Marine and freshwater micropearls: biomineralization producing strontium-rich amorphous calcium carbonate inclusions is widespread in the genus Tetraselmis (Chlorophyta)

Unicellular algae play important roles in the biogeochemical cycles of numerous elements, particularly through the biomineralization capacity of certain species (e.g., coccolithophores greatly contributing to the “organic carbon pump” of the oceans), and unidentified actors of these cycles are still being discovered. This is the case of the unicellular alga Tetraselmis cordiformis (Chlorophyta) that was recently discovered to form intracellular mineral inclusions, called micropearls, which had been previously overlooked. These intracellular inclusions of hydrated amorphous calcium carbonates (ACCs) were first described in Lake Geneva (Switzerland) and are the result of a novel biomineralization process. The genus Tetraselmis includes more than 30 species that have been widely studied since the description of the type species in 1878. The present study shows that many other Tetraselmis species share this biomineralization capacity: 10 species out of the 12 tested contained micropearls, including T. chui, T. convolutae, T. levis, T. subcordiformis, T. suecica and T. tetrathele. Our results indicate that micropearls are not randomly distributed inside the Tetraselmis cells but are located preferentially under the plasma membrane and seem to form a definite pattern, which differs among species. In Tetraselmis cells, the biomineralization process seems to systematically start with a rod-shaped nucleus and results in an enrichment of the micropearls in Sr over Ca (the Sr/Ca ratio is more than 200 times higher in the micropearls than in the surrounding water or growth medium). This concentrating capacity varies among species and may be of interest for possible bioremediation techniques regarding radioactive 90Sr water pollution. The Tetraselmis species forming micropearls live in various habitats, indicating that this novel biomineralization process takes place in different environments (marine, brackish and freshwater) and is therefore a widespread phenomenon.

The resulting lamellae were then thinned to approximately 1 μm thickness by using sequentially lower beam currents at 30 keV energy (starting at 1 nA and ending at 0.5 or 0.3 nA). The position of the lamellae was chosen to include a maximum of micropearl cross-sections. An internal micromanipulator with tungsten needle was used to lift-out the pre-thinned lamellae and to transfer them to a copper grid.
Final thinning of the sample to electron transparency (~100 to 200 nm) was carried out on both sides of the lamellae by using sequentially lower beam currents (300 to 50 pA at 30 keV energy). The lamellae underwent only grazing incidence of the ion beam at this stage of the preparation. This allows to minimize ion beam damage and surface implantation of Ga. The thinning progress was observed with SEM imaging of the lamellae at 52°. Electron beam damage was further supressed by using low electron currents and limiting electron imaging to a strict minimum.

Transmission electron microscopy (TEM)
TEM investigations were conducted with a FEI Tecnai G2 FEG transmission electron microscope operating at 200kV. In order to document the structural state of micropearls in their pristine undamaged form, selected-area electron diffraction (SAED) patterns were taken directly at the beginning of the TEM session with a broad beam. Scanning TEM (STEM) images were then acquired using a High Angle Annular Dark Field (HAADF) STEM detector (Fischione) with a camera length of 80 mm.
EDXS measurements were performed with a X-MaxN 80T SDD EDXS system (Oxford). EDXS spectra and maps were recorded in scanning TEM mode. The semi-quantitative calculation of the concentrations (including C) was obtained using the Cliff-Lorimer method using pre-calibrated k-factors and an absorption correction integrated into the Oxford software. The absorption correction is based on the principle of electroneutrality, taking into account the valence states and concentrations of cations and oxygen anions. Oxygen is thereby assumed to possess a stoichiometric concentration.

