Azoreductase Activity Research Articles

Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200 mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35 °C and pH 7.0 within 18 h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55 °C), and NaCl concentration (5–20 g/L). Further, these strains decolorize up to 800 mg/L concentrations of methyl orange within 24 h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12 h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01 µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.

Kinetic Characterization of PA1225 from Pseudomonas aeruginosa PAO1 Reveals a New NADPH:Quinone Reductase.

The pa1225 gene of Pseudomonas aeruginosa strain PAO1 was cloned, and the resulting enzyme (PA1225) was purified and revealed to be an NADPH:quinone reductase. By using kinetics, fluorescence, and mass spectrometric analyses, PA1225 was shown to utilize FAD to transfer a hydride ion from NADPH to quinones. The enzyme could also use NADH, but with an efficiency that was 40-fold lower than that of NADPH as suggested by the kcat/ Km values at pH 6.0. Similar initial rates of reaction were determined with 1,4-benzoquinone and 2,6-dimethoxy-1,4-benzoquinone in the range between 25 and 200 μM, suggesting a low Km value for the quinone-oxidizing substrate. The lack of inhibition by NADP+ versus NADPH at saturating concentrations of 1,4-benzoquinone was consistent with a ping-pong bi-bi mechanism. The reductive half-reaction at pH 6.0 had Kd values of 0.07 mM with NADPH and 1.8 mM with NADH; the kred for flavin reduction was independent of pH with values of ∼10 s-1 with NADPH and ∼5 s-1 with NADH. Thus, the enzyme specificity for the reducing substrate arises primarily from a tighter binding of NADPH than of NADH. At pH 6.0, the kcat value with NADPH and 1,4-benzoquinone was 10.1 s-1, consistent with the hydride transfer from NADPH to FAD being fully rate limiting for the overall turnover of the enzyme. The enzyme showed negligible NADPH oxidase and azoreductase activities. This study enables annotation of the pa1225 gene as NADPH:quinone reductase, elucidates the enzymatic function of PA1225 in P. aeruginosa PAO1, and establishes that PA1225 is not an azoreductase as previously proposed.

Biodegradation and Detoxification Efficiency of Azo-Dye Reactive Orange 16 by Pichia kudriavzevii CR-Y103

In consideration of the hazards associated with the presence of the textile azo-dye and their biotransformation products in the environment, the goal of this work was to study bioremediation process by the yeast strain Pichia kudriavzevii CR-Y103 related to the ability to degrade and detoxify the sulfonated Reactive Orange 16 azo-dye. In experimental conditions, the optimal inoculum/dye concentration ratio required for complete decolorization (100%) of culture medium and biomass within 24 h has been 1 g L−1 yeast cell (dry weight)/50 mg L−1 Reactive Orange 16. In the presence of 400 mg L−1 of Reactive Orange 16 (RO16), 95% of the dye was removed after 72 h of incubation. Also, the yeast strain could decolorize other eight textile dyes (56.48–99.98% decolorization within 24 h). NADH-DCIP reductase and azo reductase activities were significantly increased (ca. 5.4 times and ca. 37 times, respectively) during the decolorization process. UV-VIS spectra, high-performance liquid chromatography (HPLC), and Fourier transform infrared spectroscopy (FTIR) analysis confirmed the presence of new biotransformation products in extracted metabolites, highlighting the partial biodegradation of the dye by the new yeast isolate. The phytotoxicity evaluation strongly supported the decreased toxicity of biodegraded products as minor inhibition on germination (%), root and shoots elongation of T. pratense L. and T. aestivum L. seedlings. Increasing of mitotic index value and decreasing the frequency of chromosomal aberrations in tested plant meristem cells treated with biodegraded products, compared with RO16 treatment (500 ppm), confirmed their slightly toxic nature. A cell viability assay also confirmed the reduced toxicity of biodegraded products on healthy monkey kidney cells (Vero cells).

