# Exploring the potential of Gloeomargarita lithophora for radionuclides bioremediation

Edern Pamart, Karim Benzerara, Giovanna Rosa Fois, Stefania Viola, Franck Chauvat, Corinne Cassier-Chauvat, Virginie Chapon

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Edern Pamart, Karim Benzerara, Giovanna Rosa Fois, Stefania Viola, Franck Chauvat, et al. Exploring the potential of Gloeomargarita lithophora for radionuclides bioremediation. Journal of Hazardous Materials, 2025, 492, pp.138155. 10.1016/j.jhazmat.2025.138155. hal-05025375

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# Exploring the potential of *Gloeomargarita lithophora* for radionuclides bioremediation

Edern Pamart<sup>a,b</sup>, Karim Benzerara<sup>c</sup>, Giovanna Rosa Fois<sup>d</sup>, Stefania Viola<sup>b</sup>, Franck Chauvat<sup>a</sup>, Corinne Cassier-Chauvat<sup>a,*</sup> <sup>DED</sup>, Virginie Chapon<sup>b,*</sup> <sup>DED</sup>

## HIGHLIGHTS

* *G. lithophora* durably sequesters <sup>90</sup>Sr.

* *G. lithophora* sequestering <sup>90</sup>Sr remains viable.

* Photosynthesis is required for <sup>90</sup>Sr sequestration.

## GRAPHICAL ABSTRACT

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Nuclear effluent

Bioremediation

Cyanobacteria

<sup>90</sup>Sr

Intracellular sequestration

Photosynthesis

## ABSTRACT

Current physico-chemical techniques for remediating <sup>90</sup>Sr traces from effluents are costly and can exhibit a low selectivity for Sr over Ca. Therefore, there is an incentive to develop an alternative method. In this study, we demonstrate that the cyanobacterium *Gloeomargarita lithophora* can rapidly remove more than 90 % of the <sup>90</sup>Sr activity from a nuclear plant effluent within 24 h. This process occurs through two steps: a first rapid and passive phase of <sup>90</sup>Sr sorbition to the cell surface, followed by an active phase of <sup>90</sup>Sr accumulation within the cells, partially driven by photosynthesis. We show that <sup>90</sup>Sr sequestration within *G. lithophora* cells is stable with no release of <sup>90</sup>Sr into the surrounding medium. Furthermore, we evidence that incorporation of 2.05 × 10<sup>7</sup> Bq g<sup>-1</sup>DCW of <sup>90</sup>Sr and exposure to an estimated dose rate of 1.7 mGy h<sup>-1</sup> for 5 days did not result in cell death. To go further in the development of a bioremediation method, we established the capacity to sequester <sup>90</sup>Sr in a synthetic medium mimicking the radioactivity and mineral composition of a real industrial nuclear effluent.

<footnote><sup>a</sup> Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette 91198, France</footnote>

<footnote><sup>b</sup> Aix-Marseille Université, CEA, CNRS, BIAM, Saint Paul-Lez-Durance 13115, France</footnote>

<footnote><sup>c</sup> Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590. Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), 4 Place Jussieu, Paris 75005, France</footnote>

<footnote><sup>d</sup> Laboratoire de Physique Clermont Auvergne, CNRS-IN2P3, Université Clermont-Auvergne, Clermont-Ferrand F-63000, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: corinnne.cassier-chauvat@cea.fr (C. Cassier-Chauvat), virginie.chapon@cea.fr (V. Chapon).</footnote>
These results highlight *G. lithophora* as a promising solution for effective bioremediation of water contaminated with ⁹⁰Sr, especially from nuclear plant effluents.

# 1. Introduction

Strontium (Sr) is an alkaline earth metal commonly occurring in nature, being the 15th most abundant element in the Earth's crust, with an average concentration of 340 ppm. In aqueous environments, Sr prevails as Sr²⁺, a form that is chemically similar to Ca²⁺. This similarity leads to a nonselective sorption of Sr traces by most organisms where it behaves like Ca [43]. Strontium is generally considered to have no essential physiological function, except in few organisms, such as the *Acantharia* protozoa where Sr contributes to their skeleton [24]. In humans, Sr intake primarily comes from water and food, and Sr accounts for 0.00044 % of the body mass. It is mainly localized in bones and dental enamel, and it has been linked to the control of bone metabolism [28,49]. At low concentrations, Sr stimulates bone formation and can be beneficial against osteoporosis, whereas, at higher doses, Sr can be toxic [21,49].

Naturally occurring Sr is composed of four stable isotopes: ⁸⁴Sr (0.56 %), ⁸⁶Sr (9.86 %), ⁸⁷Sr (7.0 %) and ⁸⁸Sr (82.58 %) [44] while radioactive isotopes such as ⁸⁹Sr and ⁹⁰Sr are artificially produced by nuclear fission. ⁸⁹Sr has been used for decades in nuclear medicine for pain palliation in metastatic bone cancer [50] and has a relatively low half-life of 50.57 days. ⁹⁰Sr, a β⁻-emitter with a half-life of 28.8 years, is one of the most hazardous anthropogenic radionuclides [5]. This radioisotope has been massively released into the environment during nuclear weapons testing and nuclear reactor accidents, such as those at Chernobyl and Fukushima [12,30–32,60]. ⁹⁰Sr poses a dual threat to living organisms either through external sources or after intake. Once incorporated by plants, it enters the food chain becoming a primary concern for both environmental and human health [12]. In humans, and especially children, its accumulation in bone tissues increases the risk of cancers due to its high solubility and bio-toxicity [58]. Therefore, there is an incentive to develop effective remediation strategies to minimize its spread in the environment and mitigate its impact.

In industrial settings, ⁹⁰Sr is found in radioactive water effluents produced by nuclear power plants, requiring specific treatment before being discharged [26]. Current methods for remediating ⁹⁰Sr and other radionuclides from effluents rely on physicochemical techniques such as precipitation, ion exchange, selective adsorption, electro-coagulation and nanofiltration [29,51]. These techniques were used in the so-called Advanced Liquid Processing System (ALPS) to treat the huge volumes of contaminated water generated during the Fukushima accident [34]. However, these methods are costly and can produce large amounts of secondary waste [34]. Selectivity is a critical parameter for the efficient extraction of ⁹⁰Sr from effluents, where its concentration is much lower than that of competing ions such as calcium [1]. In this regard, the high selectivity of biological processes for the capture, transport and sequestration of metal cations makes them attractive candidates for the development of new and effective remediation techniques [36]. Few organisms capable of massive accumulation of strontium have been described. Among them are the marine heterotrophic protists *Acantharia*, which build celestite (SrSO₄) skeletons [24]; various diplomenid species, which form intracellular celestite crystals in aquatic environments [47]; several unicellular green microalgae of the genus *Tetraselmis* [37,54]; and the unicellular cyanobacterium *Gloeomargarita lithophora*, which accumulates strontium-rich calcium carbonate granules in its cytoplasm [14,20]. Many studies have been carried out for soil and water bioremediation using cyanobacteria. The possibility of cultivating cyanobacteria on a large scale in ponds, coupled with their ability to fix carbon dioxide and atmospheric nitrogen in certain genera, as well as their resistance to stresses [15] enable them to proliferate in diverse controlled or contaminated environments and serve for bioremediation

applications [16].

