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International Biodeterioration & Biodegradation 62 (2008) 51–56

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Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol

by a newly isolated Paracoccus sp. strain TRP
Gangming Xua,b, Wei Zhengc, Yingying Lic, Shenghui Wangd,
Jingshun Zhangc, Yanchun Yand,
a
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
b
Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
c
College of Life Sciences, Shandong Agricultural University, Tai’an 271018, PR China
d
Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
Received 9 April 2007; received in revised form 23 October 2007; accepted 4 December 2007
Available online 25 January 2008

Abstract

A bacterium, isolated from activated sludge and named strain TRP, could biodegrade chlorpyrifos and 3,5,6-trichloro-2-pyridinol.
Phenotypic features, physiological and chemotaxonomic characteristics, and phylogenetic analysis of 16S rRNA sequence revealed that
the isolate belongs to the genus of Paracoccus. Strain TRP could also degrade pyridine, methyl parathion and carbonfuran when
provided as sole carbon and energy sources. Native-PAGE and enzymatic degradation assay of the cell-free extracts indicated that an
alternative degradation mechanism might involve an inducible enzyme. Degradation study of chlorpyrifos by strain TRP was examined
by GC–MS and HPLC; no persistent accumulated metabolite was observed. To the best of our knowledge, this is the first report of a
bacterium that could completely mineralize chlorpyrifos. This isolate will be potentially useful in biotreatment of wastewaters and
bioremediation of contaminated soils.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Chlorpyrifos; 3, 5, 6-Trichloro-2-pyridinol; Degrading bacterium; Paracoccus sp.; Complete mineralization

1. Introduction using degrading microbes (Singh et al., 2006). The

Chlorpyrifos (CP), O,O-diethyl O-(3,5,6-trichloro-2-pyr-
idinol (TCP)) phosphorothioate, is a broad spectrum
moderately toxic organophosphorus insecticide. CP has a
high soil-absorption co-efficient, but low water solubility
(2 mg l1) (Racke, 1993). It is widely used throughout
the world, especially in recent years with the restrictions
or eliminations of highly toxic organophosphorus com-
pounds. Its wide contamination of water and soils has
aroused much public concern (Yang et al., 2005; Yu et al.,
2006).
Bioremediation has received increasing attention as an
efficient and cheap biotechnological approach to clean up
polluted environments. Several chemicals have been
successfully removed from soil and aquatic environments
Corresponding author. Tel.: +86 10 68919685; fax: +86 10 68975643.
E-mail address: yanyc_2004@hotmail.com (Y. Yan).
environmental fate of CP has been studied extensively,
and its degradation may involve a combination of
photolysis, chemical hydrolysis and microbial degradation.
The reported half-life of CP in soil varies from 10 to 120 d
(Getzin, 1981; Racke et al., 1988), with TCP as the major
degradation product (Singh and Walker, 2006). Unlike
other organophosphorus compounds, CP has been re-
ported to be resistant to enhanced degradation since its first
use in 1965. It was suggested that the accumulation of
TCP, which has anti-microbial properties, prevents the
proliferation of CP degrading microorganisms (Racke
et al., 1990). Although CP has been reported to be
degraded co-metabolically by bacteria in liquid media,
several attempts to isolate CP-degrading bacteria have not
been successful (Racke et al., 1990; Mallick et al., 1999).
Singh et al. (2004) isolated the first CP-degrading
bacterium, Enterobacter B-14, which hydrolyzed CP to
diethylthiophosphate (DETP) and TCP, and utilized

