贠娟莉，王艷芬,張洪勛

(中國科學院大學，北京 100049)

好氧甲烷氧化菌生態(tài)學研究進展

贠娟莉，王艷芬*,張洪勛

(中國科學院大學，北京 100049)

好氧甲烷氧化菌是以甲烷為碳源和能源的細菌。好氧甲烷氧化菌在自然環(huán)境中分布廣泛，人類已從土壤、淡水和海洋沉積物、泥炭沼澤、熱泉、海水和南極環(huán)境分離到甲烷氧化菌的純培養(yǎng)。好氧甲烷氧化菌可分為14個屬，包括研究較為深入的隸屬于變形菌門Alpha和Gamma綱的細菌，以及屬于疣微菌門的極端嗜熱嗜酸甲烷氧化菌。最近，好氧甲烷氧化菌還被發(fā)現(xiàn)存在于苔蘚類植物(尤其是泥炭苔蘚)共生體中，兼性營養(yǎng)好氧甲烷氧化菌也被發(fā)現(xiàn)。通過對好氧甲烷氧化菌的分類、生理生化特征、分子生物學檢測方法以及微生物生態(tài)學中的研究成果的總結(jié)與分析，以及對甲烷氧化菌研究所面臨的問題進行討論，以期為今后進一步開展好氧甲烷氧化菌及其在碳循環(huán)中的作用研究提供參考。

好氧甲烷氧化菌；微生物生態(tài)；分類學地位；多樣性；碳循環(huán)

甲烷是大氣中僅次于二氧化碳的第二號溫室氣體。雖然大氣中的甲烷的含量僅為二氧化碳的1/27，但甲烷引起的溫室效應是同等質(zhì)量二氧化碳的20—30倍[1]。造成甲烷濃度升高的主要成因有人為和自然兩種因素[2]。人為活動造成的甲烷排放約占總排放量的70%左右(圖1)，包括水稻種植、垃圾填埋以及生物質(zhì)燃燒；自然界甲烷排放的主要來源包括自然濕地、植物以及海洋[3]。

大氣中甲烷通過數(shù)量級相近的源(Source)和匯(Sink)達到平衡，隨著大氣中甲烷濃度的增加，甲烷的匯也成相應比例增加，從而一定程度上削弱了甲烷源增加造成的環(huán)境氣候影響，因而實際產(chǎn)生的甲烷量要比測得的排放量大的多[4- 5]。一直以來，甲烷匯的增長小于總甲烷源的增長，從而導致工業(yè)革命以來大氣中甲烷濃度的持續(xù)穩(wěn)定增長[3]。好氧甲烷氧化菌是重要的甲烷的匯，在環(huán)境中起著甲烷生物過濾器的作用，它們能使高達90%由產(chǎn)甲烷古菌所產(chǎn)生的甲烷在排入大氣之前被氧化[6- 7]。環(huán)境中存在兩類截然不同的甲烷氧化菌，好氧甲烷氧化菌(Methanotrophs)和厭氧甲烷氧化菌。

好氧甲烷氧化菌主要存在于甲烷與氧氣共存的微小界面空間，包括土壤-空氣、水-空氣界面、植物根際以及植物內(nèi)部。大量有關(guān)好氧甲烷氧化菌的研究工作顯示，該類微生物能適應各種環(huán)境。人類對于好氧甲烷氧化菌的研究已較為深入，這些研究不斷加深人類對全球甲烷循環(huán)的認知。本文就好氧甲烷氧化菌的生理生化及分類學特點、生態(tài)學地位以及微生物生態(tài)學研究方法進行全面深入的綜述，以此闡明好氧甲烷氧化菌在全球碳循環(huán)中的重要作用。

1 好氧甲烷氧化菌分類及生理生化特征

1.1 好氧甲烷氧化菌分類

好氧甲烷氧化菌(Aerobic methanotrophs)是以甲烷作為唯一碳源和能源的微生物，是甲基營養(yǎng)細菌(Methylotrophic bacteria)的一個分支[8]。好氧甲烷氧化菌于1906年首次被荷蘭微生物學家Sohngen分離出來[9]。1970年Whittenbury等[10]對分離和鑒定的100多種好氧甲烷氧化菌進行了分類，這些分類方法至今仍是鑒定好氧甲烷氧化菌強有力的依據(jù)。在Whittenbury等的基礎上，Bowman等采用分離方法對更多好氧甲烷氧化菌進行了保存，并進行了更加系統(tǒng)的分類和描述[11- 15]。

