全 文 ：第 35 卷第 14 期
2015年 7月
生 态 学 报
ACTA ECOLOGICA SINICA
Vol.35,No.14
Jul.,2015
http: / / www.ecologica.cn
基金项目:国家自然基金项目资助(11079053, 31200367)
收稿日期:2013鄄05鄄06; 摇 摇 网络出版日期:2014鄄09鄄09
*通讯作者 Corresponding author.E鄄mail: cuixy@ gucas.ac.cn
DOI: 10.5846 / stxb201305060936
邓永翠, 车荣晓, 吴伊波, 王艳芬, 崔骁勇.好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展.生态学报,2015,35(14):
4579鄄4591.
Deng Y C, Che R X, Wu Y B, Wang Y F, Cui X Y.A review of the physiological and ecological characteristics of methanotrophs and methanotrophic
community diversity in the natural wetlands.Acta Ecologica Sinica,2015,35(14):4579鄄4591.
好氧甲烷氧化菌生理生态特征及其在自然湿地中的群
落多样性研究进展
邓永翠1,2, 车荣晓1, 吴伊波3, 王艳芬1, 崔骁勇1,*
1 中国科学院大学, 北京摇 100049
2 南京师范大学虚拟地理环境教育部重点实验室, 南京摇 210046
3 宁波大学, 宁波摇 315211
摘要:甲烷氧化菌是一类可以利用甲烷作为唯一碳源和能源的细菌,在全球变化和整个生态系统碳循环过程中起着重要的作
用。 近年来,对甲烷氧化菌的生理生态特征及其在自然湿地中的群落多样性研究取得了较大进展。 在分类方面,疣微菌门、
NC10门及两个丝状菌属甲烷氧化菌的发现使其分类体系得到了进一步的完善;在单加氧酶方面,发现甲烷氧化菌可以利用
pMMO和 sMMO两种酶进行氧化甲烷的第一步反应,域型甲烷氧化菌中 pMMO2 的发现证实甲烷氧化菌可以利用这种酶氧化
低浓度的甲烷;在底物利用方面,已经发现了越来越多的兼性营养型甲烷氧化菌,证实它们可以利用的底物比之前认为的更广
泛,其中包括乙酸等含有碳碳键的化合物;在生存环境方面,能在不同温度、酸度和盐度的环境中生存的甲烷氧化菌不断被分离
出来。 全球自然湿地甲烷氧化菌群落多样性的研究目前主要集中在北半球高纬度的酸性泥炭湿地,域型甲烷氧化菌
Methylocystis、Methylocella和 Methylocapsa是这类湿地主要的甲烷氧化菌类群,尤其以 Methylocystis类群最为广泛,而玉型甲烷氧
化菌尤其是 Methylobacter在北极寒冷湿地中占优势。 随着高通量测序时代的到来和新的分离技术的发展,对甲烷氧化菌的现
有认识将面临更多的挑战和发展。
关键词:甲烷氧化菌; 甲烷; 甲烷单加氧酶; 湿地
A review of the physiological and ecological characteristics of methanotrophs and
methanotrophic community diversity in the natural wetlands
DENG Yongcui1,2, CHE Rongxiao1, WU Yibo3, WANG Yanfen1, CUI Xiaoyong1,*
1 University of Chinese Academy of Sciences, Beijing 100049, China
2 Key Laboratory of Virtual Geographic Environment, Ministry of Education, Nanjing Normal University, Nanjing 210046, China
3 Ningbo University, Ningbo 315211, China
Abstract: Methanotrophs are a group of bacteria that can use methane as their sole source of carbon and energy. They play a
major role in carbon cycle and global warming by controlling emissions of methane, the second most important greenhouse
gas following CO2 . In this review, we summarize recent progress on the physiology, phylogeny, and ecology of
methanotrophs, with particular focus on the diversity of methanotrophic community in natural wetlands. The traditionally
identified methanotrophs all belong to the phylum Proteobacteria. Based on intracytoplasmic membranes formation,
predominant fatty acid types, the mechanism by which carbon is assimilated into biomass and phylogenetic characteristics,
proteobacterial methanotrophs are divided into two groups, type 玉 and type 域 ( Gamma鄄 and Alpha鄄proteobacteria,
http: / / www.ecologica.cn
respectively) . Up to now, 20 methanotrophic genera have been affiliated in phylum Proteobacteria, including two
filamentous methanotrophs, Crenothrix polyspora and Clonothrix fusca. These two species have been characterized recently
and form a new branch within the family Methylococcaceae. Verrucomicrobial methanotrophs, a remarkable new finding, are
distantly related to the proteobacteria methanotrophs. They have been isolated from geothermal sites, seem to be restricted to
extreme environments and form a new genus (Methylacidiphilum). Methanotrophs are also found in a novel phylum named
NC10, which represents bacteria capable of aerobic methane oxidation coupled to denitrification under anoxic conditions.
