Biodegradation (2016) 27:3746

DOI 10.1007/s10532-015-9753-2

ORIGINAL PAPER

Multi-substrate biodegradation interaction of 1, 4-dioxane

and BTEX mixtures by Acinetobacter baumannii DD1
YuYang Zhou . Huanlin Huang . Dongsheng Shen

Received: 27 August 2015 / Accepted: 22 December 2015 / Published online: 9 January 2016
Springer Science+Business Media Dordrecht 2016

Abstract This study evaluated substrate interactions

during the aerobic biodegradation of 1, 4-dioxane and
BTEX mixtures by a pure culture, Acinetobacter
baumannii DD1, which is capable of utilizing 1,
4-dioxane for growth. A. baumannii DD1 could utilize
BTEX as a sole carbon source, but could not utilize mxylene and p-xylene. In binary mixtures, there was a
lag of about 14 h before the degradation of BTE, and
1, 4-dioxane only started to be utilized when BTE was
completely degraded by 1, 4-dioxane-grown DD1.
Furthermore, the biodegradation rate of 1, 4-dioxane
decreased from 73.33 to 40.74 mg/(h g dry weight)
after the biodegradation of benzene. 1, 4-dioxane
could not be degraded after the biodegradation of oxylene in 80 h. DD1 could also not degrade m-xylene
and p-xylene coexisting with 1, 4-dioxane. The ability
of DD1 to degrade BTEX occurred in the following
order: benzene [ ethylbenzene [ toluene [ o-xylene [ m-xylene = p-xylene. The biodegradation of 1,
4-dioxane was not activated in the mixture with

Y. Zhou
H. Huang
D. Shen (&)

School of Environmental Science and Engineering,
Zhejiang Gongshang University, Hangzhou 310012,
China
e-mail: shends@zju.edu.cn;
shends@mail.hz.zj.cn
Y. Zhou
H. Huang
D. Shen
Zhejiang Provincial Key Laboratory of Solid Waste
Treatment and Recycling, Zhejiang Gongshang
University, Hangzhou 310012, China

o-xylene, primarily because of the accumulation of the

specific toxic intermediate, 2, 3-dimethylphenol. The
lag in BTE degradation was presumably because of the
induction of enzymes necessary for BTE degradation.
Additionally, SDS-PAGE analysis demonstrated that
there were different proteins during the degradation of
benzene and 1, 4-dioxane.
Keywords Biodegradation
1, 4-dioxane
BTEX

Enzyme
Substrate inhibition

Introduction
1, 4-dioxane, a suspected carcinogen, is an industrial
solvent and a common byproduct of chemical processes. There are over 110 tons of 1, 4-dioxane
released into surface water from manufacturing and
processing facilities in the United States and Japan
annually (Sei et al. 2013; Agency for Toxic Substances
and Disease Registry (ATSDR) 2012). Because of its
high water solubility and vapor pressure (5.33 kPa/
25.2 C), 1, 4-dioxane spreads easily, and can be
detected in surface water, groundwater, landfill
leachate, and even in arctic groundwater (Li et al.
2013). Because it has a low logoctanol-water partition
coefficient (-0.27) and low Henrys Law constant
(Sun et al. 2012), 1, 4-dioxane can persist for a long
time in the environment. In the past two decades,
bioremediation has emerged as an attractive

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alternative for 1, 4-dioxane removal because of its

