典型草地土壤好氧甲烷氧化的微生物生态过程
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摘要
【目的】针对我国甘肃三个典型生态区草地土壤(玛曲MQ、临泽LZ和环县HX),研究其甲烷氧化潜力、甲烷氧化菌(methane-oxidizingbacteria,MOB)丰度及可能存在的群落分异规律。【方法】通过原位分析、室内高浓度甲烷模拟培养三种典型土壤及实时荧光定量、高通量测序的方法研究甲烷氧化菌标靶基因pmoA序列的组成及其丰度变化规律。【结果】三种典型草地土壤的原位甲烷氧化菌的丰度存在显著差异,表现为MQ>HX>LZ,其数量范围为为0.18–6.86×107g/d.w.s.;甲烷氧化潜力也表现出类似规律,其通量为109–169mg/(m2·h);甲烷氧化潜力与原位土壤中甲烷氧化菌丰度有正相关。三种草地土壤甲烷氧化菌存在明显的空间异质性,采用高通量测序的方法,发现三种草地原位土壤中的优势类群为USCγ(Upland Soil Cluster gamma,USCγ);然而,室内高浓度甲烷氧化过程中,传统的甲烷氧化菌均发生明显增加,MQ土壤中TypeⅡ的Methylocystis为优势类群,而LZ和HX土壤的优势类群均为TypeⅠ型Methylosarcina。【结论】这些研究结果表明,我国甘肃典型草地土壤中也存在难培养的大气甲烷氧化菌和经典的可培养甲烷氧化菌,这些微生物极可能氧化极低浓度的大气甲烷,也可能利用闭蓄于土壤中的高浓度甲烷生长。未来应采用先进技术原位观测大气甲烷氧化过程并分离相应微生物类群,研究草地土壤甲烷氧化菌地理分异规律及其环境驱动机制。
[Objective] The soil methane uptake potential, abundance and community structure of methaneoxidizing bacteria were investigated in three grasslands located in three different ecoregions: Maqu, Linze and Huanxian of Gansu province of China. [Methods] Eight days incubation of soil with elevated concentration of methane was carried out to measure methane uptake capacity. The methane-oxidizing bacteria abundance was quantified by real time quantitive PCR targeting particulate methane monooxygenase coding gene(pmoA) in soils.The methane-oxidizing bacteria community structure was assessed by amplicon MiSeq sequencing. [Results] The potential of methane oxidation of three grassland soils ranged from 108.53±13.12 to 168.87±18.57 mg/(m2·h). The abundance of methane-oxidizing bacteria ranged from 1.76×107 to 6.86×107 pmoA gene copies g/d.w.s. The methane oxidation potential was positively correlated with methane-oxidizing bacteria abundance at day0(R2=0.5537). MiSeq sequencing analysis revealed significant spatial heterogeneity of methane-oxidizing bacteria community within the same grassland type. The Upland Soil Cluster gamma belonging to uncultured atmospheric methane oxidizers was the dominant methanotrophic lineage within methane-oxidizing bacteria gene types found in situgrassland soils. However, the conventional methane-oxidizing bacteria increased significantly after incubated these soils under high concentration methane, such as Methylocystis in Maqu soil and Methylosarcina in Linze and Huanxian soils. [Conclusion] Both uncultured atmospheric methane-oxidizing bacteria and the conventional methane-oxidizing bacteria may play an important role in the process of methane oxidation in the typical grassland soils in Gansu province of China. These microbes are very likely to oxidize the trace methane in atmosphere, and may also grow with high concentration of methane that stored in the soil. In the future, advanced techniques should be used to observe the atmospheric methane oxidation process in situ and to isolate the corresponding microbial groups, and finally reveal the geographical differentiation of methane-oxidizing bacteria in grassland soils and the environmental driving mechanism.
