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.     

Polyanion-mediated mineralization — assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition

Summary Immunolocalization of two highly acidic polysaccharides (PS-1 and PS-2) in a calcifying algaPleurochrysis carterae is described throughout the mineralization process, from before crystal nucleation through the cessation of crystal growth. This unicellular coccolithophorid alga is a useful model for mineralization because it produces calcified scales known as coccoliths in homogeneous cell culture. PS-1 and PS-2 were localized in the crystal coats of mature coccoliths and in electron dense Golgi particles. The polyanions are synthesized in medial Golgi cisternae and co-aggregate with calcium ions into discrete 25 nm particles. Particle-laden vesicles bud from cisternal margins and fuse with a coccolith-forming saccule containing an organic oval-shaped scale which forms the base of the future coccolith. The particles are localized on the base before the onset of mineral deposition and are present in the coccolith saccule throughout the period of crystal (CaCO₃) nucleation and growth. During the final phase of coccolith formation, the particles disappear, and the mature crystals acquire an amorphous coat containing PS-1 and PS-2 polysaccharides which remain with the mineral phase after the coccoliths are extruded from the cell. Postulated mechanisms of polyanion-mediated mineralization are reviewed and their relevance to the calcification of coccoliths is addressed.Abbreviations PS-1 polysaccharide one - PS-2 polysaccharide two - BSA bovine serum albumin - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - DHA 3-deoxy-lyxo-2-heptulosaric acid - TCA trichloroacetic acid     

Incorporation of zinc into the coccoliths of the microalga <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>

The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain ‘naked’ cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.     

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.     

COVER

Ultrastructural analysis reveals how the calcifying haptophyte Scyphosphaera apsteinii engineers and secretes its polymorphic calcite coccoliths to construct the external coccosphere. [Vol. 48, No. 6, pp. 1343–1361]     

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b‐ATP4 GENES1

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool‐water group occurring in subarctic North Atlantic and Pacific and a warm‐water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE(PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

NO MECHANISTIC DEPENDENCE OF PHOTOSYNTHESIS ON CALCIFICATION IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (HAPTOPHYTA)1

There is still considerable uncertainty about the relationship between calcification and photosynthesis. It has been suggested that since calcification in coccolithophorids is an intracellular process that releases CO₂, it enhances photosynthesis in a manner analogous to a carbon‐concentrating mechanism (CCM). The ubiquitous, bloom‐forming, and numerically abundant coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was studied in nutrient‐replete, pH and [CO₂] controlled, continuous cultures (turbidostats) under a range of [Ca²⁺] from 0 to 9 mM. We examined the long‐term, fully acclimated photosynthesis‐light responses and analyzed the crystalline structure of the coccoliths using SEM. The E. huxleyi cells completely lost their coccosphere when grown in 0 [Ca²⁺], while thin, undercalcified and brittle coccoliths were evident at 1 mM [Ca²⁺]. Coccoliths showed increasing levels of calcification with increasing [Ca²⁺]. More robust coccoliths were noted, with no discernable differences in coccolith morphology when the cells were grown in either 5 or 9 mM (ambient seawater) [Ca²⁺]. In contrast to calcification, photosynthesis was not affected by the [Ca²⁺] in the media. Cells showed no correlation of their light‐dependent O₂ evolution with [Ca²⁺], and in all [Ca²⁺]‐containing turbidostats, there were no significant differences in growth rate. The results show unequivocally that as a process, photosynthesis in E. huxleyi is mechanistically independent from calcification.     

Involvement of Acidic Polysaccharide Ph-PS-2 and Protein in Initiation of Coccolith Mineralization,as Demonstrated by In Vitro Calcification on the Base Plate

Coccolithophorids, unicellular marine microalgae, have calcified scales with elaborate structures, called coccoliths, on the cell surface. Coccoliths generally comprise a base plate, CaCO₃, and a crystal coat consisting of acidic polysaccharides. In this study, the in vitro calcification conditions on the base plate of Pleurochrysis haptonemofera were examined to determine the functions of the base plate and acidic polysaccharides (Ph-PS-1, -2, and -3). When EDTA-treated coccoliths (acidic polysaccharide-free base plates) or low pH-treated coccoliths (whole acidic polysaccharide-containing base plates) were used, mineralization was not detected on the base plate. In contrast, in the case of coccoliths which were decalcified by lowering of the pH and then treated with urea (Ph-PS-2-containing base plates), distinct aggregates, probably containing CaCO₃, were observed only on the rim of the base plates. Energy dispersive X-ray spectroscopy (EDS) confirmed that the aggregates contained Ca and O, although X-ray diffraction analysis did not reveal any evidence of crystalline materials. Also, in vitro mineralization experiments performed on EDTA-treated coccoliths using isolated acidic polysaccharides demonstrated that the Ca-containing aggregates were markedly formed only in the presence of Ph-PS-2. Furthermore, in vitro mineralization experiments conducted on protein-extracted base plates suggested that the coccolith-associated protein(s) are involved in the Ca deposition. These findings suggest that Ph-PS-2 associated with the protein(s) on the base plate rim initiates Ca²⁺ binding at the beginning of coccolith formation, and some other factors are required for subsequent calcite formation.     

