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Biosurfactants in environmental biotechnology
Biosurfactants in environmental biotechnology William R Finnerty Finnerty Enterprises Inc, Athens, USA Biosurfactants are natural products derived from bacteria, yeasts, or fungi. The complex chemical structures and physical properties of biosuffactants generally result in properties equal to, or exceeding, many synthetic surfactants. Biosurfactants have low toxicity profiles to freshwater, marine, and terrestrial ecosystems, and are potential candidates for a variety of environmental applications. Research, to date, has largely been focused on the enhancement of oil biodegradation and microbial enhanced oil recovery. The solubilization and emulsification of toxic pesticides by biosurfactants has also been reported, aiding in the recovery of such hazardous materials from contaminated sites. The future success of biosurfactant technology in bioremediation initiatives will require the precise targeting of the biosurfactant system to the physical conditions and chemical nature of the pollution-affected site. Current Opinion in Biotechnology 1994, 5:291-295 Introduction Naturally occurring surface-active agents derived from microbial, plant, or animal sources are of developing interest, with respect to their efficacy and efficiency as substitutes for petrochemical-derived surfactants, particularly in the context of environmental applications. The use of surface-active systems impacts such diverse markets as bioremediation, cosmetics, food, agriculture, oil production, health care, and personal care products. Emerging interest in natural surfactants, also termed biosurfactants, relates to their perceived properties of safety and mildness, as well as their physical properties, which are equal to, if not superior to, synthetic surfactants. This review addresses solely those applications of biosurfactants in the dispersal and remediation of polluted material. I focus both on nonspecific and specific biosurfactants produced in situ by microorganisms inoculated into the target matrix, and on biosurfactants added directly to polluted material in a purified form.
General considerations Biosurfactants derived from microbial sources represent a broad spectrum of biomolecules, including normal and hydroxy fatty acids, glycolipids, cyclic lipopeptides, N-acyl amino acids, lipopeptides, monoand diglycerides, and phospholipids [1,2"',3°']. These biosurfactants exhibit physical and chemical properties that allow their concentration and physical ac-
tion at air-water, oil-water, and solid-liquid interfaces, thereby reducing the physical forces that act at such boundaries. This physical property of surface-active biomolecules produces effects including solubilization, emulsification, dispersion, wetting, foaming, and detergent capacity, as well as antimicrobial activity in some cases. Biosurfactants are generally more chemically complex and considerably bulkier than their synthetic counterparts, with single molecules occupying a larger area, while exhibiting lower critical micelle concentration values than synthetic surfactants. Table 1 lists the physical properties of selected biosurfactants and synthetic surfactants in the context of minimum surface tension, minimum interfacial tension, and critical micelle concentration values.
Biosurfactants and bioremediation Bioremediation is currently in vogue as a promising cost-effective and performance-effective technology to address numerous environmental pollution problems. These pollutants range from industrial wastes (e.g. polychlorinated biphenyls, trichloroethylene, pentachlorophenol and dioxin), polyaromatic hydrocarbons, refined petroleum products (e.g. jet fuel, gasoline, diesel fuel and the benzene, toluene, ethylbenzene and xylene cluster), acid mine drainage, pesticides, munitions compounds (e.g. trinitrotoluene), and inorganic heavy metals to crude oil. Biotechnology initiatives are in development throughout commercial and government laboratories world-
Abbreviations API--American Petroleum Institute;HLB--hydrophilic-lipophilicbalance. © Current Biology Ltd ISSN 0958-1669
291
292
Environmentalbiotechnology Table 1. Comparison of the physical properties of biosurfactants and synthetic surfactants.
Surfactant type Sophorolipid Surfactin Rhamnolipid: R1 R2 R3 R4 Trehalose lipids: Trehalose-6-mycolate Trehalose-6,6'-dimycolate
Trehalose-2,3,4,2'-tetraester Trehalose-di,tetra,hexa,octaester Glucose-6-mycolate Cellobiose-6-mycolate
Maltotriose-6,6',6"-trimycolate Ustilagic acid Sodium dodecyl sulfate Cetyltrimethylammonium bromide Tween 20 linear alkylbenzene sulfonate
Surface tension (ran m -1)
Critical micelle concentration (rag 1-1)
Interracial tension (raN m -1)
37 27
1.0-2.0 0.01
82.0 11.0
27 26 30 25
0.05 4.00 <1.00 <1.00
5.0 40.0 200.0 200.0
32 36 26 30 40 35 44 30 37 30 30 47
16.0 17.0 <1.0 0.02-10 -5 9.0 1.0 19.0 <1.0 0.02 5.00 4.80 <1.00
2.0 2.0 10.0 1500.0 20.0 4.0 10.0 20.0 2120.0 1300.0 600.0 590.0
wide to identify and develop those biotechnology strategies that will economically and successfully aid in solving pollution issues in an environmentally acceptable manner. Surfactants (both biosurfactants and synthetic surfactants) are emerging as a technology to enhance the accessibility and bioavailabiliv] of hydrophobic chemicals, thereby complementing existing (bio)remediation methods. Unfortunately, most biosurfactant work, thus far, has been laboratorybased, with large field plot treatments rare. Also, field engineers are generally not accustomed to surfactant technology, particularly biosurfactants. The literature reported, to date, is small and often indeterminate as to the effects and efficacy of these molecules in the field. The application of biosurfactants has been largely directed to the biodegradation of crude oil [4°°] and to enhanced oil recovery systems [5,6°°]. Interestingly, the extensive bioremediation studies conducted following the 1989 Alaskan oil spill failed to address possible role(s) of biosurfactants produced by the nutrient-stimulated indigenous microflora in enhancing the bioavailability and biodegradation of crude oil impacted shorelines [7°°]. As noted by Chakrabarty [8], a biosurfactant produced by Pseudomonas aeruginosa is capable of effectively dispersing oil into small droplets, and could enhance the bioremediation of oil-polluted shorelines [8].
