**2.1.1 Lipopeptides**

Microbial surface-active compounds can be roughly divided into low molecular weight molecules that efficiently reduce surface and interfacial tension (biosurfactants) (Fig. 1.), and high molecular weight polymers that stabilize emulsions but do not lower the surface tension as much (bioemulsans or bioemulsifiers) (Fig. 2.) (Neu, 1996; Rosenberg, 2006; Rosenberg & Ron, 1997; Smyth et al., 2010a, 2010c).

The most studied low-molecular-weight biosurfactant compounds are lipopeptides and glycolipids. Lipopeptides are mainly produced by members of the *Bacillus* species; they are composed of different families and each family is constituted of several variants, which can differ in their fatty acid chain and their peptide moiety (Dastgheib et al., 2008; Jacques, 2010; Thavasi et al., 2008, 2011).

foaming, cleansing, phase separation, surface activity and reduction in viscosity of heavy liquids such as crude oil, make them suitable for utilization for many industrial and domestic application purposes (Gautam & Tiagi, 2006; Franzetti et al., 2010a; Perfumo et al.,

During the past two decades biosurfactants have been under continuous investigation as a potential replacement for synthetic surfactants and are expected to have several industrial and environmental applications mainly related to detergency, emulsification, dispersion and solubilisation of hydrophobic compounds (Banat et al., 2000). In addition, biosurfactants present several advantages over surfactants of a chemical origin, particularly in relation to their biodegradability, environmental compatibility, low toxicity, high selectivity and specific activity at extreme temperatures, pH and salinity (Banat 1995a, 1995b). Due to all these properties, they have steadily gained increased significance in industrial and environmental applications such as bioremediation, soil washing, enhanced oil recovery and other general oil processing and related industries (Perfumo et al., 2010b). Furthermore, potential commercial applications in several other industries including paint, cosmetics, textile, detergent,

agrochemical, food and pharmaceutical industries begin to emerge (Banat et al., 2000).

fields of biotechnology, nanotechnology and bioengineering.

Numerous investigations in the field of biosurfactants/bioemulsifiers are leading to the discovery and description of many interesting chemical and biological properties and potential biomedical therapeutic and prophylactic applications. In this chapter we will focus on the most recent and appealing biomedical and therapeutic applications of biosurfactants and bioemulsifiers with special emphasis on the most recent results in the

**2. Classification, properties and functional mechanisms of microbial surface-**

Microbial surface-active compounds are a range of structurally diverse molecules produced by different microorganisms and are mostly therefore classified by their structural features, the producing organism and their molecular mass. Their hydrophilic moiety is mainly comprised of an acid, peptide cations, or anions, mono-, di- or polysaccharides while their hydrophobic moiety can be an unsaturated or saturated hydrocarbon chains or fatty acids. The structural orientation on the surfaces and inter phases confers the range of properties, such as the ability to lower surface and interfacial tension of liquids and the formation of micelles and microemulsions between these different phases (Chen et al., 2010a, 2010b).

Microbial surface-active compounds can be roughly divided into low molecular weight molecules that efficiently reduce surface and interfacial tension (biosurfactants) (Fig. 1.), and high molecular weight polymers that stabilize emulsions but do not lower the surface tension as much (bioemulsans or bioemulsifiers) (Fig. 2.) (Neu, 1996; Rosenberg, 2006;

The most studied low-molecular-weight biosurfactant compounds are lipopeptides and glycolipids. Lipopeptides are mainly produced by members of the *Bacillus* species; they are composed of different families and each family is constituted of several variants, which can differ in their fatty acid chain and their peptide moiety (Dastgheib et al., 2008; Jacques, 2010;

2010a; Satpute et al., 2010b).

**active compounds** 

**2.1.1 Lipopeptides** 

Thavasi et al., 2008, 2011).

**2.1 Low molecular weight compounds** 

Rosenberg & Ron, 1997; Smyth et al., 2010a, 2010c).

