02 Surf Act Ants in Microbiology and Biotechnology

Surfactants in microbiology and biotechnology:Part 2. Application aspects

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

a Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1b 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 ofimmiscible fluids and increase the solubility, mobility, bioavailability and subsequent biodegradation of hydrophobic or insolubleorganic compounds. Chemically synthesized surfactants are commonly used in the petroleum, food and pharmaceutical industriesas emulsifiers and wetting agents. Biosurfactants produced by some microorganisms are becoming important biotechnologyproducts for industrial and medical applications due to their specific modes of action, low toxicity, relative ease of preparation andwidespread applicability. They can be used as emulsifiers, de-emulsifiers, wetting and foaming agents, functional food ingredientsand as detergents in petroleum, petrochemicals, environmental management, agrochemicals, foods and beverages, cosmetics andpharmaceuticals, and in the mining and metallurgical industries. Addition of a surfactant of chemical or biological originaccelerates or sometimes inhibits the bioremediation of pollutants. Surfactants also play an important role in enhanced oil recoveryby increasing the apparent solubility of petroleum components and effectively reducing the interfacial tensions of oil and water insitu. However, the effects of surfactants on bioremediation cannot be predicted in the absence of empirical evidence becausesurfactants sometimes stimulate bioremediation and sometimes inhibit it. For medical applications, biosurfactants are useful asantimicrobial agents and immunomodulatory molecules. Beneficial applications of chemical surfactants and biosurfactants invarious 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002. Industrial and environmental applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

2.1. Oil recovery and processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012.1.1. Microbial enhanced oil recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012.1.2. Microbial de-emulsification of oil emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032.1.3. Other oil-processing operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

⁎ Corresponding author. Tel.: +1 519 888 4567x2427; fax: +1 519 746 0614.E-mail address: [email protected] (O.P. Ward).

2.2. Effects of added chemical- or biosurfactants in bioremediation . . . . . . . . . . . . . . . . . . . . . . . . 1052.2.1. Micellarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1052.2.2. Impacts on microbial adhesion/microbial mobility . . . . . . . . . . . . . . . . . . . . . . . . . . 1062.2.3. Surfactant and contaminant toxicity considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . 1062.2.4. Desorption of contaminants from soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062.2.5. Surfactant biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072.2.6. Concluding comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

2.3. Other industrial applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083. Agricultural applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084. Surfactants in bioprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

4.1. Applications in upstream and downstream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104.1.1. Surfactants and production of extracellular products . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104.1.2. Surfactants and recovery of intracellular products . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104.1.3. Applications of colloidal gas aphrons in bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . 111

4.2. Surfactants in applied biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124.2.1. Surfactants in mono-phasic organic solvent systems . . . . . . . . . . . . . . . . . . . . . . . . . . 1124.2.2. Surfactants in two-phase systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1134.2.3. Cells in microemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1134.2.4. Surfactant–substrate interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1144.2.5. Concluding comments on surfactants in biocatalysis. . . . . . . . . . . . . . . . . . . . . . . . . . . 114

5. Industrial production of microbial biosurfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146. Enzymatic synthesis of chemical surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1157. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1. Introduction

Chemical and biosurfactants are amphiphilic com-pounds which can reduce surface and interfacialtensions by accumulating at the interface of immisciblefluids and increase the solubility and mobility ofhydrophobic or insoluble organic compounds (Prince,1997; Ron and Rosenberg, 2002; Mulligan, 2005).However, bioavailability and biodegradation kinetics ofthe hydrophobic pollutants are affected variably by thesurfactants. Both stimulating and inhibiting effects ofsurfactants on bioremediation of pollutants are knowndepending on the chemical characteristics of thesurfactant, pollutant and physiology of the microor-ganism (Banat et al., 2000; Van Hamme et al., 2003).In nature, biosurfactants play a physiological role inincreasing bioavailability of hydrophobic molecules,are involved in promoting the swarming motility ofmicroorganisms and participate in cellular physiolog-ical processes of signaling and differentiation (Kearnsand Losick, 2003). They are also involved in theprocesses of biofilm formation. Surfactants can interactwith microbial proteins and can be manipulated tomodify enzyme conformation in a manner that altersenzyme activity, stability and/or specificity (Kamiya etal., 2000). Chemical surfactants can mimic the latter

effects of biosurfactants and have been exploited, forexample, as antimicrobial agents in disease control andto improve degradation of chemical contaminants. Bothchemical- and biosurfactants are potentially toxic tospecific microbes and may be exploited as antimicro-bial agents against plant, animal and human microbialpathogens (Colores et al., 2000; Boyette et al., 2002;Cameotra and Makkar, 2004). In Part 1 of this reviewthe physiological roles of biosurfactants and chemicalsurfactants in nature were examined (Van Hamme etal., 2006). In this Review a variety of industrial,environmental and agricultural applications of surfac-tants are discussed. Specific uses of surfactants inbiotechnological processes, including upstream anddownstream processes, are also reviewed and theirinnovative applications in biocatalysis are described.Finally we examine the processes for production ofmicrobial biosurfactants by fermentation and chemoen-zymatic methods for synthesis of specific chemicalsurfactants.

2. Industrial and environmental applications

Chemical and biological surfactants play an impor-tant role in oil recovery and bioremediation of pollutants.Various applications of surfactants are shown in Table 1.

2.1. Oil recovery and processing

Chemical surfactants and biosurfactants can increasethe pseudo solubility of petroleum components in water(Chu, 2003; Pekdemir et al., 2005). Surfactants areeffective in reducing the interfacial tensions of oil andwater in situ and they can also reduce the viscosity ofthe oil and remove water from the oil prior to pro-cessing (Al-Sabagh, 2000; Liu et al., 2004). Biosurfac-tants can be as effective as the synthetic chemicalsurfactants and for certain applications they have ad-vantages such as high specificity. Most of the biosur-factants and many chemical surfactants employed forbioremediation purposes are biodegradable.

2.1.1. Microbial enhanced oil recoveryPoor oil recovery in oil-producing wells may be

due to either low permeability of some reservoirs or highviscosity of the crude oil resulting in poor mobility. Theconcept of microbial enhanced oil recovery (MEOR)was first proposed nearly 80 years ago but receivedonly limited attention until the early 1980's (Stosur,1991). MEOR technology has advanced from laborato-ry-based studies in the early 1980's to field applicationsin the 1990's. The ability of indigenous or injectedmicroorganisms to synthesize useful fermentationproducts to improve oil recovery from the oil reservoirsis exploited in MEOR processes. MEOR-participatingmicroorganisms produce a variety of products such as

biosurfactants, polysaccharides, carbon dioxide, meth-ane and hydrogen (Almeida et al., 2004). Enhancedoil recovery of the residual oil in reservoirs can alsobe achieved by plugging of highly permeable watered-out regions of oil reservoirs with bacterial cells andbiopolymers (Stewart and Fogler, 2001). MEORprocesses may be implemented by direct injection ofnutrients with microbes that are capable of producingdesired products in situ for mobilization of oil or alter-natively the process may involve injection of the mic-robial products. These biological interventions arefollowed by reservoir repressurization, interfacial ten-sion/oil viscosity reduction and selective plugging of themost permeable zones to move the additional oil to theproducing wells.

Application of biosurfactants which aid oil emulsifi-cation and detachment of oil films from rocks haveconsiderable potential in MEOR processes (Desai andBanat, 1997). Microorganisms are capable of synthesiz-ing biosurfactants from crude oil, pure hydrocarbons anda variety of non-hydrocarbon substrates such as simplecarbohydrates, acids and alcohols. Any biological meth-od requires consideration of the environmental condi-tions of the reservoir in terms of salinity, pH, temperatureand pressure (Khire and Khan, 1994). Among microor-ganisms, only bacteria are considered promising candi-dates for MEOR and molds, yeasts, algae and protozoaare not suitable either due to their morphological charac-teristics and/or to the growth conditions present in

Table 1Industrial applications of chemical surfactants and biosurfactants

Industry Application Role of surfactants

Petroleum Enhanced oilrecovery

Improving oil drainage into well bore; stimulating release of oil entrapped by capillaries; wetting of solidsurfaces; reduction of oil viscosity and oil pour point; lowering of interfacial tension; dissolving of oil

De-emulsification De-emulsification of oil emulsions; oil solubilization; viscosity reduction, wetting agentEnvironmental Bioremediation Emulsification of hydrocarbons; lowering of interfacial tension; metal sequestration

Soil remediation andflushing

Emulsification through adherence to hydrocarbons; dispersion; foaming agent; detergent; soil flushing

Food Emulsification andde-emulsification

Emulsifier; solubilizer; demulsifier; suspension, wetting, foaming, defoaming, thickener, lubricating agent

Functionalingredient

Interaction with lipids, proteins and carbohydrates, protecting agent

Biological Microbiological Physiological behaviour such as cell mobility, cell communication, nutrient accession, cell–cell competition,plant and animal pathogenesis

Pharmaceuticals andtherapeutics

Antibacterial, antifungal, antiviral agents; adhesive agents; immunomodulatory molecules; vaccines;gene therapy

Agricultural Biocontrol Facilitation of biocontrol mechanisms of microbes such as parasitism, antibiosis, competition, inducedsystemic resistance and hypovirulence

Bioprocessing Downstreamprocessing

Biocatalysis in aqueous two-phase systems and microemulsions; biotransformations; recovery of intracellularproducts; enhanced production of extracellular enzymes and fermentation products

Cosmetic Health and beautyproducts

Emulsifiers, foaming agents, solubilizers, wetting agents, cleansers, antimicrobial agent, mediators ofenzyme action

reservoirs (Shennan and Levi, 1987). Three strategies arerecognized for biosurfactant application:

1 biosurfactant production in batch or continuousculture and addition to the reservoir using theconventional way of MEOR;

2 production of biosurfactant by injected microbes atthe cell–oil interface within the reservoir and

3 injection of selected nutrients into the reservoir tostimulate growth of indigenous biosurfactant pro-ducing bacteria

Microorganisms for MEOR have been isolated bysuccessively limiting the carbon sources and increasingtemperature, pressure and salinity of the media, to selectmicrobial strains capable of growing on crude oil at 70–90 °C, 2000–2500 psi, and at a salinity range of 1.3–2.5%(Premuzic and Lin, 1996). Thermophilic bacteria, withmaximum growth rates in the region 60–80 °C, have alsobeen isolated (Al-Maghrabi et al., 1999). The potentialapplication of the biosurfactants produced by the thermo-and halo-tolerant species of Bacillus licheniformis JF-2and Bacillus subtilis have been explored for enhanced oilrecoveries in laboratory columns and reservoirs with oilrecoveries from9.3–62% (Lin et al., 1994;Yakimov et al.,1997; Makkar and Cameotra, 1998). Increases in oilrecovery by about 30% have been reported fromunderground sandstone by using trehalolipids from No-cardia rhodochrus (Rapp et al., 1979). Flooding stratawith suspensions of Bacillus, Desulfovibrio, Clostri-dium, Micrococcus, Pseudomonas, Arthrobacter, Pepto-coccus, Microbacterium, and other microorganisms ofdifferent taxonomic groups has been recommended.

The National Institute of Petroleum and EnergyResearch has estimated that in the United States 27% ofthe oil reservoirs (Banat, 1995a,b) and 40% of the oil-producing carbonate reservoirs (Tanner et al., 1991)may be suitable for MEOR. Injection of biosurfactantsand bacteria such as Pseudomonas aeruginosa,Xanthomonas campestrsi, B. licheniformis and Desul-fovibrio desulfuricans along with nutrients showedincrease in oil recovery by 30–200%. More than 400MEOR tests have been conducted in the United Statesalone and the results have indicated that reservoirheterogeneity significantly affects oil recovery efficien-cy. A single-well stimulation treatment might increasethe rate of production from 0.2 to 0.4 t of oil per day andsustain the increased rate for 2–6 months withoutadditional treatments. The ecological and physiologicalfactors governing microbial activity in the oil reservoirshave been investigated (He et al., 2000). In some cases,analysis of the crude oil before and after microbial

treatment revealed degradation of long-chain alkanesand alkyl chains of aromatics, but no apparentdegradation of aromatic ring structures.

Although the usefulness of biosurfactants in theemulsification of hydrocarbons has been clearly demon-strated in the areas of bioremediation of pollutants,cleaning of oil tanks, acceleration of oil well drilling andenhancement of oil recovery, uncertainty remains regard-ing biosurfactant-based MEOR process performance andthe predictability of the results (Nazina et al., 2003).Conditions vary from one reservoir to another, and hencethere is a need to customize the MEOR process. TheMEOR process may modify the immediate reservoirenvironment in a number ways that could also causedamage to the production hardware or to the formationitself. MEOR can cause certain damage to the oil-bearingformations and field equipment. In the reservoir, some ofthe injected bacteria or some chemicals can lead to theformation plugging, biodegradation of injected chemicalsin situ and souring of the production well by activelygenerating H2S (Jenneman et al., 1986; Nazina et al.,2003). Formation of slime, fouling of internal pipesurfaces, and general cleaning problems in the oil orfuel handling hardware are other problems related to thegrowth of undesirable bacteria following MEOR applica-tions (Jack and Thompson, 1983).

Development of a universal additive mixture suitablefor extreme reservoir conditions, consisting of a combi-nation of suitable microbial strains, nutrients, biosurfac-tants and buffering agents in appropriate proportions,may represent a further productive line of research.

There are also improved oil recovery processeswhich use chemical surfactants including the micellarpolymer-, alkaline surfactant polymer-, and alkalinesurfactant foam-flooding methods (Nasr-El-Din andTaylor, 1992; Han et al., 1999; Liu et al., 2004). Thealkali/surfactant/polymer system relies on alkali andsurfactant to lower the interfacial tension between acrude oil and the displacing aqueous phase. Polymerreduces the mobility of the aqueous phase and thusincreases the sweep efficiency. Petroleum sulphonates,which have high recovery capability but low electrolytetolerance, are commonly used for enhanced oil recovery(Ng et al., 2002). Polyester surfactants have also beeneffective (Al-Sabagh, 2000). There are opportunities toachieve synergies by augmenting MEOR systemsthrough supplementation with chemical surfactants.

