Selenium Uptake and Methylation by the Microalga Chlamydomonas

Article pubs.acs.org/est

Selenium Uptake and Methylation by the Microalga Chlamydomonas reinhardtii Bas Vriens,†,‡ Renata Behra,†,‡ Andreas Voegelin,† Anze Zupanic,† and Lenny H. E. Winkel*,†,‡ †

Eawag, Swiss Federal Institute of Aquatic Science and Technology, CH-8600 Dübendorf, Switzerland Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, CH-8092 Zurich, Switzerland

‡

S Supporting Information *

ABSTRACT: Biogenic selenium (Se) emissions play a major role in the biogeochemical cycle of this essential micronutrient. Microalgae may be responsible for a large portion of these emissions via production of methylated Se compounds that volatilize into the atmosphere. However, the biochemical mechanisms underlying Se methylation in microalgae are poorly understood. Here, we study Se methylation by Chlamydomonas reinhardtii, a model freshwater alga, as a function of uptake and intracellular Se concentrations and present a biochemical model that quantitatively describes Se uptake and methylation. Both selenite and selenate, two major inorganic forms of Se, are readily internalized by C. reinhardtii, but selenite is accumulated around ten times more efficiently than selenate due to different membrane transporters. With either selenite or selenate as substrates, Se methylation was highly efficient (up to 89% of intracellular Se) and directly coupled to intracellular Se levels (R2 > 0.92) over an intracellular concentration range exceeding an order of magnitude. At intracellular concentrations exceeding 10 mM, intracellular zerovalent Se was formed. The relationship between uptake, intracellular accumulation, and methylation was used by the biochemical model to successfully predict measured concentrations of methylated Se in natural waters. Therefore, biological Se methylation by microalgae could significantly contribute to environmental Se cycling.

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Selenium has similar chemical properties to sulfur (S),1,10 and biogenic emissions of methylated S (e.g., dimethyl sulfide (DMS), up to 28.1 Tg per year)11 are very important to the global S cycle. Even though environmental Se concentrations are typically several orders of magnitude lower than S concentrations, field studies have indicated that the ratio of Se/S is up to 3 orders of magnitude higher4−6 for biogenic methylated compounds than for inorganic (nonvolatile) compounds, indicating an enrichment of Se over S in the gaseous phase of natural systems. However, it remains unclear why Se is so efficiently methylated and volatilized. Microalgae, small in size but large in number, probably exert significant control over the biogeochemical Se cycle: they actively take up various forms of aqueous Se (e.g., Emiliania,12 Scenedesmus,13 and Chlorella14,15 genera, reviewed by Gojkovic et al.16) and may perform Se methylation, as indicated by field observations in natural and constructed wetlands.5,17 Furthermore, the potential importance of microalgae to Se methylation is corroborated by positive correlations between algal biomass

INTRODUCTION

Selenium (Se) is an essential trace element for many (micro)organisms but has a relatively narrow range between beneficial and toxic concentrations.1 Health problems related to Se are due to excessive or inadequate uptake of Se and are exacerbated by an uneven Se distribution in the environment, e.g., surface water and soil.2,3 However, the processes that govern the distribution of Se in surface environments and its biogeochemical cycle are still poorly quantified. Biomethylation is a process that may play an important role in Se cycling in terrestrial and marine environments. Biomethylation of Se produces methylated, volatile compounds such as dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe), which can be emitted to the atmosphere.4 Field studies have reported substantial concentrations and fluxes of methylated Se compounds in both terrestrial4,5 and marine environments.6−8 In addition, the importance of biogenic Se emissions for the global Se cycle is implicated by atmospheric budget estimations: on a global scale, 0.4−9 × 109 g·yr−1 of biogenic Se is emitted, which may constitute up to 50% of the total atmospheric Se budget.9 Therefore, atmospheric Se deposition could be an important source of Se to surface environments, including agricultural soils.4 © XXXX American Chemical Society

