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Aug 15, 2013 - (2) Constructed wetland treatment systems have been shown to be ... Selenium removal by constructed wetlands occurs within sediments, ...
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Development of a Constructed Wetland Water Treatment System for Selenium Removal: Incorporation of an Algal Treatment Component Jung-Chen Huang, María C. Suárez, Soo In Yang,† Zhi-Qing Lin,‡ and Norman Terry* Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, United States S Supporting Information *

ABSTRACT: On the basis of the fact that algae have the ability to volatilize substantial quantities of selenium (Se), we investigated the concept of including an algal pretreatment unit into a constructed wetland system for the removal of Se from river water entering the Salton Sea. Of six different algal strains tested, the most effective in terms of Se volatilization and Se removal from the water column was a Chlorella vulgaris strain (designated Cv). Cv removed 96% of Se (supplied as selenate) from the microcosm water column within 72 h, with up to 61% being removed by volatilization to the atmosphere. X-ray absorption spectroscopy revealed that the major forms of Se likely to be accumulated in an algal−wetland system are selenomethionine, a precursor of volatile Se formation, and elemental Se. Our results suggest that the inclusion of an algal pretreatment unit within a constructed wetland water treatment system should not only enhance the efficiency of Se removal but also significantly reduce the risk of the buildup of ecotoxic forms of Se by promoting the biological volatilization of Se.

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INTRODUCTION

where it is dispersed by wind currents far from the initial location.14 Because some algae have the propensity to volatilize substantial amounts of Se,15−17 we explored the idea of incorporating an algal treatment unit within a constructed wetland water treatment system to maximize Se volatilization and minimize Se buildup. Using microcosms to investigate a number of factors that could improve the efficiency of Se removal through volatilization, our objectives in the present work were to (1) screen algal species/strains to determine those best suited for the removal and volatilization of Se; (2) determine the chemical forms of Se that are likely to accumulate within a combined algal−cattail water treatment system; (3) investigate the influence of fertilizer macronutrients, i.e., nitrogen (N) and phosphorus (P), on algal Se volatilization; and (4) determine the potential impacts on the volatilization and speciation of Se resulting from the flow of Sebearing algae into a wetland ecosystem. On the basis of this research, we propose a design for a constructed wetland water treatment system that incorporates an algal component in order to maximize Se removal through volatilization.

The Salton Sea in California is an important habitat for fish and waterfowl. Its ecosystem is threatened due to diminishing water supplies and increasing salinity. To compensate for this, the State of California has proposed to construct a Species Conservation Habitat (SCH).1 A supply of clean water for the SCH could be obtained from local rivers (e.g., the New River) provided that a water treatment system can be developed to remove selenium (Se), fertilizer nutrients, and other contaminants.2 Constructed wetland treatment systems have been shown to be effective in removing Se from oil refinery wastewater3 and from agricultural drainage water.4,5 In a greenhouse mesocosm study, Huang et al.6 showed how such wetland treatment systems might be improved to reduce Se concentrations in the water column from 15 μg Se/L to 0.1 μg Se/L within 72 h. Selenium removal by constructed wetlands occurs within sediments, principally through the microbially mediated dissimilatory anaerobic reduction of oxyanions to water insoluble elemental Se (Se0).7 Although much of the incoming Se may be trapped in the sediments in insoluble and relatively nonbioavailable forms, there is always the risk that Se buildup will eventually lead to Se ecotoxicity.8−11 One way of mitigating this risk is to enhance Se removal by biological volatilization to the atmosphere. The assimilatory reduction and methylation of Se to volatile forms, which can be carried out by both microbes12 and plants,13 is particularly beneficial because Se is removed from the aquatic ecosystem into the atmosphere © 2013 American Chemical Society

Received: Revised: Accepted: Published: 10518

April 11, 2013 August 2, 2013 August 15, 2013 August 15, 2013 dx.doi.org/10.1021/es4015629 | Environ. Sci. Technol. 2013, 47, 10518−10525

