Nitrogen consumption during batch cultivation of Neisseria meningitidis (serogroup C) in Frantz medium

Abstract

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.

Polysaccharide production in batch process of Neisseria meningitidis serogroup C comparing Frantz, modified Frantz and Cartlin 6 cultivation media

“…The detailed analysis of this phenomenon has been described in an earlier article (2). As far as the other topic related to the current article is concerned, dealing with the analysis of nitrogen consumption and its cellular assimilation in the growth kinetics of N. meningitidis serogroup C, this has also been described in detail in another reference (3).…”

“…Considering a larger scale, the cultivations in bioreactors are usually performed in Frantz medium (1,2,3,4,8). On the other hand, Catlin 6, a based in original Catlin medium formulation (5), where glycerol was replaced by glucose as the carbon source, was developed for the cultivation of N. meningitis serogroup B in bioreactor (10).…”

Optimization of Media and Feeding Strategy for Improved Capsular Polysaccharide Yield for Neisseria meningitidis W-135

“…Lactate enhances the growth of Neisseria meningitidis in presence of glucose and lactate metabolizes rapidly than glucose [19], but our study with different carbon sources showed lactate had no significant effect on bacterial growth (data not shown). Presence of phospho transacetylase acetate kinase pathway was indicated by the formation of acetate metabolite in the course of fermentation [20,21], which is responsible for pH change during fermentation [9]. In this study, pH was controlled by the addition of 0.25 M alkali, which allowed the cells to survive for a longer time in the stationary phase and hence ensured better production of capsular polysaccharide.…”

“…Reported studies showed the major part of pyruvate (80%) was synthesized through ED pathway and PP pathway corresponding to 20% of the metabolism. The activity of Embden-Meyerhof-Parnas (EMP) pathway in the metabolism has not been observed that indicates that phosphofructokinase gene is missing in Neisseria meningitidis [9,32]. A NMR and enzyme study of carbon catabolism of Neisseria meningitidis showed the presence of all the citric acid cycle enzymes in the cell extracts grown on glucose and pyruvate, whereas the specific activities for most of the enzymes were significantly higher (3-5 fold) in glucose grown cells, in which the pyruvate dehydrogenase activity was more than 10 folds higher in glucose grown cells that supported our hypothesis that glucose is one of the major growth determining factors [33].…”

“…Hence, isolate structures were studied for developing vaccines in which surface structures and bacterium extracellular products were most relevant [9]. The unique structure of the extracellular polysaccharide makes it a good target for vaccine design [5].…”