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Bio-oxidation of elemental mercury during growth of mercury resistant yeasts in simulated hydrosphere
Journal of Hazardous Materials 373 (2019) 243–249
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Bio-oxidation of elemental mercury during growth of mercury resistant yeasts in simulated hydrosphere Ganiyu Oladunjoye Oyetiboa,b,1, Keisuke Miyauchia, Hitoshi Suzukia, Ginro Endoa, a b
T
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Department of Civil and Environmental Engineering, Faculty of Engineering, Tohoku-Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537, Japan Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Yaba, Lagos, Nigeria
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Mercury dissolution Mercury oxidation Hg-resistant Yarrowia Mercury cycle Extracellular polymeric substances (EPS)
Transformation of metallic mercury (Hg°) to mercuric ion (Hg2+) in hydrosphere is the entrance of mercury cycle in water environments and leads to toxicological impact of serious global concern. Two yeast strains of Yarrowia (Idd1 and Idd2) isolated from Hg-contaminated sediments were studied for their mediating role in Hg° dissolution and oxidation. Growth of the Yarrowia cells in Hg-free liquid medium, incubated for 5 d in closed airtight systems containing Hg°, produced extracellular polymeric substances (EPS). Approximately 230 ( ± 5.7) ng and 120 ( ± 6.8) ng of the dissolved Hg° were oxidized to Hg2+ by the cultures of Idd1 and Idd2, respectively, 5 day post-inoculation. Transmission electron microscopy (TEM) and X-ray energy dispersive spectrophotometry (XEDS) analysis of the EPS and cell mass revealed the presence of extracellular Hg nanoparticles, presumably HgS, as an indication of EPS-Hg complexation that is useful for Hg° dissolution and its eventual oxidation to Hg2+ by the cells. Fourier transmission infra-red (FTIR) analyses of the EPS and cell-mass during Hg-oxidation revealed that amine and carbonyl groups were used by EPS for Hg complexation. Our findings provided information about mediatory role played by Yarrowia (Idd1 and Idd2) in hydrosphere in biogeochemical cycling of Hg.
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Corresponding author. E-mail addresses: [email protected], [email protected] (G.O. Oyetibo), [email protected] (K. Miyauchi), [email protected] (H. Suzuki), [email protected] (G. Endo). 1 Current address: Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Yaba, Lagos, Nigeria. https://doi.org/10.1016/j.jhazmat.2019.02.075 Received 26 January 2019; Accepted 20 February 2019 Available online 20 March 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
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1. Introduction
ability of yeasts to oxidize Hg° has not been explored. In this study, we sought to determine the dissolution of aerial Hg° gas into aqueous system as dissolved Hg°, and eventual oxidation to non-purgeable Hg2+. Strains of previously isolated Hg-resistant yeasts, Yarrowia spp. (Idd1 and Idd2), were used as microbial models to actualize dissolution and bio-oxidation of Hg° in simulated hydrosphere as a critical stage of biogeochemical cycling of mercury.
Mercury (Hg) is one of the most toxic trace elements on earth and equally one of the most elusive because of the volatility of elemental mercury (Hg°) and the high reactivity of the mercuric ion (Hg2+) [1]. Besides geological phenomena such as volcanic activity, the concentration of Hg in the environment has been on rise globally due to increase in anthropogenic activities including artisanal gold mining, fossil fuel combustion, smelting activities, urbanization, agricultural practices and industrial processes among others [2]. These human activities release Hg° vapor into the atmosphere, where the mercury vapor can circulate for up to a year, becoming widely dispersed due to its relatively slow reactivity with the more abundant oxidants in the atmosphere [3]. The Hg° vapor is reportedly undergone photochemical oxidation to become inorganic Hg2+ that can combine with water vapors and travel back to the Earth’s surface as rain [4]. This ‘mercurywater’ solution is deposited in soils and bodies of water from where accumulation to animate and inanimate matrixes do occur until a physical event causes it to be released again. Dissolved Hg° is a ubiquitous form of inorganic mercury in marine and terrestrial water [5]. Previous studies have shown that surface water, soil, groundwater, and ocean can accumulate high concentrations of Hg° [5–7]. In terrestrial matrix, the loss of gaseous Hg° to the atmosphere is pivotal to decreases of the Hg pool remaining in watersheds [5]. Hg° can remain dissolved in water and be mobilized by groundwater advection of saturated sediments, where gas-exchange is restricted, forming groundwater Hg° that has been reportedly discharged to drinking-water wells [8]. Bone et al. [9] suggested that such groundwater Hg° contribute to the unexpectedly low partition coefficient of Hg in confined aquifers due to its low affinity for sediment surfaces. Conversion of inorganic mercury via methylation into neurotoxic methylmercury (MeHg) in anoxic sediments during microbial processes is a critical step that governs the transfer of Hg to aquatic food webs and its biomagnification in higher trophic levels [5]. Since Hg° is generally considered to be unreactive [10], its formation and transport are thought to limit the amount of Hg available for methylation [1]. However, Hg° is oxidized to Hg2+ by physical, chemical and biological processes in aquatic environments [4,11,12]. Mercury (Hg°) oxidation to mercuric ion (Hg2+) in hydrosphere is the entrance of mercury cycle in water environments. For emphasis, Hg° oxidation is reportedly controlled by photochemical reactions in surficial waters exposed to sunlight [4]. Laboratory investigations of natural and artificial waters by Lalonde et al. [13] have shown that the oxidation of Hg° by solar irradiation is linked to photochemical production of reactive compounds such as hydroxyl radicals. Dissolved Hg° can also be oxidized in the dark by unfiltered seawater where oxygen serves as the most likely oxidant [14]. Furthermore, experimental studies have shown that presence of O2 and Cl− or thiol compounds can directly oxidize drops of liquid Hg° in water [15]. Whilst the presence of reactive ionic species (CO32- and NO3−) triggers much higher Hg° photo-oxidation rate, dissolved organic matter (DOM) decreases the oxidation rate by half [4]. The microbial oxidation of Hg° to Hg2+ and eventual transformation to MeHg is known to occur among some bacterial strains [12,16]. Thus far, aerobic and phototrophic microorganisms have been implicated as the primary microbial agents driving Hg° oxidation reactions. Smith et al. [16] showed that the catalase enzymes in Escherichia coli enhances dissolution of Hg° into water, with eventual Hg° oxidation activity associated with the cytosolic catalase/hydroperoxidase proteins (KatG and KatE). However, a double mutant lacking both the katG and katE genes found to retain the ability to oxidize Hg° suggests the existence of other bacterial oxidation pathways that are currently uncharacterized. This has been observed with aerobic soil bacteria, Bacillus and Streptomyces, exhibiting high levels of Hg° oxidizing activity [16], and consequently illustrating the potential for microbial oxidation during cycling of Hg in soils. Currently, the subsurface microbial processes involved in Hg oxidation remain poorly understood, and the
2. Materials and methods 2.1. Culture condition Stock cells of the Hg-resistant yeast Yarrowia spp that was previously isolated from polluted estuarine sediment and deposited to Japan Collection of Microorganisms under Accession no. JCM 30162 (strain Idd1), and JCM 30163 (strain Idd2) (stored at −80 °C in 1:1 glycerol:YM broth) [17] were obtained. The yeast cells were resuscitated by inoculating into sterile Bacto YM broth (Becton, Dickinson and Co, Sparks, MD, USA). After incubation (30 °C; 50×g; 72 h), 100 μl culture was spread on dried surface of sterile Bacto YM broth (containing per liter: 5 g, yeast extract; 3 g, malt extract; 5 g, peptone; and 10 g, dextrose) that was solidified with 1.5% agar (Wako, Osaka, Japan). Young, distinct colony (24 h post-inoculation, 30 °C) was inoculated into YM broth (200 ml in 1 L Erlenmeyer flask) and incubated (30 °C; 50×g; 96 h) to obtain inoculum of the resuscitated yeast strains (approx. 106 cells ml−1) for subsequent experiments.
