1686 Fig. 5 Time profile of acetate production by JCL260 PoxB− mutant and high isobutanol producer (JCL260) carrying pSA65/pSA69 in bioreactor cultures. Data shown are duplicated and single fermentation for JCL260 and JCL260 PoxB−, respectively Appl Microbiol Biotechnol (2011) 90:1681–1690 same level for all cultures, the differences in the isobutanol concentration accumulated in the fermentation broth can be attributed to differences in the isobutanol production rate (Figs. 2b and 3). Isobutanol production rate did not remain constant and decreased progressively after the growth phase. As inferred from Fig. 2b, the isobutanol production rate decreased dramatically after 22 h, for JCL260 and JCL16 cultivated at 30°C, suggesting that some events (e.g., loss of enzyme activities or substrate depletion) took place at that time. Accordingly, volumetric isobutanol production rate was calculated before and after 22 h of culture, being higher before 22 h (Fig. 3). JCL260 and JCL16 reached the maximum cell density around 10 h and SA481 at 22 h. The cell density of SA481 was constant until the end of culture, while JCL260 and JCL16 presented a slight decrease (Fig. 2c), presumably due to the differences in isobutanol tolerance. The cell growth stopped around 10 h (JCL260 and JCL16) and cells did not grow even after feeding. Initial glucose concentration was approximately 55 g/L and maintained above 2 g/L in all cultures by intermittent additions of 500 g/L glucose solution (Fig. 2d). Since isobutanol production mainly comes from glucose carbon and not from yeast extract (Atsumi et al. 2008b), glucose depletion was avoided because long starvation times (more than 1 h) diminished the isobutanol production (data not shown). Completely aerobic conditions were maintained in all cultures, and lactate, formate, and ethanol were not detected in the broth culture. Acetate was the major by-product detected, which is a well-known result of carbon imbalance during overfeeding, known as overflow metabolism (Farmer and Liao 1997; Wolfe 2005; Wong et al. 2008). Maximum acetate concen- trations reached during the growth phase (first 10 h; Fig. 2e) were 4.8 ± 0.8, 7.5 ± 2.6, and 3.5 ± 0.5 g/L for JCL260, JCL16, and SA481, respectively. JCL16 and JCL260 accumulated 13.7 ± 2.6 and 7.1 ± 3.6 g/L of acetate at 22 h, respectively. Although JCL260 accumulated less acetate than parental (JCL16), pta deletion in JCL260 was not enough to avoid acetate accumulation (Fig. 2e). Since the strain still had the PoxB path to acetate production, its contribution was tested by deleting the poxB gene (Fig. 4). Thus, JCL260 and JCL260 ΔpoxB mutant were compared in flask cultures. Isobutanol production and maximum cell density were similar in both strains (Fig. 4a, b). Glucose consumption for JCL260 was just slightly higher than JCL260 ΔpoxB. However, acetate accumulated by JCL260 at 16.5 h was twofold higher than the ΔpoxB strain. This result suggested that poxB did contribute to the acetate accumulation. Additionally, both strains were also tested in bioreactor cultures with integrated stripping out system, and the results are shown in Fig. 5. Growth and isobutanol time profile were different for JCL260 and JCL260 ΔpoxB mutant (Fig. 5a–c). Unexpectedly, JCL260 ΔpoxB did not grow as well as JCL260 and produced less isobutanol, while acetate accumulation was similar for both strains. Deletion of pta and poxB (JCL260 ΔpoxB strain) did not