Magnesium Isotope Fractionation during Synthesis of Chlorophyll a

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Magnesium isotope fractionation during synthesis of chlorophyll a and bacteriochlorophyll a of benthic phototrophs in hypersaline environments Yuta Isaji, Toshihiro Yoshimura, Daisuke Araoka, Junichiro Kuroda, Nanako O. Ogawa, Hodaka Kawahata, and Naohiko Ohkouchi ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.9b00013 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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ACS Earth and Space Chemistry

Magnesium

isotope

fractionation

during

synthesis

of

chlorophyll

a

and

bacteriochlorophyll a of benthic phototrophs in hypersaline environments Yuta Isaji1*, Toshihiro Yoshimura1, Daisuke Araoka2, Junichiro Kuroda3, Nanako O. Ogawa1, Hodaka Kawahata3, Naohiko Ohkouchi1 1Biogeochemistry

Program, Japan Agency for Marine-Earth Science and Technology

(JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan. 2Geological

Survey of Japan, National Institute of Advanced Industrial Science and

Technology, Tsukuba Central 7, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8567, Japan. 3Atmosphere

and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha,

Kashiwa, Chiba 277-8564, Japan. *Corresponding author. Email: [email protected] Abstract Magnesium is a major divalent cation in the cell and is vital for maintaining cellular function. The magnesium isotopic composition (δ26Mg) of chloropigments potentially provides detailed information on cellular physical and biochemical reactions that involve Mg, but existing data are scarce and its controlling factors remain unresolved. Here, we report δ26Mg of chlorophyll a and bacteriochlorophyll a derived from cyanobacteria and purple sulfur bacteria that inhabit benthic microbial mats and gypsum crusts formed under shallow hypersaline environments. There was substantial Mg isotope fractionation in both positive and negative directions from source Mg2+ in the brine (δ26Mg: −1.05‰ to −0.78‰) to chloropigments (chlorophyll a: −1.77‰ to −0.39‰, bacteriochlorophyll a: −2.13‰ to −0.12‰), suggesting that multiple processes are involved in the fractionation of Mg during chlorophyll biosynthesis. The relationship between δ26Mg and δ15N of bacteriochlorophyll a indicates a correlation between δ26Mg values and growth rate of phototrophs. We suggest that the extent of kinetic isotopic fractionation during the Mg-insertion step changes in response to the amounts of cellular chloropigments, resulting in the large variability in δ26Mg of chloropigments observed in natural environments.

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Keywords Magnesium isotope; chlorophyll a; bacteriochlorophyll a; benthic phototroph; hypersaline environment 1. INTRODUCTION Magnesium is a major divalent cation in the hydrosphere and lithosphere, and is the fourth most abundant cellular metal ion. It is essential for numerous processes in the cell, such as enzymatic reactions, regulation of ion channels, hydrolyzation of phosphate esters and phosphoryl transfers, and stabilization of macromolecules. Cellular Mg2+ dynamics is tightly regulated to maintain these fundamental functions of the cell.1,2 However, detailed understanding of Mg2+ influx, efflux, and intracellular movement and transformation remains incomplete, as techniques for quantifying Mg2+ on a cellular scale are insufficient.3 Fractionation of heavy and light isotopes (e.g.,

12C

and

13C; 14N

and

15N)

generally

occurs for a series of physical and biochemical reactions in a cell, with its extent reflecting the mechanisms of and providing quantitative information about each reaction.4,5 Natural stable isotopes of Mg (24Mg, 78.992%;

25Mg,

10.003%; and

26Mg,

11.005%) are also fractionated during various physical and biochemical processes, such as transport through the cell membrane6 and within the body,7 incorporation into the carbonate lattice during calcification,8,9 and formation of Mg-bearing compounds within the cell.6 Whereas bulk δ26Mg measurement of biological samples is generally applied to discussions about cellular Mg dynamics, that of Mg-containing compounds can provide more detailed insights into specific physical and biochemical reactions. Chloropigments (i.e., chlorophylls and bacteriochlorophylls) have Mg at the center of their tetrapyrrole ring. They play essential roles in photosynthesis, effectively converting enormous amounts of solar energy into chemical energy to support ecosystems. Several studies have investigated δ26Mg of chlorophyll a, b, and c of terrestrial and marine phototrophs in an attempt to understand the details of chlorophyll biosynthesis,10,11 and to determine the factors controlling their δ26Mg.12–17 Although correlations between δ26Mg of chlorophylls and temperature or growth phase (stationary or exponential) have been reported from controlled culture experiments,12,16 such relationships were not evident in a field experiment.14 It therefore remains unresolved as to which environmental and physiological factors, as well as which biosynthetic steps,


