Journal of Hazardous Materials 401 (2021) 123347

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Journal of Hazardous Materials

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Biodegradation of dioxins by Burkholderia cenocepacia strain 869T2: Role of T

2-haloacid dehalogenase
Bao-Anh Thi Nguyena,g, Ju-Liang Hsieha, Shou-Chen Loa, Sui-Yuan Wanga, Chung-Hsiung Hungd,
Eugene Huangc, Shih-Hsun Hungb, Wei-Chih Chine,f,*, Chieh-Chen Huanga,**
a
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
b
Department of Horticulture, National Chung Hsing University, Taichung, Taiwan, Republic of China
c
College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan, Republic of China
d
Department of Environmental Engineering, National Chung Hsing University, Taichung, Taiwan, Republic of China
e
General Research Service Center, National Pingtung University of Science and Technology, Pingtung, Taiwan, Republic of China
f
Department of Biological Sciences and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan, Republic of China
g
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Editor: G. Lyberatos Dioxin compounds are persistent carcinogenic byproducts of anthropogenic activities such as waste combustion
Keywords: and other industrial activities. The ubiquitous distribution of dioxins is global concerns these days. Among of
Burkholderia cenocapacia 869T2 recent techniques, bioremediation, an eco-friendly and cost-effective technology, uses bacteria or fungi to de-
Haloacid dehalogenase toxify in dioxins; however, not many bacteria can degrade the most toxic dioxin congener 2,3,7,8-tetra-
Dioxin dehalogenation chlorinated dibenzo-p-dioxin (TCDD). In this study, the endophytic bacterium Burkholderia cenocapacia 869T2
Bioremediation was capable of TCDD degradation by nearly 95 % after one-week of an aerobic incubation. Through tran-
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) scriptomic analysis of the strain 869T2 at 6 -h and 12 -h TCDD exposure, a number of catabolic genes involved in
dioxin metabolism were detected with high gene expressions in the presence of TCDD. The transcriptome data
also indicated that B. cenocepacia strain 869T2 metabolized the dioxin compounds from an early phase (at 6 h) of
the incubation, and the initial outline for a general dioxin degradation pathway were proposed. One of the
catabolic genes, L-2-haloacid dehalogenase (2-HAD) was cloned to investigate its contribution in dioxin deha-

⁎
Corresponding author at: General Research Service Center, National Pingtung University of Science and Technology, Pingtung, Taiwan, Republic of China.
⁎⁎
Corresponding author.
E-mail addresses: jason70068@mail.npust.edu.tw (W.-C. Chin), cchuang@nchu.edu.tw (C.-C. Huang).

