Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultur es with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such t hat nitrogen (NH4+) limitation terminated the exponential-growth phase. Dur ing batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chai n-length poly[(R)3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significa nt rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients there of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is con tinuously changing. Therefore, we have also investigated the effect of defi ned ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. Thes e experiments were performed at a constant D of 0.2 h(-1). The concentratio n of the nitrogen source in the inflowing medium (N-o) was kept constant, w hile its carbon concentration (C-o) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C-o/N-o ratio). The cultu re parameters and the cell composition of steady-state cultures were determ ined as a function of the C-o/N-o ratio in the feed medium. Mcl-PHA accumul ation was detected during growth with the fatty acids, and three distinct r egimes of growth limitation were discovered: In addition to carbon limitati on at low, and nitrogen limitation at high C-o/N-o ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detecte d where both substrates were used to completion. The width of this dual-nut rient-limited growth regime was dependent on the change in the yield factor s for carbon and nitrogen (Y-X/C Y-X/N) measured during single-nutrient-lim ited growth. (C) 2001 John Wiley & Sons, Inc.