Mikhail Luginin
email: mikhail.luginin@phystech.edu

Alexander Trokhimovskiy

Benjamin Taysum

Anna A Fedorova

Oleg Korablev

Kevin S Olsen

Franck Montmessin

Franck Lefèvre

Evidence of rapid hydrogen chloride uptake on water ice in the atmosphere of Mars

Keywords: IR spectroscopy, Mars, atmosphere, Atmospheres, chemistry, Atmospheres, composition

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Main

A search for trace atmospheric species of biological or volcanic origin is one of the primary goals of ExoMars Trace Gas Orbiter (TGO, [START_REF] Vago | ESA ExoMars program: The next step in exploring Mars[END_REF]. To this purpose, TGO is equipped with highly sensitive infrared spectrometers, the Atmospheric Chemistry Suite (ACS; [START_REF] Korablev | The Atmospheric Chemistry Suite (ACS) of Three Spectrometers for the ExoMars 2016 Trace Gas Orbiter[END_REF] and Nadir and Occultation for Mars Discovery (NOMAD;Vandaele et al., 2018) that mostly operate in solar occultation. The only new species detected so far is hydrogen chloride (HCl), a halide gas, whose signature were found in 2020 in the ACS spectra [START_REF] Korablev | Transient HCl in the atmosphere of Mars[END_REF] and confirmed by NOMAD [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF]. Observations reveal a seasonal increase in the volume mixing ratio (VMR) up to 5 ppbv during the perihelion season, followed by a sudden drop down to undetectable values (< 0.1 ppbv) [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF][START_REF] Olsen | Seasonal reappearance of HCl in the atmosphere of Mars during the Mars year 35 dusty season[END_REF]. The fast disappearance of HCl from the atmosphere is at variance with previous estimations of a few months HCl lifetime based on gas-phase chemistry [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF][START_REF] Krasnopolsky | Photochemistry of HCl in the martian atmosphere[END_REF].

The HCl abundance was mostly observed during Southern summer, a season characterised by warmer temperatures [START_REF] Mccleese | Structure and dynamics of the Martian lower and middle atmosphere as observed by the Mars Climate Sounder: Seasonal variations in zonal mean temperature, dust, and water ice aerosols[END_REF], greater aerosol load [START_REF] Montabone | Eight-year climatology of dust optical depth on Mars[END_REF] and highly elevated water vapor [START_REF] Maltagliati | Annual survey of water vapor vertical distribution and wateraerosol coupling in the martian atmosphere observed by SPICAM/MEx solar occultations[END_REF][START_REF] Fedorova | Stormy water on Mars: The distribution and saturation of atmospheric water during the dusty season[END_REF][START_REF] Fedorova | A Two-Martian Years Survey of the Water Vapor Saturation State on Mars Based on ACS NIR/TGO Occultations[END_REF]. [START_REF] Olsen | Seasonal reappearance of HCl in the atmosphere of Mars during the Mars year 35 dusty season[END_REF] and [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF] noticed a striking similarity between the vertical profiles of HCl and water vapor in the 5 to 25 km altitude range, suggesting that both species undergo the same atmospheric dynamics and/or that they are tied by chemistry. One possibility is that water ice could favor the heterogeneous chemical loss of HCl, which would then make the two species anticorrelated. Above the hygropause, the H2O ice clouds often form, and condensate clouds are readily detected by ACS in occultation [START_REF] Fedorova | Stormy water on Mars: The distribution and saturation of atmospheric water during the dusty season[END_REF][START_REF] Fedorova | A Two-Martian Years Survey of the Water Vapor Saturation State on Mars Based on ACS NIR/TGO Occultations[END_REF][START_REF] Luginin | Properties of Water Ice and Dust Particles in the Atmosphere of Mars During the 2018 Global Dust Storm as Inferred From the Atmospheric Chemistry Suite[END_REF][START_REF] Stcherbinine | Martian Water Ice Clouds During the 2018 Global Dust Storm as Observed by the ACS-MIR Channel Onboard the Trace Gas Orbiter[END_REF][START_REF] Stcherbinine | A Two Martian Years Survey of Water Ice Clouds on Mars With ACS Onboard TGO[END_REF]. The highest HCl abundances were detected during the Southern Hemisphere summer at altitudes below 20 km [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF]Olsen et al., 2021), that are almost free of water ice.

On the basis of the laboratory measurements [START_REF] Kippenberger | Trapping of HCl and oxidised organic trace gases in growing ice at temperatures relevant to cirrus clouds[END_REF], both [START_REF] Korablev | Transient HCl in the atmosphere of Mars[END_REF] and [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF] speculated that HCl uptake onto water ice cloud particles could play an important role. [START_REF] Kippenberger | Trapping of HCl and oxidised organic trace gases in growing ice at temperatures relevant to cirrus clouds[END_REF] found that "HCl displayed extensive, continuous uptake during ice growth". Hydrogen chloride modelling in the atmosphere of Mars by [START_REF] Krasnopolsky | Photochemistry of HCl in the martian atmosphere[END_REF] also suggested a weak irreversible uptake on water ice. Taysum et al. (in prep.) have used a 1-D atmospheric photochemistry model [START_REF] Taysum | Photochemistry of Methane and Ethane in the Martian Atmosphere[END_REF] to interpret the ACS HCl observations, considering several mechanisms, including heterogeneous uptake of chlorine species onto water ice and mineral dust aerosols. This pathway has previously been used by [START_REF] Lefèvre | Heterogeneous chemistry in the atmosphere of Mars[END_REF] to model the uptake of OH and HO2 radicals on water ice in the atmosphere of Mars. The modelling suggests that above 20 km, H2O ice becomes the most efficient sink for HCl.

In this work, we show observational evidence of a pronounced HCl and water ice anticorrelation above 20 km, manifested as particularly HCl-rich layers at altitudes where water ice was absent. This result supports the rapid HCl removal mechanism competing with atmospheric mixing at a diurnal rate. We used simultaneous measurements of atmospheric state and gaseous and aerosol components by ACS.

ACS is a set of three spectrometers [START_REF] Korablev | The Atmospheric Chemistry Suite (ACS) of Three Spectrometers for the ExoMars 2016 Trace Gas Orbiter[END_REF], operating in Martian orbit since 2018. Two of them were used for this study. Its Near-InfraRed (NIR) channel is an acousto-optical tunable filter echelle spectrometer working in the 0.76 to 1.6 µm range, well-suited to characterise aerosol continuum and where the CO2 and H2O bands can be found. The Mid-InfraRed (MIR) channel is a cross-dispersed echelle spectrometer dedicated to solar occultation measurements in the 2.2-4.4 µm range. The echelle diffraction orders are separated along the perpendicular axis by a secondary diffraction grating. The current work focuses on diffraction orders 173-192, covering the 3.09 to 3.46 μm range. This range includes a broad water ice band encompassing all orders and strong lines of HCl in diffraction orders 173-175.

