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不同类型稻田亚硝酸型CH4厌氧氧化潜力的比较研究

  • 薛梦琪1

    机 构：

    1. 1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044

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  • ，周聪1

    机 构：

    1. 1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044

    ×
  • ，饶旭东1

    机 构：

    1. 1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044

    ×
  • ，谢晴1

    机 构：

    1. 1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044

    ×
  • ，张耀鸿1

    机 构：

    1. 1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044

    ×

1.南京信息工程大学 气象灾害预报预警与评估协同创新中心/江苏省农业气象重点实验室,南京, 210044；

Comparative study on nitrite-dependent anaerobic methane oxidation in different types of paddy field

  • XUE Mengqi1

    Affiliation：

    1. 1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044

    ×
  • ，ZHOU Cong1

    Affiliation：

    1. 1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044

    ×
  • ，RAO Xudong1

    Affiliation：

    1. 1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044

    ×
  • ，XIE Qing1

    Affiliation：

    1. 1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044

    ×
  • ，ZHANG Yaohong1

    Affiliation：

    1. 1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044

    ×

1.Collaborative Innovation Center for Forecast and Evaluation of Meteorological Disasters/Jiangsu Provincial Key Laboratory of Agricultural Meteorology,Nanjing University of Information Science & Technology,Nanjing 210044；

作者简介:

薛梦琪,女,硕士生,主要从事土壤温室气体排放研究.xmq98989898@163.com;

张耀鸿(通信作者),男,博士,教授,主要从事陆地生态系统温室气体排放研究.yhzhang@nuist.edu.cn

中图分类号:S154.1;S511.061

文献标识码:A

DOI:10.13878/j.cnki.jnuist.2022.01.012

参考文献 1
Dean J F,Middelburg J J,Röckmann T,et al.Methane feedbacks to the global climate system in a warmer world[J].Reviews of Geophysics,2018,56(1):207-250
查找原文
参考文献 2
Cai Y F,Zheng Y,Bodelier P L E,et al.Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils[J].Nature Communications,2016,7:11728
查找原文
参考文献 3
Bridgham S D,Cadillo-Quiroz H,Keller J K,et al.Methane emissions from wetlands:biogeochemical,microbial,and modeling perspectives from local to global scales[J].Global Change Biology,2013,19(5):1325-1346
查找原文
参考文献 4
Conrad R,Klose M,Noll M.Functional and structural response of the methanogenic microbial community in rice field soil to temperature change[J].Environmental Microbiology,2009,11(7):1844-1853
查找原文
参考文献 5
Bodelier P L E.Toward understanding,managing,and protecting microbial ecosystems[J].Frontiers in Microbiology,2011,2:1-8
查找原文
参考文献 6
Ding J,Fu L,Ding Z W,et al.Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field[J].Applied Microbiology and Biotechnology,2016,100:439-446
查找原文
参考文献 7
Hu B L,Shen L D,Lian X,et al.Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(12):4495-4500
查找原文
参考文献 8
Deutzmann J S,Schink B.Anaerobic oxidation of methane in sediments of Lake Constance,an oligotrophic freshwater lake[J].Applied and Environmental Microbiology,2011,77(13):4429-4436
查找原文
参考文献 9
He Z F,Geng S,Cai C Y,et al.Anaerobic oxidation of methane coupled to nitrite reduction by halophilic marine NC10 bacteria[J].Applied and Environmental Microbiology,2014,81(16):5538-5545
查找原文
参考文献 10
Wang J Q,Cai C Y,Li Y F,et al.Denitrifying anaerobic methane oxidation:a previously overlooked methane sink in intertidal zone[J].Environmental Science & Technology,2019,53(1):203-212
查找原文
参考文献 11
Zhou L L,Wang Y,Long X E,et al.High abundance and diversity of nitrite-dependent anaerobic methane-oxidizing bacteria in a paddy field profile[J].FEMS Microbiology Letters,2014,360(1):33-41
查找原文
参考文献 12
Wang Y,Zhu G B,Harhangi H R,et al.Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane oxidizing bacteria in a paddy soil[J].FEMS Microbiology Letters,2012,336(2):79-88
查找原文
参考文献 13
Li X F,Gao D Z,Liu M.Composition,diversity and abundance of Candidatus M.oxyfera-like bacteria in response to the estuary salinity[J].Biogeochemistry,2019,143:1-14
查找原文
参考文献 14
Shen L D,Hu B L,Liu S,et al.Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments[J].Applied Microbiological Biotechnology,2016,100:7171-7180
查找原文
参考文献 15
张洁,宋怡轩,张鑫磊,等.不同类型稻田中全程氨氧化微生物的分异特征 [J].江苏农业学报,2020,36(3):584-590ZHANG Jie,SONG Yixuan,ZHANG Xinlei,et al.Differentiation characteristics of complete ammonia-oxidizing microorganisms in different types of paddy soils[J].Jiangsu Journal of Agriculture Science,2020,36(3):584-590
查找原文
参考文献 16
林黎,崔军,陈学萍,等.滩涂围垦和土地利用对土壤微生物群落的影响 [J].生态学报,2014,34(4):899-906LIN Li,CUI Jun,CHEN Xueping,et al.Effects of reclamation on tidal flat and land use on soil microbial community [J].Acta Ecologica Sinica,2014,34(4):899-906
查找原文
参考文献 17
Zhao W,Wang Y Y,Liu S H,et al.Denitrification activities and N2O production under salt stress with varying COD/N ratios and terminal electron acceptors[J].Chemical Engineering Journal,2013,215/216:252-260
查找原文
参考文献 18
Zhang Y H,Zhang X L,Wang F Y,et al.Exogenous nitrogen addition inhibits sulfate-mediated anaerobic oxidation of methane in estuarine coastal sediments [J].Ecological Engineering,2020,158:106021
查找原文
参考文献 19
Ettwig K F,Butler M K,Le Paslier D,et al.Nitrite-driven anaerobic methane oxidation by oxygenic bacteria [J].Nature,2010,464(7288):543-548
查找原文
参考文献 20
夏超,祝贵兵,邹雨璇,等.厌氧氨氧化细菌和反硝化厌氧甲烷氧化细菌在岸边带土壤中的分布规律 [J].环境科学学报,2015,35(12):3965-3975XIA Chao,ZHU Guibing,ZOU Yuxuan,et al.Distribution of anaerobic ammonia oxidizing bacteria and nitrite-dependent anaerobic methane oxidizing bacteria in soil profile in the riparian zone [J].Acta Scientiae Circumstantiae,2015,35(12):3965-3975
查找原文
参考文献 21
张鑫磊,宋怡轩,张洁,等.围垦植稻对崇明东滩湿地产甲烷微生物的影响 [J].农业环境科学学报,2020,39(2):411-417ZHANG Xinlei,SONG Yixuan,ZHANG Jie,et al.Effects of reclamation and cultivating rice on CH4-producing microorganisms in Chongming Dongtan wetland,China[J].Journal of Agro-Environment Science,2020,39(2):411-417
查找原文
参考文献 22
Ettwig K F,van Alen T,van de Pas-Schoonen K T,et al.Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum[J].Applied and Environmental Microbiology,2009,75(11):3656-3662
查找原文
参考文献 23
He Z F,Geng S,Shen L D,et al.The short-and long-term effects of environmental conditions on anaerobic methane oxidation coupled to nitrite reduction[J].Water Research,2015,68:554-562
查找原文
目录contents

