**2.3 Properties and functions of biosurfactants**

330 Biomedical Science, Engineering and Technology

Fig. 2. Chemical structure of most known high molecular weight microbial surface active

RAG-1 emulsan is a complex of an anionic heteropolysaccharide and protein (Rosenberg & Kaplan, 1987, as cited in Rosenberg & Ron, 1999). Its surface activity is due to the presence of fatty acids, comprising 15% of the emulsan dry weight, which are attached to the polysaccharide backbone via O-ester and N-acyl linkages (Belsky et al., 1979, as cited in

Another high molecular weight biosurfactant is alasan, a complex of an anionic polysaccharide and a protein with a molecular weight of around 1,000 kDa isolated from *Acinetobacter radioresistens* (Navon-Venezia et al., 1995, as cited in Smyth et al., 2010c). These high molecular weight biosurfactants generally possess effective emulsifying activity and are called bioemulsifiers. A large number of other polymeric compounds have been discovered but remain partially or totally uncharacterized (Smyth et al., 2010c). Little is known in general about these bioemulsifiers other than the producing organism and the overall chemical composition of the crude mixture. *Halomonas eurihalina* produces an extracellular sulfated heteropolysaccharide (Calvo et al., 1998, as cited in Rosenberg & Ron, 1999). *Pseudomonas tralucida* produced an extracellular acetylated polysaccharide that was effective in emulsifying

Several bioemulsifiers are effective at high temperature, including the protein complex from *Methanobacterium thermoautotrophium* (De Acevedo et al., 1996, as cited in Rosenberg & Ron, 1999) and the protein-polysaccharide-lipid complex of *Bacillus stearothermophilus* ATCC 12980 (Gunjar et al., 1995, as cited in Rosenberg & Ron, 1999). Yeasts produce a number of emulsifiers, which are particularly interesting because of the food-grade status of several yeasts which allows use in food related industries. Liposan is an extracellular emulsifier produced by *Candida lipolytica* (Cirigliano & Carman, 1985, as cited in Rosenberg & Ron, 1999). It is composed of 83% carbohydrate and 17% protein. Mannanprotein emulsifiers are produced by *Saccharomyces cerevisiae* (Cameron et al., 1988, as cited in Rosenberg & Ron, 1999). Many of these bioemulsifiers have been used in the food, cosmetic, and petroleum

several insecticides (Appaiah & Karanth 1991, as cited in Rosenberg & Ron, 1999).

compound; emulsan.

Rosenberg & Ron, 1999).

industries (Rosenberg & Ron, 1999).

There is a growing interest in the study of the physicochemical and biological properties of biosurfactants because of their potential industrial applications (Cameotra & Makkar, 2004; Desai & Banat, 1997; Lang, 2002; Rodrigues et al., 2006a; Singh & Cameotra, 2004). The interesting biological activities displayed by these compounds constitute an added value to their potential uses (Lang et al., 1989; Lang & Wagner, 1993; Stanghellini & Miller, 1997, as cited in Sánchez et al., 2010). Due to these reasons, an intense research activity is currently directed toward identification of new biosurfactants and characterization of their chemical and biological properties (Biria et al., 2010; Morita et al., 2009a; Satpute et al., 2010a; Singh & Cameotra, 2004; Singh et al., 2007).

The most obvious property of biosurfactants compounds is their ability to effectively lower water surface tension, and a number of approaches that measure directly the surface activity of biosurfactants can be used as screening methods for their detection. Among them, the most frequently used as quick and simple techniques are the drop collapse (Bodour and Miller-Maier, 1998) and the oil spreading tests (Morikawa et al., 2000) (Fig. 3).

Fig. 3. Oil spreading test. This technique measures the diameter of clear zones caused when a drop of a biosurfactant-containing solution is placed on an oil–water surface (Morikawa et al., 2000). Photo courtesy by Fabrizio Rivardo.

