Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: A review

Bioresource Technology 51 (1995) 1-12

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© 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/95/$9.50

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ELSEVIER

I 0 1-4

BIOSURFACTANTS PRODUCTION A N D POSSIBLE USES IN MICROBIAL E N H A N C E D OIL RECOVERY A N D OIL POLLUTION REMEDIATION: A REVIEW I. M. Banat Department of Biology, United Arab Emirates University, PO Box 17551, Al-Ain, Abu-Dhabi, UAE (Accepted 4 October 1994)

Abstract

Surfactants are widely used for various purposes in industry, but for many years were mainly chemically synthesized. It has only been in the past few decades that biological surface-active compounds (biosurfactants) have been described. Biosurfactants are gaining prominence and have already taken over for a number of important industrial uses, due to their advantages of biodegradability, production on renewable resources and functionality under extreme conditions; particularly those pertaining during tertiary crude-oil recovery. Conflicting reports exist concerning their efficacy and the economics of both their production and application. A t present, their uses are mainly in the oil and petroleum industries, where they are employed primarily for their emulsification capacity in both tertiary recovery and polluted-sites remediation. However, caution is frequently exercised with respect to their use because of possible subsequent microbial contamination of either underground oil reservoirs or products. The limited successes and applications for biosurfactants' production, recovery, use in oil pollution control, oil storage tank clean-up and enhanced oil-recovery are reviewed from the technological point of view. Key words: Biosurfactants, emulsification, biodegradation, bioremediation, microbially-enhanced oil recovery, MEOR.

INTRODUCTION

Biosurfactants are a heterogeneous group of surfaceactive molecules produced by microorganisms. These molecules reduce surface tension, critical micelle concentration (CMC) and interracial tension in both aqueous solutions and hydrocarbon mixtures. These properties create micro-emulsions in which micelle formation occurs where hydrocarbons can solubilize in water, or water in hydrocarbons. The properties of the various biosurfactants have been extensively reviewed (Cooper, 1986; Rosenberg, 1986; Haferburg et al., 1990; Fiechter, 1992a; Georgiou et al., 1992; Kosaric,

1993). Generally the structure of biosurfactants includes a hydrophilic moiety composed of amino acids or peptides, anions or cations, or mono-, di-, or polysaccharides. The hydrophobic portion is often made up of saturated, unsaturated or hydroxylated fatty acids (Georgiou et al., 1992), or composed of amophophilic or hydrophobic peptides. World-wide interest in biosurfactants has increased immensely due to their ability to meet most synthetic surfactants' requirements (Morkes, 1993). Biosurfactant(s) spontaneous release and function are often related to hydrocarbon uptake; therefore, they are predominantly synthesized by hydrocarbondegrading microorganisms. Some biosurfactants, however, have been reported to be produced o n water-soluble compounds, such as glucose, sucrose, glycerol or ethanol (Guerra-Santos, 1986; Cooper & Goldenberg, 1987; Palejwala & Desai, 1989; Passeri et al., 1992; Hommel & Huse, 1993). In some instances, these compounds have antibiotic properties which may serve to disrupt membranes of microorganisms competing for food. Examples of these include the lipopeptides of the iturin family produced by Bacillus subtilis, which have powerful anti-fungal properties (Sandrin et al., 1990; Thimon et al., 1992), Candida antarctica, which have antimicrobial activity (Kitamoto et al., 1993), and Bacillus licheniformis, which inhibit bacteria, yeast and filamentous fungi (Fiechter, 1992a). Chemically-synthesized surfactants have been used in the oil industry to aid the clean up of oil spills, as well as to enhance oil recovery from oil reservoirs. These compounds are not biodegradable and can be toxic to the environment. Biosurfactants, however, have been shown in many cases to have equivalent emulsification properties and are biodegradable. Thus, there is an increasing interest in the possible use of biosurfactants in mobilizing heavy crude oil, transporting petroleum in pipelines, managing oil spills, oil-pollution control, cleaning oil sludge from oil storage facilities, soil/sand bioremediation and microbiallyenhanced oil recovery (MEOR). M E O R offers major advantages over conventional E O R in that lower capital and chemical/energy costs are required (Sarkar et al., 1989).

2

I.M. Banat

BIOSURFACTANT-PRODUCING MICROORGANISMS A large variety of biosurfactants is known; their type, quantity and quality are influenced by the nature of the carbon substrate (Georgiou et al., 1992), the concentration of N, P, Mg, Fe and Mn ions in the medium (Atlas, 1981; Cooper et al., 1981 a, b; Guerra-Santos et al., 1984 & 1986; Haferburg et al., 1986; AbuRuwaida et aL, 199 lb) and culture conditions, including pH, temperature, agitation and dilution rate (Guerra-Santos e t al., 1984 & 1986; Abu-Ruwaida et al., 1991a; Fiechter, 1992a; Drouin & Cooper, 1992; Lin etal., 1994). When considering which microorganisms to use for MEOR, the varying conditions in which they will be used, such as temperature, pressure, pH and salinity, must be given priority (Khire & Khan, 1994b). Typically, microorganisms injected into an oil well should endure high temperatures, pressures and salinity, and be capable of growth under anaerobic or microaerophilic conditions. It has been estimated that 50-70% of oil wells in the USA could support microbial growth with pH values from 4 to 8, temperatures < 75°C and salinity < 10% (Clark et al., 1981 ).

