纤维素降解复合菌系的微生物多样性及关键功能菌解析
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摘要
为分析纤维素降解复合菌系中微生物多样性及关键功能菌,本研究通过梯度稀释法观察不同梯度下滤纸降解及酶活变化情况,确定复合菌系实现滤纸有效降解的临界点为10-5;在此基础上结合高通量测序技术对不同稀释度下的稀释液进行测序.研究结果表明,具有降解能力的稀释液(稀释度≤10-5)与无降解能力的稀释液(稀释度>10-5)微生物组成差异显著.从门水平热图看,Firmicutes和Proteobacteria为复合菌系的优势菌门;在属水平上,复合菌系中协同降解纤维素的菌株主要属于Clostridium、Petrobacter、Defluviitalea等属,Clostridium属在有降解能力(稀释度≤10-5)的稀释液中丰度较高,而随着稀释度的增大,在无降解能力(稀释度>10-5)的稀释液中几乎消失.经分析判断梭菌属Clostridium是纤维素降解复合菌系的关键功能属,它们与Petrobacter、Defluviitalea等菌属协同作用从而实现纤维素的高效降解.
In order to analyze the microbial diversity and key functional bacteria in a cellulolytic microbial consortium,a dilution-to-extinction method was used to investigate the degradation of filter paper and enzyme activities under different dilution gradients. It was found that 10-5 is the critical dilution point of the cellulolytic microbial consortium for effective filter-paper degradation. Samples with different dilutions were further sequenced with high-throughput sequencing technology. Diversity analysis results show the microbial diversity of the samples with degradation capability( dilution gradient ≤10-5) and non-degradable samples( dilution gradient > 10-5) are significantly different. Firmicutes and Proteobacteria are found as the dominant bacteria of all samples at phylum level; and the bacteria can synergically degrade cellulose mainly belong to Clostridium,Petrobacter and Defluviitalea et al in the microbial consortium. The abundance of Clostridium sp. was higher in the samples with degradation ability( dilution gradient ≤10-5),but no detected in non-degradable samples( dilution gradient >10-5). Thus,Clostridium sp. was regarded as the key functional strains in the cellulose-degrading microbial consortium,and these strains can effectively degrade cellulose with the cooperation Petrobacter and Defluviitalea sp. etc.
In order to analyze the microbial diversity and key functional bacteria in a cellulolytic microbial consortium,a dilution-to-extinction method was used to investigate the degradation of filter paper and enzyme activities under different dilution gradients. It was found that 10-5 is the critical dilution point of the cellulolytic microbial consortium for effective filter-paper degradation. Samples with different dilutions were further sequenced with high-throughput sequencing technology. Diversity analysis results show the microbial diversity of the samples with degradation capability( dilution gradient ≤10-5) and non-degradable samples( dilution gradient > 10-5) are significantly different. Firmicutes and Proteobacteria are found as the dominant bacteria of all samples at phylum level; and the bacteria can synergically degrade cellulose mainly belong to Clostridium,Petrobacter and Defluviitalea et al in the microbial consortium. The abundance of Clostridium sp. was higher in the samples with degradation ability( dilution gradient ≤10-5),but no detected in non-degradable samples( dilution gradient >10-5). Thus,Clostridium sp. was regarded as the key functional strains in the cellulose-degrading microbial consortium,and these strains can effectively degrade cellulose with the cooperation Petrobacter and Defluviitalea sp. etc.
引文
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Shiratori H,Ohiwa H,Ikeno H,et al.2008.Lutispora thermophila gen.nov.,sp.nov.,a thermophilic,spore-forming bacterium isolated from a thermophilic methanogenic bioreactor digesting municipal solid wastes[J].International Journal of Systematic and Evolutionary Microbiology,58(Pt 4):964-969
Sridhar J,Eiteman M A.2001.Metabolic flux analysis of clostridium thermosuccinogenes:effects of p H and culture redox potential[J].Applied Biochemistry and Biotechnology,94(1):51-69
田敏.2013.高效纤维素降解复合菌系RXS的微生物多样性及关键功能菌初探[D].无锡:江南大学
王林,李冰,朱健.2016.高通量测序技术在人工湿地微生物多样性研究中的研究进展[J].中国农学通报,32(5):10-15