cost-effective, energy-efficient and environmentallyfriendly properties (Bernhardt and Diekmann 1991;
Nakamiya et al. 2005). Although biodegradation of 1,
4-dioxane has been studied extensively, most studies
have focused on investigation of the degradability of
1, 4-dioxane as a single contaminant (Bernhardt and
Diekmann 1991; Parales et al. 1994; Nakamiya et al.
2005; Kim et al. 2009; Sei et al. 2013).
In fact, waste water, landfill leachate or gas
contaminated by 1, 4-dioxane is always combined
with other pollutants, especially monoaromatic hydrocarbons such as benzene (B), toluene (T), ethylbenzene (E) and xylenes (X). It is well known that BTEX
are widely used as industrial solvents for organic
synthesis and equipment cleansing (Kenneth et al.
2000). BTEX are the major aromatic components of
many petroleum products, and leaking underground
storage tanks and pipelines, improper waste disposal
practices, inadvertent spills, and leaching from landfills have resulted in their commonly being found in
groundwater and gas (Shim and Yang 1999). As a
modifier, 1, 4-dioxane can improve the thermodynamic properties of aromatic compounds in industry
(Sugiura and Ogawa 2009). The effluent discharged
from the paint and ink industry could cause cocontamination of 1, 4-dioxane and aromatic compounds in surface water and groundwater. Additionally, a recent survey (Duangduan et al. 2008) in
Thailand reported a large number of waste gases from
gas stations containing both BTEX and 1, 4-dioxane,
posing a health threat to residents in local and
neighboring communities. Based on this survey, the
lifetime cancer risks of workers exposed to the waste
gases were at least 53 times higher than normal.
Because of the cancer risks associated with cocontamination with BTEX and 1, 4-dioxane, it is
necessary to prevent the interaction between these
compounds. Several studies have investigated the
substrate interactions of benzene, toluene, ethylbenzene, and xylene (Prenafeta-Boldu et al. 2002).
However, few studies have evaluated the effects of
substrate interactions on the biodegradation rates of 1,
4-dioxane and BTEX compounds in contaminant
mixtures. Moreover, the influences of 1, 4-dioxane
and BTEX have not been demonstrated.
Biodegradation of BTEX has been studied frequently, and both aerobic and anaerobic BTEX biodegraders have been identified (Kim et al. 2014; Kunapuli

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Biodegradation (2016) 27:3746

et al. 2007; Liou et al. 2008; Noguchi et al. 2014; Oka

et al. 2008; Sun and Cupples 2012; Sun et al. 2014; Sun
et al. 2010; Vogt et al. 2011). BTEX biodegradation can
be realized by monooxygenation or dioxygenation in
different bacteria, while 1, 4-dioxane can be degraded
via monooxygenation (Mahendra and Alvarez-cohen
2006; Mahendra et al. 2007). Interestingly, strain
Mycobacterium vaccae JOB5 cometabolized dioxane
with toluene, confirming the involvement of monooxygenases (Burback and Perry 1993). These findings also
raise the possibility that 1, 4-dioxane and BTEX
biodegradation can promote each other because of the
similar function of enzymes or inhibit each other
because of the competitive inhibition in the contaminated environment. Conversely, if the biodegradation
of BTEX and 1, 4-dioxane occur via different enzymes,
the induction of the other enzyme might be inhibited,
preventing degradation from commencing at the same
time. Our previous study (Huang et al. 2014) demonstrated that Acinetobacter baumannii DD1 is an
efficient 1, 4-dioxane-degrading bacterium that acts
through monooxygenases. Furthermore, it is able to
degrade BTEX. Therefore, it is worth demonstrating
the primary enzymes and interactions during the
degradation of BTEX and 1, 4-dioxane by DD1.
This study aims to investigate the biodegradation
interaction and mechanism between 1, 4-dioxane and
BTEX. Evaluating the impact of contaminant mixtures
on the individual biotransformation rates of 1, 4-dioxane and BTEX compounds is useful to prediction of
the behavior of these compounds in both in situ and ex
situ biological treatment systems. Moreover, it will
help us better understand 1, 4-dioxane and BTEX cobiodegradation in the field.

