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1 - Original Paper 구연산 / 칼슘 / 인산염용액을이용한토양중금속안정화 : 토양미생물이미치는영향 송호철 송두섭 * 조동완 ** 박성원 최상훈 * 전병훈 ** 이장호 *** 박준홍, *** * ** *** ( , ) Stabilization of Heavy Metals using Ca-Citrate-Phosphate Solution: Effect of Soil Microorganisms Hocheol Song Doo-Sup Song* Dong-Wan Cho** Sung-Won Park Sang-Hun Choi* Byong-Hun Jeon** Jangho Lee*** Joonhong Park, *** Korea Institute of Geoscience and Mineral Resources *Department of Earth and Environmental Sciences, Choongbuk National University **Department of Environmental Engineering, Yonsei University ***School of Civil and Environmental Engineering, Yonsei University ABSTRACT : A farming area located near an abandoned copper mine in GuPo-ri, Choongchung province is heavily contaminated with heavy metals such as As, Pb, Cd, Cu and Zn of which concentrations are higher than the values typically detected in Korean soil environment. In this work, laboratory and field studies were conducted to examine feasibility of using Ca-citrate-phosphate solution in stabilizing heavy metals in the polluted soils. In laboratory batch experiments with field soil, the addition of Ca-citrate-phosphate solution resulted in decrease of aqueous phase concentration of phosphate and improvement of heavy metal stabilization, compared to those for sterilized soil samples. This indicates that microbial uptake of phosphate may have provided positive effects on availability of phosphate toward heavy metal stabilization. According to microbial community analysis for the field experiment, the use of Ca-citrate-phosphate led to increased diversity of microbial populations, and strict anaerobic microorganisms such as Anaerofilum and Treponema became the most dominant populations in the solution-amended field experiments. These findings suggest that, when Ca-citrate-phosphate is used for heavy metal stabilization in soils, microbial processes may have important roles in improving the stabilization of heavy metals by providing reducing conditions to the treatment locations or/and by making phosphate available to heavy metal stabilization. Key Words : Heavy Metal Stabilization, Phosphate Availability, Soil Microorganisms 요약 : TCLP,,,,..,,...,, Anaerofilum Treponema.,. 주제어 :,, 1. 서론 *, Corresponding author parkj@yonsei.ac.kr Tel: Fax: (Soil Washing),. (Soil Flushing) 2. (Electrokinetic Remediation) (Phytoremediation) 대한환경공학회지 31 권 4 호, 2009 년 4 월

2 242 송호철 송두섭 조동완 박성원 최상훈 전병훈 이장호 박준홍. (Ex-Situ), (In-Situ). 1),,, -,,, (complex) -. 1) (hydroxyapatite, (Ca 10(PO 4) 6(OH) 2)) (Pb/Cd/Cu/Zn/Cr/As). 2,3),,. 4 6) (Permeable Reactive Barrier). PRB.. (apatite).,. (citrate) Ca-citrate,. Ca-citrate. /.,,. 1,7). 2. 연구방법 2.1. 현장위치와특성 ( ). ( ),, 1, 2,,.,,.,,..., 45 cm 15 cm 3. (, Toxicity Characteristic Leaching Procedure) 실험실분석, TCLP (batch test). autoclave (121, 1.3 ) 1 / (GA) (GX). 8,9) 50 ml centrifuge tube 20 g 56, (Sodium Citrate) 60 %, (Ammonium Nitrate) 1 %, (Sodium Phosphate) 25 %, 30 mm, 30 mm, 20 mm Ca : Citrate : Phosphate J. of KSEE / Vol. 31, No. 4, April, 2009

3 구연산 / 칼슘 / 인산염용액을이용한토양중금속안정화 : 토양미생물이미치는영향 243 = 3:3:2, 1 mm. Ca-citrate Ca/Citrate 3, Cacitrate, apatite Ca/P 3/2 (Ca 10-xMx(PO 4) 6(OH) 2). 3) 1 M NaOH ph ) 20 g/20 ml = 1 : 1, rpm 현장실험 As As, As (red mud) 10 cm 8 kg. 10,11) ph Batch Test 30 mm, 20 mm, 30 mm, 1 mm. 2:1, 600 L. Ca-citrate cm 10 cm., 0.1 N HCl 4 ICP-AES , , mm, mm, mm 분석방법 토양의물리화학적특성, ph,,. ph (LOI, loss on ignition) XRF 토양중금속분석 (Korea Standard Test, KST), 2 mm(10 ) 1/4 100 mesh. 1 g 50 ml centrifuge tube 0.1 N 10 ml, (100 /, 10 cm) µm. (As) (1 N) (30 ). 12) (Zn) (aqua regia). 1 3, 24, soil 16± Toxicity Characteristic Leaching Procedure (TCLP) Toxicity Characteristic Leaching Procedure (TCLP) EPA SW-846 (Method 1311). 13) ph, ph 4.93± 0.05 (1), 5.7 ml glacial CH 3CH 2OOH, 500 ml 64.3 ml 1 N NaOH. (2) ph ml glacial CH 3CH 2OOH 1 L. ph 5 g 96.5 ml 5 ph. ph 5, (1), ml 1 N HCl (1) 5 (2). 5 g 100 ml Nalgen bottle 40 ml 30±2 rpm 18± µm Membrane Filter. 13) , 0.45 µm (filter paper). DR/4000 UV-VIS 토양미생물군집분석. UltraClean TM Soil Isolation Kit (MOBIO Laboratories, Inc., USA) DNA, genomic DNA 16S rdna 대한환경공학회지 31 권 4 호, 2009 년 4 월

