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International Biodeterioration & Biodegradation 62 (2008) 331–335

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Diversity of chlorpyrifos-degrading bacteria isolatedfrom chlorpyrifos-contaminated samples

Xiaohui Li, Jiandong Jiang, Lifeng Gu, Shinawar Waseem Ali, Jian He, Shunpeng Li*

Key Lab of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Science,Nanjing Agricultural University, 210095 Nanjing, Jiangsu Province, People’s Republic of China

a r t i c l e i n f o

Article history:Received 18 December 2007Received in revised form 9 March 2008Accepted 10 March 2008Available online 20 June 2008

Keywords:ChlorpyrifosBiodegradationDiversitympd gene

* Corresponding author. Tel./fax: þ86 25 84396314E-mail address: lsp@njau.edu.cn (S. Li).

0964-8305/$ – see front matter � 2008 Published bydoi:10.1016/j.ibiod.2008.03.001

a b s t r a c t

Chlorpyrifos as an agricultural insecticide has been widely used and caused great pollution. As thetoxicity of chlorpyrifos in the environment, exploration of various chlorpyrifos-degrading bacteria toclean-up the pollutant is of immense importance. Seven chlorpyrifos-degrading bacteria, named Dsp-1 toDsp-7, were isolated from organophosphate pesticide-contaminated soil and water using a culturemethod with chlorpyrifos as the sole carbon source. Comparative studies were performed to study theirmorphological, physiological and biochemical characteristics. Based on these analysis, strains Dsp-2,Dsp-4, Dsp-6 and Dsp-7 were identified as Sphingomonas sp., Stenotrophomonas sp., Bacillus sp. andBrevundimonas sp., respectively, while all other strains were members of Pseudomonas sp. ERIC (en-terobacterial repetitive intergenic consensus)-PCR fingerprints were used to distinguish strains Dsp-1and Dsp-3 which had only 1 bp difference in 16S rDNA sequences. Significant differences in the ability ofthe bacteria to degrade chlorpyrifos and other organophosphate pesticides were found. The organo-phosphate pesticide hydrolase gene mpd were cloned from strains Dsp-1 to Dsp-4 with the PCR tech-nique. Differences in amino acid sequence of mpd gene from the four bacteria strains were compared.These results showed that there were diversities of chlorpyrifos-degrading strains in the contaminatedenvironment, and the chlorpyrifos-degrading strains had the potential to clean up the organophosphatepesticide-contaminated environment.

� 2008 Published by Elsevier Ltd.

1. Introduction

Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phos-phorothioate] is used world wide as an agricultural insecticide (Choet al., 2002). Its environmental fate has been extensively studied,and its half-life in soil varies from 10 to 120 days (Getzin, 1981;Racke et al., 1988) resulting in 3,5,6-trichloro-2-pyridinol (TCP) asthe major degradation product. The extensive use of chlorpyrifoshas led to widespread environmental pollution, resulting in seriousdamage to non-target species. The control of chlorpyrifos pollut-ants is of great importance because they are toxic and recalcitrant.The use of chlorpyrifos-degrading bacteria for bioremediation ofchlorpyrifos-contaminated sites has been proved to be the mostpotential clean-up method.

Despite the widespread use of chlorpyrifos for almost 50 years,isolation of chlorpyrifos-degrading bacterial has yet been little dif-ficult. Attempts to isolate chlorpyrifos-degrading bacteria were madeby many research workers. Chlorpyrifos has been reported to bedegraded co-metabolically in liquid media by bacteria (Mallick et al.,1999; Horne et al., 2002). Till recent years, several strains, utilizing

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chlorpyrifos as the sole carbon, were isolated gradually (Singh et al.,2004; Yang et al., 2005, 2006; Li et al., 2007; Xu et al., 2007; Fanget al., 2007). Though the mpd gene (an organophosphorus hydrolaseisolated firstly from Plesiomonas sp. M6) was cloned from Sphingo-monas sp. strain Dsp-2 and Stenotrophomonas sp. strain YC-1 sepa-rately, the variety of the chlorpyrifos-degrading bacteria anddegrading mechanism of chlorpyrifos have not been studied exten-sively. In this study, isolation and identification of novel chlorpyrifos-degrading bacteria from contaminated sites will be carried out, andcomparison of their ability to degrade chlorpyrifos and the genesinvolved in chlorpyrifos degradation will also be studied.

