Enhanced biodegradation and detoxification of disperse azo dye Rubine GFL and textile industry effluent by defined fungal-bacterial consortium

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International Biodeterioration & Biodegradation 72 (2012) 94e107

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Enhanced biodegradation and detoxification of disperse azo dye Rubine GFLand textile industry effluent by defined fungal-bacterial consortium

Harshad S. Lade a, Tatoba R. Waghmode a, Avinash A. Kadamb, Sanjay P. Govindwar a,*aDepartment of Biochemistry, Shivaji University, Vidyanagar, Kolhapur, Maharashtra 416004, IndiabDepartment of Biotechnology, Shivaji University, Vidyanagar, Kolhapur, Maharashtra 416004, India

a r t i c l e i n f o

Article history:Received 30 April 2012Received in revised form28 May 2012Accepted 1 June 2012Available online

Keywords:Rubine GFLConsortium-APDecolorizationBiodegradationVeratryl alcohol oxidaseDetoxification

* Corresponding author. Tel.: þ91 231 2609152; faxE-mail address: [email protected] (S.P

0964-8305/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.06.001

a b s t r a c t

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 azodye 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 undermicroaerophilic conditions. Individual A. ochraceus NCIM-1146 showed only 46% and 5% decolorization ofthe dye and effluent. However, Pseudomonas sp. SUK1 showed 63% and 44% decolorization of the dye andeffluent respectively with the production of aromatic amines. Induction of laccase, veratryl alcoholoxidase, azo reductase and NADH-DCIP reductase in the consortium-AP suggests synergetic reactions offungal and bacterial cultures for enhanced decolorization process. Differential fate of metabolism ofRubine 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 thisconsortium may effectively be used for complete detoxification of dye and effluent and has potentialenvironmental implication in cleaning up azo dyes containing effluents.

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1. Introduction

Among the many different groups of synthetic dyes, azo (con-taining one or more azo group, R1eN NeR2) dyes are extensivelyused as raw material in textile processing industry. Azo dyes areresistant to degradation and remains persistent for long time due toits fused aromatic structure (Xu et al., 2006). The treatment ofwastewater coming from dying and textile industries becomesmost difficult due to its high chemical oxygen demand (COD) andexcess content of suspended solids such as surfactants, detergentsand dyestuff. This results in severe ecological damages whenreleased into the water resources such as rivers and lakes, whichalters its pH, increases COD and gives intense coloration. It is quiteundesirable to discharge azo dyes wastewater into the environ-ment due to its high toxicity and toxic intermediates produced(Levine, 1991). The toxicity of most of the azo dyes is one of theserious environmental concerns (Dong et al., 2003; Wang et al.,2009) as the effluents coming from dye processing andmanufacturing industries are known to be carcinogenic as well as

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mutagenic to various organisms (Mathur et al., 2005; Chen, 2006;Novotny’ et al., 2006; Mathur and Bhatnagar, 2007). This increasingtoxicity of discharged wastewater affects the human beings ina number of ways making dye contamination both, an environ-mental as well as public health issues.

A number of conventional physico-chemical wastewater treat-ment processes such as electrocoagulation, adsorption on activatedcarbon, ion exchange, flocculation, froth flotation, ozonation,membrane filtration and reverse osmosis have been suggested fordecolorization of textile effluent. However, most of the dyes formtextile effluents escape from such conventional treatmentprocesses and persist in the environment for long time as a result oftheir high stability against light, temperature and oxidizing agents.These conventional physico-chemical processes cannot be usedwidely due to their high cost, secondary pollution generated by theexcessive use of chemicals and inapplicability to a wide variety ofdyes. Compared with physical and chemical processes, bio-friendlyapproaches have been the main focus for remediation of dye-contaminated wastewater since they require lower costs, are eco-friendly and produce fewer toxic metabolites (Kobayashi andRittmann, 1982; Stolz, 2001).

A lot of research on the treatment of textile dyestuff and effluenthas been carried out using individual bacterial and fungal cultures.

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Several microorganisms belonging to different taxonomic groups offungi (Parshetti et al., 2007), bacteria (Kalyani et al., 2008) and yeast(Waghmode et al., 2011a) proved their ability to decolorize dyes bybioadsorption, biotransformation or degradation. Numerousbacterial species have been studied for degradation of azo dyes byvirtue of their rapid growth and faster degradation rates, althoughmany of them produce colorless carcinogenic and mutagenicaromatic amines (Levine, 1991; Joshi et al., 2008). On the otherhand, some bacterial cultures are able to reduce azo compoundsaerobically with the help of oxygen catalyzed azo reductase but alsoproduce aromatic amines (Lin et al., 2010). Besides bacterialcultures, diverse fungal species have been investigated forbiodegradation of textile dyestuff due to their excellence in largebiomass production, hostile growth, spacious hyphal reach andhigh surface to cell ratio. White-rot fungi Pyricularia oryzae isknown to degrade phenolic azo dyes without the formation ofaromatic amines (Chivukula and Renganathan, 1995). However, thetime consuming growth, long hydraulic retention time forcomplete decolorization and low decolorization efficiency limitsthe use of fungi for bioremediation of textile effluent (Banat et al.,1996; Chang et al., 2004).

Despite their great promise, both bacteria and fungi havesuffered certain limitations with respect to their individual abilitiesto completely degrade and detoxify azo dyes. A synergistic action offungal-bacterial consortium leads to the enhanced degradation anddetoxification of azo dyes and, thus provides an alternate way forefficient removal of contaminants (Khelifi et al., 2009; Su et al.,2009; Qu et al., 2010). Moreover the high rates of dye decoloriza-tion by fungal-bacterial synergism suggests an appropriatepowerful tool for the efficient degradation and detoxification of azodyes as well as textile effluent (Khelifi et al., 2009; Su et al., 2009;Qu et al., 2010). Eco-friendly, efficient and short degradation timesare some of the highlights of fungal-bacterial synergism overindividual cultures. Such synergisms are more effective due to theconcertedmetabolic activities, whichmight attack dyemolecules atvarious positions or utilize intermediate degradation metabolitesfor further mineralization into non-toxic form (Keck et al., 2002;Chen and Chang, 2007). It is known that, addition of intermediatemetabolites of dye decolorizing culture into another culture couldenhance the azo dye decolorization rates (Chang et al., 2004).

