and consortium GG-BL. After devel-opment of consortia (as per the method described in Section 2.3)GYHER added at 50 mg l�1 concentration and flask was incubatedfor aerobic condition (120 rpm) for first 12 h of decolorizationand after aerobic (shaking condition) treatment flask transferredto microaerophilic condition (static condition) for subsequent

Table 1Decolorization of GYHER by using G. geotrichum, B. laterosporus and consortium GG-BL at various experimental conditions.

Sr. no. Organism Condition Decolorization(%)

1. G. geotrichum Microaerophilic (24 h) 49Aerobic (24 h) 24Aerobic (12 h) tomicroaerophilic (12 h)

54

Microaerophilic (12 h)to aerobic (12 h)

26

2. B. laterosporus Microaerophilic (24 h) 68aerobic (24 h) 8Aerobic (12 h) tomicroaerophilic (12 h)

91

Microaerophilic (12 h)to aerobic (12 h)

76

3. Mixed growth ofG. geotrichum andB. laterosporusin MGYP medium

Microaerophilic (24 h) 43Aerobic (24 h) 8Aerobic (12 h) tomicroaerophilic (12 h)

80

Microaerophilic (12 h)to aerobic (12 h)

80

4. Mixed growth ofG. geotrichum andB. laterosporus innutrient medium

Microaerophilic (24 h) 77Aerobic (24 h) 31Aerobic (12 h) tomicroaerophilic (12 h)

0

Microaerophilic (12 h)to aerobic (12 h)

20

5. Consortium GG-BL(50 ml growth ofG. geotrichum þ 50 mlgrowth of B. laterosporus)

Microaerophilic (24 h) 81Aerobic (24 h) 26Aerobic (12 h) tomicroaerophilic (12 h)

35

Microaerophilic (12 h)to aerobic (12 h)

48

6. Consortium GG-BL Microaerophilic (24 h) 93Aerobic (24 h) 62Aerobic (12 h) tomicroaerophilic (12 h)

100

Microaerophilic (12 h)to aerobic (12 h)

85

decolorization for next 12 h. The same procedure was applied incase of individual strains. The optimum pH and temperature forGYHER decolorization were 9.0 and 30 �C, respectively (Fig. 1). ThepH changes from 9.0 to 8.0 during decolorization, but the decol-orization of the dye was due to microbial action and not because ofchange in pH. G. geotrichum and B. laterosporus showed 54% and91% decolorization at aerobic/microaerophilic condition at 30 �Cand pH 9.0, with 18% and 72% of COD reduction as well as 7% and51% of TOC reduction respectively. Consortium GG-BL showed 100%decolorization of GYHER (50 mg l�1) at aerobic/microaerophilicconditionwith significant reduction in COD (84%) and TOC (63%) aswell as showed 85% decolorization at microaerophilic/aerobiccondition. Table 1 showed that the formed consortium GG-BLshowed highest removal of GYHER at all experimental conditionsas compared to G. geotrichum and B. laterosporus. Consortium GG-BL also showed 91% and 70% decolorization at the concentrationof 100 mg l�1 and 250 mg l�1 within 72 h respectively (Fig. 2a).Consecutive cycles of dye decolorization were studied by therepeated additions of GYHER (50 mg l�1) in flask containing 100 mlgrowth of consortium at sequential aerobic/microaerophiliccondition (Fig. 2b). It showed effective dye decolorization up to 7tested cycles. At seventh cycle consortium GG-BL showed morethan 75% decolorization within 24 h.

3.2. Effect of various carbon, nitrogen source and agriculturalwaste on decolorization

In BHM medium (control), only 33% of decolorization of GYHERdye was observed in 72 h. Glucose and yeast extract showed 95%and 20% decolorization, respectively. BHM containing rice husk,

Fig. 2. Effect of initial dye concentration (a) and effect of repeated addition of dye ondecolorization (b) performance of consortium GG-BL.

Table 2Effect of carbon and nitrogen sources on the decolorization of GYHER byconsortium GG-BL.

Media Decolorization (%)

BHM 33BHM þ Rice husk 28BHM þ Maize husk 30BHM þ Bagasse 24BHM þ Wheat bran 36BHM þ Wood shaving 0BHM þ Glucose 95BHM þ Yeast extract 20

BHM e Bushnell Haas Medium.

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maize husk, bagasse andwheat bran showed 28%, 30%, 24% and 36%decolorization respectively, whereas BHM containing woodshaving showed no decolorization of GYHER within 72 h (Table 2).

