Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 82:687691 (2007)

Technical NoteUse of biosurfactant in the removal of oilfrom contaminated sandy soilLdia M Santa Anna,1 Adriana U Soriano,1 Absai C Gomes,1 Emerson P Menezes,2

Melissa LE Gutarra3 Denise MG Freire3 and Nei Pereira Jr41Petrobras Research Center, Centro de Pesquisas da Petrobras, Rio de Janeiro, Brazil2Gorceix Foundation, Rio de Janeiro, Brazil3Instituto de Qumica, UFRJ, Rio de Janeiro, Brazil4Escola de Qumica, UFRJ, Rio de Janeiro, Brazil

Abstract: The effectiveness of cell-free rhamnolipid biosurfactant, derived from the culture medium at the endof fermentation was investigated for the removal of two different kinds of oil from contaminated sandy soils. Thecrude cultivation medium, containing 13.2 gL1 of rhamnolipids, had a surface tension, interfacial tension andcriticalmicellar concentration of 30mNm1, 2mNm1 and 60mgL1, respectively. The evaluation of biosurfactantin the culture medium (BM) and oil concentrations in the removal of oil from different contaminated sandy soilwas performed using a statistical experimental design tool. Oil in sandy soil, containing predominantly aromaticor paraffinic hydrocarbons (5 to 10% w/w), was removed by as much as 91 and 78%, respectively, in the presenceof reduced amounts of BM (6.3 to 7.9 gL1). The progress of oil removal was monitored for 101days and resultsindicated that removal efficiency in sandy soil with aromatic characteristics was relatively stable over the entireperiod. Based on these studies, it is concluded that use of a BM was effective in reducing oil concentrations incontaminated sandy soil. 2007 Society of Chemical Industry

Keywords: biosurfactants; rhamnolipids; Pseudomonas aeruginosa; sandy soil washing; crude oil removal

NOMENCLATUREDiC10C10: 3-(3-((2 R,3 S,4 S,5 R)-4,5-dihydroxy-6-methyl-3-((2 S,3 S,4 S,5 R)-3,4,5-trihydroxy-6-methyltetrahydro-2 H-pyran-2-yloxy)tetrahydro-2 H-pyran-2-yloxy)decanoyloxy)decanoic acid

DiC10C12: 3-(3-((2 R,3 S,4 S,5 R)-4,5-dihydroxy-6-methyl-3-((2 S,3 S,4 S,5 R)-3,4,5-trihydroxy-6-methyltetrahydro-2 H-pyran-2-yloxy)tetrahydro-2 H-pyran-2-yloxy)decanoyloxy)dodecanoic acid

MonoC10C10: 3-(3-((2 R,3 S,4 S,5 R)-tetrahydro-3,5-dihydroxy-4,6-dimethyl-2 H-pyran-2-yloxy)de-canoyloxy)decanoic acid

INTRODUCTIONBiosurfactants are amphiphilic compounds that areformed on living surfaces or excreted from cells. Theyhave a variety of applications, but are used primar-ily in the oil industry. These biomolecules can beemployed both in the recovery of petroleum and inbioremediation processes and dispersal of oil spillson land and sea.1 Their structural diversity and rel-atively low toxicity allow for multiple environmentalapplications, where they may be preferable to syn-thetic surfactants.2 Biosurfactants of the rhamnolipid

(RML)-type show substantial promise due to theirexcellent surface active characteristics. Laboratorystudies examining the application of RML in theremoval of hydrocarbons in contaminated soil havetypically employed a commercial biosurfactant in itspurified form, showing better results than that usingchemical surfactants such as sodium dodecyl sulphate(SDS) and Tween 80.3,4 The treatment of crude oilsin sand using commercial biosurfactants also showedgood results.5 However, a significant barrier to thewidespread use of this biosurfactant in environmen-tal applications is the reduction of production costsand recovery of product.1 Given those challenges,the use of crude (nonpurified) biosurfactant, whichis present in the culture medium free of cells, couldbe a promising alternative, although relevant studiesare still limited. Production studies using low costsubstrates, including evaluations of toxicity and sur-face active proprieties, have been completed by SantaAnna et al.6 and Santos et al.7 The authors produceda crude fermented medium from Pseudomonas aerug-inosa PA1 containing a blend of five types of RML,which utilized a low-cost raw material (glycerol). Thegoal of this work was to investigate the solubilizationeffect of this cell-free biosurfactant, in the removal of

