Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 82:687691 (2007)

Technical Note Use of biosurfactant in the removal of oil from contaminated sandy soil
L dia 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 Jr4
1 Petrobras 2 Gorceix

Rio de Janeiro, Brazil Research Center, Centro de Pesquisas da Petrobras, Foundation, Rio de Janeiro, Brazil 3 Instituto de Qu mica, UFRJ, Rio de Janeiro, Brazil 4 Escola de Qu mica, UFRJ, Rio de Janeiro, Brazil

Abstract: The effectiveness of cell-free rhamnolipid biosurfactant, derived from the culture medium at the end of fermentation was investigated for the removal of two different kinds of oil from contaminated sandy soils. The crude cultivation medium, containing 13.2 g L1 of rhamnolipids, had a surface tension, interfacial tension and critical micellar concentration of 30 mN m1 , 2 mN m1 and 60 mg L1 , respectively. The evaluation of biosurfactant in the culture medium (BM) and oil concentrations in the removal of oil from different contaminated sandy soil was performed using a statistical experimental design tool. Oil in sandy soil, containing predominantly aromatic or parafnic hydrocarbons (5 to 10% w/w), was removed by as much as 91 and 78%, respectively, in the presence of reduced amounts of BM (6.3 to 7.9 g L1 ). The progress of oil removal was monitored for 101 days and results indicated that removal efciency in sandy soil with aromatic characteristics was relatively stable over the entire period. Based on these studies, it is concluded that use of a BM was effective in reducing oil concentrations in contaminated sandy soil. 2007 Society of Chemical Industry

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

NOMENCLATURE DiC10C10: 3-(3-((2 R,3 S,4 S,5 R)-4,5-dihydroxy6-methyl-3-((2 S,3 S,4 S,5 R)-3,4,5-trihydroxy-6methyltetrahydro-2 H-pyran-2-yloxy)tetrahydro-2 Hpyran-2-yloxy)decanoyloxy)decanoic acid DiC10C12: 3-(3-((2 R,3 S,4 S,5 R)-4,5-dihydroxy6-methyl-3-((2 S,3 S,4 S,5 R)-3,4,5-trihydroxy-6methyltetrahydro-2 H-pyran-2-yloxy)tetrahydro-2 Hpyran-2-yloxy)decanoyloxy)dodecanoic acid MonoC10C10: 3-(3-((2 R,3 S,4 S,5 R)-tetrahydro3,5-dihydroxy-4,6-dimethyl-2 H-pyran-2-yloxy)decanoyloxy)decanoic acid

INTRODUCTION Biosurfactants are amphiphilic compounds that are formed on living surfaces or excreted from cells. They have a variety of applications, but are used primarily in the oil industry. These biomolecules can be employed both in the recovery of petroleum and in bioremediation processes and dispersal of oil spills on land and sea.1 Their structural diversity and relatively low toxicity allow for multiple environmental applications, where they may be preferable to synthetic surfactants.2 Biosurfactants of the rhamnolipid

(RML)-type show substantial promise due to their excellent surface active characteristics. Laboratory studies examining the application of RML in the removal of hydrocarbons in contaminated soil have typically employed a commercial biosurfactant in its puried form, showing better results than that using chemical surfactants such as sodium dodecyl sulphate (SDS) and Tween 80.3,4 The treatment of crude oils in sand using commercial biosurfactants also showed good results.5 However, a signicant barrier to the widespread use of this biosurfactant in environmental applications is the reduction of production costs and recovery of product.1 Given those challenges, the use of crude (nonpuried) biosurfactant, which is present in the culture medium free of cells, could be a promising alternative, although relevant studies are still limited. Production studies using low cost substrates, including evaluations of toxicity and surface active proprieties, have been completed by Santa Anna et al .6 and Santos et al .7 The authors produced a crude fermented medium from Pseudomonas aeruginosa PA1 containing a blend of ve types of RML, which utilized a low-cost raw material (glycerol). The goal of this work was to investigate the solubilization effect of this cell-free biosurfactant, in the removal of

Correspondence to: Denise MG Freire, Universidade Federal do Rio de Janeiro, Depto. de Bioqu mica, Instituto de Qu mica, Laboratorio de Biotecnologia Microbiana, 549-2, Brazil E-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 parafn) from contaminated sandy soil.