The retrieval of HCl profile abundances is done similarly to work [START_REF] Trokhimovskiy | Isotopes of chlorine from HCl in the Martian atmosphere[END_REF]. We use the iterative Levenberg-Marquardt algorithm with Tikhonov regularisation developed to analyse SPICAM IR data on Mars Express [START_REF] Fedorova | Multi-Annual Monitoring of the Water Vapor Vertical Distribution on Mars by SPICAM on Mars Express[END_REF] and ACS NIR data [START_REF] Fedorova | Water vapor in the middle atmosphere of Mars during the 2007 global dust storm[END_REF][START_REF] Fedorova | Stormy water on Mars: The distribution and saturation of atmospheric water during the dusty season[END_REF]. We retrieve volume mixing ratios of HCl from the wavenumber range 2922.5-2927.4 cm -1 , the uncertainty on the retrieved quantities is given by the covariance matrix of the solution. The temperature and water vapor profiles used as a priori information are taken from available ACS NIR products [START_REF] Fedorova | Stormy water on Mars: The distribution and saturation of atmospheric water during the dusty season[END_REF][START_REF] Fedorova | A Two-Martian Years Survey of the Water Vapor Saturation State on Mars Based on ACS NIR/TGO Occultations[END_REF].

The observed spectral range covers approximately half of the water ice absorption band centred at 3230 cm -1 , which is used to determine aerosol microphysical properties [START_REF] Luginin | Properties of Water Ice and Dust Particles in the Atmosphere of Mars During the 2018 Global Dust Storm as Inferred From the Atmospheric Chemistry Suite[END_REF][START_REF] Stcherbinine | Martian Water Ice Clouds During the 2018 Global Dust Storm as Observed by the ACS-MIR Channel Onboard the Trace Gas Orbiter[END_REF]. Simultaneous measurements of continuum absorption by ACS NIR in the spectral range 0.76-1.6 μm are included in the retrieval to increase the confidence and better distinguish between dust and water ice particles. The "onion peeling" technique [START_REF] Rodgers | Inverse Methods for Atmospheric Sounding[END_REF] is used to obtain vertical profiles of the spectral dependence of aerosol extinction coefficient, which are then fitted to produce the effective radius, number density, and mass loading of water ice and dust. For details about the retrieval methodology, see [START_REF] Luginin | Properties of Water Ice and Dust Particles in the Atmosphere of Mars During the 2018 Global Dust Storm as Inferred From the Atmospheric Chemistry Suite[END_REF].

We have analysed data collected by ACS during the second half of the Martian year (MY) 34 and covering two complete MY 35 and MY 36. In all, we analysed 784 occultations providing profiles of temperature, HCl (if any), water vapor, and aerosol extinction and mass loading. The three Martian years reveal a similar HCl abundance pattern (see also [START_REF] Olsen | Seasonal reappearance of HCl in the atmosphere of Mars during the Mars year 35 dusty season[END_REF]: an increase during the perihelion season, between LS = 200° and 350° when its content reaches maximum values up to ~5.5 ppbv in the Southern Hemisphere after LS = 270°. During the same LS range in the Northern Hemisphere, the HCl abundances are several times smaller. During this season, HCl-laden air masses are transported from the Southern to Northern hemisphere at high altitudes through the Hadley cell. As the northern profiles of HCl have explicit minima in the lower atmosphere (for example, see Panel a of Fig. 3 in [START_REF] Olsen | Seasonal reappearance of HCl in the atmosphere of Mars during the Mars year 35 dusty season[END_REF], it could also point to the effective gas removal in cold conditions and substantial ice load [START_REF] Luginin | Properties of Water Ice and Dust Particles in the Atmosphere of Mars During the 2018 Global Dust Storm as Inferred From the Atmospheric Chemistry Suite[END_REF].

In some occultations we observed a characteristic pattern of local anticorrelation between HCl and water ice over 5-15 km at 30-50 km altitudes. An example of this is shown in Panels A, B, and C of Figure 1. At 40-50 km, a gap in aerosol extinction at ~3 μm (water ice band) indicates an absence of water ice, while mineral dust particles can still be detected (Panels A and B). HCl is found to be maximum at altitudes where water ice is missing. This suggests rapid hydrogen chloride uptake on water ice (Panel C). Moreover, the HCl maximum is accompanied by a maximum of water vapor, that could be caused by formation of water ice above and below the "ice-hole" or a vapor release after sublimation of water ice particles. In this particluar case, the former cause is more preferable as no clear temperature maximum was observed at the altitudes of the "ice-hole" (panel B).

Out of the 137 occultations with prominent HCl abundances, 11 were found to feature clear "HCl in ice-holes'' cases. These detections are localized around 30 and 45 km. Panel D of Figure 1 shows profiles of HCl VMR and aerosol extinction at 3.1 μm from those observations. In order to compare the various cases, all profiles have been normalized to [0, 1] and vertically shifted to align all HCl maxima at 45 km. The average of normalized profiles (bold lines) clearly demonstrates an anticorrelation between HCl and water ice. For nine of the 11 profiles, HCl and water ice have their maximum colocated in altitude. Over a period of three Martian years, no clear correlation with season or location was observed.

Figure 1. An illustration of the anticorrelation between the HCl abundance and water ice from a single observation (Panels A, B, and C) and from all "HCl in ice-holes'' cases (Panel D). Panel A. The aerosol extinction profiles at 3.09-3.46 μm (water ice band, yellow and red) and 0.76-1.6 μm (aerosol continuum, green and blue) measured by MIR and NIR, respectively. Panel B: Retrieved aerosol mass loading profiles of dust (red) and water ice (blue), bottom axes; temperature profile with uncertainties retrieved from NIR data [START_REF] Fedorova | A Two-Martian Years Survey of the Water Vapor Saturation State on Mars Based on ACS NIR/TGO Occultations[END_REF], orange area and top axes. Panel C: volume mixing ratio of HCl (black lines and bottom axes) and H2O (blue lines and top axes) retrieved from MIR and NIR, respectively. Panel D: profiles of HCl VMR (black lines) and aerosol extinction at 3.1 μm (blue lines) normalized to [0, 1] and shifted to align all HCl maxima at 45 km; mean profiles are shown in bold. Error bars of the experimental data correspond to 1-σ uncertainty level.

As mentioned above, the heterogeneous uptake of HCl onto water ice is a process known to occur in the Earth's atmosphere, acting as a major sink for chlorine in the stratosphere [START_REF] Huthwelker | The Uptake of Acidic Gases on Ice[END_REF]. Qualitatively, the reactions are known to be very efficient with uptake coefficients (probability of a HCl molecule reacting or absorbing onto the ice crystal surface) experimentally measured with values ≥ 0.1 [START_REF] Burkholder | Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies, Evaluation No. 19[END_REF]. The quantitative values and precise mechanisms acting at the surface are still debated and highly depend on the presence of other gas-phase acid species, the ice surface roughness, atmospheric temperatures, and HCl partial pressures [START_REF] Abbatt | Interaction of HNO3 with water-ice surfaces at temperatures of the free troposphere[END_REF][START_REF] Barone | Investigation of the Heterogeneous Reactivity of HCl, HBr, and HI on Ice Surfaces[END_REF][START_REF] Chu | Uptake of hydrogen chloride in water ice and nitric acid ice films[END_REF][START_REF] Fernandez | Kinetics of ClONO2 Reactive Uptake on Ice Surfaces at Temperatures of the Upper Troposphere[END_REF]. Gas-phase chemistry on Mars predicts HCl lifetimes of a few months [START_REF] Aoki | Annual Appearance of Hydrogen Chloride on Mars and a Striking Similarity With the Water Vapor Vertical Distribution Observed by TGO/NOMAD[END_REF][START_REF] Krasnopolsky | Photochemistry of HCl in the martian atmosphere[END_REF]. Hydroxyl (OH) radical reactions dominate below the hygropause, photolysis is most efficient between the hygropause and ~ 40 km, and hydrogen (H) atoms become the strongest sink in the upper (> 40 km) atmosphere. Taysum et al. (in prep.) use a temperature-dependent uptake coefficient from [START_REF] Hynes | The interaction of HCl with water-ice at tropospheric temperatures[END_REF] scaled by a non-dissociative Langmuir coefficient [START_REF] Mcneill | Interaction of Hydrogen Chloride with Ice Surfaces: The Effects of Grain Size, Surface Roughness, and Surface Disorder[END_REF] to account for saturation of uptake sites on the ice surface and find that the presence of water ice can lower this lifetime to timescales less than 1 hour. The model finds that heterogeneous loss on ice can be over two orders of magnitude greater than gas-phase processes below the hygropause. For the observation in Figure 1, calculations for altitudes 35-40 km predict the HCl lifetime to range from 1.5 to12 hours. The presence of the "ice-holes" within the clouds thus enables the build-up of HCl within these ice-free regions, and drives the development of a distinct layered structure in the HCl profiles by removing it above and below.

