Molecular Characterization of Mercury Binding Ligands Released by Freshwater Algae Grown at Three Photoperiods

Interactions between algal derived dissolved organic matter (DOM) and mercury (Hg) are crucial for understanding the fate, transport, and bioavailability of Hg to methylating microorganisms. For the first time, high resolution mass spectrometry (Q-Exactive Orbitrap) was used to examine Hg binding ligands released by Chlorella vulgaris, Chlamydomonas reinhardtii, and Scenedesmus obliquus grown at three light:dark photoperiods (i.e., 12:12, 16:8, and 20:4 h). Van Krevelen diagrams showed a significant increase in carbohydrate and protein DOM and a decrease in released lipid-like molecules as light exposure increased. Hg binding DOM were initially in the form of CHO molecular formulas whereas a shift to higher light durations prompted more Hg to be complexed to CHON and CHONS DOM structures. Despite an overall change in bulk DOM composition, molecular similarities existed in Hg binding DOM as light exposure increased. Hg binding ligands were more similar based on the exposed light duration than based algal species, suggesting growth photoperiods influence Hg binding more than algal taxa. Hg binding DOM at 16:8 and 20:4 h growth cycles were more aromatic and homologous in nature when compared to darker growth conditions that resulted in smaller, more aliphatic Hg-DOM complexes rich in sulfur and thiols. Together, these results highlight the importance of photoperiod on the composition of released DOM and its complexation with Hg.

Introduction

Anthropogenic activities have increased both atmospheric and aquatic mercury (Hg) concentrations worldwide (Andersson et al., 2008; Park et al., 2008; Lavoie et al., 2013). Mercury is of major concern due to its conversion to neurotoxin methylmercury (MeHg) that can be biomagnified and bioaccumulated in the food chain leading to adverse health effects by humans and wildlife (St. Louis et al., 2007; Lavoie et al., 2013). The conversion of Hg to MeHg was originally thought to be an exclusively anoxic process; however, studies have suggested the conversion Hg to MeHg can occur in oxic aquatic settings of polar waters (Larose et al., 2010; Lehnherr et al., 2011) and in algal biofilms (Parks et al., 2013; Leclerc et al., 2015). Studies have also explored how environments abundant in algal dissolved organic matter (DOM) yielded more MeHg formation than in environments dominated by terrestrially derived DOM (Bravo et al., 2017). In the Canadian Arctic, a subsurface maximum in MeHg concentration was found to be within metabolite enriched halocline layers abundant in lower trophic level biota (Wang et al., 2018). We are just beginning to understand that algal DOM has a profound role on MeHg formation, yet many gaps in knowledge remain around the specific role that algal DOM plays on Hg binding and bioavailability.

Algae is a diverse group of autotrophic microorganisms that has an essential role in the carbon cycle by converting abiotic to biotic energy through the production of organic substrates and dissolved organic nitrogen (DON) that largely support other prokaryotic and heterotrophic organisms (Wada and Hama, 2013; Monier et al., 2015). Algal DOM contains an abundance of nitrogen, trace amounts of sulfur, and carboxyl functional groups that can strongly bind to Hg and influence its complexation (Skyllberg and Drott, 2010). Increasing global temperatures coupled with thermal reorganization of water columns resulting from anthropogenic factors will also select for small microalgae, further amplifying algae's role in controlling metals in aquatic systems (Ansotegui et al., 2003; Winder and Sommer, 2012; Bravo et al., 2017). These lower molecular weight organic substrates present in algal DOM have been shown to play a key role in Hg bioavailability for MeHg production (Lancelot, 1984; Le Faucheur et al., 2014; Leclerc et al., 2015; Mangal and Guéguen, 2015; Mangal et al., 2016). The interactions between algal DOM and Hg remain a crucial step in understanding the fate of Hg; however, environmental variables also play a crucial role on the composition of DOM that governs Hg binding.

