Several anaerobic bioconversion technologies produce short chain volatile fatty acids and sometimes ethanol, which can together be elongated to hexanoic acid (C6 acid) by Clostridium kluyveri in a secondary fermentation process. Initiatives are needed to further optimize the process. Therefore, five strategies were tested aiming at elucidating their influence on hexanoic acid production from mixtures of acetic acid, butyric acid and ethanol. pH-regulated bioreactors, maintained at pH 7.5, 6.8 or 6.4 led to maximum C6 acid concentrations of, respectively, 19.4, 18.3 and 13.3 g L. At pH 6.8, yeast extract omission resulted in a decrease of the hexanoic acid concentration to 12.0 g L while the addition of an inorganic carbon source, such as bicarbonate, for pH control, increased the C6 acid concentration up to 21.4 g L. This research provides guidelines for efficient improved production of hexanoic acid by pure cultures of C. kluyveri, contributing to the state of art.