Surfactants in microbiology and biotechnology: Part 2.

Application aspects
Ajay Singh a , Jonathan D. Van Hamme b , Owen P. Ward a,
b

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Department of Biological Sciences, Thompson Rivers University, Kamloops, British Columbia, Canada V2C 5N3

Abstract Surfactants are amphiphilic compounds which can reduce surface and interfacial tensions by accumulating at the interface of immiscible fluids and increase the solubility, mobility, bioavailability and subsequent biodegradation of hydrophobic or insoluble organic compounds. Chemically synthesized surfactants are commonly used in the petroleum, food and pharmaceutical industries as emulsifiers and wetting agents. Biosurfactants produced by some microorganisms are becoming important biotechnology products for industrial and medical applications due to their specific modes of action, low toxicity, relative ease of preparation and widespread applicability. They can be used as emulsifiers, de-emulsifiers, wetting and foaming agents, functional food ingredients and as detergents in petroleum, petrochemicals, environmental management, agrochemicals, foods and beverages, cosmetics and pharmaceuticals, and in the mining and metallurgical industries. Addition of a surfactant of chemical or biological origin accelerates or sometimes inhibits the bioremediation of pollutants. Surfactants also play an important role in enhanced oil recovery by increasing the apparent solubility of petroleum components and effectively reducing the interfacial tensions of oil and water in situ. However, the effects of surfactants on bioremediation cannot be predicted in the absence of empirical evidence because surfactants sometimes stimulate bioremediation and sometimes inhibit it. For medical applications, biosurfactants are useful as antimicrobial agents and immunomodulatory molecules. Beneficial applications of chemical surfactants and biosurfactants in various industries are discussed in this review. 2006 Elsevier Inc. All rights reserved.
Keywords: Biosurfactant; Chemical surfactant; Emulsification; De-emulsification; Oil recovery; Toxicity; Environmental applications

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . Industrial and environmental applications . . . . 2.1. Oil recovery and processing . . . . . . . 2.1.1. Microbial enhanced oil recovery . 2.1.2. Microbial de-emulsification of oil 2.1.3. Other oil-processing operations . . . . . . . . . . . . . . . . . . . . . . . . . emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 100 101 101 103 104

Corresponding author. Tel.: +1 519 888 4567x2427; fax: +1 519 746 0614. E-mail address: opward@sciborg.uwaterloo.ca (O.P. Ward).

Effects of added chemical- or biosurfactants in bioremediation . 2.2.1. Micellarization . . . . . . . . . . . . . . . . . . . . . 2.2.2. Impacts on microbial adhesion/microbial mobility . . . 2.2.3. Surfactant and contaminant toxicity considerations. . . . 2.2.4. Desorption of contaminants from soil . . . . . . . . . 2.2.5. Surfactant biodegradation . . . . . . . . . . . . . . . . 2.2.6. Concluding comments . . . . . . . . . . . . . . . . . 2.3. Other industrial applications . . . . . . . . . . . . . . . . . . . 3. Agricultural applications . . . . . . . . . . . . . . . . . . . . . . . . 4. Surfactants in bioprocessing . . . . . . . . . . . . . . . . . . . . . . 4.1. Applications in upstream and downstream processing . . . . . . . 4.1.1. Surfactants and production of extracellular products . . . . 4.1.2. Surfactants and recovery of intracellular products . . . . 4.1.3. Applications of colloidal gas aphrons in bioprocessing. 4.2. Surfactants in applied biocatalysis . . . . . . . . . . . . . . . . 4.2.1. Surfactants in mono-phasic organic solvent systems . . . 4.2.2. Surfactants in two-phase systems . . . . . . . . . . . . 4.2.3. Cells in microemulsions . . . . . . . . . . . . . . . . 4.2.4. Surfactantsubstrate interactions . . . . . . . . . . . . 4.2.5. Concluding comments on surfactants in biocatalysis. . . . 5. Industrial production of microbial biosurfactants . . . . . . . . . . . . 6. Enzymatic synthesis of chemical surfactants . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Chemical and biosurfactants are amphiphilic compounds which can reduce surface and interfacial tensions by accumulating at the interface of immiscible fluids and increase the solubility and mobility of hydrophobic or insoluble organic compounds (Prince, 1997; Ron and Rosenberg, 2002; Mulligan, 2005). However, bioavailability and biodegradation kinetics of the hydrophobic pollutants are affected variably by the surfactants. Both stimulating and inhibiting effects of surfactants on bioremediation of pollutants are known depending on the chemical characteristics of the surfactant, pollutant and physiology of the microorganism (Banat et al., 2000; Van Hamme et al., 2003). In nature, biosurfactants play a physiological role in increasing bioavailability of hydrophobic molecules, are involved in promoting the swarming motility of microorganisms and participate in cellular physiological processes of signaling and differentiation (Kearns and Losick, 2003). They are also involved in the processes of biofilm formation. Surfactants can interact with microbial proteins and can be manipulated to modify enzyme conformation in a manner that alters enzyme activity, stability and/or specificity (Kamiya et al., 2000). Chemical surfactants can mimic the latter

effects of biosurfactants and have been exploited, for example, as antimicrobial agents in disease control and to improve degradation of chemical contaminants. Both chemical- and biosurfactants are potentially toxic to specific microbes and may be exploited as antimicrobial agents against plant, animal and human microbial pathogens (Colores et al., 2000; Boyette et al., 2002; Cameotra and Makkar, 2004). In Part 1 of this review the physiological roles of biosurfactants and chemical surfactants in nature were examined (Van Hamme et al., 2006). In this Review a variety of industrial, environmental and agricultural applications of surfactants are discussed. Specific uses of surfactants in biotechnological processes, including upstream and downstream processes, are also reviewed and their innovative applications in biocatalysis are described. Finally we examine the processes for production of microbial biosurfactants by fermentation and chemoenzymatic methods for synthesis of specific chemical surfactants. 2. Industrial and environmental applications Chemical and biological surfactants play an important role in oil recovery and bioremediation of pollutants. Various applications of surfactants are shown in Table 1.

Table 1 Industrial applications of chemical surfactants and biosurfactants Industry Petroleum Application Role of surfactants Improving oil drainage into well bore; stimulating release of oil entrapped by capillaries; wetting of solid surfaces; reduction of oil viscosity and oil pour point; lowering of interfacial tension; dissolving of oil De-emulsification of oil emulsions; oil solubilization; viscosity reduction, wetting agent Emulsification of hydrocarbons; lowering of interfacial tension; metal sequestration Emulsification through adherence to hydrocarbons; dispersion; foaming agent; detergent; soil flushing Emulsifier; solubilizer; demulsifier; suspension, wetting, foaming, defoaming, thickener, lubricating agent Interaction with lipids, proteins and carbohydrates, protecting agent

Enhanced oil recovery De-emulsification Environmental Bioremediation Soil remediation and flushing Food Emulsification and de-emulsification Functional ingredient Biological Microbiological

Physiological behaviour such as cell mobility, cell communication, nutrient accession, cellcell competition, plant and animal pathogenesis Pharmaceuticals and Antibacterial, antifungal, antiviral agents; adhesive agents; immunomodulatory molecules; vaccines; therapeutics gene therapy Agricultural Biocontrol Facilitation of biocontrol mechanisms of microbes such as parasitism, antibiosis, competition, induced systemic resistance and hypovirulence Bioprocessing Downstream Biocatalysis in aqueous two-phase systems and microemulsions; biotransformations; recovery of intracellular processing products; enhanced production of extracellular enzymes and fermentation products Cosmetic Health and beauty Emulsifiers, foaming agents, solubilizers, wetting agents, cleansers, antimicrobial agent, mediators of products enzyme action

2.1. Oil recovery and processing Chemical surfactants and biosurfactants can increase the pseudo solubility of petroleum components in water (Chu, 2003; Pekdemir et al., 2005). Surfactants are effective in reducing the interfacial tensions of oil and water in situ and they can also reduce the viscosity of the oil and remove water from the oil prior to processing (Al-Sabagh, 2000; Liu et al., 2004). Biosurfactants can be as effective as the synthetic chemical surfactants and for certain applications they have advantages such as high specificity. Most of the biosurfactants and many chemical surfactants employed for bioremediation purposes are biodegradable. 2.1.1. Microbial enhanced oil recovery Poor oil recovery in oil-producing wells may be due to either low permeability of some reservoirs or high viscosity of the crude oil resulting in poor mobility. The concept of microbial enhanced oil recovery (MEOR) was first proposed nearly 80 years ago but received only limited attention until the early 1980's (Stosur, 1991). MEOR technology has advanced from laboratory-based studies in the early 1980's to field applications in the 1990's. The ability of indigenous or injected microorganisms to synthesize useful fermentation products to improve oil recovery from the oil reservoirs is exploited in MEOR processes. MEOR-participating microorganisms produce a variety of products such as

