On-line analysis of Alcaligenes eutrophus fermentations

Abstract

Improvements in the poly(β-hydroxybutyrate) (PHB) productivity of Alcaligenes eutrophus can be achieved by using genetically engineered strains. Knowledge of their metabolic regulation can be obtained from fermentation experiments. Certain events may not be noticed owing to the typically low frequency of sampling. To avoid information gaps, a newly developed fermenter-calorimeter was used for batch experiments allowing comprehensive on-line monitoring of fermentations. PHB-producing strain HI 6 and PHB-lacking mutant PHB−4 were selected, since they are well characterized with respect to the metabolism of PHB. For both strains, the profiles of thermograms, oxygen dissolved in the medium, and carbon dioxide formation were in good agreement with the off-line analysis. The on-line measurements showed characteristic differences between strains PHB−4 and H16. During fed-batch cultivations of PHB-accumulating strain H16, rapid autolysis of the cells was observed.Key words: Alcaligenes eutrophus, PHB, fermentation, calorimetry, on-line analysis, growth efficiency.

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

“…Various techniques for monitoring process parameters of cultures have been developed, e.g. growth monitoring by thermogravimetry [Meier-Schneiders et al, 1995] and online monitoring of the PHB contents of cells by Nile Red staining [Gorenflo et al, 1999]. For heterologous production of cyanophycin, an addiction system was constructed , which may be also applicable to other polymers and proteins.…”

Bacterial Production of Poly(3-hydroxybutyrate). An Undergraduate Student Laboratory Experiment

“…In the second step, which is carried out by the students in the first laboratory session, the bacteria are transferred to a medium that stresses the cells and induces the production of PHB. In our procedure, PHB production is induced by using a nitrogen-limited medium, whose composition is modified from that reported by Schlegel (4) based on the literature reports of Repaske and co-workers (14,15) and Meier-Schneiders et al (6). Specifically, Co 2+ and Cr 3+ are added to the medium to increase PHB production as discussed in the literature (6,14,15).…”

“…In our procedure, PHB production is induced by using a nitrogen-limited medium, whose composition is modified from that reported by Schlegel (4) based on the literature reports of Repaske and co-workers (14,15) and Meier-Schneiders et al (6). Specifically, Co 2+ and Cr 3+ are added to the medium to increase PHB production as discussed in the literature (6,14,15). We note that it is important to use a chelated form of iron, specifically iron(III) ammonium citrate, so that the iron remains in solution over a longer period of time (i.e., days instead of hours).…”

“…PHB is an intracellular polymer that accumulates in the cytoplasm of bacterial cells when a carbon source is provided and at least one nutrient, such as nitrogen, is limited (1)(2)(3)(4)(5)(6). Because it is a biodegradable polyester, there is much interest in its production.…”