Oral Presentations

T5 O1

Shedding light on the nature of sea urchin pigment-bearing vesicles

Claudio R. Ferreira¹; Chakib Djediat²; Xabier Lekube³; Urtzi Izagirre³, Nerea Garcia-Veslaco³; Yael Politi⁴; Luca Bertinetti⁴; Nadine Nassif¹ and Marie Albéric¹*

1 Laboratoire de Chimie de la Matière Condensée, Sorbonne Université, Paris, France, 2 Musée Nationale d'Histoire Naturelle, Paris, France, 3 University of the Basque Country (UPV/EHU), Research Centre for Experimental Marine Biology and Biotechnology (PiE-UPV/EHU), Plentzia, Basque Country, 4 B CUBE – Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany

Red-spherule cells (RSCs) take part into the innate immune system of sea urchins likely through the release of poly-hydroxylated naphthoquinones (PHNQs) [1], initially enclosed in the cytoplasmic vesicles as dense red granules. RSCs can be very abundant in the body fluid of larvae and adult sea urchins during infection as well as in the soft tissues of regenerated spines after fracture [2]. Known for their demonstrated antimicrobial activity, PHNQs are also responsible for the multiple colors observed in sea urchin biominerals [3, 4]. However, the mechanism by which PHNQs are eventually released from the RSCs and incorporated into the biomineral structure during its growth is not known. Using complementary microscopic studies, we here investigate the biomineral pigmentation pathways in *Paracentrotus lividus* sea urchins. From FIB-SEM data on regenerated spines, we propose that pigment vesicles are released by RSCs at the vicinities of growing micro-spines during fracture healing. To go further on the structure and composition of the RSC and their pigment vesicles, we then performed TEM and histological observations of both pluteus larvae and adult coelomic fluid as well as regenerated spines. TEM observations reveal that RSCs' pigment vesicles have different PHNQ granule densities and are internally coated by a polysaccharide layer possibly acting as a cellular protective barrier against acidic pH [5]. Finally, histological staining with pH-sensitive dyes reveals a lower pH for RSCs in regenerating spines as compared to the extracellular matrix. Staining of the urchin larvae confirms the presence of acidic areas (pH ~ 6.0) where RSCs are abundant. These findings indicate that pH could play a major role upon releasing and incorporation of PHNQ molecules into the growing mineral, suggesting an intricate association between pigmentation and biomineral formation.

[1] Coates, J.; et al. Journal of Innate Immunology. 10 (2018) 119-130.

[2] Heatfield, B. M.; Travis, D. F. Journal of Morphology. 145 (1975) 13-49.

[3] Goodwin, T.; Srisukh, S. Biochemical Journal. 47 (1950) 69-76.

[4] Brasseur, L.; et al. Marine Drugs. 15 (2017) 179.

[5] Wang, X.; et al. Scientific Reports. 2 (2012) 892.

**Keywords:** red spherule cells; poly-hydroxylated naphthoquinones; pigmentation
Induced mineralization in *E. coli* biofilms: the role of extracellular matrix

Laura Zorzetto, Ernesto Scoppola, Emeline Raguin, Peter Fratzl, Cécile M. Bidan

Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Am Mühlenberg 1 -
14476 Potsdam, Germany

Biofilms are living microbial tissues emerging that occur as a survival strategy in challenging environments, after bacteria colonize a surface and synthesize extracellular matrix. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphates and calcium carbonates. Previously, we cultivated *E. coli* K-12 W3110 on an agar medium containing calcium ions and organic phosphates. This *E.coli* strain produces an extracellular matrix mainly made of amyloid curli fibers and we reported the precipitation of calcium phosphates in the form of hydroxyapatite in the [1]. Focused ion beam with scanning electron microscopy performed in cryogenic conditions (cryo-FIB-SEM) enabled us to identify both mineralized bacteria and mineralized portions of the extracellular matrix. This can indicate an interplay between different biomineralization processes related to microbial activities. With few exceptions, microbial mineralization usually results from adventitious precipitation of inorganic compounds led by their interactions with different metabolic processes [2] and with the macromolecules present in their surroundings [3]. In this context, we investigated the influence of different macromolecules in the biofilm on the formation of calcium phosphate crystals. On nutritive agar substrates inducing mineralization, we cultivated diverse *E. coli* strains, which would produce as extracellular matrix only amyloid curli fibers, only phosphoethanolamine cellulose fibers, both of them or none of them [4]. We estimated crystal dimensions using wide-angle x-ray scattering enabling us to assess crystal strains at the nanoscale. Cryo-FIB-SEM was exploited to study crystal aggregation at a larger scale. Uncovering the influence of the biomolecules present in the extracellular matrix is an important piece of knowledge to engineer living composites.

1. Zorzetto L, Scoppola E, Raguin E, et al (2023) Induced mineralization of hydroxyapatite in *Escherichia coli* biofilms and the potential role of bacterial alkaline phosphatase. Chem Mater In Press

2. Mann S (2001) Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry. Oxford University Press, New York, NY

3. Decho AW (2010) Overview of biopolymer-induced mineralization: What goes on in biofilms? Ecol Eng 36:137-144. https://doi.org/10.1016/j.ecoleng.2009.01.003

4. Thongsomboon W, Serra DO, Possling A, et al (2018) Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose. Sciece 338:334-338
# Morphology and size of mineral particles may be affected by bacteriophages

Paweł Działak¹*, Mirosław Słowakiewicz², Andrzej Borkowski¹

¹Faculty of Geology, Geophysics and Environmental Protection, AGH University of Science and Technology, Al. Mickiewicza 30, 30-059 Kraków, Poland, ²Faculty of Geology, University of Warsaw, ul. Żwirki i Wigury 93, 02-089 Warsaw, Poland

*corresponding author (dzialak@agh.edu.pl)

Until now, the properties of mineral particles have been thought to be influenced by bacteria, fungi and photosynthetic microorganisms. The latter have been recognised as one of the main factors that can influence biogenic carbonate formation in microbial mats. However, their role was limited to photic and oxic environments. On the other hand, sulphate-reducing bacteria, as well as methanogenic archaea involved in the anoxic oxidation of methane can play a role in carbonate formation in anoxic environments.

However, the abundance of these microorganisms is relatively low compared to viruses, which are an order of magnitude higher. Their role in the formation of various minerals has not been widely recognised. It is still not known how viruses affect the process of mineral formation.

In our experiments [1]-[3], we demonstrated that (i) viruses may induce framboid-like mineral formation; (ii) the electrokinetic potential of minerals can be altered during mineral precipitation in the presence of bacteriophages; (iii) viruses can alter the size of mineral particles precipitated in the presence of bacteriophages; (iv) viruses can bind to the surface of various minerals, altering their properties.

Bacteriophages are thought to affect mineral precipitation in a variety of environments. Their influence on the agglomeration or aggregation and physicochemical properties of mineral particles appears to be of particular interest. We conclude that the interaction between bacteriophages and mineral particles may play an important, but underestimated role in mineral formation in sedimentation environments.

[1] P. Działak, M. D. Syczewski, K. Kornaus, M. Słowakiewicz, Ł. Zych, and A. Borkowski, 'Do bacterial viruses affect framboid-like mineral formation?', *Biogeosciences*, vol. 19, no. 18, pp. 4533-4550, Sep. 2022, doi: 10.5194/bg-19-4533-2022.

[2] P. Działak et al., 'Surface modification of magnetic nanoparticles by bacteriophages and ionic liquids precursors', *RSC Adv.*, vol. 13, no. 2, pp. 926-936, 2023, doi: 10.1039/D2RA06661K.

[3] M. Słowakiewicz et al., 'Newly-discovered interactions between bacteriophages and the process of calcium carbonate precipitation', *Geochim. Cosmochim. Acta*, vol. 292, pp. 482-498, Jan. 2021, doi: 10.1016/j.gca.2020.10.012.
Poster Presentations

Poster 22

# What is the role of the shell organic matrix in molluscan defense against microorganisms ?

Camille Lutet-Toti¹,³, Stefano Goffredo², Giuseppe Falini³, Adeline Bidault⁴, Christine Paillard⁴, Frédéric Marin¹

¹UMR CNRS-EPHE 6282 'Biogéosciences, ' Université de Bourgogne, Dijon, France. ²Dipartimento di Scienze Biologiche Geologiche e Ambientali, Alma Mater Studiorum Università di Bologna, Bologna, Italy. ³Dipartimento di Chimica “Giacomo Ciamician”, Alma Mater Studiorum Università di Bologna, Bologna, Italy. ⁴LEMAR UMR 6539, IUEM, Brest, France.

