Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.     

Abatement of microfibre pollution and detoxification of textile dye – Indigo by engineered plant enzymes

Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%–89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution.     

Potential of combined fungal and bacterial treatment for color removal in textile wastewater

Low efficiency of dye removal by mixed bacterial communities and high rates of dye decolorization by white-rot fungi suggest a combination of both processes to be an option of treatment of textile wastewaters containing dyes and high concentrations of organics. Bacteria were able to remove mono-azo dye but not other chemically different dyes whereas decolorization rates using Irpex lacteus mostly exceeded 90% within less than one week irrespective of dye structure. Decolorization rates for industrial textile wastewaters containing 2-3 different dyes by fungal trickling filters (FTF) attained 91%, 86%, 35% within 5-12 d. Sequential two-step application of FTF and bacterial reactors resulted in efficient decolorization in 1st step (various single dyes, 94-99% within 5 d; wastewater I, 90% within 7 d) and TOC reduction of 95-97% in the two steps. Large potential of combined use of white-rot fungi and traditional bacterial treatment systems for bioremediation of textile wastewaters was demonstrated.     

Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.     

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.     

Evidence on manganese peroxidase and tyrosinase expression during decolorization of textile industry dyes by Trichosporon akiyoshidainum

Textile dyes are engineered to be resistant to environmental conditions. During recent years the treatment of textile dye effluents has been the focus of significant research because of the potentially low cost of the process. Mechanisms of biological textile dye decolorization depend greatly on the chemical structure of the dye and the microorganisms used. While basidiomycetous filamentous fungi are well recognized for dye decolorization through ligninolytic enzymes, reports on textile dye decolorization mechanisms of basidiomycetous yeasts have been scarce. Decolorization of several textile dyes by Trichosporon akiyoshidainum occurs during the first 12 h of cultivation. This fast decolorization process could not be solely related to siderophore production or dye sorption to biomass; it was shown to be a co-metabolic process. T. akiyoshidainum could use glucose, sucrose, and maltose as alternative carbon sources, and urea as an alternative nitrogen source with similar decolorization rates. The activity of two enzymes, manganese peroxidase and tyrosinase, were induced by the presence of dyes in the culture media, pointing to their potential role during the decolorization process. Manganese peroxidase titers reached 666 U l⁻¹ to 10538 U l⁻¹, while tyrosinase titers ranged between 84 U l⁻¹ and 786 U l⁻¹, depending on the dye tested. The present work provides a useful background to propose new eco-friendly alternatives for wastewater treatment in textile dying industries.     

Bacterial Indigo Reduction

We have examined bacterial indigo reduction to provide a basis for the development of a sustainable alternative to the present chemical methods used to reduce indigo for denim dyeing. Indigo was reduced by Clostridium isatidis, but not by the related Clostridium aurantibutyricum, Clostridium celatum nor Clostridium papyrosolvens. However C. papyrosolvens could, like C. isatidis, reduce the soluble dye, indigo carmine. Of the bacteria examined only the supernatant from C. isatidis cultures decreased indigo particle size. An anthraquinone-rich madder root extract, the soluble anthraquinone-2,6-disulfonic acid and humic acid all stimulated the reduction of indigo by C. isatidis, without affecting the redox potential of the cultures. C. isatidis cultures generated redox potentials from -476 to -602 mV vs SCE, which were about 100 mV more negative than those of the other bacteria examined. The mechanism of bacterial indigo reduction remains unknown, but the unique features of the indigo-reducing C. isatidis indicate possible mechanisms.     

固定化乳白耙齿菌脱色茜素红的研究

利用海藻酸钙法对乳白耙齿菌进行固定化,检测固定化乳白耙齿菌（固定化菌）对几种染料的脱色能力。同时,考察pH值、染料浓度、金属离子、碳源种类、氮源种类、盐浓度对固定化菌脱色茜素红的影响。结果表明,固定化菌的优化条件为海藻酸钠3%、氯化钙5%、固定化时间6h、接菌量10g/100mL;固定化菌对6种染料均可脱色,其中对茜素红染料的脱色效果最为明显;固定化菌对茜素红的脱色率随染料浓度的增加而下降,当染料浓度高于250mg/L时,其脱色效果明显下降;固定化菌对茜素红脱色的适宜pH为7,适宜碳源为可溶性淀粉、适宜氮源为硝酸铵。另外,固定化菌对茜素红的脱色率随盐浓度的升高,呈下降趋势,当盐浓度高于3%时,脱色率下降明显;固定化菌于生理盐水中保存10d后,脱色率维持在较高水平,达94.20%;固定化菌重复利用5次后,脱色率仍高达88.70%。     