Materials and methods

Chemicals
1, 4-Dioxane ([99 % reagent grade) was purchased
from Aladdin Company. Benzene ([99 % reagent
grade), toluene ([99 % reagent grade), ethylbenzene
([99 % reagent grade), o-xylene ([99 % reagent
grade), p-xylene ([99 % reagent grade) and mxylene([99 % reagent grade) were obtained from
Sigma Chemical Co., Ltd. SDS-PAGE sample buffer
(59) and protein markers were purchased from
Sangon Company (Shanghai, China). All other

Biodegradation (2016) 27:3746

reagents used in this study were of analytical reagent

grade and purchased from Huipu Company (Hangzhou, China).
Culture growth and conditions
A. baumannii DD1 (CCTCC M 2013560), which was
recently isolated and identified in our laboratory
(Huang et al. 2014), was used for this study. Strain
DD1 was incubated at 30 C and 130 rpm in 250 mL
sealed glass serum vials containing 50 mL of mineral
salts medium (MSM) supplemented with 1, 4-dioxane
as the sole carbon and energy source (Huang et al.
2014). Cells of A. baumannii DD1 were harvested in
the late exponential growth phase by centrifugation
(22,0009g) for 5 min at 4 C. The cells were then
washed twice with phosphate buffer solution (PBS,
140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, pH 7.4), after which they were
resuspended in MSM for further use in biotransformation.
Experimental approach
Each 250 mL bottle containing 50 mL of MSM, single
or multiple substrate, and 60 mg/L 1, 4-dioxanegrown cell suspensions were incubated at 130 rpm and
30 C in a rotary shaker. 1, 4-dioxane and BTEX
compounds were added to the cultures using high
precision 0.15 lL syringes. During the detection of
intermediates of o-xylene, 0.5 mM of 1, 4-dioxane
with lower concentrations of 2, 3-dimethylphenol
(0.05, 0.1, 0.15 and 0.2 mM) were added to the
culture. During investigation of the cause of the lag
phase of BTE and inhibition of 1, 4-dioxane, sequential addition experiments were conducted to add
0.5 mM benzene in the first three cycles. Following
the depletion of benzene, 0.5 mM toluene was added
in the 4th cycle and 0.5 mM 1, 4-dioxane was added in
the 5th cycle. Triplicate samples were collected at
various time intervals to analyze 1, 4-dioxane or
BTEX concentrations. All experiments were performed in triplicate, and each experiment included
abiotic controls and substrate-free controls.

39

secretion process during the biodegradation of benzene and 1, 4-dioxane.

DD1 cells were cultured in 50 ml MSM medium
containing 0.5 mM 1, 4-dioxane, benzene or a mixture at
30 C and 130 rpm and then harvested by centrifugation
at 22,0009g for 5 min. The supernatant was subsequently discarded, after which the strain was washed
with PBS twice and cellular lysates were obtained by
placing the sample (the phosphate buffer washings) in an
ultrasonication instrument (Sonics Ultra Cell, USA) with
a power of 97.5 W and a frequency of 20 MHz.
Ultrasound was applied for 5 s, 50 times, with a 10 s
gap between applications. The extract was centrifuged at
18,0009g for 15 min, after which the supernatant was
used for enzyme assays (Cai et al. 2011).
Microbial metabolic pathways for benzene reported
to date have shown that they are usually biodegraded
via catechols, which were identified from the absorbance at 260 and 375 nm from the intermediates
cis,cis-muconic acid [e = 16 (lM cm)-1] and 2-hydroxymucaldehyde semialdehyde [e = 12 (lM cm)-1],
respectively (Sala-trepat and Evans 1971; Zeyer et al.
1985). A mixture of 0.3 lmol catechol, 2 mL phosphate
buffer (pH 7.4), and 1 mL of the cellular lysates was
vigorously shaken, after which the absorbance was
immediately measured. Enzyme specific activities are
defined as l lmol of cis,cis-muconic acid or 2-hydroxymuconic semialdehyde produced per min per gram of
protein, and expressed as U/mg protein.
Monooxygenase activities were determined spectrophotometrically by measuring the oxidation of
NADPH at 340 nm and 30 C. The reaction mixture
(2.5 ml) contained 100 lmol PBS (pH 7.0), 5 lmol 1,
4-dioxane (substrate), 0.2 mol NADPH, and protein
(cell-free extract). The reaction was initiated after the
addition of substrate. The extinction coefficient of
NADPH was 6.22 9 103/M/cm. One unit of enzyme
was defined as the amount of enzyme required to oxidize
1 lmol NADPH per minute under these conditions (Cai
et al. 2011). The protein content was quantified by the
LowryFolin method using bovine serum albumin
(BSA) as the standard (Lowry et al. 1951).
Polyacrylamide gel electrophoresis of bacterial
protein extracts

