The nifJ gene codes for pyruvate:ferredoxin oxidoreductase, which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-CoA. A nifJ knock-out mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to wild type (WT) under light-dark cycling. This is attributed to an increase in the quantum yield of PSII charge separation as measured by photosynthetic electron turnover efficiency using fast repetition rate fluorometry (Fv/Fm). During autofermentation the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT, H2 production in vivo was 1.3-fold lower than WT. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with resulting loss of reductant and a 3-fold decrease in H2 production by nifJ cells compared to WT. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin because it decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+, while the second phase corresponded to the equilibrium NADH-mediated process.