王士强,顾春梅,赵海红,等.2010.木质纤维素生物降解机理及其降解菌筛选方法研究进展[J].华北农学报,25(b08):313-317
涂漩,薛泉宏,司美茹,等.2004.多元混菌发酵对纤维素酶活性的影响[J].工业微生物,34(1):30-34
张木明,徐振林,张兴秀,等.2006.预处理对稻草秸秆纤维素酶解产糖及纤维素木质素含量的影响[J].农产品加工·学刊,58(3):4-6
张宁.2011.高效厌氧纤维素降解菌复合菌系的构建及应用研究[D].石家庄:河北师范大学
张全国,张志萍,赵民善,等.2011.秸秆制氢过程中纤维素酶酶活测定方法研究[J].热科学与技术,10(1):35-40
Zhang Q H,He J,Tian M,et al.2011.Enhancement of methane production from cassava residues by biological pretreatment using a constructed microbial consortium[J].Bioresource Technology,102:8899-8906
赵玉萍,杨娟.2006.四种纤维素酶酶活测定方法的比较[J].食品研究与开发,(3):116-118
曹新垲,杨琦,郝春博.2012.厌氧污泥降解萘动力学与生物多样性研究[J].环境科学,33(10):3535-3541
陈娜,杨毅,张澜,等.2014.Illumina高通量测序技术分析早产儿出生后肠道菌群变化的初步研究[J].中国循证儿科杂志,9(5):359-364
陈艳琼,阮灵伟.2015.深海热液区超嗜热古菌Thermococcus sp.TVG2的培养分离与鉴定[J].微生物学通报,42(3):467-477
陈泽斌,李冰,王定康,等.2015.Illumina Mi Seq高通量测序分析核桃内生细菌多样性[J].江苏农业学报,31(5):1129-1133
陈泽斌,李冰,王定康,等.2016.应用Illumina Mi Seq高通量测序分析玉米内生细菌多样性[J].现代食品科技,32(2):113-120
骋飞,王英,葛芹玉.2010.高通量DNA测序技术及其应用进展[J].南京晓庄学院学报,26(3):1-5
崔宗均,李美丹,朴哲,等.2002.一组高效稳定纤维素分解菌复合系MC1的筛选及功能[J].环境科学,23(3):36-39
Edgar R C.2013.UPARSE:Highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,10(10):996
El-Masry H G.1983.Utilization of Egyptian rice straw in production of cellulases and microbial protein:Effect of various pretreatments on yields of protein and enzyme activity[J].Journal of the Science of Food and Agriculture,34(7):725-732
付琳琳,李海星,曹郁生,等.2004.利用变性梯度凝胶电泳分析微生物的多样性[J].生物技术通报,(2):38-40
黄根树,孔令保,王刚,等.2015.纤维素降解复合菌系中纤维结合蛋白的分离及鉴定[J].环境科学学报,35(4):1026-1031
Jauris S,Rücknagel K P,Schwarz W H,et al.1990.Sequence analysis of the Clostridium stercorarium cel Z gene encoding a thermoactive cellulase(Avicelase l):identification of catalytic and cellulosebinding domains[J].MGG-Molecular and General Genetics,223(2):258-267
李红亚,李术娜,王树香,等.2015.解淀粉芽孢杆菌MN-8对玉米秸秆木质纤维素的降解[J].应用生态学报,26(5):1404-1410
刘甲锋.2010.水稻秸秆腐解复合菌系的筛选构建[D].北京:中国农业科学院
刘爽.2009.复合菌系降解木质纤维素特性及其菌群动态[D].哈尔滨:东北农业大学
刘艺.2015.降解木质纤维素的嗜热霉菌和放线菌的筛选及其培养基的优化[D].太原:山西大学
Maccallum I,Przybylski D,Gnerre S,et al.2009.ALLPATHS 2:small genomes assembled accurately and with high continuity from short paired reads[J].Genome Biology,10(10):R103
Mbadinga S M,Li K P,Zhou L,et al.2012.Analysis of Alkanedependent methanogenic community derived from production water of a high-temperature petroleum reservoir[J].Applied and Environmental Biotechnology,96(2):531-542
Niu L L,Song L,Liu X L,et al.2009.Tepidimicrobium xylanilyticum sp.nov.,an anaerobic xylanolytic bacterium,and emended description of the genus Tepidimicrobium[J].International Journal of Systematic and Evolutionary Microbiology,59(Pt 11):2698-2701
Schuster S C.2008.Next-generation sequencing transforms today's biology[J].Nature Methods,5(1):16
Shiratori H,Ohiwa H,Ikeno H,et al.2008.Lutispora thermophila gen.nov.,sp.nov.,a thermophilic,spore-forming bacterium isolated from a thermophilic methanogenic bioreactor digesting municipal solid wastes[J].International Journal of Systematic and Evolutionary Microbiology,58(Pt 4):964-969
Sridhar J,Eiteman M A.2001.Metabolic flux analysis of clostridium thermosuccinogenes:effects of p H and culture redox potential[J].Applied Biochemistry and Biotechnology,94(1):51-69
田敏.2013.高效纤维素降解复合菌系RXS的微生物多样性及关键功能菌初探[D].无锡:江南大学
王林,李冰,朱健.2016.高通量测序技术在人工湿地微生物多样性研究中的研究进展[J].中国农学通报,32(5):10-15
王士强,顾春梅,赵海红,等.2010.木质纤维素生物降解机理及其降解菌筛选方法研究进展[J].华北农学报,25(b08):313-317
涂漩,薛泉宏,司美茹,等.2004.多元混菌发酵对纤维素酶活性的影响[J].工业微生物,34(1):30-34
张木明,徐振林,张兴秀,等.2006.预处理对稻草秸秆纤维素酶解产糖及纤维素木质素含量的影响[J].农产品加工·学刊,58(3):4-6
张宁.2011.高效厌氧纤维素降解菌复合菌系的构建及应用研究[D].石家庄:河北师范大学
张全国,张志萍,赵民善,等.2011.秸秆制氢过程中纤维素酶酶活测定方法研究[J].热科学与技术,10(1):35-40
Zhang Q H,He J,Tian M,et al.2011.Enhancement of methane production from cassava residues by biological pretreatment using a constructed microbial consortium[J].Bioresource Technology,102:8899-8906
赵玉萍,杨娟.2006.四种纤维素酶酶活测定方法的比较[J].食品研究与开发,(3):116-118
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