Nitrogen fixation and hydrogen production by photosynthetic bacteria

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The non-sulfur purple photosynthetic bacteria serve as excellent model organism for research on a series of fundamental and applied basic biological areas of interest; photosynthesis, nitrogen fixation, and biotechnological conversions. The photosynthetic bacteria are thus excellent organisms for studying both general and unique aspects of nitrogen fixation and its regulation. Besides the regulation at the transcriptional level with respect to oxygen and fixed nitrogen shared by most nitrogen fixing organisms, the photosynthetic bacteria are capable of regulating the activity of preformed enzyme through the covalent and metabolic regulation of nitrogenase. We have shown that the regulation of nitrogenase with respect to the sudden external addition of ammonium requires the membrane bound ammonia sensor/transporter AmtB. Amt/Rh family members are found in almost every sequenced organism and are present in archaea, bacteria, protists, plants, fungi and invertebrates. We will continue our investigations into the structure/function of AmtB and its role in controlling the the nitrogenase regulatory system. In particular, we will be applying site directed mutagenesis to understanding the role that specific amino acids play in ammonia sensing and/or transport. The nitrogenase enzymatic machinery is also responsible for hydrogen production by photosynthetic bacteria. These organisms, which have the potential capacity to use a variety of feedstocks, are well known for their light driven conversion of organic acids to hydrogen and carbon dioxide. Thus, they are ideal candidates for two stage or co-culture systems which derive additional hydrogen from the effluents of dark fermentations. In addition, various industrial and agricultural waste streams rich in organic acids can potentially serve as substrates for photofermentation. We have recently demonstrated a number of improvements in hydrogen production including the demonstration of the stoichiometric conversion to hydrogen of glycerol produced as a waste product of biodiesel manufacture and the generation of appreciable amounts of hydrogen from various molasses fractions. In a series of investigations we have also recently shown that significant amounts of hydrogen, up to 9 moles of H2 per mole of glucose, can be obtained in a one step, single stage fermentation of glucose. Of some interest, we have also demonstrated for the first time the production of hydrogen thought microaerobic fermentation. This process, which promises to enable the release of additional hydrogen from substrates by coupling further degradation with the generation of energy from limited respiration, will be further investigated under the present grant. This technology is of interest in the broader context of industrial biotechnology as it would allow the development of “unbalanced’ fermentations. Two approaches will be used; development of bioreactor technology using a redox stat to control the oxygen levels for achieving maximum substrate conversion, and metabolic engineering to increase metabolic flux in the desired direction. Metabolic engineering will also be used, in conjunction with metabolic modeling to further increase photofermentative hydrogen production. Thus, the current research is of both fundamental and applied interest. Further research on the molecular details of the involvement of AmtB in nitrogenase regulation should shed more light on this unique form of metabolic regulation. In addition, increasing hydrogen production through both physiological manipulation and metabolic engineering will pave the way for the production of other chemicals by these organisms as well as possibly providing a practical means of sustainable hydrogen generation from various waste materials.