微生物学通报  2020, Vol. 47 Issue (11): 3709−3718

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赵子闻, 李佳, 侯俊青, 赵曼平, 赵吉, 武琳慧
ZHAO Zi-Wen, LI Jia, HOU Jun-Qing, ZAHO Man-Ping, ZHAO Ji, WU Lin-Hui
自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展
Research progress of nitrite-dependent anaerobic methane oxidation bacteria in natural habitats
微生物学通报, 2020, 47(11): 3709-3718
Microbiology China, 2020, 47(11): 3709-3718
DOI: 10.13344/j.microbiol.china.191061

文章历史

收稿日期: 2019-12-18
接受日期: 2020-05-01
网络首发日期: 2020-05-21
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赵子闻, 李佳, 侯俊青, 赵曼平, 赵吉, 武琳慧. 自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展[J]. 微生物学通报, 2020, 47(11): 3709-3718.
ZHAO Zi-Wen, LI Jia, HOU Jun-Qing, ZAHO Man-Ping, ZHAO Ji, WU Lin-Hui. Research progress of nitrite-dependent anaerobic methane oxidation bacteria in natural habitats[J]. Microbiology China, 2020, 47(11): 3709-3718.
自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展
赵子闻1 , 李佳1 , 侯俊青1 , 赵曼平1 , 赵吉1,2 , 武琳慧1,2     
1. 内蒙古大学生态与环境学院    内蒙古  呼和浩特    010021;
2. 内蒙古自治区环境污染控制与废物资源化重点实验室    内蒙古  呼和浩特    010021
收稿日期: 2019-12-18; 接受日期: 2020-05-01; 网络首发日期: 2020-05-21
基金项目: 国家自然科学基金(41701281);内蒙古"一湖两海"科技重大专项(ZDZX2018054);内蒙古自然科学基金(2017MS0334);内蒙古自治区应用技术研究与开发类资金计划(201702111)
通信作者: 武琳慧, Tel: 0471-4991460; E-mail: imuwulinhui@163.com.
摘要: 随着功能微生物介导的亚硝酸盐型厌氧甲烷氧化(nitrite-dependent anaerobic methane oxidation，N-DAMO)过程被发现，人们对自然界的碳氮循环有了全新的认识，该过程成为自然生态系统中温室气体甲烷的汇，同时还是氮污染的消减途径。本文系统介绍了N-DAMO过程反应机理以及参与该过程的亚硝酸盐型厌氧甲烷氧化细菌(Candidatus Methylomirabilis oxyfera)的生理生化特征，并对研究该功能菌的分子微生物方法进行了汇总。通过对不同自然生境中该细菌的研究报道进行总结分析，揭示各生境中年均降水量、年均温度、所处不同自然区等大尺度宏观环境因子及碳源、氮源、pH和氧气含量等生存因子对其群落结构的潜在影响，最后在展望中提出此功能菌在未来可深入研究的方向，期望能厘清厌氧甲烷氧化过程及其功能菌在碳、氮循环中的生态学功能。
关键词: 亚硝酸盐型厌氧甲烷氧化    Candidatus Methylomirabilis oxyfera    群落结构    环境因子    
Research progress of nitrite-dependent anaerobic methane oxidation bacteria in natural habitats
ZHAO Zi-Wen1 , LI Jia1 , HOU Jun-Qing1 , ZAHO Man-Ping1 , ZHAO Ji1,2 , WU Lin-Hui1,2     
1. College of Ecology and Environment, Inner Mongolia University, Hohhot, Inner Mongolia 010021, China;
2. Inner Mongolia Key Laboratory of Environmental Pollution Control & Waste Resource Reuse, Hohhot, Inner Mongolia 010021, China
Received: 18-12-2019; Accepted: 01-05-2020; Published online: 21-05-2020
Foundation item: National Natural Science Foundation of China (41701281); Science and Technology Major Project on Lakes of Inner Mongolia Autonomous Region (ZDZX2018054); Natural Science Foundation of Inner Mongolia Autonomous Region (2017MS0334); Applied Technology Research and Development Foundation of Inner Mongolia Autonomous Region (201702111)
*Corresponding author: WU Lin-Hui, Tel: 0471-4991460; E-mail: imuwulinhui@163.com.
Abstract: The discovery of nitrite-dependent anaerobic methane oxidation (N-DAMO) process, mediated by functional microorganisms, gives us a new understanding of the natural carbon and nitrogen cycle. This process can simultaneously reduce methane and nitrogen pollution. The reaction mechanism of N-DAMO process and the physiological and biochemical characteristics of the functional bacteria Candidatus Methylomirabilis oxyfera (M. oxyfera) were systematically introduced in this paper. The molecular and microbiological methods for the study of the functional bacteria were overviewed. This paper summarized and analyzed the research reports of M.oxyfera bacteria in different natural habitats. The potential effects of macro environmental factors such as average precipitation, annual average temperature, different natural areas and growth factors, like carbon source, nitrogen source, pH and oxygen content on the structure of functional bacteria community were revealed. The future research direction of the functional bacteria is proposed, which is expected to clarify the N-DAMO process and the ecological function of the functional bacteria in carbon and nitrogen cycle.
Keywords: Nitrite-dependent anaerobic methane oxidation    Candidatus Methylomirabilis oxyfera    Community structure    Environmental factor    