One of the major draw backs of chemical- orbiosurfactant use in enhanced oil recovery is theadsorption of the surfactant on the surface of reservoirrock by the rock–oil–brine interaction (Liu et al., 2004).Adsorption is expected to increase with the surfactant's

hydrophobicity at the pre-micellar concentration rangeof surfactant, since an increase in hydrophobicity tendsto drive the surfactant from the aqueous phase to thesolid–liquid surface. This adsorption phenomenon is oneof the important factors governing the economicfeasibility of the chemical flooding processes. Theadsorption of surfactants can be affected by the surfacecharge on the rock surface and fluid interfaces wherepositively charged cationic surfactants attract to thenegatively charged surface, and negatively chargedanionic surfactants attract to positively charged surfaces.Adsorption of a surfactant (primary surfactant) could bereduced significantly by the use of another surfactant thatacts as an anti-adsorption additive (Zaitoun et al., 2003).

While technical feasibility of using surfactants inMEOR has been established variability between differ-ent well environments and also in crude oil propertieshas prevented development of a standardized MEORapproach. Every well environment will be unique withrespect to soil and rock formation characteristics as wellas physical and chemical conditions. The environmentwill also have been impacted upon by the extent towhich oil has already been recovered and the specificsof the recovery method. Oil type will vary from site tosite with respect to hydrocarbon composition andviscosity. Added to that there is a wide range ofchemical and biological surfactants from which tochoose and a wide variety of MEOR strategies may beapplied. The problem pertaining to the tendency ofsurfactants to adsorb to the soil and rock materials in thesubsurface is a serious one as it reduces surfactantefficacy, increases required surfactant loads and makes itdifficult to calculate surfactant requirements. Thuseffective MEOR application requires substantial re-search on a case-by-case basis and the associated costsand uncertainties are a major barrier to widespreadMEOR application. Clearly adopting a strategy ofavoiding such experimentation and attempting a best-guess approach has a high chance of resulting in a failedoutcome. These problems have greatly retarded theapplication of surfactants and MEOR in oil recovery andit appears very unlikely that the fundamental problemsmay be resolved to the point where technologyperformance may be predicted to a reasonable degreeof certainty.

2.1.2. Microbial de-emulsification of oil emulsionsOilfield emulsions, both oil-in-water and water-in-

oil, are formed at various stages of exploration,production and oil recovery and processing, representa major problem for the petroleum industry (Manningand Thompson, 1995). A process of de-emulsification is

required to recover oil from these emulsions. Since thepresence of water and sediments in oil causes corrosionand scaling in tanks and pipelines, a basic sediment andwater (BS & W) content of 0.5 to 2.0% has beenspecified as the maximum allowable in crude oil fortransportation through the existing pipelines (Lee,1999). Factors that influence the stability of emulsionsinclude viscosity, droplet size, phase volume ratio,temperature, pH, age of emulsion, type of emulsifyingagent present, density difference and agitation.

Traditional de-emulsification methods include centri-fugation, heat treatment, electrical treatment and chemi-cals containing soap, fatty acids and long-chain alcohols(Grace, 1992; Mohammed et al., 1994; Manning andThompson, 1995). However, physico-chemical de-emul-sification processes are capital intensive and emulsionsoften generated at the wellhead have to be transported tocentral processing facilities. Since biological processescan be carried out at non-extreme conditions, an effectivemicrobial de-emulsification process could be useddirectly to treat emulsions at the wellhead, thus savingon transport and high capital equipment costs.

Several microorganisms are known to possess de-emulsification properties (Table 2), e.g. Nocardiaamarae, Corynebacterium petrophilum, Rhodococcusauranticus, B. subtilis, Micrococcus sp., Torulopsisbombicola, Acinetobacter calcoaceticus, species of Al-teromonas, Rhodococcus, Aeromonas and mixed bacte-rial cultures (Kosaric et al., 1987; Kosaric, 1992;Nadarajah et al., 2001, 2002). Acinetobacter and Pseu-domonas species are the dominant de-emulsifiers in themixed culture.C. petrophilum, T. bombicola,N. amarae,R. auranticus and Bacillus subtilis andMicrococcus sp.,grown on non-petroleum hydrocarbon substrate can

Table 2Microbial species with de-emulsifying capability

Acinetobacter calcoaceticusAcinetobacter radioresistensAeromonas sp.Alcaligenes latusAlteromonas sp.Bacillus subtilisCorynebacterium petrophilumKingella denitrificansMicrococcus sp.Nocardia amaraePseudomonas aeruginosaPseudomonas carboxydohybrogenaRhodococcus auranticusRhodococcus globerulusRhodococcus rubropertinctusSphingobacterium thalpophilumTorulopsis bombicola

effectively de-emulsify petroleum emulsions (Kosaric,1992).

Microbes exploit the dual hydrophobic/hydrophilicnature of biosurfactants or hydrophobic cell surfaces todisplace the emulsifiers that are present at the oil–waterinterface (Kosaric et al., 1987; Nadarajah et al., 2001).Some biologically-produced agents such as acetoin,polysaccharides, glycolipids, glycoproteins, phospholi-pids and rhamnolipids exhibit de-emulsification proper-ties (Cairns et al., 1982; Das, 2001; Janiyani et al., 1994).

Elevating the incubation temperature generally accel-erates de-emulsification of emulsions by reducing theviscosity of the oil phase, increases density differencebetween the phases, weakens the stabilizing interfacialfilm, causes an increased rate of droplet collision leadingto coalescence. The ability of a mixed culture to breakemulsions was not significantly affected by lyophiliza-tion or freezing and thawing, but was completelydestroyed by autoclaving or alkaline methanolysis(Kosaric, 1992). In contrast, the de-emulsifying proper-ties of N. amarae, R. auranticus and R. rubropertinctusand microbial biomass from aerobic and anaerobicsludges were resistant to the autoclaving (Kosaric et al.,1987). Washing of cells with any lipid solubilisingsolvent such as n-pentane, n-hexane, kerosene orchloroform–methanol decreases de-emulsification capa-bility for water-in-oil (w/o) emulsions, whereas the de-emulsification rates for oil-in-water (o/w) emulsionsincreases with n-pentane and kerosene-washed cells.Generally w/o emulsions with higher water content areeasier to break than emulsions with lower water contents.

Some of the chemical de-emulsifiers are polyglycols,ethoxylated alcohols, ethoxylated nonylphenols, poly-hydric alcohols and sulfonic acid salts (Li et al., 1977;Zaki, 1997). The chemical reaches the interface of anemulsified droplet and disrupts the interfacial tensionbetween the two-phases, allowing water droplets tocoalesce 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 theresidence time for phase separation and settling are theimportant factors in emulsion breaking. A majordisadvantage with the chemical de-emulsification meth-od is the disposal of the chemical de-emulsifier in theaqueous phase and removal of the de-emulsifier fromthe oil phase, since failure to do so may prevent adesirable emulsion formation at the next processing step(Sun et al., 1998).

Due to variabilities in the properties of crude oilemulsions, inconsistencies are experienced in perfor-mance of the different de-emulsification processes in-cluding microbial processes. Further research on

microbial de-emulsification processes with field emul-sions needs to be aimed at development of more reliableand universally effective systems. Thus, while chemicalsurfactants have been effectively used to de-emulsifyproblematic oilfield emulsions disadvantages relate todisposal of the resultant surfactant-containing aqueousphase as well as surfactant removal from the oil phase.Use of microbes or biosurfactants for de-emulsificationovercomes the main disadvantages associated withchemical surfactant use, because the biosurfactantcomponent is generall readily biodegradable. However,in general, prepared biosurfactants are more costly thantheir chemical counterparts. There have also beeninconsistencies in process performance related to vari-abilities in oil, emulsion and in particular the inherentvariability of the biosystems. Nevertheless, the degree ofvariability associated with these processes is much lessthan that observed in MEOR, because the oil emulsionshave been removed from the highly variable soil/rockenvironment. Hence the major variables are confined tothe properties of the oil, properties and quantity ofparticulate matter associated with the emulsifications, theemulsion water content and the nature of the appliedbiosystem. It is our view that the inconsistencies can becharacterized through research and development leadingto development of dependable and cost effectivesbioprocessing technologies. Chemical and/or biosurfac-tants are effectively used for oil recovery from tankbottom sludges or other solid wastes and to facilitatetransport of crude oil through pipelines.

2.1.3. Other oil-processing operationsSince chemical surfactants have the properties of

solubility enhancement and surface tension reduction ofcrude oil, they also have potential application for oilrecovery from petroleum tank bottom sludges andfacilitating heavy crude transport though pipelines(Hayes et al., 1986; Banat et al., 1991).

Emulsan, an excellent bioemulsifier produced byA. calcoaceticus RAG-1, formerly Arthrobacter RAG-1,is a polyanionic heteropolysaccharide bioemulsifierwhich consists of N-acetyl-D-galactosamine, N-acetylga-lactosamine uronic acid and an amino sugar linkedcovalently with fatty acid side chains of α- and β-hydroxydodecanoic acid (Zuckerberg et al., 1979).Application of Emulsan has been found to reduce theviscosity of Boscon heavy crude oil from 200,000 to100 cP, thus facilitating pumping of heavy oil26,000 miles in a commercial pipeline (Hayes et al.,1986). Kuwait Oil Company has used biosurfactants forcrude oil storage tank clean up with up to 90% oilrecovery (Banat et al., 1991). Rhamnolipids biosurfactant

can be used to remove the soaked oil from the used oilsorbents (Wei et al., 2005).AlthoughN95%of oil removalwas achieved, with rhamnolipids JBR215 (Jeneil Biosur-factant Company, USA), concentration had little effectwhen tested at two concentrations 10 and 20 cm3/dm3 andthe main factors affecting oil removal were the sorbentpore size and washing time (Wei et al., 2005).

The main draw backs of applying biosurfactants inthe above applications rather than chemical surfactantsrelate to their significantly greater costs.

2.2. Effects of added chemical- or biosurfactants inbioremediation

Bioremediation typically involves augmentation of soilor othermedia, contaminatedwith pollutantswith nutrientsand sometimes microorganisms, to improve processesfor biodegradation of the contaminants. Biodegradationrate of a contaminant in soil depends on its bioavailabilityto the metabolizing organisms which is influenced byfactors such as desorption, diffusion and dissolution.Manyof the most persistent contaminants exhibit low watersolubility and hence, bioavailability of contaminants canoften be improved by addition of emulsifiers. By reducingsurface and interfacial tension between liquids, solidsand gases, allowing them to disperse readily as emulsions,chemical or biological surfactants may have variableeffects on contaminant biodegradation (Banat et al., 2000).Bacteria that overproduce biosurfactants may have animportant role in the biodegradation process (Ron andRosenberg, 2002). Although, substrate–surfactant interac-tions such as emulsification, pseudo solubilization andpartitioning of hydrocarbons between phases are expectedto increase the microbial accessibility to the contaminant,both improvements and reductions in bioremediationperformance have been observed.

Typical objectives in using surfactants enhancebioremediation processes relate to overcome bioavail-ability problems, due to contaminant aqueous insolu-bility and or contaminant inaccessibility due to soiladsorbtion, and to accelerate the biodegradation process.Researchers for the most part have investigated effectsof surfactants on bioremediation from an efficacy ratherthan a mechanistic standpoint such that the preciseaction of the surfactant has seldom been established. It isacknowledged that precise mechanisms are not easilyelucidated in bioremediation systems because multiplevariables are typically in play.

2.2.1. MicellarizationWith hydrophobic molecular species such as PAHs or

PCBs as predominant contaminants, surfactant promotion

of degradation is rarely achieved. At a surfactantconcentration significantly below the cmc value noenhancement or inhibitory effect on biodegradation isobserved whereas at or above the cmc value biodegrada-tion is inhibited, suggesting that the substrate, containedwithin the micelles is not bioavailable. Witconol SN70 (anonionic, alcohol ethoxylate), at a concentration below itscmc, did not affect mineralisation rates of hexadecane orphenanthrene (Colores et al., 2000) whereas above thecmc, it inhibited mineralisation of both hydrocarbons.Surfactant concentrations, greater than or equal to the cmcfor all 4 surfactants tested, inhibited mineralization ofphenanthrene by Pseudomonas aeruginosa in soil–watercultures and lower surfactant concentrations had no effect(Bramwell and Laha, 2000). Biodegradation of 4 PCBcongeners 2,4,2′,4′-chlorobiphenyl (CBP), 2,3,5,2′-CBPand 2,4,5,2′,4′,5′-CBP in aqueous media by Pseudomo-nas LB-400 was inhibited by Igepal CO-630, a nonionicsurfactant, at concentrations above its cmc (Billingsley etal., 1999a,b). The inhibitory effects were eliminated bydiluting the surfactant/PCB solution to a concentrationclose to the surfactant cmc. In contrast, at concentrationsabove the cmc, the presence of an anionic surfactantpromoted PCB transformation, compared to a controlwith no surfactant (Billingsley et al., 1999a). The effectsof surfactant on PCBbiodegradation have been studied byother research groups too (Shi et al., 1998; Golyshin et al.,1999; Ferrer et al., 2003). In some cases an increase indegradation rate was observed, whereas in other cases adecrease in degradation rates was noted after addition ofsurfactants.

There appear to be other cases where micellarizationdoes not affect degradation. Liu et al. (1995) quantifiedthe bioavailability of micelle-solubilized naphthalene toa naphthalene-degrading mixed microbial populationisolated from contaminated waste and soils using twononionic surfactants, an alkylethoxylate, Brij 30 and thealkylphenol ethoxylate, Triton X-100. Surfactant con-centrations above the cmc were not toxic to thenaphthalene-degrading bacteria and the presence ofsurfactant micelles did not inhibit mineralization ofnaphthalene. Naphthalene, solubilized by the micelles inliquid media, was bioavailable and was degraded by themixed bacterial culture.