Table 1. Variables studied and level for removal of oil

Levels Oils used Variables O (%m R (g L1 ) O (%m m1 ) R (g L1 ) m1 ) 1.41 5.0 3.6 5.0 3.3 1 5.7 4.4 5.7 3.97 0 7.5 6.3 7.5 5.6 1 9.3 8.2 9.3 7.23 1.41 10.0 9.0 10.0 7.9

MATERIALS AND METHODS Biosurfactant Production RML production was performed using a strain of Pseudomonas aeruginosa PA1 isolated from the oil production water from Sergipe, Brazil, which was grown in an optimized medium described by Santos et al 7 . Fermentation was conducted in a nitrogenfed batch mode in a 10 L bioreactor with agitation rate 120 rpm, and maintained for 7 days at 30 C.8 Afterwards the fermented medium was sterilized at 100 kPa for 1 h and centrifuged at 5523 g for 40 min to separate the cellular mass, resulting in a medium with biosurfactant (BM). Sandy soil and oil characteristics A pure sandy soil from a Brazilian beach with a granulometry of 2 mm was used in the experiments. Oils were characterized in relation to the evaluation of the n-parafnics by gaseous chromatography (GC; Agilent 6890; Agilent Technologies, Palo Alto, CA, USA) with a 30 m DB-5 column at 300 C and a 1 mL sample. The aromatic oil contained 19% saturated hydrocarbons, 69% aromatics and 12% resins and asphaltenes. The parafnic oil contained 54% saturated hydrocarbons, 23% aromatics and 23% resins and asphaltenes.8 Determination of the surface tension, interfacial tension and critical micellar concentration (CMC) and assays of pH The surface tension and interfacial tension (using nhexadecane as the organic phase) of the BM was determined using a Kruss K9 digital tensiometer at 25 C and the Du Nouy method.9 Dilutions were prepared with distilled water to make eight concentrations: 0.8, 1.6, 6.6, 26.2, 65, 84, 155 and 355 mg L1 . The CMC was calculated by stiff point determination using the graphic curve. A control experiment was also conducted where water was used instead of BM. The assays of correlation between the increase in pH and surface tension was carried out by adjusting the pH of the culture medium (BM) with a solution of NaOH 1 M to increase pH and HCl 1M to reduce pH. Washing experiments Washing tests were used to evaluate the removal of xenotibiotics of the sandy soil. The washing tests were conducted by placing 20 mL of BM with pH 6.5 and 10 g of laboratory-prepared sandy soil with oil in 500 mL Erlenmeyer asks in three replicates. The rst set of experiments was performed 72 h after oil had been placed in the sandy soil following a statistical experimental design that utilized different oil and rhamnolipid levels. The second set of experiments
688

Aromatic Parafnic

was conducted under optimized conditions after the oil/sand mix had been allowed to weather for a period of 3 to 101 days post-addition. Flasks containing the BM/sandy soil mixture were agitated on a shaker table at 250 rpm for 30 min at 30 C. After mixing, the asks remained undisturbed for 15 min to allow separation. The aqueous phase was removed by vacuum extraction. The sandy soil was dried in a vacuum stove at 60 C for 16 h to complete the gravimetric evaluation. After washing and drying, oil was extracted from 2 g of sandy soil using a dichloromethane solvent (Merck, Rio de Janeiro, Brazil 99.8% purity) and the gravimetric method for oils and grease was used.10 An extraction accelerated 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 the residual oil (g) and As is the mass of sandy soil after extraction of oil (g).

CHARACTERISTICS OF OIL AFTER THE WASHING EXPERIMENTS The extracted oil was concentrated to a volume of 1 mL in a TURBOVAP 500, double boiler (Caliper Life Sciences, Hopkinton, MA, USA) at 50 C, and the fractions were analyzed by CG (Hewlett Packard, Model 6890) under the following conditions: column 30 m DB-5 (0.25 mm inside diameter and 0.25 mm of lm); injection mode in split (5:1); sample injection volume 1 mL; injector temperature 300 C; detector temperature (FID) 340 C; Carrier gas He; carrier gas speed 30 cm s1 (1.3, mL min1 ) in constant ow; temperature programming 40 to 320 C at 2.5 C min1 , 18 min at 320 C; duration of analysis 130 min. Experimental Design A 22 factorial design was used to evaluate the percentage of oil removal from the sandy soil as a function of (1) oil concentration with aromatic and/or parafnic characteristics (O) and (2) concentration of biosurfactant in the BM (R). These variables with their real and encoded values are presented in Table 1. Statistical analysis was performed using Statistica
J Chem Technol Biotechnol 82:687691 (2007) DOI: 10.1002/jctb

Biosurfactant removal of oil from contaminated sandy soil

Statsoft Version 5.1 (Tulsa, Oklahoma, USA). Analysis of variance (ANOVA) was used to evaluate the tness of the model.

(a)

RESULTS AND DISCUSSIONS Properties of the BM The biosurfactant in the BM has a predominance of rhamnolipids with large hydroxydecanoic acid chains, which have excellent hydrophobic characteristics, such as 36.5% DiC10C10, 17.9% DiC10C12 and 37.4% MonoC10C10.8 The pH of the fermented RML medium inuences surface active properties (Fig. 1), shown by an 11% increase in surface tension when the pH rose from 4.0 to 8.0. Bai et al .4 noted that properties such as surface tension are mainly affected above the CMC. The BM containing 13.2 g L1 of rhamnolipids had surface tension, interfacial tension (n-hexadecane) and CMC of 29.4 mN m1 , 2.0 mN m1 and 60 mg L1 , respectively. Evaluation of oil removal efciency The removal of oil from sandy soils weathered for 72 h was evaluated using a statistical experimental design as a function of oil concentration and BM concentration. Oil concentration and BM concentration, in linear and quadratic terms, were statistically signicant at P < 0.1 using both aromatic and parafnic oil. The analysis of variance (ANOVA) was used to evaluate the tness of the model that was generated. The model is predictive when the value of F is above the critical F value and the coefcient of regression is close to 1. For the aromatic oil, a value of F (15.71) was observed approximately ve times greater than the critical value (3.45) and a coefcient of regression of 0.97. For the parafnic oil, a value of F (3.78) was observed one time greater that the critical value (3.45) and a coefcient of regression of 0.89. The error calculated in triplicate was also low. Using these data, it was possible to generate encoded models Removal = 88.75 + 4.61O 2.35O2 2.05R 2.40R2 + 1.85OR
33 Surface tension (mN/m) 32 31 30 29 28 27

95 90 85 80 75 70 65 60 0 0. Oi 1 9.3 lc on 5 ce 7. nt ra tio n (% )

5. 7

5.

(b)

Oi

Figure 2. Surface response analysis of the removal tests using oil with aromatic (a) and parafnic (b) characteristics, in relation to oil and rhamnolipid concentration. The points on the response surfaces indicate the experimental data.

) Oil removal(% Oil removal(% )

9.0
-1 )

Rh

am

no

( ipid

gL

90 85 80 75 70 65

8.2

6. 5

4.4 3.6

80 72 64 56 48 40

.0 10 .3 9 lc on ce nt ra t

7. 9

7.

7.5

52

ion

(%

7 5.

5.

Rh

no am

lipi

g d(

-1 )

72 64 56 48

5. 6

3. 57
3.3

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

(2 )

4.0

5.2

5.5 pH

6.5

7.0

8.0

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

and response surfaces (Fig. 2(a) and (b)) for the removal of aromatic and parafnic oils (%), respectively, where O is the encoded oil concentration variable and R is the encoded rhamnolipid concentration variable. The best oil removal observed during the washing experiment was 91 0.1% measured using 6.3 g L1 BM and 10% w/w aromatic oil. For parafnic oil, the best removal was 79.7 0.2% using 7.23 g L1 BM and 9.3% w/w parafnic oil. The control experiment, performed with water instead of BM, resulted in 38 0.2% and 13 0.2% of aromatic and parafnic oil removal, respectively. The application of BM containing rhamnolipids was very effective in removing oil from contaminated sandy soils 72 h after oil impregnation.
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J Chem Technol Biotechnol 82:687691 (2007) DOI: 10.1002/jctb

LM Santa Anna et al.

(a) Relative abundance (b) Relative abundance Retention time (minutes) (c) Relative abundance (d)

C11

C13

Retention time (minutes)

Retention time (minutes)

Relative abundance

Retention time (minutes)

