Amorphous Calcium Carbonate Research Articles

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

The response of coral skeletal nano structure and hardness to ocean acidification conditions.

Ocean acidification typically reduces coral calcification rates and can fundamentally alter skeletal morphology. We use atomic force microscopy (AFM) and microindentation to determine how seawater pCO2 affects skeletal structure and Vickers hardness in a Porites lutea coral. At 400 µatm, the skeletal fasciculi are composed of tightly packed bundles of acicular crystals composed of quadrilateral nanograins, approximately 80-300 nm in dimensions. We interpret high adhesion at the nanograin edges as an organic coating. At 750 µatm the crystals are less regular in width and orientation and composed of either smaller/more rounded nanograins than observed at 400 µatm or of larger areas with little variation in adhesion. Coral aragonite may form via ion-by-ion attachment to the existing skeleton or via conversion of amorphous calcium carbonate precursors. Changes in nanoparticle morphology could reflect variations in the sizes of nanoparticles produced by each crystallization pathway or in the contributions of each pathway to biomineralization. We observe no significant variation in Vickers hardness between skeletons cultured at different seawater pCO2. Either the nanograin size does not affect skeletal hardness or the effect is offset by other changes in the skeleton, e.g. increases in skeletal organic material as reported in previous studies.

Amorphous Calcium Carbonate Shows Anti-Cancer Properties That are Attributed to Its Buffering Capacity.

Amorphous calcium carbonate (ACC) is a non-crystalline form of calcium carbonate, and it is composed of aggregated nano-size primary particles. Here, we evaluated its anti-cancer effect postulated relative to its buffering capabilities in lung cancer. Tumors were evaluated in vivo using the Lewis lung carcinoma (LLC) mouse cell line and A549 human lung cancer carcinoma cell line. LLC and A549 cells were injected subcutaneously into the right hind leg of mice. Treatments (ACC, cisplatin, vehicle, and ACC with cisplatin, all given via daily IP injections) started once tumors reached a measurable size. Treatments were carried out for 14 days in the LLC model and for 22 and 24 days in the xenograft model (two experiments). LLC tumors were resected from ACC at the end of the study, and vehicle groups were evaluated for cathepsin B activity. Differential gene expression was carried out on A549 cells following 8 weeks of in vitro culture in the presence or absence of ACC in a culture medium. The ACC treatment decelerated tumor growth rates in both models. When tumor volumes were compared on the last day of each study, the ACC-treated animal tumor volume was reduced by 44.83% compared to vehicle-treated animals in the LLC model. In the xenograft model, the tumor volume was reduced by 51.6% in ACC-treated animals compared to vehicle-treated animals. A more substantial reduction of 74.75% occurred in the combined treatment of ACC and cisplatin compared to the vehicle (carried out only in the LLC model). Cathepsin B activity was significantly reduced in ACC-treated LLC tumors compared to control tumors. Differential gene expression results showed a shift towards anti-tumorigenic pathways in the ACC-treated A549 cells. This study supports the ACC anti-malignant buffering hypothesis by demonstrating decelerated tumor growth, reduced cathepsin B activity, and altered gene expressions to produce anti-cancerous effects.

Assembly of the Intraskeletal Coral Organic Matrix during Calcium Carbonate Formation.

Scleractinia coral skeleton formation occurs by a heterogeneous process of nucleation and growth of aragonite in which intraskeletal soluble organic matrix molecules, usually referred to as SOM, play a key role. Several studies have demonstrated that they influence the shape and polymorphic precipitation of calcium carbonate. However, the structural aspects that occur during the growth of aragonite have received less attention. In this research, we study the deposition of calcium carbonate on a model substrate, silicon, in the presence of SOM extracted from the skeleton of two coral species representative of different living habitats and colonization strategies, which we previously characterized. The study is performed mainly by grazing incidence X-ray diffraction with the support of Raman spectroscopy and electron and optical microscopies. The results show that SOM macromolecules once adsorbed on the substrate self-assembled in a layered structure and induced the oriented growth of calcite, inhibiting the formation of vaterite. Differently, when SOM macromolecules were dispersed in solution, they induced the deposition of amorphous calcium carbonate (ACC), still preserving a layered structure. The entity of these effects was species-dependent, in agreement with previous studies. In conclusion, we observed that in the setup required by the experimental procedure, the SOM from corals appears to present a 2D lamellar structure. This structure is preserved when the SOM interacts with ACC but is lost when the interaction occurs with calcite. This knowledge not only is completely new for coral biomineralization but also has strong relevance in the study of biomineralization on other organisms.

