Calcite-forming Bacillus licheniformis Thriving on Underwater Speleothems of a Hydrothermal Cave

The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO₂ levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 10⁵ colony-forming units per cm². Bacillus licheniformis appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI Bacillaceae species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the B. licheniformis isolate produced in vitro abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the B. licheniformis isolate, which was analyzed by microbiological assays.     

Non-ureolytic calcium carbonate precipitation by <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. YS11 isolated from the rhizosphere of <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis>

Although microbially induced calcium carbonate precipitation (MICP) through ureolysis has been widely studied in environmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium carbonate-precipitating bacteria that induced an alkaline environment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to precipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy dispersive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning electron microscopy analysis indicated the formation of morphologically distinct minerals around cells under these conditions. Monitoring of bacterial growth, pH changes, and Ca²⁺ concentrations under aerobic, hypoxia, and anaerobic conditions suggested that strain YS11 could induce alkaline conditions up to a pH of 8.9 and utilize 95% of free Ca²⁺ only under aerobic conditions. Unusual Ca²⁺ binding and its release from cells were observed under hypoxia conditions. Biofilm and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore-forming ability, which supports its potential application for self-healing concrete.     

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.     

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.     

Inability to demonstrate calcite precipitation by bacterial isolates from travertine

Bacteria were isolated from 10 actively depositing travertine sites in Europe and North America. Forty‐four isolates were characterized and 33 (75%) were identified as Pseudomonas. Thirty isolates produced base on glucose‐peptone media and 32 isolates produced ammonia from amino acids. Because of the low bacterial biomass in the travertines, the formation of bases, including ammonia was not considered to be significant in carbonate precipitation. None of the bacteria was observed to precipitate calcite from supersaturated calcium carbonate solutions in vitro.     

Calcium carbonate precipitation and crystal morphology induced by microbial carbonic anhydrase and other biological factors

The effect of microbial carbonic anhydrase (CA) on the calcium carbonate (CaCO₃) precipitation was systematically investigated comparing to other biological factors (bovine CA, bovine serum albumin, carboxymethyl chitosan and glutamic acid). The results showed that the precipitation rate of Ca²⁺ in the presence of either microbial CA or bovine CA was faster than that in the presence of 1% bovine serum albumin, 1% carboxymethyl chitosan or 1% glutamic acid, respectively. In addition, XRD analysis indicated that the dominant CaCO₃ crystal phase was calcite. The CaCO₃ crystal morphologies mainly showed cubic and polyhedral shapes induced by microbial CA, and became multiformity induced by other factors from FE-SEM analysis. These results suggested a novel approach for biomimetic synthesis of CaCO₃ materials by microbial CA.     

Production of nanocalcite crystal by a urease producing halophilic strain of <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis> and analysis of its properties by XRD and SEM

Cementation of salt-containing soils can be achieved by salt-tolerant or halophilic calcite precipitation bacteria. Therefore, the isolation of calcite-producing bacteria in the presence of salt is the first step in the microbial cementation of saline soils. Urease producing bacteria can cause calcite nano-crystals to precipitate by producing urease in the presence of urea and calcium. The purpose of this study was to isolate urease producing halophilic bacteria in order to make calcite precipitate in saline soil. The calcite and the properties of the strains were further analyzed by X-ray diffraction (XRD) and scanning electron microscope equipped with an energy dispersive X-ray detector. In this study, a total of 110 halophilic strains were isolated, from which 58 isolates proved to have the ability of urease production. Four strains were identified to produce nano-calcite using urease activity in the precipitation medium. The XRD studies showed that the size of these particles was in the range of 40–60 nm. Strain H3 revealed that calcite is mostly produced in the precipitation medium containing 5% salt in comparison with other strains. This strain also produced calcite precipitates in the precipitation medium containing 15% salt. Phylogenetic analysis indicated that these isolates are about 99–100% similar to Staphylococcus saprophyticus.     