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MATERIALS AND METHODS Materials. Cattail (Typha latifolia) seeds were purchased from Pacific Coast Seeds, Inc., Livermore, CA. Six freshwater algae were tested including two different strains of Chlorella vulgaris, designated Cv and Cs, two isolates of Chlorella sp. (collected from the McKendry and McDonald agricultural drains close to the Salton Sea and designated McK and McD, respectively), and C. ellipsoidea (Ce) and Scenedesmus obliquus (So). Cv, Ce, and So were obtained from the UTEX Algal Collection. 18S rRNA gene sequence analyses of the six algae revealed that Cs is closely related (more than 98%) to a C. vulgaris isolate D2 GenBank accession number JX185298. McK and McD are more closely related to Chlorella sp. (both have more than 98% identity to the 18S rRNA sequence of Chlorella sp. IFRPD1018 GenBank accession number AB260898.1). The 18S rRNA sequences of McK and McD were ∼99% identical to each other. A ClustalW alignment of a region of the 18S rRNA sequences for the different algae tested in this study is illustrated in Figure S1 of the Supporting Information. Sodium selenite (Na2SeO3), sodium selenate (Na2SeO4), Lselenomethionine (SeMet; C5H11NO2Se), and selenocystine (C6H12N2O4Se2) were purchased from Sigma-Aldrich (St. Louis, MO). Microcosms were constructed from 1 and 4 L Pyrex glass bottles. Six stainless steel tanks (75 cm × 30 cm × 20 cm) were used for mesocosm experiments in the greenhouse at UC Berkeley. Pumps (Sunterra 104506) and rubber tubing (9 mm i.d., 15 mm o.d., 3 mm w; Fisher Scientific) were set up to circulate water (3.15 × 10−5 m3 s−1) from the mesocosm outlet back to the inlet. The mesocosm studies were conducted in an environment-controlled greenhouse with 25/22 (±2) °C day/night temperatures, 16 h photoperiod, and 1000 μE (photosynthetic photon flux) m−2 s −1. Algal Species Identification and Culture. Algae were identified by 18S rRNA sequencing analyses. Total genomic DNA from each strain was isolated using the DNeasy Plant Mini kit (Qiagen, Valencia, CA). A 1790 bp fragment of the18S rRNA sequence region was amplified by polymerase chain reaction (PCR) using combinations of the primers 107F, 5′CGAATGGCTCATTAAAT-3′, 2237R, 5′-CAAATGAAGATGGGGAGGCGA-3′, and 1700R, 5′-CCGAAGTCTTCACCAGCACATC-3′ (Integrated DNA Technologies, Inc. Chicago, IL). The Phire Hot Start II DNA polymerase (Thermo Fisher Scientific, Inc. Waltham, MA) was used for the PCR under conditions specified by the manufacturer. The PCR fragments were purified and cloned using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Inc. Waltham, MA). The sequences were obtained at the UC Berkeley DNA Sequencing Facility and then compared to publicly available GenBank sequences. Individual microalgal colonies were maintained in solid agar cultures and used as inoculum for liquid cultures in 2 L Pyrex glass bottles containing 1/4strength Hoagland’s nutrient solution.18 The cultures were placed in a growth room with a 24 h photoperiod and maintained at 22 °C with a photon flux density of 400 μE m−2 s−1 for 3 weeks (late exponential growth phase). In experiments 1, 2, and 3, ampicillin (60 mg/L) was added weekly into the culture medium during growth to control microbial contamination. Selenium Uptake and Volatilization by Algae. The cultures of each algal isolate (20 L) were each centrifuged at 4500g, rinsed twice with deionized (DI) water, and tested in a 1 L Pyrex glass bottle with a two-hole stopper. Two glass tubes

served as an inlet and outlet for airflow, and the inlet airflow tube bubbled air through the culture media. The same inlet tube was connected to a small chamber in the outer end that contained a 0.22-μm filter filled with activated carbon (6−14 mesh, Thermo Fisher Scientific, Inc. Waltham, MA) to remove microbes and Se from the incoming air. During the assay of volatilization rates, each gas wash bottle was connected via short lengths of Teflon tubing (Thermo Fisher Scientific, Inc. Waltham, MA) to a second wash bottle. Volatile Se was trapped in an alkaline peroxide trap solution (40 mL, 30% H2O2, and 160 mL 0.05 M NaOH)14 contained in a series of two 500-ml gas-washing bottles. The gas-washing bottles were connected to a vacuum line via a pressure regulator (King Instrument Co., Garden Grove, CA) so that a stream of 0.22-μm filtered air was forced through both gas-washing bottles at a rate of 2.36 L min−1. Any volatile Se compounds given off by the algae were trapped in the alkaline peroxide trap solution; the solutions from the two gas-washing bottles were collected for total Se analysis and replaced every 24 h. Total Se Analysis. Total Se concentrations in water, plant tissue, and soil samples were determined according to the EPA method 200.819 using inductively coupled plasma-dynamic reaction cell-mass spectrometry (ICP-DRC-MS Agilent 7500cx; limit of quantification = 0.01 μg Se/L). All the water samples (without filtration) were acid-digested with HNO3. Plant tissue samples, including cattail shoot, rhizome, and litter, were dried at 60 °C and ground to fine powder and then wet-digested with HNO3, H2O2, and HCl. Soil samples (mixed sand and peat moss) were dried at 60 °C and then wet-digested with HNO3, H2O2, and HCl.20 NIST standard reference materials SRM2709 and SRM-1567a were used as internal quality control samples for analyses of Se in soil and plant samples. X-ray Absorption Spectroscopy (XAS). The algal biotransformation of Se was investigated using X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The collected algal samples were rinsed three times to remove any residual Se species. Plant and substrate samples were ground (solid bulky samples) using a mortar and pestle, injected into 2 mm path-length cuvettes, and then flash-frozen in iso-pentane containing liquid N. The samples, preserved at −80 °C, were later transferred to Beamline 7-3 of the Stanford Synchrotron Radiation Lightsource (SSRL). Data collection and analysis were carried out as described by Yang et al.21 XAS analysis was performed using EXAFSPAK22 and EXAFS phase, and amplitude functions were obtained using ab initio calculation code FEFF 7.23 In EXAFS fits, scale factors was set to 0.9, nominal threshold energy was 12,675 eV, and the offset to the energy (ΔE0) was determined to be −14.8 and −13.8 for SeMet (Se−C) and Se0 (Se−Se), respectively. Experiment 1. The six algal isolates were tested to characterize their efficiencies of Se volatilization and removal. The algae were grown and tested in 1 L glass bottles as described above and treated with 20 μM (1580 μg Se/L) selenate and selenite in DI water (three replicates per treatment). Volatile Se was measured daily over 72 h, after which time, water and algal samples were collected for measurement of total Se in the water column and in the algal biomass. Experiment 2. The effect of nutrients (and other environmental factors) on algal Se removal and volatilization was investigated using Scenedesmus obliquusa readily available and robust alga that has been well characterized.24 Selenium 10519

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Figure 1. Changes with time in volatile Se produced per microcosm for six different algal strains supplied with 1580 μg Se/L at day 0 as selenate or selenite (experiment 1).

volatilization and removal by Scenedesmus obliquus were compared in the presence or absence of nutrients. The initial culture was divided into three 50-mL replicates, each of which was added to 1 L bottles and raised to 1 L with Se at 40 μM Se (3160 μg Se/L) as selenate in either DI water or 1/5 Hoagland’s solution (without magnesium sulfate). Volatile Se was measured daily for four days, after which time the samples were collected for measurement of total Se. XAS speciation was carried out for Se in the water column and algal biomass. Experiment 3. The effects of excess N and P on Se volatilization and removal were determined for Scenedesmus obliquus. The three treatments were the following: control (1/5 Hoagland’s solution), high N (1/5 Hoagland’s solution with 2 × nitrate-N), and high P (1/5 Hoagland’s solution with 2 × phosphate-P), each replicated three times. The initial culture was separated into nine 25-mL lots, each of which was raised to 1 L with 1/5 Hoagland’s solution (without magnesium sulfate). Selenate was added to each replicate to yield an initial concentration of 2500 μg Se/L. Selenium volatilization was measured daily over 3 days; the total Se and XAS determination of the Se species, in the water column and in the algal biomass were obtained at the end of the experiment. Experiment 4. Six small mesocosms were planted with cattail seedlings and filled with sand and peat moss as substrate. In three of the mesocosms, a layer of dead cattail fragments was placed over the sand/peat moss substrate; the other three mesocosms served as a control. Cattails were grown for one

month with weekly 2 L additions of 1/2-strength Hoagland’s solution. The mesocosms were supplied with selenate to yield a concentration of 1580 μg Se/L. Each mesocosm was equipped with a pump and tubing to maintain constant water circulation. At the end of 10 days, measurements were made of the dry weights of cattail shoots and rhizomes. Samples of cattail shoots, rhizomes, fallen litter, and sand/peat moss substrate were taken for measurement of total Se and for XAS speciation. A mass balance for the distribution of total Se in each mesocosm component (i.e., sand/peat moss substrate, litter for the mesocosms with cattail fragments, and cattail shoots and rhizomes) was used to provide an approximate estimate of Se volatilization. Experiment 5. Three replicate 4 L microcosms were set up for each of two treatments. In one treatment, 30 g dry cattail litter was added to each microcosm. In the other treatment, an equivalent volume of sand and peat moss (mixed 1:1 v/v) was added to a depth of 7 cm. An equal volume (470 mL) of Sebearing algal culture (using Scenedesmus obliquus cultures from experiment 2) was frozen at −20 °C for 3 days to inactivate algal cells and then added to each of the six microcosms (the microcosms were covered with foil to block the light to minimize any Se volatilization from algae that were still active even after freezing). Volatile Se was measured daily over a 7-day experimental period. At the termination of the experiment, Se in the sand/peat moss, water column, and cattail litter was speciated by XAS. 10520

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Experiment 6. Three algal mesocosms (75 cm × 30 cm × 20 cm) were set up in the controlled environment greenhouse. Cultures of Cv, grown in 1/4-strength Hoagland’s solution for 3 weeks, were centrifuged and rinsed three times and then added to the mesocosms, each of which contained 30 L of DI water with an initial selenate−Se concentration of 15 μg Se/L and containing nutrients similar in composition to that of the New River, i.e., sulfate 0.5 mg/L, TN 4.1 mg/L, and TP 1 mg/ L.25 Each mesocosm was aerated using an air pump (Fusion 700 by JW Pet) at a rate of 5.6 L min−1; circulation was provided by two pumps (Hydor Koralia Evolution 750 by Hydro USA). Samples of the algal suspensions in the mesocosms were collected 5, 24, and 48 h after Se addition for measurement of total Se concentrations. Estimates of Se volatilization were obtained as the difference between the initial and final Se levels of the algal suspension.

