土壤中甲烷厭氧氧化菌多樣性的分子檢測

周京勇, 劉冬秀, 何池全, 劉曉艷, 沈燕芬, 龍錫恩, 陳學(xué)萍,*

1 上海大學(xué)環(huán)境與化學(xué)工程學(xué)院, 上海 200444 2 余姚市環(huán)境保護(hù)監(jiān)測站, 余姚 315400 3 中國科學(xué)院城市環(huán)境研究所, 廈門 361021

土壤中甲烷厭氧氧化菌多樣性的分子檢測

周京勇1, 劉冬秀1, 何池全1, 劉曉艷1, 沈燕芬2, 龍錫恩3, 陳學(xué)萍1,*

1 上海大學(xué)環(huán)境與化學(xué)工程學(xué)院, 上海 200444 2 余姚市環(huán)境保護(hù)監(jiān)測站, 余姚 315400 3 中國科學(xué)院城市環(huán)境研究所, 廈門 361021

甲烷厭氧氧化作用是減少海洋底泥甲烷釋放的重要生物地球化學(xué)過程, 然而在陸地生態(tài)系統(tǒng)中甲烷厭氧氧化作用及其功能菌群的生態(tài)功能仍然不確定。對甲烷厭氧氧化菌多樣性的研究可為減少甲烷排放提供重要科學(xué)依據(jù)。與傳統(tǒng)的分離培養(yǎng)方法比較，分子檢測方法是一種更為快速和高效的研究手段，可直接和全面的反映參與甲烷厭氧氧化作用的功能微生物。以DNA分子標(biāo)記物為研究對象，重點(diǎn)探討三類主要的分子標(biāo)記基因，即16S rRNA,mcrA 和pmoA，所采用的相關(guān)探針和引物信息，同時從定性和定量兩個角度綜述土壤甲烷厭氧氧化菌的多樣性研究的主要進(jìn)展，最后提出厭氧甲烷氧化菌多樣性研究中存在的一些問題和相應(yīng)的解決思路。

土壤; 甲烷厭氧氧化菌; 功能基因; 多樣性

甲烷(CH4)是一種溫室氣體，其溫室效應(yīng)是二氧化碳的26倍，對全球變暖的“貢獻(xiàn)率”達(dá)到 15%[1]。當(dāng)今國際重大環(huán)境科學(xué)計(jì)劃(例如IGBP、WCRP、IHDP、GCTE、IPCC)中，陸地生態(tài)系統(tǒng)碳循環(huán)是其中的核心研究內(nèi)容[2- 4]。陸地濕地甲烷排放是溫室氣體甲烷的最大排放源[5]，據(jù)估計(jì)天然濕地每年向大氣中排放110 Tg CH4，占全球CH4排放總量的15%—30%[6]。此外，IPCC報(bào)告指出全球稻田CH4排放量每年高達(dá)35—56 Tg，約占全球CH4排放總量的1/5[4]。眾所周知，甲烷厭氧氧化作用是海洋底泥中減少甲烷釋放到大氣中的重要生物地球化學(xué)過程，盡管在陸地生態(tài)系統(tǒng)中甲烷厭氧氧化作用廣泛存在，但其過程及關(guān)鍵微生物尚未清楚[7]。例如，有人應(yīng)用13C同位素估算垃圾填埋場滲濾液污染羽土壤，發(fā)現(xiàn)甲烷厭氧氧化作用消耗了80%—90% CH4[8]。呂鎮(zhèn)梅等[9]同樣也證實(shí)了水稻田土壤甲烷厭氧氧化過程的存在，但結(jié)果表明水田土壤中的甲烷厭氧氧化活性遠(yuǎn)低于甲烷好氧氧化活性，如以兩者的氧化活性作為對甲烷氧化的貢獻(xiàn)來計(jì)，則甲烷厭氧氧化作用的貢獻(xiàn)率一般都在整個甲烷氧化的10%以下。但在水田土壤淹沒的情形下，由于土壤厭氧條件的形成和甲烷擴(kuò)散受阻，甲烷厭氧氧化的速率明顯超過好氧氧化的速率，甲烷厭氧氧化在整個甲烷氧化中的貢獻(xiàn)率可到達(dá)30%以上。甲烷在這些厭氧生境中由產(chǎn)甲烷菌形成以后，經(jīng)土壤和水層，逸散至大氣，在途經(jīng)土壤和水層時可被棲息于其間的甲烷厭氧氧化菌(AOM)所氧化。因此探索土壤甲烷厭氧氧化菌的多樣性有助于深入認(rèn)知漬(淹)水土壤中甲烷厭氧消耗的微生物學(xué)機(jī)制，為減少甲烷排放通量提供科學(xué)基礎(chǔ)，對減緩因溫室效應(yīng)而帶來的氣候變暖具有重要意義。

