好氧堆肥微生物代谢多样性及其细菌群落结构
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摘要
好氧堆肥是农业废弃物无害化处理和资源化利用的一条有效途径.为了探究好氧堆肥过程中微生物群落的代谢特征和细菌群落演替现象,了解起关键作用的微生物菌群,通过筛选强降解菌种改善堆肥工艺、提高堆肥效率,采用Biolog法和宏基因组法分析了玉米秸秆和牛粪联合好氧堆肥过程中微生物的碳源代谢能力和细菌群落多样性.结果表明:在第2次翻堆(第14天)时,微生物利用碳源的能力最强,初次建堆时(0 d)和其余翻堆时(第8、20、26天)次之,发酵结束时(第34天)最弱.Simpson、Shannon-Wiener和Mc Intosh多样性指数表明,建堆时及翻堆时的菌群优势度、丰富度和均匀度均极显著优于好氧堆肥结束.不同好氧发酵时间的微生物群落对同一碳源代谢有差异,同一好氧发酵时间微生物群落对不同碳源的利用率不同.糖类、酸类和醇类是区分好氧堆肥不同时间微生物碳源利用差异的敏感碳源.好氧堆肥不同时间细菌的种类和丰度不同,共享的优势菌门有厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes),在第0、8、14、20、26、34天这6个时间内它们的相对丰度之和分别达90.27%、90.34%、94.26%、84.21%、84.31%和77.61%,且6种门类在不同发酵时间的丰度表达存在消长变化状态.研究显示,参与好氧堆肥不同时间的微生物群落在碳源代谢能力上存在多样性,在细菌菌群的种类和丰度上也存在多样性.
The aerobic fermentation composting is an effective way for the harmless disposal and resource utilization of agricultural waste.The present study is aimed at screening effectively degrading strains for improving composting process by exploring metabolic characteristics and microbial community and understanding the dominant bacteria during aerobic fermentation process. Biolog and metagenomic methods were used to analyze the carbon source metabolic capacity of microorganisms and bacterial community diversity for aerobic fermentation compost with corn stalk and cow manure. The results showed that the ability of carbon sources utilization for microorganisms was the strongest at the second turning(the 14 thday),and the early establishment of a composting(0 d) was in the second place,such as the same with the other periods of turning(the 8 th,20 thand 26 thday),and the weakest one was at the end of aerobic fermentation(the 34 thday). The diversity indices of Simpson,Shannon and Mc Intosh showed that dominance,richness and evenness for microbial community in the early establishment of a composting and the different turnover composting period were significantly better than those at the end of fermentation. The same carbon source metabolism differed to microbial communities at the different fermentation time,and the microbial community had different utilization of different carbon sources at the same fermentation period.Carbohydrates,acids and alcohols were sensitive carbon sources that could differentiate utilization variance of the microbe carbon sources from different time of aerobic compost. The species and abundances of bacteria were different at different periods of aerobic fermentation.Firmicutes,Proteobacteria,Bacteroidetes,Chloroflexi,Actinobacteria and Planctomycetes were the dominant groups on the phylum level.The sum of their relative abundances in the six periods were respectively 90. 27%,90. 34%,94. 26%,84. 21%,84. 31% and 77. 61%,and the abundances of six phyla existed changing state of growth and decline during different fermentation. It showed that the microbial communities involved in the aerobic compost at different time had diversity in carbon source metabolism,and their bacterial flora structure was different in species and abundance.
