Microb Ecol

DOI 10.1007/s00248-014-0457-7

ENVIRONMENTAL MICROBIOLOGY

Distribution of Naphthalene Dioxygenase Genes in Crude

Oil-Contaminated Soils
Yuyin Yang & Jie Wang & Jingqiu Liao & Shuguang Xie &
Yi Huang

Received: 26 May 2014 / Accepted: 27 June 2014

# Springer Science+Business Media New York 2014

Abstract Polycyclic aromatic hydrocarbons (PAHs) are

one of the major pollutants in soils in oil exploring
areas. Biodegradation is the major process for natural
elimination of PAHs from contaminated soils. Functional genes can be used as biomarkers to assess the biodegradation potential of indigenous microbial populations. However, little is known about the distribution
of PAH-degrading genes in the environment. The links
between environmental parameters and the distribution
of PAH metabolic genes remain essentially unclear. The
present study investigated the abundance and diversity
of naphthalene dioxygenase genes in the oilcontaminated soils in the Shengli Oil Field (China).
Spatial variations in the density and diversity of naphthalene dioxygenase genes occurred in this area. Four
different sequence genotypes were observed in the contaminated soils, with the predominance of novel PAHdegrading genes. Pearsons correlation analysis illustrated that gene abundance had positive correlations with
the levels of total organic carbon and aromatic hydrocarbons, while gene diversity showed a negative correlation with the level of polar aromatics. This work
could provide some new insights toward the distribution
of PAH metabolic genes and PAH biodegradation potential in oil-contaminated ecosystems.

Yuyin Yang and Jie Wang contributed equally to this study.

Electronic supplementary material The online version of this article
(doi:10.1007/s00248-014-0457-7) contains supplementary material,
which is available to authorized users.
Y. Yang : J. Wang : J. Liao : S. Xie (*) : Y. Huang (*)
State Key Joint Laboratory of Environmental Simulation and
Pollution Control, College of Environmental Sciences and
Engineering, Peking University, Beijing 100871, China
e-mail: xiesg@pku.edu.cn
e-mail: yhuang@pku.edu.cn

Introduction
A wide distribution of polycyclic aromatic hydrocarbons
(PAHs) in the environment has aroused increasing environmental concerns because of their persistence, toxicity, mutagenicity, and carcinogenicity [15, 26, 45]. Some of PAH
species have been listed by the European Union and US
Environmental Protection Agency (EPA) as the top priority
pollutants [10]. They have severe impacts on local ecosystems
and human health [42]. The release of PAHs into the environment can occur through a variety of natural and anthropogenic
sources [3, 10]. High levels of PAHs are usually found in soil
and sediment in the oil exploring and refinery areas [22, 24,
33].
Biotransformation and biodegradation are the major processes for natural elimination of PAHs from contaminated
soils [8]. In order to study the fate of PAHs in natural environments, PAH-degrading microorganisms from many bacterial genera have been isolated and characterized [13, 17].
Culture-independent methods targeting 16S ribosomal
(rDNA) genes are effective in identifying phylogenetic diversity of PAH-degrading microbial community in soil and sediment ecosystems [7, 13, 44, 45]. However, 16S methods
typically cannot directly link function to identity for environmental samples [12, 46]. Evaluating specific metabolic genes
is an option to functionally characterize microbial community,
which can provide a broader estimation of the bioremediation
potential of contaminated sites [12, 21, 35, 36, 41, 49]. Ringhydroxylating dioxygenases (RHDs) are known for catalyzing
the initial oxidation step of a range of aromatic hydrocarbons
including PAHs. These enzymes play a crucial role in the
biodegradation of these pollutants in contaminated environments [28]. Genes encoding different subunits of PAH-related
RHDs (PAH-RHDs) have been found in both Gram-negative
and Gram-positive bacteria [21, 28]. These PAH metabolic
genes can be applied as biomarkers of a PAH-degrading

Y. Yang et al.

potential in the environment [21]. To date, there have been a

few investigations on PAH metabolic genes in natural environments [4, 5, 16, 21, 25, 27, 40, 43]. However, the links
between environmental parameters and the abundance and
diversity of PAH metabolic genes still remain unclear. Moreover, information on the distribution of soil PAH-degrading
genes in oil exploring and refinery areas is still lacking.
The Shengli Oil Field, located in Dongying City (Shandong Province), is one of the largest oil fields in China. The
Shengli Oil Field has a warm temperate continental semihumid monsoon climate with a mean annual rainfall of
550 mm [24]. Crude oil contamination is a very serious
problem in the oil exploring area, as high levels of PAHs are
found in soils [24]. The knowledge of the PAH-degradation
potential in polluted soils is essential for the management of
soils for bioremediation [21]. Naphthalene dioxygenase genes
are the extensively studied PAH-degrading genes. They could
be involved in the degradation of low-molecular-weight PAHs
(two- to three-ring PAHs) [48]. Therefore, the objective of the
current study was to characterize the abundance and diversity
of naphthalene dioxygenase genes in the oil-contaminated
soils in the Shengli Oil Field. Moreover, the links between
environmental parameters and the distribution of soil PAHdegrading genes were also investigated.

Materials and Methods

amplification efficiency and coefficient (r 2 ) for the

dioxygenase genes were 97 % and 0.999, respectively.
Clone Library Analysis
For clone library analysis of the diversity of naphthalene
dioxygenase genes, the primer pairs NAH-F/NAH-R were
also used for the conventional PCR amplification [2]. PCR
was performed as follows: 5 min at 94 C; 40 cycles of 30 s at
94 C, 30 s at 57 C, 30 s at 72 C; 6 min at 72 C [4].
Triplicate amplicons for each soil sample were pooled, and the
purified PCR products were cloned. The obtained gene sequences were screened for chimeras with Mallard software
(http://www.bioinformatics-toolkit.org). Chimera-free sequences with 97 % identity were assigned as the same
operational taxonomic units (OTUs) using DOTUR program
[31]. Gene diversity indices and rarefaction curves were also
obtained using DOTUR program [31]. Phylogenetic analysis
of the dioxygenase gene composition was performed using
MEGA software version 4.0 [38]. Pearsons correlation analysis of the abundance and diversity of dioxygenase genes with
the soil physicochemical parameters was further conducted
using SPSS 20.0 software. The dioxygenase gene sequences
obtained in this study were submitted to GenBank under
accession numbers KF894843KF894870, KF956854
KF956937, KJ371585KJ371604, KJ371692KJ371761,
and KJ371852KJ371909.

Sampling
Soil samples (05 cm) in triplicate were collected at 13 different sites in the Shengli Oil Field (Fig. 1). These soil samples
(SL1SL13) were placed in a sterile plastic bag, sealed, and
kept on ice several hours before transporting to laboratory by
dry ice. All samples were passed through a 5-mm sieve before
use. The physicochemical parameters of the collected soil
samples are shown in Table 1. All the soils were alkaline,
with high salinity and heavy hydrocarbon contamination.
Quantitative PCR Assay
DNA from soil sample was extracted using the PowerSoil
DNA extraction kit (Mo Bio Laboratories, USA). Each replicate soil DNA sample was individually subjected to real-time
quantitative PCR assays. For quantitative PCR assays, the
specific primers for amplification of classical naphthalene
dioxygenase genes were according to the literature [2],
NAH-F (5-CAAAA(A/G)CACCTGATT(C/T)ATGG-3)
and NAH-R (5-A(C/T)(A/G)CG(A/G)G(C/G)GACTTCTT
TCAA-3). The PCR conditions were as previously described
[2]. Standard curve was obtained using serial dilutions of
linearized plasmids (pGEM-T, Promega) containing cloned
naphthalene dioxygenase genes amplified from soil. The

Results
Abundance of Naphthalene Dioxygenase Genes
In this study, real-time PCR assay was applied to estimate the
abundance of naphthalene dioxygenase genes in the 13 oilcontaminated soils in the Shengli Oil Field. A large variation
in the density of naphthalene dioxygenase genes occurred in
these soil samples (Fig. 2). The largest number of gene copies
was observed in sample SL2 (3.32107 gene copies per gram
of soil), followed by sample SL3 (2.41107 gene copies per
gram of soil), sample SL4 (2.39107 gene copies per gram of
soil), and sample S13 (1.63107 gene copies per gram of
soil). These four soil samples had a much higher density of
dioxygenase genes than the other ones. A relatively low
abundance of gene copies (below 1.0106 gene copies per
gram of soil) were found in samples SL1, SL6, SL9, and
SL10. In addition, Pearsons correlation analysis was conducted to illustrate the relationships between the abundance of
dioxygenase genes and the determined soil physicochemical
parameters. The results indicated that gene abundance was
positively correlated with the levels of total organic carbon
and aromatic hydrocarbons (p<0.05). However, it did not

