Subscribed Content Calcium Carbonate Precipitation by Ureolytic Subsurface Bacteria

Coprecipitation in carbonate minerals offers a means of slowing the transport of divalent radionuclides and contaminant metals (e.g.,⁹⁰Sr²⁺, UO²⁺, Co²⁺) in the subsurface. It may be possible to accelerate this process by stimulating the native microbial community to generate chemical conditions favoring carbonate precipitation. In a preliminary evaluation of this approach, we investigated the ability of ureolytic subsurface bacteria to produce alkaline conditions conducive to calcium carbonate precipitation. Groundwater samples from the Eastern Snake River Plain (ESRP) aquifer in Idaho were screened for urea-hydrolyzing microorganisms; three isolates were selected for further evaluation. Analysis of 16S rRNA gene sequences indicated that two of the ESRP isolates were of the genus Pseudomonas , and the other was a Variovorax sp. The specific urease activities of the ESRP isolates appeared to be similar to each other but less than that of Bacillus pasteurii , a known urease-positive organism. However, calcium carbonate was rapidly precipitated in all cultures that were supplied with urea and calcium, and X-ray diffraction analyses indicated that calcite was always the predominant carbonate polymorph produced. The correspondence between measured calcium concentrations and equilibrium predictions suggested that the rate of calcite precipitation was directly linked to the rate of urea hydrolysis. These results are promising with respect to the potential utility of this approach for in situ remediation and indicate that further evaluation of this approach under conditions more closely simulating environmental conditions is warranted.     

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.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.     

Bacterial Calcium Carbonate Precipitation in Cave Environments: A Function of Calcium Homeostasis

To determine if microbial species play an active role in the development of calcium carbonate (CaCO ₃ ) deposits (speleothems) in cave environments, we isolated 51 culturable bacteria from a coralloid speleothem and tested their ability to dissolve and precipitate CaCO ₃ . The majority of these isolates could precipitate CaCO ₃ minerals; scanning electron microscopy and X-ray diffractrometry demonstrated that aragonite, calcite and vaterite were produced in this process. Due to the inability of dead cells to precipitate these minerals, this suggested that calcification requires metabolic activity. Given growth of these species on calcium acetate, but the toxicity of Ca ²⁺ ions to bacteria, we created a loss-of-function gene knock-out in the Ca ²⁺ ion efflux protein ChaA. The loss of this protein inhibited growth on media containing calcium, suggesting that the need to remove Ca ²⁺ ions from the cell may drive calcification. With no carbonate in the media used in the calcification studies, we used stable isotope probing with C ¹³ O ₂ to determine whether atmospheric CO ₂ could be the source of these ions. The resultant crystals were significantly enriched in this heavy isotope, suggesting that extracellular CO ₂ does indeed contribute to the mineral structure. The physiological adaptation of removing toxic Ca ²⁺ ions by calcification, while useful in numerous environments, would be particularly beneficial to bacteria in Ca ²⁺ -rich cave environments. Such activity may also create the initial crystal nucleation sites that contribute to the formation of secondary CaCO ₃ deposits within caves.     

Spatial Patterns of Carbonate Biomineralization in Biofilms

Microbially catalyzed precipitation of carbonate minerals is an important process in diverse biological, geological, and engineered systems. However, the processes that regulate carbonate biomineralization and their impacts on biofilms are largely unexplored, mainly because of the inability of current methods to directly observe biomineralization within biofilms. Here, we present a method for in situ, real-time imaging of biomineralization in biofilms and use it to show that Pseudomonas aeruginosa biofilms produce morphologically distinct carbonate deposits that substantially modify biofilm structures. The patterns of carbonate biomineralization produced in situ were substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation should occur at the biofilm surface, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, detaching biofilm-resident cells and deforming the biofilm morphology. These findings indicate that biomineralization is a general regulator of biofilm architecture and properties.     

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.     

Kinetic Analyses of Calcium Movements in HeLa Cell Cultures : II. Calcium efflux

Calcium efflux was studied in monolayers of HeLa cells. The fast phase of exchange was studied in an open system by continuous washout. Its half-time was 1.58 min which is practically identical to the fast phase of calcium influx previously found to be 1.54 min. This suggests that the fast component of efflux represents calcium exchange from an extracellular compartment probably from calcium bound to the cell membrane surface. Dinitrophenol (DNP) and iodoacetate (IAA) do not inhibit calcium efflux from this compartment. The slow phase of calcium exchange was studied in a closed three compartment system. The half-time of calcium efflux measured under these conditions is almost identical to that obtained previously in studies of calcium influx: 33.0 and 37.0 min, respectively. This slow compartment is likely to be the intracellular exchangeable calcium pool. DNP and IAA inhibit calcium efflux from this compartment, lengthening the half-time from 33 min to 55.0 and 216 min, respectively. This suggests that calcium extrusion from the cell is an active process. Since calcium influx is not affected by metabolic inhibitors, the cellular calcium concentration increases as would be predicted under these conditions. Calcium efflux is also markedly depressed by lowering the temperature.     

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.     