Two types of enzyme, a particulate methane monooxygenase (pMMO) and a soluble methane monooxygenase (sMMO) can
be used by methanotrophs to execute the first step of methane oxidation. All known methanotrophs possess the pMMO,
except genera Methylocella and Methyloferula which only have sMMO. Some methanotrophs of type 玉 and 域 have both
pMMO and sMMO. A different pMMO (pMMO2) is discovered in some type 域 methanotrophs. pMMO2 has lower methane
oxidation kinetics and enables these methanotrophs to consume methane at atmospheric concentrations. The pmoA and mmoX
gene, encoding subunits of the pMMO and sMMO respectively, have been used as a functional marker for detecting
methanotrophs in environmental samples. However, the current public pmoA sequences database is larger than that of the
mmoX, and the sequence based pmoA phylogeny has good correlation to the 16S rRNA phylogeny. Facultative methanotrophs
have been reported in the genera Methylocella, Methylocapsa, and Methylocystis. Some species of them can use compounds
with carbon鄄carbon bonds as sole growth substrates, including acetate, large organic acids or ethanol. These findings broke
the traditional notion that methanotrophs could only use one鄄carbon compounds, indicating that broader substrate utilization
might be more common in methanotrophs. Methanotrophs have been isolated from various environments including habitats of
extreme temperature, acidity or salinity. For example, some type 玉 methanotrophs (Methylocaldum, Methylococcus, and
Methylothermus) were reported to have optimum growth temperatures above 40 益 . On the other hand there are some
methanotrophs (Methylobacter and Methylocella) adapted to cold environments and with optimum growth temperatures of 0—
30 益 . Some Methylacidiphilum species grow at extreme low pH of 2—2.5. But some Methylomicrobium species have the
optimum pH of 9. 0—9. 5. Besides, some Methylomicrobium species and Methylohalobius crimeensis are halotolerant
methanotrophs and have a growth optimum around 1—1.5 mol / L NaCl. In contrast, Methylocapsa KYG is very sensitive to
NaCl and can only grow at low NaCl concentrations. By employing the 16S rRNA gene or functional genes as molecular
markers, the methanotrophic communities have been extensively studied in many natural wetlands. A variety of molecular
biological tools, such as T鄄RFLP, DGGE, FISH, clone library and pyrosequencing, have been used to detect the
community diversity of methanotrophs in soils of these ecosystems. Most of the studies were conducted in acidic peat
wetlands at the high latitudes of the Northern Hemisphere, especially in the United Kingdom and Russian. In these
peatlands, most of the known methanotrophs belonged to type 域, such as genera Methylocystis, Methylocella and
Methylocapsa. Especially genus Methylocystis, were widely distributed in acidic peatlands. Type 玉 methanotrophs,
especially genus Methylobacter, were dominant in the cold Arctic wetlands in Norway and Finland. Furthermore, researches
revealed that type 玉 and 域 methanotrophs were widely present in natural wetlands in the United States and Japan. In
2012, the first study on methanotrophic diversity of wetland in the southern hemisphere was reported in Argentina. The high
abundance of genus Methylocystis suggests that it is probably the major contributor to the methane oxidation in this
Sphagnum wetland. Type 玉 and type 域 methanotrophs were all detected and were present with different proportion in some
natural wetlands of China. Great progress has already been made in the recent researches of the physiological and ecological
characteristics of methanotrophs and their community diversity in the natural wetlands. With the arrival of the era of high鄄
throughput sequencing and the development of new isolation and culture technology, the knowledge systems of
methanotrophs will be refreshed more frequently.