[Objective] The soil methane uptake potential, abundance and community structure of methaneoxidizing bacteria were investigated in three grasslands located in three different ecoregions: Maqu, Linze and Huanxian of Gansu province of China. [Methods] Eight days incubation of soil with elevated concentration of methane was carried out to measure methane uptake capacity. The methane-oxidizing bacteria abundance was quantified by real time quantitive PCR targeting particulate methane monooxygenase coding gene(pmoA) in soils.The methane-oxidizing bacteria community structure was assessed by amplicon MiSeq sequencing. [Results] The potential of methane oxidation of three grassland soils ranged from 108.53±13.12 to 168.87±18.57 mg/(m2·h). The abundance of methane-oxidizing bacteria ranged from 1.76×107 to 6.86×107 pmoA gene copies g/d.w.s. The methane oxidation potential was positively correlated with methane-oxidizing bacteria abundance at day0(R2=0.5537). MiSeq sequencing analysis revealed significant spatial heterogeneity of methane-oxidizing bacteria community within the same grassland type. The Upland Soil Cluster gamma belonging to uncultured atmospheric methane oxidizers was the dominant methanotrophic lineage within methane-oxidizing bacteria gene types found in situgrassland soils. However, the conventional methane-oxidizing bacteria increased significantly after incubated these soils under high concentration methane, such as Methylocystis in Maqu soil and Methylosarcina in Linze and Huanxian soils. [Conclusion] Both uncultured atmospheric methane-oxidizing bacteria and the conventional methane-oxidizing bacteria may play an important role in the process of methane oxidation in the typical grassland soils in Gansu province of China. These microbes are very likely to oxidize the trace methane in atmosphere, and may also grow with high concentration of methane that stored in the soil. In the future, advanced techniques should be used to observe the atmospheric methane oxidation process in situ and to isolate the corresponding microbial groups, and finally reveal the geographical differentiation of methane-oxidizing bacteria in grassland soils and the environmental driving mechanism.
引文
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[12]Zhou XQ,Wang YF,Huang XZ,Tian JQ,Hao YB.Effect of grazing intensities on the activity and community structure of methane-oxidizing bacteria of grassland soil in Inner Mongolia.Nutrient Cycling in Agroecosystems,2008,80(2):145-152.
[13]Knief C.Diversity and habitat preferences of cultivated and uncultivated aerobic methanotrophic bacteria evaluated based on pmoA as molecular marker.Frontiers in Microbiology,2015,6:1346
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[16]Striegl RG,McConnaughey TA,Thorstenson DC,Weeks EP,Woodward JC.Consumption of atmospheric methane by desert soils.Nature,1992,357(6374):145-147.
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[19]Knief C,Lipski A,Dunfield PF.Diversity and activity of methanotrophic bacteria in different upland soils.Applied and Environmental Microbiology,2003,69(11):6703-6714.
[20]Ma TL,Chen H,Wang YF,Kang XM,Tian JP,Zhou XQ,Zhu QA,Peng CH,Liu LF,Hu J,Zhan W,Zhu EX.Effects of enclosure time on the community composition of methanotrophs in the soils of the Inner Mongolia grasslands.Journal of Soils and Sediments,2016,16(3):1022-1031.
[21]Kou YP,Li JB,Wang YS,Li CN,Tu B,Yao MJ,Li XZ.Scale-dependent key drivers controlling methane oxidation potential in Chinese grassland soils.Soil Biology and Biochemistry,2017,111:104-114.
[22]Zheng Y,Jia ZJ.Next generation sequencing and stable isotope probing of active microorganisms responsible for aerobic methane oxidation in red paddy soils.Acta Microbiologica Sinica,2013,53(2):173-184.(in Chinese)郑燕,贾仲君.新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程.微生物学报,2013,53(2):173-184.
[23]Holm PE,Nielsen PH,Albrechtsen HJ,Christensen TH.Importance of unattached bacteria and bacteria attached to sediment in determining potentials for degradation of xenobiotic organic contaminants in an aerobic aquifer.Applied and Environmental Microbiology,1992,58(9):2016-2025
[24]Wang Q,Garrity GM,Tiedje JM,Cole JR.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267.
