互營乙酸氧化菌研究進展

吳 偉， 鄭珍珍， 麻婷婷， 承 磊， 張 輝

(1. 中國石化集團勝利油田分公司油氣開發管理中心， 山東 東營 257097； 2. 農業部沼氣科學研究所 農業部農村可再生能源開發利用重點實驗室， 四川 成都 610041)

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互營乙酸氧化菌研究進展

吳 偉1， 鄭珍珍2， 麻婷婷2， 承 磊2， 張 輝2

(1. 中國石化集團勝利油田分公司油氣開發管理中心， 山東 東營 257097； 2. 農業部沼氣科學研究所 農業部農村可再生能源開發利用重點實驗室， 四川 成都 610041)

乙酸是沼氣發酵過程中的重要中間代謝產物，可以通過乙酸裂解途徑和互營乙酸氧化產甲烷途徑代謝產生甲烷。文章主要綜述了互營乙酸氧化菌的研究歷史和最新進展，討論了影響互營乙酸氧化產甲烷代謝的環境因素，并展望了互營乙酸氧化菌的研究趨勢。

沼氣發酵； 互營乙酸氧化； 產甲烷途徑； 環境脅迫

沼氣發酵是在厭氧條件下，由多種不同類型的細菌和古菌微生物，通過互營代謝等協同作用，將復雜生物質，如秸稈、畜禽糞便、石油烴等轉化為CH4和CO2的微生物學過程(見圖1)。沼氣發酵過程不僅發生在沼氣池等人工環境中，也普遍存在于油藏等地下缺氧環境中[1-2]。參與沼氣發酵的厭氧微生物是地下油藏的主要功能微生物，在油藏生物地球化學循環和提高石油采收率中起著重要的作用[3-4]。在沼氣發酵過程中，乙酸是重要的中間代謝產物。目前已知乙酸轉化為甲烷可通過兩種代謝途徑來實施，一是乙酸裂解型產甲烷途徑，乙酸營養型產甲烷烷古菌(AM)直接降解乙酸產生CH4和CO2，其中乙酸的甲基部分轉化為CH4，羧基端轉化為CO2。迄今已發現2個屬的產甲烷古菌(Methanothrix和Methanosarcina)可以通過該途徑降解乙酸產生甲烷[5]。另一種是互營乙酸氧化產甲烷途徑(SAO-HM)[6]，乙酸先分解為H2和CO2，再通過氫營養型產甲烷古菌(HM)轉化產生CH4，這需要互營乙酸氧化菌(SAOB)和氫營養型產甲烷古菌(HM)通過互營代謝作用來完成。本文主要介紹SAOB的研究歷史和國內外研究進展，以期讀者對沼氣發酵過程有一個全面的了解。

圖1 沼氣發酵過程的一般代謝途徑

1 國內外研究現狀

1.1 互營乙酸氧化菌的研究歷史

1.2 互營乙酸氧化菌的熱力學特征

吉布斯自由能(△G)決定了一個反應是否可以自發進行，乙酸是氧化態最高的有機物之一，其進一步發酵產生H2和CO2的反應，在標準狀況下的吉普斯自由能(△G0’)為+104.6 kJ·mol-1(見表1反應2)，難以自發進行。HM可以利用H2和CO2產生CH4，其△G0’為 -135.6 kJ·mol-1。當這兩個反應耦聯，SAOB“發酵”乙酸產生的H2，可以快速的被HM轉化為CH4，整個反應體系中保持著極低的氫分壓。SAOB-HM途徑的總反應式是1 mol乙酸產生1 mol CH4和1 mol CO2，△G0’為-31.0 kJ·mol-1(見表1反應3)，這樣SAOB產生H2和CO2的反應就可以源源不斷地進行。整個反應與AM直接利用乙酸產CH4的△G0’一樣(見表1反應1)，但是這些能量需要維持SAOB和HM這2種微生物的生命代謝活動，其能量劣勢導致了SAOB的代謝速率低、生長非常緩慢[29]。

表1 乙酸降解產甲烷的代謝反應及其熱力學特征[6]