The study presented here is a first, yet robust, exploration of the active connection between hydrogen chloride and water ice in the martian atmosphere. We propose that the uptake of HCl on water ice efficiently and rapidly removes hydrogen chloride from the atmosphere and probably restricts it from reaching higher altitudes. The fact that HCl, when detected, is found to be maximum in the "holes" of the ice profiles reveals the preservation of HCl at these altitudes and offers observational evidence of chemical interactions between HCl and water ice on a diurnal scale.

Acknowledgments

This work has been funded by grant #23-12-00207 of the Russian Science Foundation. KSO acknowledges funding from the UK Space Agency (ST/T002069/1, ST/Y000196/1). FM and FL acknowledge funding from CNES.


Jihen Jalali

Pierre Gaudin

Emna Ammar

Thierry Lebeau

Bioaugmentation coupled with phytoextraction for the treatment of Cd and Sr, and reuse opportunities for phosphogypsum rare earth elements

Keywords: Bioremediation, Lanthanum, Metal trace elements, Phytostabilization, Yttrium

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Phosphogypsum (PG) is the main byproduct from phosphoric acid production (a basic step in the manufacturing of phosphate fertilizers). Approximately 1.5 tons of PG is output per ton of phosphate rock processed, for an annual worldwide production estimated at 258 Mt in 2018 (Saadaoui et al., 2017). The environmental and health impacts caused by PG make it necessary to carefully manage these wastes. Over 12 Mt of PG output every year in Tunisia are currently being discarded at sea or else stored at production sites as stockpiles (Maazoun and Bouassida, 2019). PG is primarily composed of gypsum, mixed with calcium phosphate in various forms, plus silica and a broad range of other impurities like iron oxide, magnesium and aluminum, sulfides, organic matter, metal trace elements (MTE) including heavy metals, rare earth elements (RE) and radionuclides [START_REF] Hammas-Nasri | Rare earths concentration from phosphogypsum waste by two-step leaching method[END_REF][START_REF] Masmoudi-Soussi | Study of rare earths leaching after hydrothermal conversion of phosphogypsum[END_REF]. Compared to French geochemical background values (given both the absence of Tunisian regulations on soil metal contents and available data), Cd, Ce, La, Nd, Sr and Y are all included among the most abundant MTE in PG as well as in the soils adjacent to stockpiles [START_REF] Jalali | Fate and transport of metal trace elements from phosphogypsum piles in Tunisia and their impact on soil bacteria and wild plants, Ecotoxicol[END_REF]. Cd and Sr are potentially toxic or have demonstrated real toxicity, whereas Ce, La, Nd and Y all provide the promise of some kind of economic opportunity.

PG creates an environmental and health issue due to the relatively strong MTE mobility, while at the same time a potentially reusable resource, especially for the RE they contain, given their use in high-tech products [START_REF] Balaram | Rare earth elements: A review of applications, occurrence, exploration,analysis, recycling, and environmental impact[END_REF][START_REF] Haque | Rare Earth Elements: Overview of Mining, Mineralogy, Uses, Sustainability and Environmental Impact[END_REF][START_REF] Swain | A review on the recovery and separation of rare earths and transition metals from secondary resources[END_REF]. Value can be derived from PG in several fields by means of conventional physical and chemical techniques (Saadaoui et al., 2017;[START_REF] Wang | Advanced converter sludge utilization technologies for the recovery of valuable elements: A review[END_REF]. Such techniques however are expensive to employ and highly polluting due to their use of acids. At present, the reuse of PG only involves a small proportion of the quantities produced on a global scale, i.e. around 15 % [START_REF] Tayibi | Environmental impact and management of phosphogypsum: A review[END_REF]. In order to limit the diffusion and dispersion of PG stockpiles dust and erosion, phytostabilization combined with phytoextraction may yield an attractive alternative to conventional processes, by offering a solution that's both more affordable and ecological [START_REF] Pilon-Smits | Phytoremediation[END_REF]. The choice of plant species to be used is crucial herein. When applying phytoextraction, the advantages sought are: fast growth, large quantity of biomass, high contaminant concentration in the harvestable parts, capacity to withstand contamination, and good vegetal adaptation to the cultivatable environment [START_REF] Ali | Phytoremediation of heavy metalsconcepts and applications[END_REF]. Phytoextraction constitutes the only in situ MTE pollution removal technique. Among cultivated plants, red clover (Trifolium pratense) and sunflower (Helianthus annuus) accumulate high MTE amounts of the following MTE in their aerial parts: Cd, Sr (sunflower), Ce, La, Nd, and Y (purple clover) [START_REF] Andrade | Cadmium accumulation in sunflower plants influenced by arbuscular mycorrhiza[END_REF][START_REF] Boisselet | Chemical and biological factors influencing heavy metal mobilisation in the rhizosphere: implications for phytoremediation[END_REF]. The advantage here is twofold: the commercial availability of seeds, and proven technical procedures for cultivating these plants.

The main constraint stems from the limited quantity of MTE extracted by these plants, which require several growing cycles over several years, depending on the particular plant and its growth speed, as well as on the soil properties influencing MTE availability [START_REF] Pilon-Smits | Phytoremediation[END_REF]. To shorten treatment time, adding chemical chelating agents to the soil has been successfully tested [START_REF] Alkorta | Recent findings on the phytoremediation of soil contaminated with environmentally toxic heavy metals and metalloid such as zinc, cadmium, lead and arsenic[END_REF][START_REF] Shahid | EDTA-Enhanced Phytoremediation of Heavy Metals: A Review[END_REF][START_REF] Braud | Chelate-assisted phytoextraction of lead with Fagopyrum exculentum: laboratory vs field experiments[END_REF], yet this process raised concerns over their toxicity for plants [START_REF] Nowack | Critical assessment of chelant-enhanced metal phytoextraction[END_REF] and soil microorganisms [START_REF] Lee | Effects of chelates on soil microbial properties, plant growth and heavy metal accumulation in plants[END_REF]; moreover, some chelating agents lack the requisite efficiency (case of citric acid) [START_REF] Bouquet | Brassica juncea tested on urban soils moderately contaminated by lead: Origin of contamination and effect of chelates[END_REF]. The soil cleansing technique by means of bioaugmentation-phytoextraction coupling has been raised as a promising solution [START_REF] Kuiper | Rhizoremediation: A Beneficial Plant-Microbe Interaction Bioremediation : A natural method[END_REF]. The use of bacteria capable of mobilizing MTE and/or stimulating plant development (PGPR -Plant Growth Promoting Rhizobacteria -properties) may serve to increase the quantity of MTE extracted in this manner and hence the efficiency of phytoextraction. According to the review published by [START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review[END_REF], plant-bacteria coupling on a contaminated medium has proven to be capable, in 60% of the studies conducted, of boosting plant biomass by a factor of 1.2 to 4 and/or the metal concentration in above-ground plant parts by a factor of 1.1 to 3.1. Employing this technique for the purpose of reducing the PG pollutant load entails carefully choosing the most effective bacteria-plant couples.