Fig. 1. Chemical structures of the main low molecular weight microbial surface active compounds reported; surfactin, iturin A, mono & di-rhamnolipids, mannosylerythritol lipids, dimycolates trehalose lipids, acidic and lactonic sophorolipids.

Surfactin, a cyclic lipopeptide produced by *Bacillus subtilis* is considered the most active biosurfactant discovered so far (Ron & Rosenberg, 2001). Surfactin was discovered by Arima et al., (1968) from the culture broth of *Bacillus subtilis* and it was named thus due to its exceptional surfactant activity (Peypoux et al., 1999). Natural surfactins are a mixture of isoforms A, B, C and D which are classified according to the differences in their amino acid sequences and possess various physiological properties (Shaligram & Singhal, 2010). Surfactin is composed of a seven amino-acid ring structure coupled to a fatty-acid chain via a lactone linkage. Surfactin-A has L-leucine, surfactin-B has L-valine and surfactin-C has L-isoleucine at the amino acid position involved in the lactone ring formation with the C14–C15 β-hydroxy fatty acid. The amino-acid residues may vary and the presence of these variants can be related to alterations in the culture conditions such as providing substrate containing some specific amino-acid residues in the culture media (Jacques, 2010).

Another surfactin related compound is lichenysin, a lipopeptide discovered in the supernatant of *Bacillus licheniformis* culture (Horowitz et al., 1990). Its chemical structure and physio-chemical properties are similar to surfactin (McInerney et al., 1990). In particular, lichenysin has Glutamine amino-acid in position 1 while surfactin has Glutamic acid. Other surfactin-like compounds are pumilacidin A, B, C, D, E, F and G, a complex of acylpeptide antibiotics isolated from *Bacillus pumilus* culture supernatants with interesting antiviral properties (Morikawa et al., 1992; Naruse et al., 1990). Among the lipopeptides belonging to the iturin family, iturin A is the most studied compound. It is a heptapeptide interlinked with a β-amino-acid fatty acid with carbon chain length from C14 to C17 (Peypoux, 1978, as cited in Jacques, 2010) produced by *Bacillus subtilis* strains reported to have antifungal activities (Besson et al., 1976).

Other members of the iturin family are iturin C, bacillomycin D, F, and Lc and mycosubtilin (Bonmatin et al., 2003). The family of fengycins includes fengycins A and B, lipodecapeptides which differ by their amino-acid residue in position 6 that can be Alanine or Valine and are known for their interesting fungitoxic and immunomodulating activities (Jacques, 2010). Other interesting lipopeptides are serrawettins, nonionic cyclodepsipeptide biosurfactants produced by *Serratia marcescens* (Matsuyama et al., 2010) and implicated with anti-tumor and anti-nematode activities.

### **2.1.2 Glycolipids**

Are commonly mono or disaccharides compounds acylated with long chain fatty acids or hydroxyl fatty acids. Among them, rhamnolipids, mannosylerythritol lipids (MELs), sophorolipids and trehalolipids are the best-studied structural subclasses.

Rhamnolipids are glycosides, produced mainly by *Pseudomonas aeruginosa* and by the *Burkholderia* genus, that are composed of one (for mono-rhamnolipids) or two (for dirhamnolipids) rhamnose sugar moieties linked to one or two β-hydroxyfatty acid chains (Perfumo et al., 2006; Raza, 2009). These molecules display high surface activities and many potential applications in the biomedical field due to their antibacterial, antifungal, antiviral, antiadhesive reported properties (Abalos et al., 2001; Cosson et al., 2002; Kim et al., 2000; Remichkova et al., 2008; Sotirova et al., 2008; Yoo et al., 2005). They have also been used in the preparation of nanoparticles (Palanisamy & Raichur, 2009; Xie et al., 2006) and microemulsions (Nguyen & Sabatini, 2009; Xie et al., 2007).