Mollusks are soft bodied animals that have developed a peculiar set of graded defenses against chemo-
physical and biological threats: among them, the shell constitutes the first shield, and the mucus layer,
an additional cleaning system, while hemocytes are involved in repair mechanisms and immunity at
cellular level. There are however persistent clues that the shell is not solely a physical barrier. To this
end, we investigate here the role of the calcifying organic matrix, a cocktail of macromolecules that
regulate the deposition of the crystalline units and remain occluded in the shell. Until recently, they were
thought to regulate only the mineral deposition process. High-throughput proteomics techniques backed
by transcriptomes show the complexity of this calcifying matrix and the abundance of basic peptides,
suggesting potential antimicrobial properties. This research seeks to demonstrate *in vitro* the bactericidal
capacity of calcifying matrices extracted from mollusk shells of economic interest. We intend to valorize
- through sophisticated applications - an abundant co-product of the sea which is only recycled with low
added value. For this purpose, we selected different bacterial strains: generalist bacteria (*E. coli*, *P. aeruginosa*, *B. subtilis* spp.) and common marine pathogens (*Aliivibrio salmonicida*, *Vibrio tapetis*, *V. mytili*, *V. harveyi*, *V. aestuarianus*). For each mollusk species, the effects of different acetic acid-soluble
shell fractions on bacterial growth were measured via disk diffusion and metabolism (phenol red) assays;
the insoluble fractions were also tested via the first method. In parallel, all fractions (soluble and
insoluble) were analyzed by proteomics (LC-MS-MS). Through potential applications at the interface
of aquaculture, conservation and health, this project aims to establish a virtuous circular economy and
to better understand the complex role of the mollusk shell as a protective barrier.

Keywords: mollusk, shell, biomineralization
Keynote lecture

Biological Adaptations and Blueprints for Extreme Environments

David Kisailus

*Materials Science and Engineering, University of California at Irvine*

Organisms have derived specific sets of traits in response to common selection pressures that serve as guideposts for optimal biological designs. A prime example is the evolution of toughened structures in disparate lineages within plants, invertebrates, and vertebrates¹. Extremely tough structures can function much like armor, battering rams, or reinforcements that enhance the ability of organisms to win competitions, find mates, acquire food, escape predation, and withstand high winds or turbulent flow.

Some of these natural systems have developed well-orchestrated strategies, exemplified in the biological tissues of numerous animal and plant species, to synthesize and construct materials from a limited selection of available starting materials. The resulting structures display multiscale architectures with incredible fidelity and often exhibit properties that are similar, and frequently superior to, mechanical properties exhibited by many engineering materials¹,². In specific instances, comparative analyses of multiscale structures have pinpointed which design principles have arisen convergently; when more than one evolutionary path arrives at the same solution, we have a good indication that it is the best solution. This is required for survival under extreme conditions. We describe a few of these systems that show convergent design and describe how controlled syntheses and hierarchical assembly using organic scaffolds lead to these integrated macroscale structures³⁻⁸. We describe their function and translation to biomimetic and bioinspired materials used for engineering applications⁹⁻¹¹.

1. "Multiscale toughening mechanisms in biological materials and bioinspired designs", W. Huang et al., *Adv. Mat.*, **31**, 1901561 (2019).

2. "Nano-Architected Tough Biological Composites from Assembled Chitinous Scaffolds", W. Huang et al., *Acc. Chem. Res.*, **50** (10) (2022) 1360-1371.

3. "Fibrous Anisotropy and Mineral Gradients within the Radula Stylus of Chiton: Controlled Stiffness and Damage Tolerance in a Flexible Biological Composite", J.E. Lee et al., *J. Comp. Mater.*, **57** (4) (2022).

4. "Toughening Mechanisms of the Elytra of the Diabolical Ironclad Beetle", J. Rivera et al., *Nature*, **586**, 543-548 (2020).

5. "A natural impact resistant bi-continuous composite nanoparticle coating", W. Huang et al., *Nat. Mat.* **9**, 1236-1243 (2020).

6. "Phase transformations and structural developments in the radular teeth of *Cryptochiton stelleri*", Q. Wang et al., *Adv. Funct. Mat.*, **23**, 2908-2917 (2013).

7. "The Stomatopod Dactyl Club: A Formidable Damage-Tolerant Biological Hammer", J. Weaver et al., *Science*, **336** 1275-1280 (2012).

8. "Analysis of an ultra hard magnetic biomaterial in chiton radular teeth," J. Weaver et al., *Materials Today*, **13** (2010) 42-52.

9. "Direct ink write printing of chitin-based gel fibers with customizable fibril alignment, porosity, and mechanical properties for biomedical applications", D. Montroni, et al., *J. Funct. Biomat.*, **13** (2) (2022) 83.

10. "Electrocatalytic N-Doped Graphitic Nanofiber - Metal/Metal Oxide Nanoparticle Composites", H. Tang et al., *Small*, **14**, 1703459 (2018).

11. "Bio-Inspired Impact Resistant Composites", L.K. Grunenfelder et al., *Acta Biomat.*, **10**, 3997-4008 (2014).
# Structure-function studies of intrinsically disordered proteins-iron oxide interaction

Raz Zarivach

Department of Life Sciences, The National Institute for Biotechnology in the Negev and The Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev

Zarivach@bgu.ac.il

Biomineralization is a complex process that involves specialized proteins to mediate and control mineral sedimentation. These biomineralization proteins often have intrinsically disordered regions that are critical for their function. To gain insights into this process, we investigated the Mms6 protein, an intrinsically disordered protein from magnetotactic bacteria that are involved in magnetite nucleation, using various techniques such as NMR, X-ray, and cryo-EM.

In our study, we utilized the cavity of ferritin as a nanoreactor to mimic the magnetosome lumen, where Mms6 binds to iron and participates in the nucleation event. Using these approaches, we observed the interaction between M6A, an active intrinsically disordered iron-binding domain peptide derived from Mms6, and an iron oxide particle, at high resolution. Our findings suggest that the folding of M6A correlates with detecting mineral particles in its vicinity. We also found that M6A interacts with the iron oxide particles through its C-terminal side, stabilizing a helix at its N-terminal side.

Importantly, our results demonstrate the ability of intrinsically disordered proteins to respond to signals from their surroundings by undergoing conformational changes. These findings shed light on the mechanism underlying the control of biomineralization protein over mineral microstructure, where the unstructured regions of these proteins become more ordered upon interacting with the nascent mineral particles. Our study provides crucial insights into the function of intrinsically disordered proteins and their role in biomineralization processes.
# The calcium phosphate-based biomineral clusters for rapid remineralization of tooth enamel

Nan Luo¹,², Bing-Qiang Lu¹, Xi Chen², and Feng Chen¹*

¹ Center for Orthopaedic Science and Translational Medicine, Department of Orthopedic, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, 200072, P. R. China, ² Department of Preventive Dentistry, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine

## Corresponding Authors

*E-mail: fchen@tongji.edu.cn

Using biomineral-based materials to grow hydroxyapatite (HAP) crystals on tooth enamel surfaces is a common strategy for repairing early demineralization. However, the effectiveness of these materials is usually reduced by the dynamic oral environment, which includes continuous saliva flow and friction from cheek muscles. To address this, a rapid remineralization method is needed to avoid or diminish these influences. The challenge has been finding stabilizers that do not resist transformation during remineralization. We have discovered a stable species of calcium phosphate biomineral with ultra-small size (1-2 nm) that can rapidly repair enamel via water-triggered transformation in both static and dynamic environments. The calcium phosphate biomineral can easily penetrate nano/micro-sized enamel defect sites and transform into HAP nanorods immediately, going through an intermediate phase of an amorphous calcium phosphate nanowire. The resulting nanorods stand vertically to the enamel surface and form a compact HAP repair layer within 30 minutes, which is much faster than conventional materials that take hours or days. In vitro and in vivo studies have shown that the recovers mechanical properties of samples are similar to those of sound enamel. The calcium phosphate biomineral with ultra-small size has the potential to be a widely used strategy for dental remineralization due to its rapid repair capacity, simple preparation process, low cost, and remarkable biocompatibility.
# Structural and mechanical anisotropy in crustacean claws mineralized with amorphous calcium phosphate

Miloš Vitori¹, Vesna Srot², Lidija Korat³; Birgit Bussman², Felicitas Predel², Van Aken Peter A.², Jasna Štrus¹

¹ University of Ljubljana, Biotechnical Faculty, Department of Biology, SI-1000 Ljubljana, Slovenia, ² Stuttgart Center for Electron Microscopy, Max Planck Institute for Solid State Research, Heisenbergstrasse 1, 70569 Stuttgart, Germany, ³ Slovenian National Building and Civil Engineering Institute, SI-1000 Ljubljana, Slovenia

Crustacean exoskeletons are not only fascinating from the point of view of functional morphology; their versatility, representing adaptations to a variety of functions, may inspire biomimetic materials with outstanding performance. The crustacean exoskeleton consists of the cuticle, which is secreted by epidermal cells and can be viewed as a composite material consisting of chitin fibres and a mineralized matrix. We studied the claws on the walking legs of isopods, which are minute and thin structures, yet are capable of supporting the entire animal during walking and climbing.