Petroleum hydrocarbons The addition of biosurfactants to a soil microcosm containing a 'model' oil resulted in enhanced biodegradation by shortening the time frame for greater than 90% loss of the target oil (see Table 2) [9]. The increased rate
of biodegradation required that sufficient biosuffactant be introduced into the system to reduce inteffacial tension to values of 2 - 1 6 m N m -1. In a further extension of this study, an overproducing strain of Rhodococcus erythropolis reportedly increased the rate of biodegradation of a 'model' oil in the presence of viable cells [101. Table 2. Effect of specific glycolipids on oil biedegradation.
Biosurfactant Control* Sophorolipid Rhamnolipid Trehalose-6,6'dimycola|e Cellobiose lipid
Time (h)
Oil removal (%)
114 75 77 71 79
81 97 94 93 99
f
*Containing no biosurfactant. Adapted from [4"'1.
Recently, commercial cultures have been successfully grown with either gasoline or glucose plus vegetable oil. The biosurfactant produced in the respective cultures was extracted and reconstituted in water at pH 7.0. Sand was impregnated with gasoline and eluted by either static batch or flow-through columns with the biosurfactant preparations. The eluate was monitored for the gasoline components toluene, m-xylene, 1,2,4-trimethylbenzene, and naphthalene. An increased solubiliW of gasoline components in the eluate was recorded, after which the resulting gasoline biosurfacrant eluates were examined for biodegradation by commercial cultures. The biosurfactant derived from the
Biosurfactants in environmental biotechnology Finnerty 293 glucose/vegetable oil cultures increased the solubility of gasoline, but inhibited its subsequent biodegradation. In contrast, the gasoline-derived biosurfactant solubilized the gasoline and did not inhibit biodegradation [11°]. The physical and chemical properties of the biosurfactant derived from gasoline-grown cultures differed from those of the biosurfactant derived from glucose/vegetable oil-grown cultures, and these properties accounted for the difference in ability of the respective cultures to biodegrade gasoline. In an extension of these studies, crude oil was displaced from soil and sand packs by biosurfactants produced under differing growth conditions. A pure culture was grown in a chemically defined medium of inorganic salts supplemented with glucose, hexadecane, or four different crude oil types (varying in viscosity from light to heavy oil) as the sole source of carbon and energy. The extracellular biosurfactants were recovered, and both minimum interfacial tension values and critical micelle concentrations were determined for each. All biosurfactants measured 10-2 mN m -1 or less at their critical micelle concentration. These biosurfactantts were used to displace the four crude oils from soil and sand packs at a 5% (wt/wt) oil saturation. Interestingly, the most efficient displacement systems were those biosurfactants derived from growth on the homologous crude oil, effecting greater than 90% displacement of the crude oil from the soil or sand. All heterologous biosurfactants derived from the other growth carbon sources displaced less than 20°/o of the crude oil (WR Finnerty, unpublished data). These observations suggest that biosurfactants to be used in the beneficiation of oil-impacted sites should be derived from microorganism(s) grown at the expense of the target oil. In another report, Franey et al. [12] describe the isolation of biostimulated bacteria from contaminated and uncontaminated zones at a site of an aviation fuel spill and from unleaded gasoline contaminated sites. The isolates obtained from the biostimulated aviation fuel-spill and gasoline-contaminated sites emulsified petroleum and lowered the surface tension. Mattei et al. [13] showed that the treatment of crude oil in a continuous flow bioreactor by a biosurfactant-producing mixed culture could yield 80% oil degradation. The biosurfactants, as well as the biomass, were recycled to emulsify fresh oil. A trehalose glycolipid produced by Rhodococcus species H13-A exhibits minimal interfacial tensions values in the range 0.02-0.00005 mN m -1, the lowest value requiring the presence of a co-surfactant [14,15]. This biosurfactant system reduces the relative intrinsic viscosity of heavy oils (API gravity 8-12) in excess of 90°/0, as well as promoting the microscopic displacement of heavy crude oil from rock'cores by 20% [16].
