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Photo-microbial visible light-induced magnetic mass independent fractionation of mercury in a marine microalga Kritee Kritee, Laura C. Motta, Joel D. Blum, Martin Tsz-Ki Tsui, and John R Reinfelder ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.7b00056 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017
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ACS Earth and Space Chemistry
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Photo-microbial visible light-induced magnetic mass independent fractionation of
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mercury in a marine microalga
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K. Kritee1*#, Laura C. Motta1, Joel D. Blum2, Martin Tsz-Ki Tsui3 and John R. Reinfelder1
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1
Department of Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, New
Jersey 08901; 2 Department of Earth and Environmental Sciences, University of Michigan, 1100 N. University Avenue, Ann Arbor, Michigan 48109;
3
Department of Biology, University of North Carolina at Greensboro,
321 McIver St, Greensboro, North Carolina 27402 *
To whom correspondence should be addressed; #Present address: Environmental Defense Fund, 2060
Broadway (Suite 300), Boulder CO 80303; Tel: (732) 277 8134 (Cell); Email:
[email protected] 13 14
Keywords: Mercury, Stable isotope, Mass independent fractionation, Mass independent
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fractionation, Intracellular MeHg degradation, Intracellular Hg(II) reduction, Radical pairs
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Abstract
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Methylmercury (MeHg), a highly neurotoxic substance, accumulates in aquatic food webs
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and is enriched in odd isotopes (i.e.,
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photo-degradation in surface waters. Here, we highlight the potential role of phytoplankton in
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the mass independent fractionation (MIF) of MeHg in marine food-webs by providing
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evidence of 1) degradation of intracellular MeHg and reduction of intracellular inorganic
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mercury (Hg(II)) in the marine microalga, Isochrysis galbana; 2) a large, positive MIF
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(∆199Hgreactant - ∆199Hgproduct ~5-10‰) during intracellular degradation of MeHg in cells
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exposed to visible light with no UVB, consistent with the accumulation of odd isotope-
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enriched MeHg in marine food-webs; and 3) a negative MIF (-1‰) during intracellular
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reduction of Hg(II) in the presence of UVB light. If representative of the photochemical
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reactivity of MeHg in marine phytoplankton, our results indicate that algal cell-mediated
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demethylation of MeHg by visible light could account for 20 to 55% of the total
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photochemically-driven demethylation in the open ocean and transparent freshwater
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ecosystems with deep euphotic zones.
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phytoplankton (and possibly other light permeable microorganisms) in mercury
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Hg and
201
Hg), purportedly as a result of abiotic
Thus, our results extend the importance of
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biogeochemistry beyond their role as accumulators of MeHg and/or reducers of Hg(II) at the
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base of the food chain, to include MeHg degradation and MIF of Hg in sunlit layers of the
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ocean and other aquatic systems.
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Introduction
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Phytoplankton play an important role in the speciation, mobility, bioaccumulation,
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and toxicity of Hg in aquatic ecosystems1-3. Although Hg methylation in the marine water
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column has in some cases4-6been correlated with phytoplankton biomass or productivity or
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the seasonally variable growth of pico- and nanophytoplankton (as opposed to larger (>20
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µm) cells)7; marine microalgae are not known to methylate Hg directly. They do, however,
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represent an important route for Hg entry into aquatic food webs8, 9. Methylmercury, which is
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the most biologically available organic form of Hg, is concentrated from the dissolved phase
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in phytoplankton by a factor of more than 104 from seawater10, 11. Cells enriched in MeHg are
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then consumed by zooplankton, which in turn are a primary food source of larval, juvenile,
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and some adult fish. Indeed, nearly all of the MeHg accumulated by zooplankton and fish is
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from their diet12-14. Two bacterial pathways that degrade MeHg, a reductive, enzyme-
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mediated process whose products are CH4 and Hg(0), and (an) oxidative process(es) whose
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products are CO2 and an unidentified form of inorganic Hg [ref 1 and references therein],
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have not been reported in phytoplankton. While the degradation of MeHg has been linked to
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the presence of plankton15,
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assumed to dominate MeHg degradation in the photic zone15,
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importance of dark microbial degradation of MeHg has also been discussed18.
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, UV-mediated abiotic photochemical processes have been 17
, although the potential
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The reduction of Hg(II) in marine surface waters has been primarily attributed to two
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phenomena: abiotic photochemical reactions19 (with photochemical reduction of Hg(II) being
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nearly balanced by photo-oxidation of Hg(0)20) and dark microbial reactions associated with
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Hg-resistant microorganisms1. However, it is not clear if these two phenomena can
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completely explain the reduction of inorganic Hg(II) observed in growing cultures of marine
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phytoplankton21-24.
