Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process
Highlights
► Size of mycosubtilin micelles is measured for the first time. ► Size of lipopeptides micelles changes with concentration of lipopeptides. ► Other components in the fermentation medium affect the lipopeptides micellar size. ► Surfactin and mycosubtilin were recovered applying the two-step ultrafiltration process. ► The highest MWCO membrane that could effectively retain both lipopeptides was the 100 kDa membrane.
Introduction
Lipopeptides produced by Bacillus subtilis (surfactins, iturins and fengycins) are being investigated as an alternative to chemically synthesized surfactants from petrochemical feedstocks [1] and agricultural chemicals [2]. Surfactin is a heptapeptide linked to a β-hydroxy fatty acid chain of 13–16 carbons (Fig. 1A). It is an extremely powerful surface-active compound, which is able to form micelles at and above 10 mg/L [3], [4] which is its critical micellar concentration (CMC). Mycosubtilin, a member of the iturin family, is a heptapeptide linked to a β-amino fatty acid chain of 15–17 carbons (Fig. 1B) which displays high antifungal activity [5] and forms micelles at and above 20 mg/L [3]. Mycosubtilin is often coproduced with surfactin in the culture medium, as in the fermentation of the wild type strain B. subtilis ATCC 6633 [6]. These two biosurfactants are involved in biocontrol and antifungal activities of B. subtilis through a membrane permeabilization phenomenon [7] and their combination results in synergistic effects [8]. The high production cost of these biosurfactants has been a major obstacle for their commercialisation and numerous efforts have been made to lower the cost including, increased productivity by using mutant strains [5], [6], [9] and simplification of downstream processing by the development of integrative processes [10]. Several methods have been investigated for the recovery of surfactin and mycosubtilin such as, extreme foaming [11], [12], one step ultrafiltration (UF) [13], [14], two step UF process [4], [15] and sequential ultrafiltration/diafiltration [16]. The combination of either hybrid salting out or acid precipitation and ultrafiltration processes was also studied [17], [18]. All these different techniques presented satisfactory results at laboratory scale. The membrane separation processes such as the one step or two-step ultrafiltration processes are particularly suitable for large scale processing as they do not require the use of large volumes of organic solvents as in liquid–liquid extraction [16]. In addition, relatively high flow rates can be applied since there are no mass transfer limitations as in chromatographic separations. In our previous work [4], [15], [16], we demonstrated that surfactin could be efficiently separated in the first UF step in its micellar form (rejection coefficient = 0.98–1) and partially purified from glucose and salts. In a second UF step surfactin was essentially purified from proteins (at least 93% purity). So far membranes of up to 100 kDa molecular weight cut-off (MWCO) have been used for the separation of surfactin resulting in high recoveries [14]. The use of higher MWCO membranes is particularly advantageous at large scale to increase productivity; however, this may result in reduced recovery and/or purity. Optimization of the separation of biosurfactants by a membrane based process requires good knowledge of their aggregation behaviour in the feedstock environment, e.g., fermentation culture. In ultrafiltration, the size and shape of molecules are the most important parameters affecting the separation. Grau et al. [19] investigated the aggregation behaviour of iturin A using electron microscopy techniques and found that iturin formed molecular aggregates of different size and shape depending on its concentration. This is an important feature since it has been shown that the aggregation behaviour of iturin is of relevance to its interaction with target bilayers which determines its antifungal activity [20]. Many works have reported the structure of surfactin’s micelles in aqueous solution; Ishigami et al. [21] showed that surfactin at concentrations up to its CMC was able to form β-sheet structures. Subsequently, other authors demonstrated that pH and temperature have an effect on the micellization of surfactin and the formation of β-sheet structures [22]. Moreover, univalent or bivalent counterions have also an effect on the size of surfactin micellar aggregates [23]. In summary, from the above studies it is clear that the chemical environment of the biosurfactant has an effect on their aggregation behavior and that this in turn, could have an effect on its biological activity.
The aim of the present work is to investigate the aggregation behavior of surfactin and mycosubtilin in single solutions and in fermentation culture solution in a range of concentrations, and their separation by membrane filtration using a range of MWCO membranes.
Section snippets
Lipopeptide production and purification
Surfactin was produced by the overproducer strain B. subtilis BBG131 [9]. Mycosubtilin was produced by the overproducer strain B. subtilis RFB112 [5]. Broth containing mycosubtilin and surfactin was obtained by the cultivation of the overproducer strain B. subtilis BBG100 [6]. Cultures of B. subtilis BBG131 and BBG100 were carried out in the Landy medium like described in our previous study at 160 rev/min and 37 °C for surfactin production and 30 °C for both surfactin and mycosubtilin production
Lipopeptide characterization
Surfactin and mycosubtilin were obtained from the culture supernatant of different overproducing mutant strains and characterized by LC–MS analysis (data not shown). Surfactin samples are a mixture of heptapeptides with a fatty acid chain of 13–15 carbons. Mycosubtilin samples contain heptapeptides linked to a fatty acid chain with 15–17 carbons.
Conclusions
The separation of lipopeptides by membrane filtration depends on their molecular aggregation behaviour and in particular on their ability to form micelles at above a given concentration (the CMC). This work showed that surfactin and mycosubtilin aggregation behavior changed with their concentration and their chemical environment including, the presence of proteins and other lipopeptides. Surfactin and mycosubtilin alone formed different size of micelles, the average diameter of micelles
Acknowledgements
This work received financial support from the Université Lille 1, Sciences et Technologies, the FEDER funds (ARCIR) from the Région Nord-Pas-de-Calais, the French Ministère de l’Enseignement et de la Recherche, EGIDE and the British Council as part of the Partenariat Hubert Curien “Alliance” 19406WE and from the European INTERREG IV PhytoBio project. Authors would like to thank also L. Bonneau and C. Boistel from ProBioGEM Laboratory, D. Takilt and D. Lotiquet from Polytech-Lille and Alicja
References (28)
- et al.
Bacillus lipopeptides: versatile weapons for plant disease biocontrol
Trends Microbiol.
(2008) - et al.
Interfacial and emulsifying properties of lipopeptides from Bacillus subtilis
Colloid Surf. A
(1999) - et al.
Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process
J. Membr. Sci.
(2007) - et al.
All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713
Biochim. Biophys. Acta
(2011) - et al.
Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A
Biochim.
(1992) - et al.
Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides
J. Biotechnol.
(2007) - et al.
Characterization of concentration and purification parameters and operating conditions for small-scale recovery of surfactin
Process. Biochem.
(2005) - et al.
Flux decline and membrane cleaning in cross-flow ultrafiltration of treated fermentation broths for surfactin recovery
Sep. Purif. Technol.
(2008) - et al.
A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration
Sep. Purif. Technol.
(2008) - et al.
Separation of surfactin from fermentation broths by acid precipitation and two-stage dead-end ultrafiltration processes
J. Membr. Sci.
(2007)
Recovery of surfactin from fermentation broths by hybrid salting-out and filtration process
Sep. Purif. Technol.
Aggregational behaviour of aqueous dispersions of the antifungal lipopeptide iturin A
Peptides
Significance of β-sheet formation for micellization and surface adsorption of surfactin
Colloid Surf. B
Micropolarity and microviscosity in the micelles of heptapeptide biosurfactant “surfactin”
Colloid Surf. B
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