2.2. Preparation of batch culture system for Hg° dissolution and oxidation Ability of the yeast strains to dissolve aerial Hg° into broth system as a simulation of hydrosphere was studied in a batch culture of Hg-free YM broth. A 250 ml Erlenmeyer flask containing 30 ml Hg-free YM broth with a magnetic stirrer bar was inoculated with 0.1 ml culture of the Yarrowia strains Idd1 and Idd2 in Section 2.1 above (see Fig. A1). The inoculated flask was placed in an air-tight jar, closed 1.5 ml microcentrifuge tube containing approximately 900 mg metallic Hg° was attached to the flask, the microtube was opened and the jar was immediately air-tight closed to prevent escape of Hg° from the jar. The setup was placed on a magnetic stirrer device to provide mixing of the culture at 100 rpm and altogether placed inside a dark incubator (30 °C, 5 d). For the control set-up, Hg-free YM broth was inoculated with Saccharomyces cerevisiae and subjected the same conditions as applicable to the Yarrowia strains above. Moreover, negative controls of the Yarrowia culture systems but without Hg° containing microtube attachments were set up as well. All experiments were repeated three times.
2.3. Determination of Hg° dissolution and oxidation To quantify mass of Hg° released from the micro-centrifuge tube to the aerospace, difference in the weight of micro-centrifuge tube containing Hg° before and after incubation was measured. The culture were analyzed for total Hg to determine the Hg° dissolved into the medium using Mercury Analyzer as explained in Section 2.5 below. To quantify Hg° oxidation, the culture was divided into three portions: one portion a piece was treated with 0.1 M HNO3, 0.5 M HNO3, and without acid hydrolysis. Afterwards, each portion was divided into two portions and a set was subjected to oxygen-free N2 gas sparging in a draft chamber for 10 min. at a flow rate of 0.2 l min−1, while the other portion was without N2 gas sparging. Subsequently, each portion was analyzed for total mercury using Mercury Analyzer as explained in Section 2.5 below.
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2.8. Statistical analyses
2.4. Determination dissolved/oxidized Hg° in Yarrowia cell mass and biopolymer
All statistical tests including column statistics, row statistics, and non-linear regression (curve fit) of the least squares were performed using the Prism 5 software program (GraphPad Software, San Diego, CA, USA).
This experiment was meant to establish presence of non-purgeable oxidized Hg2+ in the yeast biopolymer and cell mass. The yeast culture was centrifuged (4 °C; 6000×g; 30 min) to separate the cell mass and the supernatant. Method reported by Oyetibo et al. [18] was used in the isolation of soluble extracellular polymeric substances (EPS) from the supernatants. The supernatants were pressure-filtered through 0.45 μm pore size Minisart syringe filters (Sartorius Stedim Biotech, Gottingen, Germany) and kept overnight at 4 °C after treatment with cold 99% ethanol (ethanol:filtrate, 2:1). The EPS precipitates were recovered by centrifugation (4 °C; 6000×g; 30 min). The pellets were re-suspended in sterile water, dialyzed against sterile MilliQ water, lyophilized, and stored at 4 °C, or otherwise analyzed for Hg after resuspension with 1.0 ml MilliQ water using Mercury Analyzer as explained in Section 2.5 below. However, the cell mass precipitated after centrifugation was analyzed for Hg using the Mercury Analyzer, too.
3. Results 3.1. Hg° dissolution The deposition, mobility and cycling of mercury in natural environments are critical to global mercury pollution and significantly dependent on speciation and presence of ligands that would spell out if it remains in solution or as a dissolved gas. For emphasis, Hg° can be present in two forms in hydrosphere as liquid Hg°, which is rarely observed in uncontaminated natural waters; and aqueous Hg° that is ubiquitous and of environmental relevance. Characteristic growth of Hg-resistant Yarrowia spp. (Idd1 and Idd2) in Hg-free media produced extracellular polymeric substances (EPS) that spreads along the glassware, unlike in the control culture of Saccharomyces cerevisiae where no visible EPS production was observed 5 d post-inoculation and incubation in air-tight systems containing Hg°. Apparently, Hg° dissolution was observed with the strains of Yarrowia that was not noticeable in the culture of Saccharomyces cerevisiae as depicted in Fig. 1. Based on mass loss of the elemental mercury in the microtubes after 5 d incubation, approximately 300 ng and 500 ng Hg° was released from the microtubes into the air space of the Idd1 and Idd12 tightly closed culture systems, respectively. In the Idd1 culture, 260 ng of the aerial Hg° (constituting 88%) vividly dissolved into the culture system, while 240 ng of Hg° (constituting 49%) was observed to have dissolved into the culture system of Idd2. However, 35 ng and 250 ng of Hg° were unaccounted for in the cultures of Idd1 and Idd2, respectively.
2.5. Analyses of total mercury The total mercury in culture, EPS, and cell mass (100 μl) were determined directly without any pre-treatment using a fully automated thermal vaporization mercury analysis system, Mercury Analyzer/MA3000 (Nippon Instrument Corp., Osaka, Japan). Measurements of the atomized Hg were taken via atomic absorbance at a wavelength of 253.7 nm. The instrument was calibrated with standard Hg solution (BDH, Leicestershire, England) at concentrations ranging from 0.1 to 100.0 mg l−1. 2.6. TEM and XEDS Washed cell masses and EPS suspensions (before and after Hg2+ dissolution/oxidation) were deposited on copper Formvar-coated electron microscope grids that were placed on filter paper (to absorb excess fluid) without staining and allowed to dry. The dried specimens were covered with amorphous carbon film, and transmission electron microscope (TEM) micrographs were recorded with a JEM-2000FX II scanning transmission electron microscope (STEM) (JEOL JEM, Tokyo, Japan) that was equipped with X-ray energy dispersive spectrophotometer (XEDS) operating at an accelerating voltage of 200 kV and a magnification of 29.5 K. The images were acquired with a 2 K (2000 pixel × 2000 pixel) charged-couple device (CCD) camera (Ultrascan 1000; Gatan Inc., Pleasanton, CA, USA). The XEDS detector was used to acquire the characteristic X-ray spectra, and X-ray mapping was performed using XEDS in conjunction with a STEM module.
3.2. Hg° oxidation Dissolved gaseous mercury is highly volatile and can easily be removed from aqueous solutions via gas purging. The dissolved Hg° has
2.7. FTIR microscopy Chemical binding of Hg to cell mass and soluble EPS of the yeast strains upon Hg° dissolution and oxidation were elucidated using FTIRATR microscopy. Following Hg dissolution/oxidation experiment, EPS or cell mass pastes were mounted on a glass slide with a gold finish and analyzed in an open atmosphere using a micro FTIR spectrometer consisted of a microscope (BX51, Olympus, Tokyo, Japan) and an FTIR spectrometer (IlluminatIR, Smiths Group, London, UK). FTIR spectra were measured using attenuated transmission-reflection technique and collected from 400 to 4000 cm−1. Background spectrum was collected before mounting the biomass samples to make an average of 120 scans signals on each sample. Whereby, 10 spectra were collected from different spots on the sample slide so that heterogeneity in the sample could be evaluated. FTIR spectra were post-processed using the Grams/ 32 AO (6.00) software from Galactic Industries (Thermo Electron Corporation, Madison, WI, USA), corrected at baseline, and normalized to the height of the peak at 1240 cm−1 to account for differences in the thickness of the cells on the sample slide.
