雷竹林土壤染料降解真菌筛选与脱色效果初步研究
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摘要
应用平板分离培养和内转录间隔区(internal transcribed spacers,ITS)序列分析的方法,筛选鉴定集约栽培雷竹Phyllostachys violascens林土壤中染料降解真菌菌株,并通过摇瓶发酵研究其染料脱色效果。结果表明:土壤样品中分离频次最高的真菌种类为木霉菌属Trichoderma,对5株接种到苯胺蓝平板上能形成明显水解圈的真菌分离物,测试它们对偶氮和三苯甲烷类染料的降解能力。综合脱色效果较好的菌株为P7-2-3和P12-2-4,ITS序列比对和形态学观察表明菌株分别是卷枝毛霉Mucor circinelloides和木霉菌Trichoderma sp.,接种不超过500.0 mg·L~(-1)质量浓度结晶紫染料液体发酵脱色均可获得较高的脱色率;但染料质量浓度为300.0 mg·L~(-1)和400.0 mg·L~(-1)时分别对2菌株的脱色能力产生明显抑制,降解染料质量与菌丝生物量之间存在显著的正相关性。
This study aimed to screen high efficiency decolorizing fungi for degradation of structurally different synthetic dyes. Fungal strains were isolated and screened from soil of bamboo plantation(Phyllostachys violascens) using conventional separation, cultivation methods, and sequence alignment analysis of nuclear ribosomal internal transcribed spacers(ITS). The bamboo plantation for isolating soil fungi consisted of bamboo forests aged 1, 7, 12, and 15 years, which applied no heavy fertilization and organic mulch until year 5. Result showed that Trichoderma sp. had the highest isolation frequency in the cultured fungi from topsoil based on morphological identification and phylogenetic analysis. Decolorization capacity of the five isolated fungi((P7-2-1, P7-2-2,P1-2-2, P7-2-3, P12-2-4) to the five different synthetic dyes(Congo red, orange G, crystal violet, fuchsin acid,and methyl orange) showed that P7-2-3(identified as Mucor circinelloides) and P12-2-4(identified as Trichoderma spp.) had the strongest capacity to decolorize the five kinds of dyes. The decolorization efficiency of P7-2-3 and P12-2-4 to crystal violet was similar at different crystal violet concentrations below 500.0 mg·L-1;however, crystal violet inhibited the decolonization reaction at concentrations of 300.0 mg·L~(-1) for strain P7-2-3 and 400.0 mg·L~(-1) for strain P12-2-4. The fungal biomass and dye decolorization had a positive relationship for different concentrations of crystal violet, and strains of P7-2-3 and P12-2-4 have potential value for application in decolorization and degradation of dyes wastewater.
This study aimed to screen high efficiency decolorizing fungi for degradation of structurally different synthetic dyes. Fungal strains were isolated and screened from soil of bamboo plantation(Phyllostachys violascens) using conventional separation, cultivation methods, and sequence alignment analysis of nuclear ribosomal internal transcribed spacers(ITS). The bamboo plantation for isolating soil fungi consisted of bamboo forests aged 1, 7, 12, and 15 years, which applied no heavy fertilization and organic mulch until year 5. Result showed that Trichoderma sp. had the highest isolation frequency in the cultured fungi from topsoil based on morphological identification and phylogenetic analysis. Decolorization capacity of the five isolated fungi((P7-2-1, P7-2-2,P1-2-2, P7-2-3, P12-2-4) to the five different synthetic dyes(Congo red, orange G, crystal violet, fuchsin acid,and methyl orange) showed that P7-2-3(identified as Mucor circinelloides) and P12-2-4(identified as Trichoderma spp.) had the strongest capacity to decolorize the five kinds of dyes. The decolorization efficiency of P7-2-3 and P12-2-4 to crystal violet was similar at different crystal violet concentrations below 500.0 mg·L-1;however, crystal violet inhibited the decolonization reaction at concentrations of 300.0 mg·L~(-1) for strain P7-2-3 and 400.0 mg·L~(-1) for strain P12-2-4. The fungal biomass and dye decolorization had a positive relationship for different concentrations of crystal violet, and strains of P7-2-3 and P12-2-4 have potential value for application in decolorization and degradation of dyes wastewater.
