Clostridium carboxidivorans was grown on model syngas (CO:H-2:CO2 [70:20:10]) in a defined nutrient medium with concentrations of nitrogen, phosphate and trace metals formulated to enhance production of higher alcohols. C. carboxidivorans was successfully grown in a limited defined medium (no yeast extract, no MES buffer and minimal complex chemical inputs) using an improved fermentation protocol. Low partial pressure of CO in the headspace, coupled with restricted mass transfer for CO and H-2, was required for successful fermentation. In the absence of substrate inhibition (particularly from CO), growth limitation increased production of alcohols, especially butanol and hexanol. Concentrations of butanol (over 1.0 g/L), hexanol (up to 1.0 g/L) and ethanol (over 3.0 g/L) were achieved in bottle fermentations. Minimal medium and controlled supply of CO and H-2 should be used in characterizing candidate butanol and hexanol producing strains to select for commercial potential. (C) 2015 Elsevier Ltd. All rights reserved.