i.e., 56% of the added Se was accumulated in the algal biomass of Cs compared to 27% in Cv (Table S1). In terms of selenite removal, the most efficient algae were McK and McD: each achieved 96% removal of Se from the microcosm water column (Table S1). Although Cv and Cs had higher rates of absorption of selenite-Se per biomass than either McK or McD, their total Se accumulation was less due to their smaller biomass (Table S2). Biotransformation of Se by Algae. Using XAS, the most noninvasive and accurate way of speciating Se in vivo,26,27 we observed that the speciation of Se in the algal biomass was markedly affected by the form of Se supplied, selenate or selenite (Table S3). When the algae were supplied with selenate, only Cs was able to absorb and metabolize all of the supplied selenate; the other five algae tested retained from 14 to 60% of the Se in their biomass as selenate (Table S3). When the algae were supplied with selenite, the absorbed Se was metabolized to SeMet and Se0 (except for minor differences in Ce, Table S3). Algae, along with higher plants and bacteria, almost certainly metabolize selenate-Se and selenite-Se through the sulfurassimilation pathway generating selenocysteine (SeCys) and SeMet, the Se analogues of cysteine and methionine.13,28,29 The buildup of selenate in algal cells was most likely due to ratelimitation by ATP sulfurylase as occurs in plants.30 This rate limiting step (selenate ⇒ selenite) was eliminated when Se was supplied as selenite, so that selenite-Se was biotransformed to SeMet and Se0 (in Ce, trace amounts of selenate and selenite were also detected, Table S3). The accumulation of SeMet, which occurs in all the algae tested regardless of whether they were supplied with selenate or selenite (except for McD supplied with selenate), could be due to a second, rate-limiting enzyme in the S-assimilation pathway, methionine S-methyltransferase (MMT). MMT has been shown to be present in plants such as Arabidopsis31 and is responsible for the methylation of SeMet to precursors of volatile Se (e.g., dimethylselenide). However, as yet, no enzymes with MMT function have been reported for algae. Furthermore, there is a possibility that the SeMet fraction detected by XAS may also have included proteinaceous SeMet that is less available for volatilization. The third main species of Se present in all the algal strains tested was Se0 (Table S3). The presence of Se0 in the algae was confirmed using EXAFS (see details in Figure S2). To our knowledge, there are no published studies showing the presence of Se0 in freshwater algae. Assuming that the Se0 present in the algal biomass of the present work is truly algal, and not microbial in origin (antibiotics were used to reduce this possibility), the question arises as to the mechanism of its formation. Pilon-Smits and colleagues have shown that SeCyslyase produces Se0 and alanine from SeCys in transgenic Arabidopsis plants.32,33 One possible explanation, therefore, for the presence of Se0 is that the Chlorella strains could have enzymes with SeCys-lyase-type activity for the breakdown of SeCys to Se0. Other studies using Scenedesmus obliquus, Chlorella kessleri, and Synechococcus leopoliensis have proposed that the formation of Se0 may be necessary for the synthesis of selenocyanide; nevertheless, no direct measurements of Se0 have been reported.34,35 Another form of Se revealed by XAS was selenocystine, which was detected in McD only when treated with selenate-Se (Figure 2). Some studies have previously reported the

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RESULTS AND DISCUSSION Screening of Algae for Volatilization and Removal. In experiment 1, we compared the six different algae with respect to their ability to remove Se from the water column through the processes of volatilization and accumulation within the algal biomass. The results show that there was considerable variation in terms of their rates of volatilization. Both selenate and selenite were volatilized very rapidly after injection into the microcosms, with most of the Se being volatilized within 24−48 h (Figure 1). With respect to the volatilization of selenate, the best performing alga of the six tested was Cv, which volatilized substantially more Se per microcosm over the 72-h experimental period than the other 5 strains (Supporting Information Table S1). Since the amount of algal biomass per microcosm differed among the algal strains (Supporting Information Table S2), we recalculated volatilization as a rate per unit dry weight of algal biomass. Expressed in this way, the average rate of volatilization of selenate was much higher for Cv than for the other five strains (Supporting Information Table S2). When the algal strains were supplied with Se in the form of selenite, the amounts of Se volatilized per microcosm were greater for McK and McD (Supporting Information Table S1), but on a per unit biomass basis, Cv again volatilized at a faster rate than the other strains (Supporting Information Table S2). The results show clearly that Cv outperformed all other algae with respect to volatilization, especially when Se was supplied as selenate. The efficiency of the different algae in removing Se from the water column of the microcosms was assessed by calculating the percentage of Se removed in each case ([added Se - water column Se]/[added Se] × 100). Calculated in this way, Cv removed 96% and Cs 94% of the added selenate-Se from the water column (Table S1). The removal of Se from the water column was accomplished through two processes - Se volatilization and Se accumulation into the algal biomass, the sum of which is equivalent to the amount of Se absorbed by the algal cells. Selenium absorption varied substantially among the algal strains: Cv and Cs absorbed the most selenate-Se per microcosm (Table S1) and displayed the highest rates of absorption per unit biomass (Table S2). While Cv and Cs were the best two algal strains for removing selenate from the water column, they differed substantially in their manner of Se removal. Cv achieved its Se removal mainly through volatilization, removing 61% of the added Se by volatilization compared to 41% for Cs (Table S1). In contrast, Cs achieved its removal mostly through accumulation in the algal biomass, 10521