The aerobic fermentation composting is an effective way for the harmless disposal and resource utilization of agricultural waste.The present study is aimed at screening effectively degrading strains for improving composting process by exploring metabolic characteristics and microbial community and understanding the dominant bacteria during aerobic fermentation process. Biolog and metagenomic methods were used to analyze the carbon source metabolic capacity of microorganisms and bacterial community diversity for aerobic fermentation compost with corn stalk and cow manure. The results showed that the ability of carbon sources utilization for microorganisms was the strongest at the second turning(the 14 thday),and the early establishment of a composting(0 d) was in the second place,such as the same with the other periods of turning(the 8 th,20 thand 26 thday),and the weakest one was at the end of aerobic fermentation(the 34 thday). The diversity indices of Simpson,Shannon and Mc Intosh showed that dominance,richness and evenness for microbial community in the early establishment of a composting and the different turnover composting period were significantly better than those at the end of fermentation. The same carbon source metabolism differed to microbial communities at the different fermentation time,and the microbial community had different utilization of different carbon sources at the same fermentation period.Carbohydrates,acids and alcohols were sensitive carbon sources that could differentiate utilization variance of the microbe carbon sources from different time of aerobic compost. The species and abundances of bacteria were different at different periods of aerobic fermentation.Firmicutes,Proteobacteria,Bacteroidetes,Chloroflexi,Actinobacteria and Planctomycetes were the dominant groups on the phylum level.The sum of their relative abundances in the six periods were respectively 90. 27%,90. 34%,94. 26%,84. 21%,84. 31% and 77. 61%,and the abundances of six phyla existed changing state of growth and decline during different fermentation. It showed that the microbial communities involved in the aerobic compost at different time had diversity in carbon source metabolism,and their bacterial flora structure was different in species and abundance.
引文
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[6]INSAM H,AMOR K,RENNER M,et al.Changes in functional abilities of the microbial community during com posting of manure[J].Microbial Ecology(Historical Archive),1996,31:77-87.
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[8]杨玖,谷洁,张友旺,等.磺胺甲噁唑对堆肥过程中酶活性及微生物群落功能多样性的影响[J].环境科学学报,2014,34(4):965-972.YANG Jiu,GU Jie,ZHANG Youwang,et al.Effects of sulfamethoxazole on enzyme activity and microbial community functional diversity during pig manure composting[J].Acta Scientiae Circumstantiae,2014,34(4):965-972.
[9]陈耀宁.堆肥化中协同降解木质纤维素的混合菌筛选及其培养[D].长沙:湖南大学,2007.
[10]马海霞,张丽丽,孙晓萌,等.基于宏组学方法认识微生物群落及其功能[J].微生物学通报,2015,42(5):902-912.MA Haixia,ZHANG Lili,SUN Xiaomeng,et al.Understanding microbial communities and their functions by meta-omics approaches[J].Microbiology China,2015,42(5):902-912.
[11]MARTINS L F,ANTUNES L P,PASCON R C,et al.Metagenomic analysis of a tropical composting operation at the S2o Paulo Zoo park reveals diversity of biomass degradation functions and organisms[J].Plos One,2013,8(4):e61928.
[12]NEHER D A,WEICHT T R,BATES S T,et al.Changes in bacterial and fungal communities across compost recipes,preparation methods,and composting times[J].Plos One,2013,8(11):e79512.
[13]DE GANNES V,EUDOXIE G,HICKEY W J.Prokaryotic successions and diversity in composts as revealed by 454-yrosequencing[J].Bioresource Technology,2013,133:573-580.
[14]沈其荣,王瑞宝,王岩,等.堆肥制作中的生物化学变化特征[J].南京农业大学学报,1997,20(2):51-57.SHEN Qirong,WANG Ruibao,WANG Yan,et al.Biochemical characteristics of composting[J].Journal of Nanjing Agricultural University,1997,20(2):51-57.
[15]PARTANEN P,HULTNAN J,PAULIN L,et al.Bacterial diversity at different stages of the composting process[J].BMC Microbiology,2010.doi:10.1186/1471-21801-10-94.
[16]武爱莲,丁玉川,焦晓燕,等.玉米秸秆生物炭对褐土微生物功能多样性及细菌群落的影响[J].中国生态农业学报,2016,24(6):736-743.WU Ailian,DINGYuchuan,JIAO Xiaoyan,et al.Effect of corn-stalk biochar on soil microbial functional diversity and bacterial community in cinnamon soils[J].Chinese Journal of EcoAgriculture,2016,24(6):736-743.
