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.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Decolorization of Remazol Brilliant Blue (RBBR) and Poly R-478 dyes by <Emphasis Type="Italic">Bjerkandera adusta</Emphasis> CCBAS 930

An anamorphic Bjerkandera adusta CCBAS 930 strain isolated from soil was found to decolorize two anthraquinonic dyes: Remazol Brilliant Blue R and Poly R-478. The reduction in the level of phenolic compounds in liquid B. adusta cultures containing RBBR and Poly R-478 was correlated with decolorization of studied dyes, which suggested their biodegradation. It was shown that this process was coupled with induction of secondary metabolism (idiophase) and peak peroxidase activity in culture medium, and the appearance of aerial mycelium. Decolorization of dyes depended on the presence of glucose (cometabolism).     

Degradation of styrene by white-rot fungi

Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [¹⁴C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO₂ in 24 h and no other volatile products were found. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Evaluation of synthetic dye decolorization capacity in Ischnoderma resinosum

The little studied white rot fungus Ischnoderma resinosum was tested for its ability to decolorize seven different synthetic dyes. The strain efficiently decolorized Orange G, Amaranth, Remazol Brilliant Blue R, Cu-phthalocyanin and Poly R-478 on agar plates and in liquid culture at a relatively high concentration of 2–4 and 0.5–1 g l⁻¹, respectively. Malachite Green and Crystal Violet were decolorized to a lower extent up to the concentration of 0.1 g l⁻¹. Decolorization capacity of I. resinosum was higher than that in Phanerochaete chrysosporium, Pleurotus ostreatus or Trametes versicolor. In contrast with these thoroughly examined fungi, I. resinosum was able to degrade a wide spectrum of chemically and structurally different synthetic dyes. I. resinosum also efficiently decolorized dye mixtures. In liquid culture, Orange G and Remazol Brilliant Blue R were decolorized most rapidly; the process was not affected by different nitrogen content in the media. Shaken cultivation strongly inhibited the decolorization of Orange G.     

Isolation and selection of novel basidiomycetes for decolorization of recalcitrant dyes

Thirty wood-rotting basidiomycetes, most of them causing white rot in wood, were isolated from fruiting bodies growing on decaying wood from the Sierra de Ayllón (Spain). The fungi were identified on the basis of their morphological characteristics and compared for their ability to decolorize Reactive Black 5 and Reactive Blue 38 (as model of azo and phthalocyanine type dyes, respectively) at 75 and 150 mg/L. Only eighteen fungal strains were able to grow on agar plates in the presence of the dyes and only three species (Calocera cornea, Lopharia spadicea, Polyporus alveolaris) decolorized efficiently both dyes at both concentrations. The ligninolytic activities, involved in decolorization dyes (laccases, lignin peroxidases, Mn-oxidizing peroxidases), were followed in glucose basal medium in the presence of enzyme inducers. The results indicate a high variability of the ligninolytic system within white-rot basidiomycetes. These fungal species and their enzymes can represent new alternatives for the study of new biological systems to degrade aromatic compounds causing environmental problems.     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Complete decolorization of the anthraquinone dye Reactive blue 5 by the concerted action of two peroxidases from Thanatephorus cucumeris Dec 1

It is useful to identify and examine organisms that may prove useful for the treatment of dye-contaminated wastewater. Here, we report the purification and characterization of a new versatile peroxidase (VP) from the decolorizing microbe, Thanatephorus cucumeris Dec 1 (TcVP1). The purified TcVP1 after Mono P column chromatography showed a single band at 43 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Amino acid sequencing revealed that the N terminus of TcVP1 had the highest homology to Trametes versicolor MPG1, lignin peroxidase G (LiPG) IV, Bjerkandera adusta manganese peroxidase 1 (MnP1), and Bjerkandera sp. RBP (12 out of 14 amino acid residues, 86% identity). Mn²⁺ oxidizing assay revealed that TcVP1 acted like a classical MnP at pH ∼5, while dye-decolorizing and oxidation assays of aromatic compounds revealed that the enzyme acted like a LiP at pH ∼3. TcVP1 showed particularly high decolorizing activity toward azo dyes. Furthermore, coapplication of TcVP1 and the dye-decolorizing peroxidase (DyP) from T. cucumeris Dec 1 was able to completely decolorize a representative anthraquinone dye, Reactive blue 5, in vitro. This decolorization proceeded sequentially; DyP decolorized Reactive blue 5 to light red-brown compounds, and then TcVP1 decolorized these colored intermediates to colorless. Following extended reactions, the absorbance corresponding to the conjugated double bond from phenyl (250–300 nm) decreased, indicating that aromatic rings were also degraded. These findings provide important new insights into microbial decolorizing mechanisms and may facilitate the future development of treatment strategies for dye wastewater.     

