Direct and Indirect Effects of Microbes on Technetium Insolubilization in Paddy Fields

Direct and indirect effects of microorganisms on technetium insolubilization in water covering waterlogged soils were studied. Seven soils were waterlogged and then the water covering the soils were collected for further tracer experiments. The samples in contact with air were incubated with TcO4 at 25 ̊C for 4 to 5 days. After incubation, the samples were sequentially separated into four fractions: one insoluble fraction (> 0.2 μm) and three soluble fractions (TcO4, cationic, and other forms). The radioactivity of Tc in each fraction was measured with a NaI (Tl) scintillation counter. The insolubility of Tc was observed in the untreated samples. The maximum insolubilization radioactivity was 37% of the total radioactivity in P38, which was collected from a paddy field, gray lowland soil. Microscopic observations revealed that bacteria were the dominant species in the insoluble fraction of P38. For the other samples, less than 9% of the Tc was found in insoluble form. In order to clarify biological and nonbiological factor affecting the insolubility, a reducing agent or nutrients were added to the P38 sample. The amount of insoluble Tc was enhanced by the addition of nutrients, while the addition of the reducing agent resulted in a dramatic decrease in the amount of the insoluble Tc. Most of the TcO4 added to the filtered or autoclaved samples was present in the form of the pertechnetate anion, even in P38. The filtered and autoclaved samples contained metabolites and dead cell particles, respectively. These materials, therefore, did not affect the physicochemical changes in Tc. These results suggest that specific bacteria having the ability to render Tc insoluble even under not strictly anaerobic conditions directly contribute to the insolubility of Tc. INTRODUCTION The long-lived radionuclide, Tc, has been the object of considerable attention for its environmental behavior. This radionuclide is present in the heptavalent form as a pertechnetate anion (TcO4) under aerobic conditions. Because this species is easily available to plants (1), its transfer from plants to human is a concern, whereas several low-valence oxides of Tc are insoluble (2) and their transfer to plants is very low (3). In this way, the insolubilization of Tc influences bioavailability and thus it is important to know the insolubility of Tc in the environment. In Asia, where rice is a staple of the diet, the behavior of Tc in paddy fields has been a focal point. We previously found that most TcO4 become insoluble in water covering waterlogged soils and the Tc is deposited on the soil surface (4). It was thought that microorganisms caused the insolubility. However, it was not clear whether the insolubilization resulted from direct or indirect effects. Microorganisms in the environment have diverse capacities for interactions with metals (5). For Tc, there are many reports on its accumulation, reduction and precipitation by a variety of microorganisms, and most of these studies focused on physicochemical changes under strict anaerobic conditions (6). Although Tc released from radioactive waste disposal sites would move to paddy fields via irrigation water, the insolubilization of Tc by indigenous microbes in water on soil remain uncharacterized. In this study, we describe the possibility of direct and indirect insolubilization by microorganisms in water covering waterlogged soils. MATERIALS AMD METHODS Sample treatment Seven soils (Table I), 5 g each, were incubated for 7 days at 25 ̊C under waterlogged conditions. The water used contained glucose at a concentration of 3.3 mg l to activate microorganisms in the soil. After collection of water covering waterlogged soils, water samples were untreated or treated by autoclave or filtration with a 0.2 μm pore-size cellulose acetate membrane filter, and they were incubated with TcO4 for 4 days at 25 ̊C. The samples (1.8 ml) were in contact with about 9.8 ml of headspace air. WM’03 Conference, February 23-27, 2003, Tucson, AZ 2 For the sample of P38, an additional two sub-samples were subjected to the incubation with TcO4. A reducing agent, 2-mercaptoethanol (2-ME), was added to the first at a final concentration of 8 mM. The second was supplemented with nutrients (0.1 g of tryptone, 0.05 g of yeast extract, and 0.1 g of NaCl per liter of water). Characteristics F10 F29 F31 F47 P28 P32 P38 Andosols Andosols Andosols Dark Red Gray Lowland Andosols Gray Lowland soils soils soils Purpose Upland Upland Upland Upland Paddy Paddy Paddy Water content (air dry/110ûC dry, %) Cation Exchange Capacity (meq/100g dry) Anion Exchange Capacity (meq/100g dry) Active-Al (mg/100g dry) 5,460 4,720 4,320 536 223 6,510 284 Active-Fe (mg/100g dry) 3,060 1,920 2,830 1,170 420 3,600 1,280 Total-C (% dry) 4.31 5.31 2.81 1.11 1.87 6.66 1.54 Organic-C (% dry) 0.04 5.14 2.74 1.00 1.84 6.64 1.42 Total-N (% dry) 0.32 0.40 0.25 0.13 0.16 0.56 0.15 Soils Table I. Characteristics of soil samples 8.2 10.8 8.9 2.4 3.5 9.3 1.8 Soil groups 15.4 27.4 18.4 15.6 13.2 21.7 16.6 0.1 0.17 < 0.05 0.06 0.14 0.32 6.69 Fractionation of technetium To confirm the physicochemical form of Tc, the samples were sequentially separated into four fractions at the end of the incubation with TcO4. First, the sample was passed through a 0.2 μm pore-size cellulose acetate membrane filter to remove the insoluble form of Tc. Second, the filtrate was passed through a TEVA resin (Eichrom, Darien, IL) column to eliminate TcO4 from the solution. Third, cation forms of Tc in the eluate from the TEVA column were separated with a cation exchange resin column. The final eluate from the cation exchange column included other forms of Tc. The radioactivity of Tc (204 keV) in each solution was measured with a NaI (Tl) scintillation counter (ARC-300; Aloka, Tokyo, Japan). Characteristics of the solutions The redox potential (Eh) and pH of water samples were measured with a platinum complex electrode and pH electrode immediately after collection. RESULTS AND DISCUSSION The physicochemical form of Tc is changed directly and indirectly by microorganisms (7). Direct insolubilization is caused by bioaccumulation and biosorption. Bioaccumulation depends on cell energy metabolism, and thus cellular uptake of metals by the mechanism requires living organisms. The untreated water samples contained living microorganisms. In the samples some of the TcO4 was changed into an insoluble form (Fig. 1). The observed maximum insolubilization was 37% in P38. Observations under an epifluorescence microscope revealed that bacteria were the dominant species in the insoluble fraction of P38. Thus, bacteria would be related to the insolubility of Tc. It is known that some bacteria reduce soluble TcO4 to insoluble form, but those processes generally occur under anaerobic conditions (6, 8). In our experiment, the conditions in the culture with Tc were not strictly anaerobic. The specific bacteria in P38 seemed not to be obligatory anaerobic bacteria. For the other samples less than 9% of the Tc was found in insoluble form. Cations and other forms accounted for less than 2% in the untreated samples. These results suggest that most of the Tc is present as pertechnetate but that some of insoluble forms of Tc occurs in water WM’03 Conference, February 23-27, 2003, Tucson, AZ