Key Words: methanotrophs; methane monooxygenase (MMO); methane; wetland
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甲烷是大气中辐射强迫仅次于二氧化碳的温室气体,大气中甲烷含量仅为二氧化碳的 1 / 27,但单分子增
温效应是二氧化碳的 25倍,对全球温室效应的贡献率可达 18%[1]。 甲烷在大气中的浓度已经从工业化以前
的体数分数为 715伊10-8增长到现在的体数分数为 1770伊10-8,这种增长是由其源汇平衡决定的。 全球大气甲
烷排放量约为 500—600 Tg / a,其中自然湿地贡献总甲烷源的 23%[2],是最大的大气甲烷源。 自然湿地中厌氧
土层产甲烷菌产生的甲烷大部分并没有直接进入大气,约 90%的甲烷是通过湿地有氧土层的甲烷氧化菌消
耗掉[3鄄4]。 土壤里甲烷氧化菌的这种氧化作用是甲烷的唯一生物汇。 甲烷氧化菌(Methanotrophs)在 1906 年
首次被发现[5],它是甲基氧化菌(Methylotrophs)的一个分支,能利用甲烷(Methane,CH4)作为其唯一的碳源和
能源[6]。 甲烷氧化菌在全球变化和整个生态系统碳循环过程中起着非常重要的作用。 现已有研究证实在各
种生态环境中(湖泊底泥[7],水稻田[8],垃圾填埋场[9],泥炭湿地[10]及北极高纬度湿地[11]等)分布的甲烷氧
化菌在消耗甲烷中起着重要的作用。
甲烷氧化菌的研究一直广受关注,在 2009年之前,国际上几乎每 4年就有一篇总结甲烷氧化菌研究进展
的优秀综述发表,而在 2009—2010 年 4 篇综述论文刊载(表 1)。 这些综述主要关注甲烷氧化菌的分类、生
理、生态分布、分离培养、应用、甲烷单加氧酶(Methane Monooxygenase,MMO)的催化机理等方面的研究和所
用的研究技术等,但是缺少对甲烷氧化菌分布的重要生态系统———全球自然 湿地的详细综述;另外,关于近
两年新发现的甲烷氧化菌的相关研究也还没有归纳在内。 基于此,本文着重分析了全球自然湿地中好氧甲烷
氧化菌多样性研究的现状,综述了好氧甲烷氧化菌的种类、氧化甲烷的关键酶及底物特征方面的新进展,并展
望湿地甲烷氧化菌研究的发展趋势。
表 1摇 国内外重要的甲烷氧化菌研究综述
Table 1摇 Important reviews on researches of methanotrophs
年份 Year 题目 Title 主题 Subject 文献 References
2004 甲烷氧化菌研究进展 甲烷氧化菌的分类、生理、生态分布和研究方法 [12]
2008 甲烷氧化菌及甲烷单加氧酶的研究进展 甲烷氧化菌的分类、生态分布、MMOs 的结构与功能及基因工程研究 [13]
2011 自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测
自然湿地甲烷氧化菌的分子检测方法和研究群落多
样性的部分新成果 [14]
1996 Methnotrophic bacteria 甲烷氧化菌的生理、分类、生态、在碳循环和生物降解中的作用 [15]
2000 The methanotrophs. The families Methylococcaceaeand Methylocystaceae
甲烷氧化菌的系统发育、分类、生理、生境、分离、培
养、应用等 [16]
2005 The Leeuwenhoek Lecture 2000 The natural andunnatural history of methane鄄oxidizing bacteria 甲烷氧化菌的发展史、应用和 MMOs的作用机理等 [17]
2009 Exploring methanotroph diversity in acidic northernwetlands: molecular and cultivation鄄based studies
1995年到 2009年在北半球酸性湿地甲烷氧化菌研究
的重要进展 [18]
2010 Ecology of aerobic methanotrophs and their role inmethane cycling
甲烷氧化菌的生态分布、分子技术和环境对它的影
响等 [19]
2010 The aerobic methane oxidizing bacteria 甲烷氧化菌的分类、生理生化、生态、培养、基因组学等 [20]
2010 Methanotrophs and copper 甲烷氧化菌的分类、生理特征、多样性、应用、影响因素、Cu对其的影响和作用等 [21]
1摇 甲烷氧化菌的分类及其生理生态特征研究进展
甲烷氧化菌(Methanotrophs)是甲基氧化菌(Methylotrophs)的一个分支,于 1906 年首次被发现[5],直到 20
