Journal of Environmental Biotechnology
Vol. 13, No. 2, 151–159, 2013

Original paper (regular paper)

Comprehensive Analysis of Aerobic Benzene-Degrading Microorganisms
Using Modified and Conventional Stable-Isotope Probing Methods
Yuanbai Pan, Nobuhiko Nomura, Toshiaki Nakajima and Hiroo Uchiyama*
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan* TEL: +81–29–853–4927 FAX: +81–29–853–4927* E-mail: uchiyama.hiroo.fw@u.tsukuba.ac.jp(Received; 2 November, 2013/Accepted; 24 November, 2013)
The contamination of groundwater and soil by benzene is a serious environmental problem worldwide, and cleaning
polluted areas by bioremediation requires an understanding of the microbial community involved. To archive this purpose,
benzene-assimilating degraders were classified in the bacterial community using conventional stable-isotope probing (SIP),
whereas candidate co-metabolic degraders and non-degraders of benzene were classified with modified SIP (SIP-D) and 16S
ribosomal RNA-targeted SIP. Conventional plating method was also accompanied. Consequently, bacteria belonging Rhodococcus and Pseudomonas were mainly identified as benzene-assimilating degraders, one type of Pseudomonas was a candidate co-metabolic degrader, and Hydrogenophaga was a non-degrader, respectively. Some strains of the candidate cometabolic degraders and non-degraders of benzene that exhibited no benzene-assimilating capability displayed an active
benzene-assimilating capability, which was confirmed with the culture-dependent method. Furthermore, functional gene
analysis suggested that most benzene-assimilating degraders opened the benzene ring via a catechol ortho-cleavage
pathway. We concluded that a comprehensive analysis using a combination of the two SIP methods would be effective forunderstanding the wide bacterial community in a chemically polluted environment.
Key words: bioremediation, benzene, stable-isotope probing, bacteria community, functional genes

1. Introduction
Petroleum pollution has become a serious environmental
problem because it affects groundwater resources andcauses soil deterioration. Benzene, the main mono-aromaticcomponent in gasoline, is widely produced and used in many
applications 2). However, its volatilization and carcinogenicity
negatively influence human health 35). Bioremediation has
recently been used as a low-cost and eco-friendly method
to degrade benzene 16). The reaction-effective microorganism
community must be defined and controlled in bioremediation treatments. Head et al. 12) proposed a degradation
network of oil in marine environments, consisting of anassimilating degrader, co-metabolic degrader 14), and nondegrader. A similar microbial community is also thought
to exist and operate in soil environments. Many kinds ofbenzene-degrading bacteria have been described to date;iron-reducing 1),
nitrate-reducing 6),
sulfate-reducing 17),34)
perchlorate-reducing , and methanogenic bacteria 9) asanaerobes, and various assimilating bacteria in aerobic environments 19,37). However, these previous studies mainly focusedon assimilating degraders, and did not fully determine thebacterial community involved in benzene degradation. In thisstudy, we identified an assimilating degrader, co-metabolic
degrader, and non-degrader.

Culture-independent approaches have recently been developed to observe microbial communities and their functions.
Stable-Isotope Probing (SIP) is a particularly powerful
method used to identify assimilating degraders. This method
is based on the incorporation of stable isotopes (e.g., 13C)
into cellular components 25). SIP has been used to examine
the aerobic metabolism of methane 22), benzene 37), toluene 32),
and many other chemical compounds. However, this conventional 13C-labeling method can only detect assimilating
degraders 5). Therefore, we developed a modified SIP method
to detect dissimilating bacteria (SIP-D), and this has been
recommended for the biological dechlorination reaction 38).
Here, tetrachloroethylene (PCE) dehalorespiring bacteria
could gain energy via dehalorespiration in the presence ofPCE, and grew with the assimilation of a 13C-labeled carbon
source, which formed 13C-labeled DNA. However, dehalorespiring bacteria could neither grow nor assimilate carbon
in the absence of PCE. This physiological difference could
be investigated with polymerase chain reaction-denaturing
gradient gel electrophoresis (PCR-DGGE); differences
between specific 13C-DNA bands in the presence or absenceof a pollutant. In our previous study, we successfully identified Dehalococcoides ethenogenes 195 as a dehalorespiring
bacterium by SIP-D, subsequently applied this method to an
artificially PCE-contaminated microcosm 38), and identified

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2. Materials and Methods
2.1 Benzene degradation assay and product measurement

