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N-Demethylation of Methylene Blue by Lignin Peroxidase from Phanerochaete chrysosporium
Abstract
Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H2O2. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylene blue and azure B (AB) in reaction mixtures containing different dye:H2O2 stoichiometric relations aiming at the selective formation of N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1:1 and 1:2 MB:H2O2, respectively. Using azure B as substrate in reaction mixtures containing 1:1 AB:H2O2, a yield of 70% was also observed in azure A. Reaction mixtures containing 1:3 MB:H2O2 and 1:2 AB:H2O2, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH3)2 oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye:H2O2 relation of 1:10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500-700 nm. The product identifications were performed using a reverse-phase high-performance liquid chromatography (HPLC) C-18 column and thin-layer chromatography.
... The role of fungi and their enzymes and their potential use in degradation and detoxification of CV has been well reported and recognized (Ferreira et al. 2000; Mielgo et al. 2001; Claus et al. 2002; Assadi et al. 2003 ). Concerning the dye degradation , the most widely used fungi are the ligninolytic fungi. ...
... Whereas peroxidase enzymes such as lignin peroxidase and manganese peroxidase acts as electron acceptor, which require hydrogen peroxide or alkyl, peroxide that are not specific towards the electron donor in the redox reactions that they catalyze (Moturi and Singara 2009; Kersten et al. 1990). The role of fungi and their enzymes in degradation and detoxification of organic pollutants has been well reported globally because of their wide versatility and broad range of substrates (Assadi et al. 2003; Claus et al. 2002; Ferreira et al. 2000; Mielgo et al. 2001; Moturi and Singara 2009). Several studies have demonstrated the ability of fungal biomass and purified enzymes to degrade and decolorize a category of different types of organic pollutants (Wesenberg et al. 2003). ...
Crystal Violet (CV), a triphenylmethane dye, has been extensively used in human and veterinary medicine as a biological stain, as a textile dye in textile processing industries and also used to provide a deep violet color to paints and printing ink. CV is also used as a mutagenic and bacteriostatic agent in medical solutions and antimicrobial agent to prevent the fungal growth in poultry feed. Inspite of its many uses, CV has been reported as a recalcitrant dye molecule that persists in environment for a long period of time and pose toxic effects. It acts as a mitotic poison, potent carcinogen and a potent clastogene promoting tumor growth in some species of fish. Thus, CV is regarded as a biohazard substance. Although, there are several physico-chemical methods such as adsorption, coagulation and ion-pair extraction reported for the removal of CV, but these methods are insufficient for the complete removal of CV from industrial wastewaters and also produce large quantity of sludge containing secondary pollutants. However, biological methods are regarded as cost-effective and eco-friendly for the treatment of industrial wastewaters, but these methods also have certain limitations. Therefore, there is an urgent need to develop such eco-friendly and cost-effective biological treatment methods, which can effectively remove the dye from industrial wastewaters for the safety of environment, as well as human and animal health.
... One promising strategy for the environment is the use of the decolourising ability of ligninolytic fungi such as Phanerochaete chrysosporium , Pleurotus ostreatus , Trametes versicolor , Bjerkandera fumosa , Irpex lacteus and some others, all producing extracellular peroxidases and laccases that cooperate to degrade lignin and synthetic dyes (Fu & Viraraghavan 2001; Jarosz-Wilkolazka et al. 2002; Wesenberg et al. 2003; Liu et al. 2004). Extracellular enzymes of these fungi have been shown to decolourise a broad spectrum of structurally diverse dyes (Ollikka et al. 1993; Rodriguez et al. 1999; Ferreira et al. 2000; Hou et al. 2004), whereas the decolourisation rate and the mechanism of the dye degradation can vary among ligninolytic enzymes produced by the same fungus (Podgornik et al. 2001). Peroxidases and laccases can catalyse degradation/transformation of aromatic dyes either by precipitation or by opening the aromatic ring structure (Husain 2010 ). ...
... Anthraquinone is the main part of the molecules of synthetic and natural dyes called " the anthraquinone-type dyes. " Different authors describe the different effi ciencies in the dye decolourisation reactions catalysed by fungal peroxidases, which are dependent on the type of dyes, the source of enzyme and the addition of different additives (Ferreira et al. 2000; Wesenberg et al. 2003; Husain 2010). We investigated the catalytic activity of VPBF and VPPO towards several anthracene-derived dyes, including Acridine Orange, Neutral Red, Butyl Rhodamine B, Rhodamine 6G, Safranin T and Eosin Y. ...
The catalytic properties of a versatile peroxidase from Pleurotus ostreatus D1 (Jacquin) P. Kummer were studied in comparison with that of a typical versatile peroxidase from Bjerkandera fumosa 137 (Per.:Fr) Karst. Decolourisation activities of both enzymes towards a wide range of dyes containing condensed aromatic rings (anthraquinone- and anthracene-type) were found. The anthraquinone dyes were decolourised rapidly by both tested peroxidases. The presence of polymerisation reaction products of Acid Blue 62, Basic Blue 22 and Reactive Blue 4 oxidation, and breakdown of aromatic rings of Alizarin Red were observed. The main catalytic constants (KM and Vmax) of the decolourisation reactions of anthraquinone dyes were calculated. In the case of Alizarin Red, inhibition of the activity of versatile peroxidase from P. ostreatus D1 by an excess of the substrate was observed. Independence from Mn2+ ions of the catalytic activity of versatile peroxidase from P. ostreatus D1 towards different substrates was revealed. Finally, differences in the catalytic activity towards anthracene-type dyes and monoaromatic substrates of both peroxidases were found.
... These are some of the various extracellular types involved: H2O2 A c c e p t e d independent oxidase, laccase, and azoreductase to degrade the reactive yellow of 84A by Exiguobacterium sp. RD3 [51]. Also, Pseudomonas aeruginosa BCH is known to secrete tyrosinase, NADH-DCIP reductase, veratryl alcohol oxidase, and laccase are implicated in amaranth azo dye decomposition [52]. ...
The biotransformation and biodecolorization of methylene blue (MB) dye using the bacterium Ralstonia pickettii was investigated. This experiment was conducted in a nutrient broth (NB) medium after adding MB at 100 mg L-1 concentration. Approximately 98.11% of MB was decolourized after 18 h of incubation. In addition, the metabolic products detected by LC-TOF/MS were Azure A (AA), thionine, leuco-MB, and glucose-MB, which indicated the MB degradation through a reductase that attacked the heterocyclic central chromophore group present in the structure. Moreover, azure A and thionine fragments resulted from the N-demethylase enzyme that attacked the auxochrome group. Thus, this research was assumed to be the first scientific report suggesting the potential to use R. pickettii in the biodecolorization and biotransformation of dye waste, particularly MB.
... Table 2.2 summarizes dyes degraded by fungal peroxidases. Ferreira et al. (2000) reported the degradation of Methylene Blue and Azure B by LiP from P. chrysosporium. The study of reaction products showed that N-demethylation reactions were involved in the degradation of such dyes. ...
... Nevertheless there have been numerous attempts to develop biological processes for the treatment of textile effluents either based on partial or complete biodegradation of dyes by pure and mixed cultures of bacteria, fungi and algae (Arora & Chander 2004, Banat et al. 1996, Bhatt et al. 2000, Chen et al. 2003, Ferreira et al. 2000, Govindwar et al. 2014, Jin et al. 2007, Ma et al. 2014, Machado et al. 2006, McMullan et al. 2001, Mohan et al. 2013, Muthukumaran & Mathumitha 2013, Sandhya et al. 2005, Saratale et al. 2013, Semple et al. 1999, Wesenberg et al. 2003, Zheng et al. 1999. White rot fungi that produce ligninolytic enzymes, such as lignin peroxidase, manganese peroxides and laccase have been studied extensively because of their ability to degrade various complex organic compounds. ...
Synthetic dyes are released in the effluent from a wide variety of industries such as textile, tannery, packed food, pulp and paper and paint, thus threatening various forms of life. Bioremediation is always considered as cost effective and eco-friendly way for the treatment of recalcitrant dyes and effluents. Non-white rot fungus Aspergillus fumigatus A23 and white rot fungus Phanerochaete chrysosporium were used for comparative study of decolorization of individual dyes and simulated textile effluent (STE). Both the fungi could effectively decolorize STE under optimized conditions of medium (potato dextrose agar medium), temperature (40ºC for A. fumigatus A23 and 30ºC P. chrysosporium), pH (4.0 for A. fumigatus A23 and 5.0 for P. chrysosporium) and agitation (100 rpm for A. fumigatus A23 and P. chrysosporium). The decolorization of STE by A. fumigatus A23 and P. chrysosporium was 86% and 62% respectively after 7d incubation. The key mechanisms involved in dye removal by the fungus appeared to be adsorption and absorption and the biotransformation occurred only after absorption of the dye. Analysis of samples before and after treatment with fungus using TLC indicated the biotransformation of dye.