Other biosurfactant applications The solubilization of toxic organic chemicals by a biosurfactant-producing Pseudomonas aeruginosa
from soil has b e e n reported [17]. This biosurfactant system increased the solubility and recovery of hexo achlorobiphenyl from soil slurries b y 31%0, which was threefold greater than that achieved with synthetic surfactants. An additive effect was noted when the biosurfactant and a synthetic surfactant were used together, resulting in a recovery of 41% of the hexachlorobiphenyl. A biosurfactant produced by Bacillus strains forms stable emulsions with the pesticide fenthion. This biosurfactant formed emulsions with some other liquid organophosphorus pesticides that were immiscible in water, but failed to exhibit emulsification activity toward solid organophosphoms pesticides, organochlorine pesticides, and hydrocarbons [18]. Pseudomonas cepacia produces a biosurfactant and degrades the water-insoluble herbicide 2,4,5-trichlorophenoxyacetic acid [19]. This biosurfactant system also forms stable emulsions with herbicides other than chlorophenols, and may be beneficial in the biodegradation of other toxic chemicals. Mulligan and Cooper [20] suggested that biosurfactants can be useful in the dewatering of peat. Surfactants can be added to peat to aid in the removal of water from peat under pressure. The aqueous pressate is rich in soluble organic matter plus surfactants. Supplementation of the peat pressate with trace peptone, yeast extract, or glucose serves as a growth substrate for Bacillus subtilis, which produces biosurfactant. Synthetic surfactant systems could be substituted with this biosurfactant-containing culture broth. Recycling of the aqueous organic rich pressate as a growth medium for the production of biosurfactant would have the advantage of eliminating the environmentally polluting waste stream discharge. For the mining and paper industries, an anionic polysaccharide, termed biodispersan, produced by Acinetobacter calcoaceticus has been developed that prevents flocculation of minerals while stabilizing aqueous suspensions of minerals [21,22]. The Japanese patent literature reflects the development of biosurfactants for such applications as the stabilization of coal slurries for pipeline transportation, cosmetic and soap formulations, foods, and dermal as well as transdermal drug delivery systems.
Future developments Further development work is necessary to achieve significantly increased product yields through strain improvements, to identify alternative cheap substrates that yield consistently high quality biosurfactants, and improved bioreactor designs for efficient product removal and recovery before biosurfactant manufacturing becomes economically competitive with synthetic surfactant technology. New treatment methodologies under development are surfactant systems to improve the release of pollutants from impacted sites [23"°]. Surfactants can mediate the
294
Environmentalbiotechnology displacement of pollutants, present either as adsorbed contaminants or non-aqueous phase liquids, by two general mechanisms: solubilization and mobilization. Aqueous phase solubilization of organic solutes into surfactant micelles can generally be correlated to the octanol-water partition coefficient, where large coefficients indicate greater amounts of the solute partitioning into the surfactant micelle. An example is the aqueous solubility of trichloroethylene (980 mg 1-1) compared with its solubility in 0.5M sodium dodecyl sulfate (19 600 mg 1-1) [23°°]. Few biosurfactant studies have addressed the solubilization coefficient for various organic chemicals. Mobilization, however, requires the formulation of surfactant systems capable of generating ultralow interfacial tension values, in the range of 10-3 n~lm-1 or lower, to function in the microscopic displacement of non-aqueous phase liquids from solid surfaces. Methods used to evaluate the mobilization suitability of any (bio)surfactant system are the hydrophilic-lipophilic balance (HLB) number and phase behavior diagrams. Winsor Type III surfactant systems for balanced middle-phase microemulsions are considered optimal for non-aqueous phase liquid mobilization in subsurface environments. Few biosurfactants are characterized with respect to their HLB number or phase behaviour. One biosurfactant produced by Rhodococcus species H13-A has been characterized, with a HLB of 5.5 and low interfacial tension values for forming Winsor Type III microemulsions with various crude oils [15].
Conclusions Biosurfactants are beginning to acquire a tentative stares as potentially performance-effective molecules in environmental clean-up. The major focus in bioremediation is the inoculation of chemically contaminated sites with microorganisms having unique and specialized metabolic properties that hopefully enhance biodegradation [24°']. The toxicological data collected on biosurfactants indicate that these molecules have extremely low toxicity profiles to freshwater, marine, and terrestrial ecosystems when compared with many synthetic surfactants [25°°]. Additionally, the biodegradation of biosurfactants appears rapid and complete, in contrast to the environmental persistence of some synthetic surfactants. The economics of large-scale biosurfactant production remain to be determined [26°°]. A number of variables are important to the application of (bio)surfactants for the (bio)remediation of pollutant-impacted sites. These variables include (bio)surfactant sorption to charged surfaces present in subsurfaces, precipitation caused by various ions or pH changes, stability of the surfactant system in the subsurface matrix, chromatographic fractionation and general mobility within the immobile matrix, and toxicity and/or persistence in the ecosystem. It is critical to the success of any (bio)remediation strategy involving surface-active agents that the application of the
(bio)surfactant system be targeted both to the physical conditions of the impacted site and to the chemical nature of the polluting organic material(s). This is regardless of whether the biosurfactant is produced by inoculated microorganisms, nutrient stimulation of the indigenous microflora or added as a specialty chemical. Preliminary indications are that biosurfactants do exhibit a specificity and that this specificity is largely dictated by the method used to grow the biosurfactantmicroorganism, including the chemical nature of the carbon and energy source. Suffice it to say that surfaceactive compounds are ubiquitous in nature, and result from natural biological processes and waste disposal. For biosurfactant technology to succeed in focused (bio)remediation strategies, a more comprehensive database must be developed that defines in precise terms the physical and chemical properties of individual biosurfactants.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: * of special interest ** of outstanding interest 1.
Kosaric N, Cairns WL, Gray NCC (Eds): Biosutfactants and Biotechnology. New York: Marcel Dekker; 1987.
Kosaric N (Ed): Btosu*factants. New York: Marcel Dekker; 1993. A treatise detailing information concerning biosurfactants gathered since 1987 and following on from an earlier book [1]. Topics covered are lipopeptide and rhamnolipid production, fungal polysaccharides, genetics of microorganisms producing biosurfactants, biological activity of biosurfactants, Iipopolysaccharides, physical properties of lung surfactant systems, economics of biosurfactants, cosmetics, biosurfactants from marine microorganisms, food applications, and environmental applications. 2. ,o
Ishigami Y: Biosurfactants Face Increasing Interest. Informa 1993, 4:1156-1165. A short concise review of the better characterized biosurfactants, emphasizing physical properties and applications. 3.
oo
4.