In the present study, the potential accumulation of Cd, Ce, La, Nd, Sr and Y by Trifolium pratense and Helianthus annuus is first assessed in hydroponics. Next, the effect of inoculating a PG-compost mix by two strains of Bacillus sp., previously selected from soils extracted around PG stockpiles [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF], is determined based on phytoextraction efficiency. To the best of our knowledge, no research coupling these two techniques (bioaugmentation and phytoextraction) has yet to be implemented to treat PG. The bioaugmentation-phytoextraction association does seem to offer a rather appealing solution to help manage problems regarding PG and one that is not currently valued in Tunisia.

Material and methods

Plants cultivation

Pre-cultivation of plants

The plants used in this study are the Jonas variety of clover (Trifolium pratense) and the Kapplan variety of sunflower (Helianthus annuus) The red clover seeds were previously sterilized (9.6 % of NaClO 4 and 1 drop of Tween 80/100 mL, for 10 min and then rinsed ten times with sterile Ultra Pure Water (UPW)) before being germinated in sterile conditions in a Petri dish containing a glass filter paper soaked with sterile UPW. The sunflower seeds were washed 3 times and left stirring in sterile UPW for 1 h in order to eliminate fungicides and rinsed 10 times with sterile UPW before being germinate in a glass jar lined with vermiculite, previously humidified and sterilized by 20 min of autoclaving at 120°C. The seeds were then incubated in the dark at 25 °C for 2-5 days. After germination, seeds were transferred into an Araponics® box (36 seeds per box) with 1.7 L of sterile Hoagland medium (Table 1) set at pH 6, oxygenated by bubbling with an air pump, and placed in a growth chamber under controlled conditions (Phytotron FITOCLIMA D1200, Rio de Mouro, Portugal) under controlled conditions (16 h of daytime at 20°C and 8 h of nighttime at 18° ± 3°C) with a relative humidity of 70% and average lighting of 138 µmol of photons/m²/s over 45 days for the clover and 28 days for the sunflower. Hoagland medium was replaced once a week then every 4 days once the sunflower plants had reached the stage of 6-8 leaves and once the clover had ramified and developed secondary leaves.

Phosphogypsum-compost mix for the cultivation of plants

The PG studied herein originated from the spoil heap at the storage site located in back of the Mdhilla (Tunisia) phosphate fertilizer production facility (N 34°18.5097, E 8°46.3766). The physicochemical characteristics of phosphogypsum (PG) are listed in Table 2a. The enriched potting compost (Florentaise CTRU70-St Mars du Désert-France) used herein is a commercial product based on brown and blond peat, composted bark, Hortifibre®, manure and fertilizer.

The physicochemical characteristics of this compost are given in Table 2b.

Subsequent to a preliminary test of the phytotoxicity of two distinct ratios (w:w) for the PGcompost mix (i.e. 25%-75% and 15%-85%), the pots were filled with the 15%-85% mix. The mix humidity was calculated by applying the Standard NF ISO 11465 (AFNOR, 1994) and brought to 65% of the water-holding capacity (WHC) (as determined according to Standard ISO DIS 11269-2 (2006)): The PG-compost mix sample is saturated with water in a cylinder.

The cylinder is placed on an absorbent membrane until the excess water is drawn away by gravity. Once equilibrium is reached, the water holding capacity is calculated based on the weight of the water held in the sample vs. the sample dry weight. Next, the mix was allowed to rest for several days until pH stabilization in water (as measured according to Standard NF ISO 10390, 2005, with a soil:water (m:v) ratio of 1:5). The PG-compost mix pots (500 g capacity each) were then prepared with 250 g of mix contents per pot.

Growth of the strains and preparation of the bacterial inoculates

Description of the strains

Three bacterial strains were used for the bioaugmentation of the PG-compost mix, two of which were isolated from soils collected in the vicinity of PG stockpiles in the Tunisian regions of Sfax (34°10' north, 10°43' east) and Mdhilla (34°10' north, 8°45' east) (respectively Bacillus sp. (S3B1) and Bacillus sp. (M1B4)). These two strains were preselected for their capacity to produce metabolites involved in both MTE mobilization and plant growth promotion (the PGPR effect) [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF]. A strain of Pseudomonas xanthamarina ULPAs11 [START_REF] Koechler | Constitutive arsenite oxidase expression detected in arsenichypertolerant Pseudomonas xanthomarina S11[END_REF], originating from mining-based sediments contaminated by MTE (in particular As), was also tested.

Preparation of the inoculates

The bacterial strains, i.e. Bacillus sp. (S3B1), Bacillus sp. (M1B4) and P xanthamarina (ULPAs11), were pre-cultivated overnight with a sterile Tryptic Soy Broth medium diluted by half (TSB 1/2). One ml of each pre-culture was then transferred into 1,000 ml of Minimum Liquid Medium (MLM) (Ştefănescu et al., 2011) and incubated 48 h again at 28°C.

The bacterial biomass was harvested by centrifugation at 7,690 g for 10 min and the pellet was washed twice with a sterile KCl solution (9 g/L). This pellet was placed into the same solution so as to inoculate the bacteria found in the PG-compost mix by introducing a 1-ml volume with an optical density (OD) at 600 nm equal to 5. The OD was measured using a UV-visible VARIAN Cary 50 Scan spectrometer.

Metal trace elements phytoextraction by sunflower and red clover

Experiments in hydroponics

After 60 days of pre-culture for the clover and 28 days for the sunflower in a Hoagland medium, the plants were exposed to a mix of 6 MTE (i.e. Cd, Ce, La, Nd, Sr and Y) in an LPP (Low phosphate, low pH: see [START_REF] Fischer | Analysis of plant Pb tolerance at realistic submicromolar concentrations demonstrates the role of phytochelatin synthesis for Pb detoxification[END_REF] culture medium (composition provided in Table 1). The MTE tested were provided in the form of nitrate salts. Their concentrations, expressed in mg/L, correspond to the MTE contents of the PG extractable with CaCl 2 (A) and EDTA (B), respectively: Cd: 0.5 and 2; Ce: 0.5 and 5; La: 0.25 and 1; Nd: 0.2 and 2; Sr: 20 and 80; and Y: 0.25 and 2.5. A control without MTE was also performed under the same conditions. MTE were used as nitrate salt. A PHREEQC model served to validate the absence of precipitates at both studied concentration levels. 4 replicates of each modality, including a control, were introduced. In order to obtain a sufficient quantity of dry biomass for the mineralization step, one sunflower plant (3 for the clover) was transferred into each of the 1liter pots (washed with 2% nitric acid), covered by a high-density polyethylene (HDPE) lid and filled with 1 L of LPP medium (250 ml for the clover). The pots were then wrapped in aluminum paper to render them opaque (Fig. 1). During the exposure period, the culture medium level was readjusted every 2 to 3 days with the MTE-free LPP medium, and pH was reset to 5. The plants were harvested after 14 days of exposure.