[17]GARLAND J L,MILLS A L.Classification and characterization of heterotrophic microbial communities of on the basis of patterns of community-level sole carbon-source utilization[J].Applied Environmental Microbiology,1991,57:2351-2359.
[18]MAGURRAN A E.Ecological diversity and its measurement[M].New Jersey:Princeton University Press,1988.
[19]ZABINSKI C A,GANNON J E.Effects of recreational impacts on soil microbial communities[J].Environment Manage,1997,21:233-238.
[20]WEBER K P,GROVE J A,GEHDER M,et al.Data transformations in the analysis of community-level substrate utilization data from micro plates[J].Journal of Microbiological Methods,2007(69):461-469.
[21]ZAK J C,WILLIG M R,MOORHEAD D L,et al.Functional diversity of microbial communities:a quantitative approach[J].Soil Biology&Biochemistry,1994(26):1101-1108.
[22]魏姣皎,熊骏生,袁丽,等.好氧堆肥过程氨气释放和氮素转化影响因素研究进展[J].杭州师范大学学报(自然科学版),2017,16(1):80-86.WEI Jiaojiao,XIONG Junsheng,YUAN Li,et al.A review of influencing factors of ammonia emission and nitrogen transformation in aerobic composting[J].Journal of Hangzhou Normal University(Natural Science Edition),2017,16(1):80-86.
[23]吴艳萍,王国栋,赵明德.链霉菌对小麦秸秆堆肥中微生物群落和酶活性的影响[J].西北农林科技大学学报(自然科学版),2009,37(9):134-138.WU Yanping,WANG Guodong,ZHAO Mingde,et al.Effects of Streptomyces thermovulgaris on microbial community and enzyme activity during wheat straw composting process[J].Journal of Northwest A&F University(Natural Science Edition),2009,37(9):134-138.
[24]EL FELS L,EL OUAQOUDI FZ,BARJE F,et al.Two culture approaches used to determine the co-composting stages by assess of the total microflora changes during sewage sludge and date palm waste co-composting[J].Journal of Environmental Health Science&Engineering,2014(12):132.
[25]ANTUNES L P,MARTINS L F,PEREIRA R V,et al.Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics[J].Scientific Reports,2016,6:e38915.
[26]张丽丽.整合宏组学方法揭示天然木质纤维素堆肥中的关键功能微生物群落[D].济南:山东大学,2016.
[27]WANG Cheng,DONG Da,WANG Haoshu,et al.Metagenomic analysis of microbial consortia enriched from compost:new insights into the role of Actinobacteria in lignocellulose decomposition[J].Biotechnology for Biofuels,2016,9:22.
[28]PETERS S,KOSCHINSKY S,SCHWIEGER F,et al.Succession of microbial communities during hot composting as detected by PCRsingle-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes[J].Applied and Environmental Microbiology,2000,66(3):930-936.
[29]贾洋洋.利用宏基因组方法分析堆肥生境中微生物区系的变化[D].济南:山东大学,2012.
[30]SZKELY A J,SIPOS R,BERTA B,et al.DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP profile of mature compost[J].Microbial Ecology,2009,57(3):522-533.
[31]魏自民,王佰洁,赵越,等.堆肥低温起爆微生物筛选及其初步应用[J].环境科学研究,2015,28(6):981-986.WEI Zimin,WANG Baijie,ZHAO Yue,et al.Cold-tolerant bacteria screening and its inoculation in composting at low temperatures[J].Research of Environmental Sciences,2015,28(6):981-986.
[32]李红亚,李术娜,王树香,等.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):324-333.LI Hongya,LI Shuna,WANG Shuxiang,et al.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.
[2]田雅楠,王红旗.Biolog法在环境微生物功能多样性研究中的应用[J].环境科学与技术,2011,34(3):50-57.TIAN Yanan,WANG Hongqi.Application of Biolog to study of environmental microbial function diversity[J].Environmental Science&Technology(China),2011,34(3):50-57.