厌氧甲烷氧化过程(anaerobic methane oxidation，AMO)对减缓自然生境中温室气体甲烷的排放、调节地球气候大环境有重要作用[1]。亚硝酸盐型厌氧甲烷氧化过程(nitrite-dependent anaerobic methane oxidation，N-DAMO)自被发现以来就受到研究者们的广泛关注，此过程将氮碳循环耦合，使其作为温室气体甲烷汇的同时，还成为了氮污染的消减过程[2-3]。N-DAMO多发生在淡水及沿海湿地生态系统中，其功能菌亚硝酸盐型厌氧甲烷氧化细菌(Candidatus Methylomirabilis oxyfera，M. oxyfera)多存在于湿地生态系统上覆水-沉积物交界面及亚表层土壤中[3-4]。有关M. oxyfera生理生化特征及所参与的N-DAMO反应机理等领域的研究取得了一定进展[2-5]。在微生物生态研究领域，利用分子微生物学方法直接分析不可纯培养的N-DAMO功能细菌在不同生境中的群落组成、丰度和分布成为研究热点，且大量相关研究探讨了诸如pH、碳源、氮源、氧气含量等环境因子对其群落结构的影响[6-10]。本文简述了N-DAMO功能菌M. oxyfera的生理生化特征、N-DAMO反应机理，并整理了基于此菌的分子微生物学研究方法，同时归纳总结了近年来不同自然生境中该菌的研究报道及其与环境因子的相关性分析，进一步分析了不同生境年均降水量、年均温度、不同自然区划分等大尺度宏观环境因子对功能菌群落的潜在影响。

1 N-DAMO过程的发现

最早被发现的厌氧甲烷氧化过程是以硫酸盐为电子受体的厌氧甲烷氧化过程，其对于减缓海洋沿海等生境中甲烷的排放起着重要的作用[11]。甲烷与亚硝酸盐在自然环境中共存，由吉布斯自由能角度分析可以发现，亚硝酸盐比硫酸盐更适合作为甲烷氧化的电子受体，但长期以来研究者们并没有发现以亚硝酸盐为电子受体进行厌氧甲烷氧化过程的微生物存在[12]。2006年，Raghoebarsing等[2]通过16个多月连续向运河沉积物富集培养物中投加甲烷、亚硝酸盐和硝酸盐的实验，首次得到了以亚硝酸盐为电子受体的厌氧甲烷氧化过程发生的直接证据，随后这个过程被命名为N-DAMO过程[5]。