Sources of cultures Several types of biosurfactant have been isolated and characterized, including glycolipids, phospholipids, neutral lipids, fatty acids, peptidolipids, lipopolysaccharides and others not fully characterized. Microorganisms which produce biosurfactants, and their structures, are listed in Table 1. Certain microorganisms are likely to be found to be better adapted to particular environments, such as oil reservoirs, soil or the ocean. A strain of Pseudomonas aeruginosa isolated from crude oil-associated injection water in Venezuelan oil fields was found to be adapted to the conditions prevalent in this oil reservoir (Rocha et al., 1992). Further, the biosurfactants (rhamnolipids) produced by this strain were not inactivated by pH, temperature, salinity, calcium or magnesium at concentrations in excess of those found in many oil reservoirs in Venezuela (Rocha et al., 1992). A halotolerant Bacillus licheniformis strain JF-2, was also isolated from oil field injection water, and was found to produce biosurfactant under both aerobic and anaerobic conditions (Jenneman et al., 1983; Javaheri et al., 1985). Bacillus strain SP018 was found to tolerate anaerobic conditions at 50°C and < 10% NaCl while producing biosurfactants (Pfiffner et al., 1986). Two

Table 1. Various biosurfactants produced by microorganisms

Microorganism A rthrobacter RAG- 1 A rthrobacter MIS 38 Arthrobacter sp. Bacillus licheniformis JF-2 Bacillus licheniformis 86 Bacillus subtilis Bacillus pumilus A1 Bacillus sp. AB-2 Bacillus sp. C- 14 Candida antarctica Candida bombicola Candida tropicalis Candida lipolytica Y-917 Clostridium pasteurianum Corynebacterium hydrocarbolastus Corynebacterium insidiosum Corynebacterium lepus

Strain MM1 Nocardia erythropolis Ochrobactrum anthropii Penicillium spiculisporum Pseudomonas aeruginosa Pseudomonas fluorescens Phaffta rhodozyma Rhodococcus erythropolis Rhodococcus sp. ST-5 Rhodococcus sp. H13-A Rhodococcus sp. 33 Torulopsis bombicola

Biosuffactant hetropolysaccharides lipopeptide trehalose, sucrose and fructose lipids lipopeptides lipopeptides surfactin surfactin rhamnolipids hydrocarbon-lipid-protein mannosylerthritol lipid sophorose lipids marman-fatty acid sophoros lipid neutral lipids protein-lipid-carbohy. phospholipids fatty acids glucose, lipid and hydroxydecanoic acids neutral lipids protein spiculosporic acid rhamnolipid lipopeptide carbohydrates-lipid trehalose dicorynomycolate glycolipid glycolipid polysaccharide sophorose lipids

Reference Rosenberg et al. (1979) Morikawa et al. (1993) Suzuki et al. (1974) Itoh et al. (1974) Mclnerney et al. (1990) Horowitz et al. (1990) Arima et al. (1968) Morikawa et al. (1992) Banat (1993) Eliseev etal. (1991) Kitamoto et al. ( 1992) Gobbert et al. (1984) Kappell and Fiechter (1976) Lesik etal. (1989) Cooper et al. (1980) Zajic etal. (1977) Akit etal. (1981) Cooper etal. (1979) Passeri (1992) MacDonald et al. (1981) Wasko and Bratt (1990) Ban and Sato (1993) Robert etal. (1989) Neu etal. (1990) Lesik etal. (1991) Shulga etal. (1990) Abu-Ruwaida et al. ( 1991 a) Singer and Finnerty (1990) Neu et al. (1992) Inoue and Ito (1982)

Biosurfactants production and applications

biosurfactant-producing Bacillus strains, AB-2 and Y 12-B, were also isolated from oil sludge-mixed sand and had the ability to grow on hydrocarbon-containing medium at -<50°C (Banat, 1993). Other microorganisms have been isolated from hydrocarbon-contaminated soils, such as Rhodococcus (Singer & Finnert, 1990; Abu-Ruwaida et al., 1991a, b), Bacillus pumilus (Morikawa et al., 1992), Arthrobacter sp. strain MIS38 (Morikawa et al., 1993); others were isolated from contaminated fuels, such as Ochrobactrum anthropii (Wasko & Bratt, 1990) or from oceanic oil spillage, such as Pseudomonas aeruginosa (Shafeeq et aL, 1989).

