Birnessite-Induced Binding of Phenolic Monomers to Soil Humic

Jul 27, 2012 - ... Shandong Agricultural University, Daizong Road 61, 271018 Tai'An, China ... Institute of Agricultural Resources and Regional Planni...
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Birnessite-Induced Binding of Phenolic Monomers to Soil Humic Substances and Nature of the Bound Residues Chengliang Li,†,‡ Bin Zhang,§ Tanya Ertunc,†,▽ Andreas Schaeffer,† and Rong Ji*,†,⊥,⊗ †

Biology 5, Environmental Biology and Chemodynamics, RWTH Aachen University, D-52056 Aachen, Germany College of Resources and Environment, Shandong Agricultural University, Daizong Road 61, 271018 Tai’An, China § Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Zhongguancun 12 South Main Street, 100081 Beijing, China ⊥ State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 163 Xianlin Avenue, 210046 Nanjing, China ⊗ Institute for Climate and Global Change Research, Nanjing University, 22 Hankou Road, 210093 Nanjing, China ‡

ABSTRACT: The nature of the abiotic birnessite (δ-MnO2)-catalyzed transformation products of phenolic compounds in the presence of soil organic matter is crucial for understanding the fate and stability of ubiquitous phenolic carbon in the environment. 14C-radioactive and 13C-stable-isotope tracers were used to study the mineralization and transformation by δ-MnO2 of two typical humus and lignin phenolic monomers―catechol and p-coumaric acid―in the presence and absence of agricultural and forest soil humic acids (HAs) at pH 5−8. Mineralization decreased with increasing solution pH, and catechol was markedly more mineralized than p-coumaric acid. In the presence of HAs, the mineralization was strongly reduced, and considerable amounts of phenolic residues were bound to the HAs, independent of the solution pH. The HA-bound residues were homogeneously distributed within the humic molecules, and most still contained the unchanged aromatic ring as revealed by 13C NMR analysis, indicating that the residues were probably bound via ester or ether bonds. The study provides important information on δ-MnO2 stimulation of phenolic carbon binding to humic substances and the molecular distribution and chemical structure of the bound residues, which is essential for understanding the environmental fates of both naturally occurring and anthropogenic phenolic compounds.

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INTRODUCTION Monomeric phenolic compounds are ubiquitous in the environment,1,2 accounting for up to 10% of the dissolved organic carbon in soil.3 Phenolic compounds of natural origins are derived from microbial degradation of biomass (especially plant residues), microbial synthesis, and plant root exudation.2 Moreover, wastewater irrigation and solid-compost application of industrial waste also introduce phenolic compounds (including chlorinated and nitrified phenols) into the environment, as phenolic compounds are widely used as intermediates in production processes.4,5 Phenolic compounds are subjected to biotic and abiotic humification processes in the environment and are therefore regarded as important precursors of both soil and aquatic humic substances.2,6−9 Moreover, phenolic compounds are antioxidants, which may be key to the stability of organic matter in the soil environment.10−12 In the transformation of phenolic compounds in the environment, extracellular phenol oxidase and soil minerals (e.g., birnessite) play a catalytic role.13−16 Phenol oxidase and birnessite (δ-MnO2) are very common in the environment,16−18 and their contributions to the abiotic and enzymemediated formation of humic substances in soil have been extensively discussed in the literature.7,13,16,19,20 It has been © 2012 American Chemical Society

shown that phenol oxidase can initiate oxidative binding of both natural phenolic compounds and phenolic xenobiotics (e.g., chlorophenol) to humic substances, via formation of free phenoxyl radicals (e.g., in the case of laccase and peroxidase) or quinones (e.g., in the case of tyrosinase) that undergo coupling reactions with the existing humic substances,16,21 even though it was believed that the participation of phenol oxidases in the formation of humic substances would be of minor significance in the soil.16 Owing to its high oxidation potential (E0 = 1.2 V),22 δ-MnO2 can catalytically oxidize both phenolic and nonphenolic pollutants.7,23−34 Most of the studies on oxidative transformation of both naturally occurring and anthropogenic phenolic compounds by δ-MnO2 have been conducted in systems without humic substances (see, e.g., refs 9, 15, 20, 23, 26−30, 32, 33, and 35), even though humic substances are the major component of organic matter in the environment. Both polymerization and strong mineralization of phenolic comReceived: Revised: Accepted: Published: 8843

