Calcium isotope fractionation by intracellular amorphous calcium carbonate (ACC) forming cyanobacteria

The formation of intracellular amorphous calcium carbonate (ACC) by various cyanobacteria is a widespread biomineralization process, yet its mechanism and importance in past and modern environments remain to be fully comprehended. This study explores whether calcium (Ca) isotope fractionation, linked to ACC‐forming cyanobacteria, can serve as a reliable tracer for detecting these microorganisms in modern and ancient settings. Accordingly, we measured stable Ca isotope fractionation during Ca uptake by the intracellular ACC‐forming cyanobacterium Cyanothece sp. PCC 7425. Our results show that Cyanothece sp. PCC 7425 cells are enriched in lighter Ca isotopes relative to the solution. This finding is consistent with the kinetic isotope effects observed in the Ca isotope fractionation during biogenic carbonate formation by marine calcifying organisms. The Ca isotope composition of Cyanothece sp. PCC 7425 was accurately modeled using a Rayleigh fractionation model, resulting in a Ca isotope fractionation factor (Δ44Ca) equal to −0.72 ± 0.05‰. Numerical modeling suggests that Ca uptake by these cyanobacteria is primarily unidirectional, with minimal back reaction observed over the duration of the experiment. Finally, we compared our Δ44Ca values with those of other biotic and abiotic carbonates, revealing similarities with organisms that form biogenic calcite. These similarities raise questions about the effectiveness of using the Ca isotope fractionation factor as a univocal tracer of ACC‐forming cyanobacteria in the environment. We propose that the use of Δ44Ca in combination with other proposed tracers of ACC‐forming cyanobacteria such as Ba and Sr isotope fractionation factors and/or elevated Ba/Ca and Sr/Ca ratios may provide a more reliable approach.

However, this paradigm was challenged when several cyanobacterial strains were found capable of forming intracellular amorphous calcium carbonate (ACC) (Benzerara et al., 2014(Benzerara et al., , 2022;;Couradeau et al., 2012).The ACC-forming cyanobacteria are found in diverse environments, including soil, marine, freshwater, and brackish water (Benzerara et al., 2022;Ragon et al., 2014).Using comparative genomics, Benzerara et al. proposed that ACC formation in cyanobacteria is a genetically controlled process, involving a new gene family (Benzerara et al., 2022).These strains exhibit the highest Ca demand among cyanobacteria, potentially driven by their intracellular ACC formation, suggesting a biological role for ACC, possibly as an intracellular pH buffer or a reservoir for inorganic carbon and calcium (Cosmidis & Benzerara, 2022;De Wever et al., 2019).
The formation of intracellular ACC by cyanobacteria also holds biogeochemical implications.Among ACC-forming cyanobacteria, some can bloom and locally reach relatively high cell density, suggesting that they may produce significant amounts of ACC in these environments and perturb C and Ca cycles, although this remains to be demonstrated (Gaëtan et al., 2022).Moreover, the ACC inclusions formed by some cyanobacteria strains sequester high concentrations of alkaline earth elements such as Ba, Sr, and the radioactive 90 Sr and 226 Ra isotopes, which holds two implications: (1) these cyanobacteria may be overlooked actors in the biogeochemical cycles of these trace elements (Blondeau et al., 2018;Cam et al., 2016); (2) they may offer some potential for remediating pollution of these radioactive isotopes (Mehta et al., 2019;Mehta, Bougoure, et al., 2022).
The process of intracellular ACC formation has ancient origins, as ACC-forming cyanobacteria are deeply rooted in the cyanobacterial phylogenetic tree (Benzerara et al., 2022;Ponce-Toledo et al., 2017).
Yet, their geological record remains unexplored, in part due to a lack of reliable tracers of ACC-forming cyanobacteria.Stable isotope fractionation has emerged as one of the most valuable tools for this purpose (e.g.Johnston & Fischer, 2012).Specifically, the calcium isotope composition of geological archives has been widely utilized to identify and quantify different calcium pools in the environment.The Ca isotope compositions of marine calcifiers (e.g.corals, coccolithophores, foraminifera) have been extensively studied and debated owing to their potential relevance as proxy archives for constraining sea-surface temperature in which these organisms lived (e.g.Böhm et al., 2006;Inoue et al., 2015;Mejía et al., 2018;Roberts et al., 2018).By contrast, no studies have measured the Ca isotope composition of intracellular ACC forming cyanobacteria.Considering the significance of Ca in ACCforming cyanobacteria, if the process of ACC formation is confirmed to be associated with a substantial Ca isotope fractionation, this could serve as a valuable diagnostic tool for assessing the presence of ACCforming cyanobacteria in both modern and ancient environments.