自然生境中的厌氧甲烷氧化过程共分为三大类：亚硝酸盐型、硫酸盐型和金属离子型[2]。较多的研究关注于亚硝酸盐型厌氧甲烷氧化过程[6-9]，且该反应多发生于淡水及沿海湿地生态系统中。硫酸盐型厌氧甲烷氧化过程多发生于海洋生态系统中，为减少海洋生态系统甲烷排放方面做出了巨大的贡献[13]。金属离子型厌氧甲烷氧化过程中电子受体多为Fe3+、Mn4+和Cr5+等金属离子[14]，自从在海洋沉积物中发现此过程以来，丰富了厌氧甲烷氧化功能菌的多样性[11]。

2 M. oxyfera的生理生化特征及反应机理

氮依赖型厌氧甲烷氧化过程的功能微生物共有两种，一种是以NO2-为电子受体的亚硝酸盐型厌氧甲烷氧化细菌(M. oxyfera)，另一种则是以NO3-为电子受体的硝酸盐型厌氧甲烷氧化古菌(Candidatus Methanoperedens nitroreducens)，在自然生态环境及富集反应器中这两种菌可共存[3]，而且多项研究表明此功能细菌在N-DAMO过程功能菌群中占较大比重[2, 4]，国内外研究多集中于此类型细菌展开。目前对该功能菌的生理生化研究较多，此细菌隶属于NC10门，革兰氏阴性菌，细胞直径为0.25-0.5 μm，细胞长为0.8-1.1 μm[5, 15]。通过扫描电镜、透射电镜和电子断层扫描等手段，有研究多角度证实了其独特的多边形星形细胞形态[5]，而这一特殊细胞形态在其他细菌形态学观察报道中未曾出现[16]，Wu等[17]和Luesken等[18]发现特异性蛋白结构层，以及用于编码类骨骼物质的mreB和ftsZ基因。Kool等[19]研究多种M. oxyfera富集培养物脂质组成后发现，此细菌所独有的脂肪酸10MeC16:△7约占总脂肪酸的10%，可作为该菌的生物化学标志物[19-20]。

甲烷是亚硝酸盐型厌氧甲烷氧化细菌唯一的碳源[21]，适宜生长温度为20-35 ℃，pH为6.0-8.0[2, 22]，世代时间约为1-2周，部分超过4周[22-26]。该细菌可进行N-DAMO反应的最高氯化钠浓度为20 g/L[27]。其对甲烷的亲和力极强，亲和力常数明显低于硫酸盐型厌氧甲烷氧化菌[16]。Luesken等[18]发现此细菌在环境氧气浓度超过2%时，甲烷转化率和亚硝酸盐转化率均受到严重抑制，大部分参与甲烷氧化过程的基因不再表达，参与反硝化途径和重要细胞功能的基因表达能力显著下降。

Ettwig等[5]对两种M. oxyfera富集培养物进行宏基因组测序和同位素分析，结果显示此细菌拥有编码完整好氧甲烷氧化途径的功能基因，后续的中心分解代谢及能量代谢研究再次证实了此细菌具有独立完成甲烷氧化的能力[21, 28]。此细菌所利用的电子受体亚硝酸盐首先在亚硝酸盐还原酶(NirS)的作用下还原为NO，NO双键被未知NO歧化酶(Nod)直接转化为N2和O2，略过中间体N2O的生成过程，而细胞自身通过NO歧化反应所产生的氧气中3/4被用于甲烷氧化途径，其余被用于细胞呼吸[5, 28]。研究者进而提出这种新型甲烷氧化细菌可能利用胞内好氧化途径参与地球早期甲烷氧化，而不直接依赖光合作用进行产氧，从而提出了地球早期代谢途径演化的猜想[5]。

