Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

Modification of azo dyes by lactic acid bacteria

Aim:  The ability of Lactobacillus casei and Lactobacillus paracasei to modify the azo dye, tartrazine, was recently documented as the result of the investigation on red coloured spoilage in acidified cucumbers. Fourteen other lactic acid bacteria (LAB) were screened for their capability to modify the food colouring tartrazine and other azo dyes of relevance for the textile industry.
Methods and Results:  Most LAB modified tartrazine under anaerobic conditions, but not under aerobic conditions in modified chemically defined media. Microbial growth was not affected by the presence of the azo dyes in the culture medium. The product of the tartrazine modification by LAB was identified as a molecule 111 daltons larger than its precursor by liquid chromatography-mass spectrometry. This product had a purple colour under aerobic conditions and was colourless under anaerobic conditions. It absorbed light at 361 and 553 nm.
Conclusion:  LAB are capable of anabolizing azo dyes only under anaerobic conditions.
Impact and Significance of the Study:  Although micro-organisms capable of reducing the azo bond on multiple dyes have been known for decades, this is the first report of anabolism of azo dyes by food related micro-organisms, such as LAB.     

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris

Aims:  To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes.
Methods and Results:  A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62·8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus . Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators.
Conclusions:  Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators.
Significance and Impact of the Study:  This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.     

Degradation of sulfonated azo dyes by the purified lignin peroxidase from <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> MTCC 2298

Lignin peroxidase (EC 1.11.1.14) was purified from the Brevibacillus laterosporus MTCC 2298 by ion exchange chromatography. The K_m value of the purified lignin peroxidase (using n-propanol as substrate) was 1.6 mM. The MW of purified enzyme determined with the help of MW-standard markers was approximately 205 kDa. Purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and the activity staining using a substrate L-DOPA. Sulfonated azo dyes such as Methyl orange and Blue-2B were degraded by the purified lignin peroxidase. Degradation of the dyes was confirmed by HPLC, GC-MS, and FTIR spectroscopy. The mainly elected products of Methyl orange were 4-substituted hexanoic acid (m/z = 207), 4-cyclohexenone lactone cation (m/z = 191), and 4-isopropanal-2, 5-cyclohexa-dienone (m/z = 149) and for Blue-2B were 4-(2-hexenoic acid)-2, 5-cyclohexa-diene-one (m/z = 207; M − 1 = 206) and dehydro-acetic acid derivative (m/z = 223).     

Microaerophilic–aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31

Four different azo dyes were decolourized and biodegraded in a sequential microaerophilic–aerobic treatment by a facultative Klebsiella sp. strain VN-31, a bacterium isolated from activated sludge process of the textile industry. Dye decolourization was performed under microaerophilic conditions until no colour was observed (decolourization percentage >94%). The medium was then aerated to promote the biodegradation of the amines produced. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage demonstrate azo bond reduction and an oxidative biodegradation process, respectively. Total Organic Carbon (TOC) reduction for the growth medium plus dyes was ～50% in the microaerophilic stage and ～80% in the aerobic stage. The degradation products were also characterized by FT-IR and UV–vis techniques and their toxicity measured using Daphnia magna. The results provide evidence that the successive microaerophilic/aerobic stages, using a single Klebsiella sp. strain VN-31 in the same bioreactor, were able to form aromatic amines by the reductive break down of the azo bond and to oxidize them into non-toxic metabolites.     

Rapid decolourization of azo dyes by a new isolated higher manganese peroxidase producer: Phanerochaete sp. HSD

In the present paper, a strain of higher MnP producer, Phanerochaete sp. HSD, was screened and the important medium components influencing MnP production were optimized using fractional factorial design and central composite experimental design; statistical analysis suggested diammonium tartrate and Mn²⁺ were the important factors and under the optimum conditions, MnP activity reached 2613 ± 22 U/l, accorded with the predicted value from response surface analysis. The feasibility of using this fungus to decolourize azo dyes was examined too. Results indicated that crude enzyme solution of it could decolourize three azo dyes efficiently and speedily: for 120 and 350 mg/l of Congo red, 95% decolourization rate was observed at the 5th and 8th hour; for 200, 350 and 600 mg/l methyl orange, 95% decolourization rate was obtained at the 5th, 6th and 9th hour; furthermore, the decolourization rates of 150 and 300 mg/l of Eriochrome black T were up to 97.1% and 91.4% at the 7th and 13th hour, respectively. In addition, MnP played a crucial role in the decolourization process.     

