Redox-mediated decolorization of Direct Red 23 and Direct Blue 80 catalyzed by bioaffinity-based immobilized tomato (Lycopersicon esculentum) peroxidase

The aim of this study was to investigate the role of concanavalin A (Con A)-cellulose-bound tomato peroxidase for the decolorization of direct dyes. Cellulose was used as an inexpensive material for the preparation of bioaffinity support. Con A-cellulose-bound tomato peroxidase exhibited higher efficiency in terms of dye decolorization as compared to soluble enzyme under various experimental conditions. Both Direct Red 23 and Direct Blue 80 dyes were recalcitrant to the action of enzyme without a redox mediator. Six compounds were investigated for redox-mediating property. Immobilized peroxidase decolorized both dyes to different extent in the presence of all the used redox mediators. However, 1-hydroxybenzotriazole emerged as a potential redox mediator for tomato peroxidase catalyzed decolorization of direct dyes. These dyes were maximally decolorized at pH 6.0 and 40 degrees C by soluble and immobilized peroxidase. The absorption spectra of the untreated and treated dyes exhibited a marked difference in the absorption at various wavelengths. Immobilized tomato peroxidase showed a lower Michaelis constant than the free enzyme for both dyes. Soluble and immobilized tomato peroxidase exhibited significantly higher affinity for Direct Red 23 compared to Direct Blue 80.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

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     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decolorization of azo dyes using Basidiomycete strain PV 002

Summary Basidiomycete PV 002, a recently isolated white-rot strain from decomposed neem waste displayed high extracellular peroxidase and rapidly decolorized azo dyes. In this study, the optimal culture conditions for efficient production of ligninolytic enzymes were determined with respect to carbon and nitrogen. An additional objective was to determine the efficiency of PV 002 to degrade the azo dyes. White-rot strain PV 002 efficiently decolorized Ranocid Fast Blue (96%) and Acid Black 210 (70%) on day 5 and 9 respectively under static conditions. The degradation of azo dyes under different conditions was strongly correlated with the ligninolytic activity. The optimum growth temperature of strain PV 002 was 26 °C and pH 7.0.     

Use of bitter gourd (Momordica charantia) peroxidase together with redox mediators to decolorize disperse dyes

In this study, salt fractionated bitter gourd (Momordica charantia) peroxidase was used for the decolorization of water-insoluble disperse dyes; Disperse Red 17 and Disperse Brown 1. Effect of nine different redox mediators; bromophenol, 2,4-dichlorophenol, guaiacol, 1-hydroxybenzotriazole, m-cresol, quinol, syringaldehyde, violuric acid, and vanillin on decolorization of disperse dyes by bitter gourd peroxidase has been investigated. Among these redox mediators, 1-hydroxybenzotriazole was the most effective mediator for decolorization of both the dyes by peroxidase. Bitter gourd peroxidase (0.36 U/mL) could decolorize Disperse Red 17 maximally 90% in the presence of 0.1 mM 1-hydroxybenzotriazole while Disperse Brown 1 was decolorized 65% in the presence of 0.2 mM 1-hydroxybenzotriazole. Maximum decolorization of these dyes was obtained within 1 h of incubation at pH 3.0 and temperature 40°C. The application of such enzyme plus redox mediator systems may be extendable to other recalcitrant and water insoluble synthetic dyes using novel redox mediators and peroxidases from other new and cheaper sources.     

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 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.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.     

Decolorization of 1:2 metal complex dye Acid blue 193 by a newly isolated fungus, Cladosporium cladosporioides

Summary A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l⁻¹ in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l⁻¹) concentration of 564 mg l⁻¹ within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l⁻¹, laccase activity with 1413 U l⁻¹ and lignin peroxidase activity was negligible after day 8 of incubation.     

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.     

Glutathione and glutathione metabolizing enzymes in yeasts

Total glutathione content, glutathione peroxidase, glutathione transferase and glutathione reductase activities have been measured in 12 species of yeasts. All the strains tested contained glutathione, though in different amounts, as well as the above mentioned enzymes. To discriminate between the selenium-dependent and the selenium-independent form, glutathione peroxidase activity has been measured with both H₂O₂ and cumene hydroperoxide. Rhodotorula glutinis appeared to be the only strain in which the selenium-dependent form was not found, but this yeast exhibited the highest level of selenium-independent glutathione peroxidase activity as compared to the other strains.     

Purification and characterization of a novel peroxidase from Geotrichum candidum dec 1 involved in decolorization of dyes

A peroxidase (DyP) involved in the decolorization of dyes and produced by the fungus strain Geotrichum candidum Dec 1 was purified. DyP, a glycoprotein, is glycosylated with N-acetylglucosamine and mannose (17%) and has a molecular mass of 60 kDa and an isoelectric point (pI) of 3.8. The absorption spectrum of DyP exhibited a Soret band at 406 nm corresponding to a hemoprotein, and its Na2S2O4-reduced form revealed a peak at 556 nm that indicates the presence of a protoheme as its prosthetic group. Nine of the 21 types of dyes that were decolorized by Dec 1 cells were decolorized by DyP; in particular, anthraquinone dyes were highly decolorized. DyP also oxidized 2,6-dimethoxyphenol and guaiacol but not veratryl alcohol. The optimal temperature for DyP activity was 30 degrees C, and DyP activity was stable even after incubation at 50 degrees C for 11 h.     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.     

Evaluation of different fungal strains in the decolourisation of synthetic dyes

Of seven fungal strains tested for their ability to decolourise three structurally diverse synthetic dyes, Phanerochaete sordida, Bjerkandera sp. BOS55, Phlebia radiata, and Phanerochaete chrysosporium had average values of maximum decolourisation rates higher than 0.2 [Absorbance] d^–1. All seven fungi produced manganese peroxidase (MnP) but laccase activity was detected only in Phlebia radiata. No lignin peroxidase (LiP) activity was observed.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

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 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.