1682 In particular, Atsumi et al. (2008b) engineered an E. coli strain (JCL260) as a host for isobutanol production. Genes that code for proteins involved in by-product formation from pyruvate and acetyl-CoA were deleted in JCL260 (Table 1) with an intention to increase pyruvate availability for isobutanol synthesis. Such deletions improved the isobutanol production of JCL260 strain compared to its parental strain, JCL16, under micro-aerobic conditions in shake flasks (Atsumi et al. 2008b). Even without any process optimization, Atsumi et al. (2008b) were able to produce 22 g/L of isobutanol in 112 h with a yield of 86% of the theoretical maximum. Although this titer is 85% and 12% higher than those reported by Ezeji et al. (2003) and Qureshi and Blaschek (1999), respectively, for n-butanol produced by Clostridium in batch bioreactors, industrial-scale fermentation processes require higher final titers to be economically feasible. The final product titer can be limited by various factors depending on the particular production strain and bioprocess. Since isobutanol is toxic to the cell, isobutanol production from glucose may be limited by the toxicity of the final product itself. In this sense, improving the tolerance of the biocatalyst becomes a primary necessity to achieve a process with high product titers (Alper et al. 2006; Gonzalez et al. 2003; Jarboe et al. 2010; Miller and Ingram 2007; Yomano et al. 1998). It has been reported that E. coli was unable to grow at isobutanol concentrations >8 g/L (Brynildsen and Liao 2009). Even though the E. coli strain continued to produce isobutanol after it ceased to grow (Atsumi et al. 2008b), product toxicity may eventually damage the cell and limit the final titer (Rutherford et al. 2010). In this regard, Atsumi et al. (2010b) isolated and characterized an isobutanol-tolerant E. coli strain (SA481) able to grow at 8 g/L. SA481 was evolved from the high isobutanol producer strain (JCL260) by a sequential transfer method increasing isobutanol concentration every 15 transfers. The SA481 strain showed superior growth characteristics com- pared to the high producer (JCL260) strain when they both were cultivated at 6 and 8 g/L of isobutanol. Compared with Table 1 Strains and plasmids used in this study Appl Microbiol Biotechnol (2011) 90:1681–1690 the control condition, specific growth rate for JCL260 decreased by 35% and 74% at 6 and 8 g/L of isobutanol (calculated from data showed by Atsumi et al. 2010b), respectively, while SA481 growth was not affected at 6 g/L of isobutanol and decreased just 13% at 8 g/L. Interestingly, Atsumi et al. (2010b) also showed that SA481 did not produce more isobutanol than JCL260 under normal conditions. However, the experiments were performed in shake flasks. Accordingly, the performance of SA481 harboring pSA65/pSA69 for isobutanol production in bio- reactors will be evaluated in this work. The other approach to solve the cytotoxicity problem and its detrimental effect on the final titer from a bioprocess perspective is the in situ product removal. Several in situ solvent recovery strategies have been developed for n-butanol fermentation (Groot et al. 1992; Nielsen and Prather 2009; Roffler et al. 1987; Qureshi et al. 2005). Since gas stripping is a simple and efficient way to recover the solvent from the fermentation broth (Inokuma et al. 2010; Lee et al. 2008), in the present work, gas stripping integrated with fermentation was used to compare the isobutanol production by the high producer (JCL260), isobutanol-tolerant (SA481), and the parental (JCL16) strains carrying pSA65 and pSA69 plasmids. We hypothesize that if isobutanol could be removed from the fermentation broth, the production can be extended significantly and the final titer will surpass the 22 g/L obtained previously in flask cultures. Materials and methods Strains, plasmids, and pre-culture Strains and plasmids used are shown in Table 1. A single colony of freshly transformed cells was inoculated into a 250-mL screw-cap flask containing 25 mL of Luria-Bertani media with antibiotics (0.05 g/L kanamycin and 0.1 g/L ampicillin). Overnight pre-culture was performed at 37°C on a rotary shaker (250 rpm). Strains JCL16 JCL260 SA481 JCL260 ΔpoxB Plasmids pSA65 pSA69 Relative characteristics BW25113/F′[traD36, proAB+, lacIq ZΔM15] Same as JCL16 but with ΔadhE, ΔfrdBC, Δfnr, ΔldhA, Δpta, ΔpflB Isobutanol-tolerant strain, JCL260 derivative Same as JCL260 but with ΔpoxB ColE1 ori; AmpR; PLlacO1: kivd-adhA (Lactococcus lactis) P15 ori; KanR; PLlacO1: alsS (Bacillus subtilis)-ilvCD Reference Atsumi et al. (2008a) Atsumi et al. (2008a) Atsumi et al. (2010b) Laboratory collection Atsumi et al. (2010a) Atsumi et al. (2008b)