Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In this study, to simulate the real industry wastewaters, experiments of laccase-catalyzed decolorization of mixed dyes constituted by azo and anthraquinone dyes were carried out. The results showed that anthraquinone dyes, playing the role of mediators, dramatically promoted the degradation of azo dyes when there was no exogenous mediator in the reaction mixture. This study represents the first attempt to decolorize the mixtures of azo and anthraquinone dyes by purified T. trogii laccase, suggesting great potential for laccase to decolorize textile industry wastewaters.     

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 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.     

Production of laccase by a newly isolated deuteromycete fungus <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. and its decolorization of azo dye

The effect of various carbon and nitrogen sources on the production of laccase by newly isolated deuteromycete Pestalotiopsis sp. was tested under liquid-state fermentation. Twenty grams per liter of glucose and 10 g l⁻¹ ammonium tartrate were found to be the optimized concentrations of carbon and nitrogen sources, respectively. The influence of different inducers and inhibitors on the laccase production was also examined. Adding the Cu up to optimum concentration of 2.0 mM in medium (include 20 g l⁻¹ glucose and 10 g l⁻¹ ammonium tartrate), the highest laccase activity of 32.7 ± 1.7 U ml^−l was achieved. Cu had to be supplemented after 2 days of growth for its maximal effect, an addition after 6 days of growth, during which laccase activity was dominantly formed, resulted in distinctly reduced laccase activity. In addition, Direct Fast Blue B2RL can be effectively decolorized by crude laccase, the decolorization percentage of which was 88.0 ± 3.2% at pH 4.0 within 12 h. The results suggest that Pestalotiopsis sp. is a high potential producer of the industrially important enzyme laccase.     

Time dependent degradation of mixture of structurally different azo and non azo dyes by using Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360, a yeast species showed 88% ADMI (American dye manufacturing institute) removal of mixture of structurally different dyes (Remazol red, Golden yellow HER, Rubine GFL, Scarlet RR, Methyl red, Brown 3 REL, Brilliant blue) (70 mg l⁻¹) within 24 h at 30 °C and pH 7.0 under shaking condition (120 rpm). Glucose (0.5%) as a carbon source was found to be more effective than other sources used. The medium with metal salt (CaCl₂, ZnSO₄, FeCl₃, MgCl₂, CuSO₄) (0.5 mM) showed less ADMI removal as compared to control, but did not inhibit complete decolorization. The presence of tyrosinase, NADH-DCIP reductase and induction in laccase activity during decolorization indicated their role in degradation. HPTLC (High performance thin layer chromatography) analysis revealed the removal of individual dyes at different time intervals from dye mixture, indicating preferential degradation of dyes. FTIR (Fourier transform infrared spectroscopy) and HPLC (High performance liquid chromatography) analysis of samples before and after decolorization confirmed the biotransformation of dye. The reduction of COD (Chemical oxygen demand) (69%), TOC (Total organic carbon) (43%), and phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature.     

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.     

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.     

Simultaneous decolorization of azo dye and bioelectricity generation using a microfiltration membrane air-cathode single-chamber microbial fuel cell

Electricity generation from readily biodegradable organic substrates accompanied by decolorization of azo dye was investigated using a microfiltration membrane air-cathode single-chamber microbial fuel cell (MFC). Batch experiment results showed that accelerated decolorization of active brilliant red X-3B (ABRX3) was achieved in the MFC as compared to traditional anaerobic technology. Biodegradation was the dominant mechanism of the dye removal, and glucose was the optimal co-substrate for ABRX3 decolorization, while acetate was the worst one. Confectionery wastewater (CW) was also shown to be a good co-substrate for ABRX3 decolorization and a cheap fuel source for electricity generation in the MFC. Low resistance was more favorable for dye decolorization than high resistance. Suspended sludge (SS) should be retained in the MFC for accelerated decolorization of ABRX3. Electricity generation was not significantly affected by the ABRX3 at 300 mg/L, while higher concentrations inhibited electricity generation. However, voltage can be recovered to the original level after replacement with anodic medium not containing azo dye.     

Acceleration of azo dye decolorization by using quinone reductase activity of azoreductase and quinone redox mediator

This study demonstrated the effective application of intracellular azoreductase in mediated decolorization of azo dyes. Using the quinone reductase activity of overexpressed azoreductase AZR and quinone redox mediators, the decolorization performance of the recombinant strain Escherichia coli YB was significantly enhanced. In the presence of 0.2 mM lawsone, 75% acid red 27 (1 mM) was decolorized by E. coli YB in only 2 h, which was the highest bacterial decolorization rate ever reported. Compared to lawsone, menadione was a less effective redox mediator. Glucose was found to be the best carbon source for mediated decolorization by E. coli YB. The recombinant strain could complete four rounds of mediated decolorization repeatedly in 12 h. In addition, a 10-min pre-incubation of E. coli JM109 and activated sludge with 2-methylhydroquinone resulted in great improvement of mediated decolorization performance, which may be applied in practical treatment.     

红树林混合菌群协同降解偶氮染料活性黑5脱色性能研究

通过对福建省龙海市海门岛红树林保护区的近海沉积物样品进行富集驯化,得到了能够协同降解偶氮染料活性黑5的混合菌群HMD9-Q7。经过生化试验和16S rDNA序列分析,鉴定该混合菌群主要由越南蔷薇菌（Rossellomorea vietnamensis）、转化异源物食烷菌（Alcanivorax xenomutans）和近海生微泡菌（Microbulbifer maritimus）组成。与单一菌株相比较,混合菌群具有显著的脱色效果,可能因其菌株之间存在着相互协同促进作用。该混合菌群对含20~100 mg/L浓度的活性黑5均产生脱色作用。混合菌群HMD9-Q7可以广泛利用外加碳源和氮源,如葡萄糖、硝酸铵、牛肉膏等;NaCl浓度在5~30 g/L范围内都可对活性黑5进行脱色;在pH 7和温度30℃条件下脱色率最高。在最优条件下,该混合菌群在60 h内对活性黑5的脱色率可达到85%~90%。该新型混合菌群主要通过生物降解途径来实现对活性黑5的脱色。随着降解反应的发生,活性黑5的最大吸收峰逐渐变小直至完全消失,降解产物吸收峰位于近紫外光区。     

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.     

Simultaneous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow 3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture

The naphthalenesulfonate-oxidizing bacterium Sphingomonas sp. BN6 was immobilized in calcium alginate. These beads were incubated under aerobic conditions in a medium with the sulfonated azo dye, Mordant Yellow 3 (MY3), and glucose. The immobilized cells converted MY3, but only a marginal turnover of the dye was found under these conditions with freely suspended cells of Sphingomonas sp. BN6. Under anaerobic conditions, suspended cells of Sphingomonas sp. BN6 reductively cleaved the azo bond of MY3 to 6-aminonaphthalene-2-sulfonate (6A2NS) and 5-aminosalicylate. The turnover of MY3 by the immobilized cells under aerobic conditions resulted in the formation of more than equimolar amounts of 5-aminosalicylate, but almost no (6A2NS) was detected. Cells of Sphingomonas sp. BN6 aerobically oxidize 6A2NS to 5-aminosalicylate. It was therefore concluded that the cells in the anaerobic center of the alginate beads reduced MY3 to 6A2NS and 5-aminosalicylate and that 6A2NS was oxidized to 5-aminosalicylate by those cells that were immobilized in the outer aerobic zones of the alginate beads. The presence of oxygen gradients within the alginate beads was verified by using oxygen micro-electrodes. A coimmobilisate of Sphingomonas sp. BN6 with a 5-aminosalicylate degrading bacterium completely degraded MY3. The immobilized cells also converted the sulfonated azo dyes Amaranth and Acid Red␣1. Received: 6 May 1996 / Received revision: 6 August 1996 / Accepted: 12 August 1996     