近幾十年來，分子生態(tài)學(xué)方法已成為土壤甲烷厭氧氧化菌多樣性研究的關(guān)鍵手段，并取得了豐碩的成果[10- 12]。近幾年，國內(nèi)也開始廣泛關(guān)注甲烷厭氧氧化作用及其功能菌，多個課題組已經(jīng)對其多方面進(jìn)行了綜述[13- 15]。本文重點(diǎn)對土壤甲烷厭氧氧化菌的功能基因(16S rRNA,mcrA,pmoA)所采用的相關(guān)探針和引物序列進(jìn)行了綜述，并基于這些功能基因總結(jié)其在土壤甲烷厭氧氧化菌定性和定量分子檢測方面的應(yīng)用。

1 甲烷厭氧氧化菌的培養(yǎng)和分類

1.1 甲烷厭氧氧化菌的培養(yǎng)

由于土壤中微生物群落及環(huán)境因子極其復(fù)雜，能夠被培養(yǎng)并分離出的微生物只是非常小的一部分，因此傳統(tǒng)上依賴于培養(yǎng)的方法僅能反映不到1%的微生物種類多樣性[16]。由于在富集培養(yǎng)條件下甲烷厭氧氧化菌生長速率慢，倍增時間長達(dá)數(shù)月，并且因其生物特性須在嚴(yán)格厭氧的條件下富集培養(yǎng)、工藝條件嚴(yán)格和影響因子復(fù)雜，客觀上阻礙了對甲烷厭氧氧化菌功能和作用機(jī)理研究。許多科學(xué)家曾認(rèn)為無法富集純化依賴于硝酸根的甲烷厭氧氧化菌[17- 20]。迄今，僅獲得此類微生物的富集培養(yǎng)物。最初由Ettwig課題組從新西蘭淡水底泥中富集得到硝酸鹽甲烷厭氧氧化菌[21- 23]，此后陸續(xù)有來自其他淡水生境和人工污水處理系統(tǒng)中的富集培養(yǎng)的報(bào)道(混合培養(yǎng)活性污泥和淡水底泥富集[24- 27])。 Vecherskaya 等[28]從甲烷馴化的微氧反硝化生物反應(yīng)器中篩選純化到一株甲烷厭氧氧化菌，系統(tǒng)發(fā)育分析發(fā)現(xiàn)其屬于Methylocystisparvus。與硝酸鹽甲烷厭氧氧化菌不同，硫酸鹽甲烷厭氧氧化菌大多富集培養(yǎng)于海洋底泥，如Eckernf?rde海灣沉積物[29- 31]、Aarhus 海灣沉積物[32]、Monterey海灣沉積物[33]。國內(nèi)閔航等[34]首次報(bào)道了1株從浙江象山市郊青紫泥水稻田土壤中分離到的能獨(dú)立厭氧氧化甲烷的菌株。因此，到目前為止，僅有少數(shù)幾個課題組能富集培養(yǎng)有限生境中甲烷厭氧氧化菌，不能充分描述甲烷厭氧氧化菌的多樣性，而分子生態(tài)學(xué)方法的應(yīng)用，能從分子水平上較為客觀地揭示微生物的多樣性，有效地克服了傳統(tǒng)培養(yǎng)方法的不足，提高了分析檢測的速度及結(jié)果的準(zhǔn)確性和完整性。

1.2 甲烷厭氧氧化菌的分類

1.2.1 硫酸鹽甲烷厭氧氧化菌(SAMO)