2. Materials and methods

2.1. Samples

Water sample was collected from an industrial plant which had producechlorpyrifos for more than 15 years in NanTong, Jiangsu, China. Soil sample wastaken from an agricultural field located in the city of Nanjing, China. The soil hadbeen exposed to chlorpyrifos for more than 2 years.

2.2. Chemicals and culture medium

Chlorpyrifos, 3,5,6-trichloro-2-pyridinol (TCP), parathion, parathion-methyl,triazophos, phoxim, fenitrothion and profenofos (>99% purity) were obtained from

X. Li et al. / International Biodeterioration & Biodegradation 62 (2008) 331–335332

the Pesticide Research Institute, Shenyang, China. Methanol was chromatographicpure grade. All other reagents used in this study were of analytical grade.

Luria-bertani (LB) and mineral salts medium (MSM) that contained (in gramsper liter) 1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4$7H2O, 0.5 g NaCl and 1.5 gNH4NO3 were used for isolating bacterial strains. The initial pH value was adjusted to7.0. When necessary, 100 mg l�1 chlorpyrifos was added to MSM medium.

2.3. Strain isolation and characterization

Selective enrichment method was used for isolating chlorpyrifos-degradingbacteria and the procedure was the same as described previously by Huang et al.(2007). Strains were identified based on morphological, physiological and bio-chemical tests with reference to Bergey’s Manual of Determinative Bacteriologycombined with 16S rDNA sequence analysis.

The nucleotide sequences of the 16S rDNA of each strain were deposited in theGenBank database under the accession numbers: DQ482656 (Dsp-1), AY994060(Dsp-2), DQ482655 (Dsp-3), DQ482654 (Dsp-4), DQ115539 (Dsp-5), DQ237947(Dsp-6) and DQ676936 (Dsp-7).

2.4. ERIC-PCR fingerprinting

The ERIC-PCR fingerprints were used to select the different degrading strainfrom these isolates with the primers F1 and R1 (Hulton et al., 1991). The 50 ml re-action mixture contained 0.5 mM each of the forward and reverse primers, 0.2 mMdeoxynucleoside triphosphates, 2.0 mM MgSO4, 1� PCR buffer, 50 ng of DNA and2.5 U of rTaq DNA polymerase. The PCR reaction was performed with the followingcycling profile: initial denaturation at 95 �C for 5 min, 35 cycles of denaturation at94 �C for 10 s, 92 �C for 40 s, annealing at 49 �C for 8 s, 51.5 �C for 1 min andextension at 72 �C for 5 min, with a final extension at 72 �C for 10 min.

2.5. Degradation of chlorpyrifos by isolates

MSM medium supplemented with 100 mg l�1 chlorpyrifos was used fordegrading test. Cells were pre-cultured in LB medium, harvested by centrifugationand washed three times with sterilized water. For all experiments, cells were used ata concentration of 108 cells ml�1 and samples were incubated at 30 �C on a shaker at150 rpm unless otherwise stated. Medium without inoculation was maintained andtested in the same manner as above as controls. Removal of chlorpyrifos andaccumulation of TCP were measured at 230 and 320 nm by HPLC, respectively. Theseparation column used for HPLC (internal diameter, 4.6 mm; length, 25 cm) wasfilled with Kromasil 100-5C18. The mobile phase was methanol:water (85:15, v:v),and the flow rate was 1.0 ml min�1.

Soil samples used for degrading test were collected from the top 0–10 cm fromthe Agricultural farm, Nanjing, China. The soil has never been treated with

Fig. 1. Phylogenetic analysis of the seven isolates Dsp-1 to Dsp-7 and related species by thebars represent 0.02 substitutions per site.

chlorpyrifos. Soil samples were dried at room temperature, and then sieved to 5 mmand stored at 4 �C without sterilization. Uninoculated soil was kept as a control.

2.6. Degrading substrate range

Degradation of other organophosphate pesticides was also carried out usingsame conditions. Liquid MSM medium was supplemented with one of the followingpesticides such as parathion, parathion-methyl, triazophos, phoxim, fenitrothion orprofenofos (100 mg l�1) as carbon source.

The concentrations of other organophosphate pesticides were detected at270 nm using HPLC conditions identical to those described for chlorpyrifos.

2.7. Amplification of the mpd gene

About 1.0 kb mpd gene was amplified with previously described primers (Cuiet al., 2001). The PCR reaction mixture and procedure were the same as describedpreviously (Cui et al., 2001). Plesiomonas sp. strain M6 was used as the positivecontrol, and Escherichia. coli DH5a was used as the negative control.