Muller-Hurtig R, Wagner F, Blaszczyk R, Kosaric N: Biosurfactants for Environmental Control. In Btosu~factants. Edited by Kosaric N. New York: Marcel Dekker; 1993:447-469. A general discussion of the current status of biosurfactant technology in petroleum biodegradation. °0
5.
JaCk TR: Microbial Enhancement of Oil Recovery. Curr Op/n Biotechnol 1991, 2:444-449.
6. Finnerty WR: Fossil Resource Biotechnology: Challenges and ,,o Prospects. Curr Opin Biotechnol 1992, 3:277-282. An overview of biotechnological options for the beneficiation of fossil fuels and their development status. Bragg JR, Prince RC, Wilkenson JB, Atlas RM: Bgoremedtation f o r Shoreline Cleanup Following the 1989 Alaskan Ogl Spill. Houston, Texas: Exxon Company USA; 1992. A concise summary of the Exxon clean-up program following the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska. 7. 00
8.
Chakrabarty AM: Genetically-Manipulated Microorganisms and their Products in the Oil Service Industries. Trends
Btotechnol 1985, 3:32-38. 9.
Oberbremer A, Muller-Hurtig R, Wagner F: Effect of the Addition o f Microbial Surfactants o n Hydrocarbon Degradation in a Soil Population in a Stirred Reactor. Appl Microbtol Btotechnol 1990, 32:485-489.
Biosurfactants in environmental biotechnology Finnerty 10.
Goclik E, Muller-Hurtig, R, Wagner F: Influence of the Glycolipid-Producing Bacterium Rhodococcu$ erythropolis on the Degradation of a Hydrocarbon Mixture by an Original Soil Population. Appl Microbiol Biotechnol 1990, 34:120-126.
11. •
Falatko DM, Novak JT: Effects of Biologically Produced Biosurfactants on the Mobility and Biodegradation of Petroleum Hydrocarbons. Water Env Res 1992, 64:163-169. Commercially available cultures were grown on either gasoline or glucose/vegetable oil as the sole source of carbon and energy to produce extracellular biosurfactants. The respective biosurfactants effectively displaced gasoline from sand packs. However, biosurfactant derived from glucose/vegetable oil-grown cultures inhibited the biodegradation of gasoline, whereas biosurfactant derived from gasoline-grown cultures did not inhibit subsequent biodegradation of gasoline. 12.
Francy DS, Thomas JM, Raymond RL, Ward CH: Emulsification of Hydrocarbons by Subsurface Bacteria. J Ind Microbiol 1991, 8:237-246.
13.
Mattei G, Rambeloarisoa E, Guista G, Rontani JF, Bertrand JC: Fermentation Procedure of a Crude Oil in a Continuous Culture on Seawater. Appl Microbiol Biotechnol 1986, 23:302-304.
14.
Singer ME, Finnerty WR: Physiology of Biosurfactant Synthesis by Rhodococcus species H13-A. Can J Microbiol 1990, 30:741-745.
15.
Singer ME, Finnerty, WR, Tunelid A: Physical and Chemical Properties of a Biosurfactant Synthesized by Ri~Jdococcus species H13-A. Can J Microbiol 1990, 30:746-750.
16.
Finnerty WR, Singer ME: A Microbial Biosurfactant - - Physiology, Biochemistry and Applications. Dev lnd Microbiol 1984, 25:31-40.
17.
Berg G, Seech AF, Lee H, Trevors JT: Identification and Characterization of a Soil Bacterium with Emulsifying Activity. J Env Sct Health 1990, 7:753-764.
18.
Patel MN, Gopinathan KP: Lysozyme-Sensitive Bioemulsifiers for Immiscible Organophosphorus Pesticides. Appl Env Microbiol 1986, 52:1224-1226.
19.
Benerjee S, Duttagupta S, Chakrabarty AM: Production of Emulsifying Agent during Growth of Pseudomonas cepa-
cia with 2,4,5-Trichlorophenoxyacetic acid. Arch Microbiol 1983, 135:110-114.
20.
Mulligan C, Cooper DG: Pressate from Peat Dewatering as a Substrate for Bacterial Growth. Appl Env Microbiol 1985, 50:160-162.
21.
Rosenberg E, Rubinovitz C, Gottlieb A, Rosenhak S, Ron EZ: Production of Biodispersan by Acinetobacter caicoaceticus A2. Appl Env Mtcrobtol 1988, 54:317-322.
22.
Rosenberg E, Rubinovitz C, Legmann R, Ron EZ: Purification and Chemical Properties of Acinetobacter calcoacetlcus A2 Biodispersan. Appl Env Microblol 1988, 54:323-326.