Correspondence to: Denise MG Freire, Universidade Federal do Rio de Janeiro, Depto. de Bioqumica, Instituto de Qumica, Laboratorio de BiotecnologiaMicrobiana, 549-2, BrazilE-mail: freire@iq.ufrj.br(Received 20 December 2006; revised version received 1 March 2007; accepted 2 March 2007)Published online 6 July 2007; DOI: 10.1002/jctb.1741

2007 Society of Chemical Industry. J Chem Technol Biotechnol 02682575/2007/$30.00

LM Santa Anna et al.

two different kinds of oil (aromatic and paraffin) fromcontaminated sandy soil.

MATERIALS AND METHODSBiosurfactant ProductionRML production was performed using a strain ofPseudomonas aeruginosa PA1 isolated from the oilproduction water from Sergipe, Brazil, which wasgrown in an optimized medium described by Santoset al7. Fermentation was conducted in a nitrogen-fed batch mode in a 10 L bioreactor with agitationrate 120 rpm, and maintained for 7 days at 30 C.8Afterwards the fermented medium was sterilized at100 kPa for 1 h and centrifuged at 5523 g for 40 minto separate the cellular mass, resulting in a mediumwith biosurfactant (BM).

Sandy soil and oil characteristicsA pure sandy soil from a Brazilian beach with agranulometry of 2 mm was used in the experiments.Oils were characterized in relation to the evaluationof the n-paraffinics by gaseous chromatography (GC;Agilent 6890; Agilent Technologies, Palo Alto, CA,USA) with a 30 m DB-5 column at 300 C anda 1 mL sample. The aromatic oil contained 19%saturated hydrocarbons, 69% aromatics and 12%resins and asphaltenes. The paraffinic oil contained54% saturated hydrocarbons, 23% aromatics and 23%resins and asphaltenes.8

Determination of the surface tension, interfacialtension and critical micellar concentration (CMC)and assays of pHThe surface tension and interfacial tension (using n-hexadecane as the organic phase) of the BM wasdetermined using a Kruss K9 digital tensiometerat 25 C and the Du Nouy method.9 Dilutionswere prepared with distilled water to make eightconcentrations: 0.8, 1.6, 6.6, 26.2, 65, 84, 155 and355 mg L1. The CMC was calculated by stiff pointdetermination using the graphic curve. A controlexperiment was also conducted where water was usedinstead of BM.

The assays of correlation between the increase inpH and surface tension was carried out by adjustingthe pH of the culture medium (BM) with a solution ofNaOH 1 M to increase pH and HCl 1M to reduce pH.

Washing experimentsWashing tests were used to evaluate the removal ofxenotibiotics of the sandy soil. The washing tests wereconducted by placing 20 mL of BM with pH 6.5and 10 g of laboratory-prepared sandy soil with oil in500 mL Erlenmeyer flasks in three replicates. The firstset of experiments was performed 72 h after oil hadbeen placed in the sandy soil following a statisticalexperimental design that utilized different oil andrhamnolipid levels. The second set of experiments

Table 1. Variables studied and level for removal of oil

Levels

Oils used Variables 1.41 1 0 1 1.41Aromatic O (%m m1) 5.0 5.7 7.5 9.3 10.0

R (g L1) 3.6 4.4 6.3 8.2 9.0Paraffinic O (%m m1) 5.0 5.7 7.5 9.3 10.0

R (g L1) 3.3 3.97 5.6 7.23 7.9

was conducted under optimized conditions after theoil/sand mix had been allowed to weather for a periodof 3 to 101 days post-addition.

