Kunio Kohata

PHOSPHATE ACCUMULATION AND METABOLISM BY HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) DURING DIEL VERTICAL MIGRATION IN A STRATIFIED MICROCOSM1

Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (Pi) concentration in the upper layer and high salinity and high Pi concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using 31P‐NMR spectroscopy. At night this species migrated to the lower phosphate‐rich layer and took up inorganic phosphate (Pi) which then was accumulated as polyphosphate (PPi) by an increase in the chain length of PPi During the daytime this species migrated to the phosphate‐depleted surface water and utilized the accumulated PPi for photophosphorylation by decreasing the chain length of PPi During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as Pi But the cells did not convert Pi to PPi presumably due to their lack of ATP. After the second day of the experiment, conversion of Pi to PPi at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of Pi and conversion to PPi at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PPi in the metabolism of this species appears to be as a phosphate pool which regulates the level of Pi and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate‐rich lower layer and the photic upper layer in stratified waters. 31P‐NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms.

DIEL CHANGES IN THE COMPOSITION OF PHOTOSYNTHETIC PIGMENTS AND CELLULAR CARBON AND NITROGEN IN CHATTONELLA ANTIQUA (RAPHIDOPHYCEAE)1

An axenic clonal culture of Chattonella antiqua (Hada) Ono was grown on a 12: 12 h LD cycle in a laboratory culture tank containing 1 m3 of f/2 medium. Diel changes in mean cell volume, cellular carbon (carbon content per cell), C/N ratio, cellular Chl a, Chl a/c ratio and carotenoid composition were observed. Mean cell volume and cellular C, N and pigments increased during the light period as a result of photosynthesis and decreased with increase of cell concentration by phased cell division during the dark period. These changes indicated that carbon assimilation and pigment synthesis occurred together during the light period. However, the patterns of increase were not the same since different diel patterns were also found in the ratios of C/N and chl a/c. Photosynthetic pigments were analyzed by reversed‐phase high‐performance liquid chromatography with ion‐pairing solution. This analysis showed that the dominant carotenoids in C. antiqua were fucoxanthin, violaxanthin and β‐carotene. Diel patterns of Chls a and c were similar to that of fucoxanthin but different from those of violaxanthin and β‐carotene. The cellular contents of Chl a, fucoxanthin and carbon increased in a parallel manner during the light period. On the other hand, the increase of violaxanthin was restricted to only a few hours at the beginning of the light period during cell division cycles.

Crude oil bioremediation field experiment in the Sea of Japan.

Experimental bioremediation of crude oil was conducted for approximately 3 months in the intertidal zone of the Sea of Japan, Hyogo Prefecture. Artificial mixtures of weathered Arabian light crude oil and sand taken from the experimental site were wrapped in polyester net envelopes. The envelopes were placed in drum-shaped acrylic vessels with perforated sides to facilitate seawater exchange. The vessels were laid in the intertidal area. Slow release nitrogen and phosphorus synthetic fertilizer granules were added to the oil-sand mixtures in three different amounts. Some oil-sand mixtures were unfertilized controls. The oil-sand mixtures were periodically sampled and changes in the composition of the residual oils were monitored. Oil samples were subjected to gas chromatography coupled with mass spectrometry for analysis of some representative semi-volatile aliphatic and aromatic compounds. All values for each analyte were normalized against that of hopane to evaluate the extent of oil biodegradation. Significant increases in the concentrations of both nitrogen and phosphorus were found in the fertilized sections in accordance with the amounts of added fertilizers. Although significant natural attenuation of oil was observed in the unfertilized sections, fertilization stimulated the degradation rate of the oil in the early stage of the experimental term. The extent of the oil biodegradation increased as the amount of added fertilizer increased. However, the final degradation efficiencies for each oil component in the fertilized sections were not significantly different from those in the unfertilized sections, and the degradation of each oil component had almost ceased after 6 weeks. We conclude that excessive amounts of macronutrients are required to accelerate oil biodegradation and that fertilization is only effective in the early stages.

Phosphate metabolism during diel vertical migration in the raphidophycean alga, Chattonella antiqua

The ecological advantage of diel vertical migration on the nutrition and accumulation of Chattonella antiqua, which is one of the dominant red-tide forming phytoplankton species in the Seto Inland Sea of Japan, was examined using a large axenic culture tank, in which vertical stratification of salinity, temperature and nutrients was maintained, analogous to natural conditions observed when red tides occur. C. antiqua was capable of migrating through very sharp salinity and temperature gradients. At night the species migrated to the deep nutrient-rich water and assimilated nutrients. During the daytime it migrated to the nutrient-depleted surface water and used the accumulated nutrients for photosynthesis. Nitrogen uptake was synchronized with phosphate uptake. 31P-NMR spectroscopy during the migration experiment revealed that C. antiqua has the capability of nocturnal phosphate uptake in the deep nutrient-rich water, but no capability of synthesizing polyphosphate, which was considered to be the intracellular phosphate pool. These findings were compared with those reported for another raphidophycean, Heterosigma akashiwo. Although both species carry out vertical migration and nocturnal nutrient uptake, only H. akashiwo has the capability of making an intracellular polyphosphate pool.

