Osmotic stress restricts glycolytic flux, growth (rate and yield), D-lactate productivity, and D-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mM betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l(-1) h(-1); 0.8 to 2.3 g l(-1) h(-1)). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during D-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol D-lactate (90 g) per 100 g sugar (glucose or sucrose).