Additionally, the fractionation of Ca isotopes by ACC-forming cyanobacteria may provide a traceable record of how Ca moves from the surrounding extracellular solution to the interior of the cell.This, in turn, could offer valuable insights into the factors governing Ca regulation, uptake and storage.Accordingly, here, we experimentally determine the stable Ca isotope fractionation occurring during Ca uptake by the intracellular ACC-forming cyanobacterium Cyanothece sp.PCC 7425.

| Growth of cyanobacteria cultures
Axenic cultures of the intracellular ACC-forming cyanobacterium Cyanothece sp.PCC 7425 were incubated in the BG-11 growth medium in a batch reactor setup.We chose this strain for this study as it appears to be a good model for ACC-forming cyanobacteria.Indeed, (1) it grows reproducibly, at a relatively high rate, (2) a significant set of information about this strain is available in the literature, including about its growth and Ca uptake under different conditions (e.g.Cam et al., 2016Cam et al., , 2018)), the identity of Ca channels and transporters found in its genome (De Wever et al., 2019), and the characterization of the ACC granules formed by the strain and their relation with cell ultrastructure (e.g.Mehta, Gaëtan, et al., 2022;Mehta, Vantelon, et al., 2023), and last (3) it is genetically tractable (e.g.Chenebault et al., 2020).In the long-term compiling all these pieces of information on such a model strain may help to develop an integrative view on ACC formation and its geochemical implications.The chemical composition of BG-11 is provided in Table S1.The initial concentration of dissolved Ca in the growth medium is 300 μM.Temperature and luminosity were kept constant at 30°C and 30 μmol s −1 m −2 , respectively.The reactors were agitated at 120 rpm in a rotating shaker.Evaporation was compensated daily by adding sterile de-ionized water prior to sampling.Triplicate incubations were performed for a total duration of 15 days.The growth of the cells was monitored by measuring the optical density at 730 nm (OD 730 nm ) of the cell suspensions using a spectrophotometer.The pH of the cell suspension was measured over the growth of the cells.A control, non-inoculated experiment with the same initial conditions as in the cultures was also conducted.For measurements of dissolved Ca, 500 μL of cell suspension were filtered on a 0.22 μm polyvinylidene fluoride (PVDF) filter.The filtrate was subsequently acidified with concentrated HNO 3 (70 wt%) and analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES).

| Calcium isotope measurements
The calcium isotope compositions of Ca in both (i) the filtered solution, and (ii) the cell fraction, were measured.The cell fraction corresponds to intact cells containing ACC.At present, no protocol has been established to extract and preserve ACC outside of the cells.Therefore, here we used intact cells for the measurements.The solution fraction was collected by filtering the cell suspension.Aliquots of the samples containing 6 μg of Ca were initially acidified in conc.HNO 3 with 200 μL of ultrapure H 2 O 2 then refluxed at 80°C for ~6 h to break down all organic material in the samples.There is no isotope fractionation occurring during these steps, as the protocol ensured that all the calcium was extracted from the cells and remained in the solution.This was formerly shown by mass balance calculations (Mehta, Coutaud, et al., 2023).The samples were double spiked with equal amounts of 42 Ca and 48 Ca (a ratio of 1:1).Double spike containing 0.6 μg of calcium was added to each sample (a ratio of 1:10), before the spiked samples were dried to completion at 100°C to ensure homogenization of the double spike and sample.The spiked samples were subsequently re-dissolved in 0.5% HNO 3 and were run through a ThermoScientific Dionex 5000+ HPIC (High Performance Ion Chromatography) to separate the Ca following the methods of (Bradbury & Turchyn, 2018).
The resulting Ca was converted to a nitrate form, dried to completion at 100°C, then re-dissolved in 1 μL of 2 M HNO 3 before being loaded on zone-refined rhenium filaments with phosphoric acid activator for analysis on a ThermoScientific Triton Plus TIMS.The samples were analyzed for their Ca isotope ratios following the method detailed in (Bradbury & Turchyn, 2018).The Ca isotope values are reported using the standard delta notation δ 44 Ca (‰) = (( 44 Ca/ 40 Ca) sample / ( 44 Ca/ 40 Ca) standard −1) × 1000, where δ 44* Ca sol and δ 44* Ca bac are the relative changes in δ 44 Ca values from the initial isotope composition of the media for the filtered solution and cell fraction respectively.

