Acquirement And Electrochemical Characteristics Of Methanogenic Isolation From Coastal Riverine Sediment

Coastal riverine is an important composition of coastal zone area, connect the two carbon library of terrestrial ecosystem and ocean, which dual influenced by global climate change and human activities. This work is object to acquire iron(III) reducing bacteria with electroactivity, investigating the direct electron transfer with methanogens and lay a basis for in situ bioremediation by sediment microbial fuel cells.We selected JieHe riverine as the research object, and enrichment the Jh3 sediment sample with iron(III)-reducing medium. Apply Terminal restriction fragment length polymorphism(T-RFLP) and clone library analysis based on 16 S rRNA genes revealed the microbial community of in situ and enrichment sample of Jh3. We further employed roll-tube(Hungate) anaerobic method to get syntrophic methanogenic isolation(S6) with ethanol as the sole electron donor. Then analyze the microbial community and compare the capacity of iron(III) reduction and methane production between methanogenic isolation and co-cultured system with Geobacter metallireducens. For further study, we construct microbial fuel cells to investigate the electroactivity and verify whether existence of direct electron transfer or not in S6. At last, we further separate and purified the methanogen and iron(III) reducing bateria from S6. The main conclusions are as follows:(1) Site Jh3 has the capability of iron(III) reduction which contain iron(III) reducing bacterias such as Geobacter and Clostridium based on T-RFLP and clone libraries. After enrichment of iron reduction medium, Geobacter became the predominant bacteria of Jh3 and the relative abundance of Bacteroides was increased. Methanogens were dominant in archaea community of Jh3 and Methanobacterium/Methanosphaera became predominant archaea in enrichment samples.(2) Acquire methanogenic isolation(S6) from Jh3 iron-reducing enrichment with ethanol as the sole electron donor. Clone library analysis of 16 S rRNA gene showed that Clostridium spp.(close to C. tunisiense) and Methanosarcina barkeri was 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. These results suggested that there was direct electron transfer in the methanogenic isolation, and the dominant Gram-positive Clostridium spp. can potentially directly transfer electron to M. barkeri in a methanogenic isolation.(3) Further separate and purify the methanogenic isolation. Gain a strain of methanosarcina barkeri which can utilize acetate, methanol, trimethylamine to produce amounts of methane, named Jh-Ar. This strain presents a conglobate and general form aggregate under observation by SEM. Separate another iron reduction bacteria named F9 which can utilize acetate, ethanol, methanol and trimethylamine, belonging to Clostridiales Veillonellaceae(maximum homology of 98.82% with Selenomonadaceae str. SB90). This strain presents a long rod-shaped under observation by SEM and can not only reduces the ferric citrate but also shows electrical activity.Clostridium has the potential capabilities of extracellular electron transfer and syntrophic methane production with methanogens by interspecies direct electron transfer. This discovery expands the gram-negative microorganism to gram-positive microorganism which can participate in the interspecies direct electron transfer.