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accumulation of selenocystine in algal cells, e.g., Chlorella sp.17 and Chlorella sorokiniana.36

Figure 2. Se K near-edge X-ray absorption spectra (XANES) of Se standards (upper section of the figure) and of biomass samples of 6 algae from selenate- (broken lines) and selenite-supplied algal cultures (solid lines) (experiment 1; bottom section of the figure). The Se standards included SeO42−, SeO32−, SeMet, CysSeSeCys, and Se0, i.e., selenate, selenite, selenomethionine, selenocystine, and elemental selenium, respectively (the dotted line is used to clearly illustrate the selenite spectrum). The overall analysis based on these data is shown Supporting Information Table S3.

Figure 3. Changes with time in volatile Se produced per microcosm for Scenedesmus obliquus. (A) Alga treated with 3158 μg Se/L in deionized water (DI) or 1/5 Hoagland’s nutrient solution (1/5 H) as described in Materials and Methods, experiment 1. (B) Alga treated with 2500 μg Se/L in 1/5 Hoagland’s nutrient solution (control), and 1/5 Hoagland’s nutrient solution with double the concentration of nitrogen (2XN) or phosphorus (2XP) as described in Materials and Methods, experiment 2.

Nutrient Interactions. In experiment 2, Scenedesmus obliquus was supplied with 40 μM Se (3160 μg Se/L) in the absence (i.e., deionized water) and presence of nutrients (1/5 Hoagland’s solution). Volatilization in deionized water reached a maximum in 24−48 h and then declined (Figure 3A). The rapid increase in volatilization in the absence of added nutrients was likely due to an enhanced uptake of selenate; the concentration of Se in the algal biomass was 55% greater when Se was supplied in the absence compared to the presence of nutrients (Supporting Information Table S4). Volatilization in the presence of 1/5 Hoagland’s solution began slowly but increased progressively with time over the 4-day experimental period (Figure 3A). We further explored the effect of nutrients in experiment 3 by comparing volatilization in the presence of high levels of N and P (Figure 3B). Volatilization changed nearly linearly with time for all three nutrient treatments; the average rate of volatilization was little affected by doubling the amount of N, but was significantly increased by doubling the amount of phosphate (Figure 3B). Biomass Se in the absence of nutrients was more or less equally distributed among three Se compounds, i.e., selenate Se (37%), SeMet (36%), and Se0 (27%) (experiment 2, Supporting Information Table S4). In the presence of nutrients most of the Se was present in the form of SeMet (experiments 2 and 3, Table S4). Potential Fate of Algal-Borne Se Entering Wetlands. Experiments 4 and 5 were carried out to resolve the potential fate of algal-borne Se flowing from the algal treatment unit to the wetland unit. In experiment 4, we established a baseline for the partitioning and speciation of selenate-supplied Se in a wetland mesocosm ecosystem before the introduction of Sebearing algae, and in experiment 5, we examined the potential fate of algal-borne Se within a wetland ecosystem by adding algae to microcosms containing either fragments of dead