    摘要

    甲烷(CH4)厌氧氧化是稻田土壤中消减温室气体排放的重要过程.本试验选择内陆性南京稻田和滨海性上海崇明岛围垦稻田,比较研究稻田耕层(0~10 cm)和深层(50~60 cm)土壤中亚硝酸盐型CH4厌氧氧化(n-DAMO)潜力的差异及其微生物驱动机制.结果表明,南京稻田耕层土壤的n-DAMO速率为3.51 nmol·g-1·d-1(以13CO2计),显著高于围垦稻田耕层土壤(1.43 nmol·g-1·d-1).两种类型稻田耕层土壤的n-DAMO速率均显著高于深层土壤.南京稻田和围垦稻田M.oxyfera-like细菌的16S rRNA基因拷贝数分别为(2.31~4.82)×107和(0.89~2.12)×107 copies·g-1,与亚硝酸盐型CH4厌氧氧化速率显著正相关.相关性分析发现,土壤有机碳、总氮、无机态氮是稻田n-DAMO速率分异的重要原因.综上所述,内陆性稻田土壤n-DAMO氧化潜力较高,其主要由较高的土壤本底碳、氮水平和功能微生物丰度所致.

    Abstract

    Anaerobic methane (CH4) oxidation is an important process to reduce greenhouse gas emissions in paddy soil.The nitrite-dependent Denitrifying Anaerobic Methane Oxidation (n-DAMO) potential and its microbial mechanism in cultivated layer (0-10 cm) and deep layer (50-60 cm) of inland (Nanjing) and coastal (reclaimed shanghai Chongming Island) paddy fields were studied.The results showed that n-DAMO rate of topsoil in Nanjing paddy field was 3.51 nmol 13CO2·g-1·d-1,which was significantly higher than that of topsoil in reclaimed paddy field (1.43 nmol 13CO2·g-1·d-1).The n-DAMO rates in the cultivated layer soil were significantly higher than those in the deep layer soil in the two types of paddy field.The copy numbers of 16S rRNA gene of functional microorganism M.oxyfera-like bacteria in Nanjing paddy field and reclaimed paddy field were 2.31×107-4.82×107 copies·g-1 and 0.89×107-2.12×107 copies·g-1,respectively,which was significantly positively correlated with nitrite-dependent anaerobic CH4 oxidation rate.Regression analysis showed that soil organic carbon,total nitrogen and inorganic nitrogen were important factors for n-DAMO rate differentiation in paddy fields.In conclusion,the n-DAMO potential of inland paddy soil is higher than that in reclaimed paddy field,which is mainly caused by the different levels of soil background carbon and nitrogen and the abundance of functional microorganisms.

    关键词

    稻田 ； 甲烷厌氧氧化 ； M.oxyfera

  • 0 引言

  • 甲烷(CH4)是一种重要的温室气体,具有很强的红外吸收带,其百年时间尺度上的增温潜力是二氧化碳(CO2)的28~34倍[1],占全球变暖贡献率的20%[2].稻田是主要的CH4排放源,每年向大气释放25~300Tg[3],占全球甲烷排放量的10%~20%[4-5].氮肥的施加导致稻田产生高浓度的NO-x,为反硝化型甲烷厌氧氧化细菌和古菌提供了理想的生境[6].目前认为,亚硝酸型甲烷厌氧氧化(nitrite dependent Denitrifying Anaerobic Methane Oxidation,n-DAMO)过程由NC10门的Candidatus Methylomirabilis oxyfera细菌介导,该微生物具有内产氧功能,可单独将NO-2还原,同时将CH4氧化为CO2.n-DAMO过程多发生于淡水湿地[7]、湖泊生态系统[8]等,近期在海洋生态系统中也检测到了n-DAMO过程的存在[9].Wang等[10]报道滨海潮间带生态系统DAMO细菌与古菌可以共存,从而使潮间带生态系统成为重要的CH4汇.进一步发现n-DAMO过程在CH4循环过程中起着更重要的作用.

