纳帕海高原湿地氨氧化微生物群落结构及多样性研究
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摘要
由微生物主导的氨氧化作用是硝化过程的限速步骤,同时也是氮素循环过程中的关键步骤.本研究基于氨单加氧酶基因(amoA),对纳帕海高原湿地旱季的沼泽土(DS. YN1)、沼泽化草甸土(DS. SD1)和泥炭土(DS. NT1)中氨氧化古菌(AOA)和氨氧化细菌(AOB)群落多样性、系统发育和丰度及其与土壤理化因子的响应机制进行了初步研究.结果表明,AOA amoA基因克隆文库在DS. SD1中多样性最高,AOB amoA基因克隆文库在DS. YN1中多样性最高.系统发育分析表明,AOA菌群主要来自Thaumarchaeota Group1. 1a;,AOB菌群主要来自Nitrosomonas和Nitros-opira. AOA amoA基因拷贝数达104～105copies/g,AOB amoA基因拷贝数达105copies/g,仅DS.NT1采样区中AOA amoA基因拷贝数高于AOB amoA.理化因子相关性分析表明,在DS. YN1采样区中,AOA和AOB群落组成受p H和氨态氮(NH4+-N)影响较为显著;在DS. NT1采样区中,AOA和AOB群落组成受亚硝态氮(NO2--N)的影响较为显著. Pearson相关性表明,仅AOBamoA基因拷贝数与土壤理化因子具有显著相关性.本研究初步阐明AOA和AOB在纳帕海高原湿地氨氧化过程中的作用及其对土壤理化因子变化的响应机制,进一步为研究该高原湿地氮素循环奠定理论基础.
Ammonia oxidation is the rate-limiting step of nitrification and also the key step of nitrogen cycle carried out by ammonia oxidation microbial. Targeting the ammonia monooxygenase subunit A( amoA) genes,we investigated the community diversity,phylogeny and abundance of ammonia-oxidizing archaea( AOA) and ammonia-oxidizing bacteria( AOB) in DS. YN1,DS. SD1 and DS. NT1 soil samples of Napahai pleatu wetland.The results showed that the highest diversity of the AOA and AOB amoA clones were found in the DS. SD1 and DS. YN1 soil samples,respectively. Phylogenetic analysis revealed a considerable proportion of AOA and AOB sequences were highly affiliated with Thaumarchaeota Group1. 1 a and Nitrosomonas and Nitrosopira,respectively. The amoA gene copy numbers of AOA and AOB was104-105 copies/g and 105 copies/g respectively. In the DS. NT1 sampling area,the copy number of the AOA amoA gene was higher than in the AOB amoA. Correlation analysis indicated that pH and ammonia nitrogen( NH+4-N) were the main factors affecting the composition of AOA in DS. YN1 sampling area,whereas,the composition of AOB was much more sensitive to nitrite nitrogen( NO-2-N) in DS. NT1 sampling area. The Pearson correlation showed that soil physiochemical factors only influence on the AOB amoA gene copy number. In summary,the role of AOA and AOB in the ammonia oxidation in the Napahai Plateau wetland and their response soil physico-chemical factors,providing the theoretical basis for the study of the nitrogen cycling in the plateau wetland.
Ammonia oxidation is the rate-limiting step of nitrification and also the key step of nitrogen cycle carried out by ammonia oxidation microbial. Targeting the ammonia monooxygenase subunit A( amoA) genes,we investigated the community diversity,phylogeny and abundance of ammonia-oxidizing archaea( AOA) and ammonia-oxidizing bacteria( AOB) in DS. YN1,DS. SD1 and DS. NT1 soil samples of Napahai pleatu wetland.The results showed that the highest diversity of the AOA and AOB amoA clones were found in the DS. SD1 and DS. YN1 soil samples,respectively. Phylogenetic analysis revealed a considerable proportion of AOA and AOB sequences were highly affiliated with Thaumarchaeota Group1. 1 a and Nitrosomonas and Nitrosopira,respectively. The amoA gene copy numbers of AOA and AOB was104-105 copies/g and 105 copies/g respectively. In the DS. NT1 sampling area,the copy number of the AOA amoA gene was higher than in the AOB amoA. Correlation analysis indicated that pH and ammonia nitrogen( NH+4-N) were the main factors affecting the composition of AOA in DS. YN1 sampling area,whereas,the composition of AOB was much more sensitive to nitrite nitrogen( NO-2-N) in DS. NT1 sampling area. The Pearson correlation showed that soil physiochemical factors only influence on the AOB amoA gene copy number. In summary,the role of AOA and AOB in the ammonia oxidation in the Napahai Plateau wetland and their response soil physico-chemical factors,providing the theoretical basis for the study of the nitrogen cycling in the plateau wetland.
引文
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[23]Hu L,Weng B S,Huang F Y,et al.p H regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China[J].Applied Microbiology and Biotechnology,2015,99(14):6113-6123.
[24]Daebeler A,Abell G C J,Bodelier P L E,et al.Archaeal dominated ammonia-oxidizing communities in Icelandic grassland soils are moderately affected by long-term N fertilization and geothermal heating[J].Front Mcrobiology,2012,3:352-366.
[25]Liu Y,Zhang J,Zhao L,et al.Distribution of sediment ammonia-oxidizing microorganisms in plateau freshwater lakes[J].Applied Microbiology and Biotechnology,2015,99(10):1-10.
[26]Martens-Habbena W,Berube P M,Urakawa H,et al.Ammonia oxidation kinetics determine niche separation of nitrifying archaea and bacteria[J].Nature,2009,461(7266):976-979.
[27]Hu B,Shuai L,Shen L,et al.Effect of different ammonia concentrations on Community succession of ammonia-oxidizing microorganisms in a simulated paddy soil column[J].Plos One,2012,7(8):e44122.
[28]Glaser K,Hackl E,Inselsbacher E,et al.Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment[J].FEMS Microbiology Ecology,2010,74(3):575-591.
[29]Zhang J,Dai Y,Wang Y,et al.Distribution of ammonia-oxidizing archaea and bacteria in plateau soils across different land use types[J].Applied Microbiology and Biotechnology,2015,99(16):6899-6909.
[30]Li H,Weng B S,Huang F Y,et al.p H regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China[J].Applied Microbiology and Biotechnology,2015,99(14):6113-6123.
[31]Martin K N,Bernhard A E,Torre J R,et al.Isolation of an autotrophic ammonia-oxidizing marine archaeon[J].Nature,2005,437(7058):543-546.
[32]Hatzenpichler R,Spieck E,Richter A,et al.Frontiers A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring[J].Proceedings of the National Academy of Sciences of the United Atates of America,2008,105(6):2134-2139.
[33]Lehtovirta-Morley L E,Stoecker K,Vilcinskas A,et al.Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil[J].Applied Microbiology and Biotechnology,2011,108(38):15892-15897.
[34]Tripathi B M,Kim M,Singh D,et al.Tropical Soil Bacterial Communities in Malaysia:p H Dominates in the Equatorial Tropics Too[J].Microbiol Ecology,2012,64(2):474-484.
[35]Yao H,Campbell C D,Chapman S J,et al.Multi-factorial drivers of ammonia oxidizer communities:evidence from a national soil survey[J].Environment Microbiol,2013,15(9):2545-2556.
[36]Jiang H,Dong H,Yu B,et al.Diversity and abundance of ammonia-oxidizing archaea and bacteria in Qinghai Lake,Northwestern China[J].Geomicrobiol Journal,2009,26(3):199-211.
[37]Leininger S,Urich T,Schloter M,et al.Archaea predominate among ammonia-oxidizing prokaryotes in soils[J].Nature,2006,442(7104):806-809.
