Magnesite Enrichment with Pseudomonas oryzihabitans Isolated from Magnesite Ore

Magnesite is a primary source of magnesium and its compounds. The major problem in its practical use are the impurities such as silicon, iron and calcium carbonate. Some magnesite ores in Turkey cannot be used due to a high amount of CaCO₃ (≥3%). In this study, bacterial isolates from magnesite quarries in Mersin were tested by plate assay for their ability to decalcify magnesite. A bacterial strain producing the largest clear zones in the plate assay was identified as Pseudomonas oryzihabitans by 16S rDNA-PCR and applied to magnesite ore. It was found to be effective in decalcifying magnesite ore without significant concurrent dissolution of the magnesium carbonate.     

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).     

Role of Fungal Mycelium in the Formation of Carbonate Concretions in Growing Media—An Investigation by SEM and Synchrotron-Based X-Ray Tomographic Microscopy

Soil fungi can facilitate calcification. Mushroom Morchella sp . mycelium induced the formation of carbonate concretions on the surface of an organic-based growing media amended with sand and ground limestone. According to SEM observation and X-ray-tomographic microscopy a dense mycelial network induced calcification. The CaCO₃ content of concretions (?: 0.3–1.5 cm) was found to be at 30%. Microsparitic calcite cemented the pores between the sand grains forming a dense clogging microstructure. Besides water uptake by the mycelium, a high evaporation rate and a decrease in pCO₂ contributed to the formation of the concretions. Fungal mycelium in the concretions is surrounded by voids indicating that at the surface of the mycelium, calcification is counteracted most probably by the release of organic acids.     

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.     

New insights into the role of pH and aeration in the bacterial production of calcium carbonate (CaCO3)

Over recent years, the implementation of microbially produced calcium carbonate (CaCO₃) in different industrial and environmental applications has become an alternative for conventional approaches to induce CaCO₃ precipitation. However, there are many factors affecting the biomineralization of CaCO₃, which may restrict its application. In this study, we investigated the effects of pH and aeration as the main two influential parameters on bacterial precipitation of CaCO₃. The results showed that the aeration had a significant effect on bacterial growth and its rise from 0.5 to 4.5 SLPM could produce 4.2 times higher CaCO₃ precipitation. The increase of pH to 12 resulted in 6.3-fold increase in CaCO₃ precipitation as compared to uncontrolled-pH fermentation. Morphological characterization showed that the pH is an effective parameter on CaCO₃ morphology. Calcite was found to be the predominant precipitate during aeration-controlled fermentations, while vaterite was mainly produced at lower pH (up to 10) over controlled-pH fermentations. Further increase in pH resulted in a morphological transition, and vaterite transformed to calcite at the pH ranges between 10 and 12.

     

The Role of Autotrophic Picocyanobacteria in Calcite Precipitation in an Oligotrophic Lake

A 1-year field study monitoring depth profiles of picoplankton and physicochemical data in the oligotrophic Lake Lucerne (Switzerland) showed that picocyanobacteria play an important role in the CaCO₃ precipitation process. Laboratory experiments with Mychonastes and Chlorella, isolated from Lake Lucerne and Synechococcus using ion selective electrodes, scanning electron microscopy and X-ray powder diffraction clearly demonstrated the potential of picoplankton for fast and effective CaCO₃ precipitation. The combination of a field study with laboratory experiments confirmed the previous reports of picocyanobacteria triggering the CaCO₃ precipitation in hardwater oligotrophic lakes. Electron micrographs of particles from the water column often reveal the size and shape of picoplankton cells covered by calcite. In addition the results from the laboratory approach indicated that algae and bacteria induced different precipitation mechanisms. Experiments with Mychonastes and Chlorella produced crystalline calcite completely covering the cells. Experiments with the cyanobacteria Synechococcus, however, yielded amorphous, micritic CaCO₃, indicating a different precipitation process.     

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.     