Distribution of Naphthalene Dioxygenase Genes in Soils

Fig. 1 Schematic representation of the sampling sites

abundance of naphthalene dioxygenase genes in this sample

(Fig. 2). A total of 260 dioxygenase gene sequences were
retrieved from the other 12 soil samples (Table 3). The rarefaction curves for 12 clone libraries nearly came to a plateau
(Fig. S1), indicating that these communities were wellsampled. The number of OTUs in soil samples ranged between 1 and 4, and the values of Shannon index ranged

show a significant correlation with the level of naphthalene

(p>0.05) (Table 2).
Diversity of Naphthalene Dioxygenase Genes
In this study, sample SL9 could not be successfully amplified
under conventional PCR condition, possibly due to very low
Table 1 Physicochemical features of soil samples
Sample

pH

Salinity (g kg1)

TN (mg kg1)

TOC (g kg1)

ALH (g kg1)

ARH (g kg1)

PA (g kg1)

Asphaltenes
(g kg1)

Naphthalene
(mg kg1)

SL1
SL2
SL3
SL4
SL5
SL6
SL7
SL8
SL9
SL10
SL11
SL12
SL13

8.95
8.68
8.07
7.86
7.85
8.62
8.09
8.29
8.03
7.71
8.66
8.75
7.69

10.8
4
15.2
7.4
9.7
31.8
30.7
8.5
20
22.1
4.2
3.1
1

520
410
730
460
410
310
390
440
420
870
320
560
320

38.6
71.1
61.2
148
85.4
28.5
57.5
63.4
57.3
44.2
44.2
32.6
120

9.5
32.8
7.5
45
39.6
5.2
12
14.2
13.4
10
0.8
8
73.7

6.7
22.1
4.8
23.8
11.4
2.1
5
8.1
4.8
3.3
1.3
5
14

8.8
15.4
8.1
25.4
14.1
2.6
5
14.4
6.4
2.6
1.4
6.4
8.9

5.1
5.7
0.9
23.5
3.4
2.6
1.8
2.7
3.2
1.9
1.4
3.7
11.1

78.7
114.3
75.4
1683.6
133.8
108.3
56.2
53.9
70
43.2
63.9
135.4
77.8

TN total nitrogen, TOC total organic carbon, ALH Aliphatic hydrocarbons, ARH Aromatic hydrocarbons, PA Polar aromatics

Y. Yang et al.

Fig. 2 Abundance of naphthalene dioxygenase genes in the different soil samples. Each replicate sediment sample was individually subject to
quantitative PCR assays. Error bars represent standard deviation of mean (n=3)

between 0 and 1.22. These results suggest low diversity of

naphthalene dioxygenase genes in the oil-contaminated soils
in the Shengli Oil Field. Moreover, the diversity of
dioxygenase genes did not have an obvious link with the
abundance (Fig. 2; Table 3). For example, samples SL2,
SL3, and SL4 had very low gene diversity, but they had the
highest gene density. In contrast, sample SL13 had the highest
gene diversity despite of its high gene density. Low gene
density and diversity was found in sample SL8. Pearsons
correlation analysis shows that gene diversity was negatively
correlated with the level of polar aromatics (p<0.05). However, gene diversity did not have a significant correlation with
the level of naphthalene (p >0.05) (Table 2).
Composition of Naphthalene Dioxygenase Genes
In this study, the representative naphthalene dioxygenase gene
sequences were selected from the OTUs containing at least

two sequence members and further performed the phylogenetic analysis using MEGA software version 4.0 [38]. All of
the dioxygenase gene sequences in the soil clone libraries
could be grouped into four distinct clusters (Fig. 3). This
illustrates the existence of different PAH-degrading genotypes
in the oil exploring area. There was no soil sample that could
share all of the four dioxygenase gene clusters. The
dioxygenase gene sequences recovered from samples SL2,
SL3, SL4, SL8, and SL10 were only detected in Cluster I.
The gene sequences from each of samples SL7, SL11, and
SL13 appeared in three clusters. However, these three samples
showed a much different sequence distribution. Members of
the clone library constructed with sample SL7 were mainly
present in cluster I, but became much less abundant in clusters
II and IV. The sequences from sample SL11 existed mainly in
cluster I, and scantly in clusters III and IV. In contrast, the gene
sequences from sample SL13 were evenly distributed in clusters I, II, and III. Samples SL1 and SL12 shared clusters I and

