The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe2+) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe2+), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe3+ concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe3+ to Fe2+ ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 +/- 0.2mg IBA/L/d which is a 14-fold increase over the previously reported value. (c) 2015 Wiley Periodicals, Inc.