| Uptake of Ca and growth of Cyanothece sp. PCC 7425
The OD 730 nm of Cyanothece sp.PCC 7425 cell suspension increased continuously from 0.14 to 1.11 over 12 days (Figure 1a).The increase in OD 730 nm was accompanied by an increase in pH from 6.8 to 9.5 and a decrease in the extracellular concentration of dissolved Ca from 300 μM to 80 μM (Figure 1b).By contrast, the concentration of Ca remained constant in the non-inoculated control (Figure S1).The extracellular solution remained undersaturated with respect to carbonate phases (i.e.calcite and aragonite) as shown in Table S3.The

| Ca isotope composition and Ca fractionation factor
With the removal of dissolved Ca, the Ca isotope composition of the solution (δ 44* Ca sol ) increased by 0.75‰ (Figure 2).Throughout the experiments, the cells were enriched in 40 Ca relative to the remaining solution (Figure 2).To quantitatively assess the observed Ca isotope fractionation patterns during its uptake by Cyanothece sp.PCC 7425, the isotope data were modeled using a Rayleigh distillation equation (Equation 1).
where R t represents the ratio 44 Ca/ 40 Ca at time t, R 0 represents the initial 44 Ca/ 40 Ca ratio, F r is the fraction of the initial calcium remaining in the system and alpha (α) is the fractionation factor.
Equation 1 can be recast into equation 2 by utilizing δ 44 Ca values (instead of 44 Ca/ 40 Ca ratios) where the slope of the line between the calcium isotopic composition of the remaining medium (δ 44* Ca sol ) and -ln(F r ) is equal to the calcium isotopic fractionation factor between the cells and solution (Δ 44 Ca bac-sol ) (Equation 2) (Bradbury et al., 2020;Mariotti et al., 1981).
(1) Using this approach, the Ca isotope fractionation factor between the cyanobacteria cells and the solution (Δ 44 Ca bac−sol ) was estimated at −0.72 ± 0.05‰.This visualization approach allows for easy assessment of the error associated with calculating the isotope fractionation using a Monte Carlo simulation (Bradbury et al., 2020).A Monte Carlo simulation was conducted based on 1000 simulations using normal distributions based around the measured data points with a standard deviation of 0.05‰ for δ 44 Ca and 2% for Ca concentrations (Figure 2a).The calculated Δ 44 Ca (bac-sol) of −0.72 ± 0.05‰ is also displayed on a traditional Rayleigh distillation plot in Figure 2b, with the shaded regions representing the error on the isotope fractionation value calculated using the Monte Carlo simulation.

| Calcium isotope composition of Cyanothece sp. PCC 7425
At the end of the experiment, 73% of the total calcium (Ca) was removed from the solution (Figure 1).As shown in previous studies, this removal is due to the Ca uptake by Cyanothece sp.PCC 7425 cells (Cam et al., 2018;De Wever et al., 2019).Moreover, electron microscopy and Ca K-edge XANES analyses revealed that ACC inclusions represent 84% of the total Ca in Cyanothece sp.PCC 7425 cells (De Wever et al., 2019;Mehta, Vantelon, et al., 2023).
The origin of Ca fractionation as observed from dissolved Ca measurements can be discussed.First, the role of evaporation in Ca isotope fractionation was null since no Ca goes to the vapor fraction and we compensated for evaporation by daily addition of (Ca-free) de-ionized water prior to sampling.Second, while the formation of Ca-bearing extracellular phases could alter the dissolved Ca isotope composition, the experimental conditions did not thermodynamically allow such a precipitation, as shown by the undersaturation of the extracellular solution with respect to carbonate phases (Table S3).This is also consistent with several previous studies using electron microscopy and spectroscopy on the same strain cultured under similar conditions showing the absence of extracellular carbonate phases (e.g.Benzerara et al., 2023;De Wever et al., 2019;Mehta, Gaëtan, et al., 2022;Mehta, Vantelon, et al., 2023).Overall, the enrichment of the solution in 44 Ca relative to 40 Ca is primarily attributed to the sequestration of Ca by Cyanothece sp.PCC 7425 with a preferential uptake of the lighter Ca isotope.
