Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.     

Decolorization and degradation of reactive azo dyes by fixed bed bioreactors containing immobilized cells of Proteus vulgaris NCIM-2027

Immobilized cells of Proteus vulgaris NCIM 2027 completely decolorized C.I. Reactive Blue 172 (50 mg/L) within 8 h along with a nearly 80% reduction in TOC and COD. The dye degradation efficiency of the immobilized cells was further improved by optimizing the physicochemical conditions, including agitation, temperature, pH, dye concentration, and biomass loading. Microbial toxicity study revealed the non-toxic nature of the degraded products. Repeated-batch decolorization was conducted to evaluate the reusability of the immobilized cells. The immobilized cells were used for continuous dye decolorization in a fixed bed bioreactor under different volumetric flow rates and dye feeding concentrations. In addition, the immobilized cells were applied to decolorize a mixture of seven reactive dyes in batch and continuous modes, resulting in efficient decolorization (in terms of ADMI value) and significant reduction in TOC and COD. This suggests the potential of using immobilized cells to treat dye-containing wastewater.     

Biosorption of anionic textile dyes by nonviable biomass of fungi and yeast

The nonviable biomass of Aspergillus niger, Aspergillus japonica, Rhizopus nigricans, Rhizopus arrhizus, and Saccharomyces cerevisiae were screened for biosorption of textile dyes. The selected anionic reactive dyes were C.I. Reactive Black 8, C.I. Reactive Brown 9, C.I. Reactive Green 19, C.I. Reactive Blue 38, and C.I. Reactive Blue 3. Experiments were conducted at initial dye concentration of 50, 100, 150 and 200mg/L. The effect of initial dye concentration, dose of biosorbent loading, temperature, and pH on adsorption kinetics was studied. S. cerevisiae and R. nigricans were good biosorbents at initial dye concentration of 50mg/L, 1g% (w/v) biomass loading and 29+/-1 degrees C. R. nigricans adsorbed 90-96% dye in 15min, at 20 degrees C and pH 6.0. The data showed an optimal fit to the Langmuir and Freundlich isotherms. The maximum uptake capacity (Q(o)) for the selected dyes was in the range 112-204mg/g biomass.     

Decolorization and biodegradation of reactive dyes and dye wastewater by a developed bacterial consortium

A bacterial consortium (consortium GR) consisting of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168 could rapidly decolorize and degrade commonly-used sulfonated reactive dye Green HE4BD and many other reactive dyes. Consortium GR shows markedly higher decolorization activity than that of the individual strains. The preferable physicochemical parameters were identified to achieve higher dye degradation and decolorization efficiency. The supplementation of cheap co-substrates (e.g., extracts of agricultural wastes) could enhance the decolorization performance of consortium GR. Extent of mineralization was determined with TOC and COD measurements, showing nearly complete mineralization of Green HE4BD by consortium GR (up to 90% TOC and COD reduction) within 24 h. Oxidoreductive enzymes seemed to be involved in fast decolorization/degradation process with the evidence of enzymes induction in the bacterial consortium. Phytotoxicity and microbial toxicity studies confirm that the biodegraded products of Green HE4BD by consortium GR are non-toxic. Consortium GR also shows significant biodegradation and decolorization activities for mixture of reactive dyes as well as the effluent from actual dye manufacturing industry. This confers the possibility of applying consortium GR for the treatment of industrial wastewaters containing dye pollutants.     

Modeling the discoloration of a mixture of reactive textile dyes by commercial laccase

Degradation of a mixture of three reactive textile dyes (Reactive Black 5, Reactive Yellow 15 and Reactive Red 239), simulating a real textile effluent, by commercial laccase, was investigated in a batch reactor. The discoloration was appraised as a percentage of the absorbance reduction at the wavelength of maximum absorbance for each dye and as total color removal based in all visible spectrum. A significantly high discoloration was achieved in both cases, indicating the applicability of this method for textile wastewater treatment. Mathematical models were developed to simulate the kinetics of laccase catalyzed degradation of reactive dyes in mixtures. Like in single dye degradation, some of the reactions present an unusual kinetic behavior, corresponding to the activation of the laccase-mediator system. The kinetic constants of the models were estimated by minimizing the difference between the predicted and the experimental time courses. Although not perfect, the ability of the models in representing the experimental results suggests that they could be used in design and simulation applications.     

The toxicity of textile reactive azo dyes after hydrolysis and decolourisation

The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri. Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively). A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)). Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).     

Biosorption of reactive dyes by dried activated sludge: equilibrium and kinetic modelling

The biosorption of reactive dyes (Reactive Blue 2 - RB2 and Reactive Yellow 2 - RY2) onto dried activated sludge was investigated. The dye binding capacity of biosorbent was shown as a function of initial pH, initial dye concentration and type of dye. The equilibrium data fitted very well to both the Freundlich and Langmuir adsorption models. The results showed that both the dyes uptake processes followed the second-order rate expression.     

