Reduced calcification decreases photoprotective capability in the coccolithophorid Emiliania huxleyi

Intracellular calcification of coccolithophores generates CO? and consumes additional energy for acquisition of calcium and bicarbonate ions; therefore, it may correlate with photoprotective processes by influencing the energetics. To address this hypothesis, a calcifying Emiliania huxleyi strain (CS-369) was grown semi-continuously at reduced (0.1 mM, LCa) and ambient Ca2? concentrations (10 mM, HCa) for 150 d (>200 generations). The HCa-grown cells had higher photosynthetic and calcification rates and higher contents of Chl a and carotenoids compared with the naked (bearing no coccoliths) LCa-grown cells. When exposed to stressfull levels of photosynthetically active radiation (PAR), LCa-grown cells displayed lower photochemical yield and less efficient non-photochemical quenching (NPQ). When the LCa- or HCa-grown cells were inversely shifted to their counterpart medium, LCa to HCa transfer increased photosynthetic carbon fixation (P), calcification rate (C), the C/P ratio, NPQ and pigment contents, whereas those shifted from HCa to LCa exhibited the opposite effects. Increased NPQ, carotenoids and quantum yield were clearly linked with increased or sustained calcification in E. huxleyi. The calcification must have played a role in dissipating excessive energy or as an additional drainage of electrons absorbed by the photosynthetic antennae. This phenomenon was further supported by testing two non-calcifying strains, which showed insignificant changes in photosynthetic carbon fixation and NPQ when transferred to LCa conditions.     

ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

Structural and physiological effects of calcium and magnesium in Emiliania huxleyi (Lohmann) Hay and Mohler

In organisms which perform both photosynthesis and calcification, the fact that calcification proceeds faster in the light than in the dark has led to the long-established view that photosynthesis and calcification are closely coupled. It is now clear that calcification does not promote photosynthesis, but an enhancement of calcification by photosynthesis could still explain why calcification is faster in the light. To test this, the kinetics of the two processes were monitored over a wide range of calcium concentrations (0-50 mM) in the coccolithophore Emiliania huxleyi. The addition of 50 mM calcium strongly inhibited both processes, but when incubated in lower concentrations, rates of calcification increased up to 20 mM calcium whilst those of photosynthesis remained constant over the same range of calcium concentrations. So, rates of calcification are able to rise without a concomitant increase in photosynthetic rates. In addition, calcification rate and coccolith morphology responded similarly to changes in calcium concentrations; low calcification rates were associated with poor coccolith structure (undercalcification) and high calcification rates with perfectly formed coccoliths. Calcium concentration thus strongly influences calcification affecting both crystal structure and rate of calcite deposition. A similar structural analysis of coccoliths from cells grown in different magnesium concentrations showed that this ion is also essential for calcification, since strong signs of coccolith malformation and undercalcification were apparent at both low and high magnesium concentrations. In contrast with the calcium results, coccoliths were flawless only in the normal seawater concentration of 58 mM magnesium. We conclude that photosynthesis and calcification are not closely coupled and that calcification depends on a precise balance of both calcium and magnesium concentrations.     

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

? To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. ? Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). ? Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). ? The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.     

Cold Stress Stimulates Intracellular Calcification by the Coccolithophore, Emiliania huxleyi (Haptophyceae) Under Phosphate-Deficient Conditions

Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. (45)Ca incorporation into coccoliths was more than 97% of the total (45)Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 muM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20 degrees C than at 12 degrees C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12 degrees C than at 20 degrees C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by (45)Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.     

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.     

The mutual interplay between calcification and coccolithovirus infection

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, ‘induced media’. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host–virus interactions in the ocean and interpreting their impact on the global carbon cycle.     

GPA, A CALCIUM-BINDING PROTEIN IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)

Intracellular polysaccharide fractions were isolated from calcifying B-type cells of Emiliania huxleyi and separated by electrophoretic fractionation. In all fractions, the polysaccharide was immunologically related to the polysaccharide of (extracellular) B-type coccoliths (CP-B) and not to polysaccharides of A-type coccoliths (CP-A). Most polysaccharide fractions also contained protein material. The fraction with the largest proportion of protein was used to raise antibodies. The resulting antiserum, α-BP, contained antibodies against both CP-B- and protein-epitopes. The antibodies specific for polysaccharide-epitopes reacted with intracellular polysaccharide fractions of B-type cells only. In contrast, the antibodies specific for protein-epitopes reacted with the intracellular fractions of B-type as well as A-type cells. With immunolocalization, the presence of protein antigen in a layer surrounding both types of cells was demonstrated. A cDNA library of E. huxleyi was screened with α-BP, and a gene called gpa was isolated. The open reading frame of gpa was found to encode a protein (GPA) of 36,608 D, containing, inter alia, 24% acidic residues (18% glutamic acid and 6% aspartic acid), 12% proline, and 23% alanine. GPA has two repeats, one containing a sequence resembling the Ca²⁺-binding loop of EF-hands. Overproduction of GPA in a prokaryotic system yielded a dimeric product capable of binding Ca²⁺. The possible role of GPA in the formation of coccoliths in E. huxleyi is discussed.     

Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

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.     

Effects of elevated CO2 on growth,calcification, and spectral dependence of photoinhibition in the coccolithophore Emiliania huxleyi (Prymnesiophyceae)1

We studied the effects of elevated CO₂ concentrations on cell growth, calcification, and spectral variation in the sensitivity of photosynthesis to inhibition by solar radiation in the globally important coccolithophore Emiliania huxleyi. Growth rates and chlorophyll a content per cell showed no significant differences between elevated (800 ppmv) and ambient (400 ppmv) CO₂ conditions. However, the production of organic carbon and the cell quotas for both carbon and nitrogen, increased under elevated CO₂ conditions, whilst particulate inorganic carbon production rates decreased under the same conditions. Biometric analyses of cells showed that coccoliths only presented significant differences due to treatments in the central area width. Most importantly, the size of the coccosphere decreased under elevated CO₂ conditions. The susceptibility of photosynthesis to inhibition by ultraviolet radiation (UVR) was estimated using biological weighting functions (BWFs) and a model that predicts photosynthesis under photosynthetically active radiation and UVR exposures. BWF results demonstrated that the sensitivity of photosynthesis to UVR was not significantly different between E. huxleyi cells grown under elevated and present CO₂ concentrations. We propose that the acclimation to elevated CO₂ conditions involves a physiological mechanism of regulation and allocation of energy and metabolites in the cell, which is also responsible for altering the sensitivity to UVR. In coccolithophores, this mechanism might be affected by the decrease in the calcification rates.     

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.     

A renewable energy source — hydrocarbon gases resulting from pyrolysis of the marine nanoplanktonic alga Emiliania huxleyi

The marine coccolithophore, Emiliania huxleyi, grown in the laboratory was subjected to vacuum pyrolysis at various temperatures from 100 to 500 °C. The highest yield of pyrolytic gases (183 mL g⁻¹ dry cells) was obtained at 400 °C. The amount of total hydrocarbon gas produced at 400 °C was 129 mL, about 10 times higher than at 300 °C. CH₄ was the major component at the high gas-production stage (400–500 °C). The great increase in hydrocarbon gases at 400 °C was accompanied by a marked decrease in liquid saturates and aromatics. The results indicate that the liquid hydrocarbons (oil) produced by pyrolysis at lower temperature is a direct source for the formation of the hydrocarbon gases. Due to its large potential for the production of biomass and hydrocarbons with low energy input, E. huxleyi is suggested as one of candidates for the production of renewable fuels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Discrimination of the Cell Surface of the Coccolithophorid Pleurochrysis haptonemofera from Light Scattering and Fluorescence After Fluorescein-Isothiocyanate-Labeled Lectin Staining Measured by Flow Cytometry

We investigated the extent of calcification on the cell surface of the coccolithophorid Pleurochrysis haptonemofera using flow cytometry. Side scattering (SSC) by coccolith-bearing cells was higher than that by naked cells, suggesting the difference was due to scattering of the laser beam by the coccoliths. SSC of coccolith-bearing cells under acidic conditions corresponded well to the extracellular Ca content, although SSC could not be used to detect a delicate change in the coccolith thickness. The increase in SSC during the reproduction of coccoliths after decalcification was consistent with the increase in the number of coccoliths on the cell surface. The fluorescence after fluorescein-isothiocyanate-labeled lectin staining suggests that α-d-mannose, α-d-glucose, d-galactose, d-N-acetylgalactosamine, or derivatives of them are included in the coccoliths. Measurement of SSC and fluorescence after fluorescein-isothiocyanate-labeled lectin staining enabled rapid and quantitative determination of the status on the cell surface and isolation of desirable cells for physiological studies by cell sorting. Received May 22, 2001; accepted July 30, 2001.     

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.     