Enrichment of Cyanothece sp.PCC 7425 in 40 Ca over 44 Ca relative to the solution aligns with the kinetic isotope effects observed in Ca isotope fractionation during biogenic carbonate formation by marine calcifiers, where lighter isotopes are enriched in the precipitated biomineral (e.g.Böhm et al., 2006;Gussone et al., 2007;Inoue et al., 2015;Kisakürek et al., 2011).Among marine calcifiers, Ca isotope fractionation primarily occurs during cellular transport of Ca, possibly by dehydration of the Ca-aquocomplex at the surface of the channel or transporter, followed by a complete utilization of transported Ca for biomineral precipitation (Gussone et al., 2006;Inoue et al., 2015).The light isotope enrichment in biominerals arises from the differences in the dehydration reaction rates between light and heavy isotopes: lighter isotopes dehydrate more rapidly at the surface of the channel or transporter (DePaolo, 2011;Stevenson et al., 2014).Previously, we measured Ba and Sr isotope fractionation during their uptake by another ACC-forming cyanobacterium Gloeomargarita lithophora (Mehta, Coutaud, et al., 2023).Similar to Ca, the cells were enriched in lighter isotopes of Ba and Sr.However, the Ba and Sr isotope measurements of G. lithophora showed deviations from the Rayleigh distillation curve when the fraction of Ba and Sr remaining in the solution became very small.These deviations were attributed to a back reaction of Ba and Sr between the cells and the solution, which eventually led to an isotopic equilibrium between the cells and extracellular solution, resulting in no apparent fractionation after some time.This back reaction was hypothesized to be induced by cellular stress resulting from the accumulation of high amounts of Ba and Sr, which led to the dissolution of Ba-and Sr-enriched ACC and the subsequent release of trapped light isotopes of Ba and Sr to the extracellular solution.
Unlike Ba and Sr, the kinetic isotope effect on Ca isotope fractionation by Cyanothece sp.PCC 7425 was not overridden by isotope equilibrium effects.To further test the presence/absence of a back reaction in the case of Ca uptake, we modeled the Ca isotope composition data using the Rayleigh model that incorporates the back reaction, as used in our previous study (Mehta, Coutaud, et al., 2023).
Briefly this approach models the rate of Ca uptake into the cell versus the rate of exchange of Ca from the cell.The uptake process corresponds to the incorporation of Ca from the solution into the bacteria and is associated with a certain Ca isotope fractionation factor.The exchange process contains two equal reactions (which have an identical rate): (a) a release reaction of Ca from the bacteria to the solution (with no isotopic fractionation) and, (b) re-uptake reaction of Ca (also with no isotopic fractionation) from the solution.The uptake reaction impacts both the isotopic composition and extracellular concentration of Ca, while the exchange reaction only impacts the isotope composition of Ca (but not the concentration of Ca as the release and re-uptake reactions balance each other).
There is no isotope fractionation of Ca within the exchange term.
Both the uptake and exchange reactions are modelled using first order rate laws based on the concentration of calcium in the solution (for uptake) or cell (for exchange).For a full description of the model approach, see (Mehta, Coutaud, et al., 2023).
Figure 3 shows the evolution of the Ca (this study, A) and Ba and Sr (from the previous study; Mehta, Coutaud, et al., 2023) isotope compositions of the solution and cells as a function of the fraction of remaining element in the solution using various, yet constantwith-time ratios of uptake rate constant (k up ) over exchange rate constant (k ex ).The model with k ex /k up = 0.05 provided a reasonable fit between the data and model for both Sr and Ba in our previous study (Mehta, Coutaud, et al., 2023).The fitting of the Ca isotope data with the same ratio k ex /k up = 0.050 also agreed with the data.
However, we note that, in the case of Ca, we cannot decipher if this ratio is indeed 0.05 or is lower as the variation between these different cases would only appear for f(Ca) sol <0.26, while f(Ca) sol Cyanothece PCC 7425 contains less than 4% of Mg (defined as the Mg/(Mg + Ca) molar ratio in ACC inclusion) (Cam et al., 2018;Mehta, Vantelon, et al., 2023).This sharply contrasts with several stable biogenic ACC in eukaryotes that contain 20-40 mol % Mg in ACC, and where Mg incorporation promotes stabilization of amorphous carbonate (Levi-Kalisman et al., 2002).It also contrasts with the case of abiotic precipitation experiments, where in a solution with a high Mg:Ca ratio as, e.g., in the growth medium of Cyanothece PCC 7425, amorphous calcium magnesium carbonate should precipitate (Purgstaller et al., 2021).This is not observed within cyano-  (Evans et al., 2020), 25°C for (Mavromatis et al., 2018;Tesoriero & Pankow, 1996;Ulrich et al., 2021), and 30°C for (Couradeau et al., 2012;Dietzel et al., 2004).The data on D Ba and D Sr is not exhaustive but representative of the reported ranges in the literature.biogenic carbonates and abiotic carbonates, unless noted otherwise (Böhm et al., 2006;Bradbury et al., 2020;Gussone et al., 2005Gussone et al., , 2007;;Inoue et al., 2015;Langer et al., 2007;Niedermayr et al., 2010).While the Ca isotope fractionation factor reported for Cyanothece sp.PCC 7425 is distinctly different from those reported for biogenic aragonite, it bears resemblance with those reported for biogenic calcite.
