Azoreductase Activity Research Articles

Structural and functional changes in biofilm during adaptation towards amaranth biodegradation

In the field of the microbial ecology of biofilms and activated sludges, it is widely accepted that the microstructure of the communities depends on the environmental factors. Nevertheless, due to their complexity, the exact mechanisms are still unknown. In this study, we applied a stepwise increase of an azo-dye concentration as a selective factor for adaptation towards biodegradation. The degrading biofilm was developed in a lab-scale sand biofilter. It functioned in a semi-continuous regime for 623 h. The concentration of the azo-dye amaranth was increased from 10 to 55 mg L −1. The effectiveness was 90% and the rate of amaranth elimination was 1.136 mg h −1. The fluorescence in-situ hybridisation (FISH) revealed zones with high activity of Pseudomonas sp. Also increasing importance of the unculturable Pseudomonas sp. and the relationships in the biofilm were found. At the final stage of the experiment, a decrease of the azoreductase activity and an increase of the catechol-1,2-dioxygenase activity were established in the depth of the biofilter. The obtained results were linked with different Pseudomonas microstructures (shown by FISH). The obtained data showed that the changes in the biofilm structure occurred accordingly to the biodegradation of the toxic compound and it included the development of cooperative microbial relationships in the key genus Pseudomonas.

Synergistic effect of biological and advanced oxidation process treatment in the biodegradation of Remazol yellow RR dye

The current study investigated the efficiency of synergistic biological and Advanced Oxidation Process (AOPs) treatment (B-AOPs) using Aeromonas hydrophila SK16 and AOPs-H2O2 in the removal of Remazol Yellow RR dye. Singly, A. hydrophila and AOPs showed 90 and 63.07% decolourization of Remazol Yellow RR dye (100 mg L−1) at pH 6 and ambient temperature within 9 h respectively. However, the synergistic B-AOPs treatments showed maximum decolorization of Remazol Yellow RR dye within 4 h. Furthermore, the synergistic treatment significantly reduced BOD and COD of the textile wastewater by 84.88 and 82.76% respectively. Increased levels in laccase, tyrosinase, veratryl alcohol oxidase, lignin peroxidase and azo reductase activities further affirmed the role played by enzymes during degradation of the dye. UV–Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS) confirmed the biotransformation of dye. A metabolic pathway was proposed based on enzyme activities and metabolites obtained after GC–MS analysis. Therefore, this study affirmed the efficiency of combined biological and AOPs in the treatment of dyes and textile wastewaters in comparison with other methods.

Decolorization, degradation and detoxification of carcinogenic sulfonated azo dye methyl orange by newly developed biofilm consortia.

Metabolites of azo dyes are often carcinogenic, teratogenic, mutagenic and recalcitrant in nature. In this study, four biofilm consortia such as C1 (Vitreoscilla sp. ENSG301, Acinetobacter lwoffii ENSG302, Klebsiella pneumoniae ENSG303 and Pseudomonas fluorescens ENSG304), C2 (Escherichia coli ENSD101, Enterobacter asburiae ENSD102 and E. ludwigii ENSH201), C3 (E. asburiae ENSD102, Vitreoscilla sp. ENSG301 and Bacillus thuringiensis ENSW401), and C4 (E. coli ENSD101, E. ludwigii ENSH201 and B. thuringiensis ENSW401) were applied to degrade and detoxify methyl orange (MO), a carcinogenic, sulfonated mono azo dye, used in textile dyeing industry worldwide. The consortia of C1, C2, C3 and C4 showed 97.30, 98.75, 99.51 and 99.29% decolorization, respectively in yeast extract peptone (YEP) broth containing 200 mg L−1 MO within 60 h of incubation in static condition. The optimum pH and temperature for decolorization was 7.0 and 28 °C, respectively. Some divalent metal ions including Mg2+, Ca2+, Zn2+ and Mn2+ could stimulate MO decolorization. UV–Vis spectral analysis showed that the absorption peak at 465 nm originated from the azo (N Created by potrace 1.16, written by Peter Selinger 2001-2019 ]]> N) bond was completely disappeared within 60 h of incubation. Fourier transform infrared spectroscopy (FTIR) results also revealed that several major peaks including azo bond peak at 1602.6 cm−1 are completely or partly vanished, deformed or shifted. Activities of azoreductase, NADH-DCIP reductase and laccase were significantly increased in the bacterial cells within 60 h of incubation in comparison to that of control (0 h). The chemical oxygen demand was incredibly reduced by 85.37 to 91.44% by these consortia. Accordingly, plant (wheat seed germination) and microbial (growth of the plant probiotic bacteria such as Pseudomonas cedrina ESR12 and Bacillus cereus ESD3 on biodegraded products) toxicity studies showed that biodegraded products of MO are non-toxic. Thus, all these consortia can be utilized in bioremediation of MO from wastewater for safe disposal into environment. To our knowledge, this is the first report on degradation and detoxification of MO from wastewater by bacterial biofilm consortia.