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

These results compare favorably with those reported by Urum et al .5 who used pure biosurfactant to obtain 79% crude oil removal with a 0.5% w/w mix of commercial mono- and di-RMLs. Our data provide guidance as to the BM concentration that must be employed to maximize removal of aromatic and parafnic oils from sandy soil, depending on the soil petroleum concentration, within the studied ranges (Figs 2(a) and 1(b)). Different aromatic and parafnic fractions were removed at different rates during washing tests (Fig. 3(a)3(d)). Volatile fractions (C11 and C12) from sandy soil containing oil with parafnic characteristics were removed at the greatest rate, probably as a result of volatilization during washing and drying of the sample. However, in sandy soil containing oil with aromatic characteristics, there appeared to be less preference with regard to fractions removed. These characteristics have also been reported by other researchers.11 In the second set of experiments, where oil removal was evaluated in weathered soils, optimal BM concentrations (6.3 and 7.23 g L1 in aromatic and parafnic oil, respectively) and oil concentrations (10 and 9.3% in aromatic and parafnic oil, respectively) were used. Different behavior was observed between oils with aromatic or parafnic characteristics (Fig. 4). There was a slight decrease in the removal of aromatic oil between soil that had been weathered for 3 days (91.0%) and soils that had been weathered for longer periods of time, including 101 days (85.2%). However, removal of parafnic oil from sandy soil dropped substantially from 79.7% at 3 days to 25.1% at 101 days. These results suggest that the adsorption of parafnic oil onto sandy soil is greater than that of the
690

Aromatic oil

Paraffinic oil

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
3 17 31 Days of weathering 61 101

Figure 4. Percentage removal of oil from soil, after weathering for 3 to 101 days under optimal biosurfactant in the culture medium (BM) (6.3 and 7.23 g L1 in aromatic and parafnic oil, respectively) and oil (10 and 9.3% in aromatic and parafnic oil, respectively) concentrations.

aromatic oil, and the effects of BM on solubilization and emulsication are less pronounced for parafnic oil. Urum et al .5 reported similar conclusions. To be effective in cleaning oil spills, especially where total petroleum composition is dominated by parafn oil, soil washing should occur as quickly as possible (less than 3 days). In spite of the differences between the two types of oils, removal was generally greater than 60% when treatment occurred within 31 days of soiloil mixing. Treating with this new and promising BM, therefore, can be an efcient means of reducing concentrations of oil in sandy soils following spills.

CONCLUSIONS In this study, a culture medium free of cells containing a low concentration of rhaminolipids, BM, is shown to
J Chem Technol Biotechnol 82:687691 (2007) DOI: 10.1002/jctb

Removal of oil in washing

Biosurfactant removal of oil from contaminated sandy soil

be an excellent alternative in the removal of parafnic or aromatic oils from contaminated sand. Washing tests with oil-contaminated sand soil weathered up to 101 days showed effective removal of the oil. Moreover, the structure of aromatic and parafnic oil showed no changes after removal tests indicating the possibility of recycling of this oil. The effectiveness and reduced cost of BM suggests that it may be a preferable remediation method for the petroleum production and transport industry.

REFERENCES
1 Banat IM, Makkar RS and Cameotra SS, Potential commercial applications of microbial surfactants. Appl Microbiol Biot 5:495508 (2000). 2 Mulligan CN, Environmental applications for biosurfactants. Environ Pollut 133:183198 (2005). 3 Abdul AS and Gibson TL, Laboratorory study of surfactantenhanced washing of polychlorinated biphenil from sandy material. Environ Sci Technol 25:665671 (1991). 4 Bai G, Brusseau ML and Miller RM, Biosurfactant-enhanced removal of residual hydrocarbon from soil. J Contam Hydrol 25:157170 (1997).

5 Urum K, Pekdemir T and Copur M, Surfactants treatment of crude 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 biosurfactant from a new and promising strain of Pseudomonas aeruginosa PA1. Appl Biochem Biotechnol 9193:459467 (2001). 7 Santos AS, Sampaio PW, Sebastian GV, Santa Anna LM, Pereira NJ and Freire DMG, Evaluation of different carbon and nitrogen sources in production of rhamnolipids by a strain of Pseudomonas aeruginosa. Appl Biochem Biotechnol 98100:10251035 (2002). 8 Santa Anna LMM, Production and application of rhamnolipids in the remedying of oil-impacted soils. Doctoral Thesis. Federal University of Rio de Janeiro (2005). 9 ASTM D97199a Standard test method for interfacial tension of oil against water by the ring method. American Society for Testing and Materials (2004). 10 ASTM D4281, Standard test method for oil and grease (uorocarbon extractable substances) by gravimetric determination. American Society for Testing and Materials (2001). 11 Kyung HS and Kyoung WK, A biosurfactant-enhanced soil ushing for the removal of phenanthrene and diesel in sandy Environ Geochem Hlth 26:511 (2004).

J Chem Technol Biotechnol 82:687691 (2007) DOI: 10.1002/jctb

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