Article pubs.acs.org/est
Reductive Dehalogenation of Oligocyclic Phenolic Bromoaromatics by Dehalococcoides mccartyi Strain CBDB1 Chao Yang,† Anja Kublik,† Cindy Weidauer,‡ Bettina Seiwert,‡ and Lorenz Adrian*,† †
Department of Isotope Biogeochemistry and ‡Department of Analytics, Helmholtz Centre for Environmental Research − UFZ, Permoserstraße 15, 04318 Leipzig, Germany S Supporting Information *
ABSTRACT: Dehalococcoides mccartyi strains transform many halogenated compounds and are used for bioremediation. Such anaerobic transformations were intensively studied with chlorinated and simply structured compounds such as chlorinated benzenes, ethenes, and ethanes. However, many halogenated oligocyclic aromatic compounds occur in nature as either naturally produced materials or as part of commercial products such as pharmaceuticals, pesticides, or flame retardants. Here, we demonstrate that the D. mccartyi strain CBDB1 reductively debrominated two oligocyclic aromatic phenolic compounds, tetrabromobisphenol A (TBBPA) and bromophenol blue (BPB). The strain CBDB1 completely converted TBBPA to bisphenol A and BPB to phenol red with a stepwise removal of all bromide substituents. Debromination (but no cell growth) was detected in the cultures cultivated with TBBPA. In contrast, strain CBDB1 grew when interacting with BPB, demonstrating that this substrate was used as an electron acceptor for organobromine respiration. High doses of BPB delayed debromination and inhibited growth in the early cultivation phase. A higher toxicity of TBBPA compared with that of BPB might be due to the higher lipophilicity of TBBPA. Mass spectrometric analyses of whole-cell extracts demonstrated that two proteins encoded by the reductive dehalogenase homologous genes CbdbA1092 and CbdbA1503 were specifically induced by the used oligocyclic compounds, whereas others (e.g., CbdbA84 (CbrA)) were downregulated.
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genes were identified in their genomes.15−18 Reductive dehalogenase homologous genes (rdh genes) are particularly abundant in Dehalococcoides species with 10−36 copies in the known D. mccartyi genomes.19 Many studies have been carried out with Dehalococcoides strains on the transformation of simply structured organohalides such as chlorinated benzenes, 20,21 phenols, 22 ethenes,23−25 ethanes,26 and propanes,27 among others. Dehalococcoides strains were also able to dehalogenate di- and tricyclic aromatic compounds such as polychlorinated biphenyls,28−30 chlorinated dioxins,31−33 or brominated diphenyl ethers.34 Krzmarzick et al. proposed that Dehalococcoides strains were also growing on more complex compounds after observing growth with enzymatically chlorinated total organic carbon.13 However, the transformation of such large chemical structures contradicts the recent structural characterization of PCE-reductive dehalogenase from Sulfurospirillum multivorans, which forms a tight substrate entrance “letter box” of only 3 Å × 5.5 Å that should prevent the access of larger molecules.35 We here used brominated oligocyclic organic compounds to
INTRODUCTION Brominated aromatic compounds are naturally and anthropogenically produced. Natural production (e.g., by marine sponges) has been shown for bromophenols, bromoindoles, and brominated dibenzo-p-dioxins.1−4 Anthropogenic brominated aromatics constitute a large group of environmental contaminants and include the brominated flame retardants tetrabromobisphenol A (TBBPA), polybrominated biphenyls (PBBs), and polybrominated diphenylethers (PBDEs).5,6 Flame retardants are used as additives in commercial products such as furniture, electronic equipment, or thermal insulation material to prevent ignition. Because such flame retardants are noncovalently bound to polymeric materials, they are easily released from these products and washed to the environment, where they often resist natural transformation, persist for long periods of time, and often bioaccumulate in the food chain.7−10 Anaerobic biotransformation is a major process in environmental matrixes (e.g., soil, sediments, and anoxic groundwater) and viewed as an efficient way to remediate environmental pollutants. Dehalococcoides mccartyi strains catalyze well-known examples of such anaerobic transformations by reductively dehalogenating halogenated organics that are otherwise persistent for decades.11 The catalyzing enzymes, designated as reductive dehalogenases, have been isolated from several bacteria including D. mccartyi strains,12,14 and the encoding © XXXX American Chemical Society
Received: March 19, 2015 Revised: June 11, 2015 Accepted: June 23, 2015
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DOI: 10.1021/acs.est.5b01401 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
proteins (RdhA)) were up- or downregulation-induced by such large molecules.