Added rhamnolipids above critical micellar concentra-tion (cmc) enhanced the apparent aqueous solubility ofhexadecane, enhanced biodegradation of hexadecane,octadecane, n-paraffins, creosotes and other hydrocarbonmixtures in soil and promoted bioremediation of petro-leum sludges (Beal and Betts, 2000; Maier and Soberon-Chavez, 2000; Noordman et al., 2002; Rahman et al.,2003). Above the cmc, the formation of micelles occurs,

and hydrocarbons can partition into the hydrophobicmicellar core, increasing their apparent aqueous solubility.

Biodegradation of chlorinated hydrocarbons can beenhanced by addition of glycolipids to the medium ashas been reported even for polychlorinated biphenyls(Mata-Sandoval et al., 2001). Pesticide biodegradationwas also reported to be promoted by surfactin (Awasthiet al., 1999).

2.2.2. Impacts on microbial adhesion/microbial mobilityWhere the microbial species capable of degrading a

particular hydrophobic contaminant exploits the hydro-phobic properties of its outer membrane to access bysorbtion the contaminant in the form of individualmolecules, contaminant insoluble particles or oildroplets, application of surfactant may have a negativeaffect by counteracting the microbial–contaminantsorbtive interactions. Chen et al. (2000) observed thatTriton X-100 at low concentration (0.09 cmc) inhibitedthe rate of growth of a Mycobacterium sp. or a Pseu-domonas sp. on solid anthracene while microbial growthrate recovered by dilution of surfactants. With growth onglucose no inhibition of growth by the surfactantoccurred. Sorbtion of the surfactant onto the surfacesof both the cells and the anthracene particles couldinhibit uptake of anthracene and it was suggested thatinhibition of microbial adhesion of cells to anthracenewas responsible for the inhibition of growth by thesurfactant.

Another interesting aspect relates to a possible rolefor surfactants in promoting the mobility of candidateorganisms used to bioaugment in situ bioremediationprocesses as illustrated in the following example. Thewild-type culture of Hydrogenophaga flava ENV735,which can grow on MTBE, exhibited poor cell transportthrough aquifer sediment which was considered adisadvantage from a bioaugmentation perspective(Streger et al., 2002). An adhesion-deficient variant(H. flava ENV735:24) of the wild-type strain, thatmoved more readily through sediments, was isolated bysequential passage of cells through columns of sterilesediment. The wild-type strain is much more hydropho-bic than the adhesion-deficient variant and a nonionicsurfactant, Tween 20, enhanced cell transport of thesecells in sand columns.

2.2.3. Surfactant and contaminant toxicity considerationsWhile the microbial toxicity of surfactants is a

possible cause of bioremediation inhibition, manysurfactants are not toxic to microorganisms at concen-trations 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 toincreased toxicity of the hydrophobic contaminant dueto its increased (pseudo) solubility. Surfactants increasethe apparent aqueous solubility of hydrophobic sub-strates. Toxicity testing indicated that the presence ofsolubilized phenanthrene increased the toxicity of thesurfactant by a 100-fold (Shin et al., 2005). In addition,some surfactants or pseudosolubilised contaminantsmay exhibit selective toxicity toward specific purecultures but may have a limited inhibitory impact in aremediation system involving a diverse indigenousmicrobial population.

Cyclodextrins can form soluble complexes withhydrophobic compounds and therefore can mimic therole of surfactants. They have widespread use inmedicine and are of interest in microbial processesbecause they do not exhibit the toxicity characteristics ofmany chemical surfactants. Cyclodextrins are composedof several glucose molecules arranged in a toroidalshape, specifically the base units are composed of anumber of “chair-shaped” D(+)-glucopyranose units. Inmost application, varieties of β-cyclodextrin are mostwidely employed. Aqueous solubilities of the basecyclodextrins range from 18 to 232 g/L. Cyclodextrinhas a non-polar cavity into which the hydrophobicorganic contaminants partition to form inclusion com-plexes and a polar exterior that provides the moleculewith a relatively high aqueous solubility. This givescyclodextrin the unique property of enhancing theapparent solubility of hydrophobic organic contami-nants in aqueous solutions. Bardi et al. (2000)investigated the efficacy of cyclodextrins in hydrocar-bon degradation by a soil microbial population. β-Cyclodextrin, which did not support microbial growthwhen added alone, accelerated the degradation of allfour hydrocarbons tested (dodecane, tetracosane, an-thracene and naphthalene), as manifested by higherbiomass yields and better utilization of hydrocarbon as acarbon and energy source.

2.2.4. Desorption of contaminants from soilVery hydrophobic contaminants tend to bind very

tightly to soil particles in a manner which renders theminaccessible to degrading microbes. Chemical- andbiosurfactants can be effective in facilitating desorbtionof the contaminants from soil as a possible integral partof a bioremediation process or in an aqueous soilwashing process, where a biological or non-biologicalprocess is subsequently applied to remove the con-taminants from the recovered aqueous washings.

Traditionally, chemical surfactants are used in soilwashing. Surfactants can be used in mixture or with

additives such as an alcohol and/or salts such a sodiumchloride (Taylor et al., 2004). They have also been founduseful in displacing dense non-aqueous phase liquids(DNAPL) by reducing interfacial tension betweenDNAPL and the groundwater (Chu, 2003; Saichek andReddy, 2005). Typical surfactant concentrations forwashing of contaminant soil are 1–2%, whereas thesame contaminants may be solubilized in an aqueoussolution at a surfactant concentration of 0.1–0.2%.Nonionic surfactants can remove over 80% of totalhydrocarbons from the contaminated soil (Lee et al.,2005). Nonionic surfactants washed more of the PCBsfrom contaminated soil (up to 89%) as compared toanionic surfactants, but the latter surfactants proved tobe more effective in subsequent biodegradation tests ofthe PCBs in the washings, mediated by Pseudomonassp. LB-400 (Billingsley et al., 2002). PCBs have loweraffinity for the interior of anionic rather than nonionicmicelles with a similar non-polar chain length (Javertand Heath, 1991) and this may have promoted release ofthe PCBs from the micelles, bringing them in contactwith the degrading bacteria.

Biosurfactants have also been found useful for oilspills remediation and for dispersing oil slicks into finedroplets and converting mousse oil into oil-in-wateremulsion (Chhatre et al., 1996; Holakoo and Mulligan,2002). Shulga et al. (2000) examined the use ofbiosurfactants in cleaning oil from coastal sand. Rham-nolipid biosurfactants have also been evaluated in soilwashing applications. Rhamnolipids were effective inremoving PAHs (Poggi-Varaldo and Rinderknecht-Seijas,2003; Garcia-Junco et al., 2003) and pentachlorophenol(Mulligan and Eftekhari, 2003) for soil. Removalefficiency varies with contact time and biosurfactantconcentration but is typically about 60–80% as has beenreported by different researchers (Bai et al., 1998; Urumet al., 2003). Biosurfactants appeared to be more effectivein increasing the apparent solubility of PAHs by up to fivetimes as compared to the chemical surfactants (Vipula-nandan and Ren, 2000; Cameotra and Bollag, 2003).Sophorolipids released bitumen from tar sands (Cooperand Paddock, 1984) and surfactin was used to wash oilfrom a sand column (Makkar and Cameotra, 1997).

Biosurfactants can enhance removal of alkanes andpolycyclic aromatic hydrocarbons (PAHs) from con-taminated soils. Improved biodegradation of variousPAHs in contaminated soil by addition of rhamnolipidshave been reported by different research groups (Burdand Ward, 1996; Schippers et al., 2000; Dean et al.,2001; Straube et al., 2003).

Rhamnolipids removed heavy metals such as Ni andCd from soils due to their anionic nature, with

efficiencies of 80–100% in the lab and 20–80% in thefield samples has been reported (Neilson et al., 2003;Mulligan and Wang, 2004). The glutamate residues ofsurfactin can bind metals such as Mg, Mn, Ca, Ba, Liand rubidium (Thimon et al., 1992). Soil washing with0.25% surfactin removed 70% of the Cu and 22% of theZn (Mulligan et al., 1999). Foaming surfactant technol-ogy is a relatively new approach in soil remediationassisted by surfactants (Wang and Mulligan, 2004).Polymers or foams can also be added to control themobility of the contaminants. Metal-biosurfactant com-plexes can be removed by addition of air to causefoaming (Mulligan et al., 2001). By use of a techniquecalled micellar-enhanced ultrafiltration, 85–100% re-moval of cadmium, copper and zinc by surfactin fromcontaminated water was achieved (Mulligan et al.,1999). Role of assisted natural remediation usingsurfactants 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 biodegradationThere is a possibility that a surfactant used for

bioremediation may be utilized by the microbial popula-tion as a growth substrate resulting in increased microbialbiomass to promote a comparative increase in contami-nant removal. On the other hand preferential surfactantdegradation may some time reduce the rate of contam-inant degradation through a repression mechanism.

The mechanism of action and efficacy of a surfactanton contaminant biodegradation will vary depending onthe properties of the microbial species present. Micro-organisms have evolved different natural methods ofuptake of hydrophobic compounds, in general reflectedby their capacity to generate hydrophobic cell surfacesor to secrete biosurfactants into the surroundingmedium. The efficacy of added surfactants will relateto the extent that they enhance, antagonize or repress thenatural capabilities of the microbes in accessing thecontaminants. The beneficial or negative impacts ofadded surfactants will be more clear-cut in pure culturesystems than in indigenous mixed populations whereonly the aggregate of positive and negative effects willbe observed. It is also likely that surfactant addition willover time alter the microbial population with overallpositive or negative consequences on rates and extentsof biodegradation.

2.2.6. Concluding commentsSurfactants 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 throughemulsification, positive contributions to bioremediationhave typically been observed. With many hydrophobiccontaminants, including PAHs and PCBs, application ofsurfactants at concentrations above their cmc values hasbeen found to retard contaminant biodegradation andbiodegradation is more effective if the material isdiluted to bring the surfactant concentration below itscmc value. In contrast biosurfactants such as rhamno-lipids have enhanced apparent aqueous solubility of avariety of hydrocarbons and promoted biodegradation.Thus variable positive and negative results have beenreported presumably reflecting the variabilities inexperimental conditions applied. The tendency forsurfactants to sorb to soil will also impact effectivenessin a soil bioremediation environment. Thus it is clearthat variabilities in soil sorbtion characteristics ofsurfactants combined with variability in the propertiesof soil, including particle surface area and organiccontent, may in part be responsible for variablesurfactant performance. In addition, while micellariza-tion assists in pseudo solubilisation of hydrophobiccontaminants, this outer surfactant layer around theprospective microbial substrate often reduces access tothe substrate, thereby inhibiting degradation. In usingsurfactants in bioremediation it is important to establishthat neither the surfactant nor the contaminant whenpseudo solubilized inhibits biodegradation activity ofthe key microorganisms.

Where microbial cells have very hydrophobic cellsurfaces they often access hydrophobic contaminants bydirect adhesion to insoluble contaminant particles. Useof surfactants which pseudosolubilise the target con-taminant typically inhibit this microbial adhesion andbiodegradation. In contrast, in situ bioremediation ofaquifers contaminated with water soluble organiccontaminants, surfactants may reduce microbial adhe-sion to solids and enhance mobility through the aquiferand biodegradation effectiveness.

Since microbial cells contain membrane lipids whichare potential target for pseudo solubilisation by surfac-tants, surfactants for use in bioremediation should beselected and/or applied at non-toxic concentrations. Sur-factant properties other than lipid solubilising propertiesmay also be inhibitory. If the microbes preferentiallyuse the surfactants rather than the target contaminant,clearly that diminishes the efficacy of the surfactant.More importantly the surfactant in that case may represstranscription/translation of the enzymes required tocatabolise the contaminant, or may favour developmentof a microbial population which cannot degrade thecontaminant at all.

Thus, given the variables in soil type, microbial type,contaminant type and surfactant type, and the range ofsuccessful (promoted degradation) and unsuccessfulapplications (inhibited degradation) which have beenreported it is almost impossible to predict performanceof specific surfactants in bioremediation processes andexperimental testing is always recommended. Onbalance there are probably many more cases wheresurfactants have a negative impact on bioremediation sothe case for using them in a particular instance ought tobe clearly established in advance.

Chemical- and biosurfactants can be used to removeorganic contaminants from soil by soil washing andanionic surfactants have been employed for removal ofmetals. The effective surfactant concentration for soilwashing applications will be influenced by the amountof surfactant which adsorbs to the soil and theconsiderable extent of this adsorbtion may also resultin deciding not to apply the surfactant if costs areprohibitive. Consideration of soil sorbtion needs to takeinto account the negative environmental aspects ofleaving large amounts of surfactants in the treated soil.However, this problem may be alleviated if thesurfactant to be used is an easily biodegradable chemicalsurfactant or an intrinsically degradable biosurfactant.

2.3. Other industrial applications

Biosurfactants may also have some applications inmining and manufacturing processes. Enhanced metalextraction from the mining ores (Dahr Azma andMulligan, 2004) and partial solubilization of lignitecoal (Polman et al., 1994) has been reported. Biodis-persan from A. calcoaceticus A2 has potential use in thepaint industry (Rosenberg and Ron, 1998). Heteropoly-saccharides from Macrocystis pyrifera and Azotobactervinelandii have been successfully used as dispersants inthe ceramic industry (Pellerin et al., 1992). Species ofPseudomonas and Achromobacter capable of degradinganionic surfactants have been used as microbialbiosensors for surfactant detection and shown to havepotential for rapid evaluation of surfactants in watermedia (Taranova et al., 2002).

3. Agricultural applications

Biological control involves the exploitation ofselected microorganisms (termed antagonistic), usingnaturally occurring mechanisms, to suppress harmfulorganisms. The modes of action are parasitism, antibi-osis, competition, induced systemic resistance andhypovirulence. In many instances surfactants enhance

the effects of the microbial biocontrol agent. Typicallyonly bulk chemical surfactants, rather than biosurfac-tants, are used in these applications. Mechanisms ofsurfactant action include facilitation of penetration orinfection by the control agent or its products or co-formulated components into the cells or tissues of thetarget organism (Jazzar and Hammad, 2003; Kim et al.,2004). Surfactants may also have a direct antagonisticeffect on indigenous organisms. For example, indige-nous soil organisms may have the potential to degrade anadded chemical control agent, such as an insecticide, andsurfactants may be exploited to inhibit indigenous insec-ticide degraders. Surfactants can employ several mecha-nisms in rumen biology, when used as growth enhancers.