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are primarily responsible for determining δ26Mg of chloropigments. Here, we present δ26Mg of chlorophyll a and bacteriochlorophyll a isolated and purified from benthic phototrophs inhabiting shallow hypersaline environments. To our knowledge, this is the first report of δ26Mg of bacteriochlorophylls. There are several advantages to investigating hypersaline benthic phototrophs. One is that they are dominated by phototrophic bacteria, which have simpler cell structure than eukaryotes. Importantly, although the physiologies of these phototrophs are strongly influenced by environmental conditions fluctuating along the salinity gradient, the proportion of Mg2+ transported into the cell from the brine remains extremely small due to elevated Mg concentrations in the brines. These advantages enable us to clearly distinguish the factors determining δ26Mg of chloropigments. Moreover, carbon and nitrogen isotopic compositions (δ13C, δ15N) of these chloropigments have previously been determined, thus providing information on the physiological condition of the phototrophs.18,19 2. MATERIALS & METHODS 2.1 Sampling protocols We investigated three commercial solar salterns at Trapani, western Sicily, Italy: the Sosalt (SS), Culcasi (CU), and Chiusicella (CH) salterns (Figure S1). Benthic microbial mats were collected from three calcium carbonate-precipitating ponds (CU-6, -7, and -8; salinity 78, 108, and 146, respectively) in September 2016, and gypsum crusts, which contain layers of different colors, were collected from two gypsum-precipitating ponds (SS-1 and CH-1; salinity 159 and >300, respectively) in September 2015 (Figure 1). The yellow and green layers of the microbial mats and gypsum crusts are dominated by cyanobacteria, and the pink layers below by purple sulfur bacteria. Surface brine was collected from all ponds, and porewater from the calcium carbonate-precipitating

ponds.

For

the

porewater

sampling,

sediment

cores

approximately 50 cm in length were recovered. A Rhizon sampler connected to a 0.45-µm filter was inserted into the black layer, 2.5 cm below the deposit surface in CU-6 and 5 cm below in CU-7 and CU-8. All samples were stored in a freezer until analysis. 2.2 Magnesium isotopic composition of chloropigments and brines The analytical procedure for purification of chloropigments followed that described



previously.20 Briefly, deposits from the carbonate ponds (CU-6, -7, and -8) and gypsum crusts from the gypsum ponds (SS-1 and CH-1) were freeze-dried and ground to powder, extracted three times with acetone by sonication, and then the acetone fraction was extracted three times again with n-hexane. The n-hexane fraction was then dried completely under N2 gas and dissolved in 100 µL of N,N-dimethylformamide for high-performance liquid chromatography (HPLC) analysis. All procedures were performed in a dark room. Pigments were isolated with reverse-phase HPLC using an Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm; 5 μm silica particle size). The mobile phase was 75% acetonitrile:pyridine (100:0.5, v/v) and 25% ethyl acetate:pyridine (100:0.5, v/v) for 5 min, followed by a linear gradient of ethyl acetate:pyridine to 50% over 50 min. The flow rate was set to 1 mL min−1 and the column temperature to 30 °C. Chlorophyll a and bacteriochlorophyll a were collected with baseline resolution using a fraction collector (Figure S2). The collected chloropigments were dried under Ar gas and extracted with n-hexane three times to eliminate possible metal contamination from the HPLC system. The hexane extract was dried and re-dissolved in 1 mL of n-hexane and then reacted with 0.1 mL of 2 M HCl to liberate Mg from the tetrapyrrole ring. The liberated Mg was then extracted with Milli-Q water and dried completely on a hot plate at 90 °C. Magnesium was purified by an ion chromatograph (Metrohm 930; Metrohm, Herisau, Switzerland) coupled to an Agilent 1260 Infinity II fraction collector system.21,22 The samples were eluted through a Metrohm Metrosep C6-250/4.0 column with 8 mM ultrapure HNO3 (Tamapure AA-100, Tama Chemical, Kawasaki, Japan) at a flow rate of 0.9 mL min−1. Magnesium fractions were pooled in 7-mL Teflon vials (Savillex, Eden Prairie, Minnesota, USA), and then evaporated to dryness in a class-1000 clean bench. The Mg isotopic composition was determined with a multiple-collector inductively coupled plasma mass spectrometer (MC-ICP-MS) (Neptune Plus; Thermo Fisher Scientific, Waltham, MA, USA) at the National Institute of Advanced Industrial Science and Technology, using standard sample-bracketing methods.21,22 Standards and samples were prepared as solutions of approximately 100 ppb Mg in 0.3 M HNO3. Sample solutions were introduced at 100 L min−1 with a nebulizer (PFA MicroFlow; ESI, Omaha, NE, USA) attached to a quartz dual cyclonic spray chamber operated in free aspiration mode. We performed Mg isotope analysis with a high-sensitivity