... In MnPcatalyzed oxidation of indigo carmine, a stable reddish product, probably a dimeric condensation product, is formed instead. P. chrysosporium cultures, extracellular fluid, and purified peroxidases have been reported to degrade generally recalcitrant crystal violet and six other triphenylmethane dyes [123,124]. The degradation of these dyes follows N-demethylation reactions. ...
Extensive use of synthetic dyes and their subsequent release in industrial wastewater is a growing environmental problem. These dyes are recalcitrant in nature, and some dyes are also well established to be potentially carcinogenic and mutagenic as well as genotoxic. Research efforts have been devoted to develop new, low-cost, and eco-friendly treatments capable of reducing and even eliminating synthetic dye com‐ pounds from the environment. Enzymatic approach has attracted much interest recently in the decolorization of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical, and biological treatments, which pose serious limitations. In this chapter, the accumulated research data on the potential of the oxidoreductive enzymes—high redox potential peroxidases (lignin peroxidase [LiP] 1.7.1.6)—that have been exploited in the decolorization and degradation of synthetic dyes are presented. An overview of enzyme technology, including the importance of redox mediators for enhanced range of substrates and efficiency of degradation, current biodegradation applications, and suggestions to overcome the limitations to these proteins' large scale and efficient use, is made. Different strategies currently being used and future prospects for the potential use of genetic engineering techni‐ ques to improve the performance of these oxidoreductases in terms of stability, selectivity, and catalytic activity in dye bioremediation technologies are also explored.
... Azo dyes constitute the largest group of colorants used in industry (Maynard, 1983; Zollinger, 1987; Grag, et al., 2004 ), however, environmental fate of these compounds still needs to be understood for mitigating their affects. Dyes are removed by fungi by biosorption (Conatao and Corso, 1996; Paymann and Mehnaz, 1998; Fu and Viraraghavan, 2000), biodegradation (Nigam et al., 1996; Conneely et al., 1999) and enzymatic mineralization (Lignin peroxidase (LiP), manganese peroxidase (MnP), manganese independent peroxidase (MIP) and laccases (Lacc) (Ferreira et al., 2000; Pointing and Vrijmoed, 2000; Wesenberg et al., 2003). However, one or more of these mechanisms could be involved in color removal, depending on the fungus used. ...
Different physicochemical cultural conditions were optimized for azo dye removal by using Acid Red 151 as a model dye, being of high consumer demand and usage during the present study. The three fungal strains having the dye removal abilities, Aspergillus niger SA1, Aspergillus flavus SA2 and Aspergillus terreus SA3 were selected and processed for different optimization studies in shake flask fermentations. Aspergillus nidulans minimal media proved to be most effective for utmost decolorization of AR 151 dye. The effect of inoculum size on dye decolorization efficiency of fungal strains showed that, best level decolorization was observed with 2% inoculum in all three fungal isolates. Effect of different concentration of Acid Red 151 dye ranging from 50 to 200 ppm on the selected fungal strains showed decolorization of AR 151 dye up to their maximal limits (200 ppm) to more than 60%. However, highest percentage of decolorization was shown by A. flavus SA2 (92.56%) with lower concentration of dye (50 ppm) and increase in concentration of dye showed a negative effect on decolorization percentage of all the tested fungal strains. There was significant decolorization (>60%) from all fungal strains with different pH ranging from 3 to 10. However, optimum pH for all the three fungal strains was found to be pH 7. There was an influence of temperature on decolorization as maximum decolorization was observed by all the three strains at 30°C (>85%) and it reduced with lower and higher temperatures. Maximum decolorization was observed at 0.5 - 1 M nitrogen concentration by all the three strains, where higher concentration had a negative effect. Similar results were observed for increase in concentration of carbon source as there was increasing trend of decolorization efficiencies of all the fungal strains.
... To remove dye from waste water, the physical – chemical methods like adsorption, chemical precipitation, flocculation, photolysis, chemical oxidation and reduction, electro-chemical treatment and ion-pair extraction were extensively used (Ansari & Thakur, 2002; Zhang et al., 2002). The importance of fungi and their enzymes in the dye degradation has been well appreciated globally, because of their potential use in detoxification and degradation of dyes (Ferreira et al., 2000; Mielgo et al., 2001; Claus et al., 2002; Assadi et al., 2003), but these methods are costly. Biological methods of treatment combined with physical or chemical methods or both for colour removal are immensely useful and cost effective. ...
Decolourisation of crystal violet and malachite green by white rot fungi, Polyporus elegans, Trametes versicolor, Lenzites betulina and soil fungus Mucor mucedo isolated from dye effluent amended soils was studied. There was no toxic effect of crystal violet on the growth of the four fungi but malachite green showed retardation of growth. Mucor mucedo decolourised 78% of the crystal violet and 65% of malachite green. The white rot fungi showed more than 60% decolourisation of crystal violet and 26 to 57% decolourisation of malachite green. In the process of decolourisation, lignin peroxidase production was high at 15 days incubation by all the organisms. Manganese peroxidase was secreted more after 10 days of incubation and laccase production was high after 15 days of incubation by Polyporus elegans and Trametes versicolor and after 10 days in the case of Lengites betulina. Mucor mucedo failed to secrete manganese peroxidase and laccase in all its incubations.
... The organisms used in most of the study were Staphylococcus sp., Many studies have demonstrated that the white-rot fungi, namely Phanerochaete chrysosporium, Bjerkandera adusta, Trametes versicolor, or Phlebia radiata, are able to degrade a broad spectrum of structurally diverse dyes. Decolorization of azo, anthraquinonic, heterocyclic, triphenylmethane and polymeric dyes and their partial mineralization by enzymatic and non-enzymatic systems of these fungi have been reported by Ferreira et al. (2000). Fungal systems appear to be most appropriate biological agent in the treatment of colored and metallic effluents. ...
The present investigation focused on the isolation and characterization of fungal strains which can efficiently decolorize Red HE7B (C.I. Reactive Red 141) and Yellow FN2R (C.I. Reactive Yellow 206) textile dyes. A total of 6 indigenous fungal strains were isolated from the effluents collected around the discharge site of textile industry situated in Salem District. The fungal isolates were identified as Aspergillus niger, A. flavus, Fusarium sp., Penicillium sp., Curvularia verruciformis and Mucor racemosus. Decolorization capabilities of these fungal species against the azo dyes were carried out in potato dextrose agar medium under static invitro condition. Highest percentage of degradation was achieved against Red HE7B and Yellow FN2R by Aspergillus niger (94%) and Mucor racemosus (92%) after 5 d of incubation. This study has confirmed the potential of the test fungi in the decolorization of azo dyes and opened scope for the future analysis of their performance in the treatment of textile effluent.
... Most of these dyes are mutagenic or carcinogenic and cannot be completely removed by conventional wastewater treatment systems. Laccases from several macrofungi have shown a high capacity to decolorize a broad spectrum of structurally diverse dyes (Rodríguez et al., 1999;Ferreira et al., 2000;Eliana and Lucia, 2001;Robinson et al., 2001;Suwannawong et al., 2010;Theerachat et al., 2012). The decolorization rate depends on the structure and the redox-potential of the enzyme as well as the structure of the dye (Nyanhongo et al., 2002). ...
Wild edible macrofungi are known to produce a wide range of biologically active metabolites and enzymes. In the present study, macrofungi consumed by the mycophillic ethnic tribes of India were collected from the local markets and forest habitats and identified based on their morphology. They belonged to ten different species under nine genera and eight families. Amylase, cellulase, protease, tyrosinase and laccase enzymes of the macrofungi were investigated. Two strains of Lactarius showed higher activity of the enzyme laccase and were subjected to further purification and analysis. The partially purified laccases from these two strains showed efficient dye decolourization ability when tested against four different synthetic dyes. The present investigation suggests the potential of these wild edible macrofungi in the production of biotechnologically important enzymes for use in an array of applications from pharmaceuticals to treatment of
chemical and biological effluents
... The degradation of dyestuffs by fungi has been carried out with either whole cultures or crude enzyme preparation of extracellular ligninolytic enzymes. Decolourisation of azo, anthraquinonic, heterocyclic, triphenylmethane and polymeric dyes and their partial mineralisation by enzymatic and non-enzymatic systems of these fungi have been reported (14, 36). Several ascomycetes and basidimycetes fungi have been reported to produce the lignin-degrading enzymes laccase, Lignin Peroxidases (LiP) and Manganese Peroxidases (MnP), or at least one of these enzymes (13). ...
