Decolorization of Victoria blue by the white rot fungus, <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams. Recognizing the importance of reducing the environmental impact of these dyes, the ability of the white rot fungus Phanerochaete chrysosporium to decolorize various textile dyes was investigated. This fungus decolorized 6 of the 14 structurally diverse dyes with varying efficiency (between 14% and 52%). There was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Among the three dyes which showed >40% decolorization, Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not Lip and MnP, play a role in VB decolorization by P. chrysosporium.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

选择压力对染料脱色酵母菌活性影响的研究

一株对染料活性黑5(Reactive Black 5,RB5)具有高效降解活性的酵母菌—Debaryomyces polymor-phus,在传代培养过程中脱色能力及产锰依赖过氧化物酶(MnP)活性逐步降低。研究结果表明,采用向培养体系中反复投加新鲜染料的方法,可以对脱色酵母菌的生长形成持续性选择压力,从而使菌株的脱色和产MnP活性得以显著提高。     

Implication of <Emphasis Type="Italic">Dichomitus squalens</Emphasis> manganese-dependent peroxidase in dye decolorization and cooperation of the enzyme with laccase

Three new chromatographic forms of Dichomitus squalens manganese-dependent peroxidase (MnP) were isolated from wheat-straw cultures using Mono Q and connective interaction media (CIM) fast protein liquid chromatography. Enzymes revealed identical molar mass of 50 kDa (estimated by SDS-PAGE) and pI values of 3.5, however, they varied in Km values obtained for Mn2+ oxidation. The addition of wood and straw methanol extracts to the cultures showed that the production of MnPs in wheat-straw cultures was influenced rather by the type of cultivation than by phenolic compounds from lignocellulosic material which induced laccase production. The purified CIM1 MnP was able to decolorize selected azo and anthraquinone dyes more rapidly than laccase Lc1. In vitro dye decolorization showed a synergistic cooperation of MnP and laccase. In the case of CSB degradation MnP prevented from the production of a differently colored substance that could be produced after CSB degradation by laccase-HBT system.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings. Application to the decolorization of synthetic dyes

The production of manganese-dependent peroxidase (MnP) byPhanerochœte chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated. Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 μkat/L were obtained, respectively. Thein vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed. The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).     

Adsorption and decolorization of dyes using solid residues from Pleurotus ostreatus mushroom production

We investigated the use of solid residues from Pleurotus ostreatus mushroom production in adsorbing and decolorizing different dyes. The solid residue used in this study was composed of hemicellulose and cellulose (52.81 %), acid-insoluble lignin (25.42%), chitin (6.5%), and water extractives (14.82%). After incubating 14% (wt/vol) solid residue in distilled water for 4 h, laccase and manganese peroxidase (MnP) activities were 0.5 U/g and 12 mU/g, respectively. Enzymatic decolorization percentages were up to 100 for azure B (heterocyclic dye) and indigo carmine (indigoid dye), 74.5 for malachite green (MG) (triphenylmethane dye), and zero for xylidine (azoic dye). The optimum temperature for decolorization was in the range of 26 ∼ 36°C for all dyes. Data obtained on adsorption (enzymatic decolorization was prevented with sodium azide) at different dye concentrations and in a pH range of 3 ∼ 7 were used to plot Freundlich isotherms. The spent fungal substrate (SFS) displayed large differences in adsorption capacity, depending on the dye tested. The highest adsorption capacity was observed at pH 3 for MG, while xylidine was slightly adsorbed at pH 3 and 4 and not adsorbed at higher pH values. Laccase and MnP production were affected by the presence of the dyes. The highest enzyme levels were observed in the presence of MG, when laccase and MnP increased 1.39- and 2.13-fold, respectively. Decolorization and adsorption to SFS are both important processes in removing dyes from aqueous solutions. The application of this spent substrate for wastewater treatment will be able to take advantage of both of these dye removal processes. An important problem in bioremediation processes involving microorganisms is the amount of time required for their growth. In this report, we used the spent substrates from mushroom cultivation in wastewater treatment, thus solving the problem of waiting for microorganisms to grow.     

