Development of a Constructed Wetland Water Treatment System forSelenium Removal: Incorporation of an Algal Treatment ComponentJung-Chen Huang, María C. Suarez, 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 theability to volatilize substantial quantities of selenium (Se), weinvestigated the concept of including an algal pretreatment unitinto a constructed wetland system for the removal of Se fromriver water entering the Salton Sea. Of six different algal strainstested, the most effective in terms of Se volatilization and Seremoval 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-rayabsorption spectroscopy revealed that the major forms of Selikely to be accumulated in an algal−wetland system areselenomethionine, a precursor of volatile Se formation, andelemental Se. Our results suggest that the inclusion of an algal pretreatment unit within a constructed wetland water treatmentsystem should not only enhance the efficiency of Se removal but also significantly reduce the risk of the buildup of ecotoxic formsof Se by promoting the biological volatilization of Se.

■ INTRODUCTION

The Salton Sea in California is an important habitat for fish andwaterfowl. Its ecosystem is threatened due to diminishing watersupplies and increasing salinity. To compensate for this, theState of California has proposed to construct a SpeciesConservation Habitat (SCH).1 A supply of clean water forthe SCH could be obtained from local rivers (e.g., the NewRiver) provided that a water treatment system can bedeveloped to remove selenium (Se), fertilizer nutrients, andother contaminants.2 Constructed wetland treatment systemshave been shown to be effective in removing Se from oilrefinery wastewater3 and from agricultural drainage water.4,5 Ina greenhouse mesocosm study, Huang et al.6 showed how suchwetland treatment systems might be improved to reduce Seconcentrations in the water column from 15 μg Se/L to 0.1 μgSe/L within 72 h.Selenium removal by constructed wetlands occurs within

sediments, principally through the microbially mediateddissimilatory anaerobic reduction of oxyanions to waterinsoluble elemental Se (Se0).7 Although much of the incomingSe may be trapped in the sediments in insoluble and relativelynonbioavailable forms, there is always the risk that Se buildupwill eventually lead to Se ecotoxicity.8−11 One way of mitigatingthis risk is to enhance Se removal by biological volatilization tothe atmosphere. The assimilatory reduction and methylation ofSe to volatile forms, which can be carried out by bothmicrobes12 and plants,13 is particularly beneficial because Se isremoved from the aquatic ecosystem into the atmosphere

where it is dispersed by wind currents far from the initiallocation.14

Because some algae have the propensity to volatilizesubstantial amounts of Se,15−17 we explored the idea ofincorporating an algal treatment unit within a constructedwetland water treatment system to maximize Se volatilizationand minimize Se buildup. Using microcosms to investigate anumber of factors that could improve the efficiency of Seremoval through volatilization, our objectives in the presentwork were to (1) screen algal species/strains to determinethose best suited for the removal and volatilization of Se; (2)determine the chemical forms of Se that are likely toaccumulate within a combined algal−cattail water treatmentsystem; (3) investigate the influence of fertilizer macro-nutrients, i.e., nitrogen (N) and phosphorus (P), on algal Sevolatilization; and (4) determine the potential impacts on thevolatilization and speciation of Se resulting from the flow of Se-bearing algae into a wetland ecosystem. On the basis of thisresearch, we propose a design for a constructed wetland watertreatment system that incorporates an algal component in orderto maximize Se removal through volatilization.

Received: April 11, 2013Revised: August 2, 2013Accepted: August 15, 2013Published: August 15, 2013

Article

pubs.acs.org/est

© 2013 American Chemical Society 10518 dx.doi.org/10.1021/es4015629 | Environ. Sci. Technol. 2013, 47, 10518−10525

■ MATERIALS AND METHODS

Materials. Cattail (Typha latifolia) seeds were purchasedfrom Pacific Coast Seeds, Inc., Livermore, CA. Six freshwateralgae were tested including two different strains of Chlorellavulgaris, designated Cv and Cs, two isolates of Chlorella sp.(collected from the McKendry and McDonald agriculturaldrains 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 AlgalCollection. 18S rRNA gene sequence analyses of the six algaerevealed that Cs is closely related (more than 98%) to a C.vulgaris isolate D2 GenBank accession number JX185298. McKand McD are more closely related to Chlorella sp. (both havemore than 98% identity to the 18S rRNA sequence of Chlorellasp. IFRPD1018 GenBank accession number AB260898.1). The18S rRNA sequences of McK and McD were ∼99% identical toeach other. A ClustalW alignment of a region of the 18S rRNAsequences for the different algae tested in this study isillustrated in Figure S1 of the Supporting Information.Sodium selenite (Na2SeO3), sodium selenate (Na2SeO4), L-

