Article pubs.acs.org/est
Formation of Halogenated Polyaromatic Compounds by Laccase Catalyzed Transformation of Halophenols Junhe Lu,*,† Juan Shao,† Hui Liu,‡ Zunyao Wang,‡ and Qingguo Huang§ †
College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, China School of Environment, Nanjing University, Nanjing 210093, China § Department of Crop and Soil Sciences, University of Georgia, Griffin, 30223, United States ‡
S Supporting Information *
ABSTRACT: Laccases are a type of extracellular enzyme produced by fungi, bacteria, and plants. Laccase can catalyze one-electron oxidation of a variety of phenolic compounds using molecular oxygen as the electron acceptor. In this study, transformation of halophenols (XPs) in laccase-catalyzed oxidation processes was explored. We first examined the intrinsic reaction kinetics and found that the transformation of XPs appeared first order to the concentrations of both XPs and laccase. A numerical model was developed to describe the role of humic acid (HA) in this process. It was demonstrated that HA could reverse the oxidation of XPs by acting as the inner filtrator of XP radical intermediates formed upon reactions between the substrates and laccase. The extent of such reversion was proportional to HA concentration. MS analysis in combination with quantum chemistry computation suggested that coupling products were generated. XPs coupled to each via CC or COC pathways, generating hydroxyl polyhalogenated biphenyl ethers (OH-PCDEs) and hydroxyl polyhalogenated biphenyls, respectively. Many of these polyhalogenated products are known to be hazardous to the ecosystem and human health, but they are not synthetic chemicals. This study shed light on their genesis in the environmental media.
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INTRODUCTION Halophenols (XPs) are a group of compounds of great environmental concern due to their toxicity, mutagenicity, and carcinogenicity. They show hormone-like activity and are potential endocrine disruptors.1 Chlorophenols (CPs) are industrially produced as preservative agents for wood, fibers, and leathers. They are widely used in the manufacturing of pesticides, herbicides, fungicides, pharmaceuticals, and dyes.2 Bromophenols (BPs) are synthetic flame retardant intermediates and wood preservatives.1 Bromophenols can also be naturally produced by certain marine organisms and found in high concentrations in sponges and algae.3,4 XPs are released to the environment via industrial discharge, accidental spills, and excessive usage of related products, and can also be formed during water disinfection and pulp bleaching processes.5 XPs are generally resistant to biodegradation and difficult to remove from the environment.6,7 Due to their threat to the environment, some XPs, such as 2-dichlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol, are listed as priority pollutants by U.S. EPA.6 It was reported that XPs could be transformed through oxidative coupling reactions mediated by enzymes or metal oxides.8,9 Oxidative coupling comprises a class of reactions that facilitate the polymerization of molecules having phenolic or anilinic features. The reactions involve two stages, the first of © 2015 American Chemical Society
which is the catalyzed oxidation of the substrates to produce highly reactive intermediates, and the second is chemical coupling of the oxidation intermediates to form polymerized products.10−12 These oxidative coupling reactions can be catalyzed by a variety of naturally occurring extracellular enzymes, such as peroxidases and phenoloxidases, and by mineral oxides such as birnessite.8,13−15 These reactions are in fact central to natural humification processes, leading to the formation and growth of soil organic matter from smaller building-block moieties.16−19 Laccases are a group of phenloxidases endogenously produced in extracellular forms by a large variety of fungi and higher plants.20 Laccases catalyze the oxidation of organic substrates in the presence of molecular oxygen as the ultimate electron acceptor. A laccase contains four copper ions: one T1, one T2, and two T3 copper centers. The T2 and T3 copper centers form a trinuclear copper cluster site that is involved in the binding of oxygen during its reduction to water. The T1 copper center is involved in the oxidation of the reducing substrate.21 This type of enzymes is widely distributed in soil Received: Revised: Accepted: Published: 8550
December 24, 2014 July 5, 2015 July 6, 2015 July 6, 2015 DOI: 10.1021/acs.est.5b02399 Environ. Sci. Technol. 2015, 49, 8550−8557
Article
Environmental Science & Technology
halophenol in the samples was quantified using high performance liquid chromatography (HPLC). A total of 5 levels of enzyme dosage (0.1, 0.5, 1.0, 1.5, 2.0 unit/mL) were examined for each individual XP. The residual laccase activity after 1 h in each of the reactors was analyzed and no loss was found. Triplicate experiments were conducted for each reaction condition. Negative controls with boiled laccase were also prepared and no appreciable removal of XPs was found. The same reaction setup was used to evaluate the influence of HA on the reactions. For HA, the reaction solution initially contained 0.1 mM XP and an appropriate amount of HA. A total of four HA concentration levels (5, 10, 20, and 40 mg/L as TOC) were examined for each individual XP. The laccase dose was 1.0 unit/mL. Other conditions were exactly the same as above. Negative controls with boiled laccase were also prepared. Transformation of halophenols was further explored in soil samples (yellow brown earth) collected from Xiamafang Park in Nanjing, China. The