參與SAMO反應(yīng)的甲烷厭氧氧化古菌(ANME)往往與硫酸鹽還原細(xì)菌(SRB)形成共生體，因此甲烷氧化的同時伴隨著硫酸鹽的還原。根據(jù)系統(tǒng)發(fā)育分析，通常這類厭氧甲烷氧化菌分為三類: ANME- 1(Anaerobic methanotrophic archaea)、ANME- 2 、ANME- 3，均屬于廣古菌門[35]。其中，ANME- 1與產(chǎn)甲烷微菌目(Methanomicrobiales)和產(chǎn)甲烷八疊球菌目(Methanosarcinales)有較近的親緣關(guān)系，ANME- 2屬于產(chǎn)甲烷八疊球菌目(Methanosarcinales)，ANME- 3與甲烷擬球菌屬(Methanococcoides)親緣關(guān)系較近[35]。這三類古菌彼此間的進(jìn)化距離較遠(yuǎn)，序列相似度僅為 75%—92%。即使在ANME- 2中，分枝ANME- 2a、- 2b 與- 2c 相似度也較低。因此，雖然ANME- 1、ANME- 2 、ANME- 3屬于不同的目或科，但是都具有在各種生境厭氧氧化甲烷的能力。然而，與ANME- 2 的同源性較高的ANME的一個新的分枝GoM Arc1，試驗(yàn)表明它并不具備氧化甲烷的能力，也不與硫酸鹽還原菌(SRB)組成共生菌群[36- 38]。因此，此類甲烷厭氧氧化菌在甲烷的生物地球化學(xué)循環(huán)過程中的作用還有待進(jìn)一步研究。ANME- 1可以單細(xì)胞形式存在[39- 40]，也可以與硫酸鹽還原菌以共生體的形式存在[39]，在黑海中甚至以編繞式存在[40- 41]。ANME- 3 同樣可以單細(xì)胞形式存在，或者與硫酸鹽還原菌形成外殼型或者混合型的共生體。ANME- 2往往與硫酸鹽還原菌以外殼型或者混合型共生體存在[40,42]。

1.2.2 硝酸鹽甲烷厭氧氧化菌 (DAMO)

2 甲烷厭氧氧化菌多樣性的主要分子標(biāo)記物

鑒于自然環(huán)境中微生物群落的復(fù)雜性和傳統(tǒng)的培養(yǎng)方法的局限性，分子生物學(xué)技術(shù)的應(yīng)用越來越廣泛，它能提高分子檢測的速度和分析結(jié)果的準(zhǔn)確性。檢測甲烷氧化菌多樣性的檢測方法主要有溫度梯度凝膠電泳(TGGE)、變性梯度凝膠電泳(DGGE)、熒光原位雜交(FISH)、末端限制性片段長度多態(tài)性分析(T-RFLP)、高通量測序等新興分子生態(tài)學(xué)技術(shù)。這些分子生態(tài)學(xué)技術(shù)都需要建立在目標(biāo)微生物群落的分子標(biāo)記物的基礎(chǔ)上，目前已有的甲烷厭氧氧化菌的分子標(biāo)記物主要包括特異基因探針，16SrRNA，功能基因mcrA以及pmoA基因。

2.1 探針

基因探針(probe)又稱“寡核苷酸探針”，簡稱“探針”，是一種核酸雜交應(yīng)用。由于核酸分子雜交的高特異性及檢測方法的高靈敏性，基因探針已經(jīng)廣泛用于環(huán)境微生物學(xué)中，檢測土壤等生境中微生物多樣性，鑒別功能基因，定性、定量分析環(huán)境微生物的存在、豐度、分布等。熒光原位雜交的是熒光標(biāo)記特異核酸探針，然后與被檢測的染色體或DNA片段變性-退火-復(fù)性進(jìn)行雜交，通過熒光顯微鏡觀察熒光信號，從而對所測目標(biāo)進(jìn)行定性、定量或相對定位分析。

表1總結(jié)了鑒定甲烷厭氧氧化菌及硫酸鹽還原菌常用的一些特異性探針。這些探針中，ANME2- 712的信號比ANME- 1- 538弱，Eel-MS932同時可以檢測到ANME- 3，但是錯配的幾率較高，因此不推薦用此探針。引物ANME3- 1249幾乎可以覆蓋ANME- 3的所有序列，而且具有特異性。引物AR468f幾乎可以覆蓋ANME- 2c的所有序列，但是對于ANME- 2c沒有嚴(yán)格的特異性。