selenomethionine (SeMet; C5H11NO2Se), and selenocystine(C6H12N2O4Se2) were purchased from Sigma-Aldrich (St.Louis, MO). Microcosms were constructed from 1 and 4 LPyrex glass bottles. Six stainless steel tanks (75 cm × 30 cm ×20 cm) were used for mesocosm experiments in the greenhouseat UC Berkeley. Pumps (Sunterra 104506) and rubber tubing(9 mm i.d., 15 mm o.d., 3 mm w; Fisher Scientific) were set upto circulate water (3.15 × 10−5 m3 s−1) from the mesocosmoutlet back to the inlet. The mesocosm studies were conductedin an environment-controlled greenhouse with 25/22 (±2) °Cday/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 genomicDNA from each strain was isolated using the DNeasy PlantMini kit (Qiagen, Valencia, CA). A 1790 bp fragment of the18SrRNA sequence region was amplified by polymerase chainreaction (PCR) using combinations of the primers 107F, 5′-CGAATGGCTCATTAAAT-3′, 2237R, 5′-CAAATGAA-GATGGGGAGGCGA-3′, and 1700R, 5′-CCGAAGTCTT-CACCAGCACATC-3′ (Integrated DNA Technologies, Inc.Chicago, IL). The Phire Hot Start II DNA polymerase(Thermo Fisher Scientific, Inc. Waltham, MA) was used forthe PCR under conditions specified by the manufacturer. ThePCR fragments were purified and cloned using the CloneJETPCR Cloning Kit (Thermo Fisher Scientific, Inc. Waltham,MA). The sequences were obtained at the UC Berkeley DNASequencing Facility and then compared to publicly availableGenBank sequences. Individual microalgal colonies weremaintained in solid agar cultures and used as inoculum forliquid cultures in 2 L Pyrex glass bottles containing 1/4-strength Hoagland’s nutrient solution.18 The cultures wereplaced in a growth room with a 24 h photoperiod andmaintained at 22 °C with a photon flux density of 400 μE m−2

s−1 for 3 weeks (late exponential growth phase). In experiments1, 2, and 3, ampicillin (60 mg/L) was added weekly into theculture medium during growth to control microbial contam-ination.Selenium Uptake and Volatilization by Algae. The

cultures of each algal isolate (20 L) were each centrifuged at4500g, rinsed twice with deionized (DI) water, and tested in a 1L Pyrex glass bottle with a two-hole stopper. Two glass tubes

served as an inlet and outlet for airflow, and the inlet airflowtube bubbled air through the culture media. The same inlettube was connected to a small chamber in the outer end thatcontained a 0.22-μm filter filled with activated carbon (6−14mesh, Thermo Fisher Scientific, Inc. Waltham, MA) to removemicrobes and Se from the incoming air. During the assay ofvolatilization rates, each gas wash bottle was connected viashort lengths of Teflon tubing (Thermo Fisher Scientific, Inc.Waltham, MA) to a second wash bottle. Volatile Se was trappedin an alkaline peroxide trap solution (40 mL, 30% H2O2, and160 mL 0.05 M NaOH)14 contained in a series of two 500-mlgas-washing bottles. The gas-washing bottles were connected toa vacuum line via a pressure regulator (King Instrument Co.,Garden Grove, CA) so that a stream of 0.22-μm filtered air wasforced through both gas-washing bottles at a rate of 2.36 Lmin−1. Any volatile Se compounds given off by the algae weretrapped in the alkaline peroxide trap solution; the solutionsfrom the two gas-washing bottles were collected for total Seanalysis and replaced every 24 h.

Total Se Analysis. Total Se concentrations in water, planttissue, and soil samples were determined according to the EPAmethod 200.819 using inductively coupled plasma-dynamicreaction 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 tissuesamples, including cattail shoot, rhizome, and litter, were driedat 60 °C and ground to fine powder and then wet-digested withHNO3, H2O2, and HCl. Soil samples (mixed sand and peatmoss) were dried at 60 °C and then wet-digested with HNO3,H2O2, and HCl.20 NIST standard reference materials SRM-2709 and SRM-1567a were used as internal quality controlsamples for analyses of Se in soil and plant samples.

X-ray Absorption Spectroscopy (XAS). The algalbiotransformation of Se was investigated using X-ray absorptionnear-edge structure (XANES) and extended X-ray absorptionfine structure (EXAFS). The collected algal samples wererinsed three times to remove any residual Se species. Plant andsubstrate samples were ground (solid bulky samples) using amortar and pestle, injected into 2 mm path-length cuvettes, andthen flash-frozen in iso-pentane containing liquid N. Thesamples, preserved at −80 °C, were later transferred toBeamline 7-3 of the Stanford Synchrotron Radiation Light-source (SSRL). Data collection and analysis were carried out asdescribed by Yang et al.21 XAS analysis was performed usingEXAFSPAK22 and EXAFS phase, and amplitude functions wereobtained using ab initio calculation code FEFF 7.23 In EXAFSfits, scale factors was set to 0.9, nominal threshold energy was12,675 eV, and the offset to the energy (ΔE0) was determinedto be −14.8 and −13.8 for SeMet (Se−C) and Se0 (Se−Se),respectively.

Experiment 1. The six algal isolates were tested tocharacterize their efficiencies of Se volatilization and removal.The algae were grown and tested in 1 L glass bottles asdescribed above and treated with 20 μM (1580 μg Se/L)selenate and selenite in DI water (three replicates pertreatment). Volatile Se was measured daily over 72 h, afterwhich time, water and algal samples were collected formeasurement of total Se in the water column and in the algalbiomass.

