Establishment And Enhancement Of Direct Interspecies Electron Transfer Between Syntrophic Microorganisms During Anaerobic Methanogenesis

Conversion of organic wastes to methane via anaerobic digestion(AD)has been considered as an effective energy strategy.A major factor limiting the effectiveness of AD is the relatively slow syntrophic metabolism of the fermentative intermediates,such as organic acids and alcohols.During the past half a century,the predominant working mode for syntrophic metabolism had been considered as interspecies hydrogen trnasfer(IHT),in which hydrogen served as the electron carrier for interspecies electron and energy transport.However,IHT is a weak biological link and easily influenced by the operating conditions,such as pHs,organic loading rates(OLRs)and toxic inhibitors.When IHT is limited,it can easily cause the increase of hydrogen partial pressure of anaerobic system,break the balance of syntrophic metabolism and finally lead to a stagnation of methanogenesis.Recently,the methane-production microbial electrolysis cells(MECs)as a novel energy strategy,driven by exoelectrogenic bacteria(mainly Geobacter species),have received considerable attentions.Just inserting a pair of electrodes into an anaerobic digester to form a single-chamber methane-production MEC is expected to extend the methanogenic pathway during AD.More importantly,the methane-production MEC established by Geobacter species and methanogens is expected to form a new methanogenic pathway.Direct interspecies electron transfer(DIET)has been considered as an alternative to IHT.During DIET,the electrically conductive pili and outer surface c-type cytochromes are responsible for the extracellular electron and energy exchange.However,there are still some yet unclear problems involved in DIET during traditional AD,such as the establishment and enhancement of DIET,operating characteristics as well as the available substrates.Based on these considerations,in this study,the establishment and enhancement of DIET during traditional AD had been proposed to accelerate the decomposition of organic acids and maintain the acidic balance of anaerobic system as well as syntrophic metabolism stable,in the presence of applied electric field,conductive materials or ethanol.The main results are as follows:(1)A pair of graphite electrodes was inserted into an up-flow anaerobic sludge blanket(UASB)reactor to form a single-chamber methane-production MEC.The results showed that the MEC could substantially reduced the start-up time during the start-up stage.During the acidic feeding stage,the methanogenesis in the control reactor(without electrodes)was almost stagnant.Conversely,the MEC could maintain the methanogenic metabolism stable and to occur.Fluorescence in situ hybridization(FISH)analysis showed that the hydrogenotrophic methanogens were significantly enriched in the cathodic biofilm of MEC.The relative abundance of hydrogenotrophic methanogens enriched in the cathodic biofilm was higher than that in the suspended sludge as well as control reactor about 20-30 percentage points,suggesting that,the methanogenic pathway of biocathode could be established by the applied electric field.In the study with municipal sludge,the results showed that MEC could significantly accelerate the decomposition of complex organics contained in the municipal sludge,such as carbohydrates,proteins and organic acids,as well as improve the methane production.High-throughput sequencing showed that Geobacter species and anaerobic fermentative microorganisms were largely enriched in the anodic biofilm.The sum of abundance of Geobacter species and anaerobic fermentative microorganisms in the anodic biofilm was significantly higher than that in the suspended sludge as well as initial seed sludge.The syntrophic connection between Geobacter species and anaerobic fermentative microorganisms might be an important reason resulting in the enhancement of complex organic decpmposition in MEC.However,based on the conversation of electron,the methane production via the methanogenic pathway of biocathode only accounted for the total methane production of MEC about 13.5%.The methane production via the hydrogenotrophic methanogenesis and aceticlastic methanogenesis accounted for the total methane production about 35%.Therefore,more than 50%of the total methane might be produced from the other unknown pathway.Remarkably,the conductivity of sludge in MEC was higher than that in the control as well as initial seed sludge.Together with Methanosaeta species prevailing in the microbial communities,DIET between Geobacter and Methanosaeta species might be an important reason for the unknown methane production.It was likely that electrode materials served as the electrical conduit facilitated syntrophic metabolism between Geobacter and Methanosaeta species.