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In Situ Rhamnolipid Production at an AbandonedPetroleum RefineryDaniel P. Cassidy a , Andrew J. Hudak a , D. Dale Werkema a , Estella A. Atekwana b , SilviaRossbach c , Joseph W. Duris c , Eliot A. Atekwana d & and William A. Sauck aa Western Michigan Univ., Department of Geosciences,2 Kalamazoo, MI 49008b Univ. of Missouri-Rolla, Dept. of Geology and Geophysics, Rolla, MO 65409c Western Michigan Univ., Department of Biological Sciences, Kalamazoo, MI 49008d Indiana Univ. Purdue Univ. Indianapolis, Dept. of Geology, Indianapolis, Indiana

Available online: 24 Jun 2010

To cite this article: Daniel P. Cassidy, Andrew J. Hudak, D. Dale Werkema, Estella A. Atekwana, Silvia Rossbach, Joseph W.Duris, Eliot A. Atekwana & and William A. Sauck (2002): In Situ Rhamnolipid Production at an Abandoned Petroleum Refinery,Soil and Sediment Contamination: An International Journal, 11:5, 769-787

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Soil and Sediment Contamination, 11(5):769-787 (2002)

1532-0383/02/$.50© 2002 by AEHS

In Situ Rhamnolipid Production at anAbandoned Petroleum Refinery

KEY WORDS: biosurfactant, groundwater, hydrocarbon, rhamnolipid, surface tension.

Daniel P. Cassidy,1 Andrew J.Hudak,1 D. Dale Werkema, Jr.,1

Estella A. Atekwana,2 SilviaRossbach,3 Joseph W. Duris,3 EliotA. Atekwana,4 and William A. Sauck1

1Western Michigan Univ., Department ofGeosciences,2 Kalamazoo, MI 49008; 2Univ.of Missouri-Rolla, Dept. of Geology andGeophysics, Rolla, MO 65409; 3WesternMichigan Univ., Department of BiologicalSciences, Kalamazoo, MI 49008; 4IndianaUniv. Purdue Univ. Indianapolis, Dept. ofGeology, Indianapolis, Indiana4

A simple screening method was developedto detect in situ biosurfactant production byexploiting the relationship between surfacetension (ST) and surfactant concentration.Filtered groundwater from contaminatedwells with ST values of 60 to 70 dynes/cmdecreased to 29 dynes/cm after being con-

centrated 10 to 15 times in a rotary evapo-rator, indicating that biosurfactants in thesample reached the critical micelle con-centration (CMC). Samples from uncon-taminated groundwater concentrated 25times showed no decrease in ST below 72dynes/cm, suggesting that biosurfactantswere not present. Microorganisms from soilcores were cultured on diesel fuel and iden-tified using fatty acid methyl ester (FAME)analysis. Pseudomonas aeruginosa wasfound at very low numbers in uncontami-nated soil but was the dominant species incontaminated soil, indicating that hydro-carbon release impacted microbial diver-sity significantly. High-performance liquidchromatography (HPLC) was used to quan-tify rhamnolipids, biosurfactants producedby P. aeruginosa, in concentrated ground-water samples. Rhamnolipid concentrationsin samples from contaminated soil wereobserved equal to their CMC (50 mg/L),but were not detected in samples from un-contaminated wells. We conclude thatbiosurfactant production may be an indica-tor of intrinsic bioremediation.

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BACKGROUND

urfactants are amphiphilic molecules that partition at oil/water and air/waterinterfaces. This allows surfactants to reduce the surface tension (ST) and

interfacial tension. Figure 1 shows how ST varies with surfactant concentration. Assurfactant concentration increases, ST decreases from 72 dynes/cm (ST of purewater) to 25 to 30 dynes/cm when the critical micelle concentration (CMC) isreached. ST does not decrease substantially at surfactant concentrations above theCMC. At concentrations below the CMC surfactants partition at the air-waterinterface to maximize contact of the hydrophilic moiety with water and minimizecontact of the hydrophobic moiety with the water. At surfactant concentrationsabove the CMC all the surface sites are occupied and surfactants begin formingmicelles, with the hydrophobic moieties in the center and the hydrophilic moietieson the exterior. The hydrophobic interior of micelles can partition nonaqueousphase liquids (NAPL), which allows their emulsion in water. Above the CMC,micelle content increases linearly with increasing surfactant concentration and sodoes the capacity to emulsify NAPL.

