Research On Stoichiometrical Production Of Methane By Microbes Via Direct Interspecies Electron Transfer

For nearly half of century,interspecies H₂/formate transfer has been considered to be the main pathway for electron transfer between microbes.However,recent studies have shown that the direct interspecies electron transfer?DIET?might be alternative to interspecies H₂/formate to realize the electron transfer between some electroactive microbes and methanogens.Methanogens can directly use the electron derived from its syntrophic partner to reduce carbon dioxide to methane via DIET,which greatly improves the efficiency of methane production and the amount of methane production.Although great progress has been made in co-culture stoichiometrically producing methane via DIET,there is a lack of in-depth study on the microbes involved in methane production via DIET,the effect of conductive materials on methane production via DIET,and the mechanism for methane production by methanogens via DIET at present.In this master's thesis,the possibility of Geobacter grbiciae and Methanosarsina barkeri 800 syntrophically converting ethanol or propionate/butyrate to methane via DIET was studied firstly in order to expand the microorganisms that acts as electron donor in DIET.Then,the co-culture that might stoichiometrically produce methane via DIET was enriched from natural environment by using rice paddy sediments as inoculum and ethanol as substrate,respectively.After that,the effects of inoculation amount and the Cow dung-based biochar prepared under different conditions on the co-culture of Geobacter metallireducens GS-15and Methanosarcina barkeri 800 stoichiometrically converting ethanol to methane via DIET was studied.Finally,the mechanism for co-culture of G.metallireducens GS-15 and M.barkeri 800 stoichiometrically reducing CO₂ to methane via DIET was explored by transcriptome sequencing analysis.Main results are as follows:1.G.grbiciae was capable of forming functional co-culture with M.barkeri 800 via DIET only supplemented with granular activated carbon or nano-magnetite.The co-culture could syntrophically convert ethanol to methane,however,it couldn't convert propionate or butyrate to methane.These results expand the microorganisms acting as electron donor in co-culture that could stoichiometrically reduce CO₂ to methane via DIET.2.The enrichment culture that could stoichiometrically convert ethanol or propanol/butanol to methane was obtained with rice paddy sediments as inoculum and ethanol as substrate,respectively.No H₂ was detected in process of stoichiometrically converting ethanol or propanol/butanol to methane by the enrichment culture.In addition,the conductive material?biochar and nano-magnetite?could improve the methanogenesis rate of the enrichment culture.These results indicated that DIET might exist in the enrichment culture.Metagenomic analysis showed that Desulfovibrio aminophilus and Sedimentibacter sp.b4 were main bacteria in the enrichment culture,and the archaea were mainly unclassified-g-methanobacterium and Methanosarcina mazei.3.Compared with the high amount of G.metallireducens GS-15/M.barkeri 800 co-culture inoculation?10%?,the low amount of co-culture inoculation?5%?was favorable for methane production in G.metallireducens GS-15/M.barkeri 800 co-culture supplemented with biochar.When the carbonization time of biochar was controlled for 2 h,the biochar carbonized at 400?had the best effect on methane production in G.metallireducens GS-15/M.barkeri 800 co-culture.When the carbonization temperature of biochar was controlled at 400?,the biochar carbonized for 2.5 h had the best effect on methane production in G.metallireducens GS-15/M.barkeri 800 co-culture.The characterization's analysis of physical and chemical properties of biochar indicated that the carbonization temperature and carbonization time of biochar mainly affected the surface functional groups,pH,specific surface area,and pore size of biochar,and then affected the production of methane in G.metallireducens GS-15/M.barkeri 800 co-culture via DIET.4.Cytochrome b?Cytb?of M.barkeri 800,as well as F₄₂₀-reducing hydrogenase?Frh?,Ech hydrogenase?Ech?,heterodisulfide reductase?Hdr?,and F₄₂₀H₂ dehydrogenase?Fpo?of M.barkeri 800,plays a significant role in the co-culture of Geobacter metallireducens GS-15 and Methanosarcina barkeri 800 stoichiometrically reducing CO₂ to methane via DIET.Cytb mediated electron transfer from G.metallireducens GS-15 to M.barkeri 800 and then reduced the oxidized methanophenazine?MP?to the reduced methanophenazine?MPH₂?.Hdr catalyzed the reduction of CoM-S-S-CoB to CoM-SH and CoB-SH with MPH₂.Fpo catalyzed the reduction of oxidized coenzyme F420(F₄₂₀)to reduced coenzyme F₄₂₀(F₄₂₀H₂)with MPH₂ under the function of proton transmembrane potential.Frh catalyzed the oxidation of F₄₂₀H₂ to generate H₂,and then Ech catalyzed the reduction of oxidized ferredoxin(Fd_ox)to reduced ferredoxin(Fd_red)with H₂ under the function of proton transmembrane potential.