오염토양으로부터 분리된 미생물에 의한 Toulene으로 오염된 토양의 생물학적 복원

김 태진, 박 종만, 이 재영, 이 동완, 강 수정, 김 철경1), 고 창웅2), 김 남기

성균관대학교 화학공학과

목원대학교 화학 및 응용화학부1)

삼척대학교 화학공학과2)

Bioremediation of Soil Polluted with

Toluene by Microorganisms Isolated from Contaminated Soil

Tea Jin Kim, Jong Man Park, Jea Young Lee, Dong Wan Lee,

Soo Jung Kang, Chul Kyung Kim1), Chang Woong Koh2), Nam Ki Kim

Department of Chemical Engineering, Sungkyunkwan University

School of Chemistry and Applied Chemistry, Mokwon University1)

Department of Chemical Engineering, Samchok University2)

Tel : 031-290-7253  Fax : 031-290-7272

Introduction

Major materials of soil pollution is known oil, oil compounds and toxic inorganic․organic compounds. These pollutants have been increasingly diversified in species and toxicity. Hence their treatment methods requires more advanced technologies. This indicates that only use of the conventional physical and chemical treatment technologies cannot guarantee the ultimate and environment-friendly treatment of complex, toxic and recalcitrant pollutants. Biological methods include a number of different technologies as absorbent for oil, biosurfactants to clean oily surfaces, and the addition of materials to promote microbiological degradation of oil. The last procedure is bioremediation, which  is alternative treatment technology that can often meet the goal of achieving a permanent clean-up remedy[1].

One of promising bioremediation technology is the application of bioreactor. Bioreactor, ex-situ method, employs engineering principles in treating contaminated soil[3].

An important reason for bioremediation being the technology of choice for eliminating organic pollutants is its cost. Although any procedure for removing or destroying pollutants is expensive, biological procedures tend to be the least expensive[4].

Aromatic hydrocarbon compounds have benzene ring by a basic structure, most of them are known volatile and toxic compounds.Organic compounds are the major products of petroleum and fine chemical industries and the most frequently used organic solvents.Some of them are suspected as carcinogens and emit offensive odor to the air and pollute soil and ground-water[5]. They frequently enter soil, sediments and ground-water because of leakage from underground storage tanks pipelines.[2] Specially, toluene has been classified as priority pollutants by the EPA(Environmental Protection Agency)[6]. Toluene is present in gasoline at 5∼7 %(wt/wt)[7] and has a solubility of 515 mg/L in water 20 ℃. The EPA has recommended that drink water contain not more than 2 mg/L of toluene for lifetime exposure, and found toluene in 54 % of ground-water samples near chemical waste sites in 1988.

Bioremediation involves the use of naturally occurring microorganisms to degrade and detoxify hazardous constituents in the soil at a contaminated site to protect public health and the environment. Hence, one possible approach to bioremediation is to prepare a pure culture of microorganisms and to use context, and the screening of potential hydrocarbon degrading organisms is one of the key steps[8].

Materials and Methods

The bioreactor used in this experiment. The diameter and height of a slurry phase bioreactor was 290 mm and 225 mm, respectively. The total volume of slurry phase bioreactor was 4.5 L and the working volume was 3 L. Soil particle size distribution or soil texture is an important factor in many soil treatment technology.

The soil used in this experiment was collected from the campus area of Sungkyunkwan university, and was used soil of a fixed size between ASTM 12 mesh(1.680 mm) and ASTM 50 mesh(0.297 mm) in order to obtain general data. The bulk density of soil was 1.27 g/mL and served as a model soil for soil slurry. The soil was dried in the shade and sterilized with autoclave.

The temperature of a slurry phase bioreactor was maintained by a water jacket connected bath circulator. An air flow rate was 600 mL/min.

The soil of 1500 g was put into slurry bioreactor containing minimal medium and cultured for 32 hours at 30 ℃ and 100 rpm. The removal efficiencies of toluene in a slurry phase bioreactor were investigated by change in soil-to-water ratio. The soil was made into 1:0.7, 1:1, and 1:1.3(wt soil/ wt water) slurry with water.

This experiment was carried out in a slurry phase bioreactor at 30 ℃, 100 rpm, initial pH 7.0, and toluene of 1000 ppm in order to investigate cell growth and toluene biodegradable ability by change in soil-to-water ratio in soil slurry. Soil slurry improve mass transfer rate, and contact between contaminants and microorganisms while ensuring the rate of desorption from soil to water. Soil slurry degradation enhances control and improve retention time of treatment. The amount of contaminated soil was 1500 g and the experimental method was the same as that described previously. The culture time was 32 hours. The bulk density of soil, the weight of the soil per volume including water and voids, was 1.27 g/mL.

