Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

A multifunctional cascade enzyme system for enhanced starvation/chemodynamic combination therapy against hypoxic tumors.

Starvation therapy has shown promise as a cancer treatment, but its efficacy is often limited when used alone. In this work, a multifunctional nanoscale cascade enzyme system, named CaCO3@MnO2-NH2@GOx@PVP (CMGP), was fabricated for enhanced starvation/chemodynamic combination cancer therapy. CMGP is composed of CaCO3 nanoparticles wrapped in a MnO2 shell, with glucose oxidase (GOx) adsorbed and modified with polyvinylpyrrolidone (PVP). MnO2 decomposes H2O2 in cancer cells into O2, which enhances the efficiency of GOx-mediated starvation therapy. CaCO3 can be decomposed in the acidic cancer cell environment, causing Ca2+ overload in cancer cells and inhibiting mitochondrial metabolism. This synergizes with GOx to achieve more efficient starvation therapy. Additionally, the H2O2 and gluconic acid produced during glucose consumption by GOx are utilized by MnO2 with catalase-like activity to enhance O2 production and Mn2+ release. This process accelerates glucose consumption, reactive oxygen species (ROS) generation, and CaCO3 decomposition, promoting the Ca2+ release. CMGP can alleviate tumor hypoxia by cycling the enzymatic cascade reaction, which increases enzyme activity and combines with Ca2+ overload to achieve enhanced combined starvation/chemodynamic therapy. In vitro and in vivo studies demonstrate that CMGP has effective anticancer abilities and good biosafety. It represents a new strategy with great potential for combined cancer therapy.

Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Calcium carbonate precipitating extremophilic bacteria in an Alpine ice cave.

Extensive research has provided a wealth of data on prokaryotes in caves and their role in biogeochemical cycles. Ice caves in carbonate rocks, however, remain enigmatic environments with limited knowledge of their microbial taxonomic composition. In this study, bacterial and archaeal communities of the Obstans Ice Cave (Carnic Alps, Southern Austria) were analyzed by next-generation amplicon sequencing and by cultivation of bacterial strains at 10 °C and studying their metabolism. The most abundant bacterial taxa were uncultured Burkholderiaceae and Brevundimonas spp. in the drip water, Flavobacterium, Alkanindiges and Polaromonas spp. in the ice, Pseudonocardia, Blastocatella spp., uncultured Pyrinomonadaceae and Sphingomonadaceae in carbonate precipitates, and uncultured Gemmatimonadaceae and Longimicrobiaceae in clastic cave sediments. These taxa are psychrotolerant/psychrophilic and chemoorganotrophic bacteria. On a medium with Mg2+/Ca2+ = 1 at 21 °C and 10 °C, 65% and 35% of the cultivated strains precipitated carbonates, respectively. The first ~ 200 µm-size crystals appeared 2 and 6 weeks after the start of the cultivation experiments at 21 °C and 10 °C, respectively. The crystal structure of these microbially induced carbonate precipitates and their Mg-content are strongly influenced by the Mg2+/Ca2+ ratio of the culture medium. These results suggest that the high diversity of prokaryotic communities detected in cryogenic subsurface environments actively contributes to carbonate precipitation, despite living at the physical limit of the presence of liquid water.

The Cd immobilization mechanisms in paddy soil through ureolysis-based microbial induced carbonate precipitation: Emphasis on the coexisting cations and metatranscriptome analysis.