引文
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[11]余燚,郑平,金仁村,等.印染废水生物处理技术进展[J].化工进展,2008,27(11):1724-1727.YU Yi,ZHENG Ping,JIN Rencun,et al.Biotechnology for dyeing and printing wastewater treatment[J].Chem Ind Eng Prog,2008,27(11):1724-1727.
[12]靳奇峰,时胜男,焦庆祝,等.真菌在染料脱色中的应用及其酶学研究进展[J].辽宁师范大学学报:自然科学版,2009,32(4):480-483.JIN Qifeng,SHI Shengnan,JIAO Qingzhu,et al.Research advances in fungal enzymes and their application to dyedecolorization[J].J Liaoning Norm Univ Nat Sci,2009,32(4):480-483.
[13]袁海生,戴玉成,曹云,等.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[J].菌物学报,2010,29(3):429-436.YUAN Haisheng,DAI Yucheng,CAO Yun,et al.Screening of white-rot fungi for decolorizing synthetic dyes and investigation on factors affecting decolorizing capacity of Cerrena unicolor[J].Mycosystema,2010,29(3):429-436.
[14]梁红昌,千英花,张庆华,等.一株脱色真菌的鉴定及脱色特性的初步探讨[J].微生物学通报,2009,36(7):956-961.LIANG Hongchang,QIAN Yinghua,ZHANG Qinghua,et al.Preliminary discuss on identification and characterization of one decolorizing fungus[J].Microbiol China,2009,36(7):956-961.
[15]张培培,任随周,许玫英,等.微生物对三苯基甲烷类染料脱色的研究进展[J].微生物学通报,2009,36(9):1410-1417.ZHANG Peipei,REN Suizhou,XU Meiying,et al.Recent advances in microbial decolorization of triphenylmethane dyes[J].Microbiol China,2009,36(9):1410-1417.
[16]朱林,洪宇植,方泽民,等.真菌漆酶对蒽醌和偶氮类活性染料的脱色[J].中国科技论文在线,2010,5(3):224-227.ZHU Lin,HONG Yuzhi,FANG Zemin,et al.Decolorization of reactive dyes of anthraquinone and azo by fungal laccase[J].Sciencepaper Online,2010,5(3):224-227.
[17]王慧,郑小伟,王宾香,等.真菌对染料的脱色研究进展[J].应用与环境生物学报,2009,15(1):147-151.WANG Hui,ZHENG Xiaowei,WANG Binxiang,et al.Advance in decolorization of dye by fungi[J].Chim J Appl Environ Biol,2009,15(1):147-151.
[18]BUMPUS J A.Perspectives on the use of white rot fungi in bioremediation technologies[G]//SIKDAR S K,IRVINE R L.Bioremediation:Principles and Practices.Lancaster P A:Technomic Publishing Company,1998:67-109.
[19]可晓,陈双林,张小平,等.雷竹林存留有机覆盖物高效降解菌株分离及产酶条件优化[J].浙江农林大学学报,2012,29(2):244-250.KE Xiao,CHEN Shuanglin,ZHANG Xiaoping,et al.Selection of strains used to degrade organic mulching materials from Phyllostachys violascens forest and optimization of its enzyme production[J].J Zhejiang A&F Univ,2012,29(2):244-250.
[20]鲁如坤.土壤农业化学分析方法[M].北京:中国农业科技出版社,1999.
[21]ZHANG Tao,LI Yongfu,CHANG S X,et al.Converting paddy fields to Lei bamboo(Phyllostachys praecox)stands affected soil nutrient concentrations,labile organic carbon pools,and organic carbon chemical compositions[J].Plant Soil,2013,367(1/2):249-261.