已知的好氧甲烷營養(yǎng)菌可分為TypeⅠ型和TypeⅡ型兩類，分屬于γ-Proteobacteria綱和α-Proteobacteria綱。TypeⅠ型好氧甲烷氧化菌屬于Methylococcaceae科，包含Methylobacter，Methylomonas，Methylosoma，Methylomicrobium，Methylococcus，Methylocaldum，Methylothermus，Methylohalobius，Methylosarcina和Methylosphaera10個屬。TypeⅠ型好氧甲烷氧化菌中的Methylococcus和Methylocaldum也被稱為TypeX型好氧甲烷營養(yǎng)菌，是一類耐熱的甲烷氧化菌，其生理生化及系統(tǒng)發(fā)育學特征與其他TypeⅠ型甲烷氧化菌有所不同。TypeⅡ型好氧甲烷氧化菌歸屬于Methylocystaceae和Beijerinckiaceae 2個科，前者包含Methylocystis和Methylosinus屬，后者有Methylocapsa和Methylocella屬?？茖W家獲得的第1株兼性好氧甲烷氧化菌Methylocellasilvestris為TypeⅡ型甲烷氧化菌。該菌除能利用甲烷外，還能利用多碳化合物作為碳源[16- 17]。后來，兩種屬于TypeⅠ型好氧甲烷氧化菌的絲狀甲烷氧化菌Crenothrixpolyspora[18]和Clonothrixfusca[19]被發(fā)現(xiàn)，這2種甲烷氧化菌形成了一個獨特的TypeⅠ型甲烷氧化菌分支。值得注意的是，Nature和PNAS雜志報道了3株極端嗜酸嗜熱(pH值1.5, 65 ℃)的好氧甲烷氧化菌：Methylokorusinfernorum[20]、Acidimethylosilexfumarolicum[21]和Methyloacidakamchatkensis[22]，它們不屬于任何一類已知的好氧甲烷氧化菌，而是屬于疣微菌門(Verrucomicrobia)，研究人員將其命名為Methylacidiphilum屬[23]。圖1是依據(jù)16S rRNA序列繪制的各好氧甲烷氧化菌分支之間的系統(tǒng)進化樹。

圖1 基于16S rRNA序列的好氧甲烷氧化菌進化樹[24]Fig.1 16S rRNA gene phylogeny of the aerobic methane oxidizing bacteria

TypeⅠ和TypeⅡ型好氧甲烷氧化菌除系統(tǒng)發(fā)育學有各自特點外，在形態(tài)學上也具明顯差異，TypeⅠ型甲烷氧化菌具有成束的分布于細胞質(zhì)內(nèi)的胞質(zhì)內(nèi)膜(圖2a)，而TypeⅡ型甲烷氧化菌一般只含有緊貼外壁的胞質(zhì)內(nèi)膜(圖2b)[24]。

圖2 TypeⅠ型好氧甲烷氧化菌代表菌Methylomonas methanica電鏡照片(a)；TypeⅡ型好氧甲烷氧化菌代表菌Methylosinus trichosporium電鏡照片(b)[24]Fig.2 Electron micrograph of a cross-section of a typical TypeⅠ methanotroph Methylomonas methanica (a)； Electron micrograph of a cross-section of a typical TypeⅡ methanotroph Methylosinus trichosporium (b)[24]

1.2 好氧甲烷氧化菌生理生化特征

環(huán)境中由好氧甲烷氧化菌推動的甲烷氧化主要過程為：好氧甲烷氧化菌首先利用自身攜帶的甲烷單加氧酶(Methane monooxygenase, MMO)催化甲烷氧化為甲醇，甲醇接著被甲醇脫氫酶催化氧化生成甲醛。最后，好氧甲烷氧化菌通過絲氨酸途徑(Serine pathway)或單磷酸核酮糖途徑(RuMP pathway)將甲醛轉(zhuǎn)化為細胞物質(zhì)[25]。