cattails (to simulate fallen litter) or sand and peat moss (to simulate fine sediments). In experiment 4, most of the added Se in the mesocosms without a fallen litter layer was accumulated in rhizomes (39%) and shoots (17%), with the remainder in the sand and peat moss substrate (27%) (Supporting Information Table S5). A similar partitioning of total Se was observed in the mesocosms with fallen litter layers, except that 4.4% of the Se was present in the fallen litter layer itself (Table S5). The speciation of Se in rhizomes and shoots was similar in mesocosms with or without a fallen litter layer. Most of the Se in the plant biomass was present as selenate, which ranged from 81 to 88% (Table S5). There was no Se0 and the only other form of Se found in the plant biomass was SeMet. However, in the sand/peat moss substrate, most if not all, was in the form of Se0 (Table S5). The Se0 in the sediment was most likely formed through the microbially mediated anaerobic dissimilatory reduction of selenate.37 In the fallen litter layer, the Se was fairly evenly distributed between SeMet and Se0, indicating that both microbial assimilatory reduction through the sulfate assimilation pathway, and anaerobic dissimilatory reduction, were occurring within the fallen litter.38,39 A similar distribution between SeMet and Se0 was obtained in a field wetland study by Lin and Terry,5 who found that, in vegetated and unvegetated sediments, 41−47% of the Se was present as Se0 and 37−46% as SeMet. In experiment 5, volatilization by the microcosms containing sand/peat moss substrate increased rapidly at first, reaching a peak in 24 h, and then declined, whereas in the microcosms containing cattail litter, volatilization increased more or less progressively with time (Figure 4A). At the end of the 7-day 10522

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Figure 4. (A) Changes with time in volatile Se produced per microcosm for Scenedesmus obliquus cultures (see Materials and Methods, experiment 5) to which was added sand/peat moss or cattail fragments. (B) Table showing the amount of Se volatilized/microcosm and the percent speciation of Se among three forms of Se, selenate, SeMet, and Se0. The cattail fragments + algal mixture was separated into two components, i.e., the cattail fragments + algae and the suspending medium + algae. The speciation of Se for each of these two components was determined separately. In the case of the sand/peat moss−algal mixture, the whole mixture was subsampled for speciation.

initial Se concentration of 15 μg Se/L to 7.61 ± 0.3 at 5 h, 6.86 ± 0.22 at 24 h, and 6.57 ± 0.05 at 48 h after Se addition. Since Se levels of the algal suspension had decreased by 39.4% after 48 h, it is reasonable to assume that the remaining 60.6% was lost through volatilizationa proportion similar to that obtained for Cv at high Se concentrations (experiment 1). Furthermore, our microcosm studies with Scenedesmus obliquus show that algae are able to volatilize Se in the presence of large amounts of N and P and that it is important to optimize algal population densities in order to maximize algal growth, uptake, and volatilization (see Supporting Information Figure S3). In addition to the Se volatilized by the algal treatment unit itself, it is likely that more Se will be volatilized as the Sebearing algae enter the cattail treatment unit. This view is based on the following observations. Experiment 5 shows that Se volatilization increased in the microcosms containing Sebearing algae in the presence of cattail litter, or sand/peat moss. A second source of volatile Se will arise from the cattails themselves: 47−56% of the Se added to mesocosms was accumulated in the biomass of rhizomes and shoots (Supporting Information Table S5), both of which are capable of Se volatilization.13 Thus, our results suggest that much of the incoming Se from the river will be volatilized by algae in the algal treatment unit, by microbial decomposition of algae in the cattail unit, and by volatilization of Se accumulated in cattail tissues. We envision that the cleanup of river water contaminated with Se and nutrients from farm runoff could be achieved using a design similar to that shown in Supporting Information Figure S4. However, before implementing this design, it is essential that its benefits and limitations are thoroughly tested

