Closed-loop control of bacterial high-cell-density fed-batch cultures: production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate and cofeeding conditions.

Pseudomonas putida KT2442 is able to accumulate medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) as intracellular inclusions on a variety of fatty acids and many other carbon sources. Some of these substrates, such as octanoic acid, alkenoic acids, and halogenated derivatives, are toxic when present in excess. Efficient production of mcl-PHAs on such toxic substrates therefore requires control of the carbon source concentration in the supernatant. In this study, we develop a closed-loop control system based on on-line gas chromatography to maintain continuously fed substrates at desired levels. We used the graphical programming environment LABVIEW to set up a flexible process control system that allows users to perform supervisory process control and permits remote access to the fermentation system over the Internet. Single-substrate supernatant concentration in a high-cell-density fed-batch fermentation process was controlled by a proportional (P) controller (P = 50%) acting on the substrate pump feed rate. Na-octanoate concentrations oscillated around the setpoint of 10 mM and could be maintained between 0 and 25 mM at substrate uptake rates as high as 90 mmol L(-1) h(-1). Under cofeeding conditions Na-10-undecenoate and Na-octanoate could be individually controlled at 2.5 mM and 9 mM, respectively, by applying a proportional integral (PI) controller for each substrate. The resulting copolymer contained 43.5 mol% unsaturated monomers and reflected the ratio of 10-undecenoate in the feed. It was suggested that both substrates were consumed at similar rates. These results show that this control system is suitable for avoiding substrate toxicity and supplying carbon substrates for growth and mcl-PHA accumulation.