Fig. 1. Details of the analytical methods used in this study. Stableisotope probing (SIP) and modified SIP for dissimilating bacteria (SIP-D) are culture-independent methods (A) for identifying benzene-assimilating bacteria and co-metabolic bacteria,
respectively. [13C] Benzene and [13C] acetate were used to label
DNA, respectively. The non-degraders of benzene were also
identified in SIP-D. Therefore, RNA-based SIP was used toconfirm candidate degraders in the presence of benzene using[13C] acetate. These bacteria were identified in PCR-DGGE
analysis. Degraders were also isolated from contaminated soil(B) for comparison with the results obtained by the cultureindependent method. Functional gene analysis of the C12O
and C23O genes using real-time PCR revealed their behavior
during degradation (C).

the indigenous active dehalorespiring bacteria Dehalobacter
sp. as a candidate dissimilative PCE-degrading bacteria.
The general objective of this study was to comprehensively analyze the bacterial community involved in degrading
benzene under aerobic conditions. As shown in Fig. 1A, we
performed SIP to investigate benzene-assimilating degraders
using 13C6-labeled benzene, and SIP-D to investigate candidate co-metabolic benzene degraders using 13C2-labeledacetate. Following SIP-D, to distinguish non-degraders ofbenzene that would also be identified with SIP-D, 16S ribosomal (rRNA) gene-targeted SIP was performed. In thisstudy of SIP-D, benzene, a pollutant that can be assimilated,
was used in SIP-D instead of non-assimilative PCE, and theeffectiveness of SIP-D for assimilative chemical pollutants
was evaluated for the first time.
Accompanied by the culture-independent method, theconventional culture method was performed to investigate
whether the benzene degraders identified by the cultureindependent method were culturable or not (Fig. 1B).
Furthermore, benzene degradation begins with the addition of [OH-] to the benzene ring and proceeds via thecatechol pathway under aerobic conditions 8). Thus, the key
ring-opening functional genes, catechol 1,2-dioxygenase(C12O) 11) gene and catechol 2,3-dioxygenase (C23O) 21) gene,
were quantified using real-time PCR (qPCR; Fig. 1C).

To assess benzene removal from soil by bacteria, we usedbenzene-contaminated soil (sandy loam) collected from arefinery site, in which benzene concentration was approximately 500 μM. Benzene degradation test was performed
with 5 g soil and 35 mL minimal medium containing(per liter) 0.5 g K2HPO4, 0.5 g KH2PO4, 2 g NaCl, 0.5 g(NH4)2SO4, 0.5 g MgSO4·7H2O, 0.1 g FeSO4·7H2O, and100 mL tap water, pH 7.0, with 562.4 μM benzene as thesole carbon source in sealed glass bottles without degassing
at 30°C in darkness without shaking. Negative slurry controls
were prepared using the same conditions via autoclaving for
20 min at 120°C prior to the addition of benzene. Benzene
was measured using a flame ionization detector on a gas
chromatography system (GC-2010, Shimadzu, Japan) with
100 μL headspace gas from a bottle 26). The column was the
fused-silica capillary column DB-1 (30 m long, 0.25-mm
internal diameter, 0.25-mm film thickness; J & W ScientificCo., USA). The injector, oven, and detector temperatures
were 270°C, 100°C, and 250°C, respectively. The split:splitlessratio was 1 : 15. Nitrogen (99.99% chemical purity, 0.55 mL/min) was used as the carrier gas (detection limit of benzene,
3 μM). The liquid phase and total value of benzene in thebottle were calculated according to Henry’s law. The concentration of catechol, the intermediate product in aerobic
benzene degradation, was measured with high-performanceliquid chromatography (Waters Inc., USA) using a Symmetry
C18 column (3.5 μm, 2.1×150 mm) with 30 mM NaH2PO4
and methanol (20 : 80, v/v) as the mobile phase (0.2 mL/min) and a 239-nm fluorescence detector. All incubations
were performed in triplicate.
2.2 DNA extraction, ultracentrifugation, and fractionation
Slurry samples for culture-independent analysis werecollected using a syringe at appropriate intervals and analyzed.
The collected samples (5 mL) were centrifuged (10,000×g)
into Lysing Matrix E tubes (MP Biomedicals, Santa Ana,
CA, USA). After the samples were centrifuged and the liquid
phase was removed, DNAs were extracted directly from eachprecipitation using a FASTDNA® Spin Kit for Soil (MPBiomedicals), loaded onto CsCl gradients in centrifuge tubes(2 mL, Quick-Seal®, Polyallomer, 11×32 mm; Beckman
Coulter, Inc., USA) with CsCl gradient buffer containing
0.8 g CsCl, 775 μL TE (Tris ethylenediaminetetraacetic acid
[EDTA]) and 25 μL ethidium bromide (10 mg/mL), andcentrifuged at 354,100×g (20°C) for 20 h in a TLA 120.2
rotor (OptimaTM; Beckman Coulter). 13C-Labeled heavy
DNA and unlabeled light DNA were detected with 365-nm
ultraviolet (UV) after ultracentrifugation, and the separated
fractions (100 μL) were directly collected using a 1-mL
syringe. Ethidium bromide was removed from the samples
using TE-saturated 3-methyl-1-butanol. DNAs were purified
via ethanol precipitation, re-suspended in TE buffer, andDegrader community and functional gene analysis