基于上述探針，已經(jīng)成功地將熒光原位雜交技術(shù)(FISH)技術(shù)應(yīng)用于甲烷厭氧氧化菌種群的鑒定和種群密度的定量表達(dá)[35]。通過FISH試驗(yàn), Boetius等[42]首次從生物學(xué)角度證明甲烷氧化古菌與硫酸鹽還原菌存在共生關(guān)系，并觀察到外殼型的共生體。Raghoebarsing等[21]同樣應(yīng)用FISH方法鑒定了甲烷氧化菌與反硝化菌的共生體：甲烷氧化菌成簇存在于細(xì)胞聚集體中央, 反硝化菌則聚集在周圍。Wankel等[52]利用FISH技術(shù)檢測熱液沉積物中中溫和嗜熱厭氧甲烷氧化菌，結(jié)果發(fā)現(xiàn)所有沉積層孔隙中存在的厭氧甲烷氧化菌為ANME- 1a，并且脫硫疊球菌屬-脫硫球菌屬這類厭氧甲烷菌只有在較低溫度下才能觀察到。Maignien等[53]利用FISH技術(shù)發(fā)現(xiàn)AOM過程中總細(xì)胞的79%為厭氧甲烷氧化菌ANME- 1并且大部分ANME- 1細(xì)胞形成單一反應(yīng)鏈。隨著研究深入，F(xiàn)ISH技術(shù)和離子質(zhì)譜分析法相結(jié)合可以將AOM聯(lián)合體中古菌的系統(tǒng)發(fā)育和功能結(jié)合進(jìn)行研究。Orphan等人[54]應(yīng)用此技術(shù)直接證明了甲烷厭氧氧化偏好利用輕的碳同位素，與其他菌群的同化途徑不同。Ettwig等[22]將FISH和基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜法相結(jié)合，發(fā)現(xiàn)甲烷氧化速率增加伴隨古菌細(xì)胞數(shù)目下降，說明細(xì)菌可能在厭氧甲烷氧化過程中起主導(dǎo)作用。

2.2 16S rRNA

Woese等[55]利用16S或18S rRNA/rDNA技術(shù)比較了二百多種原核生物和真核生物的序列圖譜之后，定義并建立了古菌界，建立了真細(xì)菌(后更名為細(xì)菌)、古細(xì)菌(古菌)和真核生物三大主干。在眾多生物類群中，核糖體序列保守，結(jié)構(gòu)也保守，再加上16S rRNA相比于細(xì)菌核糖體的兩外兩種類型5S rRNA和23S rRNA，遺傳信息比較多，核苷酸數(shù)量適中，因此16S rRNA被認(rèn)為是生物系統(tǒng)發(fā)育最為合適的指標(biāo)，已成為應(yīng)用最為廣泛的標(biāo)記基因[56]。

許多研究以16S rRNA 基因作為標(biāo)記基因，對不同生境中甲烷厭氧氧化菌的多樣性進(jìn)行了表征。Girguis等[57]最先應(yīng)用古菌通用引物對Arch21F/Arch958R建立克隆文庫對富集培養(yǎng)物進(jìn)行多樣性分析，并設(shè)計(jì)專性引物對AR468f/AR736r對ANME- 2c定量分析。Miyashita[58]設(shè)計(jì)了一系列特異性擴(kuò)增甲烷厭氧氧化菌 16S rRNA 基因的一些引物對(ANME- 1, ANME- 2a, ANME- 2b, ANME- 2c and ANME- 3) (表2),并成功應(yīng)用于硫酸鹽濃度較低的厭氧生境中甲烷厭氧氧化菌多樣性的檢測，如產(chǎn)甲烷污泥，水稻土壤，蓮底泥和天然氣土壤等。硝酸鹽甲烷厭氧氧化菌是采用古菌的通用引物(如8F/1492R) 進(jìn)行 16S rRNA 基因的擴(kuò)增，并通過系統(tǒng)發(fā)育分析進(jìn)行甲烷厭氧氧化菌的分類鑒定。Ettwig等[23]利用FISH探針設(shè)計(jì)成引物對，對富集培養(yǎng)物進(jìn)行系統(tǒng)發(fā)育驗(yàn)證，并設(shè)計(jì)引物對qP1F/qP1R, qP2F/qP2R定量分析富集培養(yǎng)物的生物量。

