oil and hydrocarbon spills ii, c.a. brebbia & g.r ... · oil and hydrocarbon spills ii:...

Potentials for use of biosurfactants in oil spills

cleanup and oil bioremediation

I.M. Banat

School of Environmental Studies, University of Ulster, Northern Ireland

Abstract

Biosurfactant are environmental friendly, effective and stable compounds withmany advantages over synthetic surfactants. Their use is gaining prominence inseveral industrial applications due to their broad capabilities which includesemulsification, wetting, foaming, solubilisation and viscosity reduction. Theirmain types are glycolipids in which carbohydrates are attached to a long-chainaliphatic acids. Other more complex types such as lipopeptides, lipoproteins andheteropolysaccharides also exist. The use of biosurfactants for oil spills cleanupor enhanced oil recovery involves a reduction of the oil-water interfacial tensionleading to its emulsification. Stable emulsions are formed because biosurfactantslowers interfacial tension between interfaces and oil. Such an effect can beachieved through the direct addition of active microbial cells to the contaminatedenvironments or augmentation with the biosurfactant compounds. Laboratorystudies have shown that the addition of biosurfactant mixtures alone may beuseful for stimulating biodegradation of hydrocarbon contaminants in theenvironment. Biosurfactants are also useful in solubilisation and removal of oilfrom sand and sludge in oil storage tanks. In ecological terms, the use ofbiosurfactants is obvious in closed systems but remains speculative in the openenvironment. The precise mechanism of enhanced oil recovery in situ however,remains unclear and mainly economically unfeasible. Both productcharacterisation and production process optimisation are expected to facilitatebiosurfactants' future applications particularly in oil-related industries andenvironmental protection and by cleaning-up agencies. This article reviews thestate of the art in potential uses in biosurfactants in oil bioremediation.

Oil and Hydrocarbon Spills II, C.A. Brebbia & G.R. Rodriguez (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-828-7

178 Oil and Hydrocarbon Spills II: Modelling, Analysis and Control

Introduction

Biosurfactants are extracellular amphiphilic compounds produced bymicroorganisms. They contain both a hydrophobic and a hydrophilic moietiesthat renders them capable of reducing surface tension and interfacial tensionsbetween molecules at the surfaces and interfaces leading to the formation ofmicro-emulsions of oil in water or water in oil. The worldwide surfactantsmarkets in 1994 was estimated at around $9.4 billion per annum [1] and isexpected to steadily increase [2]. Most of the commercially available surfactantsare petroleum-derived chemical. Rapid advances in biotechnology and increasedenvironmental awareness combined with expected new legislation has providedfurther impetus for consideration of biological surfactants as possible alternativesto the existing products.

Microbial biosurfactants' spontaneous release and function are oftenrelated to hydrocarbon uptake; therefore, they are predominantly synthesized byhydrocarbon degrading microorganisms. Some, however, have been reportedproduced on water-soluble compounds such as glucose, sucrose, glycerol orethanol. In some instances, these compounds have antibiotic properties that mayserve to disrupt membranes of microorganisms competing for food. Lowtoxicity, biodegradable nature and diversity have gained them considerableinterest in recent years. The range of potential industrial applications includesenhanced oil recovery (EOR), crude oil drilling, lubricants, surfactant aidedbioremediation. Other developing areas of biosurfactants use include health care,cosmetic and foods industries.

A large variety of biosurfactants are known; their type, quantity andquality are influenced by the nature of the C, N, P, Mg, Fe and Mn ions availablein the medium and their culture conditions, including pH, temperature, agitationand dilution rate. When considering what microorganisms to use for EOR, thevarying conditions in which they will be used, such as temperature, pressure, pHand salinity must be assessed. Typically, microorganisms injected into an oilwell should be able to endure high temperatures, pressures, salinity and becapable of growth under anaerobic or microaerophilic conditions.

Several types of biosurfactants have been isolated and characterisedincluding glycolipids, phospholipids, neutral lipids, fatty acids, peptidolipids,lipopolysaccarides and others not fully characterised [2], Certainmicroorganisms are likely to be found better adapted to particular environmentssuch as oil reservoirs, soil or the ocean environment.

Several techniques have been developed to identify biosurfactant-producingmicrobes. These technique include:

1- The axisymmetric drop shape analysis by profile (ADSA-P), whichsimultaneously determines the contact angle and liquid surface tension fromthe profile of a culture droplet resting on a solid surface.

2- Coloured indicator technique for determining anionogenic bacterialpeptidolipid based on the ability of the anionic surfactants to form acoloured complex with the cationic indicator such as methylene blue [3].

3- Measurements of surface and interfacial tension reductions in culture broth.