撕裂蜡孔菌在开放体系中对甲基橙染料的静态脱色研究

为了评价撕裂蜡孔菌处理偶氮染料的应用潜力,用性能稳定的甲基橙染料为材料,采用批次试验在开放性体系中研究了染料初始浓度、菌丝生物量、温度、pH等因素对该菌脱色能力的影响,运用菌丝体反接、染液光谱扫描、菌丝体显微观察等方法探讨了菌丝体脱色的可能机制,利用植物萌发试验进行了染料和脱色后溶液的毒性测试。结果表明,撕裂蜡孔菌在开放的静止体系中能够对甲基橙高效脱色,其最适脱色温度为35℃,最佳脱色pH值在6左右。菌丝对甲基橙的脱色表现在吸附和产酶降解两个方面,脱色过程中染料对菌丝体本身的影响较少。植物毒性分析显示撕裂蜡孔菌脱色48h后的产物对植物的毒性比甲基橙本身更强,若要彻底降解可能需要较长时间。本研究可为染料脱色工艺提供新的菌种。     

Study of dye decolorization in an immobilized laccase enzyme-reactor using online spectroscopy

Decolorization of textile dyes by a laccase from Trametes modesta immobilized on gamma-aluminum oxide pellets was studied. An enzyme reactor was equipped with various UV/Vis spectroscopic sensors allowing the continuous online monitoring of the decolorization reactions. Decolorization of the dye solutions was followed via an immersion transmission probe. Adsorption processes were observed using diffuse reflectance measurements of the solid carrier material. Generally, immobilization of the laccase does not seem to sterically affect dye decolorization. A range of commercial textile dyes was screened for decolorization and it was found that the application of this enzymatic remediation system is not limited to a certain structural group of dyes. Anthrachinonic dyes (Lanaset Blue 2R, Terasil Pink 2GLA), some azo dyes, Indigo Carmine, and the triphenylmethane dye Crystal Violet were efficiently decolorized. However, the laccase displayed pronounced substrate specificities when a range of structurally related model azodyes was subjected to the biotransformation. Azodyes containing hydroxy groups in ortho or para position relative to the azo bond were preferentially oxidized. The reactor performance was studied more closely using Indigo Carmine.     

Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.     

Decolorization of textile plant effluent by Citrobacter sp. strain KCTC 18061P

Citrobacter sp. strain KCTC 18061P was found to be able to decolorize textile plant effluent containing different types of reactive dyes. Effects of physico-chemical parameters, such as aeration, nitrogen source, glucose and effluent concentrations on the color removal of real dye effluent by this strain were investigated. The observed changes in the visible spectra indicated color removal by the absorption of dye to cells during incubation with the strain. This strain showed higher decolorization ability under aerobic than static culture conditions. With 1% glucose, this strain removed 70% of effluent color within 5 days. Decolorization was not significantly dependent on the nitrogen sources tested. Chemical oxygen demand (COD) and biological oxygen demand (BOD) were decreased in proportion to incubation times, and their removal rates were about 35% and 50%, respectively, at 7 days of culture.     

Characterization of decrystallized chitosan and its application in biosorption of textile dyes

Decrystallized chitosan was produced from shrimp shells with a low degree of crystallinity (10%) and a high anionic dye binding capacity. Raw, mixed dye wastewater from a textile factory was efficiently decolorized using decrystallized chitosan that was more efficient than using normal chitosan and activated carbon. Decolorization reached 90% within 10 min and could be carried out from pH 4.5 to 8.1. Decrystallized chitosan can be regenerated by 2 M H₂SO₄ and was reusable more than 10 times. It is, therefore, an attractive candidate for the removal of dyes from textile wastewater.     

Decolorization of diazo dye Direct Red 81 by a novel bacterial consortium

Summary Samples collected from various effluent-contaminated soils in the vicinities of dyestuff manufacturing units of Ahmedabad, India, were studied for screening and isolation of organisms capable of decolorizing textile dyes. A novel bacterial consortium was selected on the basis of rapid decolorization of Direct Red 81 (DR 81), which was used as model dye. The bacterial consortium exhibited 90% decolorization ability within 35 h. Maximum rate of decolorization was observed when starch (0.6 g l⁻¹) and casein (0.9 g l⁻¹) were supplemented in the medium. Decolorization of DR 81 was monitored by high performance thin layer chromatography, which indicated that dye decolorization was due to its degradation into unidentified intermediates. The optimum dye-decolorizing activity of the culture was observed at pH 7.0 and incubation temperature of 37 °C. Maximum dye-decolorizing efficiency was observed at 200 mg l⁻¹ concentration of DR 81. The bacterial consortium had an ability to decolorize nine other structurally different azo dyes.     