determine if specific dehalogenation with respiratory coupling is still possible and to investigate the effect on the expression of rdh genes. We decided to use brominated rather than chlorinated compounds due to their wide technical use and the faster debromination versus the dechlorination rates.36,37 Because nonsubstituted oligocyclic aromatics have very low water solubility, and commercially used brominated aromatics often contain hydroxyl groups, we focused on phenolic compounds. However, phenolic groups often also induce toxicity, as has been observed with chlorophenols for strain CBDB1.22 Here we investigated the reductive transformation of TBBPA (2,2′,6,6′-tetrabromo-4,4′-isopropylidenediphenol) and bromophenol blue (BPB, 3′,3″,5′,5″-tetrabromophenolsulfonphthalein) by our model organism, D. mccartyi strain CBDB1. This strain is an obligately organohalide-respiring bacterium isolated from river sediments and can be routinely cultivated (e.g., with halogenated benzenes as the electron acceptor).37,38 It has 32 reductive dehalogenase homologous (rdh) genes, suggesting a broad dehalogenating potential.17 TBBPA is one of the most frequently used flame retardants, with an annual global consumption of about 121 000 t.5 Recently, a re-evaluation of the nonbrominated congener bisphenol A by European authorities resulted in the lowering of the safety level of bisphenol A from 50 to 4 μg kg−1 of body weight per day but at the same time concluded that bisphenol A poses no health risk to consumers at the current exposure levels (http://www.efsa. europa.eu/). The reductive transformation of TBBPA to bisphenol A has been reported by means of an enrichment culture from river sediment and a pure strain, the Comamonas sp. strain JXS-2-02, that was isolated from anaerobic sludge.39,40 Zhang et al. also proved that a Dehalobacter strain in mixed culture could debrominate TBBPA to bisphenol A.41 BPB is used as a dye in industry but also in laboratory settings for gel electrophoresis.42,43 The two compounds show structural similarity by having more than one aromatic ring, four bromine substituents, and two hydroxyl substituents (Figure 1) but
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EXPERIMENTAL SECTION Chemicals. Hexabromobenzene (HBB), TBBPA, and BPB (in its sodium salt form) were purchased from Sigma-Aldrich (Seelze, Germany) at a purity of 99%. Titanium(III) citrate was prepared as previously described.22 Cultivation. Strain CBDB1 was first cultivated in titanium(III) citrate-reduced, carbonate-buffered synthetic medium with 5 mM acetate as the carbon source, hydrogen as the electron donor, and crystalline HBB as the electron acceptor, similar to the protocol previously described for crystalline hexachlorobenzene (HCB).38 Due to its low water solubility, TBBPA was added from an acetone stock solution to a final concentration of 10 μM. BPB was added from an anoxic aqueous stock solution to a final concentration of 20 μM in the cultures. HBBdebrominating cultures with a cell density of about 108 cells mL−1 were used as the inoculum for the cultures in this study. The starting cell density in all cultures was around 6 × 106 cells mL−1. After inoculation, the headspace of the cultures was pressurized with 20% CO2 and 80% N2 (0.3 bar) and an additional 0.1 bar of H2. All cultivations were set up in triplicates and incubated in the dark at 30 °C without shaking. Positive controls fed with crystalline HBB, abiotic controls without inoculum, and negative controls without an electron acceptor were set up in parallel. To observe a color change during