Enzyme assays
The dioxygenase activities and monooxygenase activities were measured to investigate the enzyme

The extracted bacterial proteins were analyzed by

SDS-PAGE on a Mini SE260 electrophoresis cell
(Amersham) (Zhu et al. 2015). The protein samples

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were reconstituted to 3 mg/mL in 5 9 SDS-PAGE

sample buffer and heated at 100 C for 5 min. The
sample was then centrifuged at 22,0009g for 5 min,
after which the supernatant was used for SDS-PAGE.
Aliquots (10 lL) of protein were loaded into the gel
wells and electrophoresis was performed at 80 V
through the stacking gel (5 %) and 150 V through the
separation gel (10 %) until the bromophenol blue
marker dye was within 1 cm of the bottom of the gel.
The protein bands were then stained with coomassie
brilliant blue R-250. After staining, gels were
destained overnight in a solution containing 7.5 %
ethanol and 7.5 % acetic acid. The following polypeptides were used as the molecular weight markers.

Biodegradation (2016) 27:3746

silica HP-5 capillary column (30 m 9 0.25 m 9

1 lm, J&W Scientific) in splitless mode using helium
as the carrier gas at a flow rate of 1.0 mL/min. The MS
was operated in full-scan or selected ion monitoring
mode with positive ionization by electron impact. The
inlet and MS transfer line temperatures were maintained at 250 C, and the ion source temperature was
250 C. The following GC oven temperature was
applied: 40 C as the initial temperature for 5 min,
followed by an increase of 5 C/min to 150 C, then
10 C/min to 200 C, which was maintained for
3 min. The intermediates were identified based on
the mass spectra using the mass spectral library
NIST08.L.

Analytical methods
Results and discussion
Cultures were centrifuged at 22,0009g for 5 min,
after which the supernatant was analyzed for 1,
4-dioxane concentration by gas chromatography (Agilent 7890A) equipped with a silica HP-INNOWAX
capillary column (30 m 9 0.32 mm 9 0.5 lm, J&W
Scientific, USA) and a flame ionization detector. The
operation parameters and flow rates have been
described previously (Huang et al. 2014).
BTEX were measured by the headspace sampling
method. Briefly, samples were collected using a gastight syringe equipped with a side-sport needle, then
injected into the gas chromatograph. The column,
injector and detector temperature were held constant at
200, 250 and 300 C, respectively.
To analyze the intermediates during BTEX or
mixture biodegradation, cultures were centrifuged at
22,0009g for 10 min, after which the supernatant was
extracted with ethyl acetate (Vsample:Vethyl acetate =
1:1) by vigorously shaking for 30 min. After being
allowed to stand for 10 min, the organic layer was
collected and evaporated to dryness under a N2 stream.
The dry residue was subsequently derivatized by the
addition of 50 lL pyridine and 100 lL bis-(trimethylsilyl)-trifluoroacetamide (BSTFA, a kind of derivation), which were pre-heated in a heating block at
65 C for 30 min. Following derivatization, the
extracts were ready for injection into the GC/MS
(Zhou et al. 2011).
GC/MS analysis of biodegradation intermediates
samples was conducted after derivatives were prepared using a GC (Agilent 6890) equipped with an
inert mass selective detector (Agilent 5973) and a

