# Proteins and Water Control the Stability of the Intracellular Amorphous Calcium Carbonate Inclusions Formed by Cyanobacteria

Neha Mehta, Tristan Georges, Guillaume Laurent, Cristina Coelho-Diogo, Cynthia Travert, Feriel-Skouri Panet, Thierry Azaïs, Karim Benzerara

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Neha Mehta, Tristan Georges, Guillaume Laurent, Cristina Coelho-Diogo, Cynthia Travert, et al. Proteins and Water Control the Stability of the Intracellular Amorphous Calcium Carbonate Inclusions Formed by Cyanobacteria. Journal of the American Chemical Society, 2025, 147 (48), pp.44303-44312. 10.1021/jacs.5c14575 . hal-05396917

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1 Proteins and water control the stability of the intracellular amorphous carbonate (ACC)

2 inclusions formed by cyanobacteria

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4 Neha Mehta¹,²*, Tristan Georges³,⁴, Guillaume P. Laurent³, Cristina Coelho-Diogo⁵, Cynthia
5 Travert¹, Feriel-Skouri Panet¹, Thierry Azaïs³, Karim Benzerara¹

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¹Sorbonne Université, CNRS, Institut de minéralogie, de physique des matériaux et de cosmochimie, F-75005 Paris, France

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²Department of Geosciences, Université Libre de Bruxelles, Brussels, 1050, Belgium

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³Sorbonne Université, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, F-75005 Paris, France

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⁴Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland

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⁵Sorbonne Université, CNRS, Fédération de Chimie des Matériaux de Paris Centre, F-75005 Paris, France

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*Corresponding author : neha.mehta@ulb.be

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28 **Abstract**

29 The formation of stable intracellular amorphous calcium carbonate (ACC) inclusions is 30 widespread among bacteria, yet the mechanisms underlying their long-term stabilization remain 31 unresolved. In this study, we apply multinuclear solid-state NMR (ssNMR) spectroscopy to the 32 ACC-forming cyanobacterium *Cyanobacterium* sp. PCC 7425, to probe the molecular environment 33 surrounding cyanobacterial ACC and its role in promoting ACC stability. Our ¹³C and ⁴³Ca ssNMR 34 analyses confirm that the intracellular carbonate phase is composed exclusively of hydrated ACC, 35 with no detectable crystalline CaCO₃ polymorphs. The ¹H–¹³C HETCOR correlations reveal short- 36 range proximity between ACC and water molecules, but no direct interactions with organic 37 molecules. The ¹³C–¹³C DARR long-range correlations detect organic signals consistent with 38 proteins that interact with ACC indirectly, possibly via water molecules. This finding was further 39 supported by the absence of organic components in the vicinity of ACC in short-range ¹³C–¹³C 40 POST-C7 experiments. Taken together, these results are compatible with a model in which 41 cyanobacterial hydrated ACC granules are stabilized by a proteinaceous shell, with some water 42 molecules serving as molecular bridges. This configuration likely restricts water and ion mobility, 43 contributing to the kinetic stabilization of cyanobacterial ACC. 44

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# 1. Introduction

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Amorphous calcium carbonate (ACC) is widely recognized as a transient precursor to crystalline calcium carbonate (CaCO$_3$) biominerals. It functions as a temporary calcium reservoir or a metastable structural component that may persist throughout an organism's lifetime e.g. $1^{-3}$, playing a key role in controlling the final morphology, structure, and trace element composition of biomineralized structures $4-6$. While ACC readily crystallizes in aqueous solutions, biogenic ACC exists in two forms: a transient, anhydrous phase and a more stable, hydrated phase (approximately 1 mol of water per molecule of ACC) $7,8$. The best characterized biogenic transient ACC system is that of the sea urchin embryo, in which calcite spicules form from ACC within membrane-bound vacuoles $1,9$. The ACC is tightly bound by the membrane such that the system remains free of bulk water; under these conditions the initially hydrated ACC dehydrates to an anhydrous ACC phase (i.e. transient ACC), before it subsequently crystallizes to calcite $10$. A similar transformation mechanisms has been observed in synthetic ACC systems $2,11$. By comparison, stable biogenic ACC remains untransformed over the lifetime of the organisms. There are few occurrence of stable ACC that includes plant cystoliths, earthworms, ascidians, and crustaceans$^{5,12-14}$.

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Studies suggest that organisms stabilize ACC through the incorporation of various additives such as magnesium (Mg), silicon (Si), phosphorus (P), organic molecules, and structural water (i.e. incorporated into the structure of ACC) $13,15-19$. These stabilizing interactions sometimes occur within confined volumes, which further promote ACC stability $20$. Notably, all known biogenic stable ACC phases contain structural water, and most include Mg and P as well $8$.

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Bacteria are the latest addition to the group of organisms known to form stable biogenic ACC.

