Microbial surfactants and their use in field studies of soil remediation

Introduction, 915–916 Surfactants, 916–917 Microorganisms and characteristics of biosurfactants produced, 917–918 Isolation of xenobiotic-degrading microorganisms with biosurfactant-producing capabilities, 918–919 Xenobiotics in soil, 919 Sorption of xenobiotics, 919 Biodegradation, 919–920 Surfactants and bioremediation, 920 Biosurfactants and organic solubilization and degradation, 920–921 Biosurfactants and metal solubilization, 921–923 Laboratory and field studies using Rhodococcus biosurfactants, 923–925 Conclusions, 925–926 Acknowledgements, 926 References, 926–929 The degree of association of organic and inorganic pollutants is governed by the complex physico-chemical interactions at interfaces. This association involves sorption onto soil constituents, sequestration within the soil matrix (micropores) and partitioning in a nonaqueous phase liquid (NAPL) that will, ultimately, control the fate of the contaminants. NAPL represents organic substances that are relatively insoluble in water providing a long-term source of pollutant. They represent a continuous input into the water phase to replace degraded or dissipating concentrations. As a result the bioavailability of contaminants to biodegradation may be reduced (Ogram et al. 1985; Miller and Alexander 1991). The enhancement of natural biological degradation processes, in what is termed bioremediation, can be a preferred cost-effective method of removing contaminants from soil-contaminated and other contaminated environments. The role of microorganisms has been shown to be essential in the remediation of, at least, organic pollution, and the activity of microbial types naturally present can be enhanced by bioremediation techniques which include increased aeration of the polluted material and nutrient additions (Christofi et al. 1998; Ivshina et al. 1998). Supplementing the source of microorganisms capable of degrading particular pollutants (bioaugmentation) and the enhancement of the desorption of pollutants from particulates using surfactants can increase hydrocarbon degradation (Deschenes et al. 1995; Thibault et al. 1996; Christofi et al. 1998; Ivshina et al. 1998). It is only 10–15 years that the use of surfactants in increasing the availability of hydrophobic pollutants in soils and other environments has been considered (Vigon and Rubin 1989). Previous studies have revealed that surfactants can enhance pollutant desorption and availability (Oberbremer et al. 1990; Zang and Miller 1992; Volkering et al. 1995; Rahman et al. 2002). They have been applied in oil washing for secondary oil recovery and to clean oil pipes and oil reservoirs (Osipow 1962). In desorption, both synthetic and natural (biological) surfactants have been used. However, it is not always the case that increasing the bioavailability of nonpolar organic substances through the use of surfactants leads to increased degradation or removal. Synthetic surfactants have also been shown to decrease organic degradation (Bruheim et al. 1999) possibly through, for example, the toxic effects of the surfactant (Rouse et al. 1994). It can also be assumed that increasing bioavailability of a pollutant can lead to an increasing toxicity to degrading microorganisms. It has been shown that sorption of toxicants is one major factor controlling bioavailability and toxicity (Welp and Brümmer 1998). In addition, the incorporation of the substrate within surfactant micelles can decrease once again its bioavailability, and, hence, reduce degradation rates. The use of biosurfactants in the control of the bioavailability of toxicants in soils and other environments is an attractive option because of their biodegradability (Herman et al. 1995). These surface-active compounds can be diverse with novel chemical structures and characteristics; they can be produced from cheap raw materials and the organisms producing them can be modified genetically to overproduce or produce new compounds. Additionally, they are significantly less toxic than synthetic petroleum-based surfactants (Banat et al. 2000). This review deals with microbial surfactant research, placing emphasis on the use of biosurfactants in remediation of contaminated soils outside the laboratory. It does not attempt to review the general use of surfactants in soil remediation or surfactant production by microorganisms. Mulligan et al. (2001b) covers the former while Lang (2002) deals with the latter. Surfactants are SURFace ACTive AgeNTS with wide ranging properties including the lowering of surface and interfacial tensions of liquids. Surface tension is defined as the free surface enthalpy per unit area (OECD 1995) and is the force acting on the surface of a liquid leading to minimization of the area of that surface. Both synthetic and natural surfactants exist capable of reducing the surface tension of water from 72 mN m−1 to around 27 mN m−1(Table 1). Biosurfactants are biological compounds that exhibit high surface-active properties (Georgiou et al. 1992). Microorganisms, plants and animals, including humans, produce them. Surfactants have both hydrophilic and hydrophobic/lipophilic (nonpolar) portions in the