Decolorization of triphenylmethane, azo, and anthraquinone dyes by a newly isolated Aeromonas hydrophila strain

A broad-spectrum dye-decolorizing bacterium, strain DN322, was isolated from activated sludge of a textile printing wastewater treatment plant. The strain was characterized and identified as a member of Aeromonas hydrophila based on Gram staining, morphology characters, biochemical tests, and nearly complete sequence analysis of 16S rRNA gene and the gyrase subunit beta gene (gyrB). Strain DN322 decolorized a variety of synthetic dyes, including triphenylmethane, azo, and anthraquinone dyes. For color removal, the most suitable pH and temperature were pH 5.0–10.0 and 25–37°C, respectively. Triphenylmethane dye, e.g., Crystal Violet, Basic Fuchsin, Brilliant Green, and Malachite Green (50 mg l⁻¹) were decolorized more than 90% within 10 h under aerobic culture condition and Crystal Violet could be used as sole carbon source and energy source for cell growth. The color removal of triphenylmethane dyes was due to a soluble cytosolic enzyme, and the enzyme was an NADH/NADPH-dependent oxygenase; For azo and anthraquinone dyes, e.g., Acid Amaranth, Great Red GR, Reactive Red KE-3B, and Reactive Brilliant Blue K-GR (50 mg l⁻¹) could be decolorized more than 85% within 36 h under anoxic condition. This strain may be useful for bioremediation applications.     

In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor

Lignite (brown coal) can be liquefied/solubilized with several fungi by different mechanisms. When applied industrially, only catalytic mechanisms can compete with chemical methods. The well-known fungal ligninolytic peroxidases are at a disadvantage, in that the relatively expensive hydrogen peroxide must be used as a cofactor. Comparing several fungal strains, we observed that the fungus Trametes versicolor is able to decolorize coal-derived humic acids, producing a considerable amount of laccase in the process. During this reaction the amount of humic acids decreases whilst that of fulvic acids increases; this was verified by optical density measurement and GPC after the two substance classes had been separated. Received: 27 August 1998 / Received revision: 4 November 1998 / Accepted: 7 November 1998     

Isolation and evaluation of terrestrial fungi with algicidal ability from Zijin Mountain,Nanjing, China

Approximately 60 fungal isolates from Zijin Mountain (Nanjing, China) were screened to determine their algicidal ability. The results show that 8 fungi belonging to Ascomycota and 5 belonging to Basidiomycota have algicidal ability. Of these fungi, Irpex lacteus T2b, Trametes hirsuta T24, Trametes versicolor F21a, and Bjerkandera adusta T1 showed strong algicidal ability. The order of fungal chlorophyll-a removal efficiency was as follows: T. versicolor F21a > I. lacteus T2b > B. adusta T1 > T. hirsuta T24. In particular, T. versicolor F21a completely removed algal cells within 30 h, showing the strongest algicidal ability. The results also show that all 4 fungal species degraded algal cells through direct attack. In addition, most of the tested fungi from the order Polyporales of Basidiomycota exhibited strong algicidal activity, suggesting that most fungi that belong to this order have algicidal ability. The findings of this work could direct the search for terrestrial fungi for bloom control.     

Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A

Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Received: 20 May 1997 / Received revision: 17 December 1997 / Accepted: 2 January 1998     

Azo Dye Decolorization under Microaerophilic Conditions by a Bacterial Mixture Isolated from Anthropogenic Dye‐Contaminated Soil

Soil samples isolated from dye-contaminated sites were exploited for isolation of dye decolorizing microorganisms. A novel bacterial mixture, RkNb1, was selected based on its efficiency, showing maximum and faster decolorization of textile dyes. Seven bacterial strains were isolated and identified from the bacterial mixture as Ochrobactrum intermedium (HM480365), Ochrobactrum intermedium strain M16-10-4 (HM030758), Enterococcus faecalis (HM480367), Arthrobacter crystallopoietes (HM480368), Kocuria flavus (HM480369), Bacillus beijingensis (HM480370), and Citrobacter freundii (HM480371) by 16S rRNA gene sequence analysis. This bacterial mixture showed 98.17% decolorization of Reactive Violet 5 (400 mg L^?1) within 8 h. The culture exhibited good decolorization ability at pH 8 and at a temperature of 37°C. Malt extract and peptone was found to enhance the decolorization rate of Reactive Violet 5. Plackett-Burman experimental design was used for elucidation of medium components affecting Reactive Violet 5 decolorization. Dye degradation products obtained during the course of decolorization were analyzed by high-performance thin-layer chromatography (HPTLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). The potential of this bacterial mixture to decolorize Reactive Violet 5 dye from manufacturing industry effluent is to be carried out using appropriate bioreactors.     