Substrate supply for calcite precipitation in Emiliania huxleyi: assessment of different model approaches

Over the last four decades, different hypotheses of Ca²⁺ and dissolved inorganic carbon transport to the intracellular site of calcite precipitation have been put forth for Emiliania huxleyi (Lohmann) Hay & Mohler. The objective of this study was to assess these hypotheses by means of mathematical models. It is shown that a vesicle‐based Ca²⁺ transport would require very high intravesicular Ca²⁺ concentrations, high vesicle fusion frequencies as well as a fast membrane recycling inside the cell. Furthermore, a kinetic model for the calcification compartment is presented that describes the internal chemical environment in terms of carbonate chemistry including calcite precipitation. Substrates for calcite precipitation are transported with different stoichiometries across the compartment membrane. As a result, the carbonate chemistry inside the compartment changes and hence influences the calcification rate. Moreover, the effect of carbonic anhydrase (CA) activity within the compartment is analyzed. One very promising model version is based on a Ca²⁺/H⁺ antiport, CO₂ diffusion, and a CA inside the calcification compartment. Another promising model version is based on an import of Ca²⁺ and HCO₃^? and an export of H⁺.     

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.     

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ± 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ± 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ± 0.03 g of CaCO₃ g of Ca(CHOOH)₂⁻¹ calcium carbonate precipitate yield was obtained when a culture of 10⁹ cells ml⁻¹ and 5 g of calcium formate liter⁻¹ were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry.     

Biomineralization Consolidation of Fresco Wall Paintings Samples by Bacillus sphaericus

Bacterial-induced mineralization has been explored for protection and consolidation of degraded limestone, concrete and plaster by precipitation of calcium carbonate. It is the first time that Bacillus sphaericus was used for consolidating the nonsterilized decayed wall paintings samples by immersing them in sterile nutritional media. The B. sphaericus used in this study produced urease, which catalyzes the hydrolysis of urea (CO(NH₂)₂) into ammonium (NH₄) and carbonate (CO₃^?2) leading to the precipitation of calcium carbonate. The effect of B. sphaericus on wall paintings was determined by recording the evolution of culture media chemistry and examining the treated wall paintings under a scanning electron microscope to show the structural and morphological evolution of calcium carbonate that was investigated in wall paintings models.     

Enhanced Calcite Dissolution in the Presence of the Aerobic Methanotroph Methylosinus trichosporium

Microbial aerobic methane oxidation (MOx) is intrinsically coupled to the production of carbon dioxide, favoring carbonate dissolution. Recently, microbial organic polymers were shown to be able to induce carbonate dissolution. To discriminate between different mechanisms causing calcite dissolution, experiments were conducted in the presence of solid calcite with (1) actively growing cells (2) starving cells, and (3) dead cells of the methanotrophic bacterium Methylosinus trichosporium under brackish conditions (salinity 10) near calcite saturation (saturation state (Ω) 1.76 to 2.22). Total alkalinity and the amount of dissolved calcium markedly increased in all experiments containing M. trichosporium cells. After initial system equilibration, similar calcite dissolution rates, ranging between 20.16 (dead cells) and 25.68 μmol L^?1 d^?1 (actively growing cells), were observed. Although concentrations of transparent exopolymer particles declined with time in the presence of actively growing and starving cells, they increased in experiments with dead cells. Scanning electron microscopy images of calcite crystals revealed visible surface corrosion after exposure to live and dead M. trichosporium cells. The results of this study indicate a strong potential for microbial MOx to affect calcite stability negatively, facilitating calcite dissolution. In addition to CO₂ production by methanotrophically active cells, we suggest that the release of acidic or Ca²⁺-chelating organic carbon compounds from dead cells could also enhance calcite dissolution.     

Factors Controlling Microbially Induced Desaturation and Precipitation (MIDP) via Denitrification during Continuous Flow