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Several environmentally and taxonomically diverse bacteria produce intracellular ACC inclusions
(300-500 nm in diameter), considerably expanding the repertoire of organisms capable of forming
stable ACC <sup>21-23</sup>. These bacteria inhabit a range of environments including sediments, marine,
freshwater, and brackish water, and yet remain understudied in the context of ACC stabilization
<sup>21,24,25</sup>. At least 21 cyanobacterial strains affiliated to diverse genera, along with a sulfur-oxidizing
gammaproteobacterium (<i>Achromatium</i>), and magnetotactic bacteria (MTB) from both alpha- and
gammaproteobacterial lineages, are known to form intracellular ACC <sup>23,25-28</sup>. Comparative
genomics has suggested that ACC formation in cyanobacteria is a genetically controlled process,
involving a new gene family <sup>25</sup>.

Moreover, the ACC inclusions formed by some cyanobacterial strains sequester high
concentrations of alkaline earth elements such as barium (Ba), strontium (Sr) or radium (Ra),
including the radioactive <sup>90</sup>Sr and <sup>226</sup>Ra isotopes, which holds two implications: (1) these
cyanobacteria may be overlooked actors in the biogeochemical cycles of these trace elements <sup>29,30</sup>;
(2) they may offer some potential for remediating pollutions by these radioactive isotopes <sup>31,32</sup>.
Multiple biological roles for bacterial ACC include functioning as an intracellular pH buffer, and
a storage form of calcium (Ca) and carbon (C) <sup>33</sup>. The formation of bacterial ACC can occur even
in undersaturated extracellular solutions, suggesting that it costs energy to the cells, presumably in
relation with the active sequestration of Ca <sup>34</sup>.

Despite the widespread occurrence of ACC-forming bacteria and their biogeochemical
significance, little is known about the mechanisms that stabilize bacterial ACC. Like its eukaryotic
counterpart, bacterial ACC remains stable intracellularly, with no spontaneous crystallization into
phases such as calcite <sup>16</sup>. One proposed stabilizing mechanism is confinement, as bacterial ACC
granules have been shown to reside within an envelope <sup>35</sup>. Organic molecules (e.g., lipids,
proteins), inorganic additives (e.g., Mg, P) and structural water may also contribute to a stabilizing
microenvironment as observed in invertebrates<sup>20,36-38</sup>, but direct evidence in bacteria remains
lacking. Testing these hypothesis is challenging because bacterial ACC spontaneously dissolves
once extracted from cells, complicating ex-situ analyses<sup>32,39</sup>. Therefore, in order to gain insight
into the mechanisms of bacterial ACC stabilization, in-situ studies of systems are essential.

Previous investigations have employed techniques such as Ca K-edge X-ray absorption near-edge
structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopies<sup>40</sup>,
Fourier transform infrared spectroscopy (FTIR)<sup>41</sup>, scanning transmission X-ray microscopy
(STXM) <sup>23</sup>, and transmission electron microscopy (TEM) <sup>23</sup>. These techniques have provided
powerful insights into bacterial ACC structure and chemical composition but are limited in their
ability to identify the species (e.g., organics, water, phosphorus) in vicinity of bacterial ACC and
their interactions with ACC at an atomic level.

In this study, we address these questions by studying <sup>13</sup>C- or <sup>43</sup>Ca-labelled cells of ACC-forming
cyanobacteria by multinuclear (<sup>1</sup>H, <sup>13</sup>C, <sup>43</sup>Ca) solid-state NMR spectroscopy (ssNMR). The ssNMR
is a robust tool for probing the local molecular environment, and a well-established in-situ technique
for distinguishing various crystalline and amorphous calcium carbonate poly(a)morphs and
identifying the organic and inorganic species potentially involved in ACC stabilization e.g. <sup>11,42,43</sup>.

We elucidate for the first time the in-situ chemical environment in the vicinity of biogenic
cyanobacterial ACC and its relationship to cyanobacterial ACC stabilization.

## 2. Materials and Method

### 2.1. Cyanobacteria cultures and isotopic labeling

Here, we will refer to strains forming intracellular ACC as ACC (+) strains and strains not forming
them as ACC (-) strains. Two model strains, *Synechococcus* sp. PCC 7942 (ACC (-)) and
*Cyanothece* sp. PCC 7425 (ACC (+)) were cultured in BG-11 medium at 30 °C, with agitation and
continuous illumination (5-10 µmol m⁻² s⁻¹). To produce ¹³C-labeled cyanobacterial biomass, sodium bicarbonate (NaHCO₃) in the BG-11 medium was replaced with ¹³C-labelled NaH¹³CO₃ (purchased Sigma Aldrich™). To maximize ¹³C enrichment of the cyanobacterial biomass, NaH¹³CO₃ was replenished in the growth medium every two days to a final concentration of 0.04 g L⁻¹ (465 µM). In parallel, another set of cultures was conducted to produce ⁴³Ca-labeled cyanobacterial biomass. For this purpose, the calcium chloride (CaCl₂, 250 µM) in BG-11 was replaced with ⁴³CaCl₂ at the same concentration (i.e. 250 µM). The ⁴³CaCl₂ solution was prepared by dissolving ⁴³CaCO₃ (62% enrichment in ⁴³Ca; Cortecnet Inc™) in 1 mL of 3.7 wt% hydrochloric acid (HCl) followed by drying at 85°C for 2 hours and subsequently redissolving the residue in Milli-Q water. The ¹³C- or ⁴³Ca-labeled cyanobacterial cells were harvested by centrifugation and dried at 40 °C for 48 hours. The drying process did not induce extracellular precipitation, and the intracellular ACC inclusions remained intact, as shown by previous studies using SEM, FTIR spectroscopy, X-ray diffraction, and Ca K-edge XANES analyses performed on the same (but not isotopically enriched) strains ⁴⁰,⁴¹. The dried biomass was coarsely ground and packed in zirconia rotors for solid-state NMR analysis as described below.

