不同培养基对富集筛选脱色真菌菌群的效果比较

利用活性黑RB5和活性红M-3BE作为筛选因子,从染料脱色效果、菌群产酶能力以及菌群中的微生物丰富度三方面比较了酵母培养基A、产漆酶真菌培养基B和白腐真菌培养基D在脱色真菌富集筛选方面的效果。富集筛选结果共得到11组具有明显脱色效果的真菌菌群,其中5组来自于D培养基,A和B培养基各获得3组。来自A培养基的3组菌群显示出最好的脱色效果和最大的菌群丰富度,对50mg/L的活性红M-3BE和酸性红A溶液的脱色率最高达到99.53%和97.42%,从中分离到了16株真菌,初步鉴定分属于水霉科、曲霉科（红曲霉属）、节壶菌科和白粉菌科;而B和D培养基中所获得的菌群脱色效果稍差,从中仅得到3株和2株真菌,初步鉴定属于酵母和青霉。A、B两种培养基在各种染料存在下更易产生木质素过氧化物酶,产漆酶能力较弱,而D培养基产漆酶活性较高。     

不同培养基对富集筛选脱色真菌菌群的效果比较

利用活性黑RB5和活性红M-3BE作为筛选因子，从染料脱色效果、菌群产酶能力以及菌群中的微生物丰富度三方面比较了酵母培养基A、产漆酶真菌培养基B和白腐真菌培养基D在脱色真菌富集筛选方面的效果。富集筛选结果共得到11组具有明显脱色效果的真菌菌群，其中5组来自于D培养基，A和B培养基各获得3组。来自A培养基的3组菌群显示出最好的脱色效果和最大的菌群丰富度，对50mg/L的活性红M-3BE和酸性红A溶液的脱色率最高达到99.53%和97.42%，从中分离到了16株真菌，初步鉴定分属于水霉科、曲霉科（红曲霉属）、节壶菌科和白粉菌科；而B和D培养基中所获得的菌群脱色效果稍差，从中仅得到3株和2株真菌，初步鉴定属于酵母和青霉。A、B两种培养基在各种染料存在下更易产生木质素过氧化物酶，产漆酶能力较弱，而D培养基产漆酶活性较高。     

端梗霉Z45产漆酶培养基的优化及其对染料的脱色

菌株Acrophialophora sp.Z45是一株产漆酶的端梗霉属真菌,本文依据不同培养基中菌株Z45对愈创木酚的氧化圈大小探究了影响该菌株产漆酶的因素并设计正交试验优化其产漆酶培养基,进而比较了菌株Z45在土豆葡萄糖培养基和优化后的产漆酶培养基中的漆酶活力差异,最后基于漆酶与染料脱色的相关性评价了菌株Z45对8种合成染料的脱色能力。结果表明,单因素及正交试验确定了菌株Z45优化后的产漆酶培养基为：基础产酶培养基中以蔗糖为碳源、硝酸钠为氮源、C/N比为45∶1、pH为5.0;在优化后的产漆酶培养基中菌株Z45的漆酶活性显著提高;菌株Z45对溴酚蓝、中性红、亚甲基蓝、甲基蓝和结晶紫等5种染料均有一定的脱色能力,其中对三苯甲烷染料甲基蓝的脱色能力最强,菌株Z45的粗酶液能使甲基蓝快速脱色。在较低甲基蓝浓度的固体培养基上,甲基蓝完全脱色的时间不受染料浓度的影响;而较高甲基蓝浓度下,甲基蓝完全脱色的时间随染料浓度升高逐渐延长。     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.     