Azo, anthroquinone and triphenylmethane dyes are the major classes of synthetic colourants, which are difficult to degrade and have received considerable attention. Congo red, a diazo dye, is considered as a xenobiotic compound, and is recalcitrant to biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several fungi, under certain environmental conditions, are able to transfer azo dyes to non toxic products using laccases. The aim of this work was to study the factors influencing mycoremediation of Congo red. Several basidiomycetes and deuteromycetes species were tested for the decolourisation of Congo red (0.05 g/l) in a semi synthetic broth at static and shaking conditions. Poor decolourisation was observed when the dye acted as the sole source of nitrogen, whereas semi synthetic broth supplemented with fertilizer resulted in better decolourisation. Decolourisation of Congo red was checked in the presence of salts of heavy metals such as mercuric chloride, lead acetate and zinc sulphate. Decolourisation parameters such as temperature, pH, and rpm were optimized and the decolourisation obtained at optimized conditions varied between 29.25- 97.28% at static condition and 82.1- 100% at shaking condition. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis revealed bands with molecular weights ranging between 66.5 to 71 kDa, a characteristic of the fungal laccases. High efficiency decolourisation of Congo red makes these fungal forms a promising choice in biological treatment of waste water containing Congo red.
... Although the studies on the ligninolytic peroxidases were first motivated by their industrial applications in pulp and paper industries such as biochemical pulping and decolorization of bleach plant effluent (Eaton et al. 1980; Jurasek and Paice 1986; Archibald et al. 1990; Higuchi 1990; Dezotti et al. 1995; Bajpai 1999), numerous reports were published in recent years on the use of these enzymes for the degradation of xenobiotic compounds such as PAHs (Haemmerli et al. 1986b), chlorinated phenols (Hammel and Tardone 1988; Mileski et al. 1988 ), dioxins (Hammel et al. 1986; Valli et al. 1992), bisphenol A (Hirano et al. 2000), and synthetic dyes (Young and Yu 1997; Ferreira et al. 2000). A few studies on the removal of phenolic compounds using LiP and MnP though free radical coupling and polymerization , rather than the degradation, are also reported. ...
The use of enzymes for the treatment or the removal of environmental and industrial pollutants has attracted increasing attention because of their high efficiency, high selectivity, and environmentally benign reactions. Of these enzymes studied for such purposes, extracellular fungal peroxidases, such as lignin peroxidase, manganese peroxidase and Coprinus cinereus peroxidase, and fungal laccases are the two major classes of enzymes that have been evaluated for the removal of toxic phenolic compounds from industrial wastewater and the degradation of recalcitrant xenobiotics. Numerous reports have been published recently on the improvements of the production of these enzymes, such as discovery of new fungal strains, modification of growth conditions, use of inducers, and use of cheaper growth substrates such as agricultural and food wastes. In this review, these recent advances in the production of extracellular fungal peroxidases and laccases, along with brief summaries of background of these enzymes and their applications, are discussed. Key words: Arthromyces ramosus peroxidase, Coprinus peroxidase, degradation, fermentation, laccase, lignin peroxidase, manganese peroxidase, phenols, wastewater treatment, xenobiotics.
... S. Ahmed e-mail: safiamrl@yahoo.com peroxidase (LiP), manganese peroxidase (MnP), manganese independent peroxidase (MIP), laccases (Lacc s )] (Young and Yu 1997; Ollikka et al. 1998; Podgornik et al. 1999; Wong and Yu 1999; Zheng et al. 1999; Ferreira et al. 2000; Pointing and Vrijmoed 2000; Minussi et al. 2001; Wesenberg et al. 2003; Svobodova et al. 2006). However, one or more of these mechanisms could be involved in color removal, depending on the fungus used. ...
The fungal strain, Aspergillus niger SA1, isolated from textile wastewater sludge was screened for its decolorization ability for four different textile dyes.
It was initially adapted to higher concentration of dyes (10–1,000mgl−1) on solid culture medium after repeated sub-culturing. Maximum resistant level (mgl−1) sustained by fungal strain against four dyes was in order of; Acid red 151 (850)>Orange II (650)>Drimarene blue K2RL (550)>Sulfur black (500). The apparent dye removal for dyes was seen largely due to biosorption/bioadsorption into/onto
the fungal biomass. Decolorization of Acid red 151, Orange II, Sulfur black and Drimarine blue K2RL was 68.64 and 66.72, 43.23 and 44.52, 21.74 and 28.18, 39.45 and 9.33% in two different liquid media under static condition,
whereas, it was 67.26, 78.08, 45.83 and 13.74% with 1.40, 1.73, 5.16 and 1.87mgl−1 of biomass production under shaking conditions respectively in 8days. The residual amount (mgl−1) of the three products (α-naphthol, sulfanilic acid and aniline) kept quite low i.e., ≤2 in case AR 151 and Or II under shaking
conditions. Results clearly elucidated the role of Aspergillus niger SA1 in decolorizing/degrading structurally different dyes into basic constituents.
... This degradative ability has opened up new prospects for the development of biotechnological processes aimed at the degradation of complex polymers such as xenobiotics for effluent decolorization and biobleaching of the lignin in kraft pulp (Lopez et al. 2002). One promising strategy is the use of microbes including white-rot fungal and bacterial strains that possess the ability to decolorize synthetic dyes (Ferreira et al. 2000). Microbial decolorization and degradation are an environmentally friendly and costcompetitive alternative to physico-chemical decomposition processes for the treatment of industrial effluents (Verma and Madamwar 2003). ...
A newly isolated white-rot fungus, Armillaria sp. strain F022, was isolated from the decayed wood in a tropical rain forest. Strain F022 was capable of decolorizing a
variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes, with an optimal efficiency of decolorization
obtained when dyes added after 96h of culture, with the exception of Brilliant Green. All of the tested dyes were decolorized
by the purified laccase in the absence of any redox mediators, but only a few were completely removed, while others were not
completely removed even when decolorization time was increased. The laccase, with possible contributions from unknown enzymes,
played a role in the decolorization process carried out by Armillaria sp. F022 cultures, and this biosorption contributed a negligible part to the decolorization by cultures. The effect of dye
to fungal growth was also investigated. When dyes were added at 0h of culture, the maximum dry mycelium weight (DMW) values
in the medium containing Brilliant Green were 1/6 of that achieved by the control group. For other dyes, the DMW was similar
with control. The toxic tolerance of dye for the cell beads was excellent at least up to a concentration of 500mg/l. The
optimum conditions for decolorization of three synthetic dyes are at pH4 and 40°C.
Keywords
Armillaria sp. F022–Brilliant Green–Laccase activity–Microbial decolorization–Reactive Black 5–Remazol Brilliant Blue R
... Insufficient treatment of wastes of the dyestuff industries leads to dye contamination of the environment such as soil and natural water bodies (Pearce et al., 2003;Nigam et al., 1996). One promising strategy is the use of microbes that possess the ability to decolorize synthetic dyes including whiterot fungal and bacterial strains (Liu et al., 2004;Elaine and Lucia, 2001;Stolz, 2001;Robinson et al., 2001;Ferreira et al., 2000;Elizabeth et al., 1999). Microbial decolorization and degradation is an environmentally friendly and cost-competitive alternative to physico-chemical decomposition processes for the treatment of industrial effluents (Verma and Madamwar, 2003). ...
In this study, we isolated and characterized a new strain of Bacillus cereus, DC11, capable of decolorizing a broad-spectrum of dyes. Parameters including pH, temperature, oxygen concentration and carbon source were used to comparatively study the decolorizing effects on anthraquinone, triphenylmethane, and azo dyes, respectively. High decolorization efficiency (95–98%) was achieved within 6 h of incubation for 100 μM Acid Blue 25 (anthraquinone dye), 4 h for 55 μM Malachite Green (triphenylmethane dye), and 2 h for 750 μM Basic Blue X-GRRL (azo dye) at 20–45 °C and neutral pH under anaerobic conditions. Decolorizing activities, which showed NADH/NADPH-dependent traits were limited to the soluble cytosolic fractions. Among the decolorized products, there was an intermediate metabolite after the degradation of Acid Blue 25 dye. Malachite Green was degraded into 4,4′-bis(dimethylamino)benzophenone and benzophenone, and the decolorization of Basic Blue X-GRRL was probably due to the reduction of the azo bonds. Our new isolated strain DC11 is the first reported Bacillus cereus strain which can efficiently decolorize all three main group dyes, and the converging parameters for the decolorization of three group dyes may be useful for bioremediation applications.
... Nowadays other fungi have also shown some capacities to remove dyes from industrial effluents. Dyes are removed by fungi by biosorption (Contato and Corso, 1996; Tatarko and Bumpus, 1998; Payman et al., 1998; Zheng et al., 1999; Fu and Viraraghavan, 2000), biodegradation (Nigam et al., 1995; Conneely et al., 1999) and enzymatic mineralisation (LiP, MnP, manganese independent peroxidase (MIP), Lacc) (Young and Yu, 1997; Ferreira et al., 2000; Ollikka et al., 1998; Podgornik et al., 1999; Wong and Yu, 1999; Zheng et al., Coulibaly et al. 625 1999; Pointing and Vrijmoed, 2000; Wesenberg et al., 2003). However, one or more of these mechanisms could be involved in colour removal, depending on the fungus used. ...
