Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process

Abstract

The aim of this work was to investigate the lipopeptides aggregation behavior in single and mixed solutions in a wide range of concentrations, in order to optimize their separation and purification following the two-step ultrafiltration process and using large pore size membranes (up to MWCO = 300 kDa). Micelle size was determined by dynamic light scattering. In single solutions of lipopeptide both surfactin and mycosubtilin formed micelles of different size depending on their concentration, micelles of average diameter = 5–105 nm for surfactin and 8–18 nm for mycosubtilin. However when the lipopeptides were in the same solution they formed mixed micelles of different size (d = 8 nm) and probably conformation to that formed by the individual lipopeptide, this prevents their separation according to size. These lipopeptides were purified from fermentation culture by the two-step ultrafiltration process using different MWCO membranes ranging from 10 to 300 kDa. This led to their effective rejection in the first ultrafiltration step by membranes with MCWO = 10–100 kDa but poor rejection by the 300 KDa membrane. The lipopeptides were recovered at 90% purity (in relation to protein) and with 2.34 enrichment in the permeate of the second ultrafiltration step with the 100 KDa membrane upon addition of 75% ethanol.

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.