*G. lithophora* accumulates high levels of Ca in the form of intracellular amorphous calcium carbonates (iACC) granules within micro-compartments occupying ~6 % of the total cell volume [11,20,23]. Very interestingly, *G. lithophora* preferentially incorporates Ba and Sr over Ca. Hence, it can form Ba- and Sr-carbonate granules while growing in solutions containing a relatively high Ca concentration of 50 µM and Ba and Sr concentrations as low as 0.0002 and 0.001 µM, respectively [10].

This unique capability has been exploited to remove traces of ⁹⁰Sr and ²²⁶Ra from laboratory cultures with high dissolved Ca/Sr or (Ba,Sr)/Ra ratios, making *G. lithophora* an appealing candidate for further studies aiming at the bioremediation of these radionuclides [38,39]. However, this initial research did not fully explore the potential of the use of *G. lithophora* for treating industrial nuclear effluents. The tested ⁹⁰Sr concentration was 180 times lower than that found in effluents (7.3 × 10⁶ Bq L⁻¹) and the organism's tolerance to ionizing radiation was not evaluated. Furthermore, the *G. lithophora* strain used was not axenic and was contaminated by an alphaproteobacterium affiliated to the *Sandarakinorhabdus* genus [41], questioning the potential role of the bacterial contaminant in these *G. lithophora* cultures.

# 2. Materials and methods

## 2.1. Strains and growth conditions

*G. lithophora*, originally isolated from the alkaline Lake Alchichica, Mexico [20], was obtained from Karim Benzerara. It is available as a non-axenic culture from the culture collection of algae protozoa (CCAP, strain 1437/1) and the Paris Museum collection (PMC at the Museum National d'Histoire Naturelle, strain 919.15) [41]. *G. lithophora* was grown routinely at 30°C, under continuous white light (cool white neon tubes 750 lux; 9.4 µmol photons m⁻² s⁻¹) with agitation (140 rpm) in liquid mineral medium (MM). The MM medium corresponds to BG11 [57], enriched with 3.78 mM Na₂CO₃ [25] and buffered with 2 mM HEPES at pH 7.5. Growth was monitored by measuring optical density at 750 nm (OD₇₅₀). The relationships between OD₇₅₀, cell density and cell dry mass were estimated as follows: 7.9 x 10⁷ cells mL⁻¹ OD₇₅₀ unit⁻¹ and 3.2 x 10⁻⁴ g of dry cell weight (DCW) OD₇₅₀ mL⁻¹. Growth on plates was performed using MM with 1 % Bacto Agar (Difco) and 2 mM thiosulfate.

To test the capacity of *G. lithophora* to sequester the ⁹⁰Sr from a nuclear effluent, a synthetic medium mimicking the average composition of an effluent treated at the station for the treatment of liquid radioactive effluent (STEL; CEA-Marcoule, France) was prepared (Table S1). The final pH of this solution was 6. The synthetic effluent was diluted using sterile MM, moderately hard water (composition: 96 mg L⁻¹ NaHCO₃; 60 mg L⁻¹ CaSO₄·2H₂O; 60 mg L⁻¹ MgSO₄ and 4 mg L⁻¹ KCl; [62]) or reverse osmosis water, all buffered with 2 mM HEPES at pH 7.5.

## 2.2. Visualization of intracellular carbonates by electron microscopy

Scanning electron microscopy (SEM) analyses were performed as described by Benzerara et al. [9] at the IMPMC microscopy platform (Sorbonne Université, France). Briefly, cells were harvested by centrifugation, rinsed with MilliQ® water, and collected on polycarbonate filters with a pore size of 0.2 µm (Millipore). Samples were dried at room temperature and coated with a conductive carbon layer using a Leica EM SCD500 evaporator. The SEM apparatus was a Zeiss Ultra 55 microscope equipped with a field emission gun (FEG). Backscattered electron detection utilized an angle-selective backscatter (AsB) detector with a
working distance of 7.5 mm, an acceleration voltage of 15 kV, and a 60 µm aperture. Chemical mapping was conducted using energy dispersive X-ray spectrometry (EDXS). Data were processed with the ESPRIT software (Bruker).