Fungal biomasses are capable of treating metal-contaminated effluents with efficiencies several orders of magnitude superior to activated carbon (F-400) or the industrial resin Dowex-50. Additionally, fungal biomasses are susceptible to engineering improvements and regeneration of their capabilities. With regard to organic pollutants, excessive nutrients and dyes, fungi can remove them from wastewaters, leading to a decrease in their toxicities. However, the detoxification rates seem to be dependent on media and culture conditions. The postreatement by anaerobic bioprocesses of effluents that have been pretreated with fungi can lead to higher biogas than the original effluents. In addition to the degradation of organic pollutants, fungi produce added-value products such as enzymes (LiP, MnP, Lacc, amylase, etc.) and single-cell protein (SCP). Most research on fungal capacities to purify polluted effluents has been performed on a laboratory scale, hence there is a need to extend such research to pilot scale and to apply it to industrial processes.
Romanowsky staining was an important methodological breakthrough in diagnostic hematology and cytopathology during the late 19th and early 20th centuries; it has facilitated for decades the work of biologists, hematologists and pathologists working with blood cells. Despite more than a century of studying Romanowsky staining, no systematic review has been published that explains the chemical processes that produce the "Romanowsky effect" or "Romanowsky-Giemsa effect" (RGE), i.e., a purple coloration arising from the interaction of an azure dye with eosin and not due merely to their simultaneous presence. Our review is an attempt to build a bridge between chemists and biomedical scientists and to summarize the available data on methylene blue (MB) demethylation as well as the related reduction and decomposition of MB to simpler compounds by both light and enzyme systems and microorganisms. To do this, we analyze modern data on the mechanisms of MB demethylation both in the presence of acids and bases and by disproportionation due to the action of light. We also offer an explanation for why the RGE occurs only when azure B, or to a lesser extent, azure A is present by applying experimental and calculated physicochemical parameters including dye-DNA binding constants and electron density distributions in the molecules of these ligands. Finally, we discuss modern techniques for obtaining new varieties of Romanowsky dyes by modifying previously known ones. We hope that our critical literature study will help scientists understand better the chemical and physicochemical processes and mechanisms of cell staining with such dyes.
The problem of industrial dye wastewater poses a critical environmental challenge that demands urgent attention. This is because the direct release of synthetic dyes such as Methylene Blue (MB) into water bodies has been found to have adverse effects on the environment. Therefore, this study aimed to propose immobilization of a mixed Trichoderma viride and Ralstonia pickettii culture into Sodium alginate–Polyvinyl Alcohol-Bentonite (SA-PVA-Bentonite) matrix as a development method for MB decolorization and degradation. Immobilization process was carried out using the entrapment method, where bacteria and fungi cells were homogenized into the SA-PVA-Bentonite matrix. The results showed that immobilized culture (IMO Mix) outperformed the free cells in Mineral Salt Medium (MSM), achieving an impressive 97.88% decolorization rate for 48 h at 30 °C. Furthermore, a total of 3 metabolite product degradation were produced including Azure A and C, as well as Thionine by LCMS analysis. SEM-EDX analysis confirmed that culture was agglomerated within the SA-PVA-Bentonite matrix, while FTIR demonstrated the functional groups of the synthesized beads. Meanwhile, the difference in charge of bentonite facilitated the adsorption of MB onto the beads, and mixed culture supported the degradation process. This study presented a potential solution to environmental problems, particularly those related to the industrial sector. Further analysis was required to address the challenges associated with other industrial dye waste.
Methylene blue (MB) is one of synthetic dyes that is used in the textile industry which is difficult to degrade in nature. Previously, the brown-rot fungus (BRF) Daedalea dickinsii had shown a good ability to degrade MB, however, the decolorization ability was relatively still low and had a long period of incubation. Therefore, improvement of process is needed to increase the ability of D. dickinsii to decolorize MB. In this study, the effect of Ralstonia pickettii bacterium addition on MB biodecolorization by the BRF D. dickinsii in potato dextrose broth (PDB) medium was investigated. The amount of R. picketti that was added to the culture of D. dickinsii were 2, 4, 6, 8, and 10 mL (1 mL ≈ 1.39 × 10⁸ CFU). The cultures had ability to decolorize MB (100 mg/L) at 30 °C after 7 days incubation. The highest percentage of MB biodecolorization was obtained at addition of 10 mL of R. pickettii approximately 89%, while biodecolorization process by particularly D. dickinsii was approximately 17%. The MB degradation metabolites by mixed cultures of D. dickinsii and 10 mL of R. pickettii were Azure A, thionine, glucose-MB, C12H11N3SO6 and C12H13N3O6. This study indicated that the addition of R. pickettii could enhance MB biodecolorization by fungus D. dickinsii. Besides that, this study also indicated that mixed cultures of D. dickinsii and R. pickettii has great potential for high efficiency, fast and cheap dye wastewater treatment.
Dyes used in textile industry generate large amount of wastewater that needs to be treated effectively to prevent toxicity. Treatment of dye-rich wastewater by physico-chemical methods proves costly and less efficient, therefore biological methods (cost-effective and eco-friendly) have been developed. A variety of biological materials have been evaluated for their capacity to remove/treat toxic compounds such as dyes. Fungal biomass has shown high efficiency to remediate dye-rich wastewater. Fungi remove these compounds via decolorization and degradation. Reductive and oxidative enzymes assist in degradation of dyes. Many strains of filamentous fungi have shown high capacity to treat dyes. Hydrolytic enzymes secreted by filamentous fungi assist in the treatment of dyes. Lacasse enzyme produced by lignolytic fungi helps in the degradation of dyes. Enormous capacity of fungi for removal/remediation of dyes can be exploited to develop technologies for large-scale treatment of dye-containing wastewater.KeywordDecolorizationDegradationDyesLaccasesLignolytic
In the present work, one-dimensional InVO4/BiVO4 heterostructured nanobelts with the width of about 800 nm have been successfully prepared by a simple electrospinning technique followed by the subsequent calcination process. The prepared products were characterized by thermogravimetry, fourier transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, UV–Vis absorbance spectroscopy, high-performance liquid chromatography, and photoluminescence spectroscopy. The obtained InVO4/BiVO4 heterostructured nanobelts presented an admirable morphology and excellent photocatalytic properties for the degradation of methylene blue solution under visible-light irradiation.
Graphical Abstract
The electrospun precursor samples (a and b) displayed a well-defined one-dimensional (1D) belt structure. After calcined at 550 °C for 2 h (c and d), the samples can retain well the 1D morphology. And an obvious porous structure can be found from the TEM images of the calcined samples (e and f).
One can describe industries involving dye treatment as the essentials (food, beverage, and textile) simply since they involve application to entities that are used for necessity. Dye wastewater is generated from the intense application within its industry. However, it is more common to find research on the treatment and application of dyes within the textile industry, as it has been highlighted and targeted by federal regulations. Some of the more harmful components from dye wastewater include the presence of biochemical oxygen demand (BOD), chemical oxygen demand (COD), and color, to name a few. Direct discharge from the plant can cause potential harm toward a water body, impacting the lives of human health and the environment. The purpose of this chapter is to provide a survey of various treatment methods and review the literature that has been applied for the purpose of treating wastewater produced by the textile dye industry. In addition, the text will discuss some of the important preceding topics prior to treatment applications such as legislation, and definition and classification of dyes. The aim of this chapter is to be both comprehensive and concise describing some of the major components that are heavily integrated within the dyeing industry.
Microbial biotreatment of wastewaters is a concern in recent years. Discharge of toxic pollutants to wastewater collection systems has increased concurrently with society’s progressive industrialization. Although industrialization is inevitable, various devastating ecological and human disasters which have continuously occurred implicate industries as major contributors to pollution problems and environmental degradation of various magnitudes. Organic and inorganic substances which were released into the environment as a result of agricultural and industrial water activities lead to organic and inorganic pollution. It stands to reason that an effective treatment of these wastewaters is necessary. Microorganisms have been tested primarily as an approach for the removal of organic pollutants from wastewaters and have been proven effective at reducing chemical oxygen demand (COD) and toxicity. Biological treatment in the study provides some of the most viable options for the treatment of wastewaters. Microbial degradation of industrial wastewaters involving the application of a variety of microorganisms has demonstrated effective degradability of wastewaters which has attracted attention in recent time. The utilization of these microorganisms for bioremediation of toxic industrial wastewaters offers a very efficient tool for biopurification of contaminated effluents. Bacterial and fungal strains in this study have huge capability of treating wastewaters discharged from various industries. They are ubiquitous in nature and their adaptability to extreme conditions makes them good biodegraders. Their enzyme producing activity makes them effective decolorizers and they remove toxic metals by adsorption ultimately rendering the wastewaters more ecofriendly. Noteworthy, the bacterial and fungal biomasses present many assets for the biopurification of wastewaters.
