Production of biohydrogen in metabolically engineered Escherichia coli strains

Thesis (M.S.)--University of Hawaii at Manoa, 2007.Includes bibliographical references (leaves 45-49).v, 49 leaves, bound ill. 29 cmMicrobes have diverse biosynthetic pathways to produce molecular hydrogen and potentially hold the key to the viable macroscale utilization and production of hydrogen from renewable sources. However, low production yield has been a major limiting factor for large-scale biohydrogen production because of various metabolic bottlenecks. Dark fermentations seem to hold the best promise for biohydrogen production due to low costs and relatively high production yields. Because of the relative ease associated with the metabolic engineering of E. coli, this organism was chosen for this study. E. coli strains were engineered for greater production of hydrogen by using a combinatorial strategy of over-expressing the components of the hydrogen-evolving complex, and the interruption of uptake hydrogenases. This multiple-step approach, which has never been reported in E. coli, could shed light on possible bottlenecks in the hydrogen production pathway and eventually result in the engineering of a very efficient hydrogen-producing E. coli strain. Uptake hydrogenases 1 and 2 were deleted in the E. coli genome along with hycA, which is responsible for repressing the hydrogen-evolving complex. However, the deletion of hycA did not increase hydrogen yield compared to wildtype. This result was inconsistent with previous studies and may have been caused by an incorrect construction. Deletion of the uptake hydrogenases in conjunction with an over-expression of hycEG, which encodes for the large and small subunits of the hydrogen-evolving enzyme hydrogenase 3, produced about 4.5% more hydrogen than wild-type E. coli during anaerobic batch fermentations. Maximum hydrogen yield was obtained at a pH range of 5-6 for all strains. This work provides for the possible application of this knowledge towards developing a commercially efficient hydrogen-producing strain of E. col