3.3. Enzymatic analysis

3.3.1. Oxidative enzymes during decolorizationSignificant induction in the activity of veratryl alcohol oxidase by

118% and 240% was observed in consortium GG-BL (after decolor-ization) as compared to the B. laterosporus, but it was near same inconsortium GG-BL (control) (before decolorization) (Table 3). Therewas no activity observed in G. geotrichum. Laccase was inducedtremendously by 4843% in consortium GG-BL (after degradation) ascompared to control consortium GG-BL (before degradation), butit was unchanged in G. geotrichum. Laccase was absent inB. laterosporus. Intracellular tyrosinase was induced in consortiumGG-BL (after degradation) by 1030% and310% as compared to controlconsortium GG-BL (before decolorization) and B. laterosporusrespectively, but the activity was absent in G. geotrichum (afterdecolorization). Extracellular activity was reduced in controlconsortium GG-BL (before decolorization) and consortium GG-BL(after decolorization) as compared to G. geotrichum andB. laterosporus, but the activity in consortium GG-BL induced by 52%after 24 h of decolorization as compared to 12 h of decolorization.

3.3.2. Reductive enzyme during decolorizationThe enzymatic analysis of consortium GG-BL (after decoloriza-

tion) for reductive enzyme showed induction in NADH-DCIPreductase, azo reductase and riboflavin reductase as compared toconsortium GG-BL (before decolorization). The NADH-DCIP reduc-tase activity was induced by 146% and 198% in consortium GG-BL(after decolorization) as compared to G. geotrichum (after decolor-ization), but it reduced to 138% as compared to B. laterosporus (after

Table 3Enzyme activities in control cells (at 0 h of dye addition) and cells obtained after 24 h o

Enzyme G. geotrichum B. laterosporu

0 h 24 h 0 h

Laccasea 0.070 � 0.05 0.791 � 0.05# NAVeratryl alcohol oxidasea NA NA 0.276 � 0.03Tyrosinasea

Intracellular 1411.10 � 451 NA 357 � 24.00Extracellular 1217.54 � 111 726 � 6.00# 565 � 72

NADH-DCIP reductaseb 183.96 � 11.35 38.18 � 1.71# 62.67 � 4.64Azo reductasec 5.546 � 0.48 NA 3.533 � 0.90Riboflavin reductased 14.39 � 0.29 NA NA

Values are mean of three experiments � standard error mean (SEM). Significantly differeANOVA with TukeyeKramer comparison test.

a Enzyme unit’s min�1 mg protein�1.b mg of DCIP reduced min�1 mg protein�1.c mM of Methyl Red reduced min�1 mg protein�1.d mg of riboflavin reduced min�1 mg protein�1.

decolorization). Azo reductase activity was reduced in consortiumGG-BL (after decolorization) as compared to B. laterosporus.G. geotrichum showed absence of azo reductase activity afterdecolorization. Riboflavin reductase activity induced in consortiumGG-BL after decolorization as compared to control consortiumGG-BL (before decolorization) whereas it was absent inG. geotrichum and B. laterosporus (Table 3).

3.4. Nondenaturing polyacrylamide gel electrophoresis analysis

Protein profile obtained on nondenaturing polyacrylamideelectrophoresis gel after the degradation of GYHER byG. geotrichum, B. laterosporus and consortium GG-BL showeddifferential induction pattern of protein/enzyme as compared tocontrol (before decolorization) (Fig. 3a). Activity staining of non-denaturing gel showed induction of different oxidative enzymesduring the degradation GYHER by individual strains as well asconsortium GG-BL (Fig. 3b). In case of G. geotrichum no proteinband was observed in control (before decolorization) however intest (after decolorization) there was single band of oxidativeenzyme present with molecular weight 101 kDa. In case ofB. laterosporus, two bands were observed in control (beforedecolorization) and test (after decolorization) with the molecularweight of 103 and 106 kDa, respectively. Whereas in consortiumGG-BL two bands of enzyme present in control (before decolor-ization) with molecular weight 102 and 104 kDa and three bandswere present with the molecular weight of 92, 102 and 104 kDa inthe test (after decolorization). Activity staining pattern of oxidativeenzyme present on nondenaturing PAGE showed similarity withour enzyme analysis results; it indicates the confirmation of role ofoxidative enzymes in GYHER degradation by individual strains aswell as consortium GG-BL.