Quantitative mapping of calcium cell reservoirs in cyanobacteria at the submicrometer scale

Calcium plays an important role in the physiology of bacterial cells and as a free soluble form inside the cells, it is tightly regulated at a low concentration. However, much higher amounts of stored calcium exist within cells and can be remobilized at diverse occasions. As a consequence, there has been a variety of techniques developed to quantitatively map the different forms of Ca in cells. Here we show how scanning transmission x-ray microscopy (STXM), a synchrotron-based microscopy technique, conducted in the soft x-ray range at the Ca L2,3 edges (340–360 eV) offers an original and informative quantitative view of different Ca reservoirs in bacterial cells at a spatial resolution better than 100 nm. To illustrate this, we analyzed mutants of the cyanobacterium Synechococcus elongatus PCC 7942, overexpressing different versions of a gene called ccyA from two phylogenetically distant cyanobacteria: Gloeomargarita lithophora and Synechococcus sp. PCC 6312. This gene is diagnostic of the capability of some cyanobacteria to form intracellular amorphous calcium carbonate (iACC) but its function remains unknown. Here we show that the overexpression of the ccyA gene in the iACC non-forming cyanobacterium S. elongatus PCC 7942 results in an increased Ca content, especially in some Ca-rich cells. Moreover, we show that STXM can discriminate four different reservoirs of Ca in cyanobacteria and provides quantitative assessment of their relative importance.

A Comparative Study of Ultrasmall Calcium Carbonate Nanoparticles for Targeting and Imaging Atherosclerotic Plaque.

Atherosclerosis is a complex disease that can lead to life-threatening events, such as myocardial infarction and ischemic stroke. Despite the severity of this disease, diagnosing plaque vulnerability remains challenging due to the lack of effective diagnostic tools. Conventional diagnostic protocols lack specificity and fail to predict the type of atherosclerotic lesion and the risk of plaque rupture. To address this issue, technologies are emerging, such as noninvasive medical imaging of atherosclerotic plaque with customized nanotechnological solutions. Modulating the biological interactions and contrast of nanoparticles in various imaging techniques, including magnetic resonance imaging, is possible through the careful design of their physicochemical properties. However, few examples of comparative studies between nanoparticles targeting different hallmarks of atherosclerosis exist to provide information about the plaque development stage. Our work demonstrates that Gd (III)-doped amorphous calcium carbonate nanoparticles are an effective tool for these comparative studies due to their high magnetic resonance contrast and physicochemical properties. In an animal model of atherosclerosis, we compare the imaging performance of three types of nanoparticles: bare amorphous calcium carbonate and those functionalized with the ligands alendronate (for microcalcification targeting) and trimannose (for inflammation targeting). Our study provides useful insights into ligand-mediated targeted imaging of atherosclerosis through a combination of in vivo imaging, ex vivo tissue analysis, and in vitro targeting experiments.

Mg-rich amorphous to Mg-low crystalline CaCO3 pathway in foraminifera

Calcium carbonate minerals produced by marine organisms play a central role in the global carbon cycle and carbonate sedimentation, which influence the climate by regulating atmospheric CO2 levels. Foraminifera are important marine single-celled organisms that have produced calcite shells for over 300 million years. Here, we present new observations promoting our understanding for foraminiferal biocalcification by studying Amphistegina lessonii. We integrated in vivo confocal autofluorescence and dye fluorescence imaging with elemental analysis of the cell supporting the concept that the calcite shells of foraminifera are produced via deposition of intracellularly formed Mg-rich amorphous calcium carbonate (Mg-ACC) particles that transform into a stable mineral phase. This process is likely accompanied by the activity of endosymbiotic microalgae and seawater-derived endocytic vesicles that provide calcification substrates such as DIC, Ca2+, and Mg2+. The final transformation of semi-liquid amorphous nanoparticles into a crystalline shell was associated with Mg2+ liberation.

