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.     

Arthrobacter Sp. Strain MF-2 Induces High-Mg Calcite Formation: Mechanism and Implications for Carbon Fixation

To better understand the mechanism of formation of carbonate minerals by microbes, culture experiments with Arthrobacter sp. strain MF-2 were carried out using M2 medium without carbonate ions for 50 days. A series of sterile control experiments without bacteria were run simultaneously. During the incubation, cell density, the quantity of precipitate, the extracellular polysaccharide (EPS) content, the activity of carbonic anhydrase (CA), the low molecular weight organic acid concentration, the pH, the electrical conductivity, and the Ca²⁺ and Mg²⁺ concentrations of the medium were determined. The morphologies of the precipitated carbonates were observed using scanning electron microscopy, and their mineral species were determined by X-ray diffraction. The results demonstrated that the quantity of precipitate in the biotic experiments increased gradually with the incubation time; precipitate was not obtained in the abiotic experiments. The average precipitation rate correlated positively with the cell density and the EPS content, with r = 0.64 and 0.61, respectively. This suggests that bacterial cells and EPS effected carbonate precipitation. Carbonate ion incorporation into minerals results from carbon dioxide hydration, promoted by microbial secretion of CA by bacteria. These findings contribute to the ongoing search for feasible mechanisms for the sequestration of carbon dioxide in the subsurface, in this case mediated by microorganisms.     

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.     

低Mg/Ca条件下丛毛单胞菌HJ-1菌株诱导文石的形成

【目的】为了探讨细菌对碳酸盐矿物种类和形态的影响。【方法】本文利用丛毛单胞菌HJ-1菌株进行了持续50 d的培养实验。在实验过程中,对细菌数量、沉淀物重量、培养液中Ca2+和Mg2+浓度等进行了动态监测。利用扫描电子显微镜对矿物形态进行了观察,并利用X-射线衍射仪对矿物成分进行测定。【结果】丛毛单胞菌HJ-1菌株具有显著的诱导碳酸盐矿物沉淀的能力,碳酸盐矿物的重量随着培养时间的延长而逐渐增加。X-射线衍射结果表明,形成的碳酸盐沉淀主要由文石和高镁方解石组成,其中文石的最高含量达86%。上述矿物在形态上复杂多样,主要有杆状、柱状、哑铃形、球状和板状以及不规则状和鳞片状集合体。【结论】通常,在Mg/Ca≤2并且有微生物参与的条件下极少形成文石。本文在Mg/Ca为2,不含碳酸根离子的培养基中培养HJ-1菌株的过程中发现了文石。作者认为,低Mg/Ca条件下文石的形成主要与HJ-1菌株分泌较多的胞外多糖有关。     

Biomineralization of Carbonates by Marinococcus albus and Marinococcus halophilus Isolated from the Salar de Atacama (Chile)

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other Gram-positive bacteria and differs from that found in Gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10–100 μm, varying with salinity. They were of magnesium calcite (CO₃ Ca_1-x Mg_x) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates. Received: 3 November 1998 / Accepted: 9 March 1999     

Comparative studies on photosynthetic enzymes of the symbiotic Chlorella from Paramecium bursaria and other symbiotic and non-symbiotic Chlorella strains

The activities of ribulose 1,5-bisphosphate carboxylase and of carbonic anhydrase were studied in cell-free extracts of two symbiotic Chlorella strains isolated from Paramecium bursaria and from Spongilla sp., and of two nonsymbiotic strains of Chlorella (Chlorella fusca and Chlorella vulgaris) cultivated at varied CO₂-concentrations. The symbiotic Chlorella of Paramecium bursaria differs distinctly from the other Chlorella strains by a higher activity of ribulose 1,5-bisphosphate carboxylase, which is independent of the actual CO₂-concentration, and by a lack of carbonic anhydrase activity. These properties are discussed with respect to their ecological significance.Abbreviations CA carbonic anhydrase - Pbi Paramecium bursaria isolate - RuBP ribulose 1,5-bisphosphate Dedicated to Prof. Dr. André Pirson on the occasion of his 70th birthday     

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.     

Characterization of Urease and Carbonic Anhydrase Producing Bacteria and Their Role in Calcite Precipitation

Urease and carbonic anhydrase (CA) are key enzymes in the chemical reaction of living organisms and have been found to be associated with calcification in a number of microorganisms and invertebrates. Three bacterial strains designated as AP4, AP6, and AP9 were isolated from highly alkaline soil samples using the enrichment culture technique. On the basis of various physiological tests and 16S rRNA sequence analysis, these three bacteria were identified as Bacillus sp., B. megaterium, and B. simplex. Further, these Bacillus species have been characterized for the production of urease and CA in the process of biocalcification. One of the isolates, AP6 produced 553 U/ml of urease and 5.61 EU/ml CA. All the strains were able to produce significant amount of exopolymeric substances and biofilm. Further, efficacy of these strains was tested for calcite production ability and results were correlated with urease and CA. Isolate AP6 precipitated 2.26 mg calcite/cell dry mass (mg). Our observations strongly suggest that it is not only urease but CA also plays an important role in microbially induced calcium carbonate precipitation process. The current work demonstrates that urease and CA producing microbes can be utilized in biocalcification as a sealing agent for filling the gaps or cracks and fissures in constructed facilities and natural formations alike.     