Fig. 1. Mass dissolution of Hg° from the air space into the culture of Yarrowia sp. strain Idd1 and Idd2. The control was culture of Saccharomyces cerevisiae. Hg° was supplied in attached and opened microfuge tube in air-tight jars, and agitation was achieved at approx. 80 rpm. Error bars were SEM of triplicate experiments. 245
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Table 1 Distribution of dissolved/oxidized Hg° in the culture of Yarrowia spp in closed batch systems. Control
EPS Cell masses
Yield (mg/litre) Oxidized Hg° (ng) Yield (gdw/litre) Oxidized Hg° (ng)
110 ± 5.8 ND 2.4 ± 0.1 ND
Idd1
Idd2
Without Hg°
With Hg°
Without Hg°
With Hg°
290 ± 21 ND 3.8 ± 0.21 ND
220 ± 18 120 ± 2.9 3.2 ± 0.18 110 ± 3.6
240 ± 18 ND 3.3 ± 0.12 ND
170 ± 12 62 ± 1.7 2.2 ± 0.12 59 ± 1.2
been reportedly oxidized in the dark in the presence of oxygen, chloride, and a suitable surface via first-order oxidation rate constants in coastal systems reaching values around 0.1 h−1 [15]. Sparging of culture with nitrogen gas was used to free the dissolved Hg° from culture system with assumption that oxidized elemental mercury (Hg2+) and possibly aqueous Hg° chelating the ligands produced by the yeast cells would be retained in the culture. Of the dissolved Hg°, approximately 230 ( ± 5.7) ng and 120 ( ± 6.8) ng were oxidized to Hg2+ by the five-day cultivation of Idd1 and Idd2, respectively (Table 1). Separating the cell mass from the culture revealed that 51% of the oxidized Hg2+ was linked to the EPS produced during growth of the Yarrowia strains and other parts of the Hg2+ were associated with the cell masses. Biogenic organic materials produced by algae have been reportedly able to promote Hg° oxidation to Hg2+ even under dark conditions [6]. Fig. 2 depicts concentrations of elemental mercury that were oxidized by the Yarrowia species as evidenced with total Hg in culture after N2-sparging. One can reasonably presume that the non-purgeable Hg detected must be Hg° already oxidized by the Yarrowia strains since the culture did not contain suspended particulate matter that might have bound dissolved Hg° as reported earlier [19]. Further hydrolyses of the culture with HNO3 (0.1 M for example), to dislodge possible weakly bound Hg°, still confirmed at least 290 ( ± 7.6) ng of elemental mercury was oxidized per milliliter of the Idd1 culture while 270 ( ± 8.5) ng ml−1 of Hg2+ was observed in Idd2 culture after sparging with N2 (Fig. 2a). Moreover, 510 ( ± 30) ng gdw−1 and 480 ( ± 7.7) ng gdw−1 of Hg2+ was observed on the cell masses of Idd1 and Idd2, respectively, after sparging with N2. While better acid hydrolyses of bound Hg2+ from the cell masses was achieved with 0.5 M HNO3 (Fig. 2b), there was no notable difference between 0.1 M and 0.5 M HNO3 hydrolyses (Fig. 2a). The mass balance of the oxidized Hg (Hg2+) with respect to Hg°
Fig. 3. Mass balance of oxidized Hg° to Hg2+ in the culture of Yarrowia spp. Idd1 and Idd2.
dissolved into the Yarrowia culture system is presented in Fig. 3. Greater mercury oxidation was observed with strain Idd1 where 45 ( ± 0.6) % and 43 ( ± 8.5) % Hg2+ were associated with EPS and cell masses, respectively. On the contrary, approximately 50 ( ± 2.6) % of the dissolved Hg° were assumed yet to be oxidized to Hg2+ by the Idd2 culture within five-day incubation that the experiment lasted. Attempt to account for the dissolved Hg° in tandem with the oxidized Hg2+ is depicted in Fig. 3, where better Hg° oxidation was observed with strain Idd1 (88 ± 2.3% oxidation) against only 49 ± 2.9% oxidation of Hg° Fig. 2. Oxidation of dissolved Hg° to Hg2+ in the culture (a), and quantity of the Hg2+ bound to the biomasses (b) of the Yarrowia spp. N2 gas sparging in a draft chamber was achieved for 10 min. at a flow rate of 0.2 l min−1, while positive control was without N2 gas sparging, and negative control was culture of Saccharomyces cerevisiae that was subjected the same conditions but could not oxidize Hg°.
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in strain Idd2. It is worthy of note that 45 ( ± 0.5) % of the Hg2+ was associated with the EPS produced by strain Idd1while 43 ( ± 1.7) % was found bound to the cell mass and only 12 ( ± 1.2) % of the Hg° was yet to be oxidized to Hg2+. As for strain Idd2, 50 ( ± 2.6) % of the Hg° was yet to be oxidized to Hg2+, while the Hg2+ apparently distributed among the EPS and the cell mass in equal proportion.
acids, NH out of plane amides, NH2 in plane bend amines and CeO stretch esters of the extracted EPS of culture with Hg° were inferred to play vital role in the dissolution of Hg°. It is noteworthy that fewer EPS yield (190 mg per liter) and dissolution of Hg° (49 ± 1.8%) was associated with EPS from Idd2 culture with Hg°, whereby none of the functional groups in the EPS really disappeared but rather present at different wavelength of the IR absorbance. When the OH in the alcohols and carboxylic functional groups of the EPS bound to Hg2+ coordination complex, a Hg2+−OH bending mode was observed at 945–943 cm−1 unlike at 919 cm−1 where adsorption of Hg2+ to surfaces of cell masses of the Yarrowia strains was observed. Thiolate (R–S−) or disulfide (–S–S–) functional groups was conspicuously absent among the vibrational groups of the IR spectra, which can be attributed to the fact that oxic conditions were prevalent during the incubation in this study. However, thiols, akin to alcohol chemistry, form thio-ethers and thioester that are accordingly analogous to ethers and esters, were detected in the IR spectra associated with the oxidation of dissolved Hg° in this study.
3.3. Semi-quantitative evidence of Hg° oxidation to Hg2+ in Yarrowia culture systems by XEDS analysis Evidences of Hg° oxidation to Hg2+ in the culture of the yeast strains were further established with transmission electron microscopy (TEM) coupled with X-ray energy dispersive spectroscopy (XEDS) as shown in Fig. A2 for cell masses and Fig. A3 for EPS. Many rugby balllike contrasts particles were observed in the mesh containing cell mass of culture where Hg° is dissolved and oxidized to Hg2+. XEDS examination revealed the presence of Hg along with other elements (Fig. A2, Panel e and h for strain Idd1 and Idd2, respectively) unlike the cell masses of the control experiment where no Hg was detected (Fig. A2c). Sparging with N2 and subsequent XEDS analysis still revealed Hg detection as evidence of oxidized Hg2+ bound onto the surfaces of the yeast cells (Fig. A2f and A2i). Similar observations were obtained when the EPS of the cultures were analyzed (Fig. A3). The TEM micrographs and X-ray spectra of XEDS examinations of extracted EPS revealed palpable dissolution of Hg° and complexation with Hg2+ upon oxidation of Hg° as shown in (Fig. A3, panel d and e for Idd1; and panel g and h for Idd2). It can be adduced that cellular binding of Hg2+ occurs after initial metal incorporation with the cell-bound EPS and neutralization of the chemically active sites. The XEDS spectra of the cell masses and EPS reveal that oxidizing elements, notably sulfur and chlorine, drive Hg° oxidation in the Yarrowia culture. Essential deposition of mercury on the EPS as evident in the absorption dynamics and the TEM-XEDS spectra (Figs. A2 and A.3) confirmed that extracellular dissolution and eventual oxidation of dissolved Hg° by Yarrowia spp. relied on EPS.