3 基于M. oxyfera的分子微生物学研究

除使用M. oxyfera的16S rRNA基因作为目标基因研究其生态学分布外[22]，专属N-DAMO过程的pmoA功能基因也成为常见的选择[15]，也有科学家尝试用NO歧化酶(Nod)的编码基因探究此功能细菌在诺尔盖湿地的生态分布[29]。相比于使用16S rRNA基因对M. oxyfera进行研究，直接使用功能基因作为分子标记可信度更高，因为核糖体基因在许多原核生物中是多拷贝的，这通常会导致同一细胞中核糖体基因序列的异质化，而功能基因在原核生物细胞中多数不存在多拷贝，Dahllöf等[30]最早在分子层面上验证了这一现象。在现有研究未能确定亚硝酸盐型厌氧甲烷氧化细菌中核糖体拷贝数的前提下，通过功能基因进行微生物生态学调查更具优势。

常用的M. oxyfera 16S rRNA基因巢式PCR引物对为202F/1545R和qP1F/qP2R[22]。以此菌专属的pmoA为目标基因设计的巢式PCR引物对为A189_b/cmo682和cmo182/cmo568[15]。基于nod功能基因设计的PCR引物对为nod684Fv2/nod1706Rv2[31]。对于M. oxyfera丰度的研究目前还没有成熟的专属pmoA功能基因的定量PCR引物，仅有少数自然生境成功使用这一功能基因进行定量(如漳江河口[32]引物对cmo182/cmo568、香港大榄涌水塘[33]引物对HP3F1/HP3R1等)。对于功能基因nod的定量，常选用nod1446F/nod1706Rv2作为引物[31]。大部分定量研究利用该菌16S rRNA基因进行，常用的定量PCR引物对有qP1F/qP1R[22]、qP2F/qP2R[22]、qP1F/qP2R[11]、qP1mF/qP1R[34]，表 1整理了已发表的各种扩增引物及退火温度。

表 1　M. oxyfera-like的PCR扩增引物及条件 Table 1　Experimental primers and annealing temperature of PCR for M. oxyfera-like
目标基因
Target gene		 引物
Primers		 退火温度
Annealing temperature (℃)		 参考文献
References
M. oxyfera 16S rRNA 202F/1545R, qP1F/qP2R 57/63; 57/65; 63/63 [22]
M. oxyfera 16S rRNA (qPCR) qP1F/qP1R 54; 55; 63 [22]
qP2F/qP2R 63; 65 [22]
qP1F/qP2R 60 [11]
qP1mF/qP1R 62 [34]
N-DAMO pmoA A189_b/cmo682, cmo182/cmo568 56/59 [15]
N-DAMO pmoA (qPCR) cmo182/cmo568 59 [15]
HP3F1/HP3R1 54 [33]
nod nod684Fv2/nod1706Rv2 57 [31]
nod (qPCR) nod1446F/nod1706Rv2 60 [31]
表选项
4 自然生境中M. oxyfera的研究现状

目前针对于自然生境中M. oxyfera的研究主要在我国不同区域的湖泊、河流、沿海及干旱土地等生境中逐渐开展，表 2汇总分析了我国不同生境中亚硝酸盐型厌氧甲烷氧化细菌群落的分布特征。然而国外基于自然生境中该菌的研究较少，仅有康斯坦斯湖(德国)、琵琶湖(日本)、昆士兰旱地(澳大利亚)[35]等报道。从M. oxyfera 16S rRNA基因丰度上来看，丰度最低值出现在三峡小江水位波动带(4.70×102 copies/g)，这可能与此区域水位波动明显且此菌对生境中氧气含量较为敏感有关[18]。丰度最高值出现在长江口生境中(9.77×107 copies/g)，长江入海口较适宜的生境温度[22]、丰富的营养盐和碳源[21]可能导致了该生境丰度水平较高的现象。

4.1 年均降水量和年均温度对M. oxyfera的影响

Bahram等[60]对全球表层土微生物群落结构的大尺度生态研究表明，包含年均降水量在内的宏观环境因子影响着土壤细菌的功能多样性。本文通过综合我国不同生态环境中亚硝酸盐型厌氧甲烷氧化细菌群落的研究数据，将之与各生境年均降水量、年均气温、自然区划分等宏观大尺度环境因子相结合，分析其潜在的影响与关系。

