Pathways of ethanol production from sucrose by a mutant thermophilic Bacillus in continuous culture

Abstract

A mutant (strain LLD-15) of Bacillus stearothermophilus strain LLD-R, that lacks l-lactate dehydrogenase activity was studied in anaerobic single-stage continuous culture using a defined minimal medium for its capacity to produce ethanol from sucrose at 70 °C. Cultures were stable at neutral pH, dilution rates below 0·25 h−1 and sucrose concentrations below 15 g l−1, producing mainly ethanol, CO2, formate and acetate plus a little succinate. The data obtained were used to estimate the maintenance energy coefficient at 70 °C and showed that the relative fluxes through the pyruvate-formate lyase (PFL) pathway and an anaerobic pyruvate dehydrogenase (PDH) pathway were about equal, though the later predominates at low dilution rates. At higher dilution rates or at acid pH, l-lactate became the major anaerobic product due to takeover by wild-type revertants. Reversion occurred only above a critical specific sucrose consumption rate of 4·2 g sucrose h−1 (g cells)−1 at pH 7, at which point pyruvate production was also seen. This can be attributed to saturation of the PFL pathway and PDH pathways by increasing glycolytic flux and consequent accumulation of intracellular pyruvate. The growth rate then declines, but a small population of revertant LLD-R cells would always be present in the bioreactor since the mutation in the l-lactate dehydrogenase gene has a significant reversion rate. These revertants have no selective advantage below the critical sugar uptake rate, but at higher rates they divert excess pyruvate to L-lactate, so their growth is unimpaired and they take over in the culture. At lower pH, growth restriction and reversion occur at low sucrose consumption rates due to difficulty in excreting acetic and formic acids against a pH gradient.