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase

We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from Enterococcus faecalis, which cleaves the azo bonds (R-N˭N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutational spots in AzoA has not been obtained in terms of substrate binding and azoreductase activity. In this study, we further quantified, visualized, and described the pleiotropic and/or epistatic behavior of six single or double mutations at the positions, Arg-21 and Asn-121, as a further research endeavor for beneficial fine-tuning of AzoA. Based on this network-based mutational analysis, we showed that pleiotropy and epistasis are common, sensitive, and complex mutational behaviors, depending mainly on the structural and functional responsibility and the physicochemical properties of the residue(s) in AzoA.

Azoreductase kinetics and gene expression in the synthetic dyes-degrading Pseudomonas

Microorganisms have an effective capability to degrade synthetic dyestuff enzymatically. In this study, two 16S rRNA identified Pseudomonas aeruginosa bacterial isolates (ASU3 and ASU6) have been screened to enzymatically degrade both Disperse Blue 64 (DB64) and Acid Yellow 17 (AY17) which resembles 2 of the synthetic dyes that causes aquatic environmental pollution. The expression of the azoR1 gene has been detected in the strains, the degradation percentage of DB64 and AY17 by stain ASU6 was found to exceed the ASU3. This returned to the differences in the expression levels of azoR1 between the ASU6 and the ASU3 which was confirmed by RT-PCR. In addition, the specific activity (U/mg protein) of azoreductase in ASU6 was higher than ASU3 when MethylRed (MR), DB64 and AY17 were used as a substrate, separately. The azoreductase kinetics was studied to find the optimum condition for maintaining the stability of the enzyme. The degraded products separated by HPLC/MS were tested for their toxicity on two crop plants and they were nontoxic to the plants.

Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity

BackgroundNitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described.Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary.Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role.ResultsFour putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648.ConclusionsWe here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases.

Mineralization of a sulfonated textile dye Reactive Red 31 from simulated wastewater using pellets of Aspergillus bombycis

BackgroundReactive Red 31, applied extensively in the commercial textile industry, is a hazardous and persistent azo dye compound often present in dye manufacturing and textile industrial effluents. Aspergillus bombycis strain was isolated from dye contaminated zones of Gujarat Industrial Development Corporation, Vatva, Ahmedabad, India. The decolorization potential was monitored by the decrease in maximum absorption of the dye using UV–visible spectroscopy. Optimization of physicochemical conditions was carried out to achieve maximum decolorization of Reactive Red 31 by fungal pellets.ResultsPellets of A. bombycis strain were found to decolorize this dye (20 mg/L) under aerobic conditions within 12 h. The activity of azoreductase, laccase, phenol oxidase and Manganese peroxidase in fungal culture after decolorization was about 8, 7.5, 19 and 23.7 fold more than before decolorization suggesting that these enzymes might be induced by the addition of Reactive Red 31 dye, and thus results in a higher decolorization. The lab-scale reactor was developed and mineralization of Reactive Red 31 dye by fungal pellets was studied at 6, 12 and 24 h of HRT (hydraulic retention time). At 12 h of HRT, decolorization potential, chemical oxygen demand (COD) and total organic carbon reduction (TOC) was 99.02, 94.19, and 83.97%, respectively, for 20 mg/L of dye concentration.ConclusionsDye decolorization potential of A. bombycis culture was influenced by several factors such as initial dye concentration, biomass concentration, pH, temperature, and required aerated conditions. Induction of azoreductase, laccase, phenol oxidase, and Mn-peroxidase enzymes was observed during dye decolorization phase. A. bombycis pellets showed potential in mineralization of dye in the aerobic reactor system. Isolated fungal strain A. bombycis showed better dye decolorization performance in short duration of time (12 h) as compared to other reported fungal cultures.Graphical abstractDegradation of RR31 dye in developed aerobic fungal pelleted reactor.