biosurfactants, polysaccharides, carbon dioxide, methane and hydrogen (Almeida et al., 2004). Enhanced oil recovery of the residual oil in reservoirs can also be achieved by plugging of highly permeable wateredout regions of oil reservoirs with bacterial cells and biopolymers (Stewart and Fogler, 2001). MEOR processes may be implemented by direct injection of nutrients with microbes that are capable of producing desired products in situ for mobilization of oil or alternatively the process may involve injection of the microbial products. These biological interventions are followed by reservoir repressurization, interfacial tension/oil viscosity reduction and selective plugging of the most permeable zones to move the additional oil to the producing wells. Application of biosurfactants which aid oil emulsification and detachment of oil films from rocks have considerable potential in MEOR processes (Desai and Banat, 1997). Microorganisms are capable of synthesizing biosurfactants from crude oil, pure hydrocarbons and a variety of non-hydrocarbon substrates such as simple carbohydrates, acids and alcohols. Any biological method requires consideration of the environmental conditions of the reservoir in terms of salinity, pH, temperature and pressure (Khire and Khan, 1994). Among microorganisms, only bacteria are considered promising candidates for MEOR and molds, yeasts, algae and protozoa are not suitable either due to their morphological characteristics and/or to the growth conditions present in

reservoirs (Shennan and Levi, 1987). Three strategies are recognized for biosurfactant application: 1 biosurfactant production in batch or continuous culture and addition to the reservoir using the conventional way of MEOR; 2 production of biosurfactant by injected microbes at the celloil interface within the reservoir and 3 injection of selected nutrients into the reservoir to stimulate growth of indigenous biosurfactant producing bacteria Microorganisms for MEOR have been isolated by successively limiting the carbon sources and increasing temperature, pressure and salinity of the media, to select microbial strains capable of growing on crude oil at 70 90 C, 20002500 psi, and at a salinity range of 1.32.5% (Premuzic and Lin, 1996). Thermophilic bacteria, with maximum growth rates in the region 6080 C, have also been isolated (Al-Maghrabi et al., 1999). The potential application of the biosurfactants produced by the thermoand halo-tolerant species of Bacillus licheniformis JF-2 and Bacillus subtilis have been explored for enhanced oil recoveries in laboratory columns and reservoirs with oil recoveries from 9.362% (Lin et al., 1994; Yakimov et al., 1997; Makkar and Cameotra, 1998). Increases in oil recovery by about 30% have been reported from underground sandstone by using trehalolipids from Nocardia rhodochrus (Rapp et al., 1979). Flooding strata with suspensions of Bacillus, Desulfovibrio, Clostridium, Micrococcus, Pseudomonas, Arthrobacter, Peptococcus, Microbacterium, and other microorganisms of different taxonomic groups has been recommended. The National Institute of Petroleum and Energy Research has estimated that in the United States 27% of the oil reservoirs (Banat, 1995a,b) and 40% of the oilproducing carbonate reservoirs (Tanner et al., 1991) may be suitable for MEOR. Injection of biosurfactants and bacteria such as Pseudomonas aeruginosa, Xanthomonas campestrsi, B. licheniformis and Desulfovibrio desulfuricans along with nutrients showed increase in oil recovery by 30200%. More than 400 MEOR tests have been conducted in the United States alone and the results have indicated that reservoir heterogeneity significantly affects oil recovery efficiency. A single-well stimulation treatment might increase the rate of production from 0.2 to 0.4 t of oil per day and sustain the increased rate for 26 months without additional treatments. The ecological and physiological factors governing microbial activity in the oil reservoirs have been investigated (He et al., 2000). In some cases, analysis of the crude oil before and after microbial

treatment revealed degradation of long-chain alkanes and alkyl chains of aromatics, but no apparent degradation of aromatic ring structures. Although the usefulness of biosurfactants in the emulsification of hydrocarbons has been clearly demonstrated in the areas of bioremediation of pollutants, cleaning of oil tanks, acceleration of oil well drilling and enhancement of oil recovery, uncertainty remains regarding biosurfactant-based MEOR process performance and the predictability of the results (Nazina et al., 2003). Conditions vary from one reservoir to another, and hence there is a need to customize the MEOR process. The MEOR process may modify the immediate reservoir environment in a number ways that could also cause damage to the production hardware or to the formation itself. MEOR can cause certain damage to the oil-bearing formations and field equipment. In the reservoir, some of the injected bacteria or some chemicals can lead to the formation plugging, biodegradation of injected chemicals in situ and souring of the production well by actively generating H2S (Jenneman et al., 1986; Nazina et al., 2003). Formation of slime, fouling of internal pipe surfaces, and general cleaning problems in the oil or fuel handling hardware are other problems related to the growth of undesirable bacteria following MEOR applications (Jack and Thompson, 1983). Development of a universal additive mixture suitable for extreme reservoir conditions, consisting of a combination of suitable microbial strains, nutrients, biosurfactants and buffering agents in appropriate proportions, may represent a further productive line of research. There are also improved oil recovery processes which use chemical surfactants including the micellar polymer-, alkaline surfactant polymer-, and alkaline surfactant foam-flooding methods (Nasr-El-Din and Taylor, 1992; Han et al., 1999; Liu et al., 2004). The alkali/surfactant/polymer system relies on alkali and surfactant to lower the interfacial tension between a crude oil and the displacing aqueous phase. Polymer reduces the mobility of the aqueous phase and thus increases the sweep efficiency. Petroleum sulphonates, which have high recovery capability but low electrolyte tolerance, are commonly used for enhanced oil recovery (Ng et al., 2002). Polyester surfactants have also been effective (Al-Sabagh, 2000). There are opportunities to achieve synergies by augmenting MEOR systems through supplementation with chemical surfactants. One of the major draw backs of chemical- or biosurfactant use in enhanced oil recovery is the adsorption of the surfactant on the surface of reservoir rock by the rockoilbrine interaction (Liu et al., 2004). Adsorption is expected to increase with the surfactant's

hydrophobicity at the pre-micellar concentration range of surfactant, since an increase in hydrophobicity tends to drive the surfactant from the aqueous phase to the solidliquid surface. This adsorption phenomenon is one of the important factors governing the economic feasibility of the chemical flooding processes. The adsorption of surfactants can be affected by the surface charge on the rock surface and fluid interfaces where positively charged cationic surfactants attract to the negatively charged surface, and negatively charged anionic surfactants attract to positively charged surfaces. Adsorption of a surfactant (primary surfactant) could be reduced significantly by the use of another surfactant that acts as an anti-adsorption additive (Zaitoun et al., 2003). While technical feasibility of using surfactants in MEOR has been established variability between different well environments and also in crude oil properties has prevented development of a standardized MEOR approach. Every well environment will be unique with respect to soil and rock formation characteristics as well as physical and chemical conditions. The environment will also have been impacted upon by the extent to which oil has already been recovered and the specifics of the recovery method. Oil type will vary from site to site with respect to hydrocarbon composition and viscosity. Added to that there is a wide range of chemical and biological surfactants from which to choose and a wide variety of MEOR strategies may be applied. The problem pertaining to the tendency of surfactants to adsorb to the soil and rock materials in the subsurface is a serious one as it reduces surfactant efficacy, increases required surfactant loads and makes it difficult to calculate surfactant requirements. Thus effective MEOR application requires substantial research on a case-by-case basis and the associated costs and uncertainties are a major barrier to widespread MEOR application. Clearly adopting a strategy of avoiding such experimentation and attempting a bestguess approach has a high chance of resulting in a failed outcome. These problems have greatly retarded the application of surfactants and MEOR in oil recovery and it appears very unlikely that the fundamental problems may be resolved to the point where technology performance may be predicted to a reasonable degree of certainty. 2.1.2. Microbial de-emulsification of oil emulsions Oilfield emulsions, both oil-in-water and water-inoil, are formed at various stages of exploration, production and oil recovery and processing, represent a major problem for the petroleum industry (Manning and Thompson, 1995). A process of de-emulsification is

required to recover oil from these emulsions. Since the presence of water and sediments in oil causes corrosion and scaling in tanks and pipelines, a basic sediment and water (BS & W) content of 0.5 to 2.0% has been specified as the maximum allowable in crude oil for transportation through the existing pipelines (Lee, 1999). Factors that influence the stability of emulsions include viscosity, droplet size, phase volume ratio, temperature, pH, age of emulsion, type of emulsifying agent present, density difference and agitation. Traditional de-emulsification methods include centrifugation, heat treatment, electrical treatment and chemicals containing soap, fatty acids and long-chain alcohols (Grace, 1992; Mohammed et al., 1994; Manning and Thompson, 1995). However, physico-chemical de-emulsification processes are capital intensive and emulsions often generated at the wellhead have to be transported to central processing facilities. Since biological processes can be carried out at non-extreme conditions, an effective microbial de-emulsification process could be used directly to treat emulsions at the wellhead, thus saving on transport and high capital equipment costs. Several microorganisms are known to possess deemulsification properties (Table 2), e.g. Nocardia amarae, Corynebacterium petrophilum, Rhodococcus auranticus, B. subtilis, Micrococcus sp., Torulopsis bombicola, Acinetobacter calcoaceticus, species of Alteromonas, Rhodococcus, Aeromonas and mixed bacterial cultures (Kosaric et al., 1987; Kosaric, 1992; Nadarajah et al., 2001, 2002). Acinetobacter and Pseudomonas species are the dominant de-emulsifiers in the mixed culture. C. petrophilum, T. bombicola, N. amarae, R. auranticus and Bacillus subtilis and Micrococcus sp., grown on non-petroleum hydrocarbon substrate can
Table 2 Microbial species with de-emulsifying capability Acinetobacter calcoaceticus Acinetobacter radioresistens Aeromonas sp. Alcaligenes latus Alteromonas sp. Bacillus subtilis Corynebacterium petrophilum Kingella denitrificans Micrococcus sp. Nocardia amarae Pseudomonas aeruginosa Pseudomonas carboxydohybrogena Rhodococcus auranticus Rhodococcus globerulus Rhodococcus rubropertinctus Sphingobacterium thalpophilum Torulopsis bombicola

