1684 Appl Microbiol Biotechnol (2011) 90:1681–1690 Fig. 2 Typical kinetics of isobutanol production by JCL260, JCL16, and SA481 strains harboring pSA65/pSA69. a Total isobutanol production (calculated as sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L). b Isobutanol concentration in fermentation broth. c Cell growth. d, f Glucose consumption. e Acetate production. High isobutanol producer (JCL260) at 30°C (closed squares), parental (JCL16) at 30°C (closed circle), high isobutanol producer (JCL260) at 37°C (closed triangles) and high isobutanol-tolerant strain (closed diamond). Error bars correspond to the difference between duplicate cultures (a–c, e). For clarity, one set of data has been plotted in d and f (2009). KivD activity was assayed by measuring NADPH oxidation at 340 nm in 50 mM phosphate buffer (pH 7.0), 1 mM MgCl2, 1.5 mM TTP, 0.1 μM alcohol dehydroge- nase (ADH6) NADPH-dependent, 0.2 mM NADPH, and 30 mM KIV. Since NADPH oxidation was proportional to isobutiraldehyde production, it was possible to determine KivD activity. Alcohol dehydrogenase activity was detected by measuring NADH oxidation at 340 nm in 100 mM MOPS, pH 7.0, 0.2 mM NADH, and 25 mM isobutaraldehyde.