Proc. tian natn Sci Acad. B70 No. 1 pp 31-55 (2008) Types, Production and Applications of Biosurfactants CATHERINE N. MULLIGAN*! and BERNARD F. GIBBS? ‘Dept. Building, Civil and Environmental Engineering, Concordia University, 1455 de Maisonneuve Blod. W., Montreal, Quebec Canada H3G 1M8 2MDS Pharma Services, 2350 Cohen St., Ville St-Laurent, Quebec Canada H4R 2N6 (Received on 4 September 2002; Accepted after revision 24 December 2002) Biosurfactants are surfactants that are produced extracellularly or as part of the cell membrane by bacteria, yeasts and fungi. Examples include Pseudomonas aeruginosa which produces rhamnolipids, Candida (formerly Torulopsis) bombicola, one of the few yeasts to produce biosurfactants, which produces high yields of sophorolipids from vegetable oils and sugars, and Bacillus subtilis which produces a lipopeptide called surfactin. Less expensive substrates can be used for production including carbohydrates, vegetable oils and food wastes, and are required to reduce production costs. This review includes the factors influencing biosurfactant production, potential industrial applications and future research needs. Optimization of nutrients, environmental factors and process production will also be required to enable economic production of the biosurfactants. Rhamnolipids have potential as antiphytopathogenic agents and environmental applications will continue to increase. More information will be required to determine the role of biosurfactant production in substrate solubilization and bioavailability. Concerning sophorolipids, their structure is dependent on the fatty acid content of the substrate. Therefore, modifications can be performed to change the properties of these biosurfactants. In the cosmetic industry, this biosurfactant has been derivatized and used as a skin moisturizer. Applications should increase due its potential to enhance the remediation of hydrocarbon-contaminated soils. Concerning the biosynthesis of surfactin, there is still a lack of information regarding its secretion, metabolic route and primary cell metabolism. Isolation of high yield biosurfactant-producing strains and strains that can use waste substrates will be required, in addition to information on genetics and metabolism. Improved reactor design and techniques for product recovery need development. Biosurfactant applications in the food, environmental, specialty chemical, cosmetic and pharmaceutical industries are promising. Key Words: Biosurfactants, Rhamnolipids, Surfactin, Sophorolipids, Applications, Production, Metabolism, Waste substrates, Biodegradation Introduction Surfactants are amphiphilic compounds that reduce the free energy of the system by replacing the bulk molecules of higher energy at an interface. They contain a hydrophobic portion with little affinity for the bulk medium and a hydrophilic group that is attracted to the bulk medium. Surfactants have been used industrially as adhesives, flocculating, wetting and foaming agents, de-emulsifiers and penetrants (Mulligan & Gibbs 1993). They are used for these applications based on their abilities to lower surface tensions, increase solubility, detergency power, wetting ability * Correspondi and foaming capacity. The petroleum industry has traditionally been the major users, as in enhanced oil removal applications. In this application, surfactants increase the solubility of petroleum components (Falatko 1991). They have also been used for mineral flotation and in the pharmaceutical industries. Typical desirable properties include solubility enhancement, surface tension reduction, and low critical micelle concentrations (table 1). Surfactants con- centrate at interfaces (solid-liquid, liquid-liquid or vapour-liquid). An interfacial boundary exists between two immiscible phases. The hydro- phobic portion concentrates at the surface while ‘adress: Email: mulligan@ civil.concordia.ca.; Tel: (S14) 848-7925, Fax: (514) 848-280932 Catherine N Mulligan et al. Table 1. Characteristics of surfactants required for various applications (adapted from Myers 1988) Environmental remediation Lubrification Mineral flotation Petroleum recovery Pharmaceuticals Application Characteristics, Detergency Low CMC, good salt and pH stability, biodegradability, food foaming characteristics Emulsification Proper HLB, environmental safety Chemical stability, surface adsorption Low toxicity, biodegradability, affinity for contaminant Adsorption on specific ores Wetting of oil-bearing formations, microemulsion formation and solubilization, ease of emulsion breaking after recovery Biocompatibility, low toxicity the hydrophilic portion is oriented towards the solution, Surfactants are classified as cationic, anionic, zwitterionic and non-ionic and they can be made synthetically from hydrocarbons, lignosulfonates or triglycerides. Total annual sales in the United States are approximately $1.7 billion with an annual 3.5% growth rate. Major types of synthetic surfactants include linear alkyl benzenesul- phonates, alcohol sulphates, alcohol ether sulphates, alcohol glyceryl ether sulphonates, a-olefin sulphonates, alcohol ethoxylates and alkyl phenol ethoxylates (Layman 1985). Surfactants are potentially useful in every industry dealing with multiphasic systems. Sodium dodecy! sulphate (SDS, C,,H,,-SO," Na*) is an example of a widely used anionic surfactant. It contains a straight chain aliphatic hydrocarbon with a sulphate group. The effectiveness of a surfactant is determined by its ability to lower the surface tension, which is, a measure of the surface free energy per unit area or the work required to bring a molecule from the bulk phase to the surface (Rosen 1978). For example, a good surfactant can lower the surface tension of water from 72 to 35 mN/m and the interfacial tension (tension between