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X-skimmer cone in low-mass-resolution mode. Samples and standards were analyzed 40 times with an integration time of 4 seconds per cycle. Background signal intensities were determined by analysis of 0.3 M ultrapure HNO3 for one cycle with an integration time of 30 seconds per cycle, and the baselines were subsequently subtracted from standard and sample signal intensities. The isotopic data are reported as per mil (‰) deviations relative to the DSM-3 standard as follows: δxMg = [(xMg/24Mg)sample/(xMg/24Mg)DSM-3  1] 1000

,

(1)

where x is either 25 or 26. The analytical uncertainties were better than ±0.15‰ (2σ) as estimated from the reproducibility of multiple measurements of Cambridge 1 Mg standard during each analytical session: −2.65‰ ± 0.09‰ (n = 10). Within the analytical uncertainties, this value agrees well with the reported value of −2.58‰ ± 0.14‰ (2σ).23 The δ26Mg values of the seawater reference materials NASS-6 and IAPSO 　 Standard Seawater were −0.93‰ and −0.87‰ with 2σ reproducibility of 0.13‰ and 0.04‰, respectively. These values also agrees well with reported values for the same standards: −0.83‰ ± 0.07‰ (2σ) for NASS-624 and −0.87‰ ± 0.08‰ (2σ) for IAPSO Standard Seawater.25 2.3 Validity of δ26Mg measurements of chloropigments Reproducibility and blank Mg content were evaluated to validate our analytical procedure. To determine the blank Mg content, 50 µL of N,N-dimethylformamide was introduced to the HPLC system and fractions were collected at the retention times corresponding to the elution of bacteriochlorophyll a (8.5–9.7 min) and chlorophyll a (13.5–15.5 min). The collected blank fractions were extracted with n-hexane three times, extracted with Milli-Q water after addition of 0.1 mL of 2 M HCl, and then dried completely. Each dried blank fraction was dissolved with 0.3 M HNO3. Internal standards (Be, Sc, Y) were added to the solution to control for instrumental drift. Mg concentrations were measured with a quadrupole inductively coupled plasma mass spectrometer (iCAP Qc; Thermo Scientific, Bremen, Germany) at the Japan Agency for Marine-Earth Science and Technology, and calibrated using a multi-element standard reagent (Kanto Chemical Co. Inc., Tokyo, Japan). Authentic chlorophyll a standard (from Chlorella, 90% purity; Fujifilm Wako Pure



