Relation between pristinamycins production by Streptomyces pristinaespiralis, power dissipation and volumetric gas-liquid mass transfer coefficient, k_La

N. Mehmood, E. Olmos, P. Marchal, J.-L. Goergen, S. Delaunay

Process Biochemistry (2010), Vol. 45 (11), pp 1779-1786

In this work, growth and production of pristinamycins by Streptomyces pristinaespiralis in shaking flasks has been related to power dissipation. Streptomyces pristinaespiralis are filamentous bacteria, they produce the antibiotic compound as secondary metabolite under different stress conditions, especially nutritional and hydrodynamic stresses. Mechanical power dissipation impacts parameters as oxygen supply and hydrodynamics. For the first time, investigations of the relationship between volumetric mean power dissipation, gas liquid mass transfer (k_La) and production of pristinamycins were conducted in shake flasks. Different levels of power dissipation were applied on Streptomyces pristinaespiralis through a series of cultures in diverse unbaffled shake flask with different filling volumes at two agitation rates. Computational fluid dynamics (CFD) was used to describe the hydrodynamics in the system.

At first, rheograms have to be realized at various biomass concentrations to detect the impact on broth viscosity. The flow behavior of the medium appeared to be Newtonian for biomass concentrations below 14 g L^-1 and non-Newtonian otherwise. But for shear rates superior to 100 s^-1, which corresponds to the culture conditions applied in this study, a Newtonian plateau was observed even at higher biomass concentrations. Hence, a Newtonian behavior of the broth was postulated even at biomass concentrations beyond 14 g L^-1.

CFD was used to predict the volume mean power dissipation in unbaffled shake flasks from 250 to 1000 mL range with different working volumes from 10 to 200 mL and agitation rates of 250 or 350 rpm (in Newtonian conditions). The values calculated by this numerical method agreed well with those obtained by an empirical reference method.

When investigating the relationship of the maximal biomass concentration and the volumetric gas-liquid mass transfer coefficient (k_La), two phases were observed: In the first phase, corresponding to k_La values lower than 100 h^-1 and biomass concentration below 15 g L^-1, biomass increased rapidly with k_La. This behavior is characteristic for an oxygen limitation during the culture. In a second phase, when biomass titer reached values higher than 15 g L^-1, the increase of k_La beyond 100 h^-1 had almost no effect on cell density. This could be due to a higher specific oxygen consumption rate or an additional nutriment limitation.

For all cultures tested, pristinamycins production onset was observed only for maximum biomass concentrations higher than 15 g L^-1. It occurred above a clear threshold of power dissipation (increasing with total flask volume) and only for k_La values superior to 100 h^-1 (whatever type of flask used).

In this work it was shown for the first time, that the maximal pristinamycins concentration was strongly dependent on the volume mean power dissipation applied in the flask of producing cultures: For power dissipation values below 5,5 kW m^-3, a linear increase of pristinamycins production with power dissipation was observed. In this phase, the transfer of some nutriments might be limiting for antibiotic production. In a second phase, a plateau of pristinamycins concentration of about 45 mg L^-1 was reached for power dissipation values between 5,5 to 8,5 kW m^-3. Here, the increase in mass transfer might be compensated by the increase in damaging hydrodynamic stresses. For power dissipations higher than 8,5 kW m^-3, a rapid decrease in the antibiotic concentration was observed, potentially related to microbial pellet damages.

Regarding the pristinamycins on biomass yield, a very similar pattern was observed. Therefore it could be clearly shown, that power dissipation effect on pristinamycins titer was not related to a higher biomass concentration. The increase in power dissipation was not the sole parameter influencing the onset of production; it has to be coupled with a transfer of oxygen sufficient to yield biomass concentrations higher than 15 g L^-1. But when pristinamycins production occurred, its concentration was correlated with the volume mean power dissipation.

This study was advantageously performed in shake flasks because of their defined culture conditions and broad application fields.

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