생물학적 토양오염 정화시 계면활성제의 효과

강석환,조귀준,한창성,김은기

환경생물공학실, 생물공학과, 인하대

Effectiveness of Surfactant-aided

Bioremediation of Contaminated Soil

Sukwhan Kang, Kwijoon Cho, Changsung Han, Eunki Kim

Dept. Biological Engineering, Inha University, Inchon, Korea

INTRODUCTION

Surfactant has been used as an additive to increase the water solubility and biodegradation rate of nonpolar compounds in contaminated soil. Enhancing the performance of surfactant in soil bioremediation has been attempted by adding the cosolvent (paraffin oil) and by the use of biosurfactant (sophorolipid), respectively.

Part 1. Paraffin Oil

Organic solvents, such as alcohols or alkanes, can dissolve high concentrations of PAH.  The affinity of the solvent for a given PAH is typically quantified by a distribution coefficient. Among the available solvents, paraffin oil (mineral oil) is safe for human consumption and is used as an oral laxative.  Addition of this solvent to water has been shown to increase the biodegradation of PAH (Jimenze and Bartha, 1996). However, paraffin oil is not miscible with water. Therefore remedition of PAH contaminated soils by solvent augmentation with parraffin oil is presently limited to ex-situ treatment processes.

In order to use paraffin oil for in-situ treatment, this solvent must be stabilized by some method, such as the addition of a surfactant. In this work, we used a non-ionic surfactant /paraffin oil emulsion to increase the aqueous concentration of a test PAH (phenanthrene) and to enhance the mobilization of phenanthrene from a test soil.

MATERIALS AND METHODS

Materials: Polyoxyethylene 4 lauryl ether (Brij 30) was used as a non-ionic surfactant. Paraffin oil was purchased from local drug store. Radio-labelled phenanthrene was purchased from Sigma.  A bacterial consortium, as a gift of Prof. M. Alexander (Cornell University, USA), was used, which had been isolated from soil.

Determination of Surface tension: The surface tension of the solution was measured by using Fisher Tensiomet 20 (Fisher Scientific, USA). Paraffin oil and surfactant mixture was emulsified by using ultrasonicator for 1 min (Fisher Scientific, USA).

Solubility Enhancement Experiments: In the 20 mL vials with screw caps, 50 mL of mixture of [14C]-phenanthrene and non-labeled phenanthrene  dissolved in methanol was added. After the methanol was allowed to evaporate, 10 mL sodium azide solution (0.02% with 5mM CaSO4) was added. Total amount of phenanthrene was 10 ppm which was about 8 times  of the solubility (1.29 ppm) (Laha and Luthy, 1991). Vials were placed in a shaker at 30。C for 4 days. Duplicate samples were taken and passed through the 0.1 □m PTFE syringe filters to remove any undissolved phenanthrene. Predetermined amount of sample (4 mLs) was passed through filters before collecting the filtrate to minimize the adsorption during sampling. Filtrate samples were analyzed for radioactivity in a liquid scintillation counter to measure the amount of phenanthrene.

Column Experiments: 10 mL of saturated phenanthrene solution containing 5 mM CaSO4 and 0.005 % sodium azide was added with 5 grams of non-contaminated soil. The soil contains 2.4% organic matter and size of soil ranges between 0.0116 and 0.0087 inch. After equilibrating for 4 days, the soil slurry was packed into 10 cm (1 cm diameter) glass column. The slurry was slowly poured into the column and the effulent from the bottom was collected until one pore volume of the solution was left in the column. The total radioactivity in the collected effulent was measured to calculate the amount of phenanthrene in the soil column. Then the feeding solution was pumped into the column with 10 cm/hr pore velocity. The porosity of the column was determined as 42%. Effulent was collected by using a fraction collector and radioactivity of each fraction was used to measure the amount of eluted phenanthrene.