Chemical Corporation, Osaka, Japan) was used to compare δ26Mg values before and after the HPLC purification procedure. We prepared chlorophyll a standard that was purified once (n = 3) and twice (n = 1) by HPLC. Magnesium was isolated and purified from the original chlorophyll a standard (n = 1) and the purified chlorophyll a standards, and then introduced to the MC-ICP-MS for δ26Mg measurement. 3. RESULTS & DISCUSSION The total Mg procedural blanks for chlorophyll a and bacteriochlorophyll a fractions were 0.09 ng and 0.26 ng, respectively; negligible relative to the sample Mg amounts (>450 ng). The recovery of Mg through the preparation procedure—from purification of chlorophyll a with HPLC to purification of Mg with ion chromatography—was better than 90% (Table S1). Because there is 100% recovery from the purification of Mg with ion chromatography21, this loss of up to 10% is attributable to the degradation of chlorophyll a upon drying of the solvents in the collected fractions and extraction with n-hexane prior to Mg liberation. The δ26Mg values of the authentic chlorophyll a standard and the standard purified by reverse-phase HPLC were identical within analytical uncertainty (Table S2). Thus, processes such as Mg contamination from the HPLC system or partial degradation of chlorophyll a have negligible influence on the original δ26Mg values of the chloropigments. The δ26Mg values of surface brine and porewater ranged from −1.05‰ to −0.78‰ (Figure 2, Table 1), which is comparable with that of seawater (−0.83‰ ± 0.09‰).26,27 In contrast, δ26Mg of chlorophyll a from cyanobacteria and bacteriochlorophyll a from purple sulfur bacteria varied widely (chlorophyll a, −1.77‰ to −0.39‰; bacteriochlorophyll a, −2.13‰ to −0.12‰), following a mass-dependent fractionation line (Figure S3). Our results and previous studies suggest that chloropigments can be either enriched or depleted in

26Mg

relative to source Mg. This may be explained by

multiple steps fractionating Mg isotopes during chloropigment biosynthesis: Mg2+ transport through the cell membrane (and through chloroplasts, for eukaryotic phototrophs), distribution of Mg within a cell (i.e., free intracellular Mg2+ and that bound to organic compounds), binding of free intracellular Mg2+ onto Mg-chelatase, and the subsequent Mg insertion into protoporphyrin IX (discussed in detail below). Isotopic fractionation associated with one or more of these processes must have changed in response to environmental or physiological conditions in each solar saltern


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pond, resulting in the observed large δ26Mg variations of the chloropigments. Importantly, deviations of δ26Mg of chlorophyll a and bacteriochlorophyll a from the source Mg2+ (∆δ26Mgchla-brine, ∆δ26Mgbchla-brine) in each pond are similar (∆δ26Mgchla-brine − ∆δ26Mgbchla-brine < 0.70‰) with respect to their overall range of variation (Figure 3). Because the biosynthetic pathways and mechanisms for chelating Mg into chlorophylls and bacteriochlorophylls are similar,28 we infer that the observed δ26Mg variations result from environmental conditions similarly affecting the physiology of cyanobacteria and purple sulfur bacteria. Here we discuss which biosynthetic steps could potentially cause the large variability in δ26Mg of chloropigments observed in this study, and determine which environmental and physiological factors primarily control the δ26Mg value in natural environments. The first step that could fractionate Mg isotopes is Mg2+ transport through the cell membrane. The primary Mg uptake system in prokaryotes is CorA Mg2+ transporter, which takes up Mg2+, predominantly present in the environment in the form of Mg(H2O)62+.29 Although microorganisms use K+ and a variety of organic osmolytes to counter external osmotic pressure,30 Mg has not been considered an osmotically important solute.31 It therefore follows that the proportion of Mg2+ transported into the cell from the brine remains small, as the Mg2+ pool in the brine is much larger than that in the cell ([Mg2+]brine = 126−678 mM; Table 1). Whether the isotopic fractionation is kinetic or equilibrium, the range of variation is strongly influenced by the mass balance between the reactant and product. The Mg isotope ratios for the products can vary widely if the amount of the product (in this case, cellular Mg2+) is comparable to that of the reactant (brine Mg2+), but will vary little if the amount of product is small relative to that of the reactant. Thus, although neither the type of fractionation (kinetic or equilibrium) nor the isotopic fractionation factor has been determined, isotopic fractionation during Mg2+ transport is probably not responsible for the large variability in δ26Mg of chloropigments observed in this study. Over 90% of cellular Mg2+ is bound to organic components such as ribosomes, ATP, and other polyanionic cell constituents, and the concentration of free intracellular Mg2+ is regulated to several millimolar.32,33 A previous study reported equilibrium isotopic fractionation between free intracellular Mg2+ and Mg-bearing compounds, with

