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.     

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.     

海洋产电菌Shewanella marisflavi EP1的脱色特性

以一株新筛选得到的海洋产电菌Shewanella marisflavi EP1作为实验材料,研究了该菌株关于偶氮、蒽醌、三苯基甲烷等染料的脱色能力及脱色机制。结果表明,该菌株对这些染料均具有较好的脱色能力,最高脱色容量达到925 mg染料/(g细胞干重.d)。EP1能利用葡萄糖、蔗糖、木糖、乳酸、甲酸、柠檬酸等多种碳源将单偶氮染料丽春红2R脱色。脱色的pH、温度和NaCl浓度范围分别是:pH 6-10、15°C-40°C、0-8%。最优脱色条件:乳酸,pH 8、35°C、1%-2%NaCl,10 h内脱色率高达99.95%。分光光谱结果表明,在0-8%NaCl浓度范围内EP1脱色机制为降解脱色。     

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.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes

The rate and efficiency of decolorization of poly R-478- or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 μg g⁻¹) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28 °C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

An evaluation of the genotoxic properties of some chosen dyes using the micronucleus test in vivo

The frequency of micronucleated polychromatic erythrocytes and the polychromatic to normochromatic erythrocyte ratio was studied in BalbC mice treated with four azo dyes: Direct Blue 74, Direct Blue 296, Direct Blue 297 and Direct Green 98 at two (40and 80% LD₅₀/kg body weight) concentrations. None of the studied compounds revealed a genotoxic activity in the micronucleus test. However, it was found that two dyes, Direct Blue 297 at doses 40% and 80% LD₅₀ and Direct Green 98 at dose 80% LD₅₀, cause a significant decrease in the ratio of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at the doses specified above they can affect the proliferation of the blood cells.     

脱色细菌的分离和对偶氮染料的脱色研究

从印染废水中分离到8株对多种染料具有较好脱色效果的细菌,在所试验的10种染料中对其中大部分都有较好的脱色作用,尤其对三种酸性黑10B、酸性黑NG、直接湖蓝的脱色率最高;在各菌株最适条件下对这三种染料脱色率都能达到80%以上,其中有些菌株对直接湖蓝的脱色率达到100%。本实验研究了这8个菌株在不同的pH值、温度、需氧量条件下对这三种染料的脱色情况,并对有代表性的菌株脱色前后的化学需氧量(COD)值进行测定来判断染料的降解情况。     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

The demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

The significance of azo-reduction in the mutagenesis and carcinogenesis of azo dyes

Azo dyes are widely used in textile, printing, cosmetic, drug and food-processing industries. They are also used extensively in laboratories as either biological stains or pH indicators. The extent of such use is related to the degree of industrialization. Since intestinal cancer is more common in highly industrialized countries, a possible connection may exist between the increase in the number of cancer cases and the use of azo dyes. Azo dyes can be reduced to aromatic amines by the intestinal microflora. The mutagenicity of a number of azo dyes is reviewed in this paper. They include Trypan Blue, Ponceau 3R, Pinceau 2R, Methyl Red, Methyl Yellow, Methyl Orange, Lithol Red, Orange I, Orange II, 4-Phenylazo-Naphthylamine, Sudan I, Sudan IV, Acid Alizarin Violet N, Fast Garnet GBC, Allura Red, Ponceau SX, Sunset Yellow, Tartrazine, Citrus Red No. 2, Orange B, Yellow AB, Carmoisine, Mercury Orange, Ponceau S, Versatint Blue, Phenylazophenol, Evan's Blue and their degraded aromatic amines. The significance of azo reduction in the mutagenesis and carcinogenesis of azo dyes is discussed.     

Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

Summary The potential of a recently isolated wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the fungus proved to be efficiently capable of both biodegradation and biosorption as the major dye removal mechanisms. The extent of biodegradation was associated with the levels of the extracellular ligninolytic enzymes such as manganese peroxidase and laccase.     

Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (<50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.     