stored at –30°C until use.
2.3 Stable-isotope probing (SIP)
SIP was performed to identify benzene-assimilating
degraders in benzene-contaminated soil. Samples weretreated with 562.4 μM 13C6-labeled benzene (99% chemical
purity; Cambridge Isotope Laboratories, Inc., USA) as alabeled carbon source at 30°C in darkness without shaking.
Total DNA was extracted on day 2 (first degradation phase)and day 4 (second degradation phase) and fractionated via
ultracentrifugation as described above.

153

Table 1. Primers used in this study
PrimerSequence (5’–3’)

Reference

357F

CCTACGGGAGGCAGCAG

29

GC-357F

GC clamp-CCTACGGGAGGCAGCAG

29

518R

GTATTACCGCGGCTGCTGG

29

C12O-FGCGACCGTCGAHCTSTGGCA

10

C12O-RTCGCCCTCGAWGTWGAKCTG

10

C23O-FCACGTCTCGTTCDTYCTGGA

10

C23O-R

CCGACGGGTCRAAGAARTA

10GC clamp (5’-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3’)

2.4 Stable-isotope probing for dissimilation (SIP-D)
To identify candidate co-metabolic benzene degraders, we
treated the slurry samples described above with 562.4 μM
12
C6 benzene (99% chemical purity; Wako, Inc., Japan) insealed glass bottles incubated at 30°C in darkness without
shaking. Two time points (days 2 and 4) were selected forthe addition of the labeling marker: 873.4 μM [13C2] acetate(99% chemical purity; Cambridge Isotope Laboratories). As
the most abundant intermediate of organic matter degradation in anoxic soil 13), [13C] acetate was used to identify
the candidate co-metabolic benzene degraders involved inbenzene degradation. On day 2 (first phase), [13C] acetate
was added to make benzene-present (WITH-B) samples.
Total DNA was extracted from these samples after 24 h andfractionated via ultracentrifugation as described above. To
prepare day 4 DNA sample of WITH-B, another degradation assay was conducted for 4 days and the same process
was performed on day 4 (second phase) under the same
conditions. Benzene-absent (WITHOUT-B) samples addedwith [13C] acetate were also made as controls for each phase.
2.5 RNA extraction, ultracentrifugation, and fractionation
To distinguish the candidate degraders of benzene fromthe non-degraders, we extracted total RNA from WITH-B
and WITHOUT-B samples after 18 h using Lysing Matrix
E (MP Biomedicals) and an RNeasy mini kit (Qiagen,Germany) on day 4. The quantity of RNA was determinedusing a Nanodrop spectrophotometer (ND-1000; Thermo
Scientific, Inc., USA) and fractionated via equilibrium density
gradient centrifugation at 160,000×g (20°C) for 36 h in thesame rotor described above with 2 g/mL cesium trifluoroacetate, 375 μL diethylpyrocarbonate-treated water, and 75 μL
deionized formamide as described elsewhere 15). Gradientswere fractionated from the bottom into 200-μL fractions
using a glass capillary liquid pump (0.2 mL min–1; Perista,
ATTO Corporation, Tokyo, Japan) and RNAs were purified
via 2-propyl alcohol precipitation. RNAs were reverse transcribed into complementary DNAs (cDNAs) with hexamer
primers using a Superscript Kit III (Invitrogen) according to
the manufacturer’s protocol.