[22]JAROSZUK-'SCISEL J,KUREK E,SLOMKA A,et al.Activities of cell wall degrading enzymes in autolyzing cultures of three Fusarium culmorum isolates:growth-promoting,deleterious and pathogenic to rye(Secale cereale)[J].Mycologia,2011,103(5):929-945.
[23]SCHOCH C L,SEIFERT K A,HUHNDORF S,et al.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proc Nal Acad Sci,2012,109(16):6241-6246.
[24]MELO I S,FAULL J L.Parasitism of Rhizoctonia solani by strains of Trichoderma spp.[J].Sci Agric,2000,57(1):55-59.
[25]VINALE F,SIVASITHAMPARAM K,GHISALBERTI E L,et al.Trichoderma-plant-pathogen interactions[J].Soil Biol Biochem,2008,40(1):1-10.
[26]ALTOMARE C,NORVELL W,BJORKMAN T,et al.Solubilization of phosphates and micronutrients by the plantgrowth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22[J].Appl Environ Microbiol,1999,65(7):2926-2933.
[27]田晔,滕应.木霉属真菌在重金属污染土壤生物修复中的应用潜力分析[J].科学技术与工程,2013,13(36):10877-10882.TIAN Ye,TENG Ying.Application potential of Trichoderma sp.in bioremediation of soil contaminated with heavy metals[J].Sci Technol Eng,2013,13(36):10877-10882.
[28]KALRA M K,SIDHU M S,SANDHU D K,et al.Production and regulation of cellulases in Trichoderma harzianum[J].Appl Microbiol Biotechnol,1984,20(6):427-429.
[29]JORGENSEN H,MORKEBERG A,KROGH K B,et al.Production of cellulases and hemicellulases by three Penicillium species:effect of substrate and evaluation of cellulase adsorption by capillary electrophoresis[J].Enzyme Microbial Technol,2005,36(1):42-48.
[30]党宏波,马双,王秋玉.原油降解丝状真菌的分子鉴定及降解效果分析[J].环境科学与技术,2011,34(5):61-64.DANG Hong Bo,MA Shuang,WANG Qiuyu.Molecular identification of petroleum degrading filamentous fungus and analysis of the oil-contaminant degradation[J].Environ Sci Technol,2011,34(5):61-64.
[31]黄榕德,豆小敏,张书军,等.染料高效吸附菌Penicillium oxalicum表面特征解析[J].环境污染治理技术与设备,2006,7(7):34-37.HUANG Rongde,DOU Xiaomin,ZHANG Shujun,et al.Surface characteristic analysis of high effciency dye removal fungi Penicillium oxalicum[J].Tech Equip Environ Poll Control,2006,7(7):34-37.
[2]杨芳,吴家森,钱新标,等.不同施肥雷竹林土壤微生物量碳的动态变化[J].浙江林学院学报,2006,23(1):70-74.YANG Fang,WU Jiasen,QIAN Xinbiao,et al.Dynamic changes of soil microbial biomass carbon in Phyllostachys praecox stand with different fertilizers[J].J Zhejiang For Coll,2006,23(1):70-74.
[3]董林根,姜小娟,方茂盛.雷竹覆盖栽培林地土壤微生物的初步研究[J].浙江林学院学报,1998,15(3):236-239.DONG Lingen,JIANG Xiaojuan,FANG Maosheng.Primary study of soil microorganism in Lei bamboo forest of protected cultivation[J].J Zhejiang For Coll,1998,15(3):236-239.
[4]郭子武,俞文仙,陈双林,等.林地覆盖对雷竹林土壤微生物特征及其与土壤养分制约性关系的影响[J].生态学报,2013,33(18):5623-5630.GUO Ziwu,YU Wenxiang,CHEN Shuanglin,et al.Influence of mulching management on soil microbe and its relationship with soil nutrient in Phyllostachys praecox stand[J].Acta Ecol Sin,2013,33(18):5623-5630.