好氧甲烷氧化菌中存在2種甲烷單加氧酶：一種是與細胞膜結(jié)合，含銅、鐵離子的顆粒性甲烷單加氧酶(Particulate methane monooxygenase，pMMO)，存在于除Methylocella[26]及Methyloferula[27]以外的所有已發(fā)現(xiàn)的好氧甲烷氧化菌中；另一種是分泌在周質(zhì)空間中的可溶性甲烷單加氧酶(Soluble methane monooxygenase，sMMO)，存在于部分甲烷氧化菌中。由于MMO是甲烷氧化菌的功能酶系，且?guī)缀跛泻醚跫淄檠趸己衟MMO，因此利用MMO(尤其是pmoA，編碼pMMO的一段基因)作為生物標記物進行好氧甲烷氧化菌生態(tài)學研究已廣為采用。部分好氧甲烷氧化菌，如所有TypeⅡ型甲烷氧化菌、TypeⅠ型甲烷氧化菌的Methylomonas、Methylobacter和Methylococcus屬，除有氧化甲烷能力外，還有固氮能力。因此，利用nifH基因也可對該類好氧甲烷氧化菌進行分子生態(tài)學研究[28]。對于MMO的應用研究主要集中在兩方面：一是通過基因突變等手段，對好氧甲烷氧化菌編碼MMO等蛋白的基因進行改造，從而滿足不同工業(yè)催化的需要，例如提高MMO的活性、改變MMO的底物范圍、提高其對金屬離子的耐受性等；二是通過代謝工程手段，向好氧甲烷氧化菌內(nèi)部引入外源基因，利用甲烷氧化菌為載體生產(chǎn)高附加值的工業(yè)產(chǎn)品，如表達生產(chǎn)蛋白等生物產(chǎn)品[29]。

2 好氧甲烷氧化菌分子生態(tài)學研究方法

傳統(tǒng)方法是利用NMS及ANMS等無機鹽培養(yǎng)基對好氧甲烷氧化菌進行富集培養(yǎng)或者菌株分離[10]。分子微生物生態(tài)學方法的出現(xiàn)及應用極大擴展了人類對甲烷氧化菌的認知范圍。

最常用的好氧甲烷氧化菌的分子標記物是16S rRNA基因，該項應用主要基于大量的16S rRNA基因數(shù)據(jù)庫。針對好氧甲烷氧化菌各屬或菌株的特異性引物和探針已有大量報道，這些引物與以PCR技術(shù)為基礎的克隆文庫(Clone library)、變形梯度凝膠電泳(DGGE)、熒光原位雜交(FISH)等分析技術(shù)相結(jié)合，在環(huán)境微生物生態(tài)學研究中發(fā)揮重要作用[30]。但也會由于引物特異性不足，從而造成非特異性擴增，因此在以16S rRNA基因為對象研究環(huán)境中好氧甲烷氧化菌時需考慮到這一因素。除了16S rRNA基因之外，功能基因也是研究環(huán)境中好氧甲烷氧化菌的強有力工具，這些功能功能基因包括mmoX、pmoA、mxaF及nifH[28]。

DGGE和末端限制多態(tài)性研究(T-RFLP)為對比大量環(huán)境樣品中甲烷氧化菌多樣性差異提供了快速、靈敏的技術(shù)。許多針對這2種方法設計的16S rRNA基因和功能基因引物為研究環(huán)境中好氧甲烷氧化菌多樣性提供了強有力的工具[30]。另外一種研究環(huán)境中甲烷氧化菌的高通量方法是生物芯片技術(shù)，盡管生物芯片最初是作為基因組表達分析的研究工具，但基因診斷芯片已成功開發(fā)并已應用于環(huán)境中好氧甲烷氧化菌的檢測[31]。為了定量環(huán)境中好氧甲烷氧化菌的數(shù)量，可培養(yǎng)方法(最大釋然法，即MPN法)和不依賴培養(yǎng)的分子生物學方法(Real-time PCR和FHSH)均被使用[30]。這2種方法各有利弊，MPN技術(shù)依賴于特定培養(yǎng)基中甲烷氧化菌的生長情況，具有很大的偏好性；分子生物學技術(shù)雖不需培養(yǎng)，但很大程度上取決于環(huán)境樣品的類型和質(zhì)量好壞。為了檢測研究環(huán)境樣品中活躍的甲烷氧化菌菌群，穩(wěn)定同位素探針技術(shù)(SIP)應運而生。這項應用技術(shù)包括DNA-SIP[32]、RNA-SIP[33]、磷脂脂肪酸(PLFA)-SIP[34]以及最新使用的mRNA-SIP[35]。除此外，SIP技術(shù)也和宏基因組學相結(jié)合用于發(fā)現(xiàn)新的好氧甲烷氧化菌[36]。除了以上常用的分子生態(tài)學研究方法外，其他研究工具也逐漸被引入環(huán)境好氧甲烷氧化菌的研究，例如顯微鏡放射自顯影(MAR)-FISH[37- 38]、同位素芯片[39]、Raman-FISH[40]、納米二次離子質(zhì)譜(NanoSIMS)[41]和微流體數(shù)字PCR[42]。這些技術(shù)檢出限更高、可同時檢測多個樣品且能直接給出所測定菌株或菌群的功能特征。

