Environ. Sci. Technol. 1998, 32, 3185-3193
Biotransformations of Selenium Oxyanion by Filamentous Cyanophyte-Dominated Mat Cultured from Agricultural Drainage Waters TERESA W.-M. FAN* Department of Land, Air and Water Resources, University of California, One Shields Avenue, Davis, California 95616-8627 RICHARD M. HIGASHI Crocker Nuclear Laboratory, University of California, One Shields Avenue, Davis, California 95616-8627 ANDREW N. LANE Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
A filamentous cyanophyte-dominated mat was cultured from waters of a large agricultural drainage evaporation basin in California. This mat was active in volatilizing Se from selenite at 20-10 000 µg/L Se, which accounted for >60% of the Se loss from the medium. Up to 75% of the medium Se was depleted over a 50-day period. This demonstrates a potentially important mechanism contributing to the nearly 100% loss of waterborne Se persistently observed at that basin. Se volatilization was via dimethylselenide, dimethyldiselenide, and dimethylselenenylsulfide, with evidence that the precursors were methylselenomethionine and methylselenocysteine but not dimethylselenonium propionate or trimethylselenonium ion. Selenite was also incorporated into proteins primarily in the form of selenomethionine at external Se concentrations of 5000 µg/L Se or higher, which may account for the slow growth of the cyanophyte mat at these concentrations. Such Se metabolite characterization in aquatic producers is important for understanding the role of Se biogeochemistry in ecotoxicity, which is vital for development of environmentally sound in situ Se bioremediation.
incidental to the use of terminal evaporation basins for disposing the large volume of agricultural drainage waters. These basins are often arranged as multiple shallow cells (e.g., 0.7-1.5 m) where drainage waters are channeled sequentially from one cell into the next. Waterborne Se is present largely as its oxyanion forms, such that concentrations in a sequence of cells may be predicted to increase with rising salinity (5), as has been observed in several basins surveyed (5). However, it was recently brought to our attention (D. Davis, personal communication; 6) that waterborne Se concentrations in the evaporation basins of the Tulare Lake Drainage District (TLDD), CA, have been showing a decreasing trend across the sequence of cells, despite a continuous input of Se and an increase of salinity to as high as saturated brine in the terminal cell. We have since confirmed that this trend is persistent year-round, as shown in this paper, and is potentially significant as TLDD is a large facility that annually evaporates approximately 15 × 106 m3 of water. This large-scale “attenuation” of waterborne Se is likely to be biologically based for the following reasons. The TLDD basin waters are rich in microphytes (7); otherwise, the physicochemical properties of these waters are typical of evaporation basins and not expected to cause net Se depletion (5). In addition, we have obtained preliminary evidence that at least two types of microphytes isolated from TLDD basin waters are capable of Se volatilization (7). Moreover, we have shown recently that a eukaryotic microphyte (Chlorella sp.) isolated from another evaporation basin water was active in volatilizing and precipitating Se from the culture medium (8). We are therefore investigating the possible biological mechanism(s) with a focus on Se biotransformations by prokaryotic cyanophyte mats that are often dominant at the TLDD basins. If understood, these mechanism(s) may be enhanced for in situ bioremediation of Se-laden waters. At the same time, this knowledge can lead to a better understanding of the role of microphytes in the biogeochemical fate and ecotoxic effects of Se in aquatic environments, which is essential to evaluating the efficacy of any natural or in situ remediation scheme. In this paper, we describe the dependence of Se volatilization kinetics on Se treatment concentrations and light by a filamentous cyanophyte-dominated mat cultured from TLDD basin waters. We also demonstrate that this mat biotransformed selenite into volatile alkyl selenides and other organic forms while depleting selenite from the medium.
Experimental Section Introduction Selenium contamination has been an important problem in the disposal of agricultural irrigation drainage waters and coal fire-based power plant wastes in the Western and Eastern United States, respectively. Well-known examples of these are the wildlife deformities observed at the Kesterson Reservoir, California (1, 2) and at Belews Lake, North Carolina (3), respectively. In most cases, the toxic levels of selenium in the top predators of aquatic environments were aparently acquired via bioaccumulation and biotransformations through the aquatic food chain (4). However, details of biotransformation pathways through the aquatic food chain are still largely unknown. In the case of agricultural operations in the southern San Joaquin Valley of California, Se contamination has been * Corresponding author phone: (530)752-1450; (530)752-1552; e-mail:
[email protected]. S0013-936X(97)00883-3 CCC: $15.00 Published on Web 08/25/1998
1998 American Chemical Society
Culturing of Filamentous Cyanophyte Mat. Matted algal material was collected from a TLDD basin of approximately 64 ha (near Tulare, CA), inoculated into 0.22 µm filtered natural seawater (collected from Bodega Bay, CA) containing f/2 nutrients (9) and incubated at 20 °C under continuous rotary shaking and a 16/8 h fluorescent light/dark cycle. After the culture turned light green, a loopful of the water was aseptically streaked onto a plate of 1% agarose (Fisher Scientific Co., Pittsburgh, PA) in f/2 seawater medium and incubated similarly as above without shaking. Amorphous brownish-green colonies developed and dominated the plate, which was inoculated into the f/2 seawater medium and incubated with shaking as described above. This process of plating and inoculation into liquid media was repeated once more to minimize contamination. Under microscopic examination using Hoffman Modulation Contrast optics (Modulation Optics Inc., Greenvale, NY), the culture was dominated by a filamentous cyanobacterium with unVOL. 32, NO. 20, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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branched, uniformly cylindrical, and motile trichomes. No distinct morphological features such as heterocysts, gas vacuoles, or constriction between adjacent cells were observed. We tentatively assigned this bacterium to the LPP (Lyngbya, Phormidium, and Plectonema) group (10; T. Hanson, personal communication). Further species characterization is in progress, which is based on 16S ribosomal RNA typing. It should be noted that the purity of the mat culture was not extensively examined regarding the presence of heterotrophic bacteria. Therefore, the possiblity of some contribution of heterotrophic bacterial activity to Se biotransformations could not be eliminated. Measurement of Se Volatilization Kinetics. The growthdependent time courses of the Se volatilization rate was obtained as described previously (8) and briefly outlined here. Each experiment was performed with 0.8 L of f/2 seawater medium inoculated with the cyanophyte culture in exponential growth and spiked with 20, 100, 1000, 5000, or 10 000 µg/L Se (as selenite). Each experiment was performed separately twice except for the 5000 µg/L Se treatment, which was conducted once. A third experiment was performed with the 1000 µg/L Se treatment to investigate the effect of light on the Se volatilization kinetics. In this experiment, the cyanophyte culture was grown to the maximal dispersed cell density before it was split equally into two cultures with one remaining under continuous light and the other switching to dark for several days before returning to light (see Figure 3). All cultures were in air-tight glass bottles purged continuously with premoisturized sterile air (generated by purging through 0.22 µm filter and sterile doubly deionized water) at 30 °C under continuous fluorescent light with an intensity of approximately 360 cd for 15-50 days. Premoisturization of the air prevented the change in medium Se concentration due to water evaporation. The air-purged volatile Se compounds were trapped in alkaline peroxide solution or liquid nitrogen (8). Dispersed cell density was monitored by optical density at 680 nm (OD680) and by chlorophylls (8) initially. Since the time courses obtained were similar, only OD at 680 was measured subsequently. Attempts to relate OD at 680 to cell number counts was not successful since the cyanophyte filaments became long and intertwined quickly with growth. In addition, soon after the culture reached peak dispersed density, the cells aggregated to form mats and sedimented to the bottom of the flask, which made it impractical to relate OD at 680 to total cell numbers. Water column was sampled periodically, centrifuged to remove algal biomass, preserved with HNO3 (20 µL of 7.9 N HNO3 in 1 mL of medium), and stored at -20 °C before analysis of total dissolved Se (see below). Total Se in the alkaline peroxide trap was also analyzed as described below. Selenium Analysis. Total Se. Total Se was analyzed using microdigestion (8) coupled with fluorescence detection method modified from the Analytical Methods Committee (11). Digestion, reduction of selenate, derivatization of selenite by diaminonaphthalene (DAN), and extraction of the piazselenol derivative for a given sample was performed in a single vial. Briefly, the algal biomass (
