Closed-loop control of bacterial high-cell-density fed-batch cultures: Production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate andcofeeding conditions

Pseudomonas putida KT2442 is able to accumulate medium-chain-length poly(3- hydroxyalkanoates) (mcl-PHAs) as intracellular inclusions on a variety of f atty 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 substrat es therefore requires control of the carbon source concentration in the sup ernatant. In this study, we develop a closed-loop control system based on o n-line gas chromatography to maintain continuously fed substrates at desire d levels. We used the graphical programming environment LABVIEW to set up a flexible process control system that allows users to perform supervisory p rocess control and permits remote access to the fermentation system over th e internet. Single-substrate supernatant concentration in a high-cell-densi ty fed-batch fermentation process was controlled by a proportional (P) cont roller (P = 50%) acting on the substrate pump feed rate. Na-octanoate conce ntrations oscillated around the setpoint of 10 mM and could be maintained b etween 0 and 25 mM at substrate uptake rates as high as 90 mmol L-l h-l. Un der cofeeding conditions Na-10-undecenoate and Na-octanoate could be indivi dually controlled at 2.5 mM and 9 mM, respectively, by applying a proportio nal integral (PI) controller for each substrate. The resulting copolymer co ntained 43.5 mol% unsaturated monomers and reflected the ratio of 10-undece noate in the feed. It was suggested that both substrates were consumed at s imilar rates. These results show that this control system is suitable for a voiding substrate toxicity and supplying carbon substrates for growth and m cl-PHA accumulation. (C) 1999 John Wiley & Sons, Inc.