An Alternative Biotechnological Tool for Magnesite Enrichment: Lactic Acid Bacteria Isolated from Soil

Abstract

Calcium carbonate (CaCO₃) is found in different polymorph structures such as aragonite, vaterite, and calcite. The most common and stable form of CaCO₃, calcite, which is abundant in sedimentary rocks as magnesite ore. Magnesite has application areas in many industrial fields including paper, pharmaceutical and refractory materials. Magnesite is theoretically formulated MgCO₃, but contains many impurities (silicium, iron, and also calcite), that limits its usability and applicability. In this research, we aimed to investigate the decalcification possibility of the raw magnesite material through application of Enterococcus feacelis (EF) with CaCO₃ dissolution ability. The exact mechanism of CaCO₃ dissolution was investigated by carbonic anhydrase enzyme assay and HPLC analysis of organic acids produced by EF. Consequently, EF reduced the amount of CaCO₃ from 2.94% to 0.49% which means a reduction (≈83.33%) in the rate of CaCO₃ percentage. As a result of the experiments, it was observed that different organic acids produced by bacteria reacted with CaCO₃ and removed the lime of magnesite ore. The bacteria used in the study did not show any pathogenic properties in rats, thus, it can be used safely for the industrial applications.     

Isolation and Molecular Identification of Fungi with Magnesite Enrichment Potential from KÜMAŞ Quarries in Turkey

Abstract

Magnesite is an important raw material used in various industrial applications, especially the production of high-temperature resistant materials. Due to its high reactant nature, magnesite ore is not found in pure form and it contains a great variety of pollutants such as calcium compounds, which restrict its use when exceeding 1% of the ore. Thus, the development of efficient strategies for the removal of pollutants remains a crucial step for magnesite utilization. In this regard, our present work was conducted to isolate and identify active fungal strains that remove calcium pollutants without changing the main magnesium content of the ore. For this aim, magnesite ore samples were collected from two quarries (Turanoca?? and Ortaocak) of KÜMA? Magnesite Inc. and fungal isolation studies were done by using the ore’s flora. Active isolates were chosen according to their CaCO₃ and MgCO₃ dissolving capabilities and identified by using conventional light microscopy and molecular characterization techniques. 71 fungal isolates were obtained from the isolation step and 14 of them were chosen as active isolates that solve calcium compounds while not affecting the magnesium component. The data of the microscopic examination and 18S rDNA gene sequence analysis showed that 14 active strains with magnesite enrichment potential grouped in Aspergillus alliaceus (3), Aspergillus flavus (2), Aspergillus leporis (1), Aspergillus nomius (1), Fusarium tricinctum (2), Penicillium chrysogenum (1) and Penicillium sp. (4).     

Isolation and Molecular Identification of Bacteria with Magnesite Enrichment Potential from Turanocak and Ortaocak Quarries in Kütahya-Turkey

Abstract

In this study, bacteria were isolated from two different magnesite quarries in Turanocak and Ortaocak mine in Kütahya-Eski?ehir region, one of the largest processed magnesite reserves in Turkey. The obtained isolates have a potential to solve important magnesite pollutant CaCO₃ but incapable to solve magnesium that has the most crucial role in the industry. Thus, potential bacteria were identified to be used for magnesite enrichment studies. The obtained isolates were identified and characterized according to the morphological, physiological, biochemical, and molecular techniques (16S rDNA PCR). According to the gene sequencing analysis Bacillus genus bacteria have the ability to solve CaCO₃. The data of the 16S rDNA gene sequence showed that there were 13 active strains grouped in Bacillus. These active strains; Bacillus sp (3), Bacillus atrophaeus (2), Bacillus thuringiensis (1), Bacillus circulans (1), Bacillus simplex (3), Bacillus endophyticus (1) Bacillus drentensis (1) and Bacillus idriensis (1).     

Conventional and Molecular Identification of Bacteria with Magnesite Enrichment Potential from Local Quarries in Erzurum

In this study, the bacteria having ore enrichment potential were isolated from three different magnesite quarries located in Erzurum-Askale borderlines. The obtained isolates were identified and characterized according to the conventional (morphological, physiological and biochemical tests) and molecular techniques (fatty acid methyl ester profiles (FAME), BOX PCR and 16S rDNA). According to sequence analysis, they were determined as Exiguobacterium aurantiacum (4), Exiguobacterium sibiricum (2), Bacillus sp. (2), Staphylococcus epidermidis (2), Staphylococcus haemolyticus (1), Shewanella baltica (1) and Klebsiella oxytoca (1), respectively.     

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.     

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.     