https://doi.org/10.1016/j.jhazmat.2020.123347
Received 13 April 2020; Received in revised form 10 June 2020; Accepted 28 June 2020
Available online 03 July 2020
0304-3894/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
B.-A.T. Nguyen, et al.
logenation. By detecting the increasing concentration of chloride ions released from TCDD, our results indicated
that the dehalogenase played a crucial role in dehalogenation of dioxin in the aerobic condition.
1. Introduction
Dioxins are a group of halogenated aromatic compounds including
polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzo-
furans (PCDFs) and dioxin-like polychlorinated biphenyls (PCBs). These
carcinogenic xenobiotics originated from natural and anthropogenic
sources that are mostly byproducts of industrial productions (herbi-
cides, pesticides, or chlorinated compounds) and combustion processes
((EPA), E.P.A., 2004). Among of PCDDs and PCDFs congeners, 2,3,7,8 -
tetrachlorinated dibenzo-p-dioxin (TCDD) is the most toxic dioxin
compound and classified into Group I carcinogens (Van den Berg et al.,
2006). During the Vietnam - U.S war, the U.S army sprayed 45 million
liters of a toxic herbicide Agent Orange with impurities of about 150 kg
of 2,3,7,8-TCDD in Southern Vietnam (Young et al., 2004; Stone, 2007).
In recent reports, food sources, soils, blood and breast milk samples of
local people at severe dioxin hot spots in Vietnam have been found to
have high dioxin-contamination levels (Huyen et al., 2015; Tuyet-Hanh
et al., 2015). In Taiwan, a large area of soils and sediment in An-shun
Plant was contaminated by PCB, PCDDs and PCDFs (Soong et al., 1997;
Lee et al., 2006; Chen et al., 2016).
Due to low water solubility and high octanol–water partition coef-
ficients, dioxins are strongly persistent in soil and sediments, con-
taminating the food chain and causing cancer (Cole et al., 2003).
Bioremediation has been proposed as a natural and cost-effective
method which applies microorganisms to degrade or remove toxic
substances (Vidali, 2001). Since the mid-1970s, dioxin biodegradation
by isolated microorganisms had been studied (Kearney et al., 1972;
Matsumura and Benezet, 1973; Ward and Matsumura, 1978). The
higher chlorinated dioxins can be dechlorinated in anaerobic environ-
ments by reductive dehalogenases of halorespiring bacteria, such as
Dehaloccocoides genus (Bunge et al., 2003; Pöritz et al., 2015). In
aerobic conditions, a variety of microorganisms is can degrade lower
chlorinated dioxins by an initial attack of angular dioxygenases from
Sphingomonas, Pseudomonas and Burkholderia (Field and Sierra-Alvarez,
2008). Although numerous anaerobic or aerobic microorganisms are
capable of degrading dioxins, not many bacterial strains can degrade
the most toxic dioxin 2,3,7,8-TCDD (Sakaki and Munetsuna, 2010).
In bioremediation of dioxins, angular dioxygenase, cytochrome
P450, lignin peroxidase and dehalogenases are renowned as important
dioxin-smetabolic enzymes (Sakaki and Munetsuna, 2010). Dehalo-
genases catalyze dehalogenation, the key initial reaction in biode-
gradation of halogenated compounds (Janssen et al., 2001). In deha-
logenation, halogen ions are cleaved from carbon-halogen bonds of
halogenated compounds by dehalogenase. Among of more than 3800
recorded halogenated organic pollutants, dioxins are well-known as
halogenated compounds containing many chloride substituents
(Gribble, 2003). These halogen substituents in halogenated compounds
restrict catabolic enzymatic activity due to their steric effects, thereby
decrease biodegradation kinetics and block the first reaction in cata-
bolism of dioxins or other compounds (Jeon et al., 2013). Therefore,
chloride removal is critical to reduce toxicity and risks of forming toxic
intermediates during the degradation processes (Gribble, 2003). To
date, the dioxin dehalogenation by haloacid dehalogenase activity in
aerobic condition remains unknown (Sakaki and Munetsuna, 2010).
Burkholderia cenocepacia strain 869T2, a gram-negative
Proteobacterium isolated from roots of vetiver grass, is a bio-controller
and bio-fertilizer in banana plants (Ho et al., 2015). Notably, the
genome of the strain 869T2 contains many genes related to dioxin
degradation (Ho and Huang, 2015), thus the strain was selected for the
aerobic incubation. The study aimed to three main objectives: (1) to
2
Journal of Hazardous Materials 401 (2021) 123347
investigate TCDD-degradation kinetics of B. cenocepacia 869T2, (2) to
assess the transcriptome profile of the strain 869T2 under TCDD in-
cubations, which might give clues about the pathway of dioxin de-
gradation, and (3) to identify the activity and roles of the enzyme ha-
loacid dehalogenase in dioxin dehalogenation.
2. Materials and methods
2.1. Growth conditions and biodegradation of TCDD by B. cenocepacia
869T2
After 12−16 hours growth in Luria broth (LB) (10 g L−1 tryptone,
5 g L−1 yeast extract, 10 g L−1 NaCl), B. cenocepacia strain 869T2 was
added into 250 mL LB medium in 500 mL Erlenmeyer flasks at a final
OD600 nm value of 0.1. Treated mediums with three replicates contained
0.2 mg L−1 TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin; purity: 98.7 %,
CAS Number: 1746−01-6, AccuStandard, USA) and a surfactant Tween
80 (at 0.1 % (v/v)) (CAS Number: 9005−65-6; Acros Organics, Thermo
Fisher Scientific, UK). The controls with three replicates were under the
same culture but without TCDD addition. The cultures were incubated
at 28 ℃ and 200 rpm in an orbital shaker incubator (LM-570D/LM-
570RD, Yihder) in the dark. The cell density was monitored by OD600
nm using a ND-1000 spectrophotometer (Nanodrop Technology, USA)
for seven days.
2.2. TCDD Measurement in culture medium
1 mL of each sample (n = 3) was collected in 3 mL brown glass vials
during the incubation with B. cenocepacia 869T2 or recombinant
strains. TCDD from sample mediums was extracted twice with addition
of ethyl acetate. The organic layers were dried with anhydrous mag-
nesium or sodium sulfate, and the solvents containing TCDD were
transferred to new glass vials and evaporated in vacuum at 30 ℃. After
evaporation of the solvent, the residues in the vials were dissolved with
1 mL methanol and fractionated by high-performance liquid chroma-
tography (HPLC) (Fortnagel et al., 1990).
Stock standard concentrations of TCDD at 0.002; 0.005; 0.01; 0.1
and 0.2 mg L−1 were prepared in methanol with three replicates (1 mL
for each). For recovery experiments, three standard TCDDs (0.01, 0.1
and 0.2 mg L−1; three replicates for each standard) were spiked into
vials containing 1 mL growth medium and then extracted by the above-
mentioned extraction method to estimate the recovery rate. HPLC
analysis was performed using PLATINblue ultra high-performance LC
(UHPLC) system - Version A.01.05 (KNAUER, Japan) with a UV de-
tector at 254 nm and the NPE column (5 μm, 4.6 mm I.D. × 150 mm)
