Co-feeding phenolic acids with fatty acids for polyhydroxyalkanoate (PHA) synthesis in Pseudomonas putida KT2440 using continuous culture conditions.

Lignin is a promising bioeconomy feedstock for the synthesis of biofuels and biobased chemicals. Pseudomonas putida KT2440, when grown in flasks with two of the main phenolic acids present in lignin (ferulate and p-coumarate), accumulated 33 % of the cell dry weight (CDW) as the biodegradable polymer polyhydroxyalkanoate (PHA). In batch cultures (1-L stirred tank bioreactor) P. putida KT2440 accumulated 37 % of CDW as PHA when supplied with a mixture of sodium- ferulate and p-coumarate (sodium phenolate mixture). Conditions were then switched to continuous fermentation mode, where PHA accumulation (10 % of CDW) was lower compared to batch-grown culture at a dilution rate of 0.1 h^-1. However, the PHA productivity of the continuous culture was similar to the batch culture. When the sodium phenolate mixture feed was supplemented with glucose, the PHA titre increased modestly, whereas bacterial biomass remained at a similar level. After the sodium phenolate mixture was supplemented with sodium octanoate (+5 mM), the PHA content of cells increased over threefold under continuous culture growth conditions compared to the sole phenolate mixture. PHA productivity varied depending on the dilution rate of the continuous culture, increasing at lower dilution rates (0.06 h^-1). Finally, PHA monomer composition is predominantly (R)-3-hydroxydecanoic acid (C10) when only the sodium phenolate mixture is supplied. This dramatically changed when sodium octanoate was added to the sodium phenolate mixture substrate feed with (R)-3-hydroxyoctanoate (C8) monomers predominating (90 %), suggesting that sodium octanoate is prioritised for PHA synthesis while the phenolic acids are used as energy and biomass sources.