Improved Fermentation Design and Screening Devices for Biobutanol Production
Abstract
A worldwide increase in demand for renewable fuels has revived interest in fermentatively produced butanol. However, butanol fermentation suffers from low product yields and productivity. The work presented in this thesis addresses part of these research and development needs at three levels: innovative fermentation process design; genetic manipulation for strain enhancement; and the development of a new tool for anaerobic process characterization and optimization.
Product yield could be increased through traditional fermentation engineering. Co-fermentation of butyric acid with glycerol increased the butanol yield from 0.45 mol/mol (mols C in butanol / mol C in substrates) to 0.51 mol/mol. In building on this concept, and capitalizing on the unique metabolism of C. pasteurianum, an optimized glycerol to molasses (co-substrate) ratio was identified. C. pasteurianum produces butyric acid from the molasses sugars for later re-assimilation when consuming glycerol, resulting in a final product yield of 0.48 mol/mol.
A putative mutant of C. pasteurianum was generated using random mutagenesis techniques. The growth and product profile of the putative mutant was characterized, displaying higher growth rates and an altered product profile when compared to the wild-type strain. The DNA was isolated and sequenced, which confirmed that it is a novel mutant, and will allow for directed mutagenesis techniques to be used to replicate and characterize the mutations.
Finally, it was found that the gas production of the fermentation yielded valuable data only observed at the reactor scale, and not during screening in shake flasks. To remedy this gap in data acquisition, a novel screening device was developed which collected off gas data from multiple shake flasks operating in parallel. The fermentations conducted at the shake flask scale matched previously reported results at the reactor level.
In conclusion, this thesis shows possible ways to increase butanol yields through fermentation engineering, and to increase butanol production rates through strain development. It further led to development of a highly flexible screening device suitable to further optimization of this or other anaerobic fermentation processes.