Calcium carbonate precipitation and crystal morphology induced by microbial carbonic anhydrase and other biological factors

The effect of microbial carbonic anhydrase (CA) on the calcium carbonate (CaCO₃) precipitation was systematically investigated comparing to other biological factors (bovine CA, bovine serum albumin, carboxymethyl chitosan and glutamic acid). The results showed that the precipitation rate of Ca²⁺ in the presence of either microbial CA or bovine CA was faster than that in the presence of 1% bovine serum albumin, 1% carboxymethyl chitosan or 1% glutamic acid, respectively. In addition, XRD analysis indicated that the dominant CaCO₃ crystal phase was calcite. The CaCO₃ crystal morphologies mainly showed cubic and polyhedral shapes induced by microbial CA, and became multiformity induced by other factors from FE-SEM analysis. These results suggested a novel approach for biomimetic synthesis of CaCO₃ materials by microbial CA.     

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.     

Methanogenesis in Eocene Arctic soils inferred from δC of tree fossil carbonates

We report on the carbon and oxygen stable isotope composition of fossil tree material collected at the White Mountain locality of the Buchanan Lake Formation on Axel Heiberg Island in the High Arctic of Canada. The fossils are Middle Eocene in age and have been permineralized with carbonate. Microscopic examination of fossils revealed them to be the remains of Metasequoia stems, composed of secondary carbonate (calcite) and original wood intermingled at the cellular level. Because the specimens show little compression, crushing, or tissue degradation, we believe that carbonate permineralization occurred soon after burial, and therefore provides insight into Eocene carbon cycling at the locality. The carbon isotope signature of the carbonate suggests that methanogenesis resulted in a ¹³C-enriched CO₂ pool that equilibrated with soil water and gave rise to unusually ¹³C-enriched CaCO₃. Tree fossil carbonate exhibited strikingly high δ¹³C values (+4.0 to +7.4‰) compared to published Phanerozoic pedogenic carbonate δ¹³C values. These δ¹³C values, in conjunction with fractionation factors (α) previously determined for carbonate precipitation and methanogenic pathways, indicate an Eocene soil CO₂ pool containing 80-95% CO₂ produced as a by-product of acetate-fermentation methanogenesis. Because methane in the atmosphere is a powerful greenhouse gas, we suggest that methane emissions from Axel Heiberg soils contributed to the relatively warm Arctic climate during the Middle Eocene.     

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.

     

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.     

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.     

Calcium carbonate formation by Enteromorpha nana algae in a hypersaline volcanic crater lake

On the black basaltic rock surfaces in shallow water around the margin of Lake Gnotuk, and for several meters above the present lake, there is a thick white coating of aragonite marl. The water in the lake is supersaturated with CaCO₃.From close observation and experimental work, it is evident that the marl is produced by the alga Entermorpha nana which lives in the shallow lake water and grows attached to the basalt. The general mechanism of CaCO₃ precipitation by aquatic plants is outlined, and in Enteromorpha nana it can be demonstrated that the closed tubular thallus absorbs HCO-₃ from the outside water and secretes CO²-₃ inside the tube. This secretion of CO²-₃ greatly increases the ionic product of [Ca²⁺] [CO₂-₃] in the water inside the thallus and leads to the precipitation of aragonite and its accumulation at the base of the tube.     

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.     

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.     

Substrate supply for calcite precipitation in Emiliania huxleyi: assessment of different model approaches

Over the last four decades, different hypotheses of Ca²⁺ and dissolved inorganic carbon transport to the intracellular site of calcite precipitation have been put forth for Emiliania huxleyi (Lohmann) Hay & Mohler. The objective of this study was to assess these hypotheses by means of mathematical models. It is shown that a vesicle‐based Ca²⁺ transport would require very high intravesicular Ca²⁺ concentrations, high vesicle fusion frequencies as well as a fast membrane recycling inside the cell. Furthermore, a kinetic model for the calcification compartment is presented that describes the internal chemical environment in terms of carbonate chemistry including calcite precipitation. Substrates for calcite precipitation are transported with different stoichiometries across the compartment membrane. As a result, the carbonate chemistry inside the compartment changes and hence influences the calcification rate. Moreover, the effect of carbonic anhydrase (CA) activity within the compartment is analyzed. One very promising model version is based on a Ca²⁺/H⁺ antiport, CO₂ diffusion, and a CA inside the calcification compartment. Another promising model version is based on an import of Ca²⁺ and HCO₃^? and an export of H⁺.     

