Roest Kees, Altinbas Mahmut, Paulo Paula L, Heilig H G H J, Akkermans Antoon D L, Smidt Hauke, de Vos Willem M, Stams Alfons J M
Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT, Wageningen, The Netherlands.
Microb Ecol. 2005 Oct;50(3):440-6. doi: 10.1007/s00248-005-0237-5. Epub 2005 Nov 24.
To gain insight into the microorganisms involved in direct and indirect methane formation from methanol in a laboratory-scale thermophilic (55 degrees C) methanogenic bioreactor, reactor sludge was disrupted and serial dilutions were incubated in specific growth media containing methanol and possible intermediates of methanol degradation as substrates. With methanol, growth was observed up to a dilution of 10(8). However, when Methanothermobacter thermoautotrophicus strain Z245 was added for H2 removal, growth was observed up to a 10(10)-fold dilution. With H2/CO2 and acetate, growth was observed up to dilutions of 10(9) and 10(4), respectively. Dominant microorganisms in the different dilutions were identified by 16S rRNA-gene diversity and sequence analysis. Furthermore, dilution polymerase chain reaction (PCR) revealed a similar relative abundance of Archaea and Bacteria in all investigated samples, except in enrichment with acetate, which contained 100 times less archaeal DNA than bacterial DNA. The most abundant bacteria in the culture with methanol and strain Z245 were most closely related to Moorella glycerini. Thermodesulfovibrio relatives were found with high sequence similarity in the H2/CO2 enrichment, but also in the original laboratory-scale bioreactor sludge. Methanothermobacter thermoautotrophicus strains were the most abundant hydrogenotrophic archaea in the H2/CO2 enrichment. The dominant methanol-utilizing methanogen, which was present in the 10(8)-dilution, was most closely related to Methanomethylovorans hollandica. Compared to direct methanogenesis, results of this study indicate that syntrophic, interspecies hydrogen transfer-dependent methanol conversion is equally important in the thermophilic bioreactor, confirming previous findings with labeled substrates and specific inhibitors.
为深入了解实验室规模的嗜热（55摄氏度）产甲烷生物反应器中，参与甲醇直接和间接产生甲烷过程的微生物，将反应器污泥破碎，并在含有甲醇和甲醇降解可能中间体作为底物的特定生长培养基中对系列稀释液进行培养。以甲醇为底物时，稀释至10⁸仍可观察到生长。然而，当添加嗜热自养甲烷杆菌菌株Z245以去除氢气时，稀释至10¹⁰仍可观察到生长。以氢气/二氧化碳和乙酸盐为底物时，分别稀释至10⁹和10⁴仍可观察到生长。通过16S rRNA基因多样性和序列分析鉴定了不同稀释度下的优势微生物。此外，稀释聚合酶链反应（PCR）显示，除了以乙酸盐富集的样品中外，所有研究样品中古菌和细菌的相对丰度相似，在以乙酸盐富集的样品中，古菌DNA含量比细菌DNA少100倍。在含有甲醇和菌株Z245的培养物中，最丰富的细菌与甘油摩尔氏菌关系最为密切。在氢气/二氧化碳富集培养物中以及原始实验室规模生物反应器污泥中，均发现了与热脱硫弧菌亲缘关系较近且序列相似性高的菌株。嗜热自养甲烷杆菌菌株是氢气/二氧化碳富集培养物中最丰富的氢营养型古菌。在10⁸稀释度下存在的主要利用甲醇的产甲烷菌与荷兰甲烷甲基菌关系最为密切。与直接产甲烷相比，本研究结果表明，在嗜热生物反应器中，依赖种间氢转移的甲醇合成代谢转化同样重要，这证实了先前使用标记底物和特异性抑制剂的研究结果。