3 好氧甲烷氧化菌的生態(tài)學研究

過去幾十年中，培養(yǎng)及不依賴培養(yǎng)的分子生態(tài)學方法已經(jīng)被廣泛用于各種環(huán)境中好氧甲烷氧化菌的多樣性、分布及豐度研究，如稻田、垃圾填埋廠、淡水和淡水沉積物、海水、山地土壤以及極端環(huán)境。

稻田是大氣甲烷的主要來源之一，全球人口激增導致大米需求增加，故而稻田甲烷排放量呈增加趨勢。研究表明稻田中好氧甲烷氧化菌多樣性較高[43]，包括Methylomonas、Methylobacter、Methylomicrobium、Methylococcus、Methylocaldum、Methylocystis和Methylosinus屬。有關(guān)稻田中何種好氧甲烷氧化菌占據(jù)優(yōu)勢，各地研究結(jié)果并不一致。陸雅海等經(jīng)研究發(fā)現(xiàn)，水稻根部對TypeⅠ型好氧甲烷氧化菌具有選擇性，且水稻根部比根際土壤中TypeⅠ型好氧甲烷氧化菌更為豐富[44]。另有報道認為水稻根系對TypeⅠ型好氧甲烷氧化菌的偏好不受水稻物種的影響。除Methylocaldum屬的好氧甲烷氧化菌多在熱帶地區(qū)被發(fā)現(xiàn)外，稻田中的好氧甲烷氧化菌在全球范圍內(nèi)并沒有明顯的地域性特征。研究者認為TypeⅠ型好氧甲烷氧化菌成為水稻根際優(yōu)勢菌群的原因在于其能適應較廣的O2/CH4范圍。再者，水稻根系的O2濃度非常不穩(wěn)定，不適宜TypeⅡ型好氧甲烷氧化菌的生長[45]。與以上結(jié)果相反，Luke等人通過T-RFLP和基因診斷芯片技術(shù)對18種不同水稻品種進行研究，發(fā)現(xiàn)這些水稻根系中以TypeⅡ和TypeX型好氧甲烷氧化菌為主，并顯示出極大的多樣性，該研究小組還指出水稻根部的好氧甲烷氧化菌群落組成受水稻基因型影響很大[46]。鄭勇等[47]研究發(fā)現(xiàn)TypeⅡ型甲烷氧化菌在長期施肥的水稻土壤中占優(yōu)勢，定量PCR結(jié)果發(fā)現(xiàn)所有處理中TypeⅡ型甲烷氧化菌的數(shù)量是TypeⅠ型好氧甲烷氧化菌的1.88至3.32倍。不同施肥處理對甲烷氧化菌的菌群組成有所影響。長期施氮磷鉀和秸稈的處理(NPK+C)、施氮肥和鉀肥(NK)處理的稻田土壤中TypeⅡ型甲烷氧化菌的數(shù)量明顯比對照高，表明長期施肥對TypeⅡ型甲烷氧化菌的生長有促進作用。稻田中甲烷氧化菌的分布和豐度受很多因素的影響，如氧氣的可用性及水稻的生長時期等因素[48]。在稻田土壤中，高氧氣、低甲烷的環(huán)境利于TypeⅠ型好氧甲烷氧化菌的生長，反之則利于TypeⅡ型甲烷氧化菌的生長[49]。Shrestha等研究發(fā)現(xiàn)，無論施肥與否，在水稻各個生長階段，根際土壤中好氧甲烷氧化菌以TypeⅡ型為主，而水稻根部則以TypeⅠ型為主[50]。