Synchronous division and the pattern of diel vertical migration of Heterosigma akashiwo (Hada) Hada (Raphidophyceae) in a laboratory culture tank

Abstract Heterosigma akashiwo (Hada) Hada (Raphidophyceae) causes red tides in Osaka Bay (Japan). A clonal culture of the alga was grown in a 2 m tall culture tank on a 12: 12 LD cycle to determine patterns of vertical migration and cell division. A specific growth rate of 0.43 ln unit · day−1 was obtained during complete mixing conditions. Under weakly stratified conditions (≈ΔT = 3–4°C/1.5 m), H. akashiwo in the tank grew and showed a similar pattern of vertical migration to that observed in the field for at least 6 days. Cell concentration, mean cell volume, and photosynthetic capacity, estimated by DCMU-induced fluorescence increase of H. akashiwo, were monitored in the stratified tank at 2-h intervals over 24 h at three levels in the water column. Cell ascent began shortly before the light period and vertically swimming cells were smaller in size than those sampled near the bottom of the tank. The cell division cycle and the pattern of vertical migration were phased individually by the light regime and were well synchronized with each other. This synchrony must be due to the interrelation between these two processes or the existence of a clock which controlled endogenous rhythms of both processes and was entrained by a light: dark cycle. The relative increase of fluorescence with DCMU was higher for migrating cells than for non-migrating cells.

Abundance and Population Structure of Ammonia-Oxidizing Bacteria That Inhabit Canal Sediments Receiving Effluents from Municipal Wastewater Treatment Plants

ABSTRACT A polyphasic, culture-independent study was conducted to investigate the abundance and population structure of ammonia-oxidizing bacteria (AOB) in canal sediments receiving wastewater discharge. The abundance of AOB ranged from 0.2 to 1.9% and 1.6 to 5.7% of the total bacterial fraction by real-time PCR and immunofluorescence staining, respectively. Clone analysis and restriction endonuclease analysis revealed that the AOB communities influenced by the wastewater discharge were dominated by Nitrosomonas, were similar to each other, and were less diverse than the communities outside of the immediate discharge zone.

Natural water-purification system observed in a shallow coastal lagoon: Matsukawa-ura, Japan.

Field surveys and in situ experiments were conducted in the shallow Matsukawa-ura in summer to evaluate the biological efficiencies of shallow-water areas for preserving coastal ecosystems. In Matsukawa-ura (5.8 km(2)), the suspension-feeding bivalves Ruditapes philippinarum and Crassostrea gigas were the dominant animals--their total biomasses (wet weight) were estimated to be 3.4 x 10(6) and 2.3 x 10(6) kg, respectively. Ulva sp. and Zostera marina were the dominant macrophyte species during the summer, with standing crops estimated to be 0.29 x 10(6) and 0.20 x 10(6) kg, respectively. The dissolved inorganic nitrogen (DIN) uptake rates for Ulva sp. and Z. marina were determined by in situ experiments. An ecological model calculated on the basis of the observed dataset showed that, in comparison with tidal exchange, a significant amount of particulate organic matter was removed by bivalve filtration and a considerable quantity of DIN was removed by macrophyte species.

Diel changes in the composition of photosynthetic pigments and cellular carbon and nitrogen in pyramimonas parkeae (Prasinophyceae).

Diel changes in mean cell volume, cellular carbon (carbon content per cell), cellular Chl a, C/N ratio, Chl a/carbon ratio and pigment composition were determined for an axenic clonal culture of Pyramimonas parkeae Norris et Pearson through three 12:12 h LD cycles in a laboratory culture tank of 1 m3. Mean cell volume and cellular C, N and most pigments increased during the light period as a result of photosynthesis and decreased with an increase in cell density by phased cell division during the dark period. Chi a and Chi b increased in a parallel manner during the light period. Increases in the diel synthesis pattern of carotenoids varied. Violaxanthin and lutein content increased for a few hours at the beginning of the light period and preceeded that of neoxanthin. The diel synthesis pattern of neoxanthin was similar to that of Chi a. Increases of loroxanthin and its ester form were slower than that of Chi a at the beginning of the light period. A net increase of α‐carotene was observed during the dark period. Mass spectroscopy of carotenoid structure showed a new xanthophyll, loroxanthin dodecenoate, in this species.

Highly sensitive determination of photosynthetic pigments in marine in situ samples by high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographic (HPLC) method was developed by optimizing a solvent system for the highly sensitive determination of photosynthetic pigments in marine in situ samples. The proposed HPLC system can handle injection volumes greater than 400 μl without a significant loss in the resolution between chlorophyllide a and chlorophyll c 1 + c 2 . The system also reduces the sample volumes required for the analysis of typical pigments of oligotrophic sea water to 500 ml. The elution time for all pigments is 24 min at a flow-rate of 1 ml/min and 12 min at a flow-rate of 2 ml/min using a 250 mm × 4.6 mm I.D. column.

A red-tide bloom of Chattonella antiqua (Raphidophyceae) in the Seto Inland Sea, Japan, in 1987 analysed as changes in photosynthetic pigments determined by high-performance liquid chromatography

Abstract A severe red-tide bloom of Chattonella antiqua (Raphidophyceae) was observed in the Seto Inland Sea (Japan) in summer 1987. The C. antiqua bloom was monitored from beginning to end at the 0, 5, 10, 15 m and bottom layers of a fixed sampling station almost every two days. Carotenoids and chlorophyllous pigments were measured by reversed-phase high-performance liquid chromatography before and during the bloom. