Laccase-Catalyzed Oxidation of Iodide and Formation of Organically

Nov 29, 2012 - types of Japanese soil by using ABTS as a substrate. Soil samples ... is a significant correlation between laccase activity and Kd valu...
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Laccase-Catalyzed Oxidation of Iodide and Formation of Organically Bound Iodine in Soils Miharu Seki,† Jun-ichi Oikawa,† Taro Taguchi,† Toshihiko Ohnuki,‡ Yasuyuki Muramatsu,§ Kazunori Sakamoto,† and Seigo Amachi*,† †

Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba 271-8510, Japan Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan § Department of Chemistry, Gakushuin University, Mejiro 1-5-1, Toshima-ku, Tokyo 171-8588, Japan ‡

S Supporting Information *

ABSTRACT: Laccase oxidizes iodide to molecular iodine or hypoiodous acid, both of which are easily incorporated into natural soil organic matter. In this study, iodide sorption and laccase activity in 2 types of Japanese soil were determined under various experimental conditions to evaluate possible involvement of this enzyme in the sorption of iodide. Batch sorption experiment using radioactive iodide tracer (125I−) revealed that the sorption was significantly inhibited by autoclaving (121 °C, 40 min), heat treatment (80 and 100 °C, 10 min), γ-irradiation (30 kGy), N2 gas flushing, and addition of reducing agents and general laccase inhibitors (KCN and NaN3). Interestingly, very similar tendency of inhibition was observed in soil laccase activity, which was determined using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a substrate. The partition coefficient (Kd: mL g−1) for iodide and specific activity of laccase in soils (Unit g−1) showed significant positive correlation in both soil samples. Addition of a bacterial laccase with an iodideoxidizing activity to the soils strongly enhanced the sorption of iodide. Furthermore, the enzyme addition partially restored iodide sorption capacity of the autoclaved soil samples. These results suggest that microbial laccase is involved in iodide sorption on soils through the oxidation of iodide.

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INTRODUCTION Iodine is an essential trace element for vertebrates and is a constituent of the thyroid hormones, i.e. thyroxine and triiodothyronine. Insufficient iodine in the diet can cause iodine deficiency disorders such as endemic goiter and cretinism.1 On the other hand, a long-lived iodine-129 (129I, half-life: 16 million y) is one of the most persistent radionuclides released from nuclear facilities into the environment.2,3 Given its long half-life, 129I is expected to behave similarly to stable iodine (127I) over long time periods and possibly accumulates in human thyroid gland.4 Therefore, it is necessary to obtain better information on the behavior of iodine in the environment for accurate safety assessments of 129 I. The predominant chemical forms of iodine in terrestrial environments are iodate (IO3−; oxidation state: +5), iodide (I−; oxidation state: −1), and organically bound iodine.5−7 Iodate and iodide can adsorb on soils, while the former has a much higher partition coefficient (Kd) than the latter.8,9 It is widely accepted that these inorganic iodine species are immobilized in soils in the form of organically bound iodine. Several lines of evidence have suggested that iodate is reduced to molecular iodine (I2; oxidation state: 0) or hypoiodous acid (HIO; oxidation state: +1) by humic substances in soils,10−12 and that © 2012 American Chemical Society

these electrophilic iodine species react with aromatic ring of soil organic matter to form iodine−carbon covalent bond.13−15 On the other hand, abiotic oxidation of iodide to I2 or HIO may not occur or occurs very slowly in soils, since iodide is stable under pH and Eh conditions generally found in soil environments.16 A large number of studies have shown that iodide sorption on soils is influenced by soil microbial activity. Decreased sorption of iodide by autoclaving, fumigation, air-drying, γirradiation, anaerobic treatment (N2 gas flushing), heat treatment, antibiotics, and reducing agents has been reported.9,17−20 These results strongly suggest that soil microorganisms or microbial enzymes are involved in the sorption of iodide, probably through the oxidation of iodide to I2/HIO and subsequent iodination of soil organic matter. Although several peroxidases were found to iodinate humic acid in the presence of iodide and H2O2 in laboratory experiments,21 it is unclear whether such enzymes actually catalyze the oxidation of iodide in soil environments. Received: Revised: Accepted: Published: 390

August 9, 2012 November 21, 2012 November 29, 2012 November 29, 2012 dx.doi.org/10.1021/es303228n | Environ. Sci. Technol. 2013, 47, 390−397

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The fact that iodide sorption on soils is inhibited under anaerobic conditions17−19 strongly suggests the involvement of oxidases in the process. Oxidases catalyze the oxidation of various compounds using molecular oxygen as an electron acceptor. Among these, laccase (one of the phenol oxidases) is commonly produced by soil fungi and bacteria and can oxidize various compounds including methoxyphenols, diphenols, aromatic diamines, and metal ions.22 In addition, several fungal laccases have been reported to possess capacities for iodide oxidation.23 Recently, Suzuki et al.24 found that iodide-oxidizing enzyme purified from Alphaproteobacterium strain Q-1 was a laccase, since it oxidized general substrates of laccase, including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP). Furthermore, a protein encoding a major subunit of this enzyme (IoxA) showed a phylogenetically close relationship with putative laccases of soil bacteria such as Stackebrandtia nassauensis and Methylobacter tundripaludum.24 These results suggest that laccases with iodide-oxidizing activity are distributed widely among soil microorganisms. The aim of this study was to clarify whether microbial laccase is involved in iodide oxidation and subsequent incorporation into soil organic matter. We determined laccase activity in 2 types of Japanese soil by using ABTS as a substrate. Soil samples were treated by various methods to repress or inhibit laccase activity, and the partition coefficient (Kd) for iodide under the same experimental conditions was also determined by batch sorption experiment. We then examined whether there is a significant correlation between laccase activity and Kd values in these soils. Furthermore, a bacterial laccase with iodideoxidizing activity was added to untreated and sterilized soil samples to determine whether the enzyme could enhance and restore the sorption of iodide.

Kd (mL g −1) =125 I adsorbed on soil /125 I remaining in solution

Kd is generally determined at equilibrium.8,9 However, in this study, Kd was determined after the incubation of soil slurries for 2.5 h, since prolonged incubation caused auto-oxidation of reducing agents (see below) with the air. Soil slurries were subjected to various treatments that potentially repress or inhibit iodide sorption. For sterilization, the slurry was autoclaved at 121 °C for 40 min. Heat treatment was performed at 60 °C, 80 °C, or 100 °C for 10 min. Eventually after the heat treatment, the slurry was immediately transferred on ice to avoid prolonged heat treatment. 125I was then added to the slurry, and it was incubated at 30 °C for experimental assays. In cold treatment, the slurry was put on ice prior to 125I addition and then was incubated on ice. For γirradiation, a portion of each soil was irradiated with γ-ray at 30 kGy, and the slurry was prepared aseptically from the irradiated soil. A solution of reducing agents, i.e., cysteine-HCl, dithiothreitol (DTT), and sodium thiosulfate (Na2S2O3), was freshly prepared and added to obtain a final concentration of 5 mM. Similarly, general laccase inhibitors, including sodium azide (NaN3), potassium cyanide (KCN), and EDTA, were added to obtain a final concentration of 10 mM. In the preliminary experiments, reducing agents and laccase inhibitors were applied at various concentrations ranging from 0.05 to 10 mM, and appropriate concentrations at which iodide sorption is significantly repressed were determined. In anaerobic treatment, the slurry was dispensed into a 60-mL serum bottle, and it was flushed with a N2 gas stream for 5 min. The gas was passed through a column of hot reduced copper filings to remove the traces of oxygen. The bottle was then sealed using a thick butyl rubber stopper and an aluminum cap. The bottle was supplemented with 125I with a plastic syringe and was incubated at static conditions. Laccase Activity in Soils. Soil laccase activity was determined spectrophotometrically.26 Soil (0.1 to 0.4 g wet weight) was transferred to a 100-mL Erlenmeyer flask and mixed with 20 mL of distilled water. ABTS was directly added to the slurry at a final concentration of 1 mM. After static incubation at 30 °C, a portion of the slurry was centrifuged (15,000 × g, 4 °C for 3 min), and the absorbance of the supernatant was determined at 420 nm. The molar absorption coefficient (ε420) of 36.0 mM−1 cm−1 for ABTS was used. One unit of laccase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of the product (ABTS cation radical) per min. Specific activity of laccase in soil is expressed as units per gram dry soil (U g−1). Specific activity was calculated from the absorbance at 2 h incubation of the slurry. Various treatments that potentially repress or inhibit laccase activity were carried out essentially as described above. Addition of Bacterial Laccase to Soil Slurry. Alphaproteobacterium strain Q-1 is an iodide-oxidizing bacterium originally isolated from natural gas brine water in Miyazaki, Japan.27 It catalyzes the oxidation of iodide to molecular iodine by means of an extracellular iodide-oxidizing enzyme. Biochemical and molecular analyses of this enzyme have revealed that it is a laccase (multicopper oxidase).24 Although strain Q-1 was not isolated from the soil environment, its laccase (IoxA) is phylogenetically related to putative laccases of soil bacteria such as Stackebrandtia nassauensis and Methylobacter tundripaludum. We used this enzyme as a representative

