Introduction
The biogeochemical cycles of numerous elements are influenced by the
biomineralization capacities of certain unicellular organisms. This is the
case, for example, of the coccolithophores, which play an important role in
the carbon cycle through their production of biogenic calcite (Bolton et
al., 2016). Amorphous calcium carbonate (ACC) is also an important actor in
the biogenic carbonate cycle because it is a frequent precursor of calcite,
as many organisms use ACC to build biominerals with superior properties
(Albéric et al., 2018; Rodriguez-Blanco et al., 2017). For example, the
precipitation of calcium carbonate in microbial mats, the Earth's earliest
ecosystem, starts with an amorphous calcite gel (Dupraz et al., 2009), and
the formation of ACC inside tissue could make coral skeletons less
susceptible to ocean acidification (Mass et al., 2017).
In unicellular organisms, intracellular inclusions of ACC had, at first, only
been described in cyanobacteria (Couradeau et al., 2012; Benzerara et al.,
2014; Blondeau et al., 2018). More recently, similar inclusions have been
described in unicellular eukaryotes of Lake Geneva (Switzerland). Consisting
of hydrated ACCs but frequently enriched in
alkaline-earth elements (e.g., Sr or Ba) and typically displaying internal
oscillatory zonation, these inclusions have been named micropearls (Jaquet et
al., 2013; Martignier et al., 2017). The internal zonation is due to
variations in the Ba/Ca or Sr/Ca ratios.
Until now, micropearls had been observed only in two freshwater species: the
unicellular green alga Tetraselmis cordiformis (Chlorodendrophyceae,
Chlorophyta) producing micropearls enriched in Sr and a second freshwater
microorganism producing micropearls enriched in Ba, yet to be identified
(Martignier et al., 2017). Since its first description in 1878 (Stein, 1878),
the genus Tetraselmis has been much studied by biologists because
several species are economically important due to their high nutritional
value and ease of culture (Hemaiswarya et al., 2011). Tetraselmis
species are used extensively as aquaculture feed (Azma et al., 2011; Lu et
al., 2017; Park and Hur, 2000; Zittelli et al., 2006) and some have been
suggested as potential producers of biofuels (Asinari di San Marzano et al.,
1981; Grierson et al., 2012; Lim et al., 2012; Montero et al., 2011; Wei et
al., 2015). They have also served as models in algal research (Douglas, 1983;
Gooday, 1970; Kirst, 1977; Marin et al., 1993; Melkonian, 1979; Norris et
al., 1980; Regan, 1988; Salisbury et al., 1984).
The motile cells of Tetraselmis have four scale-covered flagella,
which emerge from an anterior (or apical) depression of the cell (Manton and
Parke, 1965). The Tetraselmis genus has a cell wall formation
process that is unique among green algae as the cells synthetize small
non-mineralized scales in the Golgi apparatus, which undergo exocytosis through
Golgi-derived secretory vesicles to form a solid wall (theca) composed of
fused scales (Becker et al., 1994; Domozych, 1984; Manton and Parke, 1965).
Regarding their habitat, most Tetraselmis species are free-living
(planktonic or benthic) (Norris et al., 1980), although some species live in
specialized habitats, for example as endosymbiont in flatworms (Parke and
Manton, 1967; Trench, 1979; Venn et al., 2008). Tetraselmis cordiformis is presumably the only freshwater species among the 33 species
currently accepted taxonomically in the genus Tetraselmis (Guiry and
Guiry, 2018).
However, mineral inclusions had never been described in these microorganisms
until the recent observation of micropearls in Tetraselmis cordiformis (Martignier et al., 2017). The fact that this new physiological
trait had gone unnoticed is puzzling, especially as Tetraselmis cordiformis is the type species of the genus. This can probably be explained
by the translucence of the micropearls under the optical microscope and their
great sensitivity to pH variations, leading to their alteration or
dissolution during most sample preparation techniques (Martignier et al.,
2017).
Interestingly, several Tetraselmis species (e.g., T. subcordiformis) have been mentioned as potential candidates for radioactive
Sr bioremediation due to their high Sr absorption capacities (Fukuda et al.,
2014; Li et al., 2006), but the precise process by which these microorganisms
concentrate this element has never been determined.