Microorganisms can decolorize the dyes with different enzymesystems. Fungal enzymes are non-specific towards different struc-tures of dyes and thus oxidize a wide range of them (Aust, 1990).Fungi have been extensively studied to degrade textile dyes due totheir extracellular oxidoreductive, nonspecific and non-stereoselective enzyme system, including lignin peroxidase, lac-case, manganese peroxidase and tyrosinase (Hofrichter, 2002;Kaushika and Malik, 2009). The bacterial biodegradation is asso-ciated with its intracellular and extracellular oxidoreductiveenzyme system such as azo reductase, DCIP-reductase and laccase(Chen et al., 2003; Kalyani et al., 2008; Telke et al., 2009a). Theselected pure cultures of Aspergillus ochraceus NCIM-1146 andPseudomonas sp. SUK1 are well known for the degradation ofdifferent dyes due to induction in the activities of oxidoreductiveenzymes under certain environmental conditions (Parshetti et al.,2007; Kalyani et al., 2009). In recent years, different consortialapproaches have been studied due to their enhancing degradationabilities. Consortial systems can provide advantages over individualcultures as they involve the combined and inductive effects ofvarious enzymes which can work synergistically. Few cases havebeen reported that demonstrated the potential of fungal-bacterialconsortium for enhanced degradation of textile dyestuff (Kadamet al., 2011). There is, however a great need for further researchto set up eco-friendly remediation technologies without theformation of toxicants by virtue of fungal-bacterial synergism.

Though most of the research works on dye decolorization havebeen carried out using individual fungal and bacterial cultures butthe work pertained to fungal-bacterial synergism for biodegrada-tion and detoxification of azo dyes is missing. Keeping this view aswell as to overcome the problems of partial degradation, longreaction time and formation of toxic metabolites, a developedconsortium-AP of fungal-bacterial synergism was investigated forbiodegradation of model azo dye Rubine GFL and textile effluentwithout 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) wereobtained from Hi Media Laboratories Pvt. Ltd., Mumbai, India.Chloranil, Dimethylformamide (DMF) and Aniline-2-sulfonic acidwereprocured formSigmaeAldrich,USA. Remaining chemicalswerepurchased from Sisco Research Laboratories (SRL), India. All chem-icals usedwere 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. Dispersered 78) was generously gifted by Mahesh Textile Processors,Ichalkaranji, India. The highly colored effluent of the same textileprocessing industry utilizing various dyes v.z. azoic, sulphonic,reactive and disperse dyes as rawmaterials was collected in airtightplastic can and tightly stoppered. The collected effluent wastransported to laboratory and filtered through Whatman grade no.1 filter paper to remove large suspended particles. The pH of thefiltered effluent was maintained at 7.0 and stored at 4 � 1 �Ctemperature until processing to prevent contamination by non-indigenous microbes.

2.3. Determination of aromatic amines

Sampleswere taken after decolorization, frozen and freeze-driedin Upright Freeze Dryer Model: FDU5003/8603 (Operon Co. Ltd.,Korea) and the aromatic amines formed were determined spectro-photometrically as per the method of Elisangela et al. (2009). Acalibration curve of aniline-2-sulfonic acid as a model amineproduct of azo dyes reduction was prepared and the concentrationof sample amine was calculated in mM l�1. The pre-grown cultureswithout addition of dye and effluent were used as control.

2.4. Microorganism and culture conditions

A. ochraceus NCIM-1146 culture was obtained from NationalCenter for Industrial Microorganisms, National Chemical Labora-tories (Pune, India). The stock culture was maintained on potatodextrose agar slants at 4 �C. Pseudomonas sp. SUK1 culture previ-ously isolated from textile dye contaminated site was used (Kalyaniet al., 2008). The stock culture was maintained on nutrient mediumagar slants at 4 �C. The composition of PDB used for decolorizationstudies was (g l�1); potatoes infusion 200.0, dextrose 20.0 and yeastextract 5.0. The composition of NM used for decolorization studieswas (g l�1); sodium chloride 5.0, beef extract 1.5, yeast extract 4.0and 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 oldculture were inoculated into 250 ml Erlenmeyer flasks containing

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100 ml of PDB and incubated for 96 h at 30 �C under micro-aerophilic (no agitation) as well as aerobic conditions (shaking at120 rpm). One loopful of 24 h old Pseudomonas sp. SUK1 culturewas inoculated into 100 ml of NM and incubated for 24 h at 30 �Cunder microaerophilic and aerobic conditions. Consortium-AP wasprepared by aseptically transferring the 50 ml of 96 h grown A.ochraceus NCIM-1146 culture into a 250 ml Erlenmeyer flaskscontaining 50 ml of 24 h grown Pseudomonas sp. SUK1 culture. Thepre-grown individual cultures and its developed consortium werethen used as inoculums for further degradation studies.

2.6. Optimization of media composition

Optimization of media composition for enhanced decolorizationof dye and effluent by using A. ochraceusNCIM-1146was carried outin PDB (pH 5.8) supplemented with 2.5, 5.0 and 10.0 g l�l of yeastextract and peptone as additional nitrogen source and 2.5, 5.0 and10.0 g l�l of dextrose and lactose as additional carbon source. ForPseudomonas sp. SUK1, factorial experiments were designed in NM(pH 7.0) supplemented with same concentration of additionalnitrogen and carbon sources.

2.7. Decolorization experiment and physicochemical parameters

Decolorization of Rubine GFL was carried out under micro-aerophilic conditions with 100 ml culture of A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1 pre-grown in PDB and NB sup-plemented with 5.0 and 2.5 g l�l of yeast extract respectively.Consortium-AP was prepared as per the method described inSection 2.5 and used for decolorization of dye. 100 mg l�1 of dyewas added into each 250 ml Erlenmeyer flask containing 100 ml ofindividual pre-grown cultures as well as its developed consortiumand further incubated until decolorization was observed. Aliquotsof the culture supernatant were withdrawn at regular time inter-vals during the process of decolorization. Suspended particles fromthe culture supernatant were removed by adding equal volume ofmethanol followed by centrifugation at 7500 rpm for 15 min. Thedecolorization was monitored by measuring the change in absor-bance maxima of the dye (lmax of Rubine GFL 530 nm) usinga UVevis spectrophotometer (Hitachi U-2800; Japan). All decolor-ization experiments were performed in triplicate and the % decol-orization was calculated as follows:

Decolorizationð%Þ ¼ Initial absorbance� Observed absorbanceInitial absorbance

� 100

The above mentioned protocol was followed while studying thedecolorization of textile azo dye Rubine GFL by using individualcultures as well as its consortium at wide range of pH (3e11),temperature (20, 30, 37, 40 and 50 �C) and increasing dyeconcentrations (50mg l�l to 250mg l�l). The dissolved oxygen (DO)level in the individual and consortium culture was measured withHanna HI 9146 dissolved oxygen meter (Hanna Instruments, USA).Decolorization experiments were carried out in triplicate and theabiotic (without microorganism) controls were always included tomeasure the photodecolorization or abiotic loss of dye.

2.8. Decolorization of textile effluent

Decolorization of real textile effluent was carried out in the250 ml Erlenmeyer flask containing 100 ml of pre-grown individualcultures as well as its developed consortium-AP. 100 ml of sterilizedtextile effluent (121 �C for 20 min) was added into each flask

containing 100 ml of pre-grown individual cultures as well as itsconsortium and further incubated under microaerophilic as well asaerobic conditions. Aliquots of the culture supernatant were with-drawn at regular time intervals; suspended particles were removedby adding equal volume of methanol followed by centrifugation at7500 rpm for 15 min. The obtained clear supernatant was used todetermine the decolorization of effluent. Decolorization was moni-tored using the American Dye Manufacturer’s Institute (ADMI 3WL)tristimulus filter method reported earlier (Chen et al., 2003). Thetransmittance of the sample at three differentwavelengths (590, 540and438nm)were recorded and theADMI valuewas calculatedusingthe ‘AdamseNickerson chromatic value formula’ (APHA, 1998). TheADMI value provides a true measurement of water color, indepen-dent of hue and thus gives deep insight into the more précisingdefinitionof effluent. Decolorizationwas expressed in terms of ADMIremoval ratio and calculated using the following formula:

ADMI removal ratioð%Þ ¼ Initial ADMIð0 hÞ � Observed ADMIðtÞInitial 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 underidentical conditions.