The mannosylerythritol (MELs) glycolipids are produced by the yeasts strains of the genus *Pseudozyma* sp. and *Ustilago* sp. from soybean oil or *n*-alkane (Arutchelvi & Doble, 2010). MELs are a mixture of partially acylated derivative of 4-*O*-*β*-D-mannopyranosyl-D-erythritol,

Arima et al., (1968) from the culture broth of *Bacillus subtilis* and it was named thus due to its exceptional surfactant activity (Peypoux et al., 1999). Natural surfactins are a mixture of isoforms A, B, C and D which are classified according to the differences in their amino acid sequences and possess various physiological properties (Shaligram & Singhal, 2010). Surfactin is composed of a seven amino-acid ring structure coupled to a fatty-acid chain via a lactone linkage. Surfactin-A has L-leucine, surfactin-B has L-valine and surfactin-C has L-isoleucine at the amino acid position involved in the lactone ring formation with the C14–C15 β-hydroxy fatty acid. The amino-acid residues may vary and the presence of these variants can be related to alterations in the culture conditions such as providing substrate containing some specific amino-acid residues in the culture media (Jacques,

Another surfactin related compound is lichenysin, a lipopeptide discovered in the supernatant of *Bacillus licheniformis* culture (Horowitz et al., 1990). Its chemical structure and physio-chemical properties are similar to surfactin (McInerney et al., 1990). In particular, lichenysin has Glutamine amino-acid in position 1 while surfactin has Glutamic acid. Other surfactin-like compounds are pumilacidin A, B, C, D, E, F and G, a complex of acylpeptide antibiotics isolated from *Bacillus pumilus* culture supernatants with interesting antiviral properties (Morikawa et al., 1992; Naruse et al., 1990). Among the lipopeptides belonging to the iturin family, iturin A is the most studied compound. It is a heptapeptide interlinked with a β-amino-acid fatty acid with carbon chain length from C14 to C17 (Peypoux, 1978, as cited in Jacques, 2010) produced by *Bacillus subtilis* strains reported to have antifungal

Other members of the iturin family are iturin C, bacillomycin D, F, and Lc and mycosubtilin (Bonmatin et al., 2003). The family of fengycins includes fengycins A and B, lipodecapeptides which differ by their amino-acid residue in position 6 that can be Alanine or Valine and are known for their interesting fungitoxic and immunomodulating activities (Jacques, 2010). Other interesting lipopeptides are serrawettins, nonionic cyclodepsipeptide biosurfactants produced by *Serratia marcescens* (Matsuyama et al., 2010) and implicated with

Are commonly mono or disaccharides compounds acylated with long chain fatty acids or hydroxyl fatty acids. Among them, rhamnolipids, mannosylerythritol lipids (MELs),

Rhamnolipids are glycosides, produced mainly by *Pseudomonas aeruginosa* and by the *Burkholderia* genus, that are composed of one (for mono-rhamnolipids) or two (for dirhamnolipids) rhamnose sugar moieties linked to one or two β-hydroxyfatty acid chains (Perfumo et al., 2006; Raza, 2009). These molecules display high surface activities and many potential applications in the biomedical field due to their antibacterial, antifungal, antiviral, antiadhesive reported properties (Abalos et al., 2001; Cosson et al., 2002; Kim et al., 2000; Remichkova et al., 2008; Sotirova et al., 2008; Yoo et al., 2005). They have also been used in the preparation of nanoparticles (Palanisamy & Raichur, 2009; Xie et al., 2006) and

The mannosylerythritol (MELs) glycolipids are produced by the yeasts strains of the genus *Pseudozyma* sp. and *Ustilago* sp. from soybean oil or *n*-alkane (Arutchelvi & Doble, 2010). MELs are a mixture of partially acylated derivative of 4-*O*-*β*-D-mannopyranosyl-D-erythritol,

sophorolipids and trehalolipids are the best-studied structural subclasses.

microemulsions (Nguyen & Sabatini, 2009; Xie et al., 2007).