123

Biodegradation interactions between 1, 4-dioxane

and BTEX
The bacterium used in this study, A. baumannii DD1,
is capable of utilizing 1, 4-dioxane as the sole carbon
and energy source. A previous study in our laboratory
showed that DD1 could also effectively utilize benzene, but the interaction between 1, 4-dioxane and
BTEX has not been demonstrated. In this study, the
biodegradation interactions and mechanism between
1,4-dioxane and BTEX by DD1 was further
investigated.
As shown in Fig. 1, 1, 4-dioxane, benzene, ethylbenzene and toluene were individually degraded by 1,
4-dioxane-grown cells of DD1 under the condition of
initial substrate concentrations of 0.5 mM and biomass of 60 mg/L. 1, 4-dioxane degradation started
without a lag and finished in 10 h with the biodegradation rate of 73.33 mg/(h g dry weight). Conversely,
the degradation of benzene, ethylbenzene and toluene
proceeded with a lag period of approximately 14 h. In
a mixture with 1, 4-dioxane, the biodegradation of
benzene, ethylbenzene and toluene also did not
commence in 14 h. Rather, the degradation of 1,
4-dioxane in the mixture only started when benzene,
ethylbenzene or toluene was completely degraded and
the biodegradation rate slowed significantly. Moreover, the biodegradation rate of 1, 4-dioxane in the
mixture with benzene, toluene or ethylbenzene
decreased to 40.74, 30.55 or 16.29 mg/(h g dry
weight). These observations were similar to those

Biodegradation (2016) 27:3746

41

0.55
0.50

Concentrion (mmol)

0.45

B alone
B(with D)
T alone
T(with D)
D alone
D(with B)
D(with T)
D(control)
B(control)
T(control)

0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0

10

20

30

40

50

60

70

80

Time (h)

0.55
0.50

Concentration (mmol)

0.45
0.40

E alone
E(with D)
O alone
O(with D)
D alone
D(with O)
D(with E)
D(control)
E(control)
O(control)

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0

10

20

30

40

50

60

70

80

90

100 110

Time (h)

Fig. 1 Biodegradation by 1, 4-dioxane-grown cells of DD1:

a Effect of 1, 4-dioxane (D), benzene (B) and Toluene
(T) interaction; b Effect of 1, 4-dioxane (D), ethylbenzene
(E) and o-xylene (O) interaction. The initial culture density and
substrate concentration in each bottle was respectively 60 mg/L
and 0.5 mM. The error bars represent the standard deviations of
triplicate samples

previously reported for the mixture degradation process of MTBE in the presence of benzene, where the
utilization rates of MTBE declined concurrent with the
biotransformation of benzene (Deeb et al. 2001).
In general, the degradation trends observed in
toluene/1, 4-dioxane mixtures and ethylbenzene/1,
4-dioxane mixtures were similar to those of the
benzene/1, 4-dioxane mixtures. As shown in Fig. 1a,
the degradation of toluene, which had a greater
inhibitory effect on 1, 4-dioxane removal, was slower
than that of benzene. As reported for Pseudomonas
strain (PPOI), the biodegradation of benzene was
inhibited by toluene (Young-Sook et al. 1994). It was
possible that the influence and toxicity of toluene was
more serious than that of benzene toward P. strain

