Biolech. Adv. Vol. 9, pp. 241-252,1991 0734 - 9750/91 $0.00 + .50 Printed in Great Britain. All Rishts Reserved. 1991 Pergamon Pre~ pie

APPL ICAT IONS OF MICROBIAL SURFACTANTS

MICHELE I. VAN DYKE, HUNG LEE and JACK T. TREVORS

Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

ABSTRACT Biosurfactants are amphiphilic compounds produced by microorganisms that are

capable of decreasing surface and interfacial tensions. They are useful in remediation of insoluble organic pollutants in soil and marine environments. There are also a large number of industrial uses for biosurfactants. This paper reviews recent research on applications of microbially-produced surfactants.

KEYWORDS bioemulsifier, biosurfactant, hydrocarbon, remediation

INTRODUCTtON Biosurfactants are compounds produced on microbial cell surfaces or excreted

extracellularly, which contain hydrophilic and hydrophobic portions that reduce surface and interfacial tensions. Since biosurfactants and bioemulsifiers both exhibit emulsification properties, bioemulsifiers are often categorized with biosurfactants, although emulsifiers may not lower surface tension. A biosurfactant may have one of the following structures: mycolic acid, glycolipid, polysaccharide-lipid complex, lipoprotein or lipopeptide, phospholipid, or the microbial cell surface itself (37). The identification and characterization of biosurfactants produced by various microorganisms have been extensively reviewed (16,27,37,54,58,68). However, rather than describe the numerous types of biosurfactants and emulsifiers, this article examines potential applications of biosurfactants.

The largest market for biosurfactants is the petroleum industry, where they are used in petroleum production and incorporation into oil formulations. In 1982, 300 to 400 million kg were used worldwide for these purposes. Enhanced oil recovery also represents a large

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future market for biosurfactants. The total market for surfactants totalled 1.6 billion kg in 1983, and was predicted to reach 1.8 billion kg by 1990 (41).

The second largest market for surfactants is emulsion polymerization for paints, paper coatings and industrial coatings. Layman (41) described other uses for surfactants, such as in asphalt, cement, textile and fibre manufacturing, metal treatment, mining, water treatment, coal slurries, as defoamers, and as wood preservatives. Surfactants are also used in food and cosmetic industries, in industrial cleaning products, as well as in agricultural chemicals

to dilute and disperse fertilizers and pesticides, and to enhance penetration of active

compounds into plants (37). Hydrophobic pollutants found in petroleum hydrocarbons require solubilization

before being degraded by microbial cells (32,33,64,65). Mineralization is governed by desorption of hydrocarbons from soil (1,47,48). Surfactants can increase the surface area of hydrophobic materials, thereby increasing their water solubility. Hence, the presence of surfactants may increase microbial degradation of pollutants in both soil and water.

Most industrial surfactants are chemically derived, and their replacement by

biosurfactants may be advantageous. For example, novel compounds may be produced biologically which are different and more effective for specific purposes. Also, in contrast to

some chemical surfactants, biosurfactants can be degraded by microorganisms (53,63,67). Furthermore, some biosurfactants may be less toxic than synthetic surfactants. In one study,

either the commercial surfactant Finasol OSR5 or a trehalose-lipid biosurfactant was added to oil flooded sections of tidal mud flats along the North Sea. The effect of these surfactants on diatom photosynthetic activity, on the viability of diatoms, ciliates, nematodes, copepods and halophytic plants, and on oil removal was monitored (19). The biosurfactant was non-

toxic, and the oil was removed more effectively compared to the commercial surfactant, which was highly toxic to marine organisms (20).

Biosurfactants can be produced using low cost substrates, however a low rate and yield of product formation, along with purification procedures can result in higher prices than

for chemical surfactants. The high cost of toxicity testing and the time required to have a new compound approved for use further increases the cost of biosurfactants (36). While a sophorose lipid produced in high yield by Torulopsis bombicola was estimated to cost an amount comparable to synthetic surfactant Span 60 (17), Emulsan, a heteropolysaccharide

emulsifier produced by Acinetobacter calcoaceticus RAG-1 (70), costs over three times more than Span 60 (31). Clearly, methods to improve surfactant production are important in

developing an economical product.