Flasks containing the BM/sandy soil mixture wereagitated on a shaker table at 250 rpm for 30 min at30 C. After mixing, the flasks remained undisturbedfor 15 min to allow separation. The aqueous phasewas removed by vacuum extraction. The sandy soilwas dried in a vacuum stove at 60 C for 16 h tocomplete the gravimetric evaluation. After washingand drying, oil was extracted from 2 g of sandysoil using a dichloromethane solvent (Merck, Rio deJaneiro, Brazil 99.8% purity) and the gravimetricmethod for oils and grease was used.10 An extractionaccelerated with solvent extractor (ASE) Model 300(Dionex Corpn., Sunnyvale, CA, USA) was used.After extraction, the sandy soil was dried at 50 C,cooled and weighed.9 The percentage of oil remaining(RO(%)) was calculated using the equation

RO(%) = (A0 As) 100 (1)

where A0 is the mass of sandy soil containing theresidual oil (g) and As is the mass of sandy soil afterextraction of oil (g).

CHARACTERISTICS OF OIL AFTER THEWASHING EXPERIMENTSThe extracted oil was concentrated to a volume of1 mL in a TURBOVAP 500, double boiler (CaliperLife Sciences, Hopkinton, MA, USA) at 50 C, andthe fractions were analyzed by CG (Hewlett Packard,Model 6890) under the following conditions: column30 m DB-5 (0.25 mm inside diameter and 0.25 mm offilm); injection mode in split (5:1); sample injectionvolume 1 mL; injector temperature 300 C; detectortemperature (FID) 340 C; Carrier gas He; carriergas speed 30 cm s1 (1.3, mL min1) in constantflow; temperature programming 40 to 320 C at 2.5 Cmin1, 18 min at 320 C; duration of analysis 130 min.

Experimental DesignA 22 factorial design was used to evaluate thepercentage of oil removal from the sandy soil as afunction of (1) oil concentration with aromatic and/orparaffinic characteristics (O) and (2) concentration ofbiosurfactant in the BM (R). These variables with theirreal and encoded values are presented in Table 1.Statistical analysis was performed using Statistica

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Biosurfactant removal of oil from contaminated sandy soil

Statsoft Version 5.1 (Tulsa, Oklahoma, USA). Analysisof variance (ANOVA) was used to evaluate the fitnessof the model.

RESULTS AND DISCUSSIONSProperties of the BMThe biosurfactant in the BM has a predominance ofrhamnolipids with large hydroxydecanoic acid chains,which have excellent hydrophobic characteristics, suchas 36.5% DiC10C10, 17.9% DiC10C12 and 37.4%MonoC10C10.8 The pH of the fermented RMLmedium influences surface active properties (Fig. 1),shown by an 11% increase in surface tension whenthe pH rose from 4.0 to 8.0. Bai et al.4 noted thatproperties such as surface tension are mainly affectedabove the CMC.

The BM containing 13.2 g L1 of rhamnolipids hadsurface tension, interfacial tension (n-hexadecane) andCMC of 29.4 mN m1, 2.0 mN m1 and 60 mg L1,respectively.

Evaluation of oil removal efficiencyThe removal of oil from sandy soils weathered for 72 hwas evaluated using a statistical experimental design asa function of oil concentration and BM concentration.Oil concentration and BM concentration, in linearand quadratic terms, were statistically significant atP < 0.1 using both aromatic and paraffinic oil. Theanalysis of variance (ANOVA) was used to evaluatethe fitness of the model that was generated. The modelis predictive when the value of F is above the criticalF value and the coefficient of regression is close to 1.For the aromatic oil, a value of F (15.71) was observedapproximately five times greater than the critical value(3.45) and a coefficient of regression of 0.97. Forthe paraffinic oil, a value of F (3.78) was observedone time greater that the critical value (3.45) and acoefficient of regression of 0.89. The error calculatedin triplicate was also low. Using these data, it waspossible to generate encoded models

Removal = 88.75 + 4.61O 2.35O2 2.05R 2.40R2 + 1.85OR (2)

27

28

29

30

31

32

33

4.0 5.2 5.5 6.5 7.0 8.0pH

Surfa

ce te

nsio

n (m

N/m)

Figure 1. Surface active properties of the biosurfactant in the culturemedium (BM) in relation to pH.