packed with particles of nitrophenylethyl group bonded. TCDD was
eluted by a mobile phase of methanol - water (95 % - 5%) at flow rate of
1 mL/ min. The injection volume was 10 μL, and retention time for
TCDD was 2.1 min under this condition.
Linearity of standard curves was studied in the range of
0.002−0.2 mg L−1 (0.002; 0.005; 0.01; 0.1 and 0.2 mg L−1). TCDD
concentrations were determined by using a linear equation:
23260318.125X + 21156.83 with R2 = 0.996. Limit of detection and
limit of quantification of TCDD were 0.0276 μg L−1 and 0.0835 μg L−1,
respectively. For the recovery rate, the mean recovery of TCDD com-
pounds across the entire analytical method was 91.50 ± 6.89 % in a
range of 80.27–98.50% (n = 12).
B.-A.T. Nguyen, et al.
2.3. Transcriptome analyses
The transcriptomic sample extraction, preparation, sequencing, and
data analysis of B. cenocepacia 869T2 was performed by Illumina Solexa
technology (Welgene Biotech Company, Taiwan). Treatment
(0.2 mg L−1 TCDD) and control (no TCDD) samples of B. cenocepacia
896T2 cultures at 6 h (at early log-phase) and 12 h (at middle log-
phase) of growth were collected, and total RNA was extracted from
each. The whole genome sequence of Burkholderia cenocepacia 869T2
(accession number NZ_JJOA00000000, the taxon name has been given
as JJOA_s) served as the reference genome (Ho and Huang (2015)).
All procedures were carried out according to the manufacture’s
protocol from Illumina. Total RNA was extracted by Trizol Reagent
(Invitrogen, USA) according to the instruction manual. RNA purified
was quantified at OD260 nm by using a ND-1000 spectrophotometer
(Nanodrop Technology, USA) and qualitated by using a Bioanalyzer
2100 (Agilent Technology, USA) with RNA 6000 labchip kit (Agilent
Technologies, USA). Library construction of all samples were used by
Agilent's SureSelect Strand Specific RNA Library Preparation Kit for
75SE (Single-End or Paired-End) sequencing on Solexa platform. The
sequence was directly determined using sequencing-by-synthesis tech-
nology via the TruSeq SBS Kit. Raw sequences were obtained from the
Illumina Pipeline software bcl2fastq v2.0 and expected to generate
10 M (million reads) per sample. For RNA-seq Analysis, initially, the
sequences generated went through a filtering process to obtain qualified
reads. A flexible read trimming tool for Illumina NGS data
Trimmomatic was implemented to trim or remove the reads according
to the quality score. Qualified reads after filtering low-quality data were
analyzed using TopHat/Cufflinks for gene expression estimation. The
gene expression level was calculated as FPKM (Fragments per Kilobase
of transcript per Million mapped reads) (FPKM > 0.3 and P < 0.05).
For differential expression analysis, an R package CummeRbund de-
signed to aid and simplify the task of analyzing RNA-Seq output was
employed to perform statistical analyses of gene expression profiles.
The reference genome and gene annotations were retrieved from
Ensembl database.
2.4. Construction of the recombinant strain Escherichia coli HAD
The enzyme 2-haloacid dehalogenase (2-HAD) sequence was syn-
thesized according to gene 3580 (locus_tag DT99_RS17940) of B. cen-
ocepacia 869T2 available from the National Center for Biotechnology
Information (NCBI) (Ho and Huang, 2015). Plasmid pET-24A(+) was
digested by NdeI and EcoRI, and then the 2-HAD gene sequence frag-
ments were inserted on the cloning sites on the vectors pET24A(+). To
Fig. 1. Growth of B. cenocepacia 869T2 in LB medium with and without 0.2 mg L−1 TCDD (A); TCDD concentrations after inoculation and un-inoculation with the
strain 869T2 after 7 days (B). Error bars are standard deviations (n = 3).
3
Journal of Hazardous Materials 401 (2021) 123347
check the recombinant plasmid DNA, restriction enzyme ApaI and XhoI
digestion was performed, and digested fragments were confirmed by
agarose gel electrophoresis and purified from the gel following the
manufacturer's protocols. The recombinant plasmids were transformed
into competent Escherichia coli strain BL21 (DE3) for haloacid dehalo-
genase production, and control strains were E. coli strain pET24A
(containing the plasmid pET24A(+) without 2-HAD).
2.5. Screening for enzyme activity
There were three treatments with three replicates for each treat-
ment, including one HAD-inoculated sample and two controls (pET24A-
inoculated sample and no-bacteria sample). Overnight cultures
(12−16 hours) of the target recombinant strains HAD and the control
strain pET24A were cultured in 96-well micro-titre plate containing
one-fifth-strength LB medium (without NaCl) supplemented with the
substrate ( ± )-2-chloropropionic acid (2-CPA; 92 %, CAS: 598−78-7,
Sigma, Taiwan) and kanamycin (50 μg mL−1). The 96-well plate was
incubated for two days at 37 ℃, and then 10 μL of 0.1 M AgNO3 solution
was added into each well. The haloacid dehalogenase activity was
identified by white precipitate of AgCl that formed from free Cl- ions
releasing from TCDD (Murdiyatmo et al., 1992). The formation of white
precipitation was AgCl produced by a reaction of AgNO3 solution with
Cl- ions releasing from 2-CPA. The presence of 2-CPA played a role as
an inducer of haloacid dehalogenase activity of the recombinant strain
HAD to convert the substrate to a hydroxyalkanoic acid form and re-
lease chloride ions in medium. The controls without the HAD activity
were not observed the white precipitation.
2.6. Protein preparation and extraction
Overnight cultures (12–16 hours) of the recombinant strains HAD
and pET24A were inoculated into 500 mL fresh LB medium in 1000 mL
Erlenmeyer flasks with antibiotic kanamycin (50 μg mL−1) at 37 ℃ and
200 rpm. When the OD600 nm values reached 0.8–1.0, 1 mM isopropyl β-
D-1-thiogalactopyranoside (IPTG) was added as mRNA inducer for
cloned genes, and the culture was incubated for another 3–12 hours.
After that, cells from 500 mL culture were sedimented by centrifugation
(11,000 x g, 4 ℃ and 15 min). The pellet was washed twice with 0.1 M
Tris-acetate, 1 mM EDTA, 10 % (mass/volume) glycerol, pH 7.5, and
centrifuged at 10,000 x g, 4 ℃ for 10 min. The cells were then sus-
pended in 4 mL of the same buffer and maintained at 0 ℃ for sonication.
Unbroken cells and cell wall materials were removed by centrifugation
at 22, 000 x g, 4 °C for 30 min, and the supernatants were decanted and
kept at 4 °C. 20 μL of protein samples was mixed with 5 μL Protein
B.-A.T. Nguyen, et al.
loading dye (5X), and placed on 100 ℃ for 5−10 min to prepare
samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) analysis.
2.7. Growth condition and TCDD dehalogenation by recombinant strains
Overnight cultures (12–16 hours) of the recombinant strains E. coli
strain HAD and strain pET24A were added (OD600 nm value = 0.05)
into 100 mL defined medium (Na2HPO4.12 H2O: 5.37 g/L; KH2PO4:
1.36 g/L; (NH4)2SO4: 0.5 g/L; MgSO4.7H2O: 0.2 g/L; Yeast Extract:
30 mg/L; 10 % (w/v) glucose to a final concentration: 0.5 g/L) in
250 mL Erlenmeyer flasks. Treated samples (n = 3) contained kana-
mycin (50 μg mL−1), 0.1 mg L-1 TCDD and Tween 80 (0.1 % (v/v)), and
the controls were without TCDD. After 1.5–2 hours with the bacterial
OD600nm of about 0.8–1.0, 1 mM IPTG was added into the growth cul-
tures for induction. The cultures were incubated at 37 ℃, 200 rpm in
the dark, and the cell density was monitored by the OD600nm using a
ND-1000 spectrophotometer (Nanodrop Technology, USA).
2.8. Chloride ions measurement
Samples of the recombinant strains in TCDD treatments were col-
lected every 2 h and filtered through Minisart-Plus filters with a 0.2 μm
pore size to remove bacterial cells. The mount of chlorine ions released
in the medium was measured by 883 Intelligent Ion Chromatography
(Metrohm, Switzerland) and Metrosep A Supp 5–150/4.0 (6.1006.520)
IC column (Hammer Trading Co., Taiwan). Sodium hydrogen carbonate
(84 mg L−1) and sodium carbonate (339 mg L−1) were used as carbo-
nate eluent, and the injection volume and standard flow were 1.0 μL
and 0.7 mL min−1, respectively.
3. Results and discussion
3.1. Biodegradation of TCDD by B. cenocepacia strain 869T2
After the seven-day induction with TCDD, B. cenocepacia strain
869T2 had a similar growth rate in both control and TCDD-treated
conditions, and their growths were not affected by TCDD toxicity
(Fig. 1A). In TCDD medium, the cell population exponentially increased
and reached the maximum growth with OD600 nm value of 8.1 at 21 h,
which closed to the highest point in the control medium (OD600 nm
value = 8.3). After that, the cell density of B. cenocepacia 869T2 was
gradually decreased in both two different conditions.
TCDD concentration in the 896T2-inoculated medium was de-
creased within the first day (Fig. 1B), corresponding to the exponential
growth of B. cenocepacia 869T2 in 0.2 mg L−1 TCDD (Fig. 1A). While
the bacteria reached the maximum population at 21 h, a substantial 92
% of TCDD concentration (from 0.2 mg L−1 at 0 h to 0.015 mg L−1 at
21 h) was removed at the same time. The concentration of TCDD was
slightly decreased with a reduction of the OD600 nm value, and a total
94.8 % of the TCDD concentration was declined after the one-week
exposure (Fig. 1A, B). The results indicated that B. cenocepacia 869T2
could degrade TCDD compounds with a high effectiveness.
3.2. Transcriptome profiling
To identify differentially expressed genes upon exposure to
0.2 mg L−1 TCDD, the growing cells of B. cenocepacia strain 869T2 at
the early-phase (6 h) and mid-phase (12 h) were collected and analyzed
the transcription. There were no significant differences in the tran-
scriptomic profile between the treated groups and the control groups
(Fig. S1A, B). However, in comparison with the controls, TCDD caused
upregulation of 603 and 1264 genes (≥ 2-fold change) at 6 and 12 h,
respectively (Table S1-A, B, C). Notably, numerous catabolic genes,
involved in aromatic compound and dioxin metabolism, of B. cen-
ocapacia strain 869T2 increased the expressions in response to TCDD,
4
Journal of Hazardous Materials 401 (2021) 123347
which might participate in TCDD degradation processes (Tables 1,2, S2,
S3 and S4). In Tables 1,2 and S2, related ring-cleavage dioxygenases,
hydrolase, dehalogenases and cytochrome P450 highly elevated ex-
pression levels at 6 and 12 h, which also had been found in genome-
wide gene expression of aerobic bacteria, especially a representative
Sphingomonas wittichii strain RW1 in metabolism of many PCDD, PCDF
and PCB in previous studies (Nam et al., 2006; Bünz and Cook, 1993;
Wittich et al., 1992). Sakaki and Munetsuna (2010) also indicated that
the genes cytochrome P450 and haloacid dehalogenase encode the
corresponding dioxin-metabolic enzymes in dioxin biodegradation
(Sakaki and Munetsuna, 2010). Notably, cytochrome P450 of B. cen-
ocapacia strain 869T2 was identified with up-regulation but not re-
cognized in the genomic and the proteomic studies of the S. wittichii
strain RW1 in response to PCDDs and PCDFs (Chai et al., 2016;
Coronado et al., 2012; Hartmann and Armengaud, 2014). For haloacid
dehalogenase, it is one representative of dehalogenase catalyzing for
cleaving halogen and carbon bond in halogenated compounds. How-
ever, the roles of haloacid dehalogenase in dioxin metabolism remain
unclear.
Additionally, TCDD triggered upregulation of many genes sup-
porting to cellular metabolisms and dioxin degradation, including
cross-membrane transport (TonB-dependent receptor proteins), toxicity
response and cell protection (Table S5). Many TonB-dependent re-
ceptors gene2004 and gene3111 had approximately 8-fold change at
6 h and 10-fold change for the gene3111 at 12 h in compared to controls
(Table S5). These membrane proteins play important roles in iron and
vitamin B12 uptake (Koebnik et al., 2000), particularly iron for dioxin
degradation reactions (Hartmann and Armengaud, 2014), signaling and
transportation of complex carbohydrates (Koebnik et al., 2000;
Blanvillain et al., 2007) and tolerance of aromatic compounds by
Pseudomonas putida DOT-T1E (Godoy et al., 2001) or PCDDs and PCDFs
by S. wittichii RW1 (Chai et al., 2016Hartmann and Armengaud, 2014).
With various substrates and high expressions at different times, TonB-
dependent receptor genes might take up necessary substrates for cel-
lular metabolic reactions and transport dioxins or aromatic compounds
into the cells for subsequent degradation.
TCDD also induced high regulation of toxicity response genes and
antioxidant genes involved in cell protection (Table S5). Notably, an
antioxidant alkyl hydroperoxide reductase subunit F gene increased
expression with more than 1300-fold change at 6 h under TCDD toxi-
city. The differential gene regulations at different time points might be
explained by substrate ranges and toxicity. The toxicity of TCDD, other
PCDDs and PCBs leads to some specific stresses to bacterial cells (DNA,
oxidative, and protein damages), and only DNA-related damage by
TCDD has been reported (Chai et al., 2016; Min et al., 2003). The result
indicated that the high expression of those genes might mediate stresses
of TCDD and other toxic intermediates produced within the degradation
processes, thereby facilitated the exponential growth and effective
TCDD degradation of the B. cenocapacia strain 869T2.
3.3. Haloacid dehalogenase - genes related to dioxin dehalogenation
Upon TCDD exposure, three haloacid dehalogenases (HADs)
Table 1
Expressions of haloacid dehalogenases (HADs) involving in TCDD dehalo-
genation.
Gene ID Description Expression, Fold change (TCDD/
Control)
6h 12 h TCDD (12/6 h)
gene3580 MULTISPECIES: haloacid 1.45 0.93 2.28
dehalogenase
gene6828 MULTISPECIES: dehalogenase 2.97 0.36 0.20
gene388 haloacid dehalogenase 1.24 0.62 0.55
B.-A.T. Nguyen, et al.
Table 2
Expression of genes related to the upper pathway of TCDD degradation.
