Biosurfactants for a Sustainable Future. Группа авторов

the ribbons grow with time, and after two hours, twisted ribbons with lengths of a few hundred micrometers and a width of ~5 m had formed. Ribbon formation is slowed by increasing the pH. After a time, which depends on pH, precipitates were observed. After 28 hours, at pH 4.1 dynamic light scattering measurements of the solution showed that the large aggregates coexisted with small micelles. Small‐angle X‐ray scattering (SAXS) and wide‐angle X‐ray scattering (WAXS) measurements were carried out in aqueous solutions and dried solid ribbons formed at pH 4.1. All WAXS diffractograms indicate high crystallinity of the hydrocarbon chains and the disaccharide head groups inside the ribbons. At pH 5.1 individual ribbons were rarely found. At 0.02 mg/ml and pH 7.8 (at which the carboxylic groups are in their negative carboxylate form), small aggregates are formed, the hydrodynamic radius R _h being 37 nm (measured at a scattering angle of 15°). At concentrations of 0.97–1.78 mg/ml, nearly monodisperse micellar aggregates were formed with R _h about 100 nm. An apparent radius of gyration R _g of 175 nm was estimated for the radius of gyration by measuring the scattering intensity at the angle range of 15–35° at a surfactin concentration of 1.40 mg/ml. The R _g /R _h ratio is about 1.75, a rather large value which indicates that the large micellar aggregates have a very anisotropic geometry.

      As described in the previous paragraph, the carboxylic acid group of C18:1 AS makes that its aggregation behavior is sensitive to pH. Baccile et al. [195] have studied the system by SANS at different amounts of NaOH and other bases (NH₃, KOH, and Ca(OH)₂). A core–shell prolate micelle structure with an interaction potential (which combines hard‐sphere and screened Coulomb potentials) was used to fit SANS data. The total effective cross‐radius of the micelle (core radius + shell thickness) decreases (from 21.8 to 18.2 Å) with increasing NaOH, the core radius contributing the most to this reduction as the shell is practically constant (~8 Å). The core size and the length of the oleic acid chain suggest that the chain is partially folded and that the formation of more carboxylate/Na⁺ pairs favors such a bending. The eccentricity of the prolate also reduces (from 3.3 to 2.6) and micelles become more negatively charged as the effective micellar charge varies from about −0.5 to −5.3 with increasing NaOH concentration. Baccile et al. have also noticed that in the presence of Ca(OH)₂, the system evolves toward a better surface charge screening, which has the effect of reducing the repulsive potential (between micelles) and the effective surface charge. The micellar length is also elongated.

      In a nice piece of work, Baccile et al. [26] prepared a broad range (38 new compounds) of amino derivatives of sophorolipid biosurfactant, comprising quaternary ammonium salts, amine oxides, and symmetric and asymmetric bolaamphiphiles with three or four hydrophilic centers, as well as divalent and Y‐shaped derivatives. The compounds are constituted by at least two hydrophilic head groups, different in nature and charge since sophorose is neutral and the nitrogen atom is charged, being separated by a spacer. SAXS experiments were used for the estimation of the aggregation number.

Bolaform derivatives with two (sophorose and ammonium) hydrophilic groups either do not form aggregated or exhibit a poor tendency to self‐aggregate. Correspondingly, the aggregation numbers are small (<20) and the aggregates are highly hydrated. The behavior of compounds with three hydrophilic groups (sophorose–ammonium–sophorose) depends on the nature of the ammonium group. If this is small, the compounds behave as the bolaform derivatives with two groups. If the group is charged and bulky, small hydrated aggregates are observed, coexisting with fibrilary systems. Tetra‐center compounds (sophorose–ammonium–ammonium–sophorose) with small and/or charged ammonium groups, have a poor tendency for self‐aggregation, and again small aggregation numbers and highly hydrated micelles are observed. However, divalent and Y‐shaped surfactants tend to form larger micellar aggregates as a function of the size of the hydrophobic chain and spacers between sophorose and nitrogen, the aggregation numbers usually being >50. From these experiments Baccile et al. concluded that modification of acidic sophorolipids does not necessarily improve their aggregation behavior in water.