[38]Bernhard A E,Landry Z C,Blevins A,et al.Abundance of Ammonia-Oxidizing Archaea and Bacteria along an Estuarine Salinity Gradient in Relation to Potential Nitrification Rates[J].Applied and Environmental Microbiology,2010,76(4):1285-1289.
[2]Soliman M,Eldyasti A.Development of partial nitrification as a first step of nitrite shunt process in a Sequential Batch Reactor(SBR)using Ammonium Oxidizing Bacteria(AOB)controlled by mixing regime[J].Bioresource Technology,2016,221:85-89.
[3]Wu R N,Meng H,Wang Y F,et al.A more comprehensive community of ammonia-oxidizing archaea(AOA)revealed by genomic DNA and RNA analyses of amo A gene in subtropical acidic forest soils[J].Microbial Ecology,2017,74(4):1-13.
[4]Chao X,Xu Z,Chen Z,et al.Quantitative and compositional responses of ammonia-oxidizing archaea and bacteria to longterm field fertilization[J].Scientific Reports,2016,6:28981-28992.
[5]Christman G D,Cottrell M T,Popp B N,et al.Abundance,diversity,and activity of ammonia-oxidizing prokaryotes in the coastal Arctic Ocean in summer and winter[J].Applied and Environmental Microbiology,2011,77(6):2026-2034.
[6]Erguder T H,Boon N,Wittebolle L,et al.Environmental factors shaping the ecological niches of ammonia-oxidizing archaea[J].FEMS Microbiology Reviews,2009,33(5):855-869.
[7]Qin W,Amin SA,Martens-Habbena W,et al.Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation[J].Proceedings of the National Academy of Sciences of the United Atates of America,2014,111(34):12504-12509.
[8]中科院南京土壤研究所.土壤理化分析[M].上海:上海科学技术出版社,1978.
[9]Francis C A,Roberts K J,Beman J M,et al.Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[J].Proceedings of the National Academy of Sciences of the United Atates of America,2005,102(41):14683-14688.
[10]Rotthauwe J H,Witzel K P,Liesack W.The ammonia monooxygenase structural gene amo A as a functional marker:molecular fine-scale analysis of natural ammonia-oxidizing populations[J].Applied and Environmental Microbiology,1997,63(12):4704-4712.
[11]Schloss P,Westcott S,Ryabin T,et al.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541.
[12]Tamura K,Stecher G,Peterson D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.
[13]Pester M,Rattei T,Flechl S,et al.Amo A-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amo A genes from soils of four different geographic regions[J].Environmental Microbiology,2012,14(2):525-539.
[14]Rotthauwe J H,Witzel K P,Liesack W.The ammonia monooxygenase structural gene amo A as a functional marker:molecular fine-scale analysis of natural ammonia-oxidizing populations[J].Applied and Environmental Microbiology,1997,63(12):4704-4712.
[15]Legendre P,Gallagher E D.Ecologically meaningful transformations for ordination of species data[J].Oecologia,2001,129(2):271-280.
[16]Zhou L,Wang S,Zou Y,et al.Species,Abundance and Function of Ammonia-oxidizing Archaea in Inland Waters across China[J].Scientific Reports,2015,5:1-13.
[17]Yang Y,Shan J,Zhang J,et al.Ammonia-and methane-oxidizing microorganisms in high-altitude wetland sediments and adjacent agricultural soils[J].Applied Microbiology and Biotechnology,2014,98(24):10197-10209.
[18]Damashek J,Smith J M,Mosier A C,et al.Benthic ammonia oxidizers differ in community structure and biogeochemical potential across a riverine delta[J].Front Microbiology,2013,5:743-760.
[19]Song Y,Zhang X,Ma B,et al.Biochar addition affected the dynamics of ammonia oxidizers and nitrification in microcosms of a coastal alkaline soil[J].Biology and Fertility of Soils,2014,50(2):321-332.
