秸秆降解菌株的筛选、鉴定及生物学特性研究
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摘要
为筛选优良秸秆降解菌株,解析不同菌株降解秸秆的生物学过程。本研究利用以秸秆为唯一碳源的筛选方法从土壤中获得3株具有秸秆降解能力的菌株yj1、yj2和yj3。根据8d的秸秆降解率确定菌株降解秸秆的最优温度和pH,并测定和比较参与秸秆降解的内切葡聚糖酶(EG)、β-葡萄糖苷酶(BG)、木聚糖酶、木质素过氧化物酶(LiP)、锰过氧化物酶(MnP)和漆酶(Lac)的活性。结果表明:1)筛选得到的yj1、yj2和yj3菌株分别属于假单胞菌属、芽孢杆菌属和短杆菌属。2)3种菌株分别在最适温度37、30和37℃以及最适pH 8、8和7时培养8d后,秸秆降解率达到峰值,在偏碱性条件下菌株yj2的秸秆降解率为26.61%。3)菌株yj1和yj3无木聚糖酶的活性,但LiP、MnP和Lac酶活性较高。菌株yj2的6种酶均具有较高的活性,协同完成秸秆降解。因此,菌株yj2可以作为碱性土壤条件下秸秆降解的候选菌株。
The aims of this study were to select better straw-decomposing bacteria strains and analyzing the biological processes of straw degradation. A total of three strains of straw-degrading bacteria,designated yj1,yj2 and yj3,were identified from soil samples.The optimal temperature and pH conditions for straw degradation were determined according to the 8-day straw degradation rate.The activities of six enzymes involved in EG,BG,xylanase,LiP,MnP,Lac were measured and compared.The results showed that:1)yj1,yj2 and yj3 strains were identified belonging to Pseudomonas,Bacillus and Brevibacterium,respectively.2)The straw degradation efficiency of three strains reached their peak values when the three strains were respectively cultured at 37,30 and 37℃ and the optimal pH of 8,8 and7 for 8 days.The straw degradation rate of yj2 reached 26.61% under alkaline conditions.3)The yj1 and yj3 strains were found to be xylanase-free but their LiP,MnP,Lac activities were higher,which played a key role in straw degradation.All of the six enzymes in strain yj2 showed higher activity and collaboratively completed straw degradation.In conclusion,strain yj2 could be used as a candidate strain for straw degradation under alkaline soil conditions.
The aims of this study were to select better straw-decomposing bacteria strains and analyzing the biological processes of straw degradation. A total of three strains of straw-degrading bacteria,designated yj1,yj2 and yj3,were identified from soil samples.The optimal temperature and pH conditions for straw degradation were determined according to the 8-day straw degradation rate.The activities of six enzymes involved in EG,BG,xylanase,LiP,MnP,Lac were measured and compared.The results showed that:1)yj1,yj2 and yj3 strains were identified belonging to Pseudomonas,Bacillus and Brevibacterium,respectively.2)The straw degradation efficiency of three strains reached their peak values when the three strains were respectively cultured at 37,30 and 37℃ and the optimal pH of 8,8 and7 for 8 days.The straw degradation rate of yj2 reached 26.61% under alkaline conditions.3)The yj1 and yj3 strains were found to be xylanase-free but their LiP,MnP,Lac activities were higher,which played a key role in straw degradation.All of the six enzymes in strain yj2 showed higher activity and collaboratively completed straw degradation.In conclusion,strain yj2 could be used as a candidate strain for straw degradation under alkaline soil conditions.
