On-line Characterization of Physiological State in Poly(β-Hydroxybutyrate) Production by Wautersia eutropha

Abstract

Culture fluorescence measurement technique has the potential for on-line characterization of metabolic status of fermentation processes. Many fluorophores present inside the living cells such as NADH + H⁺, tryptophan, pyridoxine, and riboflavin fluoresce at specific excitation and emission wavelength combinations. Since these key intracellular metabolites are involved in cell growth and metabolism, their concentration change at any time inside the cell could reflect the changes in cell metabolic activity. NADH + H⁺ spectrofluorometry was used for on-line characterization of physiological state during batch cultivation of poly-β-hydroxybutyric acid (PHB) production by Wautersia eutropha. The culture fluorescence increased with an increase in the biomass concentration with time. A linear correlation between cell mass concentration and net NADH + H⁺ fluorescence was established during active growth phase (13 to 38 h) of batch cultivation. The rate of change of culture fluorescence (dF/dt) exhibited a gradual increase during the predominantly growth phase of batch cultivation (till 20 h). Thereafter, a sudden drop in the dF/dt rate and its leveling was recorded indicating major changes in culture metabolism status which synchronized with the start-up of accumulation of PHB. After 48 h, yet another decrease in the rate of change of fluorescence (dF/dt) was observed primarily due to severe substrate limitation in the reactor. On-line NADH + H⁺ fluorescence signal and its rate (dF/dt) could therefore be used to distinguish the growth, product formation, and nutrient depletion stage (the metabolic state marker) during the batch cultivation of W. eutropha.