23. West CC, Harwell JH: Surfactants and Subsurface Remediao. tion. Env Sci Technol 1992, 10:2324--2330. An excellent review of the basic principles underlying the physical chemistry of surface-active molecules. Discussion is directed to the subsurface environment and to non-aqueous phase liquids. The information is applicable to biosurfactants as well as synthetic surfactants. 24. Pritchard PH: Use of Inoculation in Bioremediation. Curr ** Opin Biotechnol 1992, 3:232-243. Summarizes bioremediation strategies that involve the inoculation of specialized microorganisms into polluted sites. A good review addressing the advantages, disadvantages, and limitations of this biotechnological approach. 25. **
Lang S, Wagner F: Biological Activities of Biosurfactants. In Biosu~factants. Edited by Kosaric N. New York: Marcel Dekker, 1993:251-268. One of the few places where a concise summary of the toxicological literature concerning biosurfactants can be found. 26. **
Mulligan CN, Gibbs BF: Factors Influencing the Economics of Biosurfactants. In Biosurfactants. Edited by Kosaric N. New York: Marcel Dekker; 1993:329-371. An excellent and comprehensive treatise on the problems and potential solutk)ns to biosurfactant manufacturing on a large scale.
WR Finnerty, Finnerty Enterprises Inc, 160 Chinquapin Place, Athens, Georgia 30605, USA.
295
General considerations Biosurfactants derived from microbial sources represent a broad spectrum of biomolecules, including normal and hydroxy fatty acids, glycolipids, cyclic lipopeptides, N-acyl amino acids, lipopeptides, monoand diglycerides, and phospholipids [1,2"',3°']. These biosurfactants exhibit physical and chemical properties that allow their concentration and physical ac-
tion at air-water, oil-water, and solid-liquid interfaces, thereby reducing the physical forces that act at such boundaries. This physical property of surface-active biomolecules produces effects including solubilization, emulsification, dispersion, wetting, foaming, and detergent capacity, as well as antimicrobial activity in some cases. Biosurfactants are generally more chemically complex and considerably bulkier than their synthetic counterparts, with single molecules occupying a larger area, while exhibiting lower critical micelle concentration values than synthetic surfactants. Table 1 lists the physical properties of selected biosurfactants and synthetic surfactants in the context of minimum surface tension, minimum interfacial tension, and critical micelle concentration values.
Biosurfactants and bioremediation Bioremediation is currently in vogue as a promising cost-effective and performance-effective technology to address numerous environmental pollution problems. These pollutants range from industrial wastes (e.g. polychlorinated biphenyls, trichloroethylene, pentachlorophenol and dioxin), polyaromatic hydrocarbons, refined petroleum products (e.g. jet fuel, gasoline, diesel fuel and the benzene, toluene, ethylbenzene and xylene cluster), acid mine drainage, pesticides, munitions compounds (e.g. trinitrotoluene), and inorganic heavy metals to crude oil. Biotechnology initiatives are in development throughout commercial and government laboratories world-
Abbreviations API--American Petroleum Institute;HLB--hydrophilic-lipophilicbalance. © Current Biology Ltd ISSN 0958-1669
291
292
Environmentalbiotechnology Table 1. Comparison of the physical properties of biosurfactants and synthetic surfactants.
Surfactant type Sophorolipid Surfactin Rhamnolipid: R1 R2 R3 R4 Trehalose lipids: Trehalose-6-mycolate Trehalose-6,6'-dimycolate
Trehalose-2,3,4,2'-tetraester Trehalose-di,tetra,hexa,octaester Glucose-6-mycolate Cellobiose-6-mycolate
Maltotriose-6,6',6"-trimycolate Ustilagic acid Sodium dodecyl sulfate Cetyltrimethylammonium bromide Tween 20 linear alkylbenzene sulfonate
Surface tension (ran m -1)
Critical micelle concentration (rag 1-1)
Interracial tension (raN m -1)
37 27
1.0-2.0 0.01
82.0 11.0
27 26 30 25
0.05 4.00 <1.00 <1.00
5.0 40.0 200.0 200.0
32 36 26 30 40 35 44 30 37 30 30 47
16.0 17.0 <1.0 0.02-10 -5 9.0 1.0 19.0 <1.0 0.02 5.00 4.80 <1.00
2.0 2.0 10.0 1500.0 20.0 4.0 10.0 20.0 2120.0 1300.0 600.0 590.0
wide to identify and develop those biotechnology strategies that will economically and successfully aid in solving pollution issues in an environmentally acceptable manner. Surfactants (both biosurfactants and synthetic surfactants) are emerging as a technology to enhance the accessibility and bioavailabiliv] of hydrophobic chemicals, thereby complementing existing (bio)remediation methods. Unfortunately, most biosurfactant work, thus far, has been laboratorybased, with large field plot treatments rare. Also, field engineers are generally not accustomed to surfactant technology, particularly biosurfactants. The literature reported, to date, is small and often indeterminate as to the effects and efficacy of these molecules in the field. The application of biosurfactants has been largely directed to the biodegradation of crude oil [4°°] and to enhanced oil recovery systems [5,6°°]. Interestingly, the extensive bioremediation studies conducted following the 1989 Alaskan oil spill failed to address possible role(s) of biosurfactants produced by the nutrient-stimulated indigenous microflora in enhancing the bioavailability and biodegradation of crude oil impacted shorelines [7°°]. As noted by Chakrabarty [8], a biosurfactant produced by Pseudomonas aeruginosa is capable of effectively dispersing oil into small droplets, and could enhance the bioremediation of oil-polluted shorelines [8].
Petroleum hydrocarbons The addition of biosurfactants to a soil microcosm containing a 'model' oil resulted in enhanced biodegradation by shortening the time frame for greater than 90% loss of the target oil (see Table 2) [9]. The increased rate
of biodegradation required that sufficient biosuffactant be introduced into the system to reduce inteffacial tension to values of 2 - 1 6 m N m -1. In a further extension of this study, an overproducing strain of Rhodococcus erythropolis reportedly increased the rate of biodegradation of a 'model' oil in the presence of viable cells [101. Table 2. Effect of specific glycolipids on oil biedegradation.