effectively de-emulsify petroleum emulsions (Kosaric, 1992). Microbes exploit the dual hydrophobic/hydrophilic nature of biosurfactants or hydrophobic cell surfaces to displace the emulsifiers that are present at the oilwater interface (Kosaric et al., 1987; Nadarajah et al., 2001). Some biologically-produced agents such as acetoin, polysaccharides, glycolipids, glycoproteins, phospholipids and rhamnolipids exhibit de-emulsification properties (Cairns et al., 1982; Das, 2001; Janiyani et al., 1994). Elevating the incubation temperature generally accelerates de-emulsification of emulsions by reducing the viscosity of the oil phase, increases density difference between the phases, weakens the stabilizing interfacial film, causes an increased rate of droplet collision leading to coalescence. The ability of a mixed culture to break emulsions was not significantly affected by lyophilization or freezing and thawing, but was completely destroyed by autoclaving or alkaline methanolysis (Kosaric, 1992). In contrast, the de-emulsifying properties of N. amarae, R. auranticus and R. rubropertinctus and microbial biomass from aerobic and anaerobic sludges were resistant to the autoclaving (Kosaric et al., 1987). Washing of cells with any lipid solubilising solvent such as n-pentane, n-hexane, kerosene or chloroformmethanol decreases de-emulsification capability for water-in-oil (w/o) emulsions, whereas the deemulsification rates for oil-in-water (o/w) emulsions increases with n-pentane and kerosene-washed cells. Generally w/o emulsions with higher water content are easier to break than emulsions with lower water contents. Some of the chemical de-emulsifiers are polyglycols, ethoxylated alcohols, ethoxylated nonylphenols, polyhydric alcohols and sulfonic acid salts (Li et al., 1977; Zaki, 1997). The chemical reaches the interface of an emulsified droplet and disrupts the interfacial tension between the two-phases, allowing water droplets to coalesce and settle by gravity (Li and Wang, 1999). Type and amount of an appropriate de-emulsifier, mixing speed of de-emulsifier in the emulsion and the residence time for phase separation and settling are the important factors in emulsion breaking. A major disadvantage with the chemical de-emulsification method is the disposal of the chemical de-emulsifier in the aqueous phase and removal of the de-emulsifier from the oil phase, since failure to do so may prevent a desirable emulsion formation at the next processing step (Sun et al., 1998). Due to variabilities in the properties of crude oil emulsions, inconsistencies are experienced in performance of the different de-emulsification processes including microbial processes. Further research on

microbial de-emulsification processes with field emulsions needs to be aimed at development of more reliable and universally effective systems. Thus, while chemical surfactants have been effectively used to de-emulsify problematic oilfield emulsions disadvantages relate to disposal of the resultant surfactant-containing aqueous phase as well as surfactant removal from the oil phase. Use of microbes or biosurfactants for de-emulsification overcomes the main disadvantages associated with chemical surfactant use, because the biosurfactant component is generall readily biodegradable. However, in general, prepared biosurfactants are more costly than their chemical counterparts. There have also been inconsistencies in process performance related to variabilities in oil, emulsion and in particular the inherent variability of the biosystems. Nevertheless, the degree of variability associated with these processes is much less than that observed in MEOR, because the oil emulsions have been removed from the highly variable soil/rock environment. Hence the major variables are confined to the properties of the oil, properties and quantity of particulate matter associated with the emulsifications, the emulsion water content and the nature of the applied biosystem. It is our view that the inconsistencies can be characterized through research and development leading to development of dependable and cost effectives bioprocessing technologies. Chemical and/or biosurfactants are effectively used for oil recovery from tank bottom sludges or other solid wastes and to facilitate transport of crude oil through pipelines. 2.1.3. Other oil-processing operations Since chemical surfactants have the properties of solubility enhancement and surface tension reduction of crude oil, they also have potential application for oil recovery from petroleum tank bottom sludges and facilitating heavy crude transport though pipelines (Hayes et al., 1986; Banat et al., 1991). Emulsan, an excellent bioemulsifier produced by A. calcoaceticus RAG-1, formerly Arthrobacter RAG-1, is a polyanionic heteropolysaccharide bioemulsifier which consists of N-acetyl-D-galactosamine, N-acetylgalactosamine uronic acid and an amino sugar linked covalently with fatty acid side chains of - and hydroxydodecanoic acid (Zuckerberg et al., 1979). Application of Emulsan has been found to reduce the viscosity of Boscon heavy crude oil from 200,000 to 100 cP, thus facilitating pumping of heavy oil 26,000 miles in a commercial pipeline (Hayes et al., 1986). Kuwait Oil Company has used biosurfactants for crude oil storage tank clean up with up to 90% oil recovery (Banat et al., 1991). Rhamnolipids biosurfactant

can be used to remove the soaked oil from the used oil sorbents (Wei et al., 2005). Although N 95% of oil removal was achieved, with rhamnolipids JBR215 (Jeneil Biosurfactant Company, USA), concentration had little effect when tested at two concentrations 10 and 20 cm3/dm3 and the main factors affecting oil removal were the sorbent pore size and washing time (Wei et al., 2005). The main draw backs of applying biosurfactants in the above applications rather than chemical surfactants relate to their significantly greater costs. 2.2. Effects of added chemical- or biosurfactants in bioremediation Bioremediation typically involves augmentation of soil or other media, contaminated with pollutants with nutrients and sometimes microorganisms, to improve processes for biodegradation of the contaminants. Biodegradation rate of a contaminant in soil depends on its bioavailability to the metabolizing organisms which is influenced by factors such as desorption, diffusion and dissolution. Many of the most persistent contaminants exhibit low water solubility and hence, bioavailability of contaminants can often be improved by addition of emulsifiers. By reducing surface and interfacial tension between liquids, solids and gases, allowing them to disperse readily as emulsions, chemical or biological surfactants may have variable effects on contaminant biodegradation (Banat et al., 2000). Bacteria that overproduce biosurfactants may have an important role in the biodegradation process (Ron and Rosenberg, 2002). Although, substratesurfactant interactions such as emulsification, pseudo solubilization and partitioning of hydrocarbons between phases are expected to increase the microbial accessibility to the contaminant, both improvements and reductions in bioremediation performance have been observed. Typical objectives in using surfactants enhance bioremediation processes relate to overcome bioavailability problems, due to contaminant aqueous insolubility and or contaminant inaccessibility due to soil adsorbtion, and to accelerate the biodegradation process. Researchers for the most part have investigated effects of surfactants on bioremediation from an efficacy rather than a mechanistic standpoint such that the precise action of the surfactant has seldom been established. It is acknowledged that precise mechanisms are not easily elucidated in bioremediation systems because multiple variables are typically in play. 2.2.1. Micellarization With hydrophobic molecular species such as PAHs or PCBs as predominant contaminants, surfactant promotion

of degradation is rarely achieved. At a surfactant concentration significantly below the cmc value no enhancement or inhibitory effect on biodegradation is observed whereas at or above the cmc value biodegradation is inhibited, suggesting that the substrate, contained within the micelles is not bioavailable. Witconol SN70 (a nonionic, alcohol ethoxylate), at a concentration below its cmc, did not affect mineralisation rates of hexadecane or phenanthrene (Colores et al., 2000) whereas above the cmc, it inhibited mineralisation of both hydrocarbons. Surfactant concentrations, greater than or equal to the cmc for all 4 surfactants tested, inhibited mineralization of phenanthrene by Pseudomonas aeruginosa in soilwater cultures and lower surfactant concentrations had no effect (Bramwell and Laha, 2000). Biodegradation of 4 PCB congeners 2,4,2,4-chlorobiphenyl (CBP), 2,3,5,2-CBP and 2,4,5,2,4,5-CBP in aqueous media by Pseudomonas LB-400 was inhibited by Igepal CO-630, a nonionic surfactant, at concentrations above its cmc (Billingsley et al., 1999a,b). The inhibitory effects were eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant cmc. In contrast, at concentrations above the cmc, the presence of an anionic surfactant promoted PCB transformation, compared to a control with no surfactant (Billingsley et al., 1999a). The effects of surfactant on PCB biodegradation have been studied by other research groups too (Shi et al., 1998; Golyshin et al., 1999; Ferrer et al., 2003). In some cases an increase in degradation rate was observed, whereas in other cases a decrease in degradation rates was noted after addition of surfactants. There appear to be other cases where micellarization does not affect degradation. Liu et al. (1995) quantified the bioavailability of micelle-solubilized naphthalene to a naphthalene-degrading mixed microbial population isolated from contaminated waste and soils using two nonionic surfactants, an alkylethoxylate, Brij 30 and the alkylphenol ethoxylate, Triton X-100. Surfactant concentrations above the cmc were not toxic to the naphthalene-degrading bacteria and the presence of surfactant micelles did not inhibit mineralization of naphthalene. Naphthalene, solubilized by the micelles in liquid media, was bioavailable and was degraded by the mixed bacterial culture. Added rhamnolipids above critical micellar concentration (cmc) enhanced the apparent aqueous solubility of hexadecane, enhanced biodegradation of hexadecane, octadecane, n-paraffins, creosotes and other hydrocarbon mixtures in soil and promoted bioremediation of petroleum sludges (Beal and Betts, 2000; Maier and SoberonChavez, 2000; Noordman et al., 2002; Rahman et al., 2003). Above the cmc, the formation of micelles occurs,