Stromatolite records of environmental change in perennially ice-covered Lake Joyce,McMurdo Dry Valleys,Antarctica

Calcite-rich columnar stromatolites grew in perennially ice-covered Lake Joyce in the McMurdo Dry Valleys, Antarctica, during a period of environmental change associated with rising lake level. Stromatolite calcite contains carbon and oxygen isotope records of changes to microbial activity in response to variable light environments and water chemistry through time. The stromatolites grew synchronously with correlative calcite zones. The innermost (oldest) calcite zone has a wide range of δ¹³C_calcite values consistent with variable photosynthetic effects on local DIC ¹³C/¹²C. Subsequent calcite zones preserve a progressive enrichment in δ¹³C_calcite values of approximately + 2.6‰ through time, with δ¹³C_calcite values becoming less variable. This enrichment likely records the removal of ¹²C by photosynthesis from the DIC reservoir over decades, with photosynthetic effects decreasing as light levels became lower and more consistent through time. Mean δ¹⁸O_calcite values of the innermost calcified zone were at least 1‰ lower than those of the other calcified zones (t test p-level < 0.001). The significant difference in δ¹⁸O_calcite values between the innermost and other calcified zones could be a product of mixing source waters with different isotopic values associated with the initiation of lake stratification associated with rising lake level. Overall, Lake Joyce stromatolites record significant lateral variability in relative photosynthetic rate and long-lived lake water stratification with microbial modification of the DIC pool. Such processes provide criteria for interpreting microbial activity within polar paleolake deposits and may shed light on variability in lake environments associated with changing climate in the McMurdo Dry Valleys.     

Carbonate facies‐specific stable isotope data record climate,hydrology, and microbial communities in Great Salt Lake,UT

Organic and inorganic stable isotopes of lacustrine carbonate sediments are commonly used in reconstructions of ancient terrestrial ecosystems and environments. Microbial activity and local hydrological inputs can alter porewater chemistry (e.g., pH, alkalinity) and isotopic composition (e.g., δ¹⁸O_water, δ¹³C_DIC), which in turn has the potential to impact the stable isotopic compositions recorded and preserved in lithified carbonate. The fingerprint these syngenetic processes have on lacustrine carbonate facies is yet unknown, however, and thus, reconstructions based on stable isotopes may misinterpret diagenetic records as broader climate signals. Here, we characterize geochemical and stable isotopic variability of carbonate minerals, organic matter, and water within one modern lake that has known microbial influences (e.g., microbial mats and microbialite carbonate) and combine these data with the context provided by 16S rRNA amplicon sequencing community profiles. Specifically, we measure oxygen, carbon, and clumped isotopic compositions of carbonate sediments (δ¹⁸O_carb, δ¹³C_carb, ?₄₇), as well as carbon isotopic compositions of bulk organic matter (δ¹³C_org) and dissolved inorganic carbon (DIC; δ¹³C_DIC) of lake and porewater in Great Salt Lake, Utah from five sites and three seasons. We find that facies equivalent to ooid grainstones provide time‐averaged records of lake chemistry that reflect minimal alteration by microbial activity, whereas microbialite, intraclasts, and carbonate mud show greater alteration by local microbial influence and hydrology. Further, we find at least one occurrence of ?₄₇ isotopic disequilibrium likely driven by local microbial metabolism during authigenic carbonate precipitation. The remainder of the carbonate materials (primarily ooids, grain coatings, mud, and intraclasts) yield clumped isotope temperatures (T(?₄₇)), δ¹⁸O_carb, and calculated δ¹⁸O_water in isotopic equilibrium with ambient water and temperature at the time and site of carbonate precipitation. Our findings suggest that it is possible and necessary to leverage diverse carbonate facies across one sedimentary horizon to reconstruct regional hydroclimate and evaporation–precipitation balance, as well as identify microbially mediated carbonate formation.     

Impact of Oxygen Availability on Microbially Induced Calcite Precipitation (MICP) Treatment

Most microbially induced calcite precipitation (MICP) processes are induced by aerobic bacteria; thus, oxygen availability plays an important role in MICP treatment. To determine the effects of oxygen supply on MICP treatment catalyzed by Sporosarcina pasteurii, contrast tests under an aerated condition, air-restricted condition, and open air condition were conducted. The results showed that dissolved oxygen (DO) in the air-restricted reactor decreased with time and was almost exhausted within 7 days; DO in the open box decreased by 50% after 7 days of treatment because of the superficial air supply; and DO in the aerated box maintained an initial high level because the consumed oxygen was supplied immediately by adequate air bubbles in the treatment solution. Unconfined compressive strength (UCS) and CaCO₃ content are high under the aerated condition, moderate under the open condition, and poor under the air-restricted condition. The UCS can be 100 times different depending on the different oxygen supply conditions. The overall influence process is as follows: oxygen is dissolved to supply DO for life and activity of the aerobic urea hydrolysis bacteria; then, urea is hydrolyzed to carbonate anions for CaCO₃ precipitation in the presence of Ca²⁺; and finally, CaCO₃ precipitation results in the strengthening of sand. The results indicate that a sufficient air supply is essential to improve MICP processes catalyzed by aerobic bacteria.     

