Ethanol usage as a transportation fuel is rapidly increasing in the United States. Production of ethanol from cellulose feedstocks via gasification followed by syngas fermentation offers an environmentally friendly approach that mitigates many of the adverse effects associated with production from corn. In the syngas fermentation process, the hydrogenase enzyme of the fermentation bacterium, Clostridium P11 for this work, supplies electrons to the metabolic pathway, facilitating ethanol production. In this thesis, an assay for P11 hydrogenase activity was developed. It was determined that 1) less than 4 minutes of sparging with 50 sccm H2 is needed to reduce O2 levels to below 1 ppm in a 3 mL aqueous solution, while less than 1 minute of purging at the same rate is needed to fill an air-filled 3.5 mL cuvette to 99.9999% H2, 2) 12.5 mM DTT included in the reaction mixture at pH 6 helps scavenge O2, 3) H2 diffusion is slow compared to enzymatic reaction rates, 4) CO2 lowers media pH, 5) 0.084 atm CO causes 90% inhibition of P11 hydrogenase, 6) prolonged Triton X-100 exposure diminishes hydrogenase activity, and 7) variations in H2 pressure and electron acceptor identity and concentration affect measured hydrogenase activities. The assay developed for P11 hydrogenase activity was used to perform kinetic studies. The Okura rapid-equilibrium rate law best described this activity. A constant that regulates the effect of H2 pressure on hydrogenase activity, KH2, was determined to be independent of electron acceptor and to have a value of 0.31 atm, implying that H2 must be supplied to the syngas fermentation at ~3 atm to maximize hydrogenase activity. KBV and KMV, constants that regulate the effect of benzyl viologen and methyl viologen on hydrogenase activity, were determined to be 1.7-2.4 mM and 10.6 mM, respectively. Additionally, hydrogenase activity was temporally correlated with ethanol production in batch cultures of P11 and strongly dependent on pH. The intracellular pH of P11 was determined to be approximately 5.5.



College and Department

Ira A. Fulton College of Engineering and Technology; Chemical Engineering



Date Submitted


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ethanol, hydrogenase, syngas, fermentation, Clostridium, P11, assay