表1 靶標(biāo)甲烷厭氧氧化菌及硫酸鹽還原菌的一些特異性探針

Table 1 Oligonucleotide probes for ANME (Anaerobic Methanotroph)archaea, their sulfate-reducing partners and denitrifying methane-oxidizing bacteria

SAMO： 硫酸鹽甲烷厭氧氧化菌Sulphate-dependent anaerobic methane oxidation；SRB：硫酸鹽還原菌Sulfate Reducing Bacteria；DAMO：硝酸鹽甲烷厭氧氧化菌Denitrification-dependent anaerobic methane oxidation

表2 靶標(biāo)甲烷厭氧氧化菌的一些16S rRNA基因引物

2.3 mcrA

逆甲烷生成途徑是最早被提出, 也是研究最多的關(guān)于甲烷厭氧氧化途徑的假說。研究發(fā)現(xiàn)，產(chǎn)甲烷過程涉及的大部分酶所催化的反應(yīng)都是可逆的，即在不同反應(yīng)條件下，同一反應(yīng)在酶的催化下可向不同方向進(jìn)行，這為逆甲烷產(chǎn)生理論提供了理論支持。硫酸鹽甲烷厭氧氧化菌在酶作用下將甲烷最終轉(zhuǎn)化為CO2(反向產(chǎn)甲烷)，該過程所釋放的電子通過某種電子傳遞體轉(zhuǎn)移到 SRB中，從而使硫酸鹽發(fā)生還原作用。已有研究發(fā)現(xiàn)SAMO過程中的確存在某種酶能夠催化甲烷的氧化，這種酶非常類似產(chǎn)甲烷過程的關(guān)鍵酶-甲基輔酶 M 還原酶(Methyl-coenzyme Mreductase，MCR),該酶在產(chǎn)甲烷過程中能夠催化甲烷的形成[63]。mcrA 基因編碼甲基輔酶M 還原酶(MCR)的α亞基, 而ANME- 1和ANME- 2都有mcrA基因，在甲基輔酶M還原酶的作用下，甲烷首先被氧化為甲醇，再經(jīng)過一系列脫氫酶的作用，最終轉(zhuǎn)化為CO2。

表3 靶標(biāo)硫酸鹽型甲烷厭氧氧化菌的一些 mcrA基因引物

2.4 pmoA

對于甲烷氧化菌的研究，應(yīng)用較多的基因是編碼甲烷單加氧酶(pMMO)的pmoA基因，是好氧甲烷氧化第一步(CH4+2H++O2→CH3OH+H2O)的一個關(guān)鍵酶。M.oxyfera是一個新的甲烷氧化菌的種屬，能在缺氧條件下從亞硝酸鹽氧化甲烷的反應(yīng)中獲取能量。M.oxyfera厭氧微生物中pmoA基因的存在說明其特殊代謝過程：分子態(tài)的氧被從氮氧化物中還原出來，然后用生成的氧氣通過由pMMO開始的完整好氧途徑來氧化甲烷[70]。由于M.oxyfera的pmoA序列尤其是在反引物上有幾個關(guān)鍵堿基的錯配，所以用常用的好氧甲烷氧化菌pmoA基因引物對A189/A682[71]，Mb661[72]/A650[73]都不能擴(kuò)增pmoA基因。Luesken等[26]在前引物A189上把一個不穩(wěn)定堿基替換，就變成了一個兼并引物A189_b，它能夠匹配大多數(shù)的甲烷氧化菌。同時又設(shè)計(jì)了針對亞硝酸鹽為電子受體的厭氧甲烷氧化菌特異的nest-PCR引物，命名為cmo182和cmo568。這些新引物對最早檢測到Ooijpolder排水溝底泥中亞硝酸鹽甲烷厭氧氧化菌的DAMO多樣性[23]，并且得到了特殊脂肪酸的驗(yàn)證[74]。到目前為止，這些引物對陸續(xù)檢測了一些低濃度氧氣生境的DAMO，例如新西蘭的廢水處理(wastewater treatment plants (WWTP)[75- 76]，中國的高寒泥炭沼澤[77]，德國的污染水體[78]。