Techniques for identifying biosurfactant producers Techniques have been developed to help identify biosurfactant-producing microbes or quantify their production capabilities. One such technique is called the axisymmetric drop shape analysis by profile (ADSAP), which simultaneously determines the contact angle and liquid surface tension from the profile of a droplet resting on a solid surface (Van der Vegt et aL, 1991). Drops containing biosurfactant-producing microbes are placed on a fluoroethylene-propylene surface and the profile of the droplet determined with a contour monitor. Surface tensions are then calculated from the droplet profiles with ADSA-P. Surface tensions determined by this method were only found to be reduced by biosurfactant-producing bacteria. Shnlga et al. (1993) described a method for determining anionogenie bacterial peptidolipid biosurfactants based on the ability of the anionic surfactants to form a coloured complex with the cationic indicator methylene. Other, simpler, methods have been described, such as blood haemolysis (a known characteristic of some biosurfactant compounds) (Banat, 1993) and an emulsification index value (E-24) obtained on kerosine, as described by Cooper and Goldenberg (1987). A rapid drop-collapsing test was also employed to screen bacterial colonies for biosurfactant production (Jain et al., 1991). Drops of cell suspensions were placed on an oil-coated surface and if the drop contained biosurfactants it was observed to collapse, whereas non-surfactant-containing drops remained stable. BIOSURFACTANT PRODUCTION The fermentation of biosurfactant-producing microorganisms varies greatly and has been reviewed previously (Georgiou et al., 1992; Desai & Desai, 1993). Conditions which promote biosurfactant production have been determined for several microorganisms. Pseudomonas aeruginosa has been shown to produce rhamnolipids on C12 n-alkanes (Robert et aL, 1989), and increased production was noted on a phosphatelimited medium (Mulligan et al., 1989) or upon the exhaustion of nitrogen in the medium (Venkata Ramana & Karanth, 1989). The structure of surfactin has been shown to be influenced by amino acid con-

3

centration in the media to produce a Val-7 or Leu-7 surfactin (Peypoux & Michel, 1992). Rhodococcus sp. had maximum growth and biosurfactant production on medium containing 2% (v/v) n-paraffin and nitrate as the N source and its product was found to be a primary metabolite that could be produced in continuous culture (Abu-Ruwaida et aL, 1991 b). Other substrates, such as olive-oil mill effluent (Oome), whey and peat pressate, have also been used for biosurfactant production (Mercade et al., 1993; Mercade & Manresa, 1994). Mannosylerythritol lipid biosurfactants were produced at the highest level and accumulated by resting Candida antarctica cells in a medium containing only the carbon source (Kitamoto et aL, 1992a, b). Similar results were observed with sophorose lipids produced by resting Candida apicola (Hommel & Huse, 1993). A Pseudomonas strain Pet1006 required two carbon sources; a readily available one (glucose) and a hydrocarbon (oleic acid) to be utilized upon glucose exhaustion (Banat et aL, 1991). The results of a typical batch fermentation experiment on a biosurfactant-producing Rhodococcus ST-5 growing on paraffin oil and demonstrating biomass, surface tension, emulsification and critical micelle dilution (CMD) measurements against time, are shown in Fig. 1. Sophorose lipids were produced by Candida bombicola CBS 6009 which was grown in fed-batch fermentation (Davila et aL, 1992). Candida bombicola had previously been shown to produce sophorose lipids during a resting stage under conditions of nitrogen limitation (Gobbert et al., 1984). Davila et aL (1992) demonstrated that ethyl esters of rapeseed-oil fatty acids and glucose could be utilized to produce a high yield of sophorose lipids, with exclusion of the lipids from the aqueous phase. This lipid layer is easily separated by centrifugation and has the advantage of avoiding limitations in product concentration due to accumulation inhibition. An integrated system for biosurfactant production using P. fluorescens has also been developed (Fiechter, 1992b). In this system, HPLC is utilized to monitor biosurfactant production instead of CMC and surfacetension measurements, and a highly effective membrane technology for cell retention is employed to maximize the product formation rate (7"7 mg/1/h).

Biosurfactant recovery In many cases the recovery of biosurfactants from medium or microorganisms may be desirable. Several procedures exist: biosurfactants from Nocardia amarae, for example, have been isolated by methanol precipitation (Sutton, 1992), while rhamnolipids from P. aeruginosa have been isolated by acidification of culture media followed by extraction with chloroform/ methanol solvent (Robert et al., 1989). Improved isolation of glycolipids from Rhodococcus sp. H13A was accomplished using XM 50 diafiltration and isopropanol precipitation (Bryant, 1990). This technique has the advantage of separating the glycolipid from co-iso-

4

L M. Banat 100

~

80

-

-

60 -

100

80

--

x 0 • • • •

x~,~..- x -------.~. x

CMD -3

/x

E24 [

Surface tension

X

60

.~ 40 - ~ 4o

20 -

0 -

--

60

-

4o

--

20

20

0

I "

I-.~°~

0

8

1 5

100

Biomass CMD -l CMD -2

16

I

I

I

I

24

32

40

48

0

3 ~

.,,

--,

~_

11 0

Time (h)

Fig. 1. The results of a typical batch fermentation of n-paraffin mineral medium by Rhodococcus sp. ST-5 culture at 37°C, pH 6-8 and dissolved oxygen 30-50% saturation (Abu-Ruwaida et al., 1991 b). Symbols: x = biomass (g/l); o --critical mieelle dilution (CMD -1) (mN/m) at 1:10 culture broth to control medium; • =CMD -2 (mN/m) at 1:100 culture broth to control medium; • = CMD -3 (mN/m) at 1:1000 culture broth to medium; • =E24 (emulsification index %); • = surface tension (mN/m).

lated proteins. An aqueous two-phase fermentation system has been developed which separates suffactants on the basis of their charge (Drouin & Cooper, 1992). This two-phase system, employing polyethylene glycol and dextran, was found to partition cationic surfactants to the bottom phase and anionic surfactants to the top phase. Bacillus subtilis cells partitioned into the bottom phase, while the biosurfactant (surfactin) partitioned into the top phase. Synthesis and modification

