Enhanced Microbial Electron Transfer With Exoelectrogenic Bacteria And Carbon/Iron-based Conductive Materials For Promoting Fermentative Hydrogen And Methane Production

Biological hydrogen and methane production through anaerobic fermentation of biomass to develop clean fuels can be of great significance for achieving carbon neutrality in our country.The electron flow and interspecies interaction mechanisms within the fermentive microbial community need to be elucidated in order to design efficient biological gas production processes.In this study,the microbial electron transfer pathways in the syntrophic hydrogen production system composed of exoelectrogenic bacteria and hydrogen-producing bacteria(HPB)were investigated;the response mechanisms of the co-culture biofilms of electron-donating bacteria and electron-accepting methanogen to the addition of conductive carbon felt or applied voltage were revealed;the microbial electron transfer and syntrophic metabolism were enhanced using conductive materials,such as carbonized metal-organic frameworks(MOFs)or magnetite nanoparticles(MNPs),to boost fermnetative hydrogen and methane production from biomass.The mechanisms of hydrogen production through syntrophic interation between exoelectrogenic bacteria(Geobacter metallireducens)and HPB were revealed,and the effects of G.metallireducens serving as an external redox balance regulator on the hydrogen production metabolic pathways,electron transfer properties and microbial community structure of the hydrogen production system were elucidated.The fermentative hydrogen yield can achieve327.1 m L/g at the inoculum volume ratio of G.metallireducens culture to the hydrogen-producing sludge(HPS)of 60 m L/30 m L(the total volatile solid(TVS)ratio of 0.08),which was 65.2% higher than the control group without G.metallireducens culture addition.Soluble metabolic by-products such as acetate generated from the degradation of glucose by HPB can be oxidized by G.metallireducens to produce electrons for regulating the redox potential of the HPS system,resulting in the increased NADH/NAD+ ratio from 1.03 to 1.26.This would favor enhancing NADH-dependent hydrogen production pathway(NADH + +? + + H2).The presence of c-type cytochromes(c-Cyts)in the extracellular polymeric substance(EPS)of G.metallireducens worked as electron shuttles/redox mediators to enhance the intracellular electron transfer system(ETS)activity and extracellular electron transfer(EET)capacity of HPB.The addition of G.metallireducens culture brought about the increased relative abundance of Clostridium sensu stricto,the major hydrogen producer in the HPS system,from61.5% to 76.7%,which can be attributed to a good mutualistic symbiosis relationship formed by both of them.Therefore,the fermentative hydrogen production performance of the HPS system was significantly enhanced.The electron transfer response mechanisms of the co-culture biofilms of exoelectrogenic bacteria(Geobacter sulfurreducens)and methanogenic archaea(Methanosarcina barkeri)to the addition of conductive carbon felt or applied voltage were investigated.The methane yield of the co-culture increased from 191.5 m L/g to 358.1 m L/g with adding the conductive carbon felt,whereas which only reached 222.7 m L/g when the voltage of 0.5 V was applied to the carbon felt as the electrode.SEM analysis showed that G.sulfurreducens and M.barkeri co-adhered to the surface of the conductive carbon felt;nevertheless,G.sulfurreducens only concentrated on the anodic carbon felt,while M.barkeri only distributed on the cathodic carbon felt when the voltage of 0.5 V was applied.In the co-culure system with a voltage of 0.5 V,G.sulfurreducens was responsible for degrading acetate to produce electrons and donate to the anode,and M.barkeri was in charge of receiveing electrons from the cathode to reduce CO₂ to produce methane.When the applied voltage exceeded the hydrogen evolution potential,protons directly combined with electrons at the cathode to produce hydrogen.Electrochemical analysis indicated that the areal capacitance(Ca)of the anodic carbon felt with G.sulfurreducens attached increased by 1.1 times compared with the sterile carbon felt(blank group);while the Ca value of the cathodic carbon felt with M.barkeri attached drcreased by 19.8%;and yet the the Ca value of the carbon felt with co-culture both attached without applied voltage increased by 23.9%.These results were mainly related to the compositions of the biofilm attached to the carbon felt.Three-dimensional excitation-emission matrix(3-D EEM)spectroscopy and the polysaccharide content analysis suggested that the content of hemeprotein-like substances in anodic carbon felt biofilm