Surfactant-enhanced aquifer remediation (SEAR) is used at NAPL-contami-nated sites. Because NAPL can replace water as the wetting phase at contaminatedsites, it is difficult to remove NAPL with groundwater. Surfactants change thewetting phase from NAPL to water (Bai et al., 1997), so that NAPL can beextracted with groundwater. The accompanying decrease in ST also decreasescapillary forces, which increases the mobility of NAPL and dissolved contami-nants (Lord et al., 1997; Henry et al., 2001).

Biosurfactants are produced naturally by many microorganisms in response togrowth on hydrocarbons (Miller, 1995; Desai and Banat, 1997). Biosurfactantsconsist of common cell material (e.g., glycolipids, lipopeptides, and fatty acids)and are readily biodegradable. The emulsion of hydrocarbons in water resultingfrom the production of biosurfactants drastically increases the surface area avail-able for microbial uptake and enhances biodegradation (Zhang and Miller, 1995;Bai et al., 1998). As such, biosurfactants have the potential to aid in hydrocarbonbiodegradation, and even to serve as indicators of intrinsic bioremediation. Byemulsifying hydrocarbons in the subsurface, biosurfactants can change the wettingphase from NAPL to water, which can affect electrical resistivity and groundpenetrating radar measurements (Sauck, 2000; Cassidy et al., 2001). Biosurfactantsalso coordinate with heavy metals, increasing their subsurface mobility (Mulliganet al., 1999).

Rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa, a hy-drocarbon-degrading species that uses both oxygen and nitrate as electron acceptor(Robert et al., 1989; Bossert and Compeau, 1995). The two rhamnolipids producedmost commonly by P. aeruginosa are L-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate (C26H43O9) and L-rhamnosyl-L-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate (C32H48O13). The two differ only in the number of rhamnose

S

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FIG

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units on the lipid moiety, and are also known as monorhamnolipid (RL1) anddirhamnolipid (RL2). Both have a CMC of approximately 50 mg/L and reduce theST of water to 25 to 30 dynes/cm (Zhang and Miller, 1995). The optimal pH forrhamnolipid production by P. aeruginosa is 7.

Biosurfactants and the microorganisms that produce them have been isolatedand cultured from petroleum reservoirs and contaminated sites (Banat, 1995;Miller, 1995). However, there is a lack of information on biosurfactant productionin the subsurface, even though their in situ production has tremendous environ-mental significance. Cassidy et al. (2001) observed biosurfactant production ac-companying hydrocarbon degradation in laboratory experiments with soil from anabandoned petroleum refinery. Indigenous soil microorganisms producedbiosurfactants resulting in bulk concentrations of twice the CMC. However, theauthors did not identify biosurfactants and microorganisms.

Direct measurement of biosurfactants in natural waters is time consuming andexpensive, requiring identification of specific microorganisms and then quantify-ing the biosurfactants they produce. A goal of this study was to develop a simplescreening method to detect biosurfactants by applying the relationship between STand surfactant concentration (Figure 1). This screening method consists of measur-ing ST in groundwater samples concentrated in a rotary evaporator. Decreasing STwith increasing concentration shows that surfactants are present, and leveling offof ST values at 25 to 30 dynes/cm indicates that the surfactant concentration in thesample has reached the CMC. Careful handling and filtering is required to removeNAPL from samples. The rotary evaporator concentrates samples by applying avacuum and increasing the temperature. Thermal degradation can be avoided bycontrolling the temperature and is not likely to be a problem because mostbiosurfactants are stable at temperatures up to 100oC (Lang and Wagner, 1987).Rhamnolipids are stable up to 120oC. Stripping is minimal because biosurfactantshave high molecular weights (Lang and Wagner, 1987; Desai and Banat, 1997).For example, RL1 and RL2 have molecular weights of 499 and 640, respectively.