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depleted HA (with 99.95% of 12C atom enrichment) was isolated from a 13C-depleted compost (see below) as described for the soil HAs. 13 C-Depleted Compost. Spring wheat (Triticum aestivum L. var. ‘Horizont’) was grown under an air atmosphere containing 300 ppm 12CO2 (99.95% atomic 12CO2; Cambridge Isotope Laboratories) in a gastight plant exposure chamber for 7 weeks in a greenhouse.42 The gained plant material had a δ13C of −943‰. Plants were ground to a powder with an agate mill and mixed with quartz (0.35−2 mm) at a ratio of 8:100 (w/w). The mixture was inoculated with water extract of a peat soil from a hill moor in northern Germany and was adjusted to 40% of the maximal water capacity. The mixture was composted with a flow-through system at 20 °C, and air was drawn into the system at a rate of 6 mL min−1 through a CO2 trap (50 mL of 1 M NaOH) to remove atmospheric CO2. The mixture was composted for 25 months, and 13C-depleted HA was isolated from the obtained compost (see above). Oxidation of Phenolic Compounds by δ-MnO2. Experiments were performed in closed glass bottles (50 mL) with 2 mL of mixture containing 14C-catechol or 14C-coumaric acid (2.0 μg), δ-MnO2 (5 mg), and soil HA (1 mg C) in phosphate buffer (10 mM) under sterile conditions to avoid microbial transformation of the phenols. Released 14CO2 in the bottle was absorbed by 0.5 mL of NaOH (1 M) contained in a vial (5 mL), which was suspended from the bottom of the stopper of the bottle. At defined time intervals, the 14CO2 trap was replaced, and the radioactivity was determined by liquid scintillation counting (LSC; see below). During the experiments, the bottles were gently shaken on a rotary shaker (10 rpm). Experiments in the absence of δ-MnO2 or HA or both (i.e., the control) were performed. All experiments were carried out in duplicate. At the end of the reaction (48 h), δ-MnO2 was separated from the mixture by centrifugation, and the radioactivity of the supernatant was determined by LSC. The HA in the supernatant was then precipitated by acidification (pH 1) and separated by centrifugation. The HA was dissolved in anoxic 0.1 M NaOH and stored under N2 atmosphere. The radioactivity of HA was determined by LSC, and the molecular size distribution of 14C-catecholic and 14C-coumaric residues within the HA was analyzed by high-performance radio gel permeation chromatography (HP-14C-GPC) (see below). Preparation of HA-Bound Phenolic Residues for 13C Nuclear Magnetic Resonance (13C NMR) Spectroscopy. Solution 13C NMR was used to determine the chemical structures of the catecholic and p-coumaric residues bound to HA via the oxidation by δ-MnO2. To enhance the signal intensity, we added 13C-catechol, 13C-coumaric acid, and 13Cdepleted HA to the reaction and increased the ratio of the phenolic compounds to HA. Briefly, the reaction mixture (20 mL) contained 13C-depleted HA (20 mg of C), 13C-catechol (4.2 mg), or 13C-coumaric acid (4.9 mg) and δ-MnO2 (100 and 200 mg for catechol and p-coumaric acid, respectively). After 48 h of incubation, the HA was separated and analyzed by solution 13 C NMR spectroscopy (see below). Radioactivity Determination. Radioactivity was quantified on a liquid scintillation counter (LS 5000TD; Beckman, USA) using the Lumasafe Plus scintillation cocktail (Lumac LSC BV, Groningen, The Netherlands). For radioactivity of 14 CO2 in 1 M NaOH, 0.5 mL of the alkaline solution was mixed with 10 mL of the cocktail. For HA samples, 1 mL of suspension (about 1 mg C mL−1) was mixed with 10 mL of

pounds have been observed in these studies. The oxidative transformation products (i.e., the humic-like products) of catechol by δ-MnO2 inhibited the further degradation and mineralization of catechol in the system,7,28 suggesting that humic substances in soil would likely reduce the relative proportion of catechol mineralization produced by δ-MnO2. The presence of humic substances could affect the transformation process by acting as antioxidants,36 by interacting with phenolic compounds, especially those with two or more hydroxyl groups on a benzene ring, such as catechol,2 and by chelating Mn2+, which is released during reduction of δ-MnO2 and which significantly inhibits the oxidation potential of δMnO2 when it adsorbs to the surface of δ-MnO2.23,27 Humic substances might therefore increase δ-MnO2 oxidation efficacy through chelate formation of Mn2+ but, at the same time, might reduce δ-MnO2 oxidation efficacy by sorption on its surface active sites. Studies with pure systems have shown that reactive intermediates, including free phenoxyl radicals and quinones, are formed during oxidation of phenolic compounds by δMnO2,14,20,26,27,37,38 similar to their oxidation by phenol oxidases.16,20 It is known that the intermediates generated by phenol oxidases react with humic-like polymers and humic molecules,21 forming covalently bound residues,16,17 and therefore change the fate of phenolic compounds in the environment, as indicated by the lower mineralization of phenolic residues in humic substances.2,39 However, the effects of humic substances on the transformation and fate of phenolic compounds by δ-MnO2 are far less understood. The objectives of this study were (1) to determine the effects of humic substances on abiotic transformation of phenolic compounds by δ-MnO2 and (2) to obtain information on the nature of phenolic residues bound to humic substances, using 14 C- and 13C-labeled catechol and p-coumaric acid as representatives of typical naturally occurring phenolic compounds and metabolic intermediates of anthropogenic pollutants.2,40

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MATERIALS AND METHODS C- and 13C-Labeled Phenolic Compounds. Uniformly ring-14C-labeled catechol (14C-catechol) and p-coumaric acid (14C-coumaric acid) with specific radioactivities of 73 and 410 MBq mmol−1, respectively, were synthesized from uniformly ring-14C-labeled phenol (Hartmann Analytic, Braunschweig, Germany) according to the methods of Ji and Schaeffer40 and Ji et al.6 Uniformly ring-13C-labeled catechol (13C-catechol) and p-coumaric acid (13C-coumaric acid) with 99% 13C atom were synthesized from uniformly ring-13C-labeled phenol (Cambridge Isotope Laboratories, Andover, MA, USA).6 Birnessite and Humic Substances. Birnessite was prepared according to the method of McKenzie.18 Briefly, 2 mol of concentrated HCl was added dropwise to a boiling solution of 1 mol of KMnO4 in 2.5 L of H2O with vigorous stirring, followed by a further 10 min of boiling.18 The formed precipitate was filtered and washed with HCl (1 mM) and then with H2O until the aqueous solution reached pH 4 and then freeze-dried.41 The birnessite particles were confirmed by X-ray diffraction, showing the poorly crystalline structure. Soil humic acids (HAs) were extracted from both an agricultural and a forest soil by 0.1 M NaOH under anoxic conditions and precipitated at pH 1.2 Stock solutions of the purified HAs with 2 g C L−1 at pH 7 were prepared as previously described 2 and stored at 4 °C under N2 atmosphere until used. One 13C14

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scintillation cocktail. Quenching was corrected with external standards. The recovery of radioactivity in all experiments ranged from 96 to 105%. HP-14C-GPC. The molecular size distribution of phenolic residues within HAs was analyzed on a GPC column (PSS MCX 1000 Å, 8 mm × 300 mm; Polymer Standards Service GmbH, Mainz, Germany) using an HPLC system (HP 1100; Agilent Technology, USA), which was equipped with an autoinjector, a degasser, a diode array detector, and an online liquid scintillation radio flow detector (Ramona Star; Raytest, Straubenhardt, Germany), with a cell volume of 1300 μL. The molecules separated by the column ranged from 200 Da to 110 kDa, calibrated with molecular size standards of polysterene sulfonate sodium salts. The eluent was 0.6% K2CO3 solution (pH 11) at 0.1 mL min−1 and mixed with scintillation cocktail (Quicksafe Flow 2; Zinsser Analytic GmbH, Frankfurt, Germany) at a ratio of eluent/cocktail of 1:5 (v/v) before flowing into the radio detector. The absorbance signal of HAs was recorded at 360 nm. The detailed description has been published elsewhere.43 Solution 13C NMR. Solution 13C NMR was performed on a Bruker DMX600 spectrometer (Bruker, Rheinstetten, Germany) equipped with a 14 T magnetic field. The 13C-depleted HA with bound 13C-phenolic residues was dissolved in 0.1 M anoxic deuterated NaOH in 5 mm NMR tubes (541-PP-7; Wilmad-Labglass, Vineland, NJ, USA) under Ar gas protection, and about 20000 scans were recorded using an inverse gated method. 13C NMR spectra of pure 13C-catechol and 13C-pcoumaric acid were recorded in CDCl3 and acetone-d6 solution, respectively, containing 1% tetramethylsilane as internal standard.6

Figure 1. Mineralization of U-ring-14C-labeled catechol (A) and pcoumaric acid (B) by birnessite (δ-MnO2) in the presence and absence of humic acids (HAs) after 48 h of contact time at various pH values. Values are the mean with deviations of two individual experiments.

