Appl Microbiol Biotechnol (2011) 90:1681–1690 1687 Fig. 6 Enzyme activity of isobutanol pathway for JCL260 carrying pSA65/pSA69 plasmids in bioreactor cultures at 30°C (open symbols) and 37°C (closed symbols). a Total isobutanol production calculated as sum of isobutanol concentrations from receivers B, D (Fig. 1), and broth culture considering a working volume of 0.35 L. b Time profile of glucose concentration and cell growth. c Acetolactate synthase (AlsS) activity. d Dihydroxy-acid dehydratase activity. e 2- Ketoisovalerate decarboxylase (KivD) activity. f Alcohol dehydroge- nase (AdhA) activity. Error bars indicate the difference between duplicate assays eliminate acetate accumulation, but even a triple mutant (Δack Δpta ΔpoxB) was not enough to eliminate acetate accumulation during aerobic fermentation (Chang et al. 1999; Phue et al. 2010), suggesting that acetate might also come from other pathways. Time profile of high isobutanol producer strain (JCL260) at 30°C and 37°C Since vapor pressure increases with temperature, higher temperature should increase the gas stripping rate, and consequently, isobutanol concentration in the fermentation broth should be lower. Accordingly, the isobutanol production by JCL260 was evaluated at 37°C and compared with the production at 30°C (Fig. 2a). Unfortunately, the maximum isobutanol concentration was twofold lower at 37°C compared with that at 30°C. The isobutanol concentration in the fermentation broth reached 5.3 g/L and decreased until 0.4 g/L at the end of culture (Fig. 2b). The maximum biomass concentration (7.2 g/L) was practically the same for both temperatures (Fig. 2c). Glucose depletion was avoided by intermittent feeding in all cultures (Fig. 2d). Acetate produc-