2.6 PCR-denaturing gradient gel electrophoresis (PCRDGGE)
PCR-DGGE analysis was performed with 13C-labeled
DNAs and cDNAs as templates. The V3 regions of bacterial 16S rRNA genes were amplified using the PCR primers
GC-357F and 518R (Table 1). Amplification was performed
with the touchdown protocol, which comprised the following
steps: 94°C for 4 min; 8 cycles of 94°C for 1 min, 65°C for
15 s, 72°C for 15 s, and 20 cycles of 94°C for 1 min; 55°C for
15 s, 72°C for 15 s, with a decrease of 0.5°C per cycle, andextension at 72°C for 3 min, as described elsewhere 20,27). DNA
fragments of the expected size (0.23 kbp) were confirmedwith electrophoresis on 2% (wt/vol) agarose gel (Agarose
S; Nippon Gene, Japan) in Tris-acetate-EDTA buffer. Products were confirmed and purified using an Ultraclean PCRclean-up DNA purification kit (Mo Bio Laboratories, Inc.,
Carlsbad, CA, USA). DGGE was performed with a DCodeinstrument (Bio-Rad Laboratories, Hercules, CA, USA) witha gel acrylamide concentration of 8% and denaturing gradientof 30%–60% run at 36 V for 18 h. The gel was subsequently
stained with SYBR Gold (Molecular Probes, Eugene, OR)
and imaged using a UV transilluminator (AE-691; ATTO
Corporation, Japan). To verify the reproducibility of themethod, we performed 3 independent PCR-DGGE analyses
for each sample following a procedure described by Muyzer
et al. 23).
2.7 16S rRNA gene sequencing and phylogenetic analysis
Unique DGGE bands were excised from the gel under UV
illumination, placed in 50 μL TE buffer, and incubated overnight at 4°C. After centrifugation, the supernatant (0.5 μL)
was used as the template DNA for 16S rRNA gene amplification with the primers 357F and 518R (Table 1). The productwas subsequently purified and used for sequence analysis. The
nucleotide sequences of the DGGE bands were determinedwith the 357F and 518R primers using a BigDye® Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied
Biosystems, Tokyo, Japan). The reaction and purification
were performed according to the manufacturer’s protocol,and the products were analyzed on a 3130 Genetic Analyzer(Applied Biosystems). Sequence homology searches wereconducted using the GenBank nucleotide sequence library154

Pan et al.

and Basic Local Alignment Search Tool program throughthe DNA Data Bank of Japan website (http://blast.ddbj.
nig.ac.jp/ [accessed 31 October 2010]). Sequence data wereanalyzed with DNA Sequencing Analysis Software 4Peak(Version 1.7.2 for Mac). A phylogenetic tree was constructedwith the evolutionary distance data by the neighbor-joining
method 28) using CLUSTALX ver. 1.83 33).

mode. LightCycler software version 3.5 (Roche Diagnostics)
was used to generate the calibration curve and determine
copy numbers. The relative abundance of functional genes
was calculated by comparing the proportions of functional
genes and 16S rRNA gene in each fraction.

2.8 Colony isolation and single-strain benzene degradation
assay

3.1 Details of the study strategy

Benzene-contaminated soil was suspended in distilledwater and allowed to stand for 10 min without shaking forthe culture-dependent method. The supernatant was spread
onto 1.5% Luria-Bertani (LB) agar plates containing 1 μL/
ml of benzene, incubated at 30°C, and grown aerobicallyfor 7 days. To isolate many types of benzene degraders (e.g.
assimilating degraders, co-metabolic degraders), LB plate wasused. Single colonies were isolated when colonies appeared.
Genomic DNA was extracted using the alkali-sodium dodecyl
sulfate method, and 16S rRNA gene V3 fragments were
amplified using the described GC-357F and 518R primers.
Bacteria cells were simultaneously harvested after 18 h incubation in LB and washed with buffer (50 mM Tris-HCl and
150 mM NaCl, pH 7.5). A benzene degradation assay was
performed in sealed 5-mL vials with 5 mL minimal medium
and a density of 107 cells/mL. Degradation was initiated by
the addition of 562.4 μM benzene as the sole carbon source
at 30°C without shaking. The concentration of benzene wasdetermined using the described gas chromatography method.
2.9 Presence of C12O and C23O genes in enumeration
SIP fractions
To evaluate the behavior of the main functional genes, theC12O and C23O genes were quantified from separated 12C
and 13C gradient fractions (2 μL per fraction as the template)at the 2 phases of degradation described above. The C12O
gene was amplified using a protocol consisting of an initialdenaturation (94°C, 10 min), 40 cycles of amplification(94°C, 1 min, 60°C, 30 s, 72°C, 20 s), acquisition (92°C), anda final extension (72°C, 5 min) with the C12O-F and C12O-R
primers (Table 1). The C23O gene was amplified using the
C23O-F and C23O-R primers (Table 1) with the following
thermal conditions: initial denaturation (94°C, 10 min), 40
cycles of amplification (94°C, 1 min, 60°C, 30 s, 72°C, 5 s),
and a final extension (72°C, 5 min). To detect a wide rangeof oil-degrading bacteria, we designed the degenerate primers
with 35 sequences of the C12O gene and 137 sequences of
the C23O gene, as described by Hara et al. 10). The V3 regionof the bacterial 16S rRNA gene was also quantified using
the primers 357F and 518R (Table 1) under the conditionsof initial denaturation (94°C, 5 min), 40 cycles of amplification (94°C, 30 min, 55°C, 30 s, 72°C, 15 s), and a terminal
extension (72°C, 5 min) using the LightCycler FastStart
DNA MasterPLUS SYBER Green I kit (Roche Diagnostics,
Mannheim, Germany). Melting curves were constructed from
65°C to 95°C with the recommended setting continuous