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MATERIALS AND METHODS Soils Samples. Two types of Japanese soil collected from the surface layer (0 to 5 cm) of forest and rice paddy were used. The former was classified as light-colored Andosol, and the latter was gray lowland soil. The soils were passed through a 2mm sieve and stored in glass bottles with caps at room temperature until use. In some cases, sand-dune Regosol, dark red soil, humic Andosol, and brown forest soil were also used. The physicochemical characteristics of the soil samples are described in Table S1 (see the Supporting Information). Detailed information on sampling location and analytical methods used for determining soil properties have been described by Sakamoto and Hodono.25 Batch Sorption Experiment. Soil (0.1 to 1.0 g wet weight) was transferred to a 100-mL Erlenmeyer flask and mixed with 10 mL of distilled water. To this was added 125I (Na125I at 629 GBq mg−1; Perkin-Elmer, MA) to obtain a final concentration of approximately 25 kBq mL−1 (approximately 3 nM). In some cases, stable iodide (KI) was also added at final concentrations of 10 to 1,000 μM. After sealing with a silicone cap, the flask was incubated at 30 °C at static conditions. At various time intervals, a portion of the slurry was sampled and centrifuged at 15,000 × g at 4 °C for 3 min. The supernatant was transferred to a scintillation vial, and the activity of 125I was measured for 10 min using an NaI scintillation counter (Aloka ARC-370M). An Erlenmeyer flask containing only distilled water and 125I was also prepared for measuring the total amount of 125I in the solution. From the activity of 125I, the Kd value for iodide was calculated according to the following equation: 391

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Figure 1. Iodide sorption on forest soil (A), paddy soil (B), and forest soil under various concentrations of stable iodide (C). Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations. In most cases, standard deviation values are smaller than those denoted by the symbols.

into polyethylene bags and frozen until analysis. K-edge X-ray absorption near-edge structure (XANES) spectra of iodine were measured according to Kodama et al.28 at beamline BL14B2 at SPring-8 (Hyogo, Japan). An Si(311) double-crystal monochromator with two mirrors was used to obtain the incident X-rays. Standard samples and soil samples were measured in transmission and fluorescence modes, respectively. A 19-element Ge semiconductor detector (SSD; Ortec) was employed to collect fluorescence X-rays. The data analysis, including background subtraction, normalization, and linear combination fitting of XANES spectra, was performed with the REX2000 Ver. 2.3.0 software (Rigaku).

laccase with iodide-ozidizing activity, since kinetic study has shown that it is the most efficient iodide-oxidizing enzyme among laccases reported thus far.24 For preparation of bacterial laccase, strain Q-1 was grown in marine broth 2216 (Becton Dickinson, Sparks, MD, USA) supplemented with 40 μM CuCl2·2H2O at 30 °C. Culture broth after 48 h was centrifuged at 6,000 × g for 20 min at 4 °C, and the supernatant was used as a crude enzyme solution. One unit of iodide-oxidizing activity was defined as the amount of crude enzyme catalyzing the formation of 1 μmol of I2 per min at 30 °C. In previous studies, it was confirmed that strain Q-1 does not secrete peroxidase or haloperoxidase in the culture supernatant27 and that laccase was the main protein in the crude enzyme solution.24 Batch sorption experiment was carried out essentially as described above, but soil slurry consisted of 1 g (wet weight) of soil and 10 mL of distilled water. After autoclaving the slurry, 300 mU (as an iodideoxidizing activity) of bacterial laccase was added to the slurry. This enzyme activity corresponds with approximately 30 mU of ABTS-oxidizing activity.24 Thus, the final activities of bacterial laccase in the soil slurries were 30 to 48 mU g−1 (as ABTSoxidizing activity), which were not significantly different from those in the original soils (23 to 73 mU g−1, see Table 2), and therefore were at an environmentally relevant level. Inactivated laccase was also prepared by heating the crude enzyme solution at 121 °C for 40 min. In another experiment, untreated soil slurry (not autoclaved) was incubated with laccase. When the slurry was incubated with stable iodide (10 to 1,000 μM), significant amounts of oxidized iodine species (probably I2) were observed to be occasionally adsorbed on the silicone cap of the Erlenmeyer flask. In such cases, 125I activity adsorbed on the cap was measured, and it was added to the 125I activity remaining in solution for compensation. The maximum activity of 125I adsorbed on the cap was 1.5% (100 μM stable iodine) to 30% (1,000 μM stable iodide) of the total 125I added to the soil slurry. XANES Analysis. The slurry of forest soil was incubated with bacterial laccase and 100 μM of stable iodide for 24 h. The slurry was filtered through a 0.45 μm membrane filter and washed once with distilled water. The soil samples were packed