These differences can be interpreted in different ways as described below.The role of CaCO 3 polymorph has been shown to impact Δ 44 Ca in abiotic systems, but such impacts may not be as pronounced in biotic systems (e.g.Gussone et al., 2020).Instead, it has been suggested that the dominant source of fractionation in biogenic systems occurs during the cellular uptake of Ca as it is assumed that all the Ca entering the cell is quantitatively precipitated to carbonate mineral, and thus there is no Ca isotope fractionation during precipitation (Gussone et al., 2006;Inoue et al., 2015).In this model, the type of polymorph precipitated by the organism has negligible impact on Ca isotope fractionation provided that Ca cellular uptake pathways remains same between the organisms (Gussone et al., 2006;Inoue et al., 2015).Thus resemblance between organisms forming biogenic calcite formation and Cyanothece sp.PCC 7425 and other organisms that form calcite, may suggest similarity in Ca cellular uptake pathways.Alternatively, this similarity should be inferred carefully as the uncertainty on the Δ 44 Ca values reported in our study is very small in comparison to that reported in other studies (Table S4).Moreover, we do not know how the Ca isotope fractionation varies across different ACC-forming cyanobacteria and the impact of rate of ACC precipitation.At present we do not know the relationship between intracellular precipitation rates and Ca uptake rates.Different strains of ACC-forming cyanobacteria may precipitate ACC at varying rates due to differences in extracellular chemistry and intracellular Ca regulation mechanisms but at this point, this idea is purely speculative.While the precipitation rate dependence of Δ 44 Ca of abiotic and biotic calcium carbonate minerals is well documented (Gussone et al., 2020 and references therein), similar relationships are undocumented in the case of ACC-forming cyanobacteria.Further studies are needed to investigate how the rate of ACC precipitation compares between different strains and its relationship to Ca isotope fractionation associated with ACC forming cyanobacteria.
One motivating question for this study was to test whether the Ca isotope fractionation factor associated with ACC-forming cyanobacteria may serve as a tracer of these microorganisms in the modern and past environments.The similarity between the Ca isotope fractionation produced by ACC-forming cyanobacteria and that of the precipitation of biogenic calcite makes the application of this approach challenging.However, in the future, the use of Δ 44 Ca in combination with other proposed tracers of ACCforming cyanobacteria may provide a more reliable approach (Cam et al., 2016;Mehta, Coutaud, et al., 2023).For example, as shown in Figure 4a-c Berdón et al., 2011).As a consequence, intracellular Ca concentration is tightly regulated within a range of ~100 nM in the cells (Clapham, 2007;Dominguez, 2004).Yet, in ACC forming cyanobacteria, if Ca stored in ACC inclusions was virtually dispersed in the cells, this would represent a total Ca cytoplasmic concentration of ~0.8-1.2 mol L −1 (Li et al., 2016).
The variations in Ca isotope compositions of the ACC forming cyanobacteria cells during the formation of ACC may provide a traceable record of how Ca moves from the surrounding extracellular solution to the specific site where ACC precipitates, and therefore shedding light into the factors governing Ca regulation, uptake and storage.
Numerical modelling suggested that any back reaction that was occurring between the cells and solution had little to no impact on the observed Ca isotope values as the amount of Ca in the solution remained above 0.26.The Ca isotope fractionation factor of Cyanothece sp.PCC 7425 is different from some organisms forming biogenic aragonite but has some similarities with several organisms forming biogenic calcite.
Our results suggest that Ca isotope offsets associated with ACC formation by cyanobacteria may not be unambiguous tracers of ACC-forming cyanobacteria in past and modern environments and that the combination of this proxy together with additional ones such as Sr/Ca, Ba/Ca and/or Ba and Sr isotopic compositions would be needed to recognize former cyanobacterial ACC in the geological record.