烟管孔菌G14产锰过氧化物酶条件优化及其对染料的脱色

利用组织分离从未成熟有机蓝莓的表皮中分离出菌株G14,根据其菌落形态、ITS序列对比及系统发育树的分析,鉴定菌株G14为一株烟管孔菌Bjerkandera adusta。菌株G14可以分泌漆酶（laccase,Lac）、木质素过氧化物酶（lignin peroxidase,LiP）和锰过氧化物酶（manganese peroxidase,MnP）3种木质素降解酶,利用单因素和正交试验对活性较高的MnP进行发酵条件优化,同时检测B.adustaG14所产MnP粗酶液对5种染料的脱色能力。结果表明,B.adustaG14在培养6d时MnP活性最大,最优条件为：蔗糖10g/L、pH 7、0.5mmol/L Mn²⁺、0.1mmol/L Zn²⁺,该条件下MnP活性达17.74U/L,比优化前提高了1.42倍,B.adustaG14 MnP粗酶液对5种染料均可以脱色,对刚果红和铬黑T染料的脱色效果最好,6d后脱色率达76%和68%。     

Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants

Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.     

Purification and characterization of manganese peroxidase of the white-rot fungus Irpex lacteus

The production of manganese peroxidase (MnP) by Irpex lacteus, purified to electrophoretic homogeneity by acetone precipitation, HiPrep Q and HiPrep Sephacryl S-200 chromatography, was shown to correlate with the decolorization of textile industry wastewater. The MnP was purified 11.0-fold, with an overall yield of 24.3%. The molecular mass of the native enzyme, as determined by gel filtration chromatography, was about 53 kDa. The enzyme was shown to have a molecular mass of 53.2 and 38.3 kDa on SDS-PAGE and MALDI-TOF mass spectrometry, respectively, and an isoelectric point of about 3.7. The enzyme was optimally active at pH 6.0 and between 30 and 40 degrees C. The enzyme efficiently catalyzed the decolorization of various artificial dyes and oxidized Mn (II) to Mn (III) in the presence of H(2)O(2). The absorption spectrum of the enzyme exhibited maxima at 407, 500, and 640 nm. The amino acid sequence of the three tryptic peptides was analyzed by ESI Q-TOF MS/MS spectrometry, and showed low similarity to those of the extracellular peroxidases of other white-rot basidiomycetes.     

Decolorization and biosorption for Congo red by system rice hull- Schizophyllum sp. F17 under solid-state condition in a continuous flow packed-bed bioreactor

Synthetic dyes are important chemical pollutants from various industries. This work developed an efficient and relatively simple continuous decolorization system rice hull-Schizophyllum sp. F17 under solid-state condition in a packed-bed bioreactor, for decolorizing Congo red. In the decolorization system, two decolorization mechanisms exist, one is decolorization by Schizophyllum sp. F17, the other is biosorption by rice hull. The decolorization efficiency was greatly affected by dye concentration and hydraulic retention time (HRT), which were quantificationally analyzed and optimized through response surface methodology (RSM). A 2(2) full factorial central composite design (CCD) was performed, and three second order polynomial models were generated to describe the effects of dye concentration and HRT on total decolorization (R2=0.902), decolorization by Schizophyllum sp. F17 (R2=0.866) and biosorption by rice hull (R2=0.890). Response surface contour plots were constructed to show the individual and cumulative effects of dye concentration and HRT, and the optimum values. A maximum total decolorization 89.71% and maximum decolorization by Schizophyllum sp. F17 60.44% was achieved at dye concentration 142.63mg/L, HRT 41h, and dye concentration 110.7mg/L, HRT 29.4h, respectively. Meanwhile, the role of manganese peroxidase (MnP) in the decolorizaion process was investigated. This study proved the feasibility of continuous mode for decolorizing synthetic dyes by white-rot fungi in solid-state fermentation bioreactors.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Decolorization of practical textile industry effluents by white rot fungus Coriolus versicolor IBL-04

Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486 U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725 U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.     