淡水和淡水沉積物是大氣甲烷的又一重要來源，該類環(huán)境中好氧甲烷氧化菌主要以TypeⅠ型甲烷氧化菌中的Methylomonas、Methylobacter、Methylosarcina、Methylococcus和Methylosoma屬為主。在對華盛頓湖沉積物的研究中發(fā)現(xiàn)，TypeⅠ型比TypeⅡ型好氧甲烷氧化菌多1—2個數(shù)量級[59]。另外，在康士坦茨湖中，TypeⅠ型甲烷氧化菌占pmoA的克隆文庫序列的90%[60]。Dumont等利用DNA-SIP和mRNA-SIP相結(jié)合的方法，發(fā)現(xiàn)在Stechlin湖中也以TypeⅠ型甲烷氧化菌為主要菌群[35]。張洪勛等通過對我國兩處淡水沼澤濕地研究發(fā)現(xiàn)：我國青藏高原若爾蓋永凍土濕地中好氧甲烷氧化菌僅有Methylobacter屬和Methylocystis兩個屬，且以TypeⅠ型甲烷氧化菌為優(yōu)勢菌群，不同植被覆蓋的泥炭沼澤中好氧甲烷氧化菌數(shù)量有所不同[61- 62]；對我國東北地區(qū)松嫩平原向海濕地中好氧甲烷氧化菌多樣性進行研究，發(fā)現(xiàn)向海濕地好氧甲烷氧化菌多樣性與淡水湖泊相似，較若爾蓋高寒濕地種類多，但仍以TypeⅠ型甲烷氧化菌的Methylobacter屬為優(yōu)勢菌群[63]。研究的兩個濕地中均有與Methylococcus屬甲烷氧化菌相似度較高的新的甲烷氧化菌存在。另外，這2個濕地中Methylobacter屬的甲烷氧化菌親緣關(guān)系相近，表明我國自然濕地中甲烷氧化菌具有地域性特點。

對海水和海洋沉積物中甲烷氧化菌的研究相對較少，雖然從海水中分離到了Methylomonas和Methylomicrobium屬的甲烷氧化菌，但分子生態(tài)學方面的研究卻證明不可培養(yǎng)的好氧甲烷氧化菌Methylococcaceae科的甲烷氧化菌在海洋水體中占主導地位[64]。還有研究發(fā)現(xiàn)OPU1, OPU3和Group X是深海中占主導地位的好氧甲烷氧化菌，其中OPU1和OPU3菌群在墨西哥灣和Santa Monica海灣具有生長優(yōu)勢，且其在甲烷溪流的數(shù)量比非甲烷溪流中多50多倍，Group X則在加利福尼亞海岸中占優(yōu)勢，其生長不受甲烷溪流的影響[65]。在黑海淺海中發(fā)現(xiàn)了好氧甲烷氧化菌，TypeⅠ和Ⅱ型好氧甲烷氧化菌各占細菌總數(shù)的2.5%，在深海中參與甲烷氧化的菌群則主要是ANME-1和ANME-2厭氧甲烷氧化菌[66]。

山地和森林是大氣甲烷主要的匯，這些環(huán)境中的好氧甲烷氧化菌對大氣甲烷有很高的親和力，并以TypeⅡ型的Methylocystis屬，山地土壤菌群α(USCα)和γ(USCγ)的甲烷氧化菌為主。Kolb等[67]發(fā)現(xiàn)“USCα”型甲烷氧化菌是酸性森林土壤中的主要菌群，而“Cluster I”則在中性森林土壤中占優(yōu)勢，研究者認為這兩種甲烷氧化菌能適應低甲烷濃度環(huán)境主要是由于其細胞特異性的甲烷氧化能力。Horz等[68]在加利福尼亞山地土壤中發(fā)現(xiàn)3個不同的甲烷氧化菌分支，這些分支與已報道的RA14或VB5FH-A種群類似，這些甲烷氧化菌菌群是典型的大氣甲烷氧化菌群，其對氣候變暖的響應程度與TypeⅡ型甲烷氧化菌不同。Mohanty等[69]研究發(fā)現(xiàn)，森林中的甲烷氧化菌以TypeⅡ型的Methylocystis屬為主，且有TypeⅠ型的Methylomicrobium和Methylosarcina屬甲烷氧化菌存在。Menyailo等[70]對西伯利亞森林中不同樹種的土壤中好氧甲烷氧化菌的菌群組成及豐度進行了調(diào)查，發(fā)現(xiàn)該地土壤中以USCα為主要甲烷氧化菌群，樹種的不同并不會影響甲烷氧化菌的菌群組成，但會影響土壤甲烷氧化速率，造成這一結(jié)果的原因可能是樹種的不同會影響甲烷氧化菌的單個細胞活性，但并不會影響其周圍土壤中甲烷氧化菌的種類，這一過程是通過控制土壤中氮循環(huán)完成的。有研究者推測，USCα和USCγ為不可培養(yǎng)的可在大氣低濃度甲烷下生長的甲烷氧化菌。另一種解釋則認為在高山和森林中的好氧甲烷氧化菌以木質(zhì)素降解產(chǎn)物甲醇作為能源和碳源。直到近期，Baani和Liesack[71]發(fā)現(xiàn)在Methylocystissp. SC2中存在一種特殊的pMMO酶，該酶對甲烷的親和力不同，由此可解釋為什么Methylocystis屬的甲烷氧化菌能在山地和森林，以及其他環(huán)境中普遍存在。