Mineralization Experiments: Biodegradadation experiments were conducted in 50 mL earlenmyer flasks that contain in their headspace a glass vial in which the CO2   evolved by mineralization was absorbed into 1 mL of 1N NaOH solution. Silicone rubber stopper wrapped with aluminium foil was used to minimize the loss due to sorption. 50 mL of mixture of [14C]-phenanthrene and non-labeled phenanthrene  dissolved in methanol was added. After the methanol was allowed to evaporate, 10 mL of minimal medium was added. Total concentration of phenanthrene was 10 ppm. Minimal medium contained (per Liter) 1 g (NH4)2SO4, 3.8 g KH2PO4, 0.95 g KOH, 0.1 g NaCl, 0.2 g MgSO4 and 1 mg FeSO4.7H2O. Cells, grown for 3 days with nonlabeled phenanthrene (1 g/L), was filtered by coarse filter paper (#1 Whattman paper) to remove granular phenanthrene and was inouculated with initial cell density of 0.005 OD 600. During the cultivation at 30C in the shaking incubator, one mL NaOH solution was removed by syringe intermittently and radioactivity was measured to determine the mass of CO2 and the mineralization ratio.

RESULTS AND DISCUSSION

Determination of the ratio of surfactant and paraffin oil.  The oil and surfactant mixture can be stabilized if the surface of the oil drop was surrounded by surfactant molecule. Since the surfactant will locate in the water/air surface after surrounding the oil drop, more surfactant will be required to begin micelle formation (CMC). Therefore, by measuring the difference of CMCs between surfactant-water system and surfactant-oil-water system, the ratio of oil and surfactant could be determined.  As shown in Fig.1, with the presence of paraffin oil,  more surfactant was needed to achieve the minimum surface tension where the micelle forms. 140 ppm (v/v) of surfactant was required additionaly  for 5000ppm paraffin oil (0.5% v/v). Therefore, 2.8 ppm(v/v) of surfactant was required to surround the 100 ppm (v/v) of oil drop.

Solubility of Phenanthrene. Solubility of phenanthrene was determined at various concentrations of surfactant and surfactant/oil. As shown in Fig.2, surfactant/oil mixture can solubilize more phenanthrene than the surfactant alone. This can be attributed to the presence of oil drop in the surfactant/oil mixture. Brij 30/heptane showed similar effects.

Column experiment. In order to compare the stability of surfactant/oil mixture with surfactant only system,  column test was done. As shown in Fig.3, addition of surfactant only resulted in poor mobilization of the phenanthrene from soil. However, surfactant/oil mixture could extract phenanthrene significantly more than other solutions. Comparison of the stability of surfactant/oil system can be more clear in the column experiment than in the batch experiment, since only equilibrium data are available in the batch experiment.

Biodegradation of phenanthrene in the surfactant/oil.  Phenanthrene washed from the soil by surfactant/oil should be mineralized by microorganisms and surfactant/oil should be recycled to lower the cost of  remediation. The effects of surfactant and oil drop on the availability of PAH to the cell were investigated. It was found that  bioavailability depended on the structure of surfactant. Also the PAH dissolved in the paraffin oil was mineralized more faster than that in the water. The hydrophobicity of the cell walls of microorganisms  was changed during the mineralization.

In order to investigate the bioavailablity of phenanthrene which is dissolved in the oil drop  coated by surfactant, mineralization experimentation was performed by measuring the  C14-CO2 produced from the phenanthrene mineralization. As shown in Fig.4, phenanthrene in the surfactant/oil system was mineralized more faster than that in the water. Similar result was observed by other workers (Guha and Jaffe, 1996). For the mineralization, however, adaptation of cells to the surfactants was necessary as shown in the Fig.5. Cells grown with surfactant (16 ppm), could degrade phenanthrene more faster than non-adapted cells. These results indicate that surfactant/oil can be used successfully in the in-situ mobilization of phenannthrene in soil and biodegradation of the phenanthrene with enhanced efficiency.

Conclusion

Stabilization of paraffin oil emulsion by nonionic surfactant increased phenanthrene mobilization and biodegradation rates 5 folds, respectively, from the contaminated soil in the coulmn experiment. Effectiveness of sopholipid biosurfactant for biodegradation of pristane, hexadecane and 2-methyl naphthalene was compared with commercially available surfactants.

REFERENCES

Guha,S., and P.R. Jaffe (1996), Environ. Sci. Technol., 30, 605-611,

Jimenez,I.Y.,and R.Bartha (1996), Appl. Env. Microbiol. 62(7):2311-2316

Laha,S. and R.G., Luthy (1991), Environ. Sci. Technol., 25(11):1920-1930