Biogenic Fenton-like Reaction Involvement in Cometabolic

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Biogenic Fenton-like Reaction Involvement in Cometabolic Degradation of Tetrabromobisphenol A by Pseudomonas sp. fz Chen Gu, Jing Wang, Shasha Liu, Guangfei Liu, Hong Lu, and Ruofei Jin Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 24 Aug 2016 Downloaded from http://pubs.acs.org on August 24, 2016

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Environmental Science & Technology

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Biogenic Fenton-like Reaction Involvement in Cometabolic Degradation of Tetrabromobisphenol A by Pseudomonas sp. fz

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Chen Gu, Jing Wang,* Shasha Liu, Guangfei Liu, Hong Lu, and Ruofei Jin

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Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of

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Education), School of Environmental Science and Technology, Dalian University of

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Technology, Dalian 116024, China

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ACS Paragon Plus Environment


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ABSTRACT: Tetrabromobisphenol A (TBBPA) is a widely used brominated flame

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retardant (BFR) that has frequently been detected in various environmental

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compartments. Although TBBPA biotransformation has been observed under both

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aerobic and anaerobic conditions, knowledge of the detailed mechanism of direct

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aerobic TBBPA biodegradation still remains limited. In this study, the underlying

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mechanism of cometabolic degradation of TBBPA by Pseudomonas sp. fz under

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aerobic conditions was investigated. Two key degradation pathways (beta scission and

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debromination) were proposed based on triple quadrupole liquid chromatography-mass

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spectrometry (LC-MS) analysis. TBBPA degradation by strain fz was demonstrated to

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be an extracellular process associated with the low-molecular-mass component

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(LMMC). Moreover, LMMC was preliminarily identified as oligopeptides, mainly

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consisting of glycine, proline, and alanine in a 2:1:1 molar ratio. Quenching studies

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suggested the involvement of hydroxyl radicals (•OH) in extracellular TBBPA

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degradation. To the best of our knowledge, we provide the first evidence that TBBPA

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was degraded by a biogenic Fenton-like reaction mediated via extracellular H2O2 and

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Fe(II)−oligopeptide complexes by the genus Pseudomonas. This study provides a new

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insight into the fate and biodegradation of TBBPA and other organic pollutants in

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natural and artificial bioremediation environments.

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Tetrabromobisphenol A [4,4’-isopropylidenebis(2,6-dibromophenol), TBBPA] is one

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of the most widely used brominated flame retardants (BFRs) in the production of

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plastics, textiles, and electronic circuit boards in order to render them non-flammable.1,2

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Although TBBPA can be covalently bound to these materials, it has been frequently

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detected in various environmental compartments,3−6 and even in humans.7,8 Moreover,

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it has been regarded as an endocrine disrupting chemical due to its structural similarities

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to thyroxin and steroid estrogens.9,10 It is also associated with a myriad of toxicities to

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aquatic organisms including immunotoxicity, cytotoxicity, and neurotoxicity.11,12

INTRODUCTION

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Limited physicochemical methods have been reported to remove TBBPA,13−18

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whereas microbes in the natural environment are expected to play a major role in the

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degradation of TBBPA. TBBPA can be used as an electron acceptor by bacteria and be

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reductively debrominated to less brominated products and finally to bisphenol A (BPA)

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anaerobically.19,20 Liu et al. further pointed out that a high potential exists for the

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biotransformation of TBBPA in soil through sequential anoxic−oxic incubation.21

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Besides, TBBPA is exposed long-term to aerobic environments before it deposits into

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anaerobic environments. Thus, a comprehensive understanding of aerobic TBBPA

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biodegradation is needed.

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Under aerobic conditions, Li et al. demonstrated that TBBPA can be transformed

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along with large amounts of non-extractable bound-residue formation in sandy soil.22

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Additionally, TBBPA has been shown to be transformed aerobically by O-methylating

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enzyme(s),23,24 laccase,25 and horseradish peroxidase.26 Moreover, other researchers

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have reported TBBPA can be debrominated aerobically by microalgae and bacteria.27,28 3



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Specifically, simultaneous debromination and mineralization of TBBPA by the pure

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aerobic strain Ochrobactrum sp. T was reported,28 but the related active species

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involved in cometabolic biodegradation processes have remained obscure. Thus, to the

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best of our knowledge, a substantial knowledge gap exists regarding the role of active

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species involved in the process of aerobic biodegradation of TBBPA.

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The objectives of this study were (1) to investigate the active species responsible for

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aerobic cometabolic biodegradation of TBBPA by strain fz, and (2) to elucidate the

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underlying mechanism of TBBPA biodegradation in detail. Our results suggest that

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TBBPA is degraded by a biogenic Fenton-like reaction mediated by H2O2 and

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Fe(II)−oligopeptide complexes in the external milieu of strain fz. These results provide

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crucial information that enhances our understanding of the process by which TBBPA

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and other organic pollutants are aerobically biodegraded in natural and artificial

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bioremediation environments.