Synthetic organic dyes such as azo dyes in wastewater discharged by dye manufacturing and textile industries constitute a major environmental concern due to their poor biodegradability and toxicity; as well as affecting aesthetics and inhibiting photosynthesis and other biological activities in receiving water. Most synthetic dyes contain characteristic multiple aromatic rings that are fused and/or linked by various C-C, C-O, and C-N linkages and substituted by multiple functional groups such as amino, nitro, hydroxyl, sulfonate, carbonyl, and carboxylate groups. Because of the similarity of dye molecules and lignin, a natural polymer of aromatic alcohols, many of the synthetic dyes can be degraded and decolorized by lignin-degrading white rot fungi such as Phanerochaete chrysosporium and Trametes versicolor and their oxidoreductase systems including peroxidases and laccases. This chapter discusses the sources and application of these ligninolytic enzymes for the treatment of synthetic dyes, reaction mechanisms, and other developments and challenges associated with this enzymatic process.
Studies on decolourization of textile dyes using brown rot fungi are very scarce. In the present investigation, nine such brown rot fungi were isolated and screened for their ability to decolourize brilliant green, bromophenol blue and methyl red. Three promising isolates, A. flavus LCJ 51, A. nidulans LCJ 52 and A. niger LCJ 55 were selected on the basis of this qualitative screening and used for decolourization study in liquid medium by monitoring the decrease in absorbance of each dye. All three Aspergillus sp. (A. flavus LCJ 51, A. nidulans LCJ 52 and A. niger LCJ 55) effectively decolourized brilliant green, bromophenol blue and methyl red, but they differed in decolourization ability depending on various culture conditions which have been reported. Our result proposes that Aspergillus sp. could effectively be used as an alternative to the conventional physicochemical method of dye decolourization.
Laminated bamboo timber (LBT) are found to have a high tensile strength and modulus, good resistance to
fatigue loadings, primarily due to its fiber properties. However, it is reported that LBT production is quit
inefficient and are not well accepted due to the presence of nodes that disfigures the surface appearance of
the board. Accordingly, V-grooving method was developed to counteract these inefficiencies but still with other
problems requiring the lengthening of the short clear bamboo strips with finger-joints. The mechanical
properties of the finger-jointed clear Gigantochloa scortechinii bamboo species was therefore evaluated in
static bending test using the bamboo strips selected for strip with finger joints specimens, with node and
without node. Based on the distance between the joints, the best finger-jointed LBT strength was found to
increase from 5 to 10cm and 5 to 15cm by 30.26% and 49.12% respectively. In the case is glue spread rate,
there was an increase in MOR of 61.49% and 41.79% with phenol formaldehyde over the use of polyvinyl
acetate at 250g/m2 and 200g/m2 respectively. The structural performance of finger-jointed LBT also showed a
31.87% higher in MOR than its counterpart with nodes.
The advancement of technology throughout the world has made bamboo one of the popular raw materials in
the wood-based industry and has recently been considered as an engineering material. Malaysia has a good
number of bamboo species but few that can be utilised commercially. Unfortunately, Malaysia still lacks some
basic information about the properties of bamboo, especially on local bamboo species. This study provides a
basic and details information about some physical properties of two the most popular local bamboo species,
Gigantochloa scortechinii and Bambusa vulgaris. The study covered moisture content variation at different
heights at both nodal and internodal portions of the bamboo culm. Comparison between the portions and
between the nodes and internodes as well as between the species were carried out which showed
significantly, no difference among the MC along the bamboo culm. The shrinkage at the three perpendicular
axis directions showed that the radial shrinkage was slightly greater than tangential and was much greater
than in longitudinal direction. Nodes appeared to have lower moisture content and high percentage of shrinkage
compared to internodes. These variations were expected to be related with the anatomical structure different
between the two categories of the bamboo culms.
Chloroperoxidase (CPO) from the fungus Caldariomyces fumago is undoubtedly the most versatile member of the heme protein family. In addition to functioning as a halogenating enzyme and a classical peroxidase, CPO catalyzes the dismutation of peroxides in a catalase-type reaction and carries out cytochrome P450 oxygen insertion reactions. From the viewpoint of biocatalysis the most important CPO reactions are chiral epoxidations, hydroxylations, and sulfoxidations. CPO catalyzes a variety of chiral epoxidation reactions with high yields and high enantioselectivities. However, the industrial use of native CPO for the synthesis of chiral epoxides is limited because of its relatively low epoxidation rates in comparison to its high catalase activity, which robs the epoxidation reaction of oxidant. The use of CPO is also restricted by its poor reactivities in organic solvents. Directed evolution technology has been used to address these problems. After three rounds of PCR-based random mutagenesis, we have isolated mutants of chloroperoxidase having greatly enhanced epoxidation activity compared to the wild-type enzyme. In addition, in the screening of a first generation library of random mutation transformants, we have isolated three CPO mutant clones having improved chlorination activity and enhanced stability in a ternary solvent microemulsion comprised of toluene, isopropanol and water. Surprisingly, all three recombinant variants carry a single mutation in the cysteine residue that functions as the proximal heme ligand in the native enzyme. Two of these mutant clones are identical, having the proximal cysteine heme-ligand replaced with a tyrosine residue. The third mutant has the cysteine-29 replaced with a histidine residue. The cysteine mutation in the three mutants is the only amino acid replacement. All other mutations in the three clones were silent mutations. These data suggest that ‘‘directed evolution’’ can be successfully applied to the engineering of chloroperoxidase in the quest for a better industrial biocatalyst.
Wood white rot fungi are characterized by their capacity of degradation and mineralization of lignin by means of an enzymatic extracellular system, which mainly consists of lignin peroxidase (LiP), Manganese peroxidase (MnP) and Laccase. During the last twenty years, these fungi and their enzymatic ligninolytic system have been the focus of attention to study the degradation capacity of a wide range of xenobiotics as pesticides, dyes, explosives, etc. However, a large number of xenobiotics are not responding to ligninolytic enzymes biodegradation process. This situation has permitted the discovering of new mechanisms used by fungi as citochrome P-450 monooxygenases oxidation system, and transferases reductive system, widely identified in phase I and II of superior animals metabolism. The tree types of known degradation mechanisms used by fungi in environmental contaminants degradation and some other examples of degradation mechanisms in pesticides will be described and analyzed in this review.
The aim of this study was to compare the decolorization of several of the most utilized synthetic dyes in textile applications by immobilized white-rot fungus Trametes versicolor. Different immobilization methods for achieving maximum decolorization were explored. The initial dye concentration was 125 mg/L and the immobilized preparation concentration was 10% (w/v). Different degrees of decolorization were observed. Different operational stability of the immobilized preparations was accomplished. Increase of the immobilized preparation concentration up to 30% (w/v) was investigated. The study was performed in two stages: simulated incubation in a ‘batch’ reactor and trickle-bed continuous flow reactor During the decolorization process, laccase activity was detected. The dye-decolorizing activity of the immobilized culture was found to be associated with the processes of biodegradation, bio-oxidation and biosorption.
Methylene blue degradation has been studied spectrophotometrically in alkaline and alkaline peroxide solutions. In both systems,
the reaction proceeds via successive N-demethylation and deamination steps determined applying TLC and HPLC techniques. Disappearance of the dye obeys first-order
kinetics under the excess of all other reactants. The rate expression for the hydrolytic process (in the absence of hydrogen
peroxide) is as follows: –d[MB]/dt = (b[OH–]/(1 + c[OH–]))[MB]. Analogous forms of the reaction rate dependence on [OH–] (at constant hydrogen peroxide concentration) and on [HO2
−] (at constant OH− ion concentration) are observed: –d[MB]/dt = ((a′ + b′[OH–])/(1 + c′[OH–]))[MB] and –d[MB]/dt = ((a″+ b″[HO2
–])/(1 + c″[HO2
–]))[MB], respectively. A higher-order than linear dependence of the pseudo first-order rate constant on the nucleophiles (OH– and/or HO2
–) concentrations results from the competitive formation of the ion pair of the cationic dye with the chloride of the supporting
electrolyte and adducts of the dye with OH– and/or HO2
– anions.
Biological remediation is always envisaged as cost effective and eco-friendly for the treatment of
recalcitrant dyes and effluents. Aspergillus niger SA1, a brown rot fungi, isolated from storage pond of
textile wastewater, showed a great mineralizing ability for azo dyes, acid red (AR) 151 and orange (Or) II.
Decolorization assays were carried out for 24 h, by taking 100 ml of dye containing Simulated Textile
Effluent (STE) with 5 g of freshly grown fungal pellets. Decolorization of AR 151 was well over 95%
under different conditions, however, it reduced to 52% when treated with pre-used fungal biomass
under shaking condition. In case of Or II, results were 50 and 61% under static while 65 and 85% under
shaking condition with fresh and pre-used fungal biomass respectively. Primarily, dyes removal in STE
appeared due to biosorption/bioadsorption of the fungal biomass. However, discoloration of dyes onto
the biomass with subsequent formation and then decline in their products in STE suggested clearly that
dyes were basically metabolically degraded by the fungal strain.
