International Biodeterioration & Biodegradation 70 (2012) 141e147

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Bioremediation of polycyclic aromatic hydrocarbon-contaminated soil by

a bacterial consortium and associated microbial community changes
Jian Mao a, b, Yongming Luo a, c, *, Ying Teng a, Zhengao Li a
a
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy Sciences, Beijing east road No. 71, Nanjing 210008,
Jiangsu Province, PR China
b
Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy Sciences, Suzhou 215123, Jiangsu Province, PR China
c
Yantai Institute of Coastal Zone Research, Chinese Academy Sciences, Yantai 264003, Shandong Province, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Bioremediation of a PAH-contaminated soil was carried out with a bacterial consortium enriched from
Received 13 September 2011 the soil. The soil contained 9362.1 mg kg
1 of USEPA priority PAHs, 90.6% of which were 4- and 5-ring
Received in revised form PAHs. After incubation for 56 days, 20.2% and 35.8% of total PAHs were removed from the soil with
10 February 2012
the addition of 10% and 20% of a bacterial consortium suspension. The soil microbial population
Accepted 12 March 2012
Available online 6 April 2012
increased in the early days but decreased by the end of the experiment. Denaturing gradient gel elec-
trophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene
fragments revealed that DGGE profiles of the soil with the addition of the consortium were clustered
Keywords:
Bioremediation
together and distinct from those of control soil. Sphingobacteria and Proteobacteria were found to be the
Biodegradation dominant bacterial groups in the soil according to the sequence analysis of DGGE bands. The results
PAHs indicate that incubation with a bacterial consortium may be a promising method for bioremediation of
HMW PAH-contaminated soils.
Bacterial consortium Ó 2012 Elsevier Ltd. All rights reserved.
PCReDGGE
Soil

1. Introduction impact on the environment (Liebeg and Cutright, 1999). Microbial

consortia have been widely used in cleanup of a number of
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in laboratory and field bioremediation studies. It is
contaminants in the environment with potential mutagenicity and generally thought that microbial consortia are more effective than
carcinogenicity. They are generated from natural combustion pure cultures in biodegradation of PAHs (Mueller et al., 1989; Tam
processes as well as from human activities (Luan et al., 2006). Low- et al., 2003). This is possibly because broader enzymatic capacity
molecular-weight PAHs are acutely toxic, with some having effects is achieved (Casellas et al., 1998), and the formation of toxic inter-
on the reproduction and mortality rates of aquatic animals, and mediate metabolites is counteracted by the selection of these dead-
most high-molecular-weight (HMW) PAHs (containing four or end products formed mainly by co-metabolism processes (Vinas
more benzene rings) are mutagenic and carcinogenic (Boonchan et al., 2005a). Bouchez et al. (1999) found that a culture enriched
et al., 2000). Most of the pollutant hydrocarbons in the environ- from soil could readily mineralize a mixture of five PAHs but mixed
ment are often composed of mixtures of numerous homologous cultures of two or three pure strains possessing the capacity to
compounds and bound to particulates in soil and sediments, mineralize each of five PAHs achieved limited degradation of the
restricting their availability for biodegradation. mixture of five PAHs.
Bioremediation is one of the promising technologies to reclaim Although there have been some studies on the bioremediation
PAH-contaminated sites due to its relatively low cost and limited of PAH-contaminated soils with microbial consortia, little is known
about changes in microbial community structure during the
bioremediation process. A few studies that have been described to
* Corresponding author. Key Laboratory of Soil Environment and Pollution date have been performed with soils with single contaminants,
Remediation, Institute of Soil Science, Chinese Academy Sciences, Beijing east road spiked soils or petroleum-contaminated soils (Vinas et al., 2005a).
No. 71, Nanjing 210008, Jiangsu Province, PR China. Tel.: þ86 25 86881530;
The objectives of the present study were to enrich an efficient PAH-
fax: þ86 25 86881128.
E-mail address: ymluo@yic.ac.cn (Y. Luo). degrading bacterial consortium, to investigate the bioremediation

0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2012.03.002
142 J. Mao et al. / International Biodeterioration & Biodegradation 70 (2012) 141e147

capability of the consortium in PAH-contaminated soil, and to 2.5. Microbial analysis

analyze the changes in the microbial community during the
bioremediation process using denaturing gradient gel electropho- Heterotrophic microbial population counts in soil were deter-
resis (DGGE). mined aerobically on days 0, 14, 28, and 56 using the most probable
number (MPN) method as described previously (Taylor, 1962).
2. Materials and methods Total community DNA was extracted from 0.5 g soil using the
FastDNA Spin Kit for Soil (Q-BIOgen, Irvine, CA) according to the
2.1. Soil sampling and soil PAH extracts manufacturer’s instructions. The universal bacterial primers tar-
geting the variable V3 region of the bacterial 16S rRNA gene, F338gc
Soil contaminated by PAHs was collected from the surface layer and R518 were used to amplify fragments. The GC-clamp was added
(0e15 cm) of an industrial area in WuXi, Jiangsu Province, China. to the F338gc primer (Maarit Niemi et al., 2001). The PCR condi-
The soil with pH 6.4 contains organic matter 19.2 g kg
1, total tions have been described previously (Wu et al., 2008a).
nitrogen 1.0 g kg
1, total phosphorus 0.5 g kg
1, total potassium DGGE was performed on a DCode Universal Mutation Detection
14.2 g kg
1, and CEC 21.5 cmol kg
1. Soil was air-dried, homoge- System (Bio-Rad Laboratories, Hercules, CA). PCR products were all
nized, passed through a 2-mm sieve, and stored at 4  C in the dark separated in a 6% acrylamide gels with a denaturing gradient from
prior to use. 30% to 60% (100% denaturant corresponds to 7 M urea and 40%
Ten grams of soil were Soxhlet-extracted with dichloromethane formamide). Gels were running in 1x TAE buffer at 60  C and 80 V
for 24 h, rotary evaporated to about 2 ml, and purified by a silica gel for 13 h, and stained for 30 min in 1x TAE containing SYBR Green I
column (8 mm  220 mm). After evaporation to dryness under N2 (Roche, Mannheim, Germany), documented by Gel Doc EQ gel
streams, the extract was dissolved with 2 ml of N, N-dime- documentation system (Bio-Rad Laboratories, Hercules, CA).
thylformamide (DMF). About 200 ml of soil PAH extracts was DGGE images were digitized and processed by Gelcompar II
obtained in this way. package (Applied Maths, Inc., Austin, TX) with default values. After
normalization, bands with relative peak area intensities were
included in further analysis. The similarity in the profiles of bands
2.2. The PAH-degrading consortium
was calculated with Gelcompar II to construct a dendrogram using
the unweighted pair group method (UPGMA) based on Pearson’s
A PAH-degrading bacterial consortium was obtained from the
similarity coefficient.
PAH-contaminated soil. Enrichment cultures were established in
Predominant DGGE bands were excised and reamplified as
500-mL screw-cap Erlenmeyer flasks containing 100 mL sterile
described by Wilms et al. (2006). For purification, a second DGGE
mineral salts medium (MSM) (Wang et al., 2005) and 5 mL soil PAH
was run and bands with identical position to parent bands were
extracts. The consortium was maintained by fortnightly transfers
excised, amplified again with primer pairs without GC-clamp.
into fresh medium (20-fold dilution) for 6 months incubated at
Sequencing of PCR products was carried out by Invitrogen
28  C on a rotary shaker (200 rpm) in the dark. It was revealed by
(Shanghai, China). DNA sequences were compared to those in the
PCReDGGE analysis that the most abundant populations of the
GenBank. DGGE band sequences from this study have been
consortium were related closely to Mesorhizobium sp., Alcaligenes
deposited in the NCBI GenBank database under Accession Number
sp. and Bacillus sp. The bacterial consortium have the capability of
EU401879eEU401892. The 16S rDNA sequences were aligned using
degrading most of the 16 USEPA listed PAHs in sterile mineral salts
Clustal X software, and the phylogenetic tree was generated using
medium, especially 4- and 5-rings PAHs.
the neighbor-joining algorithms in Mega 4 software.

