好氧甲烷氧化菌生理生態(tài)特征及其在自然濕地中的群落多樣性研究進(jìn)展

鄧永翠, 車(chē)榮曉, 吳伊波, 王艷芬, 崔驍勇,*

1 中國(guó)科學(xué)院大學(xué), 北京 100049 2 南京師范大學(xué)虛擬地理環(huán)境教育部重點(diǎn)實(shí)驗(yàn)室, 南京 210046 3 寧波大學(xué), 寧波 315211

好氧甲烷氧化菌生理生態(tài)特征及其在自然濕地中的群落多樣性研究進(jìn)展

鄧永翠1,2, 車(chē)榮曉1, 吳伊波3, 王艷芬1, 崔驍勇1,*

1 中國(guó)科學(xué)院大學(xué), 北京 100049 2 南京師范大學(xué)虛擬地理環(huán)境教育部重點(diǎn)實(shí)驗(yàn)室, 南京 210046 3 寧波大學(xué), 寧波 315211

甲烷氧化菌是一類(lèi)可以利用甲烷作為唯一碳源和能源的細(xì)菌，在全球變化和整個(gè)生態(tài)系統(tǒng)碳循環(huán)過(guò)程中起著重要的作用。近年來(lái)，對(duì)甲烷氧化菌的生理生態(tài)特征及其在自然濕地中的群落多樣性研究取得了較大進(jìn)展。在分類(lèi)方面，疣微菌門(mén)、NC10門(mén)及兩個(gè)絲狀菌屬甲烷氧化菌的發(fā)現(xiàn)使其分類(lèi)體系得到了進(jìn)一步的完善；在單加氧酶方面，發(fā)現(xiàn)甲烷氧化菌可以利用pMMO和sMMO兩種酶進(jìn)行氧化甲烷的第一步反應(yīng)，Ⅱ型甲烷氧化菌中pMMO2的發(fā)現(xiàn)證實(shí)甲烷氧化菌可以利用這種酶氧化低濃度的甲烷；在底物利用方面，已經(jīng)發(fā)現(xiàn)了越來(lái)越多的兼性營(yíng)養(yǎng)型甲烷氧化菌，證實(shí)它們可以利用的底物比之前認(rèn)為的更廣泛，其中包括乙酸等含有碳碳鍵的化合物；在生存環(huán)境方面，能在不同溫度、酸度和鹽度的環(huán)境中生存的甲烷氧化菌不斷被分離出來(lái)。全球自然濕地甲烷氧化菌群落多樣性的研究目前主要集中在北半球高緯度的酸性泥炭濕地，Ⅱ型甲烷氧化菌Methylocystis、Methylocella和Methylocapsa是這類(lèi)濕地主要的甲烷氧化菌類(lèi)群，尤其以Methylocystis類(lèi)群最為廣泛，而Ⅰ型甲烷氧化菌尤其是Methylobacter在北極寒冷濕地中占優(yōu)勢(shì)。隨著高通量測(cè)序時(shí)代的到來(lái)和新的分離技術(shù)的發(fā)展，對(duì)甲烷氧化菌的現(xiàn)有認(rèn)識(shí)將面臨更多的挑戰(zhàn)和發(fā)展。

甲烷氧化菌; 甲烷; 甲烷單加氧酶; 濕地

甲烷是大氣中輻射強(qiáng)迫僅次于二氧化碳的溫室氣體，大氣中甲烷含量?jī)H為二氧化碳的1/27，但單分子增溫效應(yīng)是二氧化碳的25倍，對(duì)全球溫室效應(yīng)的貢獻(xiàn)率可達(dá)18%[1]。甲烷在大氣中的濃度已經(jīng)從工業(yè)化以前的體數(shù)分?jǐn)?shù)為715×10-8增長(zhǎng)到現(xiàn)在的體數(shù)分?jǐn)?shù)為1770×10-8，這種增長(zhǎng)是由其源匯平衡決定的。全球大氣甲烷排放量約為500—600 Tg/a，其中自然濕地貢獻(xiàn)總甲烷源的23%[2]，是最大的大氣甲烷源。自然濕地中厭氧土層產(chǎn)甲烷菌產(chǎn)生的甲烷大部分并沒(méi)有直接進(jìn)入大氣，約90%的甲烷是通過(guò)濕地有氧土層的甲烷氧化菌消耗掉[3-4]。土壤里甲烷氧化菌的這種氧化作用是甲烷的唯一生物匯。甲烷氧化菌(Methanotrophs)在1906年首次被發(fā)現(xiàn)[5]，它是甲基氧化菌(Methylotrophs)的一個(gè)分支，能利用甲烷(Methane，CH4)作為其唯一的碳源和能源[6]。甲烷氧化菌在全球變化和整個(gè)生態(tài)系統(tǒng)碳循環(huán)過(guò)程中起著非常重要的作用。現(xiàn)已有研究證實(shí)在各種生態(tài)環(huán)境中(湖泊底泥[7]，水稻田[8]，垃圾填埋場(chǎng)[9]，泥炭濕地[10]及北極高緯度濕地[11]等)分布的甲烷氧化菌在消耗甲烷中起著重要的作用。