Nucleation of Celestite and Strontianite on a Cyanobacterial S-Layer

Synechococcus strain GL24 is a unicellular cyanobacterium that was isolated from Fayetteville Green Lake, New York, a meromictic lake which has high Ca²⁺ and SO₄^2- concentrations. Epicellular mineralization of Synechococcus cells in the lake is the mechanism by which extensive calcitic bioherms (or microbial reefs) have been formed on the lake's shore and a marl sediment has been built on the lake bottom. Previous studies have shown that calcium carbonate (calcite) formation on the Synechococcus surface is dependent upon an alkaline pH, which is produced in the cellular microenvironment by the cells as their activity increases with seasonal warming of the lake water. At the circumneutral pH of bulk lake water, calcium sulfate (gypsum) is formed. In this study, we show that Synechococcus mediates a similar sulfate-to-carbonate transformation when Sr²⁺ is the major divalent cation present, forming celestite and strontianite. In experimental systems to which equimolar amounts of Ca²⁺ and Sr²⁺, Ca²⁺ or Sr²⁺ and Mg²⁺, or all three ions together were added to artificial lake water, Ca²⁺ and Sr²⁺ were incorporated equally into mineral formation to form CaSr(CO₃)₂. No Mg²⁺ -containing carbonates were formed when either or both of the other two ions were present. Mineral formation takes place on a hexagonally arranged proteinaceous template (an S-layer) which forms the outermost surface of the Synechococcus cell. Our results provide evidence that the S-layer exhibits selectivity with respect to the ions bound and subsequently incorporated into carbonate minerals and that celestite and strontianite, previously thought to be purely evaporitic minerals, can be biogenically formed.     

Photosynthesis,Respiration and Exopolymer Calcium-Binding in Biofilm Calcification (Westerhöfer and Deinschwanger Creek,Germany)

The impact of microbial activity on biofilm calcification in aquatic environments is still a matter of debate, especially in settings where ambient water has high CaCO₃ mineral supersaturation. In this study, biofilms of two CO₂-degassing karst-water creeks in Germany, which attain high calcite supersaturation during their course downstream, were investigated with regard to water chemistry of the biofilm microenvironment. The biofilms mainly consisted of filamentous cyanobacteria (Phormidium morphotype) and heterotrophic bacteria (including sulfate-reducing bacteria), which affect the microenvironment and produce acidic exopolymers. In situ and ex situ microelectrode measurements showed that a strong pH increase, coupled with Ca^{2 +} consumption, occurred in light conditions at the biofilm surface, while the opposite occurred in the dark. Calcite supersaturation at the biofilm surface, calculated from ex situ Ca^{2 +} and CO₃ ^2? microelectrode measurements, showed that photosynthesis resulted in high omega values during illumination, while respiration slightly lowered supersaturation values in the dark, compared to values in the water column. Dissociation calculation demonstrated that the potential amount of Ca^{2 +} binding by exopolymers would be insufficient to explain the Ca^{2 +} loss observed, although Ca^{2 +} complexation to exopolymers might be crucial for calcite nucleation. No spontaneous precipitation occurred on biofilm-free limestone substrates under the same condition, regardless of high supersaturation. These facts indicate that photosynthesis is a crucial mechanism to overcome the kinetic barrier for CaCO₃ precipitation, even in highly supersaturated settings.     

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.     

一株岩石放线菌对碳酸钙形貌的影响北大核心CSCD

【目的】微生物诱导成矿是近年来地质微生物学领域研究中的热点之一。采用一株分离自喀斯特地区的岩生放线菌DHS C013^T菌株,研究该菌株及其代谢产物在由NaHCO3、Ca（NO3）2.4H2O组成的成矿体系中对CaCO3生成及形貌的影响。【方法】将在葡萄糖麦芽酵母提取物（MGYP）培养液中培养的放线菌上清液、菌丝球、发酵液以及无菌的MGYP培养液和超纯水分别加入成矿体系,SEM观察不同处理成矿体系中的底部沉淀物。【结果】在超纯水成矿体系中只形成标准菱面体方解石,而在添加放线菌及其代谢产物甚至含有机质的培养液则可形成形态各异的CaCO3晶体,如球形、哑铃以及表面具有鳞片状的柱形。这些特殊形态的CaCO3晶体的形成,可能是在放线菌的菌丝球和菌丝片段以及胞外分泌物上成核和逐渐生长的结果。【结论】放线菌菌丝体及代谢产物对调控和影响CaCO3的晶体形貌有重要作用。研究结果对进一步认识放线菌及其代谢产物诱导生物成矿提供了新的证据。     