Oil and Hydrocarbon Spills II: Modelling, Analysis and Control 179

In addition to the above other simpler methods were described [4] such asblood hemolysis (a known characteristic for some biosurfactant compounds), theemulsification index value (E-24) obtained on kerosene or simply by observingdrop collapsing characteristics of culture broth suspensions placed on anoil-coated surface. Drops containing biosurfactants were observed to collapse,

whereas non-surfactant-containing drops remain stable.

Biosurfactant production and recovery

Conditions that promote biosurfactants production vary and have beendetermined for several microorganisms. On the one hand, the most commonbacteria reported (f. aeruginosd) has been shown to produce rhamnolipidsbiosurfactants on C12 n-alkanes [5]. Increased production was noted in somecases in phosphate-limited medium [6], or upon the exhaustion of nitrogen in themedium [7]. On the other hand, a Rhodococcus sp. had maximum growth andbiosurfactant production on medium containing 2%(v/v) n-paraffin using nitrateas N source and it's product was found to be a primary metabolite that could beproduced in continuous culture [8].

Other substrate such as olive oil mill effluent (Oome), whey and peatpressate have also been used for biosurfactant production. A Pseudomonasstrain Pet-1006 required two carbon sources; a readily available one (glucose)and a hydrocarbon (oleic acid) to be utilised upon glucose exhaustion thereforetriggering biosurfactant production [9].

Biosurfactants recovery from medium or microorganisms is usuallydesirable. Several procedures have been reported the most common beingmethanol or isopropanol precipitation, or acidification of culture media followedby solvent extraction with chloroform / methanol.

Biosurfactants applications

Oil industry is the largest market expected for biosurfactants use, both inpetroleum production and incorporation into oil formulations. Other applicationsrelated to the oil industries includes oil spill bioremediation/dispersion, bothinland and at sea, removal/ mobilisation of oil sludge from storage tanks andenhanced oil recovery [4,10]. The second largest market for biosurfactants isemulsion polymerisation for paints, paper and industrial coatings.

Surfactants are also used in food and cosmetic industries, industrialcleaning of products as well as in agricultural chemicals as pesticides and todilute and disperse fertilisers and enhance penetration of active compounds intoplants. Various potential applications of biosurfactants are shown in Table 1,some of which are discussed below.



Table 1. Areas of possible potential applications for biosurfactants in industry.

No

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Property

Emulsifiers and dispersant

Solubilizers and microemulsions

Wetting and penetrating agents

Detergents

Foaming agents

Thickening agents

Metal sequestering agents

Vesicle forming materials

Microbial growth enhancers

Demulsifiers

Fungicide

Viscosity reducing agents

Dispersants

Potential fields of application

Cosmetics, paints, bioremediation, oil tanks cleaning

Toiletries and pharmaceuticals

Pharmaceuticals, textile industry and paints

Household, agriculture and high tech. Products

Toiletries, cosmetics and ore floatation

Paints

Mining

Cosmetics, drug delivery system

Sewage sludge treatments for oily wastes

Waste treatment and oil recovery/separation

Biological control of some plant pathogens

Pipeline transportation

Coal -oil and coal-water slurry mixing

Biosurfactants use in hydrocarbons bioremediation

Several oil spill accidents and occasionally deliberate releases have taken placein recent years resulting in significant contamination of oceans and shorelineenvironments. Such incidents have intensified attempts to develop variousproducts, procedures and techniques for combating oil pollution both at sea andthe shorelines. Biosurfactants are one such chemical, which has been applied inparts of the Exxon Valdez oil spill [11]. The ability of biosurfactants to emulsifyhydrocarbon-water mixtures enhances the degradation of hydrocarbons in theenvironment. The presence of hydrocarbon degrading microorganisms inseawater renders biodegradation one of the most efficient methods for removingpollutants. Most biosurfactants have lower possible toxicity and persistence inthe environment in comparison to chemical surfactants [12]. The ability of asurfactant to enhance biodegradation of slightly soluble organic compounddepends on extent to which it increases the bioavailability of the compound.

Harvey et al. [11] tested a biosurfactant from P. aeruginosa for its abilityto remove oil from contaminated Alaskan gravel samples under variousconditions including concentration of surfactant, time of contact, temperature ofthe wash and presence or absence of gum. They reported increased oildisplacement (about 2-3 folds) in comparison to water alone. Necessary contacttime for the maximum effect was also reduced from 1.5-2 min. for water to 1min. In addition, the Environmental Technology Laboratory at University of



Alaska, Fairbanks reported complete removal of diesel range petroleumhydrocarbons (to the limit of 0.5mg kg"') while semi volatile petroleumhydrocarbons were reduced to 70% level, a removal of 30% [13]. These resultsdemonstrated the capacity of biosurfactants to remove oil from naturallyoccurring substrate.

Interest in biosurfactants applications in treating hydrocarbon-contaminated soils has recently intensified [10]. Hydrocarbon degradation by themicrobes present in the contaminated soil is the primary method for removal ofhydrocarbon pollutant from the soil. Partially purified biosurfactants can beeither used in bioreactors or in situ to emulsify and increase the solubility ofhydrophobic contaminants. Alternatively, either surfactant producingmicroorganisms or growth limiting factors may be added to the soil to enhancegrowth of added or indigenous microorganisms capable of producingbiosurfactants.

Biodetox (Germany) described a process to decontaminate soils,industrial sludge and waste waters [14]. The procedure involves transport ofcontaminated materials to a biopit process for microbial degradation. Biodetoxalso performs in situ bioreclamation for surface, deep ground and ground watercontamination. Microorganisms are added by means of "Biodetox foam", whichis not harmful to the environment, contains bacteria, nutrients and biosurfactantsand can be biodegraded. Jain et al. [15] found that the addition of Pseudomonasbiosurfactant enhanced the biodegradation of tetradecane, pristane, andhexadecane in a slit loam with 2.1% organic matter. Similarly Zhang & Miller[16] reported the enhanced octadecane dispersion and biodegradation by aPseudomonas rhamnolipids surfactant. Falatko & Novak [17] studiedbiosurfactant-facilitated removal of gasoline overlaid on the top of coarse grainsand packed column. Up to 15-fold increase in the effluent concentration of fourgasoline constituents; toluene, m-xylene, 1,2,4-trimethylbenzene, andnaphthalene was observed upon the addition of biosurfactant solution (600mg 1"*)

Herman et al. [18] investigated the effects of rhamnolipids biosurfactantson in situ biodegradation of hydrocarbon entrapped in porous matrix andreported a mobilisation of hydrocarbon entrapped within the soil matrix atbiosurfactants concentration higher than the critical micelle concentration(CMC). At concentrations lower than CMC they detected enhanced in-situmineralization of entrapped hydrocarbon. One of the methods of removing oilcontaminants is to add biosurfactants into soil to increase hydrocarbon mobility.The emulsified hydrocarbon can then be recovered by a production well anddegraded above ground in a bioreactor. Bai et al. [19] used an anionic monorhamnolipid biosurfactant from P. aeruginosa to remove residual hydrocarbonsfrom sand columns. They recovered approximately 84% of residual hydrocarbon(hexadecane) from sand column packed with 20/30 mesh sand and 22%hydrocarbon from 40/50-mesh sand, primarily because of increased mobilisation.They reported the optimal concentration of rhamnolipid of 500 mg 1"' and arange of possible use of 40-1500 mg 1"\



Biosurfactants and PAH & metal bioremediation

Applying surfactants as immobilizing agents might be one way to enhance thesolubility of PAHs as they may increase their solublization or emulsification, torelease hydrocarbons sorbed to soil organic matter and increase the aqueousconcentrations of hydrophobic compounds resulting in higher mass transferrates. In an investigation of the capacity of PAH utilising bacteria to producebiosurfactants using naphthalene and phenanthrene Daziel et al. [20] concludedthat biosurfactant production was responsible for an increase in the aqueousconcentration of naphthalene. This indicates a potential role for biosurfactant inincreasing the solubility of such compounds. Similarly Zhang et al. [21] testedtwo rhamnolipid biosurfactants' effects on dissolution and bioavailability ofphenanthrene and reported increased solubility and degradation rates. SimilarlyNoordman et al. [22] tested rhamnolipid (500 mg 1"*) biosurfactant solution'sability to enhanced removal of phenanthrene from contaminated soil in alaboratory columns' study and detected significant enhanced removal ofphenanthrene compared to controls.

Biosurfactant have also been reported to promote heavy metals de-sorption from soils in two ways [23]. The first is through complexation of thefree form of the metal residing in solution which decreases the solution-phaseactivity of the metal and therefore promotes de-sorption. The second occursunder reduced interfacial tension conditions; the biosurfactants will accumulateat the solid-solution interface that may allow the direct contact between thebiosurfactant and the sorbed metal.

Other workers [24] reported significant complexation / sorption of somemetals from contaminated soils using to a rhamnolipid biosurfactant. Althoughbioremediation of metal contaminated soils appear to have promise it isimportant to understand the factors affecting rhamnolipids sorption to the soil toachieve better metals removal and develop this technology. These factors includeionic strength, mineral composition and pore water chemistry within metalcontaminated soils. Future success of biosurfactant technology in bioremediationinitiatives will require targeting their use to the physical conditions and chemicalnature of the polluted sites to maximise efficiency and economical viability.