Table 2 Statistical analysis of the abundance and diversity of naphthalene dioxygenase genes with the soil physicochemical parameters
Community index

pH

Salinity

TN

TOC

ALH

ARH

PA

Asphaltenes

Naphthalene

Abundance
Shannon index

0.2
0.04

0.30
0.06

0.01
0.18

0.58*
0.22

0.48
0.17

0.75*
0.32

0.56
0.61*

0.46
0.21

0.39
0.48

TN total nitrogen, TOC total organic carbon, ALH Aliphatic hydrocarbons, ARH Aromatic hydrocarbons, PA Polar aromatics
*Correlation is significant at the 0.05 level

Distribution of Naphthalene Dioxygenase Genes in Soils

Table 3 Diversity of each clone library of naphthalene dioxygenase
genes in the different soil samples

Discussion

Sample

Number of clones

Number of OTUs

Shannon index

Spatial Variation in Gene Abundance in Oil-Polluted Soils

SL1
SL2
SL3
SL4

22
21
28
20

4
3
2
1

0.86
0.38
0.15
0.00

SL5
SL6
SL7
SL8
SL10
SL11
SL12
SL13

21
26
21
22
15
23
21
20

3
3
3
1
3
3
4
4

0.50
0.64
0.78
0.00
0.73
0.67
0.68
1.22

Functional genes can be used as biomarkers to evaluate the

biodegradation potential of indigenous microbial populations in contaminated environments [12, 21, 35, 36, 41,
49]. To date, information on the abundance of PAH metabolic genes in the natural environment is still very limited.
DeBruyn et al. reported a large spatial variation in the
density of PAH metabolic genes in coal tar-contaminated
creek sediments. They further indicated higher abundance
of nidA and nagAc genes at site with the greatest PAH
level [12]. Zhang et al. also indicated an increase in PAHrelated functional genes with a rise of PAH concentration in
soils [47]. In addition, the abundances of PAH metabolic
genes were found to be positively correlated with PAH
concentration and soil organic carbon [4]. The content of
total petroleum hydrocarbons in soil had a significant positive correlation with the number of ndoB gens, but not
with the number of nidA genes [27].
To the authors knowledge, this was the first report on
the abundance of soil PAH metabolic genes in the oil
exploring region. In this study, the NAH primers were
applied to characterize the density and diversity of naphthalene dioxygenase genes in the oil-contaminated soils in
the Shengli Oil Field. Real-time quantitative PCR assay
showed a spatial variation in the density of naphthalene
dioxygenase genes in soils. Crude oil is a complex mixture composed of mainly n-alkanes, non-hydrocarbon compounds, and various aromatic hydrocarbons including PAH
compounds [24]. Although naphthalene dioxygenase is
known for the biodegradation of naphthalene, it can catalyze the biotransformation of a wide scope of aromatic
hydrocarbons such as benzene, toluene, ethylbenzene, anthracene, phenanthrene, biphenyl, and mono-, di-, tri-, and
tetramethylated naphthalenes [30, 39]. Zhou et al. indicated that naphthalene dioxygenase could be involved in the
degradation of low-molecular-weight PAHs [48]. Moreover, the NAH primers used in this study could target a
variety of naphthalene dioxygenase genes (nahA, pahA,
ndoC, nagA, and dntA) [2, 4]. Therefore, in this study,
the gene abundance showed significant positive correlations with the levels of total organic carbon and aromatic
hydrocarbons, instead of the level of naphthalene. High
density of naphthalene dioxygenase genes usually occurred
at site with high levels of total organic carbon and aromatic hydrocarbons. However, samples SL3 and SL9
showed a large difference in the gene density, regardless
of their similar levels of total organic carbon and aromatic
hydrocarbons. Therefore, other unknown environmental
factors might affect the spatial distribution of gene density
in the oil exploring region.