研究表明M. oxyfera的pmoA功能基因多样性随气温升高而增加，低温低降水和高温高降水均有助于此菌多样性的表达[6-9, 29, 32-59]。值得注意的是在低于5 ℃的寒冷环境中此菌的多样性表达明显增加，Zhu等[25]也发现冬季样品M. oxyfera功能基因多样性表达高于夏季。随年均降水量和气温的升高，此菌16S rRNA基因丰度增大。Ettwig等[22]研究发现此菌生长适宜温度为20-35 ℃，这与其在自然生态环境中的表现相同。高降水量对应高丰度的现象可由两方面来诠释[8, 44, 49, 57]：(1)丰富的降水有助于为氧气敏感型的厌氧甲烷氧化细菌创造长期低氧无氧环境[61]；(2)高降水量也可将陆地中的碳源、氮源等营养物质通过地表及地下水径流输入湿地生态系统中[62]有助于该菌丰度增加。

4.2 不同自然区中的M. oxyfera群落

将现有M. oxyfera生态研究数据与我国自然区划分进行综合分析可发现，M. oxyfera群落在季风区中具有较为广谱的多样性和较高的丰度；其中温带湿润、半湿润区和热带湿润区多样性高于其他两类型季风区；丰度在亚热带湿润区和热带湿润区达到较高水平[6-9, 29, 32-59]。干旱、半干旱区多样性表达略高，这可能与此区内年均气温集中在10 ℃以下有关，而干旱、半干旱区和高寒区此功能菌丰度低于季风区[29, 36]。