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RESULTS AND DISCUSSION Iodide Sorption Experiment. Radioactive iodide tracer (125I−) was added to soil slurries, and a batch sorption experiment was carried out. As shown in Figure 1A, sorption of iodide on forest soil was rapid, and more than 80% of total iodide was adsorbed on the soil within 2.5 h. At 2.5 h, Kd values for iodide for 0.1, 0.2, and 1.0 g of forest soil were 698, 434, and 313 mL g−1, respectively. An inverse relationship between Kd and the soil-to-water ratio was consistent with that observed in the previous studies.9,19 Iodide adsorption on paddy soil was much slower than that on forest soil, and only 27 to 73% of total iodide adsorbed on the soil at 2.5 h (Figure 1B). Kd values for iodide to 0.1, 0.2, and 1.0 g of paddy soil were 37, 35, and 27 mL g−1, respectively. When stable iodide (127I−) was added to forest soil slurry containing 0.2 g of soil, apparent sorption of iodide decreased dramatically (Figure 1C), as has been observed in other studies.6,9,18 Kd values at 72 h in the presence of 10, 100, and 1,000 μM stable iodide decreased to 134, 32, and 11 mL g−1, respectively, while that in the absence of stable iodide was 9,497 mL g−1. When more than 10 μM of stable iodide was added, no apparent sorption of iodide was observed in the paddy soil (data not shown). Iodide sorption was then determined under various experimental conditions by using 0.2 g of soils in the absence of stable iodide (Table 1). Iodide sorption did not occur when both soil samples were autoclaved at 121 °C for 40 min (Figures 1A and 1B). Similarly, heat treatment of the soil at 80 392

dx.doi.org/10.1021/es303228n | Environ. Sci. Technol. 2013, 47, 390−397

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Table 1. Kd Values (mL g−1) for Iodide to 2 Types of Japanese Soil after Various Treatmentsa no treatment autoclaving (121 °C, 40 min) heat 100 °C, 10 min 80 °C, 10 min 60 °C, 10 min cold (reaction on ice) γ-irradiation (30 kGy) reducing agents cysteine (5 mM) DTT (5 mM) Na2S2O3 (5 mM) enzyme inhibitors NaN3 (10 mM) KCN (10 mM) EDTA (10 mM) anaerobic (N2 gas flushing)

forest soil

paddy soil

506.2 ± 93.2 (100.0) 0.0 ± 0.0 (0.0)b

20.7 ± 3.7 (100.0) 0.0 ± 0.0 (0.0)b

12.2 ± 0.2 (2.4)b 52.3 ± 0.6 (10.3)b 409.8 ± 2.5 (81.0) 277.8 ± 3.2 (54.9)b 78.4 ± 0.7 (15.5)b

2.4 ± 0.0 (11.8)b 0.8 ± 0.0 (3.9)b 27.9 ± 0.3 (134.9) 2.0 ± 0.0 (9.7)b 4.0 ± 0.1 (19.4)b

42.6 ± 2.6 (8.4)b 20.4 ± 1.2 (4.0)b 47.2 ± 0.1 (9.3)b

5.1 ± 0.0 (24.6)b 1.1 ± 0.0 (5.3)b 8.3 ± 0.0 (39.9)b

22.5 ± 0.3 (4.4)b 3.4 ± 0.1 (0.7)b 245.0 ± 10.7 (48.4)b 55.7 ± 0.9 (11.0)b

1.2 ± 0.0 (5.8)b 0.3 ± 0.0 (1.3)b 60.2 ± 0.4 (290.7)b 2.9 ± 0.0 (13.8)b

Figure 2. Laccase activity in forest soil (A) and paddy soil (B). Activity is expressed as μmol of 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonate) (ABTS) oxidized in 20 mL of reaction mixture consisting of soil (0.1 to 0.4 g), distilled water (20 mL), and 20 μmol of ABTS. Symbols represent the mean values obtained for duplicate determinations, and bars indicate range of values.

a

All the Kd values were determined after the incubation of soil slurries for 2.5 h. The slurries contained 0.2 g wet weight of soil but did not contain stable iodide. All the values except for the control value (no treatment) are the mean values obtained from triplicate determinations ± SD. The control values are the mean values obtained from 7 independent sorption experiments ± SD. Comparison of percentage Kd values with the control values are stated in parentheses. bValues are significantly (p < 0.01) different from that of the control as per Student’s t test.