The insecticidal activity of many biological systemsappears to be enhanced by use of surfactants. Forexample, the biocontrol activity of extracts of Meliaazedarach leaves and fruits when integrated with Camp-totylus reuteri, against the sweetpotato whitefly nymphswas enhanced by use of Tween-80 (Jazzar and Hammad,2003). In growth chamber and field experiments to assessthe potential of Pseudomonas syringae pv. tagetis (Pst) asa biocontrol agent for Canada thistle, Silwet L-77, anorganosilicone surfactant, was required to facilitate Pstpenetration into Canada thistle (Gronwald et al., 2002).Surfactants are often used in different formulationstogether with the entomopathogenic nematode Steiner-nema feltiae for the control of fungus gnats (Krishnayyaand Grewal, 2002). The fungus Myrothecium verrucaria(MV), when properly formulated with a surfactant (0.2%Silwet L-77), was effective as a bioherbicide incontrolling kudzu (Pueraria lobata) over a wide rangeof physical and environmental conditions and under fieldconditions (Boyette et al., 2002). It was demonstrated infield tests that when transplanted kudzu seedlings, in the2–3 leaf growth stage, were treated withM. verrucaria at2×107 conidia ml−1 in 0.2% of the surfactant, theyexhibited leaf and stem necrosis within 24 h andmortalityafter 96 h. In plots treated with the fungus/surfactantmixtures 100% of inoculated kudzu plants were killedwithin a week. Field bindweed (Convolvulus arvensis L.)is reported to be one of the 12 most important weedsworldwide (Pfirter and Defago, 1998). Stagonospora sp.LA39, isolated from diseased field bindweed plantscollected in Europe, was found to induce diseasesymptoms (i.e. lesions)mainly on leaves and less severelyon stems, of field bindweed. The application of spores inan oil emulsion (10% oil-in-water) enhanced the diseaseon field bindweed plants. The oil emulsion maintainsthe aggressiveness of the pathogen during a dew-freeperiod and provides a favourable environment during theinfection process.

Surfactants are often added as formulating agents inpesticide seed coatings. These additives can substan-tially modify pesticide fate. Addition of a nonionicmicellar surfactant (resulting from the condensation ofethylene oxide and fatty alcohol) significantly increasedthe degradation of triticonazole [5-(4-chlorophenyl)methylene)-2,2-dimethyl-1-(1H-1,2,4-triazole-1-ylmethyl)cyclopentanol] whereas sodiumalkylnaphthalensul-fate surfactants decreased degradation of triticonazolethrough inhibition of soil microbial activity (Charnayet al., 2000).

Chemical surfactants are being explored as replace-ments for antibiotics as growth enhancers in animal feeddue to their impacts on rumen microbial communities.Lee et al. (2004) found that the sorbitan trioleate SOLFA-850 increased digestion of rice straw in cow rumens byincreasing protease, amylase, carboxymethylcellulaseand xylanase activity. In addition, increased NH3-N wasdetected and the total number of viable bacteria andanaerobic fungi increased with the surfactant-supple-mented diet. A similar effect was observed in a separatestudy when Tween-80 was added alongside the iono-phore monensin to synergistically improve rumenfermentation conditions (Wang et al., 2004). Thechemical surfactants are believed to increase enzyme-substrate affinity and to increase enzyme release frommicrobial cells due to increased permeability.

Surfactants can be exploited in a wide variety ofagricultural applications, while so far biosurfactantshave not found significant usage in this area. Surfactantsact as biocontrol agents either to facilitate infection ofor to antagonize or stimulate growth of target organisms,be they microorganisms, insects or plants/weeds. Theseagricultural applications illustrate how surfactantsmay be used to selectively cause physiological changesin target organisms, thereby promoting absorbtion ofchemical agents or exhibiting selective antagonistic orstimulatory effects on one organism over another. Insome instances the interactions may be at the level ofenzyme or receptor level. Thus a focus on understandingthe causative molecular interactions involved will likelyresult in identification of further roles for surfactants inbiocontrol.

In some cases the biochemical or physiologicalmechanisms of action are known. However, these bio-control activities often take place in very complex envi-ronments containing a variety of biological species. Aswe gain greater knowledge of the species present in theseenvironments as well as the nature of the interactionswithin these communities, the contributions made bysurfactants will be more clearly established, facilitatingdevelopment of more optimized biocontrol systems.

4. Surfactants in bioprocessing

Surfactants have a variety of applications in micro-bial bioprocessing operations. Surfactants can alsopromote increased production of extracellular productsthrough interaction with cell membrane componentsduring the fermentation step. An analogous but moresevere targeting of the cell membrane by selectedsurfactants after fermentation can promote cell lysis andrecovery of intracellular products. Specific surfactantcolloidal structures (aphrons) can facilitate oxygentransfer during fermentation and may be used as partof the downstream processing train in cell or molecularseparation systems. The modulating impacts of surfac-tants on enzyme activity, specificity and stability may beexploited in industrial enzymology. These applicationshave so far been confined to chemical surfactants.

4.1. Applications in upstream and downstream processing

4.1.1. Surfactants and production of extracellular productsIn his extensive early research on Trichoderma

cellulase production and characterization, Reese andMaguire (1969) observed that incorporation of Triton-X into the culture medium increased extracellularcellulase activity. Surfactants enhanced secretion of ahyperthermostable and Ca2+-independent α-amylase ofGeobacillus thermoleovorans (Uma Maheswar Raoand Satyanarayana, 2003). Polyethyleneglycols (PEGs)caused a slight increase while Tween-20, Tween-40and Tween-60 (0.03%, w/v) significant increasedenzyme titre. With the application of SDS, Tween-80and cholic acid (0.03%, w/v), enzyme production wasnearly twice the control level. The anionic (SDS,cholic acid) and nonionic (Tweens) detergents in-creased the cell membrane permeability, and thus,enhanced α-amylase secretion. PEG 8000 and the ionicdetergents (SDS, cholic acid and Tween-80) were moreeffective in the solubilization of cell membranecomponents, and enhancing enzyme yields than thecationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Anionic surfactantsadditionally exhibited a stabilizing effect on the enzymeduring preservation at 4 °C. Below critical inhibitoryconcentrations (the concentration at which the growthwas completely inhibited) for growth of Saccharomycescerevisiae WSH-J70, the anionic surfactant sodiumdedecyl sulfate (SDS) and the cationic surfactantCTAB 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 surfac-tants such as polyoxyethylene lauryl ether (Brij 30) andpolyoxyethylene sorbitan monooleate (Tween-80) alsoaffected cell growth, but only at much higher concentra-tions. Addition of the surfactant Pluronik, a polyethox-ypolypropoxy polymer, significantly increased alkaloidbiosynthesis by immobilized Claviceps paspali myceliain a semi-continuous process (Matosic et al., 1998).

The general impact of surfactants in promotion ofprotein secretion is likely to involve interactions with thelipid components of cell membranes in a manner whichfacilitates secretion. It should be noted that most of theobservations related to the positive effects of surfactantson secretion of extracellular enzymes relate to eukaryoticorganisms which release enzymes from intracellular or-ganelles through exocytosis. This observation suggeststhat surfactants may promote this exocytosis by interac-tion with cell and organelle lipid membrane components.

Recent advances in our understanding of themolecular and cellular mechanisms involved in micro-bial protein secretion is leading to the development ofmolecular tools to enhance protein secretion (Schallmeyet al., 2004; Ward et al., 2006). In addition, research onsurfactant promoted secretion has not been on optimizedcommercial production systems, so the incrementalbenefits of surfactants in these cases are not known.Many fermentation processes use antifoaming agentswith surfactant properties to suppress foams and some ofthese may contribute to promotion of protein secretion.Thus surfactants represent a valid option to beconsidered when microbial processes for production ofextracellular proteins are being developed, especiallywhere rate of protein secretion is a barrier.

4.1.2. Surfactants and recovery of intracellular productsSurfactants have also been used to permeabilise or

lyse cells after fermentation as part of the protocol forrecovery of intracellular products. Reverse micellesolutions were used for selective permeabilization ofEscherichia coli to facilitate extraction of penicillinacylase (Bansal-Mutalik and Gaikar, 2003). The extract-ing solutions were water-in-hexane macro- and micro-emulsions stabilized by sodium bis-(2-ethylhexyl)sulfosuccinate (AOT). The recovered enzyme was freefrom contaminants due to low solubilities of AOT andaliphatic hydrocarbons in water. A possible mechanismfor cell permeabilization and enzyme purification byreverse micellar treatment was proposed which indicatesthat once the surfactant reaches the inner phospholipidlayer it increases the membrane permeability bydisorganizing the phospholipids present. This may bethe mechanism by which aqueous SDS and AOT, under

vigorously agitated conditions, can cause protein releasein the present studies.

Chen et al. (2001) used a surfactant and chelatemethod for poly-3-hydroxyalkanoate (PHAs) recoveryfrom Alcaligenes eutrophus. The purity and recoveryratewere determined by the amount of surfactant, the ratioof chelate to dry biomass, pH value, temperature andtreatment time (Chen et al., 2002). An initial washing ofbacterial cells with Tween-80 was found to improve thedegree of cell disruption in subsequent sonication orgrindingwith glass beads, resulted in a 20–200% increasein total soluble protein content from a mucous producingpsychrotrophic Gram positive bacterium (Nandakumaret al., 2000). The type of surfactant used in thepretreatment procedure before grinding strongly influ-enced the percentage lysis of tested strains, both in termsof released soluble protein and enzyme activity.

It is well known that highest efficiencies in terms ofoverall release of intracellular proteins from microbialcells are achieved through aggressive mechanical celldisintegration methods (Ward, 1989, 1991). However, inaddition to releasing intracellular proteins these methodssolubilise most of the protein components associatedwith cell walls, organelles and membranes. Moreselective permeabilization, achieved by using reagentswhich render the cell envelope more porous arebeneficial for selective release of target proteins wherethe objective is to obtain an extracted product with ahigh specific activity or where further protein purifica-tion is required. Surfactants and organic solvents are thereagents of choice for membrane permeabilization andsurfactants are preferred over solvents because of thesafety issues associated with solvent use.

Thus, in the recovery of purified intracellularproteins use of selected surfactants to permeabilisecells with selective protein release represents a prom-ising purification option. In selecting surfactants forthese applications the primary consideration is theefficiency and selectivity of the surfactant in permeabi-lising cells with the selective release of the desiredproduct. It is also important to insure the chosensurfactant has no negative impact on the stability oractivity of the product since surfactants may bind toproteins and other bioactive molecules.

4.1.3. Applications of colloidal gas aphrons inbioprocessing

Colloidal gas aphrons (CGAs) are novel structuresconsisting of microbubbles encapsulated by surfactantmultilayers (Jauregi et al., 1997). They are prepared byhigh 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 arecharacterized by having: a large surface area per unitvolume due to their small size (D=10–100 μm) andhigh gas content (∼50% v/v); relatively high stability;flow properties similar to water and easy separation byfloatation from the bulk liquid phase (Save andPangarkar, 1994; Jauregi and Varley, 1999). The largeinterfacial area can be used to adsorb charged orhydrophobic molecules in a manner dependant on thenature of the surfactant multilayer and surfactant typecan be tailored to achieve particular applications.Maximum stability requires surfactant concentrationsabove their cmc values and, with use of ionic sur-factants, ionic strength of the medium has an importanteffect on stability, being best with NaCl at 0.002–0.05 mM (Kommalapati et al., 1996).

The volumetric mass transfer co-efficient (KLa), animportant characteristic of fermenters for transferringoxygen or other gasses from the gas to the liquid phase,was enhanced 4–6 fold in aerobic fermentations ofS. cerevisiae and in a synthesis-gas(CO) fermentation, byuse of CGAs (Kaster et al., 1990; Bredwell and Worden,1998). The potential for application of CGAs to promotegas mass transfer in bioremediation processes has beensuggested by Jauregi and Varley (1999) and Jacksonet al. (1998) demonstrated that CGAs performed betterthan surfactant alone in enhancing the transport ofbacteria through the soil matrix.

Colloidal gas aphrons also have biotechnologyapplications for recovery of cellular and molecularproducts and for enhancement of gas transfer in cellbioreactor and bioremediation processes (Jauregi andVarley, 1999). Air sparging of bulk fluids, containingcells or other biological material, is often implementedto promote separation of the desired components byfloatation. Surfactants are often added to air floatationsystems to promote product aggregation or precipitationand ‘precipitate floatation’. CGAs have been used tofurther improve conventional air floatation systems inthe 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 isvery limited and our insights into their mechanisms ofinteraction with cells and the potential applications inbiotechnology are even more limited. Much moreresearch is needed on the impact of surfactant type onthe properties of colloidal gas aphrons and on theirinteractions with cells before their potential applications

in biotechnology can be properly evaluated. Furthergeneral physico-chemical and then interdisciplinarybiological research on these aspects is needed to betteridentify and exploit biotechnology applications. Part ofthe research needs to address impact of surfactant typeon structure, properties, biological interactions andapplications of CGAs.

4.2. Surfactants in applied biocatalysis

Surfactant effects in modulating enzyme activity arebeing exploited in applied biocatalysis. When enzymesare sought for use in detergent washing products,researchers concentrate on finding enzymes whichretain their activity and are stable in the presence ofthe detergent. A detergent-modified lipase from Rhizo-pus delemar was prepared using didodecyl glucosylglutamate, which was stable in organic solvent to 50 °C,and exhibited high specificity towards longer chaintriglycerides (Okazaki et al., 1997a,b). Lipase fromCandida rugosa, coated with the surfactant, glutamicacid didodecyl ester ribitol amide, exhibited consider-able activity for the esterification of lauryl alcohol andlauric acid in organic solvents such as iso-octane,whereas the native powder lipase exhibited negligibleactivity (Wu et al., 2003). The surfactant-coated enzymewas most effective in mediating esterification reactionsinvolving fatty acids and fatty alcohols of medium chainlength. Substrate specificity of the coated enzyme wassimilar to that of the native enzyme. Km of the coatedlipase was only half that of the native lipase while Vmax

was 1.4 times higher. A surfactant-resistant lipaseshowed an increased capacity to launder lipid stainsfrom fabric (Aehle et al., 1995; Frenken et al., 1996).