The effect of climbing chalk powder on the infectivity of human coronavirus OC43

There does not appear to be any studies in the published literature on the stability of SARS-CoV-2 in climbing chalk powder (magnesium carbonate and/or calcium carbonate), which has been hypothesized to pose a potential risk of fomite transmission of coronavirus disease 2019 (COVID-19) within climbing gyms. The aim of this study was to determine the infectivity of a model human coronavirus HCoV-OC43 in the presence of climbing chalk powder on a dry plastic surface. The stability of HCoV-OC43 on a plastic surface dusted with climbing chalk powders (magnesium carbonate, calcium carbonate or a blended chalk) was determined by titration on BHK-21 fibroblast cells. No chalk and no virus controls were included. HCoV-OC43 was stable on the plastic surface for 48 h. The stability of HCoV-OC43 was significantly (P ≤ 0·05) reduced in the presence of magnesium carbonate, calcium carbonate and the chalk blend; the infectivity was reduced by ≥2·29 log₁₀ 50% tissue culture infective dose (TCID₅₀) immediately upon on contact and by ≥2·46 log₁₀ TCID₅₀ within 1 h of contact. These findings suggest that the infectivity of coronaviruses is reduced by climbing chalk, limiting the risk of potential fomite transmission.     

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.     

Accumulative Characteristics of Some Plant Species to Magnesium around a Magnesite Mine Area in Northeast China

The deposition of Mg-rich dust from magnesite calcination can lead to serious soil contamination. As an efficient remediation method, phytoremediation is often used to remove contaminants from the environment. However, no information is available on phytoremediation of Mg-contaminated soils. In this study, we determined the Mg concentrations in above- and belowground parts of six dominant plant species and soils (0–20 cm layer) beneath these plants in a magnesite mining region in Northeast China. Mg was enriched in leaves of all six species. Translocation factors (TF) of all six species were far greater than 1. Enrichment factors (EF) of Kochia scoparia, Cassia nomame, and Hordeum jubatum were 1.1, 1.1, and 1.0, respectively, while those of the other three species were not greater than 0.5. The results suggest that Kochia scoparia, Cassia nomame, and Hordeum jubatum are the potential Mg-accumulators, and could be used for remediation of Mg-contaminated soils.     

Sea urchin Arbacia dufresnei (Blainville 1825) larvae response to ocean acidification

Increased atmospheric CO₂ emissions are inducing changes in seawater carbon chemistry, lowering its pH, decreasing carbonate ion availability and reducing calcium carbonate saturation state. This phenomenon, known as ocean acidification, is happening at a faster rate in cold regions, i.e., polar and sub-polar waters. The larval development of Arbacia dufresnei from a sub-Antarctic population was studied at high (8.0), medium (7.7) and low (7.4) pH waters. The results show that the offspring from sub-Antarctic populations of A. dufresnei are susceptible to a development delay at low pH, with no significant increase in abnormal forms. Larvae were isometric between pH treatments. Even at calcium carbonate (CaCO₃) saturation states (of both calcite and aragonite, used as proxies of the magnesium calcite) <1, skeleton deposition occurred. Polar and sub-polar sea urchin larvae can show a certain degree of resilience to acidification, also emphasizing A. dufresnei potential to poleward migrate and further colonize southern regions.     

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates’ antagonism against multiple fungal phytopathogens

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.     

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H₂O₂ into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H₂O₂ compared to a no H₂O₂ control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H₂O₂. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.     

PHACOTUS LENTICULARIS (CHLAMYDOMONADALES,PHACOTACEAE) ZOOSPORES REQUIRE EXTERNAL SUPERSATURATION OF CALCIUM CARBONATE FOR CALCIFICATION IN CULTURE1,2

In nature, zoospores of the chlamydophycean genus Phacotus Perty usually have a calcified lorica. The only cultured species, Phacotus lenticularis (Ehrenberg) Stein, did not readily calcify in artificial media. To overcome this deficiency, we developed an artificial culture medium (N-HS) in which Phacotus lenticularis formed mineralized loricae, as under natural conditions. Calcification of Phacotus (strain Krienitz 91/1) was achieved in a medium containing the ionic concentrations found in natural habitats (i.e. Lake Haussee and Lake Stechlin), hard-water lakes of the Baltic Lake District (Germany). The N-HS medium contained extremely low phosphate concentrations and high calcium and magnesium concentrations compared with common culture media, but the concentrations were similar to those in the lakes. Calcium carbonate in N-HS medium was dissolved up to the saturation concentration (saturation index = 1). Supersaturation was achieved by the addition of ultra-alkaline compounds (Na₂S_iO₃ or NaOH). The medium with the highest super-saturation had a saturation index of 118 and was extremely supersaturated with respect to calcium carbonate. In that medium and in modified media with less of the sodium compounds (lower supersaturations), calcification of zoospores was observed. To determine the effects of sodium and silicon compounds on calcification, various other silicon and sodium compounds were tested but were shown to be ineffective. In conclusion, calcification of Phacotus lenticularis depends directly on the degree of calcium carbonate supersaturation of the medium, but the fundamental mineralization pattern does not. Our study shows that calcification in Phacotus lenticularis can be triggered and controlled by supersaturation of calcium carbonate.     

