Production of medium-chain-length hydroxyalkanoic acids from Pseudomonas putida in pH stat

Abstract

Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA_MCL) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HA_MCL at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h⁻¹ g⁻¹ cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3–11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h⁻¹ g⁻¹ cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HA_MCL were secreted by the wild type, but only little 3HA_MCL were found for the depolymerase mutant. In summary, 3HA_MCL can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.