Calcium carbonate precipitation by two groups of moderately halophilic microorganisms at different temperatures and salt concentrations

Carbonate crystal formation by 48 strains of moderately halophilic microorganisms currently assigned to the genusFlavobacterium andAcinetobacter has been investigated. Strains were grown at different salt concentrations (2.5%, 7.5%, and 20%, wt/vol, total salts) and temperatures (22°C and 32°C). All the strains tested were capable of precipitating calcium carbonate as calcite, but onlyAcinetobacter strains formed aragonite. High temperature and low ionic strength of medium favored crystal formation. The influence of species specificity on the type of crystal precipitated by moderately halophilic microorganisms and their possible role in active precipitation in nature are discussed.     

Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species

To obtain a restoring and protective calcite layer on degraded limestone, five different strains of the Bacillus sphaericus group and one strain of Bacillus lentus were tested for their ureolytic driven calcium carbonate precipitation. Although all the Bacillus strains were capable of depositing calcium carbonate, differences occurred in the amount of precipitated calcium carbonate on agar plate colonies. Seven parameters involved in the process were examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, extracellular polymeric substances (EPS)-production, biofilm formation, ζ-potential and deposition of dense crystal layers. The strain selection for optimal deposition of a dense CaCO₃ layer on limestone, was based on decrease in water absorption rate by treated limestone. Not all of the bacterial strains were effective in the restoration of deteriorated Euville limestone. The best calcite precipitating strains were characterised by high ureolytic efficiency, homogeneous calcite deposition on limestone cubes and a very negative ζ-potential.     

A rare mineral,vaterite, acts as a shock absorber in the eggshell of a communally nesting bird

Birds’ eggshells are primarily composed of calcite, an abundant polymorph of calcium carbonate (CaCO₃). However, the eggshells of some species are coated with spherules of vaterite, a rare and thermodynamically unstable polymorph of CaCO₃, the function of which remains unknown. We experimentally tested the mechanical and physiological effects of the vaterite coating on eggshells of the Greater Ani Crotophaga major, a tropical cuckoo. Vaterite removal did not affect vapour conductance rates across the eggshell, indicating that the vaterite coating does not influence gas exchange during embryonic development. However, nanoindentation revealed that the hardness and elasticity of vaterite is similar to that of calcite, and white light interferometry showed that the vaterite layer increased the total thickness of the shell cuticle by up to 10%. Furthermore, calculations of contact mechanics found that when two eggs come into contact, the depth of the surface deformation caused by the contact is far less than the thickness of the vaterite coating. These results suggest that the layer of vaterite spherules may act as a shock absorber for the underlying calcite shell, protecting it from mechanical damage caused by collision with other eggs in the nest and reducing the risk of eggshell fracture during incubation.     

The perceptual and chemical bases of egg discrimination in communally nesting greater anis Crotophaga major

The eggshells of communally breeding greater anis Crotophaga major consist of a blue‐green pigmented calcite matrix overlaid by a chalky white layer of vaterite, both of which are polymorphs of calcium carbonate. The white vaterite layer is intact in freshly laid eggs and may function in protecting the eggs from mechanical damage, but it also abrades during incubation to reveal the blue calcite shell underneath. Previous research has shown that this color change serves a visual signaling function: nesting greater anis can discriminate between eggs that are freshly laid and those that have already been incubated, which allows them to reject asynchronous eggs laid by extra‐group parasites. Here we use avian visual modeling and pigment extraction to assess the perceptual and chemical bases of such egg recognition. We found that there was no overlap between the avian perceptual space occupied by ani eggshells with and without vaterite, and that vaterite lacked both of the pigments found in the eggshell's calcite matrix, bililverdin and protoporphyrin. The visual contrast between the unpigmented vaterite and the blue‐pigmented calcite appears to pre‐date the evolution of the signaling function, since the related guira cuckoo Guira guira, also a communal breeder, lays similarly structured and pigmented eggs but does not use the visual contrast as a signal to detect parasitism.     

Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation

The influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus, Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed.     