Decolourisation of toxic azo dye Fast Red E by three bacterial strains: process optimisation and toxicity assessment

ABSTRACTRemoval of toxic azo dyes from the industrial effluents has been posing a big challenge since many years. Degradation of azo dyes by bacteria is emerging as an eco-friendly method for the treatment of effluents from dyeing industries. This study presents the decolourisation of azo dye, Fast Red E, by three bacterial strains Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The bacterial consortium of all the three strains decolourised 93% of the 200 mg/L dye within 18 h. The strains decolourised up to 800 mg/L of the dye. The decolourisation process was optimised under different pH (5–11), temperature (25–55°C) and salt (10–30 g/L) levels. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on Fast Red E revealed the azoreductase activity of 4.0430, 3.0086 and 4.1919 µM/min/mg protein, respectively. The breakdown products of azo bond, 5-Amino-6-hydroxynaphthalene-2-sulphonic acid and 4-Aminonaphthalene-1-sulphonic acid were identified by UV-Vis and HPLC analysis. Phytotoxicity assessment indicated a significant fall in the toxicity of the decolourised medium. Hence, it could be concluded that these strains can be used effectively in the removal of azo dyes from the contaminated sites.

High Throughput Sediment DNA Sequencing Reveals Azo Dye Degrading Bacteria Inhabit Nearshore Sediments.

Estuaries and coastal environments are often regarded as a critical resource for the bioremediation of organic pollutants such as azo dyes due to their high abundance and diversity of extremophiles. Bioremediation through the activities of azoreductase, laccase, and other associated enzymes plays a critical role in the removal of azo dyes in built and natural environments. However, little is known about the biodegradation genes and azo dye degradation genes residing in sediments from coastal and estuarine environments. In this study, high-throughput sequencing (16S rRNA) of sediment DNA was used to explore the distribution of azo-dye degrading bacteria and their functional genes in estuaries and coastal environments. Unlike laccase genes, azoreductase (azoR), and naphthalene degrading genes were ubiquitous in the coastal and estuarine environments. The relative abundances of most functional genes were higher in the summer compared to winter at locations proximal to the mouths of the Hanjiang River and its distributaries. These results suggested inland river discharges influenced the occurrence and abundance of azo dye degrading genes in the nearshore environments. Furthermore, the azoR genes had a significant negative relationship with total organic carbon, Hg, and Cr (p < 0.05). This study provides critical insights into the biodegradation potential of indigenous microbial communities in nearshore environments and the influence of environmental factors on microbial structure, composition, and function which is essential for the development of technologies for bioremediation in azo dye contaminated sites.

Dual-Stimuli Responsive 2D Supramolecular Organic Framework for the Detection of Azoreductase Activity.