The present study investigates 12 species of the genus Tetraselmis, including the
freshwater Tetraselmis cordiformis, with the objective of understanding whether the
biomineralization process leading to the formation of micropearls is common
to the whole genus or is restricted to T. cordiformis. Species living in contrasting
environments have been selected to also evaluate if the formation of
micropearls is linked to their habitat. Each species is represented by one
or several strains, obtained from public algal culture collections. All
analyses were carried out on cells sampled from these cultures on the day of
their arrival in our laboratory. The micropearls were imaged by scanning
electron microscopy (SEM), and their composition was measured by
energy-dispersive X-ray spectroscopy (EDXS). The inner structure and
chemical composition of micropearls in three different species were studied
by transmission electron microscopy (TEM) on focused ion beam (FIB) cross
sections.
Samples and methods
Origin of the samples and pre-treatment methods
Culture samples of 12 different Tetraselmis species were obtained
from three different algal culture collections and were grown in different
media (Table 1). The recipe of each growth medium is available on the website
of the respective culture collections (Table S1 in the Supplement). A single
strain of each species was studied, except for T. chui (two strains)
and T. cordiformis (three strains). Table 1 lists the strain names. Most
cells in these cultures were mature at the time of observation for this
study.
Specific information for each species and their culture conditions.
Providers include the following. CCAC: Culture Collection of Algae at the University of Cologne
(Germany) – http://www.ccac.uni-koeln.de/, last access: 6 July 2018; SAG: Sammlung von Algenkulturen at the University of
Göttingen (Germany) –
https://www.uni-goettingen.de/de/184982.html, last access: 6 July 2018; AC: Algobank – culture collection of microalgae of the
University of Caen (France) –
https://www.unicaen.fr/algobank/accueil/, last access: 6 July 2018; TCC: Thonon Culture Collection of the CARRTEL of
Thonon-les-Bains (France) –
https://www6.inra.fr/carrtel-collection, last access: 6 July 2018. All culture media compositions are given on the corresponding
websites (detailed addresses in Table S1).
Origin of the sample
Approx. micropearl size
Culture medium
Provider strain no.
Abbreviation
Chlamydomonas
C. reinhardtii
FreshwaterFrance
no micropearls observed
L-C
TCC 778
–
C. intermedia
Freshwater France, Lake Geneva
no micropearls observed
L-C
TCC 113
–
Tetraselmis
T. ascus
Marine Spain, Canary Islands, Gran Canaria
no micropearls observed
ASP-12
CCAC 3902
T. chui
Marine Germany, Heligoland
0.7 µm length
ASP-H
CCAC 0014
chui_cc
MarineScotland, Millport, Clyde estuary
0.7 µm length
1/2 SWEg Ag
SAG 8–6
chui_sa
T. contracta
Marine France, Brittany, Île de Batz
1.2 µm length
ASP-H
CCAC 1405
contract_cc
T. convolutae
Marine (symbiotic in flatworm) France, Brittany, Île de Batz
0.8 µm length
ASP-H
CCAC 0100
convol_cc
T. cordiformis
FreshwaterGermany, Cologne, Lake Fühlinger
1 µm diameter
SFM
CCAC 0051
cord-F_cc
FreshwaterGermany, Münster, castle ditch
1 µm diameter
Waris – H
CCAC 0579B
cord-M_cc
Freshwaterstrain 0579B obtained from CCAC
1 µm diameter
Diat
SAG 26.82
cord-M_sa
T. desikacharyi
MarineFrance, Île de Batz, Rochigou
0.9 µm length
ASP-H
CCAC 0029
desika_cc
T. levis
MarineFrance, Saint-Gilles-Croix-de-Vie
0.6 µm length
ES
AC 257
levis_ac
T. marina
Marinestrain CA5, from L. Provasoli
no micropearls observed
Porph Ag
SAG 202.8
T. striata
MarineUK, North Wales, Conwy
0.6 µm length
SWES Ag
SAG 41.85
striata_sa
T. subcordiformis
MarineUSA, Connecticut, New Haven
0.4 µm length
Porph Ag
SAG 161-1a
subcord_sa
T. suecica
MarineUK, Plymouth
0.7 µm length
ES
AC 254
suecica_ac
T. tetrathele
Marine–
0.9 µm length
ES
AC 261
tetrath_ac
Samples for microscopic observation of each strain were prepared directly
after the organisms' arrival in our laboratory: small portions of the culture
(without any change of the original medium) were filtered under moderate
vacuum (-20 to -30 kPa) on polycarbonate filter membranes with 0.2, 1 or
2 µm pore sizes. Volumes filtered (variable depending on culture
concentration) were recorded. Species issued from SAG (Sammlung von
Algenkulturen – University of Göttingen, Germany) were grown on agar
and, therefore, cultures had to be resuspended just before filtration. Filter
membranes were dried in the dark at room temperature after filtration. A
total of 458 micropearls were analyzed by EDXS.
