Respiration of Pseudomonas fluorescens as a function of intracellular substrate concentration

A kinetic method to measure the intracellular concentration of respiratory substrates in short-term starvation-enrichment experiments is proposed. Samples of bacterial suspension from steady-state chemostat cultures were subjected to 25 min starvation, followed by pulse addition of [¹⁴C]glucose. Residual substrate utilization rates and respiration rates (uptake of dissolved O₂) before and after amendment were recorded. Increases in pool sizes (δL) during transients were calculated on the basis of C balance. The dependence of respiration rate q_resp on δL was found to obey modified Michaelis-Menten kinetics: q _resp = Q _resp (LC+δL)/(K _L+LC+δL) [Q _resp is maximal respiration rate (29.1 mmol O₂ h^-1per g biomass C), K _L = 12.14 mg C per g biomass C], where LC is the absolute value of the pool size before amendment. Direct chemical determination of LC in cold TCA extracts revealed two fractions. The first fraction was mobile and showed a close correlation with both respiration and L. The second, 'stable', fraction did not correlate with respiration dynamics and was interpreted as material formed artifactually by acid degradation of polymeric cell components.