Structural and functional analyses of organic molecules regulating biomineralization

ABSTRACT

Biomineralization by living organisms are common phenomena observed everywhere. Molluskan shells are representative biominerals that have fine microstructures with controlled morphology, polymorph, and orientation of CaCO₃ crystals. A few organic molecules involved in the biominerals play important roles in the formation of such microstructures. Analyses of structure–function relationships for matrix proteins in biominerals revealed that almost all matrix proteins have an acidic region for the binding of calcium ion in CaCO₃ crystals and interaction domains for other organic molecules. On the other hand, biomineralization of metal nanoparticles by microorganisms were also investigated. Gold nanoparticles and quantum dots containing cadmium were successfully synthesized by bacteria or a fungus. The analyses of components revealed that glycolipids, oligosaccharides, and lactic acids have key roles to synthesize the gold nanoparticle in Lactobacillus casei as reductants and dispersants. These researches about biomineralization will give new insights for material and environmental sciences in the human society.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Strain improvement of <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> for enhanced urease and calcite production

Phenotypic mutants of Sporosarcina pasteurii (previously known as Bacillus pasteurii) (MTCC 1761) were developed by UV irradiation to test their ability to enhance urease activity and calcite production. Among the mutants, Bp M-3 was found to be more efficient compared to other mutants and wild-type strain. It produced the highest urease activity and calcite production compared to other isolates. The production of extracellular polymeric substances and biofilm was also higher in this mutant than other isolates. Microbial sand plugging results showed the highest calcite precipitation by Bp M-3 mutant. Scanning electron micrography, energy-dispersive X-ray and X-ray diffraction analyses evidenced the direct involvement of bacteria in CaCO₃ precipitation. This study suggests that calcite production by the mutant through biomineralization processes is highly effective and may provide a useful strategy as a sealing agent for filling the gaps or cracks and fissures in any construction structures.     

Involvement of etfA gene during CaCO3 precipitation in Bacillus subtilis biofilm

The eftA gene in Bacillus subtilis has been suggested to be involved in the oxidation/reduction reactions during fatty acid metabolism. Interestingly etfA deletion in B. subtilis results in impairment in CaCO₃ precipitation on the biofilm. Comparisons between the wild type B. subtilis 168 and its etfA mutant during in vitro CaCO₃ crystal precipitation (calcite) revealed changes in phospholipids membrane composition with accumulation of up to 10% of anteiso-C_17:0 and 11% iso-C_17:0 long fatty acids. Ca²⁺ nucleation sites such as dipicolinic acid and teichoic acids seem to contribute to the CaCO₃ precipitation. etfA mutant strain showed up to 40% less dipicolinic acid accumulation compared with B. subtilis 168, while a B. subtilis mutant impaired in teichoic acids synthesis was unable to precipitate CaCO₃. In addition, B. subtilis etfA mutant exhibited acidity production leading to atypical flagella formation and inducing extensive lateral growth on the biofilm when grown on 1.4% agar. From the ecological point of view, this study shows a number of physiological aspects that are involved in CaCO₃ organomineralization on biofilms.     

Biogenic calcite particles from microalgae—Coccoliths as a potential raw material

Synthetic calcite (CaCO₃) particles are found in a broad range of applications. The geometry of particles produced from limestone or precipitation are versatile but limited to basic shapes. The microalga Emiliania huxleyi produces micro‐structured calcite platelets, called coccoliths. This article presents the results of an application‐orientated study, which includes characteristic values also used in the calcite industry for particle evaluation. It is demonstrated that coccoliths are significantly different from all industrial particles produced so far. Coccoliths are porous particles, mainly consisted of calcium carbonate, with further elements such as Mg, Si, Sr, and Fe often embedded in their structure. Their structure is extremely sophisticated, while the overall particle morphology and particle size distribution are homogeneous. This study gives a first inside into the potential of these exceptional objects and may set further impulses for their utilization in specific calcite particle applications.     

Deposition and cycling of carbon and nitrogen in carbonate mud of the lagoons of Arlington and Sudbury Reefs, Great Barrier Reef

The dynamics of carbon and nitrogen in carbonate mud were examined in the lagoons of Arlington and Sudbury Reefs, Great Barrier Reef. Most (89–93%) of the organic carbon and total nitrogen depositing to the carbonate mud zones was mineralized over a sediment depth of 1 m, with ∼50% of CO₂ produced during microbial decomposition involved in carbonate precipitation/dissolution reactions. There was proportionally little burial of organic carbon (10–11%) or nitrogen (7–10%). Nitrogen budgets suggest rapid turnover of porewater inorganic N pools on the order of hours to a few days. Incubation experiments indicate carbonate dissolution in surface deposits (≤20 cm depth) and carbonate precipitation in deeper sediments. Depth-integrated reaction rates indicate net carbonate precipitation of 7–10 mol CaCO₃ m² year⁻¹ over a depth of 1 m. Budget calculations at the whole-reef scale imply that deposition of CaCO₃ in the mud zones of both lagoons may equate to 50–90% of total reef carbonate production, with organic carbon fluxes equating to nearly all net primary production on each reef. These biogeochemical estimates point to the functional importance of carbonate mud zones in the lagoons of the shelf reefs of the Great Barrier Reef.     

