Environ. Sci. Technol. 1997, 31, 569-576
Selenium Biotransformations by a Euryhaline Microalga Isolated from a Saline Evaporation Pond T E R E S A W - M . F A N , * ,† ANDREW N. LANE,‡ AND RICHARD M. HIGASHI§ Department of Land, Air and Water Resources, University of California, Davis, California 95616, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K., and Crocker Nuclear Laboratory, University of California, Davis, California 95616
Selenium ecotoxicology is one of the best known examples of environmental impact resulting from toxic element biotransformations and food chain transfers. Despite this, it is becoming recognized that Se biotransformations by microbes and plants may also be a key to in situ bioremediation of Se contamination in large-scale cases such as agricultural drainage systems. We have isolated an euryhaline alga (Chlorella sp.) from Se-laden drainage pond waters and utilized GC-MS and multi-nuclear NMR to characterize its aerobic biotransformation activity for Se oxyanions. We found that this alga was active in volatilization of alkylselenides, production of putative selenonium precursor(s) of alkylselenides, and precipitation of Se, while exhibiting very low accumulation of the toxic selenomethionine in free form. Thus, such euryhaline microalgae are potentially important for in situ Se bioremediation and Se biogeochemical cycling in contaminated saline habitats.
Introduction Since the serendipitous discovery of selenium as an essential animal nutrient (1), this trace element has attracted much attention for its role in anti-oxidation processes (2-4). However, 2.5 decades later, selenium is today widely recognized as the toxic element that inflicted wildlife deformities observed in the California’s Kesterson Reservoir (5, 6), which was constructed to impound agricultural wastewaters. The severity of the problem at Kesterson forced its closure in 1985. This dichotomy of environmental Se is attributable to the extraordinarily narrow range of tolerance between nutritional requirement and toxicity from bioaccumulation and metabolism (7, 8). In California, operations of evaporation ponds that discourage primary productivity and wildlife access are currently used for disposal of agricultural drainage waters in the western San Joaquin Valley. However, concentrated by evaporative processes, Se salts unavoidably accumulate in these ponds, and Kesterson-like wildlife deformities are now occurring in some ponds (6). Long-term solutions to this problem will need to be explored, not just for California’s agriculture to achieve sustainability, but also because such large-scale cases are common throughout North America (9).
Various physical and chemical schemes have been tested but were either ineffective or uneconomical for such largescale systems. Some bioremediation schemes have also been proposed that key on Se precipitation and volatilization by bacteria and vascular plants (10-12). The anaerobic bacteriabased schemes often involve bioreactor configurations, for which the associated costs can be prohibitive for large-volume wastewaters such as agricultural drainage. In situ or intrinsic bioremediation through concerted activities of heterotrophic microbes and plants may be a viable option. Whatever the biological schemes may be, it is critically important that the Se biotransformation processes be thoroughly understood. This not only helps optimize the Se removal process, but more importantly helps to minimize the ecotoxicological risk to wildlife, the latter being the real goal of Se remediation. Although it is frequently assumed that Se metabolism follows the sulfur pathways (2, 13, 14), there are sufficient differences between the two elements (2, 13) such that the Se pathways in biota need to be independently characterized. However, the total knowledge in Se biotransformations is very limited, particularly regarding aquatic primary producers (e.g., phytoplankton) that may be major drivers of Se biogeochemical cycling in aquatic environments (15, 16). Selenium biotransformations from oxyanions into the volatile forms of dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) are generally considered as prominent processes for Se movement in the environment. These Se volatilization processes have been characterized to some extent in animals (17, 18), terrestrial plants (13, 19), and soil microbes (20-22). In addition, the release of dimethylselenone (DMSeO2) (23) has been suggested. Evidence has been presented for methylselenomethionine (CH3-Se-Met) as the biological precursor to DMSe production in terrestrial systems while methylselenocysteine was proposed to be the precursor for DMDSe release by Se-accumulator plants (13). The production of DMSe in animals is viewed as a detoxification mechanism (24) while the release of DMSe and DMDSe by plants is more likely to be a byproduct of Se metabolism along the sulfur pathways (13). Regardless of the function, the release of alkylselenides by plants and microbes has attracted recent attention as an option for reducing in situ Se contamination in wastewater such as those from agricultural drainage systems, power plants, oil refineries, and electronic industries. The present understanding of Se biotransformations by aquatic algae is predicated on the basis of incorporation into amino acids and proteins (4, 14, 25), which is likely to be important for bioaccumulation through the food web. However, virtually no information is available regarding production of alkylselenides by aquatic algae, despite the fact that this activity may be a key process by which Se is volatilized into the atmosphere from natural waters (16). In this study, we have isolated a persistent coccoid green alga from Se-contaminated agricultural evaporation pond water and examined its biotransformation activity on Se oxyanions via the production of different biochemical forms. We have focused on determining the volatile Se forms and their release as a function of algal growth, while introducing methodologies for analyzing selenonium compounds (precursors to alkylselenides) and ecotoxic forms such as selenomethionine.