2.3. Soil microcosms

2.6. Statistical analysis
Microcosms were set up to investigate the capability of the
All the data obtained were subjected to one-way analysis of
consortium to bioremediate the PAH-contaminated soil. The
variance and post hoc Tukey test using the SPSS Version 13.0 statis-
consortium cultures were centrifuged at 4000 rpm for 20 min,
tical package. The results were tested for significance at the 5% level.
washed twice with 100 mM of potassium phosphate buffer (pH 7.0),
and suspended in MSM (2.1  107 cells ml
1). 1 kg of non-sterile soil
was mixed thoroughly with 10% (v/m) (Treatment A) and 20% (v/m) 3. Results
(Treatment B) of the consortium suspension. Sterile medium
without inoculation with the consortium suspension served as 3.1. PAH concentrations and composition in contaminated soil
control. Three replicates of each treatment were set up. Soil water
content was adjusted to 60% of water holding capacity. All treat- The concentration and composition of PAHs in the contaminated
ments were incubated at 28  C in dark. After incubation for 0, 14, 28, soil and PAH extracts used in this study are shown in Table 1. The
and 56 days, soil was sampled for chemical and biological analysis. total concentration of 10 individual PAHs in the soil was
9362.1 mg kg
1 dry soil. Fluoranthene was present at the highest
2.4. Analysis of PAHs concentration among all the PAHs. The concentration of HMW-
PAHs (4- and 5-rings) accounted for 90.6% of all the PAHs.
Extraction and determination of 16 EPA priority PAHs was
carried out according to the method of Ping et al. (2007). Analysis 3.2. Changes in concentration of PAHs in the soil during
was conducted on a Shimadzu Class-VP HPLC system (Shimadzu, bioremediation
Japan), with a fluorescence detector (RF-10AXL). A reversed phase
column C18 (VP-ODS 150  4.6 mm I. D., particle size 5 mm), using Concentrations of individual and total PAHs in the soil under
a mobile phase of water and acetonitrile mixture (4:6, v/v) at different treatments during the 56-day bioremediation are pre-
a constant solvent flow rate of 0.5 ml min
1, was used to separate sented in Fig. 1. By the end of the experiment, 7.1% of total PAH
PAHs. The excitation and emission wavelengths for individual PAHs removal was detected in the control soil. Significant differences
were set separately (Wu et al., 2008b). (P < 0.05) were observed in treatment A (10% consortium) and
 J. Mao et al. / International Biodeterioration & Biodegradation 70 (2012) 141e147 143

Table 1
Concentration and composition of PAHs in the contaminated soil and removal of individual PAHs under different treatments after 56 days of bioremediation.

PAH Number of rings Concentration (mg kg
1 soil) PAH removal (%)

Control Treatment A Treatment B

Fluorene 3 36.4  8.0 15.8  5.4a 17.0  1.5a 24.3  4.6a
Phenanthrene 3 751.0  108.9 17.4  1.0a 19.7  3.6a 37.3  2.7b
Anthracene 3 88.8  23.2 5.5  1.3a 13.4  2.9b 24.1  4.5c
Fluoranthene 4 2024.6  193.4 2.8  0.7a 20.5  3.2b 34.5  2.4c
Pyrene 4 1785.4  165.0 5.1  0.5a 22.5  2.5b 27.9  3.1b
Benz[a]anthracene 4 791.0  88.5 0.7  0.7a 16.4  2.0b 30.6  3.2c
Chrysene 4 994.2  136.7 15.3  4.2a 27.6  3.3b 41.2  3.8c
Benzo[b]fluoranthene 5 1223.9  155.4 1.7  0.9a 11.8  1.7b 44.2  5.8c
Benzo[k]fluoranthene 5 476.2  57.8 7.0  1.9a 14.3  3.5b 40.7  2.7c
Benzo[a]pyrene 5 1190.8  141.8 13.6  1.9a 24.3  2.7b 36.6  3.2c
Total PAHs 9362.1  1078.5 7.1  1.5a 20.2  2.1b 35.8  3.2c