The initial products of the electrochemical polymerization of methylene blue were studied using a thin-layer flow cell coupled on-line with electrospray mass spectrometry. A potentiostatic technique was applied to initiate the polymerization while the soluble n-mers generated were monitored by mass spectrometry. Methylene blue dimers and trimers were observed at m/z values consistent with either “nitrogen-to-ring” coupling of the monomers or with “ring-to-ring” coupling of demethylated monomers during the electropolymerization. Experiments accomplished in D2O-based solutions prove that “nitrogen-to-ring” coupling is the dominant process.
The oxidative potential and low specificity of peroxidases are distinctive regarding their efficiency for recalcitrant compounds degradation. However, the usefulness of these biocatalysts for environmental biocatalysis needs a stepwise investigation on the reaction conditions that would render these biocatalysts both efficient and cost effective. In a recent work we compared the usefulness of the fungal lignin peroxidase (LiP) to that observed for the plant horseradish peroxidase (HRP) concerning the degradation of methylene blue (MB) and of its demethylated derivatives. We showed that although both enzymes are able to oxidize MB and its derivatives, HRP reactions require higher H2O2 concentrations, present a considerably lower reaction rate, and contrary to LiP, HRP is unable to achieve aromatic ring cleavage. The oxidation potential of LiP is roughly double than that of less effective HRP (∼0.7 V) and this explains relative efficacy. Thus, lignin peroxidase would be more suitable for phenothyazine dyes degradation and colour removal from waste streams. The present work shows that the use of LiP for the decolouration of MB is competitive in comparison to the majority of the reported methods, regarding reaction time, range of substrate concentration and removal efficiency. In reaction mixtures containing 50 mg/L methylene blue and carried out at 30 °C the dye was degraded within 30 min. Reaction conditions were optimized concerning H2O2 addition mode to avoid the inactivation of the enzyme by H2O2 excess, the enzyme concentration to minimize cost, and the reaction temperature. Results indicated that the use of an MB:H2O2 molar ratio of 1:5 resulted in efficient removal of 90% colour in reactions with MB concentrations up to 50 mg/mL. The enzyme stability was not affected by peroxide concentration up to 990 μM and an LiP:H2O2 molar ratio up to 1:900. The stepwise addition of the peroxide extended the possibility of using total peroxide concentrations up to 1980 μM. Lignin peroxidase was stable up to 60 °C.
The fungal strain A.niger SA1 isolated from textile wastewater pond proved to be an important source of remediation (decolorization/degradation) for
textile dye, AR 151 (Reactive diazo dye) under different physicochemical conditions. Decolorization assays of AR 151 were
carried out in Simulated textile effluent under shake flask condition for 8days. Decolorization (at 20mgl−1 of dye) and related biomass production overall decreased with increase in pH from 5 to 9, at 30°C. It was maximum (95.71%)
at pH 5 with highest amount of three residual products (36.91 (α-naphthol=5.72) (sulfanilic acid=24.81) (aniline=6.38))
besides 2.05mgml−1 of biomass production at an optimum concentration 6 and 0.1mgl−1 of glucose and urea respectively. The formation of the three products followed a quite different pattern at different pH
values, however, it was considerably low (Total=2.81mgl−1) compared to the amount of decolorization (67.26%) at pH 8. Decolorization (95–97%) was most favored under mesophilic temperature
(25–45°C). It increased i.e., 90–98% with subsequent increase in dye from 10 to 100mgl−1, kept ≥50% below 400mgl−1 and drastically declined to 17% at 500mgl−1 of dye. Apparently, decolorization is found to be associated with fungal growth and hyphal uptake mechanism (Biosorption/Bioadsorption),
however, mineralization of AR 151 and related products under different operational conditions also suggested a metabolically
mediated decolorization/degradation.
Bromophenol blue and methyl orange removal capabilities of citraconic anhydride-modified horseradish peroxidase were compared with those of native horseradish peroxidase. Citraconic anhydride-modified horseradish peroxidase showed higher decolorization efficiencies for both dyes than native horseradish peroxidase. Upon the chemical modification, the decolorization efficiencies were increased by 1.8% and 12.4% for bromophenol blue and methyl orange, respectively. The quantitative relationships between decolorization efficiencies of dyes and reaction conditions were also investigated. Experimental data revealed that aqueous phase pH, reaction time, temperature, enzyme concentration and ratio of dye and H2O2 play a significant role on the dye degradation. Lower dose of citraconic anhydride-modified horseradish peroxidase was required than that of native enzyme for the decolorizations of both dyes to obtain the same decolorization efficiencies. Citraconic anhydride-modified HRP exhibited a good decolorization of dye over a wide range of dye concentration from 8 to 24 or 32 μmol l−1 at 300 μmol l−1 H2O2, which would match industrial expectations. Kinetic constants for two different dyes were also determined. Citraconic anhydride-modified horseradish peroxidase shows greater affinity and catalytic efficiency than native horseradish peroxidase for both dyes.
One hundred and fifteen fungi from different physio-ecological groups were compared for their capacity to decolourize two structurally different dyes in agar plates. We found that the azo dye, Acid Red 183, was much more resistant to decolourization by the examined strains in both solid and liquid cultures. Among the tested fungi, 69 strains showed decolourization of the anthraquinonic dye, Basic Blue 22, within 5–14 days, and only 16 strains were able to decolourize the azo dye, Acid Red 183, within 21 days. Furthermore, the potential of selected strains for decolourization of dyes was examined with regard to their extracellular oxidative factors both enzymatic and non-enzymatic. In static aqueous culture, the three selected fungi (Bjerkandera fumosa, Kuehneromyces mutabilis, and Stropharia rugoso-annulata) formed fungal mats, which did not decolourize any dye beyond some mycelial sorption. In comparison to the static cultures, the agitated cultures (180 rpm) removed 75 to 100% of the colour of Basic Blue 22 and 20 to 100% of Acid Red 183 colour.
We have previously shown that the oxidation of methylene blue (MB) or azure B (AB) by Phanerochaete chrysosporium lignin peroxidase (LiP) (class II) occurs via stepwise N-demethylations followed by aromatic ring cleavage under selective reaction conditions related to methylene blue:H2O2 or azure B:H2O2 stoichiometry. In this work, we compare the oxidation of the same dyes by horseradish peroxidase (HRP) of plant origin (class III) and compare LiP and HRP reactions and products. Results show HRP is able to N-demethylate both dyes, but exhibits much slower reaction kinetics than LiP and requires higher H2O2 concentrations. Product yields are also different for HRP, and contrary to LiP, HRP is unable to achieve aromatic ring cleavage. Azure C (AC), which is formed by sequential oxidation of either MB or AB, was a major reaction product in HRP-mediated reactions. Yields of AC up to 60% were obtained, suggesting a potential enzymatic route for AC production that compares quite favorably to the chemical route yield of 35%.
Azo, anthroquinone and triphenylmethane dyes are the major classes of synthetic colourants, which are
difficult to degrade and have received considerable attention. Congo red, a diazo dye, is considered as a
xenobiotic compound, and is recalcitrant to biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several fungi, under certain environmental conditions, are able to transfer azo dyes to non toxic products using laccases. The aim of this work was to study the factors influencing mycoremediation of Congo red. Several basidiomycetes and deuteromycetes species were tested for the decolourisation of Congo red (0.05 g/l) in a semi synthetic broth at static and shaking conditions. Poor decolourisation was observed when the dye acted as the sole source of nitrogen, whereas semi synthetic broth supplemented with fertilizer resulted in better decolourisation. Decolourisation of Congo red was checked in the presence of salts of heavy metals such as mercuric chloride, lead acetate and zinc sulphate. Decolourisation parameters such as temperature, pH, and rpm were optimized and the decolourisation obtained at optimized conditions varied between 29.25- 97.28% at static condition and 82.1- 100% at shaking condition. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis revealed bands with molecular weights ranging between 66.5 to 71 kDa, a characteristic of the fungal laccases. High efficiency decolourisation of Congo red makes these fungal forms a promising choice in biological treatment of waste water containing Congo red.
The extracellular enzymes from Pleurotus sajor-caju were studied for lignin degrading enzyme patterns and dye decolourisation potential. Laccases are major ligninolytic enzymes excreted by the fungus. The results from a native-PAGE revealed that there were at least two isoenzymes. The crude enzyme had a pH and a temperature optimum at 6.0 and 40 degrees C, respectively when 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as substrate. The pH and thermal stability were at 5.0 and 30 degrees C. The pH optima for decolourisation of Indigo Carmine and Methyl Red were at 5.0 and 6.0, respectively. Indigo Carmine could be decolorized efficiently above 90% within 180 min, whereas Methyl Red could be decolorized only 3.5%. High efficiency decolourisation of Indigo Carmine makes this fungus to be a promise choice in biological treatment of waste water containing Indigo Carmine.