  • 湿地生态系统中n-DAMO细菌介导的CH4厌氧过程受到土壤母质、氮(N)水平、施肥、盐分以及湿地剖面深度等因素的影响,其在稻田土壤中氧化作用表现强烈.Wang等[10]发现在原位稻田湿地中,n-DAMO细菌的数量与NO-3浓度呈显著正相关.施入稻田的氮肥反硝化会产生NO-2,可以作为电子受体促进土壤厌氧微域的n-DAMO反应[6].研究还发现:湿地系统不同深度土壤中n-DAMO细菌的群落组成和活性存在明显差异[11].一般认为,相较于表层土壤,湿地深层土壤更有利于n-DAMO细菌的分布和n-DAMO反应的发生[12],可能是由深层土壤长期处于较稳定的缺氧状态所致.n-DAMO细菌群落在滨海生态系统中的分布存在较强的空间异质性,其在杭州湾和长江口滨海区中n-DAMO细菌的数量和活性均与土壤盐度呈显著负相关[13-14],其机理尚不清楚.

  • 滨海湿地处于海陆交错带,具有海洋性和陆地性双重特征,当前受人类活动的影响日益严重.随着经济的发展和人口的增长,人类对土地的需求日趋迫切,滨海滩涂围垦造田成为解决土地问题的有效途径.围垦后的湿地自然条件相对稳定,不再受海水潮汐的影响,形成与自然滩涂湿地明显不同的独特生境.在此基础上,人工灌溉、耕作、施肥、水稻种植等农业措施加速了围垦稻田的熟土化进程,即由海洋性滩涂逐渐向陆地性农田演替,土壤的物理、化学和生物特征发生了显著改变,但与内陆性稻田存在着明显差别.这种围垦植稻驱动的熟土化作用对土壤甲烷氧化过程可能会产生重要影响.那么,处于熟土化进程中的围垦稻田,其甲烷厌氧氧化潜力有何特征?与典型内陆性稻田相比,其甲烷厌氧氧化速率变化的主控因素是什么?是否与功能微生物群落结构及数量特征之间存在内在关系?

  • 鉴于此,本试验选取两个不同类型的稻田:微碱性N贫瘠的长江口崇明围垦稻田和微酸性N丰富的南京稻田,研究其表层和深层土壤的CH4厌氧氧化速率及nDAMO细菌的丰度,探明在土壤母质、pH值、N水平以及盐分等差异较大的稻田土壤环境中,n-DAMO反应速率及其微生物学机理,进一步阐明其分异规律,为稻田CH4减排提供参考依据.

  • 1 材料与方法

  • 1.1 土壤样品采集

  • 本研究选取上海市崇明岛东滩湿地(121.92°E,31.50°N)围垦16年稻田(WK)与南京信息工程大学农业气象试验站(118.86°E,32.16°N)荒地改种稻15年稻田(NJ).在每个稻田内以S形设置6个采样点,各采样点间距15m.用土钻取0~60cm深度的土柱,取出0~10cm和50~60cm的土层样品,并将相同深度的土样充分混合,放入冰盒中带回实验室冷冻保存备用.

  • 1.2 土壤理化性质测定

  • 土壤全氮(TN) 含量采用半微量凯氏定量法测定.土壤总有机碳(TOC) 含量采用浓硫酸-重铬酸钾消煮-硫酸亚铁滴定法测定.土壤铵态氮和硝态氮含量用2mol/L KCl溶液浸提后,采用AA3流动分析仪测定.土壤pH值和EC值分别采用数字酸度计和电导仪测定.

  • 1.3 厌氧CH4氧化速率测定

  • 将5g土壤样品放入40mL的培养瓶后加入10mL的超纯水,胶塞密封后抽真空-充氩气重复进行3次,以达到严格厌氧状态.室温25℃避光条件下120r/min转速预培养2d,以尽可能去除残余O2的影响.预培养结束后,再次抽真空-充氩气2次,然后将每个土样分成3组,分别进行底物添加处理:1) 添加Ar;2) 添加13CH4;3) 添加13CH4+NO-2.每个处理3次重复.添加的13CH4相对丰度为99%,瓶内顶空剩余体积以氩气补充,使得培养瓶顶空13CH4最终质量分数为10%.添加的NO-2最终浓度分别为0.5mmol.全部培养瓶在室温25℃避光条件下120r/min转速正式培养7d.

  • 培养结束后,用气相色谱仪-质谱仪连用法测定瓶中13CO2的产生量.处理1)为对照组,如果瓶中13CO2的相对丰度处于自然丰度水平(1.1%),说明试验结果可信度高;用处理3) 减去处理2)中13CO2产生量,进一步计算亚硝酸型厌氧甲烷氧化(n-DAMO)速率.

  • 1.4 荧光定量PCR分析

  • 用Fast DNA Spin Kit for Soil提取试剂盒 (MP Biomedicals,USA) 提取土壤样品中的总DNA.取部分DNA提取液用分光光度计(NanoDrop ND-1000UV-Vis)测定DNA浓度和纯度.土壤DNA保存于-80℃冰箱待用.