Calcium Oxalate Biomineralization by Piloderma fallax in Response to Various Levels of Calcium and Phosphorus

Piloderma fallax is an ectomycorrhizal fungus commonly associated with several conifer and hardwood species. We examined the formation of calcium oxalate crystals by P. fallax in response to calcium (0.0, 0.1, 0.5, 1, and 5 mM) and phosphorus (0.1 and 6 mM) additions in modified Melin-Norkrans agar medium. Both calcium and phosphorus supplementation significantly affected the amount of calcium oxalate formed. More calcium oxalate was formed at high P levels. Concentrations of soluble oxalate in the fungus and medium were higher at low P levels. There was a strong positive linear relationship between Ca level and calcium oxalate but only under conditions of phosphorus limitation. Calcium oxalate crystals were identified as the monohydrate form (calcium oxalate monohydrate [COM] whewellite) by X-ray diffraction analysis. Prismatic, styloid, and raphide forms of the crystals, characteristic COM, were observed on the surface of fungal hyphae by scanning electron microscopy. P. fallax may be capable of dissolving hyphal calcium oxalate under conditions of limited Ca. The biomineralization of calcium oxalate by fungi may be an important step in the translocation and cycling of Ca and P in soil.Many fungi from forest litter, including ectomycorrhizal fungi, exhibit calcium oxalate (CaOx) crystals on their hyphae. The ubiquity of CaOx crystals on fungal hyphae suggests that their formation may provide a selective advantage to the organism (4). CaOx formation is hypothesized to regulate intracellular pH and levels of oxalate and Ca and, hence, serves as a major sink for toxic amounts of Ca in soil and other environments (52, 53, 61). In plants, CaOx crystals have also been proposed to serve as a calcium source under conditions of calcium limitation (14, 18, 41), but such a process has yet to be established among fungi.CaOx on fungal hyphae is formed from soil-derived calcium and biologically synthesized oxalate. Oxalate released by ectomycorrhizae has been correlated with increased phosphorus bioavailability in the rhizosphere (V. Casarin, cited by Hinsinger in reference 25). The ability of oxalate to chelate metal ions makes it important in the solubilization and transport of metals in soil, the weathering and diagenesis of rocks and soil minerals (9, 23, 31, 57), and, consequently, the transport of nutrients. It is generally presumed that CaOx crystals form on the surface of fungal hyphae as a result of precipitation when released oxalic acid interacts with calcium cations (23, 43). However, the regularity of the CaOx crystals suggests that their formation is regulated and that they may be formed within the fungal hyphae at specific sites of origin (3, 5, 7).CaOx crystals vary in morphology, ranging from plates to raphides, druses, tetragonal bipyramids, and prisms. This variation in morphology can be seen among fungal genera and species (4). The crystals also usually occur either as CaOx monohydrate (COM; whewellite) (29) or CaOx dihydrate (weddellite) (3, 5, 28, 35, 60). Either crystal form or both may be present on fungal hyphae at the same time.In earlier studies (8, 9), we reported that Piloderma fallax is one of the major species of ectomycorrhizal fungi in subboreal forests. In addition, Piloderma sp. is found in temperate forest soils in association with conifer and hardwood species (34). Piloderma influences nutrient uptake and modifies mineral transformation in rock and soil systems (3, 33). In this study, we chose P. fallax because of (i) its ability to produce oxalate and form CaOx crystals (8, 56), (ii) its presence in many types of forest ecosystems, and (iii) its significant role in the breakdown and formation of soil minerals (9).The objective of this study was to quantify and characterize the formation of CaOx by P. fallax in response to various P and Ca levels in agar medium. We tested the hypothesis that P limitation will induce the production of oxalate and that increased concentrations of Ca will result in greater CaOx formation. This study also examined the dissolution of CaOx on P. fallax when it is grown on Ca-deficient medium and determined whether CaOx can serve as temporary Ca storage. Our study was conducted to add to knowledge of the ecological significance of CaOx, especially of its influence in biogeochemical cycling of P and Ca in soils.     

Process of Carbonate Precipitation by Deleya halophila

Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the formation of carbonate crystals by Deleya halophila. The formation of calcium carbonate crystals (polymorphous aragonite) by D. halophila is a sequential process that commences with a nucleus formed by the aggregation of a few calcified bacterial cells and the subsequent accumulation of more calcified cells and carbonate, which acts to weld the bacteria together. The process leads to the formation of spherical bioliths measuring approximately 50 μm in diameter. The mechanism of carbonate precipitation by D. halophila under our working conditions represents a process of induced biomineralization.     

Proteasome Inhibitors Prevent Tracheary Element Differentiation in Zinnia Mesophyll Cell Cultures

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin β-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin β-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.     

Biomediated Precipitation of Calcium Carbonate Metastable Phases in Hypogean Environments: A Short Review

Natural precipitates of metastable polymorphs of CaCO 3 , such as vaterite, are rarely found in nature however, they have been widely synthesized in laboratory under particular conditions (ie, supersaturated solutions, relative high temperatures, etc.). By SEM and XRD we recognize vaterite spherulites from culturable microbial colonies isolated from hypogean environments. Spherical bodies (∽10μin diameter), probably composed of vaterite, occur in submilimetric microbial mats and biofilms on volcanic substrates (Saint Callixtus Catacombs, Rome, Italy) and karstic caves (Altamira, Candamo, and Tito Bustillo caves, Spain, and Grotta dei Cervi, Italy) where cyanobacteria and actinomycetes are the major microbial components. These particles form beneath dense biofilms, where particular physicochemical conditions are developed by the microbial activity. Natural biofilms seems to generate microenvironments favoring the formation and preservation of metastable CaCO 3 polymorphs. This also shows a major role of microbes in processes of low-temperature alteration of different hypogean rock-substrates.     

Temperature and Food Influence Shell Growth and Mantle Gene Expression of Shell Matrix Proteins in the Pearl Oyster Pinctada margaritifera

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.