产甲烷分离物中Clostridium spp.与Methanosarcina barkeri潜在的种间直接电子传递

Potential direct interspecies electron transfer(DIET) from Clostridium spp. to Methanosarcina barkeri in methanogenic isolates

【目的】革兰氏阴性菌Geobacter metallireducens可以与乙酸型产甲烷菌Methanosaeta harundinacea或Methanosarcina barkeri通过种间直接电子传递(DIET)还原CO2产甲烷。本实验室前期的研究发现Methanosarcina mazei和Geobacteraceae在铁还原富集培养中形成团聚体,可能存在直接电子传递。然而,革兰氏阳性菌(如Clostridium spp.)与产甲烷菌是否存在种间直接电子传递尚不明确。【方法】采用Hungate厌氧滚管法,以乙醇为唯一电子供体从铁还原富集培养体系中获得产甲烷分离物(S6)。通过T-RFLP及克隆文库分析群落多样性,结合循环伏安法等电化学方法研究产甲烷分离物的电活性。【结果】Clostridium spp.(与C.tunisiense相似性最高)和M.barkeri分别在S6细菌和古菌群落中占优势。S6与G.metallireducens共培养后铁还原和产甲烷能力未明显增加,Clostridium spp.可能与G.metallireducens类似,将电子直接传递给产甲烷菌M.barkeri产甲烷。此外,电化学检测发现,在用透析袋包裹电极阻碍微生物与电极表面通过直接接触形成生物膜的条件下,电流密度显著降低,并且循环伏安扫描无明显氧化还原峰。【结论】产甲烷分离物S6中存在直接电子传递途径。本工作提出在产甲烷分离物中占优势的革兰氏阳性菌Clostridium spp.和M.barkeri之间可能存在种间直接电子传递。

[Objective] Geobacter metallireducens, a Gram-negative bacterium, can directly transfer electron to acetoclastic methanogens such as Methanosaeta harundinacea and Methanosarcina barkeri for reducing carbon dioxide to methane. Our previous results showed that Methanosarcina mazei and Geobacteraceae formed aggregates in an iron（III）-reducing enrichment culture indicating direct interspecies electron transfer. However, the capability of direct electron transfer with methanogens for Gram-positive iron（III）-reducing bacteria such as Clostridium spp. is still unknown. [Methods] In this further study, methanogenic isolates（S6） were achieved from the iron（III）-reducing enrichment by roll-tube（Hungate） method with ethanol as the sole electron donor. We used terminal restriction fragment length polymorphism（T-RFLP） and clone library analysis to investigate the community of S6 and used electrochemical method such as cyclic voltammetry（CV） to confirm the electroactivity of isolates. [Results] Clone library analysis of 16 S r RNA gene showed that Clostridium spp.（close to C. tunisiense） and Methanosarcina barkeri were predominant in the bacterial and archaeal community, respectively. Interestingly, addition of G. metallireducens into S6 did not increase the ability of both iron（III） reduction and methanogenesis, indicating that Clostridium spp. may play a similar role in direct interspecies electron transfer from G. metallireducens to M. barkeri. Furthermore, current generation of the S6 suspension dramatically decreased when contact between the organisms and the electrodes was prevented by dialysis bag, and CV showed no obvious redox peaks. [Conclusion] These results suggested that there was direct electron transfer in the methanogenic isolates. This work demonstrated that the dominant Gram-positive Clostridium spp. can potentially directly transfer electron to M. barkeri in methanogenic isolates.