Oxidative degradation of azo dyes by manganese peroxidase under optimized conditions

The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. The goal of this study is the development of a degradation system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) for the degradation of azo dyes. The experimental work also includes the optimization of the process, with the objective of determining the influence of specific physicochemical factors, such as organic acids, H(2)O(2) addition, Mn(2+) concentration, pH, temperature, enzyme activity and dye concentration. A nearly total decolorization was possible at very low reaction times (10 min) and at high dye concentration (up to 1500 mg L(-)(1)). A specific oxidation capacity as high as 10 mg dye degraded per unit of MnP consumed was attained for a decolorization higher than 90%. Among all, the main factor affecting process efficiency was the strategy of H(2)O(2) addition. The continuous addition at a controlled flow permitted the progressive participation of H(2)O(2) in the catalytic cycle through a suitable regeneration of the oxidized form of the enzyme, which enhanced both the extent and the rate of decolorization. It was also found that, in this particular case, the presence of a chelating organic acid (e.g., malonic) was not required for an effective operation. Probably, Mn(3+) was chelated by the dye itself. The simplicity and high efficiency of the process open an interesting possibility of using of MnP for solving other environmental problems.     

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degrees C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus.     

Dye decolorization by manganese peroxidase in an enzymatic membrane bioreactor

In the present work an enzymatic membrane reactor (EMR) for the oxidation of azo dyes by manganese peroxidase (MnP) has been developed. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane. The membrane allowed for most of the enzymatic activity to be recovered while both the parent dye and the degradation products could pass through. Different operational strategies (batch, fed-batch, and continuous) and parameters such as enzyme activity, H(2)O(2) feeding rate, hydraulic retention time (in continuous operation), and dye loading rate were studied. At best conditions, a continuous operation with a dye decolorization higher than 85% and minimal enzymatic deactivation was feasible for 18 days, attaining an efficiency of 42.5 mg Orange II oxidized/MnP unit consumed.     

Decolorization of Fast red by metabolizing cells of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ML34

Three different azo dyes such as Fast red, metanil yellow and Fast orange were examined for their decolorization by O. oeni ML34. Fast red (FR) was decolorized by 68%, whereas the other dyes were removed by only about 30%. The effects of glucose addition, substrate (dye) concentration and environmental factors (temperature, pH) on decolorization were investigated by two-level factorial design. The statistical analyses revealed that glucose specifically increases the extent of FR decolorization. A glucose level of 5 g/l was the optimum concentration for removal of, FR reaching a decolorization percentage of up to 93%.     

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%。     

Redox-mediated decolorization of recalcitrant textile dyes by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> WL1 laccase

The efficiency of crude and partially purified Trichoderma harzianum WL1 laccase for the decolorization of synthetic dyes (Rhodamine 6G, Erioglaucine and Trypan blue) with complex aromatic structures were evaluated. Selection of dyes was based on their extensive usage in local dyeing and textile industries around the study area. Studies on the role of redox potential of laccases on dye decolorization are rarely discussed and hence, for the first time we have shown the redox mediated dye decolorizing efficiency of T. harzianum WL1 laccase with the commonly employed redox mediator 1-hydroxybenzotriazole (HBT). The process parameters such as initial dye concentration, enzyme load and HBT concentration were studied and found that they had a great influence on dye removal process. When the dyes were treated with increased concentration of enzyme, it showed a greater percentage of decolorization. Compared to the crude laccase, partially purified laccase accounts for maximum decolorization of all the dyes studied. In addition, the rate of dye decolorization was considerably enhanced in presence of 4 mM HBT. Maximum and minimum decolorization were recorded for Rhodamine 6G and Trypan blue, respectively. The results of this study further confirmed that, T. harzianum laccase was found to be suitable with HBT and this laccase-mediator system (LMS) could be applied for the decolorization of various classes of dyes.