Amar A. Telke

Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. SUK1.

The aim of this work is to evaluate textile dyes degradation by novel bacterial strain isolated from the waste disposal sites of local textile industries. Detailed taxonomic studies identified the organisms as Pseudomonas species and designated as strain Pseudomonas sp. SUK1. The isolate was able to decolorize sulfonated azo dye (Reactive Red 2) in a wide range (up to 5 g l(-1)), at temperature 30 degrees C, and pH range 6.2-7.5 in static condition. This isolate also showed decolorization of the media containing a mixture of dyes. Measurements of COD were done at regular intervals to have an idea of mineralization, showing 52% reduction in the COD within 24h. Induction in the activity of lignin peroxidase and azoreductase was observed during decolorization of Reactive Red 2 in the batch culture, which represented their role in degradation. The biodegradation was monitored by UV-vis, IR spectroscopy, HPLC. The final product, 2-naphthol was characterized by GC-mass spectroscopy. The phytotoxicity study revealed the degradation of Reactive Red 2 into non-toxic product by Pseudomonas sp. SUK1.

Evaluation of the efficacy of a bacterial consortium for the removal of color, reduction of heavy metals, and toxicity from textile dye effluent.

A microbial consortium DAS consisting three bacterial sp. originally obtained from dye contaminated sites of Solapur, India was selected because it was capable of decolorizing textile effluent and dye faster than the individual bacteria under static conditions. Identification of the isolates by 16S rRNA techniques revealed the isolates to be Pseudomonas species. The concerted metabolic activity of these isolates led to complete decolorization of textile effluent as well as Reactive Orange 16 (100 mg l(-1)) within 48-h at pH 7 and 30 degrees C. Studies involving Reactive Orange 16 (RO16) dye were carried with the bacterial consortium DAS to elucidate the mechanism of biodegradation. Induction of the laccase and reductase enzyme during RO16 decolorization indicated their role in biodegradation. The biodegradation of RO16 was monitored by using IR spectroscopy, HPLC and GC-MS analysis. Cytotoxicity, genotoxicity and phytotoxicity studies carried out before and after decolorization of the textile effluent revealed the nontoxic nature of the biotreated sample.

Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, L-DOPA and guaiacol. The optimum pH for oxidation of o-tolidine, 2,6-dimethoxy phenol and guaiacol were 3.0, 4.0 and 5.0, respectively. The purified laccase contained 2.7 mol/mol of copper. The laccase was stable up to 40 °C and within the pH range of 7.0-9.0. Well-known inhibitors of multicopper oxidases such as, sodium azide, L-cysteine and dithiothreitol showed significant inhibition of laccase activity. The purified enzyme decolorized structurally different azo dyes with variable decolorization rates and efficiencies of 68-90%. This study is useful for understanding the precise use of Bacillus sp. ADR in the decolorization of textile dyes containing industrial wastewater.

Influence of organic and inorganic compounds on oxidoreductive decolorization of sulfonated azo dye C.I. Reactive Orange 16.

An isolated bacterial strain is placed in the branch of the Bacillus genus on the basis of 16S rRNA sequence and biochemical characteristics. It decolorized an individual and mixture of dyes, including reactive, disperse and direct. Bacillus sp. ADR showed 88% decolorization of sulfonated azo dye C.I. Reactive Orange 16 (100 mg L(-1)) with 2.62 mg of dye decolorized g(-1) dry cells h(-1) as specific decolorization rate along with 50% reduction in COD under static condition. The optimum pH and temperature for the decolorization was 7-8 and 30-40 degrees C, respectively. It was found to tolerate the sulfonated azo dye concentration up to 1.0 g L(-1). Significant induction in the activity of an extracellular phenol oxidase and NADH-DCIP reductase enzymes during decolorization of C.I. Reactive Orange 16 suggest their involvement in the decolorization. The metal salt (CaCl2), stabilizers (3,4-dimethoxy benzyl alcohol and o-tolidine) and electron donors (sodium acetate, sodium formate, sodium succinate, sodium citrate and sodium pyruvate) enhanced the C.I. Reactive Orange 16 decolorization rate of Bacillus sp. ADR. The 6-nitroso naphthol and dihydroperoxy benzene were final products obtained after decolorization of C.I. Reactive Orange 16 as characterized using FTIR and GC-MS.

