水稻秸秆堆肥发酵粗制肥料中微生物多样性研究
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摘要
研究基于Illumina平台的高通量测序技术,初步解析等量的、不同氮源添加(尿素和牛粪)的水稻秸秆经堆肥发酵后制得粗制肥料中细菌和真核微生物群落结构和多样性。添加尿素和牛粪发酵成的粗制肥料中分别得到897个和954个细菌可操纵分类单元(Operational taxonomic unit,OTU),以及508个和585个真核微生物OTU,且添加牛粪处理的细菌和真核微生物群落丰富度和多样性均高于添加尿素处理。添加尿素制得粗制肥料以放线菌门(Actinobacteria,71.9%)的Streptomyces(40.9%)和Cellulosimicrobium(22.2%)菌属为最优势的细菌类群,以真菌子囊菌门(Ascomycota,70.0%)的Pichia(46.1%)菌属为主要优势的真核微生物类群。添加牛粪制得粗制肥料以变形菌门(Proteobacteria,58.5%)的Pseudomonas(47.8%)菌属为最优势的细菌类群,以真菌子囊菌门(Ascomycota,42.5%)的Aspergillus(23.9%)菌属和接合菌门(Zygomycota,20.5%)的Mucor(9.4%)菌属为主要优势的真核微生物类群。研究结果表明,不同的氮源添加可导致水稻秸秆堆肥发酵制得粗制肥料中形成具有不同优势种群结构的细菌和真核微生物群落,并会导致腐熟各阶段发挥作用的微生物类群的差异。
Based on the high-throughput DNA sequencing of Illumina, the microbial community composition of the compost of rice straw added with urea or cow dung as the nitrogen source was entirely clarified,respectively. Totally 897 and 954 bacterial operational taxonomic unit( OTU) was obtained from the treatment applied with urea and manure,targeting the V4 region of bacterial 16 S rRNA partial gene. The richness index and diversity index of bacterial community in the treatment applied with manure were higher than that in the treatment applied with urea. In the treatment applied with urea,the bacterial phylum Actinobacteria was the most dominant group( 71. 9%), and the genus Streptomyces and Cellulosimicrobium were the main genus of 40. 9% and 22. 2%,respectively. In the treatment applied with manure,the bacterial phylum Proteobacteria was the most dominant group( 58. 5%),and the genus Pseudomonas was the main genus of 47. 8%. Totally 508 and 585 eukaryotic microbial operational taxonomic unit( OTU) was obtained from the treatment applied with urea and manure,targeting the V4 region of eukaryotic 18 S rRNA partial gene. The richness index and diversity index of eukaryotic community in the treatment applied with manure were higher than that in the treatment applied with urea.In the treatment applied with urea, the fungal phylum Ascomycota was the most dominant group( 70. 0%),and the genus Pichia was the main genus of 46. 1%. In the treatment applied with manure,the fungal phylum Ascomycota was still the most dominant group( 42. 5%),following by the fungalphylum Zygomycota( 20. 5%). The genus Aspergillus( 23. 9%) and Mucor( 9. 4%) were the main genus. The results showed that different nitrogen sources could lead to the formation of bacterial and eukaryotic microbial communities with different predominant population structures in crude fertilizers from rice straw compost fermentation,which may lead to the differences of microbial groups that played a role in the stages of composting. Isolation and compounding of the above dominant microbial populations,together with the application of appropriate urea and manure,was likely to make the process of fermenting rice straw to crude fertilizers more rapid.