注：*代表乙酸的甲基碳的流向，標準吉布斯自由能數據參考文獻[30]。

1.3 互營乙酸氧化菌的微生物多樣性研究

目前已分離的SAOB都是嚴格厭氧菌，除AOR丟失外，共有5個被鑒定為新種，它們都具有與產甲烷古菌共培養，進行互營乙酸氧化產甲烷代謝的功能(見表2)。根據文獻報道，其中有5個菌是嗜熱菌，最適生長溫度都在55℃ ～65℃，只有ClostridiumultunenseBST是中溫菌，最適生長溫度為37℃[10]。AOR,ThermacetogeniumphaeumPBT和ThermotogalettingaeTMOT不僅可以互營氧化乙酸，還具有進行同型產乙酸功能[9，11-12](見表2)。多個SAOB可以利用甜菜堿、半胱氨酸、丙酮酸、葡萄糖等有機物生長，具有不同的代謝功能。SAOB生長緩慢，分離周期長，基于分離培養的傳統方法難以全面認識SAOB。而基于未培養的微生物分子生態學方法和同位素示蹤技術的應用，科學家發現SAOB廣泛分布在人工和自然環境中。Sun[21]等發現在13個連續流攪拌(CSTR)沼氣工程中，其中有10個反應器中存在各種類型的SAOB。Lee[31]等還發現厭氧消化反應器中SpirochaetesCluster II可能代表一類新的SAOB。Ito等通過RNA-SIP和MAR-FISH等證實Synergistes group 4中也含有SAOB[32]。在高氨[33-35]、高溫[19-36]、高濃度揮發性脂肪酸(VFA)[37]和低水力停留時間(HRT)[38]等環境條件下，互營乙酸氧化產甲烷代謝通常是有機質降解產甲烷的主要途徑。在高溫水稻土中，Thermacetogenium和Thermoanaerobacteriaceae是乙酸互營氧化代謝產甲烷過程中的關鍵細菌類群[22-23]。在高溫油藏中也發現了參與互營乙酸氧化產甲烷的細菌類群。Nazina等發現中國大港油田，Thermoanaerobacteriales、Thermotogales、Nitrospirales和Planctomycetales是參與互營乙酸氧化產甲烷的主要細菌類群[28]，Mayumi發現Thermacetogenium和Methanothermobacter是日本Yabase 油藏互營乙酸氧化產甲烷的主要細菌和古菌類群[27]。Gieg[26]等推測SAO-HM途徑是原油降解過程中的主要產甲烷途徑。Conrad小組報道在以色列Kinneret湖底沉積物中(15℃ ～30℃)，互營乙酸氧化產甲烷也是乙酸代謝的主要途徑[24]，但是它們后來的同位素示蹤實驗卻發現Methanothrix是代謝乙酸產甲烷的主要功能菌[39]。這些研究表明在很多獨特的生態系統中，還存在大量未分離培養的SAOB，它們在碳素生物地球化學循環中起著重要的生理生態學功能。

表2 互營乙酸氧化菌的純培養物

注：ND為未測出。

1.4 影響互營乙酸氧化產甲烷代謝的環境因子

1.5 互營乙酸氧化菌的生物強化作用

2 研究展望

綜上所述，通過培養和未培養方法，發現SAOB廣泛參與了沼氣發酵過程，并起著重要的生理生態學功能，但是迄今為止，還不清楚SAOB響應和調控乙酸代謝的分子機理。氨,溫度,pH值,HRT和VFA等環境因子的改變，會影響SAOB的豐度和群落組成，但是，對SAOB應答環境脅迫的分子機制研究還非常有限，這些有待更深入去探討。

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Recent Advances on Syntrophic Acetate Oxidation Bacteria /

WU Wei1, ZHENG Zhen-zhen2, MA Ting-ting2, CHENG Lei2, ZHANG Hui2/

(1. Center for oil and gas development of Shengli Oifield Company, China Petroleum & Chemical Corporation， Dongying 257091,China; 2. Biogas Institute of Ministry of Agriculture, Key Laboratory of Development and Application of Rural Renewable Energy of Ministry of Agriculture, Chengdu 610041, China )

Acetate is an important intermediate during biogas fermentation, which could be converted into methane through acetoclastic methanogenesis and syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis. This paper mainly reviewed the history and progress of research on syntrophic acetate oxidation bacteria, and discussed the environmental factors affecting the pathway of syntrophic acetate oxidation coupled hydrogenotrophic methanogenesis. And the further study was prospected.

Biogas fermentation； syntrophic acetate oxidation； methanogenic pathway； environmental stress

2015-12-20

項目來源： 國家高技術研究發展計劃(2013aa064401)； 中國農業科學院基本科研業務費 (2013ZL001)； 微生物資源前期開發國家重點實驗室項目(SKLMR-20150605)

吳 偉(1968- )，男，四川簡陽人，高級工程師，主要研究方向為提高石油采收率技術，E-mail: wuwei656.slyt@sinopec

張 輝，E-mail: zhanghuits@aliyun.com

S216.4;X172

A

1000-1166(2016)02-0003-06