Phosphogypsum-compost mix pots experiment

The pots were filled with 250 g of the PG-compost mix at 65% of the water retention capacity. Once the hydroponic pre-culture in the Hoagland medium had reached stage 4 leaf pairs, i.e. approx. 21 days for sunflower and 45 days for clover, the most well-developed plants were selected and transplanted in pots. Each pot was cultivated with one plant of sunflower or four plants of purple clover (Fig. 1). The pots were then placed in phytotron with a cycle of 16 h of daytime at 20°C / 8 h of nighttime at 18° ± 3°C, 70% humidity and a lighting of 138 µmol photons/m²/s. After each weighing, the pots were randomly repositioned in the germination chamber in order to account for heterogeneity (temperature, illumination, humidity) within the enclosure. The culture period for both plants lasted 4 weeks. Bacterial suspensions were added once a week following the transplantation step. They were distributed at the surface in each pot at a rate of 1 mL per pot. The PG-compost mix was then manually homogenized around the plant and throughout the pot depth using a sterile spatula.

The non-inoculated pots were homogenized similarly after adding 1 mL of KCl. Three modalities were tested: i) PG-compost mix not planted and inoculated (bioaugmented) (NPI), for purposes of evaluating the effect of bioaugmentation on MTE speciation; ii) PGcompost mix planted with H. annuus or T. pratense and non-inoculated (PNI), to evaluate the efficiency of MTE phytoextraction by these plants; and iii) PG-compost mix planted with H. annuus or T. pratense and inoculated (PI) with each of the selected strains, so as to assess the effect of bioaugmentation on phytoextraction.

Collection and conditioning of plants and PG-compost mix

After 14 days of cultivation in hydroponic and 4 weeks of cultivation in PG-compost mix pot, the aerial parts (AP) and roots parts (RP) were collected separately and carefully rinsed several times, first with tap water, then with deionized water and lastly with UPW. For each plant, the fresh shoots and roots were weighed before drying the samples in an oven at 40 °C until obtaining a constant mass. This temperature was chosen based on the conclusions of [START_REF] Alara | Mathematical modelling and morphological properties of thin layer oven drying of Vernonia amygdalina leaves[END_REF] and [START_REF] Vyankatrao | Effect of drying methods on nutritional value of some vegetables[END_REF]. Then, the dry masses of AP and roots were weighed and milled for chemical mineralization.

In each pot, 40 g of PG-compost mix were collected, precisely weighed, and dried at 40 °C for 5 days, in order to analyze the mobility of MTE in PG-compost mix and in the presence of the plants. For the cultures in hydroponics, 10 mL of culture medium were sampled, filtered at 0.2 μm and acidified with 30 μL of 65% HNO 3 .

Metal trace elements analysis in the culture supports and plants

Total metal trace elements

Total MTE contents were determined according to Standard ISO 11464 (AFNOR, 2006) after drying and crushing to 250 µm with a German beater crusher (basic A-11 and A-11.1 IKA beater, Staufen, Germany). A 0.50 ± 0.01 g test specimen drawn from the dry PG-compost mix sample was inserted into a 55-mL Teflon reactor (CEM μWave, Orsay, France), into which 3 mL of 37% HCl (v/v) and 9 mL of 65% HNO 3 (v/v) were added. The dissolution by acid attack was performed in a CEM MARS 6 microwave oven (Microwave Accelerated Reaction System from CEM μWave, Orsay, France). Once the sample had cooled, the mineralized residue was gathered in a calibrated vial with UPW adjusted to a volume of 25 mL. The liquid was then filtered on Whatman no. 40 filter paper and recovered for conservation in acid-resistant polypropylene (PP) tubes (DigiTUBEs from SCP Science, Courtaboeuf, France).

All collected plant samples were mineralized in a microwave oven (CEM MARS 6) through acid digestion by adding a 0.5-g test specimen, 8 ml of nitric acid (65% HNO 3 ) and 3 ml of H 2 O 2 (30%; v/v). As was the case with the PG-compost mix, the mineralized residue was transferred into a calibrated vial, whose volume was adjusted to 25 mL, using UPW. The liquid was once again filtered on Whatman no. 40 paper before recovery in PP tubes.

In both the PG-compost mix and plants, MTE analyses of the hydroponic culture solution were conducted by means of ICP-AES (Inductively Coupled Plasma Atomic Emission Spectroscopy) using an iCAP 6300 DUO Radial, in association with a CETAC ASX-520 automated sampler from Thermo-Fischer SCIENTIFIC (Illkirch, France). The wavelengths used to analyze the study elements were as follows (in nm): Cd (226.502), Ce (404.076), La (379.478), Nd (378.425),Sr (346.446),and Y (242.220). The vegetal Mixed Polish Herbs Standard (INCT-MPH-2) was applied during each analytical session to validate the results obtained.

Phytoavailable metal trace elements

The so-called mobile fraction of Cd, Sr and rare earth elements (Ce, Y, La, Nd) of the PGcompost mix was extracted in accordance with Dutch Standard NEN 5704, which relies on a 0.01 M saline CaCl 2 solution (capable of extracting metals present in the soil solution as well as easily exchangeable forms), complemented into a 15-mL tube by an accurately weight 1 ± 0.01 g of the dry PG-compost mix crushed and sieved to 250 µm (ratio: 1/10 (m/v)). The tubes were then stirred for 24 h at room temperature and centrifuged for 8 min at 7690 g. The supernatant of each fraction was recovered using a 10-mL syringe, filtered on a cellulose acetate membrane to 0.22 µm and acidified with one drop (< 10 µL) of concentrated HNO 3 (65%). Each extract was stored at +4°C prior to analysis. All reagents used were of analytical grade, and the solutions were prepared in UPW.

The potentially mobilized fraction was extracted according to Standard NF X 31-120 (1992), which calls for a solution of 1 M of ammonium acetate (CH 3 COONH 4 ) plus 0.01 M of Na 2 EDTA (pH = 7), to which 1 g of the dry PG-compost mix crushed and sieved to 250 µm (1:10 ratio (w:v)) was added. This extraction step was performed just like that using CaCl 2 .

Metal trace elements accumulation indices in the plants

Both the Bioconcentration Factor (BCF) and Translocation Factor (TF) were calculated, whereby BCF is the MTE concentration in the aerial plant parts (in mg/kg of dry matter (DM)) to the total PG-compost mix concentration ratio (mg/kg DM) (vs. culture solution in mg/L), while TF is the MTE concentration in the above-ground parts (mg/kg DM) to the root part concentration ratio (mg/kg DM).