Based on the high-throughput DNA sequencing of Illumina, the microbial community composition of the compost of rice straw added with urea or cow dung as the nitrogen source was entirely clarified,respectively. Totally 897 and 954 bacterial operational taxonomic unit( OTU) was obtained from the treatment applied with urea and manure,targeting the V4 region of bacterial 16 S rRNA partial gene. The richness index and diversity index of bacterial community in the treatment applied with manure were higher than that in the treatment applied with urea. In the treatment applied with urea,the bacterial phylum Actinobacteria was the most dominant group( 71. 9%), and the genus Streptomyces and Cellulosimicrobium were the main genus of 40. 9% and 22. 2%,respectively. In the treatment applied with manure,the bacterial phylum Proteobacteria was the most dominant group( 58. 5%),and the genus Pseudomonas was the main genus of 47. 8%. Totally 508 and 585 eukaryotic microbial operational taxonomic unit( OTU) was obtained from the treatment applied with urea and manure,targeting the V4 region of eukaryotic 18 S rRNA partial gene. The richness index and diversity index of eukaryotic community in the treatment applied with manure were higher than that in the treatment applied with urea.In the treatment applied with urea, the fungal phylum Ascomycota was the most dominant group( 70. 0%),and the genus Pichia was the main genus of 46. 1%. In the treatment applied with manure,the fungal phylum Ascomycota was still the most dominant group( 42. 5%),following by the fungalphylum Zygomycota( 20. 5%). The genus Aspergillus( 23. 9%) and Mucor( 9. 4%) were the main genus. The results showed that different nitrogen sources could lead to the formation of bacterial and eukaryotic microbial communities with different predominant population structures in crude fertilizers from rice straw compost fermentation,which may lead to the differences of microbial groups that played a role in the stages of composting. Isolation and compounding of the above dominant microbial populations,together with the application of appropriate urea and manure,was likely to make the process of fermenting rice straw to crude fertilizers more rapid.
引文
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7 SARRIA-ALFONSO V,SANCHEZ-SIERRA J,AGUIRRE-MORALES M,et al.Culture media statistical optimization for biomass production of a ligninolytic fungus for future rice straw degradation[J].Indian Journal of Microbiology,2013,53(2):199-207.
8 GUO H,CHANG J,YIN Q,et al.Effect of the combined physical and chemical treatments with microbial fermentation on corn straw degradation[J].Bioresource Technology,2013,148:361-365.
9 HARUTA S,CUI Z,HUANG Z,et al.Construction of a stable microbial community with high cellulose-degradation ability[J].Applied Microbiology Biotechnology,2002,59(4-5):529-534.
10 吴金水,林启美,黄巧云,等.土壤微生物量测定方法及其应用[M].北京:气象出版社,2006.
11 SZEKELY 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.
12 TIAN W,SUN Q,XU D,et al.Succession of bacterial communities during composting process as detected by 16S rRNA clone libraries analysis[J].International Biodeterioration&Biodegradation,2013,78(2):58-66.
13 DE G V,EUDOXIE G,HICKEY W J.Prokaryotic successions and diversity in composts as revealed by 454-pyrosequencing[J].Bioresource Technology,2013,133:573-580.
14 滑留帅,王璟,徐照学,等.16S rRNA基因高通量测序分析牛粪发酵细菌多样性[J].农业工程学报,2016,32(增刊2):311-315.HUA Liushuai,WANG Jing,XU Zhaoxue,et al.Analysis ofbacterial diversity during cattle manure fermentation with 16S rRNA gene high-throughput sequencing[J].Transactions of the CSAE,2016,32(Supp.2):311-315.(in Chinese)
15 熊素敏,左秀凤,朱永义.稻壳中纤维素、半纤维素和木质素的测定[J].粮食与饲料工业,2005(8):40-41.XIONG Sumin,ZUO Xiufeng,ZHU Yongyi.Determination of cellulose,hemi-cellulose and ligin in rice hull[J].Cereal&Feed Industry,2005(8):40-41.(in Chinese)
16 CAPORASO J G,LAUBER C L,WALTERS W A,et al.Ultra-high-throughput microbial community analysis on the Illumina Hi Seq and Mi Seq platforms[J].The ISME Journal Multidisciplinary Journal of Microbial Ecology,2012,6(8):1621-1624.
17 CHEUNG M K,AU C H,CHU K H,et al.Composition and genetic diversity of picoeukaryotes in subtropical coastal waters as revealed by 454 pyrosequencing[J].The ISME Journal Multidisciplinary Journal of Micobial Ecology,2010,4(8):1053-1059.