Increased coccolith production by Emiliania huxleyi cultures enriched with dissolved inorganic carbon

Cells of Emiliania huxleyi grown on Eppley's medium enriched with dissolved inorganic carbon (DIC) developed multiple layers of coccoliths. The maximum diameter of cells grown in the presence of 13.2 mM DIC was 12.3 m, whereas that of cells grown in the presence of 1.5 mM DIC was 8.0 m. Although enrichment of Eppley's medium with DIC increased both coccolith production and cell growth, coccolith production was enhanced to a greater extent than cell growth. The enrichment of Eppley's medium with DIC was used to enhance production of coccolith particles by E. huxleyi. Repeated-batch culture, in which DIC, Ca²⁺, nitrate and phosphate concentrations in the medium were maintained by replacing the culture medium, was carried out in a closed photobioreactor. During repeated-batch culture, a maximum coccolith yield of 560 mg/l for 2 days and a maximum biomass yield of 810 mg/l for 2 days were achieved. Enrichment and maintenance of DIC is therefore an efficient method for the production of large quantities of coccoliths.     

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.     

A possible aid to survival of the marine coccolithophorid Cricosphaera and similar organisms

An examination has been made of clones of Cricosphaera from the Plymouth culture collection. These organisms are biflagellate motile coccolithophorids which, in culture, produce non-motile filamentous benthic phases similar to forms included in the Chrysotilaceae (e.g. Apistonema).

Cricosphaera carterae (Plymouth No. 17) produces, in liquid culture, motile cells invested with several layers of characteristically patterned scales and one layer of coccoliths. The scales are of three types, (I) small oval scales, (II) medium-sized round scales and (III) large oval scales which are always associated with coccoliths.

Growth on agar produces Apistonema-like cells which, although lacking scales of Types I and III, have a greatly thickened investment of closely packed Type II scales. Other Apistonema type clones, previously believed to be covered in mucilage, are similarly ensheathed by thick layers of scales which are thought to aid the benthic phases in withstanding dry conditions.     

Enhanced Calcite Dissolution in the Presence of the Aerobic Methanotroph Methylosinus trichosporium

Microbial aerobic methane oxidation (MOx) is intrinsically coupled to the production of carbon dioxide, favoring carbonate dissolution. Recently, microbial organic polymers were shown to be able to induce carbonate dissolution. To discriminate between different mechanisms causing calcite dissolution, experiments were conducted in the presence of solid calcite with (1) actively growing cells (2) starving cells, and (3) dead cells of the methanotrophic bacterium Methylosinus trichosporium under brackish conditions (salinity 10) near calcite saturation (saturation state (Ω) 1.76 to 2.22). Total alkalinity and the amount of dissolved calcium markedly increased in all experiments containing M. trichosporium cells. After initial system equilibration, similar calcite dissolution rates, ranging between 20.16 (dead cells) and 25.68 μmol L^?1 d^?1 (actively growing cells), were observed. Although concentrations of transparent exopolymer particles declined with time in the presence of actively growing and starving cells, they increased in experiments with dead cells. Scanning electron microscopy images of calcite crystals revealed visible surface corrosion after exposure to live and dead M. trichosporium cells. The results of this study indicate a strong potential for microbial MOx to affect calcite stability negatively, facilitating calcite dissolution. In addition to CO₂ production by methanotrophically active cells, we suggest that the release of acidic or Ca²⁺-chelating organic carbon compounds from dead cells could also enhance calcite dissolution.     

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.     

Light and electron microscope observations of the type species of Syracosphaera,s. pulchra (Prymnesiophyceae)

Syracosphaera pulchra, the type species of the genus was isolated into unialgal culture and studied with both the light and electron microscope. A conspicuous coiling haptonema is present containing seven microtubules in the shaft and eight in the basal region; features shared with many taxa in the order Prymnesiales. The proximal and distal coccoliths differ in shape but resemble each other structurally: the outer elements alternate to make the rim. The proximal coccoliths possess an organic base-plate scale which is absent in the distal coccoliths. The uncalcified organic scales are ornamented by a radial, more or less concentric, fibrillar pattern and are arranged in several layers between the proximal coccoliths and the plasmalemma. The ultrastructure of the cell is typical of prymnesiophycean algae. The flagellar apparatus is characterized by the absence of secondary microtubular bundles which are usually well developed in other coccolithophorids with two microtubular roots. This feature is also rather similar to that found in members of the Prymnesiales.

This investigation has indicated that S. pulchra has, in some respects, a closer affinity with members of the Prymnesiales than with the coccolithophorids.     