In the terrestrial isopod *Porcellio scaber*, the claws are only a few tens of microns in length. We analysed the architecture of the organic matrix and mineral components in the claw at the nanoscale with the use of electron microscopy. Our results showed that the isopod's claws are mineralized exclusively with amorphous calcium phosphate. Furthermore, the claw skeleton is highly structurally anisotropic, with chitin fibres aligned in a single direction, matching the principal direction of stress. The claw possesses a mineralized core enveloped by a thick layer of non-mineralized cuticle, an organization that likely confers greater fracture resistance. To understand the mechanical consequences of these features, we studied the sea slater *Ligia pallasii*. Its larger size allowed us to perform additional imaging using micro-computed X-ray tomography and the determination of the cuticle mechanical properties using nanoindentation. The organization of the matrix and the chemical nature of mineral components in the claw are similar as in *P. scaber*. Micro-CT demonstrated that the leg cuticle includes non-calcified regions that may act as shock absorbers. Nanoindentation allowed us to confirm that the claw is highly mechanically anisotropic and strengthened in the direction of predominant loading, reflecting its structural anisotropy.
# Bio-Inspired Fluorescent Consolidants for the Conservation of Gypsum Plasterwork

Miguel Burgos-Ruiz, Kerstin Elert, Carlos Rodriguez-Navarro and Encarnacion Ruiz-Agudo

Department of Mineralogy and Petrology, Faculty of Sciences, University of Granada, Av. Fuentenueva, 18002, Granada, Spain.

e-mail: miguelburgos95@ugr.es

In Nature, living organisms show an unmatched ability to originate sophisticated inorganic/organic hybrid materials and composites with a wide variety of physicochemical properties, such as enhanced mechanical strength or optical functionalities. In particular, fluorescence occurs in some species increasing their visibility, which is a powerful tool we can use in our favor for multiple purposes (e.g., drug delivery control).

Although it is still a challenge, biomimetic and/or bio-inspired strategies have been developed in order to recreate biomaterials at the synthetic scale using organic polymers that imitate the role of proteins and/or polysaccharides during biomineralization processes.

In this work we investigated the effect of calcein (i.e., an organic $Ca^{2+}$ specific fluorescent chelating agent) on the crystallization of $CaSO_4 \cdot nH_2O$ phases, and we elaborated a solvothermal procedure for the synthesis of calcein-loaded bassanite ($CaSO_4 \cdot 0.5H_2O$) nanoparticles with luminescent properties for their use as consolidants for the conservation of decayed gypsum plasterwork. Fluorescence allowed to distinguish the newly deposited nanoparticles from the original plaster, a problem that is extremely difficult to address using other imaging techniques, as both consolidant and substrate share the same composition.

<figure><img src="image_11.png" /></figure>

Fluorescence photomicrographs (a) demonstrating that $CaSO_4$ crystallizes in the presence of calcein acquiring fluorescent properties. Treated plaster specimens irradiated with UV light (b and c) showing that the synthesized luminescent bassanite nanoparticles penetrate inside the substrate and can be easily detected

## References:

[1] Magnabosco, G., Polishchuk, I., Erez, J., Fermani, S., Pokroy, B., & Falini, G. (2018). Insights on the interaction of calcein with calcium carbonate and its implications in biomineralization studies. *CrystEngComm*, 20(30), 4221-4224.

[2] Burgos-Ruiz, M., Pelayo-Punzano, G., Ruiz-Agudo, E., Elert, K., & Rodriguez-Navarro, C. (2021). Synthesis of high surface area $CaSO_4 \cdot 0.5H_2O$ nanorods using calcium ethoxide as precursor. *Chem. Commun.*, 57, 7304-7307.
# Iron-based biominerals of marine sponge origin as inspiration for biomimetics

Anita Kubiak<sup>A,B</sup>, Martyna Pajewska-Szmyt<sup>B</sup>, Martyna Kotula<sup>A,B</sup>, Bartosz Leśniewski<sup>A,B</sup>, Hermann Ehrlich<sup>B</sup>

<sup>A</sup>Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Poznańskiego 8, 61-614 Poznan, Poland; <sup>B</sup>Center of Advanced Technology, Adam Mickiewicz University, Uniwersytetu Poznańskiego 10, 61-614 Poznan, Poland;

Research into iron-based biominerals derived from skeletal spongin of marine bath sponges fits perfectly both into the fields of biomineralogy and biomimetics¹. Crystalline mineral phases of lepidocrocite (γ-FeOOH) origin on proteinaceous spongin microfibres have been already reported in 1968². Is it possible to use spongin as renewable source of unique 3D scaffolds for it functionalization using iron ions for large scale application?

For this purpose, a new bioinspired by biocorrosion of iron biomimetic method has been developed to form lepidocrocite on a spongin scaffold *in vitro* for the first time. The present study investigates the interaction of iron ions with a spongin scaffold in an artificial seawater as corrosive environment. Consequently, the novel Iron-Spongin composite was developed (Fig. 1A). Alternatively, the extreme biomimetics approach was used to form goethite on a spongin scaffold. This experiment involved mixing crystalline iodine and powdered iron with spongin to create a new FeI-Spongin biocomposite at room temperature (Fig. 1B). Obtained composites remain stable after sonication during 5 h. Furthermore, mechanisms for the formation of both iron oxides on spongin fibres were suggested. Both composites were characterised using instrumental techniques including microscopic imaging (optical, SEM/EDX, HRTEM), spectroscopic analysis (FTIR, Raman), X-Ray diffraction and thermogravimetry. For the first time we successfully used these unique 3D composites as sensors for dopamine detection. The next step will include the first attempt in the history of materials science to use the obtained iron-containing spongin composites to obtain a higher-order metallic phase by melting them at 1450°C.

<figure><img src="image_12.png" /><figcaption>**Figure 1.** Images taken with a digital microscope at 100 µm magnification. (a) Iron-Spongin; (b) FeI-Spongin.</figcaption></figure>

## Acknowledgments

This research was funded by the National Science Centre within the framework of the project Maestro 12 No. 2020/38/A/ST5/00151.

## Reference

1. Ehrlich, H., Wysokowski, M. & Jesionowski, T. The philosophy of extreme biomimetics. *Sustainable Materials and Technologies* **32**, e00447 (2022).

2. Towe, K. M. & Rützler, K. Lepidocrocite Iron Mineralization in Keratose Sponge Granules. *Science* (1979) **162**, 268–269 (1968).
Controlled crystallization of guanine nanocrystals

Yurong Ma *

School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081

E-mail: yurong.ma@bit.edu.cn

Guanine is one of the most widespread organic crystals existing in organisms, which has superior optical properties and exhibits structural colors. The excellent optical properties of biogenic guanine crystals contribute to the exquisite control on the polymorph, morphology, size and highly-ordered hierarchical microstructure assemblies of guanine crystals and cytoplasm. Controlled crystallization of guanine crystals with particular polymorphs, morphologies, exposing crystal faces was investigated in both water and organic solvents in the presence of additives. Controlled crystallization of guanine monohydrate, anhydrous guanine α and β form in pure phase were realized for the first time under mild conditions in aqueous solution without the presence of additives by changing the guanine concentrations and pH values. Amorphous guanine was synthesized by a one-step synthesis process in large scale, which transformed to anhydrous guanine β form in pure phase. Characterizations using FTIR and ss-NMR showed that anhydrous guanine β and amorphous guanine had similar short-range order structure, which might be the reason for the controlled transformation from amorphous phase to thermodynamically meta-stable anhydrous guanine β form. The controllable synthesis of twinned crystalline guanine microplatelets and dye-doped guanine microplatelets were also was realized for the first time in the presence of polymer additives.

