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Conversion of Cellulose to Ethanol by the Biofuels Microbe Clostridium Phytofermentans: Quantification of Growth and Role of an Rnf-Complex in Energy Conservation

The anaerobic mesophilic bacterium Clostridium phytofermentans grows and ferments multiple plant-based substrates into ethanol as the main product of fermentation. The capacity of C. phytofermentans to convert plant biomass into ethanol, propanol, and short-chain fatty acids is strongly attractive for industry. Specific physiological capabilities of C. phytofermentans allow the microbe to generate high amounts of ethanol compared to acetate. However, little is known about membrane energetics in C. phytofermentans, or its role in energy conservation and production of high levels of ethanol during fermentation of plant biomass substrates. In the first research project presented in this dissertation, we examined C. phytofermentans growth on three insoluble plant-based substrates: Whatman #1 filter paper, wild-type Sorghum bicolor and the S. bicolor reduced lignin double mutant, bmr-6/bmr-12. Cells were visualized and quantified employing a novel dual-fluorescent staining protocol, combining Fluorescent Brightener 28, a blue fluorescent dye that stains cellulose and chitin, and SYTO 9, a green fluorescent dye that stains nucleic acid. Our results demonstrated this protocol allows the visualization and differentiation of C. phytofermentans cells from plant debris using epifluorescence microscopy. Our results also showed greater cell growth and substrate attachment on reduced lignin substrates. Moreover, ethanol production increased over time while cell numbers decreased when cultured with either sorghum substrate. These results suggested that only a portion of the cell population remained active during growth, possibly due to sporulation and reduced metabolic activity. The second project presented in this dissertation involved the characterization of a bacterial microcompartment (BMC) in C. phytofermentans. Transmission electron microscopy (TEM) of thin sections of C. phytofermentans cells grown with fucose or rhamnose as growth substrate showed the presence of polyhedral structures, similar in appearance to known BMCs. Visualization of these intracellular structures was possible due to various modifications of a TEM sample embedding protocol. Use of picric acid and potassium ferricyanide resulted in enhanced contrast of BMC images while stabilizing cell wall structure during sample preparation. A third and final project described in this dissertation concerned the development of a model for C. phytofermentans membrane energetics and energy conservation that might provide an explanation for high levels of ethanol production by this microbe. Genomic and biochemical analyses indicated the presence of a membrane-bound sodium- and proton-translocating pyrophosphatase and three ATPases, including a Na+ V-type ATPase, a Na+ F-type ATPase and a possible H+ ATPase that had not previously been characterized. Furthermore, results showed that Rnf ferredoxin:NAD+ oxidoreductase activity in inverted membrane vesicles was driven by a proton electrochemical gradient. Supporting data showed that cell growth was inhibited by both sodium and proton ionophores. Taken together, these results suggested that C. phytofermentans is capable of conserving energy through ferredoxin oxidoreductase activity of the Rnf-complex, resulting in increased levels of NADH for ethanol production.
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