Effect of inhibitors on calcium carbonate deposition mediated by freshwater algae

Nine green algae, a diatom and three cyanobacteria were shown to precipitate CaCO₃ in batch culture, when grown in the light in a hard water medium containing 68 mg L⁻¹ soluble calcium. The composition of the medium was based on that found in a natural hardwater marina where precipitation of CaCO₃ within algal biofilms occurred. Deposition occurred as a direct result of photosynthesis which caused an increase in the pH of the medium. Once a critical pH had been reached, typically approximately pH 9.0, precipitation began evidenced by a fall in the concentration of soluble calcium in the medium. Certain characteristics of the precipitation process displayed by the diatom Navicula sp. were different to those of the other algae. All algae produced extracellular crystals of irregular morphology. Using a standardized protocol employing the green algae Chlorococcum sp. and Stigeoclonium variabile, the effects of various inhibitors of CaCO₃ nucleation or growth of crystals were studied. Fifteen compounds were screened and assessed for their performance in this context. Most materials effectively delayed deposition of CaCO₃, many decreased precipitation rates and all had a marked effect on crystal morphology. The most effective compound was HEDP (1-hydroxyethylene 1,1 diphosphonic acid), which inhibited precipitation completely at a concentration of 2.5 mg L⁻¹ The use of such compounds to reduce the precipitation of calcium salts within algal biofilms in natural hard waters is discussed.     

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.     

Seeded Growth Route to Noble Calcium Carbonate Nanocrystal

A solution-phase route has been considered as the most promising route to synthesize noble nanostructures. A majority of their synthesis approaches of calcium carbonate (CaCO₃) are based on either using fungi or the CO₂ bubbling methods. Here, we approached the preparation of nano-precipitated calcium carbonate single crystal from salmacis sphaeroides in the presence of zwitterionic or cationic biosurfactants without external source of CO₂. The calcium carbonate crystals were rhombohedron structure and regularly shaped with side dimension ranging from 33–41 nm. The high degree of morphological control of CaCO₃ nanocrystals suggested that surfactants are capable of strongly interacting with the CaCO₃ surface and control the nucleation and growth direction of calcium carbonate nanocrystals. Finally, the mechanism of formation of nanocrystals in light of proposed routes was also discussed.     

ALGAL CALCIFICATION IN SOME CODIACEAE (CHLOROPHYTA): ULTRASTRUCTURE AND LOCATION OF SKELETAL DEPOSITS1,2

The capitular filaments of Penicillus and Rhipocephalus consist of an inner tube containing the cytoplasm and an outer calcified sheath. The sheath originates at the cell wall and differentiates into several layers which form the outer filament wall. CaCO₃ is deposited between organic layers within the sheath and is not in direct contact with the seawater. Pores within the sheath, usually uncalcified, may facilitate exchange of gases and solutes. The cytoplasm is characterized by vacuolar inclusions of calcium oxalate needles 50–150 μm long. A closed cortical surface is lacking. Udotea cyathiformis Dec. and U. conglutinata (Ellis & Sol.) Lam. are similar to Penicillus and Rhipocephalus, in addition showing some CaCO₃ between filaments (ICS-calcification). Udotea flabellum (Ellis & Sol.) Lam. is different as the filaments are profusely branched giving rise to a fully developed cortical surface. Pores and vacuolar calcium oxalate inclusions are absent. CaCO₃ deposition occurs within cortical filaments in between layers of the filament wall and subcortically in intercellular spares (ICS). Cortex calcification shows primary and secondary deposits bearing some resemblance to sheath calcification and to coralline red algae. In Rhipocephalus phoenix (Ellis & Sol.) Kütz., Penicillus pyriformis A. &E. Gepp, U. cyathiformis and U. conglutinata CaCO₃ is precipitated intracellularly within the sheath, in contrast to Halimeda and Cymopolia where it is deposited extracellularly in between filaments. U. flabellum takes an intermediate position showing both intra- and intercellular calcification. The sheath compartment volume is between 12.5 and 7500 μm³and 5–3 orders of magnitude smaller than the ICS-compartment. Compartment size and location of CaCO₃may bear on calcification mechanisms. One condition for such a mechanism may be restricted exchange of solutes (CO₂, CO₃^2-, HCO₃^-, O₂, Co²⁺). Codiaceae; filament ultrastructure; Penicillus; Rhipocephalus; Udotea     