HYDROCARBON DEGRADATION IN THE SOIL ENVIRONMENT

About 2 billion metric tons per annum of petroleum are produced worldwide and 0.08 to 0.4% of the total production will eventually pollute the oceans (5). Crude oil contains mutagenic, carcinogenic and growth inhibitory compounds, which can cause severe toxic

APPLICATIONS OF MICROBIAL SURFACTANTS 243

effects to marine organisms (28). The extent of terrestrial petroleum pollution is not known,

but is estimated to equal that in the marine environment (5). Hydrocarbon degradation in soil has been extensively reviewed (2,3,5,25,42,49,66).

Degradation is dependent on the presence and species of degrading microorganisms, hydrocarbon composition, oxygen availability, water, temperature, pH and inorganic nutrients. The physical state of the hydrocarbons can also affect biodegradation. Hydrocarbons are hydrophobic and bind firmly to soil particles. They also form pockets in soil that may exclude water and nutrients. The surface area of oil can be increased by adding synthetic or biological surfactants. This results in increased mobility and solubility of hydrocarbons, which is essential for effective microbial degradation (49).

There is growing interest in biological methods to treat hydrocarbon contaminated soils. One method is to treat contaminated soil in a bioreactor, where optimal operating conditions are provided to maximize microbial degradation. In situ remediation methods have also been studied to attain an economical process resulting in a high level of restoration. Partially purified biosurfactants can be used either in bioreactors or in situ to emulsify and increase the solubility of hydrophobic contaminants. Alternatively, surfactant- producing organisms may be added, or if already present, conditions in the soil can be optimized to increase the growth and activity of these microorganisms.

There are variable results as to the utility of using biosurfactants in hydrocarbon biodegradation. Lindley and Heydeman (44) found that the fungus Cladosporium resinae

when grown on alkane mixtures produced extracellular fatty acids and phospholipids,

mainly dodecanoic acid and phosphatidylcholine. When the growth medium was supplemented with phosphatidylcholine, the alkane degradation rate was enhanced up to 30%, with the increase being dependent on alkane size. In another study, Foght et al. found the emulsifier Emulsan to inhibit alkane mineralization by pure and mixed bacterial cultures (24). This emulsifier stimulated aromatic mineralization by pure cultures, but inhibited aromatic degradation by mixed cultures.

A mixed soil population was used by Oberbremer and Muller-Hurtig to assess hydrocarbon degradation in a model oil (52). During the first phase of degradation, naphthalene was utilized. Most of the other oil components were degraded during the second phase, after production of surfactants by soil microorganisms lowered the interfacial tension. Adaptation periods for the two phases could be shortened by adding biosurfactants such as sophorose lipids, which increased both the extent of degradation and final biomass yield (53). On completion of hydrocarbon degradation, the added biosurfactants were degraded by the mixed microbial population.

Biodetox (Germany) described a process to decontaminate soils, industrial sludges and wastewaters (39). The procedure involves transport of contaminated material to a big- pit process for microbial degradation. Biodetox also uses in situ bioreclamation for surface, deep ground and ground water contamination. Microorganisms are added by means of a

244 M.I. VAN DYKE et aL

"Biodetox foam", which is not harmful to the environment, contains bacteria, nutrients and surfactants, and can be biodegraded. However the type of bacteria, surfactants, nutrients used, and other experimental details of this process have not been published.