This study describes a field investigation performed at an abandoned petroleumrefinery described previously by Cassidy et al. (2001). The purpose of this studywas to characterize in situ biosurfactant production by identifying individualsurfactant-producing microorganisms and their biosurfactants, and to develop asimple screening method to detect biosurfactants in groundwater samples from thesite.

MATERIALS AND METHODS

The Abandoned Refinery Site

The former Crystal Refinery is located in Carson City, Michigan, USA (Figure2). A large portion of the soil and groundwater at the site was contaminated

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FIGURE 2

Site map showing monitoring well location, approximate contact between contaminatedand uncontaminated areas, and the direction of groundwater flow.

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with hydrocarbons due to oil refining operations between 1935 and 1990.Millions of liters of hydrocarbons have leaked into the soil and groundwaterbeneath the property (Atekwana et al., 2000). The surficial, impacted mate-rial consists of several meters of sand underlain by a clay layer. Thehydrogeology of the site has been investigated in detail (Atekwana et al.,2000 and 2001; Hudak, 2001). The pH of the soil and groundwater at the siteranges between 6.8 and 7.5. The groundwater has nitrate concentrationsranging from 10 mg/L to 40 mg/L.

Thirteen monitoring wells (MW) were installed at the site in 1998 (Figure 2).Nine wells (MW-1 through 6, MW-8, MW-10, and MW-13) are located in areaswhere free and/or dissolved product is found. Four control (uncontaminated) wells(MW-7, MW-9, MW-11, and MW-12) are located in uncontaminated portions ofthe aquifer, upgradient from the impacted area. Extensive data were collected toidentify the impacted area (Hudak, 2001; Atekwana et al., 2000), including hydro-carbon concentrations, in situ dissolved oxygen, and microbial counts and speciesdiversity.

Soil Sampling

Six core samples were taken adjacent to six different monitoring wells in January2001 for microorganism enumeration and identification. The contaminated wellsselected for coring were MW-4, MW-5, and MW-10. The uncontaminated wellswere MW-7, MW-9, and MW-12. Cores were taken at uncontaminated sites firstto avoid contaminating the control wells with hydrocarbons or microorganisms.Coring equipment and sleeves were thoroughly decontaminated before and aftereach sampling event by liberally spraying with an 80% ethanol solution, followedby a triple rinse with deionized water.

The water table at the site has fluctuated approximately 2 m over the last5 years, which has distributed free product over a similar depth. Core sampleswere taken over a 20-cm interval, from 10 cm above the water table at thetime of sampling to 10 cm below. All sampling intervals were within thesmear zone (i.e., the zone containing free product). Once the proper samplingdepth was established from water table measurements, a 6.35-cm ID handauger was manually augured into the ground. The exact auger location was3 to 4 m from the associated monitoring well. Core samples were collectedusing a hand Geoprobe fitted with a 2.54 cm × 60 cm PVC sleeve. The entirecoring system was pulled from the ground, and the PVC sleeve containing thesediment core was removed. Sterilized vinyl end caps were used to seal thesleeve. The cores were labeled, placed in plastic bags, and stored at 4oC formicroorganism enumeration and identification.