Patterns of carbon and nitrogen uptake during blooms of Emiliania huxleyi in two Norwegian fjords

Blooms of the coccolithophorid Emiliania huxleyi were monitoredin two land-locked fjords, Fauskangerpollen and Nordåsvannet(Western Norway), in May 1993. The chemical composition of particulatematter, size-fractionated photosynthesis, calcification, nitrogenuptake rates and the patterns of macromolecular synthesis wereexamined during the peak and decline of E.huxleyi blooms. Thetemporal evolution of the bloom in Fauskangerpollen was definedby a gradual decrease in cell abundance and cell-specific calcificationrates, and by increasing numbers of empty coccospheres and theratio detached coccoliths/living cells. A large proportion ofthe nitrogen required for microplankton growth was suppliedby aminonium and dissolved organic compounds such as urea and,as a consequence, the f-ratios were low (0.2). In general, nitrogenuptake patterns were consistent with ambient concentrationsof nitrogenous species. The photosynthetic carbon metabolismof E.huxleyi dominated phytoplankton assemblages was characterizedby high carbon allocation into lipids and relatively low carbonincorporation into protein as compared with diatom-dominatedassemblages. Consequently, the organic C/nitrogen uptake ratiodetermined stoichiometrically was significantly higher (up to10.8) when coccolithophorids were dominant than in diatom-basedor mixed-phyto-plankton assemblages. These carbon incorporationpatterns were reflected in differences in the chemical compositionof particulate matter.     

Polarized distribution of Na+/H+ antiport and Na+/HCO3- cotransport in primary cultures of renal inner medullary collecting duct cells.

Primary cultures of rat renal inner medullary collecting duct cells were grown to confluence on glass coverslips and treated permeant supports, and the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein was employed to delineate the nature of the transport pathways that allowed for recovery from an imposed acid load in a HCO3-/CO2-buffered solution. The H+ efflux rate of acid-loaded cells was 13.44 +/- 0.94 mM/min. Addition of amiloride, 10(-4) M, to the recovery solution reduced the H+ efflux rate to 4.06 +/- 0.63 mM/min. The amiloride-resistant pHi recovery mechanism displayed an absolute requirement for Na+ but was Cl(-)-independent. Studies performed on permeable supports demonstrated that the latter pathway was located primarily on the basolateral-equivalent (BE) cell surface and was inhibited by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In a Na(+)-replete solution containing DIDS (50 microM) and amiloride (10(-4) M), acid-loaded cells failed to return to basal pHi. To delineate further the amiloride-inhibitable component of pHi recovery, monolayers were studied in the nominal absence of HCO3-/CO2. In 70% of monolayers studied, Na(+)-dependent, amiloride-inhibitable H+ efflux was the sole mechanism whereby acid-loaded cells returned to basal pHi. A Na(+)-independent pathway was observed in 30% of monolayers examined and represented only a minor component of the pHi recovery process. In studies performed on permeable supports, the Na(+)-dependent amiloride-inhibitable pathway was found to be confined exclusively to the BE cell surface. In summary, confluent monolayers of rat renal inner medullary collecting duct cells in primary culture possess two major mechanisms that contribute toward recovery from an imposed acid load, namely, Na+/H+ antiport and Na+/HCO3- cotransport. Na(+)-independent pHi recovery mechanisms represent a minor component of the pHi recovery process in the cultured cell. Both the Na+/H+ antiporter and Na+/HCO3- cotransporter are located primarily on the BE cell surface.     

Physiological regulation of carbon fixation in the photosynthesis and calcification of coccolithophorids

Emiliania huxleyi and Gephyrocapsa oceanica are the predominant coccolithophorid species that produce blooms in the ocean and affect the global environment. These species are capable of carbon fixation by both photosynthesis for organic matter production and by intracellular calcification for coccolith production. Both processes were strongly affected by the nutrient status in a laboratory culture. The coccolith production was stimulated by the addition of a high concentration of sodium bicarbonate and by the depletion of phosphate. Interestingly, when the calcification was stimulated, the increase in cell number during algal growth was greatly suppressed and then the cell volume increased. When the growth rate was increased under nutrient-sufficient conditions, the cells became very small in size and most of them bore few or no coccoliths. The data from laboratory experiments show that the cell growth and calcification proceeded apparently independently at different phases. We, therefore, assume that the coccolithophorid blooms in the ocean might be separated into two phases; firstly, the increase in cell population might be triggered by an adequate supply of nutrients to enhance algal growth and then the calcification might subsequently be stimulated when the nutrients become depleted by substantial algal growth.