極端環(huán)境中的好氧甲烷氧化菌研究一直備受關(guān)注。從酸性從泥炭沼澤土壤和酸性森林土壤中分離出的屬于Methylocella和Methylocapsa屬的好氧甲烷氧化菌[72- 73]；從嗜鹽和嗜堿環(huán)境中分離到的屬于Methylomicrobium屬和Methylohalobius的好氧甲烷氧化菌[74- 75]；從永凍土地區(qū)分離出的嗜冷甲烷氧化菌Methylobacterpsychrophilus、Methylosphaerahansonii和Methylomonasscandinavica[13, 76]，這些TypeⅠ型甲烷氧化菌生長于低溫環(huán)境中(5—15 ℃)并且G+C含量較低；以及從熱泉中分離的嗜熱甲烷氧化菌MethylococcuscapsulatusBath[10]、Methylocaldumspp.[77- 78]和Methylothermusthermalis[79]。最為引人矚目的是從世界不同區(qū)域火分離的疣微菌門(Verrucomicrobia)的3株極端嗜熱嗜酸甲烷氧化菌[20- 22]，這些好氧甲烷氧化菌細胞內(nèi)含有與其他菌株不同的pmoA基因，表明它們對碳的代謝和吸收可能有另外的途徑。對于疣微菌門好氧甲烷氧化菌物理化學特征及分類學地位，Op den Camp等人給予了詳細的綜述[23]。人類對極端環(huán)境中好氧甲烷氧化菌的探索有待進一步深入，相信將來還會有更多極端環(huán)境中的好氧甲烷氧化菌被發(fā)現(xiàn)。

4 苔蘚-好氧甲烷氧化菌共生體

很長一段時間內(nèi)，人們認為只有厭氧甲烷氧化菌能夠與其他生物形成共生體系，如厭氧甲烷氧化菌和硫還原細菌或硝酸還原菌共生。最新研究發(fā)現(xiàn)，好氧甲烷氧化菌也能與環(huán)境中的其他生物形成共生體系。Kip與同事研究發(fā)現(xiàn)，泥炭苔蘚可與好氧甲烷氧化菌共生，合作進行甲烷氧化(圖3)[80]。

圖3 泥炭沼澤中泥炭苔蘚-甲烷氧化菌共生甲烷氧化途徑示意圖[80]Fig.3 Methane oxidation by methanotrophs in a peat bog. Sphagnum mosses form symbioses with methane-consuming bacteria in Sphagnum-dominated peat bogs[80]

穩(wěn)定同位素標記實驗證實了泥炭苔蘚所同化的35%的CO2是通過甲烷氧化產(chǎn)生。這種共生體系的形成對于好氧甲烷氧化菌和苔蘚來說是互利的(圖3)：水下的泥炭苔蘚由于缺乏氣孔不能從大氣得到足夠的CO2進行光合作用，于是好氧甲烷氧化菌的代謝產(chǎn)物CO2就成為其絕佳的CO2來源。與此同時，水下泥炭苔蘚中的好氧甲烷氧化菌也可利用泥炭苔蘚光合作用產(chǎn)生的O2完成甲烷氧化過程。Kip等人還指出，在溫度升高時，該共生體系能更好的減少甲烷排放。當從土壤中去掉泥炭苔蘚時，土壤的甲烷排放量升至原來的4倍，表明泥炭苔蘚-好氧甲烷氧化菌共生體系比游離生長的甲烷氧化菌在甲烷氧化過程中起更重要的作用[80- 81]。

