Megumi Okazaki

Effects of calcium-binding polysaccharides from calcareous algae on calcium carbonate polymorphs under conditions of double diffusion

Abstract The effects of calcium-binding acid polysaccharides from magnesian-calcite-depositing algae, and a potent scale inhibitor, hydroxyethylidene 1,1-diphosphonic acid (HEDP), on CaCO 3 polymorphs were studied by a double diffusion technique. Magnesian calcite was formed with aragonite by adding the acid polysaccharides or HEDP to the agar-gel even in the presence of Mg 2+ in Ca 2+ solution (Ca 2+ concentration, 0.1M; Ca 2+ /Mg 2+ = 2.0), and the ratio of magnesian calcite to aragonite increased with increasing amounts of the additives. These results suggest that magnesian calcite was formed by transformation of aragonite nuclei into calcite ones with the inhibition of aragonite growth by acid polysaccharides or HEDP. The effects of the acid polysaccharides explain the occurrence of magnesian calcite in these algae.

Some observations on the Ca2+-binding phospholipid from scleractinian coral skeletons

1. 1. The lipid composition was determined of the skeletons and soft tissues of several reef-building corals. The phospholipids from these two sources were remarkable for binding Ca2+-ions in vitro. 2. 2. The Ca2+-binding capacity of skeletal phospholipid increased above pH 9.0, especially in the presence of CO32− ions. The Ca2+-binding capacity of the lipids was measured in the presence of various metals. The relative inhibition exerted by these metals was dependent on the kind of metal ions added to the reaction medium; in addition, there was a slight difference between lipids from the skeletons and the soft tissues. 3. 3. A histochemical test revealed the existence of sudanophilic material in skeletal organic matrix of a branching coral, Acropora hebes. 4. 4. These data suggest that the Ca2+-binding phospholipids of the skeletons might serve as seeding sites for CaCO3 depositions in the process of skeleton formation in scleractinians.

Calcification in Higher Plants with Special Reference to Cystoliths

The cystolith, a calcified body in the leaf of higher plants, was observed by soft X-ray microradiography using the mature leaves of nine species from five families. The microradiographs revealed very large cigar-shaped cystoliths (up to 500 μ m in length) in Pilea viridissima and Justicia procumbens, neighbor-cystoliths in Morus bombycis and Humulus scandens, and two to seven radially arranged cystoliths in Momordica charantia. The number of cystoliths (n/cm2) of various kinds of leaves vas estimated to be 850 to 4,200 by microradiography. The calcium carbonate content (mg/cm2) calculated was 0.3 to 1.1, suggesting a large reservoir of calcium or carbon dioxide. The cystoliths were isolated from leaves of nine species to analyze chemically, with electron probe, and with X-ray diffraction. The calcium carbonate content in cystoliths was about 75% on a dry weight basis. A small amount of magnesium was also found. Electron probe analysis revealed that calcium and magnesium were evenly distributed through the cystolith body except for the stalk and the basal part of the body to which the stalk is attached. In the latter parts, silicon was detected in high density, suggesting silicification of these parts. X-ray diffraction patterns showed amorphous calcium carbonate in all species tested. However, vaterite in Morus bontbycis and both vaterite and calcite in Ficus elastica were also detected in small amounts. The amorphous calcium carbonate in cystoliths changed rapidly into calcite in 0.05 M carbonate buffer (pH 9.2) or in distilled water. Then the smooth surface of the cystoliths was covered with small cubic calcite crystals. A tremendous number of cystoliths was contained in a single leaf, and the cystolith-bearing lithocyst was associated with many photosynthetic parenchyma cells in all species. These facts suggest a relationship between calcification in the cystolith and photosynthesis in the leaf. A possible mechanism of cystolith calcification coupled with bicarbonate utilization in photosynthesis is discussed.

A study of calcium carbonate deposition in the genus Padina (Phaeophyceae, Dictyotales)

Three species of the phaeophycean genus Padina were investigated with the electron microscope. Calcification is initiated in P. japonica and P. pavonica at the inrolled thallus margin which contains chloroplasts and acts as a semi-enclosed space. In these species, the apical cells are covered with a pilose layer with which small aragonite crystals are associated. The non-calcareous P. arborescens has a similar anatomy but here the apical and young cells possess a smooth cuticular layer instead of a pilose layer and it is suggested that nucleation of aragonite is inhibited by this layer. In P. pavonica, carbonate deposition continues at the thallus surface and reaches a maximum, on a percentage dry weight basis, approximately mid-way along the lamina (mean CaCO3 content 9·3% dry wt). The deposits show concentric banding, but this could not be correlated with differences in photosynthetic activity associated with tetrasporangium production.

