Degradation of reactive red 195 by selected bacteria from textile wastewater

Four selected bacterial strains coded R1, R2, Rc and Rd were successfully isolated from raw textile wastewater. They were screened for their ability to degrade an azo dye of Reactive Red 195 on solid and in liquid dye-containing media. Screening showed that decolourization was best performed under anaerobic condition with the highest colour removal (˜70%) showed by bacterium R2. The partial 16S ribosomal ribonucleic acid (16S rRNA) sequence of bacterium R2 shared 98% sequence similarity to Paenibacillus sp.. Decolourization by this bacterium in a chemically defined medium containing (gL-1) of glucose (1), NH4Cl (0.5), K2HPO4 (7), KH2PO4 (2), MgSO4.7H2O (0.1), CaCl2 (0.02), and Reactive red 195 (0.1), adjusted to pH7 and with (10% v/v) inoculum occurred under partial anaerobic condition at temperature of 37°C. Under optimized condition, bacterium R2 successfully removed more than 95% colour and up to 50% of total organic carbon. No significant change in pH was observed (pH from 7.21 to 7.25) though the anaerobiosis was found to be developed throughout the experiment (redox potential reduced from 2.5 to 0.5 mV). This bacterium produced intracellular (0.033 U/mL) and extracellular (0.026 U/mL) azoreductase enzymes which were found to be stable at pH from 6 to 8 and temperature ranging from 30 ºC to 40ºC. High performance liquid chromatography analysis revealed that biodegradation of Reactive Red 195 under partial anaerobic condition produced at least three types of sulfonated amines which were 4-aminobenzenesulphonic acid (sulphanilic acid), 4-amino-3- hydronapthalenesulphonic acid and 4-amino-5-hydronapthalene-2,7disulphonic acid. The sulphanilic acid can be further degraded to a-ketoglutaric acid, a common Krebs cycle’s intermediate in most aerobic microorganism. Therefore, it can be concluded that the Paenibacillus sp. is of good potential use for the treatment of azo dye-containing wastewater based on its ability to remove colour

Biodegradation and detoxification of textile azo dyes by bacterial consortium under sequential microaerophilic/aerobic processes

Release of textile azo dyes to the environment is an issue of health concern while the use of microorganisms has proved to be the best option for remediation. Thus, in the present study, a bacterial consortium consisting of Providencia rettgeri strain HSL1 and Pseudomonas sp. SUK1 has been investigated for degradation and detoxification of structurally different azo dyes. The consortium showed 98-99 % decolorization of all the selected azo dyes viz. Reac-tive Black 5 (RB 5), Reactive Orange 16 (RO 16), Disperse Red 78 (DR 78) and Direct Red 81 (DR 81) within 12 to 30 h at 100 mg L-1 concentration at 30 ± 0.2 °C under microaero-philic, sequential aerobic/microaerophilic and microaerophilic/aerobic processes. However, decolorization under microaerophilic conditions viz. RB 5 (0.26 mM), RO 16 (0.18 mM), DR 78 (0.20 mM) and DR 81 (0.23 mM) and sequential aerobic/microaerophilic processes viz. RB 5 (0.08 mM), RO 16 (0.06 mM), DR 78 (0.07 mM) and DR 81 (0.09 mM) resulted into the formation of aromatic amines. In distinction, sequential microaerophilic/ aerobic process doesn’t show the formation of amines. Additionally, 62-72 % reduction in total organic carbon content was observed in all the dyes decolorized broths under sequential microaerophilic/aerobic processes suggesting the efficacy of method in mineralization of dyes. Notable induction within the levels of azoreductase and NADH-DCIP reductase (97 and 229 % for RB 5, 55 and 160 % for RO 16, 63 and 196 % for DR 78, 108 and 258 % for DR 81) observed under sequential microaerophilic/aerobic processes suggested their critical involvements in the initial breakdown of azo bonds, whereas, a slight increase in the levels of laccase and veratryl alcohol oxidase confirmed subsequent oxidation of formed amines. Also, the acute toxicity assay with Daphnia magna revealed the nontoxic nature of the dye-degraded metabolites under sequential microaerophilic/aerobic processes. As biodegradation under sequential microaerophilic/aerobic process completely detoxified all the selected textile azo dyes, further efforts should be made to implement such methods for large scale dye wastewater treatment technologies