  • 在CFX96Real-Time PCR System(Bio-Rad公司)扩增仪上进行荧光定量PCR扩增.测定土壤样品中总细菌和M.oxyfera菌的16S rRNA基因拷贝数.总细菌基因扩增所用引物为细菌V4+V5高变区通用引物515F/907R;M.oxyfera菌扩增所用引物为qP1F/qP1R.反应体系为20.0 μL,包括DNA样品1.0 μL,Premix TaqTM10 μL,前后引物各1.0 μL,超纯无菌水7.0 μL.扩增条件为:预变性95℃,10min;变性95℃,30s;退火55℃,30s;延伸72℃,30s;40个循环.根据质粒梯度浓度制成的标准曲线计算目的基因的拷贝数.

  • 1.5 数据统计与分析

  • 用SPSS 19.0软件进行统计分析,通过单因素方差分析及多重比较、相关性分析(Pearson)进行土壤理化性质、氧化速率、微生物丰度的差异性以及相关性检验,显著水平α=0.05.

  • 2 结果与分析

  • 2.1 不同类型稻田土壤的理化性质

  • 南京稻田和崇明围垦稻田土壤理化性质如表1所示.南京稻田表层土壤中总有机碳(TOC)、总氮(TN)、NH+4、NO-3的含量均显著高于深层土壤,表明农业施肥、农作物生长等对耕层土壤有机质和N水平影响很大.崇明围垦稻田中表层土壤TOC显著高于深层土壤,而TN、NH+4、NO-3的含量在表层和深层之间无明显差异,且围垦稻田这些指标均显著低于南京稻田对应土层,说明围垦稻田受土壤母质、成土化进程等影响,其土壤肥力普遍低于内陆稻田.相反,围垦稻田中表层和深层土壤的SO2-4含量、EC值和pH值均显著高于南京稻田,表明围垦区稻田仍具有明显的海洋性特征.

  • 2.2 稻田土壤的13CO2丰度变化和CH4厌氧速率

  • 本试验中,培养瓶密闭气体中13CH4的起始质量分数约为10%,随着厌氧培养的进行将转化为13CO2,根据其质量分数和丰度变化与培养时间进行计算,可得出培养时间内的平均n-DAMO速率.图1结果显示,两种类型土壤在只供应Ar气体条件下厌氧培养后,其土壤13CO2丰度保持在自然丰度水平(1.08%);在供应13CH4和13CH4+NO-2条件下,土壤13CO2丰度均有不同程度的升高,表明所试两种土壤均发生了甲烷厌氧氧化反应.其中,只供应13CH4条件下13CO2丰度显著高于Ar处理,表明土壤中存在本底性电子受体可促进甲烷厌氧氧化过程.在供应13CH4+NO-2条件下,13CO2丰度显著高于13CH4处理,表明两种所试土壤均发生了以NO-2为电子受体的厌氧CH4氧化作用.在此底物供应条件下,南京稻田土壤13CO2丰度达4.57%~5.87%,明显高于围垦稻田的对应土层(4.12%~4.69%),暗示了南京稻田土壤可能具有较大的NO-2型厌氧CH4氧化潜力.图2结果显示,南京稻田表层(NJ10)和深层(NJ60)土壤的CH4厌氧氧化速率分别为3.51和2.45nmol·g-1·d-1(以13CO2计),围垦稻田表层(WK10)和深层(WK60)土壤的CH4厌氧氧化速率则分别为1.43和1.36nmol·g-1·d-1.可以看出,围垦稻田表层土壤的CH4氧化速率比南京稻田低59%,而深层土壤则低44%,表明滨海围垦稻田的亚硝酸盐型CH4厌氧速率均显著低于内陆南京稻田.

  • 2.3 稻田土壤中M.oxyfera菌和总细菌的16S rRNA基因丰度

  • 目前认为,以NO-2为电子受体的CH4厌氧氧化过程由NC10门中的M.oxyfera细菌催化进行.南京稻田表层中M.oxyfera细菌的16S rRNA基因拷贝数为4.82×107 copies·g-1,比深层土壤的基因拷贝数高出1.1倍(图3).围垦稻田表层中M.oxyfera细菌的16S rRNA基因拷贝数为2.12×107 copies·g-1,比深层土壤的基因拷贝数高出1.4倍.可以看出,南京稻田M.oxyfera细菌的16S rRNA基因拷贝数显著高于围垦稻田土壤.

  • 表1 不同类型稻田土壤的理化性质

  • Table1 Physicochemical properties of different paddy soils

  • 注:同一列不同字母表示在P ＜0.05水平差异显著.

  • 图1 南京稻田和围垦稻田土壤厌氧培养后的13CO2丰度变化

  • Fig.1 13CO2 abundance in soils of Nanjing paddy and the reclaimed paddy

  • 图2 不同类型稻田土壤的亚硝酸盐型厌氧CH4氧化速率

  • Fig.2 Potential n-DAMO rate of different paddy soils

  • 对稻田土壤总细菌的16S rRNA基因拷贝数进行了测定.其变化范围为(2.12~3.13)×108 copies·g-1,比M.oxyfera细菌的16S rRNA基因拷贝数高出1个数量级.其中,南京稻田耕层土壤的总细菌16S rRNA基因拷贝数显著高于其他三个土样.将M.oxyfera细菌与总细菌的16S rRNA基因拷贝数相比,可得出M.oxyfera细菌的相对丰度,发现南京稻田耕层和深层土壤M.oxyfera细菌相对丰度分别为1.5%和0.9%,围垦稻田则分别为0.9%和0.4%.