Biotic and abiotic factors including pH, light exposure, and microbial community structure can influence the composition of released DOM by autotrophs (Frost, 1987; Leu et al., 2015). In polar regions, the rapid increase in spring daylight hours coincides with algal growth periods and marks the beginning of fresh autochthonous DOM entering northern aquatic ecosystems (Leu et al., 2015). Photo exposure periods transition from 12 h of daylight in early spring (i.e., April) to periods of 24 h of full daylight at the summer solstice in June (LaPerriere et al., 1978; Frost, 1987). Light-induced stress can result in the formation of reactive oxygen species (ROS) for many algae in the event of prolonged or limited photo exposure (Tian and Yu, 2009). To help cope with the formation of ROS radicals, sulfur rich amino acids, glutathione (GSH), and larger peptides such as phytochelatins are generated (Tian and Yu, 2009). These changes in algal metabolism and community associated with higher light exposure during spring blooms can result in compositional changes of DOM and dissolved sulfur concentrations leading to profound implications on Hg binding (Maranon et al., 2005; Thornton, 2014).

Although techniques such as fluorescence and absorbance spectroscopy can provide an adequate preliminary assessment of DOM bulk properties, these techniques are biased to light absorbing DOM moieties (Koch and Dittmar, 2006; Chen et al., 2017). Evaluating DOM-Hg interactions has been conducted using Fourier-transform infrared spectroscopy, and X-ray absorption (Chen et al., 2002, 2017; Schartup et al., 2015), but lack the specificity of exact composition and molecular properties of DOM. High resolution mass spectrometry (HRMS) has been used to provide compound level information for humic substances (HS), algae, and DOM from a variety of temperate and Northern aquatic environments (Koch and Dittmar, 2006; Tremblay et al., 2007; Bhatia et al., 2010; D'Andrilli et al., 2013; Kellerman et al., 2015; Mangal et al., 2016; Osterholz et al., 2016). HMRS has recently become recognized for its ability to detect organometallic complexes including iron, copper and Hg (Ngu-Schwemlein et al., 2014; Waska et al., 2016; Chen et al., 2017). While earlier studies have explored Hg-HS interactions (Chen et al., 2017), algal DOM-Hg interactions have not been addressed yet. This study aims to address these gaps in knowledge by using a multi-faceted analytical approach to (1) understand how algal DOM changes in composition with varying photo exposure periods that mimic natural subpolar transitions (Van Grrest et al., 2007; Leya et al., 2009) and (2) to explore what the compositional changes in algal DOM have on Hg binding (Graphical Abstract).

GRAPHICAL ABSTRACT

Graphical Abstract. Work-flow depicting the harvesting of algal-DOM at various growth photoperiods and the identification of Hg-DOM complexes using Orbitrap high resolution mass spectrometry.

Materials and Methods

Algal Growth and DOC Concentrations

Laboratory grown cultures of Chlamydomonas reinhardtii (C. reinhardtii), Chlorella vulgaris (C. vulgaris) and Scenedesmus obliquus (S. obliquus) were obtained from the Canadian Phycological Culture Center (CPCC) in Waterloo, Ontario and grown using a high salt media (HSM) for C. reinhardtii (Sueoka, 1960) and a bold basal media (BBM) for C. vulgaris and S. obliquus (Sadiq et al., 2011). These microalgae thrive in eutrophic, nutrient rich waters and can withstand large temperature fluctuations of shallow freshwaters; two important factors to consider with warming temperatures and increasing nutrient loads in freshwater environments (Ansotegui et al., 2003; Winder and Sommer, 2012). Growth conditions included photoperiods of low, standard, and high regimes (12:12, 16:8, and 20:4 h, light:dark cycle; respectively) at a fixed light intensity of 90–100 μmol photons m⁻¹s⁻¹ and a temperature of 21°C. DOM was sampled at mid exponential growth phase as determined by hemocytometer cell counting. DOM was harvested during the fourth hour of the light phase for all light regimes and immediately filtered through a pre-combusted 0.7 μm glass fiber filter (GF/F Whatman) into acid washed and pre-combusted glassware. Biological triplicates of algal-derived DOM were collected at all three light regimes. Filtered algal-derived DOC concentrations were measured using a total organic carbon (TOC) analyzer and fixed to 1 mg C. L⁻¹ for further analyses (Table S1).

qBBr Titration

Fresh stock solution of 0.001 mol L⁻¹ qBBr (SigmaAldrich) was prepared daily in amber glass bottle covered in aluminum foil. A series of qBBr standard addition solutions was prepared by mixing 5 mL of DOM samples at increasing volumes of qBBr (final concentrations ranging from 4.98 to 152.54 μmol L⁻¹). The solutions were covered with aluminum foil and mixed for an hour at room temperature. qBBr-equivalent thiol concentrations were determined at fixed excitation and emission wavelengths (380 and 470 nm, respectively) using a Fluoromax4 spectrophotometer (Horiba Jobin Yvon) (Joe-Wong et al., 2012; Mangal and Guéguen, 2015). Since qBBr binds to thiols at a 1:1 molar ratio, the level of qBBr saturation in samples corresponded to thiol concentration. Total thiol concentrations were determined using non-linear piece-wise regression (SigmaPlot, v12.5) (0.97 < R² < 0.99; p < 0.05) (Joe-Wong et al., 2012; Mangal and Guéguen, 2015).