Gene ID Description
st
1 Step
gene2961 aromatic-ring-hydroxylating dioxygenase
gene5127 MULTISPECIES: ferredoxin-NADP(+) reductase
gene3660 MULTISPECIES: ferredoxin, 2Fe-2S type, ISC system
2nd Step
gene4676 MULTISPECIES: glyoxalase/bleomycin resistance/dioxygenase family protein
gene6989 glyoxalase/bleomycin resistance/dioxygenase family protein, partial
gene2099 dioxygenase
3rd Step
gene6609 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase
encoding the enzymes dehalogenases for dehalogenation were detected
with up-regulation at 6 h in comparison to the control condition
(without TCDD effects) (Table 1). The result indicated that haloacid
dehalogenases activated at the early phase in TCDD incubation and
might play a pivotal role in TCDD dehalogenation.
In previous studies, some haloacid dehalogenases dechlorinated
OCDD by Pseudomonas mendocina strain NSYSU in anaerobic treatment
(Tu et al., 2014) and had high gene expressions response to dioxins by
the strain RW1 in an aerobic condition (Chai et al., 2016). Haloacid
dehalogenases thus are possibly able to dehalogenate dioxin com-
pounds in both aerobic and anaerobic conditions. However, the func-
tions and activity of haloacid dehalogenase in dioxin dehalogenation
have not been characterized in aerobic conditions (Field and Sierra-
Alvarez, 2008; Sakaki and Munetsuna, 2010; Janssen et al., 2001).
Therefore, in this work, with the high up-regulation in 2 -h incubation
with 0.1 mg L−1 TCDD and in 6 -h incubation with 0.2 mg L-1 TCDD, the
gene 3580 of B. cenocapacia strain 869T2 was selected and over-
expressed in E. coli to further investigate the ability in dioxin dehalo-
genation.
3.4. Cytochrome P450 - genes related to dioxin degradation
According to the transcriptomic data of B. cenocepacia 869T2, two
detected genes cytochrome P450 expressed strongly with encoding
electron transport genes a (2Fe-2S) ferredoxin, FAD/NAD(P)-binding
oxidoreductase and a 4Fe-4S ferredoxin at 6 h (Table S3). All those
genes were only upregulated at 6 h but not at 12 h. Cytochrome P450
genes encode the corresponding proteins P450 s which are known as
one of four detected dioxin metabolizing enzymes (Sakaki and
Munetsuna, 2010). The high up-regulation of electron transport gene
transcription for the P450 system might indicate that they provided
electrons to P450 to function against TCDD toxicity.
In previous studies, cytochrome P450 BM-3 from Bacillus megaterium
was able to hydroxylate 1-MonoCDD 2,3-DiCDD, and 2,3,7-TriCDD
(Sulistyaningdyah et al., 2004) and also to oxidize polycyclic aromatic
hydrocarbons (PAHs) (Carmichael and Wong, 2001). Additionally, hy-
droxylation of camphor (Katagiri et al., 1968), and oxidation of PAHs
(Harford-Cross et al., 2000) and polychlorinated benzenes (Jones et al.,
2001; Chen et al., 2002) had been studied on cytochrome P450cam
(CYP101; Class I P450) of Pseudomonas putida which includes cyto-
chrome P450 and two electron transfer proteins, putidaredoxin, a
[2Fe–2S] ferredoxin, and a FAD-containing reductase (putidaredoxin
reductase) (Peterson et al., 1990; McLean et al., 2005).
Based on the electron transport chain, these P450 gene system of the
strain 869T2 are predictably belonged to Class I P450 systems like
CYP101 of P. putida and involved the initial processes in response to
TCDD. However, the exact role of P450 in B. cenocepacia strain 869T2
was not investigated in this study.
5
Journal of Hazardous Materials 401 (2021) 123347
Expression, Fold change (TCDD/Control)
6h 12 h TCDD (12/6 h)
4.58 0.70 0.51
2.54 3.26 1.04
21.13 2.01 0.43
1.15 0.85 0.95
1.19 1.55 0.68
1.37 0.99 1.00
3.06 1.27 0.32
3.5. Initial outline for the upper pathway of TCDD Degradation
After assessing the capacity of B. cenocepacia 869T2 in TCDD de-
gradation (Fig. 1B), the mRNA sequencing of the strain 869T2 enabled
us to identify genes involved in dioxin metabolism, and the substantial
changes of gene expression might give paramount clues about TCDD
metabolic pathway. In the aerobic biodegradation, chlorinated dioxins
metabolism is usually initiated by an angular dioxygenase system which
attacks a ring adjacent to the ether oxygen bridging the two rings,
yielding 2,2′,3-trihydroxydiphenyl ether or 2,2′,3-trihydroxybiphenyl
from dioxin or dibenzofuran respectively (Habe et al., 2001; Nojiri and
Omori, 2002). In the second step, 2-hydroxy-6-oxo-6-(2-hydro-
xyphenoxy)-hexa-2,4-dienoate (HOHPDA) was produced by the sub-
sequent dioxygenation of 2,2′,3-trihydroxydiphenyl ether performed by
2,2′,3-trihydroxybiphenyl dioxygenases (extradiol dioxygenases and
glyoxalase/bleomycin resistance protein/dioxygenases) (Bünz and
Cook, 1993; Hartmann and Armengaud, 2014). After that, the hydro-
lysis of HOHPDA by 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate
(HOPD) hydrolase produced the aliphatic and aromatic compounds
which are substrates for benzoate degradation and lower pathways to
pyruvate metabolism and TCA cycle. Many enzymes related to dioxin
degradation pathway had been studied in other aerobic bacteria (Bünz
and Cook, 1993; Happe et al., 1993; Seah et al., 2007; Bünz et al.,
1993). One of few aerobic bacteria S. wittichii strain RW1 can degrade
and utilize several dioxin congeners as carbon sources and PCDDs and
PCDFs degradation and pathway of the strain RW1 have been well
studied. According to the transcriptome data, many up-regulated genes
of the 869T2 genome have similar functions to degradative-dioxin
genes of the strain RW1, thus they were investigated following to dioxin
degradation pathway of the Kyoto Encyclopedia of Genes and Genomes
(KEGG) databases (Genomes, 2018) and the strain RW1 (Hartmann and
Armengaud, 2014) to determine key events of dioxin metabolic pro-
cesses.
Upon response to TCDD toxicity, an aromatic-ring-hydroxylating
dioxygenase (gene2961) and two electron transport genes ferredoxin-
NADP(+) reductase (gene5127) and ferredoxin, 2Fe-2S type
(gene3660) were identified with strong expression (Table 2 and Fig. 2).
Interestingly, the aromatic-ring-hydroxylating dioxygenase and ferre-
doxin gene highly increased 4.6-fold and 21.1-fold change compared to
the control at 6 h, respectively. In the initial step of dioxin degradation
pathway, the angular dioxygenases generally consist of a terminal
aromatic-ring hydroxylating dioxygenase and two electron carrier en-
coding proteins. Electrons from NAD(P)H are transferred to the dioxy-
genase via the electron supply system NAD(P)H-ferredoxin reductase
and ferredoxin to catalyze the angular oxidation of PCDDs and PCDFs
(Bünz and Cook, 1993; Habe et al., 2001; Nojiri and Omori, 2002). For
other bacteria, dibenzofuran 4,4a-dioxygenase of Sphingomonas sp.
strain RW1 (Bünz and Cook, 1993), naphthalene 1,2-dioxygenase of
Pseudomonas resinovorans (Kauppi et al., 1998), and dibenzofuran 4,4a
B.-A.T. Nguyen, et al. Journal of Hazardous Materials 401 (2021) 123347