表4 靶標(biāo)甲烷厭氧氧化菌的一些 pmoA 基因引物

3 甲烷厭氧氧化菌的分子多樣性特征

3.1 硫酸鹽甲烷厭氧氧化菌

16S rRNA的系統(tǒng)發(fā)育分析發(fā)現(xiàn)，古菌域中至少有3個不同的組代表了甲烷營養(yǎng)型古菌：ANME- 1(包括a、b兩個分支)、ANME- 2(包括a、b、c、d四個分支)、 ANME- 3。但是，根據(jù)ANME-mcrA基因的系統(tǒng)發(fā)育分析，甲烷厭氧氧化菌則歸屬于6個不同的發(fā)育型(a, b, c, d, e, f)，其系統(tǒng)發(fā)育位置離產(chǎn)甲烷八疊球菌目等產(chǎn)甲烷菌較遠(yuǎn)。然而，從16S rRNA或ANME-mcrA建立的系統(tǒng)發(fā)育關(guān)系是一致的，比如基于16S rRNA的ANME- 1，- 2c，- 2a，- 3分別對應(yīng)于ANME-mcrA的a-b，c-d，e，f分支?；谶@些分子標(biāo)記的系統(tǒng)發(fā)育分析發(fā)現(xiàn)，不同的發(fā)育類型即可以共同存在于一個海洋甲烷滲漏區(qū)[42]，也可能以某一類型優(yōu)勢存在于一個生境中，比如在黑海生物墊中主要存在ANME- 1，而在水合物脊的滲漏底泥(seep-sediment from Hydrate Ridge)主要是ANME- 2[42]。此外，即使在同一生境中也呈現(xiàn)出不同的群落結(jié)構(gòu)，比如黑海的微生物墊中同時存在ANME- 1和ANME- 2，ANME- 1聚在內(nèi)層，而ANME- 2包圍在外層，說明不同的甲烷厭氧氧化菌群落偏好不同的生態(tài)環(huán)境。ANME- 1和- 2兩大類群在研究的眾多生境中都是主要類群，ANME- 3僅在少數(shù)幾個生境中報(bào)道過。

自然環(huán)境中甲烷的厭氧氧化最早在海底沉積物中發(fā)現(xiàn)。20世紀(jì)70年代以來，開展了大量針對海底沉積物厭氧甲烷氧化古菌生理特性及其多樣性的研究工作。一般認(rèn)為海洋中SAMO與SRB形成共生體，但是陸地生態(tài)系統(tǒng)中硫酸鹽濃度較低，認(rèn)為其可能限制了SRB的生長，從而限制了共生的SAMO的生長。例如，Kadnikov等[79]發(fā)現(xiàn)了貝加爾湖底泥表層(0—20 cm)硫酸根濃度最高僅約0.17 mmol/L，而大于20 cm深度的底泥中均低于0.04 mmol/L，并且建立的古菌克隆文庫中沒有發(fā)現(xiàn)SAMO和SRB。直到2006年，Alain等人[80]首次在陸地生態(tài)系統(tǒng)(喀爾巴阡山脈的泥火山)中發(fā)現(xiàn)大量沉積有機(jī)物轉(zhuǎn)化為甲烷并釋放到大氣中，并且ANME- 2a是主要的功能古菌。之后陸續(xù)有學(xué)者在垃圾填埋場[81]、厭氧水體[82]中檢測到少量(<1%)ANME- 1和ANME- 2古菌的存在。除此以外，還在眾多土壤生境中發(fā)現(xiàn)了另一類名為 AAA 的甲烷厭氧氧化菌(表5)，此類甲烷厭氧氧化菌與 ANME- 2 有最近的親緣關(guān)系，但是與ANME- 2的任何一個分支都不同源。除了ANME- 3，在陸地生態(tài)系統(tǒng)中發(fā)現(xiàn)了其他各類甲烷厭氧氧化菌，有著較高多樣性。此外，從功能基因的定量分析的結(jié)果判斷，土壤不同生境中存在著活躍的甲烷厭氧氧化菌。例如Chang等人[83]應(yīng)用ANME- 2a的特異性引物檢測發(fā)現(xiàn)中國臺灣泥火山7 cm和29 cm深度的土壤中厭氧甲烷菌最豐富，高達(dá)1.4 × 107和 2.15 × 107copies/g 沉積物，而其他深度的土壤中約104copies/g 沉積物。Wrede等人[84]建立了古菌的克隆文庫發(fā)現(xiàn)ANME- 2a占14%，所有硝酸鹽甲烷厭氧氧化菌則占古菌克隆文庫的22%。Takeuchi等人[85]在日本的Kanto平原土壤中發(fā)現(xiàn)甲烷厭氧氧化菌的拷貝數(shù)也達(dá)到104—106copies/g濕土。但是，一般海洋中甲烷厭氧氧化菌數(shù)量>1010個/cm3，在研究最多的黑海的Hydrate Ridge中優(yōu)勢菌ANME- 2最高可達(dá)108個/cm3[35]。