<figure><img src="image_13.png" /><figcaption>Fig. 1 Controlled synthesis of single crystalline and twinned crystalline guanine nanoplatelets.</figcaption></figure>

**Keywords:** guanine; optical materials; biomimetic crystallization; functional biomimetics; nanocrystals

**References**

[1] D. Gur, Y. Politi, B. Sivan, P. Fratzl, S. Weiner, L. Addadi, *Angew. Chem. Int. Ed.* **2013**, *52*, 388.

[2] F. H. Chen‡, D. M. Guo‡, J. Gao, and Y. R. Ma*, *J. Phys. Chem. L*, **2021**, *12*, 11695.

[3] F. H. Chen, Y. N. Liu, L. Li,* L. M. Qi,* and Y. R. Ma*, *Chem. Eur. J.*, **2020**, *26*, 16228.

[4] D. M. Guo, Y. Zhang, J. Y., Hao, Y. J. Ren, X. B. Hou, J. Gao, **Y. R. Ma***, *Chem. Eur. J.*, **2023**, *10.1002/chem.202300004*.

[5] D. M. Guo, J. Y., Hao, X. B. Hou, Y. J. Ren, Y. Zhang, J. Gao, **Y. R. Ma***, *CrystEngComm*, **2023**, DOI: 0.1039/D3CE00063J.
# Role of Polyamines in the condensation of silicic acid for efficient silicification and its study with pH

Protap Biswas

Weizmann Institute of Science, Israël

Living organisms form mineral salts in highly-controlled biological processes. Biomineralization in marine organisms is a fascinating example of adapting various minerals to accommodate the essential functions of cells. Diatoms can condense soluble silicic acid into an insoluble silica barrier around their cell wall.¹ The silicification occurs within an acidic compartment called silica deposition vesicle (SDV), enriched with macromolecules like polyamines or lysine and arginine-rich.² Although these molecules effectively promote synthetic silicification, bio-silicification results in a more efficient process yielding 90% silica content. The inspired bio-silicification processes show a direction for silicification and studying the silicification in the diatoms. Since a recent study showed a role for phase separation in bioinspired silicification, in our current work, we look at liquid-liquid phase separation of ionic polymers and silicic acid, as a model system for efficient silicification.³ We are currently finding organic amines' role in silicification processes with various factors such as concentration, pH, and ionic strength. We use commercially available cationic polyallylamine (PAH), and study how neutral or basic pH conditions might produce more silica precipitation than acidic conditions. We use dynamic light scattering (DLS) to check the concentration-dependent interactions between PAH and silicic acid in various solutions. Silica precipitation was quantified in each set of experiments to analyze total silica content by FTIR, TGA and SEM microscopy. The result of this study reveals the chemistry behind the initiations of silica condensation in the presence of cationic polymers.

1. S. Weiner, L. Addadi *Annu. Rev. Mater. Res.*, **2011**, *41*, 21-40, 2. N. Kröger, R. Deutzmann, M. Sumper, *Science*, **1999**, *286*, 1129-1132, 3. H. Zhai, T. Bendikov, A. Gal, *Angew. Chem. Int. Ed.* **2022**, *61*, e202115930.
Novel Mms7-mediated biomimetic magnetite nanoparticles

Monica Jimenez-Carretero,¹ Tamara Pozo-Gualda,¹ Marina Lázaro,² Guillermo Iglesias,²
Massimiliano Perduca,³ Concepcion Jimenez-Lopez¹

¹Department of Microbiology and ²Department of Applied Physics, Faculty of Sciences, University of Granada, Spain, ²Department of Biotechnology, University of Verona, Italy

Magnetic nanoparticles (MNP) are magnetite crystals whose large surface-to-volume ratio, ability to be coupled with molecules and response to magnetic fields make them great supports to carry and concentrate a wide variety of compounds. However, commercial MNP present drawbacks that need to be improved, such as the need of post-production coating that provides functional groups to immobilize molecules, or their poor magnetic properties. These problems have been solved by the development of biomimetic magnetic nanoparticles (BMNP), which are produced by mimicking the biomineralization process carried out by magnetotactic bacteria to synthesize their magnetosomes. This biomineralization is mediated by magnetosome-associated proteins (MAPs) that bind iron and control crystal growth.¹ The addition of MAPs to chemical precipitation experiments *in vitro* enhances de nucleation and growth of magnetite crystals, providing BMNP with improved magnetic and surface properties.² MamC from *Magnetococcus marinus* MC-1 has been successfully used in the synthesis of BMNP, but it is necessary to elucidate the effect of other MAPs. In this work, Mms7 (*M. marinus* MC-1) was purified and used to synthesize BMNP. Transmission electron microscopy images show that the size of Mms7-mediated BMNP depends on the concentration of Mms7. X-ray diffraction and Fourier-transform infrared spectroscopy indicate that these magnetite nanoparticles contain Mms7, although it is unknown whether it is attached or embedded. The isoelectric point of Mms7-mediated BMNP is ~4.7, and they are less aggregated than MamC-mediated BMNP at pH values in the range 3-10. As occurs with MamC-mediated BMNP or inorganic MNP, Mms7-mediated BMNP can increase the temperature following upon laser exposure in the NIR or exposure to an alternating magnetic field. However, their response to external magnetic fields is less efficient since they present more organic matter (Mms7-mediated BMNP ~13%, MamC-mediated BMNP ~7%, MNP ~5%).

References

[1] A. Peigneux et al., *Journal of Structural Biology*, **196**: 75-84 (2016).

[2] C. Valverde-Tercedor et al., *Applied Microbiology and Biotechnology*, **99**: 5109-5121 (2015).
# Keeping cartilage covered: Quantifying growth rules in stingray tessellated cartilage

Binru Yang, Jana Ciecierska-Holmes, Jan Wölfer, David Knötel, Peter Fratzl, Daniel Baum, Mason Dean

Max Planck Institute of Colloids and Interfaces

Sharks and rays have cartilaginous skeletons covered by a continuous layer of abutting mineralized tiles (tesserae). Since these skeletons never stop growing, the presence of tesserae creates a challenging growth constraint: how can a continuous tiled covering be dynamically maintained while the volume of the underlying cartilage increases? In principle, maintaining an enlarging tiled surface could be supported either by an increase in the number of tesserae or by their growth, or by both. However, a constant mineral apposition rate at the surface of tesserae does not necessarily result in a continuous growth of the tessellation and may even lead to geometric incompatibilities. To understand how an organism has solved this conundrum, we examined the development of a stingray skeletal element (hyomandibula) in microCT datasets, which allow the first illustration of geometrical changes occurring at the level of an entire tesserae 'population', to quantify structural aspects of tesserae over 100% animal size increase. As animals age, the hyomandibula grows roughly isometrically and, although new tesserae are added, most skeletal growth is accomplished by proportional growth of individual tesserae. Tesserae are typically brick-like in cross-section (3-4x wider than they are thick), but several skeletal regions exhibited distinct trends: multilayered, thin tesserae at the cranial articulation; irregular, patchy tesserae where muscles attach; and columnar tesserae forming stout ridges. Across ontogeny, tesserae ranged from 4- to 8-sided shapes, but were predominantly hexagonal (i.e. with 6 neighbours), especially in flat regions (zero mean curvature), whereas either individual very large or multiple small tesserae were employed to tile curved surfaces. These results provide quantitative insights into how nature can craft complex shapes from tiled architectures and into the dynamic interplays governing growth in tessellated cartilage, where topological requirements (e.g. filling of gaps generated by growth) are balanced by both geometrical and mechanical constraints (e.g. neighbouring tesserae, muscular forces).
## Keynote lecture

### Invited Review: Contribution of Biomineralization Research to the Advancement of Pearl Aquaculture

Masahiko Awaji

*The Graduate School of Agricultural and Life Sciences, the University of Tokyo.*

