Characterization of the dynamics of a continuous biofilm reactor for the production of lipopeptides

Abstract

Nowadays, the increasing demand in surfactants leads the industry to find new surface-active molecules, and new production processes. Indeed, surfactants are used in multiple application fields, such as the food, pharmaceutical, cosmetic, or pest management industries. Their chemical structure bring them surface, antibacterial, antifungal, hemolytic, … properties. Since a decade, the greener trend induced the commercialization of biosurfactants. The scientific sector is thus becoming interested in these compounds. Some bacteria, such as several Bacillus or Pseudomonas, have already been frequently studied for their ability to produce lipopeptides, a major class of biosurfactants. Lipopeptides comprise compounds coming principally from the family of surfactins, fengycins, and iturins. Their production by B. subtilis is intensively studied. However, the scientific research tends also to discover other strains whose culture could be profitable. For a long time, B. subtilis has been mistaken with B. amyloliquefaciens that also proved its capacity to produce the three types of lipopeptides. The general culture for lipopeptides occurs in stirred tank reactor. Nevertheless, this production process provokes a consequent foam formation leading to implementation issues and yield losses. Several solutions concern the set-up of different kinds of reactor in order to overcome this problem. It has been demonstrated that the cells immobilization on a support allowed them to reach a higher productivity and resistance to environmental conditions. Therefore, the implementation of a biofilm reactor specific to lipopeptides production is emerging. The present study describes the scale-down of a 50 m³ reactor process for the production of lipopeptides by B. amyloliquefaciens GA1. The final reactor is composed of a 2-liters stirred tank, and an external tower containing stainless steel packings. This latter has been tested in batch mode with recirculation, and the connection to a near infra-red spectrometer (NIRS) for the on-line determination of the liquid medium composition. Therefore, the monitoring of cultures has been performed by the HPLC analysis of primary metabolites, but also with optical density, dissolved oxygen, gasses, and lipopeptides (UPLC) measures. These data showed a cellular lysis without biofilm formation in most of cases, except when the culture was submitted to a contact phase in the packing tower before recirculation, and without NIRS. However, repetitions of this process did not permit to prove its efficiency. Thus, several hypotheses concerning the cellular death have been proposed and principally questioned the external factors. The alternation of environmental conditions in the reactor seemed to prevent the adaptation of cells to these conditions and thus affect their metabolism. A qRT-PCR analysis has been effectuated on samples from cultures with and without red light. These results showed that B. amyloliquefaciens GA1 possesses red light photoreceptors. However, the impact on the stress signaling pathway of the cell could not be confirmed and needs further investigations. Moreover, the reproduction of several reactors with the presence of not of the NIRS and the contact phase should permit to draw clearer conclusions. After that, it will be possible to study the culture parameters (temperature, pH, oxygen intake, medium composition, etc) and the physiology of sessile cells before the switch in continuous phase. This project thus represents the introduction to the development of a new attractive process which has to be subsequently pursued.