An efficient biosurfactant can reduce the surface tension between pure water and air from 72mN/m to less than 30mN/m. Surfactin, as one of the most powerful biosurfactants, can reduce the surface tension of water from 72mN/m to 27mN/m (which is close to the minimum detectable value) at a concentration as low as 10 μM (Seydlová & Svobodová, 2008). Rhamnolipids can similarly achieve such level of reduction (Hisatsuka et al., 1971, as cited in Muthusamy et al., 2008; Mohammad Abdel-Mawgoud et al., 2010). The sophorolipids from *T. bombicola* have been reported to reduce the surface tension to 33mN/m (Muthusamy et al., 2008) while MELs and trehalose lipids to less than 30mN/m (Arutchelvi & Doble, 2010; Shao et al., 2010).

As surfactant monomers are added into solution, the surface or interfacial tension will decrease until the biosurfactant reaches the critical micelle concentration (CMC). The CMC is defined as the minimum concentration necessary to initiate micelle formation (Becher, 1965). Above the CMC no further reduction in surface or interfacial tension is observed. At the CMC, surfactant monomers begin to spontaneously associate into structured aggregates such as micelles, vesicles or continuous bilayers. These aggregates are produced as a result of numerous weak chemicals interactions such as hydrophobic, van der Waals and hydrogen bonding (Maier, 2003; Raza et al., 2010). Since no chemical bonds are formed, these structures are fluid-like and are easily transformed from one state to another as conditions such as electrolyte concentration and temperature are changed (Lin, 1996).

The aggregate structure depends on the polarity of the solvent in which the surfactant is dissolved. In an aqueous solution, the polar head groups of a micelle will be oriented outward toward the aqueous phase, and the hydrophobic tails will associate in the core of the micelle within an oil-in-water micelle. In contrast, in oil, the polar head groups will associate in the center of the micelle while the hydrophobic tails will be oriented toward the outside within the water–in-oil micelle (Soberón-Chávez & Maier, 2010). Efficient surfactants have a low CMC, i.e. less surfactant is necessary to decrease the surface tension (Seydlová & Svobodová, 2008).

Biosurfactants are most effective and efficient at their CMC which can be 10–40 times lower than that of chemical surfactants, i.e. less surfactant is necessary to get a maximum decrease in surface tension (Desai & Banat, 1997). Another important property for industrial and biotechnological applications is that most biosurfactants surface activities are not affected by environmental conditions such as temperature and pH (Muthusamy et al., 2008) particularly those of glycolipids composition. It has been reported that lichenysin from *B. licheniformis* JF-2 was not affected by temperature (up to 50 °C), pH (4.5–9.0) and NaCl and Ca concentrations up to 50 and 25 g/l respectively. A lipopeptide from *B. subtilis* LB5a was also stable after autoclaving (121°C/20 min) and after 6 months at –18°C; the surface activity did not change from pH 5 to 11 and NaCl concentrations up to 20% (Muthusamy et al., 2008).

Moreover, unlike synthetic surfactants, microbial-produced compounds are easily degraded and are generally considered as low or non-toxic products and therefore, appropriate for pharmaceutical, cosmetic and food uses. Although little is known about the toxicity of microbial surfactants, some data in the literature suggest that they are less toxic than synthetic surfactants (Muthusamy et al., 2008). The synthetic anionic surfactant (Corexit) for example had an LC50 (lethal concentration to 50% of test species) against *Photobacterium phosphoreum* at approximately ten times lower concentrations than that for rhamnolipids, demonstrating the higher toxicity of the chemical-derived surfactant. It was also reported that biosurfactants needed higher effective concentration to decrease 50% of test population values (EC50) and were degraded faster than commercial dispersants. In another report, biosurfactants from *P. aeruginosa* were noted to have much less toxic and mutagenic activities when compared to synthetic surfactant Marlon A-350, which is widely used in the industry.

Understanding the functional mechanisms of biosurfactants and bioemulsifiers is of great help to discover interesting applications. Surfactin, one of the most powerful biosurfactants, is known to destabilize membranes disturbing their integrity and permeability (Bernheimer et al., 1970). This is due to changes in physical membrane structure or through disrupting protein conformations which alter important membrane functions such as transport and energy generation (Ortiz et al., 2008, 2009; Sánchez et al., 2009, 2010; Sotirova et al., 2008; Van Hamme et al., 2006; Zaragoza et al., 2009).