## 2.2. Solid-State NMR spectroscopy analyses

We performed ¹³C-CP ssNMR experiments under both humid (native-state) and dried conditions (i.e. dried biomass). The resulting spectra were similar confirming that the drying of cyanobacterial cells didn't alter the native-state of intracellular ACC (Figure S1). However, the sensitivity was much limited in the humid conditions. Furthermore, we observed that the dried biomass samples remained stable over time, as verified by identical ¹³C-CP ssNMR spectra recorded after four months of storage (Figure S2). Therefore, we opted to perform analysis in dried conditions. ¹H, and ¹³C solid-state NMR experiments were conducted at room temperature using a Bruker Avance
III spectrometer operating at 7 T (i.e. corresponding to a ¹H Larmor frequency of 300.1 MHz)
using a 4 mm double resonance probe under magic-angle spinning (MAS) at a spinning frequency
$\nu_{MAS} = 4.5$ kHz. ¹H and ¹³C chemical shifts ($\delta(¹H)$ and $\delta(¹³C)$) were referenced to the ¹H and ¹³C
resonances of solid adamantane calibrated at 1.71 and 37.7 ppm, respectively. {³¹P}¹³C CP
REDOR experiments were also conducted to probe potential phosphorus environments near ACC;
however, no significant ¹³C dephasing was observed within experimental sensitivity (Figure S3).

¹³C NMR spectra were recorded using either direct excitation (DE) of a 30° ¹³C pulse at a nutation
frequency $\nu_{RF}$ of 42 kHz and a recycle delay of 100 s, or ¹H-¹³C cross-polarization (CP/MAS)
using ¹H and ¹³C radio-frequency (RF) fields of 51 and 44 kHz, respectively, and a recycle delay
of 1 s. Contact time was set to 2 ms to highlight the organic species, or to 10 ms to enhance the
ACC signal (Figure S4). Additionally, a ¹H-¹³C CP spin-echo sequence was used to enhance ACC
resonance. The spin echo block (echo duration of 48 rotor periods (~10.7 ms)) was applied after
the CP/MAS block to induce a $T_2$-filter to select the ACC signal (Figure S5) as shown in Figure
1. All ¹³C solid-state NMR experiments were recorded using spinal-64 ¹H heteronuclear
decoupling ($\nu_{RF} = 92.5$ kHz)⁴⁴. The ¹H-¹³C HETCOR spectrum was acquired using a contact time
of 2 ms, ¹H DUMBO homonuclear decoupling ($\nu_{RF} = 95$ kHz)⁴⁵, 75 kHz heteronuclear decoupling,
and by recording 48 time increments. The ¹³C-¹³C DARR experiment⁴⁶ was recorded using a
contact time of 10 ms, a mixing time of 500 ms, 96 time increments and a ¹H pulse of $\nu_{RF} = \nu_{MAS}$
= 4.5 kHz was applied during the DARR mixing block.

Although short range correlations can also be probed using shorter DARR mixing times, here we
choose to perform ¹³C-¹³C POST-C7 to probe short-range correlations for several reasons: 1) avoid
any ¹H spin diffusion effect that could prevent the probing of short distances, even at short DARR
mixing times. 2) ¹³C-¹³C DQ/SQ POST-C7 experiments have a good sensitivity for ¹³C-labelled
samples and provide enhanced resolution in the indirect dimension, which was particularly useful
in the context of our crowded ¹³C spectra. 3) extracting distance information from experiments
with shorter DARR mixing times is complicated. 4) ¹³C-¹³C DQ/SQ POST-C7 experiments are
known to probe well-defined short distances (typically on the order of several atomic bond lengths
(e.g. ⁴⁷,⁴⁸). The ¹³C-¹³C POST-C7 experiment ⁴⁹ was recorded using a contact time of 10 ms, 8 C7
cycles (3.5 ms) for both DQ excitation and reconversion, C7 recoupling pulses operating at v<sub>RF</sub> =
7 v<sub>MAS</sub> = 31.5 kHz, ¹H continuous-wave decoupling (v<sub>RF</sub> = 135 kHz) during C7 cycles and 112
time increments. For DARR and POST-C7 experiments, forward linear prediction to 256 slices
was applied in the indirect dimension, followed by cosine apodization.