Isolation of alkaliphilic calcifying bacteria and their feasibility for enhanced CaCO3 precipitation in bio-based cementitious composites

Microbially induced calcite precipitation (MICP), secreted through biological metabolic activity, secured an imperative position in remedial measures within the construction industry subsequent to ecological, environmental and economical returns. However, this contemporary recurrent healing system is susceptible to microbial depletion in the highly alkaline cementitious environment. Therefore, researchers are probing for alkali resistant calcifying microbes. In the present study, alkaliphilic microbes were isolated from different soil sources and screened for probable CaCO₃ precipitation. Non-ureolytic pathway (oxidation of organic carbon) was adopted for calcite precipitation to eliminate the production of toxic ammonia. For this purpose, calcium lactate Ca(C₃H₅O₃)₂ and calcium acetate Ca(CH₃COO)₂ were used as CaCO₃ precipitation precursors. The quantification protocol for precipitated CaCO₃ was established to select potent microbial species for implementation in the alkaline cementitious systems as more than 50% of isolates were able to precipitate CaCO₃. Results suggested 80% of potent calcifying strains isolated in this study, portrayed higher calcite precipitation at pH 10 when compared to pH 7. Ten superlative morphologically distinct isolates capable of CaCO₃ production were identified by 16SrRNA sequencing. Sequenced microbes were identified as species of Bacillus, Arthrobacter, Planococcus, Chryseomicrobium and Corynebacterium. Further, microstructure of precipitated CaCO₃ was inspected through scanning electron microscopy (SEM), X-ray diffraction (XRD) and thermal gravimetric (TG) analysis. Then, the selected microbes were investigated in the cementitious mortar to rule out any detrimental effects on mechanical properties. These strains showed maximum of 36% increase in compressive strength and 96% increase in flexural strength. Bacillus, Arthrobacter, Corynebacterium and Planococcus genera have been reported as CaCO₃ producers but isolated strains have not yet been investigated in conjunction with cementitious mortar. Moreover, species of Chryseomicrobium and Glutamicibacter were reported first time as calcifying strains.     

Calcite Solubilization by Bacteria: A Novel Method of Environment Pollution Control

Global warming due to the emission of carbon dioxide (CO₂) gas, by the anthropogenic sources is a great threat to the environment. A part of this gas is absorbed by soil bacteria as well as aquatic bacteria and it is converted into insoluble calcium carbonate (CaCO₃) or calcite. Increased calcite concentration in water and agricultural land creates many problems to the human. In this investigation, an attempt has been made to carry out some experiments to isolate some bacteria from cow dung, which have both calcite solubilization and urease activities. Isolated bacteria solubilize calcite due to the secretion of citric acid, oxalic acid and sanazine pigment. All bacterial isolates were identified by 16S rRNA gene sequencing and the phylogenetic relationship among them was also studied using MEGA 6 software.     

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats

ABSTRACT

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO₃, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO₃ and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO₃. It was concluded that MBCa is a better calcium source than CaCO₃ in terms of both calcium retention and absorption.

Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO_3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.     

The relative contributions of biological and abiotic processes to carbon dynamics in subarctic sea ice

Knowledge on the relative effects of biological activity and precipitation/dissolution of calcium carbonate (CaCO₃) in influencing the air-ice CO₂ exchange in sea-ice-covered season is currently lacking. Furthermore, the spatial and temporal occurrence of CaCO₃ and other biogeochemical parameters in sea ice are still not well described. Here we investigated autotrophic and heterotrophic activity as well as the precipitation/dissolution of CaCO₃ in subarctic sea ice in South West Greenland. Integrated over the entire ice season (71 days), the sea ice was net autotrophic with a net carbon fixation of 56 mg C m^?2, derived from a sea-ice-related gross primary production of 153 mg C m^?2 and a bacterial carbon demand of 97 mg C m^?2. Primary production contributed only marginally to the TCO₂ depletion of the sea ice (7–25 %), which was mainly controlled by physical export by brine drainage and CaCO₃ precipitation. The net biological production could only explain 4 % of this sea-ice-driven CO₂ uptake. Abiotic processes contributed to an air-sea CO₂ uptake of 1.5 mmol m^?2 sea ice day^?1, and dissolution of CaCO₃ increased the air-sea CO₂ uptake by 36 % compared to a theoretical estimate of melting CaCO₃-free sea ice. There was a considerable spatial and temporal variability of CaCO₃ and the other biogeochemical parameters measured (dissolved organic and inorganic nutrients).     