Azoreductase activity in intact cells of a non-conventional yeast strain

Poster apresentado no Annual Workshop COST 847 Textile Quality and Biotechnology, 2, Villa Olmo, Como, Itália, 10-11 Outubro 2002.Several non-conventional ascomycete yeast strains, isolated from dye-contaminated environments, display azo-reductive capabilities. Those strains can therefore decolorize azo dyes, which are widely used in the textile industries. The azoreductase activity has been described and investigated in a wide variety of anaerobic bacterial species, particularly from the intestinal microflora, and also, although more rarely, in aerobic bacteria. The present work describes some characteristics of the azoreductase present in one of the isolated yeast strains. This enzyme activity seems to be constitutive in the microorganism, as the decolourization process is not affected by pre-adaptation of the cells to the tested dyes. The azo reduction activity, measured at constant OD (optical density), has a maximum at the end of the exponential growth phase. For cells collected in this phase, the decolourization profile for the two dyes derived from N,N-dimethylaniline (I and II) presents a plateau between pH 3 and 5, while the analogue dyes derived from beta-naphtol (III and IV) have a maximum at pH 3. The azoreductase activity in resting cells shows a typical saturation kinetics in relation to the concentration of the dye. For dye II, at pH 4.0, a Km of 0,42 mM and a vmáx of 3,4 mmol*h-1*gDW-1 were obtained. Despite the low specificity of this yeast azoreductase, both the initial decolourization rate and the extent of the process are dependent on the dye structure.BIOEFTEX Project

Responsive Azobenzene-Containing Polymers and Gels

The photoviscosity effect in aqueous solutions of novel poly(4-methacryloyloxyazobenzene-co-N,N-dimethyl acrylamide) (MOAB-DMA) was demonstrated. The observed significant reduction in the zero-shear viscosity upon UV-irradiation of MOAB-DMA aqueous solutions was due to the dissociation of the interchain azobenzene aggregates. Such phenomena can be advantageously used in photoswitchable fluidic devices and in protein separation. Introduction of enzymatically degradable azo cross-links into Pluronic-PAA microgels allowed for control of swelling due to degradation of the cross-links by azoreductases from the rat intestinal cecum. Dynamic changes in the cross-link density of stimuli-responsive microgels enable novel opportunities for the control of gel swelling, of importance for drug delivery and microgel sensoric applications.Singapore-MIT Alliance (SMA

Enzymatic reduction of azo and indigoid compounds

A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40°C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N∼1∼,N∼1∼-dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H2O2) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy

Improving kinetic or thermodynamic stability of an azoreductase by directed evolution

Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (approximate to 10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50 degrees C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20 degrees C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state.European Union [BIORENEW,, FP6-2004-NMP-NI-4/026456]; Fundacao para a Ciencia e Tecnologia, Portugal [PEst-OE/EQB/LA0004/2011, PTDC/QUI-BIQ/119677/2010]; FCT, Portugal [SFRH/BPD/46808/2008

Isolation, Purification and Characterization of Oxygen Insensitive Azoreductase From Pseudomonas Aeruginosa and Biodegradation of Azo Dye - Methyl Red

A Pseudomonas aeruginosa was isolated from water sample from Industrial effluent and was tested for decolorization activity against commercially important dye of Methyl red. Percentage dye degradation by the isolated Pseudomonas aeruginosa was found to be 90%. The enzyme involved in degradation azoreductase was assayed and purified by anion-exchange chromatography. Total activity of the purified enzyme was 22.5U/mg. The enzyme gave a single band in the SDS–PAGE with a molecular weight of 29 kDa (approximately). The maximal azoreductase activity was observed at pH 7.0 and at 37°C. This activity was NADH dependent. Several metal ions inhibited the purified enzyme including Fe2+ and Hg2+

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 activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterised 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 NAD(P)H quinone oxidoreductase activities. In contrast, WrbA is the first enzyme to show NAD(P)H quinone oxidoreductase 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

EXPRESSION, PURIFICATION, AND ENZYMATIC CHARACTERIZATION OF TWO RECOMBINANT AZOREDUCTASES OF BACILLUS SP. B29

Joint Research on Environmental Science and Technology for the Eart