Figure 2A, oxidation of ABTS proceeded linearly for 2 h in the forest soil. The amount of oxidized ABTS was much higher when a much larger amount of soil was added to the enzyme assay. However, specific activity of laccase in the soil (mU g−1) calculated at 2 h showed an inverse relationship with the amounts of soil, i.e., 121, 105, and 68 mU g−1 for 0.1, 0.2, and 0.4 g of forest soil, respectively. In paddy soil, the rate of ABTS oxidation decreased with time (Figure 2B). Specific activity of laccase at 2 h was much lower than that of the forest soil, and it was 46, 39, and 28 mU g−1 for 0.1, 0.2, and 0.4 g of paddy soil, respectively. The activity in both the soils was completely inhibited by autoclaving, indicating that the oxidation of ABTS is catalyzed biologically. Generally, enzyme activity is proportional to both reaction time and enzyme concentration. The nonlinear oxidation kinetics of ABTS in paddy soil, as well as lower specific activities with much higher amount of soils, suggest that these soils contain certain compounds that interfere with the ABTS-based soil laccase activity assay. Laccase activity was then assayed under various experimental conditions by using 0.4 g of soils (Table 2). Laccase activity showed much higher heat stability than iodide sorption, but treatment at 100 °C significantly inhibited the activity in both the soils. Such a strong heat stability of laccase has also been reported in Mediterranean soils.30 Cold and γ-ray sensitivities were also apparent. More than 95% of total activity was inhibited by cysteine, DTT, and Na2S2O3. As expected, laccase activity was susceptible to the general laccase inhibitors, but EDTA did not show significant inhibitory effect in paddy soil. Under anaerobic condition, 88 to 93% of total laccase activity was inhibited in both the soils. Laccase activity in soil has been determined by using various substrates including syringaldazine, catechol, 2,6-DMP, DOPA, and ABTS.31 Among these, syringaldazine is generally considered to be a defining substrate of laccase,31 but soils often show no activity against syringaldazine.26 Floch et al.30 found that ABTS is a sensitive substrate for soil laccase assay and that the assay system is free of interference. In this study, ABTS was not oxidized by autoclaved soils, indicating that

or 100 °C for 10 min decreased the Kd values to 12% or less compared with the control (no treatment). Such heat sensitivity suggests that the sorption is, at least in part, a biological process. The sorption also showed significant cold sensitivity. Furthermore, soil samples sterilized with γ-irradiation (30 kGy) showed significantly decreased Kd values (16 to 19% of the control). Three types of reducing agents5 mM each of cysteine, DTT, and Na2S2O3inhibited the sorption of iodide. In particular, DTT decreased Kd values to 4 to 5% of the control in both the soils, although Na2S2O3 showed relatively weaker inhibitory effect. The strong inhibition of iodide sorption by reducing agents suggests that the sorption involves an iodideoxidizing process. We also tested the effect of general laccase inhibitors on the sorption of iodide. NaN3 bridges types 2 and 3 copper centers of this copper-containing enzyme, and KCN is also an irreversible inhibitor of laccase.29 Both of these compounds strongly inhibited the sorption of iodide, decreasing the Kd values to 1 to 6% of the control. EDTA, a metal chelator, showed only a weak inhibitory effect on forest soil, while it unexpectedly enhanced the sorption in paddy soil. Since there would be a substantial amount of metal ions in soil slurries, copper ions present in the active site of soil laccase might not be attacked by EDTA. Under anaerobic condition (under N2 atmosphere), significantly decreased Kd values (11 to 14% of the control) were observed in both the soils. The fact that iodide sorption is inhibited by reducing agents, laccase inhibitors, and anaerobic treatment strongly suggests that iodide is oxidized by an oxygen-dependent enzyme such as laccase, and the oxidized species (I2/HIO) is subsequently incorporated into soil organic matter. Laccase Activity in Soils. The soil slurries were incubated with ABTS for direct assay of laccase in soils. As shown in 393

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Table 2. Specific Activity of Laccase (mU g Dry Soil−1) in 2 Types of Japanese Soil after Various Treatmentsa no treatment autoclaving (121 °C, 40 min) heat 100 °C, 10 min 80 °C, 10 min 60 °C, 10 min cold (reaction on ice) γ-irradiation (30 kGy) reducing agents cysteine (5 mM) DTT (5 mM) Na2S2O3 (5 mM) enzyme inhibitors NaN3 (10 mM) KCN (10 mM) EDTA (10 mM) anaerobic (N2 gas flushing)