26Mg

preferentially coordinating to the organic phase6 because heavier isotopes are preferentially coordinated to stronger bonds at isotopic equilibrium.34,35 The free



intracellular Mg2+, which is used to chelate protoporphyrin IX,36 is therefore depleted in 26Mg

relative to total Mg in the cell. The extent of this depletion is probably essentially

constant because Mg2+ is in homeostasis within a cell.1 Taken together, although the magnitude of the isotopic fractionation associated with Mg2+ transport across the cell membrane and its distribution within the phototrophic cell may be large, they cannot account for the large observed variability in δ26Mg of chloropigments. We are thus left with the Mg-insertion step during the biosynthesis of chloropigments as the main source of chloropigment δ26Mg variability. The binding of Mg2+ onto Mg-chelatase and its chelation into protoporphyrin IX require ATP,36,37 and are thus irreversible.38 Therefore, the Mg isotopic fractionation during these processes will be kinetic. The concentration of chlorophyll a within a cell is generally several millimolar,39,40 which is comparable to that of free intracellular Mg2+. Thus, a change in the amounts of chlorophylls synthesized in a cell has the potential to produce large variations in δ26Mg of chloropigments. The concentrations of chloropigments within a cell change in response to factors such as light intensity and growth rate, with higher concentrations generally observed in low-light environments and during exponential growth.41 Indeed, previous culture experiments demonstrated that δ26Mg of chlorophyll a is lower in the stationary phase than in the late exponential phase.12,16 Previous studies suggest that phototrophic primary productivity decreases with increasing salinity.18,42 Accordingly, we speculate that the growth rates of the phototrophs decreased from CU-6 to CU-8. In determining the growth rate of phototrophs for natural samples, δ15N of chloropigments could provide valuable insights. As reported previously,19 the δ15N difference between porewater ammonium and bacteriochlorophyll a derived from purple sulfur bacteria (Δδ15Nbchla−ammonium) decreases from –8.7‰ in CU-6 to –14.7‰ in CU-8, which correlates positively with the δ26Mg difference between porewater Mg2+ and bacteriochlorophyll a (Δδ26Mgbchla−brine; Figure S4). The lowering of Δδ15Nbchla−ammonium reflects a decrease in the amount of ammonium assimilated by purple sulfur bacteria, with a possible additional contribution from the changes in the nitrogen isotopic fractionation factors for ammonium assimilation19. Because the decrease in ammonium assimilation can be interpreted as reflecting a decrease in the growth rate, we suggest that the resulting decrease in the cellular abundance of bacteriochlorophyll a from CU-6 to CU-8 caused the decrease in δ26Mg of bacteriochlorophyll a.


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There is a clear difference in δ26Mg trends from pond to pond between the microbial mat samples and the gypsum samples (Figure 3), which cannot be simply interpreted. There are no straightforward links between such a difference and environmental factors such as temperature, salinity or pH of the surface brine reported in our previous study.18 Despite being speculative, we hypothesize that the isotopic differences might result from the difference in light availability due to differences in optical properties of the microbial mats and gypsum crusts.43,44 It could also be related to the difference in available sunlight, as microbial mats and gypsums were collected in different years. We therefore propose that the large variation in δ26Mg of chloropigments observed in this study is primarily attributable to kinetic isotopic fractionation of Mg during the Mg-insertion step, which changes as a function of the cellular amounts of chloropigments varying in response to factors such as growth rate and light intensity. The Mg isotopic fractionation factor associated with the Mg-insertion step is currently unknown. However, it can be speculated that

24Mg

is preferentially chelated, because

both our field study and previous culture experiments12,16 suggest that lower cellular abundance of chloropigments results in lower δ26Mg values. Further inferences can be made about cellular Mg isotope dynamics based on our results and previous studies, with several assumptions (Figure 4). Our results suggest that the Mg-insertion step preferentially utilizes pool. This pool is depleted in preferential incorporation of