(PPOI). A similar phenomenon may also have

occurred for A. baumannii DD1.
1, 4-dioxane-grown cells of DD1 were unable to
degrade 0.5 mM m-xylene or p-xylene, either alone or
in a mixture with 1, 4-dioxane, following an incubation period of over 1 week (data not shown). Moreover, the presence of m-xylene or p-xylene in mixtures
with 1, 4-dioxane completely inhibited the biodegradation of 1, 4-dioxane. Unlike m-xylene and p-xylene,
o-xylene was degraded after a 14 h lag phase, both
alone and in a mixture with 1, 4-dioxane. However, 1,
4-dioxane was not degraded, even after o-xylene was
completely degraded.
According to the biodegradation of 1, 4-dioxane
and BTEX, all BTEX inhibited the biodegradation of
1, 4-dioxane, indicating that the inhibitory effects
were not competitive. Moreover, the inhibition of 1,
4-dioxane was irreversible for xylenes, while the
ability to transform 1, 4-dioxane recurring after
removal of BTE.
The interaction of 1, 4-dioxane and BTEX may be
attributed to enzyme competition, intermediates toxicity and reducing energy deficits (Marion et al. 2002;
Mahendra et al. 2013). Additional experiments were
performed to verify which mechanisms played the
leading role in the process.
Detection of the intermediates in BTEX
biodegradation by strain DD1
In the mixture of BTEX and 1, 4-dioxane, the
biodegradation of 1, 4-dioxane was completely inhibited by BTEX. During the biodegradation of 1,
4-dioxane and BTEX (excluding o-xylene), the color
of the culture did not change. The culture of 1,
4-dioxane and o-xylene changed from colorless to
yellow and produced an offensive odor. Moreover, the
color remained for some time after the biodegradation
of o-xylene was complete. Comparison of the chromatograms generated upon GC analysis revealed that
the biodegradation of BTEX did not result in the
accumulation of any obvious intermediates other than
o-xylene. Because biodegradation of 1, 4-dioxane did
not begin even after o-xylene was degraded, there may
have been other toxic intermediates produced during
o-xylene degradation.
Figure 2 shows the fragment ions and chromatograms of metabolites obtained upon GC/MS.
One peak with a retention time of 17.095 min was

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Biodegradation (2016) 27:3746

30000000

12 h

24 h

36 h

Peak Height

25000000
20000000

48 h

2,3-dimethylphenol

have been toxic to A. baumannii DD1. Taken together,

these phenomena implied that the accumulation of oxylene intermediates (2, 3-dimethylphenol) was toxic
to A. Baumannii DD1 and perhaps the cause of 1,
4-dioxane inhibition.

15000000

Variation in enzymes in biodegradation

of mixtures by 1, 4-dioxane-grown DD1

glycerol

10000000
2,3-dimethylphenol

5000000

2,3-dimethylphenol
2,3-dimethylphenol

0
17.095

17.095

17.095

17.095

Retention time (min)

10000
2,3-dimethylphenol (derivatized by BSTFA)

9000

Relative abundance

H3C

179.0 194.0

105.0

CH 3

8000

Si
O
CH3

7000

CH3

6000
5000

CH3

149.0

4000
3000
73.0

2000

164.0

45.0

1000

121.0

27.0

0
0

20

40

60

80

100 120 140 160 180 200

m/z

Fig. 2 The detection of the intermediates accumulation (analyzed as BSTFA derivatives) during the biodegradation of oxylene with 1, 4-dioxane

identified by MS as 2, 3-dimethylphenol in the BSTFA

form. During the degradation of o-xylene, the concentration of 2, 3-dimethylphenol increased. After oxylene was completely degraded, the concentration of
2, 3-dimethylphenol started to decline, whereas 1,
4-dioxane degradation had not started at 85 h. These
results were in agreement with the o-xylene catabolism pathway, in which it was transformed by initial
oxidation to 2, 3-dimethylphenol, then further transformed to citric acid cycle intermediates by a Pseudomonas stutzeri strain in Fig. 3.a (Grazia et al. 1987).
An additional experiment was conducted to investigate the inhibition of 1, 4-dioxane in the mixture of oxylene and 1, 4-dioxane. After 85 h of incubation, the
degradation of 1, 4-dioxane did not start, even under the
condition of 0.05 mM 2, 3-dimethylphenol. According
to a previous study (Kahru et al. 2000), the range of
L(E)C50 of 2, 3-dimethylphenol was 5.2 to 190 mg/L,
which was categorized as toxic to aquatic organisms.
These findings suggested that 2, 3-dimethylphenol may