The effect of carbonic anhydrase,urea and urease on the calcium carbonate deposition in the shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of carbonic anhydrase (CA), urea and urease on the CaCO₃ deposition in the shell-repair membrane of the snail, Helix pomatia, was studied by injection of CA separately or in combination with urease. This treatment resulted in increased deposits of CaCO₃ and apparent crystal formation within the shell-repair membranes compared with those of the controls. The reactions to CA combined with urea were not uniform. Formation of organic crystalline structures and dendritic spherulites was observed in some of these membranes, whereas the deposition of CaCO₃ crystals was suppressed. Administration of urea alone inhibited the formation of large CaCO₃ crystals, whereas urease stimulated this process. The reaction of young snails was greater compared to adults. The membranes of young snails contained tighly packed, small CaCO₃ crystals and organic crystalline structures, which indicated increase of the calcifying centra and their successive mineralization. The results support the assumption that carbonic anhydrase and urease enhance the rate of calcium carbonate deposition and crystal formation in Helix pomatia.     

Mechanism of calcium accumulation in acetate-fed aerobic granule

High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM–energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO₃). The formation of the CaCO₃ appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO₃ precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO₃ in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO₃ formation, and provided experimental evidence that a crystal CaCO₃ core was not necessarily required for granulation.     

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G_sα (pfG_sα) from the pearl oyster was isolated. The G_sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G_sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG_sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G_sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG_sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG_sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G_sα in MC3T3-E1 cells. These results indicated that G_sα may be involved in regulation of physiological function, particularly in biological biomineralization.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Effects of neutralizing agents on lactic acid production by Rhizopus oryzae using sweet potato starch

A neutralizing agent is usually employed to counteract the pH reduction during lactic acid fermentation by Rhizopus oryzae. Calcium carbonate (CaCO₃) is used as such a pH controlling agent. The low solubility of CaCO₃ in the fermentation broth could however lead to low efficiency in pH control and cause problems in the subsequent purification process. Therefore, an alternative agent in place of CaCO₃ was examined in this study. The effect of four different neutralizing agents, including CaCO₃, sodium hydroxide (NaOH), ammoniacal solution and sodium bicarbonate (NaHCO₃) on lactic acid production and the morphology of the pellets were investigated. Results indicated that CaCO₃ was still the preferred choice, because of the pellet morphology and the highest lactic acid concentration (43.3 g/L) obtained in the batch using 60 g/L of sweet potato starch as feedstock. It is noteworthy that the lactic acid purification is relatively easier when using NaHCO₃ instead of CaCO₃, due to the higher solubility of sodium lactate than calcium lactate. Therefore, even the batch with CaCO₃ had a slightly higher productivity (1.23 g/L/h) than the batch with NaHCO₃ (1.14 g/L/h), NaHCO₃ might be the first choice for process designers whenever recovery is vital.     

Changes in the cell ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium <Emphasis Type="Italic">‘Euhalothece natronophila’</Emphasis> during fossilization

The ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium ‘Euhalothece natronophila’ Z-M001 from the soda Lake Magadi was investigated during the initial stages of fossilization in a model experimental system. The cyanobacterium was cultivated in concentrated carbonate solution supplemented with calcium chloride. It was revealed that the amorphous CaCO₃ formed under these conditions could interact with the cell wall during the first stages of ‘E. natronophila’ calcification. Evidence is presented that the surface layer of the ‘E. natronophila’ envelope, presumably containing polysaccharide and/or (glyco)protein components, can be involved in the adsorption and subsequent crystallization of CaCO₃ with the formation of a massive “shell” embedding the morphologically intact cells. It was established that the ultrastructure of the cell wall and the intrathylakoid space changed during CaCO₃ mineralization. During the later fossilization stages, cells covered by the calcium-containing “shell” were apparently mummified, and mostly retained their original shape. The encapsulation of cyanobacteria in the trona globule was characterized by a different pattern. It probably involved tight binding of the growing crystal to the glycocalyx components that are anchored in the outer membrane. This may result in its detachment from the underlying peptidoglycan layer. The peptidoglycan was retained, and the protoplasts were ultrastructurally similar to the intact ones. Cyanobacteria incorporated in large trona crystals underwent degradation, deformation, and destruction. This accounts for the fact that massive trona deposits of Lake Magadi lack cyanobacterial fossils that are abundant in calcium-containing strata.     