(2)In order to investigate the mechanisms involved in DIET promoted by conductive materials during AD,the conductive carbon-based materials,such as graphite,biochar and carbon cloth,were supplemented to the traditional anaerobic digesters.The results demonstrated that,as the increase of influent OLRs.the conductive carbon-based materials with the different types could improve the methane production about 30-45%.Upon removal of the conductive carbon-based materials,methane production and COD removal declined to values comparable to the control digesters(without conductive materials),suggesting that the improved performances prior to the removal of conductive materials could be attributed to the high conductivity of conductive carbon-based materials rather than biomass retention.High-throughput sequencing showed that,Geobacter and Methanosaeta species were largely enriched on the surface of conductive carbon-based materials.The sum of relative abundance of Geobacter and Methanosaeta species was higher than that in the suspended sludge as well as control digester,suggesting that conductive carbon-based materials could facilitate DIET between Geobacter and Methanosaeta species.When IHT was limited by the stressed conditions,DIET promoted with the conductive carbon-based materials might replace IHT to become the predominant working mode of syntrophic metabolism and maintain syntrophic metabolism stable.However,the conductive carbon-based materials could not sufficiently enhance AD of complex organic wastes,in which the acidogenesis was the rate-limiting step.Magnetite(mainly Fe3O4)supplemented to the acidogenic phase could enrich the Fe(III)-reducing microorganisms that were capable of directly decomposing complex organics via dissimilatory iron reduction,accelerate the decomposition of complex organic wastes into simples as well as improve the acidification efficiency.GAC supplemented to the methanogenic phase could establish DIET and relieve the accumulation of organic acids.(3)In order to enrich Geobacter species in the traditional anaerobic digesters,ethanol,with a higher oxidizing heat,was used as the substrate to culture the UASB reactor.After the culture,the syntrophic degradation of propionate/butyrate was significantly improved in the ethanol-stimulated reactor.The ethanol-stimulated reactor could withstand a higher OLR propionate/butyrate impacts than the traditional-stimulated reactor(never ethanol fed).The conductivity of ethanol-stimulated enrichments was as 5 folds(for propionate)/76 folds(for butyrate)as that of traditional enrichments.Further experiments demonstrated that granular activated carbon(GAC)could improve the syntrophic metabolism of propionate/butyrate in the ethanol-stimulated enrichments,while had almost no effects on the traditional enrichments.Also,the high hydrogen partial pressure could inhibit the syntrophic propionate/butyrate degradation in the traditional enrichments,but its effect on that of ethanol-stimulated enrichments was negligible.Microbial community analysis revealed that Geobacter species were only detected in the ethanol-stimulated enrichments.Together with the significant increase of Methanosaeta or Methanosarcina species in these enrichments,the potential DIET between Geobacter and Methanosaeta or Methanosarcina species might be established.In order to further facilitate the established DIET,during the ethanol-stimulated culture,biochar was supplemented to the UASB reactor.The results showed that biochar could further improve the syntrophic metabolism of propionate/butyrate.In the biomass attached to the biochar,the relative abundance of Geobacter species was 15-20%,and the sum of Methanosaeta and Methanosarcina was higher than 60%.However,the continuous supply of ethanol will increase the considerable costs,and is obviously uneconomical in the application of DIET.Under the conditions employed,the ethanol-abundant acidogenic products could be produced via ethanol-type fermentation when acidogenic-phase pH was kept at 4.0-4.5.The enrichments in the methanogenic phase continuously stimulated by the ethanol-abundant acidogenic products presented a higher conductivity as well as more positive response to GAC supplemented.Microbial community analysis showed that Geobacter species were only detected in the methanogenic enrichments stimulated by the ethanol-abundant acidogenic products.Together with the significant increase of Methanosarcina species in these enrichments,the potential DIET between Geobacter and Methanosarcina species might be permanently established in the methanogenic digester to maintain the acidic balance as well as syntrophic metabolism stable.