Coccolithophore (–CaCO3) flux in the Sea of Okhotsk: seasonality, settling and alteration processes

Coccolithophore fluxes were determined in the Sea of Okhotsk using samples from a 1 year experiment (12 August 1990 to 12 August 1991) with sediment traps at 258 and 1061 m depth. A special study was made on Coccolithus pelagicus, using fragmentation and the degree of etching, as indicators of transport mechanisms. A Corrosion Index for C. pelagicus is developed. The coccolithophore flux pattern at 258 m depth was characterised by a strong seasonality, with flux peaks during autumn 1990 (late November to early December) and spring 1991 (March). The assemblage consisted almost entirely of the two species C. pelagicus and Emiliania huxleyi. During autumn, coccolithophore transportation to 258 m depth mainly occurred within cylindrical fecal pellets and marine snow aggregates of silicoflagellates, and through agglutination on tintinnids. Grazing caused severe fragmentation of coccoliths and disintegration of coccospheres. Marine snow aggregates contained many intact coccospheres of C. pelagicus. During spring, coccolithophores were probably removed from the euphotic zone by the ballast effect of sinking diatoms. The coccolithophore flux peak in spring occurred immediately after the ice had retreated from the trap station, and the trapped assemblage included coccoliths of subtropical species. These features indicate drifting from an ice-free location to the south or east.The coccolith and coccosphere flux at 1061 m was respectively 7 and 12 times lower than at 258 m depth, and maximum fluxes were recorded 2 months later. Increasing carbonate dissolution from 258 to 1061 m depth is expressed in the coccolithophore–CaCO₃ flux reduction of 82%, and in the increasing percentage of etched coccoliths of Coccolithus pelagicus from 32 to >90%.     

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO₄). Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn²⁺, Ca²⁺, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of β-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO₄, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn²⁺ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of β-hexosaminidase, ultrafine ZnO particles and ZnSO₄, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn²⁺ easily and increase intracellular Zn²⁺ concentration efficiently, thus decreasing FcεRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.     

Sr Isotope Tracing of Differences in the Effects of Microorganisms on Weathering of Calcite and Apatite

To study differences in the effects of microorganisms on weathering of calcite and apatite, one strain of Aspergillus niger (A. niger) and one strain of Penicillium glaucum (P. glaucum), which respectively contain the mixture of calcite and apatite were cultivated for 24 days in the sucrose-potato culture medium, supernatant was taken every three days from the culture medium, followed by the determination of Ca²⁺ and Sr²⁺ contents and Sr isotopic ratios. The results of measurement showed that the Sr isotope ratios in the supernatant from the culture medium are intermediate between those of the end-member constituents calcite and apatite (0.70721-0.70861). Results of isotope mixing equations to calculation showed that in the first 15 days A. niger played a dominant role in weathering of calcite in the apatite/calcite mixture. The contribution rate of apatite for Ca²⁺ in the solution increased from 39.0% on the 18th day to 61.6% on the 24th day; P. glaucum played a key role in weathering of apatite in the first 3 days. Ions dissolved from apatite account for 73.9% of the total. It is known from the results of Sr isotope tracing that in the prior period of fungus cultivation A. niger plays a key role in weathering of calcite while P. glaucum plays a key role in weathering of apatite. The ability of P. glaucum to weather calcite tends to intensify progressively over time. Therefore, Sr isotope tracing can be used to accurately recognize differences in the effects of microorganisms on weathering of minerals.     

Polyanion-mediated mineralization — mineralization in coccolithophore (Pleurochrysis carterae) variants which do not express PS2, the most abundant and acidic mineral-associated polyanion in wild-type cells

Summary Highly acidic macromolecules are postulated intermediates in biomineralization because they sequester large numbers of calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. The mineral-associated polyanions in the coccolithophorePleurochrysis carterae are acidic polysaccharides which surround calcite elements on mineralized scales called coccoliths. PS2 is the most abundant and acidicPleurochrysis polyanion, containing four carboxyl groups per disaccharide repeat. This study examines the mineralizing function of PS2 by analysis of calcium carbonate deposition in PS2-deficient cells. Mineral-deficientPleurochrysis variants were separated from wild-type cells by density gradient centrifugation and analyzed for PS2 expression. Three independent low-density variants (two spontaneous and one chemically-induced) not expressing PS2 (ps2⁻) were isolated. The mineral content of each ps2⁻ variant was less than 5% of the wild-type level. Most ps2⁻ coccoliths were unmineralized, but when crystals were present, they occurred on the coccolith rim as in wild-type cells. When ps2⁻ rim elements were adequately large and numerous, they displayed the alternating A and B anvil shapes characteristic of wild-type rim elements. Although ps2⁻ cells may have defects which affect the expression of cellular components in addition to PS2, correlation of PS2 nonexpression with mineral deficiency in three independent variants supports the hypothesis that PS2 is a functional intermediate in mineral deposition. PS2 may affect the level ofPleurochrysis mineralization by (a) controlling the amount of calcium ions available for mineralization, (b) accelerating the rate of calcite nucleation, or (c) both. However, PS2 does not influence crystal morphology, since the more mature crystals in ps2⁻ cells exhibit the anvil-shaped morphology of wild-type crystals.     

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Laboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. ¹⁴C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. ¹⁴C-labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a ¹⁴C-based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell^?1·d^?1 while net detachment rates were as high as 15 coccoliths· cell^?1·d^?1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.