⁴³Ca solid-state NMR experiments were performed at room temperature on a Bruker Avance III
spectrometer operating at 16.4 T (i.e. corresponding to a ⁴³Ca Larmor frequency of 47.1 MHz),
using a 4 mm double resonance H/X probe for low gamma nuclei under MAS at v<sub>MAS</sub> = 14 kHz.
The ⁴³Ca chemical shift (δ⁴³(Ca)) was referenced to the resonance of a 1M CaCl₂ solution (δ⁴³(Ca)
= 0 ppm). ⁴³Ca NMR spectra were recorded using a DFS enhancement scheme ⁵⁰ (DFS block of
2.8 ms sweeping from 400 to 25 kHz) followed by a 90° selective pulse of 3 µs with a recycle
delay of 0.33 s.

3. Results
## 3.1. Detection of ACC in *Cyanotheces* sp. PCC 7425 using ¹³C and ⁴³Ca ssNMR

<figure><img src="image_3.png" /><figcaption>Figure 1: (A) ¹³C CP/MAS spin-echo ssNMR spectra of *Cyanotheces* sp. PCC 7425 (ACC(+)) and *Synechococcus* sp. PCC 7942 (ACC(-)) strains. (B) ¹³C ssNMR spectra of different polymorphs of CaCO₃. The ¹³C ssNMR spectra of calcite, aragonite and vaterite spectra were recorded using ¹³C DE, whereas the ¹³C ssNMR spectra of monohydrocalcite, and synthetic ACC were recorded using ¹³C CP/MAS. Synthetic ACC refers to ACC synthesized in absence of any biological activity and is free from additives which are often present in biogenic ACC like the one produced by *Cyanotheces* sp. PCC 7425 (C). Experimental (blue line) and fitted (red line) ⁴³Ca ssNMR spectra of ACC(+) strain (*Cyanotheces* sp. PCC 7425).</figcaption></figure>
The solid state ¹³C CP/MAS NMR spectra of dried and coarsely ground biomass of ¹³C-enriched *Cyanolthece* sp. PCC 7425 (ACC (+)) and *Synechococcus* sp. PCC 7942 (ACC(-)) are shown in Figure 1A. Both strains exhibit similar spectral features in the region below δ(¹³C) < 160 ppm. This is expected as this region corresponds to common bacterial cell biomolecules such as lipids and polysaccharides. The ¹³C ssNMR spectra of both strains show an intense signal at δ(¹³C)≈ 175 ppm, which is attributed to carbonyl groups of proteins and/or lipids ⁵¹,⁵². A relatively broad signal centered at δ(¹³C) = 168.9 ppm and of full-width at half-maximum (FWHM) = 4.4 ppm was detected in the ACC-forming strain but not in the non-ACC-forming one (Figure 1A inset and Figure S6). This chemical shift is characteristic of carbonate functional groups and was therefore assigned to the presence of intracellular carbonates in *Cyanolthece* sp. PCC 7425. As shown in figure 1B, the ¹³C ssNMR fingerprints (δ¹³C, FWHM) of different CaCO₃ polymorph are as follows: synthetic ACC (168.6 ppm, 3.5 ppm); calcite (168.6 ppm, 0.15 ppm), aragonite (171 ppm, 0.3 ppm); vaterite (170.6 ppm and 169.5 ppm; 0.4 ppm and 0.6 ppm respectively), monohydrocalcite (171.7 ppm, 0.6 ppm). Thus, the carbonate signal observed in *Cyanolthece* sp. PCC 7425 is unambiguously assigned to ACC. Furthermore, the observed δ(¹³C) of cyanobacterial ACC is closest to that of calcite, showing that the local carbonate environment in cyanobacterial ACC possesses proto-calcitic structural features ⁵³.

To further support the presence of ACC, we probed the local atomic environment of Ca in cyanobacteria using ⁴³Ca ssNMR spectroscopy. Similar to ¹³C ssNMR, ⁴³Ca ssNMR is highly efficient in identifying and discriminating CaCO₃ polymorphs ⁵⁴⁻⁵⁶. Figure 1C shows the ⁴³Ca ssNMR spectrum of ⁴³Ca-enriched biomass of *Cyanolthece* sp. PCC 7425. The spectrum exhibits a single broad signal centered at δ(⁴³Ca) = -8.8 ppm displaying a Gaussian lineshape of FWHM=30.4 ppm (Figure 1C). Based on previous ⁴³Ca ssNMR studies of synthetic and naturally
occurring calcium carbonates and inorganic calcium oxides<sup>55,57,58</sup>, this signal is assigned to ACC. To the best of our knowledge, this is
the first reported <sup>43</sup>Ca ssNMR of biogenic, isotopically enriched, stable ACC.

