| Dark fermentation using Escherichia coli offers a potentially cost effective way to produce biohydrogen from renewable resources. Although E. coli is naturally capable of hydrogen production through anaerobic mixed-acid fermentation, the yields from glucose must be improved to make this process economically viable. To increase yields, E. coli strains were metabolically engineered for decreased hydrogen uptake, over-expression of the formate-hydrogen-lyase (FHL-1) complex, and increased carbon flux towards formate production. Gene knockouts were done using the Red recombinase method developed by Datsenko and Wanner. Characterizations of the strains had to be done under tightly controlled conditions, so the construction of two inexpensive bioreactor control systems was also necessary. The systems were built for maximum reproducibility by controlling for pH, temperature, and headspace pressure. Media formulation was also investigated in order to develop a rapid method of inducing hydrogen production in previously aerobically grown cells. The results indicated that rich defined media activated hydrogen production from aerobic pre-cultures with no lag time and yielded more hydrogen and biomass than the commonly used minimal media. Under these conditions, deletion of both uptake hydrogenase 1 (DeltahyaAB) and hydrogenase 2 (DeltahybABC) was shown to increase hydrogen yield from glucose by 10% over the wildtype strain BW25113. However, the deletion of the repressor for the FHL-1 complex (DeltahycA) did not further increase hydrogen production. Additional deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdBC), both belonging to the mixed-acid fermentation pathway, increased hydrogen yield by 22% and 23%, respectively. Interestingly, combined elimination of ldhA and frdBC in the uptake and hycA null strain increased hydrogen yield from 1.37 to 1.82 mol mol-1 glucose, obtaining 91% of the theoretical maximum hydrogen yield. This dissertation represents the first report of characterizing metabolically engineered E. coli strains in batch hydrogen fermentation using rich defined media under tightly controlled conditions. |