引文
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[9]聂志强.产纤维素酶真菌的筛选及其酶学性质的研究[J].中国酿造,2009,28(3):81-83Nie Z Q.Screen of the high cellulase-producing fungi and the research on its enzymatic characteristics[J].Chinese Brewing,2009,28(3):81-83(in Chinese)
[10] Zhang J,Hou H,Chen G,Wang S,Zhang J.The isolation and functional identification on producing cellulase of Pseudomonas mendocina[J].Bioengineered Bugs,2016,7(5):382-391
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[13] Bourbonnais R,Paice M G. Oxidation of non-phenolic substrates:An expanded role for laccase in lignin biodegradation[J].Febs Letters,1990,267(1):99-102
[14]黄元桐,崔杰.革兰氏染色三步法与质量控制[J].微生物学报,1996(1):76-78Hang Y T,Cui J.Three step method and quality control of gram stain[J].Acta Microbiologica Sinica.1990,267(1):99-102(in Chinese)
[15]刘晓梅,邹亚杰,胡清秀,杨小红,沈德龙.菌渣纤维素降解菌的筛选与鉴定[J].农业环境科学学报,2015,34(7):1384-1391Liu X M,Zhou Y J,Hu Q X,Yang X H,Shen D L.Screening and identification of cellulose-degrading bacteria from spent substrate of edible mushroom[J].Journal of Agro-Environment Science,2015,34(7):1384-1391(in Chinese)
[16]王洪媛,范丙全.三株高效秸秆纤维素降解真菌的筛选及其降解效果[J].微生物学报,2010,50(7):870-875Wang H Y,Fan B Q.Screening of three strains of cellulosedegrading fungi and determination of degrading effect[J].Acta Microbiologica Sinica,2010,50(7):870-875(in Chinese)
[17]张楠,杨兴明,徐阳春,沈其荣,黄启为.高温纤维素降解菌的筛选和酶活性测定及鉴定[J].南京农业大学学报,2010,33(3):82-87Zhang N,Yang X M,Xu Y C,Shen Q R,Hang Q W.Screening,cellulose activity measurement and identification of cellulose-decomposing bacteria[J].Journal of Nanjing Agricultural University,2010,33(3):82-87(in Chinese)
[18]王海滨,韩立荣,冯俊涛,张兴.高效纤维素降解菌的筛选及复合菌系的构建[J].农业生物技术学报,2015,23(4):421-431Wang H B,Han L R,Feng J T,Zhang X.Screening of highly efficient cellulose degradation microbes and construction of composite strains[J].Journal of Agricultural Biotechnology,2015,23(4):421-431(in Chinese)
[19]邹芳,赵娟,雷燕萍,祖朝龙,曹慧.一株烟草秸秆降解菌的分离、鉴定及酶学性质研究[J].土壤,2016,48(5):939-945Zuo F,Zhao J,Lei Y P,Zu C L,Cao L.Screening,identification and enzymes characteristics of tobacco strawdegrading bacteria[J].Soil,2016,48(5):939-945(in Chinese)
[20] Heredia A,Jiménez A,Guillén R.Composition of plant cell walls[J].Zeitschrift für Lebensmittel-Untersuchung und Forschung,1995,200(1):24-31
[21]Maki M,Leung K T,Qin W.The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass[J].International Journal of Biological Sciences,2009,5(5):500
[22]刘锁珠,李龙,付冠华,王利红,强巴央宗,李家奎.藏猪源高产纤维素酶菌株的筛选及鉴定[J].西北农林科技大学学报:自然科学版,2017,45(3):43-50Liu S Z,Li L,Fu G H,Wang H L,Qiang B Y Z,Li J K.Screening and identification of high-yield cellulose degrading bacteria strain from Tibetan pigs[J].Journal of Northwest A&F University:Natural Science Edition,2017,45(3):43-50(in Chinese)
[23]李立波,任晓冬,窦森.固态发酵中2种微生物降解玉米秸秆效果的对比研究[J].农业环境科学学报,2017,36(10):2136-2142Li L B,Ren X D,Dou S.Comparative study of the degradation efficiency of 2types of microorganisms on the degradation of corn stalks in solid-state fermentation[J].Journal of Agro-Environment Science,2017,36(10):2136-2142(in Chinese)
[24]刘长莉,王小芬,牛俊玲,吕育财,沈海龙,崔宗均.一组纤维素分解菌复合系NSC-7的酶活表达特性[J].微生物学通报,2008,35(5):720-724Liu C L,Wang X F,Niu J L,Lv Y C,Shen H L,Cui Z J.Expression characteristics of enzyme activity of a multifunctional bacterial community NSC-7[J]. Microbiology China,2008,35(5):720-724(in Chinese)