世纪 70年代,科学家才对其进行了广泛的分离和鉴定,使得详细的系统分类和生理研究得以进行[22]。 迄今
为止,已经发现了多种不同的甲烷氧化菌,并确定它们分别属于 3 个门:变形菌门(Proteobacteria)、疣微菌门
(Verrucomicrobia) [23]和 NC10门[24]。 其中传统分类中的甲烷氧化菌都属于变形菌门,它们广泛分布在自然
和人工生境中。 疣微菌门的甲烷氧化菌迄今只分离到了 3 个菌株,都是从极端嗜酸嗜热环境中得到的,其中
1854摇 14期 摇 摇 摇 邓永翠摇 等:好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展 摇
http: / / www.ecologica.cn
Acidimethylosilex fumarolicum来自意大利南部火山附近的沼泽土壤[25],Methylokorus infernorum 分离自新西兰
的一个地热井[23],Methyloacida kamchatkensis 是从俄罗斯的酸性温泉中分离到的[26],它们的最适生长温度都
在 55 益左右,并且能够在 65 益下生长,被统一归为一个新属———Methylacidiphilum属[27]。 NC10 门甲烷氧化
菌的代表菌株为 Methylomirabilis oxyfera,能够在厌氧环境中同时进行甲烷氧化和反硝化作用[24,28],并产生其
氧化甲烷所需要的氧气[29]。 另外,根据利用甲烷时是否需要氧气的存在,可把甲烷氧化菌分为好氧甲烷氧化
菌和厌氧甲烷氧化菌两类,本文以好氧甲烷氧化菌为对象,根据 16S rRNA基因,构建了现阶段已发现的所有
好氧甲烷氧化菌属的系统进化树(图 1),清楚地表明了各好氧甲烷氧化菌分支之间的关系。
图 1摇 利用 21个好氧甲烷氧化菌属的 16S rRNA基因构建的系统进化树
Fig.1摇 Neighbor joining phylogenetic tree of 16S rRNA gene of the current methanotrophic genera. All the sequences were from the NCBI
database and 1387 bp sequences were used to build the tree
全部 16S rRNA基因序列来自 NCBI数据库,在 ARB中用 Neighbor鄄Joining方法构建系统发育树,选择序列长度为 1387 bp
在甲烷氧化菌的 3个门中,目前发现只有变形菌门的甲烷氧化菌存在于自然湿地中,而疣微菌门和 NC10
门的甲烷氧化菌在自然湿地中仍未检测到[30]。 变形菌门的甲烷氧化菌可分为玉型甲烷氧化菌和域型甲烷氧
化菌,其中玉型甲烷氧化菌属于 酌鄄变形菌纲的Methylococcaceae科,现已发现了 15个属(表 2)。 玉型又进一步
分为玉a 型 (如 Methylobacter、Methylomicrobium、Methylomonas 和 Methylosarcina)和玉b 型 (Methylococcus 和
Methylocaldum),玉b 型也就是之前命名为 X 型的甲烷氧化菌[15]。 最近研究发现从海洋中分离到的
Methylomarinum属[31]、从森林土壤中分离的 Methylovulum属[32]以及水稻田中分离的 Methylogaea属[33]都属于
玉型甲烷氧化菌。 对存在于环境中尚不能纯培养的甲烷氧化菌,利用分子生物学手段检测其功能基因,显示
一些包含部分特定功能基因序列的甲烷氧化菌类群也可归于玉型甲烷氧化菌,如根据其提取环境而命名的
RPC(Rice paddy cluster)、FW(Fresh water)和 JRC(Japanese rice cluster)等[34]。 此外还发现两类丝状甲烷氧
化菌 Crenothrix polyspora[35]和 Clonothrix fusca[36]也是玉型甲烷氧化菌的一个独特分支。 域型甲烷氧化菌属于
琢-变形菌纲,包括 Methylocystaceae 和 Beijerinckiaceae 两个科,前者有 Methylocystis 和 Methylosinus 属,后者有
Methylocapsa、Methylocella和 Methyloferula属(表 2)。
玉型和域型甲烷氧化菌是根据其甲醛吸收和代谢途径、所含磷脂脂肪酸(Phospholipid Fatty Acid,PLFA)
的类型以及细胞膜结构的差异等进行区分的。 其中玉a 型甲烷氧化菌通过磷酸核酮糖途径(RuMP pathway)
同化甲醛;玉b型甲烷氧化菌既能通过 RuMP 途径,又同时包括低水平的丝氨酸途径(Serine pathway)同化甲