In this presentation, the relationships between pearl quality and the function of a pearl sac will be discussed, emphasizing the importance of physiological and cell biological research on pearl sac epithelial cells. 1. Quality of a pearl is determined by a pearl sac: A pearl is formed in a follicle called a pearl sac derived from the outer epithelial cells of the mantle. Changes in the pearl sac function recorded in the crystal layers cause variations in pearl quality. The initial formation of a prismatic calcite layer on the nucleus affects the size and cloudiness of a pearl. The shape of aragonite tablets in the nacre affects the color and luster of a pearl. An abnormal pearl sac formation process leads to a deformed pearl. 2. Control of the function of a pearl sac: When tissue debris is included in a pearl sac, organic substances from pearl sac epithelial cells are produced. After this step, the pearl sac cells shift their function to aragonite or calcite formation. To realize the changes in the function of a pearl sac, clarification of the molecules controlling the differentiation of pearl sac epithelial cells is essential. In the great pond snail, *Lymnaea stagnalis*, insulin-related neuropeptides control body and shell growth. Orthologous peptides in bivalves are candidates for the endocrine hormone controlling the growth of a pearl. 3. In vitro culture of pearl sac epithelial cells: Cell culture of the mantle outer epithelial cells has been tried, and transient cell proliferation is observable. But it is difficult to maintain the growth and normal functions of the cells mainly because information on the suitable culture media and substrates is insufficient. 4. Biomineralization research and pearl aquaculture: Physiological and cell biological studies on pearl sac cells will contribute to the advancement of pearl culture technologies. How do changes in environmental factors and nutritional conditions affect pearl formation? Novel research is anticipated.
# Estradiol and bisphenol A effects on early skeletogenesis in the European sea bass *D. labrax*

Martinand-Mari C¹, Potier E², Gasset E², Dutto G², Lallement S², Bourdy C², Debiais-Thibaud M¹, Farcy E²

¹Institut des sciences de l'évolution de Montpellier, ISEM, Univ. Montpellier, CNRS, IRD, Montpellier, France, ²Marine Biodiversity, Exploitation and Conservation, MARBEC, Univ. Montpellier, Ifremer, CNRS, IRD, Montpellier, France

First identified as sex hormones, oestrogens have been shown to play a key role in gonadal development, but also in skeletal development and homeostasis. In mammals, cytosolic estrogen receptors are expressed in osteoblasts, osteoclasts, osteocytes as well as in chondrocytes and chondroblasts, regulating their activity to build and maintain cartilage and bone. To extend our knowledge of estrogen signaling in skeletal development beyond mammals, we used the European sea bass Dicentrarchus labrax. Because natural and synthetic estrogens are pollutants in aquatic ecosystems, they can have important consequences for estrogen-sensitive functions, such as skeletal development, when waterborne exposure occurs during early life stages when blood estrogen concentrations are low. To better understand how (xeno)oestrogens can affect the skeleton, sea bass larvae aged 6-23 days post-hatch were experimentally exposed to two concentrations of a natural estrogen (E2), and a xenoestrogen, the bisphenol A (BPA). The levels of mineralisation of the cranium, vertebrae and fins were investigated by Alizarin Red staining. In addition, the RNA expression levels of several genes playing a key role in skeletogenesis and estrogen signaling pathways were quantified. Our results showed that E2 and BPA have contradictory effects on gene expression and mineralisation phenotype. We also show that their effects depend on the time of exposure, either before or after the initiation of bone mineralisation.

**Keywords:** estrogens, skeleton, mineralization, European sea bass
# An alternative method for extracting the organic matrix from the skeleton of the red coral *Corallium rubrum*

Philippe Ganot (1,2), Guillaume Loentgen (1,2), Frédéric Marin (4), Laurent Plasseraud (5), Denis Allemand (1,3), Sylvie Tambutté (1,2)

1 Research Unit on the Biology of Precious Corals CSM - CHANEL, Monaco, 2 Laboratory of Physiology and Biochemistry, Department of Marine Biology, Centre Scientifique de Monaco, Monaco, 3 Centre Scientifique de Monaco, Monaco, 4 Biogeosciences, UMR CNRS-EPHE 6282, Dijon, 5 Institute of Molecular Chemistry of the University of Burgundy, UMR CNRS 6302, Dijon

As in other animals, skeleton formation in corals is a biologically controlled process in which the organic matrix (OM) corresponds to the organic part of the biomineral produced and secreted by the animal. An essential step in the biochemical characterization of the OM is to isolate it, even though it is enclosed in the biomineral. Its extraction therefore requires several tedious steps usually including (cryo)grinding, demineralization, as well as multiple bleaching and rinsing steps, followed by concentration; a methodology that often results in moderate reproducibility.

Here we present an alternative and simple OM extraction method for the red coral *Corallium rubrum* that requires only a few equipment and steps. The entire calcium carbonate skeleton is directly demineralized to produce a gelatinous material called a ghost, which corresponds to the structured OM. This water-stable ghost is then rinsed and "melted" at 80 °C. To validate our approach, we compared the OM of *C. rubrum* produced by the standard method and our alternative method: The comparative analyses by electrophoretic migration, Western blot and FTIR spectroscopy show that the "alternative OM" is equal or even better in quality than the "standard OM".

Our more direct and faster method is certainly suitable for the red coral, but probably also for other OM-rich biominerals of other organisms.
# Biogenic organic crystals are solid solutions: HPLC-Quantification of purine-bases

Maximilian Bott, Frank Drescher, Susanne Machill, Anne Jantschke

¹Institute for Geosciences, Johannes Gutenberg-Universität Mainz, 55122 Mainz, Germany, ²
Bioanalytical Chemistry, Technische Universität Dresden, 01069 Dresden, Germany

Guanine is an ubiquitous molecule in nature, illustrating its essential roles by e.g., conserving genetic information in the form of DNA or RNA, acting as a high-capacity and rapid-turnover metabolite, or as a signaling molecule. Furthermore, there are numerous examples where the high refractive index of anhydrous guanine-crystals is employed to serve a light-manipulative or dyeing purpose ¹.

Jantschke et al. was the first to examine guanine deposits in the dinoflagellate *Calciodinellum operosum* *aff.* using Raman imaging and 2D/3D-cryo-(FIB)-SEM ². Later, Mojzeš et al. identified crystalline guanine in phylogenetically distinct groups of microalgae ³ that sometimes contained a significant amount of other purines ⁴. Powder-XRD and solid-state NMR analyses revealed that biogenic guanine crystals are rather solid solutions that bear the flexibility to incorporate up to 20 % of other purines, especially hypoxanthine, without altering the crystal structure significantly ⁵. Yet, a method for the precise quantification of the purine composition of those crystals remained to be found.

Herein, we present an HPLC method for the determination of various purine concentrations within cells, including uric acid, xanthine, hypoxanthine, and guanine, allowing the precise quantification of all analytes in the µM-range. Samples were prepared by acidic hydrolysis using concentrated perchloric acid. The method was optimized regarding column material, temperature, flow rate, and mobile phase. Here we found that employing an Agilent Zorbax SB-Aq column with a linear gradient using ammonium formate buffer (10 mM, pH=5) and methanol, at 30 °C, yielded clearly separated analyte peaks.

This method underlines its elegance by opening an easy and universal pathway to analyze the purine-composition of various samples and understand the purine variability in biogenic guanine crystals.

1. Gur, D., Palmer, B. A., Weiner, S. & Addadi, L. Light Manipulation by Guanine Crystals in Organisms: Biogenic Scatterers, Mirrors, Multilayer Reflectors and Photonic Crystals. *Advanced Functional Materials* **27**, 1–13 (2017).

2. Jantschke, A. et al. Anhydrous β-guanine crystals in a marine dinoflagellate: Structure and suggested function. *J Struct Biol* **207**, 12–20 (2019).

3. Mojzeš, P. et al. Guanine, a high-capacity and rapid-turnover nitrogen reserve in microalgal cells. *PNAS* **117**, 32722–32730 (2020).

4. Pilátová, J., Pánek, T., Oborník, M., Čepička, I. & Mojzeš, P. Revisiting biocrystallization: purine crystalline inclusions are widespread in eukaryotes. *ISME Journal* **16**, 2290–2294 (2022).

5. Pinsk, N. et al. Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites. *J Am Chem Soc* **144**, 5180–5189 (2022).

**Keywords:** Microalgae, HPLC, Purines, Solid Solutions
Carbonic anhydrase activity identified in the powdered nacreous layer of
Pinctada fucata.