Microbial induced carbonate precipitation (MICP) can immobilize metals and reduce their bioavailability. However, little is known about the immobilization mechanism of Cd in the presence of soil cations and the triggered gene expression and metabolic pathways in paddy soil. Thus, microcosmic experiments were conducted to study the fractionation transformation of Cd and metatranscriptome analysis. Results showed that bioavailable Cd decreased from 0.62 to 0.29 mg/kg after 330 d due to the MICP immobilization. This was ascribed to the increase in carbonate bound, Fe-Mn oxides bound, and residual Cd. The underlying immobilization mechanisms could be attributed to the formation of insoluble Cd-containing precipitates, the complexation and lattice substitution with carbonate and Fe, Mn and Al (hydr)oxides, and the adsorption on functional group on extracellular polymers of cell. During the MICP immobilization process, up-regulated differential expression urease genes were significantly enriched in the paddy soil, corresponding to the arginine biosynthesis, purine metabolism and atrazine degradation. The metabolic pathway of bacterial chemotaxis, flagellum assembly, and peptidoglycan biosynthesis and the expression of cadA gene related to Cd excretion enhanced Cd resistance of soil microbiome. Therefore, this study provided new insights into the immobilization mechanisms of Cd in paddy soils through ureolysis-based MICP process.

Sex differences in the anticompulsive-like effect of memantine: Involvement of nitric oxide pathway but not AMPA receptors.

Memantine, an N-Methyl-D-Aspartate (NMDA) antagonist, has been examined as a potential treatment for Obsessive-Compulsive Disorder (OCD). Yet, there is limited knowledge regarding how it works to reduce compulsive behaviour and whether it has different effects on individuals based on their sex. Herein, we investigated if there are sex differences in the anticompulsive-like effect of memantine in adult Swiss mice. Additionally, we explored whether the nitric oxide (NO) pathway and α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors play a role in memantine's effects. To start, we assessed the impact of a single intraperitoneal dose of memantine (at 3, 5, and 10 mg/kg) on behaviours exhibited in the open field test (OFT) and the marble-burying test (MBT), the latter being a predictive test for anticompulsive effects. All doses of memantine reduced marble-burying behaviour in both male and female mice without affecting their locomotor activity in the OFT. This anticompulsive-like effect was also confirmed in another predictive test, the nest-building test, with the highest memantine dose (10 mg/kg) reducing nest-building behaviour without significant differences between male and female mice. We observed that pre-treatment with L-arginine, a NO precursor, mitigated the anticompulsive-like effect of memantine in male mice but had no effect in female mice in the MBT. Finally, NBQX, an AMPA receptor antagonist, did not block the anticompulsive-like effect of memantine. In summary, our study suggests that the anticompulsive-like effect of memantine does not appear to be sex-specific, does not depend on AMPA receptors, and involves the NO pathway primarily in male mice.

Effect of (in)organic additives on microbially induced calcium carbonate precipitation.

AIM: This study aimed to understand the morphological effects of (in)organic additives on microbially induced calcium carbonate precipitation (MICP). METHODS AND RESULTS: MICP was monitored in real time in the presence of (in)organic additives: bovine serum albumin (BSA), biofilm surface layer protein A (BslA), magnesium chloride (MgCl2), and poly-l-lysine. This monitoring was carried out using confocal microscopy to observe the formation of CaCO3 from the point of nucleation, in comparison to conditions without additives. Complementary methodologies, namely scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction, were employed to assess the visual morphology, elemental composition, and crystalline structures of CaCO3, respectively, following the crystals' formation. The results demonstrated that in the presence of additives, more CaCO3 crystals were produced at 100 min compared to the reaction without additives. The inclusion of BslA resulted in larger crystals than reactions containing other additives, including MgCl2. BSA induced a significant number of crystals from the early stages of the reaction (20 min) but did not have a substantial impact on crystal size compared to conditions without additives. All additives led to a higher content of calcite compared to vaterite after a 24-h reaction, with the exception of MgCl2, which produced a substantial quantity of magnesium calcite. CONCLUSIONS: The work demonstrates the effect of several (in)organic additives on MICP and sets the stage for further research to understand additive effects on MICP to achieve controlled CaCO3 precipitation.

Soil microbial improvement using enriched vinasse as a new abundant waste.

