Biogas is the well-known product of Anaerobic Digestion (AD), but nowadays, the intermediate products (volatile fatty acids - VFAs) of anaerobic metabolism have gained increasing attention inside the “carboxylate platform”. However, steering and optimizing the process for selective metabolite production is still an unraveled task inside this field since it relies on the manipulation of operational parameters. The objective is to understand the conversion of glucose and glycerol in the mixed culture of anaerobic digestion to unravel possibilities to steer product formation. Glucose and glycerol are the main components in the waste streams of beverage and biodiesel industries. Regarding the degradation pathways in AD, both glucose and glycerol are oxidized to pyruvate by fermentative bacteria to obtain energy and metabolic intermediates under anaerobic conditions through the same intermediate, glyceraldehyde-3-phosphate. Pyruvate, the key branching-point, allows the process to enter different metabolic pathways which lead to the formation of various metabolites. Under the fermentation conditions, redox balance is necessary to be maintained through terminal electron transfer to internally produced compounds. Since glycerol has a higher degree of reduction than glucose (Glucose: 0.33 NADH/C-Glucose; Glycerol: 0.66 NADH/C-Glycerol), the conversion of glycerol into pyruvate generates a double amount of reducing equivalents. On the one hand, this provides the advantage of higher theoretical product yield of reduced compounds. On the other hand, half of the glucose is lost as CO2 during the fermentation, and this reduces the product yield.1 Therefore, we assume that elevated pCO2 could have a more significant detrimental effect on glucose fermentation. In this research, batch experiments at different pCO2 (0.3, 1, 3, 5, 8 bar) were performed, and different types of measurements and analyses were employed to monitor the pCO2 effect on the metabolism. We designed some of the potential pathways of glucose and glycerol conversion under elevated pCO2. The elevated pCO2 converged the product spectrum of both substrates towards propionate production but affected the degradation and production phase of propionate and acetate. Initial pCO2 of 0.3 bar and 1 bar did not cause visible inhibition on the propionate production of both substrates. However, the propionate degradation was kinetically affected under 0.3 and 1 bar initial pCO2. Although propionate was degradable, its degradation phase at 1 bar initial pCO2 was longer than 0.3 bar. On the contrary, when the pCO2 was elevated to 3, 5, and 8 bar, not only the propionate production phase became longer, but also the maximum concentration became lower on both substrates. Moreover, propionate degradation was ceased. The lower propionate production was suspected to be due to the inhibition of NADH production as a consequence of the elevated pCO2 effect. The undegradable propionate might be attributed to unfavored decarboxylation reactions under elevated pCO2. The enrichment approach was applied to examine the adaptability of the microbial consortium under the CO2-exposing environment. Therefore, not only the more predominant metabolic reaction would be favored during the enrichment, but also changes in the community due to CO2 influence were expected. Propionate degradation was achieved with this inoculum at the only tested condition (5 bar initial pCO2). From the community analysis, Smithella was enriched during the enriched and it was suspected to play a significant role in the propionate conversion. Besides, the difference between the substrates on the fermentation has been observed. Due to the higher available reducing power of glycerol than glucose, glycerol was potentially able to generate more propionate. However, the butyrate formation also needs the reducing power to proceed with the reaction, but it was not detected in the glycerol fermentation. Therefore, reducing power distribution from specific substrates with elevated pCO2 might also be affected. Moreover, the substrate was also hypothesized to influence cell viability, where glycerol fermentation increased the cell viability but glucose not. The degradation of acetate and butyrate in the non-enriched inoculum was observed to be kinetically affected by elevated pCO2, with the later becoming undegradable at 8 bar. The reason for this phenomenon needs further investigation.

In the context of steering product formation in anaerobic digestion systems, the present thesis work elaborates on the potential role of elevated CO2 partial pressures as an environmental driver that may influence end-product selectivity from methane towards compounds from the carboxylic platform. As an emerging field of research, organic acid production via mixed culture fermentation is currently in an exploratory phase and the understanding of basic functional principles driving each of the biochemical conversions of interest is of vital importance.The present investigation forms part of a series of studies conjunctively aimed at elucidating the effect of elevated CO2 partial pressures on glucose fermentation, which consists of a complex network of several metabolic routes. Specifically, this thesis work focuses on the effects of CO2 partial pressure on the degradation of two key metabolites that are central and/or highly relevant to the glucose conversion pathways, namely pyruvate and butyrate.