0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2007.12.001
 ARTICLE IN PRESS
52 G. Xu et al. / International Biodeterioration & Biodegradation 62 (2008) 51–56
DETP for growth and energy. Two recently isolated CP-
degrading bacteria, Stenotrophomonas sp. and Sphingomo-
nas sp., could also utilize CP as the sole source of carbon
and phosphorus, but they did not degrade TCP (Yang
et al., 2006; Li et al., 2007). Mineralization of TCP by a
Pseudomonas sp. has been reported previously (Feng et al.,
1997), and also a strain of Alcaligenes species was reported
capable of biodegrading both CP and TCP (Yang et al.,
2005).
In most cases reported to date, the degrading bacteria
tend to transform CP by hydrolysis to produce DETP and
TCP, which in turn accumulate in the medium. Studies on
further metabolism and identification of intermediate
products of CP have not been extensive. A co-culture of
Serratia and Trichosporon spp. that could mineralize CP
has been obtained recently in our lab (Xu et al., 2007).
However, complete mineralization of CP by a single
bacterial culture has not been described yet. The aims of
this study were to isolate and characterize new CP-
mineralizing bacteria, and investigate their degrading
enzyme(s) and the pathways of degradation.
2. Materials and methods
2.1. Media and chemicals
CP (95% purity), methyl parathion, and carbonfuran were obtained from
Shandong Huayang Pesticide Chemical Industry Group, China. TCP
standard (93.5% purity) was purchased from the Laboratories of Dr.
Ehrenstorfer, Augsburg, Germany. Pyridine and petroleum ether were
purchased from Kaitong Chemicals Co., Tianjin, China. TCP was dissolved
in methanol, while CP was dissolved in different solvents (including acetone,
methanol, ethanol, and pyridine) as stock solutions (25,000 mg l1), which
were rationed into medium to get the desired concentrations. Enrichment
medium TYC (0.5% tryptone, 0.5% yeast extract, and 0.1% KH2PO4) and
three different mineral media were used; their composition, amount and
preparation were as described in detail previously (Shelton and Somich,
1988; Karpouzas and Walker, 2000; Jia et al., 2006).
2.2. Isolation of CP and TCP degrading bacteria by enrichment
and screening
Activated sludge samples were collected from three pesticides
manufacturers of Shandong, China. Samples (10 ml) were added to
90 ml TYC media containing 25 mg l1 CP in 250 ml flasks. The suspension
was incubated at 30 1C with shaking at 100 rpm in the dark; 10 ml of the
culture broth was transferred weekly to 90 ml of fresh media, while its CP
concentration increased doubly using the stock solutions with different
solvents. After the fourth enrichment transfer, each culture (10 ml) was
centrifuged to get the pellets and was used to inoculate separate flasks
containing 100 ml of mineral media with CP (200 mg l1 in pyridine), and
then further screened weekly by TCP. After the screening transfers,
aliquots were spread on corresponding plates of mineral media (containing
1.5% agar and 100 mg l1 of CP) and incubated at 30 1C for 5–7 d. All
isolates were screened based on the formation of degrading haloes as
described previously (Cho et al., 2004). Potential isolates were obtained
and tested for their degrading ability of CP and TCP.
2.3. Characterization of isolated CP and TCP degraders
Isolates were identified with reference to Bergey’s Manual of
Determinative Bacteriology (Holt et al., 1994). Cell morphology was
examined with both light microscopy (Olympus, Japan) and transmission
electron microscopy (JEM-1200EX, Japan). Genomic DNA was extracted
as described previously (Sambrook et al., 1989). The 16S rRNA gene was
amplified by PCR using the universal primers 27f (50 -AGAGTTT-
GATCMTGGCTCAG-30 ) and 1492r (50 -TACGGYTACCTTGTTAC-
GACTT-30 ). PCR products were cloned into a pMD18-T vector
(TaKaRa) and sequenced. The determined 16S rRNA partial sequence
of strain TRP (GenBank accession no. EF070124) was compared with
those available on BLAST search of the GenBank database. Selected
sequences with the greatest sequence similarity were extracted and
compared by CLUSTAL W. Phylogenetic and molecular evolutionary
analyses were conducted using MEGA version 3.1 (Kumar et al., 2004)
with the Kimura 2-parameter model and the neighbor-joining method.
2.4. Biodegradation of CP and TCP by isolate TRP
The inoculum preparation for degradation studies was the same as
described by Yang et al. (2006). Strain TRP was pre-cultured in TYC
medium with 50 mg l1 CP and TCP, respectively. Cultures were run in
triplicate to ensure accuracy. Uninoculated media with the same
concentration of either CP or TCP were used as controls. Degradation
studies were carried out at 30 1C in mineral media containing 50 mg l1 CP
and TCP, respectively. Cultures were regularly checked for bacterial
growth as monitored by a spectrophotometer (SCINCO S-3150, Korea) at
600 nm. The CP residues were analyzed with GC-FPD as reported by Yu
et al. (2006). The concentration of TCP was measured by HPLC according
to the method of Feng et al. (1997).
2.5. Substrate range
Cross-feeding studies with other contaminants were also performed.
The liquid medium was supplemented with pyridine, methyl parathion
or carbonfuran at 50 mg l1. The pesticide residues were measured by
GC-FID (Yang et al., 2005).
2.6. Analysis of CP and TCP biodegradation metabolites by
GC-MS and HPLC
Samples and controls were extracted with petrol ether, and the
supernatant was dried over anhydrous sodium sulfate. The extracted
samples were analyzed by GC-FID as described previously with
modifications (Bakiamoh et al., 1999; Yu et al., 2006), and reconfirmed
on a Perkin-Elmer Clarus 500 GC-MS system equipped with autosampler,
an on-column, split/splitless capillary injection system, and with DB-5-MS
capillary column (300  0.25 mm2, 0.25 mm). The operating conditions
were as follows: the column was held initially at a temperature of 60 1C for
5 min, then at 10 1C min1 to 200 1C, held for 5 min, then at 15 1C min1 to
260 1C, and finally held at that temperature for 5 min. The temperatures
corresponding to the transfer line and the ion trap were 280 and 220 1C,
respectively, and the ionization energy was 70 eV. The injection volume
was 1.0 ml with a split ratio of 1:7 at 260 1C. Helium was used as a carrier
at a flow rate of 1.0 ml min1. At the same time, the remaining water phase
and aqueous samples were filtered through a 0.45 mm filter before being
analyzed by HPLC (Thermo Finnigan, USA) using a Zorbax SB-C18
column (250  4.6 mm2, 5 mm) with array detection from 200 to 600 nm
(total scan). A mixture of methanol and water (70:30, v/v, pH 3.0) was
used as the mobile phase at a flow rate of 1.0 ml min1. The injection
volumes were 10 ml. In addition, the amount of CO2 in gas samples
withdrawn from the flask headspace was analyzed by GC-TCD (Soares et
al., 2005).
2.7. Preparation of cell-free extracts
The isolate was grown at 30 1C in mineral media containing methanol,
glucose, pyridine, CP or TCP. Growth was monitored and cells were
pelleted by centrifugation (at 6000g for 10 min 4 1C). Pellets were washed
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G. Xu et al. / International Biodeterioration & Biodegradation 62 (2008) 51–56 53