[25]Cai YF,Zheng Y,Bodelier PLE,Conrad R,Jia ZJ.Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils.Nature Communications,2016,7:11728.
[26]Yang HL,Chen XJ,Hou FJ.Greenhouse gases emissions of rangeland and livestock manure in the eastern Gansu Loess Plateau in summer.Pratacultural Science,2016,33(8):1454-1459.(in Chinese)杨晗蕾,陈先江,侯扶江.陇东黄土高原草地与畜粪夏季的温室气体排放.草业科学,2016,33(8):1454-1459.
[27]Ning J.Greenhouse gas emission from growing season of sown pastures in inland arid area.Master Dissertation of Lanzhou University,2016.(in Chinese)宁娇.内陆干旱区栽培草地生长季温室气体排放.兰州大学硕士学位论文,2016.
[28]Liu Y,Yan CY,Matthew C,Wood B,Hou FJ.Key sources and seasonal dynamics of greenhouse gas fluxes from yak grazing systems on the Qinghai-Tibetan Plateau.Scientific Reports,2017,7:40857.
[29]Shrestha PM,Kammann C,Lenhart K,Dam B,Liesack W.Linking activity,composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil.The ISMEJournal,2012,6(6):1115-1126.
[30]Zheng Y,Yang W,Sun X,Wang SP,Rui YC,Luo CY,Guo LD.Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau.Applied Microbiology and Biotechnology,2012,93(5):2193-2203.
[31]Liu C.The mechanisms by grazing on grassland plant and soil spatial heterogeneity and their correlation.Doctor Dissertation of Northeast Normal University,2015.(in Chinese)刘晨.放牧对草地植被、土壤空间异质性及其相互关系的调控机制.东北师范大学博士学位论文,2015.
[32]Kolb S,Knief C,Dunfield PF,Conrad R.Abundance and activity of uncultured methanotrophic bacteria involved in the consumption of atmospheric methane in two forest soils.Environmental Microbiology,2005,7(8):1150-1161.
[33]Aronson EL,Dubinsky EA,Helliker BR.Effects of nitrogen addition on soil microbial diversity and methane cycling capacity depend on drainage conditions in a pine forest soil.Soil Biology and Biochemistry,2013,62:119-128.
[34]Borken W,Xu YJ,Beese F.Conversion of hardwood forests to spruce and pine plantations strongly reduced soil methane sink in Germany.Global Change Biology,2003,9(6):956-966.
[35]Li CN,Li JB,Li XZ.Soil methanotrophic community structure and diversity in different vegetation types at elevation gradient of Gongga Mountain,Southwest China.Chinese Journal of Applied Ecology,2017,28(3):805-814.(in Chinese)李超男,李家宝,李香真.贡嘎山海拔梯度上不同植被类型土壤甲烷氧化菌群落结构及多样性.应用生态学报,2017,28(3):805-814.
[36]Maurer D,Kolb S,Haumaier L,Borken W.Inhibition of atmospheric methane oxidation by monoterpenes in Norway spruce and European beech soils.Soil Biology and Biochemistry,2008,40(12):3014-3020.
[2]Saunois M,Jackson RB,Bousquet P,Poulter B,Canadell JG.The growing role of methane in anthropogenic climate change.Environmental Research Letters,2016,11(12):120207.
[3]Topp E,Pattey E.Soils as sources and sinks for atmospheric methane.Canadian Journal of Soil Science,1997,77(2):167-177.
[4]Hartmann AA,Buchmann N,Niklaus PA.A study of soil methane sink regulation in two grasslands exposed to drought and N fertilization.Plant and Soil,2011,342(1/2):265-275.