## 2.3. $^{90}$Sr uptake by Gloeomargarita lithophora

Cells grown in MM to an OD$_{750}$ of 1 were centrifuged for 15 min at 4415 g (i.e. 5000 rpm, SL40R Thermo Scientific centrifuge). Cell pellets were washed twice with MM and re-suspended in MM or synthetic effluent to an OD$_{750}$ of 1. Aliquots (1–3 mL depending on the experiment) were distributed in 14 mL tubes (Falcon® round bottom polystyrene tubes), and supplemented without (control) or with $^{90}$Sr (certified stock solution at $4 \times 10^7$ Bq L$^{-1}$ from LEA, Pierrelatte, France, adjusted to pH 8 with NaOH just before use). The $^{90}$Sr stock solution contained 90-Yttrium ($^{90}$Y), the decay product of $^{90}$Sr which is also a β-emitter, in secular equilibrium. The tested $^{90}$Sr activities were D1 (4 x $10^4$ Bq L$^{-1}$; 8.8 x $10^{-11}$ M; 7.9 ng L$^{-1}$), D2 (4 x $10^5$ Bq L$^{-1}$; 8.8 x $10^{-10}$ M; 7.9 ng L$^{-1}$) and D3 (7.3 x $10^6$ Bq L$^{-1}$; 1.606 x $10^{-8}$ M; 1.44 µg L$^{-1}$). The initial $^{90}$Sr activity of each cell suspension was measured by mixing aliquots (100 µL for D1 and D2, or 50 µL for D3) with 4 mL of scintillating solution (Ultima Gold™ Sigma-Aldrich) in 20 mL vial. Counting was performed 3 times for 5 min using a liquid scintillation analyzer (Tri-Carb® 3100TR PerkinElmer) in the energy range 20–310 keV. The obtained value was considered as 100% $^{90}$Sr activity. Then, the cell suspensions were incubated at 30°C under light and shaking (100 rpm). At various time point, aliquots were collected (150 µL for D1 and D2, or 100 µL for D3) and centrifuged for 10 min at 16,100 g (i.e. 13,200 rpm in an Eppendorf 5415D centrifuge). The radioactivity was measured in 100 µL (for D1 and D2) or 50 µL (for D3) of supernatant as described above. These measures define the quantity of $^{90}$Sr present in solution, i.e. the $^{90}$Sr atoms that are neither attached to nor internalized within cells. The remaining cell suspensions were centrifuged again for 10 min at 16,100 g and the residual supernatant was discarded. To remove the $^{90}$Sr atoms sorbed to the cell surface, the obtained cell pellets were resuspended in 150 µL (for D1 or D2) or 100 µL (for D3) 0.12 M NaCl and 50 mM EDTA at pH 8. The samples were centrifuged at 16,100 g for 10 min and the radioactivity was measured in 100 µL (for D1 and D2) or 50 µL (for D3) of the supernatant, corresponding to the fraction sorbed to the cell surface. The remaining cell suspensions were centrifuged again for 10 min at 16,100 g and the residual supernatant was discarded. The resulting cell pellets were resuspended in 150 µL (D1 and D2 samples) or 100 µL (D3 samples) MM and the radioactivity, corresponding to the intracellular fraction, was measured in 100 µL (for D1 and D2) or 50 µL (for D3) (see Fig. S1 summarizing the protocol). For each $^{90}$Sr concentration, a cell-free abiotic control was included to verify that $^{90}$Sr atoms did not precipitate or bind to the plastic tubes. Count per minute (CPM) values in the energy range 20–310 keV were corrected using the background activity measured on a blank MM sample. Internal standards, made with the certified $^{90}$Sr stock solution (LEA) and prepared like the samples, were systematically included in each counting sessions.

To monitor the stability of $^{90}$Sr sequestration in G. lithophora, a pulse-chase experiment was performed as follows. Cells (3 mL at OD$_{750}$=1 in MM) were exposed to $^{90}$Sr (D2) for 8 days at 30°C under light with shaking (100 rpm) until $^{90}$Sr was completely removed from the solution by the cells. The cells were pelleted, washed once with MM, resuspended in the same volume of fresh MM and incubated for 17 days at 30°C under light and agitation. To test possible release of $^{90}$Sr in the solution, an aliquot of the cell suspension (150 µL) was collected at each time point, centrifuged 10 min at 16,100 g and the radioactivity was measured in 100 µL of supernatant.

## 2.4. Calculation of $^{90}$Sr bioaccumulation

The mass of $^{90}$Sr accumulated by the cells at a time t, normalized by

cell mass, was determined as follows: $M_{Sr} = \frac{(X_0 - X_t)\alpha}{cell mass at t}$ with X: $^{90}$Sr activity (Bq L$^{-1}$); α = 2.2 x $10^{-15}$ mol Bq$^{-1}$: conversion factor from activity to mass units and t: sampling time

## 2.5. Estimation of the cell survival after exposure to $^{90}$Sr

Three 25 µL drops of cell suspensions (exposed or not to $^{90}$Sr) were spotted onto MM agar plates supplemented with 2 mM thiosulfate. Plates were incubated at 30°C under light. Photographs were taken every 5 days.

## 2.6. Estimation of the radiation dose received by Gloeomargarita lithophora

The radiation dose received by G. lithophora cells was simulated using the GATE [27,52,53] Monte-Carlo simulation toolkit (version 9.3) based on Geant4 version 11.2 [2–4]. The wall of the tube (Falcon™) measured 1 mm in thickness and was modelled with an internal diameter of 17 mm and an external diameter of 19 mm. A hemispherical shape was modeled at the bottom of the tube using a radius of 8.5 mm. Two volumes of 3.286 mL (for D1 and D2) and 1.286 mL (for D3) were simulated. The radioactive source was homogeneously distributed, and both $^{90}$Sr and $^{90}$Y continuum beta spectra were considered. Energy deposited in the volume was collected implementing a DoseActor. Microorganisms were assumed to be homogeneously distributed within the cell suspension. The energy deposited on the entire volume was considered absorbed by G. lithophora cells. Energy deposited per particle emitted and simulated by Monte Carlo was used to calculate the absorbed dose per particle emitted. The dose rate was calculated as follows:

$$DR = D_p \bullet A_v \bullet V$$

DR: Dose Rate (mGy h$^{-1}$)

Dp: Absorbed dose per particle emitted (mGy Bq$^{-1}$ h$^{-1}$)

Av: Activity concentration (Bq L$^{-1}$)

V: Volume (L)

## 2.7. Effect of DCMU on photosynthesis

Photosynthesis was inhibited by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at 50 µM 1 h prior to the $^{90}$Sr sequestration assay. To verify that DCMU completely inhibits photosystem II (PSII) activity in G. lithophora, chlorophyll fluorescence kinetics were measured and compared between treated and untreated cells, which were dark-adapted for 10 minutes before measurements. Three mL of cell suspensions at an OD$_{750}$ of 1 were used for each measurement, conducted using a laboratory-built Joliot-type spectrophotometer (JTS). Short measuring pulses (15 µs) were provided by a broad-emission white LED (COB-V6HD-THRIVE-WHT-SQ-6500K, Bridgelux) filtered via a 480 nm band-pass interference filter (Edmund Optics, 10 nm FWHM). The measuring pulses were divided between a square cuvette placed in front of the measuring diode protected by a RG665 filter (Schott) and a reference diode protected by a BG39 filter (Schott). Multi-turnover saturating pulses (500 ms, 4000 µmol photons m$^{-2}$ s$^{-1}$) and non-saturating actinic illumination (55 µmol photons m$^{-2}$ s$^{-1}$) were provided by a ring of orange-red LEDs.

## 2.8. Measurement of respiratory activity

Respiration was inhibited by the addition of 100 µM potassium cyanide (KCN) 1 h prior to the $^{90}$Sr sequestration assay. To verify that KCN inhibits the activity of the respiratory terminal oxidases in G. lithophora, we measured its effect on the rates of O$_{2}$ consumption in darkness, and on the kinetics of light-dependent oxidation and re-reduction of the reaction center chlorophylls (P$_{700}$) of photosystem I (PSI). P$_{700}$ kinetics
were measured as the difference in the light-induced absorption changes at 705 and 740 nm using the same above-described JTS apparatus used for fluorescence measurements. The measuring pulses were filtered via a 705 nm and a 740 nm band-pass interference filters (Edmund Optics, 10 nm FWHM), and both measuring and reference diodes were protected by RG695 filters (Schott). The kinetics were measured during and after 1 s illuminations at 2000 µmol photons m⁻² s⁻¹ provided by the orange-red LEDs. Three mL of cell suspensions at an OD₇₅₀ 3.5 were used for each measurement. All measurements were performed on cells treated with 50 µM DCMU, with or without 100 µM KCN. Where indicated, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was also added to a final concentration of 50 µM.