and hydrocarbons can partition into the hydrophobic micellar core, increasing their apparent aqueous solubility. Biodegradation of chlorinated hydrocarbons can be enhanced by addition of glycolipids to the medium as has been reported even for polychlorinated biphenyls (Mata-Sandoval et al., 2001). Pesticide biodegradation was also reported to be promoted by surfactin (Awasthi et al., 1999). 2.2.2. Impacts on microbial adhesion/microbial mobility Where the microbial species capable of degrading a particular hydrophobic contaminant exploits the hydrophobic properties of its outer membrane to access by sorbtion the contaminant in the form of individual molecules, contaminant insoluble particles or oil droplets, application of surfactant may have a negative affect by counteracting the microbialcontaminant sorbtive interactions. Chen et al. (2000) observed that Triton X-100 at low concentration (0.09 cmc) inhibited the rate of growth of a Mycobacterium sp. or a Pseudomonas sp. on solid anthracene while microbial growth rate recovered by dilution of surfactants. With growth on glucose no inhibition of growth by the surfactant occurred. Sorbtion of the surfactant onto the surfaces of both the cells and the anthracene particles could inhibit uptake of anthracene and it was suggested that inhibition of microbial adhesion of cells to anthracene was responsible for the inhibition of growth by the surfactant. Another interesting aspect relates to a possible role for surfactants in promoting the mobility of candidate organisms used to bioaugment in situ bioremediation processes as illustrated in the following example. The wild-type culture of Hydrogenophaga flava ENV735, which can grow on MTBE, exhibited poor cell transport through aquifer sediment which was considered a disadvantage from a bioaugmentation perspective (Streger et al., 2002). An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain, that moved more readily through sediments, was isolated by sequential passage of cells through columns of sterile sediment. The wild-type strain is much more hydrophobic than the adhesion-deficient variant and a nonionic surfactant, Tween 20, enhanced cell transport of these cells in sand columns. 2.2.3. Surfactant and contaminant toxicity considerations While the microbial toxicity of surfactants is a possible cause of bioremediation inhibition, many surfactants are not toxic to microorganisms at concentrations near their cmc values (Van Hamme and Ward, 1999). Another possible cause of a reduced rate of

bioremediation in the presence of surfactant is due to increased toxicity of the hydrophobic contaminant due to its increased (pseudo) solubility. Surfactants increase the apparent aqueous solubility of hydrophobic substrates. Toxicity testing indicated that the presence of solubilized phenanthrene increased the toxicity of the surfactant by a 100-fold (Shin et al., 2005). In addition, some surfactants or pseudosolubilised contaminants may exhibit selective toxicity toward specific pure cultures but may have a limited inhibitory impact in a remediation system involving a diverse indigenous microbial population. Cyclodextrins can form soluble complexes with hydrophobic compounds and therefore can mimic the role of surfactants. They have widespread use in medicine and are of interest in microbial processes because they do not exhibit the toxicity characteristics of many chemical surfactants. Cyclodextrins are composed of several glucose molecules arranged in a toroidal shape, specifically the base units are composed of a number of chair-shaped D(+)-glucopyranose units. In most application, varieties of -cyclodextrin are most widely employed. Aqueous solubilities of the base cyclodextrins range from 18 to 232 g/L. Cyclodextrin has a non-polar cavity into which the hydrophobic organic contaminants partition to form inclusion complexes and a polar exterior that provides the molecule with a relatively high aqueous solubility. This gives cyclodextrin the unique property of enhancing the apparent solubility of hydrophobic organic contaminants in aqueous solutions. Bardi et al. (2000) investigated the efficacy of cyclodextrins in hydrocarbon degradation by a soil microbial population. Cyclodextrin, which did not support microbial growth when added alone, accelerated the degradation of all four hydrocarbons tested (dodecane, tetracosane, anthracene and naphthalene), as manifested by higher biomass yields and better utilization of hydrocarbon as a carbon and energy source. 2.2.4. Desorption of contaminants from soil Very hydrophobic contaminants tend to bind very tightly to soil particles in a manner which renders them inaccessible to degrading microbes. Chemical- and biosurfactants can be effective in facilitating desorbtion of the contaminants from soil as a possible integral part of a bioremediation process or in an aqueous soil washing process, where a biological or non-biological process is subsequently applied to remove the contaminants from the recovered aqueous washings. Traditionally, chemical surfactants are used in soil washing. Surfactants can be used in mixture or with

additives such as an alcohol and/or salts such a sodium chloride (Taylor et al., 2004). They have also been found useful in displacing dense non-aqueous phase liquids (DNAPL) by reducing interfacial tension between DNAPL and the groundwater (Chu, 2003; Saichek and Reddy, 2005). Typical surfactant concentrations for washing of contaminant soil are 12%, whereas the same contaminants may be solubilized in an aqueous solution at a surfactant concentration of 0.10.2%. Nonionic surfactants can remove over 80% of total hydrocarbons from the contaminated soil (Lee et al., 2005). Nonionic surfactants washed more of the PCBs from contaminated soil (up to 89%) as compared to anionic surfactants, but the latter surfactants proved to be more effective in subsequent biodegradation tests of the PCBs in the washings, mediated by Pseudomonas sp. LB-400 (Billingsley et al., 2002). PCBs have lower affinity for the interior of anionic rather than nonionic micelles with a similar non-polar chain length (Javert and Heath, 1991) and this may have promoted release of the PCBs from the micelles, bringing them in contact with the degrading bacteria. Biosurfactants have also been found useful for oil spills remediation and for dispersing oil slicks into fine droplets and converting mousse oil into oil-in-water emulsion (Chhatre et al., 1996; Holakoo and Mulligan, 2002). Shulga et al. (2000) examined the use of biosurfactants in cleaning oil from coastal sand. Rhamnolipid biosurfactants have also been evaluated in soil washing applications. Rhamnolipids were effective in removing PAHs (Poggi-Varaldo and Rinderknecht-Seijas, 2003; Garcia-Junco et al., 2003) and pentachlorophenol (Mulligan and Eftekhari, 2003) for soil. Removal efficiency varies with contact time and biosurfactant concentration but is typically about 6080% as has been reported by different researchers (Bai et al., 1998; Urum et al., 2003). Biosurfactants appeared to be more effective in increasing the apparent solubility of PAHs by up to five times as compared to the chemical surfactants (Vipulanandan and Ren, 2000; Cameotra and Bollag, 2003). Sophorolipids released bitumen from tar sands (Cooper and Paddock, 1984) and surfactin was used to wash oil from a sand column (Makkar and Cameotra, 1997). Biosurfactants can enhance removal of alkanes and polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. Improved biodegradation of various PAHs in contaminated soil by addition of rhamnolipids have been reported by different research groups (Burd and Ward, 1996; Schippers et al., 2000; Dean et al., 2001; Straube et al., 2003). Rhamnolipids removed heavy metals such as Ni and Cd from soils due to their anionic nature, with

efficiencies of 80100% in the lab and 2080% in the field samples has been reported (Neilson et al., 2003; Mulligan and Wang, 2004). The glutamate residues of surfactin can bind metals such as Mg, Mn, Ca, Ba, Li and rubidium (Thimon et al., 1992). Soil washing with 0.25% surfactin removed 70% of the Cu and 22% of the Zn (Mulligan et al., 1999). Foaming surfactant technology is a relatively new approach in soil remediation assisted by surfactants (Wang and Mulligan, 2004). Polymers or foams can also be added to control the mobility of the contaminants. Metal-biosurfactant complexes can be removed by addition of air to cause foaming (Mulligan et al., 2001). By use of a technique called micellar-enhanced ultrafiltration, 85100% removal of cadmium, copper and zinc by surfactin from contaminated water was achieved (Mulligan et al., 1999). Role of assisted natural remediation using surfactants in metal-contaminated environments (Rouse et al., 1994; Adriano et al., 2005) and subsurface (Rouse et al., 2004) have also been explored. 2.2.5. Surfactant biodegradation There is a possibility that a surfactant used for bioremediation may be utilized by the microbial population as a growth substrate resulting in increased microbial biomass to promote a comparative increase in contaminant removal. On the other hand preferential surfactant degradation may some time reduce the rate of contaminant degradation through a repression mechanism. The mechanism of action and efficacy of a surfactant on contaminant biodegradation will vary depending on the properties of the microbial species present. Microorganisms have evolved different natural methods of uptake of hydrophobic compounds, in general reflected by their capacity to generate hydrophobic cell surfaces or to secrete biosurfactants into the surrounding medium. The efficacy of added surfactants will relate to the extent that they enhance, antagonize or repress the natural capabilities of the microbes in accessing the contaminants. The beneficial or negative impacts of added surfactants will be more clear-cut in pure culture systems than in indigenous mixed populations where only the aggregate of positive and negative effects will be observed. It is also likely that surfactant addition will over time alter the microbial population with overall positive or negative consequences on rates and extents of biodegradation. 2.2.6. Concluding comments Surfactants exhibit both positive and negative effects when used as additives in soil bioremediation processes. Where selected surfactants have been used to promote