  • 2.4 厌氧CH4氧化速率与基因丰度和理化性质的相关性分析

  • 对厌氧CH4氧化速率与M.oxyfera细菌的16S rRNA基因拷贝数进行相关分析,结果表明,亚硝酸型厌氧CH4氧化速率与M.oxyfera细菌的16S rRNA基因丰度呈显著正相关(图4),说明功能微生物的数量很大程度上决定了土壤CH4厌氧氧化能力.

  • 对厌氧CH4氧化速率与土壤理化性质进行回归分析,结果表明,土壤CH4厌氧氧化速率与土壤TOC、TN、NH+4、NO-3的含量的回归方程均达到了显著水平(表2),说明土壤C、N水平是影响CH4厌氧氧化潜力非常重要的环境要素.相反,土壤SO2-4含量、EC值与厌氧CH4氧化速率呈负相关,但未达到显著水平.值得注意的是,土壤pH值与厌氧CH4氧化速率呈负相关,且回归方程达到了显著水平.这些结果说明围垦稻田土壤的海洋性特征仍较明显,对厌氧CH4氧化潜力具有一定的抑制作用.

  • 图4 厌氧CH4氧化速率与功能微生物丰度的关系

  • Fig.4 Relationship between n-DAMO rate and 16S rRNA gene abundance of M.oxyfera

  • 表2 厌氧CH4氧化速率与土壤理化性质的回归方程

  • Table2 Regression relationship between n-DAMO rate (y) and soil characteristics (x)

  • 图3 南京稻田和围垦稻田中M.oxyfera菌和总细菌的16S rRNA基因拷贝数

  • Fig.3 16S rRNA gene abundance of M.oxyfera-like bacteria and total bacteria in Nanjing paddy soil and the reclaimed paddy soil

  • 3 讨论

  • 本试验采用13CH4同位素标记法证实了在滨海围垦稻田和内陆南京稻田均发生了亚硝酸盐型厌氧CH4氧化作用.本研究使用的13CH4的丰度为99%,而且起始质量分数高达10%,这样可以最大程度地降低厌氧培养过程中CH4产生过程对试验结果的影响,具有较高的可信度.而且,本厌氧培养试验设置了只加Ar的对照处理,既可测试试验过程是否保持严格厌氧状态,又可测定厌氧过程CH4产生过程特征.结果显示,所试两个土样在培养过程中CH4产生量极低,可以忽略不计.因此,本研究采用13CH4同位素标记法测出的n-DAMO速率能客观真实地反映出土壤的厌氧CH4氧化潜势.其中,围垦稻田土壤的n-DAMO速率高于杭州湾滨海自然湿地的氧化速率(0.2~1.3nmol ·g-1·d-1)[14],表明围垦植稻熟土化过程会促进亚硝酸盐型厌氧CH4氧化作用.南京稻田土壤的n-DAMO速率与内陆淡水湿地的氧化速率基本一致(1.8~3.6nmol·g-1·d-1)[7],表明内陆稻田与淡水湿地均表现出较高的n-DAMO速率.

  • 不同土壤母质、不同环境条件下发育形成的稻田,受理化性质、母质类型、农田管理等因素影响,可能对土壤CH4氧化潜力产生重要影响.本研究发现,内陆性南京稻田的亚硝酸盐型厌氧CH4氧化速率显著高于滨海围垦稻田.南京稻田的成土母质为河流沉积物,黏粒比例较高,同时也形成了较为稳定的土壤团聚体,有利于提高土壤肥力[15].在种植水稻过程中施N量和有机肥量较高,使得该所试土壤的有机碳、全氮含量较高.相反,围垦稻田的成土母质是长江口泥沙沉积物,砂粒较粗,蓄水保肥能力差[16],且在水稻生长过程中,地上部生物量小,根系生物量及分泌物量低,土壤的有机碳和N水平较低.本试验表明,土壤TOC和总N水平与厌氧CH4氧化速率显著正相关,可见土壤本底C、N水平反映了土壤的C、N转化过程的基本特征,对土壤厌氧CH4氧化潜力有着极其重要的影响.这与Shen等[14]的研究结果一致,他们认为湿地土壤硝化和反硝化作用强烈,其中间产物NO-2可以为M.oxyfera菌提供底物促进CH4氧化过程.而且南京稻田中TOC含量高,可为反硝化微生物提供较多碳源,促进反硝化作用,有利于为M.oxyfera菌提供底物NO-2.相关性分析发现,n-DAMO速率与土壤EC值、SO2-4含量均呈负相关,这与Wang等[10]的结果一致.围垦稻田土壤中SO2-4含量比南京稻田高出一个数量级,EC值也显著高于南京稻田,这些海洋性特征参数可能也是造成围垦稻田厌氧CH4氧化速率低的重要原因.这与Li等[13]的研究结果一致,他们认为,较高的盐度会导致n-DAMO细菌细胞内较高的渗透压,不利于细胞新陈代谢;而且盐度高会直接抑制亚硝酸还原酶、一氧化氮歧化酶的活性[17].不仅如此,较高的SO2-4含量本身可以作为电子供体,介导硫酸盐型厌氧CH4氧化作用,对亚硝酸盐型厌氧CH4氧化过程具有抑制效应[18].