阿魏菇与胶红酵母共培养产漆酶对活性艳蓝W-RV的脱色

利用阿魏菇与胶红酵母共培养所产漆酶对染料活性艳蓝W-RV进行脱色,同时考察不同p H、温度、染料浓度和漆酶酶活等条件对脱色的影响。结果显示,酸性范围内的p H有利于活性艳蓝W-RV的脱色,较高温度并不适于脱色反应。在研究不同染料浓度的影响时发现,不同浓度染料的脱色率在7 h反应后都达到了稳定,而漆酶酶活在达到200 U/L后脱色率不再变化。因此,最终得到的最适反应条件为p H 4.5、30℃、染料质量浓度100 mg/L、漆酶酶活200 U/L,在此条件下,活性艳蓝W-RV的最高脱色率为89.91%。     

一株产漆酶真菌新月弯孢霉JQH-100在染料脱色中的应用

从感染叶斑病的玉米叶片中分离、纯化得到一株高产漆酶的新月弯孢霉Curvularia lunata JQH-100菌株。液体培养Curvularia lunata JQH-100可产漆酶且活性较高,产酶高峰出现在第3天;以ABTS为底物粗酶液的最适反应温度是30℃,最适反应pH是2.8;染料脱色的研究表明,共培养体系对茜素红的脱色率达到了92.6%,对中性红和刚果红的脱色率也都在80%以上;Curvularia lunata JQH-100所产漆酶经纯化后对染料茜素红和刚果红有较高的脱色率,分别为82.1%和81.2%。研究结果显示Curvularia lunata JQH-100在染料废水处理中有较大应用潜力。     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

南海局部海洋沉积物中真菌多样性及产酶活性

【目的】从11份南海海洋沉积物中分离耐盐真菌,并对其物种多样性及产酶活性进行研究。【方法】利用平板涂布法分离耐盐真菌,基于形态学和ITS序列的系统进化研究耐盐真菌多样性;利用6种筛选培养基对耐盐真菌进行产酶活性筛选。【结果】分离得到1689株耐盐真菌,共41个形态种。形态学和ITS序列分析表明,这些真菌归于15个属,其中曲霉属(Aspergillus)和青霉属(Penicillium)为优势菌群。对已测序的41株耐盐真菌的产酶活性研究表明,8株产纤维素酶,9株产淀粉酶,5株产复合酶,16株产蛋白酶,3株产脂肪酶,未发现产壳聚糖酶的菌株,其中Acrodontium sp.8m和Aspergillus sp.86b产复合酶的活性相对较高,而Penicillium sp.41m产蛋白酶的活性相对较高。【结论】南海局部海洋沉积物中耐盐真菌丰富,多数菌株具有产酶活性。     

温特曲霉HD1的鉴定及其对氧蒽类染料脱色特性的研究

从土壤中分离到１株染料脱色真菌,经鉴定命名为温特曲霉ＨＤ１。该菌对氧蒽类染料虎红具有很强的脱色能力。温度在２８￣４０℃之间,ＨＤ１对虎红的脱色率为９３￣９９％,最适脱色温度为３３℃,ｐＨ值在４．０￣８．０之间,其脱色率为８９．３￣９８．８％,最适脱色ｐＨ值为６．０。培养基、碳源,氮源及接种量对其脱色率均有影响,该菌对虎红的脱色酶为组成酶,主要分布在细胞内,染料的加入能改变脱色酶在胞内外的分配比例,     

短小芽孢杆菌LC01漆酶基因在毕赤酵母中的胞外表达及重组漆酶酶学性质研究

为了获得表达量高、稳定性好及染料脱色效率高的细菌漆酶,通过PCR扩增出短小芽孢杆菌LC01的漆酶基因并构建重组表达载体pPICZαA-lac,转化毕赤酵母菌株SMD1168H后利用甲醇诱导培养重组菌获得重组漆酶,纯化并分析了重组漆酶的性质。重组菌株产漆酶活性在第7天达到最高,为1 390 U/L。纯化的重组漆酶分子量为65 kD,以丁香醛连氮为底物的最适反应温度和pH分别为70℃和6.8。在pH 9.0放置10 d活性没有下降,在70℃保温10 h后仍保留36%的酶活。Al~(3+)、Fe~(3+)和Mn~(2+)完全抑制漆酶活性。在介体乙酰丁香酮参与下该漆酶能够有效脱色RB亮蓝、活性黑5和靛红,在pH 9.0时6 h的脱色率达到了90%以上,表明该重组漆酶能有效应用于染料废水的脱色处理。     