The Roles of Mg over the Precipitation of Carbonate and Morphological Formation in the Presence of Arthrobacter sp. Strain MF-2

Microbial mineralization of carbonate is a research subject widely studied in the past decades. The magnesium ions (Mg²⁺), present in water systems, are a key determinant in biomineralization process of carbonate and they are widely found in calcium-based biominerals as an accessory component. However, the crystallization mechanism and morphological change of carbonate polymorphs in the presence of Mg²⁺ ions has not been clarified sufficiently. In this report, a series of culture experiments were performed for 50 days using the Arthrobacter sp. strain MF-2 in a M2 culture medium using Mg/Ca molar ratios (R) of 0, 1.5, 3, 6, 9, and 12 in solution. And the roles of Mg²⁺ ions on the crystal growth and morphological change of biogenic carbonate were investigated. Experimental results show: (1) MF-2 could induce aragonite, high-Mg calcite, and Ca-dolomite formation in M2 culture media with different R values. The increased stability of amorphous calcium carbonate suggests Mg²⁺ ions inhibit carbonate crystallization. (2) The mineral morphologies were varied (rod-shaped, dumbbell-shaped, cauliflower-like, spherical, etc.) in the medium with R = 1.5, but they became simple (spherical and lamellar) in high Mg²⁺ concentrations (Mg > 0.15 M, R > 3). (3) The increased ionic strength of Mg²⁺ ions in the environment has an influence on the polymorphs and morphologies of carbonate formed by controlling the metabolism of strain MF-2 and the activity of carbonic anhydrase.     

Biomineralization Abilities of Cupriavidus Strain and Bacillus subtilis Strains In Vitro Isolated from Speleothems,Rani Cave,Chhattisgarh, India

In vitro culture experiments using three bacterial strains CSJC1, CSJC2, and CSJC3 isolated from speleothems, Rani cave, Chhattisgarh, India, were studied to examine their biomineralization potential. These speleothems showed high microbial cell enumerations on nutrient agar and iron agar (9 × 10⁴ CFU/g) followed by thiosulfate agar (7 × 10⁴ CFU/g), and 60 diverse strains were isolated. The BLASTn sequence search of 16S rRNA sequences with the NCBI database to establish the identity of CSJC1, CSJC2, and CSJC3 strains yielded similarity scores of ≥99% with the respective organisms, and the strains were identified as CSJC1 – Bacillus sp., CSJC2 – Cupriavidus sp., CSJC3 – Bacillus sp. The phylogenetic analysis of CSJC2 strain suggests that it formed a separate major cluster with Cupriavidus sp. and Cupriavidus necator. The phylogenetic analysis of CSJC1 and CSJC3 strains revealed that it formed a major cluster with several strains of Bacillus sp. and Bacillus subtilis. The biominerals induced by Cupriavidus sp. CSJC2 strain imaged with an ultra high-resolution field emission scanning electron microscope (FE-SEM) were seen as calcified coccoid shells that transformed into calcified dumbbells. FE-SEM imaging of biominerals induced by B. subtilis CSJC1 and CSJC3 tested both on B4 media and sheep blood agar individually showed that the precipitates formed calcified dumbbells that were almost similar but not identical phenotypically, indicating that strain-specific morphologies and crystal formation is easier when Ca is present in the media. This is the first comprehensive report on the possible evidences about the role of Cupriavidus sp. in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains may have a role in the biogenic influences in mineral formations at Rani cave.     

Influencing factors and formation mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase

Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca²⁺ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO₃ was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca²⁺ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca²⁺ concentration would affect the generation of CaCO₃, while too high Ca²⁺ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO₂ into HCO₃⁻, and react with OH⁻ and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.     

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⁺.     