High Resolution Mass Spectrometry

The molecular composition of algal-derived DOM was determined using the Orbitrap Q Exactive (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI) source. The resolving power was 140,000 (full width half maximum @ m/z 200). Prior to injection, samples were normalized to a DOC concentration of 1 mg C L⁻¹, diluted to a 50:50 sample to MeOH ratio with ultrapure MeOH (99.9% HPLC grade; Sigma Aldrich) and the pH was adjusted to 6.8 with ultrapure NaOH (Sigma Aldrich). Samples were injected at a consistent flow rate of 50 μL min⁻¹ with an electrospray needle voltage of 4 kV and a heated capillary temperature of 300°C in positive ionization mode (Mangal et al., 2016; Sipler et al., 2017). Sodium trifluoroacetate (NaTFA, Sigma-Aldrich 98%) was added as an internal standard in each sample and externally calibrated twice a day to ensure exact mass accuracy between a mass range of 200–1,000 m/z. Samples were acquired for a minimum of 5 min to allow for 200 scans to be co-added in the data post analysis stage using the Thermo Xcalibur Qual Browser (3.0.63) software. Background signals from algal growth media and MeOH were subtracted prior to formula assignment. Elemental constraints for formula assignment were: ¹²C (0-50), ¹H (0-100), ¹⁶O (0-30), ¹⁴N (0-7), ³²S (0-4), and ²³Na (0-1) using the odd nitrogen rule. Molecular formulas were validated based carbon and sulfur isotopes (¹³C and ³⁴S) and signal to noise ratios were ≥4 within ±2 ppm exact mass error (Figures S1,S2) (Ohno and Ohno, 2013; Herzsprung et al., 2016; Mangal et al., 2017). An in-house Matlab script (SI) was used to determine the abundance of seven compound classes: lipid (0.01 ≤ O/C ≤ 0.1; 1.5 ≤ H/C ≤ 2.0), unsaturated hydrocarbons (0.01 ≤ O/C ≤ 0.1; 0.75 ≤ H/C ≤ 1.5), condensed aromatic structures (0.01 ≤ O/C ≤ 0.65; 0.25 ≤ H/C ≤ 0.75), protein (0.1 ≤ O/ C ≤ 0.65; 1.5 ≤ H/C ≤ 2.3; N ≥ 1), lignin (0.1 ≤ O/C ≤ 0.65; 0.75 ≤ H/C ≤ 1.5) with no heteroatoms (only CHO), tannins (0.65 ≤ O/C ≤ 0.85); (0.75 ≤ H/C ≤ 1.5) and carbohydrates (0.65 ≤ O/C ≤ 1.0; 1.5 ≤ H/C ≤ 2.5), similar to previous studies (Kim et al., 2003; Ohno and Ohno, 2013; Mangal and Guéguen, 2015; Mangal et al., 2017). Atomic ratios and elemental averages of O/C, H/C, S/C, %C, %H, %O, %N, %S, m/z, double bond equivalence (DBE, Equation 1) modified aromaticity index (AI_mod, Equation 2) and the nominal oxidation state of carbon (NOSC, Equation 3) were all calculated to interpret the changes in aromaticity and oxidation state of DOM (Koch and Dittmar, 2006; Riedel et al., 2013; Roth et al., 2013, 2015).