debromination, we set up cultures with BPB containing 2 mM of L-cysteine instead of titanium(III) citrate as a reducing agent. Cell densities were quantified by direct cell counting after staining with SYBR Green as previously described.44 Bromide Analysis. Sample volumes of 600 μL were transferred to a 1.5 mL Eppendorf tube and centrifuged at 20 °C at 10000g for 10 min. Afterward, 500 μL samples of the supernatant were transferred to vials with filter caps and analyzed on a Dionex-120 ion chromatograph equipped with an IonPac AS4A-SC (4 mm × 250 mm) column. The eluent used for bromide analysis was 0.7 mM Na2CO3 plus 0.7 mM NaHCO3 with a constant flow rate of 1.0 mL min−1. Calibration was done with NaBr in a range between 2 and 200 μM. The retention time of bromide was 5.4 min. Product Analysis by Liquid Chromatography−Mass Spectrometry. To determine the transformation products in active cultures, we directly injected 10 μL of each culture into a Waters ACQUITY UPLC (ultra performance liquid chromatography) system connected to a Synapt G2S time-of-flight mass spectrometer equipped with an electrospray ionization source (Waters Corp., Milford, USA) and an ACQUITY UPLC-HSST3 column (100 mm × 21 mm, 1.7 μm particle size). The mobile phases used were water (A) and acetonitrile (B), both containing 5 mM ammonium acetate and 0.05% acetic acid. The gradient was applied as follows: 100% A held for 2 min, increased to 95% B within 5 min and held for 1 min, then decreased to 100% A and held for 2 min. The flow rate was set to 0.6 mL min−1 with a column temperature of 60 °C. For mass spectrometric analysis, the electrospray ionization (ESI) was used in negative mode with the following parameters: a source temperature of 140 °C, a desolvation temperature of 550 °C, a capillary voltage of 0.7 kV, a sampling cone voltage of 35 kV, a source offset of 50 kV, and a desolvation gas flow of 950 L h−1. Mass spectra were recorded from m/z 50 to 1200 using the “resolution mode” on the machine. The transformation products were identified by their
Figure 1. Molecular structures of the three used brominated aromatic organics: tetrabromobisphenol A (TBBPA), bromophenol blue (BPB), and hexabromobenzene (HBB).
distinct hydrophobicity; of the two compounds, TBBPA has a much lower water solubility. BPB has a pKa of 4.0 (http:// pubchem.ncbi.nlm.nih.gov/), leading to the anionic form at pH 7.0. In contrast, the pKa1 of TBBPA is around 7.0, resulting in the occurrence of dissociated and nondissociated forms at neutral pH. The aim of our study was to determine if strain CBDB1 can grow by using these brominated phenolic compounds as a respiratory electron acceptor, to identify the transformation products, and to assess the possible toxic effects associated with the phenolic groups. Furthermore, we wanted to investigate whether specific rdh genes (and especially the catalytic subunit of reductive dehalogenase homologous B
DOI: 10.1021/acs.est.5b01401 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 2. Reductive debromination and growth of strain CBDB1 during the slow feeding of (A) TBBPA or (B) BPB and of (C) negative controls without an electron acceptor. Cultures were inoculated from cultures growing with HBB. Shown are the measured bromide concentration in cultures (●), the measured bromide concentration in abiotic controls without inoculation (○), and the calculated total concentration of the electron acceptor added (■). The bars show the cell densities, and the arrows indicate the time when feeding was stopped. Data are the means of the triplicate cultures ± SD.