Data are presented as means  SD. Means in the same row with a letter in common are not significantly different among treatments (P < 0.05).

treatment B (20% consortium), which lost 20.2% and 35.8% of total when the consortium suspension was added in treatments A and B at
PAHs respectively. day 0, and the highest microbial population (9.7  107 MPN g
1 soil)
The extents of degradation of individual PAHs are also shown in was detected in treatment B. However, the size of soil microbial
Table 1. In treatment A the highest PAH removal occurred in population soon decreased. After 56 days the microbial population
chrysene (27.6%), benzo[a]pyrene (24.3%) and pyrene (22.5%). In maintained in treatment B was only 1.3  107 MPN g
1 but this was
treatment B the highest degree of degradation was found in benzo still higher than that of other two treatments.
[b]fluoranthene (44.2%), chrysene (41.2%) and benzo[k]fluo-
ranthene (40.7%). In treatment B removal of benzo[b]fluoranthene,
3.4. Changes in soil bacterial community structure during the
benzo[k]fluoranthene, and benzo[a]pyrene, fluoranthene, and benz
bioremediation process
[a]anthracene were significantly different (P < 0.05) from that of
control soil and treatment A.
A DGGE analysis of PCR-amplified 16S rRNA gene fragments was
Over the 56 days the degree of degradation of 4-ring PAHs
conducted to investigate differences in the bacterial populations
(21.8%) was higher than that of 3- and 5-ring PAHs (18.7% and 17.3%)
present in soil with different treatments. Bacterial community
in treatment A. In treatment B the removal of 5-ring PAHs was the
profiles elucidated by DGGE of different soils collected at day 0 and
highest (40.5%) and the losses of 3- and 4-ring PAHs were similar to
day 56 are shown in Fig. 3. Three independent replicates per
one another (35.2% and 33.2%).
treatment and per sampling time were analyzed for DNA extrac-
tion, and there were no differences between them in DGGE profiles.
3.3. Enumeration of the heterotrophic microbial population in soil At day 0, after the addition of the consortium suspension, the
bacterial community profiles of the soils exhibited changes in
An MPN method was employed to evaluate the heterotrophic contrast to the control soil. It also can be seen that there were more
microbial population in the soil during the bioremediation process bands with deeper intensity in the soil sample of treatment B. After
and the results are shown in Fig. 2. In control soil the size of the
heterotrophic population remained constant until day 56. In contrast
to the control soil, much higher microbial counts were observed

Treatment B
Treatment A
Control
-1
MPN

0 14 28 56
Time (days)
Fig. 1. Concentration of total PAHs in soil under different treatments over the 56-day
bioremediation process (mg kg
1 soil). Each point represents the average value of Fig. 2. Heterotrophic microbial populations in the soil of treatment A, treatment B, and
triplicate samples. control soil at days 0, 14, 28 and 56 over the course of the 56-day bioremediation.
144 J. Mao et al. / International Biodeterioration & Biodegradation 70 (2012) 141e147

% Similarity
40 60 80 100

B- 1
100

67
B- 3

76
B- 2

A- 2
91
A- 3
86 C- 1
90
C- 2
88 100
C- 3

A- 4
100
A- 6
93
A- 5
87
85 B- 4
Fig. 3. DGGE profiles of bacterial community composition in soils of treatment A (lane 99
A-1 to A-6), treatment B (lane B-1 to B-6), and control (lane C-1 to C-6) at day 0 and B- 5
day 56. The bands marked with arrows were excised and sequenced (Table 2). 100

