Controlled Feeding Facilitates the Development of a Robust Production Process for Terpolymeric Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are a promising class of biodegradable and sustainable biopolymers. However, the current use of PHA is limited by its restricted availability, which is deplorable, as novel PHA variants such as copolymers or terpolymers could offer improved material properties. In this study, we report the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) (PHBVV) terpolymer with high 3-hydroxyvalerate (3HV) content using Cupriavidus necator. A fully controlled feeding strategy, based on the pH- and pO₂-signal, was successfully implemented and validated in 7.5 and 42-L bioreactors, enabling controlled delivery of the growth-inhibiting substrate levulinic acid. A maximum biomass of 40.2 ± 0.9 g L^{- 1} with 75% CDW PHA was obtained, corresponding to a yield of 0.32 ± 0.02 g_PHA g_LA ^{- 1}. Importantly, the process enabled the production of PHBVV with a high 3HV content of 38%-46% and a 4HV content of 1%-2% at both production scales. Beyond demonstrating scalable PHBVV production in stirred tank bioreactors, this work provides a comprehensive comparative dataset, including specific growth rates, substrate uptake rates, yield coefficients, and respiratory activity. The findings presented here constitute both a methodological framework for scaling and process automation of PHBVV production and a valuable reference for the future optimization of PHA bioprocesses.