Quercetin directed transformation of calcium carbonate into porous calcite and their application as delivery system for future foods

The hierarchically porous property of CaCO3 has attracted considerable attention in the field of active delivery ingredients due to its high adsorption capacity. Here, a facile and high-efficient approach to control the calcification processes of CaCO3 ending with calcite microparticles with superior porosity and stability is reported and evaluated. In this work, a series of quercetin promoted CaCO3 microparticles, using soy protein isolate (SPI) as entrapment agent, was synthesized, characterized, and their digestive behavior and antibacterial activity were evaluated. Results obtained indicated that quercetin showed good ability to direct the calcification pathway of amorphous calcium carbonate (ACC) with the formation of flower- and petal-like structures. The quercetin-loaded CaCO3 microparticles (QCM) had a macro-meso-micropore structure, which was identified to be the calcite form. The macro-meso-micropore structure provided QCM with the largest surface area of 78.984 m2g−1. The loading ratio of SPI to QCM was up to 200.94 μg per mg of QCM. The protein and quercetin composite microparticles (PQM) were produced by simply dissolving the CaCO3 core, and the obtained PQM was used for the delivery of quercetin and protein. Thermogravimetric analysis showed PQM presented with good thermal stability without the CaCO3 core. Furthermore, minor discrepancy was noted in protein conformational structures after removing the CaCO3 core. In vitro digestion revealed that approximately 80% of the loaded quercetin was released from PQM during intestinal digestion, and the released quercetin exhibited efficient transportation across the Caco-2 cell monolayer. More importantly, the PQM digesta retained enhanced antibacterial activities to inhibit growth of Escherichia coli and Staphylococcus aureus. Porous calcites show a high potential as a delivery system for food applications.

In vitro corrosion resistance and dual antibacterial ability of curcumin loaded composite coatings on AZ31 alloy: Effect of amorphous calcium carbonate

Rapid corrosion and bacterial infection are obstacles to put into use biodegradable magnesium (Mg) alloy as biomedical materials. In this research, an amorphous calcium carbonate (ACC)@curcumin (Cur) loaded poly-methyltrimethoxysilane (PMTMS) coating prepared by self-assembly method on micro-arc oxidation (MAO) coated Mg alloy has been proposed. Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy are adopted to analyze the morphology and composition of the obtained coatings. The corrosion behaviour of the coatings is estimated by hydrogen evolution and electrochemical tests. The spread plate method without or with 808 nm near-infrared irradiation is applied to evaluate the antimicrobial and photothermal antimicrobial ability of the coatings. Cytotoxicity of the samples is tested by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di- phenytetrazoliumromide (MTT) and live/dead assay culturing with MC3T3-E1 cells. Results show that the MAO/ACC@Cur-PMTMS coating exhibited favourable corrosion resistance, dual antibacterial ability, and good biocompatibility. Cur was employed as an antibacterial agent and photosensitizer for photothermal therapy. The core of ACC significantly improved the loading of Cur and the deposition of hydroxyapatite corrosion products during degradation, which greatly promoted the long-term corrosion resistance and antibacterial activity of Mg alloys as biomedical materials.

Amorphous biomineral-reinforced hydrogels with dramatically enhanced toughness for strain sensing

Ionic conductive hydrogels are promising candidates for flexible wearable strain sensors and artificial skin. However, achieving high mechanical and sensing performance concurrently remains challenging. Herein, a novel biomineral-reinforced hydrogel composed of polyacrylamide (PAM) and highly stable amorphous calcium carbonate (ACC) is reported. Benefiting from the dual ionic doping strategy (Mg2+ and PO43−), ACC nanoparticles in hybrid hydrogels show a super stable amorphous nature. The resulting mineral hydrogel displays a high stretchability (>1150% strain), a dramatically enhanced fracture toughness (9.57±1.28 vs. 0.91±0.12 kJ m−2), and a desirable linear strain sensitivity. Moreover, the novel mineral hydrogel exhibits high biocompatibility and flame retardance, making it an appealing candidate for wearable device applications.

Effect of ion and protein concentration of Ps19, a shell protein from Pteria sterna, on calcium carbonate polymorph

Calcium carbonate is present in many biological structures such as bivalve shell, which is composed mainly of two CaCO3 polymorphs: calcite and aragonite. However, exist other forms of calcium carbonate like vaterite and amorphous calcium carbonate (ACC) that are not commonly reported. Polymorph selection is influenced by salt concentration, cofactor ions, and the presence of shell matrix proteins (SMPs) which regulates calcium carbonate deposition, among other factors. In this study, calcium carbonate crystallization in vitro of four different saline solutions at two molarities was evaluated with increased concentrations of the Ps19 protein, an insoluble extracted protein from the shell of Pteria sterna, previously described as a promotor of aragonite platelet crystallization. In vitro crystallizations showed that Ps19 is capable to induce aragonite and calcite deposition in a dose-dependent manner, but also vaterite under ciertan conditions, acting as a promoter and inhibitor of crystallization. The results contribute to understand how Ps19 control precipitation of calcium polymorphs in the growth of the prismatic and nacre layer of the shell of P. sterna.

