Paenibacillus sp. Strain SB-6 Induces Weathering of Ca-montmorillonite: Illitization and Formation of Calcite

Investigation of the weathering of silicate minerals is helpful to understand the process of soil development, cycling of nutrient elements, and potential applications in fixation of carbon dioxide from the atmosphere through carbonate precipitation. In this study, weathering experiments of calcium-montmorillonite were conducted using Paenibacillus sp. strain SB-6 for 70 days. The results indicated that the Si⁴⁺, Al³⁺, Ca²⁺ and Na⁺ concentrations in the medium of the biotic experiments were evidently higher than those of the abiotic experiments, and that Paenibacillus sp. could help the transformation of partial montmorillonite into an illite–montmorillonite mixed-layer. In the process of illitization, K⁺ went into the interlayer of montmorillonite and hydrated Ca²⁺ and Na⁺ released from it. In the late stage of the experiments, the Ca²⁺ released from montmorillonite combined with carbonate ions generated by the bacterial metabolism, forming calcite.     

Exopolymeric substances of sulfate-reducing bacteria: Interactions with calcium at alkaline pH and implication for formation of carbonate minerals

Sulfate‐reducing bacteria (SRB) have been recognized as key players in the precipitation of calcium carbonate in lithifying microbial communities. These bacteria increase the alkalinity by reducing sulfate ions, and consuming organic acids. SRB also produce copious amounts of exopolymeric substances (EPS). All of these processes influence the morphology and mineralogy of the carbonate minerals. Interactions of EPS with metals, calcium in particular, are believed to be the main processes through which the extracellular matrix controls the precipitation of the carbonate minerals. SRB exopolymers were purified from lithifying mat and type cultures, and their potential role in CaCO₃ precipitation was determined from acid‐base titrations and calcium‐binding experiments. Major EPS characteristics were established using infrared spectroscopy and gas chromatography to characterize the chemical functional groups and the sugar monomers composition. Our results demonstrate that all of the three SRB strains tested were able to produce large amounts of EPS. This EPS exhibited three main buffering capacities, which correspond to carboxylic acids (pK_a = 3.0), sulfur‐containing groups (thiols, sulfonic and sulfinic acids – pK_a = 7.0–7.1) and amino groups (pK_a = 8.4–9.2). The calcium‐binding capacity of these exopolymers in solution at pH 9.0 ranged from 0.12g_Ca g_EPS^?1–0.15 g_Ca g_EPS^?1. These results suggest that SRB could play a critical role in the formation of CaCO₃ in lithifying microbial mats. The unusually high sulfur content, which has not been reported for EPS before, indicates a possible strong interaction with iron. In addition to changing the saturation index through metabolic activity, our results imply that SRB affect the rock record through EPS production and its effect on the CaCO₃ precipitation. Furthermore, EPS produced by SRB may account for the incorporation of metals (e.g. Sr, Fe, Mg) associated with carbonate minerals in the rock record.     

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。     

Bio-sequestration of carbon dioxide using carbonic anhydrase enzyme purified from <Emphasis Type="Italic">Citrobacter freundii</Emphasis>

The increase in the atmospheric concentrations of one of the vital green house gasses, carbon dioxide, due to anthropogenic interventions has led to several undesirable consequences such as global warming and related changes. In the global effort to combat the predicted disaster, several CO₂ capture and storage technologies are being deliberated. One of the most promising biological carbon dioxide sequestration technologies is the enzyme catalyzed carbon dioxide sequestration into bicarbonates which was endeavored in this study with a purified C. freundii SW3 β-carbonic anhydrase (CA). An extensive screening process for biological sequestration using CA has been defined. Six bacteria with high CA activity were screened out of 102 colonies based on plate assay and presence of CA in these bacteria was further emphasized by activity staining and Western blot. The identity of selected bacteria was confirmed by 16S rDNA analysis. CA was purified to homogeneity from C. freundii SW3 by subsequent gel filtration and ion exchange chromatography which resulted in a 24 kDa polypeptide and this is in accordance with the Western blot results. The effect of host on metal ions, cations and anions which influence activity of the enzyme in sequestration studies suggests that mercury and HCO₃ ⁻ ion almost completely inhibit the enzyme whereas sulfate ion and zinc enhances carbonic anhydrase activity. Calcium carbonate deposition was observed in calcium chloride solution saturated with carbon dioxide catalyzed by purified enzyme and whereas a sharp decrease in calcium carbonate formation has been noted in purified enzyme samples inhibited by EDTA and acetazolamide.     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