Micropearls in Tetraselmis species
SEM observations of twelve different species of Tetraselmis (culture strains) show that ten of them contained mineral inclusions ( Fig. 1, Table 1). No mineral inclusions were observed in T. ascus and T. marina. Since TEM analyses confirmed that these inclusions comply with the definition of micropearls given in Martignier et al. (2017) (see Sect. 4.1), they will be named "micropearls" hereafter.
The general shape of the micropearls in the marine species is elongated, resembling rice grains ( Fig. 1 except 1d), while it is spherical in T. cordiformis (the only freshwater species of this study) (Fig. 1d). The micropearls' size and shape differ among species. Sizes vary between 0.4 to 1.2 m in length. Detailed values for each species are given in Table 1. Backscattered electron images of dried samples. The micropearls appear in white or light grey against the darker organic matter, as elongated shapes, except for T. cordiformis (d), where they are spherical. The larger and slightly darker inclusions are polyphosphates (c, g). Pores of the filters are visible as black circles in the background (2 m of diameter except for (d): 0.2 m). Scale bars: 5m.
Micropearls do not seem to be randomly distributed inside the cells, but rather show a definite location in most species (Figs 1 and S1). Moreover, for a given species, most cells present a similar micropearl arrangement. Exceptions are cells that were damaged during sample preparation. Filtration or freshwater rinsing, for example, can disrupt the micropearl distribution pattern (Fig. S2).
In some strains, the micropearls are mostly aggregated at the one side of the cell, with "pointed" tips appearing at the center of the cell and on both sides, resulting in a "trident" shape. This is the case of T. chui, T. suecica and T. tetrathele (Figs 1a,1i,1j). T. striata shows a similar central micropearl distribution, but the lateral points of the "trident" are absent (Fig. 1g), possibly due to poorly developed micropearls at the time of observation. In T. suecica the central micropearl alignment is generally longer and not necessarily connected to the apical aggregate (Fig. 1i). T. levis (Fig. 1f) also shows a similar arrangement, but the aggregate is missing, leaving the micropearls to form three longitudinal alignments (meridians). Altogether, T. chui, T. levis, T. suecica and T. tetrathele present patterns with an approximately similar trimerous radial organization (although a tetramerous symmetry cannot be totally excluded as dried samples do not allow a definite judgement). Observations seem to indicate that, in most species, the micropearl aggregate is located at the apical side of the cell (near the apical depression from which the four flagella emerge) (Figs S1) except for T. suecica and T. convolutae where the micropearls aggregate at the distal side of the cell (Figs S1, 1c and 1i). However, the low number of preserved flagella in dried samples allowed only for few confirmed observations. In T. convolutae (Fig. 1c), the micropearls form a small aggregate at the basal extremity of the cell, while larger polyphosphate inclusions gather at the opposite (apical) side.
A different and interesting organization of the micropearls is displayed by both T. desikacharyi (Fig. 1e) and T. contracta (Fig.   1b). An apical aggregate of micropearls is generally present, while other micropearls form regularly spaced meridians, which, in T. contracta, extend from the apical pole towards the basal part of the cell (Figs 1b and S1). These meridians are not well expressed in all cells but, when they are clearly visible, there seems to be around eight or ten of them inside the cell. When well preserved, the micropearl organization in T. cordiformis also shows multiple micropearl alignments which depart from a well-developed apical aggregate, although the alignments are generally well arranged only close to the aggregate and the size of micropearls decreases quickly towards the basal end of the cell (Fig. 1d). Finally, samples observed in this study did not allow us to state if there is a definite distribution of the micropearls in T. subcordiformis (Figs 1h and S1).
Polyphosphate inclusions are frequently observed in Tetraselmis species. Their distribution seems to be random except in T. convolutae (Fig. 1c). Aggregates of small iron oxide minerals were frequently observed in dried samples at one extremity of T. desikacharyi and T. convolutae (Figs 1c and 1e)probably at the apical extremity.
In order to compare our results with members of another genus, we also analyzed other flagellate organisms (e.g. Chlamydomonas reinhardtii and Chlamydomonas intermedia) obtained from algal culture collections (Table 1). No calcium carbonate inclusions were observed in these cells. Thorough observation of samples from Lake Geneva confirms that most flagellates do not produce micropearls. This biomineralization process seems to be exclusive of a limited number of organisms.