Degradation of Remazol Red dye by <Emphasis Type="Italic">Galactomyces geotrichum</Emphasis> MTCC 1360 leading to increased iron uptake in <Emphasis Type="Italic">Sorghum vulgare</Emphasis> and <Emphasis Type="Italic">Phaseolus mungo</Emphasis> from soil

Removal of azo dyes from the effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are both mutagenic and carcinogenic. Galactomyces geotrichum MTCC 1360, a yeast species, showed more than 96% decolorization of the azo dye Remazol Red (50 mg/L) within 36 h at 30°C and pH 11.0 under static condition with a significant reduction in the chemical oxygen demand (62%) and total organic carbon (41%). Peptone (5.0 g/L), rice husk (10 g/L extract), and ammonium chloride (5.0 g/L) were found to be more significant among the carbon and nitrogen sources used. The presence of tyrosinase, NADH-DCIP reductase, riboflavin reductase and induction in azo reductase and laccase activity during decolorization indicated their role in degradation. High performance thin layer chromatography analysis revealed the degradation of Remazol Red into different metabolites. Fourier transform infrared spectroscopy and high performance liquid chromatography analysis of samples before and after decolorization confirmed the biotransformation of dye. Atomic absorption spectroscopy analysis revealed a less toxic effect of the metabolites on iron uptake by Sorghum vulgare and Phaseolus mungo than Remazol Red dye. Remazol Red showed an inhibitory effect on iron uptake by chelation and an immobilization of iron, whereas its metabolites showed no chelation as well as immobilization of iron. Phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products which had a less toxic nature.     

Biodegradation of C.I. Reactive Red 195 by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> strain YZ66

Synthetic dyes are extensively used in textile dyeing, paper, printing, colour photography, pharmaceutics, cosmetics and other industries. Among these, azodyes represents the largest and most versatile class of synthetic dyes. As high as 50% of the dyes are released into the environment during manufacture and usage. Traditional methods of treatment are found to be expensive and have operational problems. Biological decolourization has been investigated as a method to transform, degrade or mineralize azo dyes. In the present studies bacteria from soil from dye waste area, dye waste, sewage and dung were subjected to acclimatization with C.I. Reactive Red 195 an azo dye, in the basal nutrient media. The most promising bacterial isolate was used for further dye degradation studies. The 16s rRNA gene sequencing and biochemical characteristics revealed the isolated organism as Enterococcus faecalis strain YZ66. The strain showed 99.5% decolourization of the selected dye (Reactive Red 195–50 mg/l) within one and half hour in static anoxic condition. The optimum pH and temperature for the decolourization was 5.0 and 40°C respectively. The biodegradation was monitored by UV–Vis, FTIR, TLC and HPLC. The final products were characterized by Gas chromatography and Mass Spectrophotometry. Toxicity study demonstrated no toxicity of the biodegradation product. The results suggest that the isolated organism E. faecalis strain YZ 66 can be used as a useful tool to treat waste water containing reactive dyes.     

Decolourization and Detoxification of Methyl Red by Aerobic Bacteria from A Wastewater Treatment Plant

Bacterial cultures from a wastewater treatment plant degraded a toxic azo dye (methyl red) by decolourization. Complete decolourization using a mixed-culture was achieved at pH 6, 30 °C within 6 h at 5 mg/l methyl red concentration, and 16 h at 20—30 mg/l. Four bacterial species were isolated that were capable of growth on methyl red as the sole carbon source, and two were identified, namely Vibrio logei and Pseudomonas nitroreducens. The Vibrio species showed the highest methyl red degradation activity at the optimum conditions of pH 6--7, and 30—35 °C. Analysis by NMR showed that previously reported degradation products 2-aminobenzoic acid and N,N-dimethyl-1,4-phenylenediamine were not observed. The decolourized dye was not toxic to a monkey kidney cell line (COS-7) at a concentration of 250 μM. This revised version was published online in July 2006 with corrections to the Cover Date.     

