Enhanced Hydrogen Production From Biomass In Microbial Electrolysis Cells And The Environmental Responses Of Anodophilic Community Structures

Fermentation is a renewable and carbon-neutral method for biohydrogenproduction. However, photo-fermentation is limited by unstability of illuminant,dark-fermentation has a low H₂recovery from substrates. Moreover, fermentativebacteria are often constrained by substrate metabolisms and growth conditions.Microbial electrolysis cell （MEC） with the virtues of both biological andelectrochemical process is an emerging approach for biohydrogen production, and canovercome many thermodynamic barriers of fermentative H₂production. Focus on theproblems of fermentation, this thesis investigated the possibility of H₂production usingMEC from fermentative products and the substrates that can not be fermented to H₂. Ifthe MEC is more adaptable than fermentation to environment was examined. Theresponses of anodophilic microbial community structures to environmental changes andthe mechanism of microbial population interactions were studied using methodsassociated with electrochemistry and molecular biology. Metabolic process of complexsubstrates in MEC was examined. The fate of electrons, H₂and energy in reactor wasalso investigated. We offered the methods for enhancement of H₂recovery based onexperimental results.Here, we first report H₂production from molasses wastewater by combiningethanol-H₂-coproducing fermentation in continuous stirred tank reactor （CSTR） withsingle-chamber MECs. H₂was generated in CSTR at a rate of0.7m³-H₂/m³/d and arecovery of14%. After CSTR being coupled with a MEC, the whole of H₂productionrate and recovery increased to2.11m³-H₂/m³/d and81%, respectively. The energy yieldrelative to the electrical input was287%in series system with an energy consumption of1.12kWh/m³-H₂that was less than5.6kWh/m³-H₂in water electrolysis for H₂production. Ethanol-H₂-coproducing fermentation of glucose using type strain has a H₂recovery of18%, which was increased to47%by MECs. Analysis of anodophilicmicobial community using pyrosequencing and Fast UniFrac clustering indicated thatthere were three major clusters based on substrates. The micobial community fed actualfermentation effluent was separated from other two groups fed organics. Within theorganic groups, the micobial communities fed synthetic fermentation effluent （ethanoland acetic acid are major components）, ethanol, acetic acid, lactic acid were clustered and separated from those fed propionic acid, butyric acid, valeric acid. However, therewas clear difference among single community in each group. Performances of MECsreflected the differences of microbial community structures. The microbial communitystructures have undergone a change to a different degree when swicthed operation frommicrobial fuel cell （MFC） to MEC. Geobacter is the dominant exoelectrogens. Thediversity of exoelectrogens increased with rising molecular weight of substrates.H₂was first generated from proteins using MECs, which overcame the bottleneckthat H₂can not be produced by fermentation of proteins. Single-chamber MECsproduced H₂from bovine serum albumin （BSA） at a rate of0.42±0.07m³/m³/d and ayield of21.0±5.0mmol-H₂/g-COD, however, H₂production was substantially reducedusing peptone. Enrichment of anode using proteins can obtain optimal reactorperformance. Internal electron recycle likely occurred in single-chamber MECs due tooxidization of H₂and sulfide on the anode. The use of a two-chamber design can reducethis recycle. The removal of protein in MECs was8797%. Nitrogen balance analysisrevealed that most organic nitrogen was converted to ammonia nitrogen under optimalconditions. Microbial community analysis indicated that there was a distinct differencebetween microbial communities fed proteins and those fed acetate, and the former havelower biological diversity.H₂was produced from waste activated sludge （WAS） in MECs and the mechanismof enhanced H₂production was studied. H₂yields of3.89±0.39mg-H₂/g-DS from rawWAS and6.78±0.94mg-H₂/g-DS from alkaline-pretreated WAS were obtained in thetwo-chamber MECs. These yields were several times higher than that obtainedpreviously by fermentation. Single-chamber MECs with low internal resistance showedH₂production rates that13times that of two-chamber MECs and the constant H₂yieldsusing alkaline-pretreated WAS, however, methanogenesis was detected in them. A yieldbalance based on thermodynamic calculation revealed that carbohydrates and theirfermentation products were not the only substrates for MECs, protein and other organicmaterials could also be utilized by MECs. Cascade utilization of organic matter isresponsible for high yield in MECs. MEC also has the advantages of less H₂loss,insensitivity to temperature and heavy metals. Characterization of WAS bythree-dimensional excitation-emission matrix fluorescence spectroscopy with parallelfactor analysis indicated that electrohydrogenesis reacted on the extracellular polymericsubstances and intracellular substances of WAS. Pyrosequencing showed that microbial populations in MEC were more diverse than that in WAS, and there was a cleardistinction between MEC and WAS in microbial community structure. Syntrophicinteraction between acid-producing bacteria and exoelectrogens owned by anodophilicmicobial communities is the biological proof for cascade utilization of organic matter.Quantitative real-time PCR （QPCR） demonstrated that a consistent feed ofalkaline-pretreated WAS containing large amounts of acetate led to a predominance ofacetoclastic methanogens instead of hydrogenotrophic methanogens.Psychrotolerant MECs were successfully started up and operated, which overcamethe bottleneck that fermentative H₂production can not work well at low temperatures.Single-chamber MECs were enriched at4oC or9oC through inoculating reactorsfrequently using pre-acclimatized suspensions from MFCs. MECs produced H₂at ratesof0.23±0.030.53±0.04m³-H₂/m³/d and yields of2.66±0.222.94±0.02mol-H₂/mol-acetate using acetate. Clone libraries revealed that Geobacterpsychrophilus was the major exoelectrogens in psychrotolerant MECs fed acetate.MECs fed glucose were also started up at4oC and obtained the H₂production rates of0.25±0.030.37±0.04m³-H₂/m³/d and yields of6.0±0.36.1±0.5mol-H₂/mol-glucose. Pyrosequencing and electrochemical analyses showed that thesyntrophic interaction between fermentative bacteria and exoelectrogens throughfermentation products was the primary pathway for glucose degradation other thandirect glucose oxidization. Methanogenesis and homoacetogenesis could not be found inpsychrotolerant MEC fed glucose. QPCR analysis indicated that the methanogenesisobserved in MECs at mesophilic temperatures could be completely inhibited at lowtemperatures, and the total number of methanogens could be reduced by6891%.However, the methanogenesis and the number of methanogens can return to the priorlevels after the temperature is rebounded. The CH₄production and the number ofmethanogens in MEC could be restricted steadily to a negligible level by continuouslyoperating reactors at15°C when the methanogenic community has not yet beenestablished. This resulted in a H₂yield and production rate comparable to those obtainedat30°C with less CH₄production （CH₄%<1%）. The impact of temperature fluctuationson anodophilic bacterial community structures is small.