244 송호철 송두섭 조동완 박성원 최상훈 전병훈 이장호 박준홍 bacterial universal primers 27f-FAM [fluorescence labeled](agagt TTGAT CATGG CTCAG) 1492r(TACGG TTACC TTGTTA CGACTT).

4 244 송호철 송두섭 조동완 박성원 최상훈 전병훈 이장호 박준홍 bacterial universal primers 27f-FAM [fluorescence labeled](agagt TTGAT CATGG CTCAG) 1492r(TACGG TTACC TTGTTA CGACTT). PCR (Polymerase Chain Reaction), 10 µm 27f-FAM 1492r primer 0.5 µl, 5 U/ L Taqpolymerase (Fermentas, Germany) 0.25 µl, template DNA 20 ng 25 µl TM Thermal Cycler PCR, 94 3, 30 cycle 94 1, 55 30, 72 2, 5. 16S rdna QIAquick PCR purification Kit (Qiagen Inc.), DNA Tango TM buffer,, 10U HhaI 20 µl T-RFLP (terminalrestriction fragment length polymorphism) DNA (NICEM) (96-capillary 3730xl DNA Analyzer, ABI). electropherogram Gene Mapper Software v4.0 (ABI), peak height profile. Electropherogram peak Shannon index (H), Evenness (e), species richness (d). C Shannon index ( H ) = ( N log10 N nilog 10ni) N S 1 Richness ( d) = log N H Evenness () e = log S C = 2.3 N = sum of peak areas in a given T-RFLP n i = area of a T-RF(terminal-restrictionfragment) i = individual number of each T-RF in a T-RFLP chromatograph S = abundance of T-RFs in each T-RFLP chromatograph Shannon index Species richness, Evenness. 14) 3. 결과및토의 3.1. 현장의중금속오염, ph, Table 1. ph ) ph Table 1. Characteristics of Gupo-ri soil Depth (cm) Temp ( ) ph LOI (%) Fig. 1. Mineralogical composition of Gupo-ri soil.. 4.5%. Fig. 1.,,. (KST, 0.1 N HCl) (Table 2),,, ) TCLP Table 2. TCLP KST Table 2. Results of heavy metals measured from Gupo-ri soil using KST (Korea Standard Test), and TCLP (Toxicity Characteristic Leaching Procedure) Method Items As Pb Cd Cu Zn (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) A standard of soil pollution anxiety Korean average KST Content in nature Rice farm Vegetable farm Land farm in Gupo-ri TCLP Land farm in Gupo-ri J. of KSEE / Vol. 31, No. 4, April, 2009

5 구연산 / 칼슘 / 인산염용액을이용한토양중금속안정화 : 토양미생물이미치는영향 245. TCLP, TCLP 미생물의영향 Fig. 2.. ( 1 ) ( mm). 2 Fig. 2.,,.. 2 ( ). Fig. 2 TCLP Fig. 3...,... (uptake)... (121, 1.3 ) Fig. 2. Time-course changes of dissolved phosphate in laboratory batch experiments with autoclaved (GA) and non-autoclaved (GX) soil samples after adding Cacitrate-phosphate solution. Fig. 3. Time-course changes of zinc and copper (measured by TCLP) in laboratory batch experiments with autoclaved (GA) and non-autoclaved (GX) soil samples after adding Ca-citrate-phosphate solution. 대한환경공학회지 31 권 4 호, 2009 년 4 월