甲烷氧化菌的研究一直廣受關(guān)注，在2009年之前，國(guó)際上幾乎每4年就有一篇總結(jié)甲烷氧化菌研究進(jìn)展的優(yōu)秀綜述發(fā)表，而在2009—2010年4篇綜述論文刊載(表1)。這些綜述主要關(guān)注甲烷氧化菌的分類(lèi)、生理、生態(tài)分布、分離培養(yǎng)、應(yīng)用、甲烷單加氧酶(Methane Monooxygenase，MMO)的催化機(jī)理等方面的研究和所用的研究技術(shù)等，但是缺少對(duì)甲烷氧化菌分布的重要生態(tài)系統(tǒng)——全球自然 濕地的詳細(xì)綜述；另外，關(guān)于近兩年新發(fā)現(xiàn)的甲烷氧化菌的相關(guān)研究也還沒(méi)有歸納在內(nèi)?；诖?，本文著重分析了全球自然濕地中好氧甲烷氧化菌多樣性研究的現(xiàn)狀，綜述了好氧甲烷氧化菌的種類(lèi)、氧化甲烷的關(guān)鍵酶及底物特征方面的新進(jìn)展，并展望濕地甲烷氧化菌研究的發(fā)展趨勢(shì)。

表1 國(guó)內(nèi)外重要的甲烷氧化菌研究綜述

1 甲烷氧化菌的分類(lèi)及其生理生態(tài)特征研究進(jìn)展

甲烷氧化菌(Methanotrophs)是甲基氧化菌(Methylotrophs)的一個(gè)分支，于1906年首次被發(fā)現(xiàn)[5]，直到20世紀(jì)70年代，科學(xué)家才對(duì)其進(jìn)行了廣泛的分離和鑒定，使得詳細(xì)的系統(tǒng)分類(lèi)和生理研究得以進(jìn)行[22]。迄今為止，已經(jīng)發(fā)現(xiàn)了多種不同的甲烷氧化菌，并確定它們分別屬于3個(gè)門(mén)：變形菌門(mén)(Proteobacteria)、疣微菌門(mén)(Verrucomicrobia)[23]和NC10門(mén)[24]。其中傳統(tǒng)分類(lèi)中的甲烷氧化菌都屬于變形菌門(mén)，它們廣泛分布在自然和人工生境中。疣微菌門(mén)的甲烷氧化菌迄今只分離到了3個(gè)菌株，都是從極端嗜酸嗜熱環(huán)境中得到的，其中Acidimethylosilexfumarolicum來(lái)自意大利南部火山附近的沼澤土壤[25]，Methylokorusinfernorum分離自新西蘭的一個(gè)地?zé)峋甗23]，Methyloacidakamchatkensis是從俄羅斯的酸性溫泉中分離到的[26]，它們的最適生長(zhǎng)溫度都在55 ℃左右，并且能夠在65 ℃下生長(zhǎng)，被統(tǒng)一歸為一個(gè)新屬——Methylacidiphilum屬[27]。NC10門(mén)甲烷氧化菌的代表菌株為Methylomirabilisoxyfera，能夠在厭氧環(huán)境中同時(shí)進(jìn)行甲烷氧化和反硝化作用[24,28]，并產(chǎn)生其氧化甲烷所需要的氧氣[29]。另外，根據(jù)利用甲烷時(shí)是否需要氧氣的存在，可把甲烷氧化菌分為好氧甲烷氧化菌和厭氧甲烷氧化菌兩類(lèi)，本文以好氧甲烷氧化菌為對(duì)象，根據(jù)16S rRNA基因，構(gòu)建了現(xiàn)階段已發(fā)現(xiàn)的所有好氧甲烷氧化菌屬的系統(tǒng)進(jìn)化樹(shù)(圖1)，清楚地表明了各好氧甲烷氧化菌分支之間的關(guān)系。

圖1 利用21個(gè)好氧甲烷氧化菌屬的16S rRNA基因構(gòu)建的系統(tǒng)進(jìn)化樹(shù)