RESULTS AND DISCUSSION Mineralization of Phenolic Compounds by δ-MnO2. 14 C-Catechol and 14C-coumaric acid were abiotically mineralized by δ-MnO2 in the presence and absence of agricultural and forest soil HAs at pH 5, 6, 7, and 8 for 48 h. In the absence of HAs, considerable amounts of the phenolic compounds were mineralized, and the mineralization was strongly pH dependent, decreasing markedly with increasing solution pH (Figure 1). However, when HAs were present, the mineralization was significantly reduced, especially at pH 5. The mineralization of 14 C-catechol decreased from 28.5 to 6.5% at pH 5 and from 10.5 to 4.3% at pH 8. Although the two tested HAs from different land uses differed in their aromatic and aliphatic components and ash contents,2 their inhibitory effects on the mineralization of 14C-catechol and 14C-coumaric acid by δMnO2 were comparable (Figure 1). The time course of 14CO2 release from the reactions (Figure 2) showed that the mineralization was rapid and again that the two HAs had no significant differences in their inhibition of mineralization of the phenolic compounds by δ-MnO2 during the reaction time. The ion Mn2+ is released into the solution as a result of δ-MnO2 reduction during oxidation of phenolic compounds.22,38 Although released Mn2+ and reaction products of phenolic compounds may inhibit the reactivity of δ-MnO2 owing to sorption on the surface of δ-MnO2,23,27,28 the mineralization of both catechol and p-coumaric acid was still in progress after 48 h of reaction time. 14 C-Catechol and 14C-coumaric acids were mineralized at considerably different rates by δ-MnO2 (Figures 1 and 2). The mineralization rate of 14C-catechol was about 10-fold higher

than that of 14C-coumaric acid at all tested pH values (Figure 1). In addition, 14C-catechol was significantly mineralized (2.5%) within 48 h when both HA and δ-MnO2 (the control) were lacking (Figure 2A), which was attributed to autoxidation of 14C-catechol under oxic conditions. Autoxidation has been regarded as one important transformation mechanism for phenolic compounds in soil.2,9,44 The compound-specific mineralization of phenolic compounds by δ-MnO2 has also been previously observed.9,20 Together with our observations, we can conclude that the aromatic ring of phenolic compounds containing two hydroxyl groups, such as catechol, is more sensitive to mineralization than monophenols. The relative position of the two hydroxyl groups on the benzene ring determines the reactivity of the diphenols: compounds with hydroxyl groups at the ortho-position (e.g., catechol) are more rapidly oxidized than those having hydroxyl groups at the para(i.e., hydroquinone) and meta-positions (i.e., resorcinol),9 which seems to be consistent with the trend of redox potential of the diphenols.45 The pH value plays an important role in abiotic transformation of organic compounds by δ-MnO2.9,26,27 Oxidation of phenolic compounds by δ-MnO2 requires protons (MnO2 consumes four protons, and each hydroxyl group of phenols releases one proton);9 therefore, the oxidation potential of δMnO2 increases with decreasing pH.22 In the present study, the tested pH values were higher than the isoelectric point δ-MnO2 (pH 2.3).23 The strongly negatively charged surface of δ-MnO2 particles at the tested pH may have inhibited the accessibility of the phenolic compounds to δ-MnO2 and, thus, additionally attenuated the catalytic capacity of δ-MnO2 (Figure 1).

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Figure 2. Time course of the mineralization of U-ring-14C-labeled catechol (A and B) and p-coumaric acid (C, D) by birnessite (δ-MnO2) in the presence and absence of agricultural and forest soil humic acids (HAs) at pH 5 (A, C) and 8 (B, D). Values are the mean with deviations of two individual experiments.

Figure 3. Binding of residues of U-ring-14C-labeled catechol (A, B) and p-coumaric acid (C, D) to agricultural (A, C) and forest (B, D) soil humic acids (HAs) in the presence and absence of birnessite (δ-MnO2) at various pH values. Values are the mean with deviations of two individual experiments.