123

The degradation of 1, 4-dioxane was inhibited by the

accumulation of toxic intermediates from o-xylene.
However, during the degradation of BTE with 1,
4-dioxane, there was no obvious intermediate accumulation. To investigate the cause of the lag phase of
BTE and inhibition of 1, 4-dioxane, the reaction
system was supplemented with an additional 0.5 mM
benzene after the initial 0.5 mM benzene was completely depleted. As shown in Fig. 4, sequential
additions of benzene resulted in degradation without
a delay, and the time required for the 1st cycle
degradation was 34 h, whereas it was only 8 h in the
2nd cycle and 6 h in the 3rd cycle. Following the
depletion of benzene, the addition of toluene did not
induce any lag period in degradation. These findings
suggested that toluene was degraded via the similar
pathway as benzene. However, the addition of 1,
4-dioxane induced a long lag phase of about 6 h,
which was presumably because of the need for
induction of enzymes necessary for degradation. Once
these enzymes were induced, sequential additions of
benzene or toluene were degraded rapidly.
As we know, monooxygenases are the enzymes
which involved in the initial catalysis step of 1,
4-dioxane degradation in several bacteria (Mahendra
and Alvarez-cohen 2006). Moreover, we previously
confirmed that the biodegradation of 1, 4-dioxane was
completely inhibited in the presence of acetylene
[acetylene is an inhibitor of monooxygenase activity
(Mahendra and Alvarez-cohen 2006)], which supports
that the dominant enzyme in the degradation of 1,
4-dioxane by A. baumannii DD1 was monooxygenase
(Huang et al. 2014). Because 1, 4-dioxane and benzene
were degraded via independent enzymes, the primary
enzymes in the biodegradation of BTE were not
monooxygenases. Many studies have confirmed that
the catabolic pathway of BTE leads to catechol by
dioxygenase and further transformation to citric acid
intermediates in Fig. 3b (Zhang et al. 2014). During the
biodegradation of benzene, the catechol dioxygenase

Biodegradation (2016) 27:3746

43
OH

a
CH3

OH
CH3

monooxygenase

HO

CH3

monooxygenase

extra-diol
cleavage

CH3

CH3

CH3
O
OH

1,2-dioxygenase

OH
OH

O2

O
OH

TCA cycle

2,3-dioxygenase
COOH

OH

Fig. 3 a The proposed initial step for the metabolism of o-xylene by Pseudomonas stutzeri strain (Grazia et al. 1987). b The proposed
catabolic pathways of benzene by aerobic microorganism (Zhang et al. 2014)

0.6

toulene 1,4-dioxane
benzene benzene

benzene

Concentration (mmol)

0.5
0.4
0.3
0.2
0.1

B (T, dioxane)
B killed control

0.0

10

20

30

40

50

60

70

80

Time (h)

Fig. 4 Biodegradation by 1, 4-dioxane-grown cells of DD1 of

benzene. Immediately the depletion of benzene, an aliquot of
benzene or toluene or 1, 4-dioxane was added

activities were analyzed, and the specific activity of

catechol 2, 3-dioxygenase was found to be below
0.01 U/mg protein, while the specific activity of
catechol 1, 2-dioxygenase was 13 U/mg protein
(Fig. 5a). These findings showed that the aromatic ring
was opened via the ortho-cleavage pathway by DD1.
Figure 5 shows the catechol 1, 2-dioxygenase and
monooxygenase activities during the degradation of
benzene (Fig. 5a), 1, 4-dioxane (Fig. 5b) and the
mixture (Fig. 5c). The biodegradation of 1, 4-dioxane