II, while sample SL5 and sample SL6 shared clusters I and IV.
Therefore, these results illustrated a spatial variation in the
composition of naphthalene dioxygenase genes in the oilcontaminated soils in the Shengli Oil Field.
Most of the recovered naphthalene dioxygenase gene
sequences were distributed in cluster I (Fig. 3). Cluster 1
was composed of 208 gene sequences recovered from the
12 soil samples. These sequences could not be grouped
with any reported functional genes, suggesting the predominance of novel genotypes in the contaminated soils
in the Shengli Oil Field. Cluster II was a 13-member
group. The members of this cluster were from samples
SL1, SL7, SL12, and SL13, and they were related to the
naphthalene dioxygenase genes found in some cultivated
Pseudomonas species. These Pseudomonas species were
isolated from marine sediment (AJ496391), western Mediterranean region (AF306423 and AF306425) [14], and
oil-polluted intertidal sediment (HE798515 and
HE798516) [18]. Cluster III was the smallest group, containing 10 members. The dioxygenase gene sequences in
Cluster III were recovered from samples SL11 and SL13.
They were also grouped with naphthalene dioxygenase genes
and other PAH-RHD genes detected in several cultivated
Pseudomonas species. Three Pseudomonas species were isolated from contaminated soil (KF483152), and ship bilge
wastes in open lagoons (AY196827 and AY196829) [30].
Cluster IV was composed of 12 dioxygenase gene sequences.
The members of this group were from samples SL5, SL6, SL7,
and SL11, and they were related to naphthalene dioxygenase
genes and other aromatic compound-related genes from three
bacterial genera such as Comamonas, Delftia, and
Diaphorobacter. Some of these reported microorganisms were
isolated from industrial waste water treatment plant
(KC691253), and contaminated sediment (AY194931) [19].

Y. Yang et al.

SL1-19 (16)

16
14

SL8-23 (22)

22

SL2-17 (19)
SL6-9 (20)

47

SL7-7 (16)

54

SL3-14 (27)
SL10-4 (11)

31

Cluster I

SL4-20 (20)
41

SL11-12 (18)

14

SL12-13 (16)

95

SL13-15 (6)
SL5-10 (17)

48

SL12-1 (2)
SL13-3 (5)

24

SL7-5 (4)

61

nahAC Pseudomonas stutzeri (AF306425)

34

Cluster II

Nah Pseudomonas sp. (AJ496391)

SL1-7 (2)
64

nahAC Pseudomonas stutzeri (AF306423)

nahAC Pseudomonas monteilii (HE798515)
67

nahAC Pseudomonas xanthomarina (HE798516)

Nah Pseudomonas sp. (HM368649)
69

SL11-1 (2)

99

SL13-8 (8)
nahAC Pseudomonas stutzeri (AY196827)

54

Cluster III

PAH-RHD Pseudomonas sp. (KF483152)

45

SL11-9 (3)

24
23
63

nahAC Pseudomonas stutzeri (AY196829)

SL7-16 (2)
SL5-20 (2)
SL6-12 (5)
nbzR Comamonas sp. (AF379638)

99

nahAc Comamonas testosteroni (AY194931)

59

phnAc Delftia acidovorans (AY367788)

49

0.01

mntR Diaphorobacter sp. (KC691253)

Cluster IV

Distribution of Naphthalene Dioxygenase Genes in Soils

Fig. 3

Phylogenetic tree of representative naphthalene dioxygenase gene

sequences and reference sequences from GenBank. The obtained
sequences beginning with SL1SL8 and SL9SL13 were
referred to the sequences retrieved from corresponding soil sample. The
bold number in parentheses represents the numbers of the sequences in
the same OTU in a given clone library. Numbers at the nodes indicate the
levels of bootstrap support based on neighbor-joining analysis of 1,000
resampled datasets. The bar represents 1 % sequence divergence