A 2D supramolecular organic framework (SOF) based on synthetic macrocycles has been constructed in water by a self-assembly strategy. Two new organic monomers of this SOF, possessing viologen and azobenzene functional groups, form a stimuli-responsive host-guest system upon cooperatively binding with cucurbit[8]uril rings. The reversible formation and dissociation of 2D SOF can be realized by the isomerization of azobenzene under ultraviolet and visible light. The light-responsive property of the SOF is highly reversible and stable for up to four cycles. Moreover, azoreductase produced by Escherichia coli can reduce the N=N double bond of azobenzene entities, resulting in fluorescence recovery of the system. As an excellent and effective fluorescent probe, the SOF can detect azoreductase activity for real-time monitoring of the growth process of Escherichia coli. The dual-stimuli responsive 2D SOF is envisioned to drive the development of responsive devices with complex functions.

Azo dye decolorization by a halotolerant consortium under microaerophilic conditions.

As a result of the use of a large amount of salt in dye industries, azo dye decolorization is often needed under hypersaline environments and low dissolved oxygen. Consortium GG-1, which is able to decolorize azo dyes in high salt concentrations and microaerophilic conditions, can be enriched using Metanil Yellow. Consortium GG-1 is mainly composed of Zobellella (62.25%), Rheinheimera (12.4%) and Marinobacterium (9.44%) and is able to decolorize azo dyes under 1%-10% salinity. The activities of azoreductase, laccase and lignin peroxidase were also measured. Together with the detected intermediates and the results obtained from FTIR, the decolorization process of Metanil Yellow was proposed. The influences of pH, initial concentration of azo dyes and concentration of yeast extract on the decolorization rate were also detected. Meanwhile, consortium GG-1 was identified with wide substrate specificity to dyes such as Direct Blue B, Acid Black ATT, and Acid Violet 7. Therefore, consortium GG-1 was identified with potential use in azo dye elimination.

Regeneration of textile wastewater deteriorated microbial diversity of soil microcosm through bioaugmentation

Textile dye contamination is a serious concern that reduces soil productivity by destabilizing microbial community structures. Here, we investigated the influence of bioaugmentation on the degradation of a mixture of dyes (MOD) and textile industry effluent (TIE) in soil microcosms using eight different dye-degrading bacteria. The biodegradation potential improved in bioaugmented microcosms, especially in the initial phase. The bioaugmented MOD and TIE microcosms exhibited 98.33% and 94.19% decolorization, and 96.92% and 95% reduction in chemical oxygen demand, respectively, within 30 days. Activities of azoreductase, veratryl alcohol oxidase, lignin peroxidase, and tyrosinase were induced by >three-fold in bioaugmented microcosms. Changes in alpha diversity indicated significant alterations in microbial dynamics due to MOD and TIE feeding. The Rheinheimera, Kocuria, Ruminococcaceae UCG-010, Ralstonia and Pseudomonas assemblages were predominant after exposure to MOD and TIE, indicating their key role in dye degradation. The bacteria used for augmentation, namely, Staphylococcus, Bacillus, Arthrobacter and Pseudomonas dominantly survived in soil microcosms. Xenobiotic pathways including benzoate, aminobenzoate, chloroalkane and chloroalkene degradation contributed in dye’s detoxification as per illustration of functional annotation of metagenomes. This study indicates a mutualistic-symbiotic relationship between augmented bacteria and soil microflora with enhanced detoxification of xenobiotics leading to a sustainable approach for restoration of contaminated lands.

In-Situ Imaging of Azoreductase Activity in the Acute and Chronic Ulcerative Colitis Mice by a Near-Infrared Fluorescent Probe.