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Microorganism Enumeration and Identification

The six cores were individually homogenized in ethanol-disinfected stainless steelbowls, and four 1-g subsamples were taken for cell extraction and microorganismenumeration. Cells were extracted from the subsamples using the procedure de-scribed by van Elsas and Smalla (1997). Serial dilutions were plated on Noble agar(Difco) with diesel fuel as the sole carbon source as described by Walker et al.(1975). The plates were incubated for 5 weeks at 28oC, and the colony forming units(CFU) on each plate were counted. Two plates were done from each well. Microbialnumbers were determined by multiplying the total or individual species CFU by theappropriate dilution factor and were reported as CFU/g dry soil. Each distinct colonyfrom the duplicate plates was aseptically subcultured on a new plate for identifica-tion. Microcheck, Inc. (Northfield, Vermont, USA) identified the individual speciesrepresented by each subculture using fatty acid methyl ester (FAME) analysis. Thistechnique extracts the fatty acids in the cell membranes of isolated species andanalyzes them with gas chromatography. The sample chromatogram is comparedwith standard chromatograms from individual species in a database. A single matchwith a similarity index greater than 0.300, or a multiple match with a similarity indexgreater than 0.500 give a positive species identification.

Groundwater Sampling and Preparation

Groundwater samples were collected periodically from all 13 monitoring wellswith a submersible pump. The first 2 well volumes of extracted groundwater werediscarded to ensure that the samples were from the adjacent pore waters. Approxi-mately 2 L of sample was drawn from each monitoring well. The samples werebrought to the laboratory, passed through a 0.45–µm filter to remove mineralsolids, microorganisms, and nonemulsified NAPL, and were stored overnight at4oC. The concentration of the groundwater filtrate was done with a Büchi R-114Rotary Evaporator set at full vacuum and 90oC. Distillate from the rotary evapo-rator was collected for rhamnolipid analysis. The samples were concentratedincrementally by factors of 5 to a final concentration factor of 25.

Surface Tension and Total Organic Carbon

Surface tension (ST) was measured after each concentration step on duplicate 10-mLfiltrate samples using a Tantec CBVP-Z Automatic Surface Tensiometer (accuracy± 0.2 dynes/cm). The concentrated samples reached room temperature before STmeasurements. Total Organic Carbon (TOC) was measured in 2-mL filtrate samples

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according to Standard Method 5310-B (Eaton et al., 1995), with an Ionics-1270TOC Analyzer.

Rhamnolipid Analysis

Monorhamnolipid (RL1) and dirhamnolipid (RL2) concentrations were measuredin subsamples from the concentrated groundwater samples according to the methoddescribed by Schenk et al. (1995). A derivatization step was done to formp-bromophenacyl esters of RL1 and RL2. A Beckman high-performance liquidchromatograph (HPLC) with UV detection at 265 nm was used to quantify thederivitized RL1 and RL2 according to the method of Schenk et al. (1995). Sepa-ration was obtained in 24 min on a Macherey-Nagel, ET 250/8/4 Nucleosil 5 C18

column using a linear gradient of water:acetonitrile (30:70 to 0:100). Standardswere obtained from the Jeneil Biosurfactant Company (Saukville, WI). The methoddetection limit was 0.1 mg/L for both RL1 and RL2.

RESULTS AND DISCUSSION

Species Concentrations and Distribution

Each distinct colony that grew on the plates was positively identified with FAMEanalysis. Tables 1 and 2 list counts of each species identified and the totalmicroorganism counts in uncontaminated and contaminated soil, respectively. Themost abundant species in the uncontaminated soil cores were Pseudomonaschlororaphis, Bacillus sphaericus (II B), Bacillus subtilis, Arthrobacter duodecanis,and Arthrobacter oxydans (Table 1). The three genera representing most of theculturable microorganisms in the uncontaminated soil were Pseudomonas, Bacil-lus, and Arthrobacter. These common soil genera are known to degrade petroleumhydrocarbons using nitrate and oxygen as electron acceptors (Bossert and Compeau,1995; Paul and Clark, 1988). This is consistent with high nitrate concentrations ingroundwater at the site. The total microorganism concentration in the uncontami-nated soil cores ranged from approximately 5 × 106 CFU/g to 1 × 107 CFU/g.