Biosurfactant Control* Sophorolipid Rhamnolipid Trehalose-6,6'dimycola|e Cellobiose lipid
Time (h)
Oil removal (%)
114 75 77 71 79
81 97 94 93 99
f
*Containing no biosurfactant. Adapted from [4"'1.
Recently, commercial cultures have been successfully grown with either gasoline or glucose plus vegetable oil. The biosurfactant produced in the respective cultures was extracted and reconstituted in water at pH 7.0. Sand was impregnated with gasoline and eluted by either static batch or flow-through columns with the biosurfactant preparations. The eluate was monitored for the gasoline components toluene, m-xylene, 1,2,4-trimethylbenzene, and naphthalene. An increased solubiliW of gasoline components in the eluate was recorded, after which the resulting gasoline biosurfacrant eluates were examined for biodegradation by commercial cultures. The biosurfactant derived from the
Biosurfactants in environmental biotechnology Finnerty 293 glucose/vegetable oil cultures increased the solubility of gasoline, but inhibited its subsequent biodegradation. In contrast, the gasoline-derived biosurfactant solubilized the gasoline and did not inhibit biodegradation [11°]. The physical and chemical properties of the biosurfactant derived from gasoline-grown cultures differed from those of the biosurfactant derived from glucose/vegetable oil-grown cultures, and these properties accounted for the difference in ability of the respective cultures to biodegrade gasoline. In an extension of these studies, crude oil was displaced from soil and sand packs by biosurfactants produced under differing growth conditions. A pure culture was grown in a chemically defined medium of inorganic salts supplemented with glucose, hexadecane, or four different crude oil types (varying in viscosity from light to heavy oil) as the sole source of carbon and energy. The extracellular biosurfactants were recovered, and both minimum interfacial tension values and critical micelle concentrations were determined for each. All biosurfactants measured 10-2 mN m -1 or less at their critical micelle concentration. These biosurfactantts were used to displace the four crude oils from soil and sand packs at a 5% (wt/wt) oil saturation. Interestingly, the most efficient displacement systems were those biosurfactants derived from growth on the homologous crude oil, effecting greater than 90% displacement of the crude oil from the soil or sand. All heterologous biosurfactants derived from the other growth carbon sources displaced less than 20°/o of the crude oil (WR Finnerty, unpublished data). These observations suggest that biosurfactants to be used in the beneficiation of oil-impacted sites should be derived from microorganism(s) grown at the expense of the target oil. In another report, Franey et al. [12] describe the isolation of biostimulated bacteria from contaminated and uncontaminated zones at a site of an aviation fuel spill and from unleaded gasoline contaminated sites. The isolates obtained from the biostimulated aviation fuel-spill and gasoline-contaminated sites emulsified petroleum and lowered the surface tension. Mattei et al. [13] showed that the treatment of crude oil in a continuous flow bioreactor by a biosurfactant-producing mixed culture could yield 80% oil degradation. The biosurfactants, as well as the biomass, were recycled to emulsify fresh oil. A trehalose glycolipid produced by Rhodococcus species H13-A exhibits minimal interfacial tensions values in the range 0.02-0.00005 mN m -1, the lowest value requiring the presence of a co-surfactant [14,15]. This biosurfactant system reduces the relative intrinsic viscosity of heavy oils (API gravity 8-12) in excess of 90°/0, as well as promoting the microscopic displacement of heavy crude oil from rock'cores by 20% [16].
Other biosurfactant applications The solubilization of toxic organic chemicals by a biosurfactant-producing Pseudomonas aeruginosa
from soil has b e e n reported [17]. This biosurfactant system increased the solubility and recovery of hexo achlorobiphenyl from soil slurries b y 31%0, which was threefold greater than that achieved with synthetic surfactants. An additive effect was noted when the biosurfactant and a synthetic surfactant were used together, resulting in a recovery of 41% of the hexachlorobiphenyl. A biosurfactant produced by Bacillus strains forms stable emulsions with the pesticide fenthion. This biosurfactant formed emulsions with some other liquid organophosphorus pesticides that were immiscible in water, but failed to exhibit emulsification activity toward solid organophosphoms pesticides, organochlorine pesticides, and hydrocarbons [18]. Pseudomonas cepacia produces a biosurfactant and degrades the water-insoluble herbicide 2,4,5-trichlorophenoxyacetic acid [19]. This biosurfactant system also forms stable emulsions with herbicides other than chlorophenols, and may be beneficial in the biodegradation of other toxic chemicals. Mulligan and Cooper [20] suggested that biosurfactants can be useful in the dewatering of peat. Surfactants can be added to peat to aid in the removal of water from peat under pressure. The aqueous pressate is rich in soluble organic matter plus surfactants. Supplementation of the peat pressate with trace peptone, yeast extract, or glucose serves as a growth substrate for Bacillus subtilis, which produces biosurfactant. Synthetic surfactant systems could be substituted with this biosurfactant-containing culture broth. Recycling of the aqueous organic rich pressate as a growth medium for the production of biosurfactant would have the advantage of eliminating the environmentally polluting waste stream discharge. For the mining and paper industries, an anionic polysaccharide, termed biodispersan, produced by Acinetobacter calcoaceticus has been developed that prevents flocculation of minerals while stabilizing aqueous suspensions of minerals [21,22]. The Japanese patent literature reflects the development of biosurfactants for such applications as the stabilization of coal slurries for pipeline transportation, cosmetic and soap formulations, foods, and dermal as well as transdermal drug delivery systems.