$\begin{array}{cc}\text{DBE}=1+\frac{1}{2}(2\text{C}-\text{H}+\text{N}+\text{P})& (1)\end{array}$

$\begin{array}{cc}{\text{AI}}_{\text{mod}}=\frac{1+\text{C}-0.5\text{O}-\text{S}-0.5\text{H}}{\text{C}-0.5\text{O}-\text{S}-\text{N}-\text{P}}& (2)\end{array}$

$\begin{array}{cc}\text{NOSC}=4-\left[\frac{4\text{C}+\text{H}-3\text{N}-2\text{O}-2\text{S}}{\text{C}}\right]& (3)\end{array}$

Mercury Detection

Hg solutions were prepared by mixing 2.5 × 10⁻⁶ mol L⁻¹ of Hg (NO₃) (AAS grade) with 1 mg C L⁻¹ of organic ligand (i.e., algal DOM or L-Cysteine; SigmaAldrich) and kept in the dark for at least 24 h prior to analysis. A Hg concentration of 2.5 × 10⁻⁶ mol L⁻¹ was found to be suitable to confidently identify less abundant Hg isotopes required for unambiguous peak identification. It should however be noted that the molar ratio used in this study was higher than in environmental settings but comparable to that used in previous Orbitrap studies (Ngu-Schwemlein et al., 2014). The pH solution was fixed to 6.8 with ultrapure NaOH. For algal-derived DOM, blanks samples (MeOH+Hg, HSM+ Hg, and BBM+Hg) were evaluated and subtracted from Hg-Cysteine and Hg-DOM and samples, respectively. To detect Hg containing compounds, ²⁰²Hg and ²⁰⁰Hg isotopes were added to the elemental constraints for formula assignment, comparable to previous ESI HRMS Hg studies (Chen et al., 2017). The software Winnow (Doran and LeBlanc, 2016) was used to detect 2 isotopologues of Hg (²⁰⁰Hg, and ²⁰²Hg). Only peaks with a Winnow score >50% were utilized to confidently identify Hg containing DOM compounds in Xcalibur Isotope simulation (3.0.63). In addition, the ratio ²⁰⁰Hg:²⁰²Hg were calculated in organic ligand samples and compared to the theoretical ratio of 0.76 (Chen et al., 2017). For our experiments, if an experimental ratio between ²⁰⁰Hg:²⁰²Hg ratios was >0.55, within 5 ppm error based on exact mass, and scored >0.65 using Winnow, that Hg peak was considered significant.

Statistical Analyses

Multivariable linear regression model II major axis (MA) analyses were conducted when comparing qBBr reactive thiol concentrations with HRMS %S values as both variables contained intrinsic errors (Legendre, 2014). A one-tailed permutation test was conducted using the lmodel2 package in R statistical computing (3.2.2) and n = 99 at 95% confidence interval. When examining changes in algal DOM compound classes both between species and at different light regimes, a t-test was conducted where significance was evaluated at p < 0.05 and a Shapiro-Wilk normality statistic was used. Hg binding DOM molecules between algal taxa and various growth photoperiods were compared using a principal component analysis (PCA) through the FactoMineR package of R(v3.4.1) (Le et al., 2008). A correlation matrix was utilized for the PCA as HRMS variables such as %S, AI_mod, DBE and qBBr reactive thiols were all derived from a HRMS platform.

Results and Discussion

Changes in DOM Composition With Increased Light Exposure

A total of 416-1960 mass peaks were found across biological triplicates (Figures S3–S5). The mass peaks were mainly comprised of singly charged peaks and all detected sodium adducts were omitted from further analyses. As photoperiods increased, O/C ratios increased from (0.20 ± 2.09) × 10⁻³ at 12:12 to (0.21 ± 8.03) × 10⁻³ at 16:8 and then to (0.23 ± 0.01) × 10⁻³ at 20:4 for C. vulgaris (Figure 1A). A similar trend was observed for C. reinhardtii with the 20:4 growth conditions producing more oxygenated compounds than at 12:12 [p < 0.05; (0.22 ± 3.16) × 10⁻³ vs. (0.28 ± 0.02) × 10⁻³]. For S. obliquus, the average O/C ratio was maximum at light conditions of 16:8 [i.e., (0.24 ± 4.01) × 10⁻³]. Although no significant change in H/C ratios were observed between algal species, C. reinhardtii at the 16:8 photoperiod did prompt a significant maximum in hydrogenated compounds where H/C ratios peaked at 1.99 ± 0.01 (Figure 1B). N/C ratios were significantly greater at the lowest light regime for all algal species (p < 0.05; Figure 1C). S/C ratios were the lowest at 16:8 growth conditions for both C. vulgaris and S. obliquus and significantly greater at 12:12 (Figure 1D, p < 0.05). For all algal taxa, AI_mod values (Figure 1E) were the lowest at 16:8 growth conditions suggesting the preferential release of more aliphatic compounds. qBBr titrations revealed significant increases in qBBr active thiol concentrations for C. vulgaris and C. reinhardtii (p < 0.05) but these trends were not observed for S. obliquus (Table S2). A significant relationship was observed between qBBr equivalent thiol concentrations and the %S contribution reported by Orbitrap (p < 0.05; R² = 0.86) suggesting that reactive thiols were a significant fraction of S-containing compounds in algal DOM (Figure S6). NOSC values were the most negative at 16:8 growth periods for both C. vulgaris and C. reinhardtii indicating a shift in reactivity from nonpolar at 16:8 to more polar compounds at both light extremes (Figure 1F), consistent with previous studies (Riedel et al., 2012; Roth et al., 2013, 2015). Although photo-oxidative stress was not explicitly measured, a higher degree in photorespiration caused by photo-oxidative stress has been proposed as a mechanism of increased carbon loss in algae (Yamada et al., 2012; Thornton, 2014) congruent with a significant increase in DOC concentrations (Table S1) and %C from 16:8 to 20:4 (Figure S7A). Across all algal taxa, %H and %O were significantly greater at 16:8 (p < 0.05; Figures S7B,C).