Experiment 2. The effect of nutrients (and otherenvironmental factors) on algal Se removal and volatilizationwas investigated using Scenedesmus obliquusa readily availableand robust alga that has been well characterized.24 Selenium

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volatilization and removal by Scenedesmus obliquus werecompared in the presence or absence of nutrients. The initialculture was divided into three 50-mL replicates, each of whichwas 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 1/5Hoagland’s solution (without magnesium sulfate). Volatile Sewas measured daily for four days, after which time the sampleswere collected for measurement of total Se. XAS speciation wascarried 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 Scenedesmusobliquus. The three treatments were the following: control (1/5Hoagland’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 culturewas separated into nine 25-mL lots, each of which was raised to1 L with 1/5 Hoagland’s solution (without magnesium sulfate).Selenate was added to each replicate to yield an initialconcentration of 2500 μg Se/L. Selenium volatilization wasmeasured daily over 3 days; the total Se and XAS determinationof the Se species, in the water column and in the algal biomasswere 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 wasplaced over the sand/peat moss substrate; the other threemesocosms served as a control. Cattails were grown for one

month with weekly 2 L additions of 1/2-strength Hoagland’ssolution. The mesocosms were supplied with selenate to yield aconcentration of 1580 μg Se/L. Each mesocosm was equippedwith a pump and tubing to maintain constant water circulation.At the end of 10 days, measurements were made of the dryweights of cattail shoots and rhizomes. Samples of cattailshoots, rhizomes, fallen litter, and sand/peat moss substratewere taken for measurement of total Se and for XAS speciation.A mass balance for the distribution of total Se in eachmesocosm component (i.e., sand/peat moss substrate, litter forthe mesocosms with cattail fragments, and cattail shoots andrhizomes) was used to provide an approximate estimate of Sevolatilization.

Experiment 5. Three replicate 4 L microcosms were set upfor each of two treatments. In one treatment, 30 g dry cattaillitter was added to each microcosm. In the other treatment, anequivalent volume of sand and peat moss (mixed 1:1 v/v) wasadded to a depth of 7 cm. An equal volume (470 mL) of Se-bearing algal culture (using Scenedesmus obliquus cultures fromexperiment 2) was frozen at −20 °C for 3 days to inactivatealgal cells and then added to each of the six microcosms (themicrocosms were covered with foil to block the light tominimize any Se volatilization from algae that were still activeeven after freezing). Volatile Se was measured daily over a 7-dayexperimental period. At the termination of the experiment, Sein the sand/peat moss, water column, and cattail litter wasspeciated by XAS.

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 orselenite (experiment 1).

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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 for3 weeks, were centrifuged and rinsed three times and thenadded to the mesocosms, each of which contained 30 L of DIwater with an initial selenate−Se concentration of 15 μg Se/Land containing nutrients similar in composition to that of theNew 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 (Fusion700 by JW Pet) at a rate of 5.6 L min−1; circulation wasprovided by two pumps (Hydor Koralia Evolution 750 byHydro USA). Samples of the algal suspensions in themesocosms were collected 5, 24, and 48 h after Se additionfor measurement of total Se concentrations. Estimates of Sevolatilization were obtained as the difference between the initialand final Se levels of the algal suspension.

■ RESULTS AND DISCUSSIONScreening of Algae for Volatilization and Removal. In

experiment 1, we compared the six different algae with respectto their ability to remove Se from the water column through theprocesses of volatilization and accumulation within the algalbiomass. The results show that there was considerable variationin terms of their rates of volatilization. Both selenate andselenite were volatilized very rapidly after injection into themicrocosms, with most of the Se being volatilized within 24−48h (Figure 1). With respect to the volatilization of selenate, thebest performing alga of the six tested was Cv, which volatilizedsubstantially more Se per microcosm over the 72-hexperimental period than the other 5 strains (SupportingInformation Table S1). Since the amount of algal biomass permicrocosm differed among the algal strains (SupportingInformation Table S2), we recalculated volatilization as a rateper unit dry weight of algal biomass. Expressed in this way, theaverage rate of volatilization of selenate was much higher for Cvthan for the other five strains (Supporting Information TableS2). When the algal strains were supplied with Se in the form ofselenite, the amounts of Se volatilized per microcosm weregreater for McK and McD (Supporting Information Table S1),but on a per unit biomass basis, Cv again volatilized at a fasterrate than the other strains (Supporting Information Table S2).The results show clearly that Cv outperformed all other algaewith respect to volatilization, especially when Se was supplied asselenate.The efficiency of the different algae in removing Se from the