Future developments Further development work is necessary to achieve significantly increased product yields through strain improvements, to identify alternative cheap substrates that yield consistently high quality biosurfactants, and improved bioreactor designs for efficient product removal and recovery before biosurfactant manufacturing becomes economically competitive with synthetic surfactant technology. New treatment methodologies under development are surfactant systems to improve the release of pollutants from impacted sites [23"°]. Surfactants can mediate the
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Environmentalbiotechnology displacement of pollutants, present either as adsorbed contaminants or non-aqueous phase liquids, by two general mechanisms: solubilization and mobilization. Aqueous phase solubilization of organic solutes into surfactant micelles can generally be correlated to the octanol-water partition coefficient, where large coefficients indicate greater amounts of the solute partitioning into the surfactant micelle. An example is the aqueous solubility of trichloroethylene (980 mg 1-1) compared with its solubility in 0.5M sodium dodecyl sulfate (19 600 mg 1-1) [23°°]. Few biosurfactant studies have addressed the solubilization coefficient for various organic chemicals. Mobilization, however, requires the formulation of surfactant systems capable of generating ultralow interfacial tension values, in the range of 10-3 n~lm-1 or lower, to function in the microscopic displacement of non-aqueous phase liquids from solid surfaces. Methods used to evaluate the mobilization suitability of any (bio)surfactant system are the hydrophilic-lipophilic balance (HLB) number and phase behavior diagrams. Winsor Type III surfactant systems for balanced middle-phase microemulsions are considered optimal for non-aqueous phase liquid mobilization in subsurface environments. Few biosurfactants are characterized with respect to their HLB number or phase behaviour. One biosurfactant produced by Rhodococcus species H13-A has been characterized, with a HLB of 5.5 and low interfacial tension values for forming Winsor Type III microemulsions with various crude oils [15].
Conclusions Biosurfactants are beginning to acquire a tentative stares as potentially performance-effective molecules in environmental clean-up. The major focus in bioremediation is the inoculation of chemically contaminated sites with microorganisms having unique and specialized metabolic properties that hopefully enhance biodegradation [24°']. The toxicological data collected on biosurfactants indicate that these molecules have extremely low toxicity profiles to freshwater, marine, and terrestrial ecosystems when compared with many synthetic surfactants [25°°]. Additionally, the biodegradation of biosurfactants appears rapid and complete, in contrast to the environmental persistence of some synthetic surfactants. The economics of large-scale biosurfactant production remain to be determined [26°°]. A number of variables are important to the application of (bio)surfactants for the (bio)remediation of pollutant-impacted sites. These variables include (bio)surfactant sorption to charged surfaces present in subsurfaces, precipitation caused by various ions or pH changes, stability of the surfactant system in the subsurface matrix, chromatographic fractionation and general mobility within the immobile matrix, and toxicity and/or persistence in the ecosystem. It is critical to the success of any (bio)remediation strategy involving surface-active agents that the application of the
(bio)surfactant system be targeted both to the physical conditions of the impacted site and to the chemical nature of the polluting organic material(s). This is regardless of whether the biosurfactant is produced by inoculated microorganisms, nutrient stimulation of the indigenous microflora or added as a specialty chemical. Preliminary indications are that biosurfactants do exhibit a specificity and that this specificity is largely dictated by the method used to grow the biosurfactantmicroorganism, including the chemical nature of the carbon and energy source. Suffice it to say that surfaceactive compounds are ubiquitous in nature, and result from natural biological processes and waste disposal. For biosurfactant technology to succeed in focused (bio)remediation strategies, a more comprehensive database must be developed that defines in precise terms the physical and chemical properties of individual biosurfactants.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: * of special interest ** of outstanding interest 1.
Kosaric N, Cairns WL, Gray NCC (Eds): Biosutfactants and Biotechnology. New York: Marcel Dekker; 1987.
Kosaric N (Ed): Btosu*factants. New York: Marcel Dekker; 1993. A treatise detailing information concerning biosurfactants gathered since 1987 and following on from an earlier book [1]. Topics covered are lipopeptide and rhamnolipid production, fungal polysaccharides, genetics of microorganisms producing biosurfactants, biological activity of biosurfactants, Iipopolysaccharides, physical properties of lung surfactant systems, economics of biosurfactants, cosmetics, biosurfactants from marine microorganisms, food applications, and environmental applications. 2. ,o
Ishigami Y: Biosurfactants Face Increasing Interest. Informa 1993, 4:1156-1165. A short concise review of the better characterized biosurfactants, emphasizing physical properties and applications. 3.
oo
4.
Muller-Hurtig R, Wagner F, Blaszczyk R, Kosaric N: Biosurfactants for Environmental Control. In Btosu~factants. Edited by Kosaric N. New York: Marcel Dekker; 1993:447-469. A general discussion of the current status of biosurfactant technology in petroleum biodegradation. °0
5.
JaCk TR: Microbial Enhancement of Oil Recovery. Curr Op/n Biotechnol 1991, 2:444-449.