18 HAAS B J,GEVERS D,EARL A M,et al.Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J].Genome Research,2011,21(3):494-504.
19 EDGAR R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.
20 RATRI C V,KAZUO M,SUSUMU A,et al.Succession and phylogenetic composition of bacterial communities responsible for the composting process of rice straw estimated by PCR-DGGE analysis[J].Soil Science&Plant Nutrition,2003,49(4):619-630.
21 FUENTES A L,ZAFAR U,HEYWORTH A,et al.Fungal succession in an in-vessel composting system characterized using 454pyrosequencing[J].Fems Microbiology Ecology,2014,88(2):296-308.
22 El F L,HAFIDI M,OUHDOUCH Y.Date palm and the activated sludge co-composting actinobacteria sanitization potential[J].Environmental Technology,2016,37(1):129-135.
23 KAUSAR H,SARIAH M,SAUD H M,et al.Isolation and screening of potential actinobacteria for rapid composting of rice straw[J].Biodegradation,2011,22(2):367-375.
24 GIBELLO A,VELA A I,MARTIN M,et al.Pseudomonas composti sp.nov.isolated from compost samples[J].International Journal of Systematic&Evolutionary Microbiology,2011,61(12):2962-2966.
25 ERIKSEN J,GOKSOYR J.The effect of temperature on growth and cellulase(β-1,4-endoglucanase)production in the compost fungus Chaetomium thermophile,var.dissitum[J].Archives of Microbiology,1976,110(2):233-238.
26 WRIGHT C,GRYGANSKYI A P,BONITO G.Fungi in composting[M]∥PURCHASE D.Fungal applications in sustainable environmental biotechnology.Switzerland:Springer International Publishing,2016:3-28.
27 WANG S L,CHEN L G,CHEN C S,et al.Cellulase and xylanase production Aspergillus sp.G-393[J].Applied Biochemistry and Biotechnology,1994,44:231-242.
28 ZHU L,MARUYAMA J,KITAMOTO K.Further enhanced production of heterologous proteins by double-gene disruption(ΔAosed DΔAovps10)in a hyper-producing mutant of Aspergillus oryzae[J].Applied Microbiology and Biotechnology,2013,97(14):6347-6357.
2 边炳鑫,赵由才.农业固体废物的处理与综合利用[M].北京:化学工业出版社,2005.
3 毕于运,王亚静,高春雨.中国主要秸秆资源数量及其区域分布[J].农机化研究,2010,32(3):1-7.BI Yuyun,WANG Yajing,GAO Chunyu.Straw resource quantity and its regional distribution in China[J].Journal of Agricultural Mechanization Research,2010,32(3):1-7.(in Chinese)
4 孙星,刘勤,王德建,等.长期秸秆还田对剖面土壤肥力质量的影响[J].中国生态农业学报,2008,16(3):587-592.SUN Xing,LIU Qin,WANG Dejian,et al.Effect of long-term application of straw on soil fertility[J].Chinese Journal of EcoAgriculture,2008,16(3):587-592.(in Chinese)
5 LYND L R,WEIMER P J,ZYL W H V,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiology&Molecular Biology Reviews,2002,66(3):506-577.
6 OOSHIMA H,BURNS D S,CONVERSE A O.Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression[J].Biotechnology and Bioengineering,1990,36(5):446-452.
7 SARRIA-ALFONSO V,SANCHEZ-SIERRA J,AGUIRRE-MORALES M,et al.Culture media statistical optimization for biomass production of a ligninolytic fungus for future rice straw degradation[J].Indian Journal of Microbiology,2013,53(2):199-207.
8 GUO H,CHANG J,YIN Q,et al.Effect of the combined physical and chemical treatments with microbial fermentation on corn straw degradation[J].Bioresource Technology,2013,148:361-365.
9 HARUTA S,CUI Z,HUANG Z,et al.Construction of a stable microbial community with high cellulose-degradation ability[J].Applied Microbiology Biotechnology,2002,59(4-5):529-534.
10 吴金水,林启美,黄巧云,等.土壤微生物量测定方法及其应用[M].北京:气象出版社,2006.