Decolorization of Some Reactive Textile Dyes by White Rot Fungi Isolated in Pakistan

Summary Four white-rot fungi isolated in Pakistan were used for decolorization of widely used reactive textile dyestuffs. Phanerochaete chrysosporium, Coriolus versicolor, Ganoderma lucidum and Pleurotus ostreatus were grown in defined nutrient media for decolorization of Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR for 10 days in shake flasks. Samples were removed every day, centrifuged and the absorbances of the supernatants were read to determine percentage decolorization. It was observed that P. chrysosporium and C. versicolor could effectively decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR. Drimarene Orange K-GL was completely decolorized (0.2 g/l after 8 days) only by P.chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also showed good decolorization efficiencies (0.19–0.2 g/l) on all dyes except Remazol Brilliant Yellow (0.07 g/l after 10 days). G. lucidum did not decolorize any of the dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 8 days).     

Decolorization of anthraquinone dye by Shewanella decolorationis S12

A new species of genus Shewanella, Shewanella decolorationis S12, from activated sludge of a textile-printing wastewater treatment plant, can decolorize Reactive Brilliant Blue K-GR, one kind of anthraquinone dye, with flocculation first. Although S. decolorationis displayed good growth in an aerobic condition, color removal was the best in an anaerobic condition. For color removal, the most suitable pH values and temperatures were pH 6.0–8.0 and 30–37°C under anaerobic culture. More than 99% of Reactive Brilliant Blue K-GR was removed in color within 15 h at a dye concentration of 50 mg/l. Lactate was the suitable carbon source for the dye decolorization. A metal compound, HgCl₂, had the inhibitory effect on decolorization of Reactive Brilliant Blue K-GR, but a nearly complete decolorization also could be observed at a HgCl₂ concentration of 10 mg/l. The enzyme activities, which mediate the tested dye decolorization, were not significantly affected by preadaptation of the bacterium to the dye.     

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.     

Decolorizing Reactive Textile Dyes with White‐Rot Fungi by Temporary Immersion Cultivation

The decontamination of effluents from textile industries is problematic due to the fact that textile dyes are resistant to degradation in the environment. Enzymes from white rot fungi, especially laccase, are able to degrade various complex aromatic structures, and are therefore able to decolorize textile dyes. The white‐rot fungi Trametes versicolor and Phanerochaete chrysosporium were immobilized, separately, on both pine wood chips and palm oil fiber, and cultivated in the temporary immersion RITA® (Récipient à Immersion Temporaire Automatique) System, which was adapted to serve as a fungal bioreactor in a series of four experiments to determine optimal conditions for decolorizing the textile dyes Levafix Blue and Remazol Brilliant Red. The maximum rate of decolorization of both dyes occurred within 24 h of incubation, and laccase was detected in the system.     

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.     

Azo dyes decolorization under high alkalinity and salinity conditions by Halomonas sp. in batch and packed bed reactor

Biodecolorization and biodegradation of azo dyes are a challenge due to their recalcitrance and the characteristics of textile effluents. This study presents the use of Halomonas sp. in the decolorization of azo dyes Reactive Black 5 (RB5), Remazol Brilliant Violet 5R (RV5), and Reactive Orange 16 (RO16) under high alkalinity and salinity conditions. Firstly, the effect of air supply, pH, salinity and dye concentration was evaluated. Halomonas sp. was able to remove above 84% of all dyes in a wide range of pH (6–11) and salt concentrations (2–10%). The decolorization efficiency of RB5, RV5, and RO16 was found to be ≥ 90% after 24, 13 and 3 h, respectively, at 50 mg L⁻¹ of dyes. The process was monitored by HPLC-DAD, finding a reduction of dyes along the time. Further, Halomonas sp. was immobilized in volcanic rocks and used in a packed bed reactor for 72 days, achieving a removal rate of 3.48, 5.73, and 8.52 mg L⁻¹ h⁻¹, for RB5, RV5 and RO16, respectively, at 11.8 h. The study has confirmed the potential of Halomonas sp. to decolorize azo dyes under high salinity and alkalinity conditions and opened a scope for future research in the treatment of textile effluents.

     

Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity

Amaranth, Tropaeolin O, Reactive Blue 15, Congo Red, and Reactive Black 5 were completely decolorized with no dye sorption by Trametes versicolor. Cibacron Brilliant Red 3G-P, Cibacron Brilliant Yellow 3B-A, and Remazol Brilliant Blue R were partially decolorized with some dye sorbed to the biomass. The Microtox assay before decoloration showed that Amaranth and Tropaeolin O were not toxic [the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC₂₀) was greater than 100%]; Cibacron Brilliant Yellow 3B-A, Reactive Blue 15 and Cibacron Brilliant Red 3G-P were moderately non-toxic (100% > EC₂₀ > 75%); Remazol Brilliant Blue R was toxic (75% > EC₂₀ > 50%); and Congo Red and Reactive Black 5 were moderately toxic (50% > EC₂₀ > 25%). After decoloration the toxicity of the solutions containing Amaranth, Tropaeolin O and Reactive Black 5 was unchanged; Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P decreased to non-toxic levels; and Cibacron Brilliant Yellow 3B-A and Congo Red became very toxic (EC₂₀ < 25%).