3. Results
We used 2 SIP methods to investigate benzene-degrading
bacteria (Fig. 1A). SIP and SIP-D were performed todetect assimilation degraders and candidate co-metabolic
degraders, respectively. However, as SIP-D was suggested toalso detect the non-degraders of benzene, 16S rRNA-targeted
SIP was performed using [13C] acetate to overcome this challenge. In the presence of benzene, the degraders of benzene
should incorporated more 13C carbon than that of the nondegraders, and higher levels of the 16S rRNA gene should
be expressed, resulting in different proportions of expression.
Thus, bacteria that expressed higher levels of the 16S rRNA
gene in WITH-B samples (described in 2.4) were more likely
to have responded by degrading benzene and could be classified as candidate co-metabolic benzene degrader. The bands
common to both the WITH-B and WITHOUT-B samples
were classified into the candidate non-degrader of benzene.
A culture-dependent method was also used to verify theculturable strains that could be applied to bioaugmentation,and the V3 region of the 16S rRNA genes of the isolates were
amplified and analyzed with PCR-DGGE (Fig. 1B).
Finally, to evaluate the key functional genes, we quantified
the C12O and C23O genes using qPCR (Fig. 1C). The presence of these genes was assessed in the 12C and 13C gradientfractions at 2 degradation phases as described in 2.3.
3.2 Occurrence of benzene degradation and preparationof 13C-DNA
Benzene was measured by gas chromatography, and562.4 μM benzene was degraded in 6 days during the experiments (Fig. 2). The phase from benzene addition to day 2
was defined as the first phase, and days 3 to 5 composed thesecond phase. To understand the diversity of the degraders
at each phase, we selected these 2 phases for SIP and SIP-D
analyses, respectively. Only a small amount of catechol
appeared in the final stage of degradation during the entire
process. No decrease in benzene levels occurred in the autoclaved control. [13C] DNAs were successfully separated fromtotal DNA in both SIP and SIP-D using ultracentrifugation,and [13C] DNAs were directly collected under UV illumination.
3.3 Detection of benzene-assimilating degraders
Benzene degradation was initiated on day 3 (Fig. 2), andseveral DNA bands appeared in the DGGE profile of the SIP
sample from day 4 (Fig. 3, lane 2). DNA bands a–h revealed
13
C carbon-incorporating bacteria as benzene-assimilating
degraders. Non-labeled 12C samples were also analyzed andDegrader community and functional gene analysis

155

the DNA band e was detected in the second phase (data notshown). Furthermore, bands a, b, g, and h in lane 2 had thesame DNA sequences as those of bands a’, b’, g’, and h’ inlane 4, which suggested that these bacteria could assimilate
both benzene and acetate.
3.4 Detection of candidate co-metabolic benzene degrader and non-degrader of benzene

Fig. 2. Benzene degradation. Added benzene (562.4 μM) was degraded in benzene-contaminated soil (●) and negative autoclaved control (+; NC). Mixtures for the SIP method werecollected on days 2 and 4, and total DNA was extracted. [13C]
acetate was added on days 2 or 4, and DNA was extracted
after 24 h in each case for SIP-D. Catechol concentrations (■)
were determined by HPLC. Error bars correspond to the standard deviations calculated for 3 experimental replicates.