Another method of removing oil contaminants is to add biosurfactants into soil to increase hydrocarbon mobility. The emulsified hydrocarbons could then be recovered by a production well, and degraded above ground in a bioreactor. /n situ water washing of soil was studied using two synthetic surfactants, Adsee 799 and Hyonic NP-90 (22). A significant improvement was obtained in removal of PCBs and petroleum hydrocarbons from soil by adding surfactants to the wash water. It was shown that bacteria from a well contaminated with JP-5 jet fuel could emulsify the fuel if well water was supplemented with phosphate and nitrate (21). The emulsions could then be pumped to the surface for

treatment. Several strains of anaerobic bacteria are known to produce biosurfactants (18,26,40),

however the observed reduction in surface tensions (45 to 50 mN/m) was not as great as those produced by aerobic organisms (27 to 30 raN/m) (16,27). The surfactant produced by Bacil/us licheniformis JF-2 was able to reduce the surface tension of mineral medium to 28 mN/m under anaerobic conditions (34,46). The organism forms the same surfactant under both aerobic and anaerobic conditions, and may therefore be useful for biosurfactant production in anaerobic soils.

Biosurfactants may be used to enhance the solubilization of toxic organic compounds in addition to those found in the petroleum industry. Berg et al. (7) described an emulsifying agent produced by Pseudomonas aeruginosa UG2 that increased the solubility of hexachlorobiphenyl added to soil slurries, resulting in a 31% recovery of the compound in the aqueous phase. This was about three times higher than that solubilized by sodium ligninsulfonate (9.3%). When the P. aeruginosa bioemulsifier and sodium ligninsulfonate were used together, an additive effect (41.5%) on solubilization was observed. The enhanced solubilization of hexachlorobiphenyl may increase its accessibility to degrading microorganisms. Pseudomonas cepacia AC1100 degrades the insoluble, chlorinated herbicide 2,4,5-trichlorophenoxyacetic acid (4). This strain produced an emulsifier able to form a stable suspension with 2,4,5-trichlorophenoxyacetic acid, and also exhibited some emulsifying activity against chlorophenols. This emulsifier may be useful in the enhancement of bacterial degradation of chlorinated compounds.

MICROBIAL ENHANCED OIL RECOVERY (MEOR) An area of considerable potential for biosurfactant application is MEOR. Enhanced

oil recovery methods were devised to recover oil remaining in reservoirs after primary and secondary recovery procedures. In MEOR, microorganisms in reservoirs are stimulated to produce polymers and suffactants, Biosurfactants aid MEOR by lowering interfacial tension at the oil-rock interface. This reduces capillary forces that prevent oil from moving through

APPLICATIONS OF MICROBIAL SURFACTANTS 24s

rock pores. Biosurfactants can also aid in oil emulsification, and assist in the detachment of oil films from rocks (62).

For MEOR, microbial polymers and surfactants can be produced above ground and introduced into the reservoir through wells, or microorganisms within the reservoir can be stimulated to produce these compounds. The former method is more expensive, due to the capital and operating costs of bioreactors, product purification, and introduction into oil containing rock (50). To produce biosurfactants in situ, microorganisms in the reservoir are usually provided with low cost substrates such as molasses and inorganic nutrients to promote growth and surfactant production. This approach requires that the reservoir contains bacteria capable of producing sufficient amounts of surfactants. If not, a surfactant- producing strain may need to be introduced. Introduced organisms must compete with the indigenous microbial population for binding sites on rocks and for added nutrients. Another problem with inducing microbial growth in a reservoir is that the production of compounds may adversely affect oil quality. For example, sulfate-reducing bacteria, if present, can cause souring of crude oil and subsequent corrosion of equipment (11). To be useful for MEOR in situ, bacteria must be able to grow under severe environmental conditions encountered in oil reservoirs, such as high temperature, pressure, salinity and low oxygen levels. At the extreme end, temperatures and pressures have been reported to be 96oc and 2 x 104 kPa in the North Sea Forties Field, and 125oc and 5 x 104 kPa in the Ninian Field (62). Bacteria that tolerate and produce surfactants under some of the MEOR conditions have been found. For example, halobacteria capable of producing surface active agents have been isolated (56). Several anaerobic thermophiles tolerant of pressure and moderate salinity were found, which are able to mobilize crude oil in the laboratory (43). As mentioned earlier, the bacterium B./icheniformis produces the same surfactant under both aerobic and anaerobic conditions (34,46). Bubela et el. (12) described an apparatus for continuous growth of microorganisms under oil reservoir conditions of 65oc and 2 x 104 kPa. They suggested that this system may be useful for isolating surfactant producing microorganisms which can grow under some MEOR conditions.