Decolorization and degradation of Disperse Blue 79 and Acid Orange 10, by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil

The release of azo dyes into the environment is a concern due to coloration of natural waters and due to the toxicity, mutagenicity and carcinogenicity of the dyes and their biotransformation products. The dye degrading bacterial strain KMK 5 was isolated from the textile dyes contaminated soil of Ichalkaranji, Maharashtra, India. It was identified as Bacillus fusiformis based on the biochemical and morphological characterization as well as 16S rDNA sequencing. KMK 5 could tolerate and degrade azo dyes, Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) under anoxic conditions. Complete mineralization of DB79 and AO10 at the concentration of 1.5g/l was observed within 48h. This degradation potential increased the applicability of this microorganism for the dye removal.     

偶氮染料的微生物脱色研究进展

微生物法是染料废水治理的重要方法。本文综述了特异性酶作用下好氧细菌和真菌对偶氮染料的脱色以及厌氧条件下氧化还原介质作为电子穿梭体时偶氮染料的非特异性还原过程。指出厌氧偶氮还原是偶氮染料还原的主要形式, 电子供体不同脱色效率不同。对目前生物法去除偶氮染料存在的问题进行了分析, 提出了相应的对策措施。     

Biodegradation of textile azo dyes by a facultative Staphylococcus arlettae strain VN-11 using a sequential microaerophilic/aerobic process

A facultative Staphylococcus arlettae bacterium, isolated from an activated sludge process in a textile industry, was able to successfully decolourize four different azo dyes under microaerophilic conditions (decolourization percentage >97%). Further aeration of the decolourized effluent was performed to promote oxidation of the degradation products. The degradation products were characterized by FT-IR and UV–vis techniques and their toxicity with respect to Daphnia magna was measured. The amine concentrations as well as the total organic carbon (TOC) levels were monitored during the biodegradation process. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage indicated the presence of azoreductase activity and an oxidative biodegradation process, respectively. TOC reduction was ～15% in the microaerophilic stage and ～70% in the aerobic stage. The results provided evidence that, using a single Staphylococcus arlettae strain in the same bioreactor, the sequential microaerophilic/aerobic stages were able to form aromatic amines by reductive break-down of the azo bond and to oxidize them into non-toxic metabolites.     

Decolourization of textile dye Reactive Violet 5 by a newly isolated bacterial consortium RVM 11.1

Summary Soil samples collected from contaminated sites of Vatva, Gujarat, India were studied for screening and isolation of organisms capable of decolourizing textile dyes. A bacterial consortium RVM 11.1 was selected on the basis of rapid dye decolourization. Reactive Violet 5 (RV 5) was used as model dye. The consortium exhibited 94% decolourization ability within 37 h under a wide pH range from 6.5 to 8.5 and temperature ranging from 25 to 40 °C. The bacterial consortium was able to grow and decolourize RV5 under static conditions in the presence of glucose and yeast extract and also showed an ability to decolourize in the presence of starch in place of glucose. Maximum decolourization efficiency was observed at 200 ppm (mg/l) concentration of RV 5. Bacterial consortium RVM11.1 had the ability to decolourize 10 different dyes tested. The transformation and degradation products after decolourization were examined by HPTLC.     

Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01

Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5 %, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L^?1 at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV–VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography–mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4′-diamino-1,1′-biphenyl and 1,2′-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.     

In vivo and laccase-catalysed decolourization of xenobiotic azo dyes by a basidiomycetous fungus: characterization of its ligninolytic system

Bioremediation is considered a promising eco-efficient alternative for industrial wastewater treatment. Particular attention is currently being given to biological degradation of synthetic dyes and more specifically to colour removal by fungi. This work looks at the extracellular enzymatic system of strain Euc-1. Its ability to decolourize 14 xenobiotic azo dyes was evaluated and compared with the well-known species Phanerochaete chrysosporium. Strain Euc-1 is a mesophilic white-rot basidiomycete, the main secreted ligninolytic enzyme being laccase (0.38 U ml^–1). Although low manganese-dependent peroxidase activity (0.05 U ml^–1) was also detected, neither lignin peroxidase nor aryl alcohol oxidase could be found in batch culture. Optimum pH values of 4.0 and 5.0 were obtained in the laccase-catalysed oxidation of guaiacol and syringaldazine, respectively. Laccase activity increased with the temperature rise up to 50–60 °C and remarkable thermal stability was observed at 50 °C with a half-life of 12 h and no deactivation within the first 2 h. Solid-plate decolourization studies showed that basidiomycete Euc-1 decolourized 11 azo dyes whereas P. chrysosporium only two. Moreover, it is shown that purified laccase from basidiomycete Euc-1 efficiently decolourizes the azo dye acid red 88.     