表 2　各种自然生境中M. oxyfera的分布特征 Table 2　Distribution of M. oxyfera in different natural habitats
研究对象
Habitats		 OTUs
(16S rRNA gene)		 OTUs
(pmoA)		 Shannon index
(16S rRNA gene)		 Shannon index
(pmoA)		 16S rRNA丰度
16S rRNA abundance (copies/g)		 参考文献
References
诺尔盖湿地 Zoige wetland ND ND ND ND 2.80×103 [29]
漳江河口 Zhangjiang estuary 3-9[c] 0-2[c] 0.770-1.230 0.000-0.010 2.43×106-2.09×107 [32]
大榄涌水塘 Tailamchung reservoir ND 7[c] ND 1.890 ND [33]
米埔芦苇床 Maipo reed bed ND 1[c] ND 0.290 ND [33]
日喀则林地 Shigatse forestland ND 2[c] ND ND 1.50×104 [35]
巴丹湖 Badain lake 3-8[c] 6-8[c] 0.443-1.696 1.390-1.450 ND [36]
苏木吉林湖 Sumujaran lake 2-5[c] 8-10[c] 0.240-0.580 1.275-1.800 1.12×105-1.64×105 [36]
广州城市湿地 Guangzhou urban wetland 1-5[c] 1-5[c] 0.000-0.780 0.000-0.990 8.74×105-4.80×106 [37]
浑河 Hun river 3-6[c] 0-5[a] 0.520-1.400 0.000-1.570 ND [38]
大辽河 Daliao river 1-2[c] 1-3[a] 0.000-0.210 0.000-0.420 ND [38]
青藏高原玛曲 Tibet plateau Maqu 28-46[c] ND 0.160-1.390 ND 1.65×105-3.20×106 [39]
北运河 North canal 2-6[d] 2-9[c] 0.270-0.770 0.210-1.480 ND [40]
三峡白家溪 The Three Gorges Baijia stream 3-8[c] 1-4[c] 0.235-1.148 0.000-0.975 4.72×102-5.32×104 [41]
三峡小河 The Three Gorges Xiao stream 4-7[c] 4-24[c] 0.392-1.482 0.675-2.541 8.98×102-3.31×105 [41]
锡林河湿地 Xilin river wetland 2-14[c] 2-7[d] 0.080-1.270 0.290-0.970 8.92×106-4.61×107[e] [42]
东海潮间带 East China Sea intertidal zone 2-3[c] ND 0.300-0.900 ND 5.50×104-2.80×105 [43]
米埔湿地 Maipo wetland ND 3-4[b] ND 0.245-0.979 2.06×106-1.25×107 [44]
东江 Dong river ND 5-16[c] ND 1.480-2.490 ND [45]
秋香江 Xiangqiu river ND 7-18[c] ND 1.560-2.580 ND [45]
石马河 Shima river ND 2-12[c] ND 0.420-2.160 ND [45]
新丰江 Xinfeng river ND 2-10[c] ND 0.420-2.070 ND [45]
西枝江 Xizhi river ND 1-9[c] ND 0.000-1.970 ND [45]
绿水湾湿地 Green bay wetland 29-52[c] ND 1.890-2.140 ND 6.10×106-3.20×107 [46]
高州水库 Gaozhou reservoir ND 3-15[b] ND 0.320-2.500 1.37×105-8.24×105 [47]
舟山潮间带 Zhoushan intertidal zone ND 1-5[a] ND 0.000-1.330 4.90×104-3.27×105 [48]
长江口 Yangtze river estuary 4-7[c] 5-9[c] 0.470-1.440 1.300-1.940 1.03×107-9.77×107[e] [49]
杭州湾 Hangzhou bay 1-4[c] ND 0.000-1.100 ND 1.80×107-5.00×107 [50]
舟山海域 Zhoushan sea area 1-3[c] ND 0.000-0.900 ND 5.40×106-2.10×107 [50]
三峡小江 The Three Gorges Xiao river 3-7[c] 1-4[c] 0.080-0.950 0.000-0.970 4.70×102-5.30×104 [51]
广东南岭 Guangzhou Nanling ND 1-6[c] 0.000-0.900 0.000-1.290 1.69×105-5.07×106 [52]
摆龙湖 Bailong lake ND 19[c] ND 2.400 2.29×105 [53]
普者黑湖 Puzhehei lake ND 2[c] ND 0.100 ND [53]
长桥海 Changqiao lake ND 2[c] ND 0.110 ND [53]
三角海水库 Sanjiaohai reservoir ND 4[c] ND 1.170 ND [53]
清水海 Qingshuihai lake ND 9[c] ND 1.100 ND [53]
剑湖 Jian lake ND 2[c] ND 0.460 ND [53]
海西海水库 Haixihai reservoir ND 1[c] ND 0.000 ND [53]
塔里木河 Tarim river ND 2[d] ND 0.690 ND [25]
博斯腾湖 Bosten lake ND 2[d] ND 0.480 ND [25]
长白山天池 Tianchi lake ND 9[d] ND 1.810 ND [25]
吐鲁番河 Turpan river ND 3[d] ND 0.850 ND [25]
黄河(兰州市) Yellow River (Lanzhou) ND 1[d] ND 0.000 ND [25]
乌梁素海 Ulansuhai lake ND 4[d] ND 0.990 ND [25]
商丘水库 Shangqiu reservoir ND 7[d] ND 1.690 ND [25]
圆明园湖 Old summer palace lake ND 3[d] ND 0.980 ND [25]
多伦湖 Duolun lake ND 6[d] ND 1.390 ND [25]
北运河 North canal ND 2[d] ND 0.690 ND [25]
松花江 Songhua river ND 7[d] ND 1.670 ND [25]
盘锦红海滩 Panjin red beach ND 4[d] ND 1.120 ND [25]
盘锦沼泽 Panjin swamp ND 1[d] ND 0.000 ND [25]
白洋淀 Baiyangdian lake ND 1[d] ND 0.000 ND [25]
巢湖 Chao lake ND 6[d] ND 1.790 ND [25]
嘉兴湿地 Jiaxing wetland ND 8-9[d] ND 2.020-2.030 ND [25]
东湖 Dong lake ND 3[d] ND 0.8000 ND [25]
苕溪 Tiao river ND 3[d] ND 1.000 ND [25]
鄱阳湖 Poyang lake ND 2[d] ND 0.600 ND [25]
洞庭湖 Dongting lake ND 2[d] ND 0.690 ND [25]
珠江(广州市) Pearl river (Guangzhou) ND 2-6[d] ND 0.620-1.190 ND [25]
沙河 Sha river ND 5[d] ND 1.370 ND [25]
下渚湖 Xiazhu lake 0-4[c] 0-1[a] 0.000-1.430 0.000 1.80×106-5.10×107 [54]
西溪湿地 Xixi wetland 2-5[c] ND 0.620-1.410 ND 1.60×106-1.30×107 [55]
钱塘江 Qiantang river 4[c] 3[a] 0.680 0.560 1.23×106 [56]
富春江 Fuchun river 4-7[c] 1-9[a] 0.680-1.550 0.000-1.980 ND [56]
兰江 Lan river 3-4[c] 3-4[a] 0.850-1.290 0.440-0.710 1.03×107 [56]
新安江 Xin’an river 5[c] 4[a] 1.400 0.670 ND [56]
椒江河口 Jiao river estuary 1-3[c] 1-8[a] 0.000-1.000 0.000-1.890 5.80×105-8.35×107[e] [57]
舟山海岛 Zhoushan island 2-3[c] ND 0.300-0.900 ND 8.70×106-5.20×107[e] [58]
杭州西湖 Hangzhou west lake 2[c] 5[b] ND ND 2.15×105[e] [59]
注：[a]、[b]、[c]和[d]分别代表在93%、95%、97%和98%相似性水平下对基因进行OTU聚类；丰度单位默认为copies/g湿重；[e]表示此处的丰度单位为copies/g干重；ND表示未获得数据.
Note:[a], [b], [c] and [d] respectively represent OTU clustering of genes at 93%, 95%, 97% and 98% similarity levels; The default abundance unit is copies/g (wet weight); [e] represent the abundance unit of this item is copies/g (dry weight); ND represent as no data.
表选项
4.3 氮源对M. oxyfera的影响