TEM observation of FIB-cut cross-sections of micropearls
FIB-cut cross-sections of micropearls produced by T. chui and T. suecica are shown in Fig. 2, where they are compared to a similar section in a cell of the freshwater species T. cordiformis sampled in a natural environment (Lake Geneva). The choice of T. chui and T. suecica for FIB-processing and TEM observation was based on the size of the micropearls and on their strong concentration in Sr. Both features were considered to favour the observation of compositional zonation, as observed in our previous study (Martignier et al., 2017). A FIB-cut was also performed in a Tetraselmis contracta cell. This result is shown separately in Fig. 3, because the very good conservation of the organic matter in this sample allows the simultaneous observation of other intracellular constituents. Bottom TEM-HAADF images: FIB-cut sections through cells of (a) Tetraselmis chui (culture sample); (b) Tetraselmis suecica (culture sample); (c) Tetraselmis cf. cordiformis (Lake Geneva) (Martignier et al., 2017). Small bubbles inside the micropearls (particularly visible in the marine species) are due to beam damage. The contact between the cell and the filter surface is visible near the bottom in each image.
Micropearls in all four species show strong similarities. They are located inside the organic envelope, are amorphous (Figs 2 and 3) and, except for the sample with pure Ca (T. contracta in Fig. 3), they show a distinct internal concentric zonation ( Fig   2). In all observed species, the cut sections of micropearls suggest a rod-shaped nucleus in their center (Figs 2 and S3).
Moreover, all are most probably highly hydrated given their strong response under the electron beam (results no shown). The dehydration can still be observed for all micropearl types even after more than five months of conservation as dried samples at room temperature.
TEM-EDXS analyses show that the zonation observed in the marine micropearls of T. chui and T. suecica ( Fig. 2 and S4) is due to changes in the Sr/Ca concentration ratios, similarly to the zonation observed in the freshwater micropearls in Tetraselmis cf. cordiformis (Martignier et al., 2017). All micropearls within one cell do not necessarily have an identical composition. An example is shown in Fig. 2a, where one micropearl possesses a composition with a higher atomic mass than the rest (lighter grey level in STEM-HAADF image) due to a higher content of Sr. Furthermore, micropearls within one cell display variable zoning patterns ( Fig. 2a and 2c).

TEM-EDXS mapping: location of the micropearls inside a Tetraselmis contracta cell
The co-existence of micropearls with other cellular constituents and their respective positions in the cell is shown by a TEM image of a FIB-cut section through a T. contracta cell (Fig. 3). The micropearls of this species are large, numerous and nearly exclusively consist of ACC without detectable Sr (Fig. S4). They appear as round to ovoid light grey shapes with smooth surfaces (Fig. 3a). The TEM observation also reveals that most micropearls are not randomly scattered throughout the cell but are located preferentially just under the cell wall.
Although Fig. 3a is difficult to interpret because of the atypical preparation of the sample (simply dried instead of more traditional preparations for TEM-observation such as chemical fixation or cryo-sections), the identification of the visible cellular constituents can still be attempted (Fig. 3b). Side views (lower part of the section) and tangential sections of starch grains (upper part of the section) are visible, as well as a glancing view of the chloroplast, which is reticulated in this species.
Although micropearls resemble starch grains at first look, it is quite easy to differentiate them. First, they are generally more rounded than starch grains; secondly, they are not located inside the chloroplast, in particular, they are not associated with the prominent pyrenoid.  Note that, due the overlap between the P K peak and secondary Pt L peak, the Pt layer, which was deposited on top of the sample during FIB processing, is also visible in green color.
TEM-EDXS mapping provides compositional information improving the identification of the cellular constituents and organelles visible in the section ( Fig. 3c and S5). Micropearls are well visible, based on the high concentration of Ca, with small quantities of K (and sometimes Mg, not shown here). The theca, composed of fused scales, appears as a thin layer between the cell and the filter. Its composition including C, Ca, S and small amounts of K makes it apparent in Fig. 3c (in violet). The theca of these organisms is indeed known to contain 4% of Ca and 6% of S (as sulfate) by weight (Becker et al., 1994(Becker et al., , 1998. The two irregular features that are highly enriched in P (in green in Fig. 3c) are identified as being PolyP inclusions, flattened during sample preparation. Finally, the dark grey features, in the center of the section, are probably mitochondrial profiles.