Yuto Namikawa and Michio Suzuki

*Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Japan*

The Japanese pearl oyster, Pinctada fucata, is used for pearl aquaculture in Japan. The shells of P. fucata, composed of CaCO3 and several organic molecules, have the outside prismatic layer and the inside nacreous layer. The organic molecules containing chitin and proteins play significant roles in shell mineralization. Nacrein is the most abundant acid-soluble protein in the shell of P. fucata. Nacrein has a carbonic anhydrase (CA) domain that catalyzes the reversible conversion of CO2 to HCO3-. We discovered that the nacre powder (NP) directly obtained from crushed P. fucata shells displayed CA activity, with a Km of 3.44 mM and kcat/Km of 920 M-1 s-1. The addition of the NP in solutions increased the rate of CO2 uptake. The NP was also thermally stable at temperatures over 70°C and reusable after ten cycles of catalysis. The fluorescence detection study revealed that nacrein was exposed on the surface of the NP, indicating that nacrein localized on the NP's surface and contributed to the NP's CA activity. We conducted further examination of CA activity in other industrial waste shells. The powdered prismatic layer of P. fucata and the shell of Mizuhopecten yessoensis exhibited CA activity, whereas the powdered shell of Crassostrea gigas did not. CA has gained attention as a potential material for CO2 sequestration to combat global warming. As the shell of mollusks is a waste from the aquaculture, the shell powder can be produced inexpensively. Therefore, we propose usage of the shell powder as an effective, thermally stable, reusable, and low-cost biocatalyst for CO2 sequestration.

References

Namikawa, Y., Moriyasu, K., Yasumoto, K., Katsumata, S., & Suzuki, M. (2023). Carbonic anhydrase activity identified in the powdered nacreous layer of Pinctada fucata. Process Biochemistry, 128, 22-29.

**Keywords:** carbonic anhydrase, nacre powder, CO2 sequestration
Poster Presentations

Poster 25

# The very first days of cultured pearls from French Polynesia

Yannicke Dauphin<fnref n="1" />, Kadda Medjoubi², Andrea Somgyi², Jean-pierre Cuif<fnref n="3" />

The quality of natural and cultured pearls depends on the size, shape and properties of the surface (colour, lustre) of the nacreous layer. In 1893, a method allowing for production of cultured pearls was elaborated in Japan: the grafting method which involves two pearl oysters of the same species. One is used to produce the grafts, pieces of living tissues cut from the nacre producing area of the mantle. Then, each graft is deposited onto a spherical body (nucleus) in another animal. After wrapping of the nucleus by the expanding graft, the mineralization restarts. As the graft was producing nacre before being cut, it is expected that the nucleus will be covered by nacre, allowing for production of the much prized round shaped pearls.

Nevertheless, structural irregularity and heterogeneous mineralogy of pearls have long been noticed: non-nacreous material such as “aragonitic” prisms and calcite are common. Such units are not known in the shell of Pinctada. In situ characterization methods at a micro- and nanoscale on Tahitian pearls produced by P. margaritifera allow for a direct examination of samples grown during the very earliest days of the mineralization process of pearls. Correlative data on mineralogy, crystallinity, structure and elemental composition are obtained.

First day deposits show a great variability of the inner structures and composition. There are no two identical samples. Moreover, the arrangement is irregular within a single pearl, and some structures (aragonitic pseudo-prisms) do not exist in the shell. Thus, the assumption that the cultured pearl is a nucleus covered by a thin organic layer, a thin prismatic layer and a thick nacreous layer similar to what is known in the shell of the oyster is, in most cases, erroneous.

**Keywords:** nascent pearl, structure, composition, in situ analyses

<footnote n="1">¹ISYEB, museum national d'histoire naturelle, Paris, ²Nanoscopium beamline, synchrotron soleil,</footnote>

<footnote n="3">³CR2P, museum national d'histoire naturelle, Paris</footnote>
Poster 26

*In vivo* injection method to study control of calcification in Octocorals.

Clémence Forin¹,⁴, Guillaume Loentgen¹, Denis Allemand², Sylvie Tambutté³, Philippe Ganot¹

¹Research Unit on the Biology of Precious Corals CSM - CHANEL, Monaco, ²Centre Scientifique de Monaco, ³Department of Marine Biology, Monaco, ⁴Sorbonne Université – ED 515 Complexité du Vivant, 75005 Paris

Corresponding author e mail: cforin@centrescientifique.mc

Corals have evolved with the ability to build biominerals structures resulting in the formation of sclerites and/or axial skeletons made of calcium carbonate and organic molecules. The term ‘coral’ has been extensively used to describe tropical corals, or ‘reef-building corals’ defined in the class Hexacorallia, yet another class diverged 550-650 million years ago in the pre-Cambrian resulting in the Octocorallia class (soft corals, sea pens and sea fans). Our study focusses on corals from the Octocorallia class, excellent comparative models. Despite information at the cellular and physiological level, knowledge of the mechanisms controlling coral calcification remain elusive due to the lack of appropriate techniques.

Long term exposures to exogeneous molecules are very challenging in aquaria facilities because of the seawater renewal, costs of compounds, their quantity, solubility, and diffusion in seawater. Here, we provide a viable alternative method through in-vivo injection of substances of interest dissolved in a liquid vehicle which solidifies upon injection. Our experimental study evaluates slow-release and localised injection as a novel method for delivering an internal emulsion with molecules of interest. Local diffusion of the injected products in the organism was followed using visual tracers. Specifically, we followed two classes of fluorescent markers, one of which investigated the internalisation into cells, while the others were used as an application to monitor the calcification process.

This methodology was optimised on *Sarcophyton* sp. (order Malacalcyonacea) for validation of the injection procedures and characterise the diffusion of the markers, while proof of the transferability of the methodology was performed on the precious coral *Corallium rubrum* (order Scleralcyonacea).

**Keywords:** *Corallium Rubrum*, *Sarcophyton*, injection of exogeneous molecules
**Keynote lecture**

**Invited Review : Impacts of climate change on mollusc biomineralisation pathways**

Susan Fitzer

*University of Stirling, UK*

Marine molluscs are vulnerable to the impacts of ocean acidification driven by oceanic CO2 absorption, and coastal acidification driven by land run off. These drivers of environmental change have deleterious effects on shell growth. Ocean acidification and global warming, limit the ability of molluscs to produce their calcium carbonate protective shells. The mechanisms of coastal and ocean acidification are very different, however similarities occur including increases in dissolved inorganic carbon and reduced availability of carbonate. Limited carbonate under acidification has been shown to inhibit shell growth and the material properties of shells, suggesting an alteration in carbon sequestration. Molluscs control their shell growth through biomineralization, but the mechanisms behind biomineralization and their response to acidification are relatively unknown. It is unclear how much carbon is taken into the shell from the environment compared to metabolic sourcing. Shell production is energetically costly to molluscs and metabolic processes and energetic partitioning may affect their ability to perform biomineralization under environmental change. The mechanisms of shell growth through carbon uptake have been examined under both experimental and naturally occurring coastal acidification. Carbon isotopes (δ13C) have been used to examine the change in biomineralization pathway in molluscs under environmental change. The carbon uptake by δ13C tracing and deposition into mantle tissue and shell layers has been examined in several different species of mollusc with varying calcium carbonate polymorphs. These biomineralization pathways are compared in *Magallana gigas*, *Mytilus sp.* and *Saccostrea glomerata* grown under acidification and warming to assess the vulnerability of molluscs to environmental change. Are molluscan biomineralization pathways under environmental change species-specific and what can we learn from experimental versus coastal acidification?

**Keywords:** Molluscs Biomineralization ocean acidification warming
**Secondary aragonite recrystallization during otoliths diagenesis: natural and experimental evidence**

Ismael Coronado1*, Juncal A. Cruz1,, Alicja Owczarek2, Miguel Sáenz-Navajas1, J. Ricardo Mateos-Carralafuente3, Pedro Cózar4, Esperanza Fernández-Martínez1, Lurdes Fernández-Díaz3, Jarosław Stolarski2

1 Faculty of Biological and Environmental Sciences, Universidad de León, 24071 León, Spain, 2 Institute of Paleobiology, Polish Academy of Sciences, PL-00-818 Warsaw, Poland. 3 Faculty of Geology, Universidad Complutense de Madrid, 28040 Madrid, Spain. 4 Instituto de Geociencias (CSIC-UCM), 28040 Madrid, Spain.