Limited dissemination of the wastewater treatment plant core resistome.

Horizontal gene transfer is a major contributor to the evolution of bacterial genomes and can facilitate the dissemination of antibiotic resistance genes between environmental reservoirs and potential pathogens. Wastewater treatment plants (WWTPs) are believed to play a central role in the dissemination of antibiotic resistance genes. However, the contribution of the dominant members of the WWTP resistome to resistance in human pathogens remains poorly understood. Here we use a combination of metagenomic functional selections and comprehensive metagenomic sequencing to uncover the dominant genes of the WWTP resistome. We find that this core resistome is unique to the WWTP environment, with <10% of the resistance genes found outside the WWTP environment. Our data highlight that, despite an abundance of functional resistance genes within WWTPs, only few genes are found in other environments, suggesting that the overall dissemination of the WWTP resistome is comparable to that of the soil resistome.

Decolorization of adsorbed textile dyes by developed consortium of Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 under solid state fermentation.

The objective of this study was to develop consortium using Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 to decolorize adsorbed dyes from textile effluent wastewater under solid state fermentation. Among various agricultural wastes rice bran showed dye adsorption up to 90, 62 and 80% from textile dye reactive navy blue HE2R (RNB HE2R) solution, mixture of textile dyes and textile industry wastewater, respectively. Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 showed 62 and 38% decolorization of RNB HE2R adsorbed on rice bran in 24h under solid state fermentation. However, the consortium of Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 (consortium-PA) showed 80% decolorization in 24h. The consortium-PA showed effective ADMI removal ratio of adsorbed dyes from textile industry wastewater (77%), mixture of textile dyes (82%) and chemical precipitate of textile dye effluent (CPTDE) (86%). Secretion of extracellular enzymes such as laccase, azoreductase, tyrosinase and NADH-DCIP reductase and their significant induction in the presence of adsorbed dye suggests their role in the decolorization of RNB HE2R. GCMS and HPLC analysis of product suggests the different fates of biodegradation of RNB HE2R when used Pseudomonas sp. SUK1, A. ochraceus NCIM-1146 and consortium PA.

Biodegradation of hazardous triphenylmethane dye methyl violet by Rhizobium radiobacter (MTCC 8161)

Rhizobium radiobacter MTCC 8161 completely decolorized methyl violet (10 mg l–1) within 8 h both at static and shaking conditions. The decolorization time increased with increasing dye concentration. The effect of different carbon and nitrogen sources on the decolorization of methyl violet was studied. The maximum decolorization was observed in the presence of sucrose (1%) and urea (1%). UV‐Visible, HPLC and FTIR analysis of extracted products confirmed biodegradation of methyl violet. The significant increase in the activities of lignin peroxidase and aminopyrine N‐demethylase in the cells obtained after decolorization indicated involvement of these enzymes in the decolorization process. In addition to methyl violet, this strain also shows an ability to decolorize various industrial dyes, (red HE7B, yellow 4G, blue 2B, navy blue HE22, red M5B and red HE3B). (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates

The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

Biodegradation and Detoxification of Reactive Textile Dye by Isolated Pseudomonas sp SUK1

An isolated bacterium from a textile disposal site, Pseudomonas sp. SUK1, has the ability to decolorize the reactive textile dyes and methyl orange. This bacterium showed the potential to decolorize the textile dye Reactive Blue 59 at a high concentration (5 g/L(-1)), which is frequently used in the textile industry of Solapur, India. Induction in the activities of lignin peroxidase, azoreductase, and dichlorophenol indophenol reductase was observed during the decolorization of Methyl Orange and Reactive Blue 59. Methyl Orange (as model azo dye) was used to understand the mechanism of biodegradation by Pseudomonas sp. SUK1. The final product was identified as 1,4-benzenediamine, N, N-dimethyl by gas chromatography-mass spectroscopy. Microbial and phytotoxicity studies revealed the nontoxic nature of the products of Reactive Blue 59.