  • 本试验中,围垦稻田土壤M.oxyfera-like细菌的16S rRNA基因拷贝数为(0.89~2.12)×107 copies·g-1,其变化范围与杭州湾滨海和长江口滩涂湿地的数量级一致[10,14],且显著低于南京稻田.不仅如此,M.oxyfera-like细菌的相对丰度也表现为围垦稻田明显低于南京稻田.相关性分析发现,M.oxyfera-like细菌的基因拷贝数与n-DAMO速率、土壤总氮和矿质态氮均呈显著正相关,说明M.oxyfera-like细菌的数量及其活性等生物学特性是导致n-DAMO速率产生空间变异的主控因素.然而,有报道发现,内陆湖泊沉积物中,M.oxyfera-like细菌的基因拷贝数与土壤TOC、总氮和矿质态氮均呈显著负相关,暗示除了土壤理化性质之外,可能还受到其他因素的重要影响,如厌氧氨氧化菌的竞争性生长等.说明M.oxyfera-like细菌在不同类型土壤中分布规律不尽相同,受控因素较多,有待深入研究.

  • 本试验发现,两种类型的稻田中耕层土壤的厌氧CH4氧化速率均显著高于深层土壤.此前在旱地农田和西溪湿地的研究中也有类似的报道.而且,本研究还发现M.oxyfera-like细菌的丰度表现为表层远高于深层土壤.在之前的稻田土壤中也有报道M.oxyfera-like细菌仅分布在深层土壤中而在表层土壤中未能检测到[12].现已证实,M.oxyfera-like细菌是严格厌氧类型,其自身可以内产氧方式满足CH4氧化过程[19],而环境氧分压对其会有强烈抑制作用,体积分数为2%的氧气可促使n-DAMO细菌失去活性.照此推测,分布在湿地深处的土层其厌氧CH4氧化潜力应高于表层土壤.例如,白洋淀湖泊沉积物中M.oxyfera-like细菌丰度随着深度增加有明显的增加趋势[20].在杭州稻田的研究也有类似报道[7].然而,对嘉兴稻田M.oxyfera-like细菌的研究显示,其表层(0~40cm)丰度远高于深层(40~100cm)丰度[12].除了氧分压之外,亚硝酸型厌氧CH4氧化作用还受到许多其他因素的影响,如CH4底物浓度、NO-2底物浓度、反硝化速率等.稻田耕层根系分布密度大,根际分泌物多,是稻田CH4产生的主要生境[21].这种高分压CH4浓度是CH4氧化微生物的重要底物,对亚硝酸型厌氧CH4氧化过程可能具有重要的促进作用.不仅如此,施加的N肥大部分进入稻田耕层,进行强烈的硝化和反硝化作用,在耕层会产生较多的NO-x等中间产物,有利于M.oxyfera-like细菌获取NO-2底物.研究发现,从热力学角度来看,M.oxyfera-like细菌介导的甲烷厌氧化反应的标准自由能释放量远远低于反硝化菌介导的反硝化过程,在NO-x等比较丰富而耕层稻田中其反硝化菌对M.oxyfera-like细菌的抑制作用较小,有利于亚硝酸型厌氧CH4氧化作用的进行,相反,在深层土壤中NO-x等相对贫乏,M.oxyfera-like细菌会受到反硝化微生物的强烈抑制,从而使得NO-2为底物的甲烷厌氧氧化速率减小.因此,本试验中,土壤TOC、N水平对亚硝酸型厌氧CH4氧化作用起着主导作用,从而导致其耕层土壤的氧化速率显著高于深层土壤.

  • pH值是影响土壤厌氧CH4氧化作用的重要因素[22].本试验发现两者之间存在负相关关系.He等[23]考察了不同pH条件下M.oxyfera细菌的甲烷氧化活性,发现其最适pH值是7.6,暗示了该菌偏好弱碱性环境.本试验中围垦稻田土壤pH值尽管呈弱碱性,但是厌氧CH4速率远低于南京稻田,可能的原因是土壤中高浓度的SO2-4离子也可作为电子受体参与甲烷厌氧氧化,具有底物竞争效应[18].而且土壤本底N水平低,N转化产生的NO-2较少,在一定程度上也抑制了亚硝酸型厌氧CH4氧化过程.

  • 需要指出的是,稻田土壤CH4转化过程及功能微生物群落结构会受到诸多因素的影响,如水稻生育期、施肥量、农田管理等.本研究选取了两种不同类型的典型稻田进行比较,仅能代表所试稻田土壤的厌氧CH4氧化潜势.在此基础上,需要从不同气候带采集更多不同类型的稻田土壤,深入研究典型稻田厌氧CH4氧化作用的本质特征,为探索我国稻田温室气体减排措施提供科学依据.

  • 4 结论

  • 1)内陆性稻田土壤厌氧CH4氧化速率显著高于滨海围垦稻田,其土壤C、N水平和功能微生物丰度是造成两种类型稻田产生差异的主要原因.

  • 2)两种类型稻田中,耕层土壤的厌氧CH4氧化速率显著高于深层土壤,其耕层土壤中功能微生物丰度高、活性氮丰富是CH4氧化潜力较高的重要原因.