微生物对偶氮染料的脱色及其基因工程研究进展

偶氮染料广泛应用在纺织印染、造纸印刷等行业中。染料废水的排放将会导致严重的环境污染,使用微生物处理染料废水是解决此问题的有效方法。该文概述了微生物对偶氮染料的脱色的研究,包括细菌对偶氮染料的脱色,真菌对偶氮染料的脱色,脱色产生的芳香胺并进一步被降解,以及基因工程技术在微生物对偶氮染料脱色的研究进展。     

Biological Decolourization of Textile and Paper Effluents by Pleurotus florida and Agaricus bisporus (White-rot Basidiomycetes)

Summary The extracellular ligninolytic enzymes of white-rot fungi are thought to catalyse the initial steps during the degradation of highly complex compounds like lignin or polycyclic aromatic hydrocarbons. We studied the ability of Pleurotus florida isolated from the foothills of the Western Ghats, India to decolourize the three dyestuffs, Reactive Green, Yellow and Blue, which are widely used in the textile industry around Coimbatore, Tamil Nadu, India. The crude culture filtrate of Pleurotus florida when incubated with different concentrations of dye decolourized it efficiently on the third day. The highest colour removal was found in the case of Reactive Blue. However, when Agaricus bisporus extract was supplemented with Pleurotus florida filtrate, the efficiency increased. The dye decolourization was advanced to the second day and the efficiency of dye decolourization of Reactive Yellow was 89% followed by Reactive Green, which was 45% when a dye concentration of 0.5% was used. Pleurotus florida filtrate alone and in combination with Agaricus bisporus extract reduced the aromatic compounds in textile and paper industry effluents on the first day with >90% efficiency.     

白囊耙齿菌产锰过氧化物酶条件优化及其对染料的脱色

锰过氧化物酶（manganese peroxidase,MnP）是白腐真菌降解多种异生物质的主要降解酶之一。本研究对白囊耙齿菌Irpex lacteus产MnP的酶活曲线进行监测,利用单因素和正交试验对I. lacteus产MnP的发酵条件进行优化,同时检测了I. lacteus的MnP粗酶液对5种染料的脱色效果。结果显示,I. lacteus在培养5d时MnP活性较大;I. lacteus产MnP较优的条件为：可溶性淀粉20g/L、尿素1g/L、pH 6.3、CaCl₂ 1mmol/L、FeCl₃ 1mmol/L,该条件下MnP活性达29.24U/L,与优化前MnP活性相比提高了1.25倍;I. lacteus的MnP粗酶液对5种染料均可脱色,其中对直接大红和活性红的脱色效果更为明显,脱色5d后的脱色率分别达到82%和81%。     

Decolorization of Acid red 151 by Aspergillus niger SA1 under different physicochemical conditions

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 8 days. Decolorization (at 20 mg l⁻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 2.05 mg ml⁻¹ of biomass production at an optimum concentration 6 and 0.1 mg l⁻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 = 2.81 mg l⁻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 100 mg l⁻¹, kept ≥50% below 400 mg l⁻¹ and drastically declined to 17% at 500 mg l⁻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.     

Biodegradation of diazo dye Direct brown MR by Acinetobacter calcoaceticus NCIM 2890

Acinetobacter calcoaceticus was employed for the degradation of Direct brown MR (DBMR), commercially used azo dye in the textile industry in order to analyze mechanism of the degradation and role of inhibitors, redox mediators and stabilizers of lignin peroxidase during decolorization. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase and DCIP reductase represented their involvement in the biodegradation of DBMR. Decolorization and biodegradation of azo dye DBMR in broth were monitored by UV–visible spectrophotometer and TLC. The products obtained from A. calcoaceticus degradation were characterized by FTIR and identified by GC/MS as biphenyl amine, biphenyl, 3-amino 6-hydroxybenzoic acid and naphthalene diazonium. Germination (%) and growth efficiency of Sorghum vulgare and Phaseolus mungo seeds revealed the degradation of DBMR into less toxic products than original dye. A. calcoaceticus also has a potential to degrade diverse dyes present in the textile effluent, into nontoxic metabolites, hence A. calcoaceticus can be applied for the commercial application.     

Laccase production at reactor scale by filamentous fungi

Laccases have received much attention from researchers during the past decades due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases. This makes laccases highly interesting for a wide variety of processes, such as textile dye decolouration, pulp bleaching, effluent detoxification, biosensors and bioremediation.

The successful application of laccases to the above-mentioned processes requires the production of large quantities of enzyme at low cost. Filamentous fungi are able to produce laccases in high amounts, however, an efficient production system at bioreactor scale is still lacking. This is mainly due to the fact that laccase production by wild-type strains of filamentous fungi is linked to secondary metabolism, which implies that the following drawbacks must be overcome: uncontrolled fungal growth, the formation of polysaccharides around mycelia and the secretion of certain compounds (i.e. proteases) that inactivate laccases. This review summarizes the current status of laccase production by wild-type strains of filamentous fungi at the bioreactor scale.