Biochemical and phenotypical characterization of Clostridium thermocellum metabolism to identify solutions for increase ethanol yield
| dc.contributor.author | Daley, Steve | |
| dc.contributor.examiningcommittee | Levin, David (Biosystem Engineering) | |
| dc.contributor.examiningcommittee | Oresnik, Ivan (Microbiology) | |
| dc.contributor.examiningcommittee | Bressler, David (University of Alberta) | |
| dc.contributor.supervisor | Sparling, Richard | |
| dc.date.accessioned | 2024-08-30T16:39:29Z | |
| dc.date.available | 2024-08-30T16:39:29Z | |
| dc.date.issued | 2024-08-26 | |
| dc.date.submitted | 2024-08-27T01:31:09Z | en_US |
| dc.date.submitted | 2024-08-29T23:36:11Z | en_US |
| dc.date.submitted | 2024-08-30T15:10:27Z | en_US |
| dc.degree.discipline | Microbiology | |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | |
| dc.description.abstract | Clostridium thermocellum, is an anaerobic, cellulolytic bacterium (cellulose hydrolysis specialist) with the ability to ferment the released mono- or oligomeric sugars to multiple bioproducts, including ethanol. The low yield in ethanol can be attributed to three factors: the branched fermentation pathway, thermodynamic bottlenecks at key points in the glycolysis pathway, and redox balancing centered around redox cofactors. Heterologous expression of an ATP-dependent phosphofructokinase (PfkA) has been a suggested solution to overcome the upper glycolysis bottleneck, caused by the highly expressed pyrophosphate-dependent phosphofructokinase. However, C. thermocellum inherently expresses a PfkA, as well as a fructokinase, both characterized to utilize either ATP or GTP. The latter two genes show low expression levels when grown on model substrates; therefore alternate substrates were queried for potential upregulation of PfkA. C. thermocellum metabolism is generally studied using cellobiose as the sole carbon source to understand carbon and electron flux to fermentation end-products. Adaptive growth on glucose and sorbitol was performed, resulting in a ~2.5-fold and ~4.5-fold increase in ethanol yield, compared to the wild-type strain fermenting cellobiose. Initial expectations were that fermentation of sorbitol would produce higher ethanol and hydrogen concentrations by virtue of the sugar alcohol being a more reduced substrate in comparison to cellobiose, but the reason for the increase during glucose fermentation required investigation. The increase in ethanol yield was accompanied by a decrease in canonical end- products, most notably lactate for glucose adapted strains, while acetate, lactate, and formate decreased for sorbitol adapted strains. The stable fermentation profiles for adapted strains were disrupted after a single passage with cellobiose. The shift in end-products still maintained a higher ethanol yield compared to wild type strains, suggesting that potential mutations, and/or differential expression of genes or metabolites, remained constant after passage with cellobiose. Sorbitol metabolism led to an ~250-fold increase in an alcohol dehydrogenase gene, whose gene product likely oxidizes sorbitol to provide additional electrons, conferring the increase in ethanol yield, with a concomitant diversion of electrons from amino acid synthesis, as supported by the decrease in amino acids detected in the supernatant. An increase in available electrons rather than pfkA expression led to an increase in ethanol yield. | |
| dc.description.note | October 2024 | |
| dc.description.sponsorship | University of Manitoba Graduate Scholarship NSERC Discovery grant RGPIN 05878-19 | |
| dc.identifier.uri | http://hdl.handle.net/1993/38472 | |
| dc.language.iso | eng | |
| dc.rights | open access | en_US |
| dc.subject | Consolidated bioprocessing | |
| dc.subject | Bioethanol | |
| dc.subject | Fermentation | |
| dc.subject | Biochemistry | |
| dc.title | Biochemical and phenotypical characterization of Clostridium thermocellum metabolism to identify solutions for increase ethanol yield | |
| dc.type | doctoral thesis | en_US |
| local.subject.manitoba | no | |
| oaire.awardNumber | RGPIN 05878-19 | |
| oaire.awardTitle | NSERC Discovery grant | |
| project.funder.name | NSERC |