(378c) Hydrogenase Inhibition as the Mechanism of Enhanced Ethanol Production by Clostridium Thermocellum in Biphasic Continuous Culture

Clostridium thermocellum is a cellulolytic anaerobic bacterium that can directly convert abundant and inexpensive cellulosic feedstock into soluble sugars, which it uses to produce ethanol. However, the ethanol yield of this organism is low due to its low ethanol tolerance (< 1.5%). Other catabolic products of C. thermocellum include lactate, acetate, formate, hydrogen, and carbon dioxide. Our previous investigations of continuous fermentations of C. thermocellum at elevated pressure (7.0 MPa, and 13.0 MPa) demonstrate an increase of the ethanol: acetate ratio by more than 100-fold compared to cultures under atmospheric pressure. This investigation probes the basis for this dramatic increase toward ethanol selectivity, which we attribute to the increased concentration of dissolved products gas (i.e, hydrogen) in the culture broth. To test the hypothesis that the dissolved gas affects the regulation of reduced and oxidized electron carriers in the metabolism of thermophilic bacteria, the end-product synthesis patterns of C. thermocellum was measured for a series of batch and continuous cultures with the addition of exogenous hydrogen. The inhibition of hydrogenase activity, the enzyme responsible for hydrogen production, has the potential to redirect the electrons to the concurrent pathway that leads to higher ethanol production. Thus, further experiments examined the synthesis of end products under condition of hydrogenase inhibition as a function of exogenous hydrogen concentration and known hydrogenase inhibitors. From preliminary data, continuous C. thermocellum cultures sparged with hydrogen have a 2 fold increase of ethanol production relative to the same cultures sparged with nitrogen. Also, batch experiments have shown that ethanol: acetate ratio increased 3-5 folds in the presence of hydrogenase inhibitors such as carbon monoxide. Continuous fermentation carried out in a chemostat under treatments of different exogenous gases (ie. nitrogen and hydrogen) and inhibitors at various pressures demonstrates the organism's adaptation to its environment and the sensitivity of the metabolic profile to exogenous gases. The ability to control the product selectivity by manipulating carbon and electron flow via dissolved gas regulation offers novel approach to directing microbial metabolism. Furthermore, the ability to manipulate the multi-product metabolic pathway to increase ethanol yield provides measureable and varied responses of the metabolic phenotype.