Received: August 27, 2015 Revised: November 20, 2015 Accepted: December 21, 2015

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DOI: 10.1021/acs.est.5b04169 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

ducted in triplicate by exposing algae in the exponential growth phase (photosynthetic yield >0.55, cell size range 5−7.5 μm, and cell density ∼1 × 106 cells·mL−1) to either Se[VI] (Na2SeO4) or Se[IV] (Na2SeO3) that were added to the exposure media as sodium salts obtained from Sigma-Aldrich, Buchs, Switzerland, prior to inoculation. Uptake experiments with variable Se concentrations in the exposure media were done by exposing algae to 50 mL of Seenriched medium (0−100 μM Se) in 250 mL Erlenmeyer flasks for 24 h. On the basis of previous studies on Se uptake mechanisms in microalgae,12,22,47 we studied the competitive effects of SO 4 on Se[VI] uptake and of PO 4 and monocarboxylate on Se[IV] uptake. Therefore, algae were exposed to 50 mL of media enriched with 75 μM Se[VI] and increasing levels of sulfate (Na2SO4), or to media enriched with Se[IV] and increasing concentrations of either phosphate (a pH 7.5 mixture of Na2HPO4 and NaH2PO4) or lactate (as NaD,L-lactate) (all from Sigma-Aldrich, Buchs, Switzerland) for 12 h in 250 mL Erlenmeyer flasks. All uptake experiments were conducted under the controlled culturing conditions mentioned above, and control samples were obtained by exposing algae to the control growth medium (91%, respectively. Total Se and S concentrations in the digests were analyzed using ICP−MS (Thermo Element 2, Reinach, Switzerland); the analytical methods for quantifying Se and Se are described elsewhere.29 The intracellular Se speciation of four algal samples with intracellular Se concentrations ranging between 2 and 28 mM was analyzed using X-ray absorption spectroscopy at the Se Kedge (12658 eV) at the SUL-X beamline at Angströmquelle Karlsruhe (ANKA, Karlsruhe, Germany). The X-ray absorption near-edge structure (XANES) spectra of selected samples were compared to the spectra of five reference materials (gray elemental Se, Se disulfide, Se-L-methionine, Se[IV], and Se[VI]) using linear combination fitting (LCF). In addition, an extended X-ray absorption fine structure (EXAFS) spectrum of algae exposed to 75 μM Se[IV] was compared to EXAFS reference spectra of Se disulfide and gray and red elemental Se (from Sarret et al.34). The details of the XAS measurements, sample preparation and data analysis are given in the Supporting Information. Biochemical Modeling. All the experimentally determined concentrations of intra- and extracellular Se species were described with a mathematical two-compartment model in the PottersWheel toolbox for Matlab.31 The model contained terms for the enzymatic uptake of Se[VI] and Se[IV], for the reduction of intracellular Se, for the production of methylated Se, for the diffusion of methylated Se out of the cell, and for the degradation of extracellular methylated Se. A schematic of the model is given in Figure S6; the rationale behind the construction of the model is discussed in the Supporting Information. All fitting was performed under the assumptions that the algal cultures did not grow during the experiments, that Se[IV] and Se[VI] were internalized via two individual membrane transporters, and that the initial concentration of intracellular Se was zero, among others (details in the Supporting Information). The fitting was performed 100 times using iterations between the trust region algorithm and the genetic algorithm with the sum-of-squared-residuals objective function. The confidence intervals and identifiability of the model parameters were determined using the profilelikelihood method32 with relatively uninformed priors (Table S3). The model fit was assessed with the chi-squared test and the corrected Akaike Information Criterion. After calibrating the model parameters to the experimental data, we fixed the

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RESULTS AND DISCUSSION Selenium Uptake as a Function of Concentration. Exposure of C. reinhardtii to increasing concentrations of Se[IV] and Se[VI] for 24 h resulted in increasing intracellular Se concentrations, decreasing intracellular S concentrations, and a decrease in algal growth (Figure 1). The observed uptake

Figure 1. Uptake of Se by C. reinhardtii as a function of concentration. Intracellular Se and S concentrations (mmole per liter of cell volume, left axes) in C. reinhardtii after 24 h of exposure to various concentrations of Se[VI] (A) and Se[IV] (B). The growth factor (gray columns, right axes) of C. reinhardtii under variable Se exposure concentrations is defined as the ratio between the culture cell count after 24 h and the initial culture cell count (1 × 106 cells·mL−1). Error bars indicate standard deviation from triplicate experiments.