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.     

Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing effluent by using potato (Solanum tuberosum) soluble and immobilized polyphenol oxidase

Celite bound potato polyphenol oxidase preparation was employed for the treatment of wastewater/dye effluent contaminated with reactive textile and non-textile dyes, Reactive Blue 4 and Reactive Orange 86. The maximum decolorization was found at pH 3.0 and 4.0 in case of Reactive Blue 4 and Reactive Orange 86, respectively. Immobilized potato polyphenol oxidase was significantly more effective in decolorizing the individual dye and complex mixtures of dyes as compared to soluble enzyme. The absorption spectra of the treated and untreated dye mixture and dyeing effluent exhibited a marked difference in the absorption value at various wavelengths. The polluted water contaminated with an individual dye or mixtures of dyes treated with soluble and immobilized potato polyphenol oxidase resulted in the remarkable loss in total organic carbon.     

Degradation of azo dyes by oxidative processes--laccase and ultrasound treatment

Azo dyes are of synthetic origin and their environmental fate is not well understood. They are resistant to direct aerobic bacterial degradation and form potentially carcinogenic aromatic amines by reduction of the azo group. This study shows that applying the oxidative processes of enzymatic treatment with laccase and ultrasound treatment, both alone and in combination, leads to dye degradation. Laccase treatment degraded both Acid Orange and Direct Blue dyes within 1-5 h but failed in the case of Reactive dyes, whereas ultrasound degraded all the dyes investigated (3-15 h). When applied as multi-stage combinations the treatments showed synergistic effects for dye degradation compared with individual treatments. Bulk light absorption (UV-Vis) and ion pairing HPLC were used for process monitoring. Additionally, mass spectrometry was used to elucidate the structures of intermediates arising from ultrasound treatment.     

Treatment and kinetic modelling of a simulated dye house effluent by enzymatic catalysis

Biocatalytic treatment of a synthetic dye house effluent, simulating a textile wastewater containing various reactive dyestuffs (Reactive Yellow 15, Reactive Red 239 and Reactive Black 5) and auxiliary chemicals, was investigated in a batch reactor using a commercial laccase. A high decolourisation (above 86%) was achieved at the maximum wavelength of Reactive Black 5. The decolourisation at the other dyes wavelengths (above 63% for RY15 and around 41% for RR239) and the total decolourisation based on all the visible spectrum (around 55%) were not so good, being somewhat lower than in the case of a mixture of the dyes (above 89% for RB5, 77% for RY15, 68% for RR239 and above 84% for total decolourisation). Even so, these results suggest the applicability of this method to treat textile dyeing wastewaters. Kinetic models were developed to simulate the synthetic effluent decolourisation by commercial laccase. The kinetic constants of the models were estimated by minimizing the difference between the predicted and the experimental time courses. The close correlation between the experimental data and the simulated values seems to demonstrate that the models are able to describe with remarkable accuracy the simulated effluent degradation. Water quality parameters such as TOC, COD, BOD₅ and toxicity were found to be under the maximum permissible discharge limits for textile industries wastewaters.     

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.     

Optimisation for enhanced decolourization and degradation of Reactive Red BS C.I. 111 by Pseudomonas aeruginosa NGKCTS

Soil samples collected from dye contaminated sites of Vatva, Gujarat, India were studied for the screening and isolation of organisms capable of decolourizing textile dyes. The most efficient isolate, which showed decolourization zone of 48 mm on 300 ppm Reactive Red BS (C.I.111) containing plate, was identified as Pseudomonas aeruginosa. Reactive Red BS (C.I.111) was used as a model dye for the study. The isolated culture exhibited 91% decolourization of 300 ppm dye within 5.5 h over a wide pH range from 5.0 to 10.5 and temperature ranging from 30 to 40°C. The culture was able to decolourize more than 91% of Reactive Red BS under static conditions in presence of either glucose, peptone or yeast extract. Addition of 300 ppm of Reactive Red BS, in each step, in ongoing dye decolourization flask, gave more than 90% decolourization within 2 h corresponding to 136 mg l⁻¹ h⁻¹ dye removal rate. The isolate had the ability to decolourize six different reactive dyes tested as well as the actual dye manufacturing industry’s effluent. The degradation of the dye was confirmed by HPTLC.     

Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta

Eighteen fungal strains, known for their ability to degrade lignocellulosic material or lignin derivatives, were screened for their potential to decolorize commercially used reactive textile dyes. Three azo dyes, Reactive Orange 96, Reactive Violet 5 and Reactive Black 5, and two phthalocyanine dyes, Reactive Blue 15 and Reactive Blue 38, were chosen as representatives of commercially used reactive dyes. From the 18 tested fungal strains only Bjerkandera adusta, Trametes versicolor and Phanerochaete chrysosporium were able to decolorize all the dyes tested. During degradation of the nickel-phthalocyanine complex, Reactive Blue 38, by B. adusta and T. versicolor respectively, the toxicity of this dye to Vibrio fischeri was significantly reduced. In the case of Reactive Violet 5, a far-reaching detoxification was achieved by treatment with B. adusta. Reactive Blue 38 and Reactive Violet 5 were decolorized by crude exoenzyme preparations from T. versicolor and B. adusta in a H₂O₂-dependent reaction. Specific activities of the exoenzyme preparations with the dyes were determined and compared to oxidation rates by commercial horseradish peroxidase. Received: 3 February 1997 / Received revision: 9 April 1997 / Accepted: 13 April 1997     

Ecofriendly degradation of sulfonated diazo dye C.I. Reactive Green 19A using Micrococcus glutamicus NCIM-2168

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l⁻¹) within 42 h at temperature 37 °C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.     

Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases

Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds.     

In vitro studies on degradation of synthetic dye mixture by Comamonas sp. VS-MH2 and evaluation of its efficacy using simulated microcosm

Reactive azo dyes are considered as one of the most detrimental pollutants from industrial effluents and therefore their biodegradation is receiving constant scientific consideration. A bacterial isolate VS-MH2, originating from dye contaminated sites of Gujarat, India, was exploited for its ability to degrade a synthetic dye mixture (SDM) (comprising of four azo reactive dyes) under static conditions. The identification of the isolate by 16S rRNA gene sequencing revealed it to be Comamonas sp. The biodegradation of the SDM was analyzed by UV-vis spectroscopy, IR spectroscopy and GC-MS analysis. The isolate showed high metabolic activity towards SDM and degraded it completely (100 mg L(-1)) within 30 h at pH 7 and 35 °C. Simulated microcosm studies in the presence and absence of indigenous microflora confirmed the ability of Comamonas sp. VS-MH2 for dye degradation and to colonize the soil. This is the first investigation reporting the degradation of SDM by Comamonas sp. under simulated soil microcosms.     

Fixed-bed decolorization of Reactive Blue 172 by Proteus vulgaris NCIM-2027 immobilized on Luffa cylindrica sponge

Proteus vulgaris NCIM-2027 cells immobilized on Luffa cylindrica (Loofa) completely decolorized C.I. Reactive Blue 172 at 37 °C and pH 8.0 under 5-h static incubation with high total organic carbon (TOC) and chemical oxygen demand (COD) reduction. The repeated-batch decolorization experiments also indicate good reusability of the immobilized biocatalyst. Some oxidoreductive enzymes were shown to be involved in the decolorization and degradation process. Loofa immobilized cells were also able to decolorize a mixture of reactive dyes in batch mode (in terms of ADMI value) with significant reduction in TOC and COD. Loofa immobilized cells were also used for continuous decolorization of individual and mixture of reactive dyes in a fixed bed bioreactor.     

Biodegradation of azo and anthraquinone dyes in continuous systems

The purpose is to develop a complete microbiological model system for the treatment of wastewater from textile mills in developing countries. Artificial wastewater was treated by microorganisms growing on wood shavings from Norway spruce during unsterile conditions. The microorganisms were inoculated from forest residues. Mixtures of the azo dyes Reactive Black 5 and Reactive Red 2 were degraded in batch as well as continuous experiments. Reactive Red 2 mixed with the anthraquinone dye Reactive Blue 4 was also treated in the continuous system. The system consisted of three reservoirs - the first two with an anaerobic environment and the third with an aerobic. The dye concentrations were 200 mg l⁻¹ of each dye in the continuous system and the retention time was approximately 4 days and 20 h per reservoir. Samples from the process were analysed with spectrophotometer and LC/MS to monitor the degradation process. 86-90% of the colour was removed after a treatment of 4 days and 23 h in the continuous process. Two metabolites were found in the outlets of reactors one and two, but they were degraded to below the detection limit in the aerobic reactor.     

Potential of combined fungal and bacterial treatment for color removal in textile wastewater

Low efficiency of dye removal by mixed bacterial communities and high rates of dye decolorization by white-rot fungi suggest a combination of both processes to be an option of treatment of textile wastewaters containing dyes and high concentrations of organics. Bacteria were able to remove mono-azo dye but not other chemically different dyes whereas decolorization rates using Irpex lacteus mostly exceeded 90% within less than one week irrespective of dye structure. Decolorization rates for industrial textile wastewaters containing 2-3 different dyes by fungal trickling filters (FTF) attained 91%, 86%, 35% within 5-12 d. Sequential two-step application of FTF and bacterial reactors resulted in efficient decolorization in 1st step (various single dyes, 94-99% within 5 d; wastewater I, 90% within 7 d) and TOC reduction of 95-97% in the two steps. Large potential of combined use of white-rot fungi and traditional bacterial treatment systems for bioremediation of textile wastewaters was demonstrated.