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Abstract Phosphate-shelled brachiopods differ in filter-feeding lifestyle, with Lingula anatina an active infaunal burrower, and Discinisca tenuis a shallow marine epibenthic animal. The shells of these animals are built of organophosphatic constituents, the organic fibres/sheets reinforced with calcium phosphate to provide a sophisticated ultrastructural robustness. This investigation examined the nature of the organic fibres in order to improve understanding of how living organisms produce hierarchically structured biomaterials. Unlike powdered samples commonly used in previous studies, organic fibres were isolated for the first time and the shell fractions were purified, in order to study the content and nature of the biopolymer fibres. Biochemical methods including Calcofluor staining revealed a chitin matrix. Ultrastructural analysis, thermal gravimetric analysis, and spectroscopic analyses show that the core polysaccharide framework is composed of layers of β-chitin sheets and/or fibrils that are coated with a fibrous organic matrix. There is more chitin matrix in the L. anatina shells (26.6 wt.%) compared to the D. tenuis shells (12.9 wt.%). Taken together, the data show that the chitin matrix contributes to increased skeletal strength, making L. anatina highly adapted for life as an active burrower. In comparison, D. tenuis contains less chitin and lives as attached epibenthos in a shallow marine environment. Graphical abstract First spectroscopic evidence of β-chitin sheets in recent organophosphatic brachiopods

The shells of linguloid brachiopods such as Lingula and Discinisca are inorganic–organic nanocomposites with a mineral phase of calcium phosphate (Ca-phosphate). Collagen, the main extracellular matrix in Ca-phosphatic vertebrate skeletons, has not previously been clearly resolved at the molecular level in organophosphatic brachiopods. Here, modern and recently-alive linguliform brachiopod shells of Lingula and Discinisca have been studied by microRaman spectroscopy, Fourier transform infrared spectroscopy, field emission gun scanning electron microscopy, and thermal gravimetric analysis. For the first time, biomineralized collagen matrix and Ca-phosphate components were simultaneously identified, showing that the collagen matrix is an important moiety in organophosphatic brachiopod shells, in addition to prevalent chitin. Stabilized nanosized apatitic biominerals (up to ∼50 nm) permeate the framework of organic fibrils. There is a ∼2.5-fold higher wt% of carbonate (CO32−) in Lingula versus Discinisca shells. Both microRaman spectroscopy and infrared spectra show transient amorphous Ca-phosphate and octacalcium phosphate components. For the first time, trivalent moieties at ∼1660 cm−1 and divalent moieties at ∼1690 cm−1 in the amide I spectral region were identified. These are related to collagen cross-links that are abundant in mineralized tissues, and could be important features in the biostructural and mechanical properties of Ca-phosphate shell biominerals. This work provides a critical new understanding of organophosphatic brachiopod shells, which are some of the earliest examples of biomineralization in still-living animals that appeared in the Cambrian radiation.

The intertidal bivalve Katelysia rhytiphora, endemic to south Australia and Tasmania, is used here for pulsed Sr-labelling experiments in aquaculture experiments to visualize shell growth at the micro- to nanoscale. The ventral margin area of the outer shell layer composed of (i) an outermost outer shell layer (oOSL) with compound composite prismatic architecture with three hierarchical orders of prisms and (ii) an innermost outer shell layer (iOSL) with crossed-acicular architecture consisting of intersecting lamellae bundles. All structural orders in both layers are enveloped by an organic sheath and the smallest mineralized units are nano-granules. Electron backscatter diffraction reveals a strong preferred orientation of the aragonite c axes perpendicular to the growth layers, while the a and b axes are scattered within a plane normal to the local growth direction and >46 % twin grain boundaries are detected. The Young's modulus shows a girdle-like maximum of elastically stiffer orientations for the shell following the inner shell surface. For 6 d, the bivalves were subjected twice to seawater with an increased Sr concentration of 18× mean ocean water by dissolving 144 µ g g −1 Sr (159.88 Sr∕Ca mmol ∕ mol) in seawater. The pulse labelling intervals in the shell are 17× (oOSL) and 12× (iOSL) enriched in Sr relative to the Sr-spiked seawater. All architectural units in the shell are transected by the Sr label, demonstrating shell growth to progress homogeneously instead of forming one individual architectural unit after the other. Distribution coefficients, DSr ∕ Ca , for labelled and unlabelled shells are similar to shell proportions formed in the wild (0.12 to 0.15). All DSr ∕ Ca values are lower than values for equilibrium partitioning of Sr in synthetic aragonite.