Biosurfactant and microbial enhanced oil recovery

An area of considerable potential for biosurfactant application is in the field ofmicrobial enhanced oil recovery (MEOR). Biosurfactants can aid in oilemulsification and assist in the detachment of oil films from rocks [4,10]. In situremoval of oil is due to multiple effects of the microorganisms on environmentand oil. These effects include gas and acid production, reduction in oil viscosity,plugging by biomass accumulation, reduction in interfacial tension bybiosurfactants and degradation of large organic molecules. These are all factorsresponsible for decreasing the oil viscosity and making its recovery easier.

The strategies involved in the MEOR depend on the oil reservoir prevalentconditions including temperature, pressure, pH, porosity salinity, geologic make



up of the reservoir, available nutrients and the presence of indigenousmicroorganisms. These factors should be considered before devising a strategyfor use in an oil well. There are three main strategies for use of biosurfactants inEnhanced Oil Recovery (EOR) or mobilisation of heavy oils.

a- Production in batch or continuous culture under industrial conditionsfollowed by addition to the reservoir in the conventional way along with thewater flood (ex situ MEOR).

b- Production of surface active compounds by microorganisms at the cell-oilinterface within the reservoir formation implying penetration ofmetabolically active cells into the reservoir.

c- Injection of selected nutrients into a reservoir, thus stimulating the growthof indigenous biosurfactant producing microorganisms.

The first strategy is expensive due to capital required for bioreactorsoperation, product purification and introduction into oil containing rocks. Thesecond and third strategy requires that the reservoir contain bacteria capable ofproducing sufficient amounts of surfactants. For production of biosurfactants,microorganisms are usually provided with low cost substrates such as molassesand inorganic nutrients, which promote growth and surfactant production.Alternatively surfactant-producing strains may be introduced into the well. Theintroduced organism faces competition from the indigenous population ofmicrobes for the binding sites on rocks and for the added nutrients.

Another application of biosurfactants is oil storage tank cleaning.Surfactants have been studied for use in reducing the viscosity of heavy oils,thereby facilitating recovery, transportation and pipelining. In a full-scale fieldinvestigation Banat et al. [9] tested the ability of biosurfactant to clean oilstorage tanks and to recover hydrocarbon from the emulsified sludge. Two tonesof biosurfactant-containing whole cell culture were used to mobilise and clean850 m^ oil sludge. Approximately 91% (774 m^) of this sludge was recovered asre-sellable crude oil and 76 nf non-hydrocarbon materials remained asimpurities to be manually cleaned. The value of the recovered crude covered thecost of the cleaning operation ($100,000-150.000 per tank). Such a clean upprocesses is therefore economically rewarding and less hazardous to personsinvolved in the process compared to conventional process. It is also anenvironmentally sound technology leading to less disposal of oily sludge in thenatural environment. To our knowledge however, further commercialapplications of this technology has not been carried out.

Conclusion

The usefulness of biosurfactants in bioremediation is expected to gain increasingimportance in the future. To date, biosurfactants are unable to competeeconomically with the chemically synthesised compounds in the market mainlydue to their high production costs and lack of comprehensive toxicity testing.Their success in bioremediation will require precise targeting to the physicalconditions and chemical nature of the pollutant affected areas. Encouraging



results have been obtained for use of biosurfactants in hydrocarbon pollutioncontrol in marine biotypes, in closed systems (oil storage tanks) and althoughmany laboratory studies indicate potentials for use in open environment a lotremains undemonstrated in pollution treatment in marine environments or coastalareas. The possible use of biosurfactants in MEOR has many advantages, yetmore information about structures and factors such as interaction with soil,structure function analysis of surfactant solubilization, scale up and cost analysisfor ex-situ production are required.

Acknowledgement

We would like to thank the Environment and Heritage Service, DOE, for FRDFfinancial support under the N. Ireland Single Programme (Ref. WM 47/99).

References

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[9] Banat, I.M., Samarah, N., Murad, M., Home, R. & Benerjee, S. Biosurfactantproduction and use in oil tank clean-up. World Journal Microbiology &Biotechnology,!,pp. 80-84, 1991.

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[11] Harvey, S., Elashvili, I., Valdes, J.J., Kamely, D. & Chakrabarty, A.M.Enhanced removal of Exxon Valdez spilled oil from Alaskan gravel by amicrobial surfactant. Bio/ Technology. 8, pp. 228-230,1990.

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