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l^–1 within 16–48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l^–1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69–94%) and C. tropicalis (30–97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105–587 U l^–1). However, MnP activities of 198–329 U l^–1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Degradation of synthetic reactive azo dyes and treatment of textile wastewater by a fungi consortium reactor

In this paper, two microbial cultures with high decolorization efficiencies of reactive dyes were obtained and were proved to be dominant with fungi consortium in which 21 fungal strains were isolated and 8 of them showed significant decolorization effect to reactive red M-3BE. A 4.5 l continuous biofilm reactor was established using the mixed cultures to investigate the decolorization performance and the system stability under the conditions of simulated and real textile wastewater as influents. The optimal nutrient feed to this bioreactor was 0.5 g l⁻¹ glucose and 0.1 g l⁻¹ (NH₄)₂SO₄ when 30 mg l⁻¹ reactive black 5 was used as initial dye concentrations. Dye mineralization rates of 50–75% and color removal efficiencies of 70–80% were obtained at 12 h hydraulic retention time (HRT) in this case. Higher glucose concentrations in the influents could significantly improve color removal, but was not helpful for dye mineralization. Besides reactive black 5, the bioreactor could effectively decolorize reactive red M-3BE, acid red 249 and real textile wastewater with efficiency of 65%, 94% and 89%, respectively. In addition, the microbial community on the biofilm was monitored in the whole running process. The results indicated fungi as a dominant population in the decolorization system with the ratio of fungi to bacteria 6.8:1 to 51.8:1 under all the tested influent conditions. Analysis of molecular biological detection indicated that yeasts of genus Candida occupied 70% in the fungal clone library based on 26S rRNA gene sequences.     

Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases

The potential of three oxidoreductases, a laccase preparation of Pleurotus sajor-caju PS-2001, horseradish peroxidase (HRP) and a microbial peroxidase (MP) was evaluated for the decolorization of disperse textile dyes (CI Disperse Red 343, CI Disperse Red 167 and CI Disperse Blue 148) used in polyester dyeing. Decolorization was studied in aqueous solutions varying in dye concentration, pH, temperature, enzyme concentration and the addition of mediators HBT and syringaldazine. The best conditions found for Disperse Red 343 with laccase, HRP and MP were: 15 mg L^?1 dye concentration, 50°C, pH 3.0 for laccase and pH 5.0 for peroxidases. Without mediator, the highest decolorizaton results (38.5% and 58.6%) were achieved with the highest tested concentrations of laccase (10 U mL^?1) and HRP (89.7 U mL^?1), respectively, but no significant difference in decolorization was found for the tested MP concentrations (29.9–89.7 U mL^‐1). HBT or syringaldazine increased decolorization with peroxidases significantly, but no effect was observed for the laccase. Decolorization of Disperse Red 167 (up to 15%) and Disperse Blue 148 (up to 25%) was much lower than of Disperse Red 343. With respect to enzyme concentration, the use of mediator and under the selected test conditions the laccase of P. sajor-caju PS-2001 turned out to be more efficient in disperse dye decolorization, than peroxidases HRP and MP.     

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).     

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H₂O₂ resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.     

Decolorization of Alizarin Red and other synthetic dyes by a recombinant laccase from Pichia pastoris

A cDNA encoding for a laccase was isolated from the white-rot fungus Lenzites gibbosa by RT-PCR and expressed in the Pichia pastoris. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as pH, cultivation temperature, copper concentration and methanol concentration, were optimized. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a MW of ~61.5 kDa. The purified enzyme behaved similarly to the native laccase produced by L. gibbosa and efficiently decolorized Alizarin Red, Neutral Red, Congo Red and Crystal Violet, without the addition of redox mediators. The decolorization capacity of this recombinant enzyme suggests that it could be a useful biocatalyst for the treatment of dye-containing effluents. This study is the first report on the synthetic dye decolorization by a recombinant L. gibbosa laccase.     

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

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

Overexpression of a Novel Thermostable and Chloride-Tolerant Laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and Its Application in Synthetic Dye Decolorization

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2''-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.     

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H₂O₂ resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.