多孔菌Trichaptum abietinum 1302BG自然条件下对合成染料刚果红和酸性品红的高效降解

选用杭州竹林土壤分离并筛选能够降解多种类型染料的真菌。经大量筛选发现一株编号为1302BG的真菌能够在固体培养基上分解所测试的全部9种染料(苯胺蓝、刚果红、橙黄G、甲基红、甲基橙、结晶紫、酸性品红、番红花红、碱性品红、甲基紫)。经形态学和分子生物学方法鉴定, 该菌1302BG为冷杉附毛孔菌(Trichaptum abietinum)。在液体培养基中研究了pH、温度、碳源、氮源、碳氮源组合、碳氮源浓度等参数对该菌脱色效果的影响, 以寻找最适最经济的脱色条件。在液体培养基中研究表明, 冷杉附毛孔菌1302BG既能在酸性又能在碱性条件下有效分解2种测试染料(酸性品红和刚果红)。该真菌能以仅含有0.5 g/L淀粉和0.05 g/L硫酸铵的经济、环境友好的培养基为底物, 能在灭菌和非灭菌(自然)的条件下高效脱色, 在24 h内对2种染料的脱色率均在90%以上。紫外/可见光谱及微核试验分析显示, 该菌脱色主要是以生物降解为主, 2种染料经该菌分解后的毒性也同时大大降低。这些优异特点显示了该菌具有非常广阔的工业染料废水处理应用潜力。     

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%。     

白囊耙齿菌对茜素红脱色条件优化及其毒性变化检测

白囊耙齿菌Irpex lacteus是分离自倒木上的一株可以分泌漆酶和锰过氧化物酶的白腐真菌。利用I. lacteus对固体条件下的活性黑、活性红、结晶紫、茜素红和孔雀石绿进行脱色能力的检测,通过单因素和正交试验优化I. lacteus对茜素红的脱色条件,并以3种作物发芽率为指标测定茜素红被I. lacteus脱色前后的毒性变化。结果显示,I. lacteus对5种染料均可脱色,其中对茜素红染料的脱色更为彻底;单因素和正交试验优化I. lacteus对茜素红的脱色条件为：pH 7.0、葡萄糖10.0g/L、硫酸铵0.66g/L、接种量2片（Φ=8.0mm）、100.0mL三角瓶装液20.0mL,优化条件下I. lacteus对茜素红脱色10d时的脱色率为88.26%,与未优化前的脱色率相比提高了60.50%;茜素红染料被I. lacteus脱色前后毒性大小排序为：染料原液>染料脱色后>PDB培养基处理,表明茜素红染料存在一定的毒性,I. lacteus脱色茜素红后可以使其毒性减弱。通过本研究,为I. lacteus在茜素红等染料废水脱色以及降低染料废水毒性方面的应用奠定基础。     

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%。     

一色齿毛菌对刚果红的脱色优化及其毒性变化研究

一色齿毛菌Cerrena unicolor是分离自野外的一株能够降解木质素的白腐真菌。为明确一色齿毛菌对染料的脱色能力及脱色前后染料毒性的变化,本研究利用一色齿毛菌对固体条件下4种染料进行脱色能力的检测,筛选出较易脱色的染料后,对该染料的脱色条件进行优化,并以3种豆类发芽率为指标测定该染料脱色前后的毒性变化。结果表明,一色齿毛菌对4种染料均可脱色,其中对刚果红的脱色效果最为明显;一色齿毛菌对刚果红脱色条件的优化结果为：20g/L麦芽糖,1g/L硝酸铵,1mmol/L硫酸镁,接种9块直径1cm菌饼,10mg/L染料浓度,pH 7时脱色效果最好;刚果红染料脱色前后毒性测试结果显示：染料脱色前发酵液毒性>染料脱色后发酵液毒性>清水处理毒性,表明刚果红染料存在一定的毒性,但在被一色齿毛菌脱色后,染料毒性有所降低。本研究为一色齿毛菌在染料废水脱色方面的应用及降低染料废水毒性提供一定的参考依据。     

Vital Fluorochromes as Tracers for Fungal Growth Studies

Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyanines DiIC₁₈(3) and DiOC₁₈(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma. harzianum Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence with at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC₁₈(3) and DiOC₁₈(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.     

Production of laccase and manganese peroxidase by Pleurotus pulmonarius in solid-state cultures and application in dye decolorization

The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860?±?250 U/L), pineapple peel (2,450?±?230 U/L), and orange bagasse (2,100?±?270 U/L) cultures, all of them at an initial moisture level of 85 %. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200?±?205 U/L) at an initial moisture level of 75 %. In general, the condition of high initial moisture level (80–90 %) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50–70 %). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.     