Statistical analyses

Data were processed with Excel Stat for analysis of variance (ANOVA) from a multifactorial in randomization device, which allows a comparison of means by the Newman-Keuls test threshold of 5%. Homogeneous groups are were formed with the Tukey test, as represented in the figures by letters (e.g., a, b).

Results and discussion

1. Evaluation of phytoextraction potential H. annuus and T. pratense were the two commercial plants chosen in this study for their high biomass, rapid growth rate, capacity to accumulate the targeted MTE, i.e. Cd and Sr (for sunflower) (Turgut et al., 2004;[START_REF] Soudek | 137 Cs and 90 Sr uptake by sunflower cultivated under hydroponic conditions[END_REF][START_REF] Andrade | Cadmium accumulation in sunflower plants influenced by arbuscular mycorrhiza[END_REF], and Ce, La, Nd and Y (for red clover) [START_REF] Boisselet | Chemical and biological factors influencing heavy metal mobilisation in the rhizosphere: implications for phytoremediation[END_REF], and also for their commercial availability of seeds and proven technical procedures for cultivating these plants. The use of these two plants makes it possible to cover the full MTE spectrum being targeted. Moreover, the presence of a leguminous plant may limit the development of weeds and provide nitrogen to the sunflower culture as a means of reducing fertilizer addition. Eventually, these species are used in arid regions due to their deep root system.

In order to evaluate the MTE accumulation potential by these two plants, hydroponic experiments were carried out using two MTE concentrations : the first ("A") corresponding to the extract from PG with CaCl 2 , i.e. the mobile fraction (over the short term); and the second ("B") to the extract with EDTA, i.e. the fraction potentially mobilizable (over a somewhat longer term). The advantage of choosing these two extractants is to frame the minimum and maximum quantities of study elements capable of being extracted with relative ease and speed by the plant.

The concentrations of the studied MTE in the plants are presented in Table 3. The results showed that all MTE under study for both plants were found at concentrations below the limit of quantification in the culture solution without MTE (i.e. control "C"). In the presence of the MTE mix, the two plants are capable of accumulating in their root parts (RP), as well as being translocated toward the aerial parts (AP). MTE concentrations in the RP of T. pratense rise (although proportionally less than those in solution). When the exposure concentration is multiplied by a factor of 4 for Cd, La and Sr and 10 for Ce, Nd and Y in the medium, the average concentrations found in RP increase significantly, by factors of: 2.40 for Cd (from 68.7 to 165.4 mg/kg DM), 2.25 for La (51.1 to 115.1 mg/kg DM), 2.9 for Sr (645.9 to 1884.4 mg/kg DM), and 4.61 for Ce (69.8 to 321.8 mg/kg DM), 4 for Nd (35.2 to 142.2 mg/kg DM), and 1.10 for Y (42 to 46.3 mg/kg DM). Concentrations in the AP rise also (except for La) when exposure concentrations shift from A to B, significantly for Cd and Sr, and non-significantly for Ce, Nd and Y (respectively by factors of 2. 45, 2.62, 1.22, 1.29, and 11).

Similarly, for H. annuus, MTE concentrations in RP increase with medium concentration, yet also less than proportionally: 1.23 for Cd (from 95.4 to 117.5 mg/kg DM), 1.49 for La (45.7 to 68.1 mg/kg DM) and 1.67 for Sr (1,237 to 2,067 mg/kg DM), and 2.55 for Ce (61.9 to 157.9), 2.6 for Nd (31.3 to 81), and 1.2 for Y (39.8 to 47.5). Unlike T. pratense, concentrations in AP do not show a substantial increase as exposure concentrations shift from A to B, except for Sr (by a factor of 1,4). This can be explained by the rather short exposure period (14 days), with a slower MTE translocation from RP to AP in the case of T. pratense. It is noteworthy that H. annuus accumulates approx. 2 to 4 times more Cd and Sr in its AP (for concentrations B and A respectively) than T. pratense. For Ce, La and Nd, H. annuus accumulates roughly in twice as much quantity regardless of soluble concentration. Another major result is that Sr accumulated in higher concentrations in AP of H. annuus than in RP while it is the contrary for T. pratense (see explanations below with the calculation and interpretation of TF). In the former case, this agrees well with previous studies regarding H. annuus [START_REF] Wen | Uptake and distribution of 133 Cs and 88 Sr in sunflower (Helianthus annuus L.)[END_REF] and other plants [START_REF] Phieler | Phytoremediation using microbially mediated metal accumulation in Sorghum bicolor[END_REF][START_REF] Wang | Phytoremediation of strontium contaminated soil by Sorghum bicolor (L.) Moench and soil microbial community-level physiological profiles (CLPPs)[END_REF].

Table 3 shows the BCF and TF values. They are two important parameters which can describe respectively the metal accumulation ability from soil to plant and the metal transfer ability from RP to AP [START_REF] Brooks | Plants that hyperaccumulate heavy metals: their role in phytoremediation[END_REF]. Plant species are considered to exhibit removal potential for an element when the BCF value exceeds 1. The BCF values lie well above 1 for Cd, La and Sr (at the most respectively 10. 37, 11.8 and 22.8 in clover; and 45.9, 22 and 100.3 in sunflower) due to the total bioavailability of MTE in solution, which reflects the accumulation capacity of these plants. When shifting exposure concentrations from A to B, the drop in BCF is some 2 to 10-fold for sunflower, whereas clover accumulates nearly the same quantity of MTE, regardless of soluble concentration. Results showed that sunflower regulates MTE accumulation as opposed to clover, in disagreement with Dhiman et al., (2017) who showed that MTE uptake by sunflower plants was proportional to the concentration added to the nutrient solution.

The translocation factor (TF) is used to estimate the efficiency of phytoextraction [START_REF] Brooks | Plants that hyperaccumulate heavy metals: their role in phytoremediation[END_REF] since typically only the MTE of harvestable parts (AP) can offer reuse potential. A plant can efficiently phytoextract MTE from soil when TF values are higher than 1. Table 3 reveals that the highest TF values are observed for Sr (0.71 and 0.55 for red clover and 1.62 and 1.36 for sunflower at concentrations A and B, respectively). Aside from sunflower for Sr, all TF values lie below 1 and decrease with MTE concentration. Neither clover nor sunflower are renowned as hyperaccumulating plants, which has been confirmed under our study conditions.

Effect of the bioaugmentation-phytoextraction coupling on the MTE accumulation in plants

Effect of bioaugmentation on MTE mobilization in the PG-compost mix

PG are known for their toxicity in contact with plants [START_REF] Khoudi | Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum[END_REF]Ayadi et al., 2014;[START_REF] Elloumi | Effect of phosphogypsum on growth, physiology, and the antioxidative defense system in sunflower seedlings[END_REF], which makes it necessary to heavily dilute them in order to guarantee plant growth. The percentage of PG was set subsequent to a phytotoxicity test comparing two ratios: 25% PG + 75% compost, and 15% PG + 85% compost. A control test without PG was also prepared. With 25% PG, signs of plant wilting were observed as of the second day after being transplanted (data not shown). At a 15% PG concentration level, the plants were able to develop normally in spite of their slower growth.