Fewer species were cultured in the contaminated soil cores (Table 2) than in theuncontaminated cores. The most abundant species observed in the uncontaminatedsoil were not detected at all or showed very low counts in contaminated soil cores.The only two species that were detected in all three contaminated soil cores werePseudomonas aeruginosa and Pseudomonas fluorescens (C), which were found invery low numbers or not at all in uncontaminated cores. Both pseudomonads arecapable of nitrate and oxygen respiration and degrade petroleum hydrocarbons(Bossert and Compeau, 1995). P. aeruginosa was the most abundant species in allcontaminated soil cores, essentially comprising the entire microbial count for each

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TABLE 1Microbial Numbers of Species Identified with FAME Analysis in Soil Cores

from Uncontaminated Wells

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TABLE 2Microbial Numbers of Species Identified with FAME Analysis in Soil Cores

from Contaminated Wells

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core. The total microbial numbers in the contaminated soil ranged from approxi-mately 6 × 108 CFU/g to 3 × 109 CFU/g, approximately two orders of magnitudegreater than in the uncontaminated soil.

The results clearly show that contaminated soil had fewer hydrocarbon-degrad-ing species but greater total counts of hydrocarbon-degrading microorganisms thandid the uncontaminated soil. Culturing methods can misrepresent a microbialcommunity because of the enrichment bias that occurs from plating environmentalsamples onto a specific growth medium (Dunbar et al., 1997). However, even withhydrocarbons as the sole carbon source in the growth medium, the contaminatedsoil showed a lower species diversity than did nearby uncontaminated soil. Thisobservation is consistent with studies of many hydrocarbon-contaminated environ-ments (Macnaughton et al., 1999; Rosenberg and Ron, 1996; Liu et al., 1997).Clearly, the stress applied by petroleum contamination to the soil ecosystem at thesite favored the growth of P. aeruginosa and P. fluorescens (C) at the expense ofthe most abundant species in uncontaminated cores (P. chlororaphis, B. sphaericus(II B), B. subtilis, A. duodecanis, and A. oxydans).

The reason for the growth advantage enjoyed by P. aeruginosa and P. fluorescens(C) in the contaminated soil is not known. Both pseudomonads producebiosurfactants: P. aeruginosa produces rhamnolipids and P. fluorescens producesa lipoprotein biosurfactant called viscosin and a glycolipid-protein biosurfactant(Banat, 1995). Because biosurfactant production enhances rates of hydrocarbonuptake biosurfactant-producing species could experience a growth advantage rela-tive to nonproducing species. Moreover, some biosurfactants have been demon-strated to have antibiotic properties and cause antagonistic effects on other species(Desai and Banat, 1997), which could explain the predominance of the twopseudomonads in the contaminated soil. However, a predominant species in theuncontaminated soil, B. subtilis, produces surfactin. This cyclic lipopeptide is oneof the most powerful biosurfactants known and is produced by all known strainsof B. subtilis grown on hydrocarbons (Desai and Banat, 1997). This suggests thatbiosurfactant production alone cannot explain the different microbial profiles thatdeveloped in contaminated and uncontaminated soil at the site. The toxicity ofhydrocarbons toward the predominant species in the uncontaminated soil is un-likely as a reason for their reduced numbers in contaminated soil, because thesespecies are known to degrade petroleum hydrocarbons (Bossert and Compeau,1995).

These results also indicate that information on microbial numbers and distribu-tion and biosurfactant production can be used to infer that intrinsic (passive)bioremediation is occurring at the refinery site. Intrinsic bioremediation has gainedincreasing acceptance for site remediation, as stand-alone strategy or used inconjunction with other technologies (U.S. EPA, 1997). However, it is difficult toprove that NAPL biodegradation occurs intrinsically (Chapelle, 1999). No singlemeasurement has been found that is reliable as a diagnostic indicator of intrinsicbioremediation. Instead, a suite of geochemical and hydrological information is

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typically required before intrinsic bioremediation is permitted. The results fromthis and other work (Macnaughton et al., 1999; Rosenberg and Ron, 1996; Liu etal., 1997) suggest that the assessment of microbial diversity and enumeration canalso be used as an indicator of intrinsic bioremediation.

