Typical growth curves of runs 1–7 are shown in Fig. 1a and the corresponding produced OMV in Fig. 1b. The behavior of pH variation and glycerol concentrations during cultivation

are presented in Fig. 1f and e, respectively. The lactate and l-glutamic acid consumptions are shown in Fig. 1c and d, respectively. From these behaviors, it is evident that substrate consumption exerted remarkable influence on growth kinetics and OMV production. The analysis of the related dry mass and optical density indicated an average value of 0.46 g/L for each unit of O.D. (SD 0.06). This coefficient was employed for estimating dry biomass values from the O.D. values and for calculating μP. According to the kinetics parameters presented in Table 1, the assays of Series A and B (original Catlin medium and original Catlin medium with lactate and amino acids pulse at the 6th cultivation hour, respectively) presented Selleckchem DAPT BIBF 1120 cell line similar values of OMV maximum concentration (Pmax) and OMV productivity (ProdP) for these two groups. However they were the lowest ones considering the overall experimental results. Series A and B presented, respectively, average values of Pmax = 56.2 mg/L and ProdP = 3.03 mg/(L h). On the other hand, Series C experiments (Catlin medium, double initial concentrations of lactate and amino acids) presented the highest

values of these parameters, namely Pmax = 162 g/L and ProdP = 8.1 mg/(L h). In all assays, glycerol was not consumed ( Fig. 1e). In Series D (Catlin medium, without glycerol,

double initial concentrations of lactate and amino acids), the values of Pmax = 121 g/L and ProdP = 6.0 mg/(L h) were slightly better than other those from Series C. The highest OMV concentrations were obtained in Series C (where initial glycerol concentration was maintained the and the initial concentrations of amino acids and lactate doubled) ( Fig. 1c and d, run 6). Glycerol was not consumed in cultivations [25], so it has no direct influence on the OMV production. A plausible hypothesis is that glycerol could be the mechanical protector of the OMV released to the cultivation medium. Lactate is the main limiting carbon source while l-glutamic acid is the main limiting nitrogen source ( Fig. 1 and Fig. 2). l-Glutamic acid consumption contributes to ammonia formation and pH rising in the course of the cultivation ( Fig. 1f). By employing the original Catlin medium (Series A) lactate concentration decreased to zero at the 8th cultivation hour. At this moment, the cultivation reached the stationary growth phase ( Fig. 1a). Thus, its consumption is directly related to cell growth. From Series A experiments amino acids were analyzed in order to estimate the specific amino acid yield factor to conduct further assays ( Fig. 2).