SEM-EDXS: micropearl composition
The micropearls of most marine species (Fig. 4a) seem to be composed of ACC, with Ca and Sr as cations. This composition is similar to the one measured for micropearls of T. cordiformis in Lake Geneva (Martignier et al., 2017). We noted two differences with our previous observations: T. desikacharyi forms micropearls containing small amounts of Ba and micropearls of T. contracta contain low concentrations of K. However, since growth media had different compositions, these observations need to be taken with care. because the element is also present in the surrounding organic matter (Fig. S5), making it impossible to estimate the portion of the measured K that belongs to the micropearls. Magnesium was discarded for the same reason. It should be noted that the size of micropearls is close to or even below the resolution limit of the SEM-EDXS analysis technique. This means that the interaction volume of the electron beam with the sample is often larger than the micropearls themselves and that therefore the technique yields compositions that include the micropearl and the surrounding organic matter or nearby cellular constituents (e.g. polyphosphates). Relationship between the composition of the growth media and the composition of the Tetraselmis micropearls, expressed as the Sr/Ca ratio.
Each point represents the median Sr/Ca ratio measured in each species micropearls, related to the Sr/Ca ratio of the growth medium. The blue dotted lines define the values of the Sr enrichment factor of the micropearls with respect to the medium (10x, 50x, etc.). Ca concentrations of the growth media were calculated, based on media theoretical composition. Green triangles signal four samples grown in the same medium. The abbreviations and characteristics of each strain are indicated in Table 1 while Sr/Ca values appear in Table S2 (for medium) and S3 (for micropearls). Results from T. cordiformis from Lake Geneva (cord_Gen) (Martignier et al., 2017) are given as a comparison.

Sr/Ca ratio in growth media: influence on the micropearl composition
The overall composition of all culture media is rather similar. The culture media concentrations in Sr and Ba are given in Table   S2 and represented graphically in Fig. S6. Strontium concentrations range from 3.3 10 -8 M (freshwater medium SFM) to 7.1 10 -5 M (seawater SWES medium). All media have lower Sr concentrations than the average seawater (9.1 10 -5 M). SFM, used to grow T. cordiformis -the only freshwater strain under studyhas lower Sr concentrations than those measured in Lake Geneva (5.2 10 -6 M). The molar ratio Sr/Ca has been calculated for seven growth media (Table S2) and 458 micropearls (Table   S3) in order to evaluate a possible influence of the medium on the micropearls composition. Differences between the species regarding the micropearls enrichment in Sr compared to their growth medium can be observed. A Sr distribution coefficient (or enrichment factor) was calculated as the molar ratio [(Sr micropearls / Ca micropearls) / (Sr medium / Ca medium)]. These results are synthetized in Fig. 4. Figure 4b shows the relationship between the Sr/Ca ratio measured in the growth media and in the Tetraselmis micropearls.
For most of the strains, the Sr enrichment factor of the micropearls with respect to the medium varies between 10 and 100 times (see Table S3 for exact figures), with the notable exception of T. desikacharyi (more than 200 times). It is interesting to observe that both strains of T. chui -from different geographic origins (Table 1)  for Lake Fühlinger and 51 for Münster moat). Broadly speaking, Sr/Ca increases in micropearls together with its increase in the medium. However, the spread in enrichment may be large for a given medium (such as ASP-H for strains of T.contracta, T. convolutae, T. chui and T. desikacharyi).

