Published online 27 October 2006
Published in J Environ Qual 35:2017-2020 (2006)
DOI: 10.2134/jeq2006.0045
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Bioremediation and Biodegradation
Removal of Technetium from Solution by Algal Flagellate Euglena gracilis
Nobuyoshi Ishiib,* and
Shigeo Uchidaa
a Office of Biospheric Assessment for Waste Disposal, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, 263-8555 Japan
b Aquatic Radiation Ecotoxicology Research, Environmental Radiation Effects Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, 263-8555, Japan
* Corresponding author (nobu{at}nirs.go.jp)
Received for publication January 31, 2006.
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ABSTRACT
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Based on limited data for the removal of radioactive 99Tc by freshwater phytoplankton, it has been thought that phytoplankton are unsuitable for remediation of 99Tc-contaminated waters. This work sought to verify the validity of this assumption by studying the removal of 99TcO4– by freshwater and brackish water phytoplankton. The phytoplankton used were Euglena gracilis, Chlamydomonas pulsatilla, Chlorella vulgaris, and Spirulina platensis. Each of them was incubated for 63 d, and the removal of 99Tc from solution was periodically determined. Significant removal of 99Tc was observed only for E. gracilis, and the maximum removal was 70% of the total 99Tc added. The killed cells of E. gracilis, however, removed hardly any 99Tc. When E. gracilis cells were washed with fresh culture medium, only 13% of the total 99Tc was desorbed. These results suggested that intracellular uptake of 99Tc by E. gracilis occurred. These results are the first documented example of significant removal of 99Tc by planktonic microalgae.
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INTRODUCTION
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THE LONG-LIVED ß-emitter technetium-99 (99Tc) is a man-made fission product of 235U and 239Pu with a relatively high fission yield of about 6%. This radionuclide has been released to the environment from nuclear weapons testing as fallout, from U enrichment and nuclear fuel processing facilities, and from disposal sites after pharmaceutical use (Wildung et al., 1979). In aerobic environments, Tc is normally present in a heptavalent form as pertechnetate (TcO4–), and this chemical form is highly bioavailable for plants as a sulfate analog (Cataldo et al., 1989). Accumulation of Tc in plants facilitates its entry into the food chain potentially leading to humans. Removal of Tc from wastewaters is therefore required for the protection of human and environmental health.
The removal of Tc by bacteria under anaerobic conditions has been intensively studied. For instance, the sulfate-reducing bacterium Desulfovibrio desulfuricans (Lloyd et al., 1998) has the ability to reduce Tc(VII) to Tc(IV) and consequently remove the Tc from solutions. Bacteria are one of the useful organisms for removing contaminants because their large surface to volume ratio allows the maximum activity and interchange of contaminated materials in and out of the cell.
As well as bacteria, phytoplankton have a large surface to volume ratio, and they may also be useful organisms for the removal of 99Tc. However, most previous works on the removal of Tc by phytoplankton reported negative results. Fisher (1982) investigated the bioaccumulation of Tc by 7 species of marine phytoplankton and reported negligible uptake of less than 0.1% of total Tc. Garnham et al. (1992) reported that the freshwater phytoplankton Chlamydomonas reinhardtii, Chlorella emersonii, and Scenedesmus obliquus removed Tc by biosorption, but the removal amount was low. As a consequence of these results, it has been thought that phytoplankton are not suitable for the removal of 99Tc. However, these investigations on the removal of Tc by phytoplankton are insufficient, because there are various kinds of phytoplankton present in aquatic environments; in particular, the data for freshwater and brackish water phytoplankton including cyanobacteria are limited. The aim of this study is, therefore, to determine whether freshwater phytoplankton and brackish water cyanobacteria have the ability to remove 99Tc from solution.
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MATERIALS AND METHODS
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Phytoplankton and Culture Conditions
The cyanophyceae Spirulina platensis (NIES-39), the chlorophyceae Chlamydomonas pulsatilla (NIES-122) and Chlorella vulgaris (NIES-227), and the euglenophyceae Euglena gracilis (Matsui et al., 2003) were used for this study for the reasons outlined below. S. platensis usually grows in brackish water. Since increased TcO4– uptake with increased external NaCl concentration was reported in freshwater microalgae (Garnham et al., 1992), there is a possibility of 99Tc uptake by S. platensis. C. pulsatilla and C. vulgaris may remove 99Tc from solution because other species of Chlamydomonas and Chlorella removed Tc by biosorption (Garnham et al., 1992). E. gracilis does not have a cellulose cell wall. This lack of the cellulose cell wall may be advantageous for the uptake of 99Tc, and thus E. gracilis was chosen. These phytoplankton, except for E. gracilis, were obtained from the National Institute for Environmental Studies (NIES), Environmental Agency, Tsukuba, Japan. E. gracilis was kindly provided by Prof. Y. Nakano, Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Japan.
