Metabolic modeling of growth and poly-[beta]-hydroxybutyrate (PHB) production in Alcaligenes eutrophus H16

Citation

Guske, Christopher James (1990) Metabolic modeling of growth and poly-[beta]-hydroxybutyrate (PHB) production in Alcaligenes eutrophus H16. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/J4NT-HG32. https://resolver.caltech.edu/CaltechETD:etd-06072007-110221

Abstract

A physiologically based metabolic model for growth and poly-[beta]-hydroxybutyrate (PHB) production in Alcaligenes eutrophus H16 has been formulated. The model is based upon A. eutrophus' five major heterotrophic metabolic pathways: an overall biosynthesis pathway, respiration, glycolysis, the citric acid cycle, and PHB polymerization. Beginning with microscopic intracellular balances, intracellular phenomena such as pathway flux activities and intracellular balances on acetyl-CoA, ATP and NADH are related to the macroscopic time evolutions of fructose concentration, cell dry weight concentration, PHB concentration, carbon dioxide evolution rate, and oxygen consumption rate.

Using the derived model framework, Alcaligenes eutrophus H16's metabolic responses to different medium fructose, ammonium and oxygen concentrations as well as nutrient limitations and exhaustions were determined in a well-controlled, fully monitored bioreactor. Ammonium exhaustion led to the highest observed PHB synthesis rates, while the PHB polymerization rates correlated with the severity of ammonium and oxygen limitations. Surprisingly, fructose limitation led to an increase in intracellular PHB levels. Glycolysis, citric acid cycle and repiration activities were very sensitive to short-term fluctuations during fructose-limited feeding, while biosynthesis and PHB polymerization responses were sluggish. Fructose exhaustion PHB depolymerization occurred at a small absolute rate relative to polymerization during periods of fructose sufficiency. High intracellular PHB levels appeared to inhibit cell growth.

Using in vitro kinetic study data, model metabolic pathway responses were compared with the regulatory properties of the key regulatory enzymes. Glucose-6-phosphate NADH-regulation appears weaker than that exerted on isocitrate dehydrogenase as evidenced by increased ammonium-limited glycolysis activity and presumed elevated NADH levels. Increased NADH levels sharply correlated with increased PHB polymerization activity. Along this line, culture fluorescence was used as a sensitive indicator of NAD(P)H level changes. Culture fluorescence was a good indicator of metabolic shifts, particularly at the onset of increased PHB polymerization. Increases in specific culture fluorescence correlated strongly with intracellular PHB levels.

From the pathway-associated intracellular balances, average metabolic parameters for the ATP yield coefficient, Ymax/ATP, the oxidative phosphorylation ratio, P/O, and the ATP maintenance coefficient, mATp,e, were determined of 0.34 C-mol biomass/mol ATP, 2.9 mol ATP/mol H2O, and 9.8 mmol ATP/((g biomass)(hr)), respectively, which agree favorably with other work. Use of the constant metabolic parameters and the closed intracellular balances predicted balanced growth conditions well but deviated under ammonium limitation conditions. Also, the metabolic parameters seemed to shift to lower values as during the growth/PHB storage phase transition.

Incorporation of kinetic expressions for biosynthesis and PHB polymerization activities mimicked balanced growth conditions very well while deviating from ammonium limitation and ammonium exhaustion conditions.

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