6 246 송호철 송두섭 조동완 박성원 최상훈 전병훈 이장호 박준홍,. 8,9), 현장실험 (1 10 cm, cm, cm) 10 mg/kg (Fig. 4). (1 10 cm) (35 days) 10 mg/kg. 10 mg/kg (1,425 mg/kg) 0.7%. (Fig. 2) Fig. 4. Fig. 4. Time-course changes of dissolved phosphate in the field experiment. 30 cm (Fig. 5). Table 2 (553 mg/kg) ( 40 mg/kg) Table 2 ( + ), Fig N HCl. Fig. 5. Time-course changes of heavy metals (measured using 0.1 HCl) in the field experiments after applying Ca-citrate-phosphate solution. J. of KSEE / Vol. 31, No. 4, April, 2009

구연산 / 칼슘 / 인산염용액을이용한토양중금속안정화 : 토양미생물이미치는영향 247 Fig. 6. Microbial community shift in the field experiment after applying Ca-citrate-phosphate solution. Table 3.

7 구연산 / 칼슘 / 인산염용액을이용한토양중금속안정화 : 토양미생물이미치는영향 247 Fig. 6. Microbial community shift in the field experiment after applying Ca-citrate-phosphate solution. Table 3. Microbial diversity results before and after Ca-citratephosphate solution treatment in the Gupo-ri field The method of species 0 15 cm diversity analysis Before treatment After treatment Shannon Index (H) Richness (d) Evenness (e) , 1 N HCl, 0.1 N HCl. 0.1 N HCl. 62 Zn 37 47%, Cu 28 55%, Pb 29 40%, As. As As 0.1 N HCl..,, Shannon index, Richness, Evenness (Table 3). Richness Evenness Shannon index.,. Ca-citrate solution phosphate solution (Fig. 6), Azoarcus, Hyphomonas, Rhodobacter, Vibrio Anaerofilum, Treponema. 17,18),.. 4. 결론,.. 1),..,.,. 2) ( 30 cm). 3), Anaerofilum Treponema.. 대한환경공학회지 31 권 4 호, 2009 년 4 월

8 248 송호철 송두섭 조동완 박성원 최상훈 전병훈 이장호 박준홍 4),. 참고문헌 1.,,,,, 21(7), (2004). 2. Fernane, F., Mecherri, O. M., Sharrock, P., Hadioui, M., Lounici, H., and Fedoroff, M., Sorption of cadmium and copper ions on natural and synthetic hydroxylapatite particles, Mater Charact., 59(5), (2008). 3. Moore, R. C. and Sanchez, C., Formation of hydroxyapatite in soil using calcium citrate and sodium phosphate for control of strontium migration, Radiochim. Acta, 92(9-11), (2004). 4. Ma, Q. Y., Traina, S. J., Logan, T. J., and Ryan, J. A., In situ lead immobilization by apatite, Environ. Sci. Technol., 27(9), (1993). 5. Xu, Y. and Schwartz, F. W., Lead immobilization by hydroxyapatite in aqueous solutions, J. Contam. Hydrol., 15(3), (1994). 6. Xu, Y., Schwartz, F. W., and Traina, S. J., Sorption of Zn 2+ and Cd 2+ on hydroxyapatite surface, Environ. Sci. Technol., 28(8), (1994). 7.,,,, Phosphate,, 25(5), 554 (2003). 8. Andrew, V. O., Mark, L. M., James, B. H., Jeffrey, B., and Charles, A. P., Effect of DNA polymer length on its Adsorption to soils, Appl. Environ. Microbiol., 60(2), (1994). 9. Khanna, M. and Stotzky, G., Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA, Appl. Environ. Microbiol., 58(6), (1992). 10. Genc-fuhrman, H., Bregnhoj, H., McConchie, D., Arsenate removal from water using sand-red mud columns, Water Res., 39(13), (2005). 11. Bertocchi, A. F., Ghiani, M., Peretti, R., Zucca, A., Red mud and fly ash for remediation of mine sites contaminated with As, Cd, Cu, Pb and Zn, J. Hazard. Mater., 134(1-3), (2006). 12., ( ), U.S Environmental Protection Agency Home Page, /pdfs/1311(1992). 14.,,,,,, 27(6B), (2007). 15., 2007, =0063(2007). 16., Dupont, D. P., Duhamel, G. E., Carlson, M. P., and Mathiesen, M. R., Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper, Vet. Microbiol., 41(1-2), 63 73(1994). 18. Zellner, G., Stackebrandt, E., Nagel, D., Messner, P., Weiss, N., and Winter, J., Anaerofilum pentosovorans gen. nov., sp. nov., and Anaerofilum agile sp. nov., two new, strictly anaerobic, mesophilic, acidogenic bacteria from anaerobic bioreactors, Int. J. Syst. Bacteriol., 46(4), (1996). J. of KSEE / Vol. 31, No. 4, April, 2009

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