Marine micropearls
The discovery of micropearls in marine species of Tetraselmis shows that this biomineralization process can take place in organisms growing in waters of different composition, from freshwater, like Lake Geneva, to seawater (Fig. S6). This shows the capacity of these organisms to concentrate Ca and Sr from different external media.
The capacity to form micropearls is clearly not directly related to a specific habitat, since seven Tetraselmis species forming micropearls live as phytoplankton in freshwater, marine or brackish waters (Guiry et al., 2017;John et al., 2002), T. contracta and T. desikacharyi were sampled in the sand, at the bottom of a marine estuary (Marin et al., 1996) or at low tide, and T. convolutae is usually observed as a photosymbiont inside a flatworm (Muscatine et al., 1974). Regarding the only two species which did not show micropearls at the time of observation (T. ascus and T. marina), it is interesting to note that both live as stalked sessile colonies, with motile life-history stages (Norris et al., 1980). Apart from their elongated shape, "marine" micropearls have similar characteristics to micropearls formed by the freshwater species T. cordiformis (Martignier et al., 2017). Micropearls show a range of possible composition for each species (Fig. 4a and Table S3). The elemental ratio seems to be influenced by several parameters, amongst which we identified the composition of the culture medium (Fig. 4b) and the Sr concentrating capacity of each Tetraselmis species (e.g. green triangles in Fig.4b).
The enrichment capacity displayed by T. desikacharyi (219) stands well above all the others (Fig. 4b and Table S3). This could be linked to distinctive morphological features (a six-layered theca, a novel flagellar hair subtype) not found in other strains of Tetraselmis (Marin et al., 1996).

Hints about the formation process of micropearls
The biomineralization process leading to the formation of micropearls seems to start in the same way in all Tetraselmis species observed in thin sections (T. chui, T. contracta, T. cordiformis and T. suecica), with a similar rod-shaped nucleus (Figs 2, 3 and S3). These nuclei could possibly be of organic nature given their darker appearance in the STEM-HAADF images that point to a material of lower atomic mass (Fig. S3).
Internal concentric zones are observed in the micropearls formed by cells grown both in the natural environment and in cultures (Fig. 2). The presence of this concentric pattern, even when the growth media have a stable composition, may indicate that the zonation is not due to changes in the surrounding water/medium composition during micropearl growth, but rather depends on variations in the intracellular fluid composition caused by the biomineralization process itself. In the hypothesis discussed by Thien et al. (2017), it is suggested that the formation of the micropearls results from a combination of a biologically controlled process (preferential intake of specific cations inside the cell) and abiotic physical and chemical mechanisms (mineralization resulting from a non-equilibrium solid-solution growth mechanism, leading to an internal oscillatory zoning). Nevertheless, even that second part of the process does not seem to be purely abiotic, as demonstrated by the long-term amorphous state displayed by micropearls (at least five months, according to our observations). Indeed, such long-term stabilization of ACC generally implies a strong organic control through the integration of additives in the mineral (e.g. certain proteins, polyphosphonates, citrates, amino acids) (Addadi et al., 2003;Cam et al., 2015;Cartwright et al., 2012). ACC, in its pure form, is unstable and will rapidly crystallize into calcite or aragonite (Addadi et al., 2003;Bots et al., 2012;Weiner and Addadi, 2011).

A new intracellular feature in a well-known genus
Our results (Fig. 1) uncover a strong biomineralization capacity in the genus Tetraselmis, confirming that artefacts can be induced by usual biological sample preparation techniques (Martignier et al., 2017) and thus biais observations and even hide some physiological traits in otherwise well-studied organisms. Figure 3c shows that the straightforward sample preparation method used in this study (dried, with no chemical fixation) allows the preservation of the micropearls and can yield interesting data on the composition of the different elements present inside the cell, without any chemical disturbance.
Micropearls represent a new intracellular feature. Their systematic presence in most of the analysed Tetraselmis species suggests that they may have a physiological role. A possible explanation could be that micropearls increase the sedimentation rate of cells that shed their flagella upon nitrogen starvation at the end of Tetraselmis blooms. An alternative hypothesis is that micropearls represent reserves of Ca for periods when millimolar Ca is not available in the external medium. Indeed, all Chlorodendrophyceae (Tetraselmis, Platymonas and Scherffelia) require a certain concentration of Ca (mM) to survive and multiply (Melkonian, 1982). The evolutionary diversification of this class occurs in the marine habitat, where the Ca concentration is constantly around 10 mM (Table 4.1 in Pilson, 1998). The need for Ca is supported by T. cordiformis, the only freshwater species of the genus, occurring only in Ca-rich lakes, with a minimum of 1 mM of Ca (e.g. Lake Geneva (1 mM) or Fühlinger See (2mM)). Calcium is needed to support phototaxis (light-oriented movements) and for the construction and maintenance of the cell coverage (theca, flagellar scales) (Becker et al., 1994;Halldal, 1957). The Sr found in the composition of the micropearls formed by most Tetraselmis spp. (Fig. 4) could be transported by the same transporter as Ca.