Azoreductase (AzoR) is an essential reductive enzyme which is closely associated with the intestinal disease such as ulcerative colitis (UC). To date, only a few fluorescent probes for detecting AzoR activity in bacteria or cells have been constructed successfully. It is still challenging to design fluorescent probes for in situ monitoring AzoR in vivo. In this paper, a near-infrared (NIR) fluorescent probe (Cy-Azo) based on hemicyanine is designed and synthesized. The emission of the probe is located at 735 nm in the NIR region, which is favorable for its application in vivo. In addition, Cy-Azo shows high sensitivity to AzoR activity with 17-fold fluorescence enhancement and is particularly selective to AzoR over other enzymes, ions, and amino acids. Meanwhile, a possible response mechanism (the azo group in Cy-Azo is reduced by AzoR and cleaved resulting in the production of Cy-NH2) was proposed and verified by HPLC, MS, and theory calculation. In addition, based on low cell cytotoxicity, Cy-Azo is successfully applied in visualizing the activity of AzoR in two cell lines (HCT116 and HepG2 cells) and three types of bacteria (E. coli, S. aureus, and P. aeruginosa). In particular, due to its NIR emission, the probe can monitor AzoR activity in acute and chronic UC mice models. To our knowledge this is the first fluorescent probe for detecting AzoR activity in vivo, which can provide much important information for the diagnosis and treatment of UC.

Comparative static and shaking culture of metabolite derived from methyl red degradation by Lysinibacillus fusiformis strain W1B6.

This paper reports on the comparative characteristics and properties of the metabolites derived from methyl red (MR) decolorization by Lysinibacillus fusiformis strain W1B6 under static and shaking conditions. A batch culture system was used to investigate the effect of aeration on azoreductase activity in the biodegradation process, transformation of colour removal and the metabolite products. Biodegradation analysis was monitored using Fourier transform infrared spectroscopy and high-performance liquid chromatography while metabolites were determined using gas chromatography–mass spectroscopy. Phytotoxicity and anti-microbial tests were also conducted to detect the toxicity of metabolites. The results showed that this strain grew more rapidly under shaking conditions while azoreductase activity increased more rapidly under static conditions. Despite that, no significant difference in the decolorization was observed under both static and shaking conditions with up to 96% and 93.6% decolorization achieved, respectively, within 4 h of incubation. MR was degraded into two fragmented compounds, i.e. 2-aminobenzoic acid and N,N-dimethyl-1.4-benzenediamine. The concentration of 2-amino benzoic acid was higher under static conditions resulting the biotransformation of 2-amino benzoic acid into methyl anthranilate more rapidly under static conditions. Other metabolites were also detected as intermediate biotransformation products and by-products. Less or no toxic effect was found in the metabolite degradation products under both culture conditions.

Performance of a newly enriched bacterial consortium for degrading and detoxifying azo dyes.

To obtain a bacterial consortium that can degrade azo dyes effectively, a bacterial consortium was enriched that can degrade Metanil yellow effectively. After 6 h, 96.25% Metanil yellow was degraded under static conditions by the bacterial consortium, which was mainly composed of Pseudomonas, Lysinibacillus, Lactococcus, and Dysgonomonas. In particular, Pseudomonas played a main role in the decolorization process. Co-substrate increased the decolorization rate, and yeast powder, peptone, and urea demonstrated excellent effects. The optimal pH value and salinity for the decolorization of azo dyes is 4-7 and 1% salinity respectively. The bacterial consortium can directly degrade many azo dyes, such as direct fast black G and acid brilliant scarlet GR. Azo reductase activity, laccase activity, and lignin peroxidase activity were estimated as the key reductase for decolorization, and Metanil yellow can be degraded into less toxic degradation products through synergistic effects. The degradation pathway of Metanil yellow was analyzed by Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry, which demonstrated that Metanil yellow was cleaved at the azo bond, producing p-aminodiphenylamine and diphenylamine. These findings improved our knowledge of azo-dye-decolorizing microbial resources and provided efficient candidates for the treatment of dye-polluted wastewaters.

