Appl Microbiol Biotechnol (2011) 90:1681–1690 1685 Fig. 3 Volumetric isobutanol production rate calculated as slope from time profile of isobutanol production before and after 22 h for all cultures, except for JCL260 at 37°C where it was determined before and after 16 h. Error bars represent the difference between duplicate cultures Results Comparison of high isobutanol producer (JCL260), high isobutanol-tolerant (SA481), and parental (JCL16) strains at 30°C In the present work, bioreactor cultures of JCL260 were performed by controlling DO above 20%, pH at 6.8, and temperature at 30°C and were compared with JCL16 and SA481. A high aeration rate (1.2 vvm) was used for in situ isobutanol recovery. Typical kinetics of isobutanol produc- Fig. 4 Time profile of acetate production by JCL260 PoxB− mutant and high isobutanol producer (JCL260) strains harboring pSA65/pSA69 plasmids cultivated in shake flasks containing 25 mL of medium. Data shown are the results of three independent fermentations tion, cell growth, glucose consumption, and acetate pro- duction are shown in Fig. 2. Isobutanol concentration plotted in Fig. 2a represents the total isobutanol produced by the culture quantified from receivers B, D (Fig. 1), and fermentation broth. The total isobutanol concentration was calculated as a sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L. Note that we condensed isobutanol into water again for easy quantitation. One could directly condense isobutanol without redissolving into water. Isobutanol production continued even after cell growth was stopped (Fig. 2a, c). In fact, around 80% of total isobutanol accumulated by JCL260 cultivated at 30°C was produced after the growth phase. Such behavior was similar to that reported by Atsumi et al. (2008b), but 2.3-fold higher. The high isobutanol producer strain (JCL260) accumulated 50.8 ± 1.1 g/L of isobutanol in 72 h and the parental (JCL16) 41.4 ± 4.5 g/L in the same time (Fig. 2a). Surprisingly, the high isobutanol-tolerant strain (SA481) produced only 23 ± 4.4 g/L of total isobutanol in 68 h. After 22 h, the maximum isobutanol concentration in the fermentation broth for JCL260 and JCL16 was around 11 g/L and for SA481 around 6.5 g/L (Fig. 2b). The maximum cell concentrations for JCL16, SA481, and JCL260 were 8.4 ± 0.3, 7.6 ± 0.7, and 6.7 ± 0.16 g/L, respec- tively (Fig. 2c). Since the aeration rate, temperature, and pressure (hence the desorption rate) were maintained constant and at the