在甲烷氧化菌的3個(gè)門(mén)中，目前發(fā)現(xiàn)只有變形菌門(mén)的甲烷氧化菌存在于自然濕地中，而疣微菌門(mén)和NC10門(mén)的甲烷氧化菌在自然濕地中仍未檢測(cè)到[30]。變形菌門(mén)的甲烷氧化菌可分為Ⅰ型甲烷氧化菌和Ⅱ型甲烷氧化菌，其中Ⅰ型甲烷氧化菌屬于γ-變形菌綱的Methylococcaceae科，現(xiàn)已發(fā)現(xiàn)了15個(gè)屬(表2)。Ⅰ型又進(jìn)一步分為Ⅰa型(如Methylobacter、Methylomicrobium、Methylomonas和Methylosarcina)和Ⅰb型(Methylococcus和Methylocaldum)，Ⅰb型也就是之前命名為X型的甲烷氧化菌[15]。最近研究發(fā)現(xiàn)從海洋中分離到的Methylomarinum屬[31]、從森林土壤中分離的Methylovulum屬[32]以及水稻田中分離的Methylogaea屬[33]都屬于Ⅰ型甲烷氧化菌。對(duì)存在于環(huán)境中尚不能純培養(yǎng)的甲烷氧化菌，利用分子生物學(xué)手段檢測(cè)其功能基因，顯示一些包含部分特定功能基因序列的甲烷氧化菌類(lèi)群也可歸于Ⅰ型甲烷氧化菌，如根據(jù)其提取環(huán)境而命名的RPC(Rice paddy cluster)、FW(Fresh water)和JRC(Japanese rice cluster)等[34]。此外還發(fā)現(xiàn)兩類(lèi)絲狀甲烷氧化菌Crenothrixpolyspora[35]和Clonothrixfusca[36]也是Ⅰ型甲烷氧化菌的一個(gè)獨(dú)特分支。Ⅱ型甲烷氧化菌屬于α-變形菌綱，包括Methylocystaceae和Beijerinckiaceae兩個(gè)科，前者有Methylocystis和Methylosinus屬，后者有Methylocapsa、Methylocella和Methyloferula屬(表2)。

Ⅰ型和Ⅱ型甲烷氧化菌是根據(jù)其甲醛吸收和代謝途徑、所含磷脂脂肪酸(Phospholipid Fatty Acid，PLFA)的類(lèi)型以及細(xì)胞膜結(jié)構(gòu)的差異等進(jìn)行區(qū)分的。其中Ⅰa型甲烷氧化菌通過(guò)磷酸核酮糖途徑(RuMP pathway)同化甲醛；Ⅰb型甲烷氧化菌既能通過(guò)RuMP途徑，又同時(shí)包括低水平的絲氨酸途徑(Serine pathway)同化甲醛[6]，而且其生長(zhǎng)溫度比Ⅰa型和Ⅱ型高[15]；Ⅱ型甲烷氧化菌通過(guò)絲氨酸途徑同化甲醛[21]。Ⅰ型甲烷氧化菌的優(yōu)勢(shì)脂肪酸是14C和16C，而Ⅱ型甲烷氧化菌的優(yōu)勢(shì)脂肪酸是18C[37]，但也有一些甲烷氧化菌同時(shí)含有相當(dāng)比例的16C和18C兩種脂肪酸，如：Methylocystisheyeri(α-變形菌綱)、Methylohalobiuscrimeensis(γ-變形菌綱)和Methylothermusthermalis(γ-變形菌綱)同時(shí)含有Ⅰ型和Ⅱ型甲烷氧化菌的標(biāo)志性脂肪酸[38-40]。甲烷氧化菌的另一個(gè)分類(lèi)特征是胞質(zhì)內(nèi)膜的排列方式(Intracytoplasmic membrane arrangments)不同。Ⅰ型甲烷氧化菌具有成束的分布于細(xì)胞質(zhì)內(nèi)的胞質(zhì)內(nèi)膜，如表2-A中的Methylobactertundripaludum[41]，而Ⅱ型甲烷氧化菌Methylocystaceae科的Methylocystis和Methylosinus屬的胞質(zhì)內(nèi)膜平行地延伸在細(xì)胞壁的周?chē)?表2-B)[17]。Beijerinckiaceae科與Methylocystaceae科具有不同的胞質(zhì)內(nèi)膜結(jié)構(gòu)，其中Methylocapsa屬的胞質(zhì)內(nèi)膜平行分布于長(zhǎng)軸細(xì)胞膜的一側(cè)上[42](表2-C)；Methylocella屬的胞質(zhì)內(nèi)膜是由細(xì)胞質(zhì)膜內(nèi)陷形成的[43](表2-D)；而Methyloferula沒(méi)有發(fā)現(xiàn)胞質(zhì)內(nèi)膜結(jié)構(gòu)[44]。