While the conventional view has been that microbesgenerally thrive in aqueous media and that theirmetabolic machinery is better suited to transformingwater soluble substrates and metabolites the majority oforganic chemicals are not soluble in water. Recentresearch has shown that enzymes may be exploited tocarry out reactions involving hydrophobic (waterinsoluble) substrates in non-aqueous media or in two-phase aqueous-organic media. Surfactants may beexploited to pseudosolubilise these substrates and toimprove mass transfer and reaction rates.

4.2.1. Surfactants in mono-phasic organic solvent systemsLow concentrations of the nonionic surfactant, Thesit

(polyoxyethylene laurylether; C30 H62 O10), increasedthe activity of cholesterol oxidases from Streptomyceshygroscopicus (SCO) and Brevibacterium sterolicum(BCO) in aqueous media containing propanol as a

substrate solubilizer while at higher surfactant concen-trations the opposite effect occurs (Pollegioni et al.,1999). Triton X-100 (p-tertiary octylphenoxy polyethy-lalcohol) inactivated both enzymes even at lowconcentrations. The effects are considered to be relatedto surfactant–enzyme interaction rather than to micellarphenomena. BCO activity was rapidly inactivated,whereas SCO still had 70% of the initial activity after5 h in the presence of 30% propan-2-ol and hence, SCOwas concluded to be the superior catalyst for biotech-nology applications.

The oxidation of o-phenylenediamine was catalyzedin various anhydrous organic solvents by a surfactant-laccase complex, prepared by a novel preparationtechnique in water-in-oil (w/o) emulsions (Okazakiet al., 2000). In contrast, laccase, lyophilized from anaqueous buffer solution in which its activity wasoptimized, had no catalytic activity in non-aqueousmedia. A similar technique was used to prepare asurfactant-horseradish peroxidase (HRP) complex(Kamiya et al., 2000). The surfactant-HRP complex,prepared with a nonionic surfactant, exhibited a highcatalytic activity compared to those with a cationic oranionic surfactant in anhydrous benzene. The pH of theaqueous solution at the preparation step had a significanteffect on the enzymatic activity of the HRP complex inorganic media. Anionic ions present in the preparationprocess appeared to lower the catalytic activity throughcomplexation with heme iron.

Peptide synthesis from N-acetyl-L-phenylalanineethyl ester with alanineamide, catalyzed by a nonionicsurfactant complexed to various proteases, was carriedout in anhydrous hydrophilic organic solvents (Okazakiet al., 1997a,b). The surfactant-subtilisin Carlsberg(STC) complex exhibited a higher enzymatic activitythan the other protease-surfactant complexes and itsinitial reaction rate in tert-amyl alcohol was 26-fold thatof STC lyophilized from an optimum aqueous buffersolution. Water addition to the reaction mediumactivated the lyophilized STC, but the reaction rate wasmuch lower than that observed for the STC complex, andthe hydrolytic reaction preferentially proceeded. Hencethe surfactant-STC complex is a potential high utilitybiocatalyst for peptide synthesis, exhibiting high cata-lytic activity in anhydrous hydrophilic organic solvents.Since it does not need excess water undesirable sidehydrolysis reactions are not promoted.

In supercritical carbon dioxide reaction media, theα-chymotrypsin-surfactant complexes exhibited ahigher enzymatic activity than native α-chymotrypsin,for dipeptide synthesis (Mishima et al., 2003). Thenonionic surfactants, L-glutamic acid dialkyl ester

ribitol amide and sorbitan monostearate, were superiorsurfactants as compared with the anionic surfactant,sodium bis(2-ethylhexyl)sulfosuccinate. Increasing thepressure and temperature increased the maximumconversion and the enzymatic reaction rate in super-critical carbon dioxide. Maximum dipeptide synthesisby the surfactant-coated α-chymotrypsin occurred at awater content of 4%.

4.2.2. Surfactants in two-phase systemsIn 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 approachto implementing a range of enzymatic reactions inpredominantly non-aqueous environments. The enzymetypically is located in finely dispersed aqueous poolswhich are encapsulated by a surfactant within the non-polar solvent (Komives et al., 1994). Nanocapsulescontaining α-chymotrypsin in the inner aqueous cavitiescan act in both the organic solvent and the aqueousmedium (Shapiro and Pykhteeva, 1998). For suchencapsulation, the reversed hydrated micelles from N,N-diallyl-N,N-didodecyl ammonium bromide (DDAB)in cyclohexane (w0=22), including the enzyme, werepolymerized by UV initiation. The w0 is the ratio ofmolar concentration of water to that of the surfactant.The nanocapsules, precipitated with acetone, weresuspended in an aqueous medium with the aid of ionic,AOT, or nonionic, Brij-97, surfactants. The resultingunilamellar liposomes, having an inner monolayer fromthe poly-DDAB network, and the outer surfactant layerhad an average outer diameter of 20 nm. Encapsulationof α-chymotrypsin almost doubled the Km, with respectto ATEE (acetyl-L-tyrosine ethyl ester) hydrolysis,whereas the Vmax was almost halved. The characteristichigh thermostability of the encapsulated α -chymotrypsin(up to 80 °C) was attributed to the tendency for thepolymer network to block conformational transitions dueto heating in the enzyme molecule.

Because extreme halophilic enzymes have a high saltrequirement (about 4 MNaCl), their potential bioproces-sing application in organic solvents appeared to beunlikely. However, Marhuenda-Egea et al. (2002)observed that the halophilic enzyme, p-nitrophenylpho-sphate phosphatase from the archaeon Halobacteriumsalinarum, retained its catalytic properties in an organicmedium by creating a reverse micellar system with verylow salt concentration. The reverse micelles wereconstituted with an anionic surfactant AOT or with acationic surfactant CTAB in cyclohexane plus 1-butanolas co-surfactant. The enzymatic reaction appeared to

follow Michaelis-Menten kinetics only with respect tothe anionic surfactant.

The non-aqueous activity of the lipase from Mucorjavanicus was enhanced by using the anionic surfactantAOT to solubilise the enzyme in organic solvents(Altreuter et al., 2002). pH and ionic strength of theaqueous phase during solubilization had greatest impacton the extraction efficiency and specific activity of thebiocatalyst and solubility and activity response surfaceswere generated using esterification of octanoic acid with1-nonanol in isooctane as a model reaction. The resultswere shown to transfer to the acylation of doxorubicin(DOX), a potent anticancer compound, with 2-thio-phene acetic acid vinyl ester, or vinyl butyrate, intoluene. Chen et al. (2002) described the phenomenon ofprotein refolding/renaturation and interaction of ribo-nuclease A with AOT surfactant in reverse micelles.

Copolymerization of lignin with cresol can becatalyzed by peroxidase in reversed micellar systems(Liu et al., 1999). The surfactant concentration in thesystem can be manipulated to control the molecularweight of the copolymer.

Surfactant–enzyme complexes of different enzymesources have been used in transesterification processes(Okazaki et al., 1997a,b). The complexes formed bymodifying various proteases with surfactant moleculesutilizing water-in-oil (W/O) emulsions, were veryeffective in catalyzing vinyl butyrate transesterificationwith benzyl alcohol in organic media, whereas thenative proteases hardly catalyzed the above reaction.Performance of conventionally-prepared surfactant-coated protease exhibited significantly less biocatalyticactivity than the surfactant-protease complex preparedby the novel method. The method may be particularlyapplicable to enzymes that are sensitive to non-aqueousmedia and soluble in organic media.

Two membrane enzymes, fructose dehydrogenase(FDH, Gluconobacter sp.) and sarcosine dehydrogenase(SDH, Pseudomonas putida) were immobilized onto thebilayer membranes of stable vesicles (D=1–10 μm),prepared by an emulsification process involving use ofthe Span-80, Tween-80 and the lecithin (Kato et al.,2003). Enzyme activity and stability increased consid-erably as a result of immobilization.

4.2.3. Cells in microemulsionsAs has been described above for isolated enzymes,

various microorganisms (unicellular algae and cyanobac-teria) have also been dispersed inmicroemulsions withoutloss of biological activity. Each system required a definedquantity of water in the microemulsion for maximumactivity. Under optimum conditions, microbial enzymes

from the various cell sources (hydrogenases, dehydro-genases) exhibited a substantial increase in specificactivity and temperature optimum, as compared toaqueous solutions (Hopper et al., 1997). Solubilizationand growth ofCandida pseudotropicaliswas investigatedin a water-in-oil microemulsion consisting of hexadecanewith Tween 85/Span-80 (each 5%, wt/wt) as surfactantand a limited amount ofwater (up to 3%, v/v) (Pfammatteret al., 1992). The cells had a lower tendency to aggregatein the microemulsion environment and exhibited greatertime stability and a much smaller light scattering thanaqueous suspensions having the same cell concentration.A novel bioprocess using micelle biocatalysts was used toaddress disadvantages of conventional processes formicrobial desulfurization of coal (Lee and Yen, 1990).The multiphase system contained mineral oil or a mixturewith n-heptane, a surfactant and an aqueous phasecontaining Thiobacillus ferrooxidans organisms as wellas cell extracts. The reverse micelles and water-in-oilemulsion successfully removed sulfur from bituminouscoal. Cell-free enzyme extracts of T. ferrooxidans weresuperior to whole cells, and reverse micells were moreeffective than water-in-oil emulsion.

4.2.4. Surfactant–substrate interactionsWhile lignocellulose is a potential substrate for

ethanol production, high enzyme loadings are requiredto achieve high cellulose conversion rates which makethe process less economically feasible. Surfactantaddition, especially nonionic surfactants, to lignocellu-lose-enzyme reaction systems increases cellulose bio-conversion to sugars. Anionic and nonionic surfactantsreduced adsorption of the dominating cellulase of Tri-choderma reesei Cel7A (CBHI), during hydrolysis andthe improved conversion of lignocellulose with surfac-tant can be explained by the reduction of the unproduc-tive enzyme adsorption to lignin (Eriksson et al., 2002).

4.2.5. Concluding comments on surfactants in biocatalysisSurfactants may complex directly with enzymes in a

manner which alters enzyme tertiary structure, therebymodulating enzyme activity and/or specificity and/orstability and such positive modulations may beexploited. Surfactant–enzyme complexes can renderbiocatalysts resistant to denaturation in organic solventmedia, thereby facilitating catalysis of hydrophobic sub-strates dissolved in these media. Such systems canoperate where free water content is negligible which isparticularly useful in shifting the equilibrium ofhydrolase-mediated reactions in favour of synthesis.Some natural or mutated enzymes resist structural alter-ations in the presence of surfactants and these enzymes

are particularly suited to applications in detergents.Surfactants also act to promote micellarization in two-phase reaction media where typically the substrate andthe enzyme are compartmentalized in different phaseswith the reaction occurring at the interface. The enzymeused under these conditions is typically isolated but insome systems whole cells may also be employed.Finally, where there is a tendency for enzymes to sorbto insoluble material present in the reaction medium in amanner which reduces catalytic effectiveness, surfac-tants may be exploited to desorb the enzyme.

5. Industrial production of microbial biosurfactants

To date, industrial applications of biosurfactants havebeen limited due to their high costs of productionrelative to costs of chemical surfactants. For example,ethoxylate or alkyl polyglycoside synthetic surfactantshave an estimated cost of USD 1–3/kg. A wide varietyof microorganisms can synthesize biosurfactants using avariety of substrates such as sugars, oils, alkanes andagroindustrial wastes such as molasses and potatoprocessing wastes (Mulligan, 2005; Ron and Rosenberg,2002). For example lipopeptides are synthesized bymany Bacillus as well as other species. Glycolipids areproduced by Pseudomonas and Candida species whileThiobacillus thiooxidans produces phospholipids bio-surfactants. Polysaccharide–lipid complexes are syn-thesized by Acinetobacter species. Microbes oftensynthesize biosurfactants during growth on water-immiscible substrates, to facilitate uptake of thosesubstrates by the cell. However, the productivitiesexhibited by these strains are often not commerciallyviable (Van Dyke et al., 1991; Bodour and Miller-Maie,2002; Youssef et al., 2004).

The following fermentation process aspects ought tobe addressed with the objective of reducing biosurfac-tant production costs (Kosaric, 1992):

1. The producing strain should be carefully selected andstrategies should be employed to adapt or engineerthe strain to improve production.

2. The process should be engineered to minimizecapital and operating costs.

3. Process feedstock should be selected and adapted tominimize raw material costs.

4. Attention should be paid to minimizing process by-products and/or to use them as saleable productsrather than wastes.

It should be noted that these process aspects are nodifferent to those encountered with other fermentation

processes (Ward, 1989). In addition to fermentation-related costs, product recovery costs must be minimized(Ward, 1991). A typical recovery process for industrialextracellular microbial polymers would be limited to acentrifugation cell separation step followed by asupernatant (containing the biosurfactant) concentrationand/or drying step. Most likely unit processes forconcentration/drying would involve membrane filtra-tion, evaporation and/or heat mediated drying. Aconcentrated liquid product represents the most costeffective product form.

