Working volume of 0.4 L. Temperature, aeration and pH have been controlled and maintained at 28 , 1 volume per liquid volume per minute (1 vvm) and five.0 (by automatic addition of 1.5 M KOH), respectively. Dissolved oxygen was maintained at 50 saturation by manage of the stirrer speed that was initalliy set to 500 rpm, with vmax at 1200 rpm. Fermenters were inoculated from precultures to 1.0E05 cellsmL. Within the oxygen limitation research, exactly the same media and fermentation situations as for the totally aerated batch cultivations have been utilized. When cells reached a cell density of about two.0E08 cellsmL the aeration price was reduced from 1 vvm to 0.four vvm and stirring speed was maintained at 500 rpm to preserve oxygen saturation at 1 . Samples for extracellular metabolite and lipid analyses and dry weight (DW) determination were taken each 12 h following reducing the aeration. The total duration of fermentation was 72 h. For fed-batch fermentations, precultures had been inoculated into 300 mL of minimal medium containing eight.0 g L-1 glucose and 0.4 g L-1 ammonium sulfate. The feed was started following depletion of glucose, having a glucose option containing six.55 g L-1 glucose and at a continuous flow rate of 69.4 L min-1 adding a total of 200 mL of glucose solution towards the fermentor. Samples have been taken at the beginning of your fed batch phase and just after 48 h.Analytical methodsDetermination of biomass: five mL samples were withdrawn in the fermenters using a syringe and filtered through nitrocellulose filters (0.45 m Sartorius Stedim, G tingen, Germany), washed twice with deionized water and dried at 97 for 24 h and weighted. Extracellular metabolite concentrations: 1 mL in the fermentation broth was centrifuged at 16000 g at four for 1 min plus the supernatant was stored at -20 until additional analysis. Extracellular metabolites (glucose, glycerol, citrate, succinate and Danofloxacin Inhibitor acetate) have been quantified with an Agilent Technologies HP 1100 series HPLC method equipped with an Aminex HPX-87H column (Biorad, Richmond, CA, USA), Agilent autosampler, an Agilent UV detector and Knauer differential refractometer (RI detector). The column was maintained at 65 , and five mM H2SO4 at a flow rate of 0.6 mL min-1 was used as eluent. ChemStation application was utilized to determine metabolites concentration in the generated chromatograms.Determination from the obtainable nitrogen concentration in the growth medium: 450 L of sample have been mixed with 50 L D2O and adjusted to pH two.0 utilizing HCl (32 ) to quench chemical exchange in the NH+ protons. The four NH+ concentration was determined by NMR spectros4 copy on a Bruker AVIII 300 MHz spectrometer (equipped with a BBI probe head) employing a 1D 1H experiment with water suppression and (NH4)2SO4 solutions as external requirements (0.5, 0.1, 0.05 g L-1). All spectra were processed and analyzed with Topspin 2.1. Lipid analysis: about 20 mg of cell dry weight have been harvested from the fermenter and centrifuged at 2000 g for 5 min at space temperature to eliminate culture media. Pellets had been quickly frozen in liquid nitrogen and stored at -75 till additional processing. Cells were disrupted with glass beads and extracted with DOTAP custom synthesis chloroform:methanol 2:1 (vv) by shaking inside a Heidolph Multi Reax test tube shaker (Schwabach, Germany) and lipids had been extracted with chloroform:methanol two:1 [29]. Neutral lipids were quantified by thin layer chromatography as described [21]. For total FA evaluation, 200 L in the lipid extract have been employed for fatty acid methyl ester (FAME) produc.