Chemical taphonomy of biomineralized tissues

Biomineralized tissues are chemically altered after death, and this diagenetic alteration can obscure original biological chemical features or provide new chemical information about the depositional environment. To use the chemistry of fossil biominerals to reconstruct biological, environmental or taphonomic information, a solid appreciation of biomineralization, mineral diagenesis and biomineral–water interaction is needed. Here, I summarize the key recent developments in the fields of biomineralization and post‐mortem trace element exchange that have significant implications for our understanding of the diagenetic behaviour of biominerals and the ways in which biomineral chemistry can be used in palaeontological and taphonomic research.     

CRYSTALLOGRAPHY AND DIAGENESIS IN FOSSIL CRANIID BRACHIOPODS

Abstract:  One of the fundamental questions in biomineralization is how organisms control crystallographic orientation during biomineral production. The understanding of how diagenetic changes influence the preservation of original crystallographic patterns in fossilized biomineral structures provides a priori fundamental information for such an assessment. Fossil craniid brachiopods Petrocrania scabiosa (Late Ordovician) and Crania craniolaris (Late Cretaceous) are analysed using electron backscattered diffraction (EBSD) to provide crystallographic data at high spatial resolution in the structural context. EBSD analyses show that P. scabiosa maintains most of the original crystallographic signature, including data from individual calcite tablets and laminae, while C. craniolaris only retains fragmentary crystallographic data reflecting the crystallographic continuity of tablets across laminae. Data show that the preservation of the original crystallographic signature is independent of that of shell ultrastructure and geological time. In addition, results allow us to propose a series of steps in the evolution of 'crystallographic loss' due to diagenesis.     

Electron backscatter diffraction (EBSD) as a tool for detection of coral diagenesis

Fine-scale structures of intact modern and fossil coralline skeletons were analysed to determine alteration to secondary cements and phases using electron backscatter diffraction (EBSD). EBSD analysis revealed secondary aragonite cements in endolithic borings in the modern skeleton and whole dissepiments of the fossil skeleton replaced by calcite, despite X-ray diffraction (XRD) bulk analysis of the general area suggesting only aragonite was present. Non-destructive, in situ screening of coral samples by EBSD analysis provides a valuable tool for assessing the extent of alteration and can determine which areas may produce more reliable climate proxy data. Communicated by Geology Editor Dr. Bernhard Riegl     

Electron backscatter diffraction (EBSD) analysis of maniraptoran eggshells with important implications for microstructural and taphonomic interpretations

Electron backscatter diffraction (EBSD) is a useful tool for gathering crystallographic information from carbonate biominerals because it maps out the orientation of crystal grains very precisely. EBSD has become popular in invertebrate palaeontology but its application in vertebrate palaeontology remains limited. However, the study of fossil eggshells is a field where EBSD has wide potential applicability and provides a quantitative approach to fossil eggshell research as well as new qualitative data. Here we analyse fossil and extant maniraptoran dinosaur eggshells using EBSD analysis emphasizing four different aspects. The mapping imaging clarifies previously ambiguous characters such as squamatic ultrastructure and allows a more objective evaluation of avian and non‐avian maniraptoran eggshell. In particular, our results imply that the presence of an external zone in the manirpatoran eggshell is not diagnostic of avian eggshell. EBSD analysis can be also used for differentiating true pore canals from cracks in the eggshell radial section, thereby determining the biological genuineness or otherwise of a pore‐like structure. Finally, the misorientation angle distribution of the material shows a clear dichotomy that may reflect reproductive brooding strategy, although further studies on contact incubation of palaeognaths and neognaths are needed to confirm this.     

Multiscale structure of calcite fibres of the shell of the brachiopod Terebratulina retusa

The shells of rhynchonelliform brachiopods have an outer (primary) layer of acicular calcite and an inner (secondary) layer of calcite fibres which are parallel to the shell exterior. Atomic force microscopy (AFM) reveals that these fibres are composed of large triangular nanogranules of about 600-650 nm along their long axis. The nanogranules are composites of organic and inorganic components. As the shell grows, the fibres elongate with the calcite c-axis perpendicular to the fibre axis as demonstrated by electron backscatter diffraction (EBSD). Thus, despite being a composite structure comprising granules that are themselves composites, each fibre is effectively a single crystal. The combination of AFM and EBSD reveals the details of the structure and crystallography of these fibres. This knowledge serves to identify those aspects of biological control that must be understood to enable comprehension of the biological control exerted on the construction of these exquisite biomineral structures.     

In situ 3D visualization of biomineralization matrix proteins

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.     

Studies of Biominerals Relevant to the Search for Life on Mars

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO₃) and aragonite, a metastable state of calcite. We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2–25 μm wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin. Obviously, if life existed in the past on the Red Planet, we could expect to find “ancient” biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485°C. Instead, the calcite behaves like abiotic aragonite. Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and “sample-return” Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies. ^*Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005     

Will tomorrow's mineral materials be grown?

Biomineralization, the capacity to form minerals, has evolved in a great diversity of bacterial lineages as an adaptation to different environmental conditions and biological functions. Microbial biominerals often display original properties (morphology, composition, structure, association with organics) that significantly differ from those of abiotically formed counterparts, altogether defining the ‘mineral phenotype’. In principle, it should be possible to take advantage of microbial biomineralization processes to design and biomanufacture advanced mineral materials for a range of technological applications. In practice, this has rarely been done so far and only for a very limited number of biomineral types. This is mainly due to our poor understanding of the underlying molecular mechanisms controlling microbial biomineralization pathways, preventing us from developing bioengineering strategies aiming at improving biomineral properties for different applications. Another important challenge is the difficulty to upscale microbial biomineralization from the lab to industrial production. Addressing these challenges will require combining expertise from environmental microbiologists and geomicrobiologists, who have historically been working at the forefront of research on microbe–mineral interactions, alongside bioengineers and material scientists. Such interdisciplinary efforts may in the future allow the emergence of a mineral biomanufacturing industry, a critical tool towards the development more sustainable and circular bioeconomies.     

Common crystal nucleation mechanism in shell formation of two morphologically distinct calcite brachiopods

Closely related mineral-producing organisms share common biomineralisation processes. We demonstrate that, in cases of disparate mineral structures where crystal growth mechanisms are necessarily diverse, nucleation processes are the common underlying mechanism during shell formation. Detailed crystallography in the context of shell microstructure in two morphologically distinct calcite brachiopods indicates that, despite differences in shell growth and fabric, at the centre of growth, calcite crystals nucleate with the c-axis 0001 parallel to the shell surface. Such detailed contextual crystallography of biomineralisation using electron backscatter diffraction (EBSD) will have significant applications for future research in biological and medical sciences.     

Proteomics Analysis of Avian Eggshell Matrix Proteins: Toward New Advances on Biomineralization

Eggs are widely consumed all over the world. The eggshell is its protective barrier whose original function is to protect the embryo during development. Avian eggshells are made of calcium carbonate with a small amount of organic matrix (proteins and proteoglycans). During eggshell formation, the mineral precursors interact with matrix proteins to regulate the calcification of this highly resistant biomineral. In order to better characterize the functions of matrix proteins in eggshell biominerals, many proteomics studies have been performed during the last 15 years. The chicken eggshell is the main model studied in birds, but there is a need for comparative approaches in order to determine whether there is a general protein toolkits associated with calcitic biomineralization, and to determine its components. The study by Zhu et al., reported in article number 1900011, volume 19, issue 11, is a major step forward as it is the first shell proteomics survey performed on duck. Thus, it will contribute to improved knowledge of the eggshell mineralization process and will provide new insight for shell quality improvement and to guide biomimetic efforts in material sciences.     

Fine structure of the mineralized teeth of the chiton Acanthopleura echinata (Mollusca: Polyplacophora)

The major lateral teeth of the chiton Acanthopleura echinata are composite structures composed of three distinct mineral zones: a posterior layer of magnetite; a thin band of lepidocrocite just anterior to this; and apatite throughout the core and anterior regions of the cusp. Biomineralization in these teeth is a matrix-mediated process, in which the minerals are deposited around fibers, with the different biominerals described as occupying architecturally discrete compartments. In this study, a range of scanning electron microscopes was utilized to undertake a detailed in situ investigation of the fine structure of the major lateral teeth. The arrangement of the organic and biomineral components of the tooth is similar throughout the three zones, having no discrete borders between them, and with crystallites of each mineral phase extending into the adjacent mineral zone. Along the posterior surface of the tooth, the organic fibers are arranged in a series of fine parallel lines, but just within the periphery their appearance takes on a "fish scale"-like pattern, reflective of the cross section of a series of units that are overlaid, and offset from each other, in adjacent rows. The units are approximately 2 microm wide and 0.6 microm thick and comprise biomineral plates separated by organic fibers. Two types of subunits make up each "fish scale": one is elongate and curved and forms a trough, in which the other, rod-like unit, is nestled. Adjacent rod and trough units are aligned into large sheets that define the fracture plane of the tooth. The alignment of the plates of rod-trough units is complex and exhibits extreme spatial variation within the tooth cusp. Close to the posterior surface the plates are essentially horizontal and lie in a lateromedial plane, while anteriorly they are almost vertical and lie in the posteroanterior plane. An understanding of the fine structure of the mineralized teeth of chitons, and of the relationship between the organic and mineral components, provides a new insight into biomineralization mechanisms and controls.     

Oxalate production by fungi: significance in geomycology,biodeterioration and bioremediation

Oxalate is a key metabolite that plays a significant role in many metal and mineral transformations mediated by fungi. Metal and mineral transformations are central to geomycological processes including nutrient and element cycling, rock, mineral and metal transformations, bioweathering and mycogenic biomineral formation. Some fungal transformations have potential applications in environmental biotechnology, e.g. metal and radionuclide leaching, biorecovery, detoxification and bioremediation, and in the production or deposition of biominerals or metallic elements with catalytic or other properties. Metal and mineral transformations may also result in adverse effects when these processes result in biodeterioration of natural and synthetic materials, rock and mineral-based building materials (e.g. concrete), biocorrosion of metals, alloys and related substances, and adverse effects on radionuclide speciation, mobility and containment. Oxalate is ubiquitous in all these contexts. This paper seeks to draw together salient information from environmental and applied research to emphasize the importance of oxalate in geomycology, biodeterioration, environmental biotechnology and bioremediation.     

The sea urchin (<Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis>) test and spine proteomes

Background  

The organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis.     

Computer-integrated polarisation (CIP) in the analysis of fossils: a case of study in a Palaeozoic coral (Sinopora,Syringoporicae, Carboniferous)

Computer-integrated polarisation (CIP) method has been applied satisfactorily in the study of fossils skeletons of Sinopora (tabulate coral, Auloporida and Carboniferous). A previous characterisation of sample by scanning electron microscopy, atomic force microscopy and cathodoluminescence (CL) with the purpose of distinguishing the diagenetical alteration was done. Subsequently, a crystallographic comparison between CIP and electron-backscattering diffraction has been made getting a very good correlation between both methods. The CIP method allows obtaining c-axis orientation images, pole figures, and measure and mapping the misorientation of uniaxial biominerals in recent and fossil skeletons. This technique can only be used in uniaxial biominerals (calcite, quartz and hydroxylapatite), limiting its use for biaxial or bimineralic and polimineralic biominerals. CIP method has good spatial resolution (limited by camera); in our example 90 nm. The main advantage of this technique, versus other with similar properties, is the fast acquisition of data in low and high magnifications. This method does not require special treatment of samples and can be very useful for the analysis of microstructures in thin and ultra-thin sections. CIP method detects diagenetic alterations in fossil skeletons by modifications in the inner arrangement of biominerals, which combined with CL offers valuable geochemical and crystallographic information.     

Pennsylvanian sea level cycles, nutrient availability and brachiopod paleoecology

As with modern organisms, the spatial and temporal distribution of fossil communities was controlled by both the physical setting in which the organisms lived and by the organisms' physiology and interactions. By studying the sedimentological and geochemical context of fossil communities, it is possible to assess the relative importance of the physical setting and the organisms' physiology. Comparison of Pennsylvanian brachiopod associations with changing sedimentological context (water depth/facies) and nutrient availability indicates that body size is a function of water depth and nutrient availability for most spire-bearing (athyridids and spiriferids) brachiopods but rarely for productid brachiopods. Spire-bearing brachiopods dominate the associations in high-nutrient settings, and productid brachiopods dominate the associations in low-nutrient settings. This difference suggests that physiological differences between brachiopod orders, such as lophophore filtering efficiency, play an important role in controlling their distribution.     

In-depth,high-accuracy proteomics of sea urchin tooth organic matrix

Background  

The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis.     