2010).

activities (Besson et al., 1976).

**2.1.2 Glycolipids** 

anti-tumor and anti-nematode activities.

containing C2:0, C12:0, C14:0, C14:1, C16:0, C16:1, C18:0 and C18:1 fatty acids as the hydrophobic groups (Bhattacharjee et al., 1970, as cited in Arutchelvi & Doble, 2010). Based on the degree of acetylation at C4 and C6 position, and their order of appearance on the thin layer chromatography, the MELs are classified into MEL-A, -B, -C and –D (Arutchelvi & Doble, 2010). MEL-A representing the diacetylated compound while MEL-B and MEL-C are monoacetylated at C6 and C4, respectively. The completely de-acetylated structure is known as MEL-D (Rau et al., 2005, as cited in Arutchelvi & Doble, 2010).

MELs have recently gained attention due to their environmental compatibility, mild production conditions, structural diversity, self-assembling properties and versatile biochemical functions. In particular, interesting applications have been described in the biomedical field as antimicrobial, antitumor and immunomodulating molecules, in the biotechnological field for gene and drug delivery, and in cosmetic applications as skin moisturizers (Arutchelvi & Doble, 2010).

Sophorolipids are another extracellular glycolipids synthesized by some yeast species including *Candida bombicola*, *Candida apicola*, *Rhodotorula bogoriensis, Wickerhaminella domercqiae* and *Candida batistae* (Van Bogaert & Soetaert, 2010). They consist of two glucose units linked β-1,2. The 6- and 6'-hydroxyl groups are generally acetylated. The lipid portion is connected to the reducing end through a glycosidic linkage. The terminal carboxyl group of the fatty acid can be in the lactonic form or hydrolyzed to generate an anionic surfactant (Rosenberg & Ron, 1999). Sophorolipids have been reported suitable for a number of application in the biomedical field including use as antimicrobial, antiviral and anticancer. They also have been used in the synthesis of metal-bound nanoparticles in cosmetic and pharmacodermatological products (Van Bogaert & Soetaert, 2010).

Trehalose lipids are also a glycolipids containing threhalose as the sugar moiety which is a non-reducing disaccharide in which the two glucose units are linked in an α,α-1,1-glycosidic linkage. It is the basic component of the cell wall glycolipids in *Mycobacteria* and *Corynebacteria* (Franzetti et al., 2010b). The most reported trehalose lipid is trehalose 6,6' dimycolate, which is a α-branched chain mycolic acid esterified to the C6 position of each glucose. Different trehalose containing glycolipids are known to be produced by several other microorganisms belonging to mycolates group, such as *Arthrobacter*, *Nocardia*, *Rhodococcus* and *Gordonia*. *Rhodococcus* genus in particular produced several types of trehalose lipids as reported by Lang & Philp (1998). These glycolipids vary in the number and overall chain length (C20–C90) of the esterified fatty acids. Beside their known industrial applications, trehalose lipids recently attracted attention to their functions in cell membrane interaction and their potential as antitumor therapeutic agents (Aranda et al., 2007, Harland et al., 2009, Imasato et al., 1990, Isoda et al., 1995, as cited in Shao, 2010; Ortiz et al., 2008, 2009; Zaragoza et al., 2009, 2010).

### **2.2 High molecular weight biosurfactants**

These are generally grouped together as polymeric biosurfactants. They are produced by a number of different bacteria and are composed of lipoproteins, proteins, polysaccharides, lipopolysaccharides or complexes containing several of these structural types (Ron & Rosenberg, 2001; Rosenberg & Ron, 1997, 1999). The most commonly studied biopolymer is emulsan (Fig. 2.), a lipopolysaccharide isolated from *Acinetobacter calcoacetius* RAG-1 ATCC 31012 with a molecular weight of around 1,000 kDa (Rosenberg et al., 1979).

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

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 Rosenberg & Ron, 1999).

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

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 industries (Rosenberg & Ron, 1999).