<figure><img src="image_4.png" /><figcaption>Figure 2: (A) 2D<sup>1</sup>H-<sup>13</sup>C HETCOR MAS ssNMR spectrum of the ACC forming cyanobacterium Cyanothece sp. PCC 7425 (ACC (+)). Dashed lines indicate the molecular proximity between carbonyl groups and protons from aliphatic-containing biomolecules. (B) Zoom on the carbonyl and carbonate region of the spectra shown in (A). Dashed lines show the molecular proximity between carbonates from ACC and water molecules.</figcaption></figure>

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Figure 3:(A) 2D <sup>13</sup>C-<sup>13</sup>C DARR MAS ssNMR spectrum of the cyanobacterium Cyanothece sp. PCC 7425 (ACC (+)). Dashed lines highlight the correlations between different carbon environments present in the organic part of Cyanothece sp. PCC 7425 (ACC (+)). Spinning sidebands are denoted by *. (B) Zoom on the carbonyl and carbonate region of the spectra shown in (A) and highlighted by red box in (A). Dashed lines highlight the long-range correlation between some carbonyl groups and ACC. (C) 2D <sup>13</sup>C-<sup>13</sup>C POST-C7 ssNMR spectrum of the cyanobacterium Cyanothece sp. PCC 7425 (ACC (+)). Dashed lines highlight the short-range correlations found between different carbon environments present in the organic part of Cyanothece sp. PCC 7425 (ACC (+)). (D) Zoom on the carbonyl and carbonate region of the spectra shown in (A). Dashed lines highlight the lack of short-range correlations between carbonyl groups and ACC.

## 3.2. Hydrogen and carbon environment in the vicinity of cyanobacterial ACC

The proton environment in the vicinity of cyanobacterial ACC was investigated using two-dimensional (2D) heteronuclear chemical shift correlation experiment under magic angle spinning (<sup>1</sup>H-<sup>13</sup>C HETCOR MAS; Figure 2A-B). The length scale for such polarization transfers is up to approximately 10 Å for <sup>1</sup>H/<sup>13</sup>C nuclei <sup>59</sup>. Consequently, only <sup>13</sup>C nuclei in proximity to protons within ≤10 Å are detected in these cross-polarization MAS experiments. The 2D <sup>1</sup>H-<sup>13</sup>C HETCOR spectrum in the carbonate region displays two distinct correlations within the chemical shift range δ(<sup>13</sup>C) = 150-190 ppm. The first correlation is centered at the ACC resonance (δ(<sup>13</sup>C) =168.8 ppm) and a δ<sup>1</sup>H of ~4.8 ppm. The second correlation is centered in the carbonyl region (δ(<sup>13</sup>C) =175 ppm) and at a δ<sup>1</sup>H of ~0.8 ppm. Based on these <sup>1</sup>H chemical shifts, the proton environment near the cyanobacterial ACC can be assigned to water molecules<sup>60</sup>, while the protons near the carbonyl signal are associated with aliphatic carbon atoms<sup>61</sup>. Interestingly, no organic species are detected in close proximity with ACC through the 2D HETCOR experiment.

To further explore the spatial interactions between carbon atoms in cyanobacterial ACC and surrounding organic matter, both long-range and short-range <sup>13</sup>C-<sup>13</sup>C correlation ssNMR techniques were employed. Long-range spatial interactions (>10 Å <sup>59</sup>) were probed using 2D <sup>13</sup>C-<sup>13</sup>C Dipolar Assisted Rotational Resonance (DARR) MAS ssNMR spectroscopy (Figure 3A-B). This experiment generates a <sup>13</sup>C-<sup>13</sup>C correlation map in which off-diagonal signals reflect magnetization exchange allowed by <sup>1</sup>H-<sup>1</sup>H spin-diffusion during a given mixing time, thus
252 revealing long range C···C interactions relayed by ¹H spin diffusion. A strong off-diagonal signal 253 (highlighted by dashed lines in Figure 3B) indicates long range spatial proximity (>10 Å) between 254 the carbonate ions in cyanobacterial ACC (δ(¹³C) = 168.8 ppm) and some carbonyl groups (δ¹³C 255 = 175 ppm). These carbonyls also correlate with aliphatic components, consistent with the 256 observations from the ¹H-¹³C HETCOR experiment. On the other hand, the short-range 257 interactions (within <6 Å) between carbon atoms were assessed using 2D DQ/SQ ¹³C-¹³C POST- 258 C7 experiments (Figure 3C-D). In contrast with the DARR experiment, the POST-C7 spectra 259 showed no detectable correlation between the carbonyl carbons and ACC, indicating the absence 260 of short-range C···C spatial proximity between these groups, and more generally, the absence of 261 organic species dispersed inside the ACC inclusions in stark contrast with other biogenic ACC 262 such as amorphous CaCO₃ biomineral in crayfish gastroliths ¹³. 263

