Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising glucose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H, and CO2. We have begun to investigate H2-production by C. thermocellum ATCC 27405 cultured in media containing different carbon sources, including glucose, cellobiose, crystalline cellulose, a-cellulose, paper, and delignified wood fibres. We have detected formate synthesis by C. thermocellum ATCC 27405 cultured on both cellobiose and a-cellulose. While formate synthesis has been reported for one strain of Clostridium thermocellum (strain I-1-B), numerous fermentation studies of C. thermocellum 27405 have failed to detect the presence of formate. Formate production was detected throughout growth, and pyruvate:formate lyase (PFL) enzyme activity was detected in late log and stationary phase in extracts of C. thermocellum cultured on cellobiose. Formate synthesis competes with the production of hydrogen (H2) as a fermentation end-product, and thus negatively impacts H2 yields. Bioinformatic analyses of the C. thermocellum genome identified genes encoding key enzymes in pyruvate catabolism pathways, including two putative lactate dehydrogenases (LDH), one PFL. four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as "Energy-converting Hydrogenases" (Ech), or via NAD(P)H-dependent Fe-only hydrogenases which would permit H2 production from NADH generated during the glyeeraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our findings show the presence of multiple genes putatively encoding NADH:Fd oxidoreductase; suggesting a possible mechanism in which electrons could be transferred from NADH to ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H2 synthesis is the first step in developing strategies to increase hydrogen yields from biomass. My studies have outlined the likely pathways leading to hydrogen synthesis in C, thermocellum ATCC 27405. The actual functional roles of these gene products during pyruvate catabolism and in H2 synthesis remain to be elucidated and will need to be confirmed using both expression analysis and protein characterization.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/952 |
| Date | 21 May 2008 |
| Creators | Carere, Carlo R. |
| Contributors | Levin, David B. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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