Microbial traces found in microdolomite associated with seep-related shallow gas hydrate

Recently, microdolomite grains with a diameter of about 30–150 μm were discovered within seep-related shallow gas hydrate in the eastern margin of the Japan Sea, and the preliminary assumption has been that their formation is mediated by microbially secreted extracellular polymeric substances (EPS). To verify this interpretation, the present study investigates the characteristics of minerals and organic matter inside the microdolomite grains. Observation by polarizing microscopy reveals that the microdolomite grains generally consist of combinations of distinct cores and external cements. The cores are mostly dark or cloudy and their abundance does not show clear depth dependence, while the external cements are generally isopachous in the shallow part and polygonal in the deeper portions. To further clarify the characteristics of these cores and external cements, thin-foil sections were prepared from two major types of microdolomite, one with a dark core and polygonal external cement, and one with a cloudy core and isopachous external cement. These thin-foil sections were then analyzed by scanning transmission X-ray microscopy (STXM) and transmission electron microscopy (TEM). In the dark core, fibriform pores of about 100 nm diameter were observed to be closely related with amorphous calcium carbonate (ACC) and carboxy groups, likely created by heterogeneous nucleation on microbial EPS surfaces. The polygonal external cement is composed of monocrystalline dolomite, representing slow crystal growth in the deeper part where hydrate formation is less active. On the other hand, the observed cloudy core and the isopachous external cement are, respectively, composed of scattered and accumulated rhombohedral dolomite crystals, presumably resulting from homogeneous nucleation in saline pockets of rapidly growing hydrate and subsequent crystal accumulation on the surface of spherical oil films.

Effect of proteins on the mineralization, microstructure and mechanical properties of carbonation cured calcium silicate

The effect of five proteins with different physicochemical properties on the mineralization, microstructure, and mechanical properties of carbonated wollastonite pastes was examined. The negative surface charge of wollastonite particles increased as the result of protein adsorption. The flow of fresh paste was shown to increase in the pastes with proteins due to the enhanced electrostatic repulsion of the wollastonite particles as a result of the adsorption of negatively charged proteins, the ball bearing effect of the proteins in the pore solution, and formation of air bubbles. While calcite and amorphous calcium carbonate were the primary phases of calcium carbonate in all pastes, other polymorphs including aragonite and vaterite were present, to a small extent, in the microstructure of most of the pastes modified with proteins. The calcium carbonate content was shown to be higher in some of the pastes modified with proteins and this was attributed to the delayed water loss to evaporation and presence of free water due to hydrophobization of pastes modified with proteins. The X-ray micro-computed tomography (micro-CT) analysis revealed the formation of cracks in the control and some of the protein modified pastes resulting from drying shrinkage. It was demonstrated that the increased microstructure hydrophobicity was the reason for the absence of cracks in the pastes with the proteins that did not show cracks. Overall, the carbonated pastes with proteins exhibited higher compressive strength compared to the control paste. Absence of cracks, enhanced interfacial strength between carbonated products in the microstructure and organic-inorganic composite formation are suggested to be responsible for the increased compressive strength of the carbonated pastes with proteins.

Earthworm granules: A model of non-classical biogenic calcium carbonate phase transformations.

