| Some aquatic microbial oxygenic photoautotrophs (AMOPs) excrete products such as H2, lactate, ethanol, and acetate in low yields during auto-fermentation (in dark, anoxic conditions) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have utilized NMR and LC/MS metabolomics methods to characterize the marine cyanobacterium, Synechococcus sp. strain PCC 7002. In addition to identifying the WT strain, we have analyzed the phenotype of 3 mutant strains: ldhA, ldhAEx, and nifJ. In addition, we analyzed the WT strain under auto- fermentative conditions in the presence of nitrate. We have gained insight into manipulating fermentative fluxes of this bacterium, which may have future applications for fuels like bio-hydrogen.;In Chapter One, we have constructed a mutant (ldhA) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this cyanobacterium. Auto-fermentation by the ldhA mutant resulted in no D-lactate production and redistributed fluxes of the excreted acetate, alanine, succinate, and hydrogen, which was 5-fold higher during auto-fermentation. The NAD(P)H/NAD(P) + ratio was also higher in the ldhA strain and this increased ratio is the source of the increased H2, which is generated via the NADH-dependent, bidirectional [NiFe]-hydrogenase.;In Chapter 2, we overexpress the ldhA gene for increased lactate production. In addition to a 2-fold increase in fermentative lactate production, the strain we have developed, LdhAEx, also excretes lactate (up to 20 mM) during photoautotrophic growth. Surprisingly, LdhAEx, which overexpresses only the lactate dehydrogenase enzyme, excretes large concentrations of acetate (up to 80 mM) during photoautotrophic growth, likely as an energy balance for the lactate production.;In Chapter 3, we have constructed a knock-out mutant (nifJ) of Synechococcus sp. strain PCC 7002 that lacks one of two enzymes for the oxidation of pyruvate to acetyl-CoA: pyruvate: ferredoxin oxidoreductase (PFOR). During auto-fermentation of nifJ the excreted carbon flux decreased by 2-fold for acetate and 1.2-fold for lactate, while reductant flux to H2 was 1.3-fold lower than WT. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during auto-fermentation. The first phase proceeded via reduced ferredoxin, as this phase decreased 2-fold in nifJ. Measurement of the intracellular NADH/NAD + ratio revealed that the first phase of H2 production arose from a reductant source that was not in equilibrium with NADH/NAD +.;In Chapter 4, we examine the effects of nitrate during auto-fermentation in Synechococcus 7002. This cyanobacterium imports and reduces nitrate to nitrite, a majority of which is excreted rather than being further reduced and assimilated into the cells during auto-fermentation. Hydrogen production increases 11-fold, lactate increases 2.5-fold, and CO2 decreases 3-fold when nitrate is removed from fermentation media after cultures are grown in nitrate replete conditions. The cells that were fermented in the presence of nitrate seem to utilize enzymes of the oxidative pentose phosphate pathway in addition to glycolysis in order to produce more reductant equivalents per glucose equivalent catabolized as well as more CO2, while cells fermented in the absence of nitrate utilize predominantly glycolysis. |