soil has a pH of 6.62 and organic carbon content of 20.52 mg/g. None of the tested XPs was found in the collected samples. The soil was dried, ground, and sieved through a 20-mesh screen before use. Each reaction sample contained 5.0 g dry soil and sterilized before mixing with appropriate amount of halophenol dissolved in acetone to achieve a concentration of 10 mg/kg. After acetone was evaporated, 0.8 unit laccase along with 2 mL water was spiked. After incubation in 20 °C in dark for 24 h, each sample was extracted with 10 mL methylene chloride twice facilitated with sonication. The extracts were combined and blow to dryness using a gentle stream of N2. The samples were reconstituted with 1 mL methanol and filtered through 0.45 μM membrane. Control samples with boiled enzyme were prepared and treated with identical procedure. Three replicates were performed for each reaction condition. Chemical Analysis. The residual XP in the samples was quantified using a Hitachi L-2000 HPLC equipped with a photodiode array (PDA) detector. The separation was carried out on a Hitachi LaChrom C18 reverse phase column (5 μM × 250 mm × 4.6 mm). An isocratic elution consisting of 60% methanol and 40% water at a flow-rate of 1.0 mL/min was used. Quantification was based on external calibration. To characterize the reaction products, the same samples were analyzed using an Agilent G6410B Triple Quad Mass Spectrometry (MS) with negative electronspray ionization (ESI-). Instrument parameters were set as follows: capillary voltage 4.0 kV, fragmentor 135 V, desolvation gas (nitrogen, ≥ 99.995%) flow 10 mL/min, temperature 350 °C, nebulizer (nitrogen, ≥ 99.995%) pressure 40 psi. The mass analyzer was operated at scan mode (m/z 50−1000). For selected samples, HPLC/MS analysis was further performed in an attempt to separate the each individual reaction products. The separation was carried out on an Agilent 1200 HPLC equipped with a waters Symmetry C18 column (3.5 μm × 2.1 mm × 150 mm). Elution was performed at a flow rate of 0.25 mL/min with water as solvent A and acetonitrile as solvent B. The gradient lasted 60 min and was programmed as follows: 50% B increased linearly to 90% B at 30 min and 100% B at 50 min where it was held for additional 10 min. MS conditions were the same as those above. Molecular Modeling. All computations were performed using molecular and quantum mechanics algorithms available as part of the Gaussian 03 software package, Revision E.01 (Gaussian Inc., Wallingford CT, U.S.A.). Substrate molecular
and involved in humus formation and turnover of organic carbon.20,22 XPs can be effectively transformed in catalyzed oxidative coupling processes and efforts have been made to take the advantage of such processes to control XPs.23 Nonetheless, little attention has been paid to the intrinsic kinetics of the reactions and how they are affected by the ambient natural organic matter. This comprises one of the purposes of this study. Oxidative coupling reactions result in the polymerization of substrates thus diminishing their solubility, mobility, and bioavailability. It is generally considered as a detoxification process for xenobiotics.20,24 However, the story might be different for XPs because polymerization of XPs in principle generates hydroxyl polyhalobiphenyls and/or hydroxyl polyhalobiphenyl ethers. These compounds are also of great environmental concern. Thus, the other purpose is to characterize the products as well as the reaction pathways of XPs transformation in laccase-catalyzed oxidation processes. The data of this study are of great importance in predicting the environmental behavior of XPs and assess the feasibility of using such processes to control them in engineering systems.
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EXPERIMENTAL SECTION Chemicals. XPs including 2-chlorophenol (2-CP), 4chlorophenol (4-CP), 2,4-dichlophenol (2,4-DCP), 2-bromophenol (2-BP), 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), and 2,2-Azinobis(3-ehtylbenzthiazolin-6-sulfonate (ABTS) were purchased from Aladdin (Shanghai, China). Laccase from Trametes versicolor and humic acid (HA) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Analytical standard 2′-hydroxy-2,3′,4,5′-tetrabromodipheyl ether (2′-OH-BDE-68) was purchased from AccuStandard (New Haven, CT, U.S.A.). HPLC grade acetonitrile and methanol were purchased from Fisher Chemical. Other reagents were analytical grade or better and used as received without additional purification. Laccase was dissolved in Milli-Q water (>18 MΩ cm) generated by a Millipore Milli-Q system. Laccase stock solution was kept in a refrigerator and assayed prior to use. HA stock solution was prepared in Milli-Q water, and the total organic carbon (TOC) was analyzed using a Shimadzu 5050A TOC analyzer. Enzyme Assay. Laccase activity was determined spectrometrically by oxidation of 0.3 mM ABTS in citrate-phosphate buffer (pH 3.8). One unit of laccase activity is defined as the amount of enzyme that causes a unit change per minute in absorbance at 420 nm in 3 mL of this solution in a 1 cm light path cuvette.25 A Varian Cary50 spectrophotometer was used to measure the absorbance. Reaction Setup. The transformation of XPs in laccase catalyzed processes was carried out in 100 mL glass flasks as completely mixed batch reactors at ambient temperature. The reaction solution initially containing 0.1 mM one of the XPs and 0.01 M phosphate buffer was added to maintain the pH at 5.8 ± 0.02. A predetermined dosage of laccase was added to the reactor as the final reagent to initiate the reaction. Because only a tiny amount of laccase solution (