ACK N OWLED G M ENTS
We would like to thank the financial support from the Institut de science des matériaux (IMat), Sorbonne University under grant and the French Agence Nationale de la Recherche (ANR), under ANR-18-CE0-0013-02 and ANR-21-CE01-0010-02.We would also like to thank Cynthia Travert and Feriel Skouri-Panet for GEMME (geomicrobiology) facility located at L'Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC, Paris, France) that enabled streamlines measurement and analysis of datasets presented in this work.Lastly, Calcium isotope analyses were supported through NERC NE/R013519/1 (to HJB).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data reported in this manuscript are available in the supplementary information.
extracellular concentration of Mg in inoculated control remained relatively constant over time as shown in Figure S2.Intracellular concentration of Mg in Cyanothece sp.PCC 7425 is presently unknown.The time evolution of OD, pH, and Ca concentration during the growth of Cyanothece sp.PCC 7425 cells were consistent withthose observed by previous studies (e.g.Cam et al., 2018).
The pH and OD of Cyanothece sp.PCC 7425 cultures over time.(b) Dissolved concentration of Ca in the cultures of Cyanothece sp.PCC 7425 cultures.The error bars indicate standard deviation of replicates.Error bars are smaller than the symbol when not visible.Numeric data corresponding to this dataset is available in the Appendix S1.
sol = 44 * Ca sol0 + Δ 44 Ca (bac−sol) × − ln F r F I G U R E 2 (a) Ca isotope composition of the solution (δ 44* Ca sol ) plotted against -ln(F r ), where F r is the fraction of Ca remaining in the solution.(b) Ca isotope composition of the solution (solid square) and bacteria (empty square) plotted against the fraction of Ca remaining in the solution (F r ).Error bars correspond to the analytical 2 SD.The solid and dashed lines represent Rayleigh fits to δ 44* Ca values of the solution and bacteria.
remained greater than 0.26 in the present experiments.Overall, the kinetic isotope effects on Ca isotope fractionation by Cyanothece sp.PCC 7425 was well modeled based on the assumption of a Ca uptake that was mostly unidirectional, i.e. with no or very little (<5%) back reaction of Ca isotopes from the cells to the solution phase over the course of the experiments.Whether such a unidirectional Ca isotope flux would be observed when Cyanothece sp.PCC 7425 is cultured in presence of Ba, Sr and Ca remains an intriguing topic for future work.Unlike Ca, dissolved Mg concentrations remained constant over the time of the incubation, which shows that in our experiment, Mg incorporation in the cell was negligible (Figure S2).Moreover, this is supported by previous observations showing that ACC in F I G U R E 3 Visualization of the evolution of solution (filled markers) and cell fraction (empty markers) isotope values relative to the fraction of the element of interest remaining in the media for Ca (a: this study), Ba (b: Mehta, Coutaud, et al., 2023), and Sr (c: Mehta, Coutaud, et al., 2023).The black lines display the results of the uptake-exchange model utilized in the (Mehta, Coutaud, et al., 2023) study with varying ratios (0.25-dotted, 0.05-dashed, 0.01-solid) for the rate constants of exchange (k ex ) versus the rate constant for uptake (k up ).
Figure 4a summarizes the Δ 44 Ca for relevant biogenic carbonates and abiotic carbonates, re-calculated at 30°C using the relationship between Ca isotope fractionation and temperature reported for , comparing Δ 44 Ca and distribution co-efficient of Ba (D Ba ) and Sr (D Sr ) between different biogenic carbonates, abiotic carbonates, and ACC forming cyanobacteria, it becomes clear that the Δ 44 Ca + D Ba + D Sr of ACC forming cyanobacteria creates a unique fingerprint of ACC forming cyanobacteria, that may be used as a tracer to search for evidence of ACC forming cyanobacteria in the geologic rock record. 5 | CON CLUS IONS This study measured Ca isotope fractionation during Ca uptake by the ACC-forming cyanobacterium Cyanothece PCC 7425.The light isotope enrichment of Ca in Cyanothece results in a Δ 44 Ca equal to −0.72‰.Monitoring the Ca isotope composition of Cyanothece sp.PCC 7425 cells reveals that the Ca uptake by Cyanothece sp.PCC 7425 is mostly a unidirectional process.These findings could offer valuable insights into how Ca is cycled intracellularly in ACC forming cyanobacteria.While Ca is a biologically essential elements, high intracellular concentration of Ca are known to have toxic effects on the cellular functions (Barrán-