dioxygenase from Terrabacter sp. strain DBF63 (Habe et al., 2001) have
been identified. In this study, these three detected proteins might
function as the angular dioxygenase system to start a cleavage reaction
of aromatic rings in TCDD structure.
Moreover, extradiol dioxygenases (gene2099) and glyoxalase/
bleomycin resistance family protein/dioxygenases (gene4676 and
gene6989) were upregulated at 6 or 12 h upon TCDD exposure (Table 2
and Fig. 2). They might take part in the second step of the dioxin
catabolic pathway, in which dioxygenation of 2,2′,3-trihydrox-
ydiphenyl ether was carried out by those proteins to yield HOHPDA
(Fig. 2).
TCDD also caused strong gene expressions of HOPD hydrolase gene
encoding the enzyme hydrolase that catalyzes unusual CeC bond hy-
drolysis of the product HOHPDA in the third step of dioxin metabolism
(Fig. 2). Like other dioxin metabolic genes, HOPD hydrolase gene had
the highest expression at 6 h with 3.06-fold change and upregulated at
12 h. In previous studies, the ability of HOPD hydrolases to hydrolyze
polychlorinated biphenyl and dioxin metabolites were identified in the
hydrolases BphD of Burkholderia cepacia strain LB400, BphDP6 of
Rhodococcus globerulus P6 and DxnB2 of S. wittichii RW1 (Nam et al.,
2006; Wittich et al., 1992; Seah et al., 2007; Seah et al., 2001).
Therefore, the HOPD hydrolase of the strain 869T2 was predicted to
participate in hydrolysis of HOHPDA in the third step of dioxin de-
gradation (Fig. 2). In general, the dioxin degradation pathway of B.
cenocepacia strain 869T2 is suggested to be like dioxin pathways of the
strain RW1 and the KEGG databases (Fig. 2). The differential gene ex-
pression of key genes was considered as determinants of related activ-
ities in TCDD metabolism, and the degradative reactions of TCDD are
proposed to occur at the early phase of TCDD exposure due to the up-
regulation of most key genes at 6 h. However, there might have another
possible pathway related to cytochrome P450 (Fig. 6), and the actual
roles of the functional genes and proteins involved in TCDD metabolism
of B. cenocepacia 869T2 remains in need of further studies.