11 SZEKELY 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.
12 TIAN W,SUN Q,XU D,et al.Succession of bacterial communities during composting process as detected by 16S rRNA clone libraries analysis[J].International Biodeterioration&Biodegradation,2013,78(2):58-66.
13 DE G V,EUDOXIE G,HICKEY W J.Prokaryotic successions and diversity in composts as revealed by 454-pyrosequencing[J].Bioresource Technology,2013,133:573-580.
14 滑留帅,王璟,徐照学,等.16S rRNA基因高通量测序分析牛粪发酵细菌多样性[J].农业工程学报,2016,32(增刊2):311-315.HUA Liushuai,WANG Jing,XU Zhaoxue,et al.Analysis ofbacterial diversity during cattle manure fermentation with 16S rRNA gene high-throughput sequencing[J].Transactions of the CSAE,2016,32(Supp.2):311-315.(in Chinese)
15 熊素敏,左秀凤,朱永义.稻壳中纤维素、半纤维素和木质素的测定[J].粮食与饲料工业,2005(8):40-41.XIONG Sumin,ZUO Xiufeng,ZHU Yongyi.Determination of cellulose,hemi-cellulose and ligin in rice hull[J].Cereal&Feed Industry,2005(8):40-41.(in Chinese)
16 CAPORASO J G,LAUBER C L,WALTERS W A,et al.Ultra-high-throughput microbial community analysis on the Illumina Hi Seq and Mi Seq platforms[J].The ISME Journal Multidisciplinary Journal of Microbial Ecology,2012,6(8):1621-1624.
17 CHEUNG M K,AU C H,CHU K H,et al.Composition and genetic diversity of picoeukaryotes in subtropical coastal waters as revealed by 454 pyrosequencing[J].The ISME Journal Multidisciplinary Journal of Micobial Ecology,2010,4(8):1053-1059.
18 HAAS B J,GEVERS D,EARL A M,et al.Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J].Genome Research,2011,21(3):494-504.
19 EDGAR R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.
20 RATRI C V,KAZUO M,SUSUMU A,et al.Succession and phylogenetic composition of bacterial communities responsible for the composting process of rice straw estimated by PCR-DGGE analysis[J].Soil Science&Plant Nutrition,2003,49(4):619-630.
21 FUENTES A L,ZAFAR U,HEYWORTH A,et al.Fungal succession in an in-vessel composting system characterized using 454pyrosequencing[J].Fems Microbiology Ecology,2014,88(2):296-308.
22 El F L,HAFIDI M,OUHDOUCH Y.Date palm and the activated sludge co-composting actinobacteria sanitization potential[J].Environmental Technology,2016,37(1):129-135.
23 KAUSAR H,SARIAH M,SAUD H M,et al.Isolation and screening of potential actinobacteria for rapid composting of rice straw[J].Biodegradation,2011,22(2):367-375.
24 GIBELLO A,VELA A I,MARTIN M,et al.Pseudomonas composti sp.nov.isolated from compost samples[J].International Journal of Systematic&Evolutionary Microbiology,2011,61(12):2962-2966.
25 ERIKSEN J,GOKSOYR J.The effect of temperature on growth and cellulase(β-1,4-endoglucanase)production in the compost fungus Chaetomium thermophile,var.dissitum[J].Archives of Microbiology,1976,110(2):233-238.
26 WRIGHT C,GRYGANSKYI A P,BONITO G.Fungi in composting[M]∥PURCHASE D.Fungal applications in sustainable environmental biotechnology.Switzerland:Springer International Publishing,2016:3-28.
27 WANG S L,CHEN L G,CHEN C S,et al.Cellulase and xylanase production Aspergillus sp.G-393[J].Applied Biochemistry and Biotechnology,1994,44:231-242.
28 ZHU L,MARUYAMA J,KITAMOTO K.Further enhanced production of heterologous proteins by double-gene disruption(ΔAosed DΔAovps10)in a hyper-producing mutant of Aspergillus oryzae[J].Applied Microbiology and Biotechnology,2013,97(14):6347-6357.
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