  • 参考文献

    • [1] Dean J F,Middelburg J J,Röckmann T,et al.Methane feedbacks to the global climate system in a warmer world[J].Reviews of Geophysics,2018,56(1):207-250

    • [2] Cai Y F,Zheng Y,Bodelier P L E,et al.Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils[J].Nature Communications,2016,7:11728

    • [3] Bridgham S D,Cadillo-Quiroz H,Keller J K,et al.Methane emissions from wetlands:biogeochemical,microbial,and modeling perspectives from local to global scales[J].Global Change Biology,2013,19(5):1325-1346

    • [4] Conrad R,Klose M,Noll M.Functional and structural response of the methanogenic microbial community in rice field soil to temperature change[J].Environmental Microbiology,2009,11(7):1844-1853

    • [5] Bodelier P L E.Toward understanding,managing,and protecting microbial ecosystems[J].Frontiers in Microbiology,2011,2:1-8

    • [6] Ding J,Fu L,Ding Z W,et al.Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field[J].Applied Microbiology and Biotechnology,2016,100:439-446

    • [7] Hu B L,Shen L D,Lian X,et al.Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(12):4495-4500

    • [8] Deutzmann J S,Schink B.Anaerobic oxidation of methane in sediments of Lake Constance,an oligotrophic freshwater lake[J].Applied and Environmental Microbiology,2011,77(13):4429-4436

    • [9] He Z F,Geng S,Cai C Y,et al.Anaerobic oxidation of methane coupled to nitrite reduction by halophilic marine NC10 bacteria[J].Applied and Environmental Microbiology,2014,81(16):5538-5545

    • [10] Wang J Q,Cai C Y,Li Y F,et al.Denitrifying anaerobic methane oxidation:a previously overlooked methane sink in intertidal zone[J].Environmental Science & Technology,2019,53(1):203-212

    • [11] Zhou L L,Wang Y,Long X E,et al.High abundance and diversity of nitrite-dependent anaerobic methane-oxidizing bacteria in a paddy field profile[J].FEMS Microbiology Letters,2014,360(1):33-41

    • [12] Wang Y,Zhu G B,Harhangi H R,et al.Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane oxidizing bacteria in a paddy soil[J].FEMS Microbiology Letters,2012,336(2):79-88

    • [13] Li X F,Gao D Z,Liu M.Composition,diversity and abundance of Candidatus M.oxyfera-like bacteria in response to the estuary salinity[J].Biogeochemistry,2019,143:1-14

    • [14] Shen L D,Hu B L,Liu S,et al.Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments[J].Applied Microbiological Biotechnology,2016,100:7171-7180

    • [15] 张洁,宋怡轩,张鑫磊,等.不同类型稻田中全程氨氧化微生物的分异特征 [J].江苏农业学报,2020,36(3):584-590ZHANG Jie,SONG Yixuan,ZHANG Xinlei,et al.Differentiation characteristics of complete ammonia-oxidizing microorganisms in different types of paddy soils[J].Jiangsu Journal of Agriculture Science,2020,36(3):584-590

    • [16] 林黎,崔军,陈学萍,等.滩涂围垦和土地利用对土壤微生物群落的影响 [J].生态学报,2014,34(4):899-906LIN Li,CUI Jun,CHEN Xueping,et al.Effects of reclamation on tidal flat and land use on soil microbial community [J].Acta Ecologica Sinica,2014,34(4):899-906

    • [17] Zhao W,Wang Y Y,Liu S H,et al.Denitrification activities and N2O production under salt stress with varying COD/N ratios and terminal electron acceptors[J].Chemical Engineering Journal,2013,215/216:252-260

    • [18] Zhang Y H,Zhang X L,Wang F Y,et al.Exogenous nitrogen addition inhibits sulfate-mediated anaerobic oxidation of methane in estuarine coastal sediments [J].Ecological Engineering,2020,158:106021

    • [19] Ettwig K F,Butler M K,Le Paslier D,et al.Nitrite-driven anaerobic methane oxidation by oxygenic bacteria [J].Nature,2010,464(7288):543-548

    • [20] 夏超,祝贵兵,邹雨璇,等.厌氧氨氧化细菌和反硝化厌氧甲烷氧化细菌在岸边带土壤中的分布规律 [J].环境科学学报,2015,35(12):3965-3975XIA Chao,ZHU Guibing,ZOU Yuxuan,et al.Distribution of anaerobic ammonia oxidizing bacteria and nitrite-dependent anaerobic methane oxidizing bacteria in soil profile in the riparian zone [J].Acta Scientiae Circumstantiae,2015,35(12):3965-3975

    • [21] 张鑫磊,宋怡轩,张洁,等.围垦植稻对崇明东滩湿地产甲烷微生物的影响 [J].农业环境科学学报,2020,39(2):411-417ZHANG Xinlei,SONG Yixuan,ZHANG Jie,et al.Effects of reclamation and cultivating rice on CH4-producing microorganisms in Chongming Dongtan wetland,China[J].Journal of Agro-Environment Science,2020,39(2):411-417

    • [22] Ettwig K F,van Alen T,van de Pas-Schoonen K T,et al.Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum[J].Applied and Environmental Microbiology,2009,75(11):3656-3662

    • [23] He Z F,Geng S,Shen L D,et al.The short-and long-term effects of environmental conditions on anaerobic methane oxidation coupled to nitrite reduction[J].Water Research,2015,68:554-562

  • 基本信息

    中图分类号: S154.1;S511.061
    文献标识码: A
    DOI: 10.13878/j.cnki.jnuist.2022.01.012

    基金信息

    国家自然科学基金(42175138,41671247)；

    引用信息

    稿件历史

    收稿日期: 2021-11-22

  • 参考文献

    • [1] Dean J F,Middelburg J J,Röckmann T,et al.Methane feedbacks to the global climate system in a warmer world[J].Reviews of Geophysics,2018,56(1):207-250

