Capsular polysaccharide, extracted from microorganism cultivations, is the principal antigen for elaboration of vaccine against the disease caused by Neisseria meningitidis serogroup C. The final protein content allowed in this vaccine is 1%. In order to find a relationship between nitrogen consumption and cell growth, including polysaccharide production, and cell nitrogen content, cultivations were carried out in an 80 liters bioreactor (total capacity), under the following conditions: Frantz medium; temperature of 35ºC; air flow of 5 L/min (0.125 vvm); agitation frequency of 120 rpm and vessel pressure of 6 psi (kLa = 0.07 min-1). Concentrations of biomass, total polysaccharide, cellular nitrogen, residual organic and inorganic nitrogen in the medium were measured during cultivation. From five cultivations carried out under the same conditions, a mean cell nitrogen percentage of 12.6% (w/w) in respect to the dry biomass was found. The inorganic nitrogen in the medium did not change significantly along the cultivation time, whereas the organic nitrogen consumption was linearly related to cell growth, with constant yield factors (average of 8.44). Polysaccharide production kinetics followed the cell growth kinetics until the beginning of the stationary growth phase. A supplemental polysaccharide production was observed until the end of cultivation, but without cell nitrogen absorption. Thus, the results indicate that polysaccharide is produced in two phases, being the first one biomass formation followed by non-associated to growth.
Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35 degrees C, 0.5 atm, 400 rpm and air flowrate of 2 l min(-1). A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O2) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O2 control, varying glucose concentration from 1.0 to 3.0 g l(-1), nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O2 control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O2 control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.
Polysaccharide of N. meningitidis serogroup C constitutes the antigen for the vaccine against meningitis. The goal of this work was to compare three cultivation media for production of this polysaccharide: Frantz, modified Frantz medium (with replacement of glucose by glycerol), and Catlin 6 (a synthetic medium with glucose). The comparative criteria were based on the final polysaccharide concentrations and the yield coefficient cell/polysaccharide (YP/X). The kinetic parameters: pH, substrate consumption and cell growth were also determined. For this purpose, 9 cultivation runs were carried out in a 80 L New Brunswick bioreactor, under the following conditions: 42 L of culture medium, temperature 35ºC, air flow 5 L/min, agitation frequency 120 rpm and vessel pressure 6 psi, without dissolved oxygen or pH controls. The cultivation runs were divided in three groups, with 3 repetitions each. The cultivation using the Frantz medium presented the best results: average of final polysaccharide concentration = 0.134 g/L and YP/X=0.121, followed by Catlin 6 medium, with results of 0.095 g/L and 0.067 respectively. Considering the principal advantages in the use of the synthetic medium, i.e. facilitation of a cultivation and purification steps of the polysaccharide production process, there is a possibility that in the near future, Catlin 6 will replace the traditional Frantz medium.
The influences of the L-asparagine and glycerol initial concentrations in Sauton medium on the productivities of biomass and colony forming units were studied. The submerged batch cultivations of Mycobacterium bovis were carried out in a 20 L bioreactor. The L-asparagine and glycerol initial concentrations of 4.54 g/L and 25 mL/L, respectively, corresponded to the best biomass productivity, namely 2.5 g/L.day. On the other hand, the concentrations of 2.27 g/L and 25 mL/L, respectively, led to the highest productivity in terms of colony forming units, namely 2.7•10 6 colonies/mg.day. In addition, by means of the relative consumption analysis of L-asparagine and glycerol (50 and 26% respectively), it was concluded that the concentrations of such components could be reduced, with respect to the original Sauton medium composition, aiming the obtainment of an optimal BCG vaccine production in the bioreactor.
The following general equation is proposed to represent the kinetics of microbial growth \documentclass{article}\pagestyle{empty}\begin{document}$$\phi (dR/dt) + \psi R + X = 0$$\end{document}, where phi and psi depend on several parameters of the fermenting system. The values of phi and psi were calculated based on results obtained in a batch lactic acid fermentation, a batch cultivation of yeast on diesel oil, and a continuous cultivation of yeast on sugarcane molasses.
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