The molecular mechanisms of surfactin interactions with membrane structures have been described by Shaligram & Singhal (2010) and by Seydlová & Svobodová (2008). A key step for membrane destabilization and leakage is the dimerization of surfactin into the bilayer (Carrillo et al., 2003). The hypothetical mechanisms of surfactin interactions with

the CMC, surfactant monomers begin to spontaneously associate into structured aggregates such as micelles, vesicles or continuous bilayers. These aggregates are produced as a result of numerous weak chemicals interactions such as hydrophobic, van der Waals and hydrogen bonding (Maier, 2003; Raza et al., 2010). Since no chemical bonds are formed, these structures are fluid-like and are easily transformed from one state to another as conditions such as electrolyte concentration and temperature are changed (Lin, 1996). The aggregate structure depends on the polarity of the solvent in which the surfactant is dissolved. In an aqueous solution, the polar head groups of a micelle will be oriented outward toward the aqueous phase, and the hydrophobic tails will associate in the core of the micelle within an oil-in-water micelle. In contrast, in oil, the polar head groups will associate in the center of the micelle while the hydrophobic tails will be oriented toward the outside within the water–in-oil micelle (Soberón-Chávez & Maier, 2010). Efficient surfactants have a low CMC, i.e. less surfactant is necessary to decrease the surface tension

Biosurfactants are most effective and efficient at their CMC which can be 10–40 times lower than that of chemical surfactants, i.e. less surfactant is necessary to get a maximum decrease in surface tension (Desai & Banat, 1997). Another important property for industrial and biotechnological applications is that most biosurfactants surface activities are not affected by environmental conditions such as temperature and pH (Muthusamy et al., 2008) particularly those of glycolipids composition. It has been reported that lichenysin from *B. licheniformis* JF-2 was not affected by temperature (up to 50 °C), pH (4.5–9.0) and NaCl and Ca concentrations up to 50 and 25 g/l respectively. A lipopeptide from *B. subtilis* LB5a was also stable after autoclaving (121°C/20 min) and after 6 months at –18°C; the surface activity did not change from pH 5 to 11 and NaCl concentrations up to 20% (Muthusamy et al., 2008). Moreover, unlike synthetic surfactants, microbial-produced compounds are easily degraded and are generally considered as low or non-toxic products and therefore, appropriate for pharmaceutical, cosmetic and food uses. Although little is known about the toxicity of microbial surfactants, some data in the literature suggest that they are less toxic than synthetic surfactants (Muthusamy et al., 2008). The synthetic anionic surfactant (Corexit) for example had an LC50 (lethal concentration to 50% of test species) against *Photobacterium phosphoreum* at approximately ten times lower concentrations than that for rhamnolipids, demonstrating the higher toxicity of the chemical-derived surfactant. It was also reported that biosurfactants needed higher effective concentration to decrease 50% of test population values (EC50) and were degraded faster than commercial dispersants. In another report, biosurfactants from *P. aeruginosa* were noted to have much less toxic and mutagenic activities when compared to

synthetic surfactant Marlon A-350, which is widely used in the industry.

Van Hamme et al., 2006; Zaragoza et al., 2009).

Understanding the functional mechanisms of biosurfactants and bioemulsifiers is of great help to discover interesting applications. Surfactin, one of the most powerful biosurfactants, is known to destabilize membranes disturbing their integrity and permeability (Bernheimer et al., 1970). This is due to changes in physical membrane structure or through disrupting protein conformations which alter important membrane functions such as transport and energy generation (Ortiz et al., 2008, 2009; Sánchez et al., 2009, 2010; Sotirova et al., 2008;

The molecular mechanisms of surfactin interactions with membrane structures have been described by Shaligram & Singhal (2010) and by Seydlová & Svobodová (2008). A key step for membrane destabilization and leakage is the dimerization of surfactin into the bilayer (Carrillo et al., 2003). The hypothetical mechanisms of surfactin interactions with

(Seydlová & Svobodová, 2008).

membranes exhibit a complex pattern of effects such as insertion into the lipid bilayers, chelating mono- and divalent cations, modification of membrane permeability by channel formation or membrane solubilization by a detergent-like mechanism. *In vitro,* the incorporation of surfactin into the membrane gives rise to dehydration of the phospholipid polar head groups and the perturbation of lipid packing which strongly compromise the bilayer stability, leading to the disturbance of the membrane barrier properties.