water column of the microcosms was assessed by calculatingthe percentage of Se removed in each case ([added Se - watercolumn Se]/[added Se] × 100). Calculated in this way, Cvremoved 96% and Cs 94% of the added selenate-Se from thewater column (Table S1). The removal of Se from the watercolumn was accomplished through two processes - Sevolatilization and Se accumulation into the algal biomass, thesum of which is equivalent to the amount of Se absorbed by thealgal cells. Selenium absorption varied substantially among thealgal strains: Cv and Cs absorbed the most selenate-Se permicrocosm (Table S1) and displayed the highest rates ofabsorption per unit biomass (Table S2). While Cv and Cs werethe best two algal strains for removing selenate from the watercolumn, they differed substantially in their manner of Seremoval. Cv achieved its Se removal mainly throughvolatilization, removing 61% of the added Se by volatilizationcompared to 41% for Cs (Table S1). In contrast, Cs achievedits removal mostly through accumulation in the algal biomass,

i.e., 56% of the added Se was accumulated in the algal biomassof 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 themicrocosm water column (Table S1). Although Cv and Cs hadhigher rates of absorption of selenite-Se per biomass than eitherMcK or McD, their total Se accumulation was less due to theirsmaller biomass (Table S2).

Biotransformation of Se by Algae. Using XAS, the mostnoninvasive and accurate way of speciating Se in vivo,26,27 weobserved that the speciation of Se in the algal biomass wasmarkedly affected by the form of Se supplied, selenate orselenite (Table S3). When the algae were supplied withselenate, only Cs was able to absorb and metabolize all of thesupplied selenate; the other five algae tested retained from 14to 60% of the Se in their biomass as selenate (Table S3). Whenthe algae were supplied with selenite, the absorbed Se wasmetabolized to SeMet and Se0 (except for minor differences inCe, Table S3).Algae, along with higher plants and bacteria, almost certainly

metabolize selenate-Se and selenite-Se through the sulfur-assimilation pathway generating selenocysteine (SeCys) andSeMet, the Se analogues of cysteine and methionine.13,28,29 Thebuildup of selenate in algal cells was most likely due to rate-limitation by ATP sulfurylase as occurs in plants.30 This ratelimiting step (selenate ⇒ selenite) was eliminated when Se wassupplied as selenite, so that selenite-Se was biotransformed toSeMet and Se0 (in Ce, trace amounts of selenate and selenitewere also detected, Table S3).The accumulation of SeMet, which occurs in all the algae

tested regardless of whether they were supplied with selenate orselenite (except for McD supplied with selenate), could be dueto a second, rate-limiting enzyme in the S-assimilation pathway,methionine S-methyltransferase (MMT). MMT has beenshown to be present in plants such as Arabidopsis31 and isresponsible for the methylation of SeMet to precursors ofvolatile Se (e.g., dimethylselenide). However, as yet, noenzymes with MMT function have been reported for algae.Furthermore, there is a possibility that the SeMet fractiondetected by XAS may also have included proteinaceous SeMetthat 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 wasconfirmed using EXAFS (see details in Figure S2). To ourknowledge, there are no published studies showing thepresence 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 thispossibility), the question arises as to the mechanism of itsformation. Pilon-Smits and colleagues have shown that SeCys-lyase produces Se0 and alanine from SeCys in transgenicArabidopsis plants.32,33 One possible explanation, therefore, forthe presence of Se0 is that the Chlorella strains could haveenzymes with SeCys-lyase-type activity for the breakdown ofSeCys to Se0. Other studies using Scenedesmus obliquus,Chlorella kessleri, and Synechococcus leopoliensis have proposedthat the formation of Se0 may be necessary for the synthesis ofselenocyanide; 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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accumulation of selenocystine in algal cells, e.g., Chlorella sp.17

and Chlorella sorokiniana.36

Nutrient Interactions. In experiment 2, Scenedesmusobliquus was supplied with 40 μM Se (3160 μg Se/L) in theabsence (i.e., deionized water) and presence of nutrients (1/5Hoagland’s solution). Volatilization in deionized water reacheda maximum in 24−48 h and then declined (Figure 3A). Therapid increase in volatilization in the absence of added nutrientswas likely due to an enhanced uptake of selenate; theconcentration of Se in the algal biomass was 55% greaterwhen Se was supplied in the absence compared to the presenceof nutrients (Supporting Information Table S4). Volatilizationin the presence of 1/5 Hoagland’s solution began slowly butincreased progressively with time over the 4-day experimentalperiod (Figure 3A).We further explored the effect of nutrients in experiment 3

by comparing volatilization in the presence of high levels of Nand P (Figure 3B). Volatilization changed nearly linearly withtime for all three nutrient treatments; the average rate ofvolatilization was little affected by doubling the amount of N,but was significantly increased by doubling the amount ofphosphate (Figure 3B). Biomass Se in the absence of nutrientswas more or less equally distributed among three Secompounds, i.e., selenate Se (37%), SeMet (36%), and Se0

(27%) (experiment 2, Supporting Information Table S4). Inthe presence of nutrients most of the Se was present in theform 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 potentialfate of algal-borne Se flowing from the algal treatment unit tothe wetland unit. In experiment 4, we established a baseline forthe partitioning and speciation of selenate-supplied Se in awetland mesocosm ecosystem before the introduction of Se-bearing algae, and in experiment 5, we examined the potentialfate of algal-borne Se within a wetland ecosystem by addingalgae to microcosms containing either fragments of dead

cattails (to simulate fallen litter) or sand and peat moss (tosimulate 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 peatmoss substrate (27%) (Supporting Information Table S5). Asimilar partitioning of total Se was observed in the mesocosmswith fallen litter layers, except that 4.4% of the Se was presentin the fallen litter layer itself (Table S5). The speciation of Se inrhizomes and shoots was similar in mesocosms with or withouta fallen litter layer. Most of the Se in the plant biomass waspresent as selenate, which ranged from 81 to 88% (Table S5).There was no Se0 and the only other form of Se found in theplant biomass was SeMet. However, in the sand/peat mosssubstrate, most if not all, was in the form of Se0 (Table S5).The Se0 in the sediment was most likely formed through themicrobially mediated anaerobic dissimilatory reduction ofselenate.37 In the fallen litter layer, the Se was fairly evenlydistributed between SeMet and Se0, indicating that bothmicrobial assimilatory reduction through the sulfate assim-ilation pathway, and anaerobic dissimilatory reduction, wereoccurring within the fallen litter.38,39 A similar distributionbetween SeMet and Se0 was obtained in a field wetland studyby Lin and Terry,5 who found that, in vegetated andunvegetated 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 apeak in 24 h, and then declined, whereas in the microcosmscontaining cattail litter, volatilization increased more or lessprogressively with time (Figure 4A). At the end of the 7-day

Figure 2. Se K near-edge X-ray absorption spectra (XANES) of Sestandards (upper section of the figure) and of biomass samples of 6algae from selenate- (broken lines) and selenite-supplied algal cultures(solid lines) (experiment 1; bottom section of the figure). The Sestandards included SeO4

2−, SeO32−, SeMet, CysSeSeCys, and Se0, i.e.,

selenate, selenite, selenomethionine, selenocystine, and elementalselenium, respectively (the dotted line is used to clearly illustrate theselenite spectrum). The overall analysis based on these data is shownSupporting Information Table S3.

Figure 3. Changes with time in volatile Se produced per microcosmfor Scenedesmus obliquus. (A) Alga treated with 3158 μg Se/L indeionized water (DI) or 1/5 Hoagland’s nutrient solution (1/5 H) asdescribed in Materials and Methods, experiment 1. (B) Alga treatedwith 2500 μg Se/L in 1/5 Hoagland’s nutrient solution (control), and1/5 Hoagland’s nutrient solution with double the concentration ofnitrogen (2XN) or phosphorus (2XP) as described in Materials andMethods, experiment 2.

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experimental period, volatilization from the sand/peat mossmicrocosm had virtually ceased and the SeMet content haddecreased to zero; in the cattail fragments microcosm on theother 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 wasdependent on the level of the volatile Se precursor, SeMet. It isnot clear whether the volatilization was carried out by microbes,by algae, or by a combination of both. The fact that the total (7-day) volatile Se produced by the microcosm with cattailfragments was 2.7-fold greater than volatilization from thesand/peat moss microcosm, together with the fact that 75% ofthe Se in the sand/peat moss fraction was in the form ofselenate compared to only 10 and 18% in the cattail fragmentsand suspending medium, respectively, of the cattail microcosm(Figure 4 table), suggest that the presence of a cattail fallenlitter in a wetland ecosystem is likely to greatly facilitate theconversion of selenate to SeMet and volatile Se.Use of Algal−Cattail Wetland Systems for Se

Removal. On the basis of the microcosm studies of experiment1, strain Cv exhibited highly efficient characteristics withrespect to volatilizing and removing Se from the water column.Because these measurements were made at very high Seconcentrations (1580 μg Se/L) and in the absence of nutrients,we carried out experiment 6 to determine the extent of Seremoval and volatilization from water simulating Se andnutrient concentrations likely to be encountered by Cvreceiving water from rivers (e.g., the New River) supplyingthe Salton Sea. The results of experiment 6 show that the Seconcentrations of the Cv algal suspensions decreased from an

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. SinceSe levels of the algal suspension had decreased by 39.4% after48 h, it is reasonable to assume that the remaining 60.6% waslost through volatilizationa proportion similar to thatobtained for Cv at high Se concentrations (experiment 1).Furthermore, our microcosm studies with Scenedesmus obliquusshow that algae are able to volatilize Se in the presence of largeamounts of N and P and that it is important to optimize algalpopulation 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 Se-bearing algae enter the cattail treatment unit. This view is basedon the following observations. Experiment 5 shows that Sevolatilization increased in the microcosms containing Se-bearing algae in the presence of cattail litter, or sand/peatmoss. A second source of volatile Se will arise from the cattailsthemselves: 47−56% of the Se added to mesocosms wasaccumulated in the biomass of rhizomes and shoots(Supporting Information Table S5), both of which are capableof Se volatilization.13 Thus, our results suggest that much of theincoming Se from the river will be volatilized by algae in thealgal treatment unit, by microbial decomposition of algae in thecattail unit, and by volatilization of Se accumulated in cattailtissues.We envision that the cleanup of river water contaminated

with Se and nutrients from farm runoff could be achieved usinga design similar to that shown in Supporting InformationFigure S4. However, before implementing this design, it isessential that its benefits and limitations are thoroughly tested

Figure 4. (A) Changes with time in volatile Se produced per microcosm for Scenedesmus obliquus cultures (see Materials and Methods, experiment5) to which was added sand/peat moss or cattail fragments. (B) Table showing the amount of Se volatilized/microcosm and the percent speciationof Se among three forms of Se, selenate, SeMet, and Se0. The cattail fragments + algal mixture was separated into two components, i.e., the cattailfragments + algae and the suspending medium + algae. The speciation of Se for each of these two components was determined separately. In the caseof the sand/peat moss−algal mixture, the whole mixture was subsampled for speciation.

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in a pilot wetland under field conditions. The pilot wetlandshould be used to determine (1) the efficiency of Se removalfrom the water flowing through it, (2) the extent to which Se isremoved through biological volatilization versus the extent towhich it is accumulated in potentially ecotoxic forms, and (3)the effects of the different chemical forms of Se on biota withinthe 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 arelikely to be SeMet and Se0. Selenomethionine is of particularconcern because it can be easily metabolized into proteins andbecause laboratory studies indicate that it can be toxic to fishand birds.10,40 On the other hand, SeMet has been shown to bevolatilized much more rapidly by both plants41 and microbes13

than are Se oxyanions. Elemental Se is immobilized in thesediments in relatively nonbioavailable forms and is consideredto be of little toxicological significance for most organisms.42,43

The oxidation and/or potential remobilization of Se0 ispossible, especially if the wetland were to dry out, but therate of oxidation has been shown to be relatively slow.7,44

Provided the proposed design can be validated under fieldconditions, we believe that, in addition to providing clean waterfor the SCH, it might have other applications, such as removalof Se from agricultural drainage water. This would allowdrainage water to be reutilized for additional crop production,thereby making more efficient use of the rapidly dwindlingsupplies of water in California and other Western states.

■ ASSOCIATED CONTENT*S Supporting InformationTables S1 and S2 showing volatilization, biomass accumulation,and absorption rates of Se for different algal strains (experiment1); Table S3 showing percentage of the Se species observedfrom algae (experiment 1); Table S4 showing effects of nutrientsupply on rates of volatilization, biomass accumulation, andabsorption of Se by Scenedesmus obliquus (experiments 2 and3); Table S5 showing total Se and percent distribution of Seamong different Se forms in the cattail mesocosm components(experiment 4); Figure S1 illustrating the sequence similaritywith the most closely related algal species; Figure S2 showingthe Se K-edge EXAFS oscillations and phase-corrected Fouriertransform of selenite-supplied algae (experiment 1); Figure S3illustrating the relationships between Chla and Se removal/volatilization by Scenedesmus obliquus; and Figure S4 with aconceptual design for the cleanup of river water. This materialis available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +1 (510) 642 3510. E-mail: nterry@berkeley.edu.Present Addresses†Department of Geological Sciences, University of Saskatch-ewan, Saskatoon, SK, S7N 5E2, Canada.‡Department of Biological Sciences and EnvironmentalSciences Program, Southern Illinois University, Edwardsville,Illinois 62026, United States.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the greenhouse staff at the University of CaliforniaBerkeley and the Stanford Synchrotron Radiation Lightsource

(SSRL) for beam time granted to N.T. (SSRL is a Directorateof SLAC National Accelerator Laboratory and an Office ofScience User Facility operated for the US Department ofEnergy Office of Science by Stanford University). This researchwas supported by funds from the State of CaliforniaProposition 84, administered by DFG and DWR under theSpecies Conservation Habitat Project.