Different non-classical crystallization mechanisms have been invoked to explain structural and compositional properties of biocrystals. The identification of precursor amorphous nanoparticle aggregation as an onset process in the formation of numerous biominerals (crystallization via particle attachment) constituted a most important breakthrough for understanding biologically mediated mineralization. A comprehensive understanding about how the attached amorphous particles transform into more stable, crystalline grains has yet to be elucidated. Here, we document structural, biogeochemical, and crystallographic aspects of the formation as well as the further phase transformations of the amorphous calcium carbonate particles formed by cultured specimens of earthworm Lumbricus terrestris. In-situ observations evidence the formation of proto-vaterite after dehydration of earthworm-produced ACC, which is subsequently followed by proto-vaterite transformation into calcite through nanoparticle attachment within the organic framework. In culture medium spiked with trace amounts of Mn2+, the cauliflower-like proto-vaterite structures become longer-lived than in the absence of Mn2+. We propose that the formation of calcite crystals takes place through a non-classical recrystallization path that involves migration of proto-vaterite nanoparticles to the crystallization site, and then, their transformation into calcite via a dissolution-recrystallization reaction. The latter is complemented by ion-by-ion crystal growth and associated with impurity release. These observations are integrated into a new model of the biocrystallization of earthworm-produced carbonate granules which highlights the sensibility of this process to environmental chemical changes, its potential impact on the bioavailability of contaminants as well as the threat that chemical pollution poses to the normal development of its early stages. STATEMENT OF SIGNIFICANCE: Understanding the mechanisms of nucleation, stabilization and aggregation of amorphous calcium carbonate (ACC) and factors controlling its further transformation into crystalline phases is fundamental for elucidation of biogenic mineralization. Some species of earthworms are natural workbench to understand the biogenic ACC, stabilization and the transformation mechanisms, because they create millimeter-sized calcareous granules from amorphous calcium carbonate, which crystallize to a more stable mineral phase (mostly calcite). This study undergoes into the mechanisms of ACC stabilization by the incorporation of trace elements, as manganese, and the ulterior precipitation of calcareous granules by a coupled process of amorphous particle attachment and ion-by-ion growth. The study points to sensibility of this process to environmental chemical changes.

Considerations on chemical composition of psammoma bodies: Automated detection strategy by infrared microspectroscopy in ovarian and thyroid cancer tissues

Ectopic calcifications are observed in many soft tissues and are associated with several diseases, including cancer. The mechanism of their formation and the correlation with disease progression are often unclear. Detailed knowledge of the chemical composition of these inorganic formations can be very helpful in better understanding their relationship with unhealthy tissue. In addition, information on microcalcifications can be very useful for early diagnosis and provide insight into prognosis. In this work the chemical composition of psammoma bodies (PBs) found in tissues of human ovarian serous tumors was examined. The analysis using Micro Fourier Transform Infrared Spectroscopy (micro-FTIR) revealed that these microcalcifications contain amorphous calcium carbonate phosphate. Moreover, some PB grains showed the presence of phospholipids. This interesting result corroborates the proposed formation mechanism reported in many studies according to which ovarian cancer cells switch to a calcifying phenotype by inducing the deposition of calcifications. In addition, other techniques as X-ray Fluorescence Spectroscopy (XRF), Inductively Coupled Plasma Optical Emission Spectroscopy(ICP-OES) and Scanning electron microscopy (SEM) with Energy Dispersive X-ray Spectroscopy (EDX) were performed on the PBs from ovary tissues to determine the elements present. The PBs found in ovarian serous cancer showed a composition comparable to PBs isolated from papillary thyroid.Based on the chemical similarity of IR spectra, using micro-FTIR spectroscopy combined with multivariate analysis, an automatic recognition method was constructed. With this prediction model it was possible to identify PBs microcalcifications in tissues of both ovarian cancers, regardless of tumor grade, and thyroid cancer with high sensitivity. Such approach could become a valuable tool for routine macrocalcification detection because it eliminates sample staining, and the subjectivity of conventional histopathological analysis.

Flash Nanoprecipitation Fabrication of PEI@Amorphous Calcium Carbonate Hybrid Nanoparticles for siRNA Delivery.

RNA interference (RNAi) is a promising approach for disease treatments. But the development of safe and effective delivery carriers remains a major challenge. Organic-inorganic hybrid nanoparticles (NPs), with the integration of functions from distinct materials, show great potential in small interfering RNA (siRNA) delivery. Herein, pH responsive amorphous calcium carbonate NPs (ACC NPs) are prepared using flash nanoprecipitation and hybrid NPs are constructed by coating ACC NPs with polyethyleneimine (PEI) for efficient siRNA delivery. PEI/ACC NPs show robust pH responsiveness and stability as well as effective siRNA loading and protection. Furthermore, siRNA-loaded PEI/ACC NPs demonstrate enhanced cellular uptake and efficient endosomal escape, mediating improved siRNA delivery compared to pure PEI. These findings suggest that PEI/ACC NPs may have great potential in siRNA delivery for RNAi-based therapy.