Predicting Biogeochemical Calcium Precipitation in Landfill Leachate Collection Systems

Clogging of leachate collection systems within municipal solid waste landfills can result in greater potential for contaminants to breach the landfill barrier system. The primary cause of clogging is calcium carbonate (CaCO_3(s)) precipitation from leachate and its accumulation within the pore space of the drainage medium. CaCO_3(s) precipitation is caused by the anaerobic fermentation of volatile fatty acids (VFAs), which adds carbonate to and raises the pH of the leachate. An important relationship in modeling clogging in leachate collections systems is a yield coefficient that relates microbial fermentation of VFAs to precipitation of calcium carbonate. This paper develops a new, mechanistically based yield coefficient, called the carbonic acid yield coefficient (Y_H), which relates the carbonic acid (H₂CO₃) produced from microbial fermentation of acetate, propionate, and butyrate to calcium precipitation. The empirical values of Y_H were computed from the changes in acetate, propionate, butyrate, and calcium concentrations in leachate as it permeated through gravel-size material. The theoretical and empirical results show that the primary driver of CaCO_3(s) precipitation is acetate fermentation. Additionally, other non-calcium cations (e.g., iron and magnesium) precipitated with carbonate (CO_2-) when present in the leachate. A common yield between total cations bound to CO₃ ^2- and H₂CO₃ produced, called the calcium carbonate yield coefficient (Y_c), can reconcile the empirical yield coefficient for synthetic and actual leachates.     

Chemical and isotopic composition of karstic lakes in Jamaica,West Indies

Lakes in the limestone region of Jamaica exhibit a range of chemical characteristics that reflect varying inputs from precipitation, surface runoff and groundwater, together with the subsequent evolution of the water within the limnic environment. Detailed spatial and temporal sampling was conducted on one lake, Wallywash Great Pond. Chemical data, together with D/H, ¹⁸O/¹⁶O, ¹³C/¹²C and ⁸⁷Sr/⁸⁶Sr ratios confirm that the karstic spring waters entering the lake evolve chemically through degassing, mixing with rainfall and runoff, biogenic decalcification (resulting mainly from bicarbonate assimilation by the high biomass of submerged macrophytes), and evaporation. Modern carbonate sedimentation in Wallywash Great Pond is largely of high-Mg calcite. This is consistent with Mg/Ca molar ratios >2 within much of the lake. However, aragonite forms on the adaxial leaf surfaces of Potamogeton spp. This may be explained either as a result of locally elevated Mg concentrations or a high degree of supersaturation favouring very rapid carbonate precipitation. Two small lakes to the north of Wallywash Great Pond show minor influence of the Na-Cl dominated coastal aquifer, suggesting that coastal lakes are sensitive to variations in the boundary between fresh and brackish groundwater caused by changes in climate or sea level. Their ¹³C/¹²C ratios are strongly influenced by biogenic CO₂ derived from plant respiration or decay.     

Skeletal growth in the echinoderm Asterias rubens L. (Asteroidea,Echinodermata) estimated by 45Ca-labelling

The skeletal dry weight of the 4.4 ± 0.2 cm size class of Asterias rubens L. from Kiel Bay in the western Baltic is 0.34 ± 0.08 g. The sum of calcium and magnesium carbonates in the skeleton amounts to 94.0 ± 1.3% while the individual concentrations are 86.9 ± 1.3% CaCO₃ and 7.1 ± 0.7% MgCO₃. The MgCO₃ is 4% lower than expected for a magnesium calcite precipitated under Baltic temperature conditions (8°C). Strontium was not determined but strontium carbonate is known to be in the region of 0.4%. The remainder is organic matter and this gives rise to 2.9 ± 1.3% organic carbon.Animals studied were at the ‘waiting stage’ and their actual growth was minimal. Uptake of ⁴⁵Ca in the skeleton consists of a fast step followed by a slow step. The fast step is attributed to saturation of exchangeable skeletal pools while the slow step is due to net deposition of CaCO₃. Skeletal growth at the waiting stage calculated from the second rate constant was found to be 0.76 μg CaCO₃j-mg skeleton^?1 · day^?1 or 0.09% · day^?1 compared with 9.3 μg CaCO₃ · mg skeleton^t1&#x0304; · day^?1 or 1.1% · day^?1 at log phase. The isotope method is considered superior to size-frequency analysis in that it is capable of detecting differences in growth rate in individuals of the same size class and thus provides an insight into asteroid population structure.