This study proposes the use of vinasse, an inexpensive and readily available waste biopolymer, as a fundamental component of a waste culture medium that can enhance the effectiveness and cost-efficiency of the microbial-induced calcite precipitation (MICP) method for sustainable soil improvement. Vinasse enriched with urea, sodium caseinate, or whey protein concentrate is employed to optimize bacterial growth and urease activity of Sporosarcina pasteurii (S. pasteurii) bacterium. The best culture medium is analyzed using Taguchi design of experiments (TDOE) and statistical analysis, considering the concentration of vinasse and urea as effective parameters during growth time. To test the best culture medium for bio-treated soil, direct shear tests were performed on loose and bio-treated sand. The results demonstrate a substantial cost reduction from $0.455 to $0.005 per liter when using the new culture medium (vinasse and urea) compared to the conventional Nutrient Broth (NB) culture medium. Additionally, the new medium enhances soil shear strength, increasing the friction angle by 2.5 degrees and cohesion to 20.7 kPa compared to the conventional medium. Furthermore, the recycling of vinasse as a waste product can promote the progress of a circular economy and reduce environmental pollution. As ground improvement is essential for many construction projects, especially those that require high shear strength or are built on loose soil, this study provides a promising approach to achieving cost-effective and sustainable soil microbial improvement using enriched vinasse.

Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties.

Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient availability. The high content of CaCO3 leads to the increment of the soil alkalinity, which results in a lowering of the nutrient availability causing a challenge for the agriculture in these soils. In this study, the calcite-solubilizing potential of the diazotrophic Azotobacter salinestris YRNF3 was investigated in vitro as a probable bio-agent for enhancing the calcareous soils properties such as soil pH and nutrient availability. Twelve diazotrophic bacterial strains were isolated from wheat rhizosphere collected from different wheat-cultivated fields in five Egyptian governorates. Using Nessler's reagent, all isolated bacterial strains were found to have the ability to produce ammonia. By amplification of nifH gene, a PCR product of 450 bp was obtained for all isolated bacterial strains. For each isolate, three biological and three technical replicates were applied. All isolated diazotrophic bacteria were qualitatively screened for their calcite-solubilizing ability. To quantitatively investigate the calcite-solubilizing potential of A. salinestris YRNF3 in vitro, changes in the contents of soluble calcium (Ca2+), bicarbonate (HCO3-), total nitrogen (TN), total protein (TP), and pH were daily measured in its culture filtrate along 10 days of incubation. The results showed that the pH values in the culture filtrate ranged from 5.73 to 7.32. Concentration of Ca2+ and HCO3- in the culture filtrate significantly decreased with the increment in the incubation time, while concentration of TN increased along the time. The highest TN concentration (0.0807 gL-1) was observed on days 4 and 5, compared to that of the day 0 (0.0014 gL-1). Content of TP in the culture filtrate also significantly increased along the incubation period. The highest TP content was recorded in day 4 (0.0505%), while no TP content was recorded on day 0. Furthermore, data obtained revealed that A. salinestris YRNF3 produced acid phosphatase at low activity (5.4 U mL-1). HPLC analysis of the culture filtrate indicated production of different organic acids, namely lactic acid (82.57 mg mL-1), formic acid (46.8 mg mL-1), while acetic acid was detected in a low quantity (3.11 mg mL-1). For each analysis, three replicates of each treatment were analyzed. Means of the tested treatments were compared using Tukey's HSD test at p ≤ 0.05. In conclusion, findings of this work suggested that A. salinestris YRNF3 has the potential to be a probable bioagent to be used for the reclamation of the calcareous soils by solubilizing CaCO3, improving soil fertility, and promoting plant growth. However, further studies are needed to investigate its field application and their long-term effects on the soil properties and plant productivity. To the best of the author's knowledge, this is the first study reporting the calcite-solubilizing ability of a nitrogen-fixing bacteria. Having these two abilities by one microorganism is a unique feature, which qualifies it as a promising bioagent for reclamation of the calcareous soils.