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Hg(0) concentrations and phytoplankton pigments have been observed in marine
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ecosystems25, it is not clear if phytoplankton can directly and/or intracellularly demethylate
In
addition,
although
direct
positive
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relationships
between
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ACS Earth and Space Chemistry
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MeHg and/or reduce Hg(II) beyond their role in the production of extracellular dissolved
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organic matter (which may participate in abiotic photo-reduction). While Hg transformations
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within many different lineages of bacterial cells have been explored in detail, including the
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recent demonstration of the reduction of intracellular Hg in a photomixotrophic bacterium24,,
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the role of photo-microbial transformations (intracellular photochemical reactions) of MeHg
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or Hg(II) within phytoplankton cells has not been specifically examined.
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Mercury stable isotope fractionation has proven to be a useful tool in constraining the
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potential sources and transformations of different forms of Hg in the environment26-28.
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Mercury stable isotopes display both mass dependent fractionation (MDF) and mass
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independent fractionation (MIF). The magnetic moments of odd isotopes of Hg and the non-
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mass-dependent variation in the nuclear volumes of Hg isotopes, especially
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201
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MDF has been observed during both dark transformations and photochemical reactions, MIF
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has not been observed during any dark microbial Hg transformations investigated to date33-37.
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Hg and
Hg29-32, can lead to fractionation that does not scale according to isotope mass. While
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With respect to stable isotope fractionation, photo-microbial Hg transformations lie at
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the interface between biology and photochemistry. Stable isotope fractionation of Hg during
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dark microbial reduction33-35 and also during abiotic UV-mediated processes29-31 has been
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documented, but the effects of intracellular photo-reduction/degradation of Hg(II) or MeHg
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in phytoplankton on Hg stable isotopes has not been explored38. A clear understanding of the
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Hg isotopic signatures of phytoplankton-mediated transformations, which could affect the
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isotopic composition of oceanic Hg(0) in addition to that of MeHg in fish, is necessary to
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interpret stable isotope ratios of Hg occurring as Hg(II) and MeHg both at the top of the food
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web (i.e., in fish) and in the water column and sediments.
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We investigated the rates and Hg stable isotope signatures of photo-microbial
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transformations of Hg(II) and MeHg in marine phytoplankton exposed to visible light and
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varying levels of UV radiation by performing experiments with 1) sterile-filtered spent
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growth media containing extracellular exudates from cultures of Isochrysis galbana, a
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eukaryotic marine microalga of the globally important Prymnesiophyceae class ; 2) actively 3 ACS Paragon Plus Environment
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growing mono-specific cultures of I. galbana; and 3) cysteine or ocean-water washed (non-
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growing) I. galbana cells. The washed cell experiments were designed to test the ability of
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phytoplankton to transform intracellular Hg(II) and MeHg. We present our results in the
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context of known reaction mechanisms and expected ligand interactions inside and outside of
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phytoplankton cells, and discuss the general implications of the new findings with respect to
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the current understanding of aquatic Hg biogeochemistry in the ocean and Hg isotope
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systematics.
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Experimental Methods
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Phytoplankton cultures: Experiments were conducted using the unicellular, eukaryotic,
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marine microalga Isochrysis galbana (strain ISO, CCMP1323). Isochrysisis a common
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genus in temperate marine waters and represents the globally distributed class
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Prymnesiophyceae. Live cultures of I. galbana were grown in Aquil artificial seawater
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media39 with 300 µM nitrate and 10 µM phosphate and were maintained at 18°C under a
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12:12 h light:dark regime with 200 µmol quanta m−2 s−1 irradiance provided by cool white
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fluorescent low pressure Hg lamps (see more below).
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Mercury reduction experiments. Photochemical reduction of Hg(II) or MeHg was
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examined in growing whole culture, phytoplankton exudate, and washed cell experiments
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(see SI Tables 1 and 2 for details of all experiments).
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inorganic Hg(II) and MeHg stocks were made from powdered Sigma Aldrich Mercuric
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Nitrate Monohydrate and powdered Crescent Chemical Company Methylmercury Chloride,
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respectively. For growing culture and exudate experiments, the Hg-free cultures were
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acclimatized to a 24 hour light regime for 2 days. After this acclimatization, cultures (or the
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collected spent media, i.e., culture filtered through 0.2 µm polycarbonate filters) were either
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incubated with Hg(II) or MeHg for up to 20 hours in the dark, after which, lights were turned
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on and reactors were purged with Hg-free air to start the reduction experiments. In growing
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culture experiments, both intra- and extracellular Hg was present and the kinetics and
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isotopic fractionation were affected by both algal exudates and the intracellular environment.
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For intracellular experiments, phytoplankton cells in the early exponential phase (between
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4.5 x 104 and 5 x 104 cells ml-1) were exposed to Hg(II) or MeHg at the beginning of a 12h 4 ACS Paragon Plus Environment
For all experiments, 100 µg/g
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ACS Earth and Space Chemistry
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dark period and were allowed to accumulate Hg(II) or MeHg for up to 3 days. Repeated tests
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showed that, after this accumulation period, most of the added Hg(II) or MeHg was
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associated with the cells and the filtrate/exudate contained