2.9. Characterization of textile effluent

The textile effluent was characterized for reduction in chemicaloxygen demand (COD) and biological oxygen demand (BOD) beforeand after the biodegradation (APHA, 1998). The COD of the textileeffluentwasmeasuredbyusing automatedCODanalyzer (SpectralabCT 15, India). The total organic carbon (TOC) was measured usingHachDR2700 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 ðtÞInitial TOC ð0 hÞ

� 100

Where, TOC(0 h) and TOC(t) are the initial TOC value at (0 h) and theTOC value after particular reaction time (t), respectively.

2.10. Metabolites analysis

After decolorization of Rubine GFL and textile effluent, thefungal mycelium was removed by filtration; bacterial cells wereremoved by centrifugation at 10,000 rpm for 20 min while theconsortium-AP biomass was removed by filtration followed bycentrifugation at 10,000 for 20 min. The supernatant obtained wasused to extract metabolites with an equal volume of ethyl acetate;dried over anhydrous Na2SO4, dissolved in HPLC grade methanoland used for further analytical studies like HPTLC, HPLC, FTIR andGC-MS analysis.

Biodegradation of dye was confirmed by analyzing the obtainedmetabolites with HPTLC system (CAMAG, Switzerland) as reportedearlier (Kurade et al., 2011). 15 ml of control dye and obtainedmetabolites were applied on the pre-coated silica gel plates (HPTLCLichrospher silica gel 60 F254S, Merk, Germany) by micro syringeusing spray gas nitrogen sample applicator (Linomat V, CAMAG,Switzerland). The dosage parameters for plate were set as 6 mmbands, 10 mm apart from Y-axis, 10 mm from the lower edge of theplate, first application position 20 mm from left edge. The

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composition of developing solvent system used as mobile phasewas toluene: ethyl acetate: methanol (7:2:1 v/v). The twin troughchamber was pre-equilibrated with developing solvent for a periodof 20 min prior to plate development. TLC plate was developed byplacing in the trough chamber containing pre-conditioning solventuntil the desired running distance is reached and then oven dried at120 �C for 20 min. After development, densitometric evaluation ofspots was carried out at 254 and 530 nm wavelength usingdeuterium and tungsten lamp respectively with slit dimension of5 � 0.45 mm using CAMAG TLC Scanner-3 (CAMAG, Switzerland).The chromatograms were integrated using HPTLC WinCATS eval-uation 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 methanolwith a flow rate of 0.50 ml min�1 for 10 min and UV detector at280 nm (Kadam et al., 2011). 10 ml of filtered sample was manuallyinjected into the injector port.

FTIR analysis was performed in order to investigate the changesin surface functional groups of the extracted metabolites, beforeand after microbial decolorization. FTIR analysis was done on Shi-madzu 8400S spectrophotometer (Shimadzu Corporation, Japan) inthe mid IR region of 400e4000 cm�1 with 16 scan speed(Waghmode et al., 2011b). The samples were prepared usingspectroscopic pure KBr (5:95), pellets were fixed in the sampleholder and analyzed.

The identification of metabolites formed after decolorizationwas carried using GC-MS QP2010 (Shimadzu Corporation, Japan) bymodifying the procedure reported earlier (Kalyani et al., 2009). Theionization voltage was 70 eV. Gas chromatography was conductedin the temperature programming mode with a Restek column(0.25 mm id, 60 m long, nonpolar; XTI-5). The initial columntemperature was 80 �C for 2 min, then increased linearly at10 �C min�1 to 280 �C, and held for 7 min. The temperature of theinjection port was 280 �C and the GC-MS interface was maintainedat 290 �C. Helium was used as carrier gas with a flow rate of1.0 ml min�1. Degradation products were identified by comparisonof retention time and fragmentation pattern, as well as with massspectra in the NIST spectral library support stored in the GC-MSsolution software (version 1.10 beta, Shimadzu).

2.11. Enzyme extraction

The individual cultures were grown in their respective opti-mized medium while the consortium-AP was prepared as methoddescribed in Section 2.5. A. ochraceus NCIM-1146 fungal myceliumwas collected by filtration, while Pseudomonas sp. SUK1 bacterialcells were collected by centrifugation at 7500 rpm for 15 min andthe resulted fungal culture filtrate and bacterial supernatant wasused as extracellular enzyme source. The collected biomass ofindividual cultures and consortium was separately suspended in50 mM potassium phosphate buffer (pH 7.4), homogenized andsonicated (Sonics-vibracell ultrasonic processor, 7 strokes of 30 seach for 2 min interval based on 50 amplitude output) at 4 �C. Thesonicated cells were centrifuged in cold condition (4 �C, at7500 rpm for 15 min) and resulting supernatant was used as thesource of intracellular enzymes. Similar procedure was carried outto quantify enzyme activities after Rubine GFL decolorization byindividual cultures as well as its consortium.

2.12. Enzyme activities

2.12.1. Oxidative enzymes during decolorizationActivities of oxidative dye degrading enzymes such as laccase,

veratryl alcohol oxidase and tyrosinase were assayed

spectrophotometrically in the cell free extract (intracellular) as wellas culture supernatant (extracellular). Laccase activity was deter-mined in a reaction mixture of 2.0 ml containing 1.7 ml sodiumacetate buffer (20 mM, pH 4.8) and 0.1 ml 50 mM o-tolidine. Thereaction was started by adding 0.2 ml of enzyme solution and anabsorbance increase due to oxidation of o-tolidine was monitoredat 366 nm (Telke et al., 2009a). Veratryl alcohol oxidase activity wasdetermined in a reaction mixture of 2.0 ml containing 4 mMveratryl alcohol as substrate in citrate phosphate buffer (50mM, pH3.0). The reaction was started by adding 0.2 ml of enzyme solutionand an absorbance increase due to the formation of veratraldehydewas monitored at 310 nm (Jadhav et al., 2009). Tyrosinase activitywas determined by modifying the earlier reported method(Kandaswami and Vaidyanathan, 1973). The 3.0 ml reactionmixture contained 0.1 ml 50 mM catechol and 0.1 ml 2.1 mM L-ascorbic acid in potassium phosphate buffer (50 mM, pH 7.4)equilibrated at 25 �C. The DA265 nmwas 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 anddecrease in optical density was measured at 265 nm. One unit oftyrosinase activity was equal to a DA265 nm of 0.001 per min at pH7.4 at 25 �C in a 3.0 ml reaction mixture containing catechol and L-ascorbic acid.