Three continuous-flow column experiments were conducted to investigate the effects of nitrate loading rate and input concentrations of acetate and calcium on the effectiveness of denitrification to promote microbially induced desaturation and precipitation (MIDP). MIDP differs from microbially induced carbonate precipitation (MICP) in that it relies upon desaturation by biogenic gas production, along with carbonate precipitation, to improve the behavior of saturated granular soil. Denitrification is a stepwise process that is susceptible to inhibition and accumulation of intermediates, like nitrite, due to unfavorable chemical conditions (e.g., low pH). Therefore, nitrate reduction, nitrite accumulation, calcite saturation, and formation of carbonate precipitates were monitored in the columns. Factors investigated in these experiments included the effects of nitrate loading rate and input concentrations of calcium and acetate. Low nitrate-loading rates (i.e., ≤0.7?mol/m²-day) generally led to favorable outcomes (i.e., less accumulation of intermediates and more efficient carbonate precipitation). However, faster precipitation rates associated with low nitrate-loading rates led to smaller carbonate crystals and a less uniform precipitation pattern (i.e., precipitation focused near the nutrient source). Input concentrations of calcium and acetate also affected MIDP, but to a lesser extent than nitrate loading rate. The most important stimulated denitrifying bacteria, identified via 16S rDNA sequencing of suspended and dissolved DNA in the soil columns, were Bacillus species, Pseudomonas species, Brevundimonas species, and members of the Rhizobiaceae family. The results support that lower nitrate loading rates can be beneficial for MIDP although higher nitrate loading rates might be useful for some soil improvement applications.     

Efficacy of Fe3O4/Starch Nanoparticles on Sporosarcina pasteurii Performance in MICP Process

The microbial induced calcite precipitation (MICP) has been explored using well-known urease producer bacterium Sporosarcina pasteurii for many applications including soil stabilization. Urease enzyme hydrolyzes urea and in the presence of calcium chloride causes calcium carbonate precipitation between sand particles increasing sand stiffness and strength. In this study, the liquefied soil samples from Anzali coast were positioned inside injection columns by standard positioning technique. The columns were treated by injecting S. pasteurii suspension and cementation solution (CaCl₂ and urea). The effect of different conditions consisting of number of injections, injection intervals, flow rate, and ratio of injection solution on unconfined compression strength (USC) of sands formed inside the columns were evaluated. The results indicated that soil strength was increased when ratio of reactant solutions and injection time were elevated. Moreover, the maximum Ca-precipitation in MICP reaction in liquid medium was obtained while Fe₃O₄/starch concentration and time of addition of nanoparticle to culture medium were 10.8?mg/L and 1.4?h, respectively. The USC results showed that the columns injected by bacterial suspension treated by Fe₃O₄/starch under optimized conditions improved the soil strength up to 1200?kPa in comparison to the control column as 220?kPa.     

Microbially Mediated Calcium Carbonate Precipitation: Implications for Interpreting Calcite Precipitation and for Solid-Phase Capture of Inorganic Contaminants

Microbial degradation of urea was investigated as a potential geochemical catalyst for Ca carbonate precipitation and associated solid phase capture of common groundwater contaminants (Sr, UO₂, Cu) in laboratory batch experiments. Bacterial degradation of urea increased pH and promoted Ca carbonate precipitation in both bacterial control and contaminant treatments. Associated solid phase capture of Sr was highly effective, capturing 95% of the 1 mM Sr added within 24 h. The results for Sr are consistent with solid solution formation rather than discrete Sr carbonate phase precipitation. In contrast, UO₂ capture was not as effective, reaching only 30% of the initial 1 mM UO₂ added, and also reversible, dropping to 7% by 24 h. These results likely reflect differing sites of incorporation of these two elements-Ca lattice sites for Sr versus crystal defect sites for UO₂. Cu sequestration was poor, resulting from toxicity of the metal to the bacteria, which arrested urea degradation and concomitant Ca carbonate precipitation. Scanning electron microscopy (SEM) indicated a variety of morphologies reminiscent of those observed in the marine stromatolite literature. In bacterial control treatments, X-ray diffraction (XRD) analyses indicated only calcite; while in the presence of either Sr or UO₂, both calcite and vaterite, a metastable polymorph of Ca carbonate, were identified. Tapping mode atomic force microscopy (AFM) indicated differences in surface microtopography among abiotic, bacterial control, and bacterial contaminant systems. These results indicate that Ca carbonate precipitation induced by passive biomineralization processes is highly effective and may provide a useful bioremediation strategy for Ca carbonate-rich aquifers where Sr contamination issues exist.     

