Using flow cytometry to evaluate the production of biopolymers in Cupriavidus necator

Rolley, John (2023). Using flow cytometry to evaluate the production of biopolymers in Cupriavidus necator. University of Birmingham. Ph.D.

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Abstract

Polyhydroxyalkanoates offer a credible solution to the environmental problems caused by non-biodegradable plastics and also have promising medical and consumer applications due to excellent biodegradable and biocompatible properties. This study has explored the production of P(3-hydroxybutyrate) (P3HB) using Cupriavidus necator grown in meat extract (ME) media supplemented with fructose and sodium gluconate. This study utilised flow cytometry in a novel manner which allowed the analysis of C. necator P3HB content, cell size and cell counts to take place simultaneously on a single-cell basis.

Overall experiments have failed to produce maximum P3HB in a reasonable timeframe of 72 hours due to a lag in P3HB biosynthesis which extends production well beyond 72 hours. The lag phase may have been due to the action of catabolite repression. This study reports for the first time that gluconate suppresses the metabolism of fructose by catabolite repression causing diauxic growth to take place as C. necator slowly adapts to fructose metabolism once gluconate is utilised. In comparison to fructose, sodium gluconate-supplemented C. necator produce more P3HB on average per cell and per mL of culture. This may be due to gluconate being co-transported across the cell membrane with sodium ions via secondary transport proteins. Fructose-supplemented C. necator cultures appear to favour cell division while cultures containing sodium gluconate favour cell expansion. It’s likely the uptake of sodium ions also causes sodium gluconate-supplemented C. necator to expand by creating an osmotic gradient which causes an influx of H\(_2\)O into the cell, and by interacting with peptidoglycan precursors vital for cell expansion.

For the first time a P3HB\(^{++}\) population of C. necator cells which produces up to 1000 times as much P3HB as the dominant phenotype and comprises less than 5% of the total population, is reported. Evidence shows that the production of P3HB\(^{++}\) cells is sensitive to both nutrient and C concentrations. Due to this population’s small proportion in cultures, production during exponential growth and small size, it’s likely that P3HB\(^{++}\) cells are a form of persister cell produced as a survival strategy in case the main population is unable to survive.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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