Field detection of urease and carbonic anhydrase activity using rapid and economical tests to assess microbially induced carbonate precipitation

Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease-mediated ureolysis and CO₂ (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH₃) and CA (CO₂). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO₃ precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on-site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.     

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

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

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.     

Citrobacter sp. strain GW-M Mediates the Coexistence of Carbonate Minerals with Various Morphologies

To better understand the formation mechanism of carbonate minerals by microbes, culture experiments with a duration of 70 days were performed under the mediation of strain GW-M isolated from soil using modified Lagoa Vermelha (LV, a hypersaline coastal Lagoon, Rio de Janeiro, Brazil) medium with 6:1 Mg/Ca molar ratio. The results demonstrated that strain GW-M can mediate the formations of both high-Mg calcite and aragonite and that dumbbell-, cauliflower-, rhombohedra-shaped, and irregular minerals coexist in the modified LV medium. The amount of rhombohedra-shaped crystals increased significantly with culture time. A proposed mechanism for these formations is the following. Heterogeneous nucleation on the surface of the extracellular polymeric substances (EPS) always occurred, and carbonates with irregular shape existed in experimental products at any stages. The morphologies evolved from rod to dumbbell and finally to cauliflower. At the initial stage (till day 20), hydrogen ions and EPS secreted by the bacteria only influenced the microenvironment around the cells, and carbonates were precipitated on the surface of bacterial cells. At the middle and late stages (on days 45 and 70), microbes and their secretions influenced the whole medium. Under these conditions, rhombohedra-shaped crystals were formed when homogeneous nucleation occurred. In addition, the results of energy-dispersive spectrometry (EDS) showed that Mg contents in the synthesized carbonate minerals with rhombohedra-shaped were significantly lower than those of carbonates with other shapes, though relationship between morphology and species of mineral cannot be obtained by this phenomenon alone. These results shed further light on the mechanism of carbonate precipitation in the presence of microbes.     

Carbonate and Phosphate Precipitation by Saline Soil Bacteria in a Monitored Culture Medium

Carbonate and phosphate precipitation by bacteria isolated from a saline soil was studied in vitro in a liquid culture medium over 45 days. Physicochemical parameters of this medium were continuously monitored using both selective electrodes (continuous monitoring, CM) and individual measurements by other techniques on days 5, 10, 15, 20, 25, 35 and 45 (discontinuous monitoring, DM). In DM, the precipitated minerals were studied (XRD and SEM-EDX) and the saturation index of the mineral phases was analyzed (PHREEQC program). Using the CM and DM data it was possible to distinguish several temporary stages in which both the medium and the mineralogy changed: 1) 0 to 10 days: pH reaches 8.4; significant loss of Mg²⁺ (incorporated into the bacterial biomass) and Ca²⁺ (through mineral precipitation); formation of crystals, although not in sufficient quantity to be studied until day 10. 2) 10 to 25 days: pH decreases but remains above 8; appreciable loss of Mg²⁺ and Ca²⁺ due to formation of spherical carbonate bioliths with traces of phosphates occluded within these carbonates. 3) After 25 days: biomineralization slow down; pH returns to initial values and struvite is formed (idiomorphic prismatic crystals). These trends are in agreement with the findings of other workers, although with some peculiarities regarding stages and types of mineral precipitated. In some cases the struvite contained small quantities of K and Ca, possibly because these are intermediate mineral species between typic-struvite, K-struvite and Ca-struvite. The bacteria-mediated precipitation of carbonates of Ca and/or Mg and phosphates (struvite) by the bacteria from a saline soil is demonstrated. However, struvite was not found in the soils of origin of the bacteria, possibly because it is a metastable mineral in most soils.     