4. Discussion and conclusion On contrary to the natural postulates that atmospheric gaseous Hg° deposition into lithosphere and hydrosphere [20] upon aerial photochemical oxidation to Hg2+ [21], Hg° dissolution in this study was achieved via biological process. The work of Amyot et al. [15] revealed that an air-equilibrated solution at a chloride concentration near that of seawater made Hg° droplet dissolution a first-order rate constant. It is suggested that the Yarrowia strains in this study used their biomolecules in the EPS to harvest aerial Hg° independent of photochemical reactions and dissolve the Hg° into the culture system as purgeable Hg° since the incubation was in the dark. The Hg° dissolution is assumed to be maintained by efficient diffusion/advection of the purgeable dissolved Hg° that is produced. Microbial activities have been postulated to reduce levels of dissolved Hg° in lake water [12], with bacterial enzymes such as hydroperoxidase-catalase (KatG) and other unidentified catalases oxidizing Hg° to Hg2+ [16]. A number of catalase enzymes have been reportedly associated with adaptation of Yarrowia to extreme conditions [22], which might have come to play in the present study where the yeast strains might have activated mercury oxidase to transform Hg° to Hg2+. Unlike the submission of Amyot et al. [15] that the half-life of Hg° droplet in dissolution is 30 years, which get reduced to 5 years upon oxidation in oxygenated seawater; the oxidation of dissolved Hg° to Hg2+ in this study was achieved by the yeast species within five days. It has been reported that dissolved Hg° do rapidly oxidized in oxygenated solution in the presence of chloride [15,23] and thiols with electron acceptors in anoxic conditions [24]. It is worthy of note that Cl− thresholds in the XEDS spectra of cell masses and EPS with Hg° dissolution and oxidation remained more or less unchanged. The role of the chlorides may be catalytic for Hg° oxidation to Hg2+ without binding to the mercuric ion but rather being released to the culture system and made available for binding to anions such as sulfides. Increase in the threshold of XEDS spectra for sulfur in relation to Hg° dissolution and oxidation, unlike other elements indicated relevance of sulfur binding to oxidized Hg° to form micro-precipitation of Hg2+. The suggested HgS formation via interaction of Hg2+ with abundant sulfur sites that serve as ligands on the EPS and cell masses was evident by the characteristic patches of visual red colored precipitates observed in the Yarrowia biomass when the culture was centrifuged. The source of the sulphide is assumed to be compounds, like methionine, with non-thiol group containing sulfur with higher oxidation states since thiol groups do only function at anoxic conditions. It implies that the non-thiol group did not facilitate oxidation of dissolved Hg° but rather form complexation with already oxidized dissolved Hg2+, making it non-purgeable HgS micro-precipitates. Complexation of mercury species with sulfide or organo-sulfur compounds has been
3.4. Chemical analysis of Hg° oxidation by EPS and yeast biomass using FTIR-ATR microscopy The IR spectra in the cell masses (Fig. 4, Panel a and b) and EPS colloids (Fig. 4, Panel c and d) obtained from the two strains were similar, containing distinct, characteristic sharp stretching frequencies of functional chemical groups. The characteristic bands can be attributed to functional groups associated with proteins, lipids, and polysaccharides. Notable among these functional groups are amine, alcohol, amide, carbonyl, carboxylic acid, and carboxyl OeH group (see Table A1). Moreover, some weak bands indicating carboxylic groups in form of acid or basic salts were also observed in the EPS. Overall, it can be adduced from the characteristic IR spectra that the cell masses of the yeast strains and extracted EPS are polyfunctional compound as previously reported [18]. Interestingly, the organic molecules of the EPS and cell masses contained aromatic rings rather than aliphatic chains (see Table A1). The absence of RCOOH OeH bend carboxylic acid, CeO stretches of alcohols (R2CHOH) and ethers (ReOeR) and dimer OH carboxylic acids at 919, 1097, 2939 cm−1, respectively, indicated the relevance of the functional groups in cell masses of strain Idd1 during with Hg° dissolution and oxidation. Of significance is the CeO stretch of ReOeR that must have led to emergence of AreOeR at absorbance 1252 cm−1 during interaction of the cell masses with Hg° and probably adsorption of the oxidized Hg° (Hg2+). Similar disappearances of RCOOH OeH bend, CeO stretches, Ar-N stretches for amines, carboxylic acids, alcohols, ethers, and esters along with appearances of NH out of plane amides, NH2 in plane bend amines and C]O stretch esters in the cell masses of Idd2 were linked to Hg° dissolution and eventual oxidation to Hg2+. N-H wag amines, CeO stretch phenols, CeO stretch carboxylic 247
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Fig. 4. Infra-red spectra of cell masses (Idd1 in Panel a; Idd2 in Panel b) and EPS (Idd1 in Panel c; Idd2 in Panel d) after dissolution of Hg° and oxidation to Hg2+, using FTIR-ATR microscopy. FTIR spectra were collected from 400 to 4000 cm-1. The spectra of the cell-mass/EPS only (without Hg° dissolution/oxidation) are shown in blue lines while those in red lines are cell-mass/EPS + Hg (with Hg° dissolution/oxidation) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
alcohols and carboxyl as the oxidants.
reported as a mercury tolerance mechanism in Yarrowia [18,25] and other microorganisms (Glendinning et al., 2005). HgS micro-precipitates, if found in deep benthic zone of surface water, may become substrates for Hg methylation by sulfate-reducing bacteria, causing gross pollution of global concern in the environment. Presence of alcohols and aldehydes in EPS promotes higher oxidative potential that may possibly triggers oxidation of Hg°, whereby the hydroxyl radical initiates oxidation reactions to form Hg2+. Plausible explanations of the involvement of hydroxyl radicals in Hg° oxidation to Hg2+ have been reported during photolysis of dissolved organic matter [26], and photochemistry of solar UV-B radiation [13]. Moreover, it is reported that aqueous Hg° oxidation involves reaction with photochemically produced OH* that spur production of aqueous halogen radicals when reacted with marine halides or the formation of stable Hg2+ complexes that triggers greater oxidation of Hg° [23]. Our findings provided information about mediatory role played by the yeasts isolated from polluted aquatic environment during novel airto-water dissolution of atmospheric Hg°, and oxidation of dissolved Hg° to Hg2+ during global biogeochemical cycling of Hg. This is highlighted in the following synopsis:
• The dissolved Hg° may be available in the hydrosphere as HgS micro-precipitate or soluble Hg°S. • The soluble oxidized Hg and Hg°S serve as raw materials for 2+
methylation by sulfate reducing bacteria, posing potent environmental danger upon formation of neurotoxic methyl-mercury in the hydrosphere.
Conflict of interest Each of the Authors has no competing moral or financial interest in relation to the work described. Authors’ contribution GOO participated in experimental design, sample collection, experimentation, collation of data, and manuscript preparation; KM and HS participated in experimentation, and data collation; while GE designed and supervised the experiment, participated in data interpretation and manuscript preparation.
• Species of mercury resistant Yarrowia spp. (Idd1 and Idd2) facilitate • •
Acknowledgements
dissolution of aerial Hg° into aquatic system through the EPS they produced. The dissolved purgeable Hg° in the hydrosphere is further transformed into non-purgeable Hg° via complexation with biomolecules of the yeast to bioavailable Hg2+ via Yarrowia-dependent oxidation. The oxidized, non-purgeable Hg2+/Hg° bound with the cell mass and the EPS of the Yarrowia spp., using the hydroxyl radicals of the
We thank Dr. Mei-Fang Chien, Mr. Satoru Ishikawa and Dr. Atsushi Okamoto of Tohoku University, Japan, for their useful discussion and assistance during the experiment and permission to use FTIR-ATR facility. We also thank the Japan Society for the Promotion of Science for the Postdoctoral Research Fellowship FY2012-2013 to GOO. This work was funded with Grants-in-Aid (No. 24-02373) for Scientific Research 248
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from the JSPS.