Structural and functional analyses of organic molecules regulating biomineralization

ABSTRACT

Biomineralization by living organisms are common phenomena observed everywhere. Molluskan shells are representative biominerals that have fine microstructures with controlled morphology, polymorph, and orientation of CaCO₃ crystals. A few organic molecules involved in the biominerals play important roles in the formation of such microstructures. Analyses of structure–function relationships for matrix proteins in biominerals revealed that almost all matrix proteins have an acidic region for the binding of calcium ion in CaCO₃ crystals and interaction domains for other organic molecules. On the other hand, biomineralization of metal nanoparticles by microorganisms were also investigated. Gold nanoparticles and quantum dots containing cadmium were successfully synthesized by bacteria or a fungus. The analyses of components revealed that glycolipids, oligosaccharides, and lactic acids have key roles to synthesize the gold nanoparticle in Lactobacillus casei as reductants and dispersants. These researches about biomineralization will give new insights for material and environmental sciences in the human society.     

Evidences for Bioprecipitation of Pedogenic Calcite by Calcifying Bacteria from Three Different Soils of L'Aquila Basin (Abruzzi,Central Italy)

To provide further evidences on the role of bacterial soil species in the development of calcium carbonate deposits in soil, we isolated 36 heterotrophic bacterial strains from three soils of L'Aquila basin characterized by different CaCO₃ content and tested their ability to precipitate CaCO₃ when cultured on a Ca-rich medium. We found that the majority (63.89%) of these isolates could precipitate CaCO₃ minerals at 27°C. The aptitude to calcification (time and crystal amount) of each calcifying strains, morphology (SEM) and mineralogy of the formed bioliths were also investigated. X-ray diffraction confirmed the production of calcite. Crystal formation was not observed in the controls. Organic matter, total N and assimilable P, cation exchange capacity and exchangeable Ca²⁺, Mg²⁺, K⁺, Na⁺, pH, total and active calcium carbonate content, electric conductivity, skeleton, sand, silt and clay fractions of each soil sample were determined and related with its microbiological parameters. We found that the CaCO₃ content of soil was significatively related, in particular, to the percentage of calcifying bacterial strains (r = 0.95) and to the heterotrophic bacterial density (r = 0.98), which was found significatively related also with Ca²⁺ content of soil (r = ?0.97) and its CEC (r = ?0.97).     

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.

     

一株岩石放线菌对碳酸钙形貌的影响北大核心CSCD

【目的】微生物诱导成矿是近年来地质微生物学领域研究中的热点之一。采用一株分离自喀斯特地区的岩生放线菌DHS C013^T菌株,研究该菌株及其代谢产物在由NaHCO3、Ca（NO3）2.4H2O组成的成矿体系中对CaCO3生成及形貌的影响。【方法】将在葡萄糖麦芽酵母提取物（MGYP）培养液中培养的放线菌上清液、菌丝球、发酵液以及无菌的MGYP培养液和超纯水分别加入成矿体系,SEM观察不同处理成矿体系中的底部沉淀物。【结果】在超纯水成矿体系中只形成标准菱面体方解石,而在添加放线菌及其代谢产物甚至含有机质的培养液则可形成形态各异的CaCO3晶体,如球形、哑铃以及表面具有鳞片状的柱形。这些特殊形态的CaCO3晶体的形成,可能是在放线菌的菌丝球和菌丝片段以及胞外分泌物上成核和逐渐生长的结果。【结论】放线菌菌丝体及代谢产物对调控和影响CaCO3的晶体形貌有重要作用。研究结果对进一步认识放线菌及其代谢产物诱导生物成矿提供了新的证据。     

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.     

Calcium carbonate-methylene blue nanohybrids for photodynamic therapy and ultrasound imaging

Photodynamic therapy plays an important role in cancer treatment. In this work, methylene blue (MB)-embedded calcium carbonate nanorods (CaCO₃-MB NRs) have been synthesized for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO₃-MB NRs can be controlled by modulating the concentration of Na₂CO₃ aqueous solution. The generation of effective reactive oxygen species (ROS) were confirmed by 1,3-diphenylisobenzofuran (DPBF) probe. Both photodynamic therapy performance and echogenic performance of CaCO₃-MB NRs were investigated to confirm the feasibility of CaCO₃-MB nanohybrids for ultrasound image-guided photodynamic therapy.     

