Fermentative utilization of glycerol and lignocellulosic sugars and production of ethanol by Paenibacillus macerans
Doctor of Philosophy
With the recent volatility in crude oil prices and widespread concern regarding global warming, there is increased need for finding sustainable alternatives to petrochemical based fuels. One way to achieve this objective is through the production of biofuels such as ethanol by microbial fermentation. In this context, this study focuses on the anaerobic fermentation of renewable substrates; glycerol and lignocellulosic sugar mixtures for the production of ethanol by P. macerans. Further, metabolic flux analysis (MFA) was used as a systemwide tool to understand the role of various metabolic pathways in the fermentative utilization of these substrates. Glycerol is a byproduct of biodiesel and bioethanol production, which is an abundant, inexpensive and renewable substrate. In this study, we have shown that P. macerans can anaerobically ferment glycerol in the absence of external electron acceptors. Nuclear magnetic resonance (NMR) analysis of the fermentation samples identified the production of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) from glycerol. Use of U-13C glycerol as substrate demonstrated the incorporation of glycerol in the cell biomass. Glycerol fermentation was stimulated in a medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO2-enriched atmosphere. Since the consumption of reducing equivalents in the production of 1,2-PDO balances the reducing equivalents generated in the production of cell biomass, the synthesis of ethanol and 1,2-PDO are proposed to be a metabolic determinant of glycerol fermentation in P. macerans. Hexose and pentose sugars make the largest portion of lignocellulosic biomass, which is the ideal substrate for the production of biofuels. Most microorganisms can not utilize hexose and pentose sugars simultaneously. In this study, we have shown that P. macerans N234A can ferment hexose (glucose) and pentose (xylose and arabinose) sugars individually in the absence of external electron acceptors. Additionally, we have shown that P. macerans N234A can simultaneously utilize the three sugars of lignocellulosic biomass. We have also identified the factors pH and temperature, which improve the simultaneous sugar utilization by this microorganism. Metabolic flux analysis was used as an in vivo tool for systemwide study of glycerol and sugar mixture fermentations to elucidate the role of various pathways. In the case of glycerol fermentation, MFA analysis identified the role of 1,2-PDO production in achieving the redox balance by consuming the redox equivalents being generated in the production of biomass. Flux analysis also showed the role of PFL in pyruvate dissimilation in the glycerol fermentation. Similarly, in the case of sugar mixture, MFA analysis showed the role of PFL and PDH enzymes in the utilization of sugar mixtures by P. macerans.