Preliminary study on the correlation between the trace Mn2+ and the calcite polymorph in gallstones containing calcium carbonate from the northeast China via electron spin resonance

Gallstones containing calcium carbonate (GCCC) from the northeast China were analyzed using X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and electron spin resonance (ESR). The sextet signal arising from the allowed transitions of the trace Mn²⁺ ions in GCCC was found to be ESR-detectable and strong. The XRD technique revealed the crystal habit of calcite in GCCC. Of the three polymorphs of calcium carbonate, no calcite was present as a solitary crystallization form, accompanied by aragonite or vaterite or both. The sextet ESR signal and the (104) main XRD peak at 2θ = ∼29.4° were employed as two probes to explore the relationship between trace Mn²⁺ and calcite. The Mn content can be considered as an indicator of the amount of calcite in GCCC because of the existence of a correlation between Mn²⁺ and calcite. The correlation between Mn²⁺ and calcite, the relation between the levels of Mn²⁺ and the type of gallstones, the structural preference of Mn²⁺ to the calcite polymorph, and the influence of dietary habits on calcite in calcium carbonate gallstones are discussed.     

A novel role for calcite in calcium homeostasis.

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives, VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137-200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium-stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.     

Carbonate and Phosphate Precipitation by Chromohalobacter marismortui

The ability of Chromohalobacter marismortui to precipitate carbonate and phosphate minerals has been demonstrated for the first time. Mineral precipitation in both solid and liquid media at different salts concentrations and different magnesium/calcium ratios occurred whereas crystal formation was not observed in the control. The precipitated minerals were studied by X-ray diffraction, scanning electron microscopy and EDX, and were different in liquid and solid media. In liquid media aragonite, struvite, vaterite and monohydrocalcite were precipitated forming crystals and bioliths. Bioliths accreted preferentially close to organic pellicles, whereas struvite preferentially grows in microenvironments free of such pellicles. Magnesian calcite, calcian-magnesian kutnahorite, “proto-dolomite” and huntite were formed in solid media. The Mg content of the magnesian calcite and of Ca-Mg kutnahorite also varied depending on the salt concentration of the culture media. This is the first report on bacterial precipitation of Ca-Mg kutnahorite and huntite in laboratory cultures. The results of this research show the active role played by C. marismortui in mineral precipitation, and allow us to compare them with those obtained previously using other taxonomic groups of moderately halophilic bacteria.     

Crystallization of calcium carbonate salts into beta-chitin scaffold

Composites of beta-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate: aragonite, calcite and vaterite, were observed. Their location within the matrix is a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and polymorphism of the crystals.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

Vaterite Deposition During Eggshell Formation in the Cormorant,Gannet and Shag,and in ‘Shell-less’ Eggs of the Domestic Fowl.

The eggshells of the cormorant (Phalacrocorax carbo), domestic fowl (Gallus domesticus), gannet (Sula bassana), guinea fowl (Numida meleagris), greater flamingo (Phoenicopterus ruber), and shag (Phalacrocorax aristotelis) have been separated into two groups on the basis of the composition of their outer stratum. In the domestic fowl, guinea fowl and greater flamingo the outer stratum is an organic cuticle while in the sea-birds it is an inorganic cover rich in vaterite. The calcareous deposits on the membranes of eggs of the domestic fowl which are shell-less at oviposition have been shown to consist essentially of the vaterite form of calcium carbonate. Reasons for the occurrence of this polymorph of calcium carbonate are discussed with relation to the physiology of the birds.     

A mineralogical characterization of biogenic calcium carbonates precipitated by heterotrophic bacteria isolated from cryophilic polar regions

Precipitation of calcium carbonate (CaCO_3(s)) can be driven by microbial activity. Here, a systematic approach is used to identify the morphological and mineralogical characteristics of CaCO_3(s) precipitated during the heterotrophic growth of micro‐organisms isolated from polar environments. Focus was placed on establishing mineralogical features that are common in bioliths formed during heterotrophic activity, while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes. Twenty microbial isolates that precipitated macroscopic CaCO_3(s) when grown on B4 media supplemented with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning electron microscopy, high‐resolution transmission electron microscopy, and micro‐X‐ray diffraction (μ‐XRD), was used to characterize CaCO_3(s) precipitates. Scanning and transmission electron microscopy showed that complete CaCO_3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodococcus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid‐like epitaxial plates, rhomboid‐like aggregates with epitaxial rhombs, and spherulite aggregates were observed. While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either euhedral or anhedral minerals, but not both. Three anhydrous CaCO_3(s) polymorphs (calcite, aragonite, and vaterite) were identified by μ‐XRD, and calcite and aragonite were also identified based on TEM lattice‐fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin, although several mineralogical features such as crystal‐encrusted bacterial cells, or casts of bacterial cells embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the formation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylotypes consistent with certain mineralogical features is the first step toward discovering a link between these crystal features and the precise underlying molecular biology of the organism precipitating them.     