forest soil

paddy soil

72.7 ± 5.6 (100.0) 0.0 ± 0.0 (0.0)c

22.7 ± 4.1 (100.0) 0.3 ± 0.0 (1.3)c

18.3 45.4 69.3 35.0 18.3

± ± ± ± ±

0.0 0.0 0.0 5.3 0.9

(25.2)c (62.4)c (95.3) (48.1)c (25.2)c

14.1 21.6 28.5 12.1 12.7

± ± ± ± ±

0.0 0.0 0.0 0.6 0.5

values showed a significant (p < 0.001) correlation with laccase activity in forest soil with an R2 value of 0.879. In paddy soil, however, 2 data plots have been eliminated from Figure 3B, since data from these experiments (heat treatment at 80 °C and EDTA addition) strongly decreased the R2 value (less than 0.5). This was due to unexpected enhancement of the Kd value by EDTA (Table 1) and strong heat stability of laccase in the paddy soil (Table 2). In the absence of these 2 data plots, significant (p < 0.001) correlation with an R2 value of 0.786 was observed. These results indicate that the Kd value for iodide synchronizes with laccase activity in soils. The significant correlations between Kd and laccase activity supports the hypothesized role of this enzyme in the sorption of iodide. Most of the treatments listed in Tables 1 and 2 seemed to have a greater inhibitory effect on forest soil than paddy soil. It might be possible that laccases in paddy soil are localized in nonproliferating cells such as fungal spores and bacterial endospores. Alternatively, the enzyme might be bound to clay and humic colloids, which would stabilize the enzyme activity.32 The reason why EDTA stimulated both iodide sorption and laccase activity in paddy soil is unclear. EDTA might extract metal ions of soil colloids, destruct the colloidal structure, and release the colloid-bounded enzymes to the aqueous phase. Such enzymes may survive for a short period32 and would show much higher activity than those bound to soil colloids. We also determined Kd values and specific laccase activities of other 4 types of Japanese soils, i.e., Sand-dune Regosol, dark red soil, humic Andosol, and brown forest soil, and plotted these data with those of forest (light-colored Andosol) and paddy (gray lowland soil) soils (Figure 3C). However, no significant correlation was observed (p = 0.102, R2 = 0.527). Although we analyzed only a limited number of soil samples, this result suggests that laccase activity may not directly reflect the iodide sorption capacity of individual soil. Considering that laccases with and without iodide-oxidizing activity may coexist in soil environments, relative proportion of the former enzyme to the latter might vary depending on the soil type. In addition, abiotic factors such as iodide oxidation by manganese oxides

(62.1)b (95.2) (125.6) (53.3)b (55.9)b

0.1 ± 0.0 (0.1)c 0.2 ± 0.1 (0.3)c 1.4 ± 0.0 (1.9)c

0.0 ± 0.0 (0.0)c 0.0 ± 0.0 (0.0)c 1.2 ± 0.0 (5.3)c

3.1 ± 1.7 (4.3)c 5.5 ± 0.0 (7.6)c 41.7 ± 9.6 (57.4)c 4.8 ± 0.0 (6.6)c

6.2 ± 0.8 (27.3)c 1.9 ± 0.0 (8.4)c 25.1 ± 0.6 (110.6) 2.8 ± 2.1 (12.3)c

a

Soil laccase activity was determined spectrophotometrically by using ABTS as the substrate. All the specific activities were determined after the incubation of soil slurries for 2 h, and the slurries contained 0.4 g wet weight of soil. All the values except for the control value (no treatment) are the mean values obtained from duplicate determinations ± range of values. The control values are the mean values obtained from 7 independent laccase assays ± SD. Percentage values compared with the control are stated in parentheses. bValues are significantly (p < 0.05) different from the control value as per Student’s t test. cValues are significantly (p < 0.01) different from the control value as per Student’s t test.

there was no chemical or abiotic oxidation of ABTS in our soil slurries. Correlation of Kd with Laccase Activity. Figures 3A and 3B show correlation of Kd values for iodide with specific activity of laccase in forest and paddy soils, respectively. The figures consist of 20 data plots including those obtained from 7 independent control experiments. As shown in Figure 3A, Kd

Figure 3. Correlation of Kd values for iodide with specific activity of laccase in forest soil (A) and paddy soil (B). Symbols represent the mean values obtained for triplicate and duplicate determinations for Kd values and laccase activity. Horizontal and vertical bars indicate standard deviations for Kd values and range of laccase activity, respectively. (C) Correlation of Kd values for iodide with specific activity of laccase in 6 types of Japanese soil. Light-colored Andosol (●), gray lowland soil (○), sand-dune Regosol (▲), dark red soil (Δ), humic Andosol (■), and brown forest soil (□). 394

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(birnessite)33 might be involved in some soils. Further study with a large number of soil samples is needed to rigorously assess whether laccase activity could be used as a proxy for iodide sorption capacity of different soils. Enhancement of Iodide Sorption by Bacterial Laccase. Bacterial laccase prepared from a culture of Alphaproteobacterium strain Q-1 was added to soil slurries to determine if the sorption is enhanced by this enzyme. In the absence of stable iodide, no enhancement of iodide sorption was observed (data not shown). However, in the presence of 10 μM stable iodide, iodide sorption was dramatically enhanced by laccase in both forest and paddy soils (Figure 4). When bacterial laccase

Figure 5. Iodine K-edge XANES spectra of standard materials and soil slurry incubated with 100 μM of KI (stable iodide) and bacterial laccase. Dots represent the experimental spectrum, and bold line represents the linear combination of XANES spectrum from reference compounds to reproduce the experimental spectrum.