Oxygen consumption and evolution were measured with a MicroXTX3 Micro fiber optic oxygen transmitter equipped with a needle-type oxygen microsensor (PreSens). Measurements were conducted at 23°C with a sampling rate of 1 s. Two mL of cell suspensions at an OD₇₅₀ of 3.5 were used for each measurement. Samples were placed in an open square quartz cuvette and stirred continuously throughout the measurement. When indicated, illumination at 850 µmol photons m⁻² s⁻¹ was provided by a red LED.

## 2.9. Statistical analyses

Statistical analyses were performed using the non-parametric Kruskal-Wallis test (p < 0.05 *; p < 0.01 **; p < 0.001 ***; ns, not significant) with at least 3 independent experiments.

# 3. Results

## 3.1. Obtention of a nearly axenic strain of Gloeomargarita lithophora

We attempted to generate an axenic strain to better control the biological agents involved in the experiments. Moreira et al. [41] identified one contaminant as an alphaproteobacterium affiliated with the *Sandarakinorhabdus* genus, often found associated with cyanobacteria [13,56]. Contamination was easily detected on MM agar plates by

the formation of abundant white colonies as early as 9 days compared to *G. lithophora*, which was only visible after 30 days (Fig. S2). Attempts to eliminate contaminants using antibiotics were unsuccessful since they appeared to have a similar antibiotic resistance pattern than *G. lithophora*. However, three rounds of subcultivation on MM plates with 2 mM thiosulfate stimulated the growth of *G. lithophora* and greatly reduced the proportion of contaminants to an estimated abundance of 3 contaminant cells per 2 x 10⁶ *G. lithophora* cells (Fig. 1A and Fig. S3). Using this nearly axenic strain, we verified that the decontamination procedure did not affect the growth of *G. lithophora* in liquid culture, its ability to form iACC granules (Fig. 1B-C) and its capacity to sequester Sr within these granules (Fig. 1D–G). Therefore, we used this nearly axenic strain for all following experiments described herein.

## 3.2. Gloeomargarita lithophora strain is an effective ⁹⁰Sr trap

To verify that the nearly axenic *G. lithophora* strain retains its capacity to efficiently uptake and sequester ⁹⁰Sr, we exposed cells grown in liquid MM to 4 × 10⁴ Bq L⁻¹ of ⁹⁰Sr (hereafter referred to as D1), a concentration previously used by [38]. As shown in Fig. 2, ⁹⁰Sr was rapidly removed from the solution: 90 % of the initially added ⁹⁰Sr was associated with the cells within 24 h and only 2 % remained in the solution after five days. Two distinct kinetic phases of ⁹⁰Sr accumulation were observed: a first phase, lasting 4.5 h, where cells accumulated 21 ng ⁹⁰Sr g⁻¹DCW at a rate of 4.6 ng ⁹⁰Sr g⁻¹DCW h⁻¹ and a second phase, 200 times slower, where cells accumulated 3.6 ng ⁹⁰Sr g⁻¹DCW at an average rate of 0.019 ng ⁹⁰Sr g⁻¹DCW h⁻¹ (Fig. 2A and 2G). These results showed that the nearly axenic strain is still able to efficiently sequester ⁹⁰Sr provided at the D1 concentration.

In medium activity nuclear waste, ⁹⁰Sr activity often exceeds D1. Therefore, to examine the potential of *G. lithophora* for bioremediation of higher ⁹⁰Sr concentrations, cells grown in MM were exposed to ⁹⁰Sr at 4 × 10⁵ Bq L⁻¹ (D2) and 7.3 × 10⁶ Bq L⁻¹ (D3). D3 corresponds to the average ⁹⁰Sr activity in effluents treated at the STEL facility (Yves Barré personal communication). As observed for D1, ⁹⁰Sr was rapidly removed in a similar bi-phasic kinetics and 80–90 % of the initially added ⁹⁰Sr

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Growth and formation of intracellular calcium and strontium carbonate granules by the nearly axenic *Gloeomargarita lithophora*. A) Growth of *G. lithophora* spotted on MM agar plate with 2 mM thiosulfate after axenisation. B) SEM images in the secondary electron (SE) mode of the cells from almost axenic *G. lithophora* grown in MM allow detection of iACC (smaller brightest spots) together with polyphosphate granules (large bright grey). C) Overlay of Ca (red), P (blue) and Si (green) chemical maps obtained by energy dispersive X-ray spectrometry over the AsB SEM image (grey levels) shown in (B). D) SEM images in the secondary electron (SE) mode of the cells from almost axenic *G. lithophora* grown in the presence of 10 mM Sr in the mineral medium. E) Sr (yellow) chemical map obtained by EDXS F) Overlay of Ca (red) and P (blue) Sr (yellow) chemical maps obtained by EDXS and corrected from the bremsshtrahlung over the AsB SEM image (grey level) shown in (D). G) Average EDXS spectrum of the area scanned in D.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<table><caption>**Fig. 2.** The nearly axenic strain of *Gloeomargarita lithophora* is able to sequester and survive <sup>90</sup>Sr. A and D) Time evolution of <sup>90</sup>Sr residual activity (A(<sup>90</sup>Sr) in Bq L<sup>-1</sup>) in the solution (blue) or associated to the cells (red) during exposure of *G. lithophora* to D1 (A) or D3 (D) in MM. B and E) Distribution of <sup>90</sup>Sr at each sampling time (hours) in the soluble (blue) and cellular fractions (red) expressed as a percentage of the initial <sup>90</sup>Sr total activity: D1 (B) or D3 (E). C and F) Growth of *G. lithophora* after 120 h exposure to D1 (C) or 120 h exposure to D3 (F) and compared to non-exposed control cells. Cultures were spotted on MM plus 2 mM thiosulfate agar plates and incubated 28 days at 30°C with light. Error bars represent standard deviation from biological triplicates. When not visible, error bars are smaller than the symbols. G) uptake capacity and accumulation rate of <sup>90</sup>Sr by *G. lithophora* during the 2 phases of accumulation. DCW: dry cells weight.</caption><thead><tr><th rowspan="3"></th><th colspan="6">Activity of added <sup>90</sup>Sr</th></tr><tr><th colspan="2">D<sub>1</sub></th><th colspan="2">D<sub>2</sub></th><th colspan="2">D<sub>3</sub></th></tr><tr><th>4 x 10<sup>4</sup> Bq. L<sup>-1</sup></th><th>8.8 x 10<sup>-11</sup> M</th><th>4 x 10<sup>5</sup> Bq. L<sup>-1</sup></th><th>8.8 x 10<sup>-10</sup> M</th><th>7.3 x 10<sup>6</sup> Bq. L<sup>-1</sup></th><th>1.606 x 10<sup>-8</sup> M</th></tr></thead><tbody><tr><td>Duration of exposure (h)</td><td>[0-4.5]</td><td>[4.5-192]</td><td>[0-4.5]</td><td>[4.5-192]</td><td>[0-4.5]</td><td>[4.5-120]</td></tr><tr><td>Uptake capacity of <sup>90</sup>Sr<br>(ng <sup>90</sup>Sr g DCW<sup>-1</sup>)</td><td>21</td><td>3.6</td><td>189</td><td>41</td><td>2721</td><td>1523</td></tr><tr><td>Rate of <sup>90</sup>Sr<br>accumulation<br>(ng <sup>90</sup>Sr gDCW<sup>-1</sup> h<sup>-1</sup>)</td><td>4.6</td><td>0.019</td><td>42</td><td>0.22</td><td>604</td><td>13.1</td></tr></tbody></table>