and resuspended in 3 ml of phosphate buffer (0.05 M, pH 7.0). After

disruption of cells by ultrasonication on ice and centrifugation at 4 1C, the
supernatants were obtained as the cell-free extract (protein concentration
was adjusted to 1 mg ml1 before use).

2.8. Native-PAGE analysis of proteins from induced and

non-induced cultures

The proteins (cell-free extract) from induced (by CP or TCP) and non-
induced cultures were separated by native-PAGE (a total of 20 mg of
protein extract loaded per lane) followed by Comassie brilliant blue
staining as described in detail previously (Simposon, 2003).

2.9. Enzymatic degradation of CP and TCP by cell-free extract of

strain TRP Fig. 1. Phylogenetic tree based on the 16S rRNA sequences of strain TRP
and relating species. GenBank accession numbers are given in brackets.
Bootstrap values obtained with 1000 repetitions were indicated as
The reaction mixture (1.0 ml) contained phosphate buffer (0.05 M, pH percentages at all branches. A distance bar is illustrated.
7.0), CP or TCP (50 mg l1) as substrate, and cell-free extract (protein
concentration 1 mg ml1). Reactions were performed at different pH (5–9)
and temperatures (15–40 1C), and the residues were periodically quantified
as described above. All experiments were performed in triplicate and
controls lacking either substrate or enzyme were used.

3. Results

3.1. Isolation and characterization of isolated CP- and

TCP-degrading bacteria

From three activated sludge samples, eight different

bacteria were isolated and grown in high concentrations of
CP (600 mg l1) and TCP (400 mg l1). Strain TRP, selected
for further studies in this paper, was a non-motile coccoid.
It was positive in tests for oxidase, catalase, nitrate
reductase and denitrification, and utilized CP, TCP,
pyridine, methyl parathion, carbofuran, glucose, sucrose,
sodium acetate trihydrate and sodium succinate as sole
carbon sources. It also utilized CP, TCP, pyridine, and
carbofuran as sole nitrogen sources. It was negative in
Gram staining, hydrolyzation of starch and glutin, and
utilization of xylose and citrate. The 16S rRNA sequence
of strain TRP showed greatest similarity to the reference
sequences from members of the genus Paracoccus within
the GenBank database. A phylogenetic tree (Fig. 1) depicts
the position of strain TRP within the genus Paracoccus.
Based on these observations, the isolate was designated as
Paracoccus sp. TRP.