Applying the first microcapsule-based self-healing microbial-induced calcium carbonate materials to prevent the migration of Pb ions.

Lead (Pb) accumulation can lead to serious threats to surrounding environments and damage to the liver and kidneys. In the past few years, microbial-induced carbonate precipitation (MICP) technology has been widely applied to achieve Pb immobilization due to its environmentally friendly nature. However, harsh pH conditions can cause the instability of the carbonate precipitation to degrade or dissolve, increasing the potential of Pb2+ migration into nearby environments. In this study, microcapsule-based self-healing microbial-induced calcium carbonate (MICC) materials were applied to prevent Pb migration. The highest sporulation rate of 95.8% was attained at 7 g/L yeast extract, 10 g/L NH4Cl, and 3.6 g/L Mn2+. In the germination phase, the microcapsule not only prevented the bacterial spores from being threatened by the acid treatment but secured their growth and reproduction. Micro analysis also revealed that cerussite, calcite, and aragonite minerals were present, while extracellular polymeric substances (EPSs) were identified via Fourier transform infrared spectroscopy (FTIR). These results confirm their involvement in combining Pb2+ and Ca2+. The immobilization efficiency of above 90% applied to MICC materials was attained, while it of below 5% applied to no MICC use was attained. The findings explore the potential of applying microcapsule-based self-healing MICC materials to prevent Pb ion migration when the calcium carbonate degrades under harsh pH conditions.

Insight into biomolecular interaction-based non-classical crystallization of bacterial biocement.

In an attempt to draw a correlation between calcium carbonate (CaCO3) precipitation and biomacromolecules such as extracellular polymeric substances and enzyme activity in biomineralizing microbe, this report aims to elucidate the ureolytic and ammonification route in Paenibacillus alkaliterrae to explore the possible role of organic biomolecule(s) present on cell surface in mediating nucleation and crystallization of biogenic CaCO3. After 168 h of biomineralization in ureolysis and ammonification, 2.2 g/l and 0.87 g/l of CaCO3 precipitates were obtained, respectively. The highest carbonic anhydrase activity (31.8 µmoles/min/ml) was evidenced in ammonification as opposed to ureolysis (24.8 µmoles/min/ml). Highest urease activity reached up to 9.26 µmoles/min/ml in ureolytic pathway. Extracellular polymeric substances such as polysaccharides and proteins were found to have a vital role not only in the nucleation and crystal growth but also in addition direct polymorphic fate of CaCO3 nanoparticles. EPS production was higher during ammonification (3.1 mg/ml) than in ureolysis (0.72 mg/ml). CaCO3 nanoparticle-associated proteins were found to be 0.82 mg/ml in ureolysis and 0.56 mg/ml in ammonification. After 30 days of biomineralization, all the polymorphic forms stabilized to calcite in ureolysis but in ammonification vaterite predominated. In our study, we showed that organic template-mediated prokaryotic biomineralization follows the non-classical nucleation and varying proportions of these organic components causes selective polymorphism of CaCO3 nanoparticles. Overall, the findings are expected to further the fundamental understanding of enzymes, EPS-driven non-classical nucleation of CaCO3, and we foresee the design of fit-for-purpose futuristic biominerals arising from such renewed understanding of biomineralization. KEY POINTS: • Organic-inorganic interface of cell surface promote crystallization of biominerals • Carbohydrate and proteins in the interface results selective polymorphism of CaCO3 • Calcite stabilized at 30 days in ureolysis, vaterite-calcite mix in ammonification.

Digestibility of calcium in calcium-containing ingredients and requirements for digestible calcium by growing pigs.