26Mg

26Mg

24Mg

from the free intracellular Mg2+

relative to total Mg in the cell because of

into organic components.6 Thus, Mg2+ transport

through the cell membrane must be associated with

26Mg-enrichment

in the cell to

explain chloropigments with higher δ26Mg values relative to the source Mg2+ in the brine (Figure 2). The isotopic fractionation factor for Mg2+ transport has not been determined. A possible comparable example is the difference in δ26Mg between abiogenic and biogenic calcites, which is also considered primarily attributable to Mg2+ transport. Because this difference mostly ranges within ±2‰,8,9 we assumed an equilibrium isotopic fractionation factor (εcell/brine) of +2‰ for Mg2+ transport across the cell membrane in our model for the variation in δ26Mg of chloropigments in phototrophic bacteria (Figure 4). To allow the calculated δ26Mg of chloropigments to vary in the range observed in the solar salterns, we also assumed an equilibrium isotopic fractionation factor (εfree/bound) of −1‰ for Mg2+ partitioning within a cell and a kinetic isotopic fractionation factor (εC) of +3‰ for the Mg-insertion step. Note that the



εfree/bound value assumed here is smaller than values previously employed based on the calculated equilibrium isotopic fractionation factor between Mg(H2O)62+ and organic Mg compounds (−4‰ to −2‰).6,35 Although at present this discussion is still qualitative, primarily because of the limited knowledge about the Mg isotopic fractionation factors associated with these biochemical reactions, further investigations using Mg isotopes would lead to quantitative understanding of Mg dynamics at the cellular scale. 4. CONCLUSIONS We interpret systematic variations in δ26Mg of chlorophyll a and bacteriochlorophyll a of benthic phototrophs in shallow hypersaline environments to primarily reflect the cellular amounts of chloropigments, which change in response to physiological and environmental factors such as growth rate and light intensity. It is noteworthy that the extent of δ26Mg variability of chloropigments in the hypersaline environment (−2.13‰ to −0.12‰), where the proportion of Mg2+ transported through the cell membrane is minimal, is comparable to that observed in culture and in normal marine settings (−2.28‰ to +0.84‰). This implies that the extent of δ26Mg variability of chloropigments in normal marine settings is also explainable by changes in the cellular amounts of chloropigments, although additional δ26Mg variability may be introduced during Mg2+ incorporation into the cell. Our results contribute to a better understanding of chlorophyll biosynthesis and cellular Mg dynamics, and provide basic information in constraining the influence of biological processes on the Mg isotopic budget of Earth surface systems (e.g., river basins). Detailed investigations into tetrapyrrole compounds (e.g., chlorophylls, hemes, F430, vitamin B12), which catalyze crucial biological reactions, would provide unique and essential insights into Earth biogeochemistry. Acknowledgements We are grateful to SoSalt Spa and R.N.O Saline di Trapani e Paceco for permission to sample the Trapani solar works, and to S. Lugli, A. Santulli, and F. J. Jiménez-Espejo for help during the sampling. This study was partly supported by a Japan Society for the Promotion of Science (JSPS) Research Fellowship (16J07844) to Y.I., KAKENHI (16H05883, 16K21682) to T.Y. and D.A., and the JAMSTEC President Fund. All data used in this article are available from the corresponding author.


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Supporting Information Map showing the locations of the three solar salterns investigated in this study (Figure S1), sample HPLC chromatograms monitored at 660 nm and 750 nm (Figure S2), magnesium isotope ratios of chlorophyll a and bacteriochlorophyll a measured in this study and those of chlorophyll a and chlorophyll c of marine phototrophs reported in previous studies (Figure S3), the difference between δ26Mg of bacteriochlorophyll a and brine Mg2+ (∆δ26Mgbchla–brine) plotted against the difference between δ15N of bacteriochlorophyll a and porewater ammonium (∆δ15Nbchla–ammonium) in ponds CU-6, -7, and -8 (Figure S4), Mg contents in procedural blanks and samples (Table S1), comparison of the δ26Mg values of the chlorophyll a standard, the same standard purified once with HPLC, and purified twice (Table S2). References 1.

Gardner, R. C. Genes for magnesium transport. Curr. Opin. Plant Biol. 2003, 6, 263− 267.

2.

Romani, A. M. Cellular magnesium homeostasis. Arch. Biochem. Biophys. 2011, 512, 1−23.

3.

Maguire, M. E.; Cowan, J. A. Magnesium chemistry and biochemistry. Biometals 2002, 15, 203−210.