was associated with the cells within 24 h (Fig. 2D, 2E and S4). The rate of <sup>90</sup>Sr accumulation during the rapid phase and the total amount of <sup>90</sup>Sr sequestered by the cells were proportional to the initial <sup>90</sup>Sr concentration (Fig. S5), showing that the uptake system was not saturated. Moreover, 90 % of the <sup>90</sup>Sr in D3 was removed from the solution within 24 h, showing that the maximum sequestration capacity of *G. lithophora* exceeds 2.05 × 10<sup>7</sup> Bq g<sup>-1</sup>DCW. Neither the large excess of competing calcium ions (250 µM) nor the radiations emitted by <sup>90</sup>Sr altered the

ability of *G. lithophora* to sequester <sup>90</sup>Sr.

### 3.3. The viability of *Gloeomargarita lithophora* is not affected by <sup>90</sup>Sr accumulation

Growth of the nearly axenic *G. lithophora* strain on solid MM medium containing thiosulfate enabled an assessment of cell survival after <sup>90</sup>Sr exposure. Cells exposed to D1 or D2 for 72 hs, or to D3 for 120 h, i.e.
until more than 90 % of the initially added $^{90}$Sr was removed from the medium, were plated on MM agar. As shown in Fig. 2C–F and S4C, cell viability was unaffected by D1, D2 nor D3 concentrations of $^{90}$Sr. Simulations were carried out to estimate the radiation dose received by *G. lithophora* during these experiments. Since the travel distance of β-particles emitted from $^{90}$Sr and its decay product $^{90}$Y is about 500 µm, the dose received by cells (average diameter size = 3.9 µm) is mainly due to external radiations rather than internal exposure. The dose rates for *G. lithophora* cells exposed to $^{90}$Sr at D1, D2 and D3 were estimated to be of 0.025, 0.252, and 1.7 mGy h$^{-1}$ respectively.

### 3.4. $^{90}$Sr is transiently associated with the cell surface prior to its accumulation within the cells

$^{90}$Sr atoms associated with *G. lithophora* can be sorbed to cell surfaces or accumulated inside cells. To distinguish between these two localizations, radioactivity was measured after exposure to $^{90}$Sr (D2) in i) the extracellular fraction, corresponding to the supernatant obtained after centrifugation of the cell suspension; ii) the sorbed fraction, separated from the cells by washing with NaCl-EDTA and iii) the intracellular fraction represented by the NaCl-EDTA-washed cell pellet (Fig. S1). As shown in Fig. 3, $^{90}$Sr was rapidly removed from the solution. During the first 2.55 h, the radioactivity in the fraction sorbed onto cells exceeded that in the intracellular fraction. Then, from 4.62 h to 72 h, the $^{90}$Sr activity in the sorbed fraction decreased, while the radioactivity in the intracellular fraction increased up to 93 % of the total activity at 72 h. These results clearly show that the two kinetic phases of $^{90}$Sr accumulation reflects a rapid $^{90}$Sr sorption on the cell surface followed by a slower uptake and accumulation inside the cells.

### 3.5. $^{90}$Sr sequestration by Gloeomargarita lithophora is enhanced by photosynthesis

To investigate whether $^{90}$Sr uptake and accumulation inside the cells is an active process, we tested the involvement of photosynthesis. In cyanobacteria, the photosynthetic metabolism is the main source of ATP and reducing power (NAD(P)H). We measured the efficiency of $^{90}$Sr sequestration by *G. lithophora* cells exposed to D1 in the presence or absence of DCMU, an inhibitor of PSII activity [17,45]. Using measurements of fluorescence emission, we verified that 50 µM DCMU immediately and completely inhibited electron transfer from PSII to the downstream photosynthetic electron transport chain in *G. lithophora* (Fig. S6A). When DCMU was added to the cells, the bi-phasic kinetics of

$^{90}$Sr sequestration was still observed (Fig. 4A). However, $^{90}$Sr removal by the cells was significantly less efficient than in the control without DCMU. After 6.5 and 72 h, 36 % and 25 % of $^{90}$Sr remained in the soluble fraction of the DCMU-treated cell suspension respectively, compared with 5 % and 1 % in the control samples. These findings indicate that photosynthesis plays an important role in $^{90}$Sr uptake and sequestration by *G. lithophora*.

To examine whether DCMU affects $^{90}$Sr entry into the cells, we determined $^{90}$Sr distribution between extracellular, sorbed and intracellular fractions using the above-described protocol. The fractionation experiment was carried out after 30 h of incubation, when the difference in $^{90}$Sr accumulation by cells with and without DCMU reached its maximum (Fig. 4A). As shown in Fig. 4B, DCMU significantly reduced intracellular $^{90}$Sr accumulation as compared to the control, while the sorbed fraction remained unaffected. However, DCMU did not completely impair $^{90}$Sr accumulation since about 30 % of the initial $^{90}$Sr was still found to be inside the cells. This suggests that photosynthesis is involved but not directly essential for intracellular $^{90}$Sr accumulation.

Therefore, we investigated whether respiration could also play a role in $^{90}$Sr accumulation by using KCN, an inhibitor of respiratory terminal oxidases [19,48]. Respiration can produce ATP and NADH, especially in darkness when photosynthesis is not active [46]. In *G. lithophora*, KCN (100 µM) impacted both the light-dependent kinetics of oxidation and re-reduction of the reaction center chlorophylls ($P_{700}$) of PSI (Fig. S6B) and the rates of $O_2$ consumption in darkness (Fig. S6C). The data showed that $O_2$ consumption by *G. lithophora* cells in darkness is low as compared to the photosynthetic $O_2$ evolution induced by high light (Fig. S6C). Consistent with this low respiratory activity, KCN, alone or with DCMU, had no effect on $^{90}$Sr sequestration (Fig. 4A).

### 3.6. $^{90}$Sr is durably sequestered in Gloeomargarita lithophora and does not affect cell viability

Determining whether $^{90}$Sr remains durably trapped in *G. lithophora* cells is critical for its potential application in bioremediation. To address this question, *G. lithophora* cells were exposed to $^{90}$Sr (D2) for 8 days, a duration significantly exceeding the 72 h required for complete radio-nuclide trapping. Then, cells were washed and resuspended in fresh MM without $^{90}$Sr and were cultivated under standard conditions for 17 days. At various time points, $^{90}$Sr in the extracellular fraction was quantified as described earlier. At the end of the experiment, cell viability was estimated. As shown in Fig. 5A, *G. lithophora* cells stably retained their trapped $^{90}$Sr atoms, with less than 5 % of the total radioactivity released

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Distribution of $^{90}$Sr in the extracellular, adsorbed and intracellular fraction. Time evolution of the extracellular (blue), adsorbed (orange) and intracellular (green) fractions of $^{90}$Sr expressed as a % of initial concentration during exposure of *G. lithophora* cells to D2 in MM medium. Error bars represent standard deviation from biological triplicates. When not visible, error bars are smaller than the symbols.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 4. A) Time evolution of ⁹⁰Sr residual activity in the solution during exposure of *G. lithophora* to D1 in MM (open triangles) or in MM with 50 µM DCMU (open circles), 100 µM KCN (squares), or DCMU and KCN (diamonds). Error bars represent standard deviation from biological triplicates. When not visible, error bars are smaller than the symbols. B) Distribution of ⁹⁰Sr after 30 h incubation in the extracellular (blue), adsorbed (orange) and intracellular (green) fractions expressed as a percentage of the initial ⁹⁰Sr total activity. Error bars represent standard deviation from biological triplicates. Extracellular and intracellular fractions are significantly different between control and DCMU-treated samples (* P < 0.05, ** P < 0.01).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. ⁹⁰Sr is permanently trapped in the cells. A) Distribution of ⁹⁰Sr in the extracellular (blue) and cellular fractions (red) expressed as a percentage of the initial ⁹⁰Sr total activity. Cells were previously exposed to D2 for 8 days (pulse) before being resuspended in fresh MM (T = 0) and cultured for 404.5 h (chase). Error bars represent standard deviation from biological triplicates. When not visible, error bars are smaller than the symbols. B) At the end of the chase experiment, cultures were spotted on MM plus 2 mM thiosulfate agar plates and incubated at 30°C with light. A non-exposed control was performed in parallel.</figcaption></figure>

into the medium after 17 days of incubation (i.e. 22 days after complete ⁹⁰Sr incorporation). Furthermore, similar growth was observed between ⁹⁰Sr-treated and untreated cells (Fig. 5B), showing that prolonged exposure to ⁹⁰Sr does not adversely affect cell viability.

## 3.7. Toward the development of a bioremediation process: *G. lithophora* efficiently removes ⁹⁰Sr from a synthetic nuclear effluent

The chemical composition of industrial effluents varies depending on the nuclear plant. Here, we synthesized a medium mimicking the average chemical composition of effluents treated at the STEL (Y. Barré, personal communication) (Table S1). *G. lithophora* cells were resuspended in this medium and incubated for 24 h with the D3 concentration of ⁹⁰Sr. As shown in Fig. 6A, only 12 % of ⁹⁰Sr was accumulated by the cells under this condition, a much lower value than the 90 % of ⁹⁰Sr accumulation observed in MM for the same 24 h incubation duration (Fig. 2E) and (Fig. 2 F). The synthetic STEL medium appeared toxic to cells, causing discoloration of the culture, which was intensified by ⁹⁰Sr (Fig. 6B). Within 3.5 h, there was no visible change of the cell suspension incubated in the control synthetic STEL effluent, whereas, in the presence of ⁹⁰Sr, the cells suspension turned pale yellow, indicating cell

death (Fig. 6B). For longer incubation times (24 h) in the synthetic effluent without radioactivity, the cell suspension begun to discolor, whereas in MM, no visible color change was observed (Fig. 6C). Collectively these data suggest that the reduced ⁹⁰Sr sequestration efficiency observed in the synthetic STEL effluent, as compared to MM, is due to the negative influence of both the chemical composition of the STEL effluent and the radioactivity. Compared to MM, the synthetic STEL medium contains potentially detrimental concentrations of NaCl (400 mM), aluminum (0.37 mM) and boron (18 mM, approximately 400 times higher than in MM) (Table S1). To determine whether these elements were responsible for the observed toxicity, cells were incubated in modified versions of the synthetic STEL medium lacking NaCl, Al and/or B, as well as in modified MM containing one or all of these components. No toxic effect of NaCl, Al and B added individually or simultaneously to MM was observed on cells suspension incubated for 48 h (Fig. 6C). Reciprocally, removing any or all NaCl, Al and B from the synthetic STEL effluent did not eliminate toxicity (Fig. 6C). These data indicate that other factors in the synthetic STEL medium contribute to its detrimental effects.