3.2 硝酸鹽甲烷厭氧氧化菌

目前硝酸鹽/亞硝酸鹽甲烷厭氧氧化菌均屬于NC10門，經(jīng)基因組測序、蛋白表達(dá)、生理研究確定此類細(xì)菌命名為CandidatusMethylomirabilis oxygera。雖然16S rRNA 基因與此類細(xì)菌同源的細(xì)菌分布在各種生境中[23]，但是目前關(guān)于硝酸鹽/亞硝酸鹽甲烷厭氧氧化菌的富集培養(yǎng)只存在于兩個生態(tài)系統(tǒng)中：淡水沉積物和污水處理污泥。然而，CandidatusMethylomirabilis oxygera是否是唯一的硝酸鹽/亞硝酸鹽甲烷厭氧氧化菌還不得而知。根據(jù)NC10門設(shè)計(jì)的特異引物[23]，將基因庫中的序列比對之后發(fā)現(xiàn)，此類細(xì)菌可以細(xì)分為4個類群：a，b，c及 d。然而，目前所富集的細(xì)菌均歸屬于a類群，說明a類群是硝酸鹽甲烷厭氧氧化作用的主要功能群。

學(xué)者們在德國寡營養(yǎng)湖(Constance湖[78])的深水底泥表層、日本淡水湖(Biwa 湖[92])的深水底泥表層、內(nèi)陸淺水湖泊底泥表層[93]均能檢測到DAMO菌，并且，用同樣的引物定量分析發(fā)現(xiàn)，Biwa 湖和西湖中DAMO的數(shù)量分別為105—106copies/mL 沉積物及105copies/g干土。此外，在其他生境中，也發(fā)現(xiàn)了一定數(shù)量的DAMO。例如，引物的設(shè)計(jì)者Ettwig等[18]在新西蘭的一個富營養(yǎng)化的溝渠中發(fā)現(xiàn)了107—1010copies/mg DNA 的DAMO。Brunssummerheide泥炭地中維管植物(具有根際泌氧能力)的根系最深達(dá)60 cm，因此在80—100 cm深度發(fā)現(xiàn)了大量的DAMO(3.2×107個/g干土)。Wang等檢測了長期施氮肥的水稻土0—100 cm的DAMO的分布，結(jié)果發(fā)現(xiàn)表層(0—10 cm)中拷貝數(shù)最高((1.0± 0.1)×105—(7.5 ± 0.4)×104copies/g干土)，40 cm以下深度要比表層少一個數(shù)量級，70 cm以下則低于檢測限[94]。因此，在不同的土壤生境中存在豐富的DAMO。

表5 不同土壤生境中甲烷厭氧氧化菌的類型

4 總結(jié)

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Molecular detection of diversity of anaerobic methanotroph in soil

ZHOU Jingyong1, LIU Dongxiu1, HE Chiquan1, LIU Xiaoyan1, SHEN Yanfen2, LONG Xi′en3, CHEN Xueping1,*