experimental period, volatilization from the sand/peat moss microcosm had virtually ceased and the SeMet content had decreased to zero; in the cattail fragments microcosm on the other hand, both volatilization and SeMet levels were high (Figure 4). The correlation between volatilization and SeMet levels suggests that volatilization in both types of microcosms was dependent on the level of the volatile Se precursor, SeMet. It is not clear whether the volatilization was carried out by microbes, by algae, or by a combination of both. The fact that the total (7day) volatile Se produced by the microcosm with cattail fragments was 2.7-fold greater than volatilization from the sand/peat moss microcosm, together with the fact that 75% of the Se in the sand/peat moss fraction was in the form of selenate compared to only 10 and 18% in the cattail fragments and suspending medium, respectively, of the cattail microcosm (Figure 4 table), suggest that the presence of a cattail fallen litter in a wetland ecosystem is likely to greatly facilitate the conversion of selenate to SeMet and volatile Se. Use of Algal−Cattail Wetland Systems for Se Removal. On the basis of the microcosm studies of experiment 1, strain Cv exhibited highly efficient characteristics with respect to volatilizing and removing Se from the water column. Because these measurements were made at very high Se concentrations (1580 μg Se/L) and in the absence of nutrients, we carried out experiment 6 to determine the extent of Se removal and volatilization from water simulating Se and nutrient concentrations likely to be encountered by Cv receiving water from rivers (e.g., the New River) supplying the Salton Sea. The results of experiment 6 show that the Se concentrations of the Cv algal suspensions decreased from an 10523

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in a pilot wetland under field conditions. The pilot wetland should be used to determine (1) the efficiency of Se removal from the water flowing through it, (2) the extent to which Se is removed through biological volatilization versus the extent to which it is accumulated in potentially ecotoxic forms, and (3) the effects of the different chemical forms of Se on biota within the different compartments of the wetland treatment system. The results of the present work show that the major forms of Se accumulated in the proposed wetland treatment system are likely to be SeMet and Se0. Selenomethionine is of particular concern because it can be easily metabolized into proteins and because laboratory studies indicate that it can be toxic to fish and birds.10,40 On the other hand, SeMet has been shown to be volatilized much more rapidly by both plants41 and microbes13 than are Se oxyanions. Elemental Se is immobilized in the sediments in relatively nonbioavailable forms and is considered to be of little toxicological significance for most organisms.42,43 The oxidation and/or potential remobilization of Se0 is possible, especially if the wetland were to dry out, but the rate of oxidation has been shown to be relatively slow.7,44 Provided the proposed design can be validated under field conditions, we believe that, in addition to providing clean water for the SCH, it might have other applications, such as removal of Se from agricultural drainage water. This would allow drainage water to be reutilized for additional crop production, thereby making more efficient use of the rapidly dwindling supplies of water in California and other Western states.

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(SSRL) for beam time granted to N.T. (SSRL is a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University). This research was supported by funds from the State of California Proposition 84, administered by DFG and DWR under the Species Conservation Habitat Project.

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2 showing volatilization, biomass accumulation, and absorption rates of Se for different algal strains (experiment 1); Table S3 showing percentage of the Se species observed from algae (experiment 1); Table S4 showing effects of nutrient supply on rates of volatilization, biomass accumulation, and absorption of Se by Scenedesmus obliquus (experiments 2 and 3); Table S5 showing total Se and percent distribution of Se among different Se forms in the cattail mesocosm components (experiment 4); Figure S1 illustrating the sequence similarity with the most closely related algal species; Figure S2 showing the Se K-edge EXAFS oscillations and phase-corrected Fourier transform of selenite-supplied algae (experiment 1); Figure S3 illustrating the relationships between Chla and Se removal/ volatilization by Scenedesmus obliquus; and Figure S4 with a conceptual design for the cleanup of river water. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 (510) 642 3510. E-mail: [email protected]. Present Addresses

† Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK, S7N 5E2, Canada. ‡ Department of Biological Sciences and Environmental Sciences Program, Southern Illinois University, Edwardsville, Illinois 62026, United States.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the greenhouse staff at the University of California Berkeley and the Stanford Synchrotron Radiation Lightsource 10524

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