表2 好氧甲烷氧化菌的分類(lèi)及其特征(根據(jù)Lüke[49]和Reim[50]改進(jìn)，其中加粗的菌屬代表兼性營(yíng)養(yǎng)型甲烷氧化菌)

甲烷氧化菌利用甲烷的方式如下：首先由甲烷單加氧酶(MMO)將甲烷活化生成甲醇，再氧化為甲醛；然后通過(guò)絲氨酸途徑或單磷酸核酮糖途徑同化為細(xì)胞生物量，或者在氧化為甲酸后轉(zhuǎn)變?yōu)槎趸?。甲烷單加氧酶在這些過(guò)程中起關(guān)鍵性的作用，該酶存在兩種形式：與膜結(jié)合、含有銅離子和鐵離子的顆粒狀甲烷單加氧酶(pMMO)和分泌在周質(zhì)空間中的可溶性甲烷單加氧酶(sMMO)。在現(xiàn)已發(fā)現(xiàn)的好氧甲烷氧化菌中，除Methylocella和Methyloferla以外，都含有pMMO[44-45]，而只在一些Ⅱ型甲烷氧化菌(如Methylosinussp.[15])和幾種Ⅰ型甲烷氧化菌(如Methylomonassp.和Methylomicrobiumsp.)中能檢測(cè)到編碼sMMO的基因[46]。細(xì)胞中銅離子的濃度可以在轉(zhuǎn)錄水平上調(diào)節(jié)這兩種單加氧酶的表達(dá)，當(dāng)銅離子濃度小于0.8 μmol/L時(shí), sMMO可以表達(dá)，當(dāng)銅離子濃度大于4 μmol/L時(shí)，sMMO停止表達(dá)，只有pMMO表達(dá)，高濃度的銅離子濃度可以抑制sMMO基因的轉(zhuǎn)錄，而銅離子濃度的升高可以促進(jìn)pMMO基因的轉(zhuǎn)錄。另外，銅離子是合成pMMO必須的金屬元素。除了氧化甲烷外，pMMO還能氧化5個(gè)碳以內(nèi)的一些短鏈化合物，sMMO則有更廣泛的底物利用能力，能氧化種類(lèi)多樣的烷、烯和芳香族化合物[6]。

盡管16S rRNA基因是當(dāng)今微生物生態(tài)研究中最普遍使用的標(biāo)記基因，但是在研究具有特定功能的微生物類(lèi)群時(shí)，需要采用更為專(zhuān)一的編碼關(guān)鍵酶的基因(如pmoA基因和mmoX基因)替代16S rRNA基因。pmoA基因幾乎存在于所有的甲烷氧化菌中，它編碼關(guān)鍵酶pMMO的一個(gè)亞基，且基于pmoA基因和基于16S rRNA基因的甲烷氧化菌的系統(tǒng)發(fā)育關(guān)系有著很好的一致性，因此pmoA基因已經(jīng)成為甲烷氧化菌生態(tài)學(xué)研究中廣為采用的生物標(biāo)記物[47]。相對(duì)于pmoA基因，編碼sMMO的mmoX基因僅存在于少數(shù)種類(lèi)的甲烷氧化菌中，對(duì)其研究也相對(duì)較少，近期在酸性泥炭濕地中的研究檢測(cè)到了mmoX基因的表達(dá)[48]，該基因在甲烷氧化菌研究中的應(yīng)用開(kāi)始受到更多的關(guān)注。

甲烷氧化菌對(duì)甲烷濃度需求的研究近年來(lái)取得了長(zhǎng)足的進(jìn)展，早期分離到的甲烷氧化菌都是對(duì)甲烷低親和力的菌株，因此，在Baani等發(fā)現(xiàn)Methylocystissp. SC2菌除了含有pMMO外，還含有第2種甲烷單加氧酶(pMMO2)之前[51]，人們一直認(rèn)為甲烷氧化菌只能在高濃度甲烷下生存。甲烷氧化菌可在pMMO2酶的作用下氧化利用痕量的大氣甲烷，而編碼pMMO2的pmoA2基因在Ⅱ型甲烷氧化菌Methylocystis和Methylosinus屬中普遍存在，但在Ⅰ型甲烷氧化菌中還沒(méi)有發(fā)現(xiàn)[52]。上述研究結(jié)果表明大氣中甲烷生物氧化的主要貢獻(xiàn)者之一是Ⅱ型甲烷氧化菌，尤其是Methylocystissp., 而之前一直認(rèn)為山地和森林土壤中的一些未培養(yǎng)、高親和力的甲烷氧化菌USCα和USCγ是吸收大氣中低濃度甲烷的主要匯[53]。同時(shí)擁有對(duì)不同濃度甲烷源的可利用性和兼性營(yíng)養(yǎng)的特性是Methylocystis屬甲烷氧化菌在山地、森林[53]以及其它高濃度甲烷環(huán)境中廣泛分布的主要原因之一。pMMO2的發(fā)現(xiàn)是對(duì)甲烷氧化菌研究的重要進(jìn)展，對(duì)研究低甲烷濃度環(huán)境中的甲烷氧化菌有重要的指導(dǎo)作用，在這些環(huán)境中含有該酶的甲烷氧化菌可能占據(jù)優(yōu)勢(shì)。

