Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides

This work is related to the setup of overflowing exponential fed-batch cultures (O-EFBC) derived from carbon limited EFBC dedicated to the production of mycosubtilin, an antifungal lipopeptide belonging to the iturin family. O-EFBC permits the continuous removal of the product from the bioreactor achieving a complete extraction of mycosubtilin. This paper also provides a dynamical Monod-based growth model of this process that is accurate enough to simulate the evolution of the specific growth rate and to correlate it to the mycosubtilin specific productivity. Two particular and dependant phenomena related to the foam overflow are taken into account by the model: the outgoing flow rate of a broth volume and the loss of biomass. Interestingly, the biomass concentration in the foam was found to be lower than the biomass concentration in the bioreactor relating this process to a recycling one. Parameters of this model are the growth yield on substrate and the maximal specific growth rate estimated from experiments led at feed rates of 0.062, 0.071 and 0.086 h --1. The model was extrapolated to five additional experiments carried out at feed rates of 0.008, 0.022, 0.040, 0.042 and 0.062 h --1 enabling the correlation of the mean specific growth rates with productivity results. Finally, a feed rate of 0.086 h --1 corresponding to a mean specific growth rate of 0.070 h --1 allowed a specific productivity of 1.27 mg of mycosubtilin g --1 of dried biomass h --1

Étude de la productivité et de la sélectivité de la biosynthèse de mycosubtiline, un antibiotique surfactant de Bacillus subtilis : apports du génie biochimique et de la transcriptomique

The natural Bacillus subtilis strain ATCC 6633 has the ability to produce a wide range of antibacterial and antifungal antibiotics. Among these families of compounds, some are synthesized by the non-ribosomal pathway: fengycins, surfactins and mycosubtilins. The study of the biological activity of the mycosubtilins confirmed the broad spectrum of their antifungal action and showed the very strong antifungal activity of the anteisoC-17 isoform, particularly against Botrytis cinerea. A study of the environmental parameters contributed to the identification of culture conditions that made it possible to improve the productivity of mycosubtilin biosynthesis while limiting that of surfactin and to direct the cellular metabolism towards the biosynthesis of the most active isoform. The parameters tested were the following: culture media, pH, amino acid and iron additions, oxygen transfer. Several genes involved in central metabolic pathways, clearly related to the selectivity of this biosynthesis, ybgE, leuS, bfmBAA as well as the ilvBNCleuABCD operon, were then revealed through a transcriptomic study. The main genes coding for the regulators of these pathways, CodY, AbrB, ComK, CspB and CspC were also identified. The productivity of biosynthesis was finally improved by combining the use of a mycosubtilin overproducing derivative of B. Subtilis ATCC 6633 with the development of a bioreactor overflowing culture mode called O-EFBC.La souche naturelle Bacillus subtilis ATCC 6633 a la capacité de produire un large éventail d'antibiotiques antibactériens ou antifongiques. Parmi ces familles de composés, certaines sont synthétisées par la voie non ribosomale : les fengycines, les surfactines et les mycosubtilines. L'étude dans un premier temps de l'activité biologique des mycosubtilines a confirmé l'étendue du spectre d'action antifongique et a montré le très fort pouvoir antifongique de l'isoforme anteisoC-17 vis à vis de Botrytis cinerea. Une étude des paramètres environnementaux a contribué à l'identification de conditions de culture permettant, d'une part, d'améliorer la productivité de la biosynthèse en mycosubtiline tout en limitant celle de surfactine, et d'autre part, d'orienter le métabolisme cellulaire vers la biosynthèse de l'isoforme la plus active. Les paramètres testés ont été les suivants: milieux de culture, pH, ajouts d'acides aminés et de fer, transfert en oxygène. Plusieurs gènes impliqués dans des voies métaboliques centrales, manifestement liées à la sélectivité de cette biosynthèse, ybgE, leuS, bfmBAA ainsi que l'opéron ilvBNCleuABCD, ont ensuite été révélés grâce à une étude en transcriptomique. Les principaux gènes codant pour les régulateurs de ces voies, CodY, AbrB, ComK, CspB et CspC ont aussi été mis en évidence. La productivité de la biosynthèse a finalement pu être améliorée en combinant l'utilisation d'un dérivé de B. Subtilis ATCC 6633 surproducteur constitutif de mycosubtiline avec la mise au point d'un mode de culture débordant en bioréacteur appellé l'O-EFBC

In situ microscopy for in-line monitoring of hybridomal cell culture in agitated bioreactor

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In situ microscopy for in-line monitoring of hybridomal cell culture in agitated bioreactor

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Etude d’un bioprocédé en réacteur semi-discontinu accompagné de pertes par moussage : application à l’extraction de tensioactifs lipopeptidiques produits par Bacillus subtilis

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Stoechiometric modeling for physiological state changes monitoring in bioprocesses

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How can bioprocesses contribute to a reduction of global warming?

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Stoichiometric Modelling for Physiological State Changes Monitoring in Bioprocesses

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A Hybrid Approach for the Monitoring of Physiological State Changes

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