Carbonate diagenesis: A celebration of the work of John Anthony Dawson (Tony) Dickson

Carbonate diagenesis: A celebration of the work of John Anthony Dawson (Tony) Dickson

Growth dynamics and amorphous-to-crystalline phase transformation in natural nacre

Biominerals, such as nacreous bivalve shells, are important archives of environmental information. Most marine calcifiers form their shells from amorphous calcium carbonate, hypothesised to occur via particle attachment and stepwise crystallisation of metastable precursor phases. However, the mechanism of this transformation, including the incorporation of trace elements used for environmental reconstructions, are poorly constrained. Here, using shells of the Mediterranean mussel, we explore the formation of nacre from the meso- to the atomic scale. We use a combination of strontium pulse-chase labelling experiments in aquaculture and correlated micro- to sub-nanoscale analysis to show that nacre grows in a dynamic two-step process with extensional and space-filling growth components. Furthermore, we show that nacre crystallizes via localised dissolution and reprecipitation within nanogranules. Our findings elucidate how stepwise crystallization pathways affect trace element incorporation in natural biominerals, while preserving their intricate hierarchical ultrastructure.

Mechanisms of carbonate precipitation induced by two model bacteria

The investigation of microbially induced calcium carbonate precipitation is essential to understanding CO2 fixation, marine carbonate rock formation, and biogeochemical cycling of life-essential elements. In this study, two model bacteria Bacillus subtilis (possess the cell wall of G+ bacteria) and Escherichia coli (possess the cell wall of G− bacteria) were used as representatives to induce carbonate mineralization. The mineralization ability of the two bacteria and carbonate precipitation mechanisms were studied with the biochemical analysis of bacterial fermentation products, mineral characterization, and biomimetic mineralization experiments. The results showed that both bacteria induced biomineral formation by using the cell wall as a template, but B. subtilis was more capable of inducing mineralization than E. coli. Particularly, the percentage of Ca2+ removal from the culture medium by the former reached 36% in 7 days, which induced the formation of vaterite and calcite, while that of Ca2+ removal by the latter reached 28%, and the resulting carbonate was amorphous calcium carbonate. Biomimetic mineralization experiments showed that protein molecules were the key factors inducing the formation of biological vaterite, while polysaccharides tended to bind small vaterite particles to form large organo-mineral aggregates. By comparing the results of inducing calcium carbonate under the same culture conditions, the two types of bacteria with completely different cell wall structures had significant differences in the ability to induce calcium carbonate synthesis, among which that was stronger by Bacillus subtilis (G+) than by Escherichia coli (G−). It was found that factors such as bacterial cell wall structure, extracellular secretions including carbonic anhydrase, pH value, etc. all affect the microbial-induced carbonate precipitation to varying degrees, providing new insights into the theory of bacterial-induced carbonate mineralization.

Evidence for heterothermic endothermy and reptile-like eggshell mineralization in Troodon, a non-avian maniraptoran theropod

The dinosaur-bird transition involved several anatomical, biomechanical, and physiological modifications of the theropod bauplan. Non-avian maniraptoran theropods, such as Troodon, are key to better understand changes in thermophysiology and reproduction occurring during this transition. Here, we applied dual clumped isotope (Δ47 and Δ48) thermometry, a technique that resolves mineralization temperature and other nonthermal information recorded in carbonates, to eggshells from Troodon, modern reptiles, and modern birds. Troodon eggshells show variable temperatures, namely 42 and 29 ± 2 °C, supporting the hypothesis of an endothermic thermophysiology with a heterothermic strategy for this extinct taxon. Dual clumped isotope data also reveal physiological differences in the reproductive systems between Troodon, reptiles, and birds. Troodon and modern reptiles mineralize their eggshells indistinguishable from dual clumped isotope equilibrium, while birds precipitate eggshells characterized by a positive disequilibrium offset in Δ48. Analyses of inorganic calcites suggest that the observed disequilibrium pattern in birds is linked to an amorphous calcium carbonate (ACC) precursor, a carbonate phase known to accelerate eggshell formation in birds. Lack of disequilibrium patterns in reptile and Troodon eggshells implies these vertebrates had not acquired the fast, ACC-based eggshell calcification process characteristic of birds. Observation that Troodon retained a slow reptile-like calcification suggests that it possessed two functional ovaries and was limited in the number of eggs it could produce; thus its large clutches would have been laid by several females. Dual clumped isotope analysis of eggshells of extinct vertebrates sheds light on physiological information otherwise inaccessible in the fossilrecord.