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.02.075. References [1] W.F. Fitzgerald, R.P. Mason, G.M. Vandal, Atmospheric cycling and air-water exchange of mercury over mid-continental lacustrine regions, Water Air Soil Pollut. 56 (1991) 745–767. [2] S. Dominika, B. Magdalena, B. Jacek, S. Michal, G. Karolina, S. Marta, W. Agnieszka, Impact of intense rains and flooding on mercury riverine input to the coastal zone, Mar. Pollut. Bull. 127 (2018) 593–602. [3] F. De Simone, C.N. Gencarelli, L.M. Hedgecock, N. Pirrone, Global atmospheric cycle of mercury: a model study on the impact of oxidation mechanisms, Environ. Sci. Pollut. Res. 21 (2014) 4110–4123. [4] F. He, W. Zhao, L. Liang, B. Gu, Photochemical oxidation of dissolved elemental mercury by carbonate radicals in water, Environ. Sci. Technol. Lett. 1 (2014) 499–503. [5] A.L. Soerensen, R.P. Mason, P.H. Balcom, E.M. Sunderland, Drivers of surface ocean mercury concentrations and air-sea exchange in the West Atlantic Ocean, Environ. Sci. Technol. 47 (2013) 7757–7765. [6] A.J. Poulain, E. Garcia, M. Amyot, P.G.C. Campbell, F. Raofie, P.A. Ariya, Biological and chemical redox transformations of mercury in fresh and salt waters of the high arctic during spring and summer, Environ. Sci. Technol. 41 (2007) 1883–1888. [7] M.A. Walvoord, B.J. Andraski, D.P. Krabbenhoft, R.G. Striegl, Transport of elemental mercury in the unsaturated zone from a waste disposal site in an arid region, Appl. Geochem. 23 (2008) 572–583. [8] E.A. Murphy, J. Dooley, H.L. Windom, R.G. Smith, Mercury species in potable ground water in southern New Jersey, Water Air Soil Pollut. 78 (1994) 61–72. [9] S.E. Bone, M.A. Charette, C.H. Lamborg, M.E. Gonneea, Has submarine groundwater discharge been overlooked as a source mercury to coastal waters? Environ. Sci. Technol. 41 (2007) 3090–3095. [10] F.M.M. Morel, A.M.L. Kraepiel, M. Amyot, The chemical role and bioaccumulation of mercury, Annu. Rev. Ecol. Syst. 29 (1998) 543–566. [11] N.J. O’Driscoll, E. Vost, E. Mann, S. Klapstein, R. Tordon, M. Lukeman, Mercury photoreduction and photooxidation in lakes: Effects of filtration and dissolved carbon concentration, J. Environ. Sci. (2017), https://doi.org/10.1016/j.jes.2017. 12.010. [12] S.D. Siciliano, N.J. O’Driscoll, D.R.S. Lean, Microbial reduction and oxidation of
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Bio-oxidation of elemental mercury during growth of mercury resistant yeasts in simulated hydrosphere Ganiyu Oladunjoye Oyetiboa,b,1, Keisuke Miyauchia, Hitoshi Suzukia, Ginro Endoa, a b
T
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Department of Civil and Environmental Engineering, Faculty of Engineering, Tohoku-Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537, Japan Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Yaba, Lagos, Nigeria
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Mercury dissolution Mercury oxidation Hg-resistant Yarrowia Mercury cycle Extracellular polymeric substances (EPS)
Transformation of metallic mercury (Hg°) to mercuric ion (Hg2+) in hydrosphere is the entrance of mercury cycle in water environments and leads to toxicological impact of serious global concern. Two yeast strains of Yarrowia (Idd1 and Idd2) isolated from Hg-contaminated sediments were studied for their mediating role in Hg° dissolution and oxidation. Growth of the Yarrowia cells in Hg-free liquid medium, incubated for 5 d in closed airtight systems containing Hg°, produced extracellular polymeric substances (EPS). Approximately 230 ( ± 5.7) ng and 120 ( ± 6.8) ng of the dissolved Hg° were oxidized to Hg2+ by the cultures of Idd1 and Idd2, respectively, 5 day post-inoculation. Transmission electron microscopy (TEM) and X-ray energy dispersive spectrophotometry (XEDS) analysis of the EPS and cell mass revealed the presence of extracellular Hg nanoparticles, presumably HgS, as an indication of EPS-Hg complexation that is useful for Hg° dissolution and its eventual oxidation to Hg2+ by the cells. Fourier transmission infra-red (FTIR) analyses of the EPS and cell-mass during Hg-oxidation revealed that amine and carbonyl groups were used by EPS for Hg complexation. Our findings provided information about mediatory role played by Yarrowia (Idd1 and Idd2) in hydrosphere in biogeochemical cycling of Hg.
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Corresponding author. E-mail addresses: [email protected], [email protected] (G.O. Oyetibo), [email protected] (K. Miyauchi), [email protected] (H. Suzuki), [email protected] (G. Endo). 1 Current address: Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Yaba, Lagos, Nigeria. https://doi.org/10.1016/j.jhazmat.2019.02.075 Received 26 January 2019; Accepted 20 February 2019 Available online 20 March 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
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1. Introduction
ability of yeasts to oxidize Hg° has not been explored. In this study, we sought to determine the dissolution of aerial Hg° gas into aqueous system as dissolved Hg°, and eventual oxidation to non-purgeable Hg2+. Strains of previously isolated Hg-resistant yeasts, Yarrowia spp. (Idd1 and Idd2), were used as microbial models to actualize dissolution and bio-oxidation of Hg° in simulated hydrosphere as a critical stage of biogeochemical cycling of mercury.
Mercury (Hg) is one of the most toxic trace elements on earth and equally one of the most elusive because of the volatility of elemental mercury (Hg°) and the high reactivity of the mercuric ion (Hg2+) [1]. Besides geological phenomena such as volcanic activity, the concentration of Hg in the environment has been on rise globally due to increase in anthropogenic activities including artisanal gold mining, fossil fuel combustion, smelting activities, urbanization, agricultural practices and industrial processes among others [2]. These human activities release Hg° vapor into the atmosphere, where the mercury vapor can circulate for up to a year, becoming widely dispersed due to its relatively slow reactivity with the more abundant oxidants in the atmosphere [3]. The Hg° vapor is reportedly undergone photochemical oxidation to become inorganic Hg2+ that can combine with water vapors and travel back to the Earth’s surface as rain [4]. This ‘mercurywater’ solution is deposited in soils and bodies of water from where accumulation to animate and inanimate matrixes do occur until a physical event causes it to be released again. Dissolved Hg° is a ubiquitous form of inorganic mercury in marine and terrestrial water [5]. Previous studies have shown that surface water, soil, groundwater, and ocean can accumulate high concentrations of Hg° [5–7]. In terrestrial matrix, the loss of gaseous Hg° to the atmosphere is pivotal to decreases of the Hg pool remaining in watersheds [5]. Hg° can remain dissolved in water and be mobilized by groundwater advection of saturated sediments, where gas-exchange is restricted, forming groundwater Hg° that has been reportedly discharged to drinking-water wells [8]. Bone et al. [9] suggested that such groundwater Hg° contribute to the unexpectedly low partition coefficient of Hg in confined aquifers due to its low affinity for sediment surfaces. Conversion of inorganic mercury via methylation into neurotoxic methylmercury (MeHg) in anoxic sediments during microbial processes is a critical step that governs the transfer of Hg to aquatic food webs and its biomagnification in higher trophic levels [5]. Since Hg° is generally considered to be unreactive [10], its formation and transport are thought to limit the amount of Hg available for methylation [1]. However, Hg° is oxidized to Hg2+ by physical, chemical and biological processes in aquatic environments [4,11,12]. Mercury (Hg°) oxidation to mercuric ion (Hg2+) in hydrosphere is the entrance of mercury cycle in water environments. For emphasis, Hg° oxidation is reportedly controlled by photochemical reactions in surficial waters exposed to sunlight [4]. Laboratory investigations of natural and artificial waters by Lalonde et al. [13] have shown that the oxidation of Hg° by solar irradiation is linked to photochemical production of reactive compounds such as hydroxyl radicals. Dissolved Hg° can also be oxidized in the dark by unfiltered seawater where oxygen serves as the most likely oxidant [14]. Furthermore, experimental studies have shown that presence of O2 and Cl− or thiol compounds can directly oxidize drops of liquid Hg° in water [15]. Whilst the presence of reactive ionic species (CO32- and NO3−) triggers much higher Hg° photo-oxidation rate, dissolved organic matter (DOM) decreases the oxidation rate by half [4]. The microbial oxidation of Hg° to Hg2+ and eventual transformation to MeHg is known to occur among some bacterial strains [12,16]. Thus far, aerobic and phototrophic microorganisms have been implicated as the primary microbial agents driving Hg° oxidation reactions. Smith et al. [16] showed that the catalase enzymes in Escherichia coli enhances dissolution of Hg° into water, with eventual Hg° oxidation activity associated with the cytosolic catalase/hydroperoxidase proteins (KatG and KatE). However, a double mutant lacking both the katG and katE genes found to retain the ability to oxidize Hg° suggests the existence of other bacterial oxidation pathways that are currently uncharacterized. This has been observed with aerobic soil bacteria, Bacillus and Streptomyces, exhibiting high levels of Hg° oxidizing activity [16], and consequently illustrating the potential for microbial oxidation during cycling of Hg in soils. Currently, the subsurface microbial processes involved in Hg oxidation remain poorly understood, and the
2. Materials and methods 2.1. Culture condition Stock cells of the Hg-resistant yeast Yarrowia spp that was previously isolated from polluted estuarine sediment and deposited to Japan Collection of Microorganisms under Accession no. JCM 30162 (strain Idd1), and JCM 30163 (strain Idd2) (stored at −80 °C in 1:1 glycerol:YM broth) [17] were obtained. The yeast cells were resuscitated by inoculating into sterile Bacto YM broth (Becton, Dickinson and Co, Sparks, MD, USA). After incubation (30 °C; 50×g; 72 h), 100 μl culture was spread on dried surface of sterile Bacto YM broth (containing per liter: 5 g, yeast extract; 3 g, malt extract; 5 g, peptone; and 10 g, dextrose) that was solidified with 1.5% agar (Wako, Osaka, Japan). Young, distinct colony (24 h post-inoculation, 30 °C) was inoculated into YM broth (200 ml in 1 L Erlenmeyer flask) and incubated (30 °C; 50×g; 96 h) to obtain inoculum of the resuscitated yeast strains (approx. 106 cells ml−1) for subsequent experiments.