[20]Silva M C P E,Poly F,Guillaumaud N,et al.Fluctuations in ammonia oxidizing communities across agricultural soils are driven by soil structure and p H[J].Front Microbiology,2012,3:77-99
[21]Alves R J E,Wanek W,Zappe A,et al.Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea[J].ISME Journal,2013,7(8):1620-1631
[22]Wang X,Cui W,Bao L,et al.Abundance and community structure of ammonia-oxidizing microorganisms in reservoir sediment and adjacent soils[J].Applied Microbiology and Biotechnology,2014,98(4):1883-1892.
[23]Hu L,Weng B S,Huang F Y,et al.p H regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China[J].Applied Microbiology and Biotechnology,2015,99(14):6113-6123.
[24]Daebeler A,Abell G C J,Bodelier P L E,et al.Archaeal dominated ammonia-oxidizing communities in Icelandic grassland soils are moderately affected by long-term N fertilization and geothermal heating[J].Front Mcrobiology,2012,3:352-366.
[25]Liu Y,Zhang J,Zhao L,et al.Distribution of sediment ammonia-oxidizing microorganisms in plateau freshwater lakes[J].Applied Microbiology and Biotechnology,2015,99(10):1-10.
[26]Martens-Habbena W,Berube P M,Urakawa H,et al.Ammonia oxidation kinetics determine niche separation of nitrifying archaea and bacteria[J].Nature,2009,461(7266):976-979.
[27]Hu B,Shuai L,Shen L,et al.Effect of different ammonia concentrations on Community succession of ammonia-oxidizing microorganisms in a simulated paddy soil column[J].Plos One,2012,7(8):e44122.
[28]Glaser K,Hackl E,Inselsbacher E,et al.Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment[J].FEMS Microbiology Ecology,2010,74(3):575-591.
[29]Zhang J,Dai Y,Wang Y,et al.Distribution of ammonia-oxidizing archaea and bacteria in plateau soils across different land use types[J].Applied Microbiology and Biotechnology,2015,99(16):6899-6909.
[30]Li H,Weng B S,Huang F Y,et al.p H regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China[J].Applied Microbiology and Biotechnology,2015,99(14):6113-6123.
[31]Martin K N,Bernhard A E,Torre J R,et al.Isolation of an autotrophic ammonia-oxidizing marine archaeon[J].Nature,2005,437(7058):543-546.
[32]Hatzenpichler R,Spieck E,Richter A,et al.Frontiers A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring[J].Proceedings of the National Academy of Sciences of the United Atates of America,2008,105(6):2134-2139.
[33]Lehtovirta-Morley L E,Stoecker K,Vilcinskas A,et al.Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil[J].Applied Microbiology and Biotechnology,2011,108(38):15892-15897.
[34]Tripathi B M,Kim M,Singh D,et al.Tropical Soil Bacterial Communities in Malaysia:p H Dominates in the Equatorial Tropics Too[J].Microbiol Ecology,2012,64(2):474-484.
[35]Yao H,Campbell C D,Chapman S J,et al.Multi-factorial drivers of ammonia oxidizer communities:evidence from a national soil survey[J].Environment Microbiol,2013,15(9):2545-2556.
[36]Jiang H,Dong H,Yu B,et al.Diversity and abundance of ammonia-oxidizing archaea and bacteria in Qinghai Lake,Northwestern China[J].Geomicrobiol Journal,2009,26(3):199-211.
[37]Leininger S,Urich T,Schloter M,et al.Archaea predominate among ammonia-oxidizing prokaryotes in soils[J].Nature,2006,442(7104):806-809.
[38]Bernhard A E,Landry Z C,Blevins A,et al.Abundance of Ammonia-Oxidizing Archaea and Bacteria along an Estuarine Salinity Gradient in Relation to Potential Nitrification Rates[J].Applied and Environmental Microbiology,2010,76(4):1285-1289.