Fungal biotransformation of zinc silicate and sulfide mineral ores

In this work, several fungi with geoactive properties, including Aspergillus niger, Beauveria caledonica and Serpula himantioides, were used to investigate their potential bioweathering effects on zinc silicate and zinc sulfide ores used in zinc extraction and smelting, to gain understanding of the roles that fungi may play in transformations of such minerals in the soil, and effects on metal mobility. Despite the recalcitrance of these minerals, new biominerals resulted from fungal interactions with both the silicate and the sulfide, largely resulting from organic acid excretion. Zinc oxalate dihydrate was formed through oxalate excretion by the test fungi and the mineral surfaces showed varying patterns of bioweathering and biomineral formation. In addition, calcium oxalate was formed from the calcium present in the mineral ore fractions, as well as calcite. Such metal immobilization may indicate that the significance of fungi in effecting metal mobilization from mineral ores such as zinc silicate and zinc sulfide is rather limited, especially if compared with bacterial sulfide leaching. Nevertheless, important bioweathering activities of fungi are confirmed which could be of local significance in soils polluted by such materials, as well as in the mycorrhizosphere.     

The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin

The major Biomphalaria glabrata shell matrix protein of 19.6 kDa was isolated by preparative electrophoresis and sequenced. The sequence of 148 amino acids showed 32% sequence identity to mammalian dermatopontin sequences and 34-37% identity to two invertebrate dermatopontins described previously. A unique feature of the shell matrix dermatopontin was the presence of a single N-glycosylation consensus sequence, the asparagine of which was completely modified with a pentasaccharide. Sequence analysis of this short N-glycan by mass spectrometry and carbohydrate composition analysis indicated that it was the ubiquitous N-glycan core oligosaccharide with the exception that the terminal mannoses were 3-O-methylated. Dermatopontin is widespread in mammalian extracellular matrices, including the matrix of biominerals such as bone and teeth. Its occurrence in an invertebrate biomineral indicates that such phylogenetically distant biomineral-forming systems as vertebrate bone and mollusk shell share components which have undergone surprisingly few changes during a long evolution.     

Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer

The major difference between inorganic minerals and biominerals is the presence of an organic matrix consisting of proteins, glycoproteins, proteoglycans, and polysaccharides, which is synthesized by specialized cells under genetic control before or during mineralization. The organic matrix is thought to play a major role in the assembly of the biomineral and determination of its mechanical properties. The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of a biomineral using up-to-date MS-based technology. We identified 520 proteins in this matrix including the ten matrix proteins already known before. The identified proteins were divided into three abundance groups using the exponentially modified protein abundance index described recently which was roughly calibrated with the few known data on protein yield derived from Edman sequence analysis. A small group of 32 highly abundant proteins contained the presently known eggshell-specific proteins and all of the other known eggshell matrix constituents identified before with much less sensitive conventional methods. The present study, which is the first comprehensive proteomic study of a vertebrate biomineral, is intended as a starting point for the detailed molecular characterization of eggshell matrix proteins, their interactions in the matrix network and functional studies.     

Trichoderma koningii as a biomineralizing fungous agent of calcium oxalate crystals in typical Argiudolls of the Los Padres Lake natural reserve (Buenos Aires, Argentina)

The aim of the present study, performed on typical Argiudolls in a natural reserve with little or no anthropic impact, was to characterize the fungous biomineralizing process of calcium oxalate crystals in organic horizons of the soil. The chosen sites possessed different plant cover, identified as acacia woods and grassy meadows with particular micro environmental conditions that have differing effects in the process of biomineralization. The contribution of the plant material in the soil is a key factor since 1) it generates the particular composition of the organic horizons, 2) it determines the nature of decomposing organisms, and 3) it affects the presence, composition and development of biominerals. According to the results obtained, the acacia woods prove to be a site comparatively more favorable to the fungous biomineralizing process. This makes itself manifest in the greater abundance and development of crystals in the organic horizons of the soil, resulting in whewellite (CaC2O4.H2O) and weddellite (CaC2O4.(2+x) H2O) regarding biomineral species developed, the latter being the major component. The observation of both species of biominerals is noteworthy since it represents the first cited in the country. The isolated fungous organisms were Trichoderma koningii, and Absidia corymbifera. T. koningii was identified as the most active biomineralizing organism thus constituting the first reference to indicate this species as a biomineral producing agent.