Nanoparticle Accumulation in Microbial Induced Carbonate Precipitation: The Crucial Role of Extracellular Polymeric Substance

Abstract

Microbes and their secreted extracellular polymeric substance (EPS) could regulate the mineral phases, morphologies and structure of the microbial induced carbonate precipitates (MICPs). Here, two groups of mineralization experiments were carried out respectively to investigate the mechanism of the microbial induced carbonate precipitation (MICP) using Arthrobacter sp. MF-2 strain and its EPS. X-ray diffraction (XRD) were used to characterize the mineral species and scanning electron microscopy (SEM) was used to investigate the morphologies of the precipitates. Besides, focused ion beam (FIB) was used to prepare calcified bacterial slice for transmission electron microscopy (TEM) analysis. Meanwhile, the temporal change of bacterial density, pH value, conductivity, weight of precipitates, Ca²⁺ concentration and EPS content were also recorded. The results of this paper showed EPS could serve as the stabilizer of unstable phases and the scaffold for the accumulation of nanoparticles.     

Intracellular and Extracellular Biomineralization Induced by Klebsiella pneumoniae LH1 Isolated from Dolomites

Abstract

The interaction between bacteria and minerals is very complicated and has been intensively studied in the laboratory and the field in the last few decades, but the processes and mechanisms of biomineralization and mineral precipitation are still not fully understood and need to be explored further. In the present work, biomineralization experiments were undertaken using Klebsiella pneumoniae LH1, collected from a natural surface environment in an area of outcrops of Cambrian dolomite, in a culture medium with various Mg/Ca molar ratios (0, 3, 6 and 12). The mineral precipitates obtained were analyzed by X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive spectrometer (EDS), Fourier transform infrared spectrometer (FTIR), laser scanning confocal microscopy (LSCM) and X-ray photoelectron spectroscopy (XPS). Cells were analyzed with a scanning transmission electron microscope (STEM), high resolution transmission electron microscope (HRTEM) and selected area electron diffraction (SAED). The composition of amino acids in extracellular polymeric substances (EPS) was also determined. In the experiments it was found that the production of ammonia and the presence of carbonate anhydrase promoted the increase of the medium pH and that minerals are nucleated on the EPS, which consist chiefly of amino acids and negatively-charged organic functional groups. With increasing Mg/Ca ratios, the mineral phases changed, including calcite (100%) at Mg/Ca molar ratio of 0, monohydrocalcite (36.05%) + dypingite (63.95%) at Mg/Ca molar ratio of 3, monohydrocalcite (29.72%) + dypingite (15.48%) + nesquehonite (54.80%) at Mg/Ca molar ratio of 6, and monohydrocalcite (14.2%) + dypingite (1.0%) + nesquehonite (84.80%) at Mg/Ca molar ratio of 12. Some intracellular amorphous calcium- and magnesium-rich inclusions were also detected in K. pneumoniae LH1, suggesting intracellular biomineralization accompanying the extracellular mineral precipitation. This study provides further understanding of the biomineralization processes of microorganisms.     

