2.2, with a concentration of glycerol and tryptone of 30 and 20 g/L, respectively. These fermentations showed that the stationary phase of growth is reached after approximately 8 h of fermentation. Under these conditions the maximum OD attained is of about 28 (data not shown). Subsequently, the next step was to evaluate the effect of dissolved oxygen concentration VX-809 concentration on COMT production, testing three set-points for dissolved oxygen concentrations (20, 30 and 40%) and performing

recombinant COMT induction. The three different dissolved oxygen set-points (20%, 30% and 40%, Fig. 1) were tested in duplicates and the results for each set-point were averaged. All fermentations were stopped 4 h after induction, according to the experiments. For the activity assays, cell samples were retrieved at the end of the fermentation. The results from Fig. 1 show that a dissolved oxygen concentration of 20% gives better results than the other two concentrations tested in terms of maximum OD reached. The following step was the assessment of the most appropriate carbon (glycerol) and nitrogen (tryptone) source concentrations in the batch phase stage for the fed-batch process, in order to reduce time, and also to increase cell density at the end of Selleck Enzalutamide the batch phase. It is extremely relevant to reduce batch and fed-batch times in order to avoid, or at least minimize, nutrients/oxygen depletion. To achieve this, the concentration of glycerol and tryptone were varied,

according with three formulations: 1st formulation (20 g/L glycerol and 20 g/L tryptone), 2nd formulation (10 g/L glycerol and 15 g/L tryptone) and 3rd formulation

(20 g/L glycerol and 30 g/L tryptone) (growth curves were depicted in Fig. 2). The last parameters to be assessed before initiating fed-batch experiments were this strain’s growth rate and the time at which to initiate the feeding process under these conditions. The growth rates, μ (h−1), obtained for the 1st, 2nd and 3rd formulations, depicted previously, were 0.51, 0.49 and 0.55 h−1, respectively, indicating that these glycerol and tryptone concentrations allowed similar growth profiles. In theory, the fed-batch process should be initiated when the carbon source is completely depleted, to ensure nutrient limitation. Given this, it is relevant to know exactly when the carbon source is completely depleted. So, glycerol Dolichyl-phosphate-mannose-protein mannosyltransferase concentration was measured every 2 h for the three formulations mentioned in the previous subsection. Results are consistent with the initial glycerol concentrations in each fermentation. The 1st and 3rd fermentations were started at an initial glycerol concentration of 20 g/L, and the 2nd at 10 g/L, and after 4 h of fermentation, only a small amount of that initial glycerol was consumed (data not shown). Given all the previous assays, the fed-batch fermentations were initiated with a batch phase containing glycerol and tryptone at a concentration of 20 g/L and a dissolved oxygen rate of 20%.