醛[6],而且其生长温度比玉a型和域型高[15];域型甲烷氧化菌通过丝氨酸途径同化甲醛[21]。 玉型甲烷氧化菌
2854 摇 生摇 态摇 学摇 报摇 摇 摇 35卷摇
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3854摇 14期 摇 摇 摇 邓永翠摇 等:好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展 摇
http: / / www.ecologica.cn
的优势脂肪酸是 14C和 16C,而域型甲烷氧化菌的优势脂肪酸是 18C[37],但也有一些甲烷氧化菌同时含有相
当比例的 16C和 18C两种脂肪酸,如:Methylocystis heyeri (琢-变形菌纲)、Methylohalobius crimeensis (酌鄄变形菌
纲)和 Methylothermus thermalis(酌鄄变形菌纲)同时含有玉型和域型甲烷氧化菌的标志性脂肪酸[38鄄40]。 甲烷氧
化菌的另一个分类特征是胞质内膜的排列方式(Intracytoplasmic membrane arrangments)不同。 玉型甲烷氧化
菌具有成束的分布于细胞质内的胞质内膜,如表 2鄄A 中的 Methylobacter tundripaludum[41],而域型甲烷氧化菌
Methylocystaceae科的 Methylocystis 和 Methylosinus 属的胞质内膜平行地延伸在细胞壁的周围(表 2鄄B) [17]。
Beijerinckiaceae科与 Methylocystaceae科具有不同的胞质内膜结构,其中 Methylocapsa属的胞质内膜平行分布于
长轴细胞膜的一侧上[42] (表 2鄄C);Methylocella 属的胞质内膜是由细胞质膜内陷形成的[43] (表 2鄄D);而
Methyloferula没有发现胞质内膜结构[44]。
甲烷氧化菌利用甲烷的方式如下:首先由甲烷单加氧酶(MMO)将甲烷活化生成甲醇,再氧化为甲醛;然
后通过丝氨酸途径或单磷酸核酮糖途径同化为细胞生物量,或者在氧化为甲酸后转变为二氧化碳。 甲烷单加
氧酶在这些过程中起关键性的作用,该酶存在两种形式:与膜结合、含有铜离子和铁离子的颗粒状甲烷单加氧
酶(pMMO)和分泌在周质空间中的可溶性甲烷单加氧酶( sMMO)。 在现已发现的好氧甲烷氧化菌中,除
Methylocella和 Methyloferla以外,都含有 pMMO[44鄄45],而只在一些域型甲烷氧化菌(如 Methylosinus sp.[15])和几
种玉型甲烷氧化菌(如 Methylomonas sp.和 Methylomicrobium sp.)中能检测到编码 sMMO的基因[46]。 细胞中铜
离子的浓度可以在转录水平上调节这两种单加氧酶的表达,当铜离子浓度小于 0.8 滋mol / L时, sMMO可以表
达,当铜离子浓度大于 4 滋mol / L时,sMMO停止表达,只有 pMMO表达,高浓度的铜离子浓度可以抑制 sMMO
基因的转录,而铜离子浓度的升高可以促进 pMMO 基因的转录。 另外,铜离子是合成 pMMO 必须的金属元
素。 除了氧化甲烷外,pMMO还能氧化 5个碳以内的一些短链化合物,sMMO则有更广泛的底物利用能力,能
氧化种类多样的烷、烯和芳香族化合物[6]。
尽管 16S rRNA基因是当今微生物生态研究中最普遍使用的标记基因,但是在研究具有特定功能的微生
物类群时,需要采用更为专一的编码关键酶的基因(如 pmoA基因和 mmoX 基因)替代 16S rRNA 基因。 pmoA
基因几乎存在于所有的甲烷氧化菌中,它编码关键酶 pMMO的一个亚基,且基于 pmoA 基因和基于 16S rRNA
基因的甲烷氧化菌的系统发育关系有着很好的一致性,因此 pmoA 基因已经成为甲烷氧化菌生态学研究中广
为采用的生物标记物[47]。 相对于 pmoA基因,编码 sMMO的 mmoX 基因仅存在于少数种类的甲烷氧化菌中,
对其研究也相对较少,近期在酸性泥炭湿地中的研究检测到了 mmoX基因的表达[48],该基因在甲烷氧化菌研
究中的应用开始受到更多的关注。
甲烷氧化菌对甲烷浓度需求的研究近年来取得了长足的进展,早期分离到的甲烷氧化菌都是对甲烷低亲
和力的菌株,因此,在 Baani等发现 Methylocystis sp. SC2 菌除了含有 pMMO 外,还含有第 2 种甲烷单加氧酶
(pMMO2)之前[51],人们一直认为甲烷氧化菌只能在高浓度甲烷下生存。 甲烷氧化菌可在 pMMO2 酶的作用
下氧化利用痕量的大气甲烷,而编码 pMMO2的 pmoA2基因在域型甲烷氧化菌 Methylocystis 和 Methylosinus 属
中普遍存在,但在玉型甲烷氧化菌中还没有发现[52]。 上述研究结果表明大气中甲烷生物氧化的主要贡献者