3.5. Biodegradation analysis

The spectrophotometric analysis of culture supernatant afterdecolorization by consortium at 400e800 nm showed significantreduction in absorbance as compared to control GYHER,G. geotrichum and B. laterosporus (Fig. 4). The metabolites obtainedafter 24 h of decolorization of the GYHER were extracted with ethylacetate, crystallized, dissolved in HPLC grademethanol and used forthe analysis. The HPTLC analysis of degraded metabolites showeddifferent degradation patternwith different Rf values in consortiumGG-BL as compared to control GYHER, G. geotrichum andB. laterosporus (Fig. 5a, b). The difference in Rf value of control dye(0.79) and formed metabolite by G. geotrichum (0.32, 0.50, 0.60,0.65, 0.73, 0.83, 0.88), B. laterosporus (0.55, 0.63, 0.68, 0.80, 0.89)

f GYHER decolorization by G. geotrichum, B. laterosporus and consortium GG-BL.

s Consortium GG-BL

24 h 0 h 12 h 24 h

NA 0.016 � 0.00 0.365 � 0.00 0.791 � 0.09*0.179 � 0.05$$ 0.667 � 0.08 0.496 � 0.02 0.609 � 0.02

545 � 177 217.25 � 40.62 2236 � 351 NA563 � 86 239.07 � 13.98 295 � 19.00 448 � 17.00*296.28 � 6.10$ 53.62 � 0.81 94.04 � 1.14 113.88 � 0.46*53.17 � 2.82$ 2.950 � 0.55 NA 3.097 � 0.17NA 3.703 � 0.71 5.451 � 1.12 7.104 � 1.29*

nt from control cells at $$P < 0.01, #P < 0.001, $P < 0.001 and *P < 0.001 by One-way

Fig. 3. Protein profile obtained after the degradation of GYHER by using G. geotrichum, B. laterosporus and consortium GG-BL. Protein staining (Fig. 8a) was carried out by usingcoomassie brilliant blue R-250 as staining dye and activity staining (Fig. 8a) by using L-DOPA as substrate (1 mM). Nondenaturing PAGE and activity staining of proteins obtainedafter degradation of GYHER by G. geotrichum, B. laterosporus and consortium GG-BL. The lane (a) represents the marker dye, lane (b) control G. geotrichum (before decolorization),lane (c) G. geotrichum (after decolorization), lane (d) B. laterosporus (before decolorization), lane (e) B. laterosporus (after decolorization), lane (f) Consortium GG-BL (beforedecolorization), lane (g) Consortium GG-BL (after decolorization).

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and consortium GG-BL (0.51, 0.64, 0.71, 0.83, 0.88) indicate thebiodegradation of GYHER (Fig. 5a). Disappearance of dye fromcontrol sample and appearance of new metabolite spots withdifferent Rf values within HPTLC chromatogram confirmed thedegradation. HPLC chromatogram of control GYHER showed onemajor peak at retention time of 3.007 min and four minor peaks atthe retention time of 2.199, 2.683, 3.306 and 3.500 (Fig. 6a).Decolorization of GYHER by G. geotrichum showed five peaks atretention times, 2.348, 2.520, 2.783, 3.040 and 3.450 min (Fig. 6b),by B. laterosporus showed one major and three minor peaks at theretention time of 2.422, 2.595, 3.079, 3.403 (Fig. 6c) and byconsortium GG-BL showed one major peak at retention time of2.995 and three minor peaks at the retention time of 2.535, 3.530

Fig. 4. UVevisible spectra of GYHER degradation: control dye (A), G. geotrichum (-),B. laterosporus (:) and consortium GG-BL (z).

and 5.492 (Fig. 6d) suggested the further conversion of degradationproducts into various metabolites by consortium GG-BL ascompared to individual strains.

Comparison of FTIR spectrum of parent dye and the metaboliteformed after 24 h revealed the biodegradation of dye GYHER byG. geotrichum, B. laterosporus and consortium GG-BL (Fig. 7). TheFTIR analysis GYHER dye revealed the presence of aromaticcompound at 1490 cm�1, benzene ring at 828.4 cm�1, azo bond(N]N) at 1614.4 cm�1, CH3 vibration at 1365 cm�1, OeH stretchingat 3471.8 cm�1, NeH deformation at 1305 cm�1, presence ofsulfonic acid at 1041.6 cm�1 and 1190.6 cm�1 indicates that thenature of the dye is sulfonated aromatic compound. Also1114.4 cm�1 and 673.1 cm�1 indicate the presence of secondaryalcohol (CeOH stretch) and halides (CeCl) (Fig. 7a). Whereasdegraded product obtained after degradation by G. geotrichumshowed NeH stretch at 3405.61 cm�1, CeH stretch (alkenes) at2851.26e2952.96 cm�1, aromatic compound at 1516.14 cm�1, azobond at 1582.02 cm�1, S]O stretch at 1084.33e1378.57 cm�1, CeHdeformation at 820.66e897.09 cm�1, halides at 713.67 cm�1