maximum (Vmax). Km represents an enzyme’s affinity for a substrate, and a lower Km indicates much higher affinity because the rate will reach Vmax more quickly. From the rates of iodide sorption in the presence of stable iodide (Figure 1C), we can roughly estimate the Km of native laccase present in the forest soil. Assuming that the oxidation of iodide is an enzymatic step and that the following iodination is an abiotic non rate-limiting step, the Km value for iodide is calculated to be 97 μM by using the Lineweaver−Burk double reciprocal plot (Figure S1). The Km value of native laccase in paddy soil should be much lower than this value, since the sorption was more easily saturated by stable iodide (Figure 4B). On the other hand, the Km value of the laccase from Alphaproteobacterium strain Q-1 for iodide is 2,640 μM.24 Therefore, bacterial laccase would oxidize iodide only slowly under low concentration of iodide (in the absence of stable iodide), whereas native soil laccases with much lower Km values would oxidize iodide more efficiently. Since Alphaproteobacterium strain Q-1 was originally isolated from iodide-rich brine water,27 the high Km value of its laccase might reflect iodide concentration of the original bacterial habitat. Restoration of Iodide Sorption Capacity of Autoclaved Soils. We finally tested whether bacterial laccase could restore the iodide sorption capacity of autoclaved soils. As shown in Figure 6, laccase partially restored the capacity even in the absence of stable iodide in both the soils, while heatinactivated enzyme did not show any restoration effect. The partial restoration was also observed in the presence of 10 to 1,000 μM stable iodide (Figure S2). The reason why bacterial laccase did not provide full sorption capacity with autoclaved soils remains unclear. One possible explanation is that iodinesorption sites in soils were destroyed by autoclaving. However, as shown in Figure S2C, iodide sorption on autoclaved soil was not saturated even in the presence of 1,000 μM stable iodide. This clearly indicates that the autoclaved soil still maintains an excess amount of iodine-sorption sites. Considering that various soil enzymes including polyphenol oxidase are susceptible to

Figure 4. Enhanced sorption of iodide on forest soil (A) and paddy soil (B) in the presence of bacterial laccase. Soils (1 g) were incubated with 10 mL of distilled water, 33 Bq of 125I−, 10 μM of KI (stable iodide), and 300 mU of bacterial laccase. Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations.

had been heat-inactivated (121 °C, 40 min), no enhancement or only limited enhancement was observed. The addition of bacterial laccase increased Kd values to 21 and 55 times higher than those of the controls in the forest (at 52 h) and paddy (at 48 h) soils, respectively. The enhancement was also observed under 100 μM iodide, in which a Kd value 116 times higher than that of the control was observed in the forest soil (data not shown). When the slurry was incubated with laccase and stable iodide, the color of the slurry changed to yellow, indicating that iodide was actually oxidized to I2/HIO in the slurry. To confirm formation of organically bound iodine in the slurry, iodine species in the slurry was analyzed by K-edge XANES spectra. As shown in Figure 5, soil slurry incubated with 100 μM stable iodide and bacterial laccase showed a XANES spectrum that is distinct from NaI. According to the fitting of the spectra by linear combination of organic and inorganic iodine, it was estimated that 100% of iodine is in the form of organically bound iodine. When the slurry was incubated without laccase or incubated with heat-inactivated laccase, no clear XANES spectra were observed. This was probably due to leach out of iodide during washing of the soil with distilled water. As described above, the enhancement of iodide sorption was observed only in the presence of stable iodide. This was probably related to the affinity of bacterial laccase for iodide. In Michaelis−Menten kinetics, the Michaelis constant (Km) is the substrate concentration at which the reaction rate is half of the 395

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-47-308-8867. Fax: +81-47-308-8867. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by JSPS KAKENHI Grant Number 17710043 and 20780049.

