New Continuous Process for the Production of Lipopeptide Biosurfactants in Foam Overflowing Bioreactor

Abstract

In this work, an original culture process in bioreactor named overflowing continuous culture (O-CC) was developed to produce and recover continuously mycosubtilin, a lipopeptide antifungal biosurfactant of major interest. The lipopeptide production was first investigated in shake conical flasks in different culture media [ammonium citrate sucrose (ACS), Difco sporulation medium (DSM), and Landy], followed by a pH condition optimization using 3-(N-morpholino)propanesulfonic acid (MOPS) and 2-(N-morpholino)ethanesulfonic acid (MES) buffered media. A simple theoretical modeling of the biomass evolution combined with an experimental setup was then proposed for O-CC processed in stirred tank reactor at laboratory scale. Seven O-CC experiments were done in modified Landy medium at the optimized pH 6.5 by applying dilution rates comprised between 0.05 and 0.1 h–1. The O-CC allowed the continuous recovery of the mycosubtilin contained in the foam overflowing out of the reactor, achieving a remarkable in situ product removal superior to 99%. The biomass concentration in the overflowing foam was found to be twofold lower than the biomass concentration in the reactor, relating advantageously this process to a continuous one with biomass feedback. To evaluate its performances regarding the type of lipopeptide produced, the O-CC process was tested with strain BBG116, a mycosubtilin constitutive overproducing strain that also produces surfactin, and strain BBG125, its derivative strain obtained by deleting surfactin synthetase operon. At a dilution rate of 0.1 h–1, specific productivity of 1.18 mg of mycosubtilin⋅g–1(DW)⋅h–1 was reached. Compared with other previously described bioprocesses using almost similar culture conditions and strains, the O-CC one allowed an increase of the mycosubtilin production rate by 2.06-fold.

Impact of the Purification Process on the Spray-Drying Performances of the Three Families of Lipopeptide Biosurfactant Produced by Bacillus subtilis

“…Mycosubtilin was produced by B. subtilis BBG125 strain ( Béchet et al, 2013 ). BBG125 was grown for 72 h at 30°C in another modified Landy MOPS culture medium ( Guez et al, 2021 ) (composition: 20 g·L −1 of glucose, 2 g·L −1 of glutamic acid, 1 g·L −1 of yeast extract, 1 g·L −1 of K 2 HPO 4 , 0.5 g·L −1 of MgSO 4 ·7H 2 O, 0.5 g·L −1 of KCl, 2.3 g·L −1 of (NH 4 ) 2 SO 4 , 32 mg·L −1 of CuSO 4 ·5H 2 O, 100 mg·L −1 of MnSO 4 ·H 2 O, 30 mg·L −1 of Fe 2 (SO 4 ) 3 ·7H 2 O, and 21 g·L −1 of 3-morpholinopropane-1-sulfonic acid MOPS, buffered at pH 6.5 using KOH 3 M). In these culture conditions, BBG131, Bs2504, and BBG125 were able to produce respectively 1.2 ± 0.2 g·L −1 of surfactin, 0.4 ± 0.1 g·L −1 of plipastatin, and 0.05 ± 0.02 g·L −1 of mycosubtilin.…”

“…To achieve this goal, several research teams have already developed innovative processes, such as the disc bioreactor, the membrane bioreactor, or the trickle-bed biofilm reactor, enabling notably to limit the foam apparition due to the surfactant properties of LPPs ( Coutte et al, 2017 ). Conversely, some studies focused on the foaming properties of LPPs to set up overflowing culture processes ( Chen et al, 2006 ; Gong et al, 2009 ; Guez et al, 2021 ). In terms of purification, a downstream process encompassing several steps of ultrafiltration/diafiltration/evaporation has already been established and proved its worth in several studies ( Coutte et al, 2010a ; Kourmentza et al, 2021 ) ( Figure 1 ).…”

Bioinformatics Modelling and Metabolic Engineering of the Branched Chain Amino Acid Pathway for Specific Production of Mycosubtilin Isoforms in Bacillus subtilis

“…Mycosubtilin isoforms were produced by the B. subtilis BBG100 strain, using the overflowing continuous culture (O-CC) process described by Guez et al (2021) [34]. Figure S1 presents the annotated chromatogram obtained for a mixture of mycosubtilins composed of: iso-C16 (26%), Gln3 C17 (1%), n-C16 (2%), anteiso-C17 (45%), and iso-C17 (23%).…”

“…The strain BBG100 was constructed from B. subtilis ATCC 6633 by replacing the native promoter of the mycosubtilin operon by a strong and constitutive one [35]. Moreover, to intensify the mycosubtilin production, the culture medium was buffered with 100 mM MOPS at pH 7.0 [34] and cultures were grown under conditions of high oxygen limitation [36]. Finally, cultures of B. subtilis BBG100 were performed in the culture medium supplemented with 2 g/L of Ile (test) or not (control).…”

“…For amino acids testing and transcriptomic experiments, the previous medium was modified with retrieving the yeast extract. Culture media were buffered with MOPS (3-(N-morpholino)propanesulphonic acid) at 100 mM at pH 7.0 to favor the synthesis of mycosubtilins [34].…”