2.2. Preparation of batch culture system for Hg° dissolution and oxidation Ability of the yeast strains to dissolve aerial Hg° into broth system as a simulation of hydrosphere was studied in a batch culture of Hg-free YM broth. A 250 ml Erlenmeyer flask containing 30 ml Hg-free YM broth with a magnetic stirrer bar was inoculated with 0.1 ml culture of the Yarrowia strains Idd1 and Idd2 in Section 2.1 above (see Fig. A1). The inoculated flask was placed in an air-tight jar, closed 1.5 ml microcentrifuge tube containing approximately 900 mg metallic Hg° was attached to the flask, the microtube was opened and the jar was immediately air-tight closed to prevent escape of Hg° from the jar. The setup was placed on a magnetic stirrer device to provide mixing of the culture at 100 rpm and altogether placed inside a dark incubator (30 °C, 5 d). For the control set-up, Hg-free YM broth was inoculated with Saccharomyces cerevisiae and subjected the same conditions as applicable to the Yarrowia strains above. Moreover, negative controls of the Yarrowia culture systems but without Hg° containing microtube attachments were set up as well. All experiments were repeated three times.
2.3. Determination of Hg° dissolution and oxidation To quantify mass of Hg° released from the micro-centrifuge tube to the aerospace, difference in the weight of micro-centrifuge tube containing Hg° before and after incubation was measured. The culture were analyzed for total Hg to determine the Hg° dissolved into the medium using Mercury Analyzer as explained in Section 2.5 below. To quantify Hg° oxidation, the culture was divided into three portions: one portion a piece was treated with 0.1 M HNO3, 0.5 M HNO3, and without acid hydrolysis. Afterwards, each portion was divided into two portions and a set was subjected to oxygen-free N2 gas sparging in a draft chamber for 10 min. at a flow rate of 0.2 l min−1, while the other portion was without N2 gas sparging. Subsequently, each portion was analyzed for total mercury using Mercury Analyzer as explained in Section 2.5 below.
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2.8. Statistical analyses
2.4. Determination dissolved/oxidized Hg° in Yarrowia cell mass and biopolymer
All statistical tests including column statistics, row statistics, and non-linear regression (curve fit) of the least squares were performed using the Prism 5 software program (GraphPad Software, San Diego, CA, USA).
This experiment was meant to establish presence of non-purgeable oxidized Hg2+ in the yeast biopolymer and cell mass. The yeast culture was centrifuged (4 °C; 6000×g; 30 min) to separate the cell mass and the supernatant. Method reported by Oyetibo et al. [18] was used in the isolation of soluble extracellular polymeric substances (EPS) from the supernatants. The supernatants were pressure-filtered through 0.45 μm pore size Minisart syringe filters (Sartorius Stedim Biotech, Gottingen, Germany) and kept overnight at 4 °C after treatment with cold 99% ethanol (ethanol:filtrate, 2:1). The EPS precipitates were recovered by centrifugation (4 °C; 6000×g; 30 min). The pellets were re-suspended in sterile water, dialyzed against sterile MilliQ water, lyophilized, and stored at 4 °C, or otherwise analyzed for Hg after resuspension with 1.0 ml MilliQ water using Mercury Analyzer as explained in Section 2.5 below. However, the cell mass precipitated after centrifugation was analyzed for Hg using the Mercury Analyzer, too.
3. Results 3.1. Hg° dissolution The deposition, mobility and cycling of mercury in natural environments are critical to global mercury pollution and significantly dependent on speciation and presence of ligands that would spell out if it remains in solution or as a dissolved gas. For emphasis, Hg° can be present in two forms in hydrosphere as liquid Hg°, which is rarely observed in uncontaminated natural waters; and aqueous Hg° that is ubiquitous and of environmental relevance. Characteristic growth of Hg-resistant Yarrowia spp. (Idd1 and Idd2) in Hg-free media produced extracellular polymeric substances (EPS) that spreads along the glassware, unlike in the control culture of Saccharomyces cerevisiae where no visible EPS production was observed 5 d post-inoculation and incubation in air-tight systems containing Hg°. Apparently, Hg° dissolution was observed with the strains of Yarrowia that was not noticeable in the culture of Saccharomyces cerevisiae as depicted in Fig. 1. Based on mass loss of the elemental mercury in the microtubes after 5 d incubation, approximately 300 ng and 500 ng Hg° was released from the microtubes into the air space of the Idd1 and Idd12 tightly closed culture systems, respectively. In the Idd1 culture, 260 ng of the aerial Hg° (constituting 88%) vividly dissolved into the culture system, while 240 ng of Hg° (constituting 49%) was observed to have dissolved into the culture system of Idd2. However, 35 ng and 250 ng of Hg° were unaccounted for in the cultures of Idd1 and Idd2, respectively.
2.5. Analyses of total mercury The total mercury in culture, EPS, and cell mass (100 μl) were determined directly without any pre-treatment using a fully automated thermal vaporization mercury analysis system, Mercury Analyzer/MA3000 (Nippon Instrument Corp., Osaka, Japan). Measurements of the atomized Hg were taken via atomic absorbance at a wavelength of 253.7 nm. The instrument was calibrated with standard Hg solution (BDH, Leicestershire, England) at concentrations ranging from 0.1 to 100.0 mg l−1. 2.6. TEM and XEDS Washed cell masses and EPS suspensions (before and after Hg2+ dissolution/oxidation) were deposited on copper Formvar-coated electron microscope grids that were placed on filter paper (to absorb excess fluid) without staining and allowed to dry. The dried specimens were covered with amorphous carbon film, and transmission electron microscope (TEM) micrographs were recorded with a JEM-2000FX II scanning transmission electron microscope (STEM) (JEOL JEM, Tokyo, Japan) that was equipped with X-ray energy dispersive spectrophotometer (XEDS) operating at an accelerating voltage of 200 kV and a magnification of 29.5 K. The images were acquired with a 2 K (2000 pixel × 2000 pixel) charged-couple device (CCD) camera (Ultrascan 1000; Gatan Inc., Pleasanton, CA, USA). The XEDS detector was used to acquire the characteristic X-ray spectra, and X-ray mapping was performed using XEDS in conjunction with a STEM module.
3.2. Hg° oxidation Dissolved gaseous mercury is highly volatile and can easily be removed from aqueous solutions via gas purging. The dissolved Hg° has
2.7. FTIR microscopy Chemical binding of Hg to cell mass and soluble EPS of the yeast strains upon Hg° dissolution and oxidation were elucidated using FTIRATR microscopy. Following Hg dissolution/oxidation experiment, EPS or cell mass pastes were mounted on a glass slide with a gold finish and analyzed in an open atmosphere using a micro FTIR spectrometer consisted of a microscope (BX51, Olympus, Tokyo, Japan) and an FTIR spectrometer (IlluminatIR, Smiths Group, London, UK). FTIR spectra were measured using attenuated transmission-reflection technique and collected from 400 to 4000 cm−1. Background spectrum was collected before mounting the biomass samples to make an average of 120 scans signals on each sample. Whereby, 10 spectra were collected from different spots on the sample slide so that heterogeneity in the sample could be evaluated. FTIR spectra were post-processed using the Grams/ 32 AO (6.00) software from Galactic Industries (Thermo Electron Corporation, Madison, WI, USA), corrected at baseline, and normalized to the height of the peak at 1240 cm−1 to account for differences in the thickness of the cells on the sample slide.