(Fig. 7b). Degradation by B. laterosporus showed NeH deformationat 3409.99 cm�1, presence of alkane (eCH3 stretch) at2849.78e2956.78 cm�1, peak at 1516.14 cm�1 showed aromatichomocyclic compound, CeH deformation at 916.20 cm�1,9369.69 cm�1, 1305.97 cm�1 and 1455.62 cm�1, presence of sulfiteat 1187.51 cm�1, peak at 1118.72 cm�1 showed CeOH stretch, peakat 748.06 cm�1 showed benzene ring with four adjacent free Hatoms, absence of peak at 1614.4 cm�1 indicates the cleavage of azobond by reductase (Fig. 7c). Degradation by consortium GG-BLshowed NeH deformation at 3418.72 cm�1, aromatic alcohol at3247.37 cm�1, alkanes (eCH3 stretch) at 2851.45e2957.25 cm�1,peak at 1508.28 cm�1 indicates the aromatic compound, CeHdeformation at 748.76e966.69 cm�1 and at 1455.62 cm�1, CeOHstretch at 1043.20 cm�1 and at 1311.23e1333.90 cm�1, S]O stretchat 1190.52e1209.90 cm�1, peak showed CeN vibration at1118.40 cm�1, absence of peak at 1614.4 cm�1 indicates the cleavage

Fig. 5. a. HPTLC profile of control dye GYHER (a) and its metabolites obtained after 24 h degradation by G. geotrichum (b), B. laterosporus (c) and consortium GG-BL (d). b. HPTLC 3-Dgraph of control dye GYHER (a) and its metabolites obtained after 24 h of degradation by G. geotrichum (b), B. laterosporus (c) and consortium GG-BL (d).

Fig. 6. HPLC elution profile recorded at 420 nm of control GYHER (a) and its metabolites obtained after 24 h of degradation by using G. geotrichum (b), B. laterosporus (c) andconsortium GG-BL (d).

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Fig. 7. FTIR analysis of control dye GYHER (a) and its metabolites obtained after 24 h of degradation by using G. geotrichum (b), B. laterosporus (c) and consortium GG-BL (d).

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of azo bond (Fig. 7d). Proposed biodegradation pathway of azodye GYHER was shown in Fig. 8 on the basis of GCeMS analysis ofthe metabolites obtained after decolorization of GYHER byG. geotrichum (Fig. 8a), B. laterosporus (Fig. 8b) and consortium GG-BL (Fig. 8c).

3.6. Phytotoxicity analysis

The toxic nature of the dye and the textile effluent is mainlybased on the chemical nature of the dye and number of dyespresent in textile effluent. Phytotoxicity study revealed toxic natureof GYHER to the P. mungo and S. vulgare plants. Germination (%) ofseeds of these plants was less with GYHER treatment as comparedto the metabolites obtained after its decolorization and distilledwater (Table 4). The GYHER significantly affected the shoot and rootgrowth than the metabolites obtained after its decolorization.These results proved the less toxic nature of metabolites obtainedafter decolorization of GYHER.

4. Discussion

The effectiveness of decolorization depends on the structureand complexity of dye and relatively small structural differences indye can affect whole decolorization process (Verma andMadamwar, 2003). In the present study, G. geotrichum andB. laterosporus were combined as consortium GG-BL and haddemonstrated great capability in decolorization of azo dye GYHERat different experimental conditions. Synergistic action of microbes

