Environ. Sci. Technol. 2003, 37, 3885-3890
Microbial Participation in Iodine Volatilization from Soils SEIGO AMACHI,† MIZUYO KASAHARA,† SATOSHI HANADA,‡ YOICHI KAMAGATA,‡ HIROFUMI SHINOYAMA,† TAKAAKI FUJII,† AND Y A S U Y U K I M U R A M A T S U * ,§ Department of Bioresources Chemistry, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan, and National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan
The roles of microorganisms in iodine volatilization from soils were studied. Soils were incubated with iodide ion (I-), and volatile organic iodine species were determined with a gas chromatograph. Iodine was emitted mainly as methyl iodide (CH3I), and CH3I emission was sometimes enhanced by the addition of glucose. Soils were then incubated with a radioactive iodine tracer (125I), and radioiodine emitted from soils was determined. The emission of iodine was enhanced in the presence of yeast extract but was inhibited by autoclaving of soils. The addition of streptomycin and tetracycline, antibiotics that inhibit bacterial growth, strongly inhibited iodine emission, while a fungal inhibitor cycloheximide caused little effect. Forty bacterial strains were randomly isolated from soils, and their capacities for volatilizing iodine were determined. Among these, 14 strains volatilized significant amounts of iodine when they were cultivated with iodide ion. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that these bacteria are widely distributed through the bacterial domain. Our results suggest that iodine in soils is methylated and volatilized as CH3I by the action of soil bacteria and that iodine-volatilizing bacteria are ubiquitous in soil environments. The pathway of iodine volatilization by soil bacteria should be important for understanding the biogeochemical cycling of iodine as well as for the assessment of long-lived radioactive iodine (129I) in the environment.
Introduction Iodine-129 (129I, half-life: 1.6 × 107 yr), one of the most persistent anthropogenic radionuclides in the environment, has been released as a fission product from nuclear weapon tests and operation of nuclear facilities (1, 2). Enhanced levels of 129I were reported in the vicinities of nuclear spent fuel reprocessing plants (3-6). This nuclide has become widely distributed in the global environment and can be expected, * Corresponding author phone: +81 43 206 3155; fax: +81 43 251 4853; e-mail:
[email protected]. † Chiba University. ‡ National Institute of Advanced Industrial Science and Technology. § National Institute of Radiological Sciences. 10.1021/es0210751 CCC: $25.00 Published on Web 08/05/2003
2003 American Chemical Society
given its long half-life, to behave in similar ways as stable iodine over long time periods (7). In UNSCEAR-2000 Report (8), 129I is regarded as one of most important radionuclides that should be assessed from the viewpoint of global circulation. However, biogeochemical cycles of iodine are not sufficiently known, mainly due to the lack of data on its complex behavior in the environment. Thus, it is necessary to study the behavior of iodine through the biosphere to understand its biogeochemical cycles and also to ensure the safety of 129I (9). The most significant feature of the global iodine cycling is its volatilization into the atmosphere (10). In marine environments, algae (11), phytoplankton (12), and bacteria (13) are known to have capacities for volatilizing iodine. Volatilization of iodine is also important from the viewpoint of atmospheric ozone destruction. It has been suggested that volatile iodine species (mainly organic iodine) can enter the troposphere and even the lower stratosphere by convective transport (14). They are readily photolyzed to produce iodine atoms, which subsequently react with ambient ozone to form iodine oxide (IO). IO is converted back to iodine atoms by photodissociation or by reactions with halogen oxides (ClO, BrO, and IO) (15, 16). Compared with marine ecosystems, much less information is available about iodine volatilization from terrestrial environments. Iodine is usually sorbed by soil components (17-20) and can be desorbed and leached into soil waters when the soil redox potential (Eh) decreases sufficiently (21). Muramatsu and Yoshida (22) have observed that a considerable amount of iodine in soils was volatilized from the soilplant system into the atmosphere. A marked emission of iodine was observed in the soil-rice plant system under flooded conditions, and the chemical form of the gaseous iodine was identified as methyl iodide (CH3I). Redeker et al. (23) also reported CH3I emission from flooded rice fields. Several studies have demonstrated that terrestrial organisms such as plants (24) and wood-rotting fungi (25) possess the abilities to synthesize CH3I. However, CH3I production by soil microorganisms has not been studied despite their great biomass in soil environments. Recently, we have found that bacteria, including terrestrial and marine bacteria, are also capable of methylating iodide to form CH3I (13). Radiotracer experiments using resting cells indicated that they are able to methylate iodide under oligotrophic conditions in which no nutrients were supplied that support bacterial growth. Bacterial CH3I production depended greatly on the surrounding iodine levels, and the iodide-methylating reaction was mediated by an enzyme protein with S-adenosyl-Lmethionine as the methyl donor (13). In this paper, we have studied the roles of microorganisms (especially bacteria) on the iodine volatilization from soils. To change the composition and the level of microbial populations in soils, soil samples collected from different places were incubated with various substances such as nutrients and antibiotics. Gaseous iodine volatilized from soils was then determined by radiotracer technique or gas chromatography (GC). In addition, 40 bacterial strains were randomly isolated from soils, and their abilities to volatilize iodine were determined. Finally, phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences (26) was carried out to obtain a better understanding of the iodine-volatilizing bacteria.
Materials and Methods Soils. All soils used in this study were surface soils collected from the upper 20 cm and used for experiments within 2 weeks after collection. Soils were stored at room temperature VOL. 37, NO. 17, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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until use. For the experiment using GC, soils from rice paddies (soils 1-3) and a forest (soil 4) were collected from Tsukuba, Ibaraki Prefecture in September 1998. For radiotracer experiments, 53 soils (rice paddy, 20; upland field, 5; forest, 19; wetland, 9) were collected from nine prefectures in Japan (Miyagi, Fukushima, Ibaraki, Tochigi, Chiba, Saitama, Tokyo, Osaka, and Hyogo) in March and April 2002. After their abilities to volatilize iodine were tested, 8 soils that showed strong volatilizing abilities were chosen for further study. The selected soils were 5 paddy soils (paddy A-E) and 3 nonpaddy soils (upland field, forest, and wetland). The sample locations of these 8 soils were Tokaimura (Ibaraki), Matsudo (Chiba), Kashiwa (Chiba), Takarazuka (Hyogo), Inagawa (Osaka), Hitachinaka (Ibaraki), Fuchu (Tokyo), and Ikeda (Osaka), respectively. For the isolation of iodine-volatilizing bacteria, rice paddy, upland field, and forest soils were collected in May 2001 from Chino (Nagano), Chiba (Chiba), and Kawagoe (Saitama), respectively. Gas Chromatographic Determination of CH3I Emitted from Soils. To examine the production of volatile iodine from soil and its chemical species, we carried out incubation experiments followed by gas chromatographic determination. Soils were mixed with sterile water to give approximately 0.15 g of dry soils/mL. Immediately after the soil solution was prepared, 18 mL of each soil solution was dispensed into sterile 120-mL serum bottles together with 1 mL of sterile iodide solution (20 mM sodium iodide). Then the volume of the final solution was adjusted to 20 mL, which contains approximately 0.135 g of dry soils/mL, and the iodide concentration was 1 mM. The bottles were then sealed with sterile butyl rubber stoppers and incubated without shaking at 30 °C in the dark. When glucose was added, 1 mL of sterile solution (50 mM) was mixed with the solution. When soils were incubated anaerobically, 18 mL of the soil solution in serum bottles was purged under N2/CO2 (80: 20) for 5 min and sealed with butyl rubber stoppers. Iodide solution and various electron donor solutions (50 mM each of glucose, sodium acetate, sodium formate, sodium propionate, sodium lactate, sodium pyruvate, and 2% methanol) prepared in serum bottles were also purged under N2 for 5 min and sealed with butyl rubber stoppers. Finally, 1 mL of iodide solution and 1 mL of one of electron donor solutions were anaerobically added to the soil solutions by using a plastic syringe and a needle. We also added resazurin to the anaerobic samples at a final concentration of 1 mg/L to monitor whether sufficiently low redox potential is maintained in the samples. After 20 d of incubation, headspace gas was determined with a Shimadzu 14A GC equipped with an electron-capture detector (ECD) as described previously (13). Under our determination condition, not only CH3I but also ethyl iodide (C2H5I), 1-propyl iodide (1-C3H7I), 2-propyl iodide (2-C3H7I), and chloroiodomethane (CH2ClI), if produced, could be detected. CH3I was identified using a Hewlett-Packard (HP) 5890A GC coupled to a HP 5972A mass-selective detector as described previously (13). When CH3I production in serum bottles was calculated, we assumed that a Henry’s law constant of CH3I under our experimental conditions was 0.2 (27). Radiotracer Experiments. A total of 1 g wet weight of soil was suspended in 100 mL of sterile distilled water in a 100mL polypropylene vessel. The vessel was shaken gently a few times and stored for 2 min to allow large soil particles to sink. One milliliter of the supernatant was then transferred to a sterile 120-mL serum bottle containing 17 mL of sterile distilled water. To change the composition and the level of soil microbial populations, one of following sterile solutions (1 mL) was then added while 1 mL of sterile water was added in control and autoclaved (see below) samples. Yeast extract (20 g/L), which contains various amino acids and vitamins, 3886
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was used to stimulate the microbial activities. Cycloheximide (3 g/L), an antibiotic for eukaryotic microorganisms, was used to inhibit the activities of filamentous fungi or yeasts. A mixture of streptomycin sulfate and tetracycline hydrochloride (0.5 g/L each), antibiotics for prokaryotic microorganisms, was used to inhibit the bacterial activities. Finally, 1 mL of sterile iodide solution containing both 2 nmol of potassium iodide and approximately 30 kBq of Na125I (DuPont NEN Products) was added to the serum bottle. Thus, the final volume of the samples was 20 mL (with 100 mL headspace), and final concentration of stable iodine was 0.1 µM (12.7 µg/L), which is similar to the mean iodine concentration of soil pore water (28, 29). Serum bottles were sealed with sterile butyl rubber stoppers and incubated with reciprocal shaking (120 rpm) at 30 °C in the dark. When soil samples were autoclaved, serum bottles containing 18 mL of the soil solution were sealed with butyl rubber stoppers and then autoclaved at 121 °C for 20 min. After the autoclaving, the bottles were opened, and 1 mL each of sterile water and sterile iodide solution were added aseptically. In a preliminary experiment, it was confirmed that our autoclaving condition is sufficient to kill soil microorganisms completely. After the incubation (in most cases the incubation period was 7 d), volatile organic iodine was collected in an activated charcoal trap by sweeping under nitrogen gas flushing as described previously (13, 30). During this sampling, the bottle was heated in a hot bath (100 °C) to expel the dissolved CH3I into the gas phase. Volatile inorganic iodine such as I2 was also collected in a silver wool trap. The activity of 125I collected in the traps was measured with an NaI scintillation counter (Aloka ARC-380). To assess the amount of the volatile iodine production quantitatively, we used “percentage of emission”, which is defined as the total amount of 125I collected in the activated charcoal trap divided by the total amount of 125I in the serum bottle. The detection limit of this method was approximately 0.002% of emission, and more than 0.01% could be clearly detected with just small errors. Isolation and Phylogenetic Analysis of Iodine-Volatilizing Bacteria. Soils were serially diluted with sterile 0.85% NaCl and spread on both 1 × PTYG (13) and 1/10 × PTYG agar media. Forty colonies grown on these media were randomly removed and purified. Each isolate was then grown in respective PTYG liquid medium, and 1 mL of the culture was transferred to a 120-mL serum bottle containing 18 mL of the respective PTYG medium. Iodide solution (1 mL) containing both 2 nmol of potassium iodide and approximately 30 kBq of Na125I was also added. Cultivation was carried out for 48 h with reciprocal shaking (120 rpm) at 30 °C in the dark. Volatile organic iodine was collected and determined as described above. For each medium, an uninoculated medium was incubated with iodide solution as a control. Bacterial strains were judged to have capacities for volatilizing iodine when their percentage of emission were calculated to be more than 0.01%. Bacterial growth was monitored by measuring optical density of culture liquid at 660 nm. For phylogenetic analysis of iodine-volatilizing bacteria, 16S rDNA was sequenced (26). Cells grown in PTYG liquid medium were collected and were frozen-thawed twice at -80 °C. Crude lysates of the cells were then prepared by proteinase K digestion, heat treatment, and centrifugation (31). The 16S rDNA was amplified by the polymerase chain reaction (PCR) using bacterial consensus primers 8F (5′AGAGTTTGATCCTGGCTCAG-3′, Escherichia coli positions 8-27) and 1491R (5′-GGTTACCTTGTTACGACTT-3′, E. coli positions 1509-1491) (32). PCR products were purified by QIAquick PCR Purification Kit (QIAGEN), and approximately 450 bp were sequenced with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) by using the primer comple-
TABLE 1. CH3I Emission (nmol/g dry soil) from Various Soils Incubated with 1 mM Iodide Ion for 20 d additive
soil 1
soil 2
soil 3
soil 4
no iodide iodide iodide + glucose iodide (anaerobic)