*email: icorv@unileon.es

Biogeochemical signatures recorded in calcareous biominerals are used as environmental and physiological proxies. Reliability of these signatures can be compromised during the fossilization, due to diagenetic processes that affect especially aragonite biominerals, which are typically transformed into the more stable calcite during burial. Thus, aragonite is rarely preserved in fossils, and its exceptional occurrence is considered as evidence of pristine preservation. In this study, we show that fossil otoliths from several Cretaceous and Cenozoic deposits from Poland, despite of aragonite preservation, show clear evidence of some crystallographic and structural modifications such as: coarsening of aragonite crystals, generation of porosity, and lattice parameters modifications. This suggests that, although the mineralogy of fossils remains the same, the aragonite is altered. To test this hypothesis, we performed diagenetic experiments in aragonite otoliths (saggitas) of Micromesistius poutassou, in simulated burial water for 7 and 14 days (100°C and 175°C). We compared pristine and altered otolith structures, mineralogy, and biopolymer content. During the experiment, no substantial organics loss occurs, but biopolymers become more complex. Alteration of organics is suggested to slow down the aragonite transformation kinetics, making otoliths more resistant to hydrothermal alteration (containing < 2.5 wt.% calcite after 14 days at 175°C). However, secondary aragonite forms as parches via a slow dissolution - precipitation reaction facilitated by fluid migration toward the interior. The secondary aragonite keeps the original crystallographic orientation, but undergoes changes in lattice parameters, organics content, solubility, and porosity. These results suggest that aragonite "pristine" preservation of fossils requires careful reconsideration.

This work was supported by 2020/39/B/ST10/01253 and PID2021-125467NB-I00 research grants.
# Primary coral polyps responses to decreasing seawater pH: observations from cell to the complete organism

Tali Mass¹, Federica Scucchia¹, Paul Zaslansky²,

¹University of Haifa, ²Charité - Universitätsmedizin Berlin

In light of increasing stress and mass mortality of reef-building corals due to climate change, the resilience of some marine ecosystems relies on successful coral larva recruitment. Knowledge of the acclimatory and/or adaptive potential as a response to environmental challenges such as ocean acidification (OA) in the earliest life stages is limited. In this study, we report on *Stylophora pistillata* larvae and primary polyps cultured under acidic pH conditions. We investigated the response of the endosymbiotic algae and the coral host across biological levels, from cellular to organismal. By combining transcriptomic analysis with physiological and morphological measurements, we identified that while the number of survival and settlement of coral larvae were reduced under OA conditions, the surviving recruits adjusted well to the challenging conditions. Coupling synchrotron X-ray µCT with artificial intelligence, we found that OA has a morphologically-altering effect on coral skeletal features. Although skeleton growth is reduced, coral recruits possess acclimatory mechanisms allowing them to survive under OA conditions. These include the transition to a less-mineralized/increased-tissue phenotype exhibiting greater incorporation of organic matrix proteins within the skeletal fibers. Moreover, we found that the algal photosynthetic activity is stimulated under OA, as well as the transfer of photosynthates to the coral host, potentially sustaining the host energetic expenses.

**Keywords:** Coral, Ocean acidification, X-ray µCT
Investigating the impact of ocean acidification on CaCO₃ crystal growth rates in the reef coral *Stylophora pistillata*

Alexander Venn1, Margaux Gilbert 1, Eric Tambutté 1, Sylvie Tambutté 1.

1 Marine Biology Department, Centre Scientifique de Monaco (CSM), 8 Quai Antoine 1er, 98000, Monaco

Corresponding author: avenn@centrescientifique.mc

Keywords: extracellular calcifying medium, coral biomineralization, growing edge, light enhanced calcification, fluorescent markers

The aragonite skeleton of scleractinian corals forms in the extracellular calcifying medium (ECM) which is separated from seawater by the calicoblastic epithelium and the overlying tissue layers. The calicoblastic cells exert biological control on the composition of the ECM via regulation of transcellular and paracellular ion transport. Despite biological control of the ECM and its physical separation from the surrounding seawater, coral calcification is sensitive to environmental changes, such as ocean acidification. The mechanisms explaining the environmental sensitivity of coral calcification are poorly understood. Our research addresses this knowledge gap by investigating the links between cellular physiology and calcification of corals in vivo. To achieve this, we focus our investigations on the ‘growing edge’ of Stylophora pistillata, an area of the coral colony where the calicoblastic cells and the ECM can be analysed directly, and lateral growth can be observed through the formation of isolated CaCO3 crystals at the edge of the skeleton. Our previous analysis of the growing edge has provided in vivo insight into how ocean acidification disrupts pH regulation of the ECM and calicoblastic cells, and causes changes in paracellular exchange between seawater and the ECM. To understand how these physiological changes may affect calcification, we are currently characterizing the formation of the isolated crystals at the growing edge under different conditions of experimental ocean acidification. Our analysis involves using confocal microscopy and fluorescent marker dyes that allow us to quantify the growth rates of a range of crystal morphologies and sizes. Because coral calcification is sensitive to light exposure, via a poorly understood process termed ‘light enhanced calcification’, we are investigating coral crystal growth rate at the growing edge in both light and darkness in conditions of acidification.
Organization and magnetic properties of magnetic ectosymbiotic bacteria
optimize a collective magnetotaxis behavior in a microbial holobiont

Daniel M. Chevrier*, Amélie Juhin, Nicolas Menguy, Romain Bolzoni, Paul E. D. Soto-Rodriguez, Mila Kojadinovic-Sirinelli, Greig A. Paterson, Rachid Belkhou, Wyn Williams, Fériel Skouri-Panet, Artemis Kosta, Hugo Le Guenno, Eva Pereiro, Damien Faivre, Karim Benzerara, Caroline L. Monteil, and Christopher T. Lefevre*

¹Aix-Marseille Université, Centre national de la recherche scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), UMR7265, Bioscience and biotechnology institute of Aix-Marseille (BIAM), Saint-Paul-lez-Durance 13108, France, ²Sorbonne Université, UMR CNRS 7590, Muséum national d'Histoire naturelle (MNHN), Institut de recherche pour le développement (IRD), Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), 75005 Paris, France, ³Department of Earth, Ocean and Ecological Sciences, University of Liverpool, L69 7ZE Liverpool, UK, ⁴Synchrotron Soleil, L'Orme des Merisiers, 91192 Gif-sur-Yvette Cedex, France, ⁵School of GeoSciences, Grant Institute, University of Edinburgh, Edinburgh EH9 3JW, UK, ⁶Plateforme de Microscopie de l'Institut de Microbiologie de la Méditerranée, Institut de Microbiologie, FR3479, Campus CNRS, 13402 Marseille cedex 20, France, ⁷ALBA Synchrotron Light Source, Cerdanyola del Vallés, Barcelona 08290, Spain

Magnetotactic bacteria (MTB) are a diverse type of motile, single-cell prokaryote well known for their capacity to biomineralize chains or clusters of magnetosome organelles that contain magnetic nanocrystals of magnetite or greigite [1]. They are ubiquitously found in micro-/anaerobic conditions and can easily locate such habitats thanks to the collective magnetic moment of their magnetosomes, which is referred to as magnetotaxis. Though they have been known to create multicellular consortiums [2], it was only recently discovered that non-motile MTB were symbionts of a flagellated microeukaryotic host, together known as a holobiont [3]. This magnetotactic holobiont (MHB) performs a magnetic field-assisted motility guided by a chemaerotaxis system similar to individual free-living MTB. This complex behavior raises many questions regarding how magnetic properties of symbionts determine holobiont magnetism and motility. This work employed a suite of light-, electron- and X-ray-based microscopy techniques (including X-ray magnetic circular dichroism (XMCD)) to reveal that symbionts optimize the motility, the ultrastructure, and the magnetic properties of MHBs from the microscale to the nanoscale [4]. The surface organization of magnetic symbionts is explicitly presented with electron and X-ray microscopy, depicting bacterial membrane structures that ensure longitudinal alignment of cells. Magnetic dipole and nanocrystalline orientations of magnetosomes were shown to be consistently oriented in the longitudinal direction, maximizing the magnetic moment of each symbiont. The magnetic moment transferred to the host cell is in excess (100-1000X stronger than free-living MTB), well above the threshold for the host cell to gain a magnetotactic advantage. With an excessive magnetic moment given to the host cell, the benefit provided by magnetosome biomineralization beyond magnetotaxis can be questioned.