Cells of phytoplankton were precultured axenically for 14 d at 25°C under 12 h-12 h light–dark cycles in SOT medium (Ogawa and Terui, 1970) for S. platensis, P35 medium for C. pulsatilla, C medium (Ichimura, 1972) for C. vulgaris, and GM medium for E. gracilis. The composition of P35 medium was as follows: 10 mg NH4NO3; 4 mg MgSO4·7H2O; 5 mg KCl; 7.4 mg CaCl2·2H2O; 5 mg ß-Na2glycerophosphate; 100 mg sodium acetate; 0.01 µg vitamin B12; 0.01 µg biotin; 1µg thiamine HCl; 50 mg tris (hydroxymethyl) aminomethane; 58.8 µg FeCl3·6H2O; 10.8 µg MnCl2·4H2O; 6.6 µg ZnSO4·7H2O; 1.2 µg CoCl2·6H2O; 0.75 µg Na2MoO4·2H2O; 300 µg Na2EDTA·2H2O in 100 mL distilled water (pH adjusted to 8.0). The GM medium consisted of 1.0 g tryptone; 0.1 g yeast extract; 1.0 g dextrose; 0.1 µg vitamin B12, and the pH of the medium was adjusted to 3.5. The precultured cells of phytoplankton were washed three times, and then they were incubated for 63 d at 20°C under 12 h-12 h light–dark cycles. The photosynthesis photon flux density was 100 µmol m–2 s–1 under this light illumination. At the beginning of incubation, 99TcO4– was added to each culture. The NH499TcO4 was purchased from Isotope Products Laboratories (Valencia, CA) and the radioisotope was sterilized by passing its solution through a 0.2-µm pore size filter before use. The average final concentrations of 99Tc added are listed in Table 1. Four kinds of experiments listed in Table 1 were performed to determine the ability for the removal of 99Tc (removal of Tc by algae and concentration factor) and the intracellular uptake of 99Tc by phytoplankton cells (killed cells and desorption and extraction). Experiments were performed in triplicate, and results were expressed as mean values ± standard deviation.
Removal of Technetium
Removal of 99Tc from culture media was periodically determined using a Tri-Carb 2500TR liquid scintillation analyzer (Packard, Downers Grove, IL). The cultures of phytoplankton were filtered through a 0.2-µm pore size polycarbonate membrane and the obtained filtrate (100 µL) was added to a scintillation vial with 4 mL of Ultima Gold LLT scintillation fluid (PerkinElmer, Groningen, The Netherlands). Counts per minute (cpm) were recorded at between 25 and 290 keV for 10 min. Removal of 99Tc (r) was calculated by the following equation; r = (C0 – Cn)/C0 x 100, where C0 and Cn are the radioactivity of 99Tc in filtrates at Day 0 and day n of incubation, respectively.
Abundance of Phytoplankton
Growth of phytoplankton was estimated by measurement of turbidity at an optical density of 750 nm using a UV-160 spectrophotometer (Shimadzu, Kyoto, Japan). For E. gracilis, the number of cells was also enumerated by optical microscopy.
Concentration Factor
The ability for 99Tc removal was estimated as concentration factor ("kBq g–1 dry weight of cells" divided by "kBq mL–1 culture medium"). The estimation was performed using E. gracilis culture after a 35-d incubation. For the dry weight measurement, the cells were trapped onto a GF 75 glass fiber filter (Advantec Toyo Kaisha, Tokyo, Japan) and then the filter was dried for 24 h at 60°C. After drying, the weight of the cells was calculated. The initial concentration of 99Tc was 3.1 kBq mL–1 for this experiment (Table 1).
Desorption and Extraction of Technetium
The 99Tc-tagged E. gracilis cells were washed with four kinds of solutions: 7.5 N nitric acid and fresh GM medium adjusted to pH 3.5, 5.5, and 7.5 with HCl. Nitric acid was used for the extraction of 99Tc taken up by the cells. The GM medium was used for desorption of 99Tc from the cell surface. For the tagging, E. gracilis cells were incubated with an excess amount of 99Tc for 32 d. The concentrations of 99Tc on Day 0 and Day 32 of incubation are shown in Table 1. After a 32-d incubation, the culture was centrifuged at 6000 rpm for 5 min and the supernatant was removed. The obtained cell pellets were resuspended in the respective wash solution and vigorously shaken with a vortex mixer. The procedure from centrifugation to the shaking was repeated three times. The supernatants obtained during the three cell washes were collected, and then the amount of 99Tc in them was determined by liquid scintillation counting. From the determination, the relative desorption and the relative extraction amounts of 99Tc were calculated.
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RESULTS AND DISCUSSION
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Removal of Technetium by Phytoplankton
The removal of 99Tc from culture media by phytoplankton and phytoplankton growth are shown in Fig. 1. Among phytoplankton used, only E. gracilis cells removed significant amounts of 99Tc from the culture media as they grew. The removal continued for 50 d, at which time equilibrium was achieved. The maximum value of the removal was 70% of the total 99Tc added. In contrast, very small amounts of 99Tc were removed from solution by the other phytoplankton although they grew in the culture media. The low levels of removal observed agreed with previous studies for freshwater green microalgae (Garnham et al., 1992) and cyanobacteria (Garnham et al., 1993). These results demonstrate that E. gracilis has an ability to remove 99Tc from solutions. This is the first documented example of significant removal of 99Tc by planktonic microalgae.
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Fig. 1. Technetium-99 removal (closed circles) and growth (open circles) of (A) E. gracilis, (B) C. pulsatilla, (C) C. vulgaris, and (D) S. platensis. Values represent the means and standard deviations of triplicate experiments. For the removal of 99Tc, standard deviations are within closed circles.
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Ability of E. gracilis for Technetium Removal
A concentration factor was determined to estimate the ability of E. gracilis cells to remove 99Tc. After 35 d of incubation, the number of E. gracilis cells was 2.1 ± 0.1 x 106 cells mL–1, and the dry weight was 0.61 ± 0.06 mg per 106 cells. Since the activities of 99Tc left in solution and taken up by the cells were 1.3 kBq mL–1 and 1.8 kBq mL–1, respectively (Table 1), the concentration factor was approximately 1080. Gromov (1976) reported a higher concentration factor than this result; a wet weight concentration factor for a natural phytoplankton community of 7 ± 3 x 104 was recorded, but a monoculture of a marine microalga Platymonas viridis showed no removal of Tc. Although it is impossible to make a direct comparison between results by dry weight and by wet weight, the present result may be the highest concentration factor yet seen for axenic microalgae.
In contrast to most microalgae, certain marine macroalgae can remove Tc from seawater. Jeanmaire et al. (1981) reported that the brown algae Fucus serratus, Pelvetia canaliculata, and Ascophyllum nodosum accumulated Tc levels of 22 to 126 Bq kg–1 wet weight. Pentreath (1981) found an even higher Tc level of 15 kBq kg–1 wet weight in Fucus in the vicinity of the Windscale reprocessing plant on the Irish Sea. These values, however, are small in comparison with that of E. gracilis under experimental conditions, as it was calculated that 1.8 MBq of 99Tc were removed by E. gracilis cells, which existed in one kilogram of the culture medium on Day 35 of incubation. Therefore, the ability for 99Tc removal by E. gracilis has potential applications to treatment systems of 99Tc.
Intracellular Uptake of Technetium by E. gracilis
To clarify the intracellular uptake of 99Tc by E. gracilis cells, the removal of 99Tc by killed cells was performed. E. gracilis cells were killed by the addition of a protein synthesis inhibitor cycloheximid at a final concentration of 50 µg mL–1 or by heating cells at 60°C for 1 h and then added to a solution containing 2.8 kBq mL–1 99Tc (Table 1). In both cultures, E. gracilis removed negligible amounts of 99Tc (Fig. 2).
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Fig. 2. Technetium-99 removal (closed circles) and growth (open circles) for the killed cells of E. gracilis: The culture spiked with (A) cycloheximid and (B) the heat-killed culture. Values represent the means and standard deviations of triplicate experiments.