Exploring the potential of fungal-bacterial consortium for low-cost biodegradation and detoxification of textile effluent

AbstractIn the present study, the enrichment and isolation of textile effluent decolorizing bacteria were carried out in wheat bran (WB) medium. The isolated bacterium Providencia rettgeri strain HSL1 was then tested for decolorization of textile effluent in consortium with a dyestuff degrading fungus Aspergillus ochraceus NCIM 1146. Decolorization study suggests that A. ochraceus NCIM 1146 and P. rettgeri strain HSL1 alone re moves only 6 and 32% of textile effluent American Dye Manufacturing Institute respectively in 30 h at 30 ±0.2°C of microaerophilic incubation, while the fungal-bacterial consortium does 92% ADMI removal within the same time period. The fungal-bacterial consortium exhibited enhanced decolorization rate due to the induction in activities of catalytic enzymes laccase (196%), lignin peroxidase (77%), azoreductase (80%) and NADH-DCIP reductase (84%). The HPLC analysis confirmed the biodegradation of textile effluent into various metabolites. Detoxification studies of textile effluent before and after treatment with fungal-bacterial consortium revealed reduced toxicity of degradation metabolites. The efficient degradation and detoxification by fungal-bacterial consortium pre-grown in agricultural based medium thus suggest a promising approach in designing low-cost treatment technologies for textile effluent.

Functional behavior of bio-electrochemical treatment system with increasing azo dye concentrations: Synergistic interactions of biocatalyst and electrode assembly

Treatment of dye bearing wastewater through biological machinery is particularly challenging due to its recalcitrant and inhibitory nature. In this study, functional behavior and treatment efficiency of bio-electrochemical treatment (BET) system was evaluated with increasing azo dye concentrations (100, 200, 300 and 500mg dye/l). Maximum dye removal was observed at 300mg dye/l (75%) followed by 200mg dye/l (65%), 100mg dye/l (62%) and 500mg dye/l (58%). Concurrent increment in dye load resulted in enhanced azo reductase and dehydrogenase activities respectively (300mg dye/l: 39.6U; 4.96μg/ml). Derivatives of cyclic voltammograms also supported the involvement of various membrane bound redox shuttlers, viz., cytochrome-c, cytochrome-bc1 and flavoproteins during the electron transfer. Bacterial respiration during BET operation utilized various electron acceptors such as electrodes and dye intermediates with simultaneous bioelectricity generation. This study illustrates the synergistic interaction of biocatalyst with electrode assembly for efficient treatment of azo dye wastewater.

Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa.

Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity, these enzymes are also suggested to have NAD(P)H quinone oxidoreductase (NQO) activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play a role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterized recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli. The enzymes include members of the arsenic-resistance protein H (ArsH), tryptophan repressor-binding protein A (WrbA), modulator of drug activity B (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NQO activities. In contrast, WrbA is the first enzyme to show NQO activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa.

In vitro and in vivo evaluation of the prebiotic effect of raw and roasted almonds (Prunus amygdalus).

BACKGROUNDAlmonds contain considerable amounts of potential prebiotic components, and the roasting process may alter these components. The aim of this study was to compare the in vitro fermentation properties and in vivo prebiotic effect of raw and roasted almonds.RESULTS In vitro, predigested raw and roasted almonds promoted the growth of Lactobacillus acidophilus (La‐14) and Bifidobacterium breve (JCM 1192), and no significant differences were found between these two nuts. In a 4‐week animal trial, daily intake of raw or roasted almonds promoted the population of Bifidobacterium spp. and Lactobacillus spp. and inhibited the growth of Enterococcus spp. in faeces and caecal contains of rats. Compared with roasted almonds, raw almonds had a greater bifidobacteria promotion effect. Besides, significantly higher β‐galactosidase activity and lower β‐glucuronidase and azoreductase activities in faeces or caecal contents of rats were observed with raw almonds than with roasted almonds. While, in terms of metabolic effects, the ingestion of roasted almonds resulted in significantly greater intestinal lipase activities.CONCLUSIONBoth raw and roasted almonds exhibit potential prebiotic effects, including regulation of intestinal bacteria and improved metabolic activities. The roasting process may slightly reduce the prebiotic effects of almonds but significantly improve the metabolic effects.© 2016 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Yeast extract promotes decolorization of azo dyes by stimulating azoreductase activity in Shewanella sp. strain IFN4