The concentration of Ca in plant feed ingredients is low compared with the requirement for pigs and most Ca in diets for pigs is provided by limestone and Ca phosphate. To determine digestibility values for Ca that are additive in mixed diets, the standardized total tract digestibility (STTD) of Ca needs to be calculated, and the STTD of Ca by growing pigs in most Ca-containing ingredients has been reported. Although Ca is an inexpensive nutrient compared with P and amino acids, excess Ca needs to be avoided because excess dietary Ca results in reduced P digestibility, reduced feed intake, and reduced growth performance of pigs. Recent data indicate that most diets produced for pigs in the United States and Europe contain ~0.20 percentage units more Ca than formulated, which likely is because of the use of limestone as a carrier in feed additives or as a flow agent in other ingredients. An excess of this magnitude without a corresponding excess of P will result in a reduction in daily gain of growing pigs by 50 to 100 g. Greater emphasis, therefore, needs to be placed on determining the concentration of Ca in diets for pigs. Microbial phytase increases the digestibility of both Ca and P and it is, therefore, important that the release of both Ca and P by phytase is considered in diet formulation. However, due to the relationship between Ca and P in postabsorptive metabolism, diets need to be formulated based on a ratio between digestible Ca and digestible P. To maximize average daily gain, this ratio needs to be less than 1.40:1.0 in diets for weanling pigs, and the ratio needs to be reduced as the body weight of pigs increases. In contrast, to maximize bone ash, the digestible Ca to digestible P ratio needs to increase from 1.67:1.0 in 11 to 25 kg pigs to 2.33:1.0 in finishing pigs. Gestating sows have reduced STTD and retention of Ca and P compared with growing pigs and formulation of diets for sows based on digestibility values obtained in growing pigs will result in inaccuracies in the provision of Ca and P. There is, however, a lack of data for the digestibility of Ca and P by gestating and lactating sows, and responses to microbial phytase by sows are not fully understood. There is, therefore, a need for research to generate more data in this area. In the present review, a summary of data for the digestibility of Ca in feed ingredients for pigs and estimates for the requirement for digestible Ca by growing and finishing pigs are provided.

Concentration of Ca in most plant feed ingredients is low compared with the requirement for pigs and dietary Ca is, therefore, mostly provided by limestone and calcium phosphates. Although Ca is an inexpensive nutrient compared with P and amino acids, excess dietary Ca may result in reduced P digestibility, feed intake, and growth performance of pigs. Excretion of P from pigs is increased if dietary Ca is provided above the requirement, which may increase environmental pollution. Therefore, determination of the digestibility of Ca in dietary sources of Ca and formulation of diets based on the ratio between digestible Ca and digestible P are needed to reduce Ca and P excretions. This review provides a summary of values for the digestibility of Ca in feed ingredients and also provides estimates for the requirement for digestible Ca by weanling and growing-finishing pigs. Summarized data from experiments that determined the requirement for digestible Ca demonstrated that there are linear correlations between body weight of growing-finishing pigs and digestible Ca to digestible P ratios needed to maximize growth or bone ash.

Multi-omic insights into the formation and evolution of a novel shell microstructure in oysters.

BACKGROUND: Molluscan shell, composed of a diverse range of architectures and microstructures, is a classic model system to study the relationships between molecular evolution and biomineralized structure formation. The shells of oysters differ from those of other molluscs by possessing a novel microstructure, chalky calcite, which facilitates adaptation to the sessile lifestyle. However, the genetic basis and evolutionary origin of this adaptive innovation remain largely unexplored. RESULTS: We report the first whole-genome assembly and shell proteomes of the Iwagaki oyster Crassostrea nippona. Multi-omic integrative analyses revealed that independently expanded and co-opted tyrosinase, peroxidase, TIMP genes may contribute to the chalky layer formation in oysters. Comparisons with other molluscan shell proteomes imply that von Willebrand factor type A and chitin-binding domains are basic members of molluscan biomineralization toolkit. Genome-wide identification and analyses of these two domains in 19 metazoans enabled us to propose that the well-known Pif may share a common origin in the last common ancestor of Bilateria. Furthermore, Pif and LamG3 genes acquire new genetic function for shell mineralization in bivalves and the chalky calcite formation in oysters likely through a combination of gene duplication and domain reorganization. CONCLUSIONS: The spatial expression of SMP genes in the mantle and molecular evolution of Pif are potentially involved in regulation of the chalky calcite deposition, thereby shaping the high plasticity of the oyster shell to adapt to a sessile lifestyle. This study further highlights neo-functionalization as a crucial mechanism for the diversification of shell mineralization and microstructures in molluscs, which may be applied more widely for studies on the evolution of metazoan biomineralization.