Bioconservation of Deteriorated Monumental Calcarenite Stone and Identification of Bacteria with Carbonatogenic Activity

The deterioration of the stone built and sculptural heritage has prompted the search and development of novel consolidation/protection treatments that can overcome the limitations of traditional ones. Attention has been drawn to bioconservation, particularly bacterial carbonatogenesis (i.e. bacterially induced calcium carbonate precipitation), as a new environmentally friendly effective conservation strategy, especially suitable for carbonate stones. Here, we study the effects of an in situ bacterial bioconsolidation treatment applied on porous limestone (calcarenite) in the sixteenth century San Jeronimo Monastery in Granada, Spain. The treatment consisted in the application of a nutritional solution (with and without Myxococcus xanthus inoculation) on decayed calcarenite stone blocks. The treatment promoted the development of heterotrophic bacteria able to induce carbonatogenesis. Both the consolidation effect of the treatment and the response of the culturable bacterial community present in the decayed stone were evaluated. A significant surface strengthening (consolidation) of the stone, without altering its surface appearance or inducing any detrimental side effect, was achieved upon application of the nutritional solution. The treatment efficacy was independent of the presence of M. xanthus (which is known as an effective carbonatogenic bacterium). The genetic diversity of 116 bacterial strains isolated from the stone, of which 113 strains showed carbonatogenic activity, was analysed by repetitive extragenic palindromic–polymerase chain reaction (REP-PCR) and 16S rRNA gene sequencing. The strains were distributed into 31 groups on the basis of their REP-PCR patterns, and a representative strain of each group was subjected to 16S rRNA gene sequencing. Analysis of these sequences showed that isolates belong to a wide variety of phylogenetic groups being closely related to species of 15 genera within the Proteobacteria, Firmicutes and the Actinobacteria. This study shows that the abundant carbonatogenic bacteria present in the decayed stone are able to effectively consolidate the degraded stone by producing new calcite (and vaterite) cement if an adequate nutritional solution is used. The implications of these results for the conservation of cultural heritage are discussed.     

Scanning transmission X-ray microscopy study of microbial calcification

Calcium phosphates and calcium carbonates are among the most prevalent minerals involved in microbial fossilization. Characterization of both the organic and mineral components in biomineralized samples is, however, usually difficult at the appropriate spatial resolution (i.e. at the submicrometer scale). Scanning transmission X‐ray microscopy (STXM) was used to measure C K‐edge, P L‐edge, and Ca L‐edge near‐edge X‐ray absorption fine structure (NEXAFS) spectra of some calcium‐containing minerals common in biomineralization processes and to study the experimental biomineralization by the model microorganism, Caulobacter crescentus. We show that the Ca L_2,3‐edges for hydroxyapatite, calcite, vaterite, and aragonite are unique and can be used as probes to detect these different mineral phases. Using these results, we showed that C. crescentus cells, when cultured in the presence of high calcium concentration, precipitated carbonate hydroxyapatite. In parallel, we detected proteins, polysaccharides, and nucleic acids in the mineralizing bacteria at the single‐cell scale. Finally, we discussed the utility of STXM for the study of natural fossilized microbial systems.     

Conservation of Ornamental Stone by Myxococcus xanthus-Induced Carbonate Biomineralization

Increasing environmental pollution in urban areas has been endangering the survival of carbonate stones in monuments and statuary for many decades. Numerous conservation treatments have been applied for the protection and consolidation of these works of art. Most of them, however, either release dangerous gases during curing or show very little efficacy. Bacterially induced carbonate mineralization has been proposed as a novel and environmentally friendly strategy for the conservation of deteriorated ornamental stone. However, the method appeared to display insufficient consolidation and plugging of pores. Here we report that Myxococcus xanthus-induced calcium carbonate precipitation efficiently protects and consolidates porous ornamental limestone. The newly formed carbonate cements calcite grains by depositing on the walls of the pores without plugging them. Sonication tests demonstrate that these new carbonate crystals are strongly attached to the substratum, mostly due to epitaxial growth on preexisting calcite grains. The new crystals are more stress resistant than the calcite grains of the original stone because they are organic-inorganic composites. Variations in the phosphate concentrations of the culture medium lead to changes in local pH and bacterial productivity. These affect the structure of the new cement and the type of precipitated CaCO₃ polymorph (vaterite or calcite). The manipulation of culture medium composition creates new ways of controlling bacterial biomineralization that in the future could be applied to the conservation of ornamental stone.     