molecule. They are amphipathic molecules enabling the formation of specialized structures vital to their action. They function by residing at the oil–water interface. The hydrophilic (polar) part of a surfactant is usually referred to as the ‘head’, whereas the nonpolar hydrophobic portion is known as the ‘tail’. The latter is a hydrocarbon chain of varying length in different surfactants. Surfactants are classified according to the ionic charge residing in the polar part of the molecule. Hence anionic, cationic, nonionic and zwitterionic (combined presence of anionic and cationic charges) surfactants exist. The presence of surfactants can lead to an increase in the concentration of hydrophobic compounds in the water phase. This is achieved through the formation of oil/water emulsions and solubilization, where, above the Critical Micelle Concentration (CMC), biosurfactant molecules aggregate to form micelles. CMC is that concentration of surfactant favouring micelle formation. Between 50 and 100 surfactant molecules (aggregation number) form micelles. Micelles arise when the lipophilic part of the surfactant molecule that is unable to form hydrogen bonding in an aqueous phase causes an increase in the free energy of the system. One way to alleviate this free energy increase is for the hydrocarbon tail to be isolated from water by adsorption onto surfaces, absorption into an organic matrix or the formation of micelles vesicles where the hydrocarbon moiety of the surfactant become situated towards the centre with the hydrophilic part in contact with water (Haigh 1996). The presence of surfactant can lower the interfacial tension between immiscible fluids enabling them to be miscible through the creation of additional surfaces. A single interface consisting of an immiscible and miscible constituent is transformed into smaller interface of the two constituents. Micelle formation allows the partitioning of hydrophobic structures into the central hydrophobic pseudophase core enabling ‘solubility’. This can lead to increased dispersion of a compound in solution above its water solubility limit (Rouse et al. 1994). This solubilization can also lead to mobilization of sorbed and adsorbed hydrophobic soil contaminants by lowering of capillary forces (Tsomides et al. 1995). An important characteristic of surfactants relates to the relative abundance of hydrophilic and hydrophobic constituents. Surfactants can be classified according to their Hydrophile–Lipophile Balance (HLB) that affects their physico-chemical properties (Tiehm 1994). The HLB classification can be used to determine the suitability of use of surfactants. In general, a surfactant with a low HLB is lipophilic whereas a high HLB confers better water solubility (Sabatini et al. 1995). Table 2 shows the application of nonionic surfactants with different HLB values (Cross 1987). Most surfactants produced and utilized are synthetic. This is due to the present high cost that can be incurred in the production of microbial biosurfactants. There are, however, many advantages in the use of biosurfactants, including biodegradability and low toxicity (Poremba et al. 1991; Munstermann et al. 1992; see Table 3). Diverse ranges of prokaryotic and eukaryotic microorganisms are capable of producing surfactants (Lang 2002). The biosurfactants most commonly isolated are glycolipids containing the sugars rhamnose and trehalose (Fig. 1). The microorganisms produce low- and high-molecular weight biosurfactants. The low-molecular weight types are generally glycolipids or peptidyl lipids (lipopeptides). The glycolipids include trehalose tetraesters and dicorynomycolates, fructose lipids, sophorolipids and rhamnolipids. Peptidyl lipids include surfactin, viscosin and polymixin that are also effective antimicrobials. Bacillus subtilis, a common soil bacterium, produces the cyclic lipopeptide biosurfactant surfactin. Surfactin has an amphiphilic structure (Fig. 1c) associated with high surfactant activity and extensive biological properties including affecting the growth of tumours, bacteria, fungi, viruses and mycoplasmas (Bernheimer and Avigad 1970; Vollenbroich et al. 1997; Peypoux et al. 1999) and this appears to contradict with a commonly held view that biosurfactants have a low toxicity. Glycolipids and lipopeptide substances are involved in the lowering of surface and interfacial tensions in liquids. Stable emulsions are not a usual trait of these surfactants. Examples of bacterial surfactants. (a) Pseudomonas dirhamnolipid structure. R=H and R=CH3 for acid and methyl dirhamnolipids, respectively. (b) Structure of trehalose lipids from Arthrobacter sp. (c) Structure of surfactin produced by Bacillus subtilis High-molecular weight biosurfactants are amphiphilic (amphipathic) (lipo)polysaccharides (lipo)proteins or combinations of these (Karanth et al. 1999; Rosenberg and Ron 1999). These biosurfactants are associated with production of stable emulsions but do not lower the surface tension. It is interesting to note that the production of stable emulsions enables bacteria to adhere to hydrophobic surfaces very strongly (Rosenberg and Rosenberg 1981; Neu et al. 1992) with implications on biodegradation