Bio-precipitation of Calcite with Preferential Orientation Induced by Synechocystis sp. PCC6803

This article presents a research study on the deposition process of Ca²⁺ induced by Synechocystis sp. PCC6803 in BG11 liquid medium with different Ca²⁺ concentrations and different pH. The changes of Ca²⁺ concentrations were measured by using atomic absorption method and the corresponding dynamical models were studied. Minerals and cells were analyzed by high resolution transmission electron microscope, selected area electron diffraction, scanning electron microscope, energy dispersive X-ray spectroscope, X-ray diffraction. The selected area electron diffraction patterns were analyzed by Digital Micrograph 3.7 software. The result showed that Ca²⁺ concentrations decreased faster in the experimental group. The changes of calcium carbonate precipitation were fitting to an exponential model. PH 7 and Ca²⁺ concentration of 1.5 g/L were most conducive to calcium carbonate precipitation in the corresponding gradient range. The result of high-resolution transmission electron microscopy showed that minerals in the experimental group differed obviously from that of the control group in the surface morphology, but both of them were calcites. It also showed that a certain number of minute calcites adhesion to the outer surfaces of S. PCC6803 cells. The result of scanning electron microscopy displayed that many sunken holes emerged on the surfaces of the prismatic calcium carbonate minerals. The results of X-ray diffraction proved that minerals induced by S. PCC6803 were calcites with preferential orientation. This article discusses the process of carbonate formation and the possible role played by S. PCC6803. It may be useful to further study the mechanism of microbial carbonates deposition in the field of geology.     

Microbial influence on dolomite and authigenic clay mineralisation in dolocrete profiles of NW Australia

Dolomite (CaMg(CO₃)₂) precipitation is kinetically inhibited at surface temperatures and pressures. Experimental studies have demonstrated that microbial extracellular polymeric substances (EPS) as well as certain clay minerals may catalyse dolomite precipitation. However, the combined association of EPS with clay minerals and dolomite and their occurrence in the natural environment are not well documented. We investigated the mineral and textural associations within groundwater dolocrete profiles from arid northwest Australia. Microbial EPS is a site of nucleation for both dolomite and authigenic clay minerals in this Late Miocene to Pliocene dolocrete. Dolomite crystals are commonly encased in EPS alveolar structures, which have been mineralised by various clay minerals, including montmorillonite, trioctahedral smectite and palygorskite-sepiolite. Observations of microbial microstructures and their association with minerals resemble textures documented in various lacustrine and marine microbialites, indicating that similar mineralisation processes may have occurred to form these dolocretes. EPS may attract and bind cations that concentrate to form the initial particles for mineral nucleation. The dolomite developed as nanocrystals, likely via a disordered precursor, which coalesced to form larger micritic crystal aggregates and rhombic crystals. Spheroidal dolomite textures, commonly with hollow cores, are also present and may reflect the mineralisation of a biofilm surrounding coccoid bacterial cells. Dolomite formation within an Mg-clay matrix is also observed, more commonly within a shallow pedogenic horizon. The ability of the negatively charged surfaces of clay and EPS to bind and dewater Mg²⁺, as well as the slow diffusion of ions through a viscous clay or EPS matrix, may promote the incorporation of Mg²⁺ into the mineral and overcome the kinetic effects to allow disordered dolomite nucleation and its later growth. The results of this study show that the precipitation of clay and carbonate minerals in alkaline environments may be closely associated and can develop from the same initial amorphous Ca–Mg–Si-rich matrix within EPS. The abundance of EPS preserved within the profiles is evidence of past microbial activity. Local fluctuations in chemistry, such as small increases in alkalinity, associated with the degradation of EPS or microbial activity, were likely important for both clay and dolomite formation. Groundwater environments may be important and hitherto understudied settings for microbially influenced mineralisation and for low-temperature dolomite precipitation.     

Optimization of production of C-phycocyanin and extracellular polymeric substances by Arthrospira sp.