Water chemistry measurements
Elemental composition of each culture medium was measured at the IsoTraceLab
(EPFL, Lausanne, Switzerland), except for the ES medium for which we could
not obtain a sample. Blank samples of Milli-Q water were bottled at the same
time as growing medium samples and measured in the same way (Table S2).
Barium and Sr were measured by inductively coupled plasma sector field mass
spectrometry (ICP-SFMS) using a Finnigan™
Element2 high-performance high-resolution ICPMS model. The mass resolution
was set to 500 to increase analytical sensitivity. Calibration standards were
prepared through successive dilutions in cleaned Teflon bottles of
0.1 g L-1 ICPMS stock solutions (TechLab, France).
Suprapur™-grade nitric acid (65 % Merck) was
used for the acidification in the preparation of standards. Ultrapure water
was produced using Milli-Q™ ultrapure water
system (Millipore, Bedford, USA). Rhodium was used as the internal standard
for samples and standards to correct signal drift.
At this resolution mode, the sensitivity was better than 1.2×106 cps ppb-1 of 115In. The measurement repeatability
expressed in terms of relative standard deviation (RSD) was better than
5 %. The accuracy of the method was tested using a homemade standard
solution containing 5.0 ng L-1, used as a reference. Accuracy was
better than 5 %. The detection limits obtained for Sr and Ba was around
100 ng L-1 under these experimental conditions. Note that for the ES
medium (not analyzed), the concentrations were set as equivalent to standard
sea water, i.e., Sr =9×10-5 M and Ca =10-2 M, giving a
ratio of
Sr/Ca=9×10-3.
Scanning electron microscopy (SEM) and EDXS analysis
Small portions of the dried filters were mounted on aluminium stubs with
double-sided conductive carbon tape and then coated with gold (ca. 10 nm)
using
low vacuum sputter coating. A JEOL JSM-7001F scanning electron microscope
(Department of Earth Sciences, University of Geneva, Switzerland), equipped
with an EDXS detector (model EX-94300S4L1Q; JEOL), was used to perform EDXS
analyses and to obtain images of the dried samples. Semiquantitative results
were obtained using the ZAF correction method. Samples were imaged
with backscattered electrons. This method allows us to clearly locate the
micropearls inside the organisms thanks to the high difference of mean
atomic numbers between the micropearls and the surrounding organic matter.
EDXS measurements were acquired with settings of 15 kV accelerating voltage,
a beam current of ∼7 nA and acquisition times of 30 s.
Semiquantitative EDXS analyses of elemental concentrations were made without
taking carbon, nitrogen and oxygen into account. EDXS results are all
presented as mol %.
Counts and statistics lead on the Tetraselmis culture
cells
Counts were performed on the images obtained by SEM. The counts showed that
the agar medium seems to hinder the growth of micropearls. These strains were
therefore not taken into account for the statistics. Two strains of
Tetraselmis cordiformis and two strains of Tetraselmis chui
were analyzed. The samples of the two Tetraselmis cordiformis
strains taken on their first day of arrival were damaged during sample
preparation due to a too high filtration pressure, destroying the arrangement
of the micropearls in the cells. A sample obtained from one of these strains
60 days after arrival was therefore taken into account for the statistics, in
replacement.
The preservation of the pattern of micropearl arrangement in the cell is
difficult during sample preparation, as it is easily disturbed. The following
parameters directly influence the preservation of that feature: the
fragility of the cells (T. contracta cells, for example, seem very
solid while T. chui cells seem more fragile) and sample preparation
methods (e.g., pressure during filtration; see difference between e and f in
Fig. S1 in the Supplement).
Focused ion beam (FIB) preparation
Electron-transparent lamellae for TEM were prepared with a FIB–SEM
workstation (FEI Quanta 3D FEG at the Institute of Geosciences, Friedrich
Schiller University Jena, Germany). The cells were previously selected based
on SEM imaging. To protect the sample, a platinum strap of 15 to
30 µm in length, ∼3 µm wide and
∼3 µm high was deposited on the cell during lamella
preparation, via ion-beam-induced deposition using the gas injection system
(GIS). Stepped trenches were prepared on both sides of the Pt straps by Ga+
ion beam sputtering. This operation was performed at 30 keV energy and a 3 to
5 nA beam current.