72645648

90

9590858075706560

8580757065

(a)

(b) O

il removal(%)

Oil rem

oval(%)

9.3

7.5

5.75.0

3.64.4

6.5

8.29.0

Rhamno

lipid (g L

-1 )

Rhamno

lipid (g L

-1 )

Oil concentration (%)

Oil concentration (%)

80

72

64

56

48

40

10.0

10.0

9.3

7.5

5.75.0

3.33.57

5.6

7.527.9

Figure 2. Surface response analysis of the removal tests using oilwith aromatic (a) and paraffinic (b) characteristics, in relation to oil andrhamnolipid concentration. The points on the response surfacesindicate the experimental data.

Removal = 77.50 + 5.09O 7.18O2 + 2.43R 2.47R2 0.60OR (3)

and response surfaces (Fig. 2(a) and (b)) for theremoval of aromatic and paraffinic oils (%), respec-tively, where O is the encoded oil concentrationvariable and R is the encoded rhamnolipid concen-tration variable.

The best oil removal observed during the washingexperiment was 91 0.1% measured using 6.3 g L1BM and 10% w/w aromatic oil. For paraffinic oil, thebest removal was 79.7 0.2% using 7.23 g L1 BMand 9.3% w/w paraffinic oil. The control experiment,performed with water instead of BM, resulted in38 0.2% and 13 0.2% of aromatic and paraffinicoil removal, respectively. The application of BMcontaining rhamnolipids was very effective in removingoil from contaminated sandy soils 72 h after oilimpregnation.

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Rel

ativ

e ab

unda

nce

Rel

ativ

e ab

unda

nce

Rel

ativ

e ab

unda

nce

Rel

ativ

e ab

unda

nce

C11 C13

(a) (b)

(c) (d)Retention time (minutes)

Retention time (minutes) Retention time (minutes)

Retention time (minutes)

Figure 3. Chromatographic profile of the original aromatic oil (a); aromatic oil after washing with the biosurfactant in the culture medium (BM) (b);original paraffinic oil (c); paraffinic oil after washing with BM (d).

These results compare favorably with those reportedby Urum et al.5 who used pure biosurfactant to obtain79% crude oil removal with a 0.5% w/w mix ofcommercial mono- and di-RMLs. Our data provideguidance as to the BM concentration that mustbe employed to maximize removal of aromatic andparaffinic oils from sandy soil, depending on the soilpetroleum concentration, within the studied ranges(Figs 2(a) and 1(b)).

Different aromatic and paraffinic fractions wereremoved at different rates during washing tests(Fig. 3(a)3(d)). Volatile fractions (C11 and C12)from sandy soil containing oil with paraffinic charac-teristics were removed at the greatest rate, probablyas a result of volatilization during washing and dry-ing of the sample. However, in sandy soil containingoil with aromatic characteristics, there appeared tobe less preference with regard to fractions removed.These characteristics have also been reported by otherresearchers.11

In the second set of experiments, where oilremoval was evaluated in weathered soils, optimal BMconcentrations (6.3 and 7.23 g L1 in aromatic andparaffinic oil, respectively) and oil concentrations (10and 9.3% in aromatic and paraffinic oil, respectively)were used. Different behavior was observed betweenoils with aromatic or paraffinic characteristics (Fig. 4).There was a slight decrease in the removal of aromaticoil between soil that had been weathered for 3 days(91.0%) and soils that had been weathered for longerperiods of time, including 101 days (85.2%). However,removal of paraffinic oil from sandy soil droppedsubstantially from 79.7% at 3 days to 25.1% at101 days. These results suggest that the adsorption ofparaffinic oil onto sandy soil is greater than that of the