From a combined application/cost perspective rham-nolipid, produced byP. aeruginosa, represents the leadingcommercial microbial biosurfactant and hence this briefdiscourse on industrial biosurfactant production will beconfined to this product/host system. Extensive investiga-tions have been implemented at both the molecular andcell culture level aimed at understanding factors influ-encing rhamnolipid biosurfactant biosynthesis byP. aeruginosa with a view to optimising the fermentationprocess. Rhamnolipid production is dependant on thecentral metabolic pathways for fatty acid synthesis and fordTDP (thiamine diphosphate)-activated sugar formationand on enzymes which participate in the production of theexopolysaccharide alginate (Maier and Soberon-Chavez,2000). Biosynthesis is regulated by a complex geneticregulatory system which is also involved in the control ofvirulence-associated characteristics. Product transcriptionis co-ordinately regulated by at least two quorum sensingmechanisms (Fuqua and Greenberg, 1998). Supplemen-tation of the fermentation medium with vegetable oilssubstantially enhances biosurfactant yields and mediumcosts can be reduced by incorporating agroindustrialwastes into fermentation media (Banat, 1995a,b; Simet al., 1997). The product can now be produced, on anindustrial scale, to a concentration of 100 g/L at anestimated cost in the range of USD5-20/kg in fermentershaving capacities in the range 100M3–200M3 (Matsufujiet al., 1997; Lang and Wullbrandt, 1999).

As additional yield improvements are always en-countered over time through production experience theestimated costs are in general approaching costs whichare competitive with chemical surfactants. The continu-ing developments in molecular and cellular biology areincreasing our understanding of the genetics, biochem-istry and physiology underlying microbial productionprocesses and are providing us with additional tools toincrease product yields. In addition, consumers aretypically prepared to pay a premium where use ofbiosurfactants in place of chemical surfactants has aperceived advantage. It is our belief that these variousaspects will lead to more cost effective microbial

production processes and a substantial expansion ofthe overall use of biosurfactants.

6. Enzymatic synthesis of chemical surfactants

Many chemical surfactant molecules contain ester oramide linkages and are amenable to chemoenzymaticsynthesis using microbial and other lipases or proteases.These enzymes typically act in nature as biodegradativehydrolases but can be exploited in bioorganic synthesisreactions by implementing the biocatalysis in low waterenvironments which shift reaction equilibria to favoursynthesis rather than hydrolysis.

There are many commercial processes which exploithydrolases in organic solvent media for bioorganicsynthesis and some of the processes benefit from theavailability at low cost of bulk industrial hydrolyticenzymes such as proteases and lipases. Semi-syntheticpenicillins may be produced in this way from 6-aminopenicillanic acid and the appropriate acyl sidechain and an enzymatic method for conversion ofporcine to human insulin utilizes a lysine-specificprotease in organic solvent media as a step in thestrategy of replacing the terminal alanine found inporcine insulin with the threonine found in humaninsulin (Ward, 1991). An aqueous proteolytic step is firstused to remove the alanine moiety. Avariety of strategiesare available for using lipases in esterification, inter-esterification and transesterification reactions (Ward andSingh, 2005; Muralidhar et al., 2001). An interestingexample in food processing involves use of lipase ininteresterification reactions to produce high value cocoabutter substitutes from cheap palm oil (Ward, 1991).

These and other biotransformation strategies typical-ly exploit the stereo- and region-specific properties ofenzymes and their abilities to mediate reactions at non-extreme conditions with respect to pH and temperature.Hence labile substrates may be used without risk ofsubstrate or product physico-chemical denaturation andenzymes reactions may be designed to target a specificgroup on the substrate while leaving other reactivegroups unmodified, such that chemical blocking of thelatter groups is not required.

Boyat et al. (2000) described methods for usinglipases and proteases to prepare new nonionic surfac-tants from unprotected carbohydrates, amino acids, andfatty alcohols by implementing the esterification andtransesterification reactions in organic media. Dossatet al. (2002) described a continuous solvent free systemmediated by the immobilized lipase, Lipozyme, fortransesterification of sunflower oil with butan-1-ol tobutyl ester having low levels of residual mono-, di- and

triglycerides having interesting surfactant and lubricantproperties. The same immobilized enzyme was usedto transform various palm oil glyceride and fatty acidfractions with amino acids into mixed medium- to long-chained surfactants for specific applications (Soo et al.,2003). Fernandez-Perez and Otero (2003) describedmethods for lipase (Novozym 435)-mediated condensa-tions of amines (ethanolamine, diethanolamine) withfatty acides to form various amid and ester surfactants indifferent hydrophobic solvents. Product compositionand yield may be varied by manipulation of solvent andother conditions.

Papain, besides its well known protease activity, alsoexhibits esterase activity the specificity of which has beencharacterized byVilleneuve et al. (1995). Papainwas usedto mediate the formation of amide and ester bonds in thesynthesis of surfactants consisting of arginine N-alkylamide and ester derivatives (Clapes et al., 1999). Thesystem was exploited for synthesis of arginine-basedGemini surfactants, consisting of two single N-alpha-acyl-arginine structures linked through the α-carboxylicgroups of the aminoacid residues by an alpha, omega-diaminoalkane spacer chain (Piera et al., 2000).

Lipase B from Candida antarctica catalysed regio-selective acylation of β- D(+)glucose and variousglucosides at the primary hydroxyl group of the sugarmoiety with a variety of non-activated arylaliphaticcarboxylic acids to produce esters having high surfaceactivity and high water solubility for possible use aswetting agents and water-in-oil emulsions (Otto et al.,1998). Similarly, solvent free media with lipase wereused to produce surfactant-like sugar-esters containingomega-3 and omega-6 polyunsaturated fatty acids (Liand Ward, 1996; Ward et al., 1997).

The technical feasibility of using enzymes asbiocatalysts in synthesis of certain complex organicmolecules is well established. These methods have beenmost widely applied in synthesis of higher valueproducts such as food additives and pharmaceuticals.From an economic perspective this approach is unlikelyto be viable for production of low cost surfactants withenvironmental/industrial applications but may be anoption for synthesis of high value surfactant propertiesfor agricultural, food and medical applications.

7. Conclusions

Applications of chemical surfactants in desorption ofhydrophobic contaminants from soil and subsequentbiodegradation has been widely studied. The use ofbiosurfactants in remediation of contaminated sites alsohas many advantages. The biosurfactants seem to

enhance biodegradation by influencing the bioavailabil-ity of the contaminant (Van Dyke et al., 1991; Zhaoet al., 2005). Due to their biodegradability and lowtoxicity they are very promising for use in remediationtechnologies. However, more information is needed onstructure of biosurfactants, their interaction with soil andcontaminants and scale up and cost for biosurfactantproduction (Fraser, 2000). The perceived unfavorablecosts of production of biosurfactants are beingaddressed as microbial hosts and fermentation processesare developed to produce higher yields. For theproduction of biosurfactants to be commercially viable,further process optimization at the biological andengineering level are required (Sullivan, 1998; Niheiet al., 2004). Moreover, cost of downstream processingfor the recovery of any fermentation product aretypically high and these need to be minimized throughprocess development initiatives to obtain cost effectivebiosurfactant products.

For medical applications, biosurfactants are useful asantibacterial, antifungal and antiviral agents. In addition,they also have potential for use as major immunomod-ulatory molecules, adhesive agents and even in vaccinesand gene therapy. Most of the biosurfactants used inbiological applications are required in very lowconcentrations that make biosurfactants valuable bio-molecules for applications as food additives, specialtychemicals, biocontrol agents, and new generationmolecules for health and beauty care industries.

References

Adriano DC, Wenzel WW, Vangronsveld J, Bolan NS. Role of assistednatural remediation in environmental cleanup. Geoderma2005;122:121–42.

Aehle W, Gerritse G, Lenting HBM. Lipase with improved surfactantresistance. WO 95/30744; 1995.

Al-Maghrabi IMA, Bin Aqil AO, Isla MR, Chaalal O. Use ofthermophilic bacteria for bioremediation of petroleum contami-nants. Energy Sources 1999;21:17–29.

Almeida PF, Moreira RF, Almeida RCC, Guimaraes AK, CarvalhoAS, Quintella C, et al. Selection and improvement of microorgan-isms to improve oil recovery. Eng Life Sci 2004;4:319–25.

Al-Sabagh AM. Surface activity and thermodynamic properties of water-soluble polyester surfactants based on 1,3-dicarboxymethoxyben-zene used for enhanced oil recovery. Polym Adv Technol 2000;11:48–56.

Altreuter DH, Dordick JS, Clark DS. Optimization of ion-paired lipase fornon-aqueousmedia: acylation of doxorubicin based on surfacemodelsof fatty acid esterification. Enzyme Microb Technol 2002;31:10–9.

Awasthi N, Kumar A, Makkar R, Cameotra S. Enhanced biodegra-dation of endosulfan, a chlorinated pesticide in presence of abiosurfactant. J Environ Sci Health 1999;B34:793–803.

Bai G, Brusseau ML, Miller RM. Influence of cation type, ionic strengthand pH on solubilization and mobilization of residual hydrocarbonby a biosurfactant. J Contam Hydrol 1998;30:265–79.

Banat IM. Biosurfactant production and possible uses in microbialenhanced oil recovery and oil pollution remediation. BioresTechnol 1995a;51:1-12.

Banat IM. Characterization of biosurfactants and their use in pollutionremoval — state of the Art. Acta Biotechnol 1995b;15:251–67.

Banat IM, Samarah N, Murad M, Horne R, Banerjee S. Biosurfactantproduction and use in oil tank clean-up. World J MicrobiolBiotechnol 1991;7:80–8.

Banat IM, Makkar RS, Cameotra SS. Potential commercial applications ofmicrobial surfactants. Appl Microbiol Biotechnol 2000;53:495–508.

Bansal-Mutalik R, Gaikar VG. Cell permeabilization for extraction ofpenicillin acylase from Escherichia coli by reverse micellarsolutions. Enzyme Microb Technol 2003;32:14–26.

Bardi L,Mattei A, Steffan S,MarzonaM. Hydrocarbon degradation by asoil microbial population with beta-cyclodextrin as surfactant toenhance bioavailability. Enzyme Microb Technol 2000;27:709–13.

Beal R, Betts WB. Role of rhamnolipid biosurfactants in the uptakeand mineralization of hexadecane in Pseudomonas aeruginosa.J Appl Microbiol 2000;89:158–68.

Billingsley KA, Backus SM, Ward OP. Effect of surfactantsolubilization on biodegradation of polychlorinated biphenylcongeners by Pseudomonas LB400. Appl Microbiol Biotechnol1999a;52:255–60.

Billingsley KA, Backus SM, Wilson S, Singh A, Ward OP. Remediationof PCBs in soil by surfactant washing and biodegradation in thewashby Pseudomonas sp. LB400. Biotechnol Lett 1999b;24:1827–32.

Billingsley KA, Backus SM, Wilson S, Ward OP. Remediation ofPCBs in soil by surfactant washing and biodegradation in the washby Pseudomonas sp. LB400. Biotechnol Lett 2002;24:1827–32.

Bodour A, Miller-Maie RM. Biosurfactants: types, screening methods,and applications. Encyclopedia of environmental microbiology.NY: Wiley; 2002. p. 750–70.

Boyat C, Rolland-Fulcrand V, Roumestant ML, Viallefont P, Martinez J.Chemo-enzymatic synthesis of new non ionic surfactants fromunprotected carbohydrates. Prep Biochem Biotechnol 2000;30:281–94.

Boyette CD, Walker HL, Abbas HK. Biological control of kudzu(Pueraria lobata); with an isolate of Myrothecium verrucaria.Biocontrol Sci Technol 2002;12:75–82.

Bramwell DP, Laha S. Effects of surfactant addition on thebiomineralization and microbial toxicity of phenanthrene. Biode-grad 2000;11:263–77.

Bredwell MD, Worden RM. Mass-transfer properties of microbubbles.1. Experimental studies. Biotechnol Prog 1998;14:31–8.

Burd G, Ward OP. Bacterial degradation of polycyclic aromatichydrocarbons on agar plates: the role of biosurfactants. BiotechnolTech 1996;10:371–4.

CairnsWL,CooperDG, Zajic JE,Wood JM,KosaricN. CharacterizationofNocardia amarae as a potent biological coalescing agent of water-in-oil emulsions. Appl Environ Microbiol 1982;43:362–6.

Cameotra SS, Bollag J-M. Biosurfactant-enhanced bioremediation ofpolycyclic aromatic hydrocarbons. Crit Rev Environ Sci Technol2003;30:111–26.

Cameotra SS, Makkar RS. Recent applications of biosurfactants asbiological and immunological molecules. Curr Opin Microbiol2004;7:262–6.

Chaphalkar PG, Valsaraj KT, Roy D. A study of the size distributionand stability of colloidal gas aphrons using a particle size analyzer.Sep Sci Technol 1993;28:1287–302.

Charnay M, Tarabelli L, Beigel C, Barriuso E. Modifications of soilmicrobial activity and triticonazole biodegradation by pesticideformulation additives. J Environ Qual 2000;29:1618–24.

Chen P, Pickard MA, Gray MR. Surfactant inhibition of bacterialgrowth on solid anthracene. Biodegradation 2000;11:341–7.

Chen Y, Xu Q, Yang H, Gu G. Effects of cell fermentation time andbiomass drying strategies on the recovery of poly-3-hydroxyalk-anoates from Alcaligenes eutrophus using a surfactant-chelateaqueous system. Proc Biochem 2001;36:773–9.

Chen W, Lee Y, Lin S, Ho C. Renaturation and interaction ofribonuclease A with AOT surfactant in reverse micelles. Biotech-nol Prog 2002;18:1443–6.

Chhatre S, Purohit H, Shanker R, Khanna P. Bacterial consortia forcrude oil spill remediation. Water Sci Technol 1996;34:187–94.

Chu W. Remediation of contaminated soils by surfactant-aided soilwashing. Pract Period Hazard Toxic Radioact Waste Manage2003;7:19–24.

Clapes P, Moran C, Infante MR. Enzymatic synthesis of arginine-based cationic surfactants. Biotechnol Bioeng 1999;63:333–43.

Colores GM,Macur RE,Ward DM, InskeepWP.Molecular analysis ofsurfactant-driven microbial population shifts in hydrocarbon-contaminated soil. Appl Environ Microbiol 2000;66:2959–64.

Cooper DG, Paddock DA. Production of a biosurfactant from Toru-lopsis bombicola. Appl Environ Microbiol 1984;47:173–6.