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 a 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 minimization of 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 suppressed by using low electron
currents and limiting electron imaging to a strict minimum.
Transmission electron microscopy (TEM) and EDXS analysis
TEM investigations were conducted with a FEI Tecnai G2 FEG transmission
electron microscope operating at 200 kV. 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 an EDXS system, model X-MaxN 80T SDD from Oxford. EDXS spectra and maps were recorded in STEM
mode. The semiquantitative 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.
Results and interpretation
TEM analyses confirmed that the mineral inclusions observed in the
Tetraselmis species during this study comply with the definition of
micropearls given in Martignier et al. (2017) (intracellular inclusions of
hydrated ACC, frequently enriched in alkaline-earth elements (e.g., Sr or Ba)
and typically displaying internal concentric zonation linked to elemental
ratio variations). These mineral inclusions will therefore be named
micropearls hereafter.
SEM observation of micropearls in Tetraselmis species
SEM observations of 12 different species of Tetraselmis (culture
strains), on the day of their arrival from the supplier, show that 10 of
them contained micropearls (Fig. 1, Table 1). None were observed in
T. ascus and T. marina. 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
(0.4–1.2 µm in length) and shape differ among species. Detailed
values for each species are given in Table 1.
SEM images of 10 Tetraselmis species containing micropearls at the time of
observation.
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), for which they are spherical. P: the
larger and slightly darker inclusions are polyphosphates (c, g). IO: iron
oxides. Pores of the filters are visible as black circles in the background
(2 µm in diameter except for d: 0.2 µm). Strains are as follows. (a):
chui_cc; (d): cord-M_cc; (j):
tetrathele_ac. Scale bars are 5 µm.
Micropearls do not seem to be randomly distributed inside the cells, but
rather show a definite location in most species (Figs. 1 and S2). Moreover,
for a given species, most cells present a similar micropearl arrangement
(Fig. S1). Exceptions are cells that were damaged during sample preparation.
Filtration or freshwater rinsing, for example, can disrupt the micropearl
distribution pattern (Figs. S1e and f and S3).
In some species, the micropearls are mostly aggregated at 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 for T. chui, T. suecica and T. tetrathele (Fig. 1a, i, j).
T. striata shows a similar central micropearl distribution, but the
lateral points of the trident are absent (Fig. 1g). 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) (Fig. S2). 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
S2). These meridians are not well expressed in all cells but, when they are
clearly visible, there seems to be around eight or 10 of them inside the
cell. When well preserved, the micropearl organization in T. cordiformis also shows multiple micropearl alignments that 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 do not allow us to state if there is a definite
distribution of the micropearls in T. subcordiformis (Figs. 1h and
S2).
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 (Fig. 1c and e) – probably at the
apical extremity. EDXS analyses performed in both polyphosphate inclusions
and iron oxide aggregates are shown in Fig. S4.
In order to compare our results with members of another genus, we also
analyzed other flagellate species (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 not all
flagellates produce micropearls. This biomineralization process seems to be
exclusive to a limited number of species.
Table 2 shows the result of counts carried out on the species producing
micropearls. On average, 77 % of the cells contained micropearls and
amongst these, 51 % showed the pattern that is characteristic of their
species. This last value is high, considering that all cells do not fall onto
the filter with the same orientation and that the only patterns we consider
are those obtained when the cell is deposited on its lateral side. Patterns
resulting from a deposition of the cells on their apical or basal sides are not
considered because the 3-D repartition of the micropearls in the cells is
still uncertain.
Percentage of cells presenting micropearls and specific patterns of
micropearl arrangement.
Percentage of cells presenting micropearls for each strain and percentage of
these cells showing the typical micropearl arrangement pattern for that
species (see Figs. 1 and S1). Two strains have been analyzed for T. chui and T. cordiformis.
Please note that strains grown on agar generally show a much lower presence
of micropearls and were not considered for the statistics. The asterisk
marks a single sample taken 60 days after the strain's arrival in our
laboratory, while all the others were observed on the first day after
arrival from the provider. This exception allowed us to estimate the number of
cells showing the micropearl arrangement pattern of this species, as both
samples of T. cordiformis strains taken on the first day were damaged during sample
preparation by too strong of a filtration. On the first day after arrival,
strain CCAC 0579B gave results similar to those of strain CCAC 0051. mp:
micropearls. For details on providers and media, see Table 1.