(1) Hetzel, B. S.; Mano, M. T. A review of experimental studies of iodine deficiency during fetal development. J. Nutr. 1989, 119, 145− 151. (2) Moran, J. E.; Oktay, S.; Santschi, P. H.; Schink, D. R. Atmospheric dispersal of 129iodine from nuclear fuel reprocessing facilities. Environ. Sci. Technol. 1999, 33, 2536−2542. (3) Buraglio, N.; Aldahan, A.; Possnert, G.; Vintersved, I. 129I from the nuclear reprocessing facilities traced in precipitation and runoff in northern Europe. Environ. Sci. Technol. 2001, 35, 1579−1586. (4) Vandecasteele, C. M.; Van Hees, M.; Hardeman, F.; Voigt, G.; Howard, B. J. The true absorption of 131I, and its transfer to milk in cows given different stable iodine diets. J. Environ. Radioact. 2000, 47, 301−317. (5) Fuge, R.; Johnson, C. C. The geochemistry of iodine. Environ. Geochem. Health 1986, 8, 31−54. (6) Schwehr, K. A.; Santschi, P. H.; Kaplan, D. I.; Yeager, C. M.; Brinkmeyer, R. Organo-iodine formation in soils and aquifer sediments at ambient concentrations. Environ. Sci. Technol. 2009, 43, 7258−7264. (7) Otosaka, S.; Schwehr, K. A.; Kaplan, D. I.; Roberts, K. A.; Zhang, S.; Xu, C.; Li, H.-P.; Ho, Y.-F.; Brinkmeyer, R.; Yeager, C. M.; Santschi, P. H. Factors controlling mobility of 127I and 129I species in an acidic groundwater plume at the Savannah River Site. Sci. Total Environ. 2011, 409, 3857−3865. (8) Yoshida, S.; Muramatsu, Y.; Uchida, S. Adsorption of I− and IO3− onto 63 Japanese soils. Radioisotopes 1995, 44, 837−845. (9) Fukui, M.; Fujikawa, Y.; Satta, N. Factors affecting interaction of radioiodide and iodate species with soil. J. Environ. Radioact. 1996, 31, 199−216. (10) Yamaguchi, N.; Nakano, M.; Tanida, H.; Fujiwara, H.; Kihou, N. Redox reaction of iodine in paddy soil investigated by field observation and the I K-edge XANES fingerprinting method. J. Environ. Radioact. 2006, 86, 212−226. (11) Yamaguchi, N.; Nakano, M.; Takamatsu, R.; Tanida, H. Inorganic iodine incorporation into soil organic matter: evidence from iodine K-edge X-ray absorption near-edge structure. J. Environ. Radioact. 2010, 101, 451−457. (12) Zhang, S.; Du, J.; Xu, C.; Schwehr, K. A.; Ho, Y.-F.; Li, H.-P.; Roberts, K. A.; Kaplan, D. I.; Brinkmeyer, R.; Yeager, C. M.; Chang, H.-S.; Santschi, P. H. Concentration-dependent mobility, retardation, and speciation of iodine in surface sediment from the Savannah River Site. Environ. Sci. Technol. 2011, 45, 5543−5549. (13) Schlegel, M. L.; Reiller, P.; Mercier-Bion, F.; Barré, N.; Moulin, V. Molecular environment of iodine in naturally iodinated humic substances: Insight from X-ray absorption spectroscopy. Geochim. Cosmochim. Acta 2006, 70, 5536−5551. (14) Shimamoto, Y. S.; Takahashi, Y.; Terada, Y. Formation of organic iodine supplied as iodide in a soil-water system in Chiba, Japan. Environ. Sci. Technol. 2011, 45, 2086−2092. (15) Xu, C.; Zhang, S.; Ho, Y.-F.; Miller, E. J.; Roberts, K. A.; Li, H.P.; Schwehr, K. A.; Otosaka, S.; Kaplan, D. I.; Brinkmeyer, R.; Yeager, C. M.; Santschi, P. H. Is soil natural organic matter a sink or source for mobile radioiodine (129I) at the Savannah River Site? Geochim. Cosmochim. Acta 2011, 75, 5716−5735. (16) Luther, G. W., III; Wu, J.; Cullen, J. Redox chemistry of iodine in seawater: frontier molecular orbital theory considerations. In

Figure 6. Bacterial laccase partially restores iodide sorption capacity of the autoclaved soils. One gram each of the forest soil (A) and paddy soil (B) was mixed with 10 mL of distilled water, autoclaved, and supplemented with 33 Bq of 125I− and 300 mU of bacterial laccase. Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations.

humic acid,34 bacterial laccase might be inhibited by humic acid that would be extracted during autoclaving and dissolved into the liquid phase of the slurries. Since autoclaving would also dissolve part of iodine bound to the soil solid phase (Y. Muramatsu, unpublished results), laccase could provide autoclaved soils with the iodide sorption capacity without the addition of stable iodide. Microbial Oxidation of Iodide in Soils. Our results suggest that microbial laccase catalyzes the oxidation of iodide, which leads to the formation of organically bound iodine in soils. Although peroxidase may also be involved in this process,21 Sheppard et al.35 did not observe significant correlation between soil peroxidase activity and Kd values for iodide in 7 Canadian soils. Very recently, Li et al.36 determined a new pathway of bacterial iodide oxidation, in which organic acids secreted by soil bacteria react with H2O2 to form peroxy carboxylic acids under acidic condition, and these strong oxidizing agents oxidize iodide. However, it seems unlikely that iodide was oxidized mainly by this mechanism in our soil slurries, since pH of the slurries was not acidic (pH 5 to 6). Fox et al.33 demonstrated that manganese oxides, which are important mineral components of many soils and sediments, may oxidize iodide in natural waters. However, the fact that iodide sorption was completely inhibited by autoclaving (Figure 1 and Table 1) suggests that iodide oxidation and subsequent iodination of soil organic matter may proceed biologically. Further study is needed to fully understand the mechanism of microbial oxidation of iodide and subsequent sorption on soils. In particular, it is of great importance to isolate native soil microorganisms which possess laccases with high affinity for iodide.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Table S1 and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org. 396

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