[25] Hu J G, Arantes V,Pribowo A,Saddler J N. The synergistic action of accessory enzymes enhances the hydrolytic potential of a"cellulase mixture"but is highly substrate specific[J].Biotechnology for Biofuels,2013,6(1):112
[26] Zhang J, Tuomainen P, Siika-Aho M, Viikari, L.Comparison of the synergistic action of two thermostable xylanases from GH families 10and 11 with thermostable cellulases in lignocellulose hydrolysis[J]. Bioresource Technology,2011,102(19):9090
[2] Zhao S C,Li K J,Zhou W,Qiu S J,Huang S W,He P.Changes in soil microbial community,enzyme activities and organic matter fractions under long-term straw return in northcentral China[J].Agriculture Ecosystems&Environment,2016,216:82-88
[3]冯炘,裴宇航,周晓飞,解玉红.纤维素降解菌的筛选与高效混合菌群的构建[J].西北农林科技大学学报:自然科学版,2012,40(4):155-160Feng X,Pei Y H,Zhou X F,Xie H Y.Screening of stalk cellulose-degrading microorganisms and construction of a microbial community with high capacity[J].Journal of Northwest A&F University:Natural Science Edition,2012,40(4):155-160(in Chinese)
[4] Deswal D,Khasa Y P,Kuhad R C.Optimization of cellulase production by a brown rot fungus Fomitopsis sp RCK2010under solid state fermentation[J].Bioresource Technology,2011,102(10):6065-6072
[5]任大明,赵钰,白炜琪,陈红漫,阚国仕,佡思涵,张聪.一株产纤维素酶放线菌的分离鉴定及酶学性质研究[J].沈阳农业大学学报,2016,47(6):667-672Ren D M,Zhao Y,Bai W Q,Chen H M,Kan G S,Xian S H,Zhang C.Isolation,identification and enzymatic properties of a cellulose-producing actinomyces[J].Journal of Shenyang Agricultural University,2016,47(6):667-672(in Chinese)
[6] Oke M A,Annuar M S M,Simarani K.Mixed lignocellulosic biomass degradation and utilization for bacterial cellulase production[J].Waste&Biomass Valorization,2017,8(3):893-903
[7]李红亚,李术娜,王树香,王全,朱宝成.产芽孢木质素降解菌MN-8的筛选及其对木质素的降解[J].中国农业科学,2014,47(2):324-333Li H Y,Li S N,Wang S X,Wang Q,Zhu H C.Screening,identification of lignin-degradating bacillus MN-8 and its characteristics in degradation of maize straw lignin[J].Scientia Agricultura Sinica,2014,47(2):324-333(in Chinese)
[8]王全,王会,李红亚,李术娜.一株高效木质素降解菌株LG-1的筛选、鉴定及酶活测定[J].饲料工业,2016,37(12):47-52Wang Q,Wang H,Li H Y,Li S N.Isolation,identification and enzyme activity determination of a high effective lignindegradation fungi LG-1[J].Feed Industry Magazine,2016,37(12):47-52(in Chinese)
[9]聂志强.产纤维素酶真菌的筛选及其酶学性质的研究[J].中国酿造,2009,28(3):81-83Nie Z Q.Screen of the high cellulase-producing fungi and the research on its enzymatic characteristics[J].Chinese Brewing,2009,28(3):81-83(in Chinese)
[10] Zhang J,Hou H,Chen G,Wang S,Zhang J.The isolation and functional identification on producing cellulase of Pseudomonas mendocina[J].Bioengineered Bugs,2016,7(5):382-391
[11] Ming T,Kirk T K.Lignin peroxidase of phanerochaete chrysosporium[J].Methods Enzymol,1988;161:238-249
[12] Francisco Javier Ruiz-D,Francisco G,Susana C, Marta Pérez-B, María Jesús M,ángel T. M. Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii[J]. Applied&Environmental Microbiology,1999,65(10):4458
[13] Bourbonnais R,Paice M G. Oxidation of non-phenolic substrates:An expanded role for laccase in lignin biodegradation[J].Febs Letters,1990,267(1):99-102
