Journal of Environmental Biotechnology
Vol. 4, No. 2, 117–120, 2005

Original paper (regular paper)

Comparison of Dibenzothiophene-Degrading Bacteria under
Low Oxygen Conditions
FUMIYA KITAUCHI1,**, SHIN-ICHI HIRANO1, MITSURU HARUKI1,***, TADAYUKI IMANAKA2,MASAAKI MORIKAWA1* and SHIGENORI KANAYA1
Department of Material and Life Science, Graduate School of Engineering, Osaka University,
Suita, Osaka 565–0871, Japan2
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Kyoto 615–8510, Japan1* TEL: +81–6–6879–7443 FAX: +81–6–6879–7443* E-mail: morikawa@mls.eng.osaka-u.ac.jp** Present adress: Medical Products Division, UNITIKA Ltd. Chuo-ku, Osaka 541–8566, Japan*** Present adress: Department of Materials Chemistry and Engineering, College of Engineering,
Nihon University, Koriyama, Fukushima 963–8642, Japan(Received; 23 January, 2004/Accepted 20 August, 2004)
Nine dibenzothiophene (DBT) degrading bacteria were isolated from various crude oil reservoir tanks in Japan. Strains
T37 and T38 were identified as Pseudomonas cepacia and A76 and 203S as Pseudomonas stutzeri. Strains T09, 23S and
32S were suggested to be Pseudomonas flurorescens. Strains N-1 and A54 were identified as Rhizobium sp. and Shingomonas sp., respectively. Their DBT degradation abilities under low oxygen conditions were compared with that of Xanthobacterpolyaromaticivorans strain 127W, recently isolated in our laboratory. X. polyaromaticivorans strain 127W, Sphingomonas
sp. strain A54, and Rhizobium sp. strain N-1 degraded 28.5, 8.8 and 6.3 mg/l of DBT in six days, at DO 3 ppm, respectively. All the seven strains tested in the genus Pseudomonas degraded less DBT than X. polyaromaticivorans strain 127W,
Sphingomonas sp. strain A54, and Rhizobium sp. strain N-1 under this low oxygen condition.
Key words: dibenzothiophene, degradation, low oxygen condition

1.

Introduction

Polycyclic aromatic hydrocarbons (PAH) and heterocyclicaromatic compounds (HAC) are of environmental concern
because of their recalcitrant and carcinogenic behavior8,9).
Although PAH and HAC in surface water or soil particles
are susceptible to degradation by various aerobic bacteria,
large fractions of PAH and HAC stick to solid particles andsettle to the bottom of rivers or lakes where only limited
oxygen is available13). These fractions remain undegraded
for long periods because anaerobic biodegradation proceeds
slowly. Even the polluted site soon becomes anoxic due tohigh oxygen demand for hydrocarbon degradation by bacteria. Thus much interest exists in isolating and studying microorganisms that effectively degrade PAH and HAC underlimited oxygen conditions from the viewpoints of developing
bioremediation technologies and understanding natural attenuation. Dibenzothiophene (DBT) is a model compound
among sulfur-containing HAC in crude oil. Although various bacteria degrade DBT aerobically1,2,5,7,10,11,14), little
knowledge is available of DBT degradation under low oxygen conditions.
We recently isolated Xanthobacter polyaromaticivorans

strain 127W from an anoxic sludge in a crude oil reservoir
tank in Fukui4). This strain degrades significant amounts of
DBT under both aerobic and extremely low oxygen conditions. In this study, we isolated nine additional bacterial
strains that degrade DBT aerobically and compared theirdegradation ability with that of strain 127W under both
aerobic and low oxygen conditions. The DBT degradation
activity of strain 127W was the most tolerant against oxygen limitation among the strains tested. All strains tested in
the genus Pseudomonas were less active than Rhizobium
sp. strain N-1, Sphingomonas sp. strain A54, and X.
polyaromaticivorans strain 127W.

2.
2.1.

Materials and Methods

Isolation of DBT-degrading strains

Sludge samples were collected from crude oil reservoirtanks in Hokkaido, Aomori, Akita and Okinawa, Japan. An
aliquot of each sample was inoculated into a mineral salt
medium containing DBT, CSF-DBT, as a sole carbon andsulfur source. CSF-DBT contained in one liter, 4 g of
Na2HPO4, 4 g of K2HPO4, 2 g of NH4NO3, 0.1 g of
MgCl2·6H2O, 0.01 g of CaCl2·2H2O, 0.01 g of FeCl3·6H2O,

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118

0.05 g of yeast extract, and 100 mg of DBT (add 10 ml of
54 mM DBT in ethanol). The pH was adjusted to 7.0 by
HCl. Glucose minimum medium, CSF-G, containing 2%
glucose as carbon source, and Luria broth were also used if
necessary. Cultivation was at 30°C. After transferring theculture to a new medium three times, bacterial strains wereisolated on a CSF-DBT-agar plate containing 15 g/l agar tosolidify the medium. When the cells were grown on a CSFDBT-agar or on a CSF-G-agar plate overlaid with a sprayof 1% DBT-diethyl ether solution, each colonis was surrounded by a clear halo indicating DBT degradation4).
2.2.