Therefore, ⁹⁰Sr sequestration experiments were repeated in modified versions of the synthetic STEL effluent diluted 5- or 10-fold with MM. In
A

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Removal of ⁹⁰Sr from a synthetic effluent and toxic effect of the synthetic effluent and some of its components on *G. lithophora*. A) Distribution of ⁹⁰Sr in the extracellular (blue) or cellular fraction (red) after 24 h or 72 h exposure of *G. lithophora* to ⁹⁰Sr (D3 or D2) in MM or in synthetic effluent (EF) diluted or not. The effluent was diluted 5 times (1/5) in MM or 10 times (1/10) in MM, reverse osmosis water (ROW) or moderately hard water (MHW) as indicated. Error bars represent standard deviation from biological triplicates. B) Effect of the exposure medium and ⁹⁰Sr on *G. lithophora* cell suspension color at the indicated exposure time in the different conditions. For the non-diluted EF with (**) and without (*) ⁹⁰Sr at D3, the effect on cell color is shown after 3.5 h exposure. C) Monitoring of *G. lithophora* cells suspension color in MM with addition of NaCl (400 mM), H₃BO₃ (0.046 mM) and AlCl₃ (0.37 mM), or in the synthetic effluent with or without NaCl, H₃BO₃ and AlCl₃.</figcaption></figure>

C

<figure><img src="image_10.png" /></figure>

this experiment, ⁹⁰Sr was added at the D2 concentration in order to simulate effluent dilution and to compare with the previous results. In the 5-fold diluted synthetic STEL effluent, the cell color turned blue-green and only 23 % of the total ⁹⁰Sr was removed from the solution in 24 h (Fig. 6A). By contrast, when the synthetic STEL effluent was diluted 10-fold in MM, ⁹⁰Sr removal increased to 80 % and 98 % after 24 and 72 h, respectively, without cell death. Finally, we investigated whether water instead of MM could be employed to dilute the synthetic STEL effluent. Therefore, we measured ⁹⁰Sr (added at D2) accumulation by *G. lithophora* in the synthetic STEL effluent diluted 10-fold in synthetic moderately hard water (MHW) or reverse osmosis water (ROW). After 24 h, 48 % and 53 % of ⁹⁰Sr was removed from the solution when the effluent was diluted with ROW and MHW, respectively. With longer incubation times, we observed cell chlorosis in the ROW-diluted synthetic STEL effluent (data not shown), so ⁹⁰Sr accumulation was not monitored further. By contrast, no change in cell color was observed after 72 h in the MHW-diluted synthetic STEL effluent and 63 % of ⁹⁰Sr was successfully removed from the solution under this condition.

## 4. Discussion

Cyanobacteria, a diverse group of photosynthetic prokaryotes, are versatile cellular factories with high potential for industrial bio-technologies and bioremediation [7]. In a previous study, we demonstrated that the iACC-forming cyanobacterium *G. lithophora* can selectively sequester traces of ⁹⁰Sr from a medium containing high Ca concentrations, positioning it as a promising candidate for the

remediation of nuclear effluents [38]. However, the impact of ⁹⁰Sr incorporation on cell viability was not evaluated, in particular due to the non-axenic state of this strain, which complicated growth on solid medium and hindered our ability to monitor its survival after exposure to radioactivity.

In this work, improved growth and quasi-axenization of *G. lithophora* strain was achieved by adding thiosulfate in the culture medium to mitigate oxidative stress. The nearly axenic strain retained its ability to form iACC and substantially incorporate non-radioactive Sr into intracellular granules, indicating that the contaminating organism(s) were not required for Sr sequestration. Furthermore, the nearly axenic strain of *G. lithophora* was still able to efficiently sequester ⁹⁰Sr in the presence of excess Ca. Under photoautotrophic conditions, *G. lithophora* captured over 90 % of ⁹⁰Sr within 24 h when exposed to a ⁹⁰Sr concentration representing the average activity found in nuclear effluents to be treated (7.3 × 10⁶ Bq L⁻¹, i.e. the D3 concentration used in this study). This corresponds to an uptake capacity of 2.05 × 10⁷ Bq g⁻¹DCW (i.e. 4244 ng g⁻¹DCW), which is 22 times higher than that previously reported by Mehta et al. [38]. However, *G. lithophora*'s maximum uptake capacity was not reached under the tested conditions and could not be established due to laboratory regulations on the handling of radioactive substances. Nevertheless, as compared with various cyanobacteria [38] and the alga *Tetraselmis chui* (average uptake capacity of 6 ng g⁻¹DCW; [55]), *G. lithophora* appears to be the most efficient organism for ⁹⁰Sr capture described to date. It is worth noting that, in addition to ⁹⁰Sr, *G. lithophora* may also have the ability to sequester its decay product, ⁹⁰Y. In the ⁹⁰Sr stock solution used in this study, ⁹⁰Y is in secular
equilibrium with $^{90}$Sr. The radioactivity of $^{90}$Sr and $^{90}$Y, both β-emitters, can be measured by LSC within the 0–2000 keV range. In our experiments with D1, D2 or D3, approximately 10 % of the initial radioactivity in this energy range can be detected in the solution after 48 h (data not shown), suggesting that $^{90}$Y was also removed from the medium by the cells. This finding warrants further investigation.

At the three $^{90}$Sr concentrations tested here, the capture of $^{90}$Sr occurs in a bi-phasic kinetics: a rapid sorption to the cell surface within 4.5 h, followed by a slower intracellular accumulation. This two-step process mirrors mechanisms described for $^{90}$Sr accumulation by the algae *Chlorella vulgaris* [33] and *Tetraselmis chui* [55]. It is worth noting that the rate of $^{90}$Sr incorporation at the D1 concentration ($4 \times 10^4$ Bq L$^{-1}$) by the nearly axenic *G. lithophora* strain (54 % during the first hour) was slower than what was reported previously (98 % during the first hour) [38]. This two-fold difference could be due to a different abundance of contaminants cells influencing the surface area available for $^{90}$Sr sorption and/or the uptake by *G. lithophora* through a possible impact on its metabolic activity, or to some other reason yet to be understood.

Intracellular incorporation of $^{90}$Sr by *G. lithophora* is enhanced by photosynthesis, not respiration. This finding is consistent with the observation that dead *G. lithophora* cells poorly accumulate $^{90}$Sr [38]. Intracellular incorporation of $^{90}$Sr is not solely dependent of photosynthesis as DCMU does not fully inhibits the process. This indicates the potential involvement of another, yet unidentified, pathway.

Although we did not formally demonstrate that $^{90}$Sr is incorporated into iACC granules, the substantial and preferential incorporation of non-radioactive Sr (as shown by transmission electron microscopy in [14,8] and radioactive $^{226}$Ra (as shown by NanoSIMS in [39])) in these granules, strongly suggests that $^{90}$Sr exhibits a similar biodistribution within the cells. The capture of $^{90}$Sr within an intracellular biomineral likely contributes to its durable sequestration inside the cells we presently observed during 22 days. This is consistent with the recent study by [40] analyzing stable isotopes of Sr in *G. lithophora* cultures, which showed that, while some isotopic exchange could take place between the intracellular and extracellular pools of Sr by an outflux, the net flux of Sr remained toward the interior of the cells over 1 month of experiment. Last, while intracellular accumulation in *G. lithophora* shows higher selectivity for Sr over Ca [14], such a selectivity has not been suggested for alkaline earth metals sorbed onto cell surfaces. We hypothesize that any bacterium, including *G. lithophora*, may in a first stage sorb abundantly Ca and Sr with no chemical selectivity. In the second phase, the chemically-selective Sr uptake within the cells of *G. lithophora* enables uniquely high sequestration efficiency.