Tetraselmis
Strain
Medium
Total cells
% cells with mp/
% pattern/
Remarks
counted
cells
cells with mp
T. chui
CCAC 0014
ASP-H
160
93
40
T. chui
SAG 8-6
1/2 SWEg Ag
121
40
37
Resuspended from agar
T. contracta
CCAC 1405
ASP-H
103
98
79
T. convolutae
CCAC 0100
ASP-H
100
40
70
T. cordiformis
CCAC 0051
SFM
115
60
0
Strongly filtered
T. cordiformis*
CCAC 0579B
Waris-H
123
98
46
Gently filtered
T. desikacharyi
CCAC 0029
ASP-H
122
25
13
T. levis
AC 257
ES
123
94
51
T. striata
SAG 41.85
SWES (agar)
136
12
25
Resuspended from agar
T. subcordiformis
SAG 161-1a
Porph (agar)
100
1
0
Resuspended from agar
T. suecica
AC 254
ES
105
99
57
T. tetrathele
AC 261
ES
101
89
56
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 favor 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.
Comparison of FIB-cut sections of cells of three different
Tetraselmis species (dried samples). TEM–HAADF images: FIB-cut sections through cells of (a) Tetraselmis chui (culture
sample),
(b) Tetraselmis suecica (culture sample) and (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. Left top insets: SEM secondary
images of the whole cell before FIB preparation indicating the location of
the cut with a dashed line. Right top insets: SAED patterns from a single
micropearl of each FIB-cut section (broad diffraction rings are indicative
of amorphous material).
FIB-cut section through a Tetraselmis contracta cell (dried sample).
(a) TEM–HAADF image of the whole FIB-cut section. The micropearls show light
or medium grey shades and regular round or oval shapes. Left top inset: SEM
secondary image of the whole cell before it was cut, with a dashed line
indicating the location of the section. Right top inset: SAED patterns from
a single micropearl of this FIB-cut section (diffuse diffraction rings are
indicative for amorphous material). (b) Tentative identification of the
visible cellular constituents; s: starch grains; c: chloroplast; mp:
micropearls; mc: mitochondria. See Fig. S7 for a detailed image. (c) TEM–EDXS mappings – the top image shows the location of the two zones on a
TEM–HAADF image of the section. The map shows an RGB image with three
superimposed element mappings. Micropearls are mainly composed of Ca, with
small quantities of K (and Mg, not shown here). Note that, due to 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 preparation, is also visible in
green.
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 the presence of a rod-shaped nucleus in
their center (Figs. 2 and S5).
As already pointed out, the micropearls are extremely sensitive to the
action of the electron beam (Martignier et al., 2017), indicating a
vaporization of some of its components: organic matter associated
with water, water contained in the amorphous calcium carbonate
(Rodriguez-Blanco et al., 2008) or both. This ACC seems to be rather
stable, as beam sensitivity persists after more than 5 months of storage
of dry samples at room temperature.
TEM–EDXS analyses show that the zonation observed in the marine micropearls
of T. chui and T. suecica (Figs. 2 and S6) is due to
changes in the Sr/Ca concentration ratios, similar 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, in which 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, as thickness and intensity of the zones differ (Fig. 2a and
c).
TEM–EDXS mapping: location of the micropearls inside a
Tetraselmis contracta cell
The coexistence of micropearls with other cellular constituents and their
respective positions in the cell are shown by a TEM image of a FIB-cut
section through a T. contracta cell (Fig. 3). The micropearls of
this species are large and numerous and nearly exclusively consist of ACC
without detectable Sr (Fig. S6). They appear as round to ovoid light grey
shapes with smooth surfaces (Fig. 3a). TEM observations also reveal 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 cryosections),
the identification of the visible cellular constituents can still be
attempted (Figs. 3b and S7). 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, and secondly they are not located inside the chloroplast; in
particular, they are not associated with the prominent pyrenoid.
TEM–EDXS mapping provides compositional information improving the
identification of the cellular constituents and organelles visible in the
section (Figs. 3c and S8). Micropearls are very 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, 1998).
The two irregular features that are highly enriched in P (in green in
Fig. 3c) are identified as being polyphosphate (PolyP) inclusions, flattened during sample
preparation. Finally, the dark grey features, in the center of the section,
are probably mitochondrial profiles.