FIGURE 1

Figure 1. Bar graphs representing changes in atomic ratios such as O/C (A), H/C (B), N/C (C), and S/C (D) along with changes in compositional properties AI_mod (E) and NOSC (F) at 12:12 (black), 16:8 (gray), and 20:4 (red) light regimes for C. vulgaris, C. reinhardtii, and S. obliquus. Error bars for all light:dark cycles are based on biological triplicates (n = 3). *Represents a signficant difference (p < 0.05) in bulk DOM parameters between 12:12 and 20:4 h for each algae.

For each alga, the abundance of each DOM compound class based on van Krevelen diagrams differed across light durations (Figure 2; Figure S8). The lignin abundance increased by a factor of 2.8, 1.4, and 0.79 in C. reinhardtii, C. vulgaris and S. obliquus, respectively (Figure 2A). While lignin is known to provide cell wall support (Martone et al., 2009), the active secretion of these phenolic and polyphenolic compounds has been reported as a possible source of non-vascular plant derived lignin (Hernes et al., 2007). Protein content was the most abundant compound class for all algae (34.1–48.7%) and increased as photoperiods increased (Figure 2B). Across biological triplicates, the abundance of HRMS-based protein material increased for all algae as photoperiods increased ranging from 34.1 to 48.8%. Proteinaceous material in the form of amino acids, larger peptides and amino sugars serve as crucial components of the total DON and are the predominant compound class accounting for more than 50% of the material released by algae (Bronk et al., 2007; Yamada et al., 2012; Thornton, 2014). Across all algal taxa, the 20:4 growth conditions showed greater protein content compared to 12:12 conditions (p < 0.05) likely caused by a shift in algal metabolism where a change from lipid and polysaccharide synthesis to protein synthesis occurs during periods of stress (Lancelot, 1984; Guschina and Harwood, 2006). The fatty acid content of algae was higher at lower light exposure (Guschina and Harwood, 2006) where a 30–50% decrease in lipid abundance was observed with increasing photoperiods (Figure 2C). All algae in this study have been known to have high intracellular and extracellular protein and lipid content (Duong et al., 2015; Rasala and Mayfield, 2015; Schulze et al., 2016). Many sulfur containing amino acids and proteins such as cysteine and GSH have been shown to increase in concentration when marine algae are exposed to increased light intensity (Dupont et al., 2004), consistent with the increase in %S reported by HRMS at both extreme light conditions. Carbohydrate abundances increased with photoperiod for both C. reinhardtii and C. vulgaris (p < 0.05) but plateaued for S. obliquus from 16:8 to 20:4 h (Figure 2D). Across all species, a significant (p < 0.05) increase in carbohydrate material was observed at 20:4 when compared to 12:12 growth conditions. The increase in carbohydrate abundance was likely due to the degradation of algal cell walls during periods of oxidative stress induced by light (Aluwihare et al., 1997). The presence of aliphatic compounds at lower light regimes was also reinforced by the significantly greater %H contribution at 12:12 h and the greater %O contribution at 20:4 h (Figures S7B,C; p < 0.05). %N was significantly greater at 12:12 when compared to all other growth conditions (Figure S7D; p < 0.05) and when coupled to the increase in aliphatic material suggests that these constituents may contribute to the total protein and lipid pool of organic matter that have known to be released by algae as metabolic byproducts during instances of stress (Pirastru et al., 2012; Holguin and Schaub, 2013; Thornton, 2014; Mangal et al., 2016). While released DOM composition varied based on algal taxa, a higher light exposure led to an overall increase in molecules that are generally more aliphatic and polar in structure. Protein and carbohydrate-rich compounds were positively (p < 0.05) correlated whereas lipid content decreased as light exposure increased (Seyfabadi et al., 2011; Zhu et al., 2016).