Experimental Section * Corresponding author phone: (916) 752-1450; fax: (916) 7521552; e-mail address:
[email protected]. † Department of Land, Air and Water Resources. ‡ National Institute for Medical Research. § Crocker Nuclear Laboratory.
S0013-936X(96)00471-3 CCC: $14.00
1997 American Chemical Society
Isolation of Alga. The water for algal isolation was collected from the Pryse evaporation pond located in the southern San Joaquin Valley of California and stored in the laboratory at room temperature. A loopful of water was aseptically streaked
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onto a plate of 1% agarose (Fisher) in natural seawater (collected from Bodega Bay, CA) supplemented with f/2 nutrients (26) and incubated at 18 °C with a 16/8 h light/dark cycle. Predominantly small circular green colonies developed. A single well-isolated colony was inoculated into a f/2 seawater medium broth and maintained at 21-23 °C. All transfers of algal culture and algal growth were conducted under aseptic conditions. Under microscopic examination using Hoffman modulation optics plus epifluorescence to identify photosynthetic cells, the broth culture appeared to be a monoculture of non-mobile green coccoids free of non-fluorescent (e.g., bacterial) cells. The presence of distinct plastids in the coccoid alga was confirmed by epifluoresence microscopy using a dichroic B filter (Olympus). This together with the presence of budding daughter cells suggested that it is a Chlorella sp. (Dr. Norma Lang, personal communication). Total pigment profile analysis by HPLC (27) was also consistent with this initial assignment, as indicated by the major pigments including chlorophyll a, chlorophyll b, and carotenoids (including violaxanthin, neoxanthin, zeazanthin, lutein, and several carotenes) (data not shown). Growth test in media with salinity ranging from 0 to 100% of seawater indicated that this alga is a euryhaline species capable of growth on 10-100% seawater salinities. Determination of Se by GC-ECD and GC-MS. To characterize the total volatile Se and alkylselenides released as a function of growth, 1-L borosilicate bottles, each containing 0.8 L of f/2 seawater medium plus 0.1-100 ppm Se (as Na2SeO3 or Na2SeO4), were inoculated with the Chlorella stock culture in exponential growth and incubated at 30 °C under continuous fluorescent light. The culture bottle was sealed with a three-way Teflon valve cap (Omni), which provided the inlet and outlet connections for air. All connections and tubing were of Teflon or stainless steel materials to prevent adsorption or chemical reactions. The culture was aerated continuously with sterile air (filtered through activated carbon and 0.22 µm filter) at 105-110 mL/ min, which provided CO2 for growth and purged volatile compounds from the medium. Live algal density in the water column was estimated by measuring absorbance at 680 nm (A680) (14, 28), which was periodically confirmed with chlorophyll a determination of methanolic extracts of algal cells pelleted by centrifugation (29). The two methods gave comparable growth curves, so A680 was used routinely. It should be noted that, at later stages of algal growth, appreciable amounts of cell sedimentation occurred, especially for Se-treated cultures, which contributed to the decrease in A680 or chlorophyll a content of the water. For trapping volatile Se forms, the purged air was passed through a liquid nitrogen trap composed of a moisture pretrap Teflon chamber filled with Teflon chips kept at -40 °C, followed by the main trap of 6.35 mm o.d. × 4.76 mm i.d. × 61 cm length Teflon tubing immersed in liquid nitrogen. Five borosilicate beads were placed in the tube trap to equilibrate the gaseous content before GC analysis. Otherwise, the entire gas path consisted of Teflon materials, including all connections. Alternatively, the air was routed through a modified alkaline peroxide trap (12) to oxidize various Se forms to selenate. Our trap consisted of two sequential borosilicate impingers with Teflon connections (Supelco) each with 15 mL of freshly prepared (4:1, v/v) 50 mM NaOH/30% H2O2 (Fisher). The volatile Se forms from the liquid nitrogen trap were analyzed using a GC-MS system composed of a 0.18 mm i.d. × 40 m × 0.4 µm thick DB-1 coat column (J&W) in a Varian 3400 gas chromatograph coupled with a line-of-site interface to a Finnegan ITD 806 mass spectrometer. A sample of 100 µL out of a 4.7 mL trap was injected with no split flow and chromatographed with 40 cm/s H2 carrier gas velocity, a column temperature program of 30 °C for 4 min followed by ramping at 10 °C/min to 120 °C, with the injector and transfer
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line at 120 °C. There was no apparent damage to the column from air sample injection under these conditions. MS acquisition parameters were as follows: manifold ) 220 °C, electron energy ) 70 eV, emission current ) 10 µA without beam restrictor, automatic gain control set at 39 amu, full scan acquistion from 40 to 200 m/z at a rate of 9 spectra/s, which were averaged into 1 spectrum/s. Mass and ion-ratio calibration was by perfluorotributylamine, and a mass defect correction of 100 mmu/amu was applied to all spectra. DMSe, DMDSe, and DMS calibration standards were prepared by pipetting 2-5 µL each of the pure liquid standard into a 40mL brown bottle (preflushed with He) on ice and crimpsealed immediately. The liquid was allowed to vaporize completely before three lower concentrations of mixed standards were prepared by serial dilution into a 2-mL crimpsealed brown bottle (preflushed with He). The correlation coefficient of the standard curve (0-140 ppb for DMS, 0-240 ppb for DMSe, and 0-83 ppb for DMDSe) was at least 0.99. Total Se determination of the alkaline peroxide trap contents was made using a scaled-down version of existing methods (12, 30). The microscale method minimized acid wastes and sample size requirement, while improving the detection limit by sample concentration. An aliquot (0.25-1 mL) of the trap was heated in a 2-mL GC vial (Target) at 105 °C to ensure a complete oxidation to selenate and to remove H2O2 and water (10 µL of concentrated H2SO4 was added to prevent drying, which may lead to Se loss). Selenate was then reduced to selenite by heating at 105 °C in 50 µL each of concentrated HCl (trace metal grade, Fisher) and 18 MΩ water for 20-30 min (31, 32) or until the volume was less than 40 µL. The digest was diluted with 50 mN HCl or 18 MΩ water, and a 250-µL aliquot was derivatized in a crimp-sealed Teflon-lined vial with 50 µL of 0.5% 4-nitrophenylene-odiamine (4-NPD) in 1 M HCl in the presence of 250 µL of toluene (optima grade, Fisher) at 45 °C for 30 min in the dark. Extraction of the piazselenol product into the toluene layer was completed by shaking, and the piazselenol structure was confirmed by GC-MS using the conditions described below for selenomethionine analysis. Routine analysis was performed on a Varian 3300 GC with an electron capture detector (ECD). Chromatography was conducted by injecting 3 µL of toluene extract into a 0.25 mm i.d. × 30 m DB-35 column at 300 °C with a He carrier gas velocity of 30 cm/s (set at 100 °C), an injection split ratio of 8.3, and injector and detector temperatures of 250 and 320 °C, respectively. For quantification, the piazselenol peak area was normalized against that of an internal standard (a derivatization byproduct of constant peak area regardless of the Se concentration). Selenite standards (a total of 16) ranging from 2 ppb to 1 ppm as Se were subjected to the same procedure, and the resulting standard curve was trilinear over the entire range: linear from 2 to 10 ppb, from 10 to 200 ppb, and from 200 ppb to 1 ppm with correlation coefficients of greater than 0.99 for each range. Samples spiked with known amounts of selenite were also analyzed to check the precision, which was better than 10% standard deviation. To determine total Se in medium water and in algal biomass, a similar procedure as described above was employed except that the algal biomass was lyophilized and pulverized to