biodegradation of petroleum hydrocarbons through emulsification, positive contributions to bioremediation have typically been observed. With many hydrophobic contaminants, including PAHs and PCBs, application of surfactants at concentrations above their cmc values has been found to retard contaminant biodegradation and biodegradation is more effective if the material is diluted to bring the surfactant concentration below its cmc value. In contrast biosurfactants such as rhamnolipids have enhanced apparent aqueous solubility of a variety of hydrocarbons and promoted biodegradation. Thus variable positive and negative results have been reported presumably reflecting the variabilities in experimental conditions applied. The tendency for surfactants to sorb to soil will also impact effectiveness in a soil bioremediation environment. Thus it is clear that variabilities in soil sorbtion characteristics of surfactants combined with variability in the properties of soil, including particle surface area and organic content, may in part be responsible for variable surfactant performance. In addition, while micellarization assists in pseudo solubilisation of hydrophobic contaminants, this outer surfactant layer around the prospective microbial substrate often reduces access to the substrate, thereby inhibiting degradation. In using surfactants in bioremediation it is important to establish that neither the surfactant nor the contaminant when pseudo solubilized inhibits biodegradation activity of the key microorganisms. Where microbial cells have very hydrophobic cell surfaces they often access hydrophobic contaminants by direct adhesion to insoluble contaminant particles. Use of surfactants which pseudosolubilise the target contaminant typically inhibit this microbial adhesion and biodegradation. In contrast, in situ bioremediation of aquifers contaminated with water soluble organic contaminants, surfactants may reduce microbial adhesion to solids and enhance mobility through the aquifer and biodegradation effectiveness. Since microbial cells contain membrane lipids which are potential target for pseudo solubilisation by surfactants, surfactants for use in bioremediation should be selected and/or applied at non-toxic concentrations. Surfactant properties other than lipid solubilising properties may also be inhibitory. If the microbes preferentially use the surfactants rather than the target contaminant, clearly that diminishes the efficacy of the surfactant. More importantly the surfactant in that case may repress transcription/translation of the enzymes required to catabolise the contaminant, or may favour development of a microbial population which cannot degrade the contaminant at all.

Thus, given the variables in soil type, microbial type, contaminant type and surfactant type, and the range of successful (promoted degradation) and unsuccessful applications (inhibited degradation) which have been reported it is almost impossible to predict performance of specific surfactants in bioremediation processes and experimental testing is always recommended. On balance there are probably many more cases where surfactants have a negative impact on bioremediation so the case for using them in a particular instance ought to be clearly established in advance. Chemical- and biosurfactants can be used to remove organic contaminants from soil by soil washing and anionic surfactants have been employed for removal of metals. The effective surfactant concentration for soil washing applications will be influenced by the amount of surfactant which adsorbs to the soil and the considerable extent of this adsorbtion may also result in deciding not to apply the surfactant if costs are prohibitive. Consideration of soil sorbtion needs to take into account the negative environmental aspects of leaving large amounts of surfactants in the treated soil. However, this problem may be alleviated if the surfactant to be used is an easily biodegradable chemical surfactant or an intrinsically degradable biosurfactant. 2.3. Other industrial applications Biosurfactants may also have some applications in mining and manufacturing processes. Enhanced metal extraction from the mining ores (Dahr Azma and Mulligan, 2004) and partial solubilization of lignite coal (Polman et al., 1994) has been reported. Biodispersan from A. calcoaceticus A2 has potential use in the paint industry (Rosenberg and Ron, 1998). Heteropolysaccharides from Macrocystis pyrifera and Azotobacter vinelandii have been successfully used as dispersants in the ceramic industry (Pellerin et al., 1992). Species of Pseudomonas and Achromobacter capable of degrading anionic surfactants have been used as microbial biosensors for surfactant detection and shown to have potential for rapid evaluation of surfactants in water media (Taranova et al., 2002). 3. Agricultural applications Biological control involves the exploitation of selected microorganisms (termed antagonistic), using naturally occurring mechanisms, to suppress harmful organisms. The modes of action are parasitism, antibiosis, competition, induced systemic resistance and hypovirulence. In many instances surfactants enhance

the effects of the microbial biocontrol agent. Typically only bulk chemical surfactants, rather than biosurfactants, are used in these applications. Mechanisms of surfactant action include facilitation of penetration or infection by the control agent or its products or coformulated components into the cells or tissues of the target organism (Jazzar and Hammad, 2003; Kim et al., 2004). Surfactants may also have a direct antagonistic effect on indigenous organisms. For example, indigenous soil organisms may have the potential to degrade an added chemical control agent, such as an insecticide, and surfactants may be exploited to inhibit indigenous insecticide degraders. Surfactants can employ several mechanisms in rumen biology, when used as growth enhancers. The insecticidal activity of many biological systems appears to be enhanced by use of surfactants. For example, the biocontrol activity of extracts of Melia azedarach leaves and fruits when integrated with Camptotylus reuteri, against the sweetpotato whitefly nymphs was enhanced by use of Tween-80 (Jazzar and Hammad, 2003). In growth chamber and field experiments to assess the potential of Pseudomonas syringae pv. tagetis (Pst) as a biocontrol agent for Canada thistle, Silwet L-77, an organosilicone surfactant, was required to facilitate Pst penetration into Canada thistle (Gronwald et al., 2002). Surfactants are often used in different formulations together with the entomopathogenic nematode Steinernema feltiae for the control of fungus gnats (Krishnayya and Grewal, 2002). The fungus Myrothecium verrucaria (MV), when properly formulated with a surfactant (0.2% Silwet L-77), was effective as a bioherbicide in controlling kudzu (Pueraria lobata) over a wide range of physical and environmental conditions and under field conditions (Boyette et al., 2002). It was demonstrated in field tests that when transplanted kudzu seedlings, in the 23 leaf growth stage, were treated with M. verrucaria at 2 107 conidia ml 1 in 0.2% of the surfactant, they exhibited leaf and stem necrosis within 24 h and mortality after 96 h. In plots treated with the fungus/surfactant mixtures 100% of inoculated kudzu plants were killed within a week. Field bindweed (Convolvulus arvensis L.) is reported to be one of the 12 most important weeds worldwide (Pfirter and Defago, 1998). Stagonospora sp. LA39, isolated from diseased field bindweed plants collected in Europe, was found to induce disease symptoms (i.e. lesions) mainly on leaves and less severely on stems, of field bindweed. The application of spores in an oil emulsion (10% oil-in-water) enhanced the disease on field bindweed plants. The oil emulsion maintains the aggressiveness of the pathogen during a dew-free period and provides a favourable environment during the infection process.

Surfactants are often added as formulating agents in pesticide seed coatings. These additives can substantially modify pesticide fate. Addition of a nonionic micellar surfactant (resulting from the condensation of ethylene oxide and fatty alcohol) significantly increased the degradation of triticonazole [5-(4-chlorophenyl) methylene)-2,2-dimethyl-1-(1H-1,2,4-triazole-1-ylmethyl) cyclopentanol] whereas sodiumalkylnaphthalensulfate surfactants decreased degradation of triticonazole through inhibition of soil microbial activity (Charnay et al., 2000). Chemical surfactants are being explored as replacements for antibiotics as growth enhancers in animal feed due to their impacts on rumen microbial communities. Lee et al. (2004) found that the sorbitan trioleate SOLFA850 increased digestion of rice straw in cow rumens by increasing protease, amylase, carboxymethylcellulase and xylanase activity. In addition, increased NH3-N was detected and the total number of viable bacteria and anaerobic fungi increased with the surfactant-supplemented diet. A similar effect was observed in a separate study when Tween-80 was added alongside the ionophore monensin to synergistically improve rumen fermentation conditions (Wang et al., 2004). The chemical surfactants are believed to increase enzymesubstrate affinity and to increase enzyme release from microbial cells due to increased permeability. Surfactants can be exploited in a wide variety of agricultural applications, while so far biosurfactants have not found significant usage in this area. Surfactants act as biocontrol agents either to facilitate infection of or to antagonize or stimulate growth of target organisms, be they microorganisms, insects or plants/weeds. These agricultural applications illustrate how surfactants may be used to selectively cause physiological changes in target organisms, thereby promoting absorbtion of chemical agents or exhibiting selective antagonistic or stimulatory effects on one organism over another. In some instances the interactions may be at the level of enzyme or receptor level. Thus a focus on understanding the causative molecular interactions involved will likely result in identification of further roles for surfactants in biocontrol. In some cases the biochemical or physiological mechanisms of action are known. However, these biocontrol activities often take place in very complex environments containing a variety of biological species. As we gain greater knowledge of the species present in these environments as well as the nature of the interactions within these communities, the contributions made by surfactants will be more clearly established, facilitating development of more optimized biocontrol systems.