FIGURE 2

Figure 2. Compound classes based on van Krevelen diagrams protraying the normalized relative abundance (%) of lignin (A), protein (B), lipids (C), and carbohydrates (D) DOM released by C. vulgaris (blue), C. reinhardtii (green) and S. obliquus (purple) from 12 h light:12 dark to 20 h light: 4 h dark. Error bars for all light:dark cycles are based on biological triplicates (n = 3). *Represent a signficant difference (p < 0.05) in compound classes between 12:12 and 20:4 h for each algae.

Hg-DOM Interactions

Hg-Cysteine

Hg complexation was observed in the presence of model ligands, i.e., cysteine in a simple MeOH: H₂O matrix. Among the 6 Hg-containing formulas, ²⁰²HgC₆H₁₃N₂O₄S₂ was experimentally (Figure S6A) identified in the Hg-cysteine sample (1:2 molar ratio) in positive mode and consistent with the theoretical isotopic distribution natural abundances (Figure S6B). The experimentally derived spectra revealed the most abundant isotopes ²⁰²Hg and ²⁰⁰Hg differed by −2.25 and −2.26 ppm from the theoretical distribution, respectively. The peak intensity ratio between ²⁰²Hg and ²⁰⁰Hg was 0.81:1 for the detected Hg-Cys complex, comparable to the natural isotopic ratio of 0.77:1 using a 15T Fourier transform ion cyclotron mass spectrometer (FT ICR MS) (Chen et al., 2017). To further reinforce accurate formula assignment, C₁₃ and S₃₄ isotopes of the carbon-based ligand were also identified. While the exact mass of Hg isotopes in other studies incorporating a 15T FT ICR MS achieved ± 1 ppm (Herlemann et al., 2014; Chen et al., 2017), our results suggest that Hg isotopes can be reasonably identified with confidence using Orbitrap mass spectrometry.

Changes in Hg Binding DOM Composition With Varying Photoperiods

Spectra of algal-derived DOM were similar before and after Hg addition suggesting limited ion suppression using ESI Orbitrap HRMS (Figures S3–S5,S10), congruent with previous studies (Chen et al., 2017). A total of 52, 28 and 19 Hg-DOM complexes were detected for C. reinhardtii, C. vulgaris, and S. obliquus, respectively, based on exact mass, theoretical Hg isotopic distribution, Winnow score (Doran and LeBlanc, 2016), and ²⁰²Hg:²⁰⁰Hg ratios (Figure 4). A total of 37, 39 and 23 Hg-DOM complexes were found at 12:12, 16:8, and 20:4 h, respectively which accounted for 1.6–2.6% of the total assigned peaks. The addition of Hg to algal DOM allowed for the identification of Hg isotopologues while retaining similar peaks in the metal free sample (Figure S9). Experimentally detected elemental formulas of [²⁰²HgC₃₅H₆₀O₇N₂S], [²⁰²HgC₃₉H₈₂O₆NS₂], and [²⁰²HgC₃₇H₆₄O₆N] were validated based on ²⁰⁰Hg and ²⁰²Hg isotopes ratios. It can be noted that lower abundance Hg isotopes (i.e., ¹⁹⁶Hg, ¹⁹⁸Hg, ²⁰¹Hg, and ²⁰⁴Hg) were also observed (Figures 3A-F) albeit at lower absolute intensities when compared to a 15T FT ICR MS (Chen et al., 2017).

FIGURE 3

Figure 3. Identified Hg binding DOM for (A) C. vulgaris (blue), (C) C. reinhardtii (green), and (E) S. obliquus (purple), and their respective theoretical isotopic distribution (B,D,F), based on exact mass, isotopic conformation, Winnow score and ²⁰²Hg: ²⁰⁰Hg ratios.