通過進一步研究，Kip等人從泥炭苔蘚中分離出了多株嗜酸性甲烷氧化菌，所獲多數(shù)菌株屬于TypeⅡ型好氧甲烷氧化菌[82]。另外，他們還在荷蘭及巴塔哥尼亞泥炭苔蘚中發(fā)現(xiàn)Methylomonas和Methylocystis分布最為廣泛[82- 83]。Liebner等人[84]通過研究證實，苔蘚-甲烷氧化菌共生氧化甲烷的現(xiàn)象不僅存在于泥炭苔蘚和低pH值的泥炭沼澤中，還存在于褐蘚與永凍土中。該研究同樣通過穩(wěn)定同位素方法研究了西伯利亞凍土水體中的褐蘚-好氧甲烷氧化菌共生體系，發(fā)現(xiàn)該體系氧化甲烷能力在淹水條件下增強，且在光照條件下是一個純的甲烷匯，當去掉光照時，土壤就變成了強大的甲烷排放源。該研究還估測，褐蘚-好氧甲烷氧化菌共生作用使得北極多邊苔原凍土甲烷排放總量至少減少了5%，鑒于褐藻在永凍土淡水區(qū)域廣泛生長，苔蘚-甲烷氧化菌共生作用可能是該地區(qū)淡水環(huán)境主要的甲烷氧化途徑。

5 環(huán)境對好氧甲烷氧化菌的影響

影響甲烷氧化菌的環(huán)境因素可歸為化學因素和生物因素兩大類。化學因素主要包括氧氣，水分狀況，含氮化合物及重金屬等；生物因素主要有競爭和捕食。目前研究主要集中在化學因素方面。水位和氧氣會影響好養(yǎng)甲烷氧化菌的活性，尤其是在泥炭沼澤濕地和稻田中。有研究證明長期排水會影響土壤中甲烷氧化菌的群落組成，不同形式的土地利用顯著影響甲烷氧化菌的群落組成與活性[85]。一方面，水位升高會降低土壤氧氣濃度，從而導致甲烷氧化菌可用氧氣量減少；另一方面，排水會增加土壤氧氣含量從而促進甲烷氧化菌生長。

生物因素也會影響環(huán)境中好氧甲烷氧化菌對甲烷的氧化，如捕食。Murase和Frenze[89]做了一項有趣的研究，發(fā)現(xiàn)原生動物對甲烷氧化菌的捕食行為，他們從稻田中分離出了以好氧甲烷氧化菌為食的原生動物。Murase等人還發(fā)現(xiàn)原生動物偏好捕食特定的甲烷氧化菌(如Methylobacter)，這可能是由于捕食這類甲烷氧化菌后能最快速有效的進行自身的同化作用[90]。Moon等將含有蚯蚓的水稻土作為垃圾填埋場的覆蓋層能有效減少垃圾填埋場的甲烷排放，并且發(fā)現(xiàn)覆蓋層土壤中的細菌及甲烷氧化菌主要來源于水稻土及蚯蚓排泄物，TypeⅠ (主要為Methylocaldum)和TypeⅡ (主要為Methylocystis)型甲烷氧化菌都在甲烷氧化過程中起重要作用[91]。近期研究還表明在土壤中加入蚯蚓后能顯著增加好氧甲烷氧化菌的多樣性和數(shù)量[92]。

放牧對甲烷氧化菌有一定影響。周小奇等對我國青藏高原草甸土壤研究發(fā)現(xiàn)，放牧顯著影響土壤中好氧甲烷氧化菌的群落組成[93]。鄭勇等研究發(fā)現(xiàn)，放牧增加了好氧甲烷氧化菌的數(shù)量，從而促進了土壤的甲烷氧化能力[94]。

6 問題與展望

人類對好氧甲烷氧化菌及其在減少大氣甲烷排放的作用研究歷經(jīng)數(shù)十載，逐漸闡明了其在大氣碳循環(huán)中的重要作用，但研究過程中仍涉及到幾個重要問題仍值得深思，有待進一步研究[24]。

(1) 好氧甲烷氧化菌多樣性及分布在微生物世界內(nèi)究竟是怎樣？過去幾年中不斷有新的甲烷氧化菌菌株分離出來，如絲狀甲烷氧化菌Crenothrix以及嗜酸嗜低溫的疣微菌門(Verrucomicrobia)好氧甲烷氧化菌的發(fā)現(xiàn)。這些新的發(fā)現(xiàn)令人不禁想到，環(huán)境中或許還存在更多未知的、新的好氧甲烷氧化菌有待發(fā)現(xiàn)。另外，是否存在一類“大氣好氧甲烷氧化菌”？是否有好氧甲烷氧化古菌的存在？所有這些問題的解決都將取決于新的分離技術(shù)的出現(xiàn)。