4. Discussion

4.1. Carbonate mineralogy and in-situ characterization of cyanobacterial ACC

265 ¹³C CP/MAS ssNMR has proven effective for identifying carbonate minerals, and probing the local 266 (< 1 nm) structure of carbonate biominerals in various organisms ⁵⁸,⁶²⁻⁶⁴. The present study 267 represents the first application of ssNMR to detect ACC within ACC-forming bacteria. The 268 intracellular ACC in *Cyanothecе sp*. PCC 7425 is characterized by a ¹³C chemical shift centered 269 at 168.9 ppm with a FWHM of 4.4 ppm (Figure 1A). This signature is consistent with previous 270 ¹³C CP/MAS ssNMR studies of both synthetic and biogenic ACC and is diagnostic of amorphous 271 phases with a proto-calcitic structure. Notably, the ¹³C DE ssNMR spectra (Figure S7) of 272 cyanobacterial ACC did not show any signal corresponding to crystalline CaCO₃ polymorphs. 273 These results establish that ACC is the sole carbonate phase present in the *Cyanothecе sp*. PCC 274 7425. Supporting this, the ⁴³Ca ssNMR spectrum of proto-calcitic ACC inclusions from
275 *Cyanobacteriaceae* sp. PCC 7425 (Figure 1C) exhibits a single broad gaussian signal centered at -8.8 ppm in good agreement with a disordered carbonate phase <sup>58</sup>. No other <sup>43</sup>Ca resonances are observed.
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277 This is the first time that solid-state <sup>43</sup>Ca ssNMR measurements have been conducted on biogenic stable ACC. Overall, the <sup>13</sup>C and <sup>43</sup>Ca ssNMR analyses unambiguously establish that the carbonate
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279 biomineral formed by *Cyanobacteriaceae* sp. PCC 7425 is exclusively composed of ACC.

280 The <sup>43</sup>Ca ssNMR spectroscopy can also be used to gain structural insights into the local 281 coordination environment of calcium in ACC. Previous studies have shown that the <sup>43</sup>Ca isotropic 282 NMR chemical shifts (δ<sup>43</sup>(Ca)) of carbonate phases correlate with the mean Ca–O bond distance 283 in the first coordination shell: as the bond distance increases, the chemical shift decreases <sup>65</sup>. Using 284 the relationship between the Ca chemical shift and the Ca–O bond distance established by Singer 285 et al. <sup>66</sup>, we estimated the Ca-O bond distance in the first coordination shell of Ca in ACC to be 286 2.41 Å. Remarkably, this distance closely aligns with that of 2.43 Å obtained by extended X-ray 287 absorption fine structure (EXAFS) spectroscopy measurements <sup>40</sup>. The strong agreement between 288 these two independent techniques highlights their accuracy and reliability, and the usefulness of 289 <sup>43</sup>Ca ssNMR as a complementary tool for probing local structural environments in calcium-bearing 290 materials.

291 **4.2. Local water environment in cyanobacterial ACC**

292 The <sup>1</sup>H-<sup>13</sup>C HETCOR experiments revealed a single proton environment in the immediate vicinity 293 of cyanobacterial ACC with a resonance at δ<sup>1</sup>H ~ 4.8 ppm. This chemical shift closely matches 294 that of water in both synthetic and biogenic ACC <sup>11,67,68</sup>. This finding is consistent with the 295 observation that all known stable biogenic ACCs are hydrated, typically containing one equivalent 296 of structural water per formula unit <sup>69,70</sup>. Water is well-known to promote kinetic stabilization of 297 ACC and control ACC transformation dynamics. Consistently, dehydration of ACC has been
298 shown to be the first step in the transformation of both biogenic and synthetic ACC to crystalline
299 CaCO₃ poly(a)morphs 2,10,11. Molecular simulation have revealed that water interacts with ACC
300 through two primary mechanisms: the oxygen atom in water binds directly with calcium via a Ca-
301 O bond, and hydrogen atoms form hydrogen bonds with carbonate groups 71,72. These hydrogen
302 bonds cause an in-plane displacement of carbon atoms within the carbonate groups, inhibiting ion
303 rearrangement and thus kinetically stabilizing ACC 15,73.

304 Interestingly, water in cyanobacterial ACC is observed at δ¹H =4.8 ppm, whereas it is observed at
305 δ¹H =6 ppm in other stable biogenic ACC (e.g., lobster gastrolith). This difference can be due to
306 weak H-bond involved for the water molecules and/or variations in ACC composition. For
307 example, Leukel et al. showed that the presence of Mg in synthetic ACC resulted in an increase of
308 the ¹H resonance from 4.5 to 5.2 ppm and explained the shift by a stronger hydrogen bonding in
309 Mg-doped ACC 74. Comparing Mg content, ACC from lobster gastroliths contains 12-20 atom%
310 Mg, whereas ACC in *Cyanthecee* PCC 7425 contains less than 3 atom% Mg 40. Thus, the observed
311 difference in water ¹H resonances between the two systems may reflect a weaker hydrogen bonding
312 network in cyanobacterial ACC, correlated with its lower Mg content.

313 **4.3. Organics in the vicinity of cyanobacterial ACC**

314 Different types of organic components have been implicated in the stabilization of ACC across a
315 variety of systems. For example, phosphorylated acidic protein such as CAP1 associated with
316 stable crustacean ACC have been shown to inhibit calcium carbonate precipitation in-
317 vitro<sup>75</sup>. Glycoprotein GAP 65, extracted from stable ACC of crayfish gastrolith showed dual
318 functions of stabilizing synthetic ACC in-vitro and inhibiting calcium carbonate
319 crystallization<sup>76,77</sup>. Several carboxyl-containing molecules including citrate, phytic acid, acidic
320 amino acids such as glutamic acid have also been reported to strongly increase the lifetime of ACC
particles by adsorbing on the surface of ACC <sup>19,77</sup>. In cyanobacterial ACC, some organic signals were detected using long range <sup>13</sup>C-<sup>13</sup>C DARR experiments (which probe spatial proximities >10 Å), whereas they were absent in short range <sup>13</sup>C-<sup>13</sup>C DQ/SQ POST-C7 experiments (<6 Å) (Figure 3). At the same time, water signals were detected in the short-range spatial proximity of cyanobacterial ACC. In a suitable arrangement from our findings, the organics can be connected to ACC via water molecules, i.e. ACC···H<sub>2</sub>O···organic. Such interactions may reduce the mobility of water and hence increase the energy barrier to dehydration and ultimately prolong the stability of cyanobacterial ACC. This raises the question of what type of organic molecules are responsible for the ssNMR-detected signals.