Spatial Variation in Gene Diversity in Oil-Polluted Soils

To date, there have been several reports on the diversity of
PAH metabolic genes in natural environments. Vivas et al.
illustrated the greatest PAH-degrading gene diversity in the
soils with the highest levels of hydrocarbons [40]. LloydJones et al. found that genotype distribution of PAH metabolic
genes was not uniform in PAH- or oil-contaminated soils, and
some soils have more genotypes than the other ones [25]. An
uneven diversity distribution of PAH metabolic genes was
also observed in diesel oil-contaminated soils [21] and in
creosote-contaminated soils [4]. The diversity of PAH metabolic genes in chronically oil-contaminated microbial mats
was higher than that in pristine microbial mat [5]. Gomes
et al. reported that nagAc genes, instead of nah and phn genes,
were detected as the dominant naphthalene dioxygenase gene
types in oil-contaminated mangrove sediments [16]. To date,
the environmental parameters governing the diversity of PAH
metabolic genes in natural environments remain essentially
unclear. Only Gomes et al. hypothesized that the long-term
impact of PAH pollution, together with the specific environmental conditions at each site, might affect the abundance and
diversity of naphthalene dioxygenase genes in the environment [16]. Moreover, information on the diversity of soil
PAH-degrading genes in oil exploring areas is still lacking.
In this study, a spatial variation in the diversity of naphthalene
dioxygenase genes was found in the oil-contaminated soils in
the Shengli Oil Field. This was in agreement with the observation by the previous studies on other natural ecosystems. In
addition, Pearsons correlation analysis illustrated that the
diversity of naphthalene dioxygenase genes had a significant
negative correlation with the level of polar aromatics, but not
with the level of naphthalene. Different environmental factors
might shape the spatial distribution of the abundance and
diversity of PAH metabolic genes. Further work will be necessary in order to elucidate the links between environmental
parameters and the spatial distribution of PAH metabolic
genes in natural ecosystems.

with soil pH and salinity in oil exploring areas [24]. The rate
of PAH biodegradation can be influenced by environmental
conditions of pH and salinity [1, 37]. Jimenez et al. found that
salinity could affect the growth of PAH degraders [20]. Wu
et al. suggested that pH was one of the factors affecting the
distribution of PAH-degrading genes in sediments of the Pearl
River estuary [43]. Some microorganisms can function efficiently under saline and alkaline conditions [34]. In addition,
da Silva et al. found that one PAH-degrading dioxygenase
could have high activity under alkaline conditions [11]. In the
Shengli Oil Field, the saline and alkaline soils were heavily
polluted by crude oil (Table 1). The multiple selective stresses
on soil microbial community might induce the enrichment of
specific degraders and PAH-degrading genes that were
adapted to the unique habitats. This might account for the
predominance of novel PAH-degrading gene sequences in the
oil-polluted soils.
Members of genus Pseudomonas are known for the biodegradation of a variety of aromatic hydrocarbons including
PAHs, and a number of Pseudomonas species containing
naphthalene dioxygenase genes or aromatic compoundrelated functional genes have been isolated [2, 18]. In this
study, all of the 23 sequences in clusters II and III were related
to naphthalene dioxygenase genes detected in Pseudomonas
species. Most of the dioxygenase gene sequences from sample
SL13 belonged to these two clusters. Moreover, these reported
Pseudomonas species were usually isolated from saline ecosystems, such as marine sediment, intertidal sediment, seawater, and ship bilge wastes. This suggested a possibly important
contribution of two groups of Pseudomonas-related naphthalene dioxygenase gene sequences to the biodegradation of
aromatic hydrocarbons in the saline soils in the Shengli Oil
Field. Members of genera Comamonas, Delftia, and
Diaphorobacter have also been linked to the biodegradation
of PAHs and other aromatic compounds [6, 7, 9, 21, 23, 29,
32]. In cluster IV, the recovered sequences in this study were
related to a variety of aromatic compound-related functional
genes found in different genera. This suggested that multiple
dioxygenases might transform PAHs. In addition, in this
study, the recovered naphthalene dioxygenase gene sequences
were grouped into four distinct clusters. Therefore, different
types of catabolic genes could contribute to the biodegradation of PAH in the soils in the oil exploring area.

Conclusions
Composition of Naphthalene Dioxygenase Genes
in Oil-Polluted Soils
Soil structure and physicochemical and biological characteristics can be influenced by oil pollution [24]. The number of
heterotrophic bacteria was found to be negatively correlated

Spatial variations in the density and diversity of naphthalene

dioxygenase genes were observed in the oil-contaminated
soils in the Shengli Oil Field. Gene density was positively
correlated with the levels of total organic carbon and aromatic
hydrocarbons, while gene diversity showed a negative

Y. Yang et al.

correlation with the level of polar aromatics. The obtained

gene sequences could be grouped into four distinct clusters,
while the novel PAH-degrading genes predominated in soils.
Acknowledgments This work was financially supported by the Public
Welfare Project of Ministry of Environmental Protection (201309034).

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