A microplate spectrofluorometric assay for bacterial biofilms

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r ²=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.     

Occurrence of Salt, pH, and Temperature-tolerant, Phosphate-solubilizing Bacteria in Alkaline Soils

An ecological survey was conducted to characterize 4800 bacterial strains isolated from the root-free soil, rhizosphere, and rhizoplane of Prosopis juliflora growing in alkaline soils. Of the 4800 bacteria, 857 strains were able to solubilize phosphate on plates. The incidence of phosphate-solubilizing bacteria (PSB) in the rhizoplane was highest, followed by rhizosphere and root-free soil. Eighteen bacterial strains out of 857 PSB were able to produce halo at 30°C in a plate assay in the presence of 5% salt (NaCl) and solubilize tricalcium phosphate in National Botanical Research Institute's phosphate growth medium (NBRIP) broth, in the presence of various salts, pHs, and temperatures. Among the various bacteria tested, NBRI4 and NBRI7 did not produced halo in a plate assay at 30°C in the absence of salt. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in NBRIP broth assay always resulted in reliable results. The phosphate solubilization ability of NBRI4 was higher than in the control in the presence of salts (NaCl, CaCl₂, and KCl) at 30°C. Phosphate solubilization further increased in the presence of salts at 37°C as compared with 30°C. At 37°C, CaCl₂ reduced phosphate solubilization ability of NBRI4 compared with the control. The results indicated the role of calcium salt in the phosphate solubilization ability of NBRI4. Received: 9 March 1999 / Accepted: 16 April 1999     

Carbonate Mineral Precipitation for Soil Improvement Through Microbial Denitrification

Microbially induced carbonate precipitation (MICP) and associated biogas production may provide sustainable means of mitigating a number of geotechnical challenges associated with granular soils. MICP can induce interparticle soil cementation, mineral precipitation in soil pore space and/or biogas production to address geotechnical problems such as slope instability, soil erosion and scour, seepage of levees and cutoff walls, low bearing capacity of shallow foundations, and earthquake-induced liquefaction and settlement. Microbial denitrification has potential for improving the mechanical and hydraulic properties of soils because it promotes precipitation of calcium carbonate (CaCO₃) and produces nitrogen (N₂) gas without generating toxic by-products. We evaluated the potential for inducing carbonate precipitation in soil via bacterial denitrification using bench-scale experiments with the facultative anaerobe Pseudomonas denitrificans. Bench-scale experiments were conducted (1) without calcium in an N-rich bacterial growth medium in 2.0 L glass batch reactors and (2) with a source of calcium in sand-filled acrylic columns. Changes of pH, alkalinity, NO₃^? and NO₂^? in the batch reactors and columns, quantification of biogas production and observations of calcium-carbonate precipitation in the sand-filled columns indicate that denitrification led to carbonate precipitation and particle cementation in the pore water as well as a substantial amount of biogas production in both systems. These results document that bacterial denitrification has potential as a soil improvement mechanism.     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

Issues related to dust aerosols in the magnesite industry. I. Chamber exposure.

The present paper is an overview of the experimental research into the effects of flue magnesite dust in the magnesite industry in which the raw material (magnesite) is processed into refractory magnesite clinker. The issues related to dust are divided into two problem areas: a) dust aerosol arising in the process of ore mining and consisting largely of magnesite (MgCO3) and b) dust aerosol originating during ore baking in rotatory furnaces and made up mostly of MgO. Thus, larger groups of people become exposed to these aerosols as a result of solid particles escaping into the atmosphere than in the case of occupational exposure. Experimental research carried out on laboratory animals after chamber exposure provided findings on the deposition, retention and elimination of magnesite dust, on impaired balance between magnesium and calcium leading to damage of biological membranes, on how the immune profile or reproduction and embryogenesis is impacted as well as on the possible interaction with sodium salicylate as a result of an impaired acid base balance. These findings are followed up by evidence produced in the course of biological monitoring (Part II).     

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.     

Discovery of the mineral brucite (magnesium hydroxide) in the tropical calcifying alga Polystrata dura (Peyssonneliales,Rhodophyta)

Red algae of the family Peyssonneliaceae typically form thin crusts impregnated with aragonite. Here, we report the first discovery of brucite in a thick red algal crust (~1 cm) formed by the peyssonnelioid species Polystrata dura from Papua New Guinea. Cells of P. dura were found to be infilled by the magnesium‐rich mineral brucite [Mg(OH)₂]; minor amounts of magnesite and calcite were also detected. We propose that cell infill may be associated with the development of thick (> ~5 mm) calcified red algal crusts, integral components of tropical biotic reefs. If brucite infill within the P. dura crust enhances resistance to dissolution similarly to crustose coralline algae that infill with dolomite, then these crusts would be more resilient to future ocean acidification than crusts without infill.