DBT degradation under low oxygen conditions

Exponentially growing cultures in Luria broth were harvested, were washed once with sterile water, and were suspended in a small volume of water. CSF-DBT (10 ml) in a
20 ml glass vials (No. 5, Maruemu, Tokyo) was inoculatedwith the cell suspension at a final OD660 of 0.1, and 50 mg/l
(0.27 mM) DBT was added as a substrate. A low oxygen
condition was prepared as follows: vials containing CSFDBT and the cells were left in an anaerobic chamber(EAN-101, Tabai Espec, Osaka) for an defined time after
degassing twice by N2 gas and once by an anaerobic gas
mixture (CO2/H2/N2=5 : 5 : 90 with DO<0.02 ppm). Dissolved oxygen, (DO) in the medium decreased as the lengthof time kept in the anaerobic chamber increased. Afterabout 2 hr, DO decreased to 3 ppm. The vials were tightly
sealed with butyl rubber septa with aluminum crimps andwere gently shaken at 30°C for two to six days. An aerobic
degradation experiment was done in vials sealed in the atmosphere.
2.3.

Gas chromatography (GC) analysis

The reaction mixture was acidified to pH 2.0 with 6 M
HCl and DBT was extracted with one volume of ethyl acetate containing 0.12 mM of fluorene as an internal standard. To eliminate trace contamination of air and absorption of hydrocarbons into the butyl rubber stopper, we usedneither a needle to take samples from the vial nor transferred samples to another vial. Instead, the whole reaction
culture was directly subjected to extraction in the originalvial at each degradation time. Part of the ethyl acetate layer
was analyzed by using a GC system GC-14A equipped witha 30-m non-polar capillary column CBP-1 (Shimadzu,
Kyoto) and a flame ionization (FID) detector (GC/FID)
or JEOL JMS-DX303 mass spectrometer (JEOL, Tokyo,
GC/MS). The temperature and carrier gass flow conditions
were as described elsewhere4).

3.
3.1.

Results and Discussion

Isolation of DBT-degrading bacteria

We expected to isolate bacteria that actively degrade
DBT under low oxygen conditions. Bottom sludge samples
in the crude oil tank were used to isolate such bacteria, because they are insulated from air. Remaining oxygen shouldhave been consumed by aerobic bacteria. Sludge samples
from tanks, T09, T37, T38 (Hokkaido), No. 32 (Aomori),
TK-23 (Akita), #1 (Niigata), TA-54/TA-76/TK-203 (Okinawa) contained bacteria that degraded DBT. Strains thatmost effectively degraded DBT were chosen from each tank
and were named after the name of the tank: T09, T37, T38,
32S, 23S, N-1, A54, A76, and 203S.
3.2.

Identification of bacterial strains

All strains were revealed Gram-negative bacteria. All
strains, except A54 and N-1, showed similar results in thefollowing API tests: oxidase, catalase, and nitrate reductase
were positive, and indole production and the VogesProskauer test were negative. Based on these characteristics,
strains T09, T37, T38, 32S, 23S, A76, and 203S were suggested to belong to the genus Pseudomonas (15). Further
physiological characteristics were found by using an API
system (Biomerieu, Cedex, France). Strains T09, 23S, and
32S were suggested to be P. fluorescens with identification
scores 92.9%, 94.9%, and 95.6%, respectively. Strains T37
and T38 were identified as P. cepacia with identification at
99.8% and 99.9%, respectively, and strains A76 and 203S
were P. stutzeri both with identification at 99.9%. P. fluorescens, P. cepacia, and P. stutzeri are well studied bacterial groups in hydrocarbon degradation pathways. Strain A54
was identified as Sphingomonas paucimobilis with identification at 98.4%. However, strain N-1 could not be identified by using the API system because all scores were low.
The 16S rRNA gene was amplified by PCR and the nucleotide sequence was determined and analyzed by using
BLAST search program (http://www.ncbi.nlm.nih.gov/
BLAST/). The nucleotide sequence of the 16S rRNA genefrom strain N-1 (DDBJ/EMBL/GenBank; AB182639) had
the highest similarity with Rhizobium galegae at identification 97.8% and R. leguminosarum at identification 95.6%,
indicating that the strain belongs to Rhizobium.
3.3.

