A novel high-throughput method for kinetic characterisation of anaerobic bioproduction strains, applied to Clostridium kluyveri

Abstract

Hexanoic acid (HA), also called caproic acid, can be used as an antimicrobial agent and as a precursor to various chemicals, such as fuels, solvents and fragrances. HA can be produced from ethanol and acetate by the mesophilic anaerobic bacterium Clostridium kluyveri, via two successive elongation steps over butyrate. A high-throughput anaerobic growth curve technique was coupled to a data analysis framework to assess growth kinetics for a range of substrate and product concentrations. Using this method, growth rates and several kinetic parameters were determined for C. kluyveri. A maximum growth rate (μmax) of 0.24 ± 0.01 h-1 was found, with a half-saturation index for acetic acid (KS,AA) of 3.8 ± 0.9 mM. Inhibition by butyric acid occurred at of 124.7 ± 5.7 mM (KI,BA), while the final product, HA, linearly inhibited growth with complete inhibition above 91.3 ± 10.8 mM (KHA of 10.9∗10-3 ± 1.3∗10-3 mM-1) at pH = 7, indicating that the hexanoate anion also exerts toxicity. These parameters were used to create a dynamic mass-balance model for bioproduction of HA. By coupling data collection and analysis to this modelling framework, we have produced a powerful tool to assess the kinetics of anaerobic micro-organisms, demonstrated here with C. kluyveri, in order further explore the potential of micro-organisms for chemicals production.