Laboratory synthesis and modifications of biosurfactants have also been carried out. Chemical conversion of the carboxylic moiety of rhamnolipid to a non-ionic methyl ester resulted in enhanced interfacial lowering and wetting action (Ishigami et al., 1993). It was also noted that the modified rhamnolipid CMC value had increased significantly. Phospholipids were successfully modified by lipase-catalyzed transesterification to incorporate n-3 polyunsaturated fatty acids (Mutua & Akoh, 1993a). Transesterifications were also used to prepare polyunsaturated phospholipids (Totani & Hara, 1991). Candida cylindracea and Rhizopus delemar lipases were utilized to prepare polyunsaturated phospholipids from soy phospholipid and sardine oil. Alkyl glycoside fatty acid esters were also synthesized by lipase-catalyzed transesterification of methyl glucoside, methyl galactoside and octyl glucoside with methyl oleate (Mutua & Akoh, 1993b). A fixed-bed reactor has been utilized for continuous enzymatic transesterification of rapeseed oil and lauric acid (Forsell et al., 1993). These techniques may prove useful in designing biosurfactants or in producing larger quantities of relatively pure products.

BIOSURFACTANTS IN POLLUTION CONTROL Oil pollution accidents have become numerous and have caused ecological and social catastrophes (Burger, 1993; Shaw, 1992; Burns et al., 1993). The ability of biosurfactants to emulsify hydrocarbon-water mixtures has been widely reported (Broderick & Cooney, 1982; Harvey et al., 1990; Oberbremer et al., 1990; Wasko & Bratt, 1990; Francy et al., 1991; Zhang & Miller, 1992). These emulsification properties have also been demonstrated to enhance hydrocarbons degradation in the environment, hence making them potentially useful tools for oil spill pollution-control (Atlas & Bartha, 1992; Atlas, 1993; Bertrand et al., 1994). Bioremediation (laboratory experiments) Experiments with P. aeruginosa isolate $8, isolated

from oil-polluted seawater, showed its ability to degrade hexadecane, heptadecane, octadecane and nonadecane in seawater by up to 47, 58, 73 and 60%, respectively, after a 28 day incubation period (Shafeeq et al., 1989). Tensiometric studies indicated the presence of biosurfactants in the culture medium. Oil-polluted seawater, when supplemented with nitrogen and phosphate, was able to reduce the quantities of n-paraffins present in the crude oils (Vrdoljak et al., 1992). The addition of Pseudomonas aeruginosa UG2 biosurfactant to soil contaminated with a hydrocarbon mixture of tetradecane, hexadecane, pristane and 2-methylnaphthalene, followed by a 2 month incubation period, also showed enhanced degradation of all hydrocarbons except 2-methylnaphthalene (Jain et al., 1992).

Biosurfactants production and applications In another experiment, contaminated soil, packed into 8 ml solid-phase extraction columns, was inoculated with 108 cells/ml of Pseudomonas ML2 or Acinetobacter haemolyticus. Other treatments with ML2 biosurfactant product (at 41 or 82/~g/ml) were used in addition to the buffer controls. After 2 months, 39-71% reduction in hydrocarbons was achieved by A. haemolyticus, while the Pseudomonas ML2 showed 11-72% reduction (a very wide range). The treatment with the ML2-biosurfactant product gave the best results, yielding 44-46% reductions (when used at 41 /~g/ml) and 32-34% reductions (when used at 8 2 / t g / ml). Hydrocarbon reduction was believed to be due to degradation. These results suggested that using cellfree biosurfactant stimulated degradation by indigenous microorganisms in the soil, giving closely consistent results as compared to using actual microbial cells. This might have been due to poor adaptation and limited survival abilities of non-indigenous bacteria in the contaminated soil. Biosurfactants have also been demonstrated to successfully solubilize and remove hydrocarbon pollutants from contaminated substrate. Studies employing biosurfactant-containing culture broths from Rhodococcus ST-5 (Abu-Ruwaida et al., 1991a, b) and the thermophilic Bacillus AB-2 (Banat, 1993) were carried out to ascertain their effectiveness in removing residual oil from sand-packed columns. Sandpacks composed of 45-mesh acid-cleaned sand were saturated with oil and then flooded with three pore volumes of water to release unbound oil. Biosurfactant-containing culture media (ST-5, AB-2 and Pet 1006) were then compared to 0.1% SDS, 1% spolene and 1% petroleum sulfonate for their ability to remove the residual oil. Both Pet 1006 and AB-2 cultures broths were found to release 95% of the residual oil (Table 2), compared to 63 and 58% for the surfactants spolene and petroleum sulfonate, respectively (Banat, 1993). Eliseev et al. ( 1991 ) also reported the ability of a biosurfactant produced from Bacillus sp. C-14 to release oil from oily sand at a concentration of 0"04 mg/ml. These experiments demonstrated the effectiveness of biosurfactants in removing residual oil from sand and are suggestive of possible applications in EOR and in oil removal from oil-polluted sand. Numerous biosurfactant-producing microorganisms were screened for their ability to solubilize C 14 labelled