之一是域型甲烷氧化菌,尤其是Methylocystis sp., 而之前一直认为山地和森林土壤中的一些未培养、高亲和力
的甲烷氧化菌 USC琢和 USC酌是吸收大气中低浓度甲烷的主要汇[53]。 同时拥有对不同浓度甲烷源的可利用
性和兼性营养的特性是 Methylocystis属甲烷氧化菌在山地、森林[53]以及其它高浓度甲烷环境中广泛分布的主
要原因之一。 pMMO2的发现是对甲烷氧化菌研究的重要进展,对研究低甲烷浓度环境中的甲烷氧化菌有重
要的指导作用,在这些环境中含有该酶的甲烷氧化菌可能占据优势。
对甲烷氧化菌底物专一性的研究也有新进展,Dedysh 等发现从西伯利亚酸性泥炭湿地中分离到的 3 株
域型甲烷氧化菌 Methylocella palustris, Methylocella silvestris和 Methylocella tundrae[54]既能把一碳化合物甲烷和
甲醇作为其唯一的碳源和能源,又能利用多碳化合物,如有机酸(乙酸,丙酮酸,琥珀酸和苹果酸)和乙醇,作
为其生长的唯一底物,证明了兼性营养型甲烷氧化菌的存在。 属于 酌鄄变形菌纲的新丝状甲烷氧化菌
4854 摇 生摇 态摇 学摇 报摇 摇 摇 35卷摇
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Crenothrix polyspora在有甲烷存在的情况下能够利用乙酸,并且能少量吸收葡萄糖,表明该菌也是兼性营养型
甲烷氧化菌[35]。 但是另一种新近发现的丝状甲烷氧化菌 Clonothrix fusca 不能利用葡萄糖,该菌的 16S rRNA
基因序列与 C. polyspora有密切的亲缘关系[36],这类甲烷氧化菌是否具有兼性营养的特性还需要更多的实验
验证。 近期还发现 Methylocapsa属和 Methylocystis属中的一些含有 pMMO的甲烷氧化菌能利用乙酸作为唯一
碳源[55鄄56]。 这些研究结果显示甲烷氧化菌的底物利用能力并非和之前认为的那样单一,可能有更宽泛的底
物类型。 Semrau曾详细回顾了兼性营养型甲烷氧化菌的发现历史,并指出了今后可能的发展方向[57]。
Dedysh对如何分离和鉴定兼性营养型甲烷氧化菌提出了很多指导和建议[58]。
对极端环境中的甲烷氧化菌的研究也取得了令人瞩目的进展。 已发现的耐热或中度嗜热的甲烷氧化菌
主要属于 酌鄄变形菌纲的玉型甲烷氧化菌,以及新发现的疣微菌门的 3 个菌株。 在玉型甲烷氧化菌
Methylocaldum、Methylococcus和 Methylothermus属中均发现有耐高温或嗜高温的甲烷氧化菌菌株[22,39,59],其中
Methylocaldum tepidum和 Methylocaldum gracile 在 47 益下可以存活,其最适生长温度为 42 益;Methylocaldum
szegendiense的最适生长温度为 55 益 [59];从日本一个温泉里分离到的 Methylothermus thermalis 的最适生长温
度达 57—59 益 [39]。 另一方面,也有一些甲烷氧化菌适应寒冷的环境[60],如 Methylobacter属,无论是分离到的
纯菌的最适生长温度[41]还是其大量存在于各种寒冷生境中(如:西伯利亚北极永冻土[61]和高海拔湿地[62]
等)都表明它们适宜在低温环境中生存。 从瑞典一处地下水中分离到的 Methylomonas scandinavica[63]和从南
极对流湖中分离到的 Methylosphaera hansonii[64]也都属于嗜冷菌。 在域型甲烷氧化菌的 Methylocapsa 属[42]和
Methylocella属[43,45,65]中也有能在低温下生长的菌株。 这些都表明在甲烷氧化菌的多个种属中比较广泛地分
布有嗜冷或耐冷菌。
关于嗜酸性甲烷氧化菌的研究主要集中在北半球的酸性(pH 3.5—5.5)泥炭藓湿地,域型甲烷氧化菌
Methylocystis、Methylocella和 Methylocapsa属[56]是这类湿地的主要类群,它们都能在低 pH 环境下生长[18]。 焦
磷酸深度测序分析发现青藏高原的日干乔酸性泥炭湿地同样存在大量的 Methylocystis[66]。 在酸性泥炭湿地
中也检测到了玉型甲烷氧化菌 Methylomomas[67]。 新的嗜盐碱甲烷氧化菌也不断被分离到。 生存于俄罗斯高
盐碱性湖泊中的 Methylomicrobium 属甲烷氧化菌的最适生长 pH 为 9.0—9.5[68鄄69],另一株分离到的嗜盐菌
Methylohalobius crimeensis能在 2.5 mol / L NaCl下存活,其最适生长盐度为 1—1.5 mol / L NaCl[38]。 分子方法检
