Enhanced biodegradation and detoxification of disperse azo dye ...

102H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107synergistic 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 decolorizationof 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 Pseudomonassp. 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-1146fungi 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 analysisHPTLC 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 Pseudomonassp. SULK1 metabolites lanes indicates its partial degradation(Fig. 3a). Furthermore, the intensity of derivatized bands of individualcultures metabolites was found to be decreased inconsortium-AP metabolites suggesting its further biotransformation.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 degradationpattern 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 4.663 min (Fig. 4b), which were 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), whileFig. 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 Pseudomonassp. SUK1 [d] after 30 h of incubation.Fig. 7. FTIR spectrum of textile effluent [a] and its metabolites obtained after decolorizationby using consortium-AP [b] after 35 h of incubation.

H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107 103Pseudomonas 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 metabolitesby 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 theeffluent 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 retentiontimes 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 severalTable 4GC-mass spectral data of metabolites obtained after degradation of Rubine GFL by A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1.Retention time (min) m/z Mol. weight Name of metabolite Mass spectrumI] A. ochraceus NCIM-114619.356 244 241 1-(2-methyl-4-nitrophenyl)-2-phenyl diazene [I]13.029 166 165 (2-methyl-4-nitrophenyl)diazene [II]II] Pseudomonas sp. SUK115.339 256 257 4-[(2-methyl-4-nitrophenyl)diazenyl] phenol [I]14.132 154 152 2-methyl-4-nitroaniline [II]

104H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107peaks 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 new minor peaks at retention time of 3.223, 3.328, 4.125and 4.644 min (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 forNO 2 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 reductionin IR peaks was observed in the 2845.20 cm 1 to 2322.06 cm 1regions of metabolites suggests absence of charged amines in theproduced metabolites. A significant peak at 1659.73 cm 1 for NH þ 3deformation suggest the possible alkenes conjugation with C]O.Moreover, peaks at 992.89 cm 1 and 1151.77 cm 1 for CeH deformationsuggests cleavage of dye molecule. The absence of peak at1599.75 cm 1 for N]N stretching vibrations indicates the reductivecleavage 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 1for alkanes CH 3 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 1for 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 1represents alkanes CeH stretching (Fig. 6d).Analysis of FTIR results of control textile effluent showedspecific 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 OeNO 2 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 decolorizationof 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 different metabolites.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,Fig. 8. Proposed pathways for the degradation of Rubine GFL by A. ochraceus NCIM-1146 [a] Pseudomonas sp. SUK1 [b] and consortium-AP [c].

Table 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 spectrum18.964 284 284 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]

106H.S. Lade et al. / International Biodeterioration & Biodegradation 72 (2012) 94e107Table 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. mungoGermination(%)Plumule(cm)Radicle(cm)DistilledwaterRubine GFLRubine GFLmetabolitesTextileeffluentEffluentmetabolitesDistilledwaterRubine GFLRubine GFLmetabolitesTextileeffluent100 50 100 40 100 100 60 100 50 100Effluentmetabolites4.99 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 $ 0.422.29 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 $$ 0.06Values are mean of three experiments, SEM (). Seeds germinated in Rubine GFL and textile effluent are significantly different from the seeds germinated in distilled water at*P < 0.05, **P < 0.001 and the seeds germinated in metabolites are significantly different from the seeds germinated in Rubine GFL and textile effluent at $ P < 0.05, $$ P < 0.001by one-way analysis of variance (ANOVA) with TukeyeKramer comparison test.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 asymmetriccleavage 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/z ¼ 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 intermediatesnot detected by GC-MS but rationalized as necessary intermediatesduring the degradation process were labeledalphabetically.3.7. Toxicity studiesThe assessment of toxicity of dyes, effluents and its degradedproducts is often great concern as most of them exert toxic effect onplants and animals when released in stream water. 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 phytotoxicitystudy 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 forS. 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. ConclusionsA new biodegradation approach with fungal-bacterial synergismwas 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 degradationand detoxification of textile effluent containing wide range ofazo dyes.AcknowledgementsThe author Dr. Harshad S. Lade would like to acknowledgeUniversity Grant Commission, New Delhi, India for providing Dr.D.S. Kothari Postdoctoral Fellowship.ReferencesAPHA, 1998. Standard Method for the Examination of Water and Wastewater,twentieth ed. American Public Health Association, Washington, DC, USA. 2120E.Aust, S.D., 1990. Degradation of environmental pollutants by Phanerochaete chrysosporium.Microbial Ecology 20, 197e209.Banat, I., Nigam, P., Singh, D., Marchant, R., 1996. Microbial decolorization of textiledyecontaining effluents: a review. Bioresource Technology 58, 217e227.