within the consortium was found to be responsible for fasterdecolorization of GYHER than individual strains. However, a greatdrawback is the generation of potentially toxic and mutagenic endproducts (aromatic amines) by anaerobic bacteria and failure ofaerobic bacteria in azo dye reduction at aerobic condition; hencewe have constructed aerobic/microaerophilic batch process for thecomplete decolorization of azo dye. The consortium GG-BL showedcomplete decolorization of GYHER at aerobic/microaerophiliccondition with significant reduction in COD and TOC. The decol-orization potential of consortium GG-BL was higher at aerobic/microaerophilic condition as compared to G. geotrichum andB. laterosporus. The optimum pH and temperature for GYHERdecolorization were 9.0 and 30 �C, respectively that indicates thepotential use of consortium GG-BL for the decolorization of alkalineeffluent. The dye decolorization activity of our culture was found toincrease with increase in incubation temperature from 20 to 40 �Cwith maximum activity attained at 30 �C. Further increase intemperature resulted in marginal reduction in decolorization ofGYHER by consortium GG-BL. Ramya et al. (2008) reported thatPaenibacillus larvae exhibited decolorization of Indigo Carmine attemperature varying from 30 to 40 �C, whereas at 50 �C it showedmarginal reduction in decolorization capability. The time requiredfor decolorization increased with increase in dye concentration.Lower decolorization efficiency was due to the toxic effect of dye athigh dyestuff concentration on growth of microorganism or theinhibition of metabolic activity of consortium GG-BL (Junnarkaret al., 2006). Consortium GG-BL was seen to be able to repeatedlydecolorize mono azo dye up to seven cycles. This showed better

Fig. 8. Proposed pathways for the degradation of GYHER by G. geotrichum (a), B. laterosporus (b) and consortium GG-BL (c).

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adaptation of consortium GG-BL to the dye environment. Thisresult showed that the prepared consortium GG-BL was found to bemore effective in decolorization of various textile dyestuffsincluding GYHER due to concerted action of microorganism withinconsortium than individual organism used in alone. Fang et al.(2004); Senan and Abraham (2004); Khehra et al. (2005); Chenet al. (2006); Junnarkar et al. (2006); Chen and Chang (2007);Bafana et al. (2007); Kumar et al. (2007); Jadhav et al. (2008a);Saratale et al. (2009b); Yang et al. (2009); Patil et al. (2010);Phugare et al. (2011) reported concerted action of microorganismfor the biodegradation of textile dye.

Biodegradation activity of consortium GG-BL greatly variedaccording to the type of carbon and nitrogen sources used inBushnell Haas medium. In an attempt to enhance decolorization incontrol medium, the mediumwas supplemented with extra carbonand nitrogen sources and extracts of agricultural residues. The BHMcontaining yeast extract and all agricultural residues except wheatbran showed inhibitory response for the growth of microorganismas well as decolorization of the dye. Consortium GG-BL exhibitedmaximum decolorization of GYHER dye when glucose was sup-plemented in the medium. In absence of co-substrate, the consor-tium GG-BL was unable to decolorize the dye at higher rate, whichindicates that availability of supplementary carbon source seems tobe necessary for growth and decolorization of dyes. Glucose may

Table 4Phytotoxicity of GYHER (1000 ppm) and its metabolite formed after 24 h of degradation

Observation Phaseolus mungo

Distilled water GYHER GYHER met

Germination (%) 100 80 100Shoot length (cm) 10.24 � 0.086 6.40 � 0.115* 12.44 � 0.3Root length (cm) 8.55 � 0.097 2.17 � 0.100* 9.63 � 0.05

Data was analyzed by one-way ANOVA Test and mentioned values are the mean of ten gdifferent from the seeds germinated in plain water at *P < 0.001 and the seeds germinatGYHER at $P < 0.001 when compared by TukeyeKramer multiple comparison test.

act as co-metabolic substrates for increased cell growth as well asreduction of azo dye (Qingxiang et al., 2008).

The oxidoreductive enzymes responsible for dye degradationwere studied. Induction of various enzymes during decolorizationgives additional insight of decolorization mechanism and alsosupports the active role of G. geotrichum and B. laterosporusin consortium GG-BL. Individually, strain G. geotrichum showedpresence of laccase, tyrosinase and DCIP reductase whereasB. laterosporus showed presence of veratryl alcohol oxidase, tyros-inase, DCIP reductase and azo reductase. On the other handconsortium GG-BL bears all of these enzymes. The collective actionof these enzymesmight be the key of potential of consortiumGG-BLfor faster dye degradation. The role of these enzymes like laccase,veratryl alcohol oxidase, DCIP reductase and azo reductase isreported earlier in dye decolorization (Kalme et al., 2007b; Jadhavet al., 2008b, 2009; Gomare et al., 2009; Parshetti et al., 2010;Telke et al., 2010; Tamboli et al., 2010b; Phugare et al., 2011).Communal action ofmixedmicrobial cultures showed good efficacytoward rapid dye decolorization as compared to individual strains(Junnarkar et al., 2006; Chen and Chang, 2007; Phugare et al., 2011).Nondenaturing polyacrylamide electrophoresis showed differentialinduction pattern of oxidoreductive enzyme during GYHER degra-dation. Zymogram obtained after activity staining of nondenaturingPAGE confirms the induction of oxidative enzyme during dye

(1000 ppm) by consortium GG-BL for the Phaseolus mungo and Sorghum vulgare.