Insights in MICP dynamics in urease-positive Staphylococcus sp. H6 and Sporosarcina pasteurii bacterium.

Microbially induced calcite precipitation (MICP) is an efficient and eco-friendly technique that has attracted significant interest for resolving various problems in the soil (erosion, improving structural integrity and water retention, etc.), remediation of heavy metals, production of self-healing concrete or restoration of different concrete structures. The success of most common MICP methods depends on microorganisms degrading urea which leads to the formation of CaCO3 crystals. While Sporosarcina pasteurii is a well-known microorganism for MICP, other soil abundant microorganisms, such as Staphylococcus bacteria have not been thoroughly studied for its efficiency in bioconsolidation though MICP is a very important proccess which can ensure soil quality and health. This study aimed to analyze MICP process at the surface level in Sporosarcina pasteurii and a newly screened Staphylococcus sp. H6 bacterium as well as show the possibility of this new microorganism to perform MICP. It was observed that Staphylococcus sp. H6 culture precipitated 157.35 ± 3.3 mM of Ca2+ ions from 200 mM, compared to 176 ± 4.8 mM precipitated by S. pasteurii. The bioconsolidation of sand particles was confirmed by Raman spectroscopy and XRD analysis, which indicated the formation of CaCO3 crystals for both Staphylococcus sp. H6 and S. pasteurii cells. The water-flow test suggested a significant reduction in water permeability in bioconsolidated sand samples for both Staphylococcus sp. H6 and S. pasteurii. Notably, this study provides the first evidence that CaCO3 precipitation occurs on the surface of Staphylococcus and S. pasteurii cells within the initial 15-30 min after exposure to the biocementation solution. Furthermore, Atomic force microscopy (AFM) indicated rapid changes in cell roughness, with bacterial cells becoming completely coated with CaCO3 crystals after 90 min incubation with a biocementation solution. To our knowledge, this is the first time where atomic force microscopy was used to visualize the dynamic of MICP on cell surface.

Differences in carbonate chemistry up-regulation of long-lived reef-building corals.

With climate projections questioning the future survival of stony corals and their dominance as tropical reef builders, it is critical to understand the adaptive capacity of corals to ongoing climate change. Biological mediation of the carbonate chemistry of the coral calcifying fluid is a fundamental component for assessing the response of corals to global threats. The Tara Pacific expedition (2016-2018) provided an opportunity to investigate calcification patterns in extant corals throughout the Pacific Ocean. Cores from colonies of the massive Porites and Diploastrea genera were collected from different environments to assess calcification parameters of long-lived reef-building corals. At the basin scale of the Pacific Ocean, we show that both genera systematically up-regulate their calcifying fluid pH and dissolved inorganic carbon to achieve efficient skeletal precipitation. However, while Porites corals increase the aragonite saturation state of the calcifying fluid (Ωcf) at higher temperatures to enhance their calcification capacity, Diploastrea show a steady homeostatic Ωcf across the Pacific temperature gradient. Thus, the extent to which Diploastrea responds to ocean warming and/or acidification is unclear, and it deserves further attention whether this is beneficial or detrimental to future survival of this coral genus.

Microbially Induced Calcium Carbonate Precipitation by Sporosarcina pasteurii: a Case Study in Optimizing Biological CaCO3 Precipitation.

