A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation ofBucUus sfeurothermophh strain LLD-15 (NCIMB 12428) under anaerobic conditions. This mutant strah lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hex-, using a high-temperature two-stage process. The final medium contained four amino acids and five Vitamins, and growth characterhtics in this medium compared well with those in complex medium Containing yeast extract and tryptone. At 70 "C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose 1-I. High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.

Continuous fermentation with cell recycle proved very effective in increasing the ethanol volumetric productivity of the thermophilic facultative anaerobe, Bacillus stearothermophilus strain LLD-15, on sucrose at 70 degrees C. When complete cell recycle was used, cell viability decreased after a few residence times and sucrose consumption was reduced. Operation using a constant bleed rate resulted in greater stability and higher ethanol volumetric productivities. A mathematical model based on maintenance energy requirements provided an adequate description of the system.

A mutant (strain LLD-15) of Bacillus stearothevmuphihs 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-' and sucrose concentrations below 15 g 1-' , producing mainly ethanol, CO,, 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-' (g cells)-' 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.