Background: Biochemical studies and spectroscopic techniques have shown that chitin-silk fibroins are common in nacroprismatic bivalve shells. However, the nature of organic biopolymers in the less well studied shell architectures, such as crossed lamellar shells, remain unknown. Here, two venus shells, Callista disrupta and Callista kingii, with crossed lamellar ultrastructure have been studied. Methods: We employed thermal gravimetric analysis, optical-, confocal- and scanning electron-microscopes, gel-sodium dodecyl sulfate (gel-SDS), FTIR, ultra-performance liquid chromatography and high-performance anion-exchange chromatography system with pulsed amperometric detection to analyse organic macromolecules in the shells. Results: Thermal analysis showed a low concentration of organic macromolecules in C. disrupta (1.38 wt%) and in C. kingii (1.71 wt%). A combination of biochemical protocols, including Calcofluor White staining and FTIR spectroscopic assessment, indicate that amino-polysaccharide chitin together with proteins, are present in the organic scaffolding of the shells. Scanning electron microscope of insoluble acid biopolymer extracts as well as FTIR technique show that the hierarchical structural organizations of organic biopolymers consist collagen-related matrix. Our histochemical fixing and staining techniques reveal many discrete proteins and glycoproteins from soluble organic macromolecules on the gel-SDS. We show here 'singlet' and 'doublet' glycosaminoglycan bands that are far above 260 kDa. General significance/conclusions: The presence of collagen matrix in Callista shells shows promise for the new source of biomaterials. Most importantly, the structural organization of the proteinaceous motif is predominantly helical structures and not silk-fibroin unlike in nacreous bivalve shells.

Phosphate-shelled brachiopods differ in filter-feeding lifestyle, with Lingula anatina an active infaunal burrower, and Discinisca tenuis a shallow marine epibenthic animal. The shells of these biomineralized organisms are built of organophosphatic constituents, the organic fibres/sheets reinforced with calcium phosphate (Ca-phosphate) to provide a sophisticated ultrastructural robustness. This investigation studied the nature of organic fibres in order to improve our understanding of how design principles are relevant to biominerals. Unlike the powdered samples that were commonly used in previous studies, for the first time organic fibres were isolated and the shell fractions were purified, in order to study the content and nature of the biopolymer fibres. Ultrastructural analysis, thermal gravimetric analysis and spectroscopic analyses reveal that the core polysaccharide framework is composed of layers of β-chitin sheets that are coated with a fibrous protein matrix. The chitin matrix in Lingula shells, 26.6 wt.%, is about two-fold higher wt.% compared to Discinisca shells,12.9 wt.%. After thermal gravimetric analysis, skeletal imaging of these shells shows important differences. Discinisca contains just a single layer that is ~21.0 µm thick. In contrast, Lingula exhibits two separate layers: the thicknesses of the primary- and secondary-layers are ~20.3 µm and ~8.1 µm, respectively. Taken together, the data shows that the chitin matrix contributes to increased skeletal strength, making Lingula highly adapted for life as an active burrower. In comparison, Discinisca contains less chitin and lives as attached epibenthos in a shallow marine environment.

Prolonged exposure to low pH conditions affects the durability of concrete. In this work, the effect of mullite, aluminum silicate, on the strength and the acid corrosion of mortar and concrete under induced accelerated conditions in sulfuric acid solutions at pH of 0.25 and 1 was studied. The characterization of physicochemical changes was performed using techniques including compressive strength, scanning electron microscopy, micro-X-ray fluorescence spectrometry, and the Vickers hardness test. The results indicate that the addition of mullite does not have any significant effect on the overall strength of mortar and concrete samples, while it significantly increases their resistance to corrosion caused by sulfate attack by 90%, therefore, it is expected to increase the life span and decrease the maintenance costs of concrete pipes subjected to acid corrosion in sewer environments. The inhibition efficiency is observed to be sensitive to acid concentration and was improved with increase in the amount of mullite in samples.

The Perseverance rover (Mars 2020) is equipped with an instrumental and analytical payload capable of identifying a broad range of organic molecules in geological samples. To determine the efficacy of these analytical techniques in recognizing important ecological and environmental signals in the rock record, this study utilized analogous equipment, including gas chromatography/mass spectrometry, Raman spectroscopy, X-ray fluorescence (XRF), Fourier transform infrared spectroscopy, along with macroscopic and petrographic observations, to examine early-middle Cambrian microbialites from the Arrowie Basin, South Australia. Morphological and petrographic observations of these carbonate successions reveal evidence of hypersaline-restricted environments. Microbialites have undergone moderate diagenesis, as supported by XRF data that show mineral assemblages, including celestine and the illitization of smectite. Raman spectral data, carbon preference indices of ∼1, and the methylphenanthrene index place the samples in the prehnite/pumpellyite metamorphic facies. Pristane and phytane are the only biomarkers that were detected in the least thermally mature samples. This research demonstrates a multitechnique approach that can yield significant geological, depositional, paleobiological, and diagenetic information that has important implications for planning future astrobiological exploration.