As shown in Figure 3, bands i–n in lane 4 were barely labeledwith SIP (lanes 1 and 2). Compared to the WITHOUT-B
sample in lane 6, the WITH-B sample in lane 4 showed
several specific DNA bands, i, j, and n. These bands may
represent candidate co-metabolic benzene degraders. Bands
common to lane 4 (bands l, m) and lane 6 (bands l’ and m’)
may represent the non-degraders of benzene, which assimilated acetate in the presence or absence of benzene. SIP-D
may have been able to identify a non-degrader as a candidate
co-metabolic degrader in this study. To eliminate this possibility, gene expression by 16S rRNA was monitored using SIP
with [13C] acetate as follows. Fractionated [13C] RNA wasreverse transcribed into cDNA, and the V3 region of the 16S
rRNA gene was amplified and applied to DGGE. As shownin lane 7 in Figure 3, bacteria showing bands r and s, which
existed in the 13C fraction, were suggested to be active in thepresence of benzene. Moreover, as shown in Table 2, bands r
and s had the same DNA sequences as those of bands j andm, respectively. Thus, band j was concluded to be a candidate
co-metabolic benzene degrader, and bands i, m, and n werecandidate non-degraders under this condition.
3.5 Detection of culturable benzene degraders using aculture-dependent method

Fig. 3. PCR-DGGE analysis for culture-dependent and cultureindependent methods. [13C] benzene was used to detect assimilating bacteria with SIP (lanes 1 and 2). [13C] Acetate wasused to detect the other degraders with SIP-D (lanes 3–6). In
lanes 1–7, PCR-amplified DNA obtained from the heavy DNA
fraction was applied to the gel. [12C] or [13C] means the labeling carbon source, / means no additional; lanes 1, 3, and 5:
day 2; lanes 2, 4, and 6: day 4; lane 7: reverse transcriptionPCR products of the heavy fraction from [13C] acetate plus
the benzene-added sample; lanes 8–11: profile of the isolated
benzene-degrading strains; M: marker.

The strains identified in the culture-independent method
were culturable or non-culturable bacteria. This finding
may be valuable when bioremediation is performed because
culturable degraders may be feasible in bioaugmentation andnon-culturable degraders in biostimulation. Therefore, theconventional colony isolation method was performed, and 12
colonies were observed on LB agar containing benzene after
incubation for 7 days. Four of 12 colonies were selected basedon different types of colony morphology (data not shown),
and their benzene degradation capabilities were determinedwith the same concentration of 562.4 μM benzene as thesole carbon source (Table 3). The initial concentration ofbenzene was almost degraded by these strains, suggesting thatthese isolates exhibited benzene-assimilating capabilities in
the pure cultivation condition. These isolates were detectedusing SIP-D (Fig. 3, lanes 8–11), and sequencing analysis
showed that the DNA bands r and s in lane 7 were the sameas z(x) and y (100% sequence similarity), respectively (Table2). However, strain III (bands m, s, y) and IV (bands j, r, z)were not detected with SIP (lane 2). These results suggest thatthe isolated strains I, II, and IV were co-metabolic benzene
degraders and strain III was a non-degrader in this benzenecontaminated soil.

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Table 2. Sequence analysis of the major DGGE bands in the 13C fractions
Band

Organism with the closest sequence (accession No.)

Homology (%)

Phylogenetic group

a, a’
b, b’
d

Simplicispira metamorpha (AB680538)
Hydrogenophaga palleronii (AF078769)
Rhodococcus wratislaviensis strain J7 (AY940038)

Betaproteobacteria
Betaproteobacteria
Actinobacteria

e
f
g, g’
h, h’
i
j, k, r, x, z
l, l’
m, m’, s, ynpq

Rhodococcus erythropolis (AB177887)
Rhodococcus sp. A2Y26 (AY512637)
Frankia sp. strain Agb1-9 (L40611)
Pseudomonas oleovorans DT4 (GQ387664)
Pseudomonas pseudoalcaligenes strain H16 (JN571066)
Pseudomonas sp. LYBRD3-7 (HM246142)
Xenophilus azovorans (AF285414)
Pseudomonas sp. c1 (2011) (HQ652598)
R. wratislaviensis strain C6-1 (AY940037)
Pseudomonas sp. SC13 (EU939717)
Rhodococcus rhodochrous strain SR-6 (JQ040005)

98
99
100
100
99
100
93
100
100
93
100
97
100
99

Actinobacteria
Actinobacteria
Actinobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Betaproteobacteria
Gammaproteobacteria
Actinobacteria
Gammaproteobacteria
Actinobacteria

See Figure 3 for details of the DGGE analysis.

Table 3. Benzene degradation test for isolated strains
I
Decreased rate of benzene (%)
Residual absolute concentration (μM)

II

III

IV

NC

73.2

66.5

75.1

75.8

0.2

150.7±1.4

188.5±2.1

140.0±1.4

136.3±3.2

561.2±0.4

Benzene (562.4 μM) was added as the sole carbon source in minimal medium containing 107 cells/mL of each strain. The concentration was determined after 65 h using gas chromatography, and these samples were decreased to the detection limit after 80 h. NC: autoclaved control. Standard deviations were calculated for 3 experimental replicates.