白腐真菌染料脱色菌株的筛选及一色齿毛菌脱色条件的研究

以平皿培养方式对采集自中国和芬兰的白腐真菌菌株降解6种不同结构的人工染料的能力进行了筛选研究。在40株菌株中,黑管孔菌Bjerkandera adusta Y5012,一色齿毛菌Cerrena unicolor Y5002,硬毛粗盖孔菌Funalia trogii Y4997,香栓孔菌Trametes suaveolens D8325和云芝栓孔菌Trametes versicolor Y4946对刚果红、橙黄G、茜素红、结晶紫、中性红和亚甲基蓝均显示出较强的脱色能力。对一色齿毛菌Cerrena unicolor Y5002的液体培养脱色条件进行了研究,其最适碳源和氮源分别为蔗糖和麦芽浸粉;在不同橙黄G浓度下均获得较高的脱色率,因此浓度为500mg/L的橙黄G未对该菌的脱色能力产生抑制作用,而浓度为400mg/L茜素红则对其脱色作用产生明显抑制。对菌丝生物量和染料脱色率的研究表明,在不同碳源和氮源条件下,两者之间具有明显的正相关性。     

Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes

A total of 37 strains of aquatic hyphomycetes and 95 fungal isolates derived from diverse freshwater environments were screened on agar plates for the decolourisation of the disazo dye Reactive Black 5 and the anthraquinone dye Reactive Blue 19. The decolourisation of 9 azo and 3 anthraquinone dyes by 9 selected aquatic fungi was subsequently assessed in a liquid test system. The fungi were representatives of mitosporic anamorphs, and 6 strains had proven ascomycete affiliations. For comparison, 5 white rot basidiomycetes were included. The majority of dyes were decolourised by several mitosporic aquatic isolates at rates essentially comparable to those observed with the most efficient white rot fungus. Under certain conditions, particular aquatic strains decolourised dyes even more efficiently than the best performing white rot basidiomycete. Upon fungal treatment of several dyes, new absorbance peaks appeared, indicating biotransformation metabolites. All together, these results point to the potential of fungi occurring in freshwater environments for the treatment of dye-containing effluents.     

Laccase-mediator system in the decolorization of different types of recalcitrant dyes

Phloroglucinol, thymol, and violuric acid (VIO) were selected as laccase mediators after screening 14 different compounds with indigo carmine (indigoid dye) as a substrate. With the presence of these three mediators, a nearly complete decolorization (90-100%) was attained in 1 h. Thus, these three compounds were used as mediators for the decolorization of other four dyes. The results indicated that VIO was effective mediator in decolorization of Remazol brilliant blue R (RBBR, anthraquinoid dye) and Coomassie brilliant blue G-250 (CBB, triphenylmethane dyes), and Acid red (diazo dye). In presence of VIO, the four dyes described above attained 70% decolorization. Thymol was able to mediate decolorization of RBBR and Azure A (heterocyclic dye). Phloroglucinol has no mediating capability in decolorization of the four dyes analyzed. Mediator concentration, pH, and copper ion have an effect on the decolorization of the RBBR. Our data suggested that the decolorization capabilities of laccase/mediator system were related to the types of mediator, the dye structure and decolorization condition.     

Decolorisation of artificial dyes by peroxidase from the white-rot fungus, Pleurotus ostreatus

The Remazol Brilliant Blue R (RBBR) decolorising peroxidase of Pleurotus ostreatus decolorised several recalcitrant dyes. Eight different types of dyes, including triphenyl methane, heterocyclic, azo, and polymeric dyes, were decolorised to some extent. The best decolorisation was obtained for Bromophenol blue (98%). The enzyme oxidised triphenyl methane and azo dyes effectively. However, heterocyclic dyes, Methylene Blue and Toluidine Blue O were decolorised only by 10%. © Rapid Science Ltd. 1998     

Decolorization of simulated textile dye baths by crude laccases from Trametes hirsuta and Cerrena unicolor

In this study crude laccases from the white‐rot fungi Cerrena unicolor and Trametes hirsuta were tested for their ability to decolorize simulated textile dye baths. The dyes used were Remazol Brilliant Blue R (RBBR) (100 mg/L), Congo Red (12.5 mg/L), Lanaset Grey (75 mg/L) and Poly R‐478 (50 mg/L). The effect of redox mediators on dye decolorization by laccases was also assessed. C. unicolor laccase was able to decolorize all the dyes tested. It was especially effective towards Congo Red and RBBR with 91 and 80% of color removal in 19.5 h despite the fact that simulated textile dye baths were used. Also Poly R‐478 and Lanaset Grey were partially decolorized (69 and 48%, respectively). C. unicolor laccase did not need any mediators for removing the dyes. However, T. hirsuta laccase was only able to decolorize simulated Congo Red and RBBR dye baths (91 and 45%, respectively) in 19.5 h without mediators. When using mediators the decolorization capability was enhanced substantially, e.g. Poly R‐478 was decolorized by 78% in 25.5 h. On the whole, both laccases showed potential to be used in industrial applications.     