[5]陈晓,刘勇,李国雷,等.土壤真菌研究方法及人为干扰对森林土壤真菌群落影响研究进展[J].世界林业研究,2011,24(5):7-12.CHEN Xiao,LIU Yong,LI Guolei,et al.Progress on research method of soil fungi and human disturbances to forest soil fungi[J].World For Res,2011,24(5):7-12.
[6]韩国民,何兴兵,张鹏,等.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[J].微生物学通报,2011,38(4):603-614.HAN Guomin,HE Xingbing,ZHANG Peng,et al.Efficient degradation of polyporus fungus Trichaptum abiet inum1302BG on the synthetic dyes congo red and fuchsin acid under non-sterile condition[J].Microbiol China,2011,38(4):603-614.
[7]SENAN R C,ABRAHAM T E.Bioremediation of textile azo dyes by aerobic bacterial consortium aerobic degradation of selected azo dyes by bacterial consortium[J].Biodegradation,2004,15(4):275-280.
[8]NIGAM P,ARMOUR G,BANAT I M,et al.Physical removal of textile dyes from effluents and solid-state fermentation by dye-absorbed agricultural residues[J].Bioresour Technol,2000,72(3):219-226.
[9]FU Yuzhu,VIRARAGHAVAN T.Fungal decolorization of dye wastewaters:a review[J].Bioresour Technol,2001,79(3):251-262.
[10]尹亮,陈章和,赵树进.微生物对偶氮染料的脱色及其基因工程研究进展[J].生物技术,2007,17(6):86-89.YIN Liang,CHEN Zhanghe,ZHAO Shujin.Progress of studies in decolorization of azo dyes by microorganisms and gene engineering for decoloring bacteria of azo dyes[J].Biotechnology,2007,17(6):86-89.
[11]余燚,郑平,金仁村,等.印染废水生物处理技术进展[J].化工进展,2008,27(11):1724-1727.YU Yi,ZHENG Ping,JIN Rencun,et al.Biotechnology for dyeing and printing wastewater treatment[J].Chem Ind Eng Prog,2008,27(11):1724-1727.
[12]靳奇峰,时胜男,焦庆祝,等.真菌在染料脱色中的应用及其酶学研究进展[J].辽宁师范大学学报:自然科学版,2009,32(4):480-483.JIN Qifeng,SHI Shengnan,JIAO Qingzhu,et al.Research advances in fungal enzymes and their application to dyedecolorization[J].J Liaoning Norm Univ Nat Sci,2009,32(4):480-483.
[13]袁海生,戴玉成,曹云,等.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[J].菌物学报,2010,29(3):429-436.YUAN Haisheng,DAI Yucheng,CAO Yun,et al.Screening of white-rot fungi for decolorizing synthetic dyes and investigation on factors affecting decolorizing capacity of Cerrena unicolor[J].Mycosystema,2010,29(3):429-436.
[14]梁红昌,千英花,张庆华,等.一株脱色真菌的鉴定及脱色特性的初步探讨[J].微生物学通报,2009,36(7):956-961.LIANG Hongchang,QIAN Yinghua,ZHANG Qinghua,et al.Preliminary discuss on identification and characterization of one decolorizing fungus[J].Microbiol China,2009,36(7):956-961.
[15]张培培,任随周,许玫英,等.微生物对三苯基甲烷类染料脱色的研究进展[J].微生物学通报,2009,36(9):1410-1417.ZHANG Peipei,REN Suizhou,XU Meiying,et al.Recent advances in microbial decolorization of triphenylmethane dyes[J].Microbiol China,2009,36(9):1410-1417.
[16]朱林,洪宇植,方泽民,等.真菌漆酶对蒽醌和偶氮类活性染料的脱色[J].中国科技论文在线,2010,5(3):224-227.ZHU Lin,HONG Yuzhi,FANG Zemin,et al.Decolorization of reactive dyes of anthraquinone and azo by fungal laccase[J].Sciencepaper Online,2010,5(3):224-227.
