Identification of the growth-limiting step in continuous cultures from initial rates measured in response to substrate-excess conditions

Abstract

When steady state chemostat cultures are abruptly exposed to substrate-excess conditions, they exhibit long lags before adjusting to the new environment. The identity of the rate-limiting step for this slow response can be inferred from the initial yields and specific growth rates measured by exposing steady state cultures at various dilution rates to substrate-excess conditions. We measured these parameters for glucose-limited cultures of E. coli ML308 growing at various dilution rates between 0.03 and 0.6 1/hr. In all the cases, the initial yields were 20-30% less than the steady state yields. The decline of the yield implies that overflow metabolism is triggered in response to excess glucose. It is therefore unlikely that the initial response of the cells is limited by substrate uptake. The initial specific growth rates of cultures growing at low dilution rates (D = 0.03, 0.05, 0.075, 0.1, 0.3 1/hr) were significantly higher than the steady state specific growth rates. However, the increment in the specific growth rate decreased with the dilution rate, and at D=0.6 1/hr, there was no improvement in the specific growth rate. The initial specific growth rates varied hyperbolically with the dilution, decreasing sharply at dilution rates below 0.1 1/hr and saturating at D=0.6 1/hr. This is consistent with a picture in which the initial response is limited by the activity of glutamate dehydrogenase.