3.6 Phylogenetic analysis of detected bacteria
The phylogenetic tree showed that most SIP-identified
bacterial strains were related to Actinobacteria and Betaproteobacteria in benzene-contaminated soil (Fig. 4).
Rhodococcus, detected as bands d–f (Fig. 3, lane 2), hasbeen implicated in the degradation of benzene, toluene,
and xylene 7,24). Several species of Hydrogenophaga (bandb), a genus of Comamonad bacteria, were formerly classified in the genus Pseudomonas 36), and band h was highly
related to Pseudomonas oleovorans strain DT4, which has
been described as a benzene degrader 39). In contrast, SIP-Didentified strains mainly belonged to Gammaproteobacteria.
In lane 4 (Fig. 3), band i was closely related to Pseudomonas pseudoalcaligenes (100% sequence similarity), which
exhibited a highly enhanced benzene-degrading capability 31).
Bands m and j were related to Pseudomonas sp. C1 andPseudomonas sp. strain LYBRD3-7 (100% sequence similarity), respectively. This study is the first to show that the
Pseudomonas strain (band j) is a benzene degrader (marked
with red dots in Fig. 4). The most closely related strain toband n in lane 4 (Fig. 3) was a non-cultured bacterium,
Rhodococcus wratislaviensis (97% sequence similarity),
which has been reported to degrade a mixture of hydrocarbons, gasoline, and diesel oil 3).
3.7 Quantification of C12O and C23O genes with qPCR
Benzene was aerobically degraded via the catechol cleavage

pathway, and its key genes, the C12O and C23O genes, andthe 16S rRNA gene were quantified using qPCR to determine
which gene was functioning in benzene-ring opening (Fig. 5).
The quantity of the 16S rRNA gene increased from the firstto the second phase (from ~105 to ~106 copies per μL). In
the SIP assay, most of the C12O gene was detected in the 13C
fraction, suggesting that most benzene-assimilating bacteria
harbor the C12O gene and metabolize benzene with it. And,
the C23O gene was detected only in the 12C fraction of thesecond phase.

4. Discussion
Bacterial communities differ from site to site, andinformation regarding pollutant-degraders existing in theenvironment is important in bioremediation. Therefore,
identifying functional bacteria is an essential component ofeffective bioremediation. To contribute our findings to thesuccess of bioremediation, we considered the conditions ofrefinery sites and performed this study with approximately
500 μM of benzene, a minimal medium (without a carbon
source), and mesophilic conditions.
Our results demonstrated the possibility of identifying thefollowing bacteria. Xenophilus, a non-benzene-degrading
bacterium, and the acetate-assimilating types of Pseudomonas were classified in the first degradation phaseof benzene because their DNA bands appeared in both
samples (Fig. 3, lanes 3 and 5). In the second phase, theDegrader community and functional gene analysis

157

Fig. 4. Phylogenetic relationships derived from 16S rRNA gene V3 region analysis (150 bp). SIP- and SIP-D-identified DNA bands are shownin blue and red, respectively. Red dots indicate candidate benzene co-metabolic degraders. Phylogenetic positions traced strains related to
Rhodococcus, Hydrogenophaga, or Pseudomonas strains based on the 16S rRNA gene V3 region sequence. Accession numbers of thereference organisms are indicated in parentheses. Bootstrap values for branches are based on 1000 replicates. Bar=2% nucleotide substitution rate (Knuc).

benzene-assimilating bacteria belonging to Pseudomonas,
Rhodococcus, Frankia, and Simplicispira, were directly
identified by the SIP method (Fig. 3, lane 2). The candidate
co-metabolic types of Pseudomonas, and the non-benzenedegrading types of Pseudomonas and Hydrogenophaga weremainly identified by the SIP-D method (Fig. 3, lanes 4 and 6).
These results suggested that this combination of SIP methods
may possible to compensate the comprehensive analysis in
the microbe ecology field and delineate a comprehensive
bacterial community that may contribute to biodegradation.
Benzene-assimilating degraders could be identified by
the SIP method. Unfortunately, SIP-D covered a group of
bacteria that assimilated both benzene and acetate in thisstudy because bands were detected in both SIP (bands a, b, g,
and h in lane 2) and SIP-D (bands a’, b’, g’, and h’ in lane 4)
samples. 16S rRNA-targeted SIP (lane 7, Fig. 3) was used toaddress this question, and the results suggested that the mostactive bacteria in the second degradation phase belonged to
Pseudomonas sp. (bands j and m) and were considered to bepotential benzene degraders (Fig. 3).
These 2 strains displayed active benzene-assimilating
capabilities, which were clarified using the culture-dependent
method (Table 3). They could use acetate in benzene-contaminated soil as shown in lane 4, Figure 3, and also the LB brothas their carbon source in the culture-dependent method.