[17]王慧,郑小伟,王宾香,等.真菌对染料的脱色研究进展[J].应用与环境生物学报,2009,15(1):147-151.WANG Hui,ZHENG Xiaowei,WANG Binxiang,et al.Advance in decolorization of dye by fungi[J].Chim J Appl Environ Biol,2009,15(1):147-151.
[18]BUMPUS J A.Perspectives on the use of white rot fungi in bioremediation technologies[G]//SIKDAR S K,IRVINE R L.Bioremediation:Principles and Practices.Lancaster P A:Technomic Publishing Company,1998:67-109.
[19]可晓,陈双林,张小平,等.雷竹林存留有机覆盖物高效降解菌株分离及产酶条件优化[J].浙江农林大学学报,2012,29(2):244-250.KE Xiao,CHEN Shuanglin,ZHANG Xiaoping,et al.Selection of strains used to degrade organic mulching materials from Phyllostachys violascens forest and optimization of its enzyme production[J].J Zhejiang A&F Univ,2012,29(2):244-250.
[20]鲁如坤.土壤农业化学分析方法[M].北京:中国农业科技出版社,1999.
[21]ZHANG Tao,LI Yongfu,CHANG S X,et al.Converting paddy fields to Lei bamboo(Phyllostachys praecox)stands affected soil nutrient concentrations,labile organic carbon pools,and organic carbon chemical compositions[J].Plant Soil,2013,367(1/2):249-261.
[22]JAROSZUK-'SCISEL J,KUREK E,SLOMKA A,et al.Activities of cell wall degrading enzymes in autolyzing cultures of three Fusarium culmorum isolates:growth-promoting,deleterious and pathogenic to rye(Secale cereale)[J].Mycologia,2011,103(5):929-945.
[23]SCHOCH C L,SEIFERT K A,HUHNDORF S,et al.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proc Nal Acad Sci,2012,109(16):6241-6246.
[24]MELO I S,FAULL J L.Parasitism of Rhizoctonia solani by strains of Trichoderma spp.[J].Sci Agric,2000,57(1):55-59.
[25]VINALE F,SIVASITHAMPARAM K,GHISALBERTI E L,et al.Trichoderma-plant-pathogen interactions[J].Soil Biol Biochem,2008,40(1):1-10.
[26]ALTOMARE C,NORVELL W,BJORKMAN T,et al.Solubilization of phosphates and micronutrients by the plantgrowth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22[J].Appl Environ Microbiol,1999,65(7):2926-2933.
[27]田晔,滕应.木霉属真菌在重金属污染土壤生物修复中的应用潜力分析[J].科学技术与工程,2013,13(36):10877-10882.TIAN Ye,TENG Ying.Application potential of Trichoderma sp.in bioremediation of soil contaminated with heavy metals[J].Sci Technol Eng,2013,13(36):10877-10882.
[28]KALRA M K,SIDHU M S,SANDHU D K,et al.Production and regulation of cellulases in Trichoderma harzianum[J].Appl Microbiol Biotechnol,1984,20(6):427-429.
[29]JORGENSEN H,MORKEBERG A,KROGH K B,et al.Production of cellulases and hemicellulases by three Penicillium species:effect of substrate and evaluation of cellulase adsorption by capillary electrophoresis[J].Enzyme Microbial Technol,2005,36(1):42-48.
[30]党宏波,马双,王秋玉.原油降解丝状真菌的分子鉴定及降解效果分析[J].环境科学与技术,2011,34(5):61-64.DANG Hong Bo,MA Shuang,WANG Qiuyu.Molecular identification of petroleum degrading filamentous fungus and analysis of the oil-contaminant degradation[J].Environ Sci Technol,2011,34(5):61-64.
[31]黄榕德,豆小敏,张书军,等.染料高效吸附菌Penicillium oxalicum表面特征解析[J].环境污染治理技术与设备,2006,7(7):34-37.HUANG Rongde,DOU Xiaomin,ZHANG Shujun,et al.Surface characteristic analysis of high effciency dye removal fungi Penicillium oxalicum[J].Tech Equip Environ Poll Control,2006,7(7):34-37.
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