4.

Ohkouchi, N.; Ogawa, N. O.; Chikaraishi, Y.; Tanaka, H.; Wada, E. Biochemical and physiological bases for the use of carbon and nitrogen isotopes in environmental and ecological studies. Progress in Earth and Planetary Science 2015, 2, 1.

5.

Fogel M.L.; Cifuentes L.A. In Organic Geochemistry; Engel M. H.; Macko S. A., Eds.; Springer: Boston, 1993; pp 73−98.

6.

Pokharel, R.; Gerrits, R.; Schuessler, J. A.; Floor, G. H.; Gorbushina, A. A.; von Blanckenburg, F. Mg Isotope Fractionation during Uptake by a Rock-Inhabiting, Model Microcolonial Fungus Knufia petricola at Acidic and Neutral pH. Environ. Sci. Technol. 2017, 51, 9691−9699.

7.

Bolou-Bi, E. B.; Poszwa, A.; Leyval, C.; Vigier, N., Experimental determination of magnesium isotope fractionation during higher plant growth. Geochim. Cosmochim. Acta 2010, 74, 2523−2537.



8.

Saenger, C.; Wang, Z. Magnesium isotope fractionation in biogenic and abiogenic carbonates: implications for paleoenvironmental proxies. Quaternary Sci. Rev. 2014, 90, 1−21.

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Pogge von Strandmann, P. A.; Forshaw, J.; Schmidt, D. Modern and Cenozoic records of seawater magnesium from foraminiferal Mg isotopes. Biogeosciences 2014, 11, 5155−5168.

10. Black, J. R.; Yin, Q.-z.; Rustad, J. R.; Casey, W. H. Magnesium isotopic equilibrium in chlorophylls. J. Am. Chem. Soc. 2007, 129, 8690−8691. 11. Moynier, F.; Fujii, T. Theoretical isotopic fractionation of magnesium between chlorophylls. Sci. Rep. 2017, 7, 6973. 12. Black, J. R.; Yin, Q.-z.; Casey, W. H. An experimental study of magnesium-isotope fractionation in chlorophyll-a photosynthesis. Geochim. Cosmochim. Acta 2006, 7, 4072−4079. 13. Black, J. R.; Epstein, E.; Rains, W. D.; Yin, Q.-z.; Casey, W. H. Magnesium-isotope fractionation during plant growth. Environ. Sci. Technol. 2008, 42, 7831−7836. 14. Ra, K. Determination of Mg isotopes in chlorophyll a for marine bulk phytoplankton from the northwestern Pacific Ocean. Geochem. Geophys. Geosyst. 2010, 11, Q12011. 15. Ra, K.; Kitagawa, H. Magnesium isotope analysis of different chlorophyll forms in marine phytoplankton using multi-collector ICP-MS. J. Anal. At. Spectrom. 2007, 22, 817−821. 16. Ra, K.; Kitagawa, H.; Shiraiwa, Y. Mg isotopes in chlorophyll-a and coccoliths of cultured coccolithophores (Emiliania huxleyi) by MC-ICP-MS. Mar. Chem. 2010, 122, 130−137. 17. Pokharel, R.; Gerrits, R.; Schuessler, J. A.; Frings, P. J.; Sobotka, R.; Gorbushina, A. A.; von Blanckenburg, F. Magnesium stable isotope fractionation on a cellular level explored by cyanobacteria and black fungi with implications for higher plants. Environ. Sci. Technol. 2018, 52, 12216−12224. 18. Isaji, Y.; Kawahata, H.; Kuroda, J.; Yoshimura, T.; Ogawa, N. O.; Suzuki, A.; Shibuya, T.; Jiménez-Espejo, F. J.; Lugli, S.; Santulli, A.; Manzi, V.; Roveri, M.; Ohkouchi, N. Biological and physical modification of carbonate system parameters along the salinity gradient in shallow hypersaline solar salterns in Trapani, Italy.


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Figures and Tables Table 1. δ26Mg and δ25Mg of chloropigment and brine samples. The 2σ values were determined from triplicate measurements of each sample. Concentrations of Mg2+ were reported previously.20,21 CU, Culcasi saltern; SS, Sosalt saltern; CH, Chiusicella saltern; Chl a, chlorophyll a; BChl a, bacteriochlorophyll a.

