Effect of pps disruption and constitutive expression of srfA on surfactin productivity, spreading and antagonistic properties of Bacillus subtilis 168 derivatives.

AIMS: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. METHODS AND RESULTS: The srfA native promoter was replaced by the constitutive promoter P(repU) in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P(repU), than that found with P(srfA), led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1.47 g g(-1) of biomass), spreading behaviour and haemolytic activity of the strains. CONCLUSIONS: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.

Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633.

The effect of oxygen transfer rate (OTR) on the synthesis of mycosubtilin, a non ribosomal lipopeptide antifungal biosurfactant, was investigated in the respiration activity monitoring system (RAMOS) for two Bacillus subtilis strains. These cultures were performed under definite oxygen-limited conditions without the adding of any anti-foam in the culture medium. By using four different filling volumes (FV) in the shaken bioreactors, different levels (20, 14, 9 and 7 mmol O(2)l(-1)h(-1)) of oxygen-limited growth could be obtained. A 25-fold increase of the specific productivity of mycosubtilin was observed for B. subtilis ATCC6633 in the case of the most severe oxygen limitation. But nearly no effect could be found with strain BBG100 carrying the constitutive P(repU) promoter instead of the natural P(myc) promoter. Transcript analysis of the fenF gene belonging to the myc operon indicated that the P(myc) promoter regulation could be slightly oxygen sensitive. Additionally, different patterns of the synthetised mycosubtilin homologues were obtained for different level of oxygen-limited growths. At the present state of investigation, oxygen regulation was thus shown to act at different levels suggesting the existence of a complex regulatory system of NRPS lipopeptide synthesis in the natural B. subtilis ATCC6633 strain.

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 set-up 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 0.086h(-1). The model was extrapolated to five additional experiments carried out at feed rates of 0.008, 0.022, 0.040, 0.042 and 0.062h(-1) enabling the correlation of the mean specific growth rates with productivity results. Finally, a feed rate of 0.086h(-1) corresponding to a mean specific growth rate of 0.070h(-1) allowed a specific productivity of 1.27mg of mycosubtiling(-1) of dried biomassh(-1).

Real time in situ microscopy for animal cell-concentration monitoring during high density culture in bioreactor.

An in situ microscope (ISM) device is utilised in this study to monitor hybridoma cells concentration in a stirred bioreactor. It generates images by using pulsed illumination of the liquid broth synchronised with the camera frame generation to avoid blur from the cell's motion. An appropriate image processing isolates the sharp objects from the blurred ones that are far from the focal plane. As image processing involves several parameters, this paper focuses on the robustness of the results of the cells counting. This stage determines the applicability of the measuring device and has seldom been tackled in the presentations of ISM devices. Calibration is secondly performed for assessing the cell-concentration from the cell automated numeration provided by the ISM. Flow cytometry and hemacytometer chamber were used as reference analytical methods. These measures and the output of the image processing allow estimating a single calibration parameter: the reference volume per image equal to 1.08 x 10(-6) mL. In these conditions, the correlation coefficient between both reference and ISM data sets becomes equal to 0.99. A saturation of this system during an ultrasonic wave perfusion phase that deeply changes the culture conditions is observed and discussed. Principal component analysis (PCA) is used to undergo the robustness study and the ISM calibration step.