6. Finnerty WR: Fossil Resource Biotechnology: Challenges and ,,o Prospects. Curr Opin Biotechnol 1992, 3:277-282. An overview of biotechnological options for the beneficiation of fossil fuels and their development status. Bragg JR, Prince RC, Wilkenson JB, Atlas RM: Bgoremedtation f o r Shoreline Cleanup Following the 1989 Alaskan Ogl Spill. Houston, Texas: Exxon Company USA; 1992. A concise summary of the Exxon clean-up program following the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska. 7. 00
8.
Chakrabarty AM: Genetically-Manipulated Microorganisms and their Products in the Oil Service Industries. Trends
Btotechnol 1985, 3:32-38. 9.
Oberbremer A, Muller-Hurtig R, Wagner F: Effect of the Addition o f Microbial Surfactants o n Hydrocarbon Degradation in a Soil Population in a Stirred Reactor. Appl Microbtol Btotechnol 1990, 32:485-489.
Biosurfactants in environmental biotechnology Finnerty 10.
Goclik E, Muller-Hurtig, R, Wagner F: Influence of the Glycolipid-Producing Bacterium Rhodococcu$ erythropolis on the Degradation of a Hydrocarbon Mixture by an Original Soil Population. Appl Microbiol Biotechnol 1990, 34:120-126.
11. •
Falatko DM, Novak JT: Effects of Biologically Produced Biosurfactants on the Mobility and Biodegradation of Petroleum Hydrocarbons. Water Env Res 1992, 64:163-169. Commercially available cultures were grown on either gasoline or glucose/vegetable oil as the sole source of carbon and energy to produce extracellular biosurfactants. The respective biosurfactants effectively displaced gasoline from sand packs. However, biosurfactant derived from glucose/vegetable oil-grown cultures inhibited the biodegradation of gasoline, whereas biosurfactant derived from gasoline-grown cultures did not inhibit subsequent biodegradation of gasoline. 12.
Francy DS, Thomas JM, Raymond RL, Ward CH: Emulsification of Hydrocarbons by Subsurface Bacteria. J Ind Microbiol 1991, 8:237-246.
13.
Mattei G, Rambeloarisoa E, Guista G, Rontani JF, Bertrand JC: Fermentation Procedure of a Crude Oil in a Continuous Culture on Seawater. Appl Microbiol Biotechnol 1986, 23:302-304.
14.
Singer ME, Finnerty WR: Physiology of Biosurfactant Synthesis by Rhodococcus species H13-A. Can J Microbiol 1990, 30:741-745.
15.
Singer ME, Finnerty, WR, Tunelid A: Physical and Chemical Properties of a Biosurfactant Synthesized by Ri~Jdococcus species H13-A. Can J Microbiol 1990, 30:746-750.
16.
Finnerty WR, Singer ME: A Microbial Biosurfactant - - Physiology, Biochemistry and Applications. Dev lnd Microbiol 1984, 25:31-40.
17.
Berg G, Seech AF, Lee H, Trevors JT: Identification and Characterization of a Soil Bacterium with Emulsifying Activity. J Env Sct Health 1990, 7:753-764.
18.
Patel MN, Gopinathan KP: Lysozyme-Sensitive Bioemulsifiers for Immiscible Organophosphorus Pesticides. Appl Env Microbiol 1986, 52:1224-1226.
19.
Benerjee S, Duttagupta S, Chakrabarty AM: Production of Emulsifying Agent during Growth of Pseudomonas cepa-
cia with 2,4,5-Trichlorophenoxyacetic acid. Arch Microbiol 1983, 135:110-114.
20.
Mulligan C, Cooper DG: Pressate from Peat Dewatering as a Substrate for Bacterial Growth. Appl Env Microbiol 1985, 50:160-162.
21.
Rosenberg E, Rubinovitz C, Gottlieb A, Rosenhak S, Ron EZ: Production of Biodispersan by Acinetobacter caicoaceticus A2. Appl Env Mtcrobtol 1988, 54:317-322.
22.
Rosenberg E, Rubinovitz C, Legmann R, Ron EZ: Purification and Chemical Properties of Acinetobacter calcoacetlcus A2 Biodispersan. Appl Env Microblol 1988, 54:323-326.
23. West CC, Harwell JH: Surfactants and Subsurface Remediao. tion. Env Sci Technol 1992, 10:2324--2330. An excellent review of the basic principles underlying the physical chemistry of surface-active molecules. Discussion is directed to the subsurface environment and to non-aqueous phase liquids. The information is applicable to biosurfactants as well as synthetic surfactants. 24. Pritchard PH: Use of Inoculation in Bioremediation. Curr ** Opin Biotechnol 1992, 3:232-243. Summarizes bioremediation strategies that involve the inoculation of specialized microorganisms into polluted sites. A good review addressing the advantages, disadvantages, and limitations of this biotechnological approach. 25. **
Lang S, Wagner F: Biological Activities of Biosurfactants. In Biosu~factants. Edited by Kosaric N. New York: Marcel Dekker, 1993:251-268. One of the few places where a concise summary of the toxicological literature concerning biosurfactants can be found. 26. **
Mulligan CN, Gibbs BF: Factors Influencing the Economics of Biosurfactants. In Biosurfactants. Edited by Kosaric N. New York: Marcel Dekker; 1993:329-371. An excellent and comprehensive treatise on the problems and potential solutk)ns to biosurfactant manufacturing on a large scale.
WR Finnerty, Finnerty Enterprises Inc, 160 Chinquapin Place, Athens, Georgia 30605, USA.
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