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

Abstract

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 (NH(4)(+)) 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 (here 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()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() 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()/N() 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.

Physiological and Proteomic Adaptation of “Aromatoleum aromaticum” EbN1 to Low Growth Rates in Benzoate-Limited, Anoxic Chemostats

“…However, other factors, which have not been studied in much detail yet, also have an impact on PHA synthesis (52,53). Pseudomonas oleovorans has been demonstrated to accumulate PHA also under carbon-limited conditions (17,58). In the present study, residual NH 4 ϩ concentrations (provided as a nitrogen source) were Ͼ5 mM across the investigated range (data not shown).…”

Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review

“…The main difference is in the final polymer precipitation step where ethanol is replaced with ice-cold methanol [150]. Apart from chloroform, dichloromethane is a less hazardous choice as an extraction solvent and together with ice-cold methanol as precipitant, up to 98% purity of mcl-PHA could be attained [151]. Solvent extraction however, tends to result in significant polymer degradation [144].…”

Tailored Synthesis of Poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) inRalstonia eutrophaDSM 428

“…The carbon to nitrogen ratio (C/N) of the feed was set by the pump rates of the ammonium containing medium and the carbon sources. The use of two syringe pumps allowed the setting of different ratios of butyric to valeric acid in the feed, and at the same time maintaining the C/N ratio constant at 17 mol/mol, which has been found to permit dual (C, N) limited growth in previous studies [10,11].…”

“…However, recently it has been shown in chemostat cultures of Pseudomonas putida GPo1 that PHA production can also take place when nitrogen and carbon are limiting growth simultaneously. In the growth of P. putida GPo1, this dual (C, N) limited growth regime was investigated for such parameters as the kind of C substrate, the C/N ratio in the feed medium, and the growth rate of the cells [10,11]. It was shown that the extension of the dual (C, N) limited growth regime correlated with the capacity of P. putida GPo1 to accumulate PHA and hence the PHA content.…”