Sorghum vulgare

abolite Distilled water GYHER GYHER metabolite

90 40 8004$ 1.89 � 0.037 0.73 � 0.014* 1.76 � 0.201$

1$ 6.64 � 0.034 0.94 � 0.020* 8.24 � 0.005$

erminated seeds of three sets SEM (�). Seeds germinated in GYHER are significantlyed in degradation products are significantly different from the seeds germinated in

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degradation in individual strains as well as in consortium GG-BL.Oxidative enzymes obtained after degradation showed molecularweight between 92 and 104 kDa.

HPLC analysis of metabolites formed after degradation byG. geotrichum, B. laterosporus and consortium GG-BL showedmineralization of parent dye into different metabolites. FTIR spec-trumof GYHERobtained after the degradation by B. laterosporus andconsortium GG-BL showed absence of peak at 1614.4 cm�1 (eN]Ne stretching vibrations) suggested that the breakdown of azobond by azo reductase, while in case of G. geotrichum no azo bondreduction due to the absence of azo reductase enzyme. The GCeMSanalysis showed the probable metabolites produced during theGYHER biotransformation process (Fig. 8). In case of G. geotrichumasymmetric cleavage of GYHER mediated by laccase yields twometabolites, one of them is identified as 4 (5-hydroxy, 4-aminocyclopentane) sulfobenzene (M/Z ¼ 256). In B. laterosporus asym-metric cleavage of GYHER by veratryl alcohol enzyme (first 12 h ofincubation at aerobic condition) yields two intermediates [I] and[II]. Intermediate [II] is identified as 4 (5-hydroxyamino cyclo-pentane) sulfobenzene (M/Z ¼ 244) while the intermediate [I]reduces by reductase (next 12 h of incubation at microaerophiliccondition) into 2-amino benzene sulphonic acid (M/Z ¼ 154) [III].Whereas, in case of consortium GG-BL, significant induction oflaccase (first 12 h of incubation at aerobic condition) and formationof two intermediates, 5-sulfone diazonium (M/Z ¼ 168) [I] and4-methyl-2-m-tolylamino-cyclopentanol (M/Z¼ 256) [II] suggestedthe asymmetric cleavage of GYHER by laccase. Further reduction ofintermediate [I] by reductase enzyme (12 h of incubation atmicroaerophilic condition) give 2-amino benzene sulphonic acid(M/Z ¼ 154) [III], which undergo deamination gives 5-sulfonebenzene (M/Z ¼ 141) [IV]. Induced status of veratryl alcoholoxidase and formation of 2-aminocyclopentanol (M/Z¼ 113) [V] and5-sulfone benzene (M/Z ¼ 141) [VI] suggested asymmetric cleavageof intermediate [II], which undergo deamination gives cyclo-pentanol (M/Z¼ 97) [VII]. Phytotoxicity study revealed the less toxicnature of metabolites formed after degradation as compared to theparent dye (Kalyani et al., 2009; Phugare et al., 2010).

5. Conclusions

In the present study, the decolorization activity of consortiumGG-BL was studied in a batch system under aerobic/micro-aerophilic conditions, in order to determine the optimal conditionsrequired for the highest decolorization activity. Combined activitiesof oxidoreductive enzymes in consortium GG-BL resulted in anincreased decolorization of GYHER as compared to individualstrains. The consortium system was found to be better alternativeas compared to the individual microbial strains. Nondenaturing gelelectrophoresis showed differential induction pattern of oxidor-eductive enzymes obtained after decolorization of GYHER andzymogram obtained after activity staining of nondenaturing PAGEconfirms the induction of oxidase enzyme during GYHER degra-dation. Studies presented here demonstrate the success ofconsortium GG-BL for the decolorization of dye and simultaneousreduction of phytotoxicity.

Acknowledgments

T. R. Waghmode and M. B. Kurade wish to thanks Department ofBiotechnology, New Delhi, India for providing Junior ResearchFellowship. Mr. R. V. Khandare wishes to thank Department ofBiotechnology, Shivaji University, Maharashtra, India for providingfinancial support. Prof. S. P. Govindwar wishes to thank Departmentof Biotechnology, New Delhi for financial support to this project.

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