Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha

Ralstonia eutropha ATCC 17699 was grown to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from fructose and γ-butyrolactone. Through flask cultures, it was found that R. eutropha cells were inhibited by high concentrations of fructose (more than 20 g/L) and γ-butyrolactone (more than 6 g/L). The 4HB fraction could be increased from 37.9 to 53.7 mol% by adding small amount (2 g/L) of acetate or propionate as a stimulator. Fed-batch cultures (DO-stat and constant feeding) were carried out to improve cell and PHA concentration. In the first step, cells were grown to 38-48 g/L by DO-stat feeding of fructose. Nitrogen limitation was applied and the fructose feed was replaced with the mixture of fructose and γ-butyrolactone. The effects of γ-butyrolactone to fructose ratio in the second step feeding solution were examined. As the ratio increased, 4HB fraction increased, while cell and PHA concentration, PHA content, and productivity decreased. The results obtained were in the range of 33.6-49.1 g/L of cell concentration, 13.8-24.4 g/L of PHA concentration, 38.8-50.2% of PHA content, 1.64-25.2 mol% of 4HB fraction, and 0.32-0.55 g/L/h of productivity.