Results and Discussion

The effects of water ratio on the cell growth and toluene biodegradable ability by P strain in a slurry phase bioreactor were indicated in Figure 1, 2, 3 respectively. In the result, the optimum water ratio was 1:1. As shown in Figure 1, initial concentration of toluene was rapidly decreased in the water ratio of 1:0.7, but subsequent toluene degradation was slow by 24 hours.

The cell growth and toluene degradation by P strain at water ratio of 1:1 was indicated in Figure 2. Toluene was not detected after 16 hours culture.

In this experiment results, toluene concentration after 5 minutes experiment was indicated 757 ppm in soil-to-water ratio 1:1, and toluene concentration after 36 hours was reduced 161 ppm by volatilization. The reason lowly indicated toluene concentration of experiment beginning was shown to absorb most toluene in soil or to volatilize a little toluene, and absorbed toluene was completely metabolized by biodegradation of microorganisms.

As shown in Figure 3, the water ratio of 1:1.3 was completely degraded before 16 hours culture, and P strain was rapidly grown without lag phase. and initial degradation rate of toluene was the most high.

Time course of cell growth and toluene concentration by change in water ratio by P strain was shown in Figure 4. In a slurry phase bioreactor containing soil contaminated with toluene, toluene was rapidly metabolized below detection and regulatory. The toluene degradable ability was shown a little difference, but toluene was not detected after 24 hours culture. Specially, at the water ratio of 1:1 and 1:1.3, toluene was not detected after 16 hours culture. The toluene degradable ability at water ratio of 1:1 was higher than that at water ratio of 1:1.3. In growth of microorganisms, the water was very important substance, but water of too much amount diminished quantity of adsorptive microorganisms at soil, and left a state of free cell. Hence water of too much amount was not suitable because it got more shear stress.

The results of this study suggest that while it is possible to biodegrade the toluene in soils using a slurry phase bioreactor. It was shown that could be applied to actual bioremediation technology of contaminated soil because total treatment cost of bioremediation was decreased by reduction amount of water including many parts in treatment cost of slurry phase bioreactor.

References

1. J. L. Sims, R. C. Sims, and J. E, Matthews, "Approach to                     Bioremediation of Contaminated soil", Hazardous Waste ＆ Hazardous           Materials, 7, 117～148(1990)

2. Choi, D. H., Lee, D. H., "Biodegradation of crude oil hydrocarbons by          Acinetobacter sp. isolated from activated sludge", J. KoSES., 5, 97～            108(2000)

3. Ko, B. J., Yang, J. W., "Biodegradation of crude oil in soil slurry phase by     Nocardia" M. S. Thesis, KAIST(1997)

4. Chung, J. C., Bark, C. H. and Lee, S. T., "The Principle and Application of     Bioremediation", J. KoSES., 1, 3～13(1996)

5. Sung, h. Y., Young, J. Y., "Removal of benzene in a hybrid bioreactor",        Proc. Biochem., 34, 281～288(1999)

6. 이 창호, 오 희목, 권 태종, 안 성구, 권 기석, 고 영희 , 윤 병대, “Toluene을      분해하는 Acinetobacter sp. GEM63의 분리 및 특성”, Kor. J. Microbiol., 32,      329～335(1994)

7. Patrick, J. E., Dzung, T. M., Kim, K. S. and L. Y. Young, "Anaerobic          Degradation of toluene by a denitrifying bacterium", Appl. Environ.             Microbiol., 57, 1139～1145(1991)

8. Lee, S. W., Lee, J. M. and Jahng, D. J., "Degradation of BTEX and            trichloroethylene by Pseudomonas Putida F1 and Burkholderia cepacia G4",     Kor. J. Biotechnol., 13, 561～568(1998)

Fig. 1. Effect of soil-to-water ratio 1:0.7 on the cell growth and toluene degradation in a slurry phase bioreactor

Fig. 2. Effect of soil-to-water ratio 1:1 on the cell growth  and toluene degradation in a slurry phase       bioreactor

Fig. 4. Time course change of cell growth and toluene concentration by soil-to-water ratio in a slurry   phase bioreactor

(-▲- :1 : 0.7  -●- : 1 : 1  -■- : 1 : 1.3 )

Fig. 3. Effect of soil-to-water ratio 1:1.3 on the ell growth and toluene degradation in a slurry phase   bioreactor