Differences in DOM composition at various growth periods can have an impact on the speciation of Hg by DOM. The number of Hg binding CHO, CHON, CHOS and CHONS varied with photoperiod and taxa (Figure 4). For example, the number of CHO binding Hg compounds was maximum at 16:8 across all taxa whereas the number of CHONS increased with photo exposure for C. reinhardtii and S. obliquus but decreased for C. vulgaris. The average m/z of Hg-DOM complexes increased with photoperiod from 520 to 842 m/z and from 613 to 800 m/z for C. reinhardtii and S. obliquus, respectively, whereas smaller m/z values were found at intermediate photoperiods (i.e., 16:8 h) for C. vulgaris (612 vs. 867 and 729 m/z).

FIGURE 4

Figure 4. Line and scatter plots portraying trends for generic Hg binding DOM formulas such as CHO (solid line, circles), CHON (long dash, circles), CHOS (short dash, downward triangles) and CHONS (dotted dash, triangles) for C. reinhardtii (A), C. vulgaris (B), and S. obliquus (C) as the duration of light exposure increases.

To assess how Hg binding DOM varies based on algal taxa and growth conditions, a PCA was conducted using %S, AI_mod, DBE, and qBBr equivalent thiol concentrations (Figure 5). The first principal component (PC1) indicative of DBE, AI_mod driven variables explained 49% of variance whereas the second principal component (PC2) explained 34% of the variance and was strongly influenced by %S and qBBr equivalent thiol concentrations. Three distinct groups were observed that follow along a similar vector as DBE, suggesting homologous Hg binding structures along a double bond gradient. Clusters 1 and 2 followed this pattern along a DBE vector and were comprised of 38 and 20 Hg binding compounds, respectively. A final third cluster spanning from PC2 1 to 3 was less robust along the DBE gradient and had a greater contribution and influence of %S and qBBr reactive thiols. Sixteen Hg binding DOM compounds were associated with Cluster 3. No specific trends were observed between algal taxa, as each cluster contained a similar proportion of each species and overall species distributions were quite heterogenous (Figure 5A). When the PCA was organized based on light exposure (Figure 5B), the majority of 16:8 Hg binding compounds were found in the first cluster (57%), where the second cluster is mainly comprised of compounds originating from 20:4 light dark samples (70%). Finally, the 12:12 Hg binding DOM molecules in the final cluster influenced by %S and qBBr concentrations included 50% of the total DOM compounds.

FIGURE 5

Figure 5. Principal component analysis (PCA) combining bivariate and factor plots depicting compositional changes in Hg-DOM complexes based on algal species (A: green= C. reinhardtii, blue= C. vulgaris, and purple = S. obliquus). The second PCA (B) also outlines Hg-DOM complexes, but colors indicate the light regime when Hg-DOM compounds were found (black = 12:12, gray = 16:8, red = 20:4). In both plots, three distinct clusters were observed corresponding to similar Hg-DOM compounds along a DBE gradient.

A shift in light exposure from 16:8 h to either 12:12 h or 20:4 h showed an increase in %S, qBBr equivalent thiols, and the number of S-containing Hg binding compounds. With the availability of more sulfhydryl binding sites at both extremes of the photoperiods (Figure S7), a shift from amine and carboxyl functional groups to thiols may be observed, even at higher DOM:Hg ratios (Haitzer et al., 2002; Le Faucheur et al., 2014). Structural properties of released DOM are governed by the specific algal taxa as the only conserved Hg binding structure between algal taxa was C₉H₁₉O₂SHg found during the 12:12 photoperiod of both C. reinhardtii and S. obliquus. As %S content increased, we observed more Hg-S structures at 20:4 and 12:12 that tend to have stronger binding affinities (log K > 22) than the carboxyl functional groups (log K >10) at 16:8 (Skyllberg and Drott, 2010; Chen et al., 2017). While assessing present thiol and sulfhydryl binding sites allows for predicting Hg complexation, the structure of the side chain attached to the sulfur containing group has important implications for the bioavailability of the complex (Le Faucheur et al., 2014; Leclerc et al., 2015). It has been shown that algal DOM abundant in low molecular weight thiols may act as a shuttle for Hg to bacteria, larger sinking algal DOM may facilitate Hg mobility to larger colloidal DOM fractions where Hg methylation readily occurs (Leclerc et al., 2015; Schartup et al., 2015; Gascón Díez et al., 2016). A greater proportion of lower molecular weight compounds coupled to the increase in %S at 12:12 and to a lesser extent 20:4, suggests that early spring and later into the summer months may prompt periods of increased Hg bioavailability to smaller microorganisms whereas optimal light conditions of 16:8 for most algae may reduce Hg bioavailability. Smaller compounds are generally more easily uptaken by microorganisms, suggesting that Hg-binding DOM compounds produced by C. reinhardtii and S. obliquus at 12:12 h can be internalized due to their smaller size coupled to the increase in %S and reactive thiols at 12:12 when compared to other light durations (Leclerc et al., 2015; Bravo et al., 2017).

