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The Science and Engineering for a Biobased Industry and Economy
S.E. Nokes, C. Lee, C. Crofcheck, M. Montross
Department of Biosystems and Agricultural Engineering
The Land Grant University System, Resource Limitation, and the Impending Biological Revolution. A need for biofuels and other biobased products has been recognized as a national priority. The objectives of this project address research relating directly to SAAESD Goal 1 F (biobased products) and H (processing agricultural coproducts); research will influence Goal 5 B (rural community development and revitalizing rural economies) indirectly.
The importance and extent of the problem is characterized by the fact that the U.S. must drastically reduce its dependence on petroleum. This is not the fetish of a small proportion of the population; the U.S. society as a whole recognizes the need to reduce its dependence on petroleum as a source of fuels, chemicals and other materials. If this research work is not conducted, the technical capability necessary to switch from a petroleum-based economy to a bioresource-based one will not be developed.
The technical feasibility of the research is reinforced by the fact that this research will be conducted by professional researchers who currently are part of the Land Grant University system. As outlined in this project description, the Land Grant University system provides a unique capability to enable research for biobased products by providing a world class research network. Replacing petroleum products with those originating from biological sources will require significant fundamental and applied research efforts.
Outcomes or projected Impacts: -The committee has served and will continue to serve as a resource for: Bioresearch and Development Initiative (BRDI), Biomass, Research and Development Board working groups, SBIR panel Biofuels 8.8, USDA/DOE Biomass Initiative Project Review Teams, NRI 71.2 panel and reviewers for the Sun Grant Initiative. -The multi-state membership will contribute to the implementation of the REE energy science strategic plan. -Multi-state membership will contribute to identification of funding priorities and shaping policy of Federal agencies -Research as a result of this project will create technology adopted by industry with at least two licensed technology per year. -Research will enable reduced dependency on foreign-based fuels and chemicals.
2011 Project Description
Clostridium thermocellum is a cellulolytic anaerobic bacterium that can directly convert cellulosic feedstock into ethanol. However, ethanol yield of this organism is low due to the reallocation of carbon to other fermentation products (lactate, acetate, formate), as well as gases (carbon dioxide, and hydrogen).
C. thermocellum at elevated pressure (7.0 MPa, and 13.0 MPa) increased the ethanol: acetate ratio by more than 100-fold compared to that under atmospheric pressure. The observed effect has been attributed to the increased concentration of hydrogen in the culture broth. Hydrogen is hypothesized to inhibit hydrogenase.
To separate the increased hydrogen inhibition effect from the additional effect seen at pressure, this project focused on the effect of exogenous hydrogen and other hydrogenase inhibitors on the product formation of C. thermocellum.
Objective 2: Incorporate metabolic flux model into whole-cell model that accounts for growth (i. e., dilution) rate and dissolved gas effects on product selectivity. Experimental observations from literature have shown increased ethanol production (the target product) and decreased acetate production (the by-product) under conditions of elevated pressures and/or pH and the presence of increasing dissolved hydrogen gas. These conditions continue to influence the control of NADH/NAD pairs towards target product formation.
The model predicted the flux spectrum of metabolic distribution to account for ethanol and acetate yields as functions of dissolved hydrogen gas and pressure alongside the NADH/NAD effect. Acetate and ethanol yields mostly agreed with the corresponding values reported. Product selectivity mainly shifted from acetate to ethanol at elevated pressures. The role of NADH/NAD is significant for controlling product selectivity in fermentation processes.
It is expected that more NADH consumption will continuously produce hydrogen in the oxidation-reduction which will in turn inhibit acetate formation and cause acetylcoA to move towards ethanol. This explains the linear relationship observed between NADH consumption flux and hydrogen flux under conditions of elevated pressures which confirms NADH's effect on the metabolic flux distribution.
It is also useful to note that the observed linear relationship between NADH consumption and hydrogen flux is analogous to the relationship between NADH/NAD ratio and hydrogen under pressure reported in the literature.
For this reason, it was useful to incorporate an NADH flux due to the reversible NADH reaction into the model in order to quantify and interpret the effect of relative changes in NADH/NAD ratio due to hydrogen and pressure on the metabolic flux distribution end-products, especially ethanol. It was determined that ethanol is brought up to an approximate ratio of 1.4:1 of NADH flux due to the reversible NADH reaction when pressure is elevated.
We showed that the ethanol: acetate ratio increased in the presence of exogenous hydrogen and hydrogenase inhibitors, but only under certain conditions. Further experiments and metabolic modeling resulted in the determination that two conditions must be satisfied to have increased ethanol production: hydrogenase must be inhibited such that acetate product is severely impaired, and the substrate uptake mechanism must be impaired such that the cellobiose feed rate into the cell is below a threshold level.
The ability to control product selectivity by environmentally manipulating carbon and electron flows offers a novel approach to directing microbial metabolism. The pattern of acetate and ethanol production at different dilutions rates were similar to previous results. The assumed zero flux of glycogen had little or no impact on the predicted flux spectrum and consequently the effect dissolved hydrogen gas and pressure. However, based on model predictions, varying hydrogen flux across dilution rates for each pressure versus keeping hydrogen flux constant for each pressure significantly influenced ethanol yields.
These observations provide important information for conditions under which ethanol yields may be highest. It was determined that ethanol yields are at their highest when hydrogen flux is maintained at about 4.81 coupled with pressure at 7 MPa and above.
Hsin-Fen Li, Barbara L. Knutson, Sue E. Nokes, Bert C. Lynn, Michael D. Flythe. 2011. Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate. Applied Microbiology and Biotechnology.
Adotey, B., Nokes, S.E., Knutson, B.L., Lynn, B.C., and M.A. Flythe. 2011. Metabolic Flux and Control Analyses of Wild Type and Ethanol Adapted Clostridium thermocellum . Presented at the 2011 ASABE Annual Meeting, Louisville, KY. August 7-10, 2011.
Adotey, B., Nokes, S.E., Knutson, B.L., Lynn, B.C., and M.A. Flythe. 2011. Metabolic Flux and Control Analyses of Wild Type and Ethanol Adapted Clostridium thermocellum . Presented at the 2011 Symposium on the Thermochemical Conversion of Biomass to Fuels. Advanced Technology and Research Center, Oklahoma State University, August 2, 2011.