對(duì)甲烷氧化菌底物專(zhuān)一性的研究也有新進(jìn)展，Dedysh等發(fā)現(xiàn)從西伯利亞酸性泥炭濕地中分離到的3株Ⅱ型甲烷氧化菌Methylocellapalustris,Methylocellasilvestris和Methylocellatundrae[54]既能把一碳化合物甲烷和甲醇作為其唯一的碳源和能源，又能利用多碳化合物，如有機(jī)酸(乙酸，丙酮酸，琥珀酸和蘋(píng)果酸)和乙醇，作為其生長(zhǎng)的唯一底物，證明了兼性營(yíng)養(yǎng)型甲烷氧化菌的存在。屬于γ-變形菌綱的新絲狀甲烷氧化菌Crenothrixpolyspora在有甲烷存在的情況下能夠利用乙酸，并且能少量吸收葡萄糖，表明該菌也是兼性營(yíng)養(yǎng)型甲烷氧化菌[35]。但是另一種新近發(fā)現(xiàn)的絲狀甲烷氧化菌Clonothrixfusca不能利用葡萄糖，該菌的16S rRNA基因序列與C.polyspora有密切的親緣關(guān)系[36]，這類(lèi)甲烷氧化菌是否具有兼性營(yíng)養(yǎng)的特性還需要更多的實(shí)驗(yàn)驗(yàn)證。近期還發(fā)現(xiàn)Methylocapsa屬和Methylocystis屬中的一些含有pMMO的甲烷氧化菌能利用乙酸作為唯一碳源[55-56]。這些研究結(jié)果顯示甲烷氧化菌的底物利用能力并非和之前認(rèn)為的那樣單一，可能有更寬泛的底物類(lèi)型。Semrau曾詳細(xì)回顧了兼性營(yíng)養(yǎng)型甲烷氧化菌的發(fā)現(xiàn)歷史，并指出了今后可能的發(fā)展方向[57]。Dedysh對(duì)如何分離和鑒定兼性營(yíng)養(yǎng)型甲烷氧化菌提出了很多指導(dǎo)和建議[58]。

對(duì)極端環(huán)境中的甲烷氧化菌的研究也取得了令人矚目的進(jìn)展。已發(fā)現(xiàn)的耐熱或中度嗜熱的甲烷氧化菌主要屬于γ-變形菌綱的Ⅰ型甲烷氧化菌，以及新發(fā)現(xiàn)的疣微菌門(mén)的3個(gè)菌株。在Ⅰ型甲烷氧化菌Methylocaldum、Methylococcus和Methylothermus屬中均發(fā)現(xiàn)有耐高溫或嗜高溫的甲烷氧化菌菌株[22,39,59]，其中Methylocaldumtepidum和Methylocaldumgracile在47 ℃下可以存活，其最適生長(zhǎng)溫度為42 ℃；Methylocaldumszegendiense的最適生長(zhǎng)溫度為55 ℃[59]；從日本一個(gè)溫泉里分離到的Methylothermusthermalis的最適生長(zhǎng)溫度達(dá)57—59 ℃[39]。另一方面，也有一些甲烷氧化菌適應(yīng)寒冷的環(huán)境[60]，如Methylobacter屬，無(wú)論是分離到的純菌的最適生長(zhǎng)溫度[41]還是其大量存在于各種寒冷生境中(如：西伯利亞北極永凍土[61]和高海拔濕地[62]等)都表明它們適宜在低溫環(huán)境中生存。從瑞典一處地下水中分離到的Methylomonasscandinavica[63]和從南極對(duì)流湖中分離到的Methylosphaerahansonii[64]也都屬于嗜冷菌。在Ⅱ型甲烷氧化菌的Methylocapsa屬[42]和Methylocella屬[43,45,65]中也有能在低溫下生長(zhǎng)的菌株。這些都表明在甲烷氧化菌的多個(gè)種屬中比較廣泛地分布有嗜冷或耐冷菌。

關(guān)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)泥炭蘚濕地，Ⅱ型甲烷氧化菌Methylocystis、Methylocella和Methylocapsa屬[56]是這類(lèi)濕地的主要類(lèi)群，它們都能在低pH環(huán)境下生長(zhǎng)[18]。焦磷酸深度測(cè)序分析發(fā)現(xiàn)青藏高原的日干喬酸性泥炭濕地同樣存在大量的Methylocystis[66]。在酸性泥炭濕地中也檢測(cè)到了Ⅰ型甲烷氧化菌Methylomomas[67]。新的嗜鹽堿甲烷氧化菌也不斷被分離到。生存于俄羅斯高鹽堿性湖泊中的Methylomicrobium屬甲烷氧化菌的最適生長(zhǎng)pH為9.0—9.5[68-69]，另一株分離到的嗜鹽菌Methylohalobiuscrimeensis能在2.5 mol/L NaCl下存活，其最適生長(zhǎng)鹽度為1—1.5 mol/L NaCl[38]。分子方法檢測(cè)也證明在一些高鹽堿湖泊(如：Mono湖和Transbaikal湖)中有很多耐鹽堿性或抗鹽堿的甲烷氧化菌生存[70-71]。與此相對(duì)的是，Methylocella和Methylocapsa屬的甲烷氧化菌只適宜在低鹽濃度下生長(zhǎng)[45,56]，尤其是MethylocapsaKYG菌對(duì)鹽濃度非常敏感，0.1% NaCl就可以抑制其生長(zhǎng)速率的90%，0.2%—0.3% NaCl 完全抑制其生長(zhǎng)[56]。

2 自然濕地甲烷氧化菌的研究進(jìn)展

近年來(lái)，利用分子檢測(cè)方法或者純培養(yǎng)方法在全球自然濕地中開(kāi)展了甲烷氧化菌的深入研究，總體情況見(jiàn)表3。

第一個(gè)用分子生物學(xué)方法研究濕地甲烷氧化菌的是英國(guó)Murrell小組的McDonald及其同事[72-73]，他們使用16S rRNA和pmoA基因構(gòu)建克隆文庫(kù)，發(fā)現(xiàn)Ⅱ型甲烷氧化菌，尤其是Methylocystis及Methylosinus，是酸性泥炭濕地主要的甲烷氧化菌。之后，該組的Chen等在酸性泥炭濕地甲烷氧化菌多樣性方面的研究工作突出。他們使用DNA穩(wěn)定性同位素與微陣列的方法，或者通過(guò)檢測(cè)pmoA基因表達(dá)以及構(gòu)建mmoX基因文庫(kù)，進(jìn)一步證實(shí)Ⅱ型甲烷氧化菌，特別是其中的Methylocystis屬，在酸性泥炭濕地的甲烷氧化菌中的優(yōu)勢(shì)地位[74-75]，另外Methylocella和Methylocapsa也是泥炭濕地常見(jiàn)的類(lèi)群[75]。雖然酸性泥炭濕地是以Ⅱ型甲烷氧化菌為主的，但是在Moorhouse自然保護(hù)區(qū)濕地中也發(fā)現(xiàn)有Ⅰ型甲烷氧化菌(如Methylobacter)存在[74]。

位于俄羅斯西伯利亞地區(qū)的酸性(pH 3.5—5.5)泥炭蘚濕地是另一處甲烷氧化菌研究很多的地點(diǎn)[18,76-78]。該濕地主要的甲烷氧化菌屬于α-變形菌綱，甲烷氧化菌的數(shù)量介于106—108個(gè)/g濕土之間[18,76-78]。在該濕地上還開(kāi)展了大量的甲烷氧化菌分離培養(yǎng)的工作，已經(jīng)分離得到了Methylocystis、Methylocella和Methylocapsa三個(gè)屬的甲烷氧化菌菌株，這些也是該酸性泥炭濕地的主要甲烷氧化菌類(lèi)群[42,45,54-56,79]，它們都可以在pH<6的環(huán)境下生長(zhǎng)[18]。