Trametes versicolor, a white-rot basidiomycete, degrades cellulose and lignin as well as many recalcitrant chemicals. There have been many reports about the cloning of laccase and peroxidase genes of T. versicolor which are involved in lignin degradation. In order to analyze a gene function and introduce foreign genes into an organism, genetic transformation is required. Here we have successfully transformed T. versicolor to hygromycin B resistance using pAN 7-1 plasmid by restriction enzyme mediated integration and have obtained many mutants in peroxidase activity and growing patterns. The transformation frequency was 25-50 transformants (microg plasmid DNA)(-1). The transformants were quite stable after 10 consecutive transfers in non-selectable medium.
One laccase-secreting engineered strain and four white-rot fungi were tested for their capacity to decolorize nine dyes that could be classified as azo, anthraquinonic and triphenylmethane dyes. Trametes versicolor was the most efficient of the tested strains under these experimental conditions. Anthraquinonic dyes were decolorized more easily than the other two types. Small structural differences among the dyes could significantly affect decolorization. None of the strains showed lignin peroxidase or veratryl alcohol oxidase activity. None of the dyes were decolorized completely by laccase alone. It is likely that other phenoloxidases, such as Mn-dependent and versatile peroxidase, were also involved in decolorization of the dyes.
The decolorization of six xanthene dyes (conc. 100 microM) by a white rot fungus, Coriolus versicolor (C. versicolor), was investigated in liquid culture. The decolorization of Fluorescein, 4-Aminofluorescein, and 5-Aminofluorescein by the fungus was 85.0, 95.0, and 91.9% after 14 days incubation, respectively. However, no decolorization of Rhodamine B, Rhodamine 123 hydrate, and Rhodamine 6G was observed. The first three dyes also were decolorized with cell-free extracts from C. versicolor. The decolorization activity was 10.2, 6.7, and 7.2 microM min(-1)mg(-1), respectively. Thin layer chromatography (TLC) analyses indicated that degradation of Fluorescein was occurring with the detection of three degradation products.
The aim of the current work was to assess the removal of direct and reactive dyes using biotic and abiotic agents. Removal of dyes and their derivatives from aqueous solutions was investigated using sugarcane bagasse, sawdust, rice straw, charcoal and fungal biomass as dye removing agents. Seven fungal strains known to have high capacity in removing textile dyes were used. Results of this study indicated that Penicillium commune, P. freii, and P. allii removed 96, 64 and 65%, respectively, of direct violet dye after two hours of incubation. In addition, the use of rice straw was shown to be more efficient in dye removal, than was bagasse or sawdust. Rice straw was effective in removing 72% of direct violet dye within 24 hours. However, with reactive dyes, removal activity was reduced to 27%. Similar trends were recorded with the other tested biotic agents, fast removal of reactive dye was not found after 48 hours of contact time. Results of this study indicate that low-cost, renewable, bioadsorption agents are relatively effective in removing textile dyes from solution.
Decolorization of 100 microM malachite green (MG) by Coriolus versicolor f. antarcticus using a two-phase bioreactor, was investigated. In the first phase the decolorization ability of this fungus, growing under conditions of solid-state fermentation (SSF), was proved; in the second phase the capacity of the enzymes present in extracts from the solid residues was exploited. During the first phase using the same culture in the bioreactor, five consecutive charges were made, each with 75 ml of 100 microM MG solution, at 28 degrees C. Each cycle ended when MG solution reached a decolorization of 50%, at this time the bioreactor was discharged to a stainless steel coil at 50 degrees C, initiating the second phase of decolorization. Time required in order to reach 50% decolorization during the first phase varied between 25 and 65 min, with an average retention time of 48 min. The second stage had a retention time of 120 min. Residual MG after this phase varied from 0% to 6.3%. The role of laccase and Mn-peroxidase in MG decolorization is discussed. Toxicity of MG solutions before and after decolorization treatments was assayed using Lumbriculus variegatus as test organism.
Dyes released by the textile industries pose a threat to the environmental safety. Recently, dye decolourization through biological means has gained momentum as these are cheap and can be applied to wide range of dyes. This review paper focuses on the decolourization of dye wastewaters through fungi via two processes (biosorption and bioaccumulation) and discusses the effect of various process parameters like pH, temperature, dye concentration etc. on the dye removing efficiency of different fungi. Various enzymes involved in the degradation of the dyes and the metabolites thus formed have been compiled. Genetic manipulations of microorganisms for production of more efficient biological agents, various bioreactor configurations and the application of purified enzymes for decolourization, which constitute some of the recent advances in this field, have also been reviewed. The studies discussed in this paper indicate fungal decolourization has a great potential to be developed further as a decentralized wastewater treatment technology for small textile or dyeing units. However, further research work is required to study the toxicity of the metabolites of dye degradation and the possible fate of the utilized biomass in order to ensure the development of an eco-friendly technology.
Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor, AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona, AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium.
The degradation of methylene blue in crude extracellular medium of the white rot fungus Phanerochaete chrysosporium was demonstrated by changes of the visible spectra of absorption and emission. Such a reaction is H2O2-dependent and peroxide concentration near 500 μM allowed the maximum reaction rate. The optimum pH was found to be 3.5. The visible spectrum of absorption of the peroxidation derivative (absorption maximum at 611 nm) and a thin-layer chromatographic method using ethanol-concentrated HCl (99:1 v/v) indicated the formation of Azure C as the main or unique product of reaction. The enzymatic reaction was not enhanced by 100 μM Mn2+ which precludes any measurable role to Mn-peroxidase. Derivative appearance could be monitored by the increase of the absorbance at 550 nm. This reaction could be very useful as an analytical method for lignin peroxidase detection and may serve as a method of production of Azure C.
A colorimetric method for determination of lignin peroxidase activity has been developed which is based on the demethylation of methylene blue. The use of this dye shows the practical advantage of using a wavelength in the visible region of spectrum. The method can be efficiently used for the enzyme quantification as its sensitivity is close to the veratryl alcohol assay.
Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high nitrogen medium with glucose, could decolorize two azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). When starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12mM NH4Cl, whether or not veratric acid was present. In medium containing 1.2mM NH4Cl and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2mM NH4Cl, decoloration by the local isolate was enhanced but not that by the other strains.
Lignin peroxidase, cytochrome c and haemoglobin were tested for oxidation of polycyclic aromatic hydrocarbon (PAH) in the presence of hydrogen peroxide. The reaction mixture Contained water-miscible organic solvents in order to reduce the mass transfer limitation of hydrophobic substrates. The reaction products from all three haemoproteins were mainly quinones, suggesting the same oxidation mechanism for the three biocatalysts. The haeme prosthetic group must have located in a protein environment for it to catalyze these reactions, and only certain types of protein environment are able to induce this type of haemebased catalytic activity. The solvent hydrophobicity is a factor affecting the biocatalysis in organic media. Substrate partitioning between the active site (haeme) and the bulk solvent is the main factor of the biocatalytic behaviour in organic solvent mixtures. Site-directed mutagenesis of yeast cytochrome c significantly altered the kinetic behaviour of the protein. The Gly82;Thr 102 variant was 10 times more active and showed a catalytic efficiency 10-fold greater than the wild-type iso-1-cytochrome c. These results suggest that it is possible to design a new biocatalyst for environmental purposes.
The oxidative degradation of 3,4-dimethoxybenzyl alcohol and 3,4-dimethoxybenzyl methyl ether by the lignin peroxidase (ligninase, LiP) of Phanerochaete chrysosporium was studied. In addition to previously isolated products of 3,4-dimethoxybenzyl alcohol oxidation (veratraldehyde, two quinones, γ-lactones, and a δ-lactone) three new products - 4,5-dimethoxy-3,5-cyclohexadiene-1,2-dione, (E)-δ-lactone, and 2,5-dihydroxy-4-methoxybenzaldehyde - were now identified as oxidation products. The relative quantities of the products were determined. Six products formed in the oxidation of 3,4-dimethoxybenzyl methyl ether by LiP in the presence of oxygen, veratraldehyde, veratric acid methyl ester, three quinones, and an aromatic ring cleavage product, 3-(methoxymethyl)-(Z,Z)-muconic acid dimethyl ester, were identified and their relative quantities determined. Under anaerobic conditions only trace amounts of products other than veratraldehyde were formed. With cerium(IV) ammonium nitrate as the oxidant comparable results were obtained. The influence of pH and of manganese(II) ion on the LiP reaction was also studied. The major oxidation product at pH 3.0 was veratraldehyde and at pH 5.0 veratric acid methyl ester. This is the first time that this kind of compound has been identified in lignin peroxidase catalyzed reactions. Possible mechanisms for the formation of these products which indicate involvement of activated oxygen species are presented, and the results are discussed in relation to lignin degradation by P. chrysosporium.