氧气对混合菌群脱色降解偶氮染料效果的影响

【背景】偶氮染料及其中间产物具有一定的环境毒性,利用混合菌群降解偶氮染料是一种环境友好型方法,但降解过程中氧气的存在起到至关重要的作用,可以促进或抑制偶氮染料的微生物降解作用。【目的】探讨氧气对偶氮染料微生物脱色液的影响,分析氧气对混合菌群脱色降解偶氮染料效果的影响。【方法】利用混合菌群DDMY1在3种培养条件(好氧、厌氧、兼氧)下,对7种偶氮染料进行脱色降解,探讨偶氮染料脱色液对氧气的响应情况,利用紫外可见分光光度法(ultraviolet visible spectrophotometry,UV-vis)和傅里叶变换红外光谱法(Fourier transform infrared spectroscopy,FTIR)对脱色产物进行分析。【结果】在兼氧和厌氧条件下反应48 h后的染料脱色液,与氧气充分接触后,部分偶氮染料微生物脱色液发生较为明显的复色现象,如活性黑5、直接黑38;UV-vis分析结果表明,这种复色现象是由于脱色液与氧气接触之后产生新物质所致;FTIR分析结果表明,混合菌群对发生复色反应的偶氮染料仍然具有一定脱色降解效果,但是脱色尚不够完全。【结论】兼氧和厌氧条件下,氧气对部分偶氮染料微生物脱色液具有较为明显的影响,从而影响混合菌群对偶氮染料的整体脱色效果,这可为今后研究偶氮染料彻底生物降解提供理论基础。     

Cytotoxicity and genotoxicity evolution during decolorization of dyes by White Rot Fungi

White Rot Fungi (WRF) are able to decolorize dyes through the use of relatively non-specific extracellular oxidative enzymes. Nevertheless, decolorization does not imply that the resulting metabolites are less toxic than the parent molecules. The aim of the present study was to evaluate the detoxification potential of six strains (Pycnoporus sanguineus, Perenniporia tephropora, Perenniporia ochroleuca, Trametes versicolor, Coriolopsis polyzona and Clitocybula dusenii) during decolorization of dyes. Cytotoxicity assays were carried out on human Caco-2 cells, which are considered as a validated model for the human intestinal epithelium, and the results were compared with those obtained on classical bacterial cells. Genotoxic character was monitored through VITOTOX^® assays. The biotransformation of an anthraquinonic dye (CI Acid Blue 62, ABu62) was studied. All tested strains were able to decolorize extensively ABu62 (between 83 and 95% decolorization), however, different cytotoxicity reduction levels were reached (from 44 to 99%). Best results were achieved with P. sanguineus strain and the major role of laccases in cytotoxicity reduction was underlined. Based on this result, efficiency of P. sanguineus strain was further studied. Four azo and two anthraquinonic dyes were treated by this strain. After WRF treatment, two dyes were found to be more toxic in one or both toxicity assays. Genotoxic character appeared during biotransformation of one dye, however, it was removed by the addition of hepatic rat extract to mimic liver transformation. These results stress the importance of monitoring several parameters, such as colour, toxicity and mutagenicity, to ensure the efficiency of the bioremediation process.     

Efficient decolorization and detoxification of the sulfonated azo dye Reactive Orange 16 and simulated textile wastewater containing Reactive Orange 16 by the white-rot fungus Ganoderma sp. En3 isolated from the forest of Tzu-chin Mountain in China