5

3,3', 4,4', 5,5'-hexachlorobiphenyl in hexane (Van Dyke et al., 1993 b). Their filtered biosurfactant-containing culture broths were used to solubilize the hydrocarbon mixed with soil in centrifuge tubes which were incubated with shaking for 2 h at 22°C, centrifuged and tested for solubilized hexachlorobiphenyl. Of the organisms tested, Acinetobacter calcoaceticus RAG-1 and Pseudomonas aeruginosa UG2 demonstrated the best solubilization, at 41-9 and 48"0%, respectively, compared to controls. Rhanmolipid biosurfactants from Pseudomonas aeruginosa were characterized for their ability to remove hydrocarbons from sandy-loam soil and siltloam soil (Van Dyke et al., 1993a). Soil containing hydrocarbon mixtures (naphthalene, anthracene, phenanthrene, fluorene, 2,2',5,5'-tetrachlorobiphenyl, 3,3',4,4',5,5'-hexachiorobiphenyl) plus partially-purified biosurfactant solutions were incubated before testing hydrocarbon removal. Distilled water was found, in most cases, to remove < 10% of the hydrocarbons from the soil. The rhamnolipids at a concentration of 5 g/1 were found to increase recovery of the hydrocarbons to 25-70% in silt-loam soil and 40-80% in sandy-loam soil. However, substantial adsorption of the rhamnolipid to the soil was observed and such a high concentration of biosurfactant (5 g/l) was used as to be highly impractical. Similar experiments with a mixture of aliphatic and aromatic hydrocarbons, mixed with sandy-loam soil yielded similar results (Scheibenbogen et al., 1994). In this study, a 0"08% mixture of rhamnolipids removed 36 and 40% of the aliphatic and aromatic hydrocarbons, respectively, compared to 8.9 and 7.2% for water. The addition of 0"1% pyrophosphate to the rhamnolipid mixture was found to enhance aliphatic and aromatic hydrocarbon removal to 56 and 73%, respectively. These results compared favourably with 2% Triton X-100 (43 and 71%) and 2% Tween 60 (7.9 and 32%), where the Tween 60 had the disadvantage of clogging the column after several total pore-volume passages. Bioremediation (field experiment) Oil-contaminated soil is a common problem and its treatment techniques, including excavation, incineration, landfarming and landfilling, can be difficult or economically prohibitive. The other most economical

Table 2. Effectiveness of various surfactant solutions in releasing residual light crude oil from sand (Abu-Ruwaida et aL, 1991; Banat etaL, 1991; Banat, 1993)

Surfactant

ST (mN/m)

RO (%)

RRO (%)

0"1% SDS 1% Spolene 1% Petroleum sulfonate ST-5 (from Abu-Ruwaida et aL, 1991a) Pet 1006 (from Banat etal., 1991) AB-2 (from Banat, 1993)

27"1 28"0 27"5 27-6 29"0 29"0

35 33 33 35 35 35

0"0 63"0 58"0 80"0 95"0 95"0

ST = surface tension; RO = residual oil; RRO = recovered residual oil.

6

I.M. Banat

methods include in situ bioremediation. Machine-oilcontaminated soil has been shown to be remediated by microbial inoculation and by biosurfactant treatment (Fry et al., 1993b). Furthermore, Fry et al. (1993a) demonstrated the successful bioremediation of oil-contaminated soil and groundwater from a US Army engineering plant using natural surfactants produced by indigenous microorganisms. The addition of biosurfactant can increase the bioavailability of hydrophobic compounds to receptive bacteria. Biosurfactants from Pseudomonas aeruginosa SB30 were tested for their abilities to remove oil from the Exxon Valdez Alaskan contaminated gravel in the laboratory (Harvey et al., 1990). A 1% biosurfactant solution was found to consistently yield three-times higher oil removal at temperatures > 40°C and 1 min contact time compared to water controls. These results demonstrate the capacity of biosurfactants to remove environmental pollutants, such as oil, from naturallyoccurring substrates. In a recent large-scale investigation, Bragg et at (1994) reported the effectiveness of bioremediation activities on the Exxon Valdez oil spill in situ. This was carried out through treatment of the contaminated shore lines with an oleophilic liquid fertilizer containing N and P to accelerate the growth of the natural ~ hydrocarbon-degrading microorganisms; chemical surfactant agents, in comparison, were found to be ineffective in removing oil from sediments. Other extensive bioremediation studies were successfidly carried out on oil-contaminated desert sand in Kuwait, both in situ and on-site (AI-Awadhi et al., 1994). All these techniques directly involved utilizing indigenous microbial populations, through the introduction of specific nutrients and oxygen to encourage biosurfactant production and hydrocarbon utilization (Miiller-Hurtig et al., 1993).

BIOSURFACTANTS IN OIL STORAGE TANK CLEAN-UP Field tests, utilizing biosurfactants produced from a proprietary bacterial strain (Pet 1006), were performed to test their ability to clean oil storage tanks and to recover hydrocarbons from the emulsified sludge (Banat et al., 1991). Pilot-plant-scale production of the biosurfactant using a 1500 1up-lift fermenter produced 2 tonnes of culture broth. The biosurfactant-containing broth was used as a substitute for chemical surfactants in a test carried out on an oil storage tank belonging to Kuwait Oil Company, Kuwait. Basal salt medium containing 2% w/v glucose as a readily available carbon source was used and oleic acid, a hydrocarbon source (2% v/v), was added after glucose consumption. Biosurfactant production reached a maximum after 18-19 h, as measured by reductions in the surface and interracial tension in the broth (Banat et al., 1991). At the end of the production run, the culture broth was sterilized in the fermenter and stored in 200 1 sterile drums.