capabilities. Biosurfactants can be more ecologically acceptable than synthetic surfactants in the bioremediation of contaminated soil and subsurface environments. The high production costs for biosurfactants currently prohibits their large-scale utilization, although organisms involved in their production can use a wide range of cheap substrates including industrial (oils/fats) and agricultural waste products (Haba et al. 2000; Makkar and Cameotra 1999; Veenanadig et al. 2000). Bacterial surfactant-producing organisms include Pseudomonas aeruginosa (mono- and di-rhamnolipid biosurfactants), Corynebacterium, Nocardia and Rhodococcus, spp. (phospholipids, trehalose dimycolates/dicorynomycolates, glycolipids, etc.), Bacillus subtilis (surfactin), Bacillus licheniformis (lipopeptide similar to surfactin), Arthrobacter paraffineus (trehalose and sucrose lipids) and others (see Table 4). Fungi involved in surfactant production include the yeasts Torulopsis spp. (sophorolipids) and Candida spp. (liposan, phospholipids). For more on the types and properties of biosurfactants produced by microorganisms refer to Desai and Banat (1997), Vardar-Sukar and Kosaric (2000) and Lang (2002). Factors controlling the production of biosurfactants include the quality and quantity of carbon and nitrogen constituents in culture and natural physical environment of soil systems (Desai and Banat 1997; Lang and Philp 1998). The type of nitrogen present (whether NH4+, NO3–, urea or amino acid) influences the biosurfactant produced (Duvnjak et al. 1983; Robert et al. 1989; Haba et al. 2000). Interesting observations relate to the effect of nitrogen limitation that appears to stimulate biosurfactant production and overproduction by some microorganisms (Suzuki et al. 1974; Guerra-Santos et al. 1984). This has implications for bioremediation as systems are often supplemented with nitrates and phosphates to alleviate N and P limitation and enhance microbial activity. This may affect in-situ biosurfactant production and requires further investigation. Biosurfactant production has been demonstrated in the presence of water-soluble substrates, hydrocarbons and oils. The type of surfactant formed when growing on these carbon sources can be influenced (Duvnjak and Kosaric 1985; Robert et al. 1989; Makkar and Cameotra 1999). Generally, the inclusion of a hydrophobic organic substrate in culture media can affect the isolation of microorganisms capable of its degradation. Hydrocarbon-degrading microorganisms, for example, can easily be isolated from oil-contaminated sites and many are capable of producing biosurfactants. This is generally achieved by adding aliphatic and aromatic hydrocarbons as the sole carbon sources. Studies have revealed large populations of Rhodococcus species including R. ruber, R. erythropolis, R. ‘longus’ and R. opacus (see Section 11) and Acinetobacter, Pseudomonas and other genera (Martin et al. 1996, Huy et al. 1999). Desai and Banat (1997) have reviewed a wide range of techniques to determine the presence of biosurfactants in culture media. The methods include colorimetric analyses for the detection of anionic surfactants (Shulga et al. 1992) and rhamnolipids (Hansen et al. 1993; Siegmund and Wagner 1991), Emulsification Index determination over a 24-h period (Cooper and Goldenberg 1987), drop-collapsing test (Jain et al. 1991), direct TLC technique (Matsuyama et al. 1991) and axisymmetric drop shape analysis (ADSA; Van der Vegt et al. 1991). Extraction and purification of biosurfactants from culture media can then be identified structurally and compared to previously identified types using a range of techniques and surfactant type. Techniques include one or a combination of TLC, HPLC, FT-IR, NMR (H-1 and C-13) and MS, LC-MS (Deziel et al. 1999; Esch et al. 1999; Kim et al. 1999; Makkar and Cameotra 1999; Mata-Sandoval et al. 1999; Nielsen et al. 1999; Gartshore et al. 2000). Organics and metal pollutants are of concern in soil environments and their removal is of paramount importance. Organic hydrophobic (nonpolar) pollutants include polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons, polychlorinated biphenyls (PCB) and a range of biocides. PAHs are common environmental contaminants due to industries involved in coal and crude oil processing. These include low molecular weight compounds (e.g. naphthalene) with adequate solubility in water for use by microorganisms, to high molecular weight components (pyrene, fluorene) that have very low water solubility and are strongly bound to soils (McElroy et al. 1989). Heavy metals are also associated with soils and these can be removed by surfactants. Heavy metals mainly enter soils through atmospheric deposition and application of domestic and industrial waste effluents. Other inputs include natural geological formations, mining activity and remnants of industrial activity. Metals of particular concern to health are arsenic, cadmium, chromium, copper, lead, mercury and zinc. Mulligan et al. (2001a) have recently evaluated remediation technologies for metal-contaminated soils. The transport and fate of many pollutants in soils and groundwaters can be a function of nonlinear, rate-limited sorption and biodegradation (Hu and Brusseau 1998). Many of the hydrophobic organic contaminants finding their way into soils become rapidly and irreversibly associated with the organic fractions in soils. This association generally involves sorption. Constituents involved in sorption include organic matter such as humics (humic acids, fulvic acids and humin) and nonhumics (proteins, waxes and lipids, carbohydrates and others). The humics significantly affect the fate of hydrophobic soil contaminant and humin is an important constituent, accounting more than 50% of the organic carbon fraction of soil. An amalgam of biocides, PAH, PCB and other pollutants become quickly and irreversibly associated with humin (Stevenson 1976; Aiken et al. 1985; Wang et al. 1995). Hydrophobic organic compounds can also exist as NAPL. The removal of pollutants from soils depends on complex interaction of the physical, chemical and biological factors within the environment. Microbial degradation of hydrophobic compounds is a function of their structure, availability to microorganisms and the physico-chemical conditions affecting the metabolic capability of the microorganisms. The latter includes important factors such as pH, water, aeration status (redox potential) and presence of oxygen, nutrients and temperature. Biodegradation of organic pollutants can be attained realistically but according to some authors there is a need to understand fully the microorganisms and their ecology. Holden and Firestone (1997) suggest a need to determine the factors controlling the desired metabolic capability; the distribution of degrading communities relative to contaminant source and the overall activity of relevant communities. The majority of work, by far, carried out on enhancing the solubility of organic hydrophobic contaminants in soils and other environments utilizes synthetic surfactants. Synthetic surfactants have been shown to remove nonpolar compounds from surfaces but problems can be associated with their use, such as reduced availability of compounds sequestered into micelles, their toxicity, and ultimate resistance to biodegradation leading to increased pollution (Cort et al. 2002; Mulligan et al. 2001b). Evidence exists that suggests synthetic surfactants such as Triton X-100, Tween 80, Afonic 1412–7 (a nonionic alkyl ethoxylate) and others can enhance the concentration of PAHs in the aqueous phase (Grasso et al. 2001; Cuypers et al. 2002; Prak and Pritchard 2002). In a survey of eight synthetic surfactants, Tiehm (1994) found that the ability to solubilize PAH was variable. The more hydrophobic surfactants exhibited better solubilizing properties. Contradictory results have been obtained from tests on biodegradability of PAH solubilized by synthetic surfactants (Liu et al. 1995). Tiehm (1994) showed that bacteria were less tolerant to the more hydrophobic surfactants. The experiments of Liu et al. (1995) showed that Brij 30 was degraded along with naphthalene, but that Triton X-100 was not. However, in both cases the addition of surfactant did not significantly affect either the rate of naphthalene degradation or the eventual amount degraded, despite improved bioavailability. In contrast, surfactant-solubilized alkanes show substantially increased biodegradation rates (Bury and Miller 1993). Therefore the current knowledge has created an apparent quandary. Studies with nonionic and anionic surfactant additions have indicated that they can enhance the biodegradation of soil xenobiotics including phenanthrene, biphenyl and a range of other hydrocarbons (Aronstein et al. 1991; Bury and Miller 1993; Bruheim et al. 1999; Margesin and Schinner 1999). Non-ionic surfactants have also been shown to inhibit biodegradation at concentrations above their CMC (Aronstein et al. 1991; and 1992; et al. 1998). many synthetic surfactants are known to an effect on microorganisms (Tsomides et al. 1995). This may be a function of the toxicity of the surfactant to degrading microorganisms through increased and of et al. toxicity of increased fractions of the hydrophobic of microbial contact with the of micelle or substrate (Tiehm 1994). The formation of stable emulsions by microorganisms can enhance oil degradation, as with water is increasing nutrient availability et al. 1998). In natural systems including this can be utilized by the biosurfactant populations to produce such emulsions through of growth with biosurfactant-producing microorganisms is also as they are to in the environment as years many studies have shown that biosurfactants can solubilize and sorbed onto soil constituents and Miller 1992; et al. Brusseau et al. 1995; et al. 1995; et al. 1996; et al. 1997; and 1998; Ivshina et al. 1998; et al. 1998; et al. 1999). Brusseau et al. (1995) used acid for the removal of and at a concentration of around of was removed from Ivshina et al. used crude biosurfactant from Rhodococcus to remove oil from and oil et al. also surfactant production in a of the Nocardia and showed solubilization of hydrophobic The of crude oil removed in the by Ivshina et al. on the relative abundance of and in the crude used. produced surfactant that removed of oil sorbed onto whereas the of removal for other was low The growth conditions