[3]闫冰,齐月,付刚,等.莠去津对野生植物群落下土壤微生物功能多样性的影响[J].环境科学研究,2017,30(8):1246-1254.YAN Bing,QI Yue,FU Gang,et al.Effects of atrazine on soil microbial functional diversity of wild weed communities[J].Research of Environmental Sciences,2017,30(8):1246-1254.
[4]徐万里,唐光木,葛春辉,等.长期施肥对新疆灰漠土土壤微生物群落结构与功能多样性的影响[J].生态学报,2015,35(2):468-477.XU Wanli,TANG Guangmu,GE Chunhui,et al.Effects of long-term fertilization on diversities of soil microbial community structure and function in grey desert soil of Xinjiang[J].Acta Ecologica Sinica,2015,35(2):468-477.
[5]孙雪,隋心,韩冬雪,等.原始红松林退化演替后土壤微生物功能多样性的变化[J].环境科学研究,2017,30(6):911-919.SUN Xue,SUI Xin,HAN Dongxue,et al.Changes of soil microbial functional diversity in the degraded and successional primitive Korean pine forest in Lesser Khingan Mountain,northern China[J].Research of Environmental Sciences,2017,30(6):911-919.
[6]INSAM H,AMOR K,RENNER M,et al.Changes in functional abilities of the microbial community during com posting of manure[J].Microbial Ecology(Historical Archive),1996,31:77-87.
[7]ATKINSON C F,JONES D D,GAUTHIER J J.Microbial activities during composting of pulp and paper-mill primary solids[J].World Journal of Microbiology&Biotechnology,1997,13:519-525.
[8]杨玖,谷洁,张友旺,等.磺胺甲噁唑对堆肥过程中酶活性及微生物群落功能多样性的影响[J].环境科学学报,2014,34(4):965-972.YANG Jiu,GU Jie,ZHANG Youwang,et al.Effects of sulfamethoxazole on enzyme activity and microbial community functional diversity during pig manure composting[J].Acta Scientiae Circumstantiae,2014,34(4):965-972.
[9]陈耀宁.堆肥化中协同降解木质纤维素的混合菌筛选及其培养[D].长沙:湖南大学,2007.
[10]马海霞,张丽丽,孙晓萌,等.基于宏组学方法认识微生物群落及其功能[J].微生物学通报,2015,42(5):902-912.MA Haixia,ZHANG Lili,SUN Xiaomeng,et al.Understanding microbial communities and their functions by meta-omics approaches[J].Microbiology China,2015,42(5):902-912.
[11]MARTINS L F,ANTUNES L P,PASCON R C,et al.Metagenomic analysis of a tropical composting operation at the S2o Paulo Zoo park reveals diversity of biomass degradation functions and organisms[J].Plos One,2013,8(4):e61928.
[12]NEHER D A,WEICHT T R,BATES S T,et al.Changes in bacterial and fungal communities across compost recipes,preparation methods,and composting times[J].Plos One,2013,8(11):e79512.
[13]DE GANNES V,EUDOXIE G,HICKEY W J.Prokaryotic successions and diversity in composts as revealed by 454-yrosequencing[J].Bioresource Technology,2013,133:573-580.
[14]沈其荣,王瑞宝,王岩,等.堆肥制作中的生物化学变化特征[J].南京农业大学学报,1997,20(2):51-57.SHEN Qirong,WANG Ruibao,WANG Yan,et al.Biochemical characteristics of composting[J].Journal of Nanjing Agricultural University,1997,20(2):51-57.
[15]PARTANEN P,HULTNAN J,PAULIN L,et al.Bacterial diversity at different stages of the composting process[J].BMC Microbiology,2010.doi:10.1186/1471-21801-10-94.