These structural fluctuations may well explain the primary mode of the antibiotic action and the other important biological effects of this lipopeptide (Carrillo et al., 2003). The extent of perturbation of the phospholipid bilayer correlates with the concentration of surfactin. At low concentrations surfactin penetrates readily into the cell membrane, where it is completely miscible with the phospholipids and forms mixed micelles. At moderate concentrations, the lipopeptide forms domains segregated within the phospholipid bilayer that may contribute to the formation of ion-conducting pores in the membrane leading to membraine disruption and permeabilization at high concentrations, showing a stronger activity than that of Triton (Heerklotz et al., 2007, as cited in Seydlová & Svobodová, 2008).

As biological amphiphilic molecules, biosurfactants naturally tend to self-assemble into hierarchically ordered structures using hydrogen bonding, hydrophobic and van der Waals interactions (Kitamoto et al., 2009). Glycolipids, and in particular MELs, are well known for their self-assembling properties, that are influenced by the stereochemistry of the saccharide head groups (Kitamoto et al., 2005). Some of glycolipid type surfactants, which possess relatively large hydrophilic head groups as compared to the hydrophobic parts, generally form micelles in a dilute aqueous solution. Other than spherical micelles, they also form oblate (disk-like) and prolate (rod-like) structures (Söderman, 2000, as cited in Kitamoto et al., 2009). As the surfactant concentration further increases, glycolipid/water systems start to form a range of liquid crystalline phases. In particular, glycolipid biosurfactants spontaneously self-assemble into a variety of molecular assemblies with well-defined and/or unique structures, such as sponge (L3), cubic (V2), hexagonal (H2), or lamellar (Lα) configurations (Imura et al., 2007, as cited in Kitamoto et al., 2009).

Among these molecular assemblies, vesicles are one of the most intensively studied ones. MELs in particular, due to their efficient molecular orientation property and effective balance between hydrophilic and hydrophobic groups, are able to form giant vesicles of diameter larger than 10 µm (Kitamoto et al., 2002). In comparison, rhamnolipids show a pHsensitive conversion of molecular assemblies due to the presence of a carboxyl group on the side chain (Kitamoto et al., 2002). This leads rhamnolipids to form micelles at pH more than 6.8, lipid particles at pH 6.6-6.2, lamella structures at pH 6.5-6.0, and finally vesicles in the size of 50-100 nm at pH 5.8-4.3. Glycolipid biosurfactant-based vesicles or bilayer membranes appear, thus, to be very promising for exploiting useful nanostructured materials and/or systems.

Another function of microbial surface-active molecules with interesting biotechnological potential is the ability to form stable emulsions (Fig. 4.). High molecular-mass biosurfactants are in general better emulsifiers than low-molecular-mass biosurfactants and are thus called bioemulsifiers. Bioemulsifiers, can form and stabilize oil in water or water in oil emulsions, but are not necessarily efficient detergents that are able to demonstrate remarkable surface tension reduction (Dastgheib et al., 2008). Emulsions can be produced with prolonged lifespan of months and years. Liposan, for example, does not reduce surface tension, but has been used successfully to emulsify edible oils (Cirigliano & Carman, 1985). Emulsan is an effective emulsifier at low concentrations (0.01-0.001%) representing emulsan-tohydrocarbon ratios of 1:100 to 1:1000, while exhibiting considerable substrate specificity (Ron & Rosemberg, 2001). Polymeric surfactants offer additional advantages because they coat droplets of oil, thereby forming stable emulsions. This property is especially useful for making oil/water emulsions for cosmetics and food industries (Muthusamy et al., 2008).

Fig. 4. Example of emulsion produced by the bioemulsifier extracted from the bacterial strain 7bCT5, isolated from a Cambodian soil. This emulsion is stable since 2009.