[1] Faivre, D. & Schüler, D. Magnetotactic bacteria and magnetosomes. Chem. Rev. 108, 4875-4898 (2008)

[2] Farina M., Debarros H. L., Esquivel D. M. S., Danon J. Ultrastructure of a magnetotactic microorganism. Biol. Cell 48 85-88 (1983)

[3] Monteil, C. L. et al. A symbiotic origin of magnetoreception in unicellular eukaryotes. Nat. Microbiol. 4, 1088-1095 (2019)

[4] Chevrier, D. M. et al. Collective magnetotaxis of microbial holobionts is optimized by the three-dimensional organization and magnetic properties of ectosymbionts. Proc. Natl. Acad. Sci. U. S. A. 120, e2216975120 (2023)

**Keywords:** Magnetosome, magnetite, X-ray microscopy, magnetotaxis
Cellular pH regulatory systems underlying calcification in the sea urchin
larva

Marian Hu

Institute of Physiology, Christian-Albrechts-University, Hermann-Rodewaldstrasse 5, 24114 Kiel,
Germany

e-Mail: m.hu@physiologie.uni-kiel.de

To generate their skeletons, sea urchin larvae like many other marine calcifyers acquire carbonate ions that largely derive from the hydration of metabolic CO2. In this process protons are liberated that need to be removed from the calcification front to promote calcification and to defend cellular acid-base balance. Thus, biological calcification and pH regulation are intrinsically linked processes that remain relatively unexplored.

Using a wide range of techniques ranging from life cell imaging and intracellular pH recordings over
protein-biochemical methods to molecular technologies our work identified a set of PMC specific ion
transporters and channels that mediate a carbon concentration mechanism (CCM) and removal of
protons liberated by the calcification process. During skeleton regeneration pH₁ and intracellular HCO₃⁻
levels are increased accompanied by a change in proton transport pathways. During active
mineralization, resting PMCs are recruited into the calcifying state seen in their physiology and
increased expression of of key acid-base transporters including HCO₃⁻-transporters (Sp-Slc4a10),
Carbonic anhydrases (Cara7) and the proton channel Otop2l.

Using the sea urchin larva as a model organism we are able to resolve fundamental mechanisms of
acid-base transport in calcifying systems. Energy efficient mechanisms of intracellular pH regulation
to generate CaCO₃ from a metabolic waste product – carbon dioxide- demonstrates the elegance of
nature in utilizing resources in the most sustainable manner. In this way, insights into the cellular
calcification mechanisms may help to develop novel approaches for biology-inspired technologies to
sequestrate carbon in times of rapid climate change.

**Keywords:** Cell physiology, membrane transport, pH regulation, carbon concentration, environmental change
Poster Presentation

Poster 27

Bone as a bioadsorbent for bacteria removal

T. Pozo-Gualda¹, A. B. Rodríguez-Navarro², C. Jiménez-López¹

¹Department of Microbiology, Faculty of Sciences, ²Department of Mineralogy and Petrology, University of Granada, Granada 18071, Spain.

e-mail of the presenter: tamapg25@ugr.es

Contamination of domestic water by potentially dangerous bacteria and other microbes is a common problem, which may cause gastrointestinal and short-term diseases. The development of procedures that use environmentally friendly materials (preferably residues) with high bacterial retention capabilities is an interesting approach. In this context, chicken bone is a residue from the food industry, mainly composed of an inorganic phase (nanocrystalline carbonated apatite) mineralizing an organic matrix (mainly type I collagen). Thermal treatments can change the characteristics of the mineral and the organic matrix, resulting in a material with increased microporosity and enhanced surface reactivity. Thus, thermally treated bone can be used as an efficient adsorbent for different compounds and/or microorganisms. With increasing temperature bone properties change due to dehydration, loss of organic components and recrystallization of the mineral. To obtain deproteinate bone mineral, bones are treated at 600°C, since combustion of most organic components occurs up to 600°C. This calcination process eliminates all pathogens and creates additional microporosity and a large surface area. Our results show that calcined bones placed in a bacterial culture, have a higher adsorption capacity of microorganisms than untreated bone. Also, we have studied the bacterial adsorption ability of different types of bone (cortical and medullary), subjected to different calcination temperatures (400°C, 600°C and 800°C) that differ in their porosity and reactivity, as a proof of concept of green and sustainable technology involving bone waste recycling for bacterial adsorption from aqueous samples.

References

[1] Peigneux, Ana; Puentes-Pardo, Jose D; Rodríguez-Navarro, Alejandro B; Hincke, Maxwell T; Jimenez-Lopez, Concepción. Development and characterization of magnetic eggshell membranes for lead removal from wastewater, *Ecotoxicology and Environmental Safety*. 2020

[2] Dominguez-Gasca, Nazaret. Benavides-Reyes, Cristina. Sánchez-Rodríguez, Estefanía. Rodríguez-Navarro, Alejandro B. Changes in avian cortical and medullary bone mineral composition and organization during acid-induced demineralization, *Eur. J. Mineral*. 2019

**Keywords:** Chicken bone, thermal treatments, microporosity, bacterial adsorption
Keynote lecture

Invited review : Few thoughts on the evolution of calcifying matrix proteins
in metazoans

Frédéric Marin

UMR CNRS-EPHE 6282 Biogeosciences, Université de Bourgogne, Dijon

frederic.marin@u-bourgogne.fr

In metazoans, the deposition of calcium carbonate biominerals is regulated by an organic matrix, secreted at the interface between the mineralizing cells and the forming calcified tissue (skeleton, or non-skeletal tissue). This matrix – or a part of it - remains occluded in the calcified tissue once formed.

Recent high-throughput screening techniques (i.e., combination of transcriptomics and proteomics) have
revealed that this matrix is a complex system composed of dozens to hundreds of proteins that belong
to several families of elusive molecular functions. Similarly to proteins of the extracellular matrix
(ECM) of vertebrates, many of these proteins are modular, i.e., their primary structure is organized in a
succession of different functional domains. While some domains are identified by sequence similarity
(like enzymatic domains), the function of others is unknown, or ‘guessed’ but not experimentally tested.
Of particular importance are Low Complexity Domains (LCDs) that can account for a large proportion
of all domains in a matrix, but also domains putatively involved in protective functions sensu lato (like
immune or protease inhibitor functions). Transmembrane domains and domains of cytoplasmic / nuclear
origin are also of notice. Today, the matrix is considered as a molecular toolbox for calcifying, with a
high evolutionary plasticity. Through few examples, this talk will sketch how functional domains were
recruited for calcification, and how some calcifying matrix proteins are lineage-specific while others are
ancient and may have been recruited multiple times. This talk will also address the question of the
emergence of complex and stable-through-times forms under the control of ‘molecular tools’ (proteins)
that evolve fast and, consequently, are unstable-through-time. It will also reemphasize the central role
of the calcifying cells/tissues and the likely dialog between these living entities and the growing mineral.

Keywords: Proteomics - Metazoan - Skeleton - Evolution - Matrix
# The vertebrate skeleton through the eye of a cartilaginous fish:
Evolutionary perspectives raised by the study of the small spotted catshark
*Scyliorhinus canicular*

L. Nicolas¹, C. Martinand-Mari¹, M Debiais-Thibaud¹,

¹Institut des Sciences de l'Evolution de Montpellier, ISEM, Univ Montpellier, CNRS, IRD, EPHE,
Montpellier, France

Bone together with other mineralized tissues evolved early in the vertebrate lineage (more than 415
Myr ago), even before the evolution of jaws. Endochondral bone though has evolved as a general rule
in the bony fish lineage only, to which most model organisms for developmental biology belong
(tetrapods or teleost fishes). In the cartilaginous fish lineage however, bone is considered secondarily
lost despite conspicuous mineralisation of the skeleton in the form of tessellated cartilage. Recently, an
integrative set of data has informed the developmental dynamics, the cellular characteristics and the
genetic actors of skeletal mineralisation in sharks and rays, in particular through the study of a non-
model organism, the small spotted catshark *Scyliorhinus canicula*. These data have highlighted several
aspects of cartilaginous fish-specific evolutionary events, including gene duplication, diverged
developmental processes and probably cell type identity. Despite prolonged independent evolution in
the bony fish and cartilaginous fish lineages, the comparative analysis of these data also helps identify
an ancestral genetic toolkit, still to be discussed regarding other mineralised tissues in extant jawed
vertebrates.

**Keywords:** EvoDevo; vertebrate skeleton; shark; genetics