DBT degradation under aerobic and low oxygen
conditions

Isolated strains were tested for DBT degradation underboth aerobic and low oxygen (DO=3.0 ppm) conditions (Fig.
1). X. polyaromaticivorans strain 127W and P. stutzeristrain T102, that were previously isolated strains4), wereused as comparative strains. Fig. 1a shows that strains A76,
A54, and 127W degraded 50 mg/l DBT completely in threedays under aerobic conditions. When the DO was reduced
to 3.0 ppm, strains A76, 203S, T102, T37, 23S, and T09
lost almost all their degradation abilities. But strains N-1,
A54, and 127W degraded 6.3, 8.8, and 28.5 mg/l of DBT,
respectively. Strain 32S also showed a degradation ability,
but at a lower level than strains N-1, A54, and 127W.
3.4.

Classification of isolates based on the DBT-degrading activity

Our experimental results clearly show that all strains ingenus Pseudomonas require more oxygen than Rhizobium,Degradation of dibenzothiophene under low oxygen conditions

119

Fig. 1. Comparison of DBT degradability of strains under aerobic and a low oxygen conditions. Each value is an average of three independent experiments.
(a) Degradation of DBT after 3 days under aerobic conditions (DO was continuously over 7 ppm). (b) Degradation of DBT after 6 days
under a low oxygen condition (DO=3 ppm).

Fig. 2. GC/FID and GC/MS analyses of the degradation products from DBT.
DBT and its degradation products were analyzed after 3 days cultivation of strains 32S and A54. Because the chromatograms are almost
identical for these two strains, only the data of strain 32S is shown in the figure. Cultivation was aerobic and extraction of DBT and its
related compounds was done with ethyl acetate without fluorene in this case. Column temperature increased from 100°C to 200°C at a
gradient of 5°C/min and from 200°C to 290°C at 10°C/min gradient.

KITAUCHI et al.

120

Sphingomonas, and Xanthobacter. Toluene degradation
under limited oxygen condition (DO=2 ppm) by P. picketti
strain PKO1, P. fluorescens strain CFS215, and Pseudomonas sp. strain W31 increased by adding 10 mM nitrate to
the medium, but degradation by P. cepacia and P. putida
was not affected by nitrate6). This event was attributed tohigher oxygen affinity of the enzyme responsible, catechol
2,3-dioxygenase, from strains PKO1, CF215, and W31, andan increased gene expression level in response to denitrification under oxygen limitation. In this study, because the
CSF-DBT medium contained 25 mM NH4NO3, the low
degradation ability by Pseudomonas strains should nothave been due to nitrate starvation. When Pseudomonas
strains are used, considerable aeration is required for theeffective degradation of aromatic hydrocarbons. That is,
Rhizobium, Sphingomonas and especially X. polyaromaticivorans could be more cost effective than Pseudomonas for DBT degradation under low oxygen conditions. Thisknowledge may be informative for constructing an effective
bioremediation system to clear up accidental spillage of hydrocarbons.
3.5.

DBT-degradation pathway of the strains

To understand the DBT-degradation pathway of thestrains, the structure of the degradation products was analyzed by using GC/FID and GC/MS (Fig. 2, Table 1). Molecular ion peaks for benzothiophene-2,3-dione (BT-dione,A), 3-hydroxy-2-formyl benzothiophene (3H2FBT, B), DBT(C), and dibenzothiophene-5-oxide (DBTO, D) were clearlyobserved at m/z=164, 178, 184, and 200, respectively. The
differences in the m/z of the peaks, such as the eliminationof the CO group at 28 and at 32 for S, are also used to
identify the compounds12). The most abundant and common
product was 3H2FBT, suggesting that all strains degraded
DBT by the popular Kodama pathway5). Strains 32S andA54 produced DBTO as well as 3H2FBT. These strains
should have sulfoxidase-like activity, as suggested for other
Pseudomonas isolates12). Several bacterial strains in RhodoTable 1.

T102
T09
T23
32S
A76
A54
N-1
127W

Degradation products of DBT.
Cultivation was at 30°C for 3 days.
Amounts were estimated by the peak area in GC/FID.
3H2FBT
++
++
+
++
+
+
++
+++

BT-dione
+/–
+/–
+/–
+/–
+/–
+/–
–
–

DBTO
–
–
–
++
–
+
–
–

T102, T09, T23, 32S, A76, A54, N-1, 127W are the names ofthe strains.
3H2FBT, 3-hydroxy-2-formylbenzothiophene; BT-dione, benzothiophene-2,3-dione; DBTO, dibenzothiophene-5-oxide.
–, not detectable; +/–, <0.002 mM; +, 0.002~0.05 mM;++, 0.05~0.1 mM; +++, 0.1 mM<.

coccus, Mycobacterium, Phaebacillus desulfurize DBT bycleaving the C-S bond3). DBTO and dibenzothiophene
5-dioxide are common intermediates in hydroxyl biphenyl
and sulfate formation. However, the desulfurization activity
is less probable for strains 32S and A54, because neither
reduction in amount of DBTO nor production of dibenzothiophene 5-dioxide and hydroxyl biphenyls was observed
even after incubation for a long time.

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