with an assigned monoisotopic m/z and a charge state of two to seven were selected for MS/MS analysis and isolated by the quadrupole of the machine (1.6 m/z isolation window). Dynamic exclusion was enabled over 45 s with an exclusion mass width of ±10 ppm. Precursor fragmentation was performed by higher-energy collisional dissociation (HCD) with a normalized collision energy of 30% followed by an MS/ MS scan of the fragment ions using the Orbitrap mass analyzer at a resolution of 60 000 (AGC target 5 × 104, maximum ion injection time of 100 ms). LC−MS/MS data were processed with Proteome Discoverer v1.4.1.14 (Thermo Scientific). Peptide identification was performed using the Mascot database search engine as described previously.37 Data were searched against the UniProt D. mccartyi strain CBDB1 database using the following parameters: trypsin as the cleavage enzyme; a maximum of two missed cleavages; and precursor and fragment mass tolerances of 10 ppm and 0.05 Da, respectively. Carbamidomethylation on cysteine was included as static modification, and oxidation on methionine was set as dynamic modification. Proteins were considered to be expressed when at least two peptides were identified at a false discovery rate (FDR, Target Decoy PSM Validator) of 0.05. Proteins expressed in at least two cultures of the respective analyzed substrate triplicate were ranked according to their mean emPAI values.37,46
retention time in combination with the mass spectra of the molecule and of its main fragments (Supplementary Tables 1 and 2 and Supplementary Figures 1 and 2 in the Supporting Information). The quantification of TBBPA and bisphenol A was carried out by external calibration between 0.1 and 10 μM with a detection limit of 0.03 μM. Because the standards of other intermediates were unavailable, the peak areas are reported instead of the concentrations. RdhA Protein Expression Analysis. Cultures of strain CBDB1 grown with TBBPA or BPB were harvested by centrifugation at 6000g and 16 °C for 60 min under anoxic conditions. For each sample, around 1.5 × 108 cells were disrupted by three repeated cycles of freezing and thawing as described previously.45 As a control, strain CBDB1 was cultured with HBB as an electron acceptor, and the cultures were analyzed analogously. The obtained protein lysates were reduced (60 mM dithiothreitol for 1 h at 30 °C), alkylated (120 mM iodacetamide for 1 h at 30 °C in the dark), and digested (0.6 μg trypsin at 37 °C overnight). Tryptic peptides were desalted using ZipTip-C18 material (Merck Millipore, Darmstadt, Germany) and reconstituted in 0.1% formic acid prior to nLC−MS/MS analysis using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoUPLC system (Dionex Ultimate 3000 RSLCnano System, Thermo Fisher Scientific). Samples of 0.6 μL were loaded on a trap column (Acclaim PepMap100 C18, 75 μm × 2 cm, film thickness 3 μm, Thermo Scientific) using 96% eluent A (0.1% formic acid) and 4% eluent B (0.08% formic acid, 80% acetonitrile) at a flow rate of 5 μL min−1. After 6 min, in which the contaminating salts were washed off of the loading column, peptides were eluted onto the analytical column (Acclaim PepMap100 C18, 75 μm × 25 cm, film thickness 3 μm, Thermo Scientific) and separated at 40 °C by reversed-phase chromatography using a constant flow rate of 300 nL min−1. Peptide separation was achieved by applying a linear gradient of eluent B from 4% to 55% within 120 min. Eluted peptides were analyzed using an Orbitrap Fusion mass spectrometer equipped with a nano electrospray ion source (TriVersa NanoMate, Advion). The Orbitrap Fusion was operated in a data-dependent top speed mode with a maximum cycle time of 3 s. Precursor scans were acquired in the Orbitrap at a resolution of 120 000 between 350 and 2000 m/z with an automatic gain control (AGC) target of 4 × 105 and a maximum ion injection time of 50 ms. Instrument lock mass correction was applied using a contaminant ion at 445.12003 m/z. The most abundant precursor ions (intensity of ≥5 × 104)
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RESULTS Debromination of TBBPA and BPB. D. mccartyi strain CBDB1 was first cultivated with 10 μM of TBBPA or 20 μM of BPB using a starting cell density of 2 × 106 cells mL−1. After 78 days of incubation, only a small amount of bromide (