Biological treatment of azo dyes commonly requires a combined anaerobic–aerobic process in which initial decolorization is achieved by reductive cleavage of azo bonds on the parent molecule. The present study was conducted to examine the relative importance of co-substrates for driving reductive decolorization of azo dyes by Shewanella sp. strain IFN4 using whole cells and enzyme assays. Results showed that the dye decolorization by strain IFN4 was faster in medium containing 1gL−1 yeast extract (YE) as compared to nine other co-substrates. Moreover, only YE stimulated azoreductase activity (increased from 1.32 to 4.19U/mg protein). Increasing the level of YE up to 8gL−1 resulted into 81% decolorization of the dye in 1h along with an increase in azoreductase activity up to 6.16U/mg protein. Among the components of YE, only riboflavin stimulated the decolorization process as well as enzyme activity. Moreover, strain IFN4 demonstrated flavin reductase activity, and a significant correlation (r2=0.98) between flavin reduction and dye reduction by this strain emphasized the involvement of flavin compounds in the decolorization process. The results of this study show that YE serves both as a source of reducing equivalents and an electron shuttle for catalyzing dye reduction.

Removal of Remazol Blue by azoreductase from newly isolated bacteria

The removal of an azo dye, namely Remazol Blue, by wastewater-isolated bacteria (Bacillus megaterium, Micrococcus luteus and Bacillus pumilus) has been investigated in media containing molasses as a carbon and energy source. The dye removal properties of bacteria were studied in a batch system where the initial pH values (6, 7, 8, 9) and dye concentrations are the variables (28.7, 57.9, 78.3, 97.9mg/L) throughout three days of incubation time. Optimal pH value was 7 for the tested bacteria in media containing 25mg/L dye. All of the isolated bacteria had capacity for removing Remazol Blue in the range from 23.7% to 69.9%. Moreover, experiments reveal notable results even 97.9mg/L dye concentrations in a short incubation time such as 3 days in the liquid media that contains only molasses as a carbon and energy source. The highest azoreductase activity was obtained as 39.9U/mL for B. pumilus at 2nd day while M. luteus did not show any azoreductase activity.

English

The initial critical step of reduction of azo bond during the metabolism of azo dyes is catalysed by a group of NADH and FAD dependant enzyme called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis of the substrate specificity and the nature of catalysis. Azoreductase enzyme of Pseudomonas putida has a wider broad spectrum of substrate specificity and capable of degrading a wide variety of azo dyes. In the present study, the crystal structure of the enzyme from PDB and 10 azo dyes from NCBI PubChem compound were retrieved and their interactions were studied. These azo dyes were then docked with the FMN-dependent NADH-azoreductase enzyme to analyze the binding affinity of the azo dyes with the enzyme and predict the catalytic sites. Consequently, the catalytic residues of FMN-dependent and NADH dependent enzyme were then analysed in terms of properties including function, hydrogen bonding and flexibility. The results suggest that Ala-114, Phe-172 and Glu-174 play a predominant role as catalytic site residues in the enzyme. Furthermore, the approach emphasis on  predicting the active sites of this enzyme where substrates can bind in order to give a better understanding of the biodegradation of some of the commercially important azodyes mediated by azoreductase. These results will pave way for further increase in azoreductase activity and for better understanding of the dye degradation pathway.   Key words: Azoreductase, NADH, FMN, chemical properties, docking, active sites.  &nbsp