测也证明在一些高盐碱湖泊(如:Mono 湖和 Transbaikal 湖)中有很多耐盐碱性或抗盐碱的甲烷氧化菌生
存[70鄄71]。 与此相对的是,Methylocella和 Methylocapsa属的甲烷氧化菌只适宜在低盐浓度下生长[45,56],尤其是
Methylocapsa KYG菌对盐浓度非常敏感,0.1% NaCl就可以抑制其生长速率的 90%,0.2%—0.3% NaCl 完全抑
制其生长[56]。
2摇 自然湿地甲烷氧化菌的研究进展
近年来,利用分子检测方法或者纯培养方法在全球自然湿地中开展了甲烷氧化菌的深入研究,总体情况
见表 3。
第一个用分子生物学方法研究湿地甲烷氧化菌的是英国 Murrell小组的 McDonald及其同事[72鄄73],他们使
用 16S rRNA和 pmoA基因构建克隆文库,发现域型甲烷氧化菌,尤其是 Methylocystis及 Methylosinus,是酸性泥
炭湿地主要的甲烷氧化菌。 之后,该组的 Chen 等在酸性泥炭湿地甲烷氧化菌多样性方面的研究工作突出。
他们使用 DNA稳定性同位素与微阵列的方法,或者通过检测 pmoA基因表达以及构建 mmoX 基因文库,进一
步证实域型甲烷氧化菌,特别是其中的 Methylocystis属,在酸性泥炭湿地的甲烷氧化菌中的优势地位[74鄄75],另
外 Methylocella和 Methylocapsa也是泥炭湿地常见的类群[75]。 虽然酸性泥炭湿地是以域型甲烷氧化菌为主
的,但是在 Moorhouse自然保护区湿地中也发现有玉型甲烷氧化菌(如 Methylobacter)存在[74]。
位于俄罗斯西伯利亚地区的酸性 ( pH 3. 5—5. 5)泥炭藓湿地是另一处甲烷氧化菌研究很多的地
点[18,76鄄78]。 该湿地主要的甲烷氧化菌属于 琢鄄变形菌纲,甲烷氧化菌的数量介于 106—108个 / g 湿土之
5854摇 14期 摇 摇 摇 邓永翠摇 等:好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展 摇
http: / / www.ecologica.cn
间[18,76鄄78]。 在该湿地上还开展了大量的甲烷氧化菌分离培养的工作,已经分离得到了 Methylocystis、
Methylocella和 Methylocapsa三个属的甲烷氧化菌菌株,这些也是该酸性泥炭湿地的主要甲烷氧化菌类
群[42,45,54鄄56,79],它们都可以在 pH<6的环境下生长[18]。
除了英国和俄罗斯之外,在欧洲大陆的西班牙、芬兰、荷兰、德国以及挪威的北极地区都有对甲烷氧化菌
群落结构的研究。 这些研究大部分是在泥炭藓湿地上开展的,只有少数关注到森林泥炭湿地和河流湿地土壤
中的甲烷氧化菌。 研究表明位于寒冷地区的挪威和芬兰湿地玉型甲烷氧化菌比例更高[11,80],尤其是
Methylobacter,在北极湿地中用变形梯度凝胶电泳(DGGE)和稳定性同位素探针(SIP)的方法都检测到该菌的
存在[11,81],当然在这些研究地点还同时检测到了域型甲烷氧化菌[48,80鄄83]。 另外,Kip 从荷兰泥炭藓湿地植物
中分离到了一株 Methylomomas属的甲烷氧化菌,这是第一株嗜酸的 酌鄄变形菌纲的甲烷氧化菌[67],在这之前,
从没有在酸性泥炭湿地中分离到属于 酌鄄变形菌纲的甲烷氧化菌。
除欧洲大陆以外,在美国和日本的自然湿地中也开展了甲烷氧化菌的研究,研究集中在泥炭湿地上,植被
类型除泥炭藓外,还包括莎草科植被和森林湿地。 与英国和俄罗斯的泥炭藓湿地以域型甲烷氧化菌为主不
同,在这些地区玉型和域型甲烷氧化菌都广泛存在,有的样地主要是玉型甲烷氧化菌,有的样地域型甲烷氧化
菌占优。 直到 2012年,在自然湿地上开展的甲烷氧化菌群落多样性的所有研究都集中在北半球,该年 Kip 小
组将这项工作推进到了南半球,他们用分子生物学方法,研究发现高丰度的 Methylocystis属甲烷氧化菌可能是
南半球泥炭藓湿地甲烷氧化的主要贡献者[84]。
我国幅员辽阔,自然湿地类型多样。 但对我国自然湿地甲烷氧化菌的研究一直是空白,直到 2010 年才在
位于青藏高原的若尔盖湿地开展了初步的研究[62]。 青藏高原作为全球海拔最高的一个独特地域,其湿地众
多,面积约为 13.3伊l04 km2,每年从青藏高原湿地排放的甲烷约为 0.56—1 Tg[85鄄86],除了若尔盖湿地之外,青藏
高原的其它自然湿地也是甲烷排放的主要源。 近期在位于红原的日干乔湿地对甲烷氧化菌群落多样性和活
性甲烷氧化菌类进行了较系统的研究[66]。 另外,在位于松嫩平原的向海湿地上也有关于甲烷氧化菌的丰度
和多样性随土壤深度变化的研究发表[87]。
3摇 结论与展望
随着分子生物学和分离培养技术的发展,对甲烷氧化菌的认识不断深入。 研究表明甲烷氧化菌的多样性