Biosurfactant Screening Method

The biosurfactant screening method consisted of measuring ST in groundwatersamples subjected to increasing degrees of concentration in the rotary evaporator.Figure 3 shows ST vs. concentration factor in groundwater samples obtained fromMW-5 (contaminated) and MW-9 (uncontaminated) at three different times. STvalues in fresh samples from MW-9 were near 72 dynes/cm and did not decreasenoticeably, even after being concentrated 25 times. Identical results were obtainedin groundwater samples from all the uncontaminated wells. These results show thatbiosurfactants did not accumulate in pore waters from uncontaminated areas of thesite, even though the biosurfactant-producing B. subtilis was abundant (Table 1).In order for biosurfactants to accumulate in pore waters, the rate of production bymicroorganisms must exceed the rate of removal via biodegradation and sorptionto soil. It is possible that biosurfactants were produced in the uncontaminated areasof the site but did not accumulate in groundwater because rates of biodegradationand sorption kept pace with production.

In contrast to MW-9, ST in groundwater samples from MW-5 decreased pro-gressively with increasing concentration factor until a minimum value of 29 dynes/cm was reached (Figure 3). This is a clear indication that surfactants accumulatedin groundwater at MW-5, and that concentration increased surfactant levels to theCMC. The initial ST in groundwater sampled in June (63 dynes/cm) was consid-erably lower than in groundwater sampled in December and March (70 dynes/cm).The lower initial ST value indicates that groundwater sampled in June had a higherinitial surfactant concentration than groundwater sampled in December and March.This explains why June samples required only a concentration factor of 10 to reachthe CMC compared with December and March samples, which required a concen-tration factor of 15. Regardless of initial ST values, the slope of the decrease in STwith concentration factor was nearly identical for all three sampling dates in MW-5.Similar results were obtained for all monitoring wells that sampled contaminatedgroundwater at the site. Initial ST values in contaminated groundwater varied from60 dynes/cm to 71 dynes/cm, but ST decreased to a minimum of 29 dynes/cm ata slope nearly identical to that observed for MW-5. A concentration factor of 10to 20 was required to reach the CMC in groundwater samples from all thecontaminated wells.

It is highly unlikely that anything other than biosurfactants could reduce ST tothe values observed in the screening studies. Other naturally occurring organiccompounds (e.g., volatile organic acids, alcohols, fulvic, and tannic acids) have

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surfactant properties but are not nearly as powerful as biosurfactants. Moreover,biosurfactants have a low volatility, whereas volatile organic acids and alcoholsvolatilize readily under the conditions in the rotary evaporator. The CMC ofbiosurfactants ranges from 1 to 200 mg/L (most are below 100 mg/L) (Lang andWagner, 1987). A low CMC makes it easy to concentrate samples sufficiently toreach the CMC and lower the ST to 30 dynes/cm. Other naturally occurring organiccompounds require much higher concentrations to reduce ST measurably. Forexample, a 9% n-butanol solution is required to reduce ST to 30 dynes/cm, and a5.8% propionate concentration only reduces ST to 49 dynes/cm (Zajic and Seffens,1984). Humic and fulvic acids have similar surfactant properties. Such highorganic concentrations are not found in groundwater from hydrocarbon-contami-nated sites. TOC in groundwater samples from MW-5 and other contaminatedwells ranged from only 3 to 5 mg/L, and volatile organic acid concentrationsranged from 5 to 12 mg/L. These data strongly indicate that biosurfactants were themajor contributor to surfactant activity observed in the screening studies.