Application of a downflow microaerophilic fixed film (DFMFF) reactor for the treatment of dye house effluents using a developed bacterial consortium

The present study describes a downflow microaerophilic fixed film (DFMFF) reactor based treatment of dye house effluents. The major characteristics of the two effluents E-2 and E-5 under study were; chemical oxygen demand (COD) 4680-16,266 mg L−1, biological oxygen demand (BOD) 1575–3151 mg L−1 and American Dye Manufacturers' Institute (ADMI) color index of 10,794–21,413. DFMFF reactor was packed with ultra-high molecular weight polyethylene (UHMWPE) shredding. The developed consortium was allowed to grow and form bio-film on packed UHMWPE. Degradation of the effluents at a various hydraulic retention time (HRT) and organic loading rate (OLR) was studied. Batch mode studies in the reactor showed > 80% removal of BOD and COD at OLR of 4.3 kg L−1 day−1 of E-2 and 2.4 kg L−1 day−1 of E-5 at 48 h HRT. Fourier Transform Infrared Spectroscopy (FTIR) and High Performance Liquid Chromatography (HPLC) analysis confirmed biodegradation and obtained results indicated significant induction of oxidoreductive enzyme activities of azoreductase, NADH–DCIP reductase and laccase enzymes. Reduction in phytotoxicity and microbial toxicity was observed after the treatment of both the effluents. The reactor was also operated on continuous mode for treatment of E-2 under sequential anaerobic–aerobic phase and charcoal treatment. Results of sequential effluent treatment resulted in a reduction of > 99% of COD, BOD and ADMI along with > 88% of solids removal from the effluent. The resulting effluent was clear and meets the discharge criteria so as to release into the sewers. .

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:Quinone Reductases.

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.

Remarkable diversification of bacterial azoreductases: primary sequences, structures, substrates, physiological roles, and biotechnological applications.

Azoreductases reductively cleave azo linkages by using NAD(P)H as an electron donor. The enzymes are widely found in bacteria and act on numerous azo dyes, which allow various unique applications. This review describes primary amino acid sequences, structures, substrates, physiological roles, and biotechnological applications of bacterial azoreductases to discuss their remarkable diversification. According to primary sequences, azoreductases were classified phylogenetically into four main clades. Most members of clades I-III are flavoproteins, whereas clade IV members include flavin-free azoreductases. Clades I and II prefer NADPH and NADH, respectively, as electron donors, whereas other members generally use both. Several enzymes formed no clades; moreover, some bacteria produce azoreductases with longer primary structures than those hitherto identified, which implies further diversification of bacterial azoreductases. The crystal structures commonly reveal the Rossmann folds; however, ternary structures are moderately varied with different quaternary conformation. Although physiological roles are obscure, several azoreductases have been shown to act on metabolites such as flavins, quinones, and metal ions more efficiently than on azo dyes. Considering that many homologs exclusively act on these metabolites, it is possible that azoreductases are actually side activities of versatile reductases that act on various substrates with different specificities. In parallel, this idea raises the possibility that homologous enzymes, even if these are already defined as other types of reductases, widely harbor azoreductase activities. Although azoreductases for which their genes have been identified are not abundant, it may be simple to identify azoreductases of biotechnological importance that have novel substrate specificities.

Azoreductase activity of dye-decolorizing bacteria isolated from the human gut microbiota

The gut microbiota enriches the human gene pool and contributes to xenobiotic metabolism. Microbial azoreductases modulate the reduction of azo-bonds, activating produgs and azo polymer-coated dosage forms, or degrading food additives. Here, we aimed to screen the healthy human gut microbiota for food colorant-reducing activity and to characterize factors modulating it. Four representative isolates from screened fecal samples were identified as E. coli (AZO-Ec), E. faecalis (AZO-Ef), E. avium (AZO-Ev) and B. cereus (AZO-Bc). Both AZO-Ef and AZO-Ev decolorized amaranth aerobically and microaerophilically while AZO-Ec and AZO-Bc had higher aerobic reduction rates. The isolates varied in their activities against different dyes, and the azo-reduction activity mostly followed zero-order reaction kinetics, with a few exceptions. Additionally, the isolates had different pH dependence, e.g., AZO-Ec was not affected by pH variation while AZO-Bc exhibited variable degradation kinetics at different pH levels. Cell-free extracts showed NADH-dependent enzymatic activities 14–19 times higher than extracellular fractions. FMN did not affect the reducing activity of AZO-Ef cell-free extract, whereas AZO-Ec, AZO-Ev and AZO-Bc had significantly higher reduction rates in its presence (P values = 0.02, 0.0001 and 0.02, respectively). Using Degenerate primers allowed the amplification of azoreductase genes, whose sequences were 98–99% similar to genes encoding FMN-dependent-NADH azoreductases.

