Decolorization Screening of Synthetic Dyes by Anaerobic Methanogenic Sludge Using a Batch Decolorization Assay

The nonspecific ability of anaerobic sludge bacteria obtained from cattle dung slurry was investigated for 17 different dyes in a batch assay system using sealed serum vials. Experiments using Reactive Violet 5 (RV 5) showed that sludge bacteria could effectively decolorize solutions having dye concentrations up to 1000 mg l⁻¹ with a decolorization efficiency of above 75% during 48 h of incubation. Headspace gas composition of anaerobic batch systems for varying dye concentration revealed that lower concentrations of RV 5 (upto 500 mg l⁻¹) were found to be stimulatory to the methanogenic activity of sludge bacteria. However at higher dye concentrations, the headspace gas composition was found to be similar to batch assay controls without dye, indicating that dye at higher concentrations was inhibitory to methanogenic bacteria of sludge. The optimum inoculum and incubation temperature for maximum decolorization of RV 5 was found to be 9.0 g l⁻¹(in terms of total solids) and 37°C, respectively. Of sixteen other dyes tested, nine (Reactive Black 5, Reactive Blue 31, Reactive Blue 28, Reactive Red HE8B, Reactive Yellow, Reactive Golden Yellow, Mordant Orange, Novatic Olive R S/D & Navilan Yellow GL) were decolorized with more than 88% efficiency; three (Orange II, Navy Blue HER & Novatic Blue BC S/D) were decolorized with about 50–65% efficiency, whereas other three dyes (Procion Orange H2R, Procion Brilliant Blue HGR & Novatic Blue BC S/D) were decolorized with less than 40% efficiency. Though Ranocid Fast Blue was decolorized with about 92.5% efficiency, this was merely due to sorption, whereas the other dyes were decolorized due to biotransformation.     

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.     

Chemical characterization of a dye processing plant effluent--identification of the mutagenic components

This work shows the chemical characterization of a dye processing plant effluent that was contributing to the mutagenicity previously detected in the Cristais river, S?o Paulo, Brazil, that had an impact on the quality of the related drinking water. The mutagenic dyes Disperse Blue 373, Disperse Orange 37 and Disperse Violet 93, components of a Black Dye Commercial Product (BDCP) frequently used by the facility, were detected by thin layer chromatography (TLC). The blue and orange dyes were quantified by high performance liquid chromatography (HPLC/DAD) in a raw and treated effluent samples and their contribution to the mutagenicity was calculated based on the potency of each dye for the Salmonella YG1041. In the presence of S9 the Disperse Blue 373 accounted for 2.3% of the mutagenic activity of the raw and 71.5% of the treated effluent. In the absence of S9 the Disperse Blue 373 accounted for 1.3% of the mutagenic activity of the raw and 1.5% of the treated effluent. For the Disperse Orange 37, in the presence of S9, it contributed for 0.5% of the mutagenicity of the raw and 6% of the treated effluent. In the absence of S9; 11.5% and 4.4% of the raw and treated effluent mutagenicity, respectively. The contribution of the Disperse Violet 93 was not evaluated because this compound could not be quantified by HPLC/DAD. Mutagenic and/or carcinogenic aromatic amines were also preliminary detected using gas chromatograph/mass spectrometry in both raw and treated and are probably accounting for part of the observed mutagenicity. The effluent treatment applied by the industry does not seem to remove completely the mutagenic compounds. The Salmonella/microsome assay coupled with TLC analysis seems to be an important tool to monitor the efficiency of azo dye processing plant effluent treatments.     

The role of Mn-dependent peroxidase in dye decolorization by static and agitated cultures ofIrpex lacteus

Dye decolorization capacity of two white-rot fungi, Irpex lacteus and Phanerochaete chrysosporium, was compared in N-limited liquid cultures. The agitated cultures showed lower ability to decolorize azo dyes Reactive Orange 16 and Naphthol Blue Black than static cultures. Similar effect was also observed with other structurally different synthetic dyes. The effect of surfactants on the decolorization process is discussed. A significant increase in the Reactive Orange 16 decolorization by the agitated I. lacteus cultures was observed after adding 0.1% Tween 80, following a higher Mn-dependent peroxidase production. The in vitro dye decolorization using the purified enzyme proved its decolorization ability.     

