Poly-3-hydroxybutyrate production from used cooking olive oil by Cupriavidus necator DSM 545: Bioprocess development and OTR-ka kinetic modelling in continuous fed-batch fermentation

Large-scale production of poly-3-hydroxybutyrate (PHB) is hindered by high operational costs, mainly due to raw material expenses and fermentation requirements. Waste plant oils offer a sustainable, low-cost alternative, supporting residue valorisation. In particular, used cooking olive oil (UCOO), a challenging waste stream in the Mediterranean, requires efficient recycling and bioconversion strategies. In this study, we investigate the production of PHB from UCOO as the sole carbon source by Cupriavidus necator DSM 545. Operating in continuous fed-batch regime, we reached 125.5 g/L of total biomass and 95.3 g PHB/L, tripling the maximum productivity up to 1.8 g PHB/(L·h) and achieving a 99% of waste conversion. Moreover, we identified oxygen transfer rate (OTR) as a key modulator of carbon flux distribution: PHB accumulation was maximized at 0.05 mol O₂/(L·h), while lower values of this variable boosted cell growth. Based on these observations, we developed a kinetic model to predict residual biomass, oil consumption, oxygen dynamics and PHB accumulation, by using experimental profiles of volumetric mass transfer coefficient (k_La) and culture dilution rate (D) as independent variables. Our findings validate UCOO as an effective alternative substrate for PHB production and highlight the critical influence of OTR in optimizing high-yield PHA fermentation strategies.