Dahr Azma B, Mulligan CN. Extraction of copper from miningresidues by rhamnolipids. Prac Per Haz Tox Radioact WasteManag 2004;8:166–72.

Das M. Characterization of de-emulsification capabilities of aMicrococcus sp. Biores Technol 2001;79:15–22.

Dean SM, Jin Y, Cha DK, Wilson SV, Radosevich M. Phenanthrenedegradation in soils co-inoculated with phenanthrene-degradingand biosurfactant-producing bacteria. J Environ Qual 2001;30:1126–33.

Desai JD, Banat IM. Microbial production of surfactants and theircommercial potential. Microbiol Mol Biol Rev 1997;61:47–64.

Dossat V, Combes D, Marty A. Efficient lipase catalysed production ofa lubricant and surfactant formulation using a continuous solvent-free process. J Biotechnol 2002;97:117–24.

Eriksson T, Boerjesson J, Tjerneld F. Mechanism of surfactant effect inenzymatic hydrolysis of lignocellulose. Enzyme Microb Technol2002;31:353–64.

Ferrer M, Golyshin P, Timmis KN. Novel maltotriose esters enhancebiodegradation of Aroclor 1242 by Burkholderia cepacia LB400.World J Microbiol Biotechnol 2003;19:637–43.

Fernandez-Perez M, Otero C. Selective enzymatic synthesis of amidesurfactants from diethanolamine. Enzyme Microb Technol2003;33:650–60.

Fraser L. Innovations: lipid lather removes metals. Environ HealthPerspect 2000;108:320.

Frenken LG, Peters H, Suerbaum HMU, DeVlieg J, Verrips CT.Modified Pseudomonas lipases and their use. WO 96/00292; 1996.

Fuqua WC, Greenberg EP. Self perception in bacteria: molecularmechanisms of stimulus-response coupling. Curr Opin Microbiol1998;1:183–9.

Garcia-Junco M, Gomez-Lahoz C, Niqui-Arroyo J-L, Ortego-CalvoJ-J. Biosurfactant- and biodegradation-enhanced partitioning ofpolycyclic aromatic hydrocarbons from nonaqueous- phaseliquids. Environ Sci Technol 2003;37:2988–96.

Golyshin PM, Fredrickson HL, Giuliano L, Rothmel R, Timmis KN,Yakimov MM. Effect of novel biosurfactants on biodegradation ofpolychlorinated biphenyls by pure and mixed bacterial cultures.Microbiologica 1999;22:257–67.

Grace R. Commercial emulsion breaking. In: Schramm LL, editor.Emulsions: fundamentals and applications in the petroleum industry.Washington, DC: American Chemical Society; 1992. p. 313–39.

Gronwald JW, Plaisance KL, Ide DA, Wyse DL. Assessment ofPseudomonas syringae pv. tagetis as a biocontrol agent for Canadathistle. Weed Sci 2002;50:397–404.

Han DK, Yang C-Z, Zhang Z-Q, Zhu-Hong Lou Z-H, Chang Y-I.Recent development of enhanced oil recovery in China. J PetrolSci Eng 1999;22:181–8.

Hashim MA, Sengupta B, Kumar SV. Clarification of yeast bycolloidal gas aphrons. Biotechnol Tech 1995a;9:403–8.

Hashim MA, Sengupta B, Subramanian MB. Investigations on thefloatation of yeast cells by colloidal gas aphron (CGA) dispersions.Bioseparation 1995b;5:167–73.

Hayes ME, Nestaas E, Hrebenar KR. Microbial surfactants. Chemtech1986;4:239–43.

He Z, She Y, Xiang T, Xue F, Mei B, Li Y, et al. MEOR pilot seesencouraging results in Chinese oil field. Oil Gas J 2000;98:4.

Holakoo L, Mulligan CN. On the capability of rhamnolipids for oilspill control of surface water. Proc Ann Confe Can Soc Civil EngJune 5–8, Montreal; 2002.

Hopper M, Mlejnek K, Seiffert B, Mayer F. Activities of microorgan-isms and enzymes in water-restricted environments: biologicalactivities in aqueous compartments at μm-scale. SPIE Proceed1997;3111:501–9.

Jack TR, Thompson BG. Patents employing microorganisms in oilproduction. In: Zajic JE, Cooper DG, Jack TR, Kosaric N, editors.Microbial enhanced oil recovery. Tulsa: PennWell Publishing Co;1983. p. 14–25.

Jackson A, Kommalapati R, Roy D, Pardue J. Enhanced transport ofbacteria through a soil matrix using colloidal gas aphronsuspensions. J Environ Sci Health Part A 1998;33:369–84.

Janiyani KL, Purohit HJ, Shanker R, Khanna P. De-emulsification ofoil-in-water emulsions by Bacillus subtilis. World J MicrobiolBiotechnol 1994;10:452–6.

Jauregi P, Varley J. Colloidal gas aphrons: potential applications inbiotechnology. Trends Biotechnol 1999;17:389–95.

Jauregi P, Gilmour S, Varley J. Characterisation of colloidal gas aphronsfor subsequent use for protein recovery. Chem Eng J 1997;65:1-11.

Javert CT, Heath JK. Sediment and saturate-soil-associated reactionsinvolving an anionic surfactant (dodecyl sulfate). Environ SciTechnol 1991;25:1031–8.

Jazzar C, Hammad EA. The efficacy of enhanced aqueous extracts ofmelia azedarach leaves and fruits integrated with the camptotylusreuteri releases against the sweetpotato whitefly nymphs. BullInsectol 2003;56:269–75.

JennemanGE,McInerneyMJ, Knapp RM. Effect of nitrate on biogeneicsulfide production. Appl Environ Microbiol 1986;51:1205–11.

Kamiya N, Inoue M, Goto M, Nakamura N, Naruta Y. Catalytic andstructural properties of surfactant-horseradish peroxidase complexin organic media. Biotechnol Prog 2000;16:52–8.

Kaster JA, Michelsen DL, Velander WH. Increased oxygen-transfer ina yeast fermentation using a microbubble dispersion. ApplBiochem Biotechnol 1990;24:469–84.

Kato K, Walde P, Mitsui H, Higashi N. Enzymatic activity and stabilityof D-fructose dehydrogenase and sarcosine dehydrogenase immo-bilizd onto giant vesicles. Biotechnol Bioeng 2003;84:415–23.

Kearns DB, Losick R. Swarming motility in undomesticated Bacillussubtilis. Mol Microbiol 2003;49:581–90.

Khire JM, Khan MI. Microbially enhanced oil recovery (MEOR). Part1. Importance and mechanisms of MEOR. Enzyme MicrobTechnol 1994;16:170–2.

Kim PI, Bai H, Bai D, Chae H, Chung S, Kim Y, et al. Purification andcharacterization of a lipopeptide produced by Bacillus thuringiensisCMB26. J Appl Microbiol 2004;97:942–9.

Komives CF, Osborne DE, Russel AJ. Characterization of a nonionicsurfactant reversed micellar system for enzyme catalysis. J PhysChem 1994;98:369–76.

Kommalapati RR, Roy D, Valsaraj KT, Constant WD. Characterizationof colloidal gas aphron suspensions generated from plant-basednatural surfactant solutions. Sep Sci Technol 1996;31:2317–33.

Kosaric N. Biosurfactants in industry. PureAppl Chem 1992;64:1731–7.Kosaric N, Cairns WL, Gray NCC. Microbial de-emulsifiers. In:

Kosaric N, Cairns WL, Gray NCC, editors. Biosurfactants andbiotechnology. New York: Marcel Dekker; 1987. p. 247–320.

Krishnayya PV, Grewal PS. Effect of neem and selected fungicides onviability and virulence of the entomopathogenic nematodesteinernema feltiae. Biocontrol Sci Technol 2002;12:259–66.

Lang S, Wullbrandt D. Rhamnose lipids — biosynthesis, microbialproduction and application potential. Appl Microbiol Biotechnol1999;51:22–32.

Lee RF. Agents which promote and stabilize water-in-oil emulsions.Spill Sci Technol Bull 1999;5:117–26.

Lee K, Yen TF. Sulfur removal from coal through multiphasemedia containing biocatalysts. J Chem Technol Biotechnol1990;48:71–9.

Lee SS, Kim HS, Moon YH, Choi NJ, Ha JK. The effects of a non-ionic surfactant on the fermentation characteristics, microbialgrowth, enzyme activity and digestibility in the rumen of cows.Anim Feed Sci Technol 2004;115:37–50.

Lee M, Kang H, DoW. Application of nonionic surfactant-enhanced insitu flushing to a diesel contaminated site. Water Res 2005;39:139–46.

Li Z-Y, Ward OP. Enzymatically solvent-free synthesis of sugar estercontaining ω-3 polyunsaturated fatty acids. Chin Chem Lett1996;7:611–4.

Li X, Wang J. Effects of mixed anionic–cationic surfactants andalcohol on solubilization of water-in-oil microemulsions. J DispersSci Technol 1999;20:993-1007.

Li, N.N., Bucal, T., Cahn, R.P., Demulsification Process. US PatentNo. 4,001,109; 1977.

Lin S-C, Carswell KS, Sharma MM, Georgiou G. Continuousproduction of the lipopeptide biosurfactant of Bacillus lichenifor-mis JF-2. Appl Microbiol Biotechnol 1994;41:281–5.

Liu J, Weiping Y, Lo T. Copolymerization of lignin with cresolcatalyzed by peroxidase in reversed micellar systems. Electron JBiotechnol 1999;2:120–5.

Liu Z, Jacobson AM, Luthy RG. Biodegradation of naphthalene inaqueous nonionic surfactant systems. Appl Environ Microbiol1995;61:145–51.

Liu Q, Dong M, Zhoua W, Ayub M, Zhang YP, Huang S. Improved oilrecovery by adsorption–desorption in chemical flooding. J PetrolSci Eng 2004;43:75–86.

Maier RM, Soberon-Chavez G. Pseudomonas aeruginosa rhamnoli-pids: biosynthesis and potential applications. Appl MicrobiolBiotechnol 2000;54:625–33.

Makkar RS, Cameotra SS. Utilization of molasses for biosurfactantproduction by two Bacillus strains at thermophilic conditions.J Am Oil Chem Soc 1997;74:887–9.

Makkar RS, Cameotra SS. Production of biosurfactant at mesophilicand thermophilic conditions by a strain of Bacillus subtilis. J IndMicrobiol Biotechnol 1998;20:48–52.

Manning FC, Thompson RE. Oilfield processing. Crude oil, vol. 2.Tulsa, Oklahoma: PennWell; 1995.

Marhuenda-Egea FC, Piera-Velazquez S, Cadenas C, Cadenas E. Anextreme halophilic enzyme active at low salt in reversed micelles.J Biotechnol 2002;93:159–64.

Mata-Sandoval JC, Karns J, Torrents A. Influence of rhamnolipids andTriton X-100 on the biodegradation of three pesticides in aqueousand soil slurries. J Agric Food Chem 2001;49:3296–303.

Matosic S, Mehak M, Ercegovic L, Brajkovic N, Suskovic J. Effect ofsurfactants on the production of ergot-alkaloids by immobilizedmycelia of Claviceps paspali. World J Microbiol Biotechnol1998;14:447–50.

Matsufuji M, Nakata K, Yoshimoto A. High production ofrhamnolipids by Pseudomonas aeruginosa growing on ethanol.Biotechnol Lett 1997;19:1213–5.

Matsushita K, Mollah H, Stuckey DC, Delcerro C, Bailey AI.Predispersed solvent-extraction of dilute products using colloidalliquid aphrons — aphron preparation, stability and size. ColloidsSurf 1992;69:65–72.

Mishima K, Matsuyama K, Baba M, Chidori M. Enzymatic dipeptidesynthesis by surfactant-coated alpha -chymotrypsin complexes insupercritical carbon dioxide. Biotechnol Prog 2003;19:281–4.

Mohammed RA, Baily AI, Luckham PF, Taylor SE. Dewatering ofcrude oil emulsions 3. Emulsion resolution by chemical means.Colloids Surf, A Physicochem Eng Asp 1994;83:261–71.

Mulligan CN. Environmental applications of biosurfactants. EnvironPollut 2005;133:183–98.

Mulligan CN, Eftekhari F. Remediation with surfactant foam of PCPcontaminated soil. Eng Geol 2003;70:269–79.

Mulligan CN, Wang S. Remediation of a heavy metal contaminatedsoil by a rhamnolipid foam. In: Yangt RN, Thomas HR, editors.Geoenvironmental engineering. Integrated management ofgroundwater and contaminated land. London: Thomas Telford;2004. p. 544–51.

Mulligan CN, Yong RN, Gibbs BF. Metal removal from contaminatedsoil and sediments by the biosurfactant surfactin. Environ SciTechnol 1999;33:3812–20.

Mulligan CN, Yong RN, Gibbs BF. Heavy metal removal fromsediments by biosurfactants. J Hazard Mater 2001;85:111–25.

Muralidhar RV, Chirumamilla RR, Marchant R, Ramachandran VN,Ward OP, Nigam P. Understanding lipase stereoselectivity. WorldJ Microbiol Biotechnol 2001;18:81–97.

Nadarajah N, Singh A, Ward OP. De-emulsification of petroleum oilemulsion by a mixed bacterial culture. Proc Biochem 2001;37:1135–41.

Nadarajah N, Singh A, Ward OP. Evaluation of a mixed bacterialculture for de-emulsification of water-in-oil petroleum oil emul-sions. World J Microbiol Biotechnol 2002;18:435–40.

Nandakumar R, Gounot AM, Mattiasson B. Gentle lysis of mucousproducing cold-adapted bacteria by surfactant treatment combinedwith mechanical disruption. J Biotechnol 2000;83:211–7.

Nasr-El-Din HA, Taylor KC. Dynamic interfacial tension of crude oil/alkali/surfactant, systems. Colloids Surf 1992;66:23–37.

Nazina TN, Sokolova DS, Grigor'yan AA, Xue Y-F, Belyaev SS, IvanovMV. Production of oil-releasing compounds bymicroorganisms fromthe Daqing oilfield, China. Mikrobiologiya 2003;72:206–11.

