ABSTRACT Thermoanaerobacterium thermosaccharolyticum is an anaerobic and thermophilic bacterium that has been genetically engineered for ethanol production at very high yields. However, the underlying reactions responsible for electron flow, redox equilibrium, and how they relate to ethanol production in this microbe are not fully elucidated. Therefore, we performed a series of genetic manipulations to investigate the contribution of hydrogenase genes to high ethanol yield, generating evidence for the importance of hydrogen-reacting enzymes in ethanol production. Our results indicate that a high ethanol yield, >85% of the theoretical maximum, only occurs when the hfsD, hydAB , and nfnAB genes are all present together, while the hfsB gene is absent. We propose that the products of these three gene clusters facilitate an NADPH-generating reaction via hydrogen cycling, with a stoichiometry comparable with a canonical ferredoxin:NADP + oxidoreductase (FNOR; EC 1.18.1.2) reaction. The hypothesized mechanism provides a balance of nicotinamide cofactors and facilitates ferredoxin recycling, leading to progress in optimizing the energy conversion of biomass-derived sugars to ethanol. IMPORTANCE Our study elucidates the crucial role of electron flow and redox balancing mechanisms in improving ethanol yields from renewable biomass. We delve into the mechanism of electron transfer, highlighting the potential of key genes to be leveraged for enhanced ethanol production in anaerobic microbial species. We suggest by genetic investigation the existence of a novel Ferredoxin:NADP+ Oxidoreductase (FNOR) reaction, comprising HfsD, HydAB, and NfnAB enzymes, as a promising avenue for achieving balanced stoichiometry and efficient ethanol synthesis. Our findings not only advance the understanding of microbial metabolism but also offer practical insights for developing strategies to improve bioenergy production and sustainability.