亚硝态氮作为N-DAMO过程的电子受体，在自然生境中多由硝态氮通过硝化作用还原而来，近期东江底泥的研究发现，底泥中丰富的氨氧化古菌及细菌使氨氮迅速向亚硝态氮转化[63]，提升底泥中亚硝态氮含量，增加了M. oxyfera丰度[45]，因此不同形态氮均有可能对其产生影响。多项研究表明氨氮和亚硝氮浓度的提升有利于此菌丰度的增加[9, 32, 44-45]，总氮、硝态氮浓度的升高使此菌在OTU水平上的物种多样性增加[54]，N-DAMO速率也随着亚硝态氮和硝态氮浓度增高而增大[44]。Shen等[46]发现氨氮、硝态氮和pH 3个环境因子协同影响了此菌群落的空间分布。但硝化功能菌与M. oxyfera间的协同或竞争关系还有待进一步的研究。

4.4 碳源对M. oxyfera的影响

甲烷是亚硝酸盐型厌氧甲烷氧化细菌唯一的碳源[21]，M. oxyfera通过好氧甲烷氧化途径氧化甲烷为二氧化碳并获得能量[28]。大量的研究发现高浓度有机碳有助于提高此菌丰度和N-DAMO速率[32, 55-56]，但有研究表明在高浓度甲烷引发该细菌丰度增加的同时，往往伴随着M. oxyfera基因多样性的降低[32]。这也表明充足的碳源会加剧竞争，导致群落结构演替，但针对M. oxyfera的多环境因子联合相关性分析仍然较少，且产甲烷古菌、厌氧甲烷氧化古菌、好氧甲烷氧化细菌等微生物与此菌的种间关系还有待于更深入的研究。

