1688 Fig. 7 Comparison of specific growth rate (μ, liters per hour), volumetric productivity (grams per liter per hour) and product yield on glucose (YP/S, gram per gram) of the high isobutanol producer (JCL260), high isobutanol-tolerant (SA481), and parental (JCL16) strains cultured at 30°C and JCL260 at 37°C. The μ was calculated during the exponential growth phase. Error bars indicate the difference between replicate cultures tion was higher at 37°C (Fig. 2e), reaching a maximum concentration of 15.8±1.6 g/L at 10.5 h. Initially, it was hypothesized that the lower isobutanol production at 37°C was due to the poor quality of heterologous protein expression. To test this hypothesis, activities of four (AlsS, IlvD, KivD, and AdhA) enzymes involved in the biosynthetic isobutanol pathway were determined in bioreactor cultures performed at 30°C and 37°C (Fig. 6). Isobutanol, growth, and glucose time profile are shown in Fig. 6a, b, and in concordance with the results of Fig. 2a, maximum isobutanol concentration reached at 30°C was twofold higher than at 37°C. Enzyme activities of AlsS, IlvD, and KivD were roughly similar in both 30°C and 37°C cultures (Fig. 6c–e). However, the AdhA activity (Fig. 6f) in the 37°C culture was significantly lower than that in the 30°C culture from 17 h to the end of cultures, suggesting that this step may contribute to the difference between the two temperatures. Kinetic and stoichiometric comparison of the strains The specific growth rate (μ), volumetric productivity, and isobutanol yield on glucose were determined (Fig. 7). The specific growth rate for JCL260 was similar to the parental strain (JCL16) and was 5% lower for SA480. As expected, the specific growth rate for JCL260 at 37°C was higher (around 26%) than at 30°C. In agreement, the acetate production was higher at 37°C than at 30°C because the acetate production rate is directly related to the specific growth rate (Eiteman and Altman 2006). The volumetric productivity of JCL260 at 30°C was 0.7 g L−1 h−1, 93% higher than the productivity of Clostridium beijerinckii BA101 for 1-butanol production estimated from data reported by Ezeji et al. (2003) using fermentation cultures with similar feeding and in situ product removal strategies Appl Microbiol Biotechnol (2011) 90:1681–1690 (gas stripping integrated with fermentation process). In contrast, the volumetric productivity for JCL16 and the isobutanol-tolerant strain (SA481) was lower than JCL260. The maximum isobutanol yield on glucose (determined at the end of fermentation) for JCL260 cultivated at 30°C was 0.29 ±0.003 g/g, which is 68% of the theoretical maximum. A lower isobutanol yield on glucose was obtained for JCL260 at 37°C and for SA481. Discussion Batch fermentations were integrated with gas stripping to evaluate the isobutanol production of the high- producing (JCL260), high isobutanol-tolerant (SA481), and parental (JCL16) strains. For the first time, we were able to produce more than 50 g/L of isobutanol in 72 h. We found that with in situ product removal, E. coli was able to surpass the 22 g/L produced in flask culture (Atsumi et al. 2008b). Although SA481 has shown superior isobutanol tolerance to JCL260 (Atsumi et al. 2010b), it was not reflected in the final titer of isobutanol obtained in the integrated process. These results suggest that under these culture conditions, isobutanol production was not limited by growth or the product toxicity during growth phase. Atsumi et al. (2010b) found that isobutanol production, cell growth, and glucose consumption rate of SA481 tested in flask cultures were similar to those of JCL260 and that improved tolerance of SA481 did not increase the final titer. In this study, glucose consumption of SA481 was lower than that of JCL260; consequently, isobutanol production rate was also lower (Figs. 2a and 3). Cell growth of SA481 stopped around 22 h, reaching practi- cally the same cellular concentration as JCL260. At this time, isobutanol concentration in the broth was around 7 g/L and cells did not grow even though there was no apparent nutrient limitation. On the other hand, temperature should be an important factor in isobutanol production since the final titer at 37°C was 53% lower than at 30°C. The observed difference might be caused by a lower Adh activity at 37°C toward the end of the culture (Fig. 6e) In addition, it has been reported that a shift-up in growth temperature decreases the proportion of unsaturated fatty acids in the membrane (Balamurugan 2010; Casadei et al. 2002; Ingram and Buttke 1984). Such changes in membrane composition may determine the tolerance level of cells to a particular stress. For example, Ingram and Buttke (1984) observed that alcohol tolerance of Zymomonas mobilis reduces with increasing growth temperatures. If such behavior will be the same for E. coli, poor isobutanol production at 37°C could be justified by a reduced alcohol tolerance compared with 30°C.