3. Results

3.1. Isolation and identification of chlorpyrifos-degrading strains

In our study, seven degrading bacteria were found to be capableof degrading chlorpyrifos in liquid culture. These strains weredesignated as Dsp-1 to Dsp-7, respectively. Strains Dsp-1 to Dsp-4were isolated from water sample, however, strains Dsp-5 to Dsp-7were from soil sample. These isolated strains were identified basedon morphological, physiological and biochemical tests with refer-ence to Bergey’s Manual of Determinative Bacteriology combinedwith 16S rDNA sequence analysis. Based on these characteristics,strains Dsp-2, Dsp-4, Dsp-6 and Dsp-7 were identified as Sphingo-monas sp., Stenotrophomonas sp., Bacillus sp. and Brevundimonas sp.,respectively, while other strains were identified as members ofPseudomonas sp.

To identify the phylogeny of the isolates, strains from differentgenera were chosen to construct the phylogenetic tree based on 16SrDNA sequences (Fig. 1). Results revealed a phylogenetic diversity ofthe chlorpyrifos-degrading bacteria in the chlorpyrifos-contaminated

neighbor-joining approach. Bootstrap values (%) are indicated at the nodes. The scale

X. Li et al. / International Biodeterioration & Biodegradation 62 (2008) 331–335 333

environment. Even though the seven isolated bacteria were all clus-tered into the Proteobacteria group, they belonged to six genera. Dsp-6 was gram positive which was different from the other isolates.

3.2. Distinguishing Dsp-1 and Dsp-3 using ERIC-PCR fingerprinting

Dsp-1 and Dsp-3 were both identified as Pseudomonas sp. Theyhad only 1 base pair difference in 16S rDNA sequences, and themain physiological and biochemical characteristics tested werealmost the same. ERIC-PCR technique which had been successfullyapplied to study bacterial diversity (Judd et al., 1993; Ventura et al.,2003; Yamamura et al., 2004) was used to further distinguish Dsp-1and Dsp-3. Strains Dsp-1 and Dsp-3 had two distinctive ERIC-PCRpatterns indicating that they were different (Fig. 2).

Fig. 3. Degradation of chlorpyrifos by strains Dsp-1 to Dsp-7 in mineral salts medium.Standard error was within 5% of the mean.

3.3. Chlorpyrifos degradation by the seven isolates

The degrading abilities of the seven strains were tested in cul-ture media. The degradation rates of seven strains varied from37 mg l�1 d�1 to 100 mg l�1 d�1(Fig. 3). Strain Dsp-2 showed thefastest degradation rate among these isolates and 100 mg l�1

chlorpyrifos was degraded to an undetectable level in 24 h. TCP wasobserved to accumulate during chlorpyrifos-degrading processbecause its degradation rate was much slower than chlorpyrifos.Strains Dsp-1 to Dsp-4 could degrade a low concentration of TCP(<30 mg l�1). Added high concentration of TCP was found to reducethe degradation rate of chlorpyrifos. This supports the previousreport of a possible toxic effect of this compound on the microbialcommunity (Racke et al., 1990).

Addition of these four strains to soil with chlorpyrifos resultedin a more rapid rate of degradation of chlorpyrifos compared to theuninoculated control. Strain Dsp-1 instead of Dsp-2 had the highestdegrading rate, it might be due to Dsp-1 belongs to Pseudomonasgenus, which has the finer environmental applicability. From Fig. 4

Fig. 2. Enterobacterial repetitive intergenic consensus (ERIC)-PCR profiles of strainsDsp-1 and Dsp-3. Lane A, DL2000 marker; lane B, strain Dsp-1; lane C, strain Dsp-3.

we could see that approximately 98% of chlorpyrifos was degradedwithin 10 days. It suggested that the strains worked well in this soil.

3.4. Degrading substrate range

The liquid medium supplemented with 100 mg l�1 parathion,parathion-methyl, triazophos, phoxim, fenitrothion or profenofos,respectively, was used to test the degrading substrate range ofthese bacteria. The results indicated that there were differences interms of the types of organophosphate pesticides degradation. Onlystrain Dsp-2 was found to be capable of hydrolyzing profenofos,however, triazophos and phoxim were not observed to be degradedin 3 days by any isolates. Strains Dsp-5 and Dsp-7 cannot degradeany supplied pesticides except chlorpyrifos. We deduced that theside chain of the substrate has a significant effect on the bio-degradation of organophosphate insecticides, even though theyhave the same P–O–C linkage.