was highest,which was favourable to the improvement in electrochemical performance;while the hemeprotein-like substances content in cathodic carbon felt biofilm was relatively low but polysaccharides content was high,which was unfavourable for the electron reception of M.barkeri from cathode;the carbon felt biofilm without applied voltage had an intermediate content of hemeprotein-like substances.Therefore,M.barkeri attached to the carbon felt without applied voltage was more efficient to receive electrons for CO₂ reduction compared with cathodic carbon felt biofilm,resulting in a higher methane yield.Zeolite imidazolate framework-67(ZIF-67)-derived porous carbon(PC)was hypothesized to act as a microbial electron transfer highway and assessed with respect to understanding the fluorimetric and electrochemical responses of multilayered EPS.The highest biomethane yield(614.0 m L/g)from ethanol was achieved in the presence of 100 mg/L PC prepared at a carbonization temperature of 800 °C(PC-800),which was 28.2% higher than that without PC addition.Electrochemical analysis revealed that both the redox peak currents and conductivity of the methanogenic sludge increased,while the free charge transfer resistance decreased with PC-800 addition.The conductive PC-800 potentially functioned as an abiotic electron conduit to promote direct interspecies electron transfer,thereby resulting in decreased expression of functional genes associated with electrically conductive pili(e-pili)and hemeproteins.Additionally,PC-800 stimulated the secretion of redox-active humic substances(HSs).3-D EEM analysis indicated that the largest increase in percent fluorescence response of HSs occurred in the tightly bound EPS(TB-EPS)with addition of PC-800,and which was attributed to the strong complexation ability of PC-800 particles to hydroxyl/carboxylic/phenolic moieties of HSs contained in the TB-EPS.Microbial community analysis revealed that syntrophic/exoelectrogenic bacteria as well as hydrogenotrophic/electrotrophic methanogens were enriched with adding PC-800.These favorable impacts resulted in the enhanced methanogenic performance.Conductive magnetite nanoparticles(MNPs)were added into a cascading dark fermentation and anaerobic digestion system that was inoculated with Enterobacter aerogenes ZJU1 and methanogenic activated sludge(MAS),respectively.During the hydrogen-producing stage,the ratio of NADH/NAD+ and the activities of hydrogenase and ETS of E.aerogenes ZJU1 were all increased by dosing 200 mg/L MNPs,which was conducive to hydrogen production through the NADH-dependent pathway.In the presence of 200 mg/L MNPs,hydrogen production increased by 21.1%,while subsequent methane production improved by22.9%.Electrochemical analysis demonstrated the improvement in EET capacity of MAS after adding MNPs,which can be ascribed to the contribution of MNPs and electrochemically active extracellular polymeric substances(EPS)induced by MNPs,such as humic acid-like and fulvic acid-like substances.Microbial community analysis suggested that bacteria Syntrophomonas and archaea Methanosarcina were the dominating enriched syntrophic partners,and the expression of functional genes involved in CO₂ reduction to methane pathway was found to increase.Therefore,a more efficient fermentative hydrogen and methane co-production system was established by improving microbial electron transfer with the addition of MNPs.A Geobacter-magnetite biohybrid was used to promote the co-production of hydrogen methane with the anaerobic fermentation of biomass feedstock.Magnetite nanoparticles were formed by biological reduction of ferrihydrite with Geobacter species,and which interacted with bacterial cells to form a Geobacter-magnetite biohybrid.Electrochemical analysis showed increases in the peak redox current and Ca value(from 25.3 to 27.5 m F/cm²) of the Geobacter-magnetite biohybrid system compared with the pure culture.This hybrid system was used in the fermentative hydrogen and methane co-prodution system with real biomass(alligator weed)as the feedstock.The alligator weed harvested in September had a suitable C/N molar ratio(26.8),high cellulose content(20.3%)and a low weight ratio of ash to volatile(0.1),which were favorable for biological gas production.The hydrogen and methane co-production were achieved by two-stage anaerobic fermentation with alligator weed harvested in September as feedstock.The hydrogen and methane yield was 48.4 m L/g VS and 209.9 m L/g VS,respectively,and the total energy conversion efficiency(ECE)was 44.8%.With addition of the Geobacter-magnetite biohybrid,the total ECE of fermentative hydrogen and methane co-production from alligator weed increased to 64.9%.