We present an in-depth characterisation of shells from two bivalve species with crossed lamellar microstructure, namely Tridacna gigas and Fulvia tenuicostata. High-resolution scanning electron microscopy and confocal microscopy imaging reveal a fine structure of nanogranular particles that are inorganic–bioorganic nanocomposites for both shells. In F. tenuicostata, inorganic–organic components are arranged in a polycrystalline fibre-like fabric. T. gigas consists of up to four hierarchical lamellar structural orders and the second-order lamellae consist of elongated nanometre-sized laths. The inorganic matrix is intimately intergrown with the total amount of organic matter (1.8 and 1.5 wt%), and the composition of the shell macromolecules is variable between the two calcareous biominerals. This work shows for the first time the presence of polysaccharide-based compounds that could be essential for the construction of bio-organics as well as many prominent protein bands, glycoproteins and/or glycosaminoglycans of unknown sizes far above 260 kDa in bivalve shells with crossed lamellar microstructure. Chitosan (deacetylated chitin) with apparent molecular weights from 18 to 110 kDa for T. gigas and from 12 kDa till above 110 kDa for F. tenuicostata are detected in gel electrophoresis after Calcofluor staining. In each of the shell extracts, the infrared spectroscopy shows polysaccharides, proteins and lipids. Our findings from two crossed lamellar shells representing two genera of Mollusca: Cardiidae indicate that chitin–protein complexes and lipid–lipoproteins are not restricted only to bivalves with nacroprismatic shells.

We use pulsed Sr-labelling experiments to visualize growth of aragonitic Katelysia rhytiphora (Mollusca, Bivalvia) shells. The outer compound composite prismatic structure is organized into three orders of prisms, and the inner crossed acicular structure consists of intersecting lamellae. Electron Backscatter Diffraction (EBSD) reveals substantial twinning of the aragonite crystals (> 46 %) and an overall reduced and strategically oriented anisotropy of the Young’s modulus in the whole shell compared to that of monolithic aragonite. All structural orders in both layers are enveloped by an organic sheath and the smallest mineralized units are nanogranules. Total organic contents are 2.2 (outer) and 1.4 wt. % (inner layer) and are, thus, intermediate between those of nacreous and crossed-lamellar shells. Prisms in the outer structure can be correlated to yearly, daily and sub-daily growth rates. Average daily growth rates at the ventral margin for the outer structure are 17 % higher than for the inner crossed acicular structure. The calcification front runs evenly across all structures and architectural orders independently of the current growth rate. Sharply defined transitions from labelled to unlabelled areas in the shell indicate that physiological processes driving calcification have no lag. This suggests that the extrapallial fluid cannot be very voluminous. Narrow increments of varying Sr content within labelled shell, despite constant Sr concentrations in seawater, suggest cyclic metabolic activity during calcification. Micro-Raman spectroscopy maps validate a low impact of high Sr-conditions on the aragonite crystal structure. Identical Sr-enrichment factors for labelled and ambient conditions support models of ion transport via a passive selective pathway to the mantle epithelium followed by calcification via amorphous calcium carbonate.

Bivalve shells are inorganic-organic nanocomposites whose material properties outperform their purely inorganic mineral counterparts. Most typically the inorganic phase is a polymorph of CaCO3, while the organic phase contains biopolymers which have been presumed to be chitin and/or proteins. Identifying the biopolymer phase is therefore a crucial step in improving our understanding of design principles relevant to biominerals. In this work we study seven shells; four are examples of nacroprismatic shells (Alathyria jacksoni, Pinctada maxima, Hyriopsis cumingii and Cucumerunio novaehollandiae), one homogeneous (Arctica islandica), and two are crossed lamellar (Callista kingii, Tridacna gigas). Both intact shells, their organic extracts as isolated after decalcification in acid, and the periostracum overlay have been studied by solid-state CP-MAS NMR, FTIR, SEM and chemical analysis. In none of the shells examined in this work do we find a significant contribution to the organic fraction from chitin or its derivatives despite popular models of bivalve biomineralization which assume abundant chitin in the organic fraction of mollusk bivalve shells. In each of the nacroprismatic extracts the 13C NMR spectra represent similar proteinaceous material, Ala and Gly-rich and primarily organized as β-sheets. A different, yet highly conserved protein was found in the periostracum covering each of the three nacreous shells studied. The Arctica islandica shells with homogeneous microstructure contained proteins which do not appear to be silk-like, while in the crossed lamellar shells we extracted too little organic matter to characterize. STATEMENT OF SIGNIFICANCE: Hydrophobic macromolecules are structural components within the calcareous inorganic matrix of bivalve shells and are responsible for enhanced materials properties of the biominerals. Prevalent models suggest that chitin is such major hydrophobic component. Contrary to that we show that chitin is rare within the hydrophobic biopolymers which primarily consist of proteinaceous matter with structural motifs as silk-like β-sheets, or others yet to be determined. Recognizing that diverse proteinaceous motifs, devoid of abundant chitin, can yield the optimized mechanical properties of bivalve shells is critical both to understand the mechanistic pathways by which they regulate biomineralization and for the design of novel bioinspired materials.

