Hydrogen yields of dark fermentation are limited due to the need to also produce reduced side products, and photofermentation, an alternative, is limited by the need for light. A relatively new strategy, dark microaerobic fermentation, could potentially overcome both these constraints. Here, application of this strategy demonstrated for the first time significant hydrogen production from lactate by a single organism in the dark. Response surface methodology (RSM) was used to optimize substrate and oxygen concentration as well as inoculum using both (1) regular batch and (2) O-2 fed batch cultures. The highest hydrogen yield (HY) was observed under regular batch (1.4 +/- 0.1 mol H-2/mol lactate) and the highest hydrogen production (HP) (173.5 mu mol H-2) was achieved using O-2 fed batch. This study has provided proof of principal for the ability of microaerobic fermentation to drive thermodynamically difficult reactions, such as the conversion of lactate to hydrogen.