Effect of calcium carbonate and iron on the availability and uptake of iron,manganese, phosphorus and calcium in pea (Pisum sativum L.)

Summary The effect of CaCO₃ and iron on the availability of iron, manganese phosphorus and calcium was studied in the greenhouse on pea (Pisum sativum L.) crop on a light textured soil, which was marginal in exchangeable calcium. Addition of calcium carbonate caused significant increase in dry matter yield with no added iron at both the stages of crop growth. But yeild intended to decrease with 8% CaCO₃ at 75 days of crop growth. Dry matter yield also increased with the addition of iron upto 10 ppm at 45 days and upto 5 ppm at 75 days. The iron concentration and uptake decreased with the increase in CaCO₃ and increased with the application of iron at both the stages of crop growth. The application of iron and CaCO₃ decreased concentration and uptake of phosphorus significantly at both the stages.Like phosphorus, concentration and uptake of manganese also decreased with the increase in added CaCO₃ upto 8% and iron upto 20 ppm at 45 and 75 days. The concentration of calcium increased with the addition of CaCO₃ to the extent of 50 and 40% with 8% CaCO₃ at 45 and 75 days, while the uptake of calcium increased more than 3 folds at 45 days and more than 2 folds at 75 days. The concentration of calcium decreased with the application of iron upto 20 ppm but the uptake at 45 days increased upto 10 ppm and at 75 days upto 5 ppm and then decreased.The concentration of Fe, P and Ca decreased at 75 days and that of Mn increased while the uptake of all these nutrients increased at 2nd stage due to higher dry matter.     

The effect of adenosine triphosphate,magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl₂) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO₃) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0mM ATP. The inhibitory effect of Mg²⁺ on CaCO₃ crystal formation was diminished by reduction of the concentration of MgCl₂ in the incubation solution. Thus, after incubation for only 24h, 1.0mM MgCl₂ promoted the formation of birefringent CaCO₃ crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl₂ and phospholipids in the recalcification of shell-repair membrane is discussed.The author wishes to thank Mrs. E. Hellmén for valuable technical assistance     

Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation

During a study of ureolytic microbial calcium carbonate (CaCO₃) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO₃ crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K_m) and maximum hydrolysis rates (V_max) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.     

Isolation of alkaliphilic calcifying bacteria and their feasibility for enhanced CaCO3 precipitation in bio-based cementitious composites

Microbially induced calcite precipitation (MICP), secreted through biological metabolic activity, secured an imperative position in remedial measures within the construction industry subsequent to ecological, environmental and economical returns. However, this contemporary recurrent healing system is susceptible to microbial depletion in the highly alkaline cementitious environment. Therefore, researchers are probing for alkali resistant calcifying microbes. In the present study, alkaliphilic microbes were isolated from different soil sources and screened for probable CaCO₃ precipitation. Non-ureolytic pathway (oxidation of organic carbon) was adopted for calcite precipitation to eliminate the production of toxic ammonia. For this purpose, calcium lactate Ca(C₃H₅O₃)₂ and calcium acetate Ca(CH₃COO)₂ were used as CaCO₃ precipitation precursors. The quantification protocol for precipitated CaCO₃ was established to select potent microbial species for implementation in the alkaline cementitious systems as more than 50% of isolates were able to precipitate CaCO₃. Results suggested 80% of potent calcifying strains isolated in this study, portrayed higher calcite precipitation at pH 10 when compared to pH 7. Ten superlative morphologically distinct isolates capable of CaCO₃ production were identified by 16SrRNA sequencing. Sequenced microbes were identified as species of Bacillus, Arthrobacter, Planococcus, Chryseomicrobium and Corynebacterium. Further, microstructure of precipitated CaCO₃ was inspected through scanning electron microscopy (SEM), X-ray diffraction (XRD) and thermal gravimetric (TG) analysis. Then, the selected microbes were investigated in the cementitious mortar to rule out any detrimental effects on mechanical properties. These strains showed maximum of 36% increase in compressive strength and 96% increase in flexural strength. Bacillus, Arthrobacter, Corynebacterium and Planococcus genera have been reported as CaCO₃ producers but isolated strains have not yet been investigated in conjunction with cementitious mortar. Moreover, species of Chryseomicrobium and Glutamicibacter were reported first time as calcifying strains.