4. Surfactants in bioprocessing Surfactants have a variety of applications in microbial bioprocessing operations. Surfactants can also promote increased production of extracellular products through interaction with cell membrane components during the fermentation step. An analogous but more severe targeting of the cell membrane by selected surfactants after fermentation can promote cell lysis and recovery of intracellular products. Specific surfactant colloidal structures (aphrons) can facilitate oxygen transfer during fermentation and may be used as part of the downstream processing train in cell or molecular separation systems. The modulating impacts of surfactants on enzyme activity, specificity and stability may be exploited in industrial enzymology. These applications have so far been confined to chemical surfactants. 4.1. Applications in upstream and downstream processing 4.1.1. Surfactants and production of extracellular products In his extensive early research on Trichoderma cellulase production and characterization, Reese and Maguire (1969) observed that incorporation of TritonX into the culture medium increased extracellular cellulase activity. Surfactants enhanced secretion of a hyperthermostable and Ca2+-independent -amylase of Geobacillus thermoleovorans (Uma Maheswar Rao and Satyanarayana, 2003). Polyethyleneglycols (PEGs) caused a slight increase while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) significant increased enzyme titre. With the application of SDS, Tween-80 and cholic acid (0.03%, w/v), enzyme production was nearly twice the control level. The anionic (SDS, cholic acid) and nonionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced -amylase secretion. PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,Ntrimethyl ammonium bromide). Anionic surfactants additionally exhibited a stabilizing effect on the enzyme during preservation at 4 C. Below critical inhibitory concentrations (the concentration at which the growth was completely inhibited) for growth of Saccharomyces cerevisiae WSH-J70, the anionic surfactant sodium dedecyl sulfate (SDS) and the cationic surfactant CTAB increased the extracellular glutathione (GSH) concentration by 10 and 15%, respectively (Wei et al., 2003). Above their critical inhibitory concentrations, these surfactants increased extracellular GSH concen-

tration by 35 and 60%, respectively. Nonionic surfactants such as polyoxyethylene lauryl ether (Brij 30) and polyoxyethylene sorbitan monooleate (Tween-80) also affected cell growth, but only at much higher concentrations. Addition of the surfactant Pluronik, a polyethoxypolypropoxy polymer, significantly increased alkaloid biosynthesis by immobilized Claviceps paspali mycelia in a semi-continuous process (Matosic et al., 1998). The general impact of surfactants in promotion of protein secretion is likely to involve interactions with the lipid components of cell membranes in a manner which facilitates secretion. It should be noted that most of the observations related to the positive effects of surfactants on secretion of extracellular enzymes relate to eukaryotic organisms which release enzymes from intracellular organelles through exocytosis. This observation suggests that surfactants may promote this exocytosis by interaction with cell and organelle lipid membrane components. Recent advances in our understanding of the molecular and cellular mechanisms involved in microbial protein secretion is leading to the development of molecular tools to enhance protein secretion (Schallmey et al., 2004; Ward et al., 2006). In addition, research on surfactant promoted secretion has not been on optimized commercial production systems, so the incremental benefits of surfactants in these cases are not known. Many fermentation processes use antifoaming agents with surfactant properties to suppress foams and some of these may contribute to promotion of protein secretion. Thus surfactants represent a valid option to be considered when microbial processes for production of extracellular proteins are being developed, especially where rate of protein secretion is a barrier. 4.1.2. Surfactants and recovery of intracellular products Surfactants have also been used to permeabilise or lyse cells after fermentation as part of the protocol for recovery of intracellular products. Reverse micelle solutions were used for selective permeabilization of Escherichia coli to facilitate extraction of penicillin acylase (Bansal-Mutalik and Gaikar, 2003). The extracting solutions were water-in-hexane macro- and microemulsions stabilized by sodium bis-(2-ethylhexyl) sulfosuccinate (AOT). The recovered enzyme was free from contaminants due to low solubilities of AOT and aliphatic hydrocarbons in water. A possible mechanism for cell permeabilization and enzyme purification by reverse micellar treatment was proposed which indicates that once the surfactant reaches the inner phospholipid layer it increases the membrane permeability by disorganizing the phospholipids present. This may be the mechanism by which aqueous SDS and AOT, under

vigorously agitated conditions, can cause protein release in the present studies. Chen et al. (2001) used a surfactant and chelate method for poly-3-hydroxyalkanoate (PHAs) recovery from Alcaligenes eutrophus. The purity and recovery rate were determined by the amount of surfactant, the ratio of chelate to dry biomass, pH value, temperature and treatment time (Chen et al., 2002). An initial washing of bacterial cells with Tween-80 was found to improve the degree of cell disruption in subsequent sonication or grinding with glass beads, resulted in a 20200% increase in total soluble protein content from a mucous producing psychrotrophic Gram positive bacterium (Nandakumar et al., 2000). The type of surfactant used in the pretreatment procedure before grinding strongly influenced the percentage lysis of tested strains, both in terms of released soluble protein and enzyme activity. It is well known that highest efficiencies in terms of overall release of intracellular proteins from microbial cells are achieved through aggressive mechanical cell disintegration methods (Ward, 1989, 1991). However, in addition to releasing intracellular proteins these methods solubilise most of the protein components associated with cell walls, organelles and membranes. More selective permeabilization, achieved by using reagents which render the cell envelope more porous are beneficial for selective release of target proteins where the objective is to obtain an extracted product with a high specific activity or where further protein purification is required. Surfactants and organic solvents are the reagents of choice for membrane permeabilization and surfactants are preferred over solvents because of the safety issues associated with solvent use. Thus, in the recovery of purified intracellular proteins use of selected surfactants to permeabilise cells with selective protein release represents a promising purification option. In selecting surfactants for these applications the primary consideration is the efficiency and selectivity of the surfactant in permeabilising cells with the selective release of the desired product. It is also important to insure the chosen surfactant has no negative impact on the stability or activity of the product since surfactants may bind to proteins and other bioactive molecules. 4.1.3. Applications of colloidal gas aphrons in bioprocessing Colloidal gas aphrons (CGAs) are novel structures consisting of microbubbles encapsulated by surfactant multilayers (Jauregi et al., 1997). They are prepared by high speed intensive stirring of surfactant solutions (nonionic and ionic surfactants) in a regime which

causes gas entrainment and formation of microbubbles (Subramaniam et al., 1990; Matsushita et al., 1992; Chaphalkar et al., 1993). The resulting CGAs are characterized by having: a large surface area per unit volume due to their small size (D = 10100 m) and high gas content ( 50% v/v); relatively high stability; flow properties similar to water and easy separation by floatation from the bulk liquid phase (Save and Pangarkar, 1994; Jauregi and Varley, 1999). The large interfacial area can be used to adsorb charged or hydrophobic molecules in a manner dependant on the nature of the surfactant multilayer and surfactant type can be tailored to achieve particular applications. Maximum stability requires surfactant concentrations above their cmc values and, with use of ionic surfactants, ionic strength of the medium has an important effect on stability, being best with NaCl at 0.002 0.05 mM (Kommalapati et al., 1996). The volumetric mass transfer co-efficient (KLa), an important characteristic of fermenters for transferring oxygen or other gasses from the gas to the liquid phase, was enhanced 46 fold in aerobic fermentations of S. cerevisiae and in a synthesis-gas(CO) fermentation, by use of CGAs (Kaster et al., 1990; Bredwell and Worden, 1998). The potential for application of CGAs to promote gas mass transfer in bioremediation processes has been suggested by Jauregi and Varley (1999) and Jackson et al. (1998) demonstrated that CGAs performed better than surfactant alone in enhancing the transport of bacteria through the soil matrix. Colloidal gas aphrons also have biotechnology applications for recovery of cellular and molecular products and for enhancement of gas transfer in cell bioreactor and bioremediation processes (Jauregi and Varley, 1999). Air sparging of bulk fluids, containing cells or other biological material, is often implemented to promote separation of the desired components by floatation. Surfactants are often added to air floatation systems to promote product aggregation or precipitation and precipitate floatation. CGAs have been used to further improve conventional air floatation systems in the separation of various cell types (Save and Pangarkar, 1995; Hashim et al., 1995a,b) and for extraction, concentration and/or recovery of proteins and enzymes (Save et al., 1993). Information on the physical make-up of CGAs is very limited and our insights into their mechanisms of interaction with cells and the potential applications in biotechnology are even more limited. Much more research is needed on the impact of surfactant type on the properties of colloidal gas aphrons and on their interactions with cells before their potential applications

in biotechnology can be properly evaluated. Further general physico-chemical and then interdisciplinary biological research on these aspects is needed to better identify and exploit biotechnology applications. Part of the research needs to address impact of surfactant type on structure, properties, biological interactions and applications of CGAs. 4.2. Surfactants in applied biocatalysis Surfactant effects in modulating enzyme activity are being exploited in applied biocatalysis. When enzymes are sought for use in detergent washing products, researchers concentrate on finding enzymes which retain their activity and are stable in the presence of the detergent. A detergent-modified lipase from Rhizopus delemar was prepared using didodecyl glucosyl glutamate, which was stable in organic solvent to 50 C, and exhibited high specificity towards longer chain triglycerides (Okazaki et al., 1997a,b). Lipase from Candida rugosa, coated with the surfactant, glutamic acid didodecyl ester ribitol amide, exhibited considerable activity for the esterification of lauryl alcohol and lauric acid in organic solvents such as iso-octane, whereas the native powder lipase exhibited negligible activity (Wu et al., 2003). The surfactant-coated enzyme was most effective in mediating esterification reactions involving fatty acids and fatty alcohols of medium chain length. Substrate specificity of the coated enzyme was similar to that of the native enzyme. Km of the coated lipase was only half that of the native lipase while Vmax was 1.4 times higher. A surfactant-resistant lipase showed an increased capacity to launder lipid stains from fabric (Aehle et al., 1995; Frenken et al., 1996). While the conventional view has been that microbes generally thrive in aqueous media and that their metabolic machinery is better suited to transforming water soluble substrates and metabolites the majority of organic chemicals are not soluble in water. Recent research has shown that enzymes may be exploited to carry out reactions involving hydrophobic (water insoluble) substrates in non-aqueous media or in twophase aqueous-organic media. Surfactants may be exploited to pseudosolubilise these substrates and to improve mass transfer and reaction rates. 4.2.1. Surfactants in mono-phasic organic solvent systems Low concentrations of the nonionic surfactant, Thesit (polyoxyethylene laurylether; C30 H62 O10), increased the activity of cholesterol oxidases from Streptomyces hygroscopicus (SCO) and Brevibacterium sterolicum (BCO) in aqueous media containing propanol as a