89
B- 6
56 days of incubation, in the lanes of the different treatments,
a significant shift in community structure was noted by the pres-
C- 4
100
ence and absence of some bands.
The dendrogram of the UPGMA analysis of the DGGE patterns
C- 5
for the soil with different treatments is presented in Fig. 4. This
C- 6
shows that the soil samples at different days (days 0 and 56) are 100
clustered into two main groups. Furthermore, the DGGE profiles of A- 1
treatment A and treatment B were clearly different from the control
soil at both day 0 and day 56. Fig. 4. Cluster analysis of community composition for different soil samples.
Prominent bands were excised, PCR-amplified, and sequenced
(indicated by arrows in Fig. 3). The closest relatives of DGGE bands
degraders. In previous studies pure PAH compounds (Boonchan
are shown in Table 2 and most of the sequences exhibited levels of
et al., 2000; Jacques et al., 2007) or creosotes (Mueller et al.,
similarity greater than 95%. Bands 3, 9, 12, 13, and 14 appeared at the
1989; Vinas et al., 2005a) were most commonly used to enrich
same positions in all treatments, which was the most abundant band
PAH degraders. In our study a bacterial consortium was enriched
in the soil, were detected as Sphingobacteria. Several of the domi-
from the soil with the PAH extracts as an energy source, which
nant bands observed in the soil of day 0, such as band 2 (Pedobacter
contained 28779.2 mg l
1 of total PAHs. The PAH extracts might
sp.), band 5 (uncultured Sphingobacteriales), band 6 (Bacillus sp.),
contain other organic matters besides PAHs, but their composition
band 7 (uncultured Sphingobacteriales), and band 8 (Paenibacillus
was more similar to the soil than simple addition of pure PAHs. The
sp.) were not present or present at relatively reduced levels on day
consortium cannot utilize DMF (data not shown), so this method is
56. On the other hand, band 11 (Uncultured Proteobacterium) was
feasible for the enrichment of PAH-degraders.
a new dominant band which appeared at day 56 in treatment B.
Microbial consortia have been used in several studies to reme-
A phylogenetic tree was constructed to show the relationship of
diate PAH-contaminated soils. Ruberto et al. (2006) found that
all the partial 16S rDNA sequences representing the respective
a combination of bioaugmentation with a consortium and bio-
excised DGGE bands (Fig. 5). The neighbor-joining analysis revealed
stimulation with organic nutrients caused a significant removal
that most of 14 the bacterial sequences were affiliated with
(46.6%) of phenanthrene after 56 days of incubation in Antarctic
Sphingobacteria (9 sequences, 64.3%), especially uncultured
soil. Vinas et al. (2005b) found that in a bioremediation of heavily
Sphingobacteriales (7 sequences, 50%). The remaining sequences
creosote-contaminated soil for 200 days, PAHs were significantly
were primarily related to Proteobacteria (3 sequences, 21.4%) and
degraded (83e87%).
Firmicutes (2 sequences, 14.2%).
Compared to previous studies, our study showed that after
bioremediation for 56 days, 20.2% and 35.8% of total PAH removal
4. Discussion was detected in the soil with addition of 10% and 20% consortium.
Observed losses of PAHs were probably the result of both primary
As far as microbial biodegradation is concerned, the most utilization and co-metabolism (Mueller et al., 1989). This indicates
important problem is the method for enrichment or isolation of the that the consortium can survive in non-native soil and compete
 J. Mao et al. / International Biodeterioration & Biodegradation 70 (2012) 141e147 145

Table 2
Sequence analysis of the major DGGE bands appearing in Fig. 3.