The key factors influencing the production of C-phycocyanin (C-PC) and extracellular polymeric substances (EPS) by photoautotrophic culture of Arthrospira sp. were optimized using Taguchi method. Six factors were varied at either three or two levels as follows: light intensity at three levels; three initial culture pHs; two species of Arthrospira; three concentrations of Zarrouk’s medium; three rates of aeration of the culture with air mixed with 2% v/v carbon dioxide; and two incubation temperatures. All cultures ran for 14 days. The optimal conditions for the production of C-PC and EPS were different. For both products, the best cyanobacterium proved to be Arthrospira maxima IFRPD1183. The production of C-PC was maximized with the following conditions: a light intensity of 68 µmol photons m⁻² s⁻¹ (a diurnal cycle of 16-h photoperiod and 8-h dark period), an initial pH of 10, the full strength (100%) Zarrouk’s culture medium, an aeration rate of 0.6 vvm (air mixed with 2% v/v CO₂) and a culture temperature of 30 °C. The concentration of Zarrouk’s medium was the most important factor influencing the final concentration of C-PC. The optimal conditions for maximal production of EPS were as follows: a light intensity of 203 µmol photons m⁻² s⁻¹ with the earlier specified light–dark cycle; an initial pH of 9.5; a 50% strength of Zarrouk’s medium; an aeration rate of 0.2 vvm (air mixed with 2% v/v CO₂); and a temperature of 35 °C. Production of C-PC and EPS in raceway ponds is discussed.

     

Manganese Carbonate Precipitation Induced by Calcite-Forming Bacteria

Abstract

Several dissimilatory metal-reducing bacteria and a halophilic bacterium are able to induce manganese carbonate (rhodochrosite) precipitation. In this study, it was revealed that Ensifer adhaerens JCM 21105^T, Microbacterium testaceum JCM 1353^T, Pseudomonas protegens DSM 19095^T, and Rheinheimera texasensis DSM 17496^T, which are calcite-forming bacteria, were able to aerobically induce the precipitation of manganese carbonate crystals on an agar medium. In the case of all four strains, the principal morphology of the precipitated manganese carbonate crystals was that of micro-sized spheres, when they were aerobically cultivated over the entire surface of the agar medium at 28?°C for 7?days.     

Characteristics and turnover of exopolymeric substances in a hypersaline microbial mat

The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO₃) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c . 80% of the EPS were turned over in the upper 15–20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5–8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO₃. We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO₃ to precipitate. Therefore, this model explains interactions between EPS and CaCO₃ precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.     

Biomineralization of carbonate and phosphate by moderately halophilic bacteria

We investigated the precipitation of carbonate and phosphate minerals by 19 species of moderately halophilic bacteria using media with variable Mg(2+)/Ca(2+) ratios. The precipitated minerals were calcite, magnesium (Mg) calcite, and struvite (MgNH(4)PO(4) x 6H(2)O) in variable proportions depending on the Mg(2+)/Ca(2+) ratio of the medium. The Mg content of the Mg-calcite decreased with increasing Ca(2+) concentration in the medium. According to the saturation indices, other minerals could also have precipitated. We observed important differences between the morphology of carbonate and phosphate, which may help us to recognize these minerals in natural systems. We studied the growth and pH curves of four bacteria in media specific for carbonate and struvite precipitation. We consider the biomineralization processes that produce carbonate and phosphate minerals, and propose a hypothesis for the lack of struvite in natural environments and ancient rocks.     

Development of an improved procedure for isolation and purification of exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483

A method was developed for the isolation and purification of exopolysaccharide (EPS) produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 that can be adapted for industrial-scale operation. Hydrolyzed milk medium, which was ultrafiltered to remove molecular species larger than 2.5×10⁵ Da, was found to be a suitable growth medium for the bacteria, which produced approximately 400 mg EPS/l . Optimal isolation of EPS was achieved using centrifugation, filtration and ethanol precipitation methods. Insoluble and soluble EPS fractions were obtained. The soluble fraction was purified using a series of ethanol precipitations to achieve approximately 98% (w/w) purity. This fraction consisted of galactose, glucose, rhamnose and mannose in the ratio of approximately 5:1:0.6:0.5, with traces of glucosamine.     

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.