non-polar and polar liquids) for water against n-hexadecane from 40 to 1 mN/m. The surface tension correlates with the concentration of the surface- active compound until the critical micelle concentration (CMC) is reached (Scheme 1). Efficient surfactants have a low critical micelle concentration (i.e. less surfactant is necessary to decrease the surface tension). The CMC is defined as the minimum concentration necessary to initiate micelle formation (Becher 1965). In practice, the CMC is also the maximum con- centration of surfactant monomers in water and is influenced by pH, temperature and ionic strength. Scheme 1 shows how other parameters vary as a function of surfactant concentration. An emulsion is defined as a “heterogeneous system, consisting of at least one immiscible liquid dispersed in another in the form of droplets, whose diameters, in general, exceed 0.1 mum. Such systems possess a minimal stability, which may be accented by such additives as surface active agents, finely defined solids, etc.” (Becher 1965). It is necessary to specify water-in- oil (w/o) or oil-in-water (0/w) phases. The term hydrophilic-lipophilic balance (HLB) is used to classify which type of emulsion the emulsifier will favour. Emulsifiers and deemul-sifiers stabilize or destabilize the emulsion. Many of these products are polymeric and decrease surface tension insignificantly. owe Soubity = a g : b> — - — suttace tension 3 2 é Interfacial tension ‘Surfactant concentration Scheme 1. Surface tension, interfacial tension and solubilization as a function of surfactant concentration (CMC represents critical micelle concentration).Types, Production and Applications of Biosurfactants The choice of surfactant is primarily based on product cost (Mulligan & Gibbs 1993). In general, surfactants are used to save energy and consequently energy costs (such as the energy required for pumping in pump and treat techniques). Charge-type, physicochemical behaviour, solubility and adsorption behaviour are some of the most important selection criteria for surfactants. New markets are currently being, developed for use in the bioremediation of contaminated land sites (Oberbremer et al. 1990, Samson et al. 1990). Surfactants, in addition to organic solvents, chelating agents, acids and bases, have been used to enhance metal removal (Holden et al. 1989). Some surfactants, known as biosurfactants, are biologically produced by yeast or bacteria from various substrates including sugars, oils, alkanes and wastes (Lin 1996). For example, Pseudomonas aeruginosa can produce rhamnolipids from substrates including C,, and C,, alkanes, succinate, pyruvate, citrate, fructose, glycerol, olive oil, giucose and mannitol (Robert et al.1989). Composition and yields depend on the fermentor design, pH, nutrient composition, substrate and temperature used (Mulligan & Gibbs 1993). They can be potentially as effective with some distinct advantages over the highly used synthetic surfactants including high specificity, bio- degradability and biocompatibility (Cooper 1986). For example, glycolipids from Rhodococcus species 413A were 50% less toxic than Tween 80 in naphthalene solubilization tests (Kanga et al. 1997). Biosurfactants are grouped as glycolipids, lipopeptides, phospholipids, fatty acids, neutral lipids, polymeric and particulate compounds (Bierman et al. 1987). Most of these compounds are either anionic or neutral. Only a few are cationic such as those containing amine groups. The hydrophobic part of the molecule is based on. long-chain fatty acids, hydroxy fatty acids or o-alkyl-a-hydroxy fatty acids. The hydrophilic portion can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol. A. wide variety of microorganisms can produce these compounds (table 2). The CMCs of the biosurfactants generally range from 1 to 200 mg/L and their molecular mass from 500 to 1500 Daltons (Lang & Wagner 1987). Biosurfactants have been tested in enhanced oil recovery and the transportation of crude oils (Hayes et al. 1986). They were demonstrated to be Log number of bacteria 33 effective in the reduction of the interfacial tension of oil and water in situ, the viscosity of the oil, the removal of water from the emulsions prior to processing, and in the release of bitumen from tar sands. The high molecular weight Emulsan® has been commercialized for this purpose (Anonymous 1984). It contains a poly-saccharide with fatty acids and proteins attached. Other high molecular mass biosurfactants are reviewed by Ron and Rosenberg (2002). Although most biosurfactant-producing organisms are aerobic, a few examples of anaerobic producers exist. Bacillus licheniformis JF-2 is an example, which would be well suited for in situ studies for enhanced oil recovery or soil decontamination (Javaheri et al. 1985). A 70% bioremediation and bioreclamation rate of a slop oil-contaminated soil was achieved with oil degrading cultures (Dave et al. 1994). Most biosurfactants are produced from hydrocarbon substrates (Syldatk et al. 1987) Production can be growth associated. In this case, they can either use the emulsification of the substrate (extracellular) or facilitate the passage of the substrate through the membrane (cell membrane associated). Biosurfactants, however, are also produced from carbo-hydrates, which are very soluble. They are usually secondary metabolites, produced during the late logarithmic and /or stationary growth phases (Scheme 2). In this review, we will emphasize three types of very effective and well-studied low molecular mass biosurfactants, rhamnolipids, sophorolipids and surfactin. In each case, we will examine substrates used for production, nutrient and environmental factors affecting biosurfactant Stationary phase Growth limited a Time > Scheme 2. Typical production of biosurfactants during growth,