Biodegradation of Reactive Blue 59 by isolated bacterial consortium PMB11

Morphologically different, three bacterial strains, capable of decolorizing Reactive Blue 59 were isolated from dye effluent contaminated soil sample, collected from Ichalkaranji, India. The individual bacterial strains viz. Bacillus odysseyi SUK3, Morganella morganii SUK5 and Proteus sp. SUK7 decolorized Reactive Blue 59 (50 mg l(-1)) completely within 60, 30, 24 h, respectively, while the bacterial consortium PMB11 of these strains required 3 h for the complete decolorization. The decolorization was confirmed by UV-Vis spectroscopy. Further, the biodegradation of Reactive Blue 59 in to different metabolites was confirmed by High performance liquid chromatography and Fourier transform infrared spectroscopy analysis. Significant increase in the activity of aminopyrine N-demethylase (AND) in the individual as well consortium cells, obtained after decolorization showed involvement of AND in the decolorization process. Phytotoxicity studies, revealed the nontoxic nature of the degraded metabolites of Reactive Blue 59 indicating effectiveness of bacterial consortium PMB11 for the treatment of textile effluent containing Reactive Blue 59.     

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     

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.     

Decolorization of synthetic and real textile wastewater by the use of white-rot fungi

Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor.     

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.     

Chitosan-coated Lentinus polychrous Lév.: Integrated biosorption and biodegradation systems for decolorization of anionic reactive dyes

Research and development of an effective color removal system is needed to reduce the severity of water pollution caused by effluent that contains dyes. In this study, the integrated biosorption and biodegradation system of chitosan coated Lentinus polychrous Lév. was developed and evaluated for its decolorization efficiency with regard to anionic reactive dye mixtures of Reactive Blue 19, 160, and 198. The fungi were coated with 0.1, 0.5, and 1.0% w/v of low molecular weight chitosan. The scanning electron micrographs confirmed that chitosan was successfully coated on the surface of the fungi. Studies of changes in UV–visible absorption spectra, dye desorption, ligninolytic enzyme activity, and Fourier transform infrared spectroscopy showed that within 6 h, the biosorption was the control mechanism and the dyes were reduced to 91.50, 77.66, 37.39, and 26.93% by the fungi coated with 0, 0.1, 0.5, and 1.0% w/v chitosan, respectively. From the 36th hour to the end of colorization at the 72nd hour, the fungal biodegradation by laccase and manganese peroxidase was dominant and all treatments had 5–8% of the dye remaining. Therefore, the chitosan coat acted as an efficient biosorbent for the anionic reactive dyes, thereby effectively improving the decolorization efficiency of the white rot fungus.     

一株高效广谱染料降解细菌的分离鉴定及脱色特性研究

通过梯度驯化,从印染废水长期污染土壤中分离筛选出能以4种不同结构类型的染料(刚果红、美蓝、孔雀绿和活性艳蓝KN-R)为唯一碳源的菌株XSMR,根据其形态学特征和生理生化鉴定及16S rDNA序列分析,初步鉴定为无色杆菌属(Achromobacter sp.)的菌株。菌株XSMR对4种染料均具有强的脱色降解能力,且对染料脱色的同时,自身能够生长繁殖,培养24h菌体干重超过不加染料的对照。在振荡培养条件下对该菌株的脱色反应条件进行研究,结果表明,当刚果红、美蓝、孔雀绿及活性艳蓝KN-R的初始浓度分别小于200mg/L、200mg/L、150mg/L及150mg/L时,在pH7.5、温度35℃、接种量4%(V/V)条件下,接种菌株XSMR脱色14h对4种染料的脱色率均可达到98%以上。通过对降解产物的紫外-可见光谱分析,进一步证明了菌株XSMR能彻底降解染料。菌株XSMR对染料脱色的机理包括生物降解和菌株吸附两方面。     

Implication of mycelium-associated laccase from Irpex lacteus in the decolorization of synthetic dyes

The white rot fungus Irpex lacteus is able to decolorize such synthetic dyes as Reactive Orange 16 and Remazol Brilliant Blue R. Here, we demonstrate that this type of dye decolorization is mainly related to a laccase-like enzyme activity associated with fungal mycelium. In its bound form, the enzyme detected showed a pH optimum of 3.0 for the oxidation of ABTS, DMP and guaiacol, and a pH of 7.0 for syringaldazine. The highest enzymatic activity was obtained with ABTS as substrate. Enzyme activity was fully inhibited with 50mM NaN(3). Depending on the chemical structure of dyes, redox mediators had a positive effect on the dye decolorization by fungal mycelium. Enzyme isolated from fungal mycelium was able to decolorize synthetic dyes in vitro.     