Fig. 1. Mass dissolution of Hg° from the air space into the culture of Yarrowia sp. strain Idd1 and Idd2. The control was culture of Saccharomyces cerevisiae. Hg° was supplied in attached and opened microfuge tube in air-tight jars, and agitation was achieved at approx. 80 rpm. Error bars were SEM of triplicate experiments. 245
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Table 1 Distribution of dissolved/oxidized Hg° in the culture of Yarrowia spp in closed batch systems. Control
EPS Cell masses
Yield (mg/litre) Oxidized Hg° (ng) Yield (gdw/litre) Oxidized Hg° (ng)
110 ± 5.8 ND 2.4 ± 0.1 ND
Idd1
Idd2
Without Hg°
With Hg°
Without Hg°
With Hg°
290 ± 21 ND 3.8 ± 0.21 ND
220 ± 18 120 ± 2.9 3.2 ± 0.18 110 ± 3.6
240 ± 18 ND 3.3 ± 0.12 ND
170 ± 12 62 ± 1.7 2.2 ± 0.12 59 ± 1.2
been reportedly oxidized in the dark in the presence of oxygen, chloride, and a suitable surface via first-order oxidation rate constants in coastal systems reaching values around 0.1 h−1 [15]. Sparging of culture with nitrogen gas was used to free the dissolved Hg° from culture system with assumption that oxidized elemental mercury (Hg2+) and possibly aqueous Hg° chelating the ligands produced by the yeast cells would be retained in the culture. Of the dissolved Hg°, approximately 230 ( ± 5.7) ng and 120 ( ± 6.8) ng were oxidized to Hg2+ by the five-day cultivation of Idd1 and Idd2, respectively (Table 1). Separating the cell mass from the culture revealed that 51% of the oxidized Hg2+ was linked to the EPS produced during growth of the Yarrowia strains and other parts of the Hg2+ were associated with the cell masses. Biogenic organic materials produced by algae have been reportedly able to promote Hg° oxidation to Hg2+ even under dark conditions [6]. Fig. 2 depicts concentrations of elemental mercury that were oxidized by the Yarrowia species as evidenced with total Hg in culture after N2-sparging. One can reasonably presume that the non-purgeable Hg detected must be Hg° already oxidized by the Yarrowia strains since the culture did not contain suspended particulate matter that might have bound dissolved Hg° as reported earlier [19]. Further hydrolyses of the culture with HNO3 (0.1 M for example), to dislodge possible weakly bound Hg°, still confirmed at least 290 ( ± 7.6) ng of elemental mercury was oxidized per milliliter of the Idd1 culture while 270 ( ± 8.5) ng ml−1 of Hg2+ was observed in Idd2 culture after sparging with N2 (Fig. 2a). Moreover, 510 ( ± 30) ng gdw−1 and 480 ( ± 7.7) ng gdw−1 of Hg2+ was observed on the cell masses of Idd1 and Idd2, respectively, after sparging with N2. While better acid hydrolyses of bound Hg2+ from the cell masses was achieved with 0.5 M HNO3 (Fig. 2b), there was no notable difference between 0.1 M and 0.5 M HNO3 hydrolyses (Fig. 2a). The mass balance of the oxidized Hg (Hg2+) with respect to Hg°
Fig. 3. Mass balance of oxidized Hg° to Hg2+ in the culture of Yarrowia spp. Idd1 and Idd2.
dissolved into the Yarrowia culture system is presented in Fig. 3. Greater mercury oxidation was observed with strain Idd1 where 45 ( ± 0.6) % and 43 ( ± 8.5) % Hg2+ were associated with EPS and cell masses, respectively. On the contrary, approximately 50 ( ± 2.6) % of the dissolved Hg° were assumed yet to be oxidized to Hg2+ by the Idd2 culture within five-day incubation that the experiment lasted. Attempt to account for the dissolved Hg° in tandem with the oxidized Hg2+ is depicted in Fig. 3, where better Hg° oxidation was observed with strain Idd1 (88 ± 2.3% oxidation) against only 49 ± 2.9% oxidation of Hg° Fig. 2. Oxidation of dissolved Hg° to Hg2+ in the culture (a), and quantity of the Hg2+ bound to the biomasses (b) of the Yarrowia spp. N2 gas sparging in a draft chamber was achieved for 10 min. at a flow rate of 0.2 l min−1, while positive control was without N2 gas sparging, and negative control was culture of Saccharomyces cerevisiae that was subjected the same conditions but could not oxidize Hg°.
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in strain Idd2. It is worthy of note that 45 ( ± 0.5) % of the Hg2+ was associated with the EPS produced by strain Idd1while 43 ( ± 1.7) % was found bound to the cell mass and only 12 ( ± 1.2) % of the Hg° was yet to be oxidized to Hg2+. As for strain Idd2, 50 ( ± 2.6) % of the Hg° was yet to be oxidized to Hg2+, while the Hg2+ apparently distributed among the EPS and the cell mass in equal proportion.
acids, NH out of plane amides, NH2 in plane bend amines and CeO stretch esters of the extracted EPS of culture with Hg° were inferred to play vital role in the dissolution of Hg°. It is noteworthy that fewer EPS yield (190 mg per liter) and dissolution of Hg° (49 ± 1.8%) was associated with EPS from Idd2 culture with Hg°, whereby none of the functional groups in the EPS really disappeared but rather present at different wavelength of the IR absorbance. When the OH in the alcohols and carboxylic functional groups of the EPS bound to Hg2+ coordination complex, a Hg2+−OH bending mode was observed at 945–943 cm−1 unlike at 919 cm−1 where adsorption of Hg2+ to surfaces of cell masses of the Yarrowia strains was observed. Thiolate (R–S−) or disulfide (–S–S–) functional groups was conspicuously absent among the vibrational groups of the IR spectra, which can be attributed to the fact that oxic conditions were prevalent during the incubation in this study. However, thiols, akin to alcohol chemistry, form thio-ethers and thioester that are accordingly analogous to ethers and esters, were detected in the IR spectra associated with the oxidation of dissolved Hg° in this study.
3.3. Semi-quantitative evidence of Hg° oxidation to Hg2+ in Yarrowia culture systems by XEDS analysis Evidences of Hg° oxidation to Hg2+ in the culture of the yeast strains were further established with transmission electron microscopy (TEM) coupled with X-ray energy dispersive spectroscopy (XEDS) as shown in Fig. A2 for cell masses and Fig. A3 for EPS. Many rugby balllike contrasts particles were observed in the mesh containing cell mass of culture where Hg° is dissolved and oxidized to Hg2+. XEDS examination revealed the presence of Hg along with other elements (Fig. A2, Panel e and h for strain Idd1 and Idd2, respectively) unlike the cell masses of the control experiment where no Hg was detected (Fig. A2c). Sparging with N2 and subsequent XEDS analysis still revealed Hg detection as evidence of oxidized Hg2+ bound onto the surfaces of the yeast cells (Fig. A2f and A2i). Similar observations were obtained when the EPS of the cultures were analyzed (Fig. A3). The TEM micrographs and X-ray spectra of XEDS examinations of extracted EPS revealed palpable dissolution of Hg° and complexation with Hg2+ upon oxidation of Hg° as shown in (Fig. A3, panel d and e for Idd1; and panel g and h for Idd2). It can be adduced that cellular binding of Hg2+ occurs after initial metal incorporation with the cell-bound EPS and neutralization of the chemically active sites. The XEDS spectra of the cell masses and EPS reveal that oxidizing elements, notably sulfur and chlorine, drive Hg° oxidation in the Yarrowia culture. Essential deposition of mercury on the EPS as evident in the absorption dynamics and the TEM-XEDS spectra (Figs. A2 and A.3) confirmed that extracellular dissolution and eventual oxidation of dissolved Hg° by Yarrowia spp. relied on EPS.