[14]黄元桐,崔杰.革兰氏染色三步法与质量控制[J].微生物学报,1996(1):76-78Hang Y T,Cui J.Three step method and quality control of gram stain[J].Acta Microbiologica Sinica.1990,267(1):99-102(in Chinese)
[15]刘晓梅,邹亚杰,胡清秀,杨小红,沈德龙.菌渣纤维素降解菌的筛选与鉴定[J].农业环境科学学报,2015,34(7):1384-1391Liu X M,Zhou Y J,Hu Q X,Yang X H,Shen D L.Screening and identification of cellulose-degrading bacteria from spent substrate of edible mushroom[J].Journal of Agro-Environment Science,2015,34(7):1384-1391(in Chinese)
[16]王洪媛,范丙全.三株高效秸秆纤维素降解真菌的筛选及其降解效果[J].微生物学报,2010,50(7):870-875Wang H Y,Fan B Q.Screening of three strains of cellulosedegrading fungi and determination of degrading effect[J].Acta Microbiologica Sinica,2010,50(7):870-875(in Chinese)
[17]张楠,杨兴明,徐阳春,沈其荣,黄启为.高温纤维素降解菌的筛选和酶活性测定及鉴定[J].南京农业大学学报,2010,33(3):82-87Zhang N,Yang X M,Xu Y C,Shen Q R,Hang Q W.Screening,cellulose activity measurement and identification of cellulose-decomposing bacteria[J].Journal of Nanjing Agricultural University,2010,33(3):82-87(in Chinese)
[18]王海滨,韩立荣,冯俊涛,张兴.高效纤维素降解菌的筛选及复合菌系的构建[J].农业生物技术学报,2015,23(4):421-431Wang H B,Han L R,Feng J T,Zhang X.Screening of highly efficient cellulose degradation microbes and construction of composite strains[J].Journal of Agricultural Biotechnology,2015,23(4):421-431(in Chinese)
[19]邹芳,赵娟,雷燕萍,祖朝龙,曹慧.一株烟草秸秆降解菌的分离、鉴定及酶学性质研究[J].土壤,2016,48(5):939-945Zuo F,Zhao J,Lei Y P,Zu C L,Cao L.Screening,identification and enzymes characteristics of tobacco strawdegrading bacteria[J].Soil,2016,48(5):939-945(in Chinese)
[20] Heredia A,Jiménez A,Guillén R.Composition of plant cell walls[J].Zeitschrift für Lebensmittel-Untersuchung und Forschung,1995,200(1):24-31
[21]Maki M,Leung K T,Qin W.The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass[J].International Journal of Biological Sciences,2009,5(5):500
[22]刘锁珠,李龙,付冠华,王利红,强巴央宗,李家奎.藏猪源高产纤维素酶菌株的筛选及鉴定[J].西北农林科技大学学报:自然科学版,2017,45(3):43-50Liu S Z,Li L,Fu G H,Wang H L,Qiang B Y Z,Li J K.Screening and identification of high-yield cellulose degrading bacteria strain from Tibetan pigs[J].Journal of Northwest A&F University:Natural Science Edition,2017,45(3):43-50(in Chinese)
[23]李立波,任晓冬,窦森.固态发酵中2种微生物降解玉米秸秆效果的对比研究[J].农业环境科学学报,2017,36(10):2136-2142Li L B,Ren X D,Dou S.Comparative study of the degradation efficiency of 2types of microorganisms on the degradation of corn stalks in solid-state fermentation[J].Journal of Agro-Environment Science,2017,36(10):2136-2142(in Chinese)
[24]刘长莉,王小芬,牛俊玲,吕育财,沈海龙,崔宗均.一组纤维素分解菌复合系NSC-7的酶活表达特性[J].微生物学通报,2008,35(5):720-724Liu C L,Wang X F,Niu J L,Lv Y C,Shen H L,Cui Z J.Expression characteristics of enzyme activity of a multifunctional bacterial community NSC-7[J]. Microbiology China,2008,35(5):720-724(in Chinese)
[25] Hu J G, Arantes V,Pribowo A,Saddler J N. The synergistic action of accessory enzymes enhances the hydrolytic potential of a"cellulase mixture"but is highly substrate specific[J].Biotechnology for Biofuels,2013,6(1):112
[26] Zhang J, Tuomainen P, Siika-Aho M, Viikari, L.Comparison of the synergistic action of two thermostable xylanases from GH families 10and 11 with thermostable cellulases in lignocellulose hydrolysis[J]. Bioresource Technology,2011,102(19):9090