Our findings support presence of a proteinaceous matrix in the vicinity of ACC granules. Evidence of a proteinaceous environment comes from multiple ssNMR experiments. Specifically, the carbonyl signal displays correlations with aliphatic signals in the <sup>1</sup>H-<sup>13</sup>C HETCOR (Fig. 2A), <sup>13</sup>C-<sup>13</sup>C DARR (Figure 3A), and <sup>13</sup>C-<sup>13</sup>C POST-C7 (Figure 3C) experiments. Importantly, this carbonyl signal shows no correlation with the <sup>13</sup>C sharp resonances at ~130 ppm, which are usually characteristic of lipids and unsaturated fatty acids <sup>78,79</sup>(Figure 3A). In lipids, carbonyl group is esterified to long saturated or unsaturated acyl chain; the latter consistently produce <sup>13</sup>C resonance at ~130 ppm<sup>78,79</sup>. In proteins, by contrast, carbonyl group is part of the peptide backbone, directly attached to an amide nitrogen, and surrounded by alkyl chains that can be charged/uncharged aliphatic/aromatic. Thus, the ssNMR data points to a carbonyl environment consistent with either protein or saturated lipid. To further distinguish between these possibilities, we considered prior cryo-electron microscopy of cyanobacterial ACC, which revealed an electron dense monolayer surrounding the granules<sup>35</sup> (Figure 4). A lipid-based origin of this monolayer is unlikely, because it would require the hydrophilic headgroups to face the cytoplasm and the hydrophobic chains
inward—an orientation incompatible with ACC precipitation, which requires the opposite arrangement. Taken together, the spectral correlations found in ¹H-¹³C HETCOR, ¹³C-¹³C DARR, and ¹³C-¹³C POST-C7 combined with the ultrastructural evidence of a monolayer, strongly support the interpretation that the carbonyl group reside in a proteinaceous environment rather than a lipid matrix. Protein-encased microcompartments are well established in bacteria. Carboxysomes, which encapsulate enzymes such as the RubisCO and carbonic anhydrases, serve as a notable example⁸⁰. Similar protein-bound compartments, such as the Pdu and Eut microcompartments in bacteria, support specialized metabolic functions and resemble carboxysomes by their architecture⁸¹. Some cyanobacteria also contain gas vesicles surrounded by a protein layer measuring approximately 2 nm in thickness⁸⁰.
<figure><img src="image_9.png" /><figcaption>Figure 4: (A) Cryo-electron microscopy image of a Cyanothece sp. 7425 cell showing a central ACC inclusion. (B) Close up of the ACC inclusion, showing an envelope around, marked by white arrow. (C, D). Schematic showing the role of water as a “molecular bridge” between ACC and proteins. For details on cryo-electron microscopy imaging of Cyanothece sp. 7425, refer to ³⁵. Scale bars in (A) and (B) is 200 nm and 1 nm respectively.</figcaption></figure>

In eukaryotes, it is known that biogenic ACC frequently incorporates acidic proteins rich in carboxylated amino acids ⁸². For example, Aizenberg et al. identified proteins rich in glutamic acid and hydroxyamino acids (serine, glycine, threonine) in stable ACC of sponges and ascidians ¹⁷. These proteins exhibit high affinity for Ca²⁺ ions due to their negatively charged carboxyl groups present in the amino acid side chains (C₂H₄COO⁻ in glutamic acid). This interaction not only facilitates Ca²⁺ transport to the site of ACC formation but also suppresses ion rearrangement, thereby stabilizing the amorphous phase. Based on our ssNMR data, proteins may be present as
shell or occluded within the cyanobacterial ACC, as observed in other biogenic ACC. However, when considered together with cryo-electron microscopy observations of presence of monolayer enveloping cyanobacterial ACC, the most parsimonious interpretation is that proteins in vicinity of cyanobacterial ACC forms a spatially separated domain, possibly hydration shells, engaging in hydrogen bonding networks that modulate water dynamics. Such interactions can reduce the mobility of water molecules, suggesting that water could acts as a “molecular bridge” between ACC and protein (Figure 4). This bridging may also contribute to the kinetic stabilization of ACC by increasing the energy barrier for dehydration, an essential step in ACC crystallization. The precise identity of these proteins remains an interesting topic for future biochemical studies.