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Materials. TBBPA (98% purity) was purchased from Tokyo Chemical Industry Co.,

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Ltd. (Tokyo, Japan). A stock solution of TBBPA (20,000 mg L−1) was prepared in

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NaOH (1 mol L−1) and stored at 4 C in the dark prior to use. HPLC grade methanol

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and ethyl acetate were purchased from Tedia Company Inc. (Fairfield, USA). All

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biochemicals were of the highest purity commercially available. All other chemicals

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were reagent grade or better. Ultrapure water from a Milli-Q water purification system

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(Millipore, Bedford, USA) was used throughout the experiments. All flasks were acid

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cleaned prior to use.

MATERIALS AND METHODS

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Bacterial Strain and Culture Conditions. Isolation and identification of the strain

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Pseudomonas sp. fz (GenBank accession number JX195653) are described in Text S1

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of the Supporting Information (SI). The conditions for aerobic biodegradation of

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TBBPA were optimized, as described in Text S2. Mineral salt medium (MSM)

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consisted of 2.00 g L−1 NH4NO3, 3.65 g L−1 K2HPO4·3H2O, and 0.54 g L−1 KH2PO4.

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TBBPA biodegradation experiments were conducted in optimized MSM containing 8

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g L−1 glucose and 0.5 g L−1 beef extract. The MSM and optimized MSM (pH 7.2 ±0.1)

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media were autoclaved at 115 C for 30 min prior to use. All TBBPA degradation

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experiments were conducted in the dark to avoid photolysis.13,14

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Oxidative Activity. Oxidative activity was determined by measuring the oxidation of

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2,2’-azinobis-(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS, 500 μmol L−1) in sodium

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acetate buffer (50 mmol L−1, pH 4.5) at 420 nm (ε = 3.6×104 mol−1 cm−1) by using a

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UV−Vis spectrophotometer (V-560, JASCO, Tokyo, Japan). Oxidative activity is

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expressed in units defined as 1 μmol of product formed per min.29

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Preparation of Active Species. Strain fz cells were precultured in Luria−Bertani (LB)

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broth until they reached the exponential growth phase and harvested by centrifugation

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at 10,280 × g for 10 min at 4 C. Cell pellet was washed twice with sterile phosphate-

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buffered saline solution (pH 7.2), and inoculated at an optical density at 600 nm (OD600)

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of 0.4 in optimized MSM until they reached the stationary growth phase. For the sake

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of comparison, a parallel experiment was also conducted under the same conditions in

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the presence of TBBPA (final concentration, 2 mg L−1). After centrifugation at 3,700 ×

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g for 10 min at 4 C, the supernatant was collected, and the cell pellet was washed twice 5



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with freezing ultrapure water and resuspended in 10 mL Tris-HCl buffer (10 mmol L−1,

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pH 8.0) and centrifuged (3,700 × g, 10 min, 4 C). Then the supernatants were pooled

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as the extracellular fraction. Osmotic shock and lysozyme/EDTA methods were used

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to isolate the periplasmic fraction, which was collected by centrifugation at 17,370 × g

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for 10 min at 4 C.30 Then, the cell pellet was lysed via a freezing and thawing process

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prior to sonication (225 W, at 4 C for 30 min, Ultrasonic processor CPX 750). Cell

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debris was removed by centrifugation at 23,120 × g at 4 C for 20 min, and the

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supernatant was intracellular fraction.31 The extracellular, periplasmic, and intracellular

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fractions were separately filtered through 0.22 μm syringe filters (Millipore) prior to

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starting the following experiments. TBBPA was added to the three fractions to achieve

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a final concentration of 2 mg L−1 in each, and the samples were incubated at 35 C on

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a rotary shaker at 150 r min−1 for 5 h. The optimized MSM without strain fz was used

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for control assay. To further confirm whether the extracellular fraction contained

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proteins capable of degrading TBBPA, the effect of protease treatment was examined.

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A protease-treated sample was incubated with proteinase K (final concentration, 20 U

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mL−1) for 30 min at 30 C. Then TBBPA was added to the sample to achieve a

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concentration of 2 mg L−1, and the sample was incubated at 35 C on a rotary shaker at

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150 r min−1 for 5 h. Each experiment was carried out in triplicate.

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The extracellular fraction was further isolated by ultrafiltration (UF) using a

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MINITAN UF system with a 50 kDa cutoff membrane (Millipore). Briefly, the

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extracellular fraction was isolated by centrifugation at 3,500 ×g for 8 min at 4 C. Then

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TBBPA (final concentration, 2 mg L−1) and glucose (final concentration, 1 g L−1) were

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added to the filtrate and retentate, respectively, and the samples were incubated at 35

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C on a rotary shaker at 150 r min−1 for 5 h. The residual TBBPA concentration and 6


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oxidation activity of the two fractions (>50 kDa and

Biogenic Fenton-like Reaction Involvement in Cometabolic

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