Binding of Residual Carbons of Phenolic Compounds to HAs. The reduced mineralization of the phenolic compounds by δ-MnO2 in the presence of HAs was accompanied by binding of the phenolic compounds to the HAs (Figure 3). As previously observed by Vinken et al.,2 binding of 14C-catechol and 14C-coumaric acid to HAs in the absence of δ-MnO2 was compound specific. 14C-Catechol, with

two hydroxyl groups on the benzene ring, bound to a great extent to both the agricultural and forest soil HAs, and the binding was pH dependent, whereas 14C-coumaric acid did not significantly bind in the absence of δ-MnO2 (Figure 3). This was because catechol has a low redox potential (E0 = 0.20 V at pH 7 vs Ag/AgCl)46 and, therefore, high autoxidation potential, whereas the redox potential of p-coumaric acid as a para8846

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substituted phenol is too high45,46 for it to be spontaneously oxidized in the presence of O2. However, when δ-MnO2 was present, the binding of 14C-coumaric acid at all tested pH values and of 14C-catechol at pH 5 and 6 was markedly enhanced (Figure 3). The binding of the phenolic compounds to the HAs in the presence of δ-MnO2 was not significantly pH dependent and was not compound specific, as opposed to the mineralization (Figure 1). This indicates that δ-MnO2 actively catalyzes the transformation of phenolic compounds even at higher pH values, although it was not able to completely mineralize the compounds. The binding of both 14C-catechol and 14C-coumaric acid to the agricultural soil HA (approximately 70% of total 14C applied to the system) was higher than that to the forest soil HA (approximately 60%) (Figure 3). This difference can probably be attributed to the higher aromaticity of the agricultural soil HA,2 as phenoxyl free radicals may easily react with the aromatic moieties of HAs. Molecular Size Distribution of Bound Residues within HAs. Oxidation of phenolic compounds by δ-MnO2 forms, in addition to CO2, small polar fragments and active intermediates, which condense to humic-like polymers with high aliphaticity, especially under oxic conditions.9,28,34,37,47 However, the active intermediates may also react with humic molecules, if present in the system, and are incorporated into the humic molecules, forming bound residues. We analyzed the molecular size distribution of the bound residues of 14Ccatechol and 14C-coumaric acids within the agricultural and forest HAs by HP-14C-GPC. The distribution of catecholic residues within the forest soil HA in the presence of δ-MnO2 at pH 5 is shown in Figure 4 as an example. Significant amounts

molecules. The other residues were homogeneously distributed within the soil HAs (Figure 4). Chemical Structure of Bound Residues. We used solution 13C NMR to analyze the chemical structure of the residues of catechol and p-coumaric acid bound to HAs. 13CCatechol, 13C-coumaric acid, and 13C-depleted HA were used in the study. By this means, the 13C signal intensity was enhanced approximately 2000-fold as compared to using phenolic compounds and HAs with natural 13C abundance, and therefore the identification of residues of catechol and pcoumaric acid within the HAs was greatly facilitated. 13 C NMR spectra of the catechol and p-coumaric acid residues in humic acids are shown in Figure 5. The 13C-

Figure 5. Solution 13C NMR of 13C-depleted humic acid (HA) (A), free U-ring-13C-labeled catechol (C) and free U-ring-13C-labeled pcoumaric acid (E), and their bound residues to the humic acid formed in the absence (B) and presence (D, F) of δ-MnO2 at pH 5. Signals at 77.0 ppm of catechol (C) and at 204.2 and 27.8 ppm of p-coumaric acid (E) are attributed to solvents of chloroform and acetone, respectively. Assignments of signals of pure 13C-catechol and 13Ccoumaric acid have been described by Ji et al.6

Figure 4. Molecular size distribution of 14C-catecholic residues within forest soil humic acid after oxidation by δ-MnO2 at pH 5 for 48 h. UV signals represent the distribution of the humic molecules, whereas 14C signals represent that of 14C-labeled residues. Shadows are attributed to oligomers of catechol self-condensation by autoxidation.

depleted HA had a very low 13C-background, with only one signal at 160 ppm (probably from carbonate, Figure 5A), which was in agreement with its low δ13C value (−943‰). Therefore, the signals in spectra B, D, and F represent the HA-bound residual carbons of 13C-catechol and 13C-coumaric acid. By comparing the spectra of the bound residues of 13C-catechol (Figure 5D) and 13C-coumaric acid (Figure 5F) formed in the presence of δ-MnO2 with those of pure 13C-catechol (Figure 5A) and 13C-coumaric acid (Figure 5E), respectively, it becomes apparent that most of the 13C signals of the residues had the same chemical shifts as their parent compounds. This clearly indicates that the majority of the HA-bound residues still contained the intact benzene ring. Moreover, the residues seem to be bound to the HA mainly via mechanisms that did not

of bound residues of 14C-catechol and 14C-coumaric acid of small molecular mass (400−900 Da) were observed on the 14C signal, accounting for 2−8% of the total HA-bound residues. Because the carbon content of the residues was