■ REFERENCES(1) DWR California Department of Water Resources. Salton SeaSpecies Conservation Habitat project; Sacramento, CA, 2010; p 1018.(2) The California Resources Agency (CRA). Final Report onSelenium at the Salton Sea and Summary of Data Gaps; Sacramento,CA, 2005.(3) Hansen, D.; Duda, P. J.; Zayed, A.; Terry, N. Selenium removalby constructed wetlands: role of biological volatilization. Environ. Sci.Technol. 1998, 32 (3), 591−597.(4) Gao, S.; Tanji, K. K.; Lin, Z. Q.; Terry, N.; Peters, D. W.Selenium removal and mass balance in a constructed flow-throughwetland system. J. Environ. Qual. 2003, 32 (4), 1557−1570.(5) Lin, Z.-Q.; Terry, N. Selenium removal by constructed wetlands:quantitative importance of biological volatilization in the treatment ofselenium-laden agricultural drainage water. Environ. Sci. Technol. 2003,37 (3), 606−615.(6) Huang, J. C.; Passeport, E.; Terry, N. Development of aconstructed wetland water treatment system for selenium removal: useof mesocosms to evaluate design parameters. Environ. Sci. Technol.2012, 46 (21), 12021−12029.(7) Masscheleyn, P. H.; Patrick, W. H., Jr. Biogeochemical processesaffecting selenium cycling in wetlands. Environ. Toxicol. Chem. 1993,12 (12), 2235−2243.(8) Saiki, M. K.; Lowe, T. E. Selenium in aquatic organisms fromsubsurface agricultural drainage water, San Joaquin Valley, California.Arch. Environ. Contam. Toxicol. 1987, 16 (6), 657−670.(9) Lemly, A. D. A Procedure for Setting Environmentally Safe TotalMaximum Daily Loads (TMDLs) for Selenium. Ecotoxicol. Environ.Safety 2002, 52, 123−127.(10) Hamilton, S. J. Review of selenium toxicity in the aquatic foodchain. Sci. Total Environ. 2004, 326, 1−31.(11) Ohlendorf, H. M.; Heinz, G. H. 2011. Selenium in birds. InEnvironmental contaminants in biota: interpreting tissue concentrations;Beyer, W. N., Meador, J. P., Eds.; CRC Press: FL, 2011; p 669−701.(12) Dungan, R. S.; Frankenberger, W. T. Microbial transformationsof selenium and the bioremediation of seleniferous environments.Bioremed. J. 1999, 3 (3), 171−188.(13) Terry, N.; Zayed, A. M.; de Souza, M. P.; Tarun, A. S. Seleniumin higher plants. Ann. Rev. Plant Physiol. Plant Molecular Biol. 2000,51, 401−432.(14) Lin, Z.-Q.; Cervinka, V.; Pickering, I. J.; Zayed, A.; Terry, N.Managing selenium-contaminated agricultural drainage water by theIntegrated on-Farm Drainage Management system: role of seleniumvolatilization. Water Res. 2002, 36 (12), 3150−3160.(15) Oyamada, N.; Takahashi, G.; Ishizaki, M. Methylation ofinorganic selenium compounds by freshwater green algae Ankistro-desmus sp., Chlorella vulgaris and Selenastrum sp., Japanese. J. Toxicol.Environ. Health 1991, 37 (2), 83−88.(16) Fan, T. W. -M.; Lane, A. N.; Higashi, R. M. Seleniumbiotransformations by a euryhaline microalga isolated from a salineevaporation pond. Environ. Sci. Technol. 1997, 31 (2), 569−576.(17) Neumann, P. M.; de Souza, M. P.; Pickering, I. J.; Terry, N.Rapid microalgal metabolism of selenate to volatile dimethylselenide.Plant Cell Environ. 2003, 26 (6), 897−905.(18) Hoagland, D. R.; Arnon, D. I. The Water-Culture Method forGrowing Plants without Soil. California Agricultural Experimental StationCircular; College of Agriculture, University of California: Berkeley, CA,1950; Vol. 347, pp 1−32.