Current production of traditional concrete requires enormous energy investment that accounts for approximately 5 to 8% of the world's annual CO2 production. Biocement is a building material that is already in industrial use and has the potential to rival traditional concrete as a more convenient and more environmentally friendly alternative. Biocement relies on biological structures (enzymes, cells, and/or cellular superstructures) to mineralize and bind particles in aggregate materials (e.g., sand and soil particles). Sporosarcina pasteurii is a workhorse organism for biocementation, but most research to date has focused on S. pasteurii as a building material rather than a biological system. In this review, we synthesize available materials science, microbiology, biochemistry, and cell biology evidence regarding biological CaCO3 precipitation and the role of microbes in microbially induced calcium carbonate precipitation (MICP) with a focus on S. pasteurii. Based on the available information, we provide a model that describes the molecular and cellular processes involved in converting feedstock material (urea and Ca2+) into cement. The model provides a foundational framework that we use to highlight particular targets for researchers as they proceed into optimizing the biology of MICP for biocement production.

In situ mapping of biomineral skeletal proteins by molecular recognition imaging with antibody-functionalized AFM tips.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

Unique evolution of foraminiferal calcification to survive global changes.

Foraminifera, the most ancient known calcium carbonate-producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidification, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca2+) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.

A promising approach for simultaneous removal of ammonia and multiple heavy metals from landfill leachate by carbonate precipitating bacterium.

The effective and cheap remediation of ammonia (NH+4) and multiple heavy metals from landfill leachate is currently a grand challenge. In this study, Paracoccus denitrificans AC-3, a bacterial strain capable of heterotrophic nitrification aerobic denitrification (HNAD) and carbonate precipitation, exhibited good tolerance to a variety of heavy metals and could remove 99.70% of NH+4, 99.89% of zinc (Zn2+), 97.42% of cadmium (Cd2+) and 46.19% of nickel (Ni2+) simultaneously after 24 h of incubation. The conversion pathway of NH+4 by strain AC-3 was dominated by assimilation (84.68%), followed by HNAD (14.93%), and the increase in environmental pH was mainly dependent on assimilation rather than HNAD. Calcium (Ca2+) primarily played four roles in heavy metal mineralization: (ⅰ) improving bacterial tolerance to heavy metals; (ⅱ) ensuring the HNAD capacity of strain AC-3; (ⅲ) co-precipitating with heavy metals; and (ⅳ) precipitating into calcite to adsorb heavy metals. The heavy metals removal mechanisms were mainly calcite adsorption and formation of carbonate and hydroxide precipitation for Zn2+, co-precipitation for Cd2+, and adsorption for Ni2+. The Zn2+, Cd2+, and Ni2+ precipitates displayed unique morphologies. This research provided a promising biological resource for the simultaneous remediation of NH+4 and heavy metals from landfill leachate.

Outer fold is sole effective tissue among three mantle folds with regard to oyster shell colour.

Molluscs constitute the second largest phylum of animals in the world, and shell colour is one of their most important phenotypic characteristics. In this study, we found among three folds on the mantle edge of oyster, only the outer fold had the same colour as the shell. Transcriptome and mantle cutting experiment indicated that the outer fold may be mainly reflected in chitin framework formation and biomineralisation. There were obvious differences in SEM structure and protein composition between the black and white shell periostraca. The black shell periostraca had more proteins related to melanin biosynthesis and chitin binding. Additionally, we identified an uncharacterized protein gene (named as CgCBP) ultra-highly expressed only in the black outer fold and confirmed its function of chitin-binding and CaCO3 precipitation promoting. RNAi also indicated that CgCBP knockdown could change the structure of shell periostracum and reduce shell pigmentation. All these results suggest that the mantle outer fold plays multiple key roles in shell periostraca bioprocessing, and shell periostracum structure affected by chitin-binding protein is functionally correlated with shell pigmentation. The investigation of oyster shell periostracum structure and shell colour will provide a better understanding in pigmentation during biological mineralisation in molluscs.