(2) 不同種類的好氧甲烷氧化菌菌群之間是怎樣相互競爭最基本的生存物質(zhì)，如氧氣和氮素？隨著新近發(fā)明的可測定單個細胞的Raman熒光原位雜交及納米二次離子質(zhì)譜(NanoSIMS)技術(shù)的問世，相信這一問題也會迎刃而解。

(3) 兼性甲烷氧化菌是否只存在于Methylocella屬，如果不是，那么還有哪些？它們何時以及怎樣從異養(yǎng)型微生物轉(zhuǎn)換為甲烷營養(yǎng)型的？作為兼性營養(yǎng)的好氧甲烷氧化有哪些生長優(yōu)勢？比較基因組學和蛋白質(zhì)組學在定義代謝途徑及兼性營養(yǎng)的基因調(diào)控機制方面具有重要作用，這些研究的應用將為以上問題的解決帶來可能。

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Ecologyofaerobicmethaneoxidizingbacteria(methanotrophs)

YUN Juanli, WANG Yanfen*， ZHANG Hongxun

GraduateUniversityofChineseAcademyofSciences,Beijing100049,China

Aerobic methane oxidizing bacteria (methanotrophs) are a fascinating group of bacteria that have the unique ability to grow on methane as their sole carbon and energy source. They appear to be widespread in nature and have been isolated from a number of different environments. There are now 14 recognized genera of methanotrophs belong to two phyla, Proteobacteria and thermoacidiphilic Verrucomicrobia. The former was well studied and separated into two classes, TypeⅠ and TypeⅡ methanotrophs, which belong to Alpha and Gamma Proteobacteria. Extremely thermophilic, acidophilic methanotrophs from the phylum Verrucomicrobia have been isolated, thus expanding both the taxonomic diversity and physiological range of aerobic methanotrophy.

The discovery of the facultative methanotrophMethylocellasilvestrishas changed the view that methanotrophs were obligate organism. They can cooxidize a considerable number of organic compounds and also have considerable potential in biotechnology. A wide variety of methanotrophic symbionts in and on the mosses were recently detected, and showing the global prevalence of this symbiosis. Traditional way used cultivation to enrichment or isolation to study methanotrophs in the environment. Molecular ecology techniques applied in the last few decades have greatly expanded our knowledge of methanotroph ecology. The most obvious marker for detecting methanotrophs in various environments is the 16S rRNA gene, due to the large database of sequences available. Primers and probes targeting different genera or species have been designed and used extensively in combination with polymerase chain reaction (PCR) based clone library analysis, denaturing gradient gel electrophoresis (DGGE) analysis, and fluorescent in situ hybridization (FISH) analysis. Several functional genes have also been used for the detection of methanotrophs in environmental samples, includingpmoA(encoding the key subunits of particulate methane monooxygenase),mmoX(encoding the key subunits of soluble methane monooxygenase),mxaF(encoding the key subunits of methanol dehydrogenase),nifH(encoding the dinitrogenase reductase), and genes involved in C1 transfer pathways. To understand the active community of methanotrophs in the environment, stable isotope probing (SIP) techniques have been developed, including DNA-SIP, RNA-SIP, mRNA-SIP, and phospholipid fatty acid (PLFA)-SIP. SIP has also been combined with metagenomics to discover novel methanotrophs. Other very powerful molecular techniques have been developed in the last few years, including microautoradiography (MAR)-FISH, isotope array, Raman-FISH, nano-secondary ion mass spectrometry (NanoSIMS), and microfluidic digital PCR, these techniques can now be used in the analyses of methanotrophs. Both cultivation and cultivation independent molecular methods have been used intensively in last few decades to study the diversity, distribution, and abundance in environments of methanotrophs, such as soils, freshwater, marine sediments, acid peat bogs, hot springs, seawater and extreme environments. In the microcosm of soil, the growth and diversity of methanotrophs are also influenced by several environmental factors. This review highlights recent progress in the research of the taxonomy, of the discovery of novel aerobic methanotrophs, of the biochemistry, of the molecular techniques and the environment impacts on methanotrophs, we also emphasize deficiencies and issues need to be solved in future studies. This review will provide theoretical foundation for future methanotrophic ecology study and explain the key role methanotrophs play in carbon cycle.

methanotrophs; microbial ecology; taxonomy; diversity; carbon-cycle

國家自然科學基金資助項目(41271277/D010504)

2012- 07- 17;

2013- 04- 24

*通訊作者Corresponding author.E-mail: yfwang@ucas.ac.cn

10.5846/stxb201207171013

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