Ng WL, Rana D, Neale GH, Hornof V. Physico-chemical behavior ofmixed surfactant systems: petroleum sulfonate and lignosulfonate.J Appl Polym Sci 2002;88:860–5.

Neilson JW, Artiola JF, Maier RM. Characterization of lead removalfrom contaminated soils by non-toxic soil-washing agents. JEnviron Qual 2003;32:899–908.

Nihei K-I, Nihei A, Kubo I. Molecular design of multifunctional foodadditives: antioxidative antifungal agents. J Agric Food Chem2004;52:5011–20.

Noordman WH, Wachter JJJ, de Boer GJ, Janssen DB. Theenhancement by biosurfactants of hexadecane degradation by

Pseudomonas aeruginosa varies with substrate availability.J Biotechnol 2002;94:195–212.

Okazaki S, Kamiya N, Goto M. Application of novel preparationmethod for surfactant-protease complexes catalytically active inorganic media. Biotechnol Prog 1997a;13:551–6.

Okazaki S, Kamiya N, Goto M, Nakashio F. Enantioselectiveesterification of glycidol by surfactant-lipase complexes in organicmedia. Biotechnol Lett 1997b;19:541–3.

Okazaki S, Goto M, Furusaki S. Surfactant-protease complex as anovel biocatalyst for peptide synthesis in hydrophilic organicsolvents. Enzyme Microb Technol 2000;26:159–64.

Otto RT, Bornscheuer UT, Syldatk C, Schmid RD. Lipase-catalyzedsynthesis of arylaliphatic esters of beta-D(+)-glucose, n-alkyl- andarylglucosides and characterization of their surfactant properties.J Biotechnol 1998;64:231–7.

PekdemirT,CopurM,UrumK.Emulsification of crude oil–water systemsusing biosurfactants. Process Saf Environ Prot 2005;83(B1):38–46.

Pellerin NB, Staley JT, Ren T, Graf GL, Treadwell DR, Aksay IA.Acidic biopolymers as dispersants for ceramic processing. MaterRes Soc Symp 1992;218:123–8.

Pfammatter N, Hochkoeppler A, Luisi PL. Solubilization and growthof Candida pseudotropicalis in water-in-oil microemulsions.Biotechnol Bioeng 1992;40:167–72.

Pfirter HA, Defago G. The potential of Stagonospora sp. as amycoherbicide for field bindweed. Biocon Sci Technol 1998;8:93-101.

Piera E, Infante MR, Clapes P. Chemo-enzymatic synthesis of arginine-based gemini surfactants. Biotechnol Bioeng 2000;70:323–31.

Poggi-Varaldo HM, Rinderknecht-Seijas N. A differential availabilityenhancement factor for the evaluation of pollutant availability insoil treatments. Acta Biotechnol 2003;23:271–80.

Pollegioni L, Gadda G, Ambrosius D, Ghisla S, Pilone MS.Cholesterol oxidase from Streptomyces hygroscopicus and Brevi-bacterium sterolicum: effect of surfactants and organic solvents onactivity. Biotechnol Appl Biochem 1999;30:27–33.

Polman JK, Miller KS, Stoner DL, Brakenridge CR. Solubilization ofbituminous and lignite coals by chemically and biologicallysynthesized surfactants. J ChemTechnol Biotechnol 1994;61:11–7.

Premuzic, E.T., Lin, M., Process for producing modified microorgan-isms for oil treatment at high temperatures, pressures and salinity.US Patent 5,492,828; 1996.

Prince RC. Bioremediation of marine oil spills. Trends Biotech1997;15:158–60. Pritchard PH, Mueller JG, Rogers JC, KremerFV, Glaser JA. Oil spill bioremediation: experiences, lessons andresults from the Exxon Valdez oil spill in Alaska. Biodegradation1997;3:315–35.

Rahman KSM, Rahman TJ, Kourkoutoas Y, Petsas I, Marchant R,Banat IM. Enhanced bioremediation of n-alkane in petroleumsludge using bacterial consortium amended with rhamnolipids andmicronutrients. Biores Technol 2003;90:159–68.

Rapp P, Bock H, Wray V, Wagner F. Formation, isolation andcharacterization of trehalose dimycolates from Rhodococcus ery-thropolis grown on n-alkanes. J Gen Microbiol 1979;115:491–503.

Reese ET, Maguire A. Surfactants as stimulants of enzyme productionby microorganisms. Appl Microbiol 1969;17:242–9. Ron EZ,Rosenberg E. Biosurfactants and oil bioremediation. Curr OpinBiotechnol 1969;13:249–52.

Rosenberg E, Ron EZ. Surface active polymers from the genus Aci-netobacter. In: Kaplan DL, editor. Biopolymers from renewableresources. Berlin: Springer; 1998. p. 281–91.

Rouse JD, Sabatini DA, Suflita JM, Harwell JH. Influence ofsurfactants on microbial degradation of organic compounds. CritRev Microbiol 1994;24:325–70.

Rouse JD, Bjornen KK, Taylor RW, Shiau B-J. Surfactant-basedtechnologies applicable to remediation of mercury pollution in thesubsurface. Environ Pract 2004;6:157–64.

Saichek RE, Reddy KR. Electrokinetically enhanced remediation ofhydrophobic organic compounds in soils: a review. Crit RevEnviron Sci Technol 2005;35:115–92.

Save SV, Pangarkar VG. Characterization of colloidal gas aphrons.Chem Eng Commun 1994;127:35–54.

Save SV, Pangarkar VG. Harvesting of Saccharomyces cerevisiaeusing colloidal gas aphrons. J Chem Technol Biotechnol 1995;62(170):192–9.

Save SV, Pangarkar VG, Kumar SV. Intensification of mass-transfer inaqueous 2-phase systems. Biotechnol Bioeng 1993;41:72–8.

Schallmey M, Singh A, Ward OP. Developments in the use of Ba-cillus species in industrial production. Can J Microbiol 2004;50:1-17.

Schippers C, Geßner K, Muller T, Scheper T. Microbial degradation ofphenanthrene by addition of a sophorolipid mixture. J. Biotechnol2000;83:89-198.

Shapiro YE, Pykhteeva EG. Immobilization of alpha-chymotrypsin intothe poly(N,N-diallyl-N,N-didodecyl ammonium bromide)/surfactantnanocapsules. Appl Biochem Biotechnol 1998;74:67–84.

Shennan JL, Levi JD. In situ microbial enhanced oil recovery. In:Kosaric N, Cairns WL, Gray NCC, editors. Biosurfactants andbiotechnology. New York: Marcel Dekker; 1987. p. 163–80.

Shi Z, LaTorre KA, GhoshMM, Layton AC, Luna SH, Bowles L, et al.Biodegradation of UV-irradiated polychlorinated biphenyls insurfactant micelles. Water Sci Technol 1998;38:25–32.

Shulga A, Karpenko E, Vildanova-Martishin R, Turovsky A, SoltysM. Biosurfactant-enhanced remediation of oil contaminatedenvironments. Adsorp Sci Technol 2000;18:171–6.

Shin KH, Ahn Y, Kim KW. Toxic effect of biosurfactant addition onthe biodegradation of phenanthrene. Environ Toxicol Chem2005;24:2768–74.

Sim L, Ward OP, Li Z-Y. Production and characterisation of abiosurfactant isolated from Pseudomonas aeruginosa UW-1. J IndMicrobiol Biotechnol 1997;19:232–8.

Soo EL, Salleh AB, Basri M, Rahman RNZRA, Kamaruddin K.Optimization of the enzyme-catalyzed synthesis of amino acid-basedsurfactants from palm oil fractions. J Biosci Bioeng 2003;95:361–7.

Stewart TL, Fogler HS. Biomass plug development and propagation inporous media. Biotechnol Bioeng 2001;72:353–63.

Stosur GJ. Unconventional EOR concepts. Crit Rev Appl Chem1991;33:341–73.

Straube WL, Nestler CC, Hansen LD, Ringleberg D, Pritchard PJ,Jones-Meehan J. Remediation of polyaromatic hydrocarbons(PAHs) through landfarming with biostimulation and bioaugmen-tation. Acta Biotechnol 2003;23:179–96.

Streger SH, Vainberg S, Dong H, Hatzinger PB. Enhancing transportof hydrogenophaga flava ENV735 for bioaugmentation of aquiferscontaminated with methyl tert-butyl ether. Appl EnvironMicrobiol2002;68:5571–9.

Subramaniam MB, Blakebrough N, Hashim MA. Clarification ofsuspensions by colloidal gas aphrons. J Chem Technol Biotechnol1990;48:41–60.

Sullivan ER. Molecular genetics of biosurfactant production. CurrOpin Biotechnol 1998;9:263–9.

Sun D, Duan X, LiW, Zhou D. Demulsification of water-in-oil emulsionby using porous glass membranes. Membr Sci 1998;146:65–72.

Tanner RS, Udegbunam EO, McInerney MJ, Knapp RM. Microbiallyenhanced oil recovery from carbonate reservoirs. Geomicrobiol J1991;9:169–95.

Taranova L, Semenchuk I, Manolov T, Iliasov P, Reshetilov A. Bacteria-degraders as the base of an amperometric biosensor for detection ofanionic surfactants. Biosens Bioelectron 2002;17:635–40.

Taylor TP, Rathfelder KM, Pennell KD, Abriola LM. Effects of ethanoladdition on micellar solubilization and plume migration duringsurfactant enhanced recovery of tetrachloroethene. J ContamHydrol 2004;69:93–9.

Thimon L, Peypoux F, Michel G. Interactions of surfactin, abiosurfactant from Bacillus subtilis with inorganic cations.Biotechnol Lett 1992;14:713–8.

Uma Maheswar Rao J, Satyanarayana T. Enhanced secretion and lowtemperature stabilization of a hyperthermostable and Ca2+-independent α-amylase of Geobacillus thermoleovorans bysurfactants. Lett Appl Microbiol 2003;36:191–6.

Urum K, Pekdemir T, Gopur M. Optimum conditions for washing ofcrude oil-contaminated soil with biosurfactant solutions. Trans InstChem Eng 2003;81B:203–9.

Van DykeMI, Gulley SL, Lee H, Trevors JT. Applications of microbialbiosurfactants. Biotechnol Adv 1991;9:241–52.

Van Hamme J, Ward OP. Influence of chemical surfactants on thebiodegradation of crude oil by a mixed bacterial culture. CanJ Microbiol 1999;45:130–7.

Van Hamme JD, Singh A, Ward OP. Recent advances in petroleummicrobiology. Microbiol Mol Biol Rev 2003;67:503–49.

Van Hamme JD, Singh A, Ward OP. Surfactants in microbiologyand biotechnology: Part 1. physiological aspects. Biotech Adv2006;24:604–20.

Villeneuve P, PinaM,Montet D, Graille J.Carica papaya latex lipase: sn-3 stereospecificity or short chain selectivity? Model chiral triglycer-ides are removing the ambiguity. J Am Oil Chem Soc 1995;72:753.

Vipulanandan C, Ren X. Enhanced solubility and biodegradationof naphthalene with biosurfactant. J Environ Eng 2000;126:629–34.

Wang S, Mulligan CN. An evaluation of surfactant foam technology inremediation of contaminated soil. Chemosphere 2004;57:1079–89.

Wang Y, Alexander TW, McAllister TA. In vitro effects of Monensinand Tween 80 on ruminal fermentation of barley grain: barleysilage-based diets for beef cattle. Animal Feed Sci. Technol2004;16:197–209.

Ward OP. Fermentation biotechnology. Milton Keynes, UK: OpenUniversity Press; 1989. p. 1-225.Ward OP. Bioprocessing. MiltonKeynes, UK: Open University Press; 1991. p. 1-198.

Ward OP, Singh A. Omega-3/6 fatty acids: alternative sources ofproduction. Process Biochem 2005;40:3627–52.

Ward OP, Fang J-W, Li Zu-Yi. Lipase-catalyzed synthesis of sugar estercontaining arachidonic acid. Enzyme Microb Technol 1997;20:52–6.

Ward OP, Qin QM, Dhanjoon J, Ye J, Singh A. Physiology andBiotechnology of Aspergillus. Adv Appl Microbiol 2006;58:1-75.

Wei G, Li Y, Chen GCJ. Effect of surfactants on extracellularaccumulation of glutathione by Saccharomyces cerevisiae. ProcBiochem 2003;38:1133–8.

Wei QF, Mather RR, Fotheringham AF. Oil removal from usedsorbents using a biosurfactant. Biores Technol 2005;96:331–4.

Wu J, Ding H, Song B, Hayashi Y, Talukder MMR, Wang S.Hydrolytic reactions catalyzed by surfactant-coated candida rugosalipase in an organic-aqueous two-phase system. Proc Biochem2003;39:233–8.

Yakimov MM, Amor MM, Bock M, Bodekaer K, Fredrickson HL,Timmis KN. The potential of Bacillus licheniformis for in situenhanced oil recovery. J Petrol Sci Eng 1997;18:147–60.

Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM,McInerney MJ. Comparison of methods to detect biosurfactant

production by diverse microorganisms. J Microbiol Methods2004;56:339–47.

Zaitoun A, Fonseca C, Berger P, Bazin B, Monin N. New surfactant forchemical flood in high salinity reservoir. Paper SPE 80237, SPEInternational Symposium on Oilfield Chemistry, Houston, Texas;2003.

Zaki NN. Surfactant stabilized crude oil-in-water emulsions forpipeline transportaion of viscous crude oils. Colloids Surf,A Physicochem Eng Asp 1997;83:261–71.

Zhao B, Zhu L, Li W, Chen B. Solubilization and biodegradation ofphenanthrene in mixed anionic-nonionic surfactant solutions.Chemosphere 2005;58:33–40.

Zuckerberg A, Diver A, Perry Z, Gutnick DL, Rosenberg E. Emulsifierof Arthrobacter RAG-1, chemical and physical properties. ApplEnviron Microbiol 1979;37:414–20.