Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH4+) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant 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 continuously changing. Therefore, we have also investigated the effect of defined 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. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N-o) was kept constant, while 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 culture parameters and the cell composition of steady-state cultures were determined as a function of the C-o/N-o ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C-o/N-o ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y-X/C Y-X/N) measured during single-nutrient-limited growth. (C) 2001 John Wiley & Sons, Inc.