SEM–EDXS analysis of micropearl composition
The micropearls of most marine species (Fig. 4a) are composed of ACC, with Ca
and Sr as cations. This composition is similar to that measured for
micropearls of T. cordiformis in Lake Geneva (Martignier et al.,
2017). We noted two differences from 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 differences
need to be taken with care.
Composition of the Tetraselmis micropearls and their relation with the growth
media composition.
(a) Distribution of the Sr/Ca ratio for each Tetraselmis
strain (EDXS analyses), ranked according to the median value of Sr/Ca.
At least 20 SEM–EDXS analyses were performed on micropearls of each strain.
Asterisks highlight freshwater strains. The range between the minimum and
maximum data is shown by black lines. The blue boxes represent the 25–75 %
inter-quartiles, while the black horizontal line in the boxes shows the median
value. (b) 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. Points with blue stars highlight freshwater strains. The
blue dotted lines define the values of the Sr enrichment factor of the
micropearls with respect to the medium (10×, 50×, etc.). Calcium
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.
Figure 4a compiles the composition of the micropearls for each
Tetraselmis strain (SEM–EDXS analyses), ranked in increasing order
of Sr/Ca median values. Even if low concentrations of K are present in
micropearls of T. contracta, it was not considered because this
element is also present in the surrounding organic matter (Fig. S8), 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. Therefore the technique yields compositions that include the
micropearl and the surrounding organic matter or nearby cellular constituents
(e.g., polyphosphates).
ICP-SFMS analysis of Sr/Ca ratio in growth media: data and
interpretation
The concentrations of Sr and Ba in the culture media are given in Table S2 and
represented graphically in Fig. S9. 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 study – has 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 among
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)]. 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) – have rather similar Sr distribution
coefficients (around 30), while the three strains of T. cordiformis
show slightly different enrichment factors (25 for Lake Geneva water, 33 for
Lake Fühlingen and 51 for Münster castle 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).
Discussion
Micropearls had been previously interpreted as a feature specifically
related to freshwater environments (Martignier et al., 2017). The present
results show that the biomineralization process leading to the formation of
micropearls can take place in very different environments. The following
paragraphs aim to discuss our present knowledge on micropearls in general and
on their formation process as well as the newly discovered widespread
biomineralization capacity in the Tetraselmis genus, involving high concentration
capacities of these organisms regarding Sr.
Marine and freshwater micropearls
The discovery of micropearls in marine species of Tetraselmis shows
that this biomineralization process can take place in organisms living in
waters of different composition, from freshwater, like Lake Geneva, to
seawater (Fig. S9). This highlights the capacity of these organisms to
integrate Ca and Sr from different external media.
The production of 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 and Guiry,
2018; 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 that 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 characteristics
similar 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 Sr/Ca 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).
Indeed, the general trend seen in this diagram is an adaptation of the ACC
precipitation to the medium composition. However, more relevant information
is provided by the enrichment factor (E factor; see Table S4 and dotted
isolines in Fig. 4b), which allows us to rank species (Table S4) from low values
(12–16) to more than 200. This ranking would need to be confirmed by
cultivating the species in different media (e.g., T. convolutae
group in ES and T. tetrathele group in ASP-H) and comparing the new
enrichment factor with the current values. The very high E factor for
T. desikacharyi can tentatively 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).
The pattern drawn by the arrangement of the micropearls in the cell is
clearly more homogeneous within a strain compared to among strains.
Statistics show that these patterns are characteristic for a given species
(Table 2 and Fig. S1), which means that the organisms can probably exert a
strong control on the number, size and organization of the micropearls in the
cells.
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
FIB sections (T. chui, T. contracta, T. cordiformis and T. suecica),
with a similar rod-shaped nucleus (Figs. 2, 3 and S5). These nuclei could
possibly be of an organic nature given their darker appearance in the STEM–
HAADF images that point to a material of lower atomic mass (Fig. S5).
It is important to note that there are many parameters which seem to
influence the presence and absence of micropearls in the cells: the state of the
culture (fully healthy or suffering from the transport, for example), the pH
of the medium and probably other parameters we are not yet aware of. For
example, the use of agar as a culture medium seems to hinder the development of
micropearls (Table 2 and Fig. 1g and h). Nevertheless, the composition of the
medium does not seem to influence the arrangement of the micropearls in the
cell, as demonstrated by T. chui, T. contracta and
T. convolutae (respectively Fig. 1a, c and d), which have different
patterns, although all were cultured in the ASP-H medium.