2.12.2. Reductase enzymes during decolorizationActivities of reductive dye degrading enzymes such as azo

reductase and NADH-DCIP reductase were determined spectro-photometrically in cell free extracts using the procedure reportedearlier. Azo reductase assay was performed in a reaction mixture of2.0 ml containing 25 mM of methyl red and 0.2 ml of enzymesolution in potassium phosphate buffer (50 mM, pH 7.4). Thereaction mixture was pre-incubated for 4 min at room temperaturefollowed by the addition of 1000 mM NADH. The decrease in colorabsorbance due to enzymatic cleavage of azo dye methyl red (2-[4-(dimethylamino)phenylazo] benzoic acid) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid was monitored at430 nm. Methyl red reduction was calculated by using its molarextinction coefficient of 0.023 mM�1 cm�1 (Chen et al., 2005).Activity of NADH-DCIP reductase was determined bymodifying theprocedure reported earlier (Salokhe and Govindwar, 1999). Briefly,5.0 ml reaction mixture contained 25 mMDCIP (2,6-dichlorophenolindophenol) and 0.1 ml enzyme solution in potassium phosphatebuffer (50 mM, pH 7.4). From this, 2.0 ml reaction mixture wasassayed at 590 nm by adding 250 mM NADH. The DCIP reductionwas calculated using the extinction coefficient of 0.019 mM�1 cm�1.One unit of reductase enzyme activity was defined as amount ofenzyme required to reduce 1 mM of substrate min�1 mg ofprotein�1.

All enzyme assays were carried at room temperature wherereference blank run along the test. All enzyme assays were run intriplicate, average rates were calculated and one unit of enzymeactivity was defined as a change in absorbance unit min�1 mg ofprotein�1. The protein content was determined by using themethod of Lowry et al. (1951) with bovine serum albumin as thestandard.

2.13. Toxicity studies

It is known that, the colouration of water due to presence oftextile dyes, even in small concentration may have inhibitory effecton the process of photosynthesis and thus affects its growth. Inorder to assess the toxicity of dye Rubine GFL, textile effluent andit’s produced metabolites after decolorization by consortium-AP;phytotoxicity tests were carried out on two kinds of commonIndian agricultural crops: Sorghum vulgare (monocot) and Phaseolus

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mungo (dicot) as described earlier (Telke et al., 2010). The 1000 ppmsolution of dye Rubine GFL and ethyl acetate extracted degradationmetabolites of dye and effluent were prepared in distilled waterand applied for toxicity testing. The filtered real textile effluent wasdirectly used to assess it toxicity. Ten healthy seeds of each cropwere separately sowed into the plastic pot containing 15.0 g ofwashed and oven dried sand. The toxicity study was carried out atroom temperature i.e. 27 � 3 �C by daily watering 5 ml of RubineGFL (1000 ppm), real textile effluent and its degradation metabo-lites (1000 ppm). Control set was carried out at the same time bywatering the seeds with distilled water (daily 5 ml). Germination(%) and the length of plumle (shoot) and radicle (root) was recordedafter 13 days. Germination % was calculated as follows:

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

� 100

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 enhanceddecolorization of dye Rubine GFL and textile effluent by usingA. ochraceus NCIM-1146 and Pseudomonas sp. SUK1; effect ofadditional nitrogen and carbon sources was studied at micro-aerophilic conditions as no considerable decolorization perfor-mance was observed in aerobic conditions. A total of 46%decolorization of Rubine GFL (100 mg l�1) in 30 h and 5% ADMIremoval of textile effluent color in 35 h was observed usingA. ochraceus NCIM-1146 grown in PDB supplemented with5.0 g l�1 yeast extract as additional nitrogen source, while lessdecolorization with other supplements of nitrogen source(peptone) was observed (Data not shown). NM containing 2.5 g l�1

yeast extract was found to be better additional nitrogen source forenhanced decolorization of Rubine GFL (63% in 30 h) and textileeffluent (44% ADMI removal) using Pseudomonas sp. SUK1, whileless decolorization with peptone as nitrogen source was observed(Data not shown). There are some evidences to suggest that theazo dye decolorization by pure as well as mixed culture requiresadditional complex organic sources. For example nitrogen sourcessuch as yeast extract or peptone could enhance the decolorizationefficiency of Aeromonas hydrophila for dye Red RBN (Chen et al.,2003). Moreover, Pseudomonas aeruginosa NBAR 12 was able to

Table 1Determination of aromatic amines produced and % decolorization of Rubine GFL andconsortium-AP under microaerophilic conditions.

Cultures Enrichment conditions Rubine GFL

% Decolorizatio

A. ochraceus NCIM-1146 Microaerophilic 43 � 0.58Aerobic 46 � 1.00

Pseudomonas sp. SUK1 Microaerophilic 63 � 1.53Aerobic 34 � 0.58

Consortium-APA. ochraceus NCIM-1146 Aerobic 95 � 1.00Pseudomonas sp. SUK1 MicroaerophilicA. ochraceus NCIM-1146 Microaerophilic 78 � 1.53Pseudomonas sp. SUK1 Microaerophilic

ND ¼ Not detected. Values are mean of three experiments, � standard deviation (SD).

decolorize diazo dye Reactive blue rapidly when supplied withadditional yeast extract in the medium (Bhatt et al., 2005). Theorganic nitrogen sources can regenerate NADH, which acts as anelectron donor for the reduction of azo dyes which ultimatelyenhances decolorization (Hu, 1994). Furthermore, addition ofdextrose and lactose as additional carbon source decreases thedecolorization rate of dye Rubine GFL as well as textile effluent.The negative effect of carbon sources like glucose on micro-aerophilic decolorization has been ascribed either due to decreasein pH by acid formation or to catabolic repression (Chen et al.,2003). The accelerating effect of consortium-AP of A. ochraceusNCIM-1146 and Pseudomonas sp. SUK1 pre-grown in respectivemedium with additional nitrogen sources showed greatimprovement in the dye Rubine GFL (95% in 30 h) and textileeffluent (98% ADMI removal in 35 h) decolorization efficiency inmicroaerophilic condition (Table 1).