of Se[IV] was over 10-fold higher than the uptake of Se[VI] at similar exposure concentrations, with intracellular Se concentrations up to 55 mM (11 fmoles·cell−1) and 4 mM (900 amoles·cell−1) when C. reinhardtii was exposed for 24 h to 100 μM Se[IV] or Se[VI], respectively (Figure 1). Exposure to increasing Se[IV] or Se[VI] concentrations decreased the intracellular S content from 450 to 250 mM and from 500 to 200 mM within 24 h, respectively (Figure 1). The average cellular volumes of the cultures after 24 h of exposure ranged from 128 fL in the control cells to up to 215 fL in the cultures exposed to 100 μM Se[VI] or Se[IV] and increased with increasing Se exposure concentrations (see Figure S8). The algal biomass accrual (see the growth factor in Figure 1, defined as the ratio between the cell count after 24 h and the initial cell count) decreased with increasing Se exposure concentrations: growth factors decreased from 2.5 to 1.3 and from 2.3 to 1.5 between the control and 100 μM exposure concentrations of Se[VI] and Se[IV], respectively. Prolonged exposure (>24 h) of C. reinhardtii to Se concentrations exceeding 75 μM induced C


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Environmental Science & Technology toxic effects such as cell bloating, formation of starch-granules, and loss of chlorophyll a (Supporting Information). The observed intracellular Se concentrations are in the same order of magnitude as those previously reported for C. reinhardtii exposed to Se: up to 700 amoles Se·cell−1 after 96 h of exposure to 38.1 μM Se[IV]24 and up to 400 amoles Se· cell−1 after 96 h of exposure to 2.5 μM Se[VI].22 Similar to our findings, a higher accumulation of Se[IV] than Se[VI] has been observed for the marine microalgae Cricosphaera elongata,33 the green algae Scenedesmus quadricauda,13 and the bacterium Ralstonia metallidurans.34 The observed decrease in intracellular S content with increasing Se exposure may be partially due to competition of Se[VI] with SO4 that is internalized from the exposure media via similar transporters (see below) or to the replacement of S by Se in biomolecules, as was indicated in microalgae13 and higher plants.35 Because the decreases in intracellular S were larger than increases in intracellular Se, additional processes (e.g., stress-induced degradation of S biomolecules and active S efflux transport36) may have also contributed to the decrease in intracellular S. The progressive growth inhibition of C. reinhardtii with increasing Se exposure concentrations is related to a delayed cell division (hence, the increased average cell volumes in Figure S8) and agrees with previously reported EC50 values (between 0.4−4.5 μM for Se[VI]22,23 and 65−94 μM for Se[IV]24,25), particularly because Se exposure concentrations used in this study were above these values.37 Thus, despite the effects of Se[IV] and Se[VI] on the growth and intracellular S content of C. reinhardtii, both Se species are readily internalized by C. reinhardtii. Selenium Uptake in the Presence of Competing Species. Average Se uptake rates between 0−24 h of exposure were calculated using the intracellular Se concentrations given in Figure 1 and assuming that intracellular Se concentrations were zero at the start of exposure. Accordingly, the uptake of Se[VI] appears to follow Michaelis−Menten kinetics, indicating that a single enzymatic transport mechanism governs Se[VI] uptake. Fitting the Se[VI] uptake rates to the Michaelis− Menten equation (R2 = 0.98, fit not shown) showed a Vmax of 73 (amoles·cell−1·h−1) and a Km of 89 μM. Furthermore, the competition experiments show that Se[VI] uptake by C. reinhardtii was suppressed by increasing concentrations of sulfate in the growth medium: a doubling of the SO 4 concentration in the exposure media led to a 30% decrease in Se[VI] uptake, and a 5-fold increase in SO4 concentration led to a 55% decrease in Se[VI] uptake (Figure 2). In contrast, the uptake of Se[IV] was not affected by SO4 concentrations (Figure 2) and followed sigmoidal kinetics (Figure 1). Instead, Se[IV] uptake was reduced by increasing PO4 concentrations (15% and 50% decrease in Se[IV] uptake with a doubling and 10-fold increase in PO4 concentration in the exposure media, respectively; Figure 2). Furthermore, Se[IV] uptake was enhanced by 50% after the addition of 150 μM lactate to the growth medium but only 5% after the addition of 750 μM lactate (Figure 2). To our knowledge, no specific Se influx transporter has been identified in microalgae; the internalization of Se oxyanions probably occurs via shared proteins that do not discriminate between Se and structurally similar anions.38,39 The calculated Km value for Se[VI] uptake in this study (89 μM) is comparable to reported Km values for Se[VI] uptake (12 μM)42 and for SO4 reduction (180 μM)43 via ATP-sulfurylase,44 an enzyme involved in the reduction of both SO4 and Se[VI], which is a