3.6. Dioxygenases – genes related to dioxin degradation

In response to TCDD toxicity, many related ring-cleavage dioxy-

genases participating in aromatic-ring-opening reactions were detected
Fig. 2. The predicted upper pathway of dioxin degradation by the strain 869T2.
Upregulated genes are indicated in bold. with high expression (Table S4). Most of these genes had high upre-
gulation at 6 h, but some were downregulated at 12 h depending on its

Fig. 3. SDS-PAGE of haloacid dehalogenase.

Lane pET24A (=Control 1): BL21(DE3) with pET24A induced by IPTG; Lane HAD- (Control 2): HAD strain without IPTG induction; Lane HAD + IPTG: HAD strain
induced by IPTG.

6
B.-A.T. Nguyen, et al.
specific functions and substrate specificities. Based on the KEGG data-
bases, hydroxyquinol 1,2-dioxygenase (gene1246), catechol 1,2-dioxy-
genase (gene104), benzene 1,2-dioxygenase (gene4841), benzoate 1,2-
dioxygenase (gene4842), protocatechuate 3,4-dioxygenase (gene6557;
gene6558) and phenylpropionate dioxygenase (gene285) participated
in metabolic pathways of aromatic pollutants: chlorocyclohexane and
chlorobenzene degradation, benzoate degradation, fluorobenzoate de-
gradation, toluene degradation, microbial metabolism in diverse en-
vironments, degradation of aromatic compounds and xylene degrada-
tion (Table S4). Although those aromatic metabolic pathways are not
starting processes of dioxin metabolism, benzoate degradation is in-
volved in subsequent steps in dioxin, biphenyl and furan biodegrada-
tions as indicated on KEGG dioxin degradation pathway. Prior to pyr-
uvate metabolism and TCA cycle, benzoate degradation of intermediate
products from the hydrolysis of 2HOHPDA might occur to produce
aliphatic compounds (Field and Sierra-Alvarez, 2008; Wittich et al.,
1992; Seah et al., 2007). Altogether, the strong upregulation of the
related ring-cleavage dioxygenases indicated their participation in di-
oxygenation in the lower pathway of TCDD metabolic processes.
3.7. Construction and identification of recombinant strain E. coli HAD
The DNA fragment (789 bps) of L-2-haloacid dehalogenase (2-HAD)
(gene 3580) was cloned in a vector pET24A (+) to construct the re-
combinant plasmid DNA 2-HAD in pET24A (+) with 6048 base pairs
(Fig. S2-A). The recombinant plasmid was then introduced into a host E.
coli strain BL21(DE3) to form the recombinant strain E. coli HAD with
the target gene 2-HAD, and further confirmed by restriction enzyme
digestion of ApaI and XhoI and observation via agarose gel electro-
phoresis (Fig. S2-B).
To screen dehalogenation activity, only the 2-CPA-treated medium
inoculated with the recombinant strain HAD formed white precipitation
after addition of AgNO3 solution (Fig. S3). The formation of white
precipitation was AgCl produced by a reaction of AgNO3 solution with
Cl− ions releasing from 2-CPA due to the haloacid dehalogenase ac-
tivity of the recombinant strain HAD. In contrast, under the same
conditions, the control strain pET24A (without target gene 2-HAD) and
the non-bacteria controls did not have HAD activity, thus white pre-
cipitation was not detected.
Through SDS-PAGE assay of total proteins produced by the re-
combinant strain HAD, the bands of the enzyme HAD with the esti-
mated molecular weight of 27.8 kDa were clearly shown in the poly-
acrylamide gel (Fig. 3). No band of enzyme HAD was observed in the
control strain BL21 (DE3) with pET24A vector under IPTG induction
and the target strain without IPTG induction.
Fig. 4. Growth of the recombinant train HAD and control strain pET24A in 0.1 mg L−1 TCDD (B) and without TCDD (A). Error bars are standard deviations (n = 3).
7
Journal of Hazardous Materials 401 (2021) 123347
3.8. Growth of recombinant strains
Both the control strains (only the plasmid pET24A) and strains HAD
had similar growth in defined medium without TCDD (Fig. 4A). In
contrast, the presence of TCDD differentiated the growth of two
transformants (Fig. 4B). During first 12 h, the population of the strain
HAD exponentially was around two time higher than that of the control
strain and reached the peak at 33 h (OD600 nm value = 2.49) before
steadily declining, in opposition to the weak growth of the control
strain. Therefore, these results indicated that the strain HAD could grow
in 0.1 mg L−1 TCDD condition.
3.9. TCDD Dehalogenation
After 12 h, a significant 65 % of TCDD was removed by uptake and
dehalogenation of the target E. coli strain HAD (Fig. 5A). While TCDD
compounds in the control medium without bacteria was disappeared a
negligible 2%, there was 59 % of TCDD concentration decreased in
culture medium with the control strain (with only plasmid pET24A)
that might take up a certain amount of TCDD inside the cells lead to a
negative influence on the growth of the control strain (Fig. 4B).
In addition, the haloacid dehalogenase activity was further esti-
mated by the amount of chloride ions released from TCDD in culture
medium during the incubation (Fig. 5B). TCDD compounds were de-
halogenated by the activity of the target enzyme haloacid dehalogenase
with a significant increase of chloride ions in medium from 0 h to 12 h
(Fig. 5B) and a substantial decrease of TCDD (Fig. 5A). It is assumed
that all chloride ions released were cleaved from Cl-C bonds in TCDD
compounds at the medium, thus the total amount of released chloride
ions should be 0.124 μmol after HAD activity after 12 h. However, the
detected amount of chloride ions was 0.25 μmol after 12 -h incubation,
which was two times higher than the assumption. At the beginning of
the incubation (0 h), the samples inoculated with the strain HAD were
detected the certain amount of chloride ions (4.27 μmol ± 0.045 on
average). Perhaps, the impurities of higher chlorinated dioxins (octa-
chlorodibenzodioxin, hexachlorodibenzo-p-dioxin, hepta-
chlorodibenzo-p-dioxin) or background concentration of medium might
affect the amount of chloride ions.
The experimental evidence of the enzyme activity demonstrated
that haloacid dehalogenase played a crucial role in dioxin dehalo-
genation. The removal of halogen substituents facilitates the biode-
gradation of dioxins or other halogenated aromatic compounds (Jeon
et al., 2013), thus TCDDs metabolism was predictably initiated by the
dehalogenation reaction of haloacid dehalogenases. As a result, the
initial outline of TCDD degradation processes was proposed for B.
cenocepacia 869T2 (Fig. 6). In hypothesis, TonB-dependent receptors
B.-A.T. Nguyen, et al. Journal of Hazardous Materials 401 (2021) 123347