## 4.4. Conclusions

This study provides the first in situ ssNMR characterization of intracellular ACC in an ACC-forming cyanobacterium. Using ¹³C and ⁴³Ca ssNMR, we confirm that the intracellular carbonate phase in *Cyanothecе* sp. PCC 7425 consists exclusively of hydrated ACC of proto-calcitic arrangement, with no evidence of crystalline CaCO₃ polymorphs. ¹H–¹³C HETCOR reveals that water is intimately associated with the ACC phase while ¹³C–¹³C DARR evidence organic components—most likely proteins—located at longer spatial ranges (>10 Å). Together, these findings support a model in which ACC granules are confined within a proteinaceous shell, and interactions between ACC and the protein matrix could be mediated by water molecules. Water molecules within the ACC granules reduce ionic mobility, creating a microenvironment that suppresses dehydration and inhibits crystallization. These structural insights provide a foundation for understanding ACC stability in biological systems and may inform biomimetic approaches to calcium carbonate synthesis and stabilization.

## 4.5 Acknowledgments
389 Financial support for this project was provided by the Institute of Materials Science (iMAT) of the
390 Alliance Sorbonne University and the Agence Nationale de la Recherche (ANR Harley, ANR-19-
391 CE44-0017-01). Neha Mehta acknowledges iMAT for a Post-Doc grant. Tristan Georges and
392 Thierry Azaïs acknowledge NTU-CNRS "Excellence Science" Joint Research Program for
393 funding. We also thank the GEMME (geomicrobiology) facility located at the Institut de
394 Minéralogie, Physique des Matériaux et Cosmochimie (IMPMC, Paris, France) for microbiology
395 support. The French Region Ile-de-France – SESAME program is acknowledged for financial
396 support (700 MHz spectrometer).

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Supplementary information

Proteins and water control the stability of the intracellular amorphous carbonate (ACC) inclusions

formed by cyanobacteria

Neha Mehta<fnref n="1" />, <fnref n="2" />, Tristan Georges<fnref n="3" />,<fnref n="4" />, Guillaume P. Laurent<fnref n="3" />, Cristina Coelho-Diogo<fnref n="5" />, Cynthia Travert<fnref n="1" />,
Feriel-Skouri Panet<fnref n="1" />, Thierry Azaïs<fnref n="3" />, Karim Benzerara<fnref n="1" />

<footnote n="1">¹Sorbonne Université, CNRS, IRD, MNHN, Institut de minéralogie, de physique des matériaux et de cosmochimie, F-75005 Paris, France</footnote>

<footnote n="2">²Department of Geosciences, Université Libre de Bruxelles, Brussels, 1050, Belgium</footnote>

<footnote n="3">³ Sorbonne Université, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, F-75005 Paris,
France</footnote>

<footnote n="4">⁴ Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland</footnote>

<footnote n="5">⁵ Sorbonne Université, CNRS, Fédération de Chimie des Matériaux de Paris Centre, F-75005 Paris,
France</footnote>
Figure S1: ¹³C CP/MAS spectra of ACC(+) before (bottom, blue) and after (top, red) drying recorded at vMAS = 4.5 kHz using a contact time of 2 ms. The drying process does not seem to affect the stabilization mechanism of ACC here, as no additional signal is observed upon drying.

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>Figure S2: ¹³C CP/MAS spectra of ACC(+) right after drying (red) and after 4 months (blue) recorded at vMAS = 4.5 kHz using a contact time of 10 ms. The sample remains very stable under dried conditions since no significant difference is observed between the two spectra.</figcaption></figure>
Figure S3: {$^{31}$P}$^{13}$C CP REDOR ssNMR spectra of cyanobacterial ACC, showing the spectrum without dipolar recoupling S0 (blue) and the spectrum with $^{31}$P-$^{13}$C dipolar recoupling S (7.5 ms, red). The difference spectrum ΔS (purple) should show significant dephasing of the $^{13}$C resonances which are close in space to $^{31}$P species. Note the absence of dephasing for the aliphatic region and carbonate region suggesting that phosphorous-containing species are inorganic and do not stabilize ACC.

<figure><img src="image_12.png" /></figure>
Figure S4: ¹³C CP/MAS spectra of cyanobacterial ACC acquired with contact times of 2 ms (bottom, blue) and 10 ms (top, red). (A) Carbonate/carbonyl region; (B) aliphatic region. In (A), spectra are normalized to the carbonyl peak to highlight the improved selectivity of the ACC carbonate signal at longer contact times. In (B), spectra are displayed without normalization to illustrate the enhanced sensitivity to organic components at shorter contact time (2 ms).

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>Figure S5: ¹³C CP/MAS spectra of cyanobacterial ACC recorded with (top, red) and without (bottom, blue) a spin-echo following the cross-polarization.</figcaption></figure>
Figure S6: Peak fit of the ACC peak detected in the 13C CP/MAS spin echo ssNMR spectra of *Cyanothecë* PCC 7425 (ACC+). The ACC peak was simulated as a pure gaussian signal centered at $\delta_{iso} = 168.67$ ppm with FWHM 4.42 ppm.

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /><figcaption>Figure S7: ¹³C standard CP MAS ssNMR spectra (red) and direct excitation ¹³C ssNMR spectra of *Cyanothecе* PCC 7425 (ACC+).</figcaption></figure>