[16]武爱莲,丁玉川,焦晓燕,等.玉米秸秆生物炭对褐土微生物功能多样性及细菌群落的影响[J].中国生态农业学报,2016,24(6):736-743.WU Ailian,DINGYuchuan,JIAO Xiaoyan,et al.Effect of corn-stalk biochar on soil microbial functional diversity and bacterial community in cinnamon soils[J].Chinese Journal of EcoAgriculture,2016,24(6):736-743.
[17]GARLAND J L,MILLS A L.Classification and characterization of heterotrophic microbial communities of on the basis of patterns of community-level sole carbon-source utilization[J].Applied Environmental Microbiology,1991,57:2351-2359.
[18]MAGURRAN A E.Ecological diversity and its measurement[M].New Jersey:Princeton University Press,1988.
[19]ZABINSKI C A,GANNON J E.Effects of recreational impacts on soil microbial communities[J].Environment Manage,1997,21:233-238.
[20]WEBER K P,GROVE J A,GEHDER M,et al.Data transformations in the analysis of community-level substrate utilization data from micro plates[J].Journal of Microbiological Methods,2007(69):461-469.
[21]ZAK J C,WILLIG M R,MOORHEAD D L,et al.Functional diversity of microbial communities:a quantitative approach[J].Soil Biology&Biochemistry,1994(26):1101-1108.
[22]魏姣皎,熊骏生,袁丽,等.好氧堆肥过程氨气释放和氮素转化影响因素研究进展[J].杭州师范大学学报(自然科学版),2017,16(1):80-86.WEI Jiaojiao,XIONG Junsheng,YUAN Li,et al.A review of influencing factors of ammonia emission and nitrogen transformation in aerobic composting[J].Journal of Hangzhou Normal University(Natural Science Edition),2017,16(1):80-86.
[23]吴艳萍,王国栋,赵明德.链霉菌对小麦秸秆堆肥中微生物群落和酶活性的影响[J].西北农林科技大学学报(自然科学版),2009,37(9):134-138.WU Yanping,WANG Guodong,ZHAO Mingde,et al.Effects of Streptomyces thermovulgaris on microbial community and enzyme activity during wheat straw composting process[J].Journal of Northwest A&F University(Natural Science Edition),2009,37(9):134-138.
[24]EL FELS L,EL OUAQOUDI FZ,BARJE F,et al.Two culture approaches used to determine the co-composting stages by assess of the total microflora changes during sewage sludge and date palm waste co-composting[J].Journal of Environmental Health Science&Engineering,2014(12):132.
[25]ANTUNES L P,MARTINS L F,PEREIRA R V,et al.Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics[J].Scientific Reports,2016,6:e38915.
[26]张丽丽.整合宏组学方法揭示天然木质纤维素堆肥中的关键功能微生物群落[D].济南:山东大学,2016.
[27]WANG Cheng,DONG Da,WANG Haoshu,et al.Metagenomic analysis of microbial consortia enriched from compost:new insights into the role of Actinobacteria in lignocellulose decomposition[J].Biotechnology for Biofuels,2016,9:22.
[28]PETERS S,KOSCHINSKY S,SCHWIEGER F,et al.Succession of microbial communities during hot composting as detected by PCRsingle-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes[J].Applied and Environmental Microbiology,2000,66(3):930-936.
[29]贾洋洋.利用宏基因组方法分析堆肥生境中微生物区系的变化[D].济南:山东大学,2012.
[30]SZKELY A J,SIPOS R,BERTA B,et al.DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP profile of mature compost[J].Microbial Ecology,2009,57(3):522-533.
[31]魏自民,王佰洁,赵越,等.堆肥低温起爆微生物筛选及其初步应用[J].环境科学研究,2015,28(6):981-986.WEI Zimin,WANG Baijie,ZHAO Yue,et al.Cold-tolerant bacteria screening and its inoculation in composting at low temperatures[J].Research of Environmental Sciences,2015,28(6):981-986.
[32]李红亚,李术娜,王树香,等.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):324-333.LI Hongya,LI Shuna,WANG Shuxiang,et al.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.