The Escherichia coli K-12 strain TG1 was grown at 28 °C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0·044 to 0·415 h−1. The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0·3 h−1. At dilution rates between 0·3 h−1 and 0·4 h−1, a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0·4 h−1. An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0·4 h−1, close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.
THE RÔLE OF THE TRICARBOXYLIC ACID CYCLE IN ACETATE OXIDATION IN ESCHERICHIA COLI