Albeit important, the duration of photo exposure is not the only variable to consider when determining Hg complexation with algal DOM. Salinity, light intensity, pH, and DOM concentration can all impact biogenic DOM composition and Hg binding to various functional groups within DOM. For example, an increase in DOM released by algae during changing photoperiods is likely due to an increased carbon fixation rate when compared to optimal diurnal growing conditions (Cherrier et al., 2015). Relationships between DOC concentration released by algae and light exposure have been reported in temperate regions (Mueller et al., 2016) wherein longer photoperiod exposures have been shown to prompt an increase in the hydrogen metabolism cycle leading to greater hydrogen exudation, and higher DOC concentrations in C. reinhardtii cultures (Oncel and Sukan, 2011). In natural systems, these changes in DOC concentrations, natural variation in light intensity, and additional physiochemical properties may further impact the composition of released DOM and thus metal binding properties (McIntyre and Guéguen, 2013).

Conclusion

In this study, we applied HESI Orbitrap HRMS to explore compositional differences between various algae cultivated across a photo exposure gradient and how these compositional changes can impact DOM-Hg complexation. Increasing the growth photoperiod led to an overall increase in protein and carbohydrate DOM being released by all algae. Conversely, the lipid content significantly decreased as photo exposure increased, in line with previous studies (Zhu et al., 2016). Of all cultivated algae, S. obliquus produced the greatest %S and potential thiol structures when cultivated at a 12:12 h light:dark photoperiod, reinforced by both HRMS and qBBr titrations. While taxonomical differences across algae played an important role in Hg binding, the duration of light exposure significantly altered algal derived DOM composition and thus influenced Hg complexation. Lower photoperiods enhanced %S and qBBr reactive thiol production allowing for the production of unique CHONS and CHOS compounds more aliphatic in nature that may result in the increased bioavailability of Hg-DOM compounds. Our results emphasize that Orbitrap HRMS can be used for both (1) the untargeted characterization of complex algal biomolecules as cultivation methods vary and (2) the identification of Hg isotopes within a complex matrix to determine Hg-NOM speciation information.

Although further studies incorporating MS/MS are needed to accurately quantify and confirm definitive Hg-DOM structures, the use of Orbitrap HRMS can be utilized for the preliminary identification of Hg binding DOM molecules. We suggest that a similar methodological approach using Orbitrap HRMS be utilized to explore how other cultivation variables such as pH, salinity, light intensity, and other algal species can all impact Hg-DOM complexation. The role of algal DOM in controlling Hg fate is continuously advancing and the approaches utilized in this study provide novel insights into the profound role that algal DOM has on the Hg complexation in aquatic systems.

Author Contributions

VM, TP, and TN conducted the experiments. VM, TP, and CG analyzed the data. All authors contributed to writing the manuscript.

Funding

This work was funded by the Canada Research Chairs Program (CG), the Natural Sciences and Engineering Research Council of Canada (NSERC; CG, VM), the Ontario Graduate Scholarship (VM), the Weston Garfield Award for Northern Research (Doctoral) (VM), and the Trent University Symons Trust Fund (VM).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors would like to thank Dr. Naomi Stock and Dr. Michael Doran for their assistance with Orbitrap and Winnow troubleshooting. We are also thankful for Antoine Perroud for his assistance with DOC analyses and Dr. Paul-Philippe Champagne for his constructive feedback. Finally, many thanks to the two reviewers for their constructive feedback during the peer review process.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fenvs.2018.00155/full#supplementary-material

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