After 4 weeks of culture, the MTE contents (Cd, Ce, La, Nd, Sr and Y) extractable with CaCl 2 and EDTA from the PG-compost mix are presented in Figure 2 (H. annuus) and Figure F1 of the Supplemental Data (T. pratense), as well as the total concentrations in MTE. Results for NPNI indicate that irrespective of the plant, practically no MTE are extracted (< LOD) by the CaCl 2 extraction solution, except for Sr (approx. 30 mg/kg MS PG-compost mix due to its highest total concentration among all MTE). This is not surprising that the LOD was reached given the dilution of the PG (only 15%) in the mix. It is possible that the addition of compost to PG may nevertheless have altered the MTE speciation (e.g., theoretical value of 3 mg/L for Sr in hydroponics, by applying the percentage of PG in the mix while the experimental value is approx. 30 mg/kg g PG-compost mix). Similar conclusions can be drawn for MTE extracted with EDTA, in particular for La, Nd, Sr and Y with concentrations for the PGcompost mix approx. 10-fold the theoretical ones.

No significant effect due to bioaugmentation is apparent on MTE contents regardless the extractant (CaCl 2 and EDTA), except for Cd trends with clover (Supplemental Data, see Fig. F1): in the NPI modalities, as compared to NPNI, bioaugmentation increases the average contents of the various PG-compost mix MTE to an insignificant extent. In modality PI, bioaugmentation has non-significantly raised the average concentrations of the PG-compost mix MTE extracted with EDTA (although, e.g., the Cd content extracted with EDTA equals 0.27 mg/kg for NPNI and 0.28 mg/kg for PNI; it reaches 0.36 mg/kg when inoculating the mix with PIS2). When the PG-compost mix has been bioaugmented, this zero (or only minimal) increase of MTE concentrations in the CaCl 2 and EDTA extracts can be explained, in case NPI is compared to NPNI, by a resorption of MTE (mobilized by the bacteria) on the carrying phases [START_REF] Bouquet | A new assay of bacterial selection with Pb reveals an unexpected effect of Pb on bacterial behavior. Implications for remediation[END_REF]. When PI is compared to PNI, one can assume that the additional amount of MTE extracted by the effect of bacteria is immediately taken up by the plants.

Effect of bioaugmentation on the MTE concentrations in plants

Bacillus sp. S3B1 and M1B4 stem from PG deposit zones in Tunisia [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF].

Pseudomonas xanthamarina ULPAs1 originates from an arsenic-rich mining zone [START_REF] Koechler | Constitutive arsenite oxidase expression detected in arsenichypertolerant Pseudomonas xanthomarina S11[END_REF] and is known for its tolerance to MTE. These three strains shall be denoted respectively PIS1, PIS2 and PIS3 in the following discussion (and in Figures 2 and3 and Table 4). They were inoculated in pots containing the PG-compost mix and cultivated with both T. pratense and H. annuus.

Total MTE concentrations in AP and RP cultivated on a PG-compost mix whether bioaugmented or not are shown in Table 4. Overall, the MTE accumulation levels by plants cultivated in pots with a PG-compost mix are much lower than in hydroponics, even at the smallest concentration corresponding to the MTE extracted with CaCl 2 (concentration A), i.e., a fraction assumed to be mobile over the short term (Table 3). These diminished plant-based extraction levels, compared to those of hydroponic culture, can be explained by the limited availability of metals over the relatively short duration of the experiment (4 weeks).

Sequential extractions of Cd, Ce, La, Nd, Sr and Y were performed on pure PG and soils contaminated by PG in order to evaluate their mobilities [START_REF] Jalali | Fate and transport of metal trace elements from phosphogypsum piles in Tunisia and their impact on soil bacteria and wild plants, Ecotoxicol[END_REF]. Such extractions have revealed that Ce, La, Nd, Sr and Y are in fact relatively non-exchangeable since they were basically found in the carbonate-bound fraction. In contrast, the more mobile Cd was found in the exchangeable fraction. MTE were accumulated in different parts of H. annuus and T. pratense. These amounts significantly vary among plant species, MTE and treatments (bioaugmentation). Table 4 indicates that MTE concentrations in H. annuus and T. pratense cultivated on a nonbioaugmented PG-compost mix (PNI) are approximately similar, except for Ce with a concentration differential in the clover AP (vs. RP) of approx. 38.9 times (7.5 times) greater than that of sunflower. The concentrations in Cd, La, Nd and Y are all less than 1 mg/kg; they reach roughly 14.8 mg/kg DM for Ce (T. pratense) and in the range of 34-91.6 mg/kg DM for Sr in both RP (T. pratense) and AP (H. annuus) due to the high Sr concentration in the substrate PG-compost mix (up to 229.74 mg/kg). Despite being a non-essential element for plants, Sr is easily extracted, mobilized and accumulated in plants due to its chemical similarities with Ca [START_REF] Camps | Quantitative assessment of the effects of agricultural practices designed to reduce 137Cs and 90Sr soil-plant transfer in meadows[END_REF]. Sr uptake by terrestrial plants was investigated by several authors. A summary of these studies are reported by [START_REF] Burger | Strontium in the environment: Review about reactions of plants towards stable and radioactive strontium isotopes[END_REF].

Regardless of the bacterial isolate employed (PIS1, PIS2, PIS3), bioaugmentation has on average raised the concentrations of all MTE (except Cd) in the AP of H. annuus and T. pratense, which after all are the plant parts being collected. On the other hand, bioaugmentation does not on the whole increase concentrations in RP. Unlike roots, the Cd concentration in the AP for both plants were below the detection limit. This may be due to the reciprocal effects of Ce and Cd on the plant uptake. [START_REF] Rossi | Mutual effects and in planta accumulation of coexisting cerium oxide nanoparticles and cadmium in hydroponically grown soybean (Glycine max (L.) Merr.)[END_REF] evaluate the mutual effects of Cd and Ce on their uptake and accumulation by Glycine max (L.) in a hydroponic system. They demonstrated that although Ce did not affect the total Cd accumulated in roots, the translocation of Cd from roots to shoots was reduced by 70%.

The jump in AP concentrations is noteworthy regardless of the strain used during bioaugmentation for both La (T. pratense and H. annuus) and Sr (T. pratense). For Ce, PIS1, weight (e.g. siderophores, organic acids). In the present study, the bacteria used for bioaugmentation had stemmed from extractions conducted around PG storage sites in Sfax (Tunisia) and were selected for, among other reasons, their capacity to produce siderophores [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF]. When the plant is not regulating metal extraction, several reviews have reported higher MTE concentrations in various plants [START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review[END_REF][START_REF] Rajkumar | Potential of siderophore-producing bacteria for improving heavy metal phytoextraction[END_REF]Sessitch et al., 2013;[START_REF] Dimkpa | Microbial siderophores: Production, detection and application in agriculture and environment[END_REF]. Table 4 notes that the TF value of the non-bioaugmented modality varies between 1.72 (Ce) and 2.43 (Sr) for T. pratense and between 0.25 (Ce) and 1.27 (Sr) for H. annuus. For all MTE studied herein, given the lack of data in the literature, our results provide an initial estimation of the potential of these two plants for phytoextraction based on PG.

On the whole, bioaugmentation raises TF values, and even sharply in some cases. For PIS2, the TF values systematically rose by a good amount, for all MTE, whenever the PG-compost mix was cultivated with H. annuus. These values varied by factors of: 1.14 for Sr, 2.73 for La, 6.24 for Ce, 6.35 for Nd, and 6.81 for Y. The fact that the TF values exceed 1 means that MTE are accumulating more readily in AP, which would support the phytoextraction premise.