Efficient decolorization and detoxification of sulphonated azo dye Ponceau 4R by using single and mixed bacterial consortia

The decolorization of toxic azo dye Ponceau 4R by three strains of bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1 individually and in consortia was studied. At optimal conditions, up to 95%, 93% and 87% of the dye was decolorized by the strains AK1, AK2 and VKY1, respectively, in 24 h at 200 mg/L of the dye. Decolorization of the dye was optimized for different parameters such as the concentration of dye, pH, temperature and NaCl concentration. These strains were able to decolorize Ponceau 4R up to an initial concentration of 800 mg/L in the pH range of 5–10, temperature 25–55 °C and NaCl concentration up to 30 g/L. The dye decolorization efficiency of these strains was further enhanced by using different consortia of AK1, AK2 and VKY1 in various combinations. The complete decolorization of the dye by a consortium was achieved within 18 h at 200 mg/L. The cell-free extract of these strains grown on this dye exhibited a remarkable activity of azoreductase which is involved in the breakage of the azo bond. The steady-state kinetics of azoreductase, validated the ping pong Bi-Bi mechanism of enzyme action. UV–Vis spectra, HPLC, FTIR and LC-MS analysis of the dye decolorized samples showed the formation of 4-aminonaphthalene-1-sulphonic acid and 5-amino-6-hydroxynaphthalene-2, 4-disulphonic acid as the products of azo bond breakage. The phytotoxicity test of decolorized sample revealed a considerable reduction in the toxicity in comparison with the parent dye.

The Impact of Cholecystectomy on the Gut Microbiota: A Case-Control Study.

Cholecystectomy alters the bile flow into the intestine and the enterohepatic circulation of the bile acids; this may affect the gut microbiota. We assessed the gut microbiota composition of patients who had undergone cholecystectomy and compared with those who had not. From a cohort of 1463 adult participants who underwent comprehensive health screening examinations, 27 subjects who had undergone cholecystectomy (cholecystectomy group) and 81 age- and sex-matched subjects who had not (control group) were selected. Clinical parameters were collected and compared. Microbial composition was determined by 16S rRNA gene sequencing of DNA extracted from fecal samples. We evaluated differences in the overall microbial composition and in the abundance of taxa. The two groups were comparable with respect to clinical characteristics and laboratory results. The actual number of taxa observed in a sample (observed features) was significantly lower in the cholecystectomy group than in the control group (p = 0.042). The beta diversity of Jaccard distance index was significantly different between the two groups (p = 0.027). Blautia obeum and Veillonella parvula were more abundant in the cholecystectomy group. The difference in the diversity of the gut microbiota between the cholecystectomy and control groups was subtle. However, B. obeum and V. parvula, which have azoreductase activity, were more abundant in the cholecystectomy group. The impact of such changes in the gut microbiota on health remains to be determined.

