In-vitro synthesis of amorphous Mg-, Ca-, Sr- and Ba-carbonates: what do we learn about intracellular calcification by cyanobacteria? Nithavong Cam 1, 2 Jean-François Lambert 1, Karim Benzerara 2, Thomas Georgelin 1, Maguy Jaber 1 Laboratoire de Réactivité des Surfaces, UMR 7197, CNRS & UPMC 2 Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, UMR 7590, CNRS & UMPC, Muséum National d’Histoire Naturelle, IRD UMR 206

Cyanobacteria and mineralization  Photosynthetic bacteria Riding R. (2006) Geobiology (4); 299: CO H 2 O → C 6 H 12 O O 2 used to be considered as exclusively extracellular  important mineralization activity

Some cyanobacteria form intracellular carbonates K. Benzerara et al. (2014) PNAS (111); 10933:10938 Cyanothece sp. PCC 7425 Synechococcus sp. PCC µm 3

Particularities of intracellular carbonates Inclusions Ø: nm Amorphous E. Couradeau et al. (2012) Science (336); 6080, 459:462 Enriched in Sr and Ba = 86 = 1370 Mg 0.18 Ca 0.53 Sr 0.08 Ba 0.21 CO 3 Sr/Ca solid Sr/Ca solution Ba/Ca solid Ba/Ca solution 4

Some properties of calcium carbonates Partitioning 0.021(X=Sr) 0.012(X=Ba) Amorphous (ACC)  How does the cell stabilize the amorphous phase? Calcite Aragonite Dietzel M. et al.(2004) Tesoriero A. J. and Pankow J. F. (1996) 1.19(X=Sr) 1.5(X=Ba) X/Ca solid X/Ca solution ?  Where does come from the partitioning? 5 25 °C 3.31 x10 -9 M x10 -9 M 2 M. Kellermeier et al. (2014) 2.32 x10 -8 M 2 Solubility Plummer L. N. and Busenberg E. (1982) <<

CO 3 2- Mg 2+ Sr 2+ Ca 2+ Ba 2+ + CO 3 2- Abiotic carbonates [Mg 2+ ]=10 mM [Ca 2+ ]=5.3 mM [Sr 2+ ]=0.8 mM [Ba 2+ ]=2.1 mM Same Sr/Ca and Ba/Ca ratios as observed in cyanobacteria carbonates Initial pH = 10.6 SI ACC = SI = log (IAP/Ks) [CO 3 2- ]=10 mM 6 Procedure for carbonate precipitation

Similarities: - size - poor crystallinity - composition Elemental ratios in precipitates are same as in the solution 7 Mg 0.16 Ca 0.51 Sr 0.05 Ba 0.28 CO 3 Ø ≈ 200 nm Poorly crystalline Cam N. et al. (2015) Geochimica et Cosmochimica Acta (161); 36:49. Abiotic carbonates like bacterial ones

Association of Mg with Sr or Ba helps amorphous phase stability without Mg without Srwithout Ba without Sr and Ba Calcite Poorly crystalline Monohydrocalcite SEM TEM 8 Poorly crystalline Substituants effect in carbonates precipitation Cam N. et al. (2015) Geochimica et Cosmochimica Acta (161); 36:49.

H2OH2OMgCO 3 -> CO 2 + MgOCaCO 3 -> CO 2 + CaO ~ 1 H 2 O per 1 CO 3 unit As observed in biotic and abiotic ACC L.Brečević and A.E. Nielsen (1989) For further in abiotic ACC characterization 9 Mg 0.16 Ca 0.51 Sr 0.05 Ba 0.28 CO 3 ·1.03H 2 O Thermogravimetric analysis Cam N. et al. (2015) Geochimica et Cosmochimica Acta (161); 36:49. CO 2 DTG Heat flow (derivative of the weight loss)

CO 2 DTG Heat flow (derivative of the weight loss) 325°C 375°C  Exothermic event = Crystallization Calcite 10 XRD Thermogravimetric analysis For further in abiotic ACC characterization Cam N. et al. (2015) Geochimica et Cosmochimica Acta (161); 36:49.

 Reproduce similar carbonates in an abiotic way is possible Conclusions  no partitioning between Ca, Sr and Ba ≈ 1 Sr/Ca solid Sr/Ca solution Ba/Ca solid Ba/Ca solution ≈ 1 Amorphous calcium carbonate 11 enrichment in Sr and Ba inside the cells  carbonates formation solution has same elemental ratios as the minerals – size – poor crystallinity – composition

– pH = – [Ca] = 5.3 mM – [inorganic carbon] = 10 mM About precipitation solution composition  Known chemistry of cyanobacteria intracellular medium is very different – pH < 7.9 – [Ca]< 2.6µM – [inorganic carbon] < 30 mM Cyanobacteria intracellular medium A. L. Barrán-Berdón et al. (2011) M. R. Badger and T. J. Andrews (1982) H.-B.Jiang et al. (2013) SI ACC = no precipitation SI ACC =  what we know about cyanobacteria intracellular composition can not explain the precipitation of calcium carbonate Abiotic synthesis medium 12

Thank you for attention This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX , and more specifically within the framework of the Cluster of Excellence MATISSE 13 G. Morin, F. Skouri-Panet and M. Poinsot (IMPMC) Acknowledgment: K.Benzerara was supported by ERC grant (Calcyan)