4. Discussion and conclusion On contrary to the natural postulates that atmospheric gaseous Hg° deposition into lithosphere and hydrosphere [20] upon aerial photochemical oxidation to Hg2+ [21], Hg° dissolution in this study was achieved via biological process. The work of Amyot et al. [15] revealed that an air-equilibrated solution at a chloride concentration near that of seawater made Hg° droplet dissolution a first-order rate constant. It is suggested that the Yarrowia strains in this study used their biomolecules in the EPS to harvest aerial Hg° independent of photochemical reactions and dissolve the Hg° into the culture system as purgeable Hg° since the incubation was in the dark. The Hg° dissolution is assumed to be maintained by efficient diffusion/advection of the purgeable dissolved Hg° that is produced. Microbial activities have been postulated to reduce levels of dissolved Hg° in lake water [12], with bacterial enzymes such as hydroperoxidase-catalase (KatG) and other unidentified catalases oxidizing Hg° to Hg2+ [16]. A number of catalase enzymes have been reportedly associated with adaptation of Yarrowia to extreme conditions [22], which might have come to play in the present study where the yeast strains might have activated mercury oxidase to transform Hg° to Hg2+. Unlike the submission of Amyot et al. [15] that the half-life of Hg° droplet in dissolution is 30 years, which get reduced to 5 years upon oxidation in oxygenated seawater; the oxidation of dissolved Hg° to Hg2+ in this study was achieved by the yeast species within five days. It has been reported that dissolved Hg° do rapidly oxidized in oxygenated solution in the presence of chloride [15,23] and thiols with electron acceptors in anoxic conditions [24]. It is worthy of note that Cl− thresholds in the XEDS spectra of cell masses and EPS with Hg° dissolution and oxidation remained more or less unchanged. The role of the chlorides may be catalytic for Hg° oxidation to Hg2+ without binding to the mercuric ion but rather being released to the culture system and made available for binding to anions such as sulfides. Increase in the threshold of XEDS spectra for sulfur in relation to Hg° dissolution and oxidation, unlike other elements indicated relevance of sulfur binding to oxidized Hg° to form micro-precipitation of Hg2+. The suggested HgS formation via interaction of Hg2+ with abundant sulfur sites that serve as ligands on the EPS and cell masses was evident by the characteristic patches of visual red colored precipitates observed in the Yarrowia biomass when the culture was centrifuged. The source of the sulphide is assumed to be compounds, like methionine, with non-thiol group containing sulfur with higher oxidation states since thiol groups do only function at anoxic conditions. It implies that the non-thiol group did not facilitate oxidation of dissolved Hg° but rather form complexation with already oxidized dissolved Hg2+, making it non-purgeable HgS micro-precipitates. Complexation of mercury species with sulfide or organo-sulfur compounds has been
3.4. Chemical analysis of Hg° oxidation by EPS and yeast biomass using FTIR-ATR microscopy The IR spectra in the cell masses (Fig. 4, Panel a and b) and EPS colloids (Fig. 4, Panel c and d) obtained from the two strains were similar, containing distinct, characteristic sharp stretching frequencies of functional chemical groups. The characteristic bands can be attributed to functional groups associated with proteins, lipids, and polysaccharides. Notable among these functional groups are amine, alcohol, amide, carbonyl, carboxylic acid, and carboxyl OeH group (see Table A1). Moreover, some weak bands indicating carboxylic groups in form of acid or basic salts were also observed in the EPS. Overall, it can be adduced from the characteristic IR spectra that the cell masses of the yeast strains and extracted EPS are polyfunctional compound as previously reported [18]. Interestingly, the organic molecules of the EPS and cell masses contained aromatic rings rather than aliphatic chains (see Table A1). The absence of RCOOH OeH bend carboxylic acid, CeO stretches of alcohols (R2CHOH) and ethers (ReOeR) and dimer OH carboxylic acids at 919, 1097, 2939 cm−1, respectively, indicated the relevance of the functional groups in cell masses of strain Idd1 during with Hg° dissolution and oxidation. Of significance is the CeO stretch of ReOeR that must have led to emergence of AreOeR at absorbance 1252 cm−1 during interaction of the cell masses with Hg° and probably adsorption of the oxidized Hg° (Hg2+). Similar disappearances of RCOOH OeH bend, CeO stretches, Ar-N stretches for amines, carboxylic acids, alcohols, ethers, and esters along with appearances of NH out of plane amides, NH2 in plane bend amines and C]O stretch esters in the cell masses of Idd2 were linked to Hg° dissolution and eventual oxidation to Hg2+. N-H wag amines, CeO stretch phenols, CeO stretch carboxylic 247
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Fig. 4. Infra-red spectra of cell masses (Idd1 in Panel a; Idd2 in Panel b) and EPS (Idd1 in Panel c; Idd2 in Panel d) after dissolution of Hg° and oxidation to Hg2+, using FTIR-ATR microscopy. FTIR spectra were collected from 400 to 4000 cm-1. The spectra of the cell-mass/EPS only (without Hg° dissolution/oxidation) are shown in blue lines while those in red lines are cell-mass/EPS + Hg (with Hg° dissolution/oxidation) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
alcohols and carboxyl as the oxidants.
reported as a mercury tolerance mechanism in Yarrowia [18,25] and other microorganisms (Glendinning et al., 2005). HgS micro-precipitates, if found in deep benthic zone of surface water, may become substrates for Hg methylation by sulfate-reducing bacteria, causing gross pollution of global concern in the environment. Presence of alcohols and aldehydes in EPS promotes higher oxidative potential that may possibly triggers oxidation of Hg°, whereby the hydroxyl radical initiates oxidation reactions to form Hg2+. Plausible explanations of the involvement of hydroxyl radicals in Hg° oxidation to Hg2+ have been reported during photolysis of dissolved organic matter [26], and photochemistry of solar UV-B radiation [13]. Moreover, it is reported that aqueous Hg° oxidation involves reaction with photochemically produced OH* that spur production of aqueous halogen radicals when reacted with marine halides or the formation of stable Hg2+ complexes that triggers greater oxidation of Hg° [23]. Our findings provided information about mediatory role played by the yeasts isolated from polluted aquatic environment during novel airto-water dissolution of atmospheric Hg°, and oxidation of dissolved Hg° to Hg2+ during global biogeochemical cycling of Hg. This is highlighted in the following synopsis:
• The dissolved Hg° may be available in the hydrosphere as HgS micro-precipitate or soluble Hg°S. • The soluble oxidized Hg and Hg°S serve as raw materials for 2+
methylation by sulfate reducing bacteria, posing potent environmental danger upon formation of neurotoxic methyl-mercury in the hydrosphere.
Conflict of interest Each of the Authors has no competing moral or financial interest in relation to the work described. Authors’ contribution GOO participated in experimental design, sample collection, experimentation, collation of data, and manuscript preparation; KM and HS participated in experimentation, and data collation; while GE designed and supervised the experiment, participated in data interpretation and manuscript preparation.
• Species of mercury resistant Yarrowia spp. (Idd1 and Idd2) facilitate • •
Acknowledgements
dissolution of aerial Hg° into aquatic system through the EPS they produced. The dissolved purgeable Hg° in the hydrosphere is further transformed into non-purgeable Hg° via complexation with biomolecules of the yeast to bioavailable Hg2+ via Yarrowia-dependent oxidation. The oxidized, non-purgeable Hg2+/Hg° bound with the cell mass and the EPS of the Yarrowia spp., using the hydroxyl radicals of the
We thank Dr. Mei-Fang Chien, Mr. Satoru Ishikawa and Dr. Atsushi Okamoto of Tohoku University, Japan, for their useful discussion and assistance during the experiment and permission to use FTIR-ATR facility. We also thank the Japan Society for the Promotion of Science for the Postdoctoral Research Fellowship FY2012-2013 to GOO. This work was funded with Grants-in-Aid (No. 24-02373) for Scientific Research 248
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from the JSPS.
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