    • [2] Cai Y F,Zheng Y,Bodelier P L E,et al.Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils[J].Nature Communications,2016,7:11728

    • [3] Bridgham S D,Cadillo-Quiroz H,Keller J K,et al.Methane emissions from wetlands:biogeochemical,microbial,and modeling perspectives from local to global scales[J].Global Change Biology,2013,19(5):1325-1346

    • [4] Conrad R,Klose M,Noll M.Functional and structural response of the methanogenic microbial community in rice field soil to temperature change[J].Environmental Microbiology,2009,11(7):1844-1853

    • [5] Bodelier P L E.Toward understanding,managing,and protecting microbial ecosystems[J].Frontiers in Microbiology,2011,2:1-8

    • [6] Ding J,Fu L,Ding Z W,et al.Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field[J].Applied Microbiology and Biotechnology,2016,100:439-446

    • [7] Hu B L,Shen L D,Lian X,et al.Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(12):4495-4500

    • [8] Deutzmann J S,Schink B.Anaerobic oxidation of methane in sediments of Lake Constance,an oligotrophic freshwater lake[J].Applied and Environmental Microbiology,2011,77(13):4429-4436

    • [9] He Z F,Geng S,Cai C Y,et al.Anaerobic oxidation of methane coupled to nitrite reduction by halophilic marine NC10 bacteria[J].Applied and Environmental Microbiology,2014,81(16):5538-5545

    • [10] Wang J Q,Cai C Y,Li Y F,et al.Denitrifying anaerobic methane oxidation:a previously overlooked methane sink in intertidal zone[J].Environmental Science & Technology,2019,53(1):203-212

    • [11] Zhou L L,Wang Y,Long X E,et al.High abundance and diversity of nitrite-dependent anaerobic methane-oxidizing bacteria in a paddy field profile[J].FEMS Microbiology Letters,2014,360(1):33-41

    • [12] Wang Y,Zhu G B,Harhangi H R,et al.Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane oxidizing bacteria in a paddy soil[J].FEMS Microbiology Letters,2012,336(2):79-88

    • [13] Li X F,Gao D Z,Liu M.Composition,diversity and abundance of Candidatus M.oxyfera-like bacteria in response to the estuary salinity[J].Biogeochemistry,2019,143:1-14

    • [14] Shen L D,Hu B L,Liu S,et al.Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments[J].Applied Microbiological Biotechnology,2016,100:7171-7180

    • [15] 张洁,宋怡轩,张鑫磊,等.不同类型稻田中全程氨氧化微生物的分异特征 [J].江苏农业学报,2020,36(3):584-590ZHANG Jie,SONG Yixuan,ZHANG Xinlei,et al.Differentiation characteristics of complete ammonia-oxidizing microorganisms in different types of paddy soils[J].Jiangsu Journal of Agriculture Science,2020,36(3):584-590

    • [16] 林黎,崔军,陈学萍,等.滩涂围垦和土地利用对土壤微生物群落的影响 [J].生态学报,2014,34(4):899-906LIN Li,CUI Jun,CHEN Xueping,et al.Effects of reclamation on tidal flat and land use on soil microbial community [J].Acta Ecologica Sinica,2014,34(4):899-906

    • [17] Zhao W,Wang Y Y,Liu S H,et al.Denitrification activities and N2O production under salt stress with varying COD/N ratios and terminal electron acceptors[J].Chemical Engineering Journal,2013,215/216:252-260

    • [18] Zhang Y H,Zhang X L,Wang F Y,et al.Exogenous nitrogen addition inhibits sulfate-mediated anaerobic oxidation of methane in estuarine coastal sediments [J].Ecological Engineering,2020,158:106021

    • [19] Ettwig K F,Butler M K,Le Paslier D,et al.Nitrite-driven anaerobic methane oxidation by oxygenic bacteria [J].Nature,2010,464(7288):543-548

    • [20] 夏超,祝贵兵,邹雨璇,等.厌氧氨氧化细菌和反硝化厌氧甲烷氧化细菌在岸边带土壤中的分布规律 [J].环境科学学报,2015,35(12):3965-3975XIA Chao,ZHU Guibing,ZOU Yuxuan,et al.Distribution of anaerobic ammonia oxidizing bacteria and nitrite-dependent anaerobic methane oxidizing bacteria in soil profile in the riparian zone [J].Acta Scientiae Circumstantiae,2015,35(12):3965-3975

    • [21] 张鑫磊,宋怡轩,张洁,等.围垦植稻对崇明东滩湿地产甲烷微生物的影响 [J].农业环境科学学报,2020,39(2):411-417ZHANG Xinlei,SONG Yixuan,ZHANG Jie,et al.Effects of reclamation and cultivating rice on CH4-producing microorganisms in Chongming Dongtan wetland,China[J].Journal of Agro-Environment Science,2020,39(2):411-417

    • [22] Ettwig K F,van Alen T,van de Pas-Schoonen K T,et al.Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum[J].Applied and Environmental Microbiology,2009,75(11):3656-3662

    • [23] He Z F,Geng S,Shen L D,et al.The short-and long-term effects of environmental conditions on anaerobic methane oxidation coupled to nitrite reduction[J].Water Research,2015,68:554-562

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