3.2. Bacterial growth and degrading ability of Paracoccus

sp. strain TRP
Strain TRP could utilize CP and TCP as the sole carbon,
nitrogen and phosphate source, and the cell yields (dry
weight biomass) associated with growth were about
0.1 g l1 d1. In biodegradation assay, complete degrada-
tion of 50 mg l1 CP was observed within 4 d (Fig. 2a).
Degradation of CP was accompanied by transient accu-
mulation of TCP, but it was subsequently degraded rapidly
and disappeared finally. Furthermore, 50 mg l1 TCP
was also completely degraded by strain TRP within 4 d
Fig. 2. Degradation of CP (a) and TCP (b) by Paracoccus sp. strain TRP
and bacterial growth monitored by measuring OD at 600 nm (The OD
value of CP was deducted). &, CP control; ’, CP inoculated; W, TCP
control; m, TCP inoculated; K, OD600 nm. Values are means7SD of
three replicates.
(Fig. 2b). All degradation was accompanied by bacterial
growth as shown in Fig. 2. Addition of other carbon
sources led to a lag phase followed by accelerated
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54 G. Xu et al. / International Biodeterioration & Biodegradation 62 (2008) 51–56

degradation (data not shown). All contaminants tested the presence of glucose, methanol or pyridine. The novel
in the cross-feeding experiment, including pyridine protein band may correspond to an enzyme involved in
(12.5 mg l1 d1), methyl parathion (9.6 mg l1 d1), and degrading CP and TCP, suggesting that a responsible
carbonfuran (9.4 mg l–1 d–1) were degraded by strain TRP. degrading enzyme might be inducible. Also, there were
In controls without incubation, abiotic degradation was more protein bands in lanes that contained pyridine,
negligible throughout all studies. suggesting that another enzyme was produced for its
biodegradation.
3.3. Analysis of CP and TCP biodegradation metabolites
3.5. Enzymatic degradation of CP and TCP by cell-free
Under the conditions described above, the retention time extracts of strain TRP
for CP by GC–MS was about 22.3 min, and the retention
time for TCP by HPLC was about 3.7 min. Complete The degradation patterns of CP and TCP by cell-free
degradation of CP and TCP were confirmed by GC–MS extracts of strain TRP are shown in Fig. 4a. Both CP and
and HPLC analysis. Throughout the present study, only TCP (50 mg l1) were degraded rapidly in the first 3 h by
TCP was detected, which was transiently accumulated in the cell-free extracts pre-cultured with CP or TCP. The
the medium but was subsequently degraded by the enzymatic degradation rates specific for CP and TCP were
bacterium. In the biodegradation assay of TCP, no
accumulative metabolites were observed to be persistent
in the system. Moreover, the CO2 levels increased with
increase in culture growth (data not shown). Therefore, CP
was hydrolyzed by this bacterium to DETP and TCP, and
then both intermediates were utilized as the sources of
carbon, phosphorus or nitrogen, finally resulting in
complete mineralization.

3.4. Native-PAGE analysis of proteins from induced and

non-induced cultures

The protein patterns of the crude extracts from strain

TRP cells cultivated under various conditions were
investigated by native-PAGE (Fig. 3). In mineral media
containing CP or TCP as the sole carbon source, a strong
protein band appeared when compared with cells grown in

Fig. 4. Enzymatic degradation of CP and TCP by cell-free extract of

Fig. 3. Native-PAGE protein patterns of strain TRP under induced and
non-induced conditions. Cells were cultured in mineral media with
different carbon sources: lane 1, with methanol; lane 2, with CP (in
methanol); lane 3, with CP (in pyridine); lane 4, with glucose; lane 5, with
TCP (in methanol); and lane 6, with pyridine. The new protein band that
appeared upon induction is indicated by the arrow.
strain TRP. (a) Enzymatic degradation of CP or TCP. &, CP control
without induction; W, TCP control without induction; ’, CP induced for
CP degradation; E, CP induced for TCP degradation; m, TCP induced
for TCP degradation; K, TCP induced for CP degradation. (b) Enzymatic
degradation study of CP under different temperatures (B, 20–40 1C) and
pH (J, 5–9). Values are means7SD of three replicates.
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G. Xu et al. / International Biodeterioration & Biodegradation 62 (2008) 51–56
about 13.4 and 15.1 mg l–1 h–1, respectively. Only slight
degradation was observed in the cell-free extracts without
induction.
The bacterial cell-free extract was able to degrade CP
and TCP at temperatures ranging from 15 to 40 1C and the
most rapid degradation was at 35 1C. It was capable of
degrading CP and TCP in the pH range from 5 to 9, with
the most rapid degradation rate at pH 8 (Fig. 4b).
4. Discussion
It is assumed that the extremely low solubility of CP in
water caused the limited supply of the carbon source (Singh
and Walker, 2006). For the first time, pyridine was
employed to exclude solvent-dependent strains in this
study. Unlike other organic solvents, when CP was
dissolved in pyridine and rationed into medium, the water
solubility of CP was greatly enhanced, which might
increase the supply of the carbon source for bacterial
growth. Moreover, pyridine was a commonly reported
waste (Mohan et al., 2003). Only those bacteria capable of
degrading both CP and pyridine could survive. TCP was
also used in the screening process to isolate bacteria for the
complete mineralization of CP and TCP. It is believed that
the conditions under which environmental microbes are
enriched and screened are crucial in selecting for isolates
not only with the desired degrading enzymes systems but
also with the specific regulation mechanisms for the
degradation pathways (Kertesz et al., 1994).
In this research, the isolate TRP showed greatest
similarity to members of the genus Paracoccus. Recent
interest in this biochemically versatile genus has focused on
its wide range of diverse degrading capabilities and
potential applications in bioremediation (Baker et al.,
1998). A few Paracoccus species have been reported to
degrade halobenzoate (Song et al., 2000), n-alkanes (Zhang
et al., 2004), and monocrotophos (Jia et al., 2006).
However, no CP or TCP degrading bacterium has been
reported from the genus Paracoccus. Also in this work,
another strain DM was identified to be of the same genus,
although they were from different sampling origins. These
indicate that further study of this genus may help to
understand the ecological diversity, metabolic versatility
and potential applications in bioremediation.
CP has been reported to be degraded co-metabolically by
bacteria, which needs extra carbon sources (Richins et al.,
1997; Mallick et al., 1999). Singh et al. (2004) reported
isolation of Enterobacter sp. B-14, which can use CP for the
supply of carbon and phosphorus sources, while it stopped
degrading CP in the presence of other carbon sources.
However, strain TRP shows a more rapid degradation in
the presence of an additional carbon source. This indicates
that CP could also be degraded co-metabolically by strain
TRP, which might signify the environmental adaptation of
this bacterium. Moreover, native-PAGE and enzymatic
degradation assay of cell-free extracts under inductions
suggest that the degrading enzyme for CP and TCP might
55
be inducible. The present results are not completely
consistent with those from previous studies (Singh et al.,
2003; Yang et al., 2005). However, this alternative
degradation mechanism involving inducible enzyme might
be an economical strategy, which indicates its bioenergetic
flexibility and provides a substantial competitive advantage
over other microorganisms.
Previous researchers reported that the removal of CP
took place with the formation of metabolites like CP-oxon,
3,5,6-trichloro-2-methoxypyridine, 2-chloro-6-hydroxypyr-
idine (Singh and Walker, 2006; Yu et al., 2006), and TCP,
which was the major degradation product accumulated in
pure cultures, water and soils (Mallick et al., 1999; Liu
et al., 2001; Singh et al., 2003). In the present study, only
transient accumulation of TCP was observed after incuba-
tion, without any other persistent metabolites detected. No
accumulative products were detected in the biodegradation
of TCP; the metabolites might have formed and been
immediately degraded by the bacterium. Furthermore,
Paracoccus sp. TRP could utilize pyridine as the sole
carbon and nitrogen source, indicating that the pyridinyl
ring of CP might be cleaved. Release of CO2 in medium
containing CP or TCP as a sole source of carbon indicated
the complete mineralization. Although a pure culture of
Pseudomonas sp. capable of mineralizing TCP has been
isolated (Feng et al., 1997), three other CP-degrading
bacteria, Enterobacter strain B-14, Stenotrophomonas sp.
YC-1, and Sphingomonas sp. Dsp-2, failed to utilize
TCP for growth and energy (Singh et al., 2004; Yang
et al., 2006; Li et al., 2007). A strain of Alcaligenes
species and a co-culture of Serratia and Trichosporon spp.
capable of biodegrading both CP and TCP were also
reported (Yang et al., 2005; Xu et al., 2007), but without
attempts to identify any intermediate metabolites. To the
best of our knowledge, this is the first report of
isolation and characterization of a bacterium that could
completely mineralize CP and TCP. Paracoccus sp. strain
TRP may prove to be a promising bacterium in the
biotreatment of wastewaters and bioremediation of con-
taminated soils. The bacterial cell-free extracts might be a
promising way to detoxify CP residues rapidly on the
surface of fruits and vegetables. Further investigation is in
progress to identify the genes and enzymes involved in the
degradation process. For practical application, further
research on degradation in soils and aqueous environments
is necessary.
Acknowledgments
This work was supported by the Hi-tech Research and
Development Program of China (no. 2006AA10Z401). We
are indebted to Min Xia, Xinxin Wang and Xiaoman
Zhang (Beijing Center for Physical and Chemical Analysis,
China) for providing facilities and helping with GC and
HPLC. We are thankful to John Loux for his help in the
manuscript’s preparation.
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56 G. Xu et al. / International Biodeterioration & Biodegradation 62 (2008) 51–56
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