[5]Unwin D.Climate change 1995:the science of climate change:JT Houghton,LG Meira Filho,BA Callander,NHarris,N Kattenberg,K Maskell(eds).Cambridge University Press,Cambridge(1996),572 pp.￡65.00 hardback:￡22.95paperback.Applied Geography,1997,17(2):163.
[6]Wang YF,Chen H,Zhu QA,Peng CH,Wu N,Yang G,Zhu D,Tian JQ,Tian LX,Kang XM,He YX,Gao YH,Zhao XQ.Soil methane uptake by grasslands and forests in China.Soil Biology and Biochemistry,2014,74:70-81.
[7]Ishizuka S,Sakata T,Ishizuka K.Methane oxidation in Japanese forest soils.Soil Biology and Biochemistry,2000,32(6):769-777.
[8]Nanba K,King GM.Response of atmospheric methane consumption by maine forest soils to exogenous aluminum salts.Applied and Environmental Microbiology,2000,66(9):3674-3679.
[9]Schnell S,King GM.Mechanistic analysis of ammonium inhibition of atmospheric methane consumption in forest soils.Applied and Environmental Microbiology,1994,60(10):3514-3521.
[10]Hu MJ,Tong C,Zou FF.Effects of nitrogen input on CH4production,oxidation and transport in soils,and mechanisms:a review.Acta Prataculturae Sinica,2015,24(6):204-212.(in Chinese)胡敏杰,仝川,邹芳芳.氮输入对土壤甲烷产生、氧化和传输过程的影响及其机制.草业学报,2015,24(6):204-212.
[11]Ma G,Wang P,Wang DX,Xu SQ.Response of soil greenhouse gas emissions to different forms of nitrogen in alpine shrub ecosystems.Acta Prataculturae Sinica,2015,24(3):20-29.(in Chinese)马钢,王平,王冬雪,徐世权.高寒灌丛土壤温室气体释放对添加不同形态氮素的响应.草业学报,2015,24(3):20-29.
[12]Zhou XQ,Wang YF,Huang XZ,Tian JQ,Hao YB.Effect of grazing intensities on the activity and community structure of methane-oxidizing bacteria of grassland soil in Inner Mongolia.Nutrient Cycling in Agroecosystems,2008,80(2):145-152.
[13]Knief C.Diversity and habitat preferences of cultivated and uncultivated aerobic methanotrophic bacteria evaluated based on pmoA as molecular marker.Frontiers in Microbiology,2015,6:1346
[14]Costello AM,Lidstrom ME.Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments.Applied and Environmental Microbiology,1999,65(11):5066-5074.
[15]Steudler PA,Bowden RD,Melillo JM,Aber JD.Influence of nitrogen fertilization on methane uptake in temperate forest soils.Nature,1989,341(6240):314-316.
[16]Striegl RG,McConnaughey TA,Thorstenson DC,Weeks EP,Woodward JC.Consumption of atmospheric methane by desert soils.Nature,1992,357(6374):145-147.
[17]Holmes AJ,Costello A,Lidstrom ME,Murrell JC.Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related.FEMSMicrobiology Letters,1995,132(3):203-208.
[18]Holmes AJ,Roslev P,McDonald IR,Iversen N,Henriksen K,Murrell JC.Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake.Applied and Environmental Microbiology,1999,65(8):3312-3318.
[19]Knief C,Lipski A,Dunfield PF.Diversity and activity of methanotrophic bacteria in different upland soils.Applied and Environmental Microbiology,2003,69(11):6703-6714.
[20]Ma TL,Chen H,Wang YF,Kang XM,Tian JP,Zhou XQ,Zhu QA,Peng CH,Liu LF,Hu J,Zhan W,Zhu EX.Effects of enclosure time on the community composition of methanotrophs in the soils of the Inner Mongolia grasslands.Journal of Soils and Sediments,2016,16(3):1022-1031.
[21]Kou YP,Li JB,Wang YS,Li CN,Tu B,Yao MJ,Li XZ.Scale-dependent key drivers controlling methane oxidation potential in Chinese grassland soils.Soil Biology and Biochemistry,2017,111:104-114.