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.     

猴头菌和金针菇漆酶对不同染料的降解

利用漆酶（laccase）处理染料废水是近年来研究的热点。本研究以猴头菌Hericium erinaceus和金针菇Flammulina filiformis的发酵液为试验材料,通过硫酸铵沉淀、离子交换层析和超滤等方法,对发酵液中的漆酶进行了初步的分离纯化,然后分别研究了两种初提纯漆酶及其与小分子介体组成的漆酶介体系统（laccase-mediator system,LMS）对12种常用染料的降解效果。结果表明,猴头菌的初提纯漆酶对甲基红、铬黑T、孔雀绿和活性蓝R都具有很好的降解效果,反应24h后,降解率分别为79.6%、66.8%、80.3%和64.6%,且表现出时间依赖性,而对其他染料的降解效果不明显。当反应体系中加入介体2,2′-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐[2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphate),ABTS]后,可增加猴头菌漆酶对考马斯亮蓝、结晶紫和孔雀绿的降解率,分别提高到33.7%、45.6%和94.2%。金针菇初提纯漆酶对孔雀绿、活性艳橙K-7R和活性红KD-8B都具有一定的降解效果,降解率分别为37.8%、39.9%和49%,也表现出了明显的时间依赖性。当反应体系中也加入ABTS后,金针菇漆酶对活性红KM-8B的降解率明显增加,由9.7%增加到58.2%。以上研究为猴头菌和金针菇漆酶在染料废水处理领域的应用提供了科学依据。     

Decolorization of triphenylmethane and azo dyes by Citrobacter sp.

A Citrobacter sp., isolated from soil at an effluent treatment plant of a textile and dyeing industry, decolorized several recalcitrant dyes except Bromophenol Blue. More than 90% of Crystal Violet and Methyl Red at 100 M were reduced within 1 h. Gentian Violet, Malachite Green and Brilliant Green lost over 80% of their colors in the same condition, but the percentage decolorization of Basic Fuchsin and Congo Red were less than the others, 66 and 26%, respectively. Decolorization of Congo Red was mainly due to adsorption to cells. Color removal was optimal at pH 7–9 and 35–40 °C. Decolorization of dyes was also observed with extracellular culture filtrate, indicating the color removal by enzymatic biodegradation.     

Ligninolytic activity of tropical rainforest basidiomycetes

A total of 116 strains of Brazilian tropical rainforest basidiomycetes were evaluated in terms of their ability to oxidize the dye rhemazol brilliant blue R (RBBR) and guaiacol. Laccase and peroxidase activities were detected by the drop test using solutions of α-naphthol and pyrogallol, respectively. RBBR and guaiacol oxidation occurred in 96.6 and 87.1% of the strains tested, respectively. One hundred strains oxidized both substrates. In the drop test, most strains presented laccase (96.6%) and peroxidase (92.2%) activity. The quick screening method used here can be useful to identify ligninolytic fungal strains to be used in various biotechnological applications. This revised version was published online in November 2006 with corrections to the Cover Date.     

Grape seeds: the best lignocellulosic waste to produce laccase by solid state cultures of Trametes hirsuta

Grape seeds were used by Trametes hirsuta as a substrate for laccase production giving 23 kU l^–1, which was 10-fold the value attained in the cultures with no lignocellulosic waste addition. The dyes, Indigo Carmine and Bromophenol Blue, were easily decolourised (100% in 24 h) by the extracellular liquid obtained in such cultures, whereas Methyl Orange (65% in 24 h) and Phenol Red (36% in 24 h) were more resistant to degradation. This shows the specificity of laccase towards different dye structures.     

Enhanced dye decolorization efficiency by citraconic anhydride-modified horseradish peroxidase

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.     

毛木耳对孔雀石绿染料降解条件的优化

随着我国印染工业的发展,废水对生态环境的危害日趋严重,亟需开发一种脱色明显且成本低廉的降解方法。本研究发现毛木耳Auricularia cornea菌株AC5对不同结构的染料均具有一定的降解作用,尤其是三苯甲烷类染料。利用26℃、160r/min振荡培养7d的粗酶液对染料（75.0mg/L）进行12h降解,结果显示三苯甲烷染料孔雀石绿、结晶紫,蒽醌染料活性蓝19和偶氮染料活性蓝222的降解效率分别为83.27%、71.77%、67.81%和63.92%。染料降解实验和酶活力测定结果表明,毛木耳对孔雀石绿的降解率达到最高时漆酶活性最高,为321.0U/mL,木质素过氧化物酶和锰过氧化物酶活性较低。因此,推测在降解过程中漆酶起到主要作用。研究表明利用毛木耳菌丝发酵液降解染料废水成本低且操作方便,为染料废水的降解研究提供了前期基础。     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.