Band Accession no. Closest organisms in GenBank database (Accession no.) Phylum or class Similarity (%)
1 EU401890 Pedobacter sp. MSCB-14 (EF103211) Sphingobacteria 100
2 EU401885 Pedobacter sp. hp16 (JN637320) Sphingobacteria 100
3 EU401886 Uncultured Sphingobacteriales bacterium clone CCF88 (JN541184) Sphingobacteria 100
4 EU401880 Uncultured proteobacterium clone GASP-WC2W2-B05 (EF075246) Proteobacteria 96
5 EU401881 Uncultured Sphingobacteriales bacterium clone 366 (FJ645651) Sphingobacteria 100
6 EU401882 Bacillus sp. JC46 (FN430771) Firmicutes 96
7 EU401884 Uncultured Sphingobacteriales bacterium clone CG77 (JN541173) Proteobacteria 99
8 EU401883 Paenibacillus sp. MP7 (AJ582394) Firmicutes 93
9 EU401879 Uncultured Sphingobacteriales bacterium clone DMS07 (FJ536877) Sphingobacteria 99
10 EU401887 Uncultured proteobacterium clone GASP-KC1W1_F10 (EU299390) Proteobacteria 98
11 EU401888 Uncultured Proteobacterium clone GASP-MA1W2_A08 (EF662734) Proteobacteria 100
12 EU401891 Uncultured Sphingobacteriales bacterium clone DMS05 (FJ536875) Sphingobacteria 99
13 EU401889 Uncultured Sphingobacteria bacterium clone BF-48 (HM238164) Sphingobacteria 100
14 EU401892 Uncultured Sphingobacteriales bacterium clone DMS11 (FJ536881) Sphingobacteria 100

with indigenous microbial populations. It was also shown that the conditions. In the bioremediation of an aged PAH-contaminated
bioremediation efficiency was in proportion to the biomass of the soil by a microbial consortium, Li et al. (2009) observed that the
consortium added to the soil. There was 7.1% of total PAH loss biodegradation of 5e6 rings PAHs was significantly higher than
observed in the control soil, which may have been because the soil that of 2e4 rings PAHs. In our study the degradation of 3-ring PAHs
was non-sterile, and the addition of mineral salts medium stimu- was less than that of 4- or 5-ring PAHs, perhaps due to the bacterial
lated the biodegradation capability of indigenous microorganisms consortium being enriched from a soil containing more than 90% of
in the soil (Li et al., 2008). HMW-PAHs, and it had a greater capability to degrade HMW-PAHs.
It is generally accepted that the biodegradation of low- Bioremediation of PAH-contaminated soil with specific isolates
molecular-weight PAHs occurs much more rapidly and exten- of bacteria or fungi has been monitored in other studies showing
sively than that of HMW-PAHs (Nam et al., 2001). However, high opposite results. Some successful examples have shown signifi-
degradation of the latter would have been obtained in some cantly enhanced degradation of HMW-PAHs (Kotterman et al.,

Fig. 5. Phylogenetic tree based on 16S rDNA-V3 sequences representing the respective DGGE bands in Fig. 3. Bootstrap analysis is based on 500 replicates. Scale indicates 5%
sequence divergence.
146 J. Mao et al. / International Biodeterioration & Biodegradation 70 (2012) 141e147

1998; Boonchan et al., 2000), in a similar fashion to in our experi- Yucheng Wu of Nanjing Institute of Geography and Limnology,
ment. These results were especially important for HMW-PAHs and Chinese Academy of Sciences, for his helpful discussions. We also
could be promising for bioremediation of PAH-contaminated soils. thank Dr. Peter Christie of Queen’s University Belfast, School of
Monitoring the microbial community by DGGE analysis Biology and Biochemistry, Belfast, UK, for language revision of this
provided an effective way of visualizing the microbial changes manuscript.
occurring during the bioremediation process. In the present study
the DGGE analysis showed that the incubation of the consortium
had an impact on the microbial community structure of the soil.
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