was complete in 10 h when 1, 4-dioxane was used as

the sole carbon source. In addition, the monooxygenase activity was induced without any lag and
continuously enhanced up to a maximum of 3.5 U,
after which it decreased. During the degradation of
benzene by 1, 4-dioxane-grown-cells, 1, 2-dioxygenase activity was not induced until approximately
14 h. The activity of the enzyme increased quickly,
reaching the maximum at 25 h. Interestingly, the
tendency for induction of the 1, 2-dioxygenase
corresponded to the regulation of biodegradation of
benzene. During biodegradation of the mixture with
benzene and 1, 4-dioxane, the degradation pathway of
1, 4-dioxane was inhibited and the secretion of
monooxygenase was restrained. After a lag of 14 h,
1, 2-dioxygenase secretion was induced and the
activity increased rapidly. Meanwhile, the rate of
benzene degradation peaked at 30 h, with no benzene
detected at 34 h. The concentration of 1, 2-dioxygenase declined after benzene was no longer detected.
The biodegradation of 1, 4-dioxane did not begin for
6 h due to the inhibitory effects. Monooxygenase was
not expressed during the early biodegradation stage,
but increased at a later time point (about 38 h) to a
maximum of 2.2 U. However, the maximum activity
of monooxygenase in the mixture was lower than the
maximum (3.5 U) observed when 1, 4-dioxane was
the sole carbon source. Moreover, the biodegradation

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Biodegradation (2016) 27:3746

15

0.3
0.2

9
6
3

0.1
0.0
0

10

15

20

25

30

0
0

35

10

15

Time (h)

20

25

30

35

40

Time (h)
4.0

0.5

Monooxygenase

3.5

1,4-dioxane

0.4

3.0

U/mg

Concentrion (mmol)

Catechol 1,2 dioxygenase

12

Benzene

0.4

U/mg

Concentrion (mmol)

0.5

0.3
0.2

2.5
2.0
1.5
1.0

0.1

0.5
0.0

0.0 2

10

10 12 14 16 18 20 22

Time (h)

Time (h)

c
14

0.5

Catechol-1,2-dioxygenase
Monooxygenase

0.4
10
0.3

U/mg

Concentrion (mmol)

12

0.2

6
4

Benzene
1,4-dioxane

0.1

0.0
0

10 15 20 25 30 35 40 45 50 55

Time (h)

0
0 5 10 15 20 25 30 35 40 45 50 55 60

Time (h)

Fig. 5 a The catechol dioxygenase activity and biodegradation

profiles during degradation of benzene. b The monooxygenase activity and biodegradation profiles during degradation
of 1, 4-dioxane. c The catechol dioxygenase activities,

monooxygenase activities, and biodegradation profiles during

degradation of 1, 4-dioxane and benzene mixtures. The error
bars represent the standard deviations of triplicate samples

rate of 1, 4-dioxane was reduced from 73.33 mg/(h g

dry weight) to 40.74 mg/(h g dry weight), suggesting
that benzene or the intermediates of benzene may also
influence the activity of DD1. The induction of
different enzymes during degradation of the mixture
implied that the induction of dioxygenase occurred
prior to that of monooxygenase under aerobic conditions, and that dioxygenase might affect the secretion
of monooxygenase.

To investigate the molecular weight of proteins

during benzene degradation and 1, 4-dioxane degradation, polyacrylamide gel electrophoresis was
applied to the bacterial protein extracts. The molecular
weights of proteins in DD1 grown in 1, 4-dioxane or
benzene primarily ranged from 29.0 to 66.2 kDa
(Fig. 6). There were many common protein bands
observed between the two samples, indicating that
most of these proteins were not unique during

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45

the secreted proteins were found to be different by

polyacrylamide gel electrophoresis. These findings
will contribute to improvement of the available
approaches for engineered bioremediation.
Acknowledgments This work was financially supported by
the Fundamental Research Funds for Zhejiang Gongshang
University [3100XJ1514154].

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Fig. 6 SDS-PAGE of proteins from cells. L1 ,L2: proteins

extract of the benzene-grown DD1; L3, L4: proteins extract of
the 1, 4-dioxane-grown DD1; L5: the molecular weight markers

degradation of the contaminant. There were only four

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Fig. 6, while the diverse proteins induced by 1,
4-dioxane were b and d. These different proteins
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Conclusion
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