0%10%20%30%40%50%60%70%80%90%

100%

3 17 31 61 101Days of weathering

Rem

oval

of o

il in

was

hing

Aromatic oil Paraffinic oil

Figure 4. Percentage removal of oil from soil, after weathering for 3 to101 days under optimal biosurfactant in the culture medium (BM) (6.3and 7.23 g L1 in aromatic and paraffinic oil, respectively) and oil (10and 9.3% in aromatic and paraffinic oil, respectively) concentrations.

aromatic oil, and the effects of BM on solubilizationand emulsification are less pronounced for paraffinicoil. Urum et al.5 reported similar conclusions. To beeffective in cleaning oil spills, especially where totalpetroleum composition is dominated by paraffin oil,soil washing should occur as quickly as possible (lessthan 3 days).

In spite of the differences between the two typesof oils, removal was generally greater than 60% whentreatment occurred within 31 days of soiloil mixing.Treating with this new and promising BM, therefore,can be an efficient means of reducing concentrationsof oil in sandy soils following spills.

CONCLUSIONSIn this study, a culture medium free of cells containinga low concentration of rhaminolipids, BM, is shown to

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Biosurfactant removal of oil from contaminated sandy soil

be an excellent alternative in the removal of paraffinicor aromatic oils from contaminated sand. Washingtests with oil-contaminated sand soil weathered upto 101 days showed effective removal of the oil.Moreover, the structure of aromatic and paraffinic oilshowed no changes after removal tests indicating thepossibility of recycling of this oil. The effectiveness andreduced cost of BM suggests that it may be a preferableremediation method for the petroleum production andtransport industry.

REFERENCES1 Banat IM, Makkar RS and Cameotra SS, Potential commercial

applications of microbial surfactants. Appl Microbiol Biot5:495508 (2000).

2 Mulligan CN, Environmental applications for biosurfactants.Environ Pollut 133:183198 (2005).

3 Abdul AS and Gibson TL, Laboratorory study of surfactant-enhanced washing of polychlorinated biphenil from sandymaterial. Environ Sci Technol 25:665671 (1991).

4 Bai G, Brusseau ML and Miller RM, Biosurfactant-enhancedremoval of residual hydrocarbon from soil. J Contam Hydrol25:157170 (1997).

5 Urum K, Pekdemir T and Copur M, Surfactants treatment ofcrude oil contaminated soils. J Colloid Interf Sci 276:456464(2004).

6 Santa Anna LMM, Sebastian GV, Pereira NJ, Alves TLM,Menezes EP and Freire DMG, Production of biosurfactantfrom a new and promising strain of Pseudomonas aeruginosaPA1. Appl Biochem Biotechnol 9193:459467 (2001).

7 Santos AS, Sampaio PW, Sebastian GV, Santa Anna LM,Pereira NJ and Freire DMG, Evaluation of different carbonand nitrogen sources in production of rhamnolipids by astrain of Pseudomonas aeruginosa. Appl Biochem Biotechnol98100:10251035 (2002).

8 Santa Anna LMM, Production and application of rhamnolipidsin the remedying of oil-impacted soils. Doctoral Thesis.Federal University of Rio de Janeiro (2005).

9 ASTM D97199a Standard test method for interfacial tensionof oil against water by the ring method. American Society forTesting and Materials (2004).

10 ASTM D4281, Standard test method for oil and grease (fluoro-carbon extractable substances) by gravimetric determination.American Society for Testing and Materials (2001).

11 Kyung HS and Kyoung WK, A biosurfactant-enhanced soilflushing for the removal of phenanthrene and diesel in sandyEnviron Geochem Hlth 26:511 (2004).

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