The higher TF values could be explained by the effect of microbial complexing agents (siderophores), whose positive effect on this parameter had already been proven in previous studies [START_REF] Hazotte | Phytoextraction of cesium by red clover in hydroponics and soil systems[END_REF]. In the present study, the bacterial isolates utilized for bioaugmentation were selected for, among other reasons, their production of siderophores including pyoverdine [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF]. This rise in TF may be explained by disrupted root cell membranes, as shown for EDTA [START_REF] Schaider | Uptake of EDTA-complexed Pb, Cd and Fe by solution-and sand-cultured Brassica juncea[END_REF], with as a result a purely apoplastic pathway of siderophore-MTE complexes.

BCF values obtained for all MTE under study lie below 1 for clover, except for Ce (2.24 with the PNI modality). These values increase significantly regardless of the bacterial isolate introduced (by a factor of 3.2 to 3.86). For sunflower, BCF values are less than those of clover and all remain under 1.

Effect of bioaugmentation on the MTE quantity accumulated in plants

To reach solid conclusions regarding the potential of a given plant species for phytoextraction, the total amount of metal extracted, i.e., metal concentration in shoots × shoot biomass, must be calculated. Data are collected in Table 4.

As noted for the MTE concentrations in plants (section 2.2.), the MTE quantities recorded by plants cultivated on the PG-compost mix (Table 4) are also much lower than recordings by those cultivated in hydroponics.

Regardless of the plant, these quantities in the AP of bioaugmented modalities (PIS1, PIS2, PIS3) are significantly greater than those of the control (non-bioaugmented) modality (PNI).

Whether for PIS1, PIS2 or PIS3, an almost doubling of La, Nd and Y content in the sunflower AP can also be observed. In particular, the strain P. xanthamarina increases more the extraction of Sr through sunflower by a factor of 2.54 in AP. Bioaugmentation also raises the quantities of all MTE studied in clover, yet this increase tends to be more readily observed in AP. The inoculation of T. pratense with Bacillus sp. (PIS1) serves to extract more Ce, La, Nd, Y and Sr than the control respectively by factors of 6.4, 0.6, 0.93, 0.58 and 5.5 in AP. These quantities rise significantly but to a lesser extent, in the presence of PIS2 and PIS3.

Regardless of the strain the average amount found in RP increase significantly, by factors up of 0.4 for Cd, 5 for Ce, 6.7 for Nd and 8.5 for Sr with PIS2. In contrast, bioaugmentation does not boost the concentrations of La and Y in RP and values remain below the detection limit.

Overall, this increase in the quantity of MTE accumulated in AP with bioaugmentation has already been cited in reviews [START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review[END_REF]Sessitch et al., 2013). [START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review[END_REF] showed that on average over the entire set of studies and MTE examined, the MTE quantity in plant AP increases by a factor of 5 (2 for the concentration), with wide variability from one study to the next. Despite the growing concern on the environmental impact of an increasing use of the RE elements in agriculture and their effects on plant growth and productivity [START_REF] Balaram | Rare earth elements: A review of applications, occurrence, exploration,analysis, recycling, and environmental impact[END_REF][START_REF] Maheswaran | Impact of rare earth elements on plant physiology and productivity: a report for the Rural Industries Research and Development[END_REF][START_REF] Tyler | Rare earth elements in soil and plant systems -A review[END_REF] there is no information concerning the effect of bacteria on the extraction of RE elements.

Effect of the bioaugmentation-phytoextraction coupling on plant growth

Figure 3 illustrates the effect of bioaugmentation with PIS1, PIS2 and PIS3 on the gains in both the above-ground and root biomasses of the two plants. A major increase is observed in the above-ground (AP) biomass of H. annuus for all three strains by factors of 3.36, 3.40 and 2.16, respectively. An equivalent significant rise was shown for T. pratense, with respective factors of 3.7, 2.5 and 2. The root biomass trend (Fig. 3) remains flat, regardless of the plant except for PIS2 with T. pratense (by a factor of 2.9). As regards plant height, results show a significant increase subsequent to bioaugmentation with PIS1, PIS2 and PIS3 (i.e. factors of 5, 4.03 and 3.22 for AP, and 3.1, 2.25 and 1.85 for RP with respect to the non-bioaugmented control (data not shown)). In their review, [START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: a review[END_REF] reported that in most cases, PGPR bacteria boost biomass production, by a factor of 1.2 to 4 for AP and 1.1 to 2.6 for RP. These results therefore are not surprising since the strains used in our study (PIS1 and PIS2) do possess PGPR (Plant Growth Promoting Rhizobacteria) properties [START_REF] Jalali | Isolation and screening of indigenous bacteria from phosphogypsum-contaminated soils for their potential in promoting plant growth and trace elements mobilization[END_REF].

Conclusion

Soil remediation using coupled bioaugmentation-phytoextraction processes aims at enhancing the efficiency of accumulating plants, increasing either their biomass and/or the concentration of MTE accumulated within the plants. The present study has evaluated the association of two commercial plants, namely Trifolium pratense and Helianthus annuus, with Bacillus sp.

(strains PIS1 and PIS2) and with P. xanthamarina for their capability to improve phytoextraction efficiency. Results have demonstrated that this association does serve to stimulate the growth of T. pratense while increasing the concentrations of studied MTE for both strains PIS1 and PIS2 and, to a lesser extent, for P. xanthamarina, even though this latter strain does not stem from PG. The PIS2 strain is especially promising, in association with H. annuus, for Ce, La, Nd and Y, with plant concentrations being raised by a maximum factor of 21 for Y. The follow-up to this work will focus on the selection of pioneer plants from PG stockpiles from among those recently preselected (Zygophyllum album and Kochia indica: [START_REF] Jalali | Fate and transport of metal trace elements from phosphogypsum piles in Tunisia and their impact on soil bacteria and wild plants, Ecotoxicol[END_REF]. In conjunction with the aptitude of plants to extract MTE (more specifically rare earth elements), their capacity to phytostabilize PG stockpiles will also be tested in order to reduce airborne particulates, which has proven to be a real public health risk due to their inhalation by PG storage site personnel and the neighboring population.

  PIS2 and PIS3 associated with T. pratense have significantly raised concentrations in AP. For Y, it turns out that PIS2 and PIS3 also significantly boost the concentrations in H. annuus. Concentration enhancement factors in AP are highest when the PG-compost mix is being bioaugmented with PIS2 and cultivated with H. annuus. These values vary from 1.13 for Sr, to 3.43 for Nd, 4.36 for Ce, 21 for Y and 38.3 for La. For PIS1 associated with T. pratense, concentrations in AP climb by factors of 1.70 for Sr and 1.72 for Ce. By means of various mechanisms (complexation, mineral dissolution), many microorganisms are known to be capable of mobilizing MTE in the soil through releasing organic compounds of low molecular

Acknowledgments

This work was financed by the Tunisian Ministry of Higher Education and Scientific Research as well as by France's Pays de la Loire Regional Council (under the POLLUSOLS-OSUNA Project). The authors would like to thank the Tunisian Chemical Group for their interest in this study and technical assistance, in addition to Mr. Didier Lièvremont (Strasbourg University, France) for providing the S11 strain.