were used for et al. but it is known that different can form structurally different surfactants (Lang and Philp with different desorption capabilities. Zang and Miller and showed that biosurfactant structure has an effect on solubilization and degradation of In cases the type of surfactant produced is to the carbon source in growth media (Suzuki et al. 1974; et al. and nutrient In addition, some are formed (Lang and Philp 1998). It is in bioremediation studies that the biosurfactants and surfactant-producing microorganisms be used to the particular is on the pollutants and the ability of the biosurfactant to solubilization with degradation by or microorganisms in environments. The biosurfactant surfactin, from Bacillus subtilis, is considered to be the most biosurfactant known et al. and can lower the surface tension of water to 27 mN Studies on the Rhodococcus species et al. have similar values 1). R. was more effective in of interfacial tension in than R. et al. 1998). is known the biosurfactants from R. but on produced by other studies have shown that R. produces glycolipids with different structures to of R. interfacial contact of degrading microorganisms can of hydrophobic pollutants the need for et al. hydrocarbon from micelles produced by biosurfactant or from interfaces. et al. used biosurfactant produced by subtilis in soils and on systems to the biodegradation of They found enhanced degradation in both and soil systems with the and biosurfactant although soil degradation was a need to fully understand conditions for mobilization and degradation of the pollutant. et al. used systems to and the of surfactants on the enhancement of and degradation of et al. used the produced by et al. 1995) to enhance PAH and degradation results showed and in the of phenanthrene, and respectively. results on PAH solubilization have been obtained with the important rhamnolipids produced by Pseudomonas aeruginosa and other (Deschenes et al. 1996; et al. 1998; Mulligan et al. 2001b). et al. found that a biosurfactant from Rhodococcus was to more effective than the synthetic Tween in increasing the of PAHs into the aqueous phase. There is a of on the use of biosurfactants in soil The use of biosurfactants in remediation experiments is on the that they are to be less to the microorganisms the This is by the that biosurfactants to have a lower CMC (see Table 1). it is that microbial biosurfactants have been over and are in the solubilization of hydrophobic compounds. This is not the case for synthetic surfactants. In the use of biosurfactants, there is less need for et al. and are from in-situ production by or microbial As many organic pollutants in soil can be the of a bioremediation may more on increasing their bioavailability by desorption from soil et al. the role of biosurfactants on substrate availability in soil. They that surfactants at increase the bioavailability to nutrients and can be used at low concentrations to enhance In the field studies carried addition of biosurfactants biosurfactant-producing microorganisms can be used in in-situ and bioremediation techniques and soil In soil the recovery and of biosurfactants be a preferred option because of the in although they are very this may not be an studies for bioremediation techniques have been to enhance PAH degradation et al. 1999). experiments have been with nutrient to bioremediation of oil-contaminated et al. 1999). includes a biosurfactant and its use enhanced oil degradation over an although in using the surfactant were not results were obtained by Christofi et al. using of soil with additions of surfactants produced by Rhodococcus (see also Section The of this and other et al. is in the production and use of crude surfactant for bioremediation of contaminated The of soils can be high and is a function of the presence of organic material and that have a ionic a weight it is that organic matter is more effective in removing than from a of techniques are used to clean soils contaminated with including methods such as soil and to techniques also include the use of plants to and of plants the have also been used to the metals and their to the pollution may be The use of microorganisms to remove metals from soils is due to the complex association of the contaminants with and organic constituents. In with soil constituents and are involved affecting of the metal to the microorganisms. organic microorganisms for metals include their to through, for example, (e.g. can lead to the of bacteria involved in production can lead to insoluble metal Surfactants can remove metals from surfaces in a of metals in a nonionic form can complex with biosurfactants, enhancing surface removal by 1995). Additionally, the use of anionic surfactants which contact metals can lead to their desorption from surfaces. The then need to be removed from the soil surfactants can also to reduce the association of metals by for some but not surfaces. and a range of surfactants and their effects on sorbed anionic surfactants, there to be an increase in association of metal with surfaces sorption of the combination formed or of the A by et al. on the formation of biosurfactant produced by aeruginosa and metal showed that and stable