Comparison of 7 azo dyes and their azo reduction products in the rat and hamster hepatocyte primary culture/DNA-repair assays

Pursuant to the characterization of species differences in the effects of chemical carcinogens, several studies have demonstrated that hamster hepatocytes are more effective than rat hepatocytes in mediating the metabolic activation of certain chemicals to their genotoxic (i.e., mutagenic) derivatives. In the present investigation, a comparison of the amount of DNA repair induced in rat and hamster hepatocytes by 7 azo dyes and 7 aromatic amine azo reduction products of the dyes was performed using the primary hepatocyte culture/DNA repair (HPC/DR) assay. Congo Red and its azo reduction product, benzidine, were more potent inducers of DNA repair in hamster than in rat hepatocytes, whereas Trypan Blue and its reduction product, o-tolidine, were equipotent in the 2 hepatocyte systems. Evans Blue, another o-tolidine-based dye, elicited a greater DNA-repair response in hamster hepatocytes. The absolute potency of these dyes, however, was much less than their reduction products. o-Aminoazotoluene was the most potent of the dyes tested, and its DNA repair-inducing activity was much greater than that of its azo reduction products, o-toluidine and 2,5-diaminotoluene. Ponceau SX, which is carcinogenic in hamsters, but not in rats, was inactive in both hepatocyte systems. Dimethylaminobenzeneazo-1-naphthalene and its 2-naphthalene congener, as well as the 1- and 2-naphthylamine azo reduction products of these dyes, were more potent in hamster than in rat hepatocytes. However, the DNA repair-inducing activities of the parent dyes could not be entirely accounted for by the potencies of their respective naphthylamine derivatives. Taken together, these findings extend previous observations of the superior metabolic activation capabilities of hamster, relative to rat hepatocytes, and further demonstrate the utility of testing chemicals in both the hamster and rat HPC/DR assays.     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005     

Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Brevibacillus laterosporus MTCC 2298: a potential azo dye degrader

Aims:  To evaluate the potential of Brevibacillus laterosporus MTCC 2298 for the decolourization of different textile azo dyes including methyl red, mechanism of biotransformation and the toxicity of products.
Methods and Results:  Brevibacillus laterosporus showed decolourization of thirteen different azo dyes including methyl red. Decolourization of methyl red was faster (93% within 12 h) under static condition at the concentration 0·2 g l⁻¹. Induction in the activities of lignin peroxidase, laccase, aminopyrine N-demethylase, NADH-DCIP reductase and malachite green reductase was observed in the cells obtained after decolourization. Fourier transform infra-red spectral analysis of products indicated conversion of methyl red into secondary aryl amines and nitrosamines, which further transformed into the aromatic nitro compounds. Gas chromatography–mass spectroscopy analysis suggested conversion of methyl red into high molecular weight complex derivatives. The heterocyclic substituted aryl amine ( m / z 281), p -(N,N di formyl)-substituted para -di amino benzene derivative ( m / z 355) and p -di-amino benzene derivative ( m / z 282) are the mainly elected biotransformation products. Microbial and phytotoxicity studies suggested nontoxic nature of the biotransformation products.
Conclusions:  Brevibacillus laterosporus has potential for the decolourization of different textile azo dyes.
Significance and Impact of the Study:  Brevibacillus laterosporus decolourized different azo dyes including methyl red and can be utilized for textile dye decolourization.     

Decolorization of textile azo dyes by aerobic bacterial consortium

The decolorization potential of two bacterial consortia developed from a textile wastewater treatment plant showed that among the two mixed bacterial culture SKB-II was the most efficient in decolorizing individual as well as mixture of dyes. At 1.3 g L^?1 starch supplementation in the basal medium by the end of 120 h decolorization of 80–96% of four out of the six individual azo dyes Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC (10 mg L^?1) was noted. The culture exhibited good potential ability in decolorizing 50–60% of all the dyes (Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC) when present as a mixture at 10 mg L^?1. The consortium SKB-II consisted of five different bacterial types identified by 16S rDNA sequence alignment as Bacillus vallismortis, Bacillus pumilus, Bacillus cereus, Bacillus subtilis and Bacillus megaterium which were further tested to decolorize dyes. The efficient ability of this developed consortium SKB-II to decolorize individual dyes and textile effluent using packed bed reactors is being carried out.