A series of eleven p-aminotriphenylmethane dyes have been studied by high-performance liquid chromatography (HPLC). The combined use of HPLC and spectrophotometry permits specific detection of these compounds in the visible range around 600 nm. As the high affinity of the imminium cations for the active sites of the hydrocarbonaceous stationary phase has presented difficulties for reversed-phase HPLC with pure solvents, organic electrolytes were added to the mobile phase to facilitate the elution of the components with improved selectivity, sensitivity (minimum detection limit, 0.1 μg/ml), and peak symmetry. The effects of chromatographic variables on the component retentivity were investigated. Retention times of the dye analytes decreased with increasing concentration of the added ionic reagent and with decreasing number of the hydrophobic alkyl substituents on the nitrogen atom. The influence of pH on the retention parameters appears to parallel that observed previously for cationic quaternary ammonium compounds. Among the acidic reagents employed, naphthalenesulfonic acid yielded the most satisfactory results. The use of binary electrolyte systems invariably improved the chromatographic behavior of the imminium solutes analyzed. Results obtained with two different octadecylsilica columns have been compared.
The white-rot fungus Phanerochaete chrysosporium produces extracellular peroxidases (ligninase and Mn-peroxidase) believed to be involved in lignin degradation. These extracellular enzymes have also been implicated in the degradation of recalcitrant pollutants by the organism. Commercial application of ligninase has been proposed both for biomechanical pulping of wood and for wastewater treatment. In vitro stability of lignin degrading enzymes will be an important factor in determining both the economic and technical feasibility of application for industrial uses, and also will be critical in optimizing commercial production of the enzymes. The effects of a number of variables on in vitro stability of ligninase and Mn-peroxidase are presented in this paper. Thermal stability of ligninase was found to improve by increasing pH and by increasing enzyme concentration. For a fixed pH and enzyme concentration, ligninase stability was greatly enhanced in the presence of its substrate veratryl alcohol (3,4-dimethoxybenzyl alcohol). Ligninase also was found to be inactivated by hydrogen peroxide in a second-order process that is proposed to involve the formation of the unreactive peroxidase intermediate Compound III. Mn-peroxidase was less susceptible to inactivation by peroxide, which corresponds to observations by others that Compound III of Mn-peroxidase forms less readily than Compound III of ligninase.
Contamination of soils and aquifers by aliphatic halocarbons is a serious environmental pollution problem. We report here the novel observation that the halocarbons trichloroethylene (TCE) and CCl4 were mineralized by Phanerochaete chrysosporium under aerobic conditions. Ligninolytic cultures of this white rot fungus mineralized 20.3% of 10 ppm TCE and 18.8% of 10 ppm CCl4 in 9 days. These chemicals were not mineralized by nonligninolytic cultures of P. chrysosporium, indicating that lignin peroxidases play an important role in the mineralization of these chemicals. In a previous study, we reported lignin peroxidase-catalyzed reductive dehalogenation of CCl4 with the resultant formation of trichloromethyl radical. We have extended this study and report here reductive dehalogenation of CHCl3, CH2Cl2, TCE, and 1,1,1-trichloroethane. Dehalogenation was catalyzed by a reductive reaction system containing lignin peroxidase, veratryl alcohol, EDTA or oxalate, H2O2, and the halocarbon with phenyl N-tert-butylnitrone as a spin trap for electron spin resonance detection of the resulting radicals. Since all the components of the reductive system with oxalate as an electron donor are excreted by P. chrysosporium, we propose that this mechanism may be involved in the degradation of these halocarbons by the fungus.
Contrary to earlier literature reports, the impurities in toluidine blue O were shown by column chromatography, TLC, and mass spectrometry to be N-methyl homologs of 2-methylthionine rather than N-methyl homologs of thionine. Small amounts of 2-methyl-3-amino-7-methylaminophenothiazine and 2-methyl-3,7-diaminophenothiazine were identified in commercial samples of toluidine blue O. However, sample handling and a warm alkaline environment can cause rapid demethylation of the dye.
Lignin peroxidase (LiP) is an extracellular enzyme produced by the lignin-degrading fungus Phanerochaete chrysosporium and is involved in azo dye degradation by this organism. In this study, LiP oxidation of the sulfonated azo dyes 4-(4'-sulfophenylazo)-2,6- dimethylphenol (I), Orange II [1-(4'-sulfophenylazo)-2-naphthol] (II), a dimethyl analog of Orange II [1-(2',6'-dimethyl-4'-sulfophenylazo)-2-naphthol] (III), and 4-(4'-sulfonamidophenylazo)-2,6-dimehtylphenol (IV) was examined. Azo dye I was oxidized to 2,6-dimethyl-1,4-benzoquinone and 4-sulfophenyl hydroperoxide. Orange II (II) was oxidized to 1,2-naphthoquinone and 4-sulfophenyl hydroperoxide. The dimethyl analog of Orange II (III) was oxidized to 1,2-naphthoquinone and 2,6-dimethyl-4-sulfophenyl hydroperoxide. Azo dye IV was oxidized predominantly to 2,6-dimethyl-1,4-benzoquinone and another product, tentatively characterized as 4-sulfonamidophenyl hydroperoxide. In the 18O-labeling studies with 18O2, oxygen incorporation into the phenyl hydroperoxides from the oxidation of I and III was observed. A mechanism for azo dye degradation consistent with product identification and the 18O-labeling studies is proposed. Two successive one-electron oxidations of the phenolic ring of an azo dye by the H2O2-oxidized forms of LiP produces a carbonium ion. Then water attacks the phenolic carbon bearing the azo linkage, producing an unstable hydroxy intermediate which breaks down to yield a quinone and a sulfo- or sulfonamidophenyldiazene. The phenyldiazene is oxidized by O2 to generate the corresponding phenyldiazene radical, which eliminates N2 to yield a sulfo- or sulfonamidophenyl radical. O2 scavenges the latter to yield the corresponding hydroperoxide.(ABSTRACT TRUNCATED AT 250 WORDS)
The stabilities of the cation radicals of veratryl alcohol, 3,4-dimethoxytoluene and 1,4-dimethoxybenzene were compared by monitoring the formation of dimeric products during the oxidation of these substrates by lignin peroxidase (LiP). LiP oxidized veratryl alcohol to generate veratraldehyde as the major product. Several other monomeric products were obtained in low yield. Dimeric products resulting from the coupling of two cation radicals, or a cation radical with a neutral molecule, were obtained only in trace amounts or not at all. This suggests that the cation radical of veratryl alcohol rapidly loses a benzylic proton to form a benzylic radical which undergoes further reactions to form veratraldehyde. In contrast, the LiP oxidation of 3,4-dimethoxytoluene generated the dimeric product 3-(2,3-dimethoxy-6-methylphenyl)-4-methyl-1,2-benzoquinone as the major product. Several other monomeric and dimeric products were produced in lower yields. The generation of these dimeric products indicates that the cation radical of 3,4-dimethoxytoluene is considerably more stable than that of veratryl alcohol. This suggests that the electronegative benzylic oxygen of veratryl alcohol increases the acidity of the benzylic protons, destabilizing the veratryl alcohol cation radical. LiP oxidized 1,4-dimethoxybenzene to generate 1,4-benzoquinone and 2-(2,5-dimethoxyphenyl)-1,4-benzoquinone as the major products. The formation of these products indicates that the cation radical of 1,4-dimethoxybenzene also is relatively stable, as previously demonstrated by ESR. All of these results indicate that the veratryl alcohol cation radical generated by LiP oxidation is unstable, suggesting that it would not act as a diffusable radical mediator in LiP-catalyzed reactions.
An extracellular lignin-degrading enzyme from the basidiomycete Phanerochaete chrysosporium Burdsall was purified to homogeneity by ion-exchange chromatography. The 42,000-dalton ligninase contains one protoheme IX per molecule. It catalyzes, nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds: C(alpha)-C(beta) cleavage in lignin-related compounds of the type aryl-C(alpha)HOH-C(beta)HR-C(gamma)H(2)OH (R = -aryl or -O-aryl), oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in phenylglycol structures, and hydroxylation of benzylic methylene groups. It also catalyzes oxidative coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. All reactions require H(2)O(2). The C(alpha)-C(beta) cleavage and methylene hydroxylation reactions involve substrate oxygenation; the oxygen atom is from O(2) and not H(2)O(2). Thus the enzyme is an oxygenase, unique in its requirement for H(2)O(2).
The production of lignin peroxidase from Phanerochaete chrysosporium was studied using immobilized mycelia in nylon-web cubes in semicontinuous fermentation using glucose pulses or ammonium tartrate pulses. Consistent enzyme production was achieved when glucose pulses were used, leading to an average activity of 253 U/L. The crude enzyme was added to eucalyptus kraft pulp before conventional and ECF bleaching sequences. Optimization of the enzymatic pretreatment led to the following operational conditions: enzyme load of 2 U/g of pulp, hydrogen peroxide addition rate of 10 ppm/h, and reaction time of 60 min. Pulp final characteristics were dependent on the chemical treatment sequence that followed enzymatic pretreatment. The chief advantage of enzymatic pretreatment was pulp viscosity preservation, which was observed in most of the experiments carried out with seven different chemical treatment sequences.