Accumulation of Acid Orange 7, Acid Red 18 and Reactive Black 5 by growing Schizophyllum commune

The effect of Acid Orange 7, Acid Red 18 and Reactive Black 5 on the growth and decolorization properties of Schizophyllum commune was studied with respect to the initial pH varying from 1 to 6 and initial dye concentration (10-100 mg/L). The optimum pH value was found to be 2 for both growth and color removal of these azo dyes. Increasing the concentration of azo dyes inhibited the growth of S. commune. It was observed that S. commune was capable of removing Acid Orange 7, Acid Red 18 and Reactive Black 5 with a maximum specific uptake capacity of 44.23, 127.53 and 180.17 (mg/g) respectively for an initial concentration of 100 mg/L of the dye. Higher decolorization was observed at lower concentrations for all the dyes. Finally it was found that the percentage decolorization was more in the case of Reactive Black 5 dye compared to the other two dyes used in the present investigation.     

Studies on phytoremediation potentiality of Typhonium flagelliforme for the degradation of Brilliant Blue R

In vitro culture plants of Typhonium flagelliforme were found to decolorize a variety of dyes, including Malachite Green, Red HE 8B, Methyl Orange, Reactive Red 2, Direct Red 5B (DR5B), Red HE 7B, Golden Yellow HER, Patent Blue, and Brilliant Blue R (BBR), to varying extents within 4 days. The enzymatic analysis of plant roots of aseptically raised plantlets performed before and after degradation of the dye BBR by these plantlets showed a significant induction in the activities of peroxidase, laccase, tyrosinase, and 2,6-dichlorophenol-indophenol reductase, which indicated the involvement of these enzymes in the metabolism of the dye. Comparative study of the enzyme status of the plants Typhonium flagelliforme and Blumea malcolmii during the degradation of DR5B and BBR showed marked variations in the enzyme profile with respect to the use of different sources of the enzyme. Phytoremediation of BBR using Typhonium flagelliforme was confirmed with high performance liquid chromatography and Fourier transform infrared spectroscopy analysis performed before and after the degradation of the dye. One of the products of the metabolism of the dye was identified as 4-(4-ethylimino-cyclohexa-2,5-dienylidinemethyl)-phenylamine with the aid of gas chromatography–mass spectroscopy (GC–MS) analysis. Significant decrease in the American Dye Manufacturer’s Institute, biological oxygen demand, and chemical oxygen demand values of synthetic mixture of textile dyes and industrial effluent confirmed the decolorization and detoxification. Phytotoxicity studies also revealed the nontoxic nature of the metabolites of BBR.     

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.     

Degradation analysis of Reactive Red 198 by hairy roots of <Emphasis Type="Italic">Tagetes patula</Emphasis> L. (Marigold)

Tagetes patula L. (Marigold) hairy roots were selected among few hairy root cultures from other plants tested for the decolorization of Reactive Red 198. Hairy roots of Tagetes were able to remove dye concentrations up to 110 mg L^−l and could be successively used at least for five consecutive decolorization cycles. The hairy roots of Tagetes decolorized six different dyes, viz. Golden Yellow HER, Methyl Orange, Orange M2RL, Navy Blue HE2R, Reactive Red M5B and Reactive Red 198. Significant induction of the activity of biotransformation enzymes indicated their crucial role in the dye metabolism. UV–vis spectroscopy, HPLC and FTIR spectroscopy analyses confirmed the degradation of Reactive Red 198. A possible pathway for the biodegradation of Reactive Red 198 has been proposed with the help of GC–MS and metabolites identified as 2-aminonaphthol, p-aminovinylsulfone ethyl disulfate and 1-aminotriazine, 3-pyridine sulfonic acid. The phytotoxicity study demonstrated the non-toxic nature of the extracted metabolites. The use of such hairy root cultures with a high ability for bioremediation of dyes is discussed.     

Removal of basic and reactive dyes using ethylenediamine modified rice hull

Wastewaters from textile industries may contain a variety of dyes that have to be removed before their discharge into waterways. Rice hull, an agricultural by-product, was modified using ethylenediamine to introduce active sites on its surface to enable it to function as a sorbent for both basic and reactive dyes. The sorption characteristics of Basic Blue 3 (BB3) and Reactive Orange 16 (RO16) by ethylenediamine modified rice hull (MRH) were studied under various experimental conditions. Sorption was pH and concentration dependent. Simultaneous removal of BB3 and RO16 occurred at pH greater than 4. The kinetics of dye sorption fitted a pseudo-second order rate expression. Increase in agitation rate had no effect on the sorption of BB3 but increased uptake of RO16 on MRH. Decreasing particle size increased the uptake of dyes in binary dye solutions. Equilibrium data could be fitted into both the Langmuir and Freundlich isotherms. Maximum sorption capacities calculated from the Langmuir model are 14.68 and 60.24 mg/g for BB3 and RO16, respectively in binary dye solutions. This corresponds to an enhancement of 4.5 and 2.4 fold, respectively, compared to single dye solutions. MRH therefore has the potential of being used as an efficient sorbent for the removal of both dyes in textile wastewaters.     

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.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

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.