토양 박테리아에 의한 2,4-Dichlorophenoxyacetic acid의 분해

한명희, 유선균, 김승재, 조성용

전남대학교 환경공학과

Microbial Degradation of 2,4-Dichlorophenoxyacetic acid by Soil Isolated Bacteria

Myung Hee Han, Sun Kyun Yoo, Seung Jai Kim, Sung Young Cho

Department of Environmental Engineering, Chonnam National University

Introduction

A short decomposition process after harvest of the crops could diminish the negative effects of pesticides on the environment caused by residues remaining in the soil and subsequently leaching to the ground water.  Mineralization with microorganisms, inoculated in bulk soil or in the rhizosphere of resistant plants with dense roots, can be used to increase the degradation rate of the pesticides [1,2].  Xenobiotic compound 2,4-Dichlorophenoxyacetic acid (2,4-D) is a widely used herbicide. Since 2,4-D is a recent origin compound, the characteristics of catabolism of this compound are sure of an interesting field. The degradation of 2,4-D by bacteria has been studied intensively at the biochemical, genetic, and ecological level [3,4].  However, a few studies about mineralization of 2,4-D by biological treatment have been reported in Korea.  In our study, we have isolated bacteria capable of utilizing 2,4-D as a sole carbon and energy sources from 2,4-D soaked soil and evaluated degradation of 2,4-D by resting and growing cells.

Materials and Methods

Enrichment cultures of microorganisms capable of utilizing 2,4-D a sole carbon sources was established with samples from several different plots of an orchard at Gwang-Ju in Korea.

Samples (ca. 1g) will be aseptically transferred into 250ml flasks containing 50ml of single strength mineral medium (pH 7.0) containing 1000ppm 2,4-D.  Mineral medium consisted of 4g KH2PO4, 1g Na2HPO4, 2g (NH4)2SO4, 0.2g MgSO4·7H2O, 0.003g FeSO4·7H2O, 0.003g ZnSO4·7H2O, 0.1g MnSO4·H2O, and 0.025g CaCl2 per liter of deionized water.  Medium was adjusted to pH 7.0.  Five ml aliquots from each suspension broth was transferred to new 250ml culture flasks containing 50ml of sterile mineral medium containing 1000ppm 2,4-D.  The procedure was repeated until consistent growth observed.  Pure cultures were isolated by repeated streaking onto petri-dish plates containing mineral medium agar plus 2,4-D.  Culture purity was determined after plating on tryptic soy agar (Difco Laboratories, Detroit, MI).  Pure cultures was maintained through biweekly transfers and storage in at 5℃.  Working cultures were maintained at room temperature with weekly transfers.  Other strains, Alcaligenes eutrophus ATCC 17699, Pseudomonas alcaligenes ATCC 14909, Pseudomonas aureofaciens ATCC 13985, Pseudomonas putida ATCC 11250, Pseudomonas fluorescens ATCC 11541, Alcaligenes sp. ATCC 31371, and Pseudomonas putida ATCC 12633 were purchased from Korean Federation of Culture Collection. Fore resting cells preparation, bacteria grew in 50ml tryptic soy broth of 250ml culture flasks at 30℃ for 48hr. Cells were harvested by centrifuging the culture broth for 20min at 15,000 × g.  After aseptically removing supernant, cell pellets were washed twice.  Finally, the supernant was decanted. The cell pellets were resuspended with mineral medium. Transformation treatments were prepared using test tubes (25 × 150mm) containing 10ml of cell suspension plus 500ppm 2,4-D. Reaction was performed in shaker with 200rpm and 30℃ for 6days.  Fermentation with growing cells was prepared by adding 20ml of mineral medium and 500ppm 2,4-D to 250ml culture flasks.  Culture flasks were incubated in shaker at 200rpm and 30℃ for 6days.  Five ml of all culture tubes and flasks were extracted with 5ml of ethyl acetate. The extract containing 2,4-D were transferred to a spectrophotometer cuvette, of which absorbance was measured at 280nm. The amount of 2,4-D was estimated by a calibration curve (Fig.1).

Results and Discussion

About 10 bacterial colonies capable of utilizing 2,4-D as a sole carbon and energy source on mineral medium agar plates were preliminary isolated.  Four strains that showed notable difference in their morphology on TSA and MMA were selected finally and designated as NOH 1, 2, 3, and 4, respectively.  The growth of all strains was repressed beyond about 2000ppm 2,4-D as previous studies have been reported [5,6]. We have reacted washed resting cells prepared from soil-isolated and supplied strains with 500ppm of 2,4-D, of which this concentration could be comparatively dissolved with ease in mineral medium (Fig.2).  Degradation of 2,4-D by growing cells was also performed and compared with that by resting cells (Fig.3).  In our study, we have suffered the determination of quantity of 2,4-D by a spectrophotometer because bacterial metabolites media components such as nucleic and amino acids were absorbed at similar wave length. To solve this problem, several solvents were used to extract 2,4-D.  Ethyl acetate of those selected solvents was most effective (about 98% 2,4-D moved to ethyl acetate layer from water, data not shown) because not much difference was found in calibration curve between water and ethyl acetate (Fig.1).

All bacteria isolated from soil and supplied could degrade 2,4-D within 6days by either resting or growing cells.  In both cases, the degradation efficiency varied dependent on bacterial strain.  As a whole, soil-isolated bacteria showed lower degradation efficiency than supplied bacteria when resting cells of those strains were applied.  Not much different efficiency of 2,4-D degradation was found with growing cells.  However, the rate of 2,4-D degradation with resting cells of soil-isolated bacteria was lower than that with growing cells.  Not much difference between soil isolated and supplied bacteria with growing cells was found to degrade 2,4-D except NOH 4.  NOH 1, 2, and 3 except 4 with growing cells showed about 80 to 90% degradation of 2,4-D.  The degradation efficiency of 2,4-D by NOH 1 and NOH 2 growing cells was 93 and 92% of total supplied 2,4-D, respectively.  In resting cells, NOH 2  showed the highest degradation of 2,4-D, about 76%.  P. alcaligenes ATCC 14909 showed the highest degradation of 2,4-D, that was about 96% with growing cells.  However, A. eutrophus ATCC 17699 showed the highest degradation of 2,4-D, that was about 94% with resting cells.  Our results were compared to previous results showing that fungi, C. elegans degrade about 45% of 100ppm of 2,4-D within 7days [7], microbial consortium consisting of soil bacteria mixture breaks down 216ppm of 2,4-D to about 55ppm for about 16days [5], and bacteria BKME isolated from bleached kraft mill effluents records about 50 to 60% degradation efficiency of 400ppm of 2,4-D for 6days [6].  To elucidate the degradation kinetics, we will select two strains NOH 2 and P. alcaligenes ATCC 14909 that show relatively higher efficiency degradation of 2,4-D.

Fig.1. Calibration curve of the amount of 2,4-D versus absorbance at 280nm.

Fig.2. Reaction of resting cells of bacteria with 500 ppm 2,4-D.

Fig.3. Degradation of 2,4-D by growing cells of bacteria.

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