比之前想象的要多,但是大部分环境中主要的好氧甲烷氧化菌属于变形菌门,可根据其生理特性分为玉型和
域型两类甲烷氧化菌。 甲烷氧化菌可以利用 pMMO 和 sMMO 两种酶进行氧化甲烷的第一步反应。 pMMO2
的发现表明域型甲烷氧化菌中有些类群可以利用这种酶氧化低浓度的甲烷。 新研究证实甲烷氧化菌可以利
用的底物比之前认为的更广泛,包括乙酸等含有碳碳键的化合物。 另外,从极端环境中发现的各种嗜冷、嗜
热、嗜酸、嗜碱和嗜盐的甲烷氧化菌表明甲烷氧化菌广泛分布在各种生态环境中。
目前对甲烷氧化菌多样性的研究主要集中在酸性泥炭湿地,尤其是位于北半球高纬度的酸性泥炭藓湿
地。 研究表明域型甲烷氧化菌 Methylocystis、Methylocella 和 Methylocapsa 是这类湿地主要的甲烷氧化菌类群,
尤其是 Methylocystis,广泛分布在酸性泥炭湿地中。 在北极寒冷的湿地中,玉型甲烷氧化菌占优势,其中
Methylobacter大量存在。 我国对自然湿地甲烷氧化菌的研究还处于起步阶段,但也取得了一些有价值的研究
结果。
随着高通量测序时代的到来和纯培养技术的不断发展,更多的在极端环境中生存的甲烷氧化菌会被发
现,将不断完善人们对甲烷氧化菌生理和生态适应性的认识。 同时,新的甲烷氧化菌类群的不断发现,会挑战
现有甲烷氧化菌的分类方法;特别是随着海量序列数据的出现,必将发现更多新的甲烷氧化菌序列和新的甲
烷氧化菌菌株,整个分类系统是否需要做较大幅度的修改还属未知。 另外,先进的分子生物学技术也给甲烷
氧化菌的生物地理学研究提供了有力的工具,随着在全球更多生态环境中对甲烷氧化菌研究的积累,其群落
结构及其多样性特征是否存在有规律的地理格局等重要生物地理学问题的答案将变得逐渐清晰。
6854 摇 生摇 态摇 学摇 报摇 摇 摇 35卷摇
http: / / www.ecologica.cn
表
3摇
自
然
湿
地
甲
烷
氧
化
菌
多
样
性
研
究
概
况
Ta
bl
e
3摇
Su
m
m
ar
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of
th
e
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on
m
et
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no
tr
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di
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ity
in
na
tu
ra
lw
et
la
nd
s
研
究
地
点
Re
sea
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sit
e
国
家
Co
un
ty
湿
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类
型
We
tla
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typ
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植
被
Ve
get
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实
验
方
法
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tho
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化
菌
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型
Me
tha
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tro
ph
ic
typ
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文
献
Re
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3]
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[8
4]
7854摇 14期 摇 摇 摇 邓永翠摇 等:好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展 摇
http: / / www.ecologica.cn
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