Fig. 5. Quantification of the C12O and C23O gene proportions indegradation processes.
□, C12O gene; ■, C23O. Targeting assimilating bacteria detected by SIP. Quantitative analysis of the 12C- and 13C-DNA
fractions from SIP data. Evaluation of the functional genes of
12
C and 13C gradient fractions from SIP data. (1st), first phase;(2nd), second phase. The absolute values of all samples exhibited similar trends. Error bars correspond to the standard deviations calculated for 3 experimental replicates.

158

Pan et al.

However, one bacterium (band j) showed co-metabolic
degrading potential, while another (band m) exhibited nobenzene-assimilating capability. For these strains could not
be labeled by [13C] benzene in lane 2, Figure 3, they wereclassified as the candidate co-metabolic degrader (band j)
and candidate non-degrader (band m) of benzene under
this condition. These results suggested that a bacterium,
even if it has an active benzene degradation capability, is
not necessarily directly associated with degradation. And,an interaction between bacteria in the contaminated soil
might inhibit the assimilating ability of this isolated strain.
This important characteristic must be considered duringbioremediation treatment. Furthermore, bands p and q inlane 6 were not detected in the WITH-B sample (Fig. 3,
lane 4), suggesting that several acetate-assimilating bacteria,
which may not have benzene-degrading capabilities, could
be substituted in the WITH-B condition even in the presenceof acetate.
Our findings also demonstrated the potential to trace
uncultured bacteria (bands i, l, a’, b’, n, g’, and h’) by
a comparison with a culture-independent method, whichsuggests that most degraders are rarely isolated under thiscondition, but could be detected with our combination of
SIP methods. These results suggested that biostimulation(but not bioaugmentation) may be suitable at this benzenecontaminated site because many benzene degraders may be
uncultured suggested by this conventional isolation method.
An interesting challenge for the future will be to improve
the SIP-D method by finding an appropriate labeling carbon
source with which to target a group of bacteria, labeling theposition, and optimizing the environmental pollutant concentration for a comprehensive analysis.
Previous studies have shown that some Pseudomonas
could co-metabolize aromatic compounds coexistent withorganic acids 4,18), and our study shows the possibility of identifying this kind of co-metabolizing bacteria. The existenceof electron acceptors or donors also has a large impact on
carbon flux in ecosystems, and for the selection of labelingcarbon source to optimize SIP-D.
The relationship observed between the C12O and C23O
gene is similar to that described by a previous study thatanalyzed genes associated with aromatic compound degradation 30). Although acetate was added, the bacteria (bands c,
d, e, and f ) continued to express the C12O gene because noband shifting of these bacteria was observed between lanes
2 and 4 (Fig. 3). The functional gene quantification experiment detected the C12O gene in both the light and heavy
DNA fractions (Fig. 5), suggesting that the most benzene
assimilating-degraders were processing the C12O gene andused this pathway to open the benzene ring. Although the
C23O gene was processed in the second phase of the 12C
fraction, they may not use this pathway because the C23O
gene was not detected in the 13C fraction.
In conclusion, understanding the structure of thedegradation community including assimilating degraders,co-metabolic degraders, and non-degraders, is essential for

improving bioremediation. In this study, a comprehensive
analysis was performed to classify the bacterial members
related to benzene degradation under mesophilic aerobic
conditions. Benzene-assimilating degraders were classified
with SIP, whereas the candidate co-metabolic degraders andcandidate non-degraders of benzene were classified using
SIP-D and RNA-based SIP. Furthermore, the qPCR results
showed that the assimilating degraders used ortho-cleavage
for benzene degradation. This combination of SIP methods ison the start line to be able to perform comprehensive analysisof mixed culture roughly, and could be used to confirm thestructure of the bacterial community in the presence ofchemical pollutants such as benzene.

Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research (B) (no.21310050, to H.U.), CREST program ofJapan Science and Technology Corporation (JST), and the
Monbukagakusho Scholarship (no.114042, to Y.P.) of the
Ministry of Education, Culture, Sports, Science and Technology. Japan. We thank Eri Hara for help with the primer
construction, and Shouhei Yamasaki for helpful technicaladvice.

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