Pond

Sample

Chloropigments CU-6

Chl a BChl a Chl a BChl a Chl a BChl a Chl a BChl a Chl a BChl a

　

CU-7 CU-8 SS-1 CH-1

Brines

CU-6 CU-7 CU-8

　

SS-1 CH-1

[Mg2+] (mM)

Surface Porewater Surface Porewater Surface Porewater Surface Surface

126.2 177.8 260.7 282.1 677.6

δ26Mg (‰)

2σ (‰)

δ25Mg (‰)

2σ (‰)

–0.53 –0.12 –0.81 –0.69 –1.77 –2.13 –0.49 –1.18 –0.60 –1.11

0.10 0.09 0.07 0.21 0.10 0.10 0.09 0.10 0.13 0.03

–0.29 –0.09 –0.44 –0.36 –0.92 –1.11 –0.26 –0.62 –0.31 –0.58

0.07 0.06 0.06 0.11 0.06 0.06 0.05 0.07 0.10 0.03

–0.86 –0.94 –0.92 –1.05 –0.78 –0.88 –0.81 –0.87

0.12 0.07 0.08 0.09 0.04 0.13 0.08 0.12

–0.42 –0.48 –0.48 –0.54 –0.39 –0.46 –0.42 –0.46

0.10 0.02 0.05 0.07 0.05 0.05 0.04 0.09


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A

1 cm

B

Figure 1. (A) Bottom deposit from a carbonate pond, with a slimy surface layer several millimeters thick composed of thin yellow, green, and pink layers, and underlying loose black deposits. (B) Representative gypsum crust containing transparent yellowish, green, and pink layers (from the surface downward), and loose black deposits buried beneath. Modified after Isaji et al. (2017).18



Figure 2. Depth profiles showing δ26Mg of chloropigments, and of surface brine and porewater Mg2+, in (A) the microbial mats of the carbonate ponds (CU-6, -7, -8) and (B) the gypsum crusts in the gypsum ponds (SS-1, CH-1). The error bars correspond to ±2σ. Blue lines indicate the δ26Mg value of seawater.26,27 CU, Culcasi saltern; SS, Sosalt saltern; CH, Chiusicella saltern; Chl a, chlorophyll a; BChl a, bacteriochlorophyll a.


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1.5 Chlorophyll a Bacteriochlorophyll a

1.0

Δδ26Mgchls−brine (‰)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 0.0 -0.5 -1.0 -1.5

CU-6

CU-7

CU-8

SS-1

CH-1

Figure 3. Deviations of δ26Mg of chlorophyll a and bacteriochlorophyll a from that of the brine Mg2+ in each pond.



Figure 4. (A) Schematic diagram of the proposed model for Mg isotope fractionation associated with chloropigment biosynthesis by phototrophic bacteria. The equilibrium (or kinetic) Mg isotopic fractionation factor associated with Mg2+ transport through the cell membrane is hypothesized to be positive, as discussed in the text (εcell/brine > 0), with only a small proportion of the cell quota assimilated from the environment (fA ≈ 1). Magnesium in a cell (Mg2+cell) is largely present as bound-Mg2+ (fB = 0.90–0.95),32,33 and is in isotopic equilibrium with free-Mg2+ (εfree/bound < 0).6 Our results suggest that the kinetic isotopic fractionation factor during Mg insertion (εC) is negative, and δ26Mg of chloropigments varies primarily as a function of the proportion of free Mg2+ utilized for Mg insertion (fC). The three panels below show an example of a combination of (B) εcell/brine and (C) εfree/bound assuming a reversible-reaction model, and (D) εC assuming a Rayleigh distillation model, that could explain the range of δ26Mg of chloropigments found in this study. Blue bars in (B) and (C) indicate the probable ranges of fA (≈1) and fB (=0.90–0.95), and the green and red circles in (D) indicate δ26Mg values of chlorophyll a and bacteriochlorophyll a, respectively, determined in this study.


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For TOC Only


Magnesium Isotope Fractionation during Synthesis of Chlorophyll a

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