Phytoremediation of sulfonated Remazol Red dye and textile effluents by Alternanthera philoxeroides: An anatomical, enzymatic and pilot scale study

Alternanthera philoxeroides Griseb. a macrophyte was found to degrade a highly sulfonated textile dye Remazol Red (RR) completely within 72 h at a concentration of 70 mg L−1. An induction in the activities of azoreductase and riboflavin reductase was observed in root and stem tissues; while the activities of lignin peroxidase, laccase and DCIP reductase were induced in leaf tissues. Some enzymes namely tyrosinase, veratryl alcohol oxidase, catalase and superoxide dismutase displayed an increase in their activity in all the tissues in response of 72 h exposure to Remazol Red. There was a marginal reduction in contents of chlorophyll a (20%), chlorophyll b (5%) and carotenoids (16%) in the leaves when compared to control plants. A detailed anatomical study of the stem during uptake and treatment revealed a stepwise mechanism of dye degradation. UV–vis spectrophotometric and high performance thin layer chromatographic analyses confirmed the removal of parent dye from solution. Based on the enzymes activities and gas chromatography-mass spectroscopic analysis of degradation products, a possible pathway of phytotransformation of RR was proposed which revealed the formation of 4-(phenylamino)-1,3,5-triazin-2-ol, naphthalene-1-ol and 3-(ethylsulfonyl)phenol. Toxicity study on Devario aequipinnatus fishes showed that the anatomy of gills of fishes exposed to A. philoxeroides treated RR was largely protected. The plants were further explored for rhizofiltration experiments in a pilot scale reactor. A. philoxeroides could decolorize textile industry effluent of varying pH within 96 h of treatment which was evident from the significant reductions in the values of American dye manufacturers' institute color, chemical oxygen demand, biological oxygen demand, total dissolved and total suspended solids.

Decolorization of Reactive Black-5 by Shewanella sp. in the Presence of Metal Ions and Salts.

In this study, effect of various metal ions and salts on biodecolorization of Reactive black-5, azoreductase activity, and growth of Shewanella sp. strain IFN4 was evaluated. Among the tested metals, Cr²⁺, Pb(²⁺, Ni²⁺, Fe²⁺, and Mn²⁺ did not inhibit the biodecolorization of reactive black-5, azoreductase activity and bacterial growth. Three metals (Cu²⁺, Zn²⁺, and Co²⁺) delayed the decolorization process without completely inhibiting the reaction and also suppressed the bacterial growth. However, no dye decolorization was observed in the presence of Cd²⁺ (10 mg L⁻¹). Furthermore, bacterium decolorized the dye at high concentration (15 mg L⁻¹) of mixed metal ions. Strain IFN4 was also able to decolorize the dye at 50 g NaCl L⁻¹ and 60 g Na₂SO₄ L⁻¹. NaCl was found to be more inhibitory to bacterial growth than Na₂SO₄and the reverse was observed for azoreductase activity. These findings suggest that strain IFN4 could be used in designing a bioreactor for the treatment of textile effluent.

English

Biodegradation of acid blue 113 has been studied using two strains-Staphylococcus aureus and Escherichia coli. The effect of pH, time, initial dye concentration, the inoculum size, the co-substrate, static and shaking on decolourisation was studied. The optimum condition for decolourisation was found to be at pH 7 for an initial dye concentration of 200 mg/L at 37 C under static condition. The removal of dye by S.aureus and E.coli at optimum condition was 90% and 88% within 30 h and 42 h respectively. Addition of fructose and starch as carbon source increased the removal of colour by S.aureus and E.coli respectively. Similarly addition of ammonium formate and glycine as nitrogen source under static condition increased the colour removal by S.aureus and E. coli. The enzyme activity of azoreductase for S. aureus and E. coli was found to be 7.33 U/ml and 7.24 U/ml respectively. The COD reduction, FTIR and TLC analysis of the sample before and after decolourisation confirmed the biodegradation of dye and the formation of new metabolite. Phytotoxicity studies indicated that the microbial treated effluent is safe for disposal into the environment.

