Converting Glycerol To1,3-propanediol Using Mixed-culture Microbial Bioelectrochemical Systems

Biofuel is regarded as an effective alternative to reduce the global carbon emmission. Among all types of biofuel used today, biodiesel is the most common one, with global production of more than20million MT annually. However, currently biodiesel production delivers approximately10w/w%glycerol as a by-product, which amounts to65-90w/w%of the biodiesel waste stream, and becomes a nuisance pollutant that poses significant problem and extra cost to the biodiesel industry. To reach a healthy development for this industry, recycling glycerol for valuable chemicals production is crucial. Given that in nature, glycerol is the substrate for bioproduction of1,3-propanediol (1,3-PDO), which has a larger market and added value, converting glycerol to1,3-PDO can be an appealing option to recover the chemical energy in waste glycerol. In this thesis,1,3-PDO production by mixed bacterial populations was stimulated by means of microbial electrochemical technology, which holds great potentials for renewable chemical production in the future. By constructing bioelectrochemical systems (BESs), the mechanism of current driven1,3-PDO biosynthesis from glycerol was demonstrated, bioelectrocatalytic activity of the cathodic biofilm was reported, syntrophic interactions of the cathodic bacterial populations were elucidated, the robustness of mixed culture1,3-PDO production in BES against pH changes was unraveled, in addition, the long-term performance of BES was provided. Contents and results are as follows:(1) Glycerol reduction with external electron supply was investigated, which showed that microbial electrochemical technology can significantly promote1,3-PDO production. Relative to open circuit condition, when polarizing the cathodic potential at-0.9V (vs standard hydrogen electrode, SHE), glycerol metabolism was redirected from the NADH neutral conversion of propionate fermentation to1,3-PDO production. With BES connected to a titration and off-gas analysis (TOGA) sensor, carbon and electron flux analysis indicated that the cumulative NADH flux inside the cell was enhanced, hence, the1,3-PDO yield. The production was also enhanced by hydrogen gas likely through suppressing hydrogen fermentation. Moreover, significantly more hydrogen was detected with-0.6V (vs SHE) cathodic potential, which indicates that hydrogen evolution competed with1,3-PDO production, and posed as the major electron sink under this condition. Investigations to the syntrophic interactions showed that interspecies hydrogen transfer took place, which encouraged hydrogenotrophic methanogenesis and represented carbon and electron losses.(2) The long-term performance of the BES was evaluated, and the productivity declined over time. To investigate the reason, bioelectrocatalytic activity and microbial community were characterized with voltammetric method and16S rRNA gene amplicon sequencing. Linear sweep voltammetry (LSV) demonstrated that at the first stage of operation, the biofilm was bioelectrocatalytically active and that the cathodic current was greatly enhanced only in the presence of both biofilm and glycerol. This indicates that glycerol or its degradation products effectively served as the cathodic electron acceptor. In addition, the selectivity of polarized electrodes to its electrode-associated biofilm was revealed by community analysis, which demonstrated that the cathode-associated microbial community was dominated by an operational taxonomic unit closely related to Citrobacter sp., with relative abundance of80.3%, and the relative abundance of which was significantly lower in the planktonic community (29.4%). However, with lower1,3-PDO production at the second stage, LSV showed that the electron sink shifted to hydrogen evolution almost exclusively, with approximately90%electrons used for hydrogen production under different glycerol conditions. Moreover, the abundance of Gammaproteobacteria (including Citrobacter) reduced dramatically by visualizing the biofilm with fluorescence in situ hybridization (29.2%), further explained the decreased1,3-PDO yield. It is thus shown here that in a process where substrate conversion can occur independently of the electrode, electroactive microorganisms can be outcompeted and effectively disconnected from the substrate.(3) pH is one of the most important operational factors in the glycerol-fed BES reactor. Protons migrated from the anode and generated from acid fermentation cannot sufficiently neutralize the hydroxide ions delivered from the cathodic reaction. Therefore, the cathodic pH would increase without manual control. This is not only deleterious to the electrochemical performance of BES reactors, the activity of bacteria may also be affected. In order to investigate the robustness of the mixed-culture glycerol-fed BES reactors for1,3-PDO production against pH changes, pH tests were conducted. With a continuous supply of glycerol, pH of5.5or6.5has marginal effect to the performance of reactors during or after acclimation period. In addition, when the reactors reached stable after acclimation, pH tests were further conducted with a wide window (pH=4.5,5.5,6.5,7.5and8.5) at both continuous and batch mode operation. Results suggested that the electricity driven1,3-PDO production by mixed-culture glycerol fermentation is resilient to pH changes, with a wide pH window of pH=5.5to8.5. Especially, when the cathodic pH went high, acid fermentation was stimulated, which favored the accumulation of reducing equivalent, hence, promoted1,3-PDO production. This showed the robust nature of the mixed culture BES for recycling glycerol to produce1,3-PDO against pH changes.