Internal concentric zones are observed in the micropearls formed by cells
grown in both the natural environment and 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 or 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 nonequilibrium 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 5 months, according to our observations). Indeed, synthetic ACC with
no additives is unstable and rapidly crystallizes into calcite or aragonite
(Addadi et al., 2003; Bots et al., 2012; Weiner and Addadi, 2011;
Purgstaller, 2016), often through the intermediate form of vaterite
(Rodriguez-Blanco et al., 2011). In contrast, long-term stabilization of ACC
implies the presence of mineral or organic additives (Aizenberg et al., 2002;
Sun et al., 2016). Magnesium is known to play a key role in the stabilization
of ACC (Politi et al., 2010). This might well be the case for the
Tetraselmis-hosted micropearls, in which Mg content is around
2 mol %. Although the phosphate ion has also been reported to inhibit ACC
crystallization (Albéric et al., 2018), that does not seem to be the case
here since the phosphorus concentration of the micropearls is below the
detection level of EDXS. Stabilization of ACC is also enhanced by certain
proteins, polyphosphonates, citrates and amino acids (Levi-Kalisman et al.,
2002; Addadi et al., 2003; Cam et al., 2015; Cartwright et al., 2012). The
presence of these molecules inside the micropearls is suggested by their
observed sensitivity to beam damage. As for the possible role of Sr in the
ACC long-term stability, we did not find any reference
thereof in the literature. However, in an in vitro experiment, Littlewood et al. (2017)
found, in the presence of Mg, a correlation between added Sr and the reaction
time to transform ACC into calcite (2 h to a maximum of 24 h).
A new intracellular feature in a well-known genus
Our results (Fig. 1) confirm that artifacts can be induced by the usual
biological sample preparation techniques (Martignier et al., 2017) and thus
introduce bias in 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 yields useful 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 analyzed Tetraselmis species suggests that they
probably play a physiological role. A possible explanation could be that
micropearls increase the sedimentation rate of cells that shed their flagella
upon N 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, most
Chlorodendrophyceae are known to require the presence of Ca++ 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 Lake Fühlinger (2 mM)), and tests on cultures
showed that T. cordiformis cannot develop normally in an environment
with 0.42 mM of Ca (Melkonian, 1982). Calcium is needed to support
phototaxis (light-oriented movements) and for the construction and
maintenance of 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. Indeed, Chlorodendrophyceae have very
efficient light-gated Ca channels (channelrhodopsins), which are also
essential for phototaxis of these flagellates (Govorunova et al., 2013;
Halldal, 1957).
Bioremediation possibilities
The capacity of some organisms to concentrate Sr is of great interest
regarding bioremediation. Strontium (90Sr) 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 sludge linked with nuclear activities (Bradley et al., 1996).
Its relatively long half-life of ∼30 years and high water solubility
cause persistent water pollution (Thorpe et al., 2012; Yablokov et al.,
2009). For example, the desmid green alga Closterium moniliferum,
which can incorporate 45 mol % of Sr in barite crystals, is considered to
be
a potential candidate as a bioremediation agent (Krejci et al., 2011). The
high Sr absorption capacity of several Tetraselmis species also led
to their mention as potential candidates for radioactive Sr bioremediation
(Fukuda et al., 2014; Li et al., 2006). In our experiments, T. suecica, for instance, produced a high number of micropearls that contained
more than 50 mol % of Sr when cultured in the ES medium (data not shown).
Nevertheless, the process allowing these microorganisms to concentrate Sr had
not yet been investigated and 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 including freshwater, brackish
lakes, open sea and hypersaline lagoons (Table 1).
Conclusions
Until recently, nonskeletal intracellular inclusions of calcium carbonate
were considered 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 and 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 in the past, possibly destroyed by the usual
sample preparation techniques for electron microscopy. Thus other
microorganisms could have similar capacities and intracellular inclusions of
ACCs may be more widespread than currently known.
Micropearls represent a new intracellular feature. This study shows that
they can be clearly distinguished from other cellular constituents and are
not randomly distributed in the cell. On the contrary, micropearls seem to
be essentially located just under the cell wall and they draw a pattern
that seems to be characteristic for each species. Strong correlations
hint that this might have a link with the species habitat.
It appears that, for most of the observed Tetraselmis species, 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.