3.5. Comparison of the mpd gene amplified

mpd Gene were amplified from strains Dsp-1 to Dsp-4 and noamplification products were detected in strains Dsp-5, Dsp-6 andDsp-7 even by using different dilutions of DNA and differentannealing temperatures (Fig. 5). This result might indicate thatthere was no conserved mpd gene existed in strains Dsp-5 to Dsp-7.However, further investigation is needed.

The complete nucleic acid sequence of the amplified mpd genewas 996 bp and the predicted hydrolase contained 331 amino acid

Fig. 4. Degradation of chlorpyrifos by strains Dsp-1 to Dsp-4 in soil. Standard error waswithin 5% of the mean.

Fig. 5. Amplification of mpd gene from the seven degrading strains. Lane A, DL2000marker; lane B, strain M6 as positive control; lanes C–D, strains Dsp-1 to Dsp-7; lane J,E. coli as negative control.

X. Li et al. / International Biodeterioration & Biodegradation 62 (2008) 331–335334

residues. When compared at the amino acids level, there wereseven sites with different amino acids and the four hydrolasescould be divided into three groups. Hydrolases from strains Dsp-1and Dsp-3 were identical.

4. Discussion

In the present study, we used selective enrichment methods toisolate chlorpyrifos-degrading bacteria from polluted samples, andseven distinct strains were obtained finally. Although the culturemethods used are likely to only isolate a small fraction of degradingbacteria present in the original sample, the results in our study stillsuggest that the method is well-suited for the isolation of numbersof physiologically and genetically different strains.

The diversity of isolated chlorpyrifos-degrading bacteria wasanalyzed by five different approaches including 16S rDNA genesequences, ERIC-PCR technique, physiological characterization,degrading characters and mpd gene sequence comparison. Withthese techniques, the diversity of these isolates could be resolved toa much higher degree as compared to single approach. Strains Dsp-1 to Dsp-4, which were isolated from long time polluted watersample, were very effective in chlorpyrifos degradation, they coulddegrade 100 mg l�1 chlorpyrifos to an undetectable level in 48 h.Moreover, these bacteria could also degrade other organophos-phate pesticides such as parathion, parathion-methyl, fenitrothionand profenofos. The degrading abilities of TCP also made themoutstanding compared with the isolates from soil sample. Additionof these four strains to soil with chlorpyrifos resulted in a morerapid rate of degradation of chlorpyrifos compared to the un-inoculated control. All these revealed that they had the high po-tential in the bioremediation of contaminated sites.

Even though the mpd genes amplified from strains Dsp-1 toDsp-4 were highly conserved, several key amino acid substitutionswere found. These variations contributed to delightful changes inthe specificity between hydrolase and substrates of strain Dsp-2

have been reported (Li et al., 2007). Structural changes may stabi-lize MPH in a more active conformation, thereby increasing notonly its hydrolytic efficiency for chlorpyrifos, but also the globalhydrolysis rate of other tested substrates such as profenofos. Thempd gene sequences of this research would provide valuable in-formation for elucidating the relationships between function andstructure of organophosphorus hydrolase. It was reported that mpdgene cluster constituted a catabolic transposon and it contributedto the dispersion of this gene among indigenous bacteria in a pol-luted environment (Zhang et al., 2006). Similar phenomenon hadbeen confirmed in the opd gene. (Siddavattam et al., 2003). Thediversity of the chlorpyrifos-degrading bacteria in the contami-nated water and the conservation of the hydrolase gene suggestedthat mpd gene could be transferred horizontally and expressedamong a variety of bacterial hosts.

In conclusion, we describe here seven new isolates capable ofutilizing chlorpyrifos as the sole carbon. They were identified to sixgenera, which could enrich the resource of chlorpyrifos-degradingbacteria. To our knowledge, this is the first report of isolatingchlorpyrifos-degrading bacteria from the genus Brevundimonas.Moreover, this study confirms the diversity of chlorpyrifos-degrading bacteria isolated from chlorpyrifos-contaminated sites inChina and could be used efficiently for remediation of contami-nated soils. Presence of mpd gene indicated possible presence ofbacterial community with more efficient and novel enzymatic ca-pabilities. Polymorphisms of the mpd gene sequence will help us inthe better understanding of gene transfer and evolution.

Acknowledgements

This work was supported by grants from Chinese NationalNatural Science Foundation (30600016) and National Programs forHigh Technology Research and Development of China(2007AA10Z405).

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