substrate solubilizer while at higher surfactant concentrations the opposite effect occurs (Pollegioni et al., 1999). Triton X-100 (p-tertiary octylphenoxy polyethylalcohol) inactivated both enzymes even at low concentrations. The effects are considered to be related to surfactantenzyme interaction rather than to micellar phenomena. BCO activity was rapidly inactivated, whereas SCO still had 70% of the initial activity after 5 h in the presence of 30% propan-2-ol and hence, SCO was concluded to be the superior catalyst for biotechnology applications. The oxidation of o-phenylenediamine was catalyzed in various anhydrous organic solvents by a surfactantlaccase complex, prepared by a novel preparation technique in water-in-oil (w/o) emulsions (Okazaki et al., 2000). In contrast, laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, had no catalytic activity in non-aqueous media. A similar technique was used to prepare a surfactant-horseradish peroxidase (HRP) complex (Kamiya et al., 2000). The surfactant-HRP complex, prepared with a nonionic surfactant, exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. The pH of the aqueous solution at the preparation step had a significant effect on the enzymatic activity of the HRP complex in organic media. Anionic ions present in the preparation process appeared to lower the catalytic activity through complexation with heme iron. Peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alanineamide, catalyzed by a nonionic surfactant complexed to various proteases, was carried out in anhydrous hydrophilic organic solvents (Okazaki et al., 1997a,b). The surfactant-subtilisin Carlsberg (STC) complex exhibited a higher enzymatic activity than the other protease-surfactant complexes and its initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Water addition to the reaction medium activated the lyophilized STC, but the reaction rate was much lower than that observed for the STC complex, and the hydrolytic reaction preferentially proceeded. Hence the surfactant-STC complex is a potential high utility biocatalyst for peptide synthesis, exhibiting high catalytic activity in anhydrous hydrophilic organic solvents. Since it does not need excess water undesirable side hydrolysis reactions are not promoted. In supercritical carbon dioxide reaction media, the -chymotrypsin-surfactant complexes exhibited a higher enzymatic activity than native -chymotrypsin, for dipeptide synthesis (Mishima et al., 2003). The nonionic surfactants, L-glutamic acid dialkyl ester

ribitol amide and sorbitan monostearate, were superior surfactants as compared with the anionic surfactant, sodium bis(2-ethylhexyl)sulfosuccinate. Increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in supercritical carbon dioxide. Maximum dipeptide synthesis by the surfactant-coated -chymotrypsin occurred at a water content of 4%. 4.2.2. Surfactants in two-phase systems In water-in-oil microemulsions, microdroplets of water, surrounded by a layer of surfactant molecules (reversed micelles), are dispersed in an organic solvent. Reversed micellar systems represent a popular approach to implementing a range of enzymatic reactions in predominantly non-aqueous environments. The enzyme typically is located in finely dispersed aqueous pools which are encapsulated by a surfactant within the nonpolar solvent (Komives et al., 1994). Nanocapsules containing -chymotrypsin in the inner aqueous cavities can act in both the organic solvent and the aqueous medium (Shapiro and Pykhteeva, 1998). For such encapsulation, the reversed hydrated micelles from N, N-diallyl-N,N-didodecyl ammonium bromide (DDAB) in cyclohexane (w0 = 22), including the enzyme, were polymerized by UV initiation. The w0 is the ratio of molar concentration of water to that of the surfactant. The nanocapsules, precipitated with acetone, were suspended in an aqueous medium with the aid of ionic, AOT, or nonionic, Brij-97, surfactants. The resulting unilamellar liposomes, having an inner monolayer from the poly-DDAB network, and the outer surfactant layer had an average outer diameter of 20 nm. Encapsulation of -chymotrypsin almost doubled the Km, with respect to ATEE (acetyl-L-tyrosine ethyl ester) hydrolysis, whereas the Vmax was almost halved. The characteristic high thermostability of the encapsulated -chymotrypsin (up to 80 C) was attributed to the tendency for the polymer network to block conformational transitions due to heating in the enzyme molecule. Because extreme halophilic enzymes have a high salt requirement (about 4 M NaCl), their potential bioprocessing application in organic solvents appeared to be unlikely. However, Marhuenda-Egea et al. (2002) observed that the halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, retained its catalytic properties in an organic medium by creating a reverse micellar system with very low salt concentration. The reverse micelles were constituted with an anionic surfactant AOT or with a cationic surfactant CTAB in cyclohexane plus 1-butanol as co-surfactant. The enzymatic reaction appeared to

follow Michaelis-Menten kinetics only with respect to the anionic surfactant. The non-aqueous activity of the lipase from Mucor javanicus was enhanced by using the anionic surfactant AOT to solubilise the enzyme in organic solvents (Altreuter et al., 2002). pH and ionic strength of the aqueous phase during solubilization had greatest impact on the extraction efficiency and specific activity of the biocatalyst and solubility and activity response surfaces were generated using esterification of octanoic acid with 1-nonanol in isooctane as a model reaction. The results were shown to transfer to the acylation of doxorubicin (DOX), a potent anticancer compound, with 2-thiophene acetic acid vinyl ester, or vinyl butyrate, in toluene. Chen et al. (2002) described the phenomenon of protein refolding/renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles. Copolymerization of lignin with cresol can be catalyzed by peroxidase in reversed micellar systems (Liu et al., 1999). The surfactant concentration in the system can be manipulated to control the molecular weight of the copolymer. Surfactantenzyme complexes of different enzyme sources have been used in transesterification processes (Okazaki et al., 1997a,b). The complexes formed by modifying various proteases with surfactant molecules utilizing water-in-oil (W/O) emulsions, were very effective in catalyzing vinyl butyrate transesterification with benzyl alcohol in organic media, whereas the native proteases hardly catalyzed the above reaction. Performance of conventionally-prepared surfactantcoated protease exhibited significantly less biocatalytic activity than the surfactant-protease complex prepared by the novel method. The method may be particularly applicable to enzymes that are sensitive to non-aqueous media and soluble in organic media. Two membrane enzymes, fructose dehydrogenase (FDH, Gluconobacter sp.) and sarcosine dehydrogenase (SDH, Pseudomonas putida) were immobilized onto the bilayer membranes of stable vesicles (D = 110 m), prepared by an emulsification process involving use of the Span-80, Tween-80 and the lecithin (Kato et al., 2003). Enzyme activity and stability increased considerably as a result of immobilization. 4.2.3. Cells in microemulsions As has been described above for isolated enzymes, various microorganisms (unicellular algae and cyanobacteria) have also been dispersed in microemulsions without loss of biological activity. Each system required a defined quantity of water in the microemulsion for maximum activity. Under optimum conditions, microbial enzymes

from the various cell sources (hydrogenases, dehydrogenases) exhibited a substantial increase in specific activity and temperature optimum, as compared to aqueous solutions (Hopper et al., 1997). Solubilization and growth of Candida pseudotropicalis was investigated in a water-in-oil microemulsion consisting of hexadecane with Tween 85/Span-80 (each 5%, wt/wt) as surfactant and a limited amount of water (up to 3%, v/v) (Pfammatter et al., 1992). The cells had a lower tendency to aggregate in the microemulsion environment and exhibited greater time stability and a much smaller light scattering than aqueous suspensions having the same cell concentration. A novel bioprocess using micelle biocatalysts was used to address disadvantages of conventional processes for microbial desulfurization of coal (Lee and Yen, 1990). The multiphase system contained mineral oil or a mixture with n-heptane, a surfactant and an aqueous phase containing Thiobacillus ferrooxidans organisms as well as cell extracts. The reverse micelles and water-in-oil emulsion successfully removed sulfur from bituminous coal. Cell-free enzyme extracts of T. ferrooxidans were superior to whole cells, and reverse micells were more effective than water-in-oil emulsion. 4.2.4. Surfactantsubstrate interactions While lignocellulose is a potential substrate for ethanol production, high enzyme loadings are required to achieve high cellulose conversion rates which make the process less economically feasible. Surfactant addition, especially nonionic surfactants, to lignocellulose-enzyme reaction systems increases cellulose bioconversion to sugars. Anionic and nonionic surfactants reduced adsorption of the dominating cellulase of Trichoderma reesei Cel7A (CBHI), during hydrolysis and the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to lignin (Eriksson et al., 2002). 4.2.5. Concluding comments on surfactants in biocatalysis Surfactants may complex directly with enzymes in a manner which alters enzyme tertiary structure, thereby modulating enzyme activity and/or specificity and/or stability and such positive modulations may be exploited. Surfactantenzyme complexes can render biocatalysts resistant to denaturation in organic solvent media, thereby facilitating catalysis of hydrophobic substrates dissolved in these media. Such systems can operate where free water content is negligible which is particularly useful in shifting the equilibrium of hydrolase-mediated reactions in favour of synthesis. Some natural or mutated enzymes resist structural alterations in the presence of surfactants and these enzymes

