Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H2 plus CO2, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day−1 for methanol, 0.38 mM day−1 for acetate, 0.90 mM day−1 for H2, and 1.85 mM day−1 for formate. Substrate consumption and CH4 production during all tests suggested that at least three different physiologic types of methanogens were present: H2 plus CO2 or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H2 and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO2-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.
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机译：在石油烃污染的含水层中，通过一系列的四次推拉试验对产甲烷活性进行了研究，这些试验采用乙酸盐，甲酸盐，H2加CO2或甲醇，以不同组的产甲烷古生菌为目标。此外，通过分子分析探索了产水和含水层物质中产甲烷菌的群落组成，即荧光原位杂交（FISH），用古细菌特异性引物对ARCH915和UNI扩增的16S rRNA基因的变性梯度凝胶电泳（DGGE）。 -b-rev，以及从主要DGGE带进行DNA测序。随后将分子分析与推挽测试数据进行比较。除单独的试验中加入2-溴乙烷磺酸盐（一种产甲烷菌的特异性抑制剂）外，所有试验均产生甲烷。甲醇的底物消耗率为0.11 mM day -1 ，乙酸盐为0.38 mM day -1 ，H2为0.90 mM day -1 ，甲酸盐1.85 mM天 -1 。在所有测试中，底物消耗和CH4的产生表明存在至少三种不同生理类型的产甲烷菌：H2 + CO2或甲酸盐，乙酸盐和甲醇利用物。 DGGE图谱中存在15至20个条带，表明古细菌种群多样化。高H2和甲酸的消耗率与甲烷化古细菌消耗这些底物（与Methanomicrobiaceae的几个成员相关的16S rRNA基因序列）和使用FISH检测Methanomicrobiaceae的多样性高度一致（占DAPI的1.4％[4'，6-在一个水样中用二mid基-2-苯基吲哚]染色的微生物；探针MG1200）。醋酸盐的大量消耗与存在与专性醋酸盐降解物Methanosaeata concilii相关的序列以及通过FISH检测该物种（占总微生物的5至22％；探针Rotcl1）相一致。结果表明，回弹碎屑和消耗CO2型底物的产甲烷菌都可能参与了烃降解的最终步骤，而甲醇的产甲烷作用则微不足道。 DGGE剖面进一步表明水和含水层材料中的古细菌群落组成相似。本研究中采用的水文地质和分子方法相结合，提供了有关石油烃污染的含水层中群落和产甲烷菌潜在活性的改进信息。
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