Bioremediation of wastewaters from local textile industries

The present study evaluates the bioremediation potential of indigenous bacterial species isolated from dye-contaminated soil samples from small dyeing outlet located in Ilorin. The water pollution index was estimated based on the physicochemical characteristics and heavy metal concentrations of the raw (Day 0) and treated textile wastewater such as pH, biochemical oxygen demand-5, chemical oxygen demand, total suspended solids and total dissolved solid with mean values of 8.85±0.45 mg/L, 1200±21.3 mg/L, 2440±31.3 mg/L, 1660±17.2 mg/L and 2650±28.1 mg/L respectively, similarly, Lead was the most abundant heavy metal detected in the sample while Cadmium concentration was the lowest with the mean values of 3.52±0.00 mg/L and 2.18±0.00 mg/L respectively. The bacterial strain with highest dye decolorization capacity was screened and identified as Bacillus licheniformis ZUL012.The isolate was consequently used for the bioremediation of the wastewater over a period of 10 days. The results showed an incredible reduction in the physiochemical characteristics and heavy metal concentrations of the textile wastewater in the following ranges (8.85-6.55), (1200-300) mg/L, (2440-518) mg/L, (1660-666) mg/L and (2650-920) mg/L with the highest removal efficiency of 75 %, 78 %, 60%, 65%, recorded for biochemical oxygen demand, chemical oxygen demand, total suspended solid, total dissolved solid, respectively while that of heavy metals such as lead, cadmium, chromium and nickel were 80 %, 60 %, 67 %, 72 % reduction, respectively. Laccase and Azoreductase activities tend to decrease as the pH gradually moved towards acidic condition during the bioremediation process. Toxicity of the treated effluent was assessed using Maize and Bean seed germination test. Conclusively, these research findings can serve as a framework for the outlet design of wastewater treatment plant for local textile outlets.

Isolation, cloning and characterization of an azoreductase and the effect of salinity on its expression in a halophilic bacterium

Understanding the molecular mechanisms of azo dye decolorization is important for the development of effective bioremediation for textile-colored wastewater. A halophilic bacterium Halomonas sp. strain GT was isolated, which could degrade the azo dye Acid Brilliant Scarlet GR at 10% NaCl. The complete genome sequence of this strain was obtained using the PacBio RS II platform. Genome annotation revealed that four proteins are related to decolorization of azo dyes, such as azoreductase, laccases, benzene 1,2-dioxygenase, and catechol 1,2-dioxygenase. The putative azoreductase gene of Halomonas sp. strain GT responsible for the decolorization of azo dye in high salt environment was isolated. Phylogenetic tree analysis showed that the azoG (azoreductase gene of Halomonas sp. strain GT) and its homologs constituted a new branch of the NADH depending azoreductases, with all the homologous sequence of the protein from halophilic bacteria. At high NaCl concentrations, azoreductase gene expression and azoreductase activity were restrained in Halomonas sp. strain GT, which resulted in low a decolorization rate.

Development of an efficient recombinant bacterium and its application in the degradation of environmentally hazardous azo dyes

In the present work, azoreductase gene, azoK, from a bacterium Klebsiella pneumoniae, isolated from the sludge of a local dyeing industry was cloned and expressed in Escherichia coli. The wild-type K. pneumoniae was previously observed to efficiently decolorize mono-, di- and tri-substituted azo dyes. The recombinant E. coli was utilized for the degradation of a model azo dye, methyl orange. azoK was amplified through polymerase chain reaction and subsequently cloned in pGEM-T vector followed by sequencing of a cloned gene to check its integrity. Further, the gene was overexpressed in E. coli BL21 (DE3) using recombinant pET-28b-azoK to produce the protein of interest. A strong band with an approximate molecular mass of 23 kDa was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant azoK gene encoded a protein consisting of 201 amino acids with azoreductase activity. The cell fractionation method was used to determine the location of azoreductase within the periplasmic fraction of the cells and, thus, involved in the extracellular reduction pathway of azo dyes. Some amount of the enzyme was also present in the cytoplasmic fraction. The enzyme from recombinant strain could successfully decolorize the model azo dye. The decolorization efficiency of the enzyme was observed to increase in the presence of a redox mediator. Thus, the viability of the recombinant azoK for the abatement of azo dyes from industrial wastewater was established. It was noted that azoK was the first azoreductase gene to be cloned from K. pneumoniae and expressed in E. coli.