Microbially Induced Calcium Carbonate Precipitation on Surface or in the Bulk of Soil

Microbial precipitation of calcium carbonate takes place in nature by different mechanisms. One of them is microbially induced carbonate precipitation (MICP), which is performed due to bacterial hydrolysis of urea in soil in the presence of calcium ions. The MICP process can be adopted to reduce the permeability and/or increase the shear strength of soil. In this paper, a study on the use of Bacillus sp., which was isolated from tropical beach sand, to perform MICP either on the surface or in the bulk of sand is presented. If the level of calcium salt solution was below the sand surface, MICP took place in the bulk of sand. On the other hand, if the level of calcium salt solution was above the sand surface, MICP was performed on the sand surface and formed a thin layer of crust of calcium carbonate. After six sequential batch treatments with suspension of urease-producing bacteria and solutions of urea and calcium salt, the permeability of sand was reduced to 14 mm/day (or 1.6×10^?7 m/s) in both cases of bulk and surface MICP. Quantities of precipitated calcium after six treatments were 0.15 and 0.60 g of Ca per cm² of treated sand surface for the cases of bulk or surface MICP, respectively. The stiffness of the MICP treated sand also increased considerably. The modulus of rupture of the thin layer of crust was 35.9 MPa which is comparable with limestone.     

Impact of Oxygen Availability on Microbially Induced Calcite Precipitation (MICP) Treatment

Most microbially induced calcite precipitation (MICP) processes are induced by aerobic bacteria; thus, oxygen availability plays an important role in MICP treatment. To determine the effects of oxygen supply on MICP treatment catalyzed by Sporosarcina pasteurii, contrast tests under an aerated condition, air-restricted condition, and open air condition were conducted. The results showed that dissolved oxygen (DO) in the air-restricted reactor decreased with time and was almost exhausted within 7 days; DO in the open box decreased by 50% after 7 days of treatment because of the superficial air supply; and DO in the aerated box maintained an initial high level because the consumed oxygen was supplied immediately by adequate air bubbles in the treatment solution. Unconfined compressive strength (UCS) and CaCO₃ content are high under the aerated condition, moderate under the open condition, and poor under the air-restricted condition. The UCS can be 100 times different depending on the different oxygen supply conditions. The overall influence process is as follows: oxygen is dissolved to supply DO for life and activity of the aerobic urea hydrolysis bacteria; then, urea is hydrolyzed to carbonate anions for CaCO₃ precipitation in the presence of Ca²⁺; and finally, CaCO₃ precipitation results in the strengthening of sand. The results indicate that a sufficient air supply is essential to improve MICP processes catalyzed by aerobic bacteria.     

Precipitation of calcium carbonate byDeleya halophila in media containing NaCl as sole salt

The precipitation of calcium carbonate by 27 strains ofDeleya halophila using solid and liquid media containing different NaCl concentrations (2.5, 7.5, or 20%, wt/vol) as sole salt, and two incubation temperatures (22° and 32°C) have been studied. All the strains tested were able to precipitate calcium carbonate under the different environmental conditions assayed. Crystals formed were calcite and vaterite; the ratio of calcite to vaterite was dependent on total salts and on the type of medium.     

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.     

Remediation of copper-contaminated soil by Kocuria flava CR1, based on microbially induced calcite precipitation

An indigenous calcifying bacterial strain CR1, identified as Kocuria flava, was isolated from soil of a mining area, Urumqi, China. An extensive copper bioremediation capacity of this isolate was studied based on microbially induced calcite precipitation (MICP). K. flava CR1 removed 97% of copper when initial Cu concentration was 1000 mg L⁻¹. The isolate produced significant amount of urease (472 U mL⁻¹), an enzyme that leads to calcite precipitation. The isolate removed 95% of copper from contaminated soil. The MICP process in bioremediation was further confirmed by FTIR and XRD analyses. FTIR analysis showed two different forms of calcium carbonate, i.e., calcite and aragonite, and the results were well supported by XRD. For the first time, the ability of K. flava has been documented in the bioremediation of polluted soil. This study showed that MICP-based bioremediation by K. flava is a viable, environmental friendly technology for cleaning-up the copper-contaminated site.