Optimized cell growth and poly(3-hydroxybutyrate) synthesis from saponified spent coffee grounds oil

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C₀) and the specific growth rate (µ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C₀ = 2.9-23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C₀ = 19.1 g/L) saponified SCG oil. Peak µ-values of 0.139 h^-1 and 0.145 h^-1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates (µ = 0.35 h^-1) than on the crude SCG oil (µ = 0.23 h^-1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA. KEY POINTS: • The bioavailability of plant oil substrates can be improved via saponification. • Cell growth and inhibition were accurately described by the Han-Levenpsiel model. • Mixing crude and saponified oils enable variation of free fatty acid content.