Functions of Calcium-bound Phosphorus in Relation to Characteristics of Phosphorus Releasing Bacteria in Sediment of a Chinese Shallow Lake (Lake Wabu)

Sediment phosphorus (P) release accelerates lake eutrophication, while retention capacity and release potential of different P fractions, calcium-bound P (CaCO₃～P) in particular, still remains unclear. Fractionation and sorption behaviors of phosphorus were studied in sediment of a Chinese shallow lake (Lake Wabu) and two inflowing rivers from December 2011 to December 2012. Abundance of P releasing bacteria was analyzed, and their main species were isolated using a culture-dependent method and identified by their 16S rDNA sequences. CaCO₃～P release abilities of these bacteria were also tested. In sediments of both the lake and rivers studied, the rank order of the different P extracts was CaCO₃～P > iron-bound P > acid-soluble organic P > hot NaOH-extractable organic P. At the same time, CaCO₃～P content and equilibrium P concentration (EPC₀) values in river sediments were significantly higher than those in the lake. Additionally, EPC₀ changes non-monotonically with increasing CaCO₃～P content, forming a V-shaped curve, with the lowest EPC₀ at an intermediate CaCO₃～P content (around 180 mg kg^?1). Below this threshold, CaCO₃～P was a component strengthening P retention; moreover, CaCO₃～P became an active species responsible for P release. Noticeably, between the two parts divided by this threshold, the differences in abundance of inorganic phosphorus solubilizing bacteria (IPB) and organic phosphorus mineralizing bacteria (OPB) were insignificant and the dominant IPB species clustered together. By contrast, OPB was distinguished from each other, whose dominant species isolated from the part with higher CaCO₃～P content, namely Novosphingobium sp., exhibited a stronger ability to solubilize CaCO₃～P. Shortly, with lower content, CaCO₃～P tends to stabilize P in sediment; while with higher content or under eutrophic condition, it shifted into P source, with some OPB species becoming the main factors to drive its release.     

The impacts of a watershed CaCO3 treatment on stream and wetland biogeochemistry in the Adirondack Mountains

Temporal and longitudinal variations in the chemistry of two tributary streams of Woods Lake in the Adirondack Mountains of New York were monitored before and after a watershed CaCO₃ application. One subcatchment of the lake had a large beaver pond and wetland at its headwaters, while the second was free-flowing. Treatment of both subcatchments with CaCO₃ resulted in an immediate increase in acid neutralizing capacity (ANC) associated with Ca²⁺ release. The extent and duration of the response to the treatment were greater in the wetland-impacted stream. Aluminum was retained and complexed with organic solutes generated within the beaver-pond. In the free-flowing stream, NO ₃ ^– concentration increased significantly after the manipulation; this pattern was not evident in the wetland-impacted stream. Net retention of SOkinf4/sup2– was evident in the beaver pond prior to and following treatment, and this response was enhanced after the watershed liming. Comparisons of beaver pond inlet/outlet concentrations, mass balance calculations, and in-pond profiles of chemical parameters revealed patterns of retention of SO ₄ ^2– , NO ₃ ^– and Al, and release of Fe²⁺, dissolved organic carbon (DOC) and NH ₄ ⁺ in the wetland during the summer before CaCO₃ treatment. Post-treatment releases of Ca²⁺ from the near-sediment zone in the beaver pond corresponded to anoxic periods in mid- to late-summer and under ice in winter. These findings demonstrate the importance of increased microbial processing of organic matter, along with high partial pressure Of CO₂ (Pco₂) in facilitating the dissolution of the applied CaCO₃. Dissolved silica (H₄SiO₄) was retained in the wetland during the summer prior to treatment but was released after the manipulation. This phenomenon may reflect the dissolution of diatom frustules or silicate minerals in the wetland at higher pH and DOC concentrations. Within two years of the CaCO₃ treatment 60% of the CaCO₃ applied to the beaver pond and surrounding wetland was dissolved and transported from the pond, in contrast to only 2.2% of the CaCO₃ applied to the upland subcatchment draining into the wetland. These results, coupled with high quantities of exchangeable Ca²⁺ found in sediments and onSphagnums mosses in the pond, demonstrate the importance of hydrologic source areas and wetlands in facilitating the dissolution of added CaCO₃ and in regulating the production of chemical species important in ANC generation.