Figure 2. Competition of anions with Se uptake by C. reinhardtii. Intracellular Se concentrations in C. reinhardtii after 12 h of exposure to 75 μM Se[VI] (green columns) and variable levels of SO4 (4:1 or 10:1 molar excess to Se[VI]) or to 75 μM Se[IV] (orange columns) and variable levels of PO4 (2:1 or 6.7:1 molar excess to Se[IV]) or lactate (2:1 and 10:1 molar excess to Se[IV]). Control experiments (no additions; the original culture medium contained 150 μM SO4, 50 μM PO4, and 0 μM lactate) are indicated by an asterisk. Error bars indicate standard deviation from triplicate experiments.

rate-limiting step in the assimilation of Se[VI] in higher plants.54,58 The comparable Km values and the observed competition between Se[VI] and SO4 indicate that Se[VI] is internalized via the SO4 pathway in C. reinhardtii, which has been previously reported for other algae and higher plants.22,38,40,41 The observed sigmoidal uptake kinetics for Se[IV] and the competition effects of both PO4 and lactate indicate that multiple pathways may be involved in the uptake of Se[IV]. The reduction of Se[IV] uptake with increasing PO 4 concentrations suggests a direct competition between Se[IV] and PO4 . Indeed, previous studies have identified PO4 transporters to be involved in Se[IV] uptake in green algae,39 marine algae,12 yeasts,45 and higher plants.38 The observed effects of an increasing lactate concentration on Se[IV] uptake are somewhat inconclusive. We suggest that upon exposure to lactate (containing a monocarboxylate group), the mixotrophic C. reinhardtii switched from autotrophic (C. reinhardtii was initially cultured autotrophically) to heterotrophic growth, which activated monocarboxylate uptake mechanisms, as previously reported for Chlamydomonas humicola.46 Because a H+-monocarboxylate transporter may be involved in Se[IV] uptake, and because this transporter poorly discriminates between Se[IV] and monocarboxylate,47 the consequence of lactate addition could be, paradoxically, an initial increase in Se[IV] uptake. Although more knowledge on the Se membrane transporters for these different Se species is required (e.g., Si and SO3 transporters48,49), our results illustrate that distinct mechanisms underlie the uptake of Se[VI] and Se[IV] by C. reinhardtii. Selenium Uptake as a Function of Time. The timeresolved uptake experiments (Figure 3C,D) illustrate that the uptake of Se[IV] by C. reinhardtii is more rapid than that of Se[VI], with maximum uptake rates of 1000 amoles·cell−1·h−1 for Se[IV] and 90 amoles·cell−1·h−1 for Se[VI], resulting in a higher accumulation of Se from Se[IV] than from Se[VI] (up to 30 mM versus 3 mM of Se within 15 h, respectively). This time-resolved Se accumulation quantitatively agrees with Se uptake observed in the 24 h uptake experiments discussed previously (Figure 1 and Figure 2). The rate of Se[VI] uptake D


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Figure 3. Uptake and methylation of Se by C. reinhardtii as a function of time. Time-resolved concentrations of Se[VI] and Se[IV] in the exposure media (μM, A and B), intracellular Se (mmole per liter cell volume, C and D) and total produced methylated Se (nM, E and F) in C. reinhardtii cultures exposed to two concentrations (30 and 75 μM Se) of Se[VI] and Se[IV], respectively. All parameters were analyzed synchronously. The dashed lines in each frame illustrate the fit of the biochemical model to the corresponding experiment. Error bars indicate standard deviation from triplicate experiments.

bioconcentration factors (defined as the molar ratio of internal Se concentrations over Se concentrations in the exposure media) ranged from 38−73 for Se[VI] to 403−429 for Se[IV]. Intracellular Se Speciation. XANES and EXAFS spectroscopy was conducted to analyze intracellular Se speciation in C. reinhardtii cells exposed to Se[VI] and Se[IV]. For C. reinhardtii exposed to 30 or 75 μM Se[VI] for 15 h, the XANES data indicated that intracellular Se speciation was dominated by organo-Se compounds (86−92%, using Se-L-methionine as a reference for organo-Se; Supporting Information) and smaller fractions of Se[VI] (Figure 4 and Table S2). Cells that were exposed to 30 μM Se[IV] for 15 h also contained mainly organo-Se species (92%) together with a small fraction of Se[0]. However, in cells exposed to 75 μM Se[IV], only a minor fraction of organo-Se remained. Evaluation of EXAFS spectra revealed that in cells exposed to 75 μM Se[IV], 83% of total intracellular Se was present as Se[0], mainly in the form of red Se[0] consisting of Se8 rings (Supporting Information and Figure S5). In all samples, the organo-Se fractions derived from

was highest immediately after exposure, while Se[IV] uptake was initially relatively slow (lag phase) and reached maximum rates after approximately 6 h of exposure. It should be noted that Se[IV] internalization also took place during the lag phase, although only in comparatively small amounts (i.e., up to 2 mM and 7 mM intracellular Se after 5 h exposure to 30 μM and 75 μM Se[IV], respectively). The lag in Se[IV] uptake was not related to the sensitivity of our Se measurements (the method detection limit for determining intracellular Se (e.g., including cell rinsing and digestion) was 80 μmole per liter of cell volume) or to culture growth (the culture cell counts remained relatively constant in the time-resolved exposure experiments (Figure S12)). The different dynamics of Se[VI] and Se[IV] uptake may similarly be the result of different uptake mechanisms for Se[VI] and Se[IV], as discussed above. Intracellular Se concentrations reached steady state after approximately 10 h of exposure to both Se[VI] and Se[IV], which was not related to depletion of Se (Figure 3A,B) or to changes in Se speciation in the exposure media (Figure S1). After 15 h of exposure, the E


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production of total methylated Se is shown in Figure 3E,F, and the production of the individual volatile Se and S species is given in Figure S13. DMDSe and DMSe were the major methylated Se species produced (up to 525 nM and 70 nM after 15 h of exposure, respectively), with DMDSe being 3.5 and 10 times more abundant on average than DMSe with exposure to Se[VI] and Se[IV], respectively. The prevalence of DMDSe over DMSe may be explained by the fact that DMDSe, which contains two Se atoms, is a more efficient volatile “detoxification product” than DMSe,10 as hypothesized after finding DMDSe to be the major volatile species produced by Se hyperaccumulator plants.35 C. reinhardtii also produced methylated S species, up to 2.4 nM dimethyl sulfide (DMS) and up to 3 nM dimethyl disulfide (DMDS) after 15 h of exposure to Se[IV] (Figure S13). Interestingly, the production of DMS and DMDS did not follow the same dynamics observed for the production of methylated Se compounds, and methylated S species were less abundant than methylated Se species in the methylation experiments, despite higher concentrations of S in the exposure media and inside the cells (Figure 1). Furthermore, volatile mixed Se−S compounds, of which DMSeS (up to 29 nM) was the main species, were found in the algal cultures (Figure S13). Maximum methylation rates attained after approximately 6 h of exposure to Se[IV] were 160 amole·cell−1·h−1 DMDSe and 12 amole·cell−1·h−1 DMSe. Because methylated Se compounds are potentially unstable in exposure medium,9 and an unknown fraction may have decomposed prior to analysis, these production rates represent minimum rates. Nonetheless, our results indicate that the methylation of Se was very efficient: up to 89% and 67% of intracellular Se was methylated and excreted during the Se[IV] and Se[VI] methylation experiments, respectively (percentages calculated as the molar ratio of methylated Se over the sum of methylated and intracellular Se). Combined with the bioconcentration factors mentioned above, this means that within 15 h, C. reinhardtii metabolized and methylated up to 4% and 0.17% of the total aqueous Se[IV] and Se[VI], respectively. Production of Methylated Se as a Function of Intracellular Se. The time-resolved uptake and methylation of Se display similar trends (compare Figure 3C,D and Figure 3E,F). When the time-resolved measurements of intracellular Se and methylated Se from Figure 3 are combined and the production of methylated Se is plotted as a function of intracellular Se, strong and positive linear correlations (R2 > 0.92, p < 0.05) are observed (Figure 5A,B). The x-axis intercepts of these correlations suggest that Se methylation becomes particularly active after approximately 1 mM intracellular Se is accumulated from either Se[VI] or Se[VI] (Figure 5A,B and inset). When the relationships between intracellular Se and external methylated Se from the Se[IV] and Se[VI] experiments (Figure 5A,B) are combined in a log/log plot, the production of methylated Se is positively correlated to intracellular Se over a wide range of intracellular Se concentrations (Figure 5C). This finding suggests that, even though different mechanisms may underlie the uptake and metabolism of Se[IV] and Se[VI], internalized Se[VI] is reduced to Se[IV] and subsequently methylated via similar mechanisms. A relative decrease in the production of methylated Se is observed only at high intracellular Se concentrations. This decrease is most likely due to the fact that a large fraction of intracellular Se is present as Se[0] (symbols 3 and 4 in Figure 5C) at high intracellular Se