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In addition to the killed cells experiment, 99Tc-tagged E. gracilis cells were washed with the GM medium to estimate the sorption amount of 99Tc on the cell surface or with nitric acid to estimate the leaching amount of 99Tc from intracellular stores (Fig. 3). The total activities in 99Tc-tagged E. gracilis cells before each desorption and extraction treatment were 1.7 kBq mL–1 (Table 1). As the pH of the GM medium was increased from 3.5 to 7.5, a slight increase in desorption amounts of the 99Tc was observed. The relative amount of desorption by the GM medium, however, was, at a maximum, 14% of the total 99Tc removed. E. gracilis was cultured in the GM medium, and thus there would be little leaching of 99Tc from the inside of the cells by washing with fresh GM medium. Therefore the results suggest that a slight amount of 99Tc had been adsorbed onto the cell surface. It was expected that intracellular 99Tc would be completely extracted by nitric acid extraction, but the relative extraction amount of 99Tc by nitric acid was 74% of the total 99Tc removed (Fig. 3). The other 26% of 99Tc bound strongly to intracellular components. Results of both the killed cell experiments and the washed cell experiments show that the removal of 99Tc by E. gracilis cells was primarily the result of intracellular uptake.
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Fig. 3. Relative extraction and desorption amounts of 99Tc. Bars represent the means and standard deviations of triplicate experiments.
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In addition to E. gracilis, some bacteria remove significant amounts of 99TcO4– from solutions (Lloyd, 2003). In the case of E. coli, the cells have been found to accumulate Tc(VII)O4– and reduce it to a black insoluble precipitate (Lloyd et al., 1997). However, no black precipitates were observed in cultures for the present study of E. gracilis. The authors thought that the concentrations of 99Tc used here would be enough to give a visible black precipitate if 99Tc(IV) were being formed because they had observed black precipitate-like materials when E. coli HB101 ATCC33694 was used in other experiments (data not shown). Mechanisms of Tc removal, therefore, must differ between E. coli and E. gracilis.
Technetium is highly accumulated by brown algae (Jeanmaire et al., 1981; Pentreath, 1981) but not by green and red algae (Topcuoglu and Fowler, 1984), and this accumulation is a metabolically controlled process (Topcuoglu and Fowler, 1984). The strong bioaccumulation of Tc by brown algae seems to be unique among macroalgae. This situation is similar to that of the significant removal of 99Tc only by E. gracilis among microalgae used in this study. In the case of E. gracilis, the active uptake of 99Tc by the cells is also suggested by the results of Fig. 2 and 3. It thus appears that E. gracilis may be behaving in the same way as the brown algae for the uptake of 99Tc although the detail mechanism of the 99Tc uptake by brown algae is still unclear. The uptake of 99Tc by E. gracilis cells could be caused by mistaking 99Tc for another essential nutrient such as sulfate that they actively acquire because there is no biological requirement for this element. In addition, the lack of the cellulose cell wall could be advantageous for the uptake process of 99Tc by E. gracilis cells. It is necessary to study the underlying mechanisms of 99Tc removal by E. gracilis.
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CONCLUSION
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In conclusion, the present results may point to a potential course for the removal of 99Tc from freshwater using E. gracilis. This is the first documented example of significant removal of 99Tc by phytoplankton. E. gracilis can be easily grown and it removes 99Tc under aerobic conditions. Since 99Tc-reducing bacteria remove 99Tc only under anaerobic conditions, an effective treatment for removal of 99Tc is expected to be a combination of E. gracilis and 99Tc-reducing bacteria.
If the ability for 99Tc removal is common to Euglena species, the present results will be important for understanding the behavior of 99Tc in aquatic environments, because Euglena species are widely present in such environments and are not limited to freshwater, and because microalgae such as E. gracilis play important roles in the entry of many pollutants into aquatic food chains.
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ACKNOWLEDGMENTS
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The authors wish to thank Mr. H. Koiso at Tokyo Nuclear Services Co., Ltd. for his experimental support. They also thank Dr. H. Takeda, NIRS, for his support of this study. This work has been partially supported by a Grant-in-Aid from Radiation Effects Association Foundation and by the Agency for Natural Resources and Energy, the Ministry of Economy, Trade and Industry (METI) Japan.
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REFERENCES
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ABSTRACT
INTRODUCTION