4.5 pH对M. oxyfera的影响

生理研究表明M. oxyfera最适pH为7.60[27]。对米埔湿地的研究发现，在沉积物pH背景值(5.98-6.47)低于7.60时，此菌丰度随着pH的升高而增大[44]。当自然生境沉积物中pH大于7.60时，N-DAMO反应速率和此菌多样性均呈现出负相关[32]，Zhu等[25]也研究认为pH是影响此菌多样性的重要因素。因而适宜的pH有利于M. oxyfera的丰度增加、多样性表达和N-DAMO速率提高。

4.6 生境中氧气对M. oxyfera的影响

亚硝酸盐型厌氧甲烷氧化细菌是一种对生境中氧气含量极为敏感的厌氧菌，外部生境中氧气浓度增加会抑制M. oxyfera的内产氧机制，进而对甲烷和亚硝酸盐转化率产生明显影响[18]。Hu等[3]研究发现低氧气含量的深层土壤中M. oxyfera丰度较高于表层土壤，而此菌在OTU水平上各项多样性指数也存在氧气含量较低的深层样点要高于表层和浅层样点的现象[8]。但关于此功能菌在应对环境中氧气含量变化时菌种及群落应激反应的研究仍然较少，有待进一步深入探究。

5 结论与展望

亚硝酸盐型厌氧甲烷氧化过程广泛存在于自然生境中，在作为温室气体甲烷的汇的同时消减了日益严重的氮污染[1-3]。此过程中重要的功能微生物M. oxyfera的研究也逐渐深入，当前研究多基于N-DAMO过程甲烷氧化途径中pmoA功能基因展开，但关于独特的亚硝酸盐还原途径中将NO直接转化为N2和O2的NO歧化酶(Nod)的研究较少，在分子微生物学领域已有对nod功能基因进行定量及系统发育树分析的研究[29]。同时关于Nod酶的空间结构、生化反应机理等方面还有待于更深入的探究。

使用高通量和宏基因组测序技术探究不可纯培养的N-DAMO过程功能微生物M. oxyfera群落结构、丰度及生态学价值成为近年来的研究热点。通过分析宏观环境因子对M. oxyfera的影响中发现，较高的年均气温有利于此菌在OTU水平上物种多样性的提升，同时较高的年均降水和气温均有利于此菌的丰度增加。值得注意的是，在低温(0-5 ℃)生境中M. oxyfera存在多样性表达激增的现象，而其中的具体应激机理还有待于进一步的泛基因组学探析。

在三大自然区中，季风区是此菌丰度最高、多样性表达较为广谱的区域。在高寒区和干旱、半干旱区中对于N-DAMO过程的研究仍然较少，仅有巴丹吉林沙漠湖泊[36]、青藏高原[39]、锡林郭勒湿地[42]和塔里木河[25]等几个自然生境的研究报告，丰富这两大自然区的研究空白，将对分析亚硝酸盐型厌氧甲烷氧化细菌在我国自然生境中的生态学价值及现状提供帮助。

碳源、氮源、pH和氧气浓度都对M. oxyfera的群落存在影响，多环境因子协同作用也成为了关注度较高的研究方向。硝化细菌、厌氧氨氧化细菌、厌氧产甲烷古菌等与此细菌或协同或竞争的种间关系研究日益兴起，而更深入地研究碳氮循环中多种功能微生物种间关系，将有助于多维立体地分析自然生境中M. oxyfera参与各个营养循环的生态学意义。

当今研究N-DAMO过程的重点除了计算和控制水稻田和湿地等自然生态环境中甲烷排放潜力外，人类活动所导致的各种形态氮、碳源以及抗生素等物质，通过径流输入湿地等生态系统中对厌氧甲烷氧化过程所产生的影响也应当被重视。同时随着各种人类活动的进行，原本自然状态下湿地生态系统中的水量、盐量、热量平衡被打破，而由此引发的M. oxyfera群落应激反应及其机理还有待更深入的研究。同时有望借助此菌建立微生物标尺，应用于评价环境污染程度、判断湿地富营养化治理成效以及精准农业选址等众多领域。

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