Appl Microbiol Biotechnol (2011) 90:1681–1690 Culture media for bioreactor fermentations Culture media for bioreactor cultures contained the following composition, in grams per liter: glucose, 55; (NH4)2SO4, 3; K2HPO4, 14.6; KH2PO4, 4; sodium citrate, 2.2; yeast extract, 25; MgSO4⋅7H2O, 1.25; ampicillin, 0.1; kanamycin, 0.05; and 1 mL/L of trace metal solution. Trace metal solution contained, in grams per liter: EDTA, 14.1; CoCl2⋅6H2O, 2.5; M nC l 2 ⋅4 H 2 O , 1 5 ; C u C l 2. 2 H 2 O , 1 .5 ; H 3 B O 3 , 3; Na2MoO4⋅2H2O, 2.1; Zn(CH3COO)2⋅2H2O, 33.8; Fe(III) citrate 100.8. The feeding solution contained, in grams per liter: glucose, 500; MgSO4⋅7H2O, 1.25; ampicillin, 0.1; kanamycin, 0.05, 0.1 mM of isopropyl-B-D-thio-galactoside (IPTG); and 1 mL/L of trace elements. Bioreactor culture conditions Cultures of E. coli were performed in 1-L stirred tank bioreactor (Applikon Biotechnology, Schiedam, the Netherlands) using a working volume of 0.35 L. The bioreactor was inoculated with 2% of overnight pre- culture and the cells grown at 37°C. After 2 h, 0.1 mM IPTG was added and the temperature was decreased to 30°C to start isobutanol production. The induction time represents the beginning of fermentation described in Fig. 2. Isobutanol production was tested at two temperatures, 30°C and 37°C, for JCL260. The pH was controlled at 6.8 by automatic addition of 2 M NaOH solution. Dissolved oxygen (DO) was maintained above 20% with respect to air saturation by raising stirrer speed (from 200 to 600 rpm). Air was bubbled in the bioreactor with two goals: (1) provide oxygen and (2) in situ isobutanol stripping out. Air flow rate was maintained at 0.5 vvm the first 3 h of isobutanol production and then was increased to 1.2 vvm in order to remove isobutanol from the culture broth. The evaporated isobutanol was condensed using two Graham condensers connected in series (Fig. 1). The exhaust gases from the fermentor were bubbled in a trap (picker B containing 800 mL of water; Fig. 1) cooled with ice and then circulated through condenser 1 (L 300 mm and cooling coil surface area 400 cm2; Fig. 1) maintained at 4°C. After that, gas continued circulating through a second equal loop (D receiver and condenser 2; Fig. 1). Fermentation was allowed to proceed in batch mode until entry into the stationary phase (between 9 and 10 h), and then an intermittent manual feeding was started to avoid glucose depletion. Fermentation samples were collected to determinate growth, isobutanol production, and organic acid and glucose concentrations. Analytical methods Cell growth was followed by optical density measurements at 600 nm and converted to dry cell weight concentration 1683 Fig. 1 Schematic diagram of fermentation/gas stripping process for isobutanol production by E. coli. A and C are ice containers to cool recipients B and D. B and D are receivers containing 0.8 L of water where the evaporated isobutanol was collected by calibration plots of samples dried to constant weight in an oven at 80°C. Glucose concentration was determined with YSI biochemical analyzer (YSI Instruments, Yellow Springs, OH, USA). Isobutanol concentration was quanti- fied by a gas chromatograph (GC) model 6850 (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector 6850 series (Agilent Technologies). A 30-m, 0.32-mm i.d., 0.25-μm DB-FFAP capillary column was used. GC oven temperature was initially held at 85°C for 3 min and raised with a gradient 45°C/min until 225°C and held for 3 min. Helium at 1.7 mL/min was used as the carrier gas with 9.52-psi inlet pressure. A volume of 1 μL supernatant of the culture broth was injected using 1-pentanol as internal standard. Acetate concentration was determined by high-pressure liquid chromatography (Agilent Technologies) with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) maintained at 35°C. A mobile phase of 5 mM H2SO4 was used at 0.6 mL/min. Preparation of cell-free extracts and enzyme activity assays Cells were harvested by centrifugation at 10,000×g for 10 min at 4°C from bioreactor cultures, washed twice with 50 mM potassium phosphate buffer (pH 7.0), and stored frozen at −80°C. Frozen cells were suspended in the same washing buffer, blended with glass beads, and disrupted in series 4 × 1 min. Crude extract was centrifuged at 10,000×g at 4°C for 30 min and the supernatant used for enzyme assays. The concentration of the protein prepara- tion was determined by the Bradford method. Acetolactate synthase (AlsS) and dihydroxy-acid dehydratase (IlvD) assays were performed as described by Atsumi et al.