A detailed characterisation of the organic composition of Arctica islandica (Linnaeus, 1767) shells with homogenous microstructure is compared with nacroprismatic shells of Pinctada fucata martensii (Dunker, 1872), Hyriopsis cumingii (Lea, 1852) and Diplodon chilensis patagonicus (d’Orbigny, 1835). Thermogravimetric analysis shows lowest total organic contents of 1.65 wt% for A. islandica shells, while all nacroprismatic shells are higher (3.14–4.13 wt%). Macromolecules extracted from the nacroprismatic shells are dominated by hydrophobic amino acids (~ 54%) in the acid extracts, while EDTA-extracts are moderately rich in aspartate and glutamate (16% in total) and in glycine–alanine (42%). In comparison, A. islandica shells have higher concentrations of proline, glycine and aspartate (ca 40%). Infrared spectroscopy shows some acidic protein bands in A. islandica, which cannot be found in the nacroprismatic shells. Alcian Blue and/or modified silver staining methods reveal many prominent bands. Protein bands at around 10, 14, 17, 21, 26, 31, 40 and 55 kDa are detected in A. islandica shells for the first time, thus may constitute a new set of proteins in mollusc shells. SDS-PAGE exhibits apparent molecular weights from 5 to 63 kDa in nacroprismatic shells. Distinct protein bands at around 17 and 26 kDa in A. islandica shells may correspond to a post-translational modification of proteins; these prominent bands, however, are absent in the nacroprismatic samples. Contrarily, SDS-PAGE of both, homogeneous and nacroprismatic shell microstructures show nonacidic-matrix-proteins.

The terpenoid composition of fossil resin from the Cape York Peninsula, Australia has been analysed by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) to determine its origin. The pyrolysis products were dominated by cadalene-based C15 bicyclic sesquiterpenoids including some C30–C31 bicadinanes and bicadinenes typical of Class II resin derived from angiosperm plants of Dipterocarpaceae. This observation contrasts with the Araucariaceae (Agathis sp.) source previously suggested for the resin based on Fourier transform infrared (FTIR) analyses. Dipterocarpaceae are not known in Australian vegetation but grow abundantly in Southeast Asia including New Guinea, indicating that the geological origin of the amber is not the Australian mainland but could be traced to Southeast Asia.

Tridacna derasa shells show a crossed lamellar microstructure consisting of three hierarchical lamellar structural orders. The mineral part is intimately intergrown with 0.9 wt% organics, namely polysaccharides, glycosylated and unglycosylated proteins and lipids, identified by Fourier transform infrared spectrometry. Transmission electron microscopy shows nanometre-sized grains with irregular grain boundaries and abundant voids. Twinning is observed across all spatial scales and results in a spread of the crystal orientation angles. Electron backscatter diffraction analysis shows a strong fibre texture with the [001] axes of aragonite aligned radially to the shell surface. The aragonitic [100] and [010] axes are oriented randomly around [001]. The random orientation of anisotropic crystallographic directions in this plane reduces anisotropy of the Young's modulus and adds to the optimization of mechanical properties of bivalve shells., Royal Society Open Science, 4 (9), ISSN:2054-5703

The calcium carbonate shells of planktic foraminifera provide our most valuable geochemical archive of ocean surface conditions and climate spanning the last 100 million years, and play an important role in the ocean carbon cycle. These shells are preserved in marine sediments as calcite, the stable polymorph of calcium carbonate. Here, we show that shells of living planktic foraminifers Orbulina universa and Neogloboquadrina dutertrei originally form from the unstable calcium carbonate polymorph vaterite, implying a non-classical crystallisation pathway involving metastable phases that transform ultimately to calcite. The current understanding of how planktic foraminifer shells record climate, and how they will fare in a future high-CO2 world is underpinned by analogy to the precipitation and dissolution of inorganic calcite. Our findings require a re-evaluation of this paradigm to consider the formation and transformation of metastable phases, which could exert an influence on the geochemistry and solubility of the biomineral calcite., Understanding foraminifera mineralisation pathways is essential to correctly decipher the geochemical climate signals recorded in their shells. Here, the authors identify a non-classical crystallization pathway via metastable phases for Orbulina universa and Neogloboquadrina dutertrei.