(639f) Demonstration of a Viable Hydrogenase Assay for Syngas Fermentation to Ethanol: Quantitative Accounting for Competing Reactions, Enzyme Inhibition, and Diffusion Limitations

Rising fuel prices, concern about long term crude oil supply, and increasing anthropogenic carbon dioxide emissions have increased motivation to find alternatives to traditional oil-derived transportation fuels. Among the most promising alternative fuels is ethanol, which in the United States is produced almost entirely from corn. The simple-sugar fermentation process that is currently used is inefficient in that it wastes much of the corn plant and costs almost as much energy to produce as is generated by its combustion.(Shapouri, 2004) A more promising alternative is to utilize the entire plant and expand the spectra of feedstocks that can be used for ethanol production into the non-food sector. Syngas (a mixture composed primarily of CO, CO₂, and H₂) is easily created by gasifying any carbon-based feedstock (e.g. switchgrass, municipal waste, forestry residues) and has been demonstrated to be able to be fermented by specialized microorganisms to make ethanol. (Datar, 2004)

As part of the fermentation process of converting syngas to ethanol, the hydrogenase enzyme(s) plays a critical role in converting H₂ to electrons for use in the metabolic process. If hydrogenase activity is diminished, CO is oxidized to CO₂ to provide the needed electrons. Thus, CO used in this aspect cannot be used to produce ethanol. Therefore, properly assessing the hydrogenase activity in the presence of syngas constituents is critical in understanding how to maximize ethanol productivity.

Many assays exist for determining the hydrogenase activity although numerous studies show varying results. One of the most prevalent methods is using an artificial electron acceptor (usually a color changing dye such as methyl or benzyl viologen) and a spectrophotometer to monitor the rate of hydrogen oxidation. Hydrogenase activity can vary depending upon the environmental conditions and the type of bacteria. However, the activity can be incorrectly measured as a result of experimental protocols. Previous research has shown that oxygen scavengers used to keep the assay anoxic can result in an additional reaction of the scavenger with the electron acceptor, leading to an overestimated measurement of the hydrogenase activity (Van Dijk, 1979). Additionally, it has been shown that hydrogenase activity in some assays may be limited by the rate at which H₂ can diffuse to the surface of the cells (Tatsumi, 2000). Finally, CO (a component of syngas) is a known inhibitor of hydrogenase activity. (Chen, 1974)

As a result of potential side reactions interfering with an accurate hydrogenase assay, this work quantified the reaction rate between one common oxygen scavenger (dithiothreitol) and one common electron acceptor (benzyl viologen). Also, rate data from CO inhibited hydrogenase assays was fit to a modified Michaelis-Menton equation and an inhibition constant (K_i) was obtained. Finally, diffusional analysis was applied to show that H₂ in the assay solution cannot be replenished by H₂ in the headspace during the analysis time. The diffusional analysis, inhibition constant, and knowledge of the side reaction rate provided the background to develop a viable hydrogenase activity assay. This work will describe the hydrogenase assay and demonstrate how to accurately account for diffusional effects, inhibition, and side reactions.

References

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Datar R, Shenkman R, Cateni B, Huhnke R, Lewis R. Fermentation of bio-mass generated producer gas to ethanol. Biotechnology and Bioengineering 2004;86:587-594.

Shapouri H, Duffield J, Mcaloon A. The 2001 net energy balance of corn-ethanol. Proceedings of the Conference on Agriculture as a Producer and Consumer of Energy, Arlington, VA., June 24-25, 2004.

Tatsumi H, Kano K, Ikeda T. Kinetic Analysis of Fast Hydrogenase Reaction of Desulfovibrio vulgaris Cells in the Presence of Exogenous Electron Acceptors. J. Phys. Chem. B 2000;104:12079-12083.

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