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Keywords:

  • Continuous culture;
  • Medium feed gradient;
  • PHA;
  • Process analytical technology;
  • Pseudomonas putida

Abstract

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between Cf/Nf ≤ 6.4 g/g and Cf/Nf >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the Cf/Nf ratio was changed continuously through a convex increase of Cf, a convex increase of Nf, or a linear decrease of Cf (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g · for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.