Bacterial Degradation of Chlorpyrifos by Bacillus cereus Zhiyuan Liu 1,2,3, a, Xin Chen 1,2,b*,Yi Shi 1,2,c ZhenCheng Su 1d

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1 Advanced Materials Research Online: ISSN: , Vols , pp doi:1428/ 212 Trans Tech Publications, Switzerland Bacterial Degradation of Chlorpyrifos by Bacillus cereus Zhiyuan Liu 1,2,3, a, Xin Chen 1,2,b*,Yi Shi 1,2,c ZhenCheng Su 1d 1. State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, 1116, China 2. Key laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, 1116, China 3. Graduate University of Chinese Academy of Sciences, Beijing, 139, China a @163.com, b chenxin@iae.ac.cn, c shiyi@iae.ac.cn, d zhenchengsu@yahoo.com.cn Keywords: Organophosphorus pesticides, Bioremediation, Chlorpyrifos. Abstract. Eleven chlorpyrifos-degrading bacterium were isolated from a Chinese soil and compared by microbiological and molecular methods. The strains were significantly different in the ability of degradation efficiency, and one strain (Bacillus cereus) was selected for further analysis. The ability of Bacillus cereus to degrade chlorpyrifos was investigated under different culture conditions, such as ph, temperature, chlorpyrifos concentration and so on. Experimental results obtained in different conditions show that the optimum temperature, ph, concentration of chlorpyrifos were 3 C, 7., less than 1 mg L -1, respectively. Introduction Pesticides are applied to lower damages on crops and forestry products from pests, pathogens, weeds, mites, nematodes, rodents and regulating plant growth [1,2]. They are used to lower the cost of agricultural products, and greatly reduce the losses caused by pests and diseases, creating great economic benefits. However, pesticide residues can adversely affect ecosystems and human health and also cause serious environmental pollution. Chlorpyrifos, the active ingredient of which is O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, is one of the most widely used pesticides in the US and accounts for 11% of total pesticide use [3]. Chlorpyrifos was developed by the U.S. chemical company Dow AgroSciences in 1965, and since then it has been widely used in the cultivation of rice, wheat, sugarcane, corn, cotton, tea, fruit trees, vegetables, flowers, livestock, etc [4]. It has a significant effect through the mode of contacting and stomach poison on leaf worms, armyworms, scale insects, aphids, cotton bollworm, leafhoppers and mites and other pests. It is considered to be a better choice to replace high toxic organophosphate pesticides such as methamidophos, parathion and methyl parathion [5]. For that reason, Chlorpyrifos is more and more widely used in recent years [6-8], and the accumulation of residual Chlorpyrifos in soil also become more and more serious. Chlorpyrifos-degrading bacteria may be applied either directly or indirectly in the bioremediation of Chlorpyrifos-contaminated soils. In this study, we isolated chlorpyrifos-degrading bacteria from soil and their capacities of degrading chlorpyrifos were investigated. Materials and Methods Chemicals Samples of chlorpyrifos (97.%, pure analytical grade) were obtained from Jiangsu Jinghong Chemical Co., Ltd, China. All other Chemicals were of analytical grade. Isolation and culture conditions The mineral salt medium (MSM, ph 7.2) was prepared by adding 1.5 g K 2 HPO 4, 5 KH 2 PO 4, 1. g (NH4) 2 SO 4, 5 g NaCl, 2 g MgSO 4, 2 g FeSO 4 into 1L of distilled water. The LB medium was prepared with distilled water containing 1% tryptone, 5% yeast, 1% NaCl, with ph at 7. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (# , Pennsylvania State University, University Park, USA-18/9/16,17:2:22)

2 Advanced Materials Research Vols One gram of organophosphorus pesticides contaminated soil was added into the flask containing 1mL MSM with chlorpyrifos at 25mg L -1 and cultivated for 3 days under shaking at 15 rpm at 3. Then 1 ml of medium was removed from the flask and inoculated into fresh MSM with chlorpyrifos (5, 75, 1 or 125 mg L -1 ). The colonies on the MSM plate with 1 mg L -1 chlorpyrifos at 3 C were observed in about 2 days. Then the colonies were selected and purified and their degrading capability was further tested by inoculation in liquid medium. Chlorpyrifos residue was measured by High-performance liquid chromatography according to the method of National Standard GB/T "Measurement of organic phosphorus pesticides in water and soil by Chromatography." Identification of degrading bacteria The isolates were discriminated by comparison of their 16S rdna genes. Bacterial DNA was extracted by the method of pyrolysis. The 16S rdna gene of the strain was amplified by PCR using two universal primers, 27F (5 -AGAGTTTGATCCTGGCTCA-3 ), and 1492R (5 -GGTTACCTTGTTACGACTT-3 ), respectively. The PCR was performed with a PTC-2 thermal cycler (MJ Research, USA). The amplification program was operated as below: denaturation at 94 C for 5 min, 35cycles at 94 C for 1 min, at 55 C for 1 min, and at 72 C for 1.5 min, followed by a final extension at 72 C for 1 min; PCR fragments were purified by agarose gel electrophoresis. The gene was sequenced by the Genomics Institute Co., Ltd. The gene sequences were compared with most similar sequence available in the Genbank nucleotide sequence database using the NCBI blast program. Inoculum preparation for degradation study. The insolate DH was cultured in the 25 ml flask containing 1mL LB medium supplemented with 1 mg L -1 chlorpyrifos. Flasks were shaken at 15 rpm and at 3 C for 3 days. The insolate DH was collected by centrifugation and the cells were washed twice with MSM medium and then re-suspended. The strain in the same fresh medium was prepared for the following degradation experiment. For the whole experiment, 16 cells per ml were used and samples were incubated at 15 rpm at 3 C unless otherwise stated. Bacterial growth and degradation of chlorpyrifos. The strain DH was isolated from the MSM supplemented with 1 mg L -1 Chlorpyrifos as the sole nitrogen source. Degradation experiments were conducted at 3 C and at 15 rpm in MSM supplemented with 1 mg L -1 chlorpyrifos. The fresh MSM medium without inoculating any insolate was used as control. The degradation efficiency of the strain DH was determined and estimated by the removal percentage of chlorpyrifos from the liquid culture. Determination of optimum conditions of chlorpyrifos degradation by DH. Effects of temperature, medium ph, chlorpyrifos concentration and inoculation on bacterial degradation of chlorpyrifos were studied in this experiment, with temperature of 2, 25, 3 or 35 C, with medium ph of 5., 6., 7., 8. or 9., with chlorpyrifos concentration of 2, 5, 1, 15 or 2 mg L -1. Each treatment was tri-replicated, with a non-inoculation treatment as the control. Quantification of the chlorpyrifos by HPLC. Chlorpyrifos residue was measured by High-performance liquid chromatography according to the method of National Standard GB/T "Measurement oforganic phosphorus pesticides in water and soil by Chromatography." The quantitative analysis was performed using a gas chromatography HP 589 A fused silica capillary column (3 mm 25 mm 25µm) (Shenyang Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China) was employed. The temperatures of the injection port and detector were set at 24 and 3 C, respectively. The oven temperature was increased linearly from 15 to 26 C at 5 C min -1. The detection limit of gas chromatography for chlorpyrifos was 1mg L -1. Volume of 1 ml was injected and the peak area was used to quantify the insecticide residue.

3 678 Progress in Environmental Science and Engineering Results and Discussion Strain identification and characterization. Eleven chlorpyrifos-degrading bacteria were isolated from the soil samples in the MSM medium containing 1 mg L -1 chlorpyrifos by the enrichment procedure, after 3-day incubation at 3 C, several microbial colonies became visible. DH was chosen for further investigation because it showed the highest degrading capability in the liquid medium. DH on the plate medium was cream-colored and the surfaces of colonies of DH were glossy with clear margins. (Fig.1) The 16S rdna gene sequence of DH was obtained,and a phylogenetic tree based on the partial l 6S rdna gene sequence of DH was constructed.the sequence of DH was compared with that of referred strains gene sequences in the Genbank and it was most related to Bacillus cereus with 98% similarity. Based on these observations, the isolate DH was finally identified as Bacillus cereus sp. Fig.1 The colony of DH on the Plant chlorpyrifos concentration(mg L time(d) Fig.2 The bacterial growth and chlorpyrifos degradation OD 56nm Strain growth and degradation by DH To obtain the best degradation of chlorpyrifos in the liquid culture,the temperature was set 3 C, ph was set 7. with 1 mg L -1 chlorpyrifos and 2ml L -1 bacterium inoculation. The degradation of chlorpyrifos and the growth of isolate in MSM are illustrated in Fig.2. Fig. 2 shows that, the delay period of the isolate DH in the medium occurred on the 1st day, the logarithmic growth period occurred since the 2nd day, and the decline phase observed on the 4th day. In contrast with the growth pattern, the bacterial degradation of chlorpyrifos is quite obvious. The residue of chlorpyrifos in the medium was decreased from 1 mg L-1 to 26.1 mg L -1 in 7 days. The effect of temperature on bacterial degradation of chlorpyrifos The temperature is one of the most important factors on microbial survival and growth [9]. In this experiment, 2, 25, 3 and 35 C were selected to evaluate the degradation capacity of the strain DH. The other conditions were as follow: ph was set 7. with 1 mg L-1 chlorpyrifos and 2 ml L-1 bacterium inoculation. Fig.3 indicates that the maximum degradation rate occurred at 3 C (78.85%), and followed was the degradation rate at 25 C (75.76%), while the degradation capability of the isolate DH in liquid at 2 or 35 C was relatively weak.

4 Advanced Materials Research Vols d e g rad atio n ra te % temperature ( ) Fig.3 Bacterial degradation of chlorpyrifos at different temperature The effect of ph on bacterial degradation of chlorpyrifos The degradation experiment was conducted with the ph of the culture medium at 5., 6., 7., 8. or 9., respectively. The degradation of chlorpyrifos was evaluated after 5-day cultivation at 3 C and 12 rpm. Fig.4 shows that the optimum ph was 7., and the bacterial degradation of chlorpyrifos was still considerable at ph 6. or 8., but became significantly inhibited at ph below 6. or above 8. d e g ra d a tio n ra te % Fig.4 Bacterial degradation of chlorpyrifos at different ph ph The effect of chlorpyrifos concentration on its bacterial degradation The experiment was conducted with chlorpyrifos at 2, 5, 1, 15 or 2 mg L -1 in the MSM liquid medium. The degradation of chlorpyrifos was evaluated after 5-day cultivation at 3 C and 12 rpm. Fig.5 demonstrates that the degradation rate was decreased slowly with the concentration of chlorpyrifos being increased from 1 to 15 mg L -1, and dropped down quickly to 37.59% with the concentration of chlorpyrifos being increased to 2 mg L -1.

5 68 Progress in Environmental Science and Engineering d eg rad atio n rate % concentration (mg L -1 ) Conclusions Fig.5 Effect of chlorpyrifos concentration on its Bacterial degradation An enrichment method was allowed to isolate bacteria for efficient biodegradation of the organophosphorus insecticide chlorpyrifos. Results on the bacterial degradation of chlorpyrifos in liquid medium indicated that it was ideally degraded under the condition of 3 C, ph 7., concentration of chlorpyrifos below 15 mg L -1, with a degradation rate up to 78.85%. However, the use of pesticide-degrading bacteria for removal of organophosphorus compounds from contaminated environments requires a better understanding of ecological requirements of chlorpyrifos-degrading bacteria. Further research is needed on biochemical and genetic aspects of chlorpyrifos-degradating bacteria for bioremediation of chlorpyrifos-contaminated environments. Acknowledgements This work was supported by the open foundation of Key Laboratory of Terrestrial Ecological Process Institute of Applied Ecology, Chinese Academy of Sciences ( ), and the National Natural Science Foundation of China (No ) References [1] W.S.Lan, J.D.Gu and J.L.Zhang. Inter Biodeterioration & Biodegradation vol. 58 (26), p [2] S.H.Wang, C.Zhang, Y.C.Yan : Biote (Harbin),vol.16 (26), p (in Chinese) [3] EPA(24)on [4] Q.K.Liu. New pesticide manual. Shanghai: science and technology publish. 1993, [5] X.H. Li, K.Z.Jia, J. He. Acta pedologica sinica, 27.44(4): (in Chinese) [6] K.Mallick, K.Bharati, A.Banerji.bacteria.bull.environ.contam.toxicol. vol. 62 (1999), p [7] C.Yang, N.Liu, X.M.Guo,C.L. Qiao: FEMS Microbiol Lett vol.265(26), p (in Chinese) [8] B.K.Singh, A.Walker, J.A.W.Morgan, D.J.Wright: Apply and envir microb, agu. (24), p [9] J.L. Wang, S.p. Li, Z.Huang in: Environmental Microbiology (second Edition) Beijing:Higher Education Press.(24)(in Chinese)