are particularly suited to applications in detergents. Surfactants also act to promote micellarization in twophase reaction media where typically the substrate and the enzyme are compartmentalized in different phases with the reaction occurring at the interface. The enzyme used under these conditions is typically isolated but in some systems whole cells may also be employed. Finally, where there is a tendency for enzymes to sorb to insoluble material present in the reaction medium in a manner which reduces catalytic effectiveness, surfactants may be exploited to desorb the enzyme. 5. Industrial production of microbial biosurfactants To date, industrial applications of biosurfactants have been limited due to their high costs of production relative to costs of chemical surfactants. For example, ethoxylate or alkyl polyglycoside synthetic surfactants have an estimated cost of USD 13/kg. A wide variety of microorganisms can synthesize biosurfactants using a variety of substrates such as sugars, oils, alkanes and agroindustrial wastes such as molasses and potato processing wastes (Mulligan, 2005; Ron and Rosenberg, 2002). For example lipopeptides are synthesized by many Bacillus as well as other species. Glycolipids are produced by Pseudomonas and Candida species while Thiobacillus thiooxidans produces phospholipids biosurfactants. Polysaccharidelipid complexes are synthesized by Acinetobacter species. Microbes often synthesize biosurfactants during growth on waterimmiscible substrates, to facilitate uptake of those substrates by the cell. However, the productivities exhibited by these strains are often not commercially viable (Van Dyke et al., 1991; Bodour and Miller-Maie, 2002; Youssef et al., 2004). The following fermentation process aspects ought to be addressed with the objective of reducing biosurfactant production costs (Kosaric, 1992): 1. The producing strain should be carefully selected and strategies should be employed to adapt or engineer the strain to improve production. 2. The process should be engineered to minimize capital and operating costs. 3. Process feedstock should be selected and adapted to minimize raw material costs. 4. Attention should be paid to minimizing process byproducts and/or to use them as saleable products rather than wastes. It should be noted that these process aspects are no different to those encountered with other fermentation

processes (Ward, 1989). In addition to fermentationrelated costs, product recovery costs must be minimized (Ward, 1991). A typical recovery process for industrial extracellular microbial polymers would be limited to a centrifugation cell separation step followed by a supernatant (containing the biosurfactant) concentration and/or drying step. Most likely unit processes for concentration/drying would involve membrane filtration, evaporation and/or heat mediated drying. A concentrated liquid product represents the most cost effective product form. From a combined application/cost perspective rhamnolipid, produced by P. aeruginosa, represents the leading commercial microbial biosurfactant and hence this brief discourse on industrial biosurfactant production will be confined to this product/host system. Extensive investigations have been implemented at both the molecular and cell culture level aimed at understanding factors influencing rhamnolipid biosurfactant biosynthesis by P. aeruginosa with a view to optimising the fermentation process. Rhamnolipid production is dependant on the central metabolic pathways for fatty acid synthesis and for dTDP (thiamine diphosphate)-activated sugar formation and on enzymes which participate in the production of the exopolysaccharide alginate (Maier and Soberon-Chavez, 2000). Biosynthesis is regulated by a complex genetic regulatory system which is also involved in the control of virulence-associated characteristics. Product transcription is co-ordinately regulated by at least two quorum sensing mechanisms (Fuqua and Greenberg, 1998). Supplementation of the fermentation medium with vegetable oils substantially enhances biosurfactant yields and medium costs can be reduced by incorporating agroindustrial wastes into fermentation media (Banat, 1995a,b; Sim et al., 1997). The product can now be produced, on an industrial scale, to a concentration of 100 g/L at an estimated cost in the range of USD5-20/kg in fermenters having capacities in the range 100 M3200 M3 (Matsufuji et al., 1997; Lang and Wullbrandt, 1999). As additional yield improvements are always encountered over time through production experience the estimated costs are in general approaching costs which are competitive with chemical surfactants. The continuing developments in molecular and cellular biology are increasing our understanding of the genetics, biochemistry and physiology underlying microbial production processes and are providing us with additional tools to increase product yields. In addition, consumers are typically prepared to pay a premium where use of biosurfactants in place of chemical surfactants has a perceived advantage. It is our belief that these various aspects will lead to more cost effective microbial

production processes and a substantial expansion of the overall use of biosurfactants. 6. Enzymatic synthesis of chemical surfactants Many chemical surfactant molecules contain ester or amide linkages and are amenable to chemoenzymatic synthesis using microbial and other lipases or proteases. These enzymes typically act in nature as biodegradative hydrolases but can be exploited in bioorganic synthesis reactions by implementing the biocatalysis in low water environments which shift reaction equilibria to favour synthesis rather than hydrolysis. There are many commercial processes which exploit hydrolases in organic solvent media for bioorganic synthesis and some of the processes benefit from the availability at low cost of bulk industrial hydrolytic enzymes such as proteases and lipases. Semi-synthetic penicillins may be produced in this way from 6aminopenicillanic acid and the appropriate acyl side chain and an enzymatic method for conversion of porcine to human insulin utilizes a lysine-specific protease in organic solvent media as a step in the strategy of replacing the terminal alanine found in porcine insulin with the threonine found in human insulin (Ward, 1991). An aqueous proteolytic step is first used to remove the alanine moiety. A variety of strategies are available for using lipases in esterification, interesterification and transesterification reactions (Ward and Singh, 2005; Muralidhar et al., 2001). An interesting example in food processing involves use of lipase in interesterification reactions to produce high value cocoa butter substitutes from cheap palm oil (Ward, 1991). These and other biotransformation strategies typically exploit the stereo- and region-specific properties of enzymes and their abilities to mediate reactions at nonextreme conditions with respect to pH and temperature. Hence labile substrates may be used without risk of substrate or product physico-chemical denaturation and enzymes reactions may be designed to target a specific group on the substrate while leaving other reactive groups unmodified, such that chemical blocking of the latter groups is not required. Boyat et al. (2000) described methods for using lipases and proteases to prepare new nonionic surfactants from unprotected carbohydrates, amino acids, and fatty alcohols by implementing the esterification and transesterification reactions in organic media. Dossat et al. (2002) described a continuous solvent free system mediated by the immobilized lipase, Lipozyme, for transesterification of sunflower oil with butan-1-ol to butyl ester having low levels of residual mono-, di- and

triglycerides having interesting surfactant and lubricant properties. The same immobilized enzyme was used to transform various palm oil glyceride and fatty acid fractions with amino acids into mixed medium- to longchained surfactants for specific applications (Soo et al., 2003). Fernandez-Perez and Otero (2003) described methods for lipase (Novozym 435)-mediated condensations of amines (ethanolamine, diethanolamine) with fatty acides to form various amid and ester surfactants in different hydrophobic solvents. Product composition and yield may be varied by manipulation of solvent and other conditions. Papain, besides its well known protease activity, also exhibits esterase activity the specificity of which has been characterized by Villeneuve et al. (1995). Papain was used to mediate the formation of amide and ester bonds in the synthesis of surfactants consisting of arginine N-alkyl amide and ester derivatives (Clapes et al., 1999). The system was exploited for synthesis of arginine-based Gemini surfactants, consisting of two single N-alphaacyl-arginine structures linked through the -carboxylic groups of the aminoacid residues by an alpha, omegadiaminoalkane spacer chain (Piera et al., 2000). Lipase B from Candida antarctica catalysed regioselective acylation of - D(+)glucose and various glucosides at the primary hydroxyl group of the sugar moiety with a variety of non-activated arylaliphatic carboxylic acids to produce esters having high surface activity and high water solubility for possible use as wetting agents and water-in-oil emulsions (Otto et al., 1998). Similarly, solvent free media with lipase were used to produce surfactant-like sugar-esters containing omega-3 and omega-6 polyunsaturated fatty acids (Li and Ward, 1996; Ward et al., 1997). The technical feasibility of using enzymes as biocatalysts in synthesis of certain complex organic molecules is well established. These methods have been most widely applied in synthesis of higher value products such as food additives and pharmaceuticals. From an economic perspective this approach is unlikely to be viable for production of low cost surfactants with environmental/industrial applications but may be an option for synthesis of high value surfactant properties for agricultural, food and medical applications. 7. Conclusions Applications of chemical surfactants in desorption of hydrophobic contaminants from soil and subsequent biodegradation has been widely studied. The use of biosurfactants in remediation of contaminated sites also has many advantages. The biosurfactants seem to

enhance biodegradation by influencing the bioavailability of the contaminant (Van Dyke et al., 1991; Zhao et al., 2005). Due to their biodegradability and low toxicity they are very promising for use in remediation technologies. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production (Fraser, 2000). The perceived unfavorable costs of production of biosurfactants are being addressed as microbial hosts and fermentation processes are developed to produce higher yields. For the production of biosurfactants to be commercially viable, further process optimization at the biological and engineering level are required (Sullivan, 1998; Nihei et al., 2004). Moreover, cost of downstream processing for the recovery of any fermentation product are typically high and these need to be minimized through process development initiatives to obtain cost effective biosurfactant products. For medical applications, biosurfactants are useful as antibacterial, antifungal and antiviral agents. In addition, they also have potential for use as major immunomodulatory molecules, adhesive agents and even in vaccines and gene therapy. Most of the biosurfactants used in biological applications are required in very low concentrations that make biosurfactants valuable biomolecules for applications as food additives, specialty chemicals, biocontrol agents, and new generation molecules for health and beauty care industries. References
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