The sulfonated azo dye Reactive Orange 16 is the commonly used representative of reactive dyes, but is hard to be degraded by some conventional treatment methods. In order to develop more efficient and more cost-effective treatment methods for degrading this recalcitrant dye, the capability of the white-rot fungus Ganoderma sp. En3 isolated by our laboratory to decolorize and detoxify Reactive Orange 16 was investigated in this study. Ganoderma sp. En3 had a strong ability to decolorize high concentrations of Reactive Orange 16 and simulated textile wastewater containing Reactive Orange 16 in submerged cultures. Decolorization of Reactive Orange 16 and its simulated dye effluents by this fungus resulted in the decrease of phytotoxicity. Ganoderma sp. En3 had strong adaptability and tolerance to high concentrations of Reactive Orange 16. Compared with some previous research, Ganoderma sp. En3 was superior to some other fungal strains reported previously in the rate and extent of decolorizing Reactive Orange 16. It was also found that the real textile wastewater could be efficiently decolorized by Ganoderma sp. En3 in submerged cultures. The crude enzyme produced by Ganoderma sp. En3 could also efficiently decolorize Reactive Orange 16 and simulated textile wastewater under in vitro conditions.     

Eco-friendly biodegradation of a reactive textile dye Golden Yellow HER by Brevibacillus laterosporus MTCC 2298

Brevibacillus laterosporus MTCC 2298 showed 87% decolorization of Golden Yellow HER within 48 h under static condition at the concentration 50 mg l^?1; however no significant change in the decolorization performance was observed under shaking condition. Decolorization performance was maximum (74%) at the pH 7.0 and 30 °C. TLC and HPLC analysis confirmed the biodegradation of Golden Yellow HER. Biodegradation pathway was proposed using GC–MS and FTIR spectral analysis. Mainly elected metabolites are the 2,5-Dichloro-4 (3-hydrazino-2-hydroxy cyclopentylamino-) dibenzene-sulfonic acid (peak 1, m/z = 526), 4-(3-hydrazino-2-hydroxy cyclopentylamino)-benzene-sulfonic acid (peak 2, m/z = 455), 4-(3-amino-2-hydroxy-cyclopentylamino)-benzene-sulfonic acid and 5-amino-cyclohex-3-ene-sulfonic acid (peak 3, m/z = 183). Phytotoxicity results suggested that degradation products of Golden Yellow HER are non-toxic to the common crops such as Sorghum vulgare and Phaseolus mungo. Also, degradation products are non-toxic to B. laterosporus as well as ecologically important bacteria like Pseudomonas aeruginosa and Azotobacter vinelandii.     

Microbial decolorization of azo dyes and dye industry effluent by Fomes lividus

The white rot fungus, Fomes lividus, was isolated from the logs of Shorea robusta in the Western Ghats region of Tamil Nadu, India. The fungus was tested for decolorization of azo dyes such as orange G (50 M) congo red (50 M) amido black 10B (25 M) and also for colour removal from dye industry effluents. The results revealed that the fungus could remove only 30.8% of orange G in the synthetic solution, whereas congo red and amido black 10B were removed by 74.0 and 98.9% respectively. A dye industry effluent was treated by the fungus in batch and continuous mode. In batch mode treatment, a maximum decolorization of 84.4% was achieved on day 4, and in continuous mode a maximum decolorization of 37.5% was obtained on day 5. The colour removal by the basidiomycete fungus might be due to adsorption of the dyes to the mycelial surface and metabolic breakdown. These results suggested that the batch mode treatment of Fomes lividus is one of the most efficient ways for colour removal in dye industry effluents.     

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

Abstract

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.     

Decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated Trametes sp. SQ01 and its laccase

A white-rot fungus, strain SQ01, was isolated from decayed wood in a temperate forest. The strain was identified as a member of genus Trametes, based on the morphological characteristics and a complete sequence analysis of its 18S rRNA gene and ITS region. Strain SQ01 was capable of decolorizing a variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes, with an optimal efficiency of decolorization obtained when dyes added after 5 days of culture, with the exception of Cresol Red, showing that the point of dye addition was an important influencing factor for decolorization by this fungus. All of the tested dyes were decolorized by the purified laccase in the absence of any redox mediators, but only a few were completely removed, while others were not completely degraded even with increased decolorization time.     

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

Abstract

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.     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.