Decolourization of recalcitrant dyes with a laccase from Streptomyces coelicolor under alkaline conditions

Colored wastewater from textile industries is a consequence of dye manufacturing processes. Two percent of dyes that are produced are discharged directly in aqueous effluent and more than 10% are subsequently lost during the textile coloration process. It is not surprising that these compounds have become a major environmental concern. In that context, we have evaluated the potential use of Streptomyces coelicolor laccase for decolourization of various dyes with and without a mediator. Results showed that in all cases the combination of laccase and the mediator acetosyringone was able to rapidly decolourize, to various degrees, all the dyes tested. In 10 min, decolourization was achieved at 94% for acid blue 74, 91% for direct sky blue 6b and 65% for reactive black 5. Furthermore, decolourization was achieved at 21% for reactive blue 19 and at 39% for the direct dye Congo red in 60 min. These results demonstrate the potential use of this laccase in combination with acetosyringone, a natural mediator, for dye decolourization.     

Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters

The azoreductase PpAzoR from Pseudomonas putida shows a broader specificity for decolourization of azo dyes than CotA-laccase from Bacillus subtilis. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We show that addition of CotA-laccase to PpAzoR reaction mixtures lead to a significant drop in the final toxicity. A sequential enzymatic process was validated through the use of 18 representative azo dyes and three model wastewaters that mimic real dye-containing effluents. A heterologous Escherichia coli strain was successfully constructed co-expressing the genes coding for both PpAzoR and CotA. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourization levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.     

Simple and easy method for the determination of fungal growth and decolourative capacity in solid media

A technique was developed for studying the biodegradative ability of white rot fungi in different solid media. This technique enables the gravimetric determination of fungal growth (increase of biomass) and the spectrometric measurement of fungal decolourization ability (both by the determination of the production of the extracellular enzyme manganese-dependent peroxidase (MnP) and by the rate of decolourization of dyes). Bjerkandera sp., strain BOS55, was grown in different solid media. Its growth rate, decolourization of solophenil blue 2BL (azoic dye), neutral red (eurhodin dye), methyl green and crystal violet (triphenylmethane dyes) and the production of MnP were determined. Application of this technique enabled a spectrometric quantification of enzymatic activity. Assays indicate that greater amounts of MnP were present in agar plate cultures of Bjerkandera sp. than in liquid cultures.     

Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes

Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg⁻¹ protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58±5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80–92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k _cat/K _M) on guaiacol followed by 2,2′-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5–5.4 days.     

POTENTIAL OF BRASSICA JUNCEA IN ORDER TO TREAT TEXTILE—EFFLUENT—CONTAMINATED SITES

Three plant species (Brassica juncea, Sorghum vulgare, and Phaseolus mungo) of different agronomic consequence were evaluated for the decolorization of the dyes from textile effluent. B. juncea, S. vulgare, and P. mungo showed textile effluent decolorization up to 79, 57, and 53%, respectively. A significant decrease in shoot and root height, but no significant injury, was observed in the case of P. mungo and S. vulgare. B. juncea (Indian mustard), the most tolerant and more effective metals accumulator than other tested agricultural plant species, showed enhanced growth with respect to the height of the shoot and root, 129 and 178%, respectively, when grown using original textile effluent. Textile effluent induced intracellular nicotinamide adenine dinucleotide reduced (NADH)–dichlorophenol indophenol reductase significantly in the case of S. vulgare and B. juncea with 209 and 194%, respectively. The extracellular riboflavin reductase activity was induced by 223% in the case of P. mungo as compared to control plants. Significant induction of intracellular laccase (266%) was observed in the case of B. juncea, indicating their crucial role for a potential metabolism and further degradation of the textile effluent. The metabolites were identified as napthalenesufamide (m/z 372) and 2-amino-4, 6-dichlorotriazine (m/z 167), when B. juncea was used to degrade a model dye, Reactive red 2.