In Situ Observation of the Internal Structure and Composition of Biomineralized Emiliania huxleyi Calcite by Solid-State NMR Spectroscopy
Ronen Gertman, Ira Ben Shir, Shifi Kababya, and Asher Schmidt*
Biomineralization, particularly the formation of calcium carbonate structures by organisms under ambient conditions, is of vast fundamental and applied interest. Organisms finely control all aspects of the formation of the biomaterials: composition, polymorph, morphology, and macroscopic properties. While in situ molecular-level characterization of the resulting biominerals is a formidable task, solid-state magic angle spinning NMR is one of the most powerful analytical techniques for this purpose. It is employed in this study to elucidate the structure and composition of biogenic calcite formed by Emiliania huxleyi, a unicellular alga distinguished by its exquisitely sculptured calcite cell coverings known as coccoliths. Strain 371 (CCMP) was grown and harvested from 15N- and 13C-enriched growth medium, with biosynthetic labeling to enhance the sensitivity of the NMR measurements. Crystalline and interfacial calcite environments were selectively probed using direct and indirect (cross-polarized) 13C excitation, respectively. Different crystalline environments, in particular structural defect sites at concentrations of up to 1.4% with P and N moieties incorporated, were identified using 13C rotational-echo double-resonance (REDOR) NMR. REDOR-derived geometrical constraints show that the P and N atoms at the defect sites are 3.2 and 2.3 (0.2) Å apart from a crystalline carbon carbonate. The phosphorus and nitrogen moieties within the biogenic calcite are identified as small, non-protonated moieties, attributed to inorganic ions such as PO43− and NO3−. The carbonates adjacent to these defects are chemically indistinguishable from bulk crystalline carbonates, yet their immediate environments experience reduced rigidity, as reflected by substantial T1(13CO32−) shortening. Interfacial carbonates, on the other hand, reside in structurally/chemically perturbed environments, as reflected by heterogeneous line broadening. This study is the first to directly unravel evidence on the incorporation of P/N moieties as structural defects within E. huxleyi biogenic calcite, and on the state of the adjacent crystalline carbonates.