Fig. 5. TCDD concentrations (A) and chloride ion amounts (B) in the defined mediums after HAD activity. HAD and pET24A indicate the amount of chloride ions
released from TCDD compounds to the medium inoculated with the strain HAD and the control strain, respectively. Significant differences are indicated by letters (a,
b, c, d) (ANOVA, LSD test, P < 0.05). Error bars are standard deviations (n = 3).

Fig. 6. The proposed cellular degradation process of TCDD by B. cenocepacia 869T2. Ton B and 2-HAD indicate TonB-dependent siderophore receptor and 2-haloacid
dehalogenase.

with other active cross-membrane transport proteins might transport 4. Conclusion

TCDD into the cells. Inside the cells, TCDD compounds were initially
dehalogenated by haloacid dehalogenases, and then metabolized by the
dioxygenation and hydrolysis reactions in the upper TCDD degreada-
tion pathway. There is another possible pathway in which the break-
down of TCDD was started by the P450 s activity; however, the reac-
tions and function of the P450 systems are still open questions.
Afterwards, the products of the upper pathway became substrates for
subsequent reactions in the lower pathway. The roles of key genes with
their encoded enzymes still need to be confirmed.
In conclusion, we demonstrated the great capacity and efficiency of
B. cenocepacia 869T2 in the degradation of 0.2 mg L−1 TCDD com-
pounds. More importantly, based on high expressions of many key di-
oxin-catabolic genes, we proposed the important outline of TCDD de-
gradation pathway by the strain 869T2, which orientates the future
findings and further investigations in the degradation pathway of di-
oxin compounds. Our result further highlighted that the haloacid de-
halogenases played a crucial role in dioxin dehalogenation in response

8
B.-A.T. Nguyen, et al.
to TCDD. Therefore, the strain 869T2 and haloacid dehalogenases are
promising to apply for dichlorination and bioremediation of other di-
oxin and halogenated aromatic compounds in future. Further studies
will be necessary to decipher TCDD metabolic pathways and have
deeper insights into the dioxin degradative process.
Availability of data and materials
The datasets supporting the conclusions of this article are included
within the manuscript and its additional files. The data set supporting
the results of this article are available in the NCBI Sequence Read
Archive (http://www.ncbi.nlm.nih.gov/sra/) repositories,
SRR9829639, SRR9829638, SRR9829637 and SRR9829636.
Credit author statement
All authors read and approved the manuscript.
CRediT authorship contribution statement
Bao-Anh Thi Nguyen: Conceptualization, Methodology, Data
curation, Writing - original draft. Ju-Liang Hsieh: Data curation,
Formal analysis, Writing - original draft. Shou-Chen Lo: Data curation,
Formal analysis, Writing - original draft. Sui-Yuan Wang: Data cura-
tion, Investigation. Chung-Hsiung Hung: Resources. Eugene Huang:
Investigation. Shih-Hsun Hung: Investigation. Wei-Chih Chin:
Writing - review & editing, Supervision, Project administration. Chieh-
Chen Huang: Writing - review & editing, Supervision, Project admin-
istration.
Declaration of Competing Interest
No conflict of interest exits in the submission of this manuscript, and
manuscript is approved by all authors for publication.
Acknowledgement
This work was supported by Ministry of Science and Technology,
R.O.C. (MOST 104-2622-E-006-037-CC2). We thank Professor Simon
Silver, University of Illinois at Chicago (United States), for helps in
preparation of the manuscript.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.123347.
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