Environmental Science & Technology Article

dx.doi.org/10.1021/es4015629 | Environ. Sci. Technol. 2013, 47, 10518−1052510524

(19) U.S. EPA. Determination of trace Elements in Waters and Wastesby Inductively Coupled Plasma-Mass Spectrometry (EPA 200.8), rev. 5.4;US EPA: Cincinnati, OH, 1994.(20) U.S. EPA. Acid digestion of sediments, sludges, and soils.Method 3050B. http://www.epa.gov/wastes/hazard/testmethods/sw846/online/3_series.htm (accessed August 26, 2013).(21) Yang, S. I.; Lawrence, J. R.; Pickering, I. J. Biotransformation ofselenium and arsenic in multi-species biofilm. Environ. Chem. 2011, 8(6), 543−551.(22) George, G. N.; Pickering, I. J. EXAFSPAK: A Suite of computerprograms for analysis of X-ray absorption spectra available at http://ssrl.slac.stanford.edu/exafspak.html (accessed August 26, 2013).(23) Zabinsky, S. I.; Rehr, J. J.; Ankudinov, A.; Albers, R. C.; Eller, M.J. Multiple-scattering calculations of X-ray-absorption spectra. Phys.Rev. B. 1995, 52, 2995.(24) Luhrling, M. Phenotypic plasticity in the green algaeDesmodesmus and Scenedesmus with special reference to induction ofdefensive morhpology. Ann. Limnol.−Int. J. Lim. 2003, 39 (02), 85−101.(25) TetraTech, Inc. Performance evaluation of the New Riverdemonstration wetlands. Citizen’s congressional task office on the NewRiver; Brawley, CA, 2006; p37.(26) Pickering, I. J.; Brown, G. E., Jr.; Tokunaga, T. K. Quantitativespeciation of selenium in soils using X-ray absorption spectroscopy.Environ. Sci. Technol. 1995, 29 (9), 2456−2459.(27) Lee, A.; Lin, Z.-Q.; Pickering, I. J.; Terry, N. X-ray absorptionspectroscopy study shows that the rapid selenium volatilizer,pickleweed (Salicornia bigelovii Torr.) reduces selenate to organicforms without the aid of microbes. Planta 2001, 213 (6), 977−980.(28) Sors, T. G.; Ellis, D. R.; Na, G. N.; Lahner, B.; Lee, S.; Leustek,T.; Pickering, I. J.; Salt, D. E. Analysis of sulfur and seleniumassimilation in Astragalus plants with varying capacities to accumulateselenium. Plant J. 2005, 42 (6), 785−797.(29) Takahashi, H.; Kopriva, S.; Giordano, M.; Saito, K.; Hell, R.Sulfur assimilation in photosynthetic organisms: molecular functionsand regulations of transporters and assimilatory enzymes. Annu. Rev.Plant Biol. 2011, 62, 157−184.(30) Pilon-Smits, E. A. H.; Hwang, S.; Lytle, C. M.; Zhu, Y.; Tai, J.C.; Bravo, R. C.; Chen, Y.; Leustek, T.; Terry, N. Overexpression ofATP sulfurylase in Indian mustard leads to increased selenate uptake,reduction and tolerance. Plant Physiol. 1999, 119 (1), 123−132.(31) Tagmount, A.; Berken, A.; Terry, N. An essential role of S-adenosyl-L-methionine S-methyltransferase in selenium volatilizationby plants. Methylation of selenomethionine to selenium-methyl-L-selenium-methionine, the precursor of volatile selenium. Plant Physiol.2002, 130 (2), 847−856.(32) Pilon-Smits, E. A. H.; Garifullina, G.; Abdel-Ghany, S.; Kato, S.;Mihara, H.; Hale, K.; Burkhead, J.; Esaki, N.; Kurihara, T.; Pilon, M.Characterization of a NifS-like chloroplast protein from Arabidopsis:implications for its role in sulfur and selenium metabolism. PlantPhysiol. 2002, 130 (3), 1309−1318.(33) Pilon, M.; Owen, J. D.; Garifullina, G. F.; Kurihara, T.; Mihara,H.; Esaki, N.; Pilon-Smits, E. A. H. Enhanced selenium tolerance andaccumulation in transgenic Arabidopsis expressing a mouse selenocys-teine lyase. Plant Physiol. 2003, 131 (3), 1250−1257.(34) Simmons, D. B. D.; Wallschlager, D. Release of reducedinorganic selenium species into waters by the green fresh water algaeChlorella vulgaris. Environ. Sci. Technol. 2011, 45, 2165−2171.(35) LeBlanc, K. L.; Smith, M. S.; Wallschlager, D. Production andrelease of selenocyanate by different green freshwater algae inenvironmental and laboratory samples. Environ. Sci. Technol. 2012,46, 5867−5875.(36) Gomez-Jacinto, V.; García-Barrera, T.; Garbayo-Nores, I.;Vilchez-Lobato, C.; Gomez-Ariza, J. Metal-metabolomics of microalgaChlorella sorokiniana growing in selenium- and iodine-enriched media.Chem. Pap. 2012, 66 (9), 821−828.(37) Oremland, R. S.; Hollibaugh, J. T.; Maest, A. S.; Presser, T. S.;Miller, L. G.; Culbertson, C. W. Selenate reduction to elementalselenium by anaerobic bacteria in sediments and culture: Bio-

geochemical significance of a novel sulfate-independent respiration.Appl. Environ. Microbiol. 1989, 55 (9), 2333−2343.(38) Dungan, R. S.; Frankenberger, W. T. Microbial transformationsof selenium and the bioremediation of seleniferous environments.Bioremed. J. 1999, 3 (3), 171−188.(39) Siddique, T.; Okeke, B. C.; Zhang, Y. Q.; Arshad, M.; Hans, S.K.; Frankenberger, W. T. Bacterial diversity in selenium reduction ofagricultural drainage water amended with rice straw. J. Environ. Qual.2005, 34 (1), 217−226.(40) Fan, T. W.; Teh, S. J.; Hinton, D. E.; Higashi, R. M. Seleniumbiotransformations into proteinaceous forms by foodweb organisms ofselenium-laden drainage waters in California. Aquat Toxicol. 2002, 57(1−2), 65−84.(41) Zayed, A. M.; Lytle, C. M.; Terry, N. Accumulation andvolatilization of different chemical species of selenium by plants. Planta1998, 206, 284−292.(42) Combs, G. F.; Garbisu, C.; Yee, B. C.; Yee, A.; Carlson, D. E.;Smith, N. R.; Magyarosy, A. C.; Leighton, T.; Buchanan, B. B.Bioavailability of selenium accumulated by selenite-reducing bacteria.Biol Trace Elem. Res. 1996, 52, 209−225.(43) Schlekat, C. E.; Dowdle, P. R.; Lee, B. G.; Luoma, S. N.;Oremland, R. S. Bioavailability of particle-associated Se to the bivalvePotamocorbula amurensis. Environ. Sci. Technol. 2000, 34, 4504−4510.(44) Hibbs, B.; Lee, M.; Walker, J. Selenium remobilization due todestruction of wetlands in the Irvine subbasin, Orange County.California Environ. Geosci. 2000, 7 (4), 211.

Environmental Science & Technology Article

dx.doi.org/10.1021/es4015629 | Environ. Sci. Technol. 2013, 47, 10518−1052510525