Bioremediation possibilities
The capacity of some organisms to concentrate Sr is of great interest regarding bioremediation. Strontium ( 90 Sr) is one of the radioactive nuclides released in large quantities by accidents such as Chernobyl or Fukushima (Casacuberta et al., 2013) and a major contaminant in wastewater and sludges linked with nuclear activities (Bradley and Frank, 1996). Its relatively long half-life of ~30 years and high water solubility cause persistent water pollutions (Thorpe et al., 2012;Yablokov et al., 2009).
The high Sr absorption capacities of several Tetraselmis species previously led to their mention as potential candidates for radioactive Sr bioremediation (Fukuda et al., 2014;Li et al., 2006) although the process allowing these microorganisms to concentrate Sr had not yet been investigated . Further studies of micropearl formation processes could therefore lead to new bioremediation techniques. The genus Tetraselmis presents the additional advantage of including species living in diverse habitats, which might offer interesting bioremediation applications in different aquatic environments (e.g. freshwater, brackish lakes, open sea, hypersaline lagoons).

Conclusions
Until recently, non-skeletal intracellular inclusions of calcium carbonate were considered as nonexistent in unicellular eukaryotes (Raven and Knoll, 2010). After the first observation of at least two micropearl-forming organisms in Lake Geneva (Martignier et al., 2017), the present study shows that these amorphous calcium carbonate (ACC) inclusions are widespread in a common phytoplankton genus (Tetraselmis), not only in freshwater, but also in seawater and brackish environments. This newly discovered biomineralization process therefore takes place in media of very different composition but our results suggest that it is similar in all studied species: an oscillatory zoning process that starts from an organic rod-shaped nucleus. Although frequent in this well-studied genus, these mineral inclusions had been overlooked to date, possibly obliterated by the usual sample preparation techniques for electron microscopy. Thus other microorganisms could have similar capacities and intracellular inclusions of amorphous calcium carbonates may be more widespread than currently known.
Micropearls represent a new intracellular feature. This study shows that they are not randomly distributed in the cell. On the contrary, the distribution of micropearls within the cell seems to be characteristic for each species, and we suggest that this might have a link with the species habitat. Observations of a cell cross-section showed that micropearls are essentially located just under the cell wall, and can be clearly distinguished from other organelles.
In the genus Tetraselmis, the biomineralization process leading to the formation of micropearls enables a selective concentration of Sr. The elements concentrated in the micropearls, as well as their degree of enrichment seem to be characteristic for each species. Selecting the species with the highest concentration capacities could be of high interest for bioremediation, especially regarding radioactive Sr contaminations linked with nuclear activities.

Competing interests
The authors declare that they have no conflict of interest.   Table S1). Figure     STEM -HAADF image. The location of the EDXS analyses (right hand-side table) is indicated by the corresponding numbers.

Supplementary Materials
Results are normalized to 100 at%. O is calculated stoichiometrically based on the cation concentrations. Notice the low but significant presence of K in the micropearl composition of T. contracta. However its analysis n°1 does not fulfil carbonate stoichiometry, which may be due to the excess C from organic matter. Note that the calculation mode for the analyses presented in this figure differ from those presented in the rest of the manuscript, as C and O are included in the composition. Top image shows the location of the two mappings on a TEM-HAADF image of the section. The maps show the concentration of the different elements: the lighter the color, the more the element is concentrated in that point. Micropearls are mainly composed of Ca, with small quantities of K (and Mg, not shown here). The ACC appears to contain less carbon than the surrounding organic matter, because calcite is known to contain 12 wt% of carbon while the biomass contains 40-50%. Note that, due to the overlap between the P K peak and secondary Pt L peak, the Pt which was deposited on top of the sample during FIB processing is also visible in green.

Figure S6: Growth media concentrations in Sr and Ba.
Black dots: culture media; white squares: natural waters. Lake Geneva (Jaquet et al. 2013) and oceans (Bruland and Lohan, 2003    and Diat (SAG) were not available for analysis.