除了英國(guó)和俄羅斯之外，在歐洲大陸的西班牙、芬蘭、荷蘭、德國(guó)以及挪威的北極地區(qū)都有對(duì)甲烷氧化菌群落結(jié)構(gòu)的研究。這些研究大部分是在泥炭蘚濕地上開(kāi)展的，只有少數(shù)關(guān)注到森林泥炭濕地和河流濕地土壤中的甲烷氧化菌。研究表明位于寒冷地區(qū)的挪威和芬蘭濕地Ⅰ型甲烷氧化菌比例更高[11,80]，尤其是Methylobacter，在北極濕地中用變形梯度凝膠電泳(DGGE)和穩(wěn)定性同位素探針(SIP)的方法都檢測(cè)到該菌的存在[11,81]，當(dāng)然在這些研究地點(diǎn)還同時(shí)檢測(cè)到了Ⅱ型甲烷氧化菌[48,80-83]。另外，Kip從荷蘭泥炭蘚濕地植物中分離到了一株Methylomomas屬的甲烷氧化菌，這是第一株嗜酸的γ-變形菌綱的甲烷氧化菌[67]，在這之前，從沒(méi)有在酸性泥炭濕地中分離到屬于γ-變形菌綱的甲烷氧化菌。

除歐洲大陸以外，在美國(guó)和日本的自然濕地中也開(kāi)展了甲烷氧化菌的研究，研究集中在泥炭濕地上，植被類(lèi)型除泥炭蘚外，還包括莎草科植被和森林濕地。與英國(guó)和俄羅斯的泥炭蘚濕地以Ⅱ型甲烷氧化菌為主不同，在這些地區(qū)Ⅰ型和Ⅱ型甲烷氧化菌都廣泛存在，有的樣地主要是Ⅰ型甲烷氧化菌，有的樣地Ⅱ型甲烷氧化菌占優(yōu)。直到2012年，在自然濕地上開(kāi)展的甲烷氧化菌群落多樣性的所有研究都集中在北半球，該年Kip小組將這項(xiàng)工作推進(jìn)到了南半球，他們用分子生物學(xué)方法，研究發(fā)現(xiàn)高豐度的Methylocystis屬甲烷氧化菌可能是南半球泥炭蘚濕地甲烷氧化的主要貢獻(xiàn)者[84]。

我國(guó)幅員遼闊，自然濕地類(lèi)型多樣。但對(duì)我國(guó)自然濕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爾蓋濕地開(kāi)展了初步的研究[62]。青藏高原作為全球海拔最高的一個(gè)獨(dú)特地域，其濕地眾多，面積約為13.3×l04km2，每年從青藏高原濕地排放的甲烷約為0.56—1 Tg[85-86]，除了若爾蓋濕地之外，青藏高原的其它自然濕地也是甲烷排放的主要源。近期在位于紅原的日干喬濕地對(duì)甲烷氧化菌群落多樣性和活性甲烷氧化菌類(lèi)進(jìn)行了較系統(tǒng)的研究[66]。另外，在位于松嫩平原的向海濕地上也有關(guān)于甲烷氧化菌的豐度和多樣性隨土壤深度變化的研究發(fā)表[87]。

3 結(jié)論與展望

隨著分子生物學(xué)和分離培養(yǎng)技術(shù)的發(fā)展，對(duì)甲烷氧化菌的認(rèn)識(shí)不斷深入。研究表明甲烷氧化菌的多樣性比之前想象的要多，但是大部分環(huán)境中主要的好氧甲烷氧化菌屬于變形菌門(mén)，可根據(jù)其生理特性分為Ⅰ型和Ⅱ型兩類(lèi)甲烷氧化菌。甲烷氧化菌可以利用pMMO和sMMO兩種酶進(jìn)行氧化甲烷的第一步反應(yīng)。pMMO2的發(fā)現(xiàn)表明Ⅱ型甲烷氧化菌中有些類(lèi)群可以利用這種酶氧化低濃度的甲烷。新研究證實(shí)甲烷氧化菌可以利用的底物比之前認(rèn)為的更廣泛，包括乙酸等含有碳碳鍵的化合物。另外，從極端環(huán)境中發(fā)現(xiàn)的各種嗜冷、嗜熱、嗜酸、嗜堿和嗜鹽的甲烷氧化菌表明甲烷氧化菌廣泛分布在各種生態(tài)環(huán)境中。

目前對(duì)甲烷氧化菌多樣性的研究主要集中在酸性泥炭濕地，尤其是位于北半球高緯度的酸性泥炭蘚濕地。研究表明Ⅱ型甲烷氧化菌Methylocystis、Methylocella和Methylocapsa是這類(lèi)濕地主要的甲烷氧化菌類(lèi)群，尤其是Methylocystis，廣泛分布在酸性泥炭濕地中。在北極寒冷的濕地中，Ⅰ型甲烷氧化菌占優(yōu)勢(shì)，其中Methylobacter大量存在。我國(guó)對(duì)自然濕地甲烷氧化菌的研究還處于起步階段，但也取得了一些有價(jià)值的研究結(jié)果。