Surveys conducted of the 44 m diameter floatingroof tank showed the presence of approximately 750 m 3 of sludge. After installation of circulation pumps, hoses, circulation boxes and connections to manholes, 1"5 tonnes biosurfactant were added to the tank, in addition to a 1 : 1 crude oil to sludge ratio and a 1.25:1 brackish water to total hydrocarbon (sludge + fresh crude) ratio. Circulation of the mixture was carried out for 5 days, at ambient temperatures of between 40 and 50°C, after which most sludge had been resuspended. An emulsion breaker was added to separate the water from the crude oil and the two layers were easily extracted from the tank. After cleaning, the total sludge was determined to be 850 m 3, and nearly 91% (774 m 3) was recovered as crude oil, whereas 76 m 3 remained as impurities at the tank bottom and consisted mainly of non-hydrocarbon materials. The characteristics of the recovered crudeoil samples are shown in Table 3. The crude oil extracted after cleaning was found to have API values ranging between 27.6 and 29.8, similar to the API range for the standard Kuwaiti crude, and a total hydrocarbon content of 100%. Approximately 5550 barrels of saleable crude oil were produced. This oil may be sold to cover costs of the cleaning at approximately S100 000-150 000 (US) per storage tank. Such a clean-up process is highly desirable as it is economically rewarding, environmentally sound and is less hazardous for the persons involved than the conventional process (Lillienberg et al., 1992). In addition it is a limited application for biosurfactants that can be easily controlled and, therefore, would be a suitable way for progressing towards involving the oil companies who are usually reluctant to adopt this technology.

MICROBIALLY-ENHANCED OIL RECOVERY (MEOR) M E O R is an important tertiary recovery technology utilizing microorganisms and/or their metabolic endproducts for recovery of residual oil. It is generally accepted that approximately 30% of the oil present in a reservoir can be recovered using current EOR technology (Singer & Finnerty, 1984). Poor oil recovery in existing producing wells may be due to several factors. The main factor is the low permeability of some reservoirs or the high viscosity of the oil which results in poor mobility. High interfadal tensions between the water and oil may also result in high capillary forces retaining the oil in the reservoir rock (Bubela, 1987). Since most of the oil remains in the reservoir following primary and secondary recovery techniques, interest has evolved in tertiary recovery techniques (Morkes, 1993). Techniques involving the use of chemical or physical processes such as pressurization, waterflooding or steaming, however, are generally unapplicable to most oil reservoirs. The use of chemical surfactants for cleaning-up oil reservoirs is an unfavourable practice

Biosurfactants production and applications

7

Table 3. Characteristics of biosurfaetant-recovered crude oil samples before and after blending with fresh crude and after long storage (Banat etat, 1991)

Property

Specific gravity API gravity Viscosity kinematic at 20°C Water content (A) Sediment (B) Water & sediment (BS&W) (A+ B) Asphaltic material Oil content 100 - (A+ B)

Test method

Unit

Sample 1

2

3

IP 160/D1298 IP 200/D1250 IP 71/D445 IP 74/D95 IP 53

g/ml API cSt % vol % vol

0-883 28.6 166.6 ND ND

0"877 29.8 54.1 ND ND

0"888 27"6 196.6 ND ND

-IP 143

% vol % wt

ND 2.09

ND 2-14

ND 1"05

--

% vol

100

100

100

ND = None detected. Sample 1; biosurfactant-extracted crude oil before blending. Sample 2; biosurfactant-extracted crude oil after blending. Sample 3; biosurfactant-extracted crude oil after blending and 2 months storage.

that is hazardous, costly and will leave undesirable residues which are difficult to dispose of without adversely affecting the environment. Strategies and factors affecting M E O R

The appropriate remedy for any given oil reservoir will vary and be based on the conditions present. Temperature, pressure, pH, porosity, salinity, geologic make-up of the reservoir, available nutrients and the presence of indigenous flora must all be taken into consideration. It is estimated, based on criteria developed by the National Institute for Petroleum & Energy Research, that 27% of the oil reservoirs in the major oil-producing states in the USA may be suitable for M E O R (Bryant, 1991 ). It has also been estimated that 40% of the oil-producing carbonate reservoirs in the USA may also be suitable for M E O R (Tanner et al., 1991 ). The mechanisms of MEOR's action in situ are most probably due to multiple effects of the microorganisms on the environment and oil. These mechanisms include: gas formation and pressure increases; acid production and degradation of limestone matrices; reduction in oil viscosity and interfacial tension by biosurfactant; solvent production; plugging by biomass accumulation or polymer formation; and degradation of large organic molecules in oil, resulting in decreases in viscosity (Jack, 1988; Khire & Khan, 1994a). The presence of different types of microorganisms with varying growth properties and metabolite production will have different effects on the reservoir environment. Thus, it is important to consider all aspects of M E O R when trying to influence oil production by one mechanism, such as the use of biosurfactants. There are several strategies involving the use of biosurfactants in M E O R (Shennan& Levi, 1987): 1. The first involves injection of biosurfactant-producing microorganisms into a reservoir through the well, with subsequent propagation in situ through the reservoir rock (Bubela, 1985).