1SchoolofEnvironmentalandChemicalEngineering,ShanghaiUniversity,Shanghai200444,China2YuyaoEnvironmentalProtectionMonitoringStation,Yuyao315400,China3InstituteofUrbanEnvironment,ChineseAcademyofSciences,Xiamen361021,China

Anaerobic oxidation of methane is the most important biogeochemical process to reduce methane released into the atmosphere from marine sediments, however, the anaerobic oxidation of methane and related functional microorganisms in soil still remain uncertain. Therefore, the studies on the diversity of anaerobic methanotrophs may be able to assist with reducing methane emissions from soil. Compared with traditional culture-dependent methods, molecular methods independent of culture techniques has vastly improved the knowledge on microbial diversity. This review mainly focused on the recent progress surrounding abundance and diversity of anaerobic methanotrophs in soils with emphasis on the molecular gene markers including 16S rRNA,mcrA andpmoA used for detecting anaerobic methanotrophs. Furthermore, the questions existing in the present research as well as the related resolution were also discussed. Methane oxidation in anoxic environments is microbially mediated and of global significance. In the last decade, the diversity of anaerobic methane oxidation populations has been studied intensively. Initially, most studies concerning environmental AOM were carried out in anaerobic marine waters and sediments where AOM was coupled to sulfate reduction. It is now known that there are also some microorganisms capable of coupling AOM to denitrification. Fluorescence in situ hybridization with target probes firstly showed that the sulfate dependent AOM archaea were in the absence of close physical association with sulfate reducing bacteria. With the development of probes, different types of AOM consortia were visualized. In addition, most investigations on the diversity of AOM archaea involved in the consortia were based on the 16S rRNA ormcrA gene phylogeny. Three lineages of the sulfate dependent AOM have been identified that are referred to as ANME- 1, ANME- 2, ANME- 3. The first nitrate dependent methane oxidation cultures were initially enriched anaerobically, which contained a bacterium belonging to the candidate division NC10. “Candidatus, Methylomirabilis oxyfera,” a member of the uncultured NC10 phylum, forms a novel taxonomic group of bacterial methanotrophs. Recently, special primers targeting methane monooxygenase (pMMO) for detection of anaerobic methanotrophs were developed. Based on these probes and primers, culture independent approaches were used to screen samples from several oxygen-limited habitats for the presence of both sulfate and nitrate dependent methane oxidation bacteria and archaea, e.g. quantitating the abundance of anaerobic methanotrophs by quantity PCR, detecting the community structure by clone library. Although methane oxidation occurs in a variety of different habitats and appears to be performed by different organisms, the distribution of AOM organisms in aquatic and terrestrial ecosystems remains to be fully revealed. Thus, several suggestions for future research on AOM processes and related microorganisms are put forward as follows: 1) to investigate more diverse terrestrial environments where AOM may occur or is known to occur based on genomic and biomarker -related methods. 2) to combine the enrichment culture with molecular method to better understand the mechanism of AOM and related microorganisms. The enrichment or isolation of these organisms will allow for a variety of novel physiological, biochemical, and genomic studies of AOM one or more key organisms. 3) to detect the environmental factors affecting the AOM process or organisms. Future biogeochemical studies also hold the potential to further our understanding of this process. 4) to explore new types of AOM microorganisms coupled with SO2-4, Mn4+, Fe3+, NO-3acting as the electron acceptors. Understanding AOM communities and the environmental conditions under which they consume methane may help to refine computational models for methane cycling on earth and should improve the accuracy of long-term climate change projections.

soil; anaerobic methanotrophs; functional gene; diversity

國家自然科學(xué)基金項(xiàng)目(41101230)

2013- 07- 23;

2014- 06- 12

10.5846/stxb201307231936

*通訊作者Corresponding author.E-mail: xpchen@shu.edu.cn

周京勇, 劉冬秀, 何池全, 劉曉艷, 沈燕芬, 龍錫恩, 陳學(xué)萍.土壤中甲烷厭氧氧化菌多樣性的分子檢測.生態(tài)學(xué)報(bào),2015,35(11):3491- 3503.

Zhou J Y, Liu D X, He C Q, Liu X Y, Shen Y F, Long X E, Chen X P.Molecular detection of diversity of anaerobic methanotroph in soil.Acta Ecologica Sinica,2015,35(11):3491- 3503.