Doctor of Philosophy
Chemical and Biochemical Engineering
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.
Summary for Lay Audience
While the most commonly known biofuel is currently ethanol, research has been ongoing to develop ways to produce another biofuel known as butanol. Butanol is similar to ethanol in that it is also an alcohol; however, butanol is a far more suitable biofuel for use with current engines and fuel infrastructure. In order to produce butanol in an environmentally friendly fashion, most research has turned to using bacteria to convert inexpensive wastes through fermentation. This approach comes with several obstacles though, as butanol fermentations will generally be slow, taking a long time to convert the waste to butanol, and require extremely large amounts of raw material in order to make the process worthwhile.
The research conducted for this thesis addressed these shortcomings in two different ways: increasing how much butanol can be made from the wastes by adding specific compounds to the fermentation, and by using mutant strains of the bacteria that can produce butanol at a faster rate. For the first, an acid was added in small amounts to the fermentation, which had the effect of “pushing” the bacteria towards making more butanol and less by-products. The acid was made from difference sources to try and find an inexpensive method for generating the acid. Regardless of the source, the presence of the acid increased how much butanol was produced. For the second shortcoming, a mutant strain of the bacteria was created by forcing random mutations to a large number of the bacteria, and allowing the ‘winners’, that is, the strains that could grow faster, to populate the fermentation. To prove these were indeed mutants, DNA sequencing was done to pinpoint where the mutations happened, and attempt to explain why they improved the bacteria.
For both of these approaches, many test fermentations were conducted. To help with this, a new device was created that allowed for multiple tests to be conducted simultaneously, using small flasks containing a small amount of materials while still collecting large amounts of data. This prevented the need for large, expensive tests to be done one-at-a-time, and greatly increased the rate at which tests were conducted.
Munch, Garret Christopher, "Improved Fermentation Design and Screening Devices for Biobutanol Production" (2020). Electronic Thesis and Dissertation Repository. 7099.
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