2. The second involves the injection of selected nutrients into a reservoir, thus stimulating the growth of indigenous biosurfactant-producing microorganisms. 3. The third mechanism involves the production of biosurfactants in bioreactors ex situ and subsequent injection into the reservoir. Laboratory studies on M E O R

Laboratory studies on M E O R have typically utilized core samples and columns containing the desired substrate. These substrates have been utilized to demonstrate the usefulness of biosurfactants in oil recovery from sand and limestone. Similarly, core samples have been used to model the movement of microorganisms and nutrients through substrates to ascertain their usefulness after injection into oil reservoirs. In an investigation in which B. subtilis was injected through sand-packed columns (2-5 cm diameter x 28 cm length) with a permeability of 4000 md, a release of 35% residual oil, compared to 21% using the nutrient solution control, was observed. Similar experiments with C. acetobutylicum using molasses (4%) and 0"5% ammonium diphosphate nutrient medium yielded 66 and 50% oil recovery in the presence and absence of pyrophosphate, respectively. It was suggested that C. acetobutylicum has an advantage in M E O R because of its anaerobic growth and gas-producing capabilities (Chang, 1987). The added oil was not sterile, however, and the role of indigenous bacteria in the oil recovery was not monitored. Vibrio aspartigenicus strain GSP-1 and Bacillus licheniformis JF-2 were also tested for their ability to recover residual oil from crushed unconsolidated Viola limestone (20-50 mesh) cores (Adkins et al., 1992). Vitrio aspartigenicus (an acid-producing, halophilic, anaerobic, motile bacterium) was observed to recover 32-36% more oil from saturated cores than the control columns after three treatments (injection of nutrients,

8

I.M. Banat

incubation, flooding with brine). In this experiment a 27-38% increase in dissolved calcium concentration was observed, due to acid production which can degrade the limestone matrix and cause release of the residual oil. In comparison, B. licheniformis increased oil recovery by 27%, with a 13% increase in dissolved calcium which was also attributed to biosurfactant production (Adkins et al., 1992).

Plugging and clogging investigations One of the methods of MEOR involves using microorganisms in in situ selective plugging through the introduction of viable bacteria in the aqueous displacing-fluid injected into the oil well water-swept zones. Investigations involving high-pressure consolidated systems were employed using 1:1 mixtures of a cement-sand mixture with powdered aluminum and shaped into cores. These oil-saturated cores were mounted in a Hessler cell and subjected to high pressure sweeps (250-350 kPa). Water resulted in a 15% recovery of OIP, whereas injections of microorganisms resulted in plugging of the cores. Experiments using brine-saturated Berea sandstone cores showed that injecting nutrients and viable bacterial cells resulted in permeability reductions (clogging) of 60-80% (Jenneman et al., 1984). To facilitate microbial movements and formation of plugs deep within the reservoir, strata studies examining the injection of nutrient-starved ultramicrobacteria (UMB) into three-dimensional sandpack-reservoir simulations have been carried out (Cusack et al., 1992). Starved Pseudomonas sp. FC3 cells were found to penetrate a sandpack 45 cm in diameter and 38 cm in length. The UMB were then resuscitated with nutrients to grow in situ and form a confluent bacterial plug. Scanning electron microscopy and carbohydrate assays revealed glycocalyx formations in all sections examined. UMB are effective for deeper penetration into oil reservoirs as they are small in size, have high shear-stress tolerance and are useful for microbial plugging in reservoirs with a permeability range of 200 mD-5.9D (Cusack et al., 1992) which can facilitate secondary oil-recovery techniques involving water sweeps. Studies to determine the factors influencing microbial movement through anaerobic, nutrient-saturated, unconsolidated Ottawa sand-packed cores were carried out under static conditions (Reynolds et al., 1989). Gas-producing motile Escherichia coli were found to penetrate cores saturated with galactose-peptone up to six times faster than non-gas-producing motile or nonmotile mutants. Similarly, motile strains of E. coli were found to penetrate the core four times faster than mutant strains defective in flagellar formation. Furthermore, motile strains with faster growth-rates penetrated the cores at a more rapid rate. Chemotaxis was not found to influence the penetration rate of these bacteria. Such factors are important to consider in the choice of bacterium to employ in in situ MEOR.

