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.
Highlights
IntroductionGrowth rates and several kinetic parameters were determined for C. kluyveri
Using this method, growth rates and several kinetic parameters were determined for C. kluyveri
The validity of the novel, 96-well plate (96-WP) based experimental method was demonstrated by comparing product output of the pooled replicates in a 96-WP with that of a culture grown by a traditional method i.e. in a Balch tube
Summary
Growth rates and several kinetic parameters were determined for C. kluyveri. 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 exerts toxicity These parameters were used to create a dynamic mass-balance model for bioproduction of HA. The limited data on the kinetic properties of C. kluyveri – or microbial MCCA-production processes in general – requires expansion to obtain detailed information on growth rates, substrate affinity, substrate inhibition and product inhibition. This knowledge is of value to enable microbial production of HA, and ideally towards more sustainable carbon sources for HA. Working with strictly anaerobic, axenic cultures is labour-intensive and often difficult to manage in large batch experiments
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