Microrespirometric Validation of a Two-stage Process for Polyhydroxyalkanoates Production from Peanut Oil and Propionate with Cupriavidus necator

Poly(3-hydroxyalkanoates) (PHA) is a biodegradable polyester synthesized by various bacteria, including Cupriavidus necator. The composition of PHA is influenced by the type of microorganism, cultivation conditions, and carbon substrate. The selection of a carbon source is critical for PHA production, significantly impacting process costs. Therefore, the common goal is scalability, with the design and optimization of PHA production relying on experimental determination of constitutive parameters. In this work, a two-stage process for PHA production was conducted in shaking flasks functioning as mini reactors with the bacteria Cupriavidus necator. During the first stage, fructose was used as the sole carbon source. Once fructose was exhausted, a second stage commenced with a new carbon source, either peanut oil or propionate, to enhance PHA production. Ex-situ pulse respirometry approach was employed during the two-stage process to follow the kinetics of substrate consumption. The results indicated that the use of peanut oil would be advantageous over propionate, resulting in 12.2% more biomass and 13.9% more PHA. Additionally, the growth rate was 88.9% higher with peanut oil. The characterization by pulse respirometry applied in microreactors, i.e., micro respirometry, allowed for the performance of up to 216 biological experiments to determine four important kinetic and stoichiometric parameters, namely maximum oxygen uptake (rO2max), substrate affinity constant (KS), growth yield (YX/S), and substrate oxidation yield (YO2/S). The values of these parameters indicated that peanut oil would be the best carbon source to promote PHA production during the second stage. The implementation of the microrespirometry technique during the screening of carbon sources for PHA production provided reliable information within a short period and with significantly less experimental effort.