Field studies of MEOR One of the early field studies on MEOR involved an unconsolidated waterflooded sandstone reservoir in Union County, Arkansas, USA (Yarbrough & Coty, 1983; Hitzman, 1988). Molasses was injected along with an inoculum of Clostridium acetobutylicum into the reservoir. After treatment, carbon dioxide production increased and the production of oil increased by 250% (Tanner et al., 1991). Metabolite production, however, was found to match that of an indigenous microorganism, C. kluyveri, detected in the reservoir water. The mechanisms by which these microorganisms acted were believed to result from acid production. Reports of successful MEOR use in carbonaceous reservoirs have been reviewed (Tanner et al., 1991). Injection of molasses and anaerobic acid- and gasproducing microorganisms resulted in increased oil recovery. A decrease in water pH and an increase in carbon dioxide production indicated active growth of microorganisms in the reservoir. Acid and gas production were believed to be important elements in the oil recovery (Lazar et al., 1988; Tanner et al., 1991). Other M E O R investigations in carbonate reservoirs showed an increase of 60-126% in oil production in Hungary (Hitzman, 1983) and a 200% increase in Germany (Wagner, 1991). Injection of nutrients and Desulfovibrio desulfuricans (Hitzman, 1988) obtained from sewage sludge, followed by a shut-down of oil production for approximately 6 months, resulted in a 60-200% increase in oil recovery. After production resumed, a reduction in pH, an increase in carbon dioxide and a decrease in oil viscosity were observed. Increased numbers of D. desulfuricans in the water produced from the reservoir were also detected. It is the author's belief, however, that the use of sulphatereducing bacteria such as D. desulfuricans would not be favoured in, or recommended to, the oil industries, due to the adverse effects an increase in sulphur content could have on the oil quality. Three wells on the Burnett J lease (USA) were used in a pilot investigation of M E O R which involved the injection of 80 1of kerosene, followed by 15 I of a commercial biosurfactant product (RAM Biochemicals' WelPrep 5). The wells were then flushed with saltwater and shut down for 48-96 h. Oil production increased five-fold from 0.3 barrel/day before treatment, to 1"6 barrel/day after treatment (Nelson & Launt, 1991). In another set of experiments involving the injection of microorganisms and nutrients into the wells, followed by a 3 day shut-down period; an 11-25% increase in weekly oil production was detected (Nelson & Launt, 1991; Bryant etaL, 1993). Similarly, injections of P. aeruginosa, Xanthomonas campestris and B. licheniformis through the well casing, followed by longer shut-down periods of 40 and 64 days for two wells at the Daqing oil field in China, resulted in an increase in the numbers of injected bacteria, organic acids and carbon dioxide and a slight

Biosurfactants production and appfications

decrease in the interfacial tension in the oil-water mixtures. The two wells tested showed an increase from 3"5 to 5"5 t/day in the first and from 7"6 to 10-11 t/day in the second (Zhang & Zhang, 1993). In all the previously-mentioned studies, however, it is not known what effect the shut-down of the oil production well itself may have had on the increase in oil production, which is a serious drawback in the methodology. Furthermore, a field experiment carried out at the Romashkino oil field in Russia, where wells were injected with phosphate and nitrogen to stimulate the growth of native microflora (Ivanov et al., 1993) resulted in an increase of 32"9% in oil production, the mechanisms of which are not yet clear. Nevertheless, carefully controlled field studies of M E O R at the Alton Field in Queensland, Australia were carried out (Sheehy, 1990). The M E O R strategy used was to introduce microorganisms that were prescreened in sandpacks designed to simulate the physical parameters found in the reservoir. Laboratory analyses of nutrient requirements, metabolite production and interactions with the indigenous microbiota were carried out. A mixture of microorganisms and nutrients was filtered through 10 and 28 ~m filters and injected into the well followed by a shut-down of 20 days. Control experiments were initiated in which the natural baseline production was determined before and after the trial shut-down period, as well as after a control injection of water. An approximately 40% ino ease in oil production was observed, compared to the control baseline (Sheehy, 1990). Additionally, a decrease in the level of the base sediment and water, with an increased percentage of oil content was observed. Increased gas produced in the reservoir was found to be the result of increased carbon dioxide and methane. Microbial numbers were found to rise from = 103/ml in pre-injection water to > 105/ml after microbial injection. Sulphate-reducing bacteria were not observed to be stimulated and H2S production was not detected. In all the previous reports involving microorganisms, no changes in any physical characteristics of the oil were reported or observed, however, occasionally the interfacial tension of the water-oil interface decreased compared to the control values. These results are indicative of the effectiveness of M E O R in obtaining oil with the same desired characteristics as before the treatment. CONCLUSION AND FUTURE O U T L O O K The usefulness of biosurfactants in the emulsification of aqueous hydrocarbon mixtures has been clearly demonstrated. Biosurfactants in many cases have proved to be more effective than chemical surfactants and have the added benefit of being biodegradable. Studies of oil- or hydrocarbon-contaminated sand or soil have also indicated that microorganisms which produce biosurfactants, when stimulated properly, can aid bioremediation. Laboratory studies have shown

9

that the addition of biosurfactant mixtures alone may be useful for stimulating biodegradation of these contaminants in the environment. Biosurfactants have also demonstrated their usefulness in the solubilization and removal of oil from sand and sludge in oil storage tanks. Therefore, in ecological terms, the use of biosurfactants is obvious for closed systems but remains speculative in the open environment. The utility of M E O R has not been conclusively documented in the field at this stage. Preliminary findings from the few investigations carried out to date, however, seem promising. The precise mechanism of enhanced oil recovery in situ is unclear due to the lack of controls in some cases, with the unforseen difficulties usually encountered in situ and insufficient analyses in other cases. It would appear that in certain circumstances M E O R could be a viable alternative, which, if carefully applied, could prove to be an economically-feasible method of enhancing oil recovery. However, the technologies involved require an interdisciplinary effort among microbiologists, biochemists, geologists and petroleum engineers. An evaluation of the different criteria for the application of the various microbial methods needs to be extensively investigated. Future efforts in strain improvement and development with the aid of genetic engineering are expected to help progress in this emerging technology.

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