岩溶洞穴微生物沉积碳酸钙——以贵州石将军洞为例

为探讨洞穴微生物沉积碳酸钙作用对洞穴沉积物的影响,利用传统生物学方法,采集贵州中西部地区石将军洞洞穴沉积物表面的微生物样品,结合洞穴监测数据和理化背景资料,利用B-4培养基和B-4C培养基对洞穴细菌进行筛选和纯化,分离出能沉积碳酸钙的菌种,观察和了解洞穴细菌形成的CaCO3晶体,应用X射线衍射分析仪(XRD)分析细菌形成的CaCO3晶体成分,并利用扫描电子显微镜(SEM)观察晶体结构特征。结果表明:1)在B-4培养基下微生物产生的碳酸钙晶体主要为方解石、球霰石和方解石混合物、球霰石,这种变化与培养基pH值的增幅相关;同时,在添加Mg离子的B-4C培养基下形成的碳酸钙晶体主要为方解石,此外,研究中并未检测到文石晶体。2)通过SEM扫描,发现微生物作用形成的碳酸钙晶体存在不规则六方体、柱状体、四方体层状、半球状等,这些晶体形态在化学作用系统下少见,多见于微生物作用形成的方解石。此外,晶体中微生物作用痕迹明显,微生物作用贯穿于整个沉积过程。     

Purification and functional analysis of a 40 kD protein extracted from the Strombus decorus persicus mollusk shells

A 40 kD protein has been extracted from the biomineral matrix of the calcium carbonate gastropod shell of Strombus decorus persicus. The protein was isolated by decalcification and ion exchange HPLC. We have named this protein ACLS40, i.e., aragonite crossed-lamellar structure protein. A partial sequence of the isolated ACLS40 and amino acid analysis both indicate that it does not belong to the family of very acidic proteins, i.e., rich in aspartic and glutamic residues. The shell-extracted protein shows the ability to stabilize calcium carbonate in vitro, in the form of thermodynamically unstable vaterite polymorph, and to inhibit the growth of calcite.     

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

Summary The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.     

Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization

Increasing environmental pollution in urban areas has been endangering the survival of carbonate stones in monuments and statuary for many decades. Numerous conservation treatments have been applied for the protection and consolidation of these works of art. Most of them, however, either release dangerous gases during curing or show very little efficacy. Bacterially induced carbonate mineralization has been proposed as a novel and environmentally friendly strategy for the conservation of deteriorated ornamental stone. However, the method appeared to display insufficient consolidation and plugging of pores. Here we report that Myxococcus xanthus-induced calcium carbonate precipitation efficiently protects and consolidates porous ornamental limestone. The newly formed carbonate cements calcite grains by depositing on the walls of the pores without plugging them. Sonication tests demonstrate that these new carbonate crystals are strongly attached to the substratum, mostly due to epitaxial growth on preexisting calcite grains. The new crystals are more stress resistant than the calcite grains of the original stone because they are organic-inorganic composites. Variations in the phosphate concentrations of the culture medium lead to changes in local pH and bacterial productivity. These affect the structure of the new cement and the type of precipitated CaCO(3) polymorph (vaterite or calcite). The manipulation of culture medium composition creates new ways of controlling bacterial biomineralization that in the future could be applied to the conservation of ornamental stone.     

New insights into the role of pH and aeration in the bacterial production of calcium carbonate (CaCO3)

Over recent years, the implementation of microbially produced calcium carbonate (CaCO₃) in different industrial and environmental applications has become an alternative for conventional approaches to induce CaCO₃ precipitation. However, there are many factors affecting the biomineralization of CaCO₃, which may restrict its application. In this study, we investigated the effects of pH and aeration as the main two influential parameters on bacterial precipitation of CaCO₃. The results showed that the aeration had a significant effect on bacterial growth and its rise from 0.5 to 4.5 SLPM could produce 4.2 times higher CaCO₃ precipitation. The increase of pH to 12 resulted in 6.3-fold increase in CaCO₃ precipitation as compared to uncontrolled-pH fermentation. Morphological characterization showed that the pH is an effective parameter on CaCO₃ morphology. Calcite was found to be the predominant precipitate during aeration-controlled fermentations, while vaterite was mainly produced at lower pH (up to 10) over controlled-pH fermentations. Further increase in pH resulted in a morphological transition, and vaterite transformed to calcite at the pH ranges between 10 and 12.