Figure 4. Intracellular Se speciation. (A) Se K-edge XANES spectra of five reference compounds (Se[0] in Se[0]S2, Se[0] in gray Se, organoSe (L-Se-methionine), and sodium salts of Se[IV] and Se[VI]) and four samples of C. reinhardtii cells exposed to 30 or 75 μM of Se[VI] or Se[IV] for 15 h. (B) the Se speciation derived by linear combination fitting of the XANES spectra. Error bars reflect fit uncertainties related to potential small energy shifts in incident photon energy (Supporting Information). Italics indicate the total intracellular Se concentration of each sample. An extended X-ray absorption fine structure (EXAFS) spectrum of algae exposed to 75 μM Se[IV] (sample 4) was compared to EXAFS reference spectra of SeS2 and gray and red Se[0] (Supporting Information).

linear combination fitting (LCF) corresponded to 2−9 mM of the intracellular Se. The observed intracellular Se speciation indicates that the metabolic processing of internalized Se by C. reinhardtii involves a series of reduction steps (Se[VI] to Se[IV] to Se[−II]) and subsequent incorporation of Se[−II] into organoSe compounds (e.g., as Se[II] in Se-methionine and Secysteine). Similar mechanisms of reduction and organo-Se synthesis have been observed in higher plants10,35,41,50 and the green algae Chlorella.18 Intracellular red Se[0] has also been observed in bacteria exposed to mM-range levels of Se,34,51 but to our knowledge, its presence has not been observed in microalgae. Production of Methylated Se as a Function of Time. Immediately upon exposure to either Se[IV] or Se[VI], C. reinhardtii produced volatile Se compounds. The time-resolved F