隨著高通量測(cè)序時(shí)代的到來(lái)和純培養(yǎng)技術(shù)的不斷發(fā)展，更多的在極端環(huán)境中生存的甲烷氧化菌會(huì)被發(fā)現(xiàn)，將不斷完善人們對(duì)甲烷氧化菌生理和生態(tài)適應(yīng)性的認(rèn)識(shí)。同時(shí)，新的甲烷氧化菌類(lèi)群的不斷發(fā)現(xiàn)，會(huì)挑戰(zhàn)現(xiàn)有甲烷氧化菌的分類(lèi)方法；特別是隨著海量序列數(shù)據(jù)的出現(xiàn)，必將發(fā)現(xiàn)更多新的甲烷氧化菌序列和新的甲烷氧化菌菌株，整個(gè)分類(lèi)系統(tǒng)是否需要做較大幅度的修改還屬未知。另外，先進(jìn)的分子生物學(xué)技術(shù)也給甲烷氧化菌的生物地理學(xué)研究提供了有力的工具，隨著在全球更多生態(tài)環(huán)境中對(duì)甲烷氧化菌研究的積累，其群落結(jié)構(gòu)及其多樣性特征是否存在有規(guī)律的地理格局等重要生物地理學(xué)問(wèn)題的答案將變得逐漸清晰。

表3 自然濕地甲烷氧化菌多樣性研究概況

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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,*

1UniversityofChineseAcademyofSciences,Beijing100049,China2KeyLaboratoryofVirtualGeographicEnvironment,MinistryofEducation,NanjingNormalUniversity,Nanjing210046,China3NingboUniversity,Ningbo315211,China

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 phylumProteobacteria. 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 Ⅰ and type Ⅱ (Gamma-andAlpha-proteobacteria, respectively). Up to now, 20 methanotrophic genera have been affiliated in phylumProteobacteria, including two filamentous methanotrophs,CrenothrixpolysporaandClonothrixfusca. These two species have been characterized recently and form a new branch within the familyMethylococcaceae. 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 generaMethylocellaandMethyloferulawhich only have sMMO. Some methanotrophs of type Ⅰ and Ⅱ have both pMMO and sMMO. A different pMMO (pMMO2) is discovered in some type Ⅱ methanotrophs. pMMO2 has lower methane oxidation kinetics and enables these methanotrophs to consume methane at atmospheric concentrations. ThepmoAandmmoXgene, encoding subunits of the pMMO and sMMO respectively, have been used as a functional marker for detecting methanotrophs in environmental samples. However, the current publicpmoAsequences database is larger than that of themmoX, and the sequence basedpmoAphylogeny has good correlation to the 16S rRNA phylogeny. Facultative methanotrophs have been reported in the generaMethylocella,Methylocapsa, andMethylocystis. Some species of them can use compounds with carbon-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-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 Ⅰ methanotrophs (Methylocaldum,Methylococcus, andMethylothermus) were reported to have optimum growth temperatures above 40 ℃. On the other hand there are some methanotrophs (MethylobacterandMethylocella) adapted to cold environments and with optimum growth temperatures of 0—30 ℃. SomeMethylacidiphilumspecies grow at extreme low pH of 2—2.5. But someMethylomicrobiumspecies have the optimum pH of 9.0—9.5. Besides, someMethylomicrobiumspecies andMethylohalobiuscrimeensisare halotolerant methanotrophs and have a growth optimum around 1—1.5 mol/L NaCl. In contrast,MethylocapsaKYG 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-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 Ⅱ, such as generaMethylocystis,MethylocellaandMethylocapsa. Especially genusMethylocystis, were widely distributed in acidic peatlands. Type Ⅰ methanotrophs, especially genusMethylobacter, were dominant in the cold Arctic wetlands in Norway and Finland. Furthermore, researches revealed that type Ⅰ and Ⅱ 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 genusMethylocystissuggests that it is probably the major contributor to the methane oxidation in this Sphagnum wetland. Type Ⅰ and type Ⅱ 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-throughput sequencing and the development of new isolation and culture technology, the knowledge systems of methanotrophs will be refreshed more frequently.

methanotrophs; methane monooxygenase (MMO); methane; wetland

國(guó)家自然基金項(xiàng)目資助(11079053, 31200367)

2013-05-06;

2014-09-09

10.5846/stxb201305060936

*通訊作者Corresponding author.E-mail: cuixy@gucas.ac.cn

鄧永翠, 車(chē)榮曉, 吳伊波, 王艷芬, 崔驍勇.好氧甲烷氧化菌生理生態(tài)特征及其在自然濕地中的群落多樣性研究進(jìn)展.生態(tài)學(xué)報(bào),2015,35(14):4579-4591.

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):4579-4591.