Clark et al. (14) conducted a computer search and estimated that about 27% of oil reservoirs in the United States have conditions which are amenable to microbial growth and hence MEOR. The effectiveness of MEOR has been reported in field studies carded out in the United States, Czechoslovakia, Romania, USSR, Hungary, Poland and The Netherlands, with significant increases in oil recovery noted in some cases. However the data may be unreliable due to lack of proper controls, insufficient data accumulation and failure to take into account of factors which can affect the reservoir, especially considering the long time span for such experiments (11).

246 M.I. VAN DYKE et al.

HYDROCARBON DEGRADATION IN AQUATIC ENVIRONMENTS

When oil is spilled in aquatic environments, the lighter components volatilize while the polar, soluble components dissolve in water. Because of low solubility (

APPLICATIONS OF MICROBIAL SURFACTANTS 247

oils and enhance oil recovery (6). Emulsan was used to stabilize a 70% emulsion of

Venezuelan crude oil which was then pumped through a pipeline under high stress. The

viscosity of the oil was lowered, and inversions of oil-in-water emulsions, which can occur

with classical surfactants, did not result. Emulsan may also be used to stabilize water-in-oil

emulsions in fuel. Small amounts of water are added to fuel to improve its combustion characteristics (29).

OTHER APPLICATIONS

Biosurfactants have also been used in the formulation of poorly soluble

organophosphorus pesticides. For example, two Baci//us strains which produce an emulsifier, possibly a glycolipopeptide, able to form a stable emulsion in the presence of the

pesticide fenthion (55) were found to possess some activity against other liquid immiscible

organophosphorus pesticides, but not solid organophosphorus pesticides, organochlorine

pesticides, or hydrocarbons.

Mulligan and Cooper (51) have suggested that biosurfactants can be used as dewatering agents in pressing peat. Peat has a water content of 90% which is normally removed by pressing or thermal drying. When surfactants are added to the peat before

pressing, the amount of water released is enhanced. However, the water pressed out of peat contains organic matter, and may present a pollution problem. One way to address this

problem is to use the pressate as a substrate for suffactant production by microorganisms.

When small amounts of peptone, yeast extract or glucose were added to peat water, B.

subti/is produced a surfactant, which could be used to enhance water release from peat (51). Biosurfactants may be used for the dispersion of inorganic minerals in mining and

manufacturing processes. Rosenberg eta/. (60,61) described the production by A. ca/coaceticus A2 of an anionic polysaccharide called biodispersan, which prevented flocculation and dispersed a 10% limestone in water mixture. They suggested that for a

microbially produced compound to act as a dispersant, it must have low molecular weight and high charge density, as opposed to a hydrocarbon emulsifier which must be

amphipathic and have high molecular weight. Similarly, Kosaric eta/. (38) have isolated surface active materials produced by pure and mixed cultures of Pseudomonas sp. and A/caligenes sp. that could be used for the flotation separation of scheelite and calcite. The recoveries obtained were 95% for CaWO4 and 30% for CaCO 3. Conventional chemical

reagents were unable to separate these two minerals.

KAO Chemical Co. (Japan) used Pseudomonas, Corynebacterium, Nocardia, Arthrobacter, Baci//us, and A/ca/igenes sp. to produce surfactants for the stabilization of coal slurries (35) which could aid in the transportation of coal. The Research institute of Synthetic Fibers in USSR described a suffactant produced by Candida yeasts for which there are uses in textile, pharmaceutical and cosmetics industries (57).

248 M.I. VAN DYKE et al.

ACKNOWLEDGEMENTS Research in the authors' laboratories on biosurfactants was supported by Imperial Oil

Limited, the Institute for Chemical Science and Technology, the Natural Sciences and Engineering Research Council of Canada, and the Ontario Ministry of the Environment. The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the Ontario Ministry of the Environment, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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