Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals

Marine calcium carbonate (CaCO₃) biomineralization is a crucial process in the global carbon cycle, removing atmospheric CO₂ and forming solid minerals that constitute a significant portion of sedimentary rocks. Marine biominerals, such as mollusk shells and coral skeletons, exhibit superior properties compared to their individual components, inspiring the design of novel materials. While previous research has established the role of amorphous calcium carbonate (ACC) precursors in biomineral formation, the mechanisms involved remain debated, particularly concerning their resilience to climate change. This study aims to investigate the complexity of biomineralization pathways by analyzing the mineral phases present during the formation of various biominerals using advanced nanoscale techniques.

Extensive research has been conducted on the formation of marine calcium carbonate biominerals. Studies have shown that several biominerals form via amorphous calcium carbonate (ACC) precursor phases. However, the precise mechanisms of biomineral formation, especially the role of different precursor phases and their transformation pathways, remain a subject of ongoing debate. Previous work has identified ACC as a key intermediary, but lacked sufficient resolution to reveal the presence of potential other crystalline precursors. The present study addresses this gap using a combination of high-resolution spectroscopy and advanced image processing techniques.

The researchers employed a multifaceted approach combining soft X-ray spectromicroscopy and synchrotron infrared nanospectroscopy (SINS). The soft X-ray spectromicroscopy, performed at the Advanced Light Source (ALS), allowed for the identification of mineral phases at the nanoscale using the calcium L-edge spectra. A novel method, Myriad Mapping (MM), was developed to quantitatively display the spatial distribution of multiple mineral phases (up to five) simultaneously within a single image. SINS-FTIR, performed at the ALS as well, provided corroborating evidence using a surface-sensitive infrared technique. This analysis included synthetic reference minerals and freshly deposited samples of coral skeletons, nacre, and sea urchin spines, prepared using a rigorous protocol to avoid artifacts. The method involves meticulous sample preparation including careful fixation and dehydration to protect the metastable precursor phases. Both X-ray absorption spectroscopy and SINS-FTIR methods were utilized, providing complementary data. Statistical analysis, including reduced χ² values and comparisons between different component fits, was used to assess the accuracy of the phase identification and mapping. The analysis was carried out on a large sample set of 86 areas which contains more than 200 million spectra in total.

Using Myriad Mapping (MM), the researchers identified two previously unknown mineral phases in forming biominerals: calcium carbonate hemihydrate (CCHH) and monohydrocalcite (MHC). These phases, along with amorphous calcium carbonate hydrated (ACCH₂O) and anhydrous (ACC), were found to be present on the surfaces of freshly deposited coral skeletons and nacre, but not on sea urchin spines. CCHH was the most abundant precursor phase in both nacre and coral, comprising more than 50% of the precursor pixels in a significant proportion of the analyzed areas. ACC was the second most abundant, followed by MHC, which always co-occurred with CCHH. ACCH₂O was the least abundant precursor phase detected. The spatial distribution of these phases suggests a possible sequence of phase transitions, such as ACCH₂O → CCHH → aragonite. Synchrotron infrared nanospectroscopy (SINS) provided independent confirmation of the presence of CCHH, MHC, ACCH₂O, and ACC at the surface of coral skeletons. The study suggests that CCHH and MHC are transient precursors to aragonite but not calcite, making the biomineralization process more complex than previously understood. Phase transitions were observed in repeat acquisitions, confirming the transient nature of the identified metastable phases. The researchers constructed a hypothetical energy landscape including all observed phases, proposing several possible pathways for aragonite and calcite formation. These pathways depend on the organism and vary significantly between aragonite and calcite producing animals.

The discovery of CCHH and MHC as transient crystalline precursors significantly expands our understanding of biomineralization. The presence of multiple metastable phases, both amorphous and crystalline, suggests a more complex energy landscape than previously envisioned. This complexity may provide organisms with greater biological control over the crystallization process, allowing them to fine-tune the properties of their biominerals for optimal function. The differing abundances and distribution of these phases among various biominerals (corals, nacre, sea urchins) highlight the organism-specific control over the biomineralization process. The observation that CCHH and MHC are primarily associated with aragonite-producing organisms supports the conclusion that these phases serve as intermediates specifically in the formation of aragonite, not calcite. The study's findings raise questions about the mechanisms of phase transitions, including whether they occur via solid-state transformations or dissolution-reprecipitation, and the impact of these mechanisms on the elemental and isotopic composition of biominerals.

This research significantly advances our understanding of biomineralization by revealing the presence of previously unknown crystalline intermediate phases (CCHH and MHC). The findings highlight the complexity of biomineralization pathways and the organism-specific control over crystal polymorph selection. Further research utilizing techniques such as electron diffraction and nano-SIMS, which are more widely accessible, are encouraged to validate these findings and explore the mechanisms of phase transitions in greater detail. Investigating biomineral formation in organisms that produce both aragonite and calcite simultaneously is proposed as a future research direction.

While the study employed rigorous methods and a large sample size, some limitations exist. The potential for artifacts during sample preparation or data acquisition, such as post-mortem crystallization, cannot be entirely ruled out. The interpretation of phase transition mechanisms (solid-state vs. dissolution-reprecipitation) requires further investigation. The precise role of organic molecules in mediating phase transitions also warrants further exploration. The study focuses primarily on aragonite and calcite, limiting the generalizations about broader biomineralization processes.