Development of a rapid isolation procedure for coccolith ultrafine particles produced by coccolithophorid algae

A rapid procedure for effective purification of large quantities of coccolith ultrafine particles from marine algae is reported. Coccoliths are detached from cells by optimized sonication in the presence of 50 mM NaHCO3. Contaminating cell debris is then removed from coccoliths by cycles of washing and floatation. Coccolith particles were purified fromEmiliania huxleyi andPleurochrysis carterae. The surface area of these particles is three to five times greater than synthetic calcite particles. Glucose oxidase and uricase have been immobilized onto purified coccolith ultrafine particles to illustrate their potential as a support material for biotechnological application.

Production of ultrafine calcite particles by coccolithophorid algae grown in a biosolar reactor supplied with sunlight

Ultrafine calcite particle production by coccolithophorid algae using a biosolar reactor system was carried out. Solar light was collected by Fresnel lenses and transmitted to a bundle of light diffusing optical fibers (LDOFs) that distributed light through the algal culture. The irradiance spectrum of light emitted from the LDOF surface was the same as that of solar light.Emiliania huxleyi was cultured for 8 d, and 43 mg/L of calcite were produced. These results demonstrate the potential of the biosolar reactor system for CO2 fixation, and ultrafine calcite particle production using coccolithophorid algae and sunlight.

Application of soft X-ray microradiography to observation of cystoliths in the leaves of various higher plants.

Soft X-ray microradiography was applied to observation of the cystoliths, calcified bodies of higher plants, in the leaves ofMorus bombycis, Humulus scandens, Ficus elastica, F. retusa (Moraceae),Boehmeria platanifolia, Pilea viridissima (Urticaceae) andMomordica charantia (Cucurbitaceae). It was proved that this technique is useful for examination of the shape, size, distribution and number of cystoliths in fresh leaves. The microradiographs revealed large cigar-shaped cystoliths in the leaf ofP. viridissima, and neighbor-cystoliths in somewhat restricted areas of the leaves ofM. bombycis andH. scandens, and two to seven radially arranged cystoliths in the leaf ofM. charantia. The number of cystoliths per unit area of leaf (nos./cm2) was estimated to be from 1,090 to 3,900 by means of the microradiographs, varying from species to species. The CaCO3 content of the leaf calculated from the volume and number of cystoliths was approximately 0.4 mg/cm2 in all species exceptF. retusa. InF. retusa, it was about 1.06 mg/cm2, the highest value among all species tested. Hand-sections of the leaves showed that the lithocysts were localized in the upper and/or lower epidermis, and they were associated with many photosynthetic cells in all species, suggesting some relationship between CaCO3 deposition in cystoliths and photosynthesis.

Continuous production of extracellular ultrafine calcite particles by the marine coccolithophorid alga Pleurochrysis carterae

A new procedure for the production of ultrafine calcite particles by the marine coccolithophorid alga Pleurochrysis carterae is reported. During continuous culture, calcite particles (coccoliths) were detached from the cell surface by optimized air-bubbling, which greatly reduced the damage associated with previous sonication methods. Detached calcite particles could be continuously recovered directly from the culture medium using a nylon mesh membrane filtration module. Cells remained viable and continued to produce coccoliths during culture. The optimum productivity of ultrafine calcite particles was 18 mg/l per day. These results demonstrate the potential for a continuous system for the photosynthetically driven removal of CO2 and its fixation into ultrafine inorganic calcite particles.

Silicon-accumulating idioblasts in leaves of Cecropiaceae (Urticales)

A survey of the structure and mineral composition of leaf idioblasts in the Cecropiaceae was conducted. In all six genera of the family, idioblasts usually occur as trichomes or enlarged epidermal cells and nearly always accumulate Si, in marked contrast to the idioblasts of other members of the Urticales, which mostly possess cystoliths containing abundant Ca and Si. The horizontally elongate, mineralized structures ofPoikilospermum, reported formerly as “cystoliths” also contain mainly Si and little Ca. Six genera of Cecropiaceae share a common character in accumulating abundant Si in idioblasts of the leaf epidermis, while lacking cystoliths composed of abundant Ca and Si.

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two‐phase partitioning. Its purity was examined with marker enzymes, Mg2+‐dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH‐cytochrome c reductase, as well as the sensitivity of Mg2+‐dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid‐chromic acid. Mg2+‐ or Ca2+‐dependent ATPases were associated with the plasma membrane. Ca2+‐dependent ATPase was activated at physiological cytoplasmic concentrations of Ca2+ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg2+‐dependent ATPase. The isolated plasma membrane vesicles were mostly right side‐out. To test for H+‐translocation, right side‐out vesicles were inverted; 27% of vesicles were inside‐out after treatment with Triton X‐100. The inside‐out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca2+. The reduced fluorescence was recovered with the addition of 10 mmol/L NH4Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca2+‐dependent ATPase might pump H+ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.