1.7 Surfactin

In 1968, Arima et al. [196] reported the isolation and characterization of a potent clotting inhibitor produced in the culture fluids of several strains of Bacillus subtilis. As its surface activity was much stronger than that of sodium lauryl sulfate they named the product surfactin. Figure 1.9 shows the structure of a typical surfactin. Other structures are also known [20].

Figure 1.9 Schematic structure of a surfactin.

      According to Arima et al., surfactin is a peptide lipid composed of L‐aspartic acid, L‐glutamic acid, L‐valine, L‐leucine, D‐leucine (1 : 1 : 1 : 2 : 2) and unidentified fatty acids. The complete elucidation of the structure was published by Kakinuma et al. in a series of papers one year later [197–200]. Bonmatin et al. [201] studied surfactin by two‐dimensional ¹H‐NMR in DMSO and observed two conformations characterized by a saddle‐shape topology with polar Glu and Asp side chains oppositely oriented to that of the C11–13 aliphatic chain. The conformation of surfactin was reinvestigated by FTIR spectroscopy by Vass et al. [202]. Circular dichroism (CD) and FTIR spectroscopic data in different solvents with or without Ca²⁺ ions indicate that surfactin has a unique ability of adopting strongly different conformations depending on the conditions. The carboxyl groups of Glu¹ and Asp⁵ are responsible for Ca²⁺ binding at low concentration (ratio of calcium/lipopeptide < 1). The NMR structure of surfactin has been determined in sodium dodecyl sulfate and dodecylphosphocholine micellar solutions [203]. pKa values of Asp and Glu are around 4.3 and 4.5 [204, 203]. When comparing results for surfactins from different authors it is important to notice that different hydrophobic alkyl chains may be involved. Razafindralambo et al. [205] have isolated a homologous series of surfactins containing β‐hydroxy fatty acids having different alkyl chain lengths (13, 14, or 15 carbon atoms). These authors have investigated their dynamic surface properties and found a dependence with both bulk concentration and hydrophobic character of the alkyl chain. The cmc values depend on the length of the alkyl chain and the tendency is the same as that observed for classical alkyl chain surfactants, i.e. the values are lower the larger is the alkyl chain. The surface tension at cmc changes in the same direction. These authors also observed that at low concentrations, the longer the alkyl chain, the faster is the decrease of the surface with time, but at high concentrations the maximum rate was observed for n _c = 14. Surfactin reduces to 27 mN/m at a concentration as low as 2 × 10⁻⁵ M. For surfactin, several authors have found two break points in the plot surface tension vs log (concentration), both being dependent on the experimental conditions [177]. The first one of 1.89 × 10⁻⁶ M (20 mM phosphate buffer) has been ascribed to a premicellization phenomenon, while the second one was assigned to the cmc (Table 1.2).

The effect of monovalent and divalent cations on surfactin was studied by Thimon et al. [206]. The experiments were performed at pH 9.5 (5 mM Tris), and Mg²⁺, Ca²⁺, and Mn²⁺ chloride salts were added at 0.5 mM concentration and monovalent ions (Li⁺, Na⁺, K⁺, and Rb⁺) were added at 0.1 M. Both type of cations, as well their concentrations, had a strong effect on cmc. For instance, values of 2.0 × 10⁻⁵ M (Ca²⁺), 5.7 × 10⁻⁵ M (Mn²⁺), 6.0 × 10⁻⁷ M (Li⁺) and 1.47 × 10⁻⁶ M (Rb⁺) were found. Molecular areas (determined from the analysis of the premicellar surface tension‐log (concentration) plot) are also affected and the authors concluded that the micellization of surfactin is highly favored in the presence of divalent cations. Later Thimon et al. [207] calculated the association constants of some cations, the obtained values being K = 1.5 × 10⁵/M and 1.9 × 10⁴/M for Ca²⁺ and Mg²⁺, respectively. The