Incorporation of $2.05 \times 10^7$ Bq g$^{-1}$DCW and exposure to an estimated dose rate of 1.7 mGy h$^{-1}$ during 5 days (cumulative dose = 204 mGy) did not dramatically impair *G. lithophora* viability. While gamma radiations effects on microorganisms, including radioresistant species, are well-documented [18,22,35,59], little is known about the biological effects of $^{90}$Sr or other β-emitters in microorganisms. Considering the literature, the $^{90}$Sr concentrations and associated dose rates (from 0.025 to 1.7 mGy.h$^{-1}$) investigated in this study are expected to impact cellular components. For example, a dose rate of 6.5 mGy h$^{-1}$ was shown to induce DNA damages and functional changes in mesenchymal stromal cells exposed to $^{90}$Sr during 7 days [42] and exposure of *Escherichia coli* to $^{3}$H (a low-energy β-emitter) at an estimated dose rate of 0.42 mGy.h$^{-1}$ induced a broad transcriptional response after 15 days (more than 1000 induced genes) [61]. The algae *Chlorella vulgaris* was able to survive for 6 days of exposure to $^{90}$Sr at $2 \times 10^6$ Bq L$^{-1}$ [33], while the growth of *Tetraselmis chui* was inhibited by $^{90}$Sr at $1.242 \times 10^5$ Bq L$^{-1}$ [55]. While complementary experiments are needed to determine how long *G. lithophora* could survive and remain active in contaminated effluents, our findings suggest that the $^{90}$Sr concentration should not be a limiting factor for its potential use in a bioremediation process.

To advance toward practical application, we tested the capacity of a microorganism to sequester $^{90}$Sr in a synthetic mineral medium

mimicking the average composition of nuclear effluents treated at the STEL in Marcoule. This synthetic effluent proved to be toxic to *G. lithophora*, for an unknown reason which was not due to its high concentration of NaCl, $H_3BO_3$ or AlCl$_3$. However, when this synthetic effluent was diluted 10-fold with MM or ROW, *G. lithophora* was able to sequester respectively 98 % and 63 % of $^{90}$Sr provided at $4 \times 10^5$ Bq L$^{-1}$ in 72 h, demonstrating that *G. lithophora* has the potential for the bioremediation of $^{90}$Sr-contaminated nuclear plant effluents.

A conceptual two steps bioremediation process based on this organism can be proposed: i) large-scale photoautotrophic cultivation of *G. lithophora* in open-ponds followed by ii) exposure of cells to industrial effluents eventually diluted with a cheap mineral medium or industrial water to allow a complete extraction and storage of the radionuclide until its complete decay. After careful verification, the decontaminated effluent will be discharged. This process will allow the concentration of the radionuclide within the biomass and thus a considerable reduction of waste volumes. After several passages of radioactive effluent, the *G. lithophora* cells will be saturated with $^{90}$Sr. At this stage, the biomass will be collected (sedimentation, centrifugation), dried (and even burned), and stored in closed containers as solid nuclear wastes (Fig. 7). Because all the effluents to be treated do not have the same chemical composition, it will be necessary in the future to test the ability of *G. lithophora* to remove $^{90}$Sr from various types of nuclear effluents. Moreover, close relatives of *G. lithophora*, also able to incorporate Sr selectively over Ca but adapted to distinct environmental conditions [6] may be of interest in this context. In addition, a better understanding of the molecular mechanisms involved in the active sequestration of the radionuclide will help to improve the remediation process. The development of genetic tools to manipulate *G. lithophora* will be a future challenge to increase its performance, e.g., by overexpressing the genes involved in the Sr transport pathway. We can also envisage that the identification of genes and mechanisms involved in efficient $^{90}$Sr remediation could be transfer to others iACC-forming cyanobacteria with various capacities to grow under distinct conditions.

## 5. Conclusion

In the present study, we developed a nearly axenic strain of *G. lithophora* and tested its ability to bioremediate a synthetic nuclear effluent containing a typical $^{90}$Sr concentration. This work has revealed the very interesting potential of this unicellular cyanobacterium to stably incorporate and withstand the hazardous radionuclide $^{90}$Sr. We showed that photosynthesis is important for the efficient sequestration of $^{90}$Sr inside the cells, which is likely an active process requiring energy. Furthermore, we demonstrated that *G. lithophora* can efficiently remove $^{90}$Sr from a simulated nuclear effluent containing a variety of cations (Na, K, Ca...), paving the way for the development of a bioremediation process.

## Environmental implication

In the present study, we describe the capacity of the unicellular cyanobacterium *G. lithophora* to stably sequester and withstand $^{90}$Sr and to efficiently remove this hazardous radionuclide from aqueous solutions, including a synthetic nuclear effluent. Therefore, the cyanobacterium *G. lithophora* could be used for a cheap and effective bioremediation of $^{90}$Sr contamination, thereby safeguarding the well-being of our ecosystems.

## CRediT authorship contribution statement

**Benzerara Karim:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Conceptualization. **Pamart Edern:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Conceptualization. **Chauvat Franck:** Writing – review & editing, Writing – original draft, Validation, Investigation,
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Proposed bioremediation process of <sup>90</sup>Sr based on *G. lithophora*. Radioactive effluent from nuclear plant containing <sup>90</sup>Sr and calcium are added to *G. lithophora* cells grown in a photobioreactor. After 24 h, <sup>90</sup>Sr is incorporated into intracellular carbonate granules and trapped in biomass. Decontaminated effluent may be discharged in the environment.</figcaption></figure>

**Conceptualization.** **Cassier-Chauvat Corinne:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Investigation, Funding acquisition, Conceptualization. **Fois Giovanna Rosa:** Writing – review & editing, Writing – original draft, Investigation. **Viola Stefania:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation. **Chapon Virginie:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

**Declaration of Competing Interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgements**

This work and the PhD grant of Edern Pamart were funded by the FOCUS-DEM program of the CEA. We would like to thank Catherine Berthomieu for her contribution to the FOCUS-DEM program and helpful discussions, Yves Barré for providing us with the average composition of effluents treated at the STEL, Renaud Bouillanne and Audrey Beyly for the management of the laboratory in controlled area at the BIAM, Daniel Orjollet for his advice on LSC, Cynthia Travert and Feriel Skouri-Panet for the support from the GEMME facility and Imène Esteve for the support from the SEM facility, both at IMPMC.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2025.138155.

**Data availability**

Data will be made available on request.

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