Isolation of Bacterial Strain from Sediment Core of Pulp and Paper Mill Industries for Production and Purification of Lignin Peroxidase (LiP) Enzyme

Five bacterial strains were isolated and purified (CSA101 to CSA105) from the sediment core of the effluent released from the Century Pulp and Paper Mill Ltd., India. These strains were grown in minimal salt medium (MSM) containing pulp (10% as a carbon source). The production of lignin peroxidase, CMCase, Fpase, and xylanase together with protein and reducing sugar by all bacterial strains was observed. All of the bacterial isolates responded differently with respect to growth and ligninocellulolytic enzyme production. The maximum lignin peroxidase (LiP) was obtained from the cell extract of Bacillus sp. (CSA105) strain, which was used for purification, fractionation and characterization. The culture filtrate from Bacillus sp. (CSA105) was purified with ammonium sulfate precipitation. Crude protein was desalted by dialyzing with Tris buffer. The lignolytic enzyme produced in the liquid medium was fractionated by gel filtration on Sephadex G-100. In the present study, 12.4-fold purification of LiP enzyme was obtained and 35.85% yield of lignin peroxidase was achieved in the cell extract of Bacillus sp. (CSA105). Lignin peroxidase enzyme plays an important role in lignin degradation process. The ligninolytic enzymes were produced by all of the bacterial strains but maximum lignin peroxidase activity was found in cell extract of CSA105. On the basis of the results obtained, the bacterial strain (CSA105) was found most suitable for the purification of the LiP enzyme.     

Distribution of carbonic anhydrase in British marine macroalgae

Summary Thirty-four species of marine macroalgae from around St. Andrews, Scotland, have been assayed for their external activity and thirty-three species for their total activity of carbonic anhydrase. Activity was detected in all the Rhodophyta tested apart from Chondrus crispus, but was absent in Codium fragile, Enteromorpha sp. and Monostroma fuscum (Chlorophyta), and Alaria esculenta, Laminaria digitata, L. saccharina and L. hyperborea (Phaeophyta). Total activity of carbonic anhydrase per unit fresh weight tended to be higher in the Rhodophyta than in the Chlorophyta or Phaeophyta. External activity was present in two of the six Chlorophyta, four of the twelve Phaeophyta and four of the sixteen Rhodophyta tested. On average, when present, external carbonic anhydrase activity represented 2.7% of the total activity. A relationship was found between total carbonic anhydrase activity and habitat. Species from the high intertidal and the low-light subtidal habitats had significantly higher activity than species from the mid and low intertidal, rockpools, or high-light region of the subtidal. External carbonic anhydrase activity did not vary significantly with habitat. There appeared to be no strong relationship between carbonic anhydrase activity and the ability of a species to use HCO _- ³ in photosynthesis under water.     

Carbonic anhydrase activity of Prochloron sp. isolated from an ascidian host

The prokaryotic algal symbiont of ascidians, Prochloron sp., was found to exhibit carbonic anhydrase activity which is largely associated with the cell surface. This extracellular carbonic anhydrase activity was inhibited, while the intracellular activity was not affected, by chloride or bromide. Acetazolamide and ethoxyzolamide inhibited carbonic anhydrase activity with I₅₀ values of 7×10^-4 and 3×10^-4M, respectively. These I₅₀ values are similar to those observed for intracellular carbonic anhydrases of Synechococcus sp. PCC7942, Chlamydomonas reinhardii and spinach.Abbreviations AZA acetazolamide - CA carbonic anhydrase - chl chlorophyll - EZA ethozyzolamide - I₅₀ concentration of an inhibitor required to cause 50% inhibition - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - U unit     

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.     

Precipatation of calcium carbonate by Vibrio spp. from an inland saltern

Abstract Calcium carbonate precipitation by 63 strains of moderately halophilic bacteria ( Vibrio spp.) isolated from water of a saltern pond has been studied, taking into account the influence of salinity and temperature on the quantity and type of crystals precipitated. The bacteria formed crystals under all the conditions tested. All the crystals were magnesium calcite, with a variable Mg content, depending upon the medium provided. No aragonite was detected, even in a media with high Mg contents. Whether these microorganisms play an active role in precipitation is discussed, as well as the possibility that they may contribute to CaCO₃ precipatation in their natural habitats.     

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium-rich groundwater and carbonate-rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa-inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO₂ outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.     

梭菌MH18 菌株诱导碳酸盐矿物的形成

【目的】研究细菌作用下碳酸盐矿物的形成过程有助于了解微生物成矿的机理。【方法】在LagoaVermelha培养基中(Mg/Ca为6:1)对一株分离自土壤样品的梭菌MH18菌株进行了为期35天的碳酸盐矿物培养实验,同时还完成了一组无菌对照实验。利用X-射线衍射技术对沉淀物的矿物成分进行了测定,利用光学显微镜和扫描电子显微镜对沉淀物的形态进行了系统的观察。【结果】MH18菌株在Lagoa Vermelha培养基中诱导形成了以高镁方解石为主的碳酸盐矿物;这些矿物起初具有哑铃状的外形,后来发展为球状;无菌对照实验中出现少量沉淀物,但X-射线衍射技术图谱显示它们是非晶态物质。【结论】MH18菌株具有促进碳酸盐矿物结晶的功能;碳酸盐矿物的特殊形态(哑铃状和球状)可能与细菌形态存在着某种成因上的联系。     

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil–cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.