Experimental Exploration on Degradation of Orange G 16 an Azo Dye by Novel Pseudoalteromonas sp. and Its Enzyme Activity

Increasing textile industries in the last few decade has resulted in the discharge of very large quantities of complex chemical dye to the water system. This has affected the ecological and biological balance causing immense destruction to the nature. In this work, azo dye (Orange G 16) degradation studies by Pseudoalteramonas sp. were investigated. From the experiments, Pseudoalteromonas species degraded the Orange G 16 azo dye at a short time span within 3 h. The assay time for azoreductase activity was standardized, and maximum activity was observed at 24th. The protein concentration was estimated at three different stages of dye degradation. First stage is before addition of dye, second stage is 3 h after addition of the dye during which the dye had completely decolourized, and the third stage is 18 h after the addition of dye. From the study, it was observed that protein concentration was higher in the 24-h sample which has been induced with Orange G 16 azo dye having a concentration of 0.92 mg/ml. While comparing the total activity and specific activity of the enzyme for three samples, it was observed that the activity was higher at the 24th s and then subsided gradually. Thus, Pseudoalteromonas sp. is found to be efficient in degrading azo dyes because of its potential to degrade the dyes rapidly which also has a higher azoreductase activity.

Biomineralization of azo dye bearing wastewater in periodic discontinuous batch reactor: Effect of microaerophilic conditions on treatment efficiency

The present study illustrates the influence of microaerophilic condition on periodic discontinuous batch reactor (PDBR) operation in treating azo dye containing wastewater. The process performance was evaluated with the function of various dye load operations (50–750mg/l) by keeping the organic load (1.6kg COD/m3-day) constant. Initially, lower dye operation (50mg dye/l) resulted in higher dye [45mg dye/l (90%)] and COD [SDR: 1.29kg COD/m3-day (92%)] removal efficiencies. Higher dye load operation (750mg dye/l) also showed non-inhibitory performance with respect to dye [600mg dye/l (80%)] and COD [1.25kg COD/m3-day (80%)] removal efficiencies. Increment in dye load showed increment in azo reductase and dehydrogenase activities (39.6U; 4.96μg/ml; 750mg/l). UV–Vis spectroscopy (200–800nm), FTIR and 1H NMR studies revealed the disappearance of azo bond (–NN–). First derivative cyclic voltammogram supported the involvement of various membrane bound redox shuttlers, viz., cytochrome-C, cytochrome-bc1 and flavoproteins (FAD (H)).

Improved biodegradation of textile dye effluent by coculture

The present study demonstrates the de-colorization and degradation of textile effluent by coculture consisting of three bacterial species isolated from textile effluent contaminated environment with an aim to reduce the treatment time. The isolates were identified as Ochrobactrum sp., Pseudomonas aeruginosa and Providencia vermicola by 16S rRNA analysis. Their secondary structure was predicted and GC content of the sequence was found to be 54.39, 52.10, and 52.53%. The co-culture showed a prominent increase in the degradation activity due to the action of oxidoreductase enzymatic mechanism of laccase, NADH–DCIP reductase and azoreductase activity. The biodegradability index of 0.75 was achieved with 95% chemical oxygen demand (COD) reduction in 16h and 78 and 85% reduction in total organic carbon (TOC) and total solids was observed. Bioaccumulation of metals was identified by X-ray diffraction (XRD) analysis. The effective decolorization was confirmed from the results of UV–vis spectroscopy, high performance liquid chromatography and Fourier transformed infrared spectrometer analyzes. The possible degradation pathway was obtained from the analysis of liquid chromatography–mass spectroscopy analysis and the metabolites such as 2-amino naphthalene and N-phenyl-1.3,5 triazine were observed. The toxic nature of the effluent was analyzed using phyto-toxicity, cell-death assay and geno-toxicity tests.