[22]Zheng Y,Jia ZJ.Next generation sequencing and stable isotope probing of active microorganisms responsible for aerobic methane oxidation in red paddy soils.Acta Microbiologica Sinica,2013,53(2):173-184.(in Chinese)郑燕,贾仲君.新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程.微生物学报,2013,53(2):173-184.
[23]Holm PE,Nielsen PH,Albrechtsen HJ,Christensen TH.Importance of unattached bacteria and bacteria attached to sediment in determining potentials for degradation of xenobiotic organic contaminants in an aerobic aquifer.Applied and Environmental Microbiology,1992,58(9):2016-2025
[24]Wang Q,Garrity GM,Tiedje JM,Cole JR.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267.
[25]Cai YF,Zheng Y,Bodelier PLE,Conrad R,Jia ZJ.Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils.Nature Communications,2016,7:11728.
[26]Yang HL,Chen XJ,Hou FJ.Greenhouse gases emissions of rangeland and livestock manure in the eastern Gansu Loess Plateau in summer.Pratacultural Science,2016,33(8):1454-1459.(in Chinese)杨晗蕾,陈先江,侯扶江.陇东黄土高原草地与畜粪夏季的温室气体排放.草业科学,2016,33(8):1454-1459.
[27]Ning J.Greenhouse gas emission from growing season of sown pastures in inland arid area.Master Dissertation of Lanzhou University,2016.(in Chinese)宁娇.内陆干旱区栽培草地生长季温室气体排放.兰州大学硕士学位论文,2016.
[28]Liu Y,Yan CY,Matthew C,Wood B,Hou FJ.Key sources and seasonal dynamics of greenhouse gas fluxes from yak grazing systems on the Qinghai-Tibetan Plateau.Scientific Reports,2017,7:40857.
[29]Shrestha PM,Kammann C,Lenhart K,Dam B,Liesack W.Linking activity,composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil.The ISMEJournal,2012,6(6):1115-1126.
[30]Zheng Y,Yang W,Sun X,Wang SP,Rui YC,Luo CY,Guo LD.Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau.Applied Microbiology and Biotechnology,2012,93(5):2193-2203.
[31]Liu C.The mechanisms by grazing on grassland plant and soil spatial heterogeneity and their correlation.Doctor Dissertation of Northeast Normal University,2015.(in Chinese)刘晨.放牧对草地植被、土壤空间异质性及其相互关系的调控机制.东北师范大学博士学位论文,2015.
[32]Kolb S,Knief C,Dunfield PF,Conrad R.Abundance and activity of uncultured methanotrophic bacteria involved in the consumption of atmospheric methane in two forest soils.Environmental Microbiology,2005,7(8):1150-1161.
[33]Aronson EL,Dubinsky EA,Helliker BR.Effects of nitrogen addition on soil microbial diversity and methane cycling capacity depend on drainage conditions in a pine forest soil.Soil Biology and Biochemistry,2013,62:119-128.
[34]Borken W,Xu YJ,Beese F.Conversion of hardwood forests to spruce and pine plantations strongly reduced soil methane sink in Germany.Global Change Biology,2003,9(6):956-966.
[35]Li CN,Li JB,Li XZ.Soil methanotrophic community structure and diversity in different vegetation types at elevation gradient of Gongga Mountain,Southwest China.Chinese Journal of Applied Ecology,2017,28(3):805-814.(in Chinese)李超男,李家宝,李香真.贡嘎山海拔梯度上不同植被类型土壤甲烷氧化菌群落结构及多样性.应用生态学报,2017,28(3):805-814.
[36]Maurer D,Kolb S,Haumaier L,Borken W.Inhibition of atmospheric methane oxidation by monoterpenes in Norway spruce and European beech soils.Soil Biology and Biochemistry,2008,40(12):3014-3020.