Rhamnolipid and TOC Measurements

FAME analyses showed that P. aeruginosa was the predominant hydrocarbon-degrading species in contaminated wells (Table 2), and the screening methodshowed that surfactants were present in groundwater from contaminated wells(Figure 3). Therefore, it was deemed likely that rhamnolipids were produced incontaminated areas of the site, and that they (wholly or partly) reduced ST in thescreening studies. To test this hypothesis, RL1, RL2, and TOC were quantified inthe samples that were concentrated in the rotary evaporator for the screeningstudies (Figure 3). TOC measurements in the subsamples were compared withTOC contributed by RL1 and RL2 in order to identify other sources of surfactantsin the pore waters. Rhamnolipid TOC was calculated using values of 0.625 for RL1and 0.60 for RL2, which can be determined from their empirical formulas. Table3 lists concentrations of RL1 and RL2, TOC calculated from RL1 and RL2, andmeasured TOC in the same concentrated groundwater samples of MW-5 and MW-9shown in Figure 3. Neither RL1 nor RL2 was detected in rotary evaporatordistillate. This is consistent with the chemical properties of RL1 and RL2. Prelimi-nary experiments with purified rhamnolipids obtained from Jeneil BiosurfactantCompany (Saukville, WI) also verified that RL1 and RL2 volatilize only slightlyin the rotary evaporator.

Neither RL1 nor RL2 was detected in groundwater concentrated 25 times fromMW-9 (Table 3). These results are consistent with ST measurements (Figure 3) andshow that rhamnolipids did not accumulate in groundwater from MW-9. Identicalresults were obtained from rhamnolipid analyses for the three other uncontami-nated wells at the site. Measured TOC values in the concentrated groundwatersamples from MW-9 ranged from 1.2 to 1.4 mg/L and could have been from

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TABLE 3Concentrations of Monorhamnolipid (RL1), Dirhamnolipid (RL2), TOCCalculated from Rhamnolipid Concentrations, and Measured TOC in

Concentrated Groundwater Samples from MW-5 and MW-9

naturally occurring organic compounds. The concentration factor at whichrhamnolipids were quantified in groundwater samples from MW-5 correspondedto that required to reach the CMC in the screening studies (Figure 3). Concentra-tions of RL1 and RL2 in groundwater from MW-5 ranged from 27 to 30 mg/L and22 to 26 mg/L, respectively. Measured TOC values in groundwater from MW-5were greater than the rhamnolipid TOC values by an amount ranging from 0.3 to1.6 mg/L. This difference can be explained by experimental error in the TOCanalyses or by the presence of other organic compounds. Total rhamnolipid con-centrations (RL1+RL2) ranged from approximately 49 to 56 mg/L, which is nearthe CMC value of 50 mg/L reported for rhamnolipids (Zhang and Miller, 1995).TOC measurements showed that rhamnolipids constituted nearly all of the organiccarbon in the concentrated groundwater samples from MW-5. Very similar resultswere obtained from all the contaminated wells.

The results from this study provided clear evidence that rhamnolipids wereproduced in situ in contaminated soils of the abandoned Crystal Refinery. Thisconclusion is supported by the FAME analyses that identified P. aeruginosa as thepredominant hydrocarbon-degrading species in the contaminated portion of the

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site (Table 2), by the results from the screening studies that showed low ST valuesin fresh and concentrated groundwater samples (Figure 3), and by the chemicalanalyses showing that rhamnolipid concentrations had reached the CMC in con-centrated samples (Table 3). The results also show that rhamnolipids accumulatedin contaminated pore waters to sufficient levels that they could be detected with thebiosurfactant screening method. This is possible when rates of biosurfactant pro-duction are greater than rates of biodegradation and sorption to the aquifer solids.The results indicate that P. aeruginosa produced excess rhamnolipids to enhanceits uptake of hydrocarbons, and that doing so provided P. aeruginosa a growthadvantage relative to other petroleum-degrading species at the site.

The biosurfactant screening method offered a reliable means of detectingbiosurfactants in the groundwater and provided a bulk measure of average in situconcentrations. If a concentration factor of 10 is required to reach the CMC withthe screening method, the average in situ biosurfactant concentration in the ground-water was one-tenth the CMC. However, the screening method results can saynothing about the spatial distribution of biosurfactant concentrations in groundwa-ter. Some pores may have very low concentrations, whereas others may have levelsabove the CMC. Also, the screening method provides only a bulk concentration ofa single biosurfactant or a mixture containing more than one. Individualbiosurfactants must be quantified with chemical analyses. This is difficult andexpensive because there are a number of biosurfactants produced by a variety ofdifferent species. Identifying biosurfactants requires microorganism screeningstudies which require considerable time and cost.

The screening method is a simple and cost-effective way to detect in situbiosurfactant production and to determine whether further microbial and chemicalanalyses are warranted. The method is easy, only requiring the use of a rotaryevaporator and a surface tensiometer. The rotary evaporator effectively concen-trates biosurfactants, which are highly nonvolatile and stable at high temperatures(Desai and Banat, 1997; Lang and Wagner, 1987) and can remove other organiccompounds from the sample via stripping. Even when biosurfactants are present atlow concentrations, they can be concentrated sufficiently to measure ST reduction.This was illustrated with groundwater samples from MW-5 from December andMarch. These samples had low biosurfactant concentrations, as evidenced byinitial ST values over 70 dynes/cm, but even slight concentration in the rotaryevaporator reduced the ST significantly. Some samples from the contaminatedwells showed reduced ST values (as low as 60 dynes/cm) without being concen-trated at all. The greater the concentration factor required to measure ST reductionsthe greater the initial sample volume required. Because organic compounds otherthan biosurfactants exhibit surfactant properties only at extremely high concentra-tions, supplemental TOC measurements can be used to evaluate the likelihood thatthese compounds contribute to reduced ST.

We conclude from this research that biosurfactant production can be used as anindicator of intrinsic bioremediation at many NAPL-contaminated sites. Microor-

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ganisms produce biosurfactants in order to enhance rates of NAPL biodegradation.Therefore, the presence of biosurfactants in the subsurface at NAPL-contaminatedsites can be used as a positive indication of intrinsic bioremediation, especiallywhen nearby uncontaminated areas show no biosurfactant production, as was thecase in this study. There are dozens of known biosurfactants produced by manydifferent and widespread bacterial and fungal species (Miller, 1995; Desai andBanat, 1997) and possibly many biosurfactants that are yet unidentified. Thescreening method used in this study allows biosurfactants to be detected whethertheir chemical composition or producing species is known or not. Biosurfactantproduction that is incidental to (i.e., not directly induced by) NAPL biodegradationis highly unlikely, especially when they are present above the CMC. However, thisdirect link must first be established if biosurfactant production is to be used as anindicator of intrinsic bioremediation.

SUMMARY AND CONCLUSIONS

Rhamnolipids were produced in situ by P. aeruginosa in hydrocarbon-contami-nated soils at the former Crystal Refinery and accumulated in groundwater suchthat they could be detected by ST measurements and measured by HPLC. A simplescreening method was developed and tested for detection of in situ biosurfactantproduction. The screening method consisted of measuring ST in filtered ground-water subjected to increasing degrees of concentration in a rotary evaporator. STvalues in groundwater from contaminated wells decreased to 29 dynes/cm afterbeing concentrated 10 to 15 times, indicating that biosurfactants in the samplereached the CMC. Groundwater from uncontaminated wells concentrated 25 timesshowed no decrease in ST from 72 dynes/cm, showing that biosurfactants were notpresent. The biosurfactant screening method shows promise for detecting in situbiosurfactant production at other sites. Microorganism plating and FAME identi-fication showed that contamination with petroleum hydrocarbons resulted in asignificant shift in the distribution of hydrocarbon-degrading species at the site.The most abundant species in the uncontaminated soil were P. chlororaphis,B. sphaericus (II B), B. subtilis, A. duodecanis, and A. oxydans. Hydrocarboncontamination favored the growth of P. aeruginosa and P. fluorescens at theexpense of the predominant species in the uncontaminated soil. Biosurfactantproduction, used alone or in conjunction with other measurements, could be usefulat many NAPL-contaminated sites as an indicator of intrinsic bioremediation.

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