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In the Se[VI] exposure experiments, excess intracellular Se was probably sufficiently mitigated by efficient methylation of intracellular organo-Se. In the Se[IV] exposure experiments, however, the intracellular Se concentrations increased so quickly (up to 30 mM within 15 h) that the methylation activity of C. reinhardtii may have been insufficient to balance the large increase in intracellular Se; this is illustrated by the fact that from 8 h onward, the production of methylated Se after exposure to 75 μM Se[IV] was less than or equal to its production after exposure to 30 μM Se[IV] (Figure 3F). Thus, although C. reinhardtii methylated Se efficiently, its ability to methylate Se appears to be limited, e.g., by limitation of methyldonor compounds or by saturation of an enzyme catalyzing a rate-limiting step in the Se methylation pathway (e.g., methyl transferases54,55 or cystathionine synthases56). In addition to methylation, the formation of nonbioactive intracellular Se[0] may have contributed to the detoxification of high intracellular Se levels. Although intracellular Se[0] constituted a significant fraction of the intracellular Se pool at high intracellular Se concentrations (Figure 4), the overall contribution of Se[0] to the detoxification of internalized Se was minor: after 15 h of exposure to Se[IV], approximately 1200 nM of all Se present in the system was transformed into extracellular methylated Se, while only 5 nM of Se was transformed into intracellular Se[0]. The sum of total intracellular Se and total methylated Se corresponded to 93 ± 19% of the measured decreases in aqueous inorganic Se in the exposure media on average, indicating that adsorption of inorganic Se onto cell walls did not play a major role for the Se mass balance in these experiments. The role of efflux transport as a regulation mechanism of intracellular Se concentrations requires further investigation. Modeling of the Experimental Data. A biochemical model describing Se[VI] and Se[IV] uptake, intracellular metabolism of Se[VI], Se[IV], and Se[0], and production of methylated Se among others (details in the Supporting Information) could quantitatively fit the experimental Se uptake and methylation data (Figure 3 and Supporting Information). The model confirms that Se[VI] uptake is well described by Michaelis−Menten type enzymatic uptake via SO4 transporters: the parameter value for the Michaelis constant Km for Se[VI] uptake (18−220 μM) agrees with the Km value obtained from the experimental data (89 μM) and with previous literature (Supporting Information Results section and Table S4). Furthermore, the model illustrates that (i) the observed lag phase in Se[IV] uptake may be explained by competition between Se[IV] and PO4 for membrane-transporters, (ii) the transformation of Se[IV] to Se[0] may be enzymatically catalyzed (e.g., a NifS-like enzyme57), and (iii) the formation of Se[0] becomes significant at an intracellular Se[IV] concentration exceeding 1 mM (Supporting Results). Finally, obtained model parameter values (Table S4) imply that the reduction of Se[VI] to Se[IV] is slower than the metabolism of Se[IV] to methylated Se, suggesting that Se[VI] reduction is a rate-limiting step for the production of methylated Se, as was previously proposed for higher plants.58 Thus, the biochemical model provided a good quantitative fit to all experimental data, plausible parameter values for the biochemical processes and insights into the mechanisms underlying the experimentally observed metabolism of Se in microalgae. Extrapolation of the Model. Experiments in this study were conducted with Se concentrations orders of magnitude

Figure 5. Selenium methylation as a function of intracellular Se. Production of methylated Se species by C. reinhardtii as a function of the intracellular Se concentration, obtained by combining timeresolved uptake- and methylation data from Figure 3. The relationships are plotted for (A) Se[VI], (B) Se[IV], and (C) Se[VI] and Se[IV] combined (double-logarithmic axis) for 30 and 75 μM Se. Trends are indicated by dashed lines with their corresponding regression coefficients. The axes of the inset in (B) have the same units as the main axis. The gray shading in (C) illustrates the overlap of the relationships between Se[VI] and Se[IV] experiments, and the numbers 1−4 correspond to the samples analyzed with XAS in Figure 4. Error bars indicate standard deviation from triplicate experiments.

concentrations and to the fact that Se[0] is insoluble in the cytosol and thus more difficult to access for enzymatic methylation.52 However, further study of the cellular localization of the various forms of internalized Se in microalgae, e.g., using SEM, is required. Selenium Detoxification Mechanisms. Methylation and the subsequent volatilization of Se is often regarded as a detoxification mechanism for excreting excess intracellular Se,4,10,53 which explains the high methylation efficiencies reported above. The time-resolved uptake and methylation experiments (Figure 3) show that the uptake of Se continues when Se methylation is already active, suggesting that Se uptake mechanisms were decoupled from Se methylation mechanisms under our experimental conditions. G


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methylate Se[IV] more rapidly and efficiently than Se[VI]. In freshwater environments, Se[IV] is usually less bioavailable than Se[VI] because it is more strongly adsorbed to mineral and organic-matter surfaces.1 However, Se[IV] is readily available in the ocean and reported to be the most actively internalized form of Se in a marine calcifying algae.12 High (∼28 mM) concentrations of competing SO4 in the ocean render it unlikely that Se[VI] uptake via SO4 transporters is how microalgae acquire substantial amounts of Se. Marine Se[IV] may be readily acquired through active PO4 transporters, particularly in oligotrophic regions.12 This efficient uptake of Se[IV] may enrich intracellular Se over S (SO4 is by far the major source of S for most microorganisms) and thereby explain the relatively higher enrichment of Se over S in the volatile phase, as observed in field studies in marine and freshwater environments.6,29 However, further investigation of Se uptake and methylation by marine algae is required. In conclusion, this study illustrates that microalgae can actively internalize Se and may produce significant amounts of biogenic methylated Se, both of which could play an important role in the global Se cycle.

higher than typical Se concentrations in the natural environment (

Selenium Uptake and Methylation by the Microalga Chlamydomonas

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