Better growth of A. ochraceus NCIM-1146 (Dry weight 6.41 g l�1

in 96 h at 30 �C) and Pseudomonas sp. SUK1 (Dryweight 1.35 g l�1 in24 h at 30 �C) was observed under aerobic condition whencompared with microaerophilic condition (Dry weight 4.41 g l�1

and 0.55 g l�1 respectively). The aerobic grown A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1 showed 46% and 34% decoloriza-tion of dye Rubine GFL in 30 h and 5% and 35% ADMI removal oftextile effluent within 35 h in microaerophilic conditions respec-tively (Table 1). On the other hand, individual cultures pre-grown atmicroaerophilic conditions showed 43% and 63% decolorization ofdye and 4% and 44% ADMI removal of textile effluent when incu-bated at microaerophilic conditions (Table 1). The DO levels undermicroaerophilic condition were found to be 0.04, 0.02 and0.01 mg l�1 for A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 andconsortium-AP respectively. Decolorization efficiency ofconsortium-AP for dye (95% in 30 h) and effluent (98% ADMIremoval in 35 h) of aerobic grown A. ochraceus NCIM-1146 andmicroaerophilic grown Pseudomonas sp. SUK1 was appreciablyimproved in microaerophilic conditions suggest the involvement ofoxygen sensitive reductases in the process of decolorization(Table 1). This is similar to previous report where nearly zero DOlevel was observed in static decolorization of azo dye with Escher-ichia coli NO3 (Chang and Kuo, 2000). In contrast, only 5% decol-orization of dye and 2% ADMI removal of effluent was observedunder aerobic condition by using the same consortium-AP (data notshown). The presence of oxygen may inhibit the enzymaticreduction of azo bond (eN]Ne), since aerobic condition may ruleover the utilization of NADH, thus preventing the electron transferfrom NADH to azo bonds (Stolz, 2001). The results obtained in thepresent study are in agreement with the reports recorded duringdecolorization of Reactive red 120 and Direct red 81 by A. niger aswell as decolorization of Reactive red 2 by Pseudomonas sp. SUK1,where microaerophilic conditions were used in the best possible

textile effluent by using A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 and its

Effluent

n Amines (mM) % ADMI removal Amines (mM)

ND 4 � 1.00 NDND 5 � 1.53 ND0.14 � 0.01 44 � 1.00 0.18 � 0.010.06 � 0.01 35 � 1.53 0.07 � 0.01

ND 98 � 1.00 ND

ND 82 � 1.53 ND

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107 99

manner than aerobic (Husseiny, 2008; Kalyani et al., 2009). Thus,further decolorization study of dye and effluent was carried out inmicroaerophilic conditions only.

3.2. Decolorization experiment and physicochemical parameters

Microbial decolorization of model azo dye Rubine GFL, which issuspected to be recalcitrant, was investigated at different physi-cochemical conditions by using pure cultures as well as itsconsortium-AP. It is important to study the effect of pH on decol-orization process, as transport of dye molecule into the cell is pHdependent and thought to be rate limiting step for decolorization ofdyes (Lourenco et al., 2000). Both the individual cultures were ableto decolorize the dye at broad range of pH, however optimum pH

0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

(%)

pH

a

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

(%)

Temperature (oC)

b

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

(%)

Dye concentration (mg l-1)

c

Fig. 1. Effect of pH [a], temperature [b] and initial dye concentration [c] on decolor-ization of Rubine GFL by using consortium-AP (-), A. ochraceus NCIM-1146 (:) andPseudomonas sp. SUK1 (�). Data points represents the mean of three independentreplicates, standard error of mean (SEM) is indicated by error bars. Decolorization (%)was measured after 30 h of incubation.

for dye decolorization was found to be 8.5 for consortium-AP and8.0 for Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 (Fig. 1a).Decrease in % decolorization was observed at lower pH (5e7) aswell as higher pH (9e12) for both the cultures. It is thought thatmetabolites formed during the process of decolorization by indi-vidual and consortium cultures may significantly increase the pH ofculture medium towards alkaline. An incubation temperature of37 �C was found to be optimum for enhanced degradation of dyeRubine GFL by using consortium-AP (Fig. 1b). Further increase inthe temperature decreased the extent of degradation for bothconsortium-AP and individual cultures. Decolorization perfor-mance at increasing dye concentrations suggest its potential forcomplete decolorization of 100 mg l�1 of dye Rubine GFL (95% in30 h), whereas individual A. ochraceus NCIM-1146 (46% in 30 h) andPseudomonas sp. SUK1 (63% in 30 h) showed less decolorization forthe same concentration of dye Rubine GFL (Fig. 1c). Waghmodeet al. (2012) reported the enhanced decolorization and degrada-tion of azo dye Rubine GFL (50 mg l�1 within 30 h) using definedconsortium GG-BL of Galactomyces geotrichum MTCC 1360 yeastand Brevibacillus laterosporus MTCC 2298 bacterium, whereasindividual 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 byindividual cultures as well as its consortium-AP. The spectropho-tometric analysis of culture supernatant after decolorization byconsortium-AP showed significant reduction in absorbance thanboth of the individual cultures (Fig. 2). As expected, the rate ofdecolorization of consortium-AP was significantly higher than thatof individual cultures. The increased decolorization rate might bedue to the synergistic enzymes actions of both the organisms in theconsortium. As previously reported, the degradation of interme-diates metabolites by bacteria could decline the fungal inhibitionand thus enhances the decolorization efficiency of consortium (Gouet al., 2009). It is also known that the degradation products of oneculture in the consortiummay 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 werereported by Kadam et al. (2011), who observed higher decoloriza-tion rate of azo dye Navy blue HE2R in solid state fermentation bydeveloped consortium-PA of A. ochraceus NCIM-1146 and Pseudo-monas sp. SUK1. However, such studies are limited up to thedecolorization of water soluble dyes as dye must adsorb on solidsubstrate. Hence, to overcome the problem of adsorption, thesubmerged cultures of same organisms were used to study thedecolorization of disperse azo dye Rubine GFL.

0

0.05

0.1

0.15

0.2

0.25

0.3

400 450 500 550 600 650 700 750 800

Abs

orba

nce

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 Pseudomonassp. SUK1 (�).

Table 2Environmental parameters of untreated and treated textile industry effluent by using consortium-AP, A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1.

Environmental parameters Untreated effluent Treated effluenta

A. ochraceus NCIM-1146 Pseudomonas sp. SUK1 Consortium-AP

BOD (mg l�1) 260 � 4.0 221 � 2.5 63 � 5.0 47 � 3.5COD (mg l�1) 3920 � 20.0 3489 � 19.0 391 � 6.0 157 � 5.0TOC (mg l�1) 4175 � 22.0 3966 � 20.0 2631 � 16.0 2171 � 19.0Color (% ADMI removal) 100 � 0.0 5 � 1.5 44 � 2.0 98 � 1.5

Values are mean of three experiments, � SD.a Treated effluent samples were analyzed after 35 h of incubation.

Table 3Enzyme status during decolorization of Rubine GFL by A. ochraceus NCIM-1146, Pseudomonas sp. SUK1 and consortium-AP.

Enzymes Enzyme activity

A. ochraceus NCIM-1146 Pseudomonas sp. SUK1 Consortium-AP

Control Test Control Test Control Test

Laccasea 2.11 � 0.2 1.74 � 0.3 1.68 � 0.3 1.89 � 0.4** 0.80 � 0.2 0.91 � 0.3**Veratryl alcohol oxidasea ND 0.28 � 0.3*** 1.41 � 0.7 0.36 � 0.3 0.60 � 0.4 0.81 � 0.6***Tyrosinasea Intracellular 566 � 5.0 708*** � 7.0 ND ND 1038 � 8.0 768 � 7.0

Extracellular 692 � 6.0 775 � 7.0*** ND ND 657 � 5.0 543 � 4.0Azoreductaseb ND ND 1.32 � 0.3 1.80 � 0.4** 1.95 � 0.3 3.20 � 0.5***NADH-DCIP reductasec 23 � 2.0 27 � 3.0* 227 � 6.0 147 � 4.0 52 � 2.0 156 � 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 ofvariance (ANOVA) with Tukey Kramer comparison test.

a Enzyme unit’s min�1 mg protein�1.b mM of methyl red reduced min�1 mg protein�1.c mg of DCIP reduced min�1 mg protein�1.

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107100

3.3. Biodegradation of textile effluent

Various azo dyes with fused aromatic structures are commonlyused in the textile processing industry and thus their waste streamhas marked variation in its composition. The physicochemicalstatus of an untreated textile effluent showed considerably highvalues of BOD (260 mg l�1), COD (3920 mg l�1), TOC (4175 mg l�1)and color above the prescribed fresh water limits (Table 2).However, a considerable decline in almost all studied parameterssuch as BOD (82%), COD (96%), TOC (48%) and ADMI color removal(98%) was observed after treatment with consortium-AP undermicroaerophilic conditions within 35 h (Table 2). The higher

Fig. 3. a. HPTLC profile of control dye Rubine GFL [a] and its metabolites obtained afterconsortium-AP [d] after 30 h of incubation. b. HPTLC 3-D chromatogram of control dye Rubin1146 [b], Pseudomonas sp. SUK1 [c] and consortium-AP [d].

decolorization performance of consortium-AP at alkaline pH (8.5)suggests the sign of its suitability for degradation of most of thetextile effluents as it have alkaline pH. In contrast, using individualcultures of A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1a lower reduction in COD (11% and 90%), BOD (15% and 76%), TOC(5% and 37%) and ADMI color removal ratio (5% and 44%) wasachieved within same time (Table 2). Since the fungi and bacteriaalone cannot completely decolorize this textile effluent, it is sus-pected that the fungal-bacterial consortium could cooperativelydecolorize the effluent. This is consistent with the observation thatconsortium-AP of fungal-bacterial synergism used in this studyshowed considerably better decolorization performance than any

decolorization by using A. ochraceus NCIM-1146 [b], Pseudomonas sp. SUK1 [c] ande GFL [a] and its metabolites obtained after decolorization by using A. ochraceus NCIM-

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107 101

individual culture. These results are better than a previous reportwhich showed that individual Pseudomonas sp. SU-EBT decolorized90% effluent within 60 h with 50% and 45% reduction in COD andBOD respectively (Telke et al., 2010). Our results suggest thatfungal-bacterial synergisms could be used as a better alternative forbioremediation of textile effluent than individual cultures.

3.4. Aromatic amine determination

The focus on azo dyes degradation by fungal-bacterial syner-gism in recent years has attributed due to its higher ability tocomplete mineralizes the dye without the formation of toxicaromatic amines. Our studies have demonstrated that partialdecolorization of dye and effluent by individual Pseudomonas sp.SUK1 culture produce aromatic amines, while the samples treatedwith consortium-AP achieved complete removal of amines undermicroaerophilic conditions. The microaerophilic pre-grown Pseu-domonas sp. SUK1 showed amine concentration of 0.14 and

Fig. 4. HPLC chromatogram of control dye Rubine GFL [a] and its metabolites obtainedafter decolorization by using consortium-AP [b], A. ochraceus NCIM-1146 [c] andPseudomonas sp. SUK1 [d] after 30 h of incubation.

0.18 mM for Rubine GFL and textile effluent under microaerophilicdegradation conditions respectively. At the same time aerobic pre-grown Pseudomonas sp. SUK1 culture showed presence of aminesfor both the samples dye (0.06 mM) and effluent (0.07 mM) undermicroaerophilic conditions (Table 1). Bacterial azo reductases areknown to be key enzymes responsible for reductive azo dyesdegradation and are capable of transforming them into aromaticamines. This is consistent with a number of previous reports thatsuggest the reductive cleavage of azo dye by bacterial cultures inmicroaerophilic conditions which leads to the formation ofaromatic amines (Joshi et al., 2008). On the other hand, under sameconditions no presence of amines were detected in the dye andeffluent samples treated with aerobic and microaerophilic pre-grown A. ochraceus NCIM-1146 fungal culture (Table 1). This isprobably due to the absence of reductase enzyme systems such asazo reductase in the A. ochraceusNCIM-1146 fungal culture. Further,the fungal-bacterial consortium used in our study suggestsincreased dye and effluent degradation rates without the formationof toxic aromatic amines.

3.5. Enzyme activities

Several microorganisms including bacteria and fungi have beenreported to decolorize azo dyes with its highly versatile enzymesystems. In the present study, significant induction in the activity ofveratryl alcohol oxidase by 35% and 28% was observed inconsortium-AP and A. ochraceus NCIM-1146 cells respectively afterdecolorization as compared to control (cultures without dye);however therewas no activity in Pseudomonas sp. SUK1 cells for thesame enzyme. In addition to this, laccasewas also induced by 14% inconsortium-AP and 12% in Pseudomonas sp. SUK1 cells (afterdecolorization) as compared to control, but it was reduced inA. ochraceus NCIM-1146 cells. Intracellular and extracellular tyros-inase activity was induced in A. ochraceus NCIM-1146 cells by 25%and 12% respectively after decolorization, but the same activity wasabsent in Pseudomonas sp. SUK1. Reduced tyrosinase activity wasobserved in consortium-AP after decolorization as compared tocontrol (Table 3). The higher induction of oxidoreductive enzymesduring decolorization of dye by consortium-AP might be due to

Fig. 5. HPLC chromatogram of textile effluent [a] and its metabolites obtained afterdecolorization by using consortium-AP [b].

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107102

synergistic effect of both cultures which supports their vigorousrole in the consortium. The role of oxidoreductive enzymes in thedecolorization of azo dye Reactive red 2 have been previouslycharacterized in Pseudomonas sp. SUK1 (Kalyani et al., 2009).

Available literature on degradation of dyes shows that reductivecleavage of azo bond is the initial step in bacterial metabolism ofazo dyes under microaerophilic conditions. In our study, significantinduction of azo reductase (64%) and NADH-DCIP reductase (200%)activities in consortium-AP suggests their involvement in decol-orization of dye molecule. No consequential change was seen inNADH-DCIP reductase activity of A. ochraceus NCIM-1146 culturecells after decolorization, while it was reduced to 65% in Pseudo-monas sp. SUK1 cells. Moreover, induction in azo reductase activityup to 36% was observed in individual Pseudomonas sp. SUK1 cellsafter decolorization, whereas it was absent in A. ochraceus NCIM-1146 cells (Table 3). In the same contest, the inductive pattern ofreductase was reported during the decolorization of azo dye Navyblue HE2R by developed consortium-PA of A. ochraceus NCIM-1146

Fig. 6. FTIR spectrum of control dye Rubine GFL [a] and its metabolites obtained afterdecolorization by using consortium-AP [b], A. ochraceus NCIM-1146 [c] and Pseudo-monas 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 completelydegrade the dye molecule is not clear, but in the consortium it maybe 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 ofdye Rubine GFL was carried out to provide an additional insight tothe biotransformation of dye molecule. The HPTLC chromatogramshowed the absence of control dye band in the consortium-APmetabolites lane, which indicates its complete mineralization,whereas it was present in A. ochraceus NCIM-1146 and Pseudo-monas sp. SULK1 metabolites lanes indicates its partial degradation(Fig. 3a). Furthermore, the intensity of derivatized bands of indi-vidual cultures metabolites was found to be decreased inconsortium-AP metabolites suggesting its further biotransforma-tion. With respect to Rf values, control dye Rubine GFL showed twopeaks (0.84, 0.94), where as individual A. ochraceus NCIM-1146showed six peaks (0.13, 0.16, 0.38, 0.64, 0.84, 0.94), Pseudomonassp. 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 degra-dation pattern of dye by individual cultures and its consortium-AP(Fig. 3b).

HPLC analysis of the control dye Rubine GFL showed single peakat retention time of 2.971 min (Fig. 4a), while formed metaboliteafter decolorization by consortium-AP showed the disappearanceof the major peak as seen in case of control dye Rubine GFL and theformation of two major peaks at retention time of 3.047 and3.317 min and three minor peaks at retention times of, 2.265, 4.123and 4.663min (Fig. 4b), whichwere not seen in the control dye. Theappearance of five new peaks and disappearance of the single peakin the metabolites formed after decolorization by consortium-APsupport the more mineralization of parent dye Rubine GFL intodifferent metabolites. In case of individual cultures, decolorizedproduct of Rubine GFL by A. ochraceus NCIM-1146 showed twomajor 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 decol-orization by using consortium-AP [b] after 35 h of incubation.

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107 103

Pseudomonas sp. SUK1 showed two major and two minor peaks atthe retention time of 2.106, 2.462, 2.858 and 3.001 min (Fig. 4d).This suggested the conversion of parent dye into various metabo-lites by individual cultures.

It is well known that textile industry consume large volume ofwater for various dyeing processes and thus releases large volumesof wastewater with numerous pollutants are discharged. Since the

Table 4GC-mass spectral data of metabolites obtained after degradation of Rubine GFL by A. och

Retention time (min) m/z Mol. weight Name of metabolite

I] A. ochraceus NCIM-1146

19.356 244 241 1-(2-methyl-4-nitroph2-phenyl diazene [I]

13.029 166 165 (2-methyl-4-nitrophendiazene [II]

II] Pseudomonas sp. SUK1

15.339 256 257 4-[(2-methyl-4-nitropdiazenyl] phenol [I]

14.132 154 152 2-methyl-4-nitroanilin

effluent is a complex mixture of dyes, it showed different peakswhen characterized by HPLC. The HPLC chromatogram of the realtextile effluent showed the presence of four major peaks at reten-tion times of 3.199, 3.325, 4.122 and 5.098 min and four minorpeaks at retention times of 3.758, 4.706, 4.516 and 7.895 min(Fig. 5a). The degraded products of textile effluent by consortium-AP after 35 h of incubation showed the disappearance of several

raceus NCIM-1146 and Pseudomonas sp. SUK1.

Mass spectrum

enyl)-

yl)

henyl)

e [II]

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107104

peaks as seen in case of real textile effluent and the formation ofthree major peaks at retention times of 3.461, 3.553 and 3.774 min,while four newminor peaks at retention time of 3.223, 3.328, 4.125and 4.644min (Fig. 5b). The difference in the retention times of realtextile effluent and metabolites formed after degradation byconsortium-AP confirms the biodegradation of effluent intodifferent metabolites.

FTIR spectra obtained from control dye Rubine GFL showedspecific peaks at 779.766e910.90 cm�1 and 1173.17 cm�1 for CeHdeformation, 1202.15 cm�1 for CeN vibrations, 1341.18 cm�1 forNO2 stretching of aromatic nitro compound,1520.82 cm�1 for N]Ostretching of aromatic nitro compound, 1599.45 cm�1 for N]Nstretching in azo group, 2248.70 cm�1 for C^N stretching insaturated nitriles and 2926.58 cm�1 for CeH stretching in alkanes(Fig. 6a). After the consortium decolorization, a significant reduc-tion in IR peaks was observed in the 2845.20 cm�1 to 2322.06 cm�1

regions of metabolites suggests absence of charged amines in theproduced metabolites. A significant peak at 1659.73 cm�1 for NHþ

3deformation suggest the possible alkenes conjugation with C]O.Moreover, peaks at 992.89 cm�1 and 1151.77 cm�1 for CeH defor-mation suggests cleavage of dye molecule. The absence of peak at1599.75 cm�1 for N]N stretching vibrations indicates the reduc-tive cleavage of azo bond (Fig. 6b). Vanishing of major peaks andformation of new peaks in the IR spectrum of consortium-APmetabolites suggests the biotransformation of dye into distinctmetabolites.

Metabolites obtained after partial decolorization of Rubine GFLby A. ochraceus NCIM-1146 showed peaks at 756.51 cm�1 to942.51 cm�1 and 1151.94 cm�1 for CeH deformations,1384.28 cm�1

for alkanes CH3 deformation, 1456.49 cm�1 for alkanes CeHdeformation, 1531.93 cm�1 for N]O stretching and peaks at2872.33, 2926.58 and 2958.63 cm�1 for alkanes CeH stretching(Fig. 6c). Metabolites obtained after the partial decolorization ofRubine GFL by Pseudomonas sp. SUK1 showed peak at 810.76 cm�1

Fig. 8. Proposed pathways for the degradation of Rubine GFL by A. ochrac

for CeH deformation and 1333.90 cm�1 for formation of primaryaromatic amine which has also been additionally confirmed by GC-MS analysis. The peak at 1450.16 cm�1 represents alkanes CeHdeformation while that at 2849.08, 2917.59 and 2961.46 cm�1

represents alkanes CeH stretching (Fig. 6d).Analysis of FTIR results of control textile effluent showed

specific peaks at 2925.65 cm�1 for alkanes CeH stretching,2862.31 cm�1 for alkanes CeH stretching,1637.41 cm�1 for urea C]N stretching, 1458.04 cm�1 for alkanes CeH deformation,1400.72 cm�1 for phenols OeH deformation, 1261.09 cm�1 fornitrates OeNO2 vibration, 1097.15 cm�1 for aliphatic ethersstretching, 805.60 cm�1 for benzene ring containing two adjacent Hatoms eCeH deformation and 601.68 cm�1 for alkynes CeHdeformation (Fig. 7a). Metabolites obtained after complete decol-orization of effluent by consortium-AP showed disappearance ofmajor peaks and formation of new peak at 2922.08 cm�1 foralkanes CeH stretching, 1650.88 cm�1 for acyclic C]N stretching,1461.65 cm�1 for alkanes CeH deformation, 1400.54 cm�1 forketones CeH deformation and 1109.02 cm�1 for secondary alcoholsCeOH stretching (Fig. 7b). Considerable difference between theFTIR spectrum of control textile effluent and the metabolitesobtained after complete decolorization by consortium-APconfirmed the biodegradation of effluent into differentmetabolites.

GC-MS analyses of the metabolites raised from the degradationof dye Rubine GFL by A. ochraceus NCIM-1146 demonstrated theasymmetric cleavage of dye Rubine GFL mediated by veratrylalcohol enzyme to yields two metabolites, one of them is identifiedas 1-(2-methyl-4-nitrophenyl)-2-phenyl diazene (m/z ¼ 244).Further asymmetric cleavage of intermediate metabolite [I] byfungal 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-4-nitrophenyl) diazenyl] phenol (m/z ¼ 256) [I] indicates the initialoxidative cleavage of parent dye Rubine GFL by bacterial laccase,

eus NCIM-1146 [a] Pseudomonas sp. SUK1 [b] and consortium-AP [c].

Table 5GC-MS spectral data of metabolites obtained after degradation of Rubine GFL by consortium-AP.

Retention time (min) m/z Mol. weight Name of metabolite Mass spectrum

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-phenyldiazene [III]

13.030 165 165 (2-methyl-4-nitrophenyl) diazene [IV]

14.137 152 152 2-methyl-4-nitrophenol [V]

Table 6Phytotoxicity of Rubine GFL, textile effluent and its metabolites formed after degradation by consortium-AP for the S. vulgare and P. mungo.

Parameters S. vulgare P. mungo

Distilledwater

Rubine GFL Rubine GFLmetabolites

Textileeffluent

Effluentmetabolites

Distilledwater

Rubine GFL Rubine GFLmetabolites

Textileeffluent

Effluentmetabolites

Germination(%)

100 50 100 40 100 100 60 100 50 100

Plumule(cm)

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

Radicle(cm)

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< 0.001by one-way analysis of variance (ANOVA) with TukeyeKramer comparison test.

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107106

which was further cleaved at azo position by azo reductase to gave2-methyl-4-nitroaniline (m/z ¼ 154) [II] as identified aromaticamine (Table 4; Fig. 8b). This is in agreement with a previous reportwhich supports the involvement of bacterial reductases in thereductive cleavage of azo dyes to yield aromatic amines (Levine,1991). In addition, with the cleavage of azo bonds by bacterial azoreductase, most azo dyes get reduced microaerophilically to thecorresponding amines (Zimmerman et al., 1982). Pseudomonas sp.SUK1 laccase is known for oxidative as well as asymmetric cleavageof dye molecules, where as reductase is known for reductivecleavage of azo dyes (Kalyani et al., 2009; Kadam et al., 2011).

In case of consortium-AP, enzymes from both bacteria and fungifacilitates dye metabolism, as there was significant induction inveratryl alcohol activity which results in asymmetric cleavage ofparent dye molecule to form an intermediate N-ethyl-4-[(2-methyl-4-nitrophenyl) diazenyl] aniline (m/z ¼ 284) [I] (Table 5).It is reported that veratryl alcohol oxidase brings about the asym-metric cleavage of azo dyes (Jadhav et al., 2009). Further oxidativecleavage of intermediate [I] by laccase gives 4-[(2-methyl-4-nitrophenyl) diazenyl] phenol (m/z ¼ 256) [II], which undergoesdehydroxylation to form 1-(2-methyl-4-nitrophenyl)-2-phenyldiazene (m/z ¼ 244) [III]. Furthermore, asymmetric cleavage ofintermediate [III] by veratryl alcohol enzyme leads to the formationof (2-methyl-4-nitrophenyl) diazene (m/z ¼ 165) [IV], whichundergoes azo bond cleavage by azo reductase to form 2-methyl 4-nitroaniline as unidentified aromatic amine. The earlier reportconfirms the role of azo reductase in direct cleaves of azo bond(Chen et al., 2003). This aromatic amine further get deaminated andoxidised by laccase to gave 2-methyl-4-nitrophenol (m/z¼ 152) [V]as final metabolite (Table 5; Fig. 8c). The ability of consortium-AP tocompletely decolorize the dye without forming aromatic aminessuggested its applicability over individual cultures. The interme-diates not detected by GC-MS but rationalized as necessary inter-mediates during the degradation process were labeledalphabetically.

3.7. Toxicity studies

The assessment of toxicity of dyes, effluents and its degradedproducts is often great concern asmost of them exert toxic effect onplants and animals when released in streamwater. Use of bioassayssuch as phytotoxicity for monitoring the toxic effect of dyes as wellas its metabolites on plants was suggested by many researchers(Valerio et al., 2007; Jadhav et al., 2011). Plant bioassays have beenused to establish the toxicity levels of dye, effluent and its degradedproducts on common agricultural crops. In this case, the phyto-toxicity study revealed that there is an inhibition of germination insolutions containing 1000 ppm of the dye Rubine GFL for bothS. vulgare and P. mungo by 50 and 40% respectively (Table 6).Moreover the inhibition of germination in real textile effluent for

S. vulgare and P. mungo was 60 and 50% respectively (Table 6). Onthe other hand, complete germination (100%) as well as significantgrowth in the plumule and radical was observed for both the plantsgrown in consortium-AP metabolites as compared to that of dyeand effluent (Table 6). In addition to this, the length of plumule andradicle was found to be lower in seeds germinated with dye andeffluent samples than those germinated in distilled water as well asdye and effluent metabolites. This study suggest that the dye andeffluent was toxic to these plants, while the metabolites formedafter consortium degradation was less toxic, which signifies thedetoxification of dye and effluent by consortium-AP. These resultsunderline the importance of fungal-bacterium synergism forbioremediation of textile effluent in terms of both decolorizationand detoxification.

4. Conclusions

A new biodegradation approach with fungal-bacterial syner-gism was first applied for degradation of disperse azo dye RubineGFL and textile effluent in submerged conditions. Overall studiesrevealed that the combined metabolic activities of A. ochraceusNCIM-1146 and Pseudomonas sp. SUK1 in the consortium led tocomplete decolorization and detoxification of dye and effluent. Incontrast, individual cultures showed lesser decolorization rate withthe formation of toxicants. The enhanced decolorization efficiencyof consortium-AP could be due to the induced synergetic reactionsof oxidoreductases viz. laccase, veratryl alcohol oxidase, azoreductase and NADH-DCIP reductase. Deep insight into thedifferent aspects presented here strongly supports its applicabilityfor enhanced biodegradation and detoxification of azo dyes whichare recalcitrant to degradation by individual cultures. With a betterunderstanding, this fungal-bacterium synergism would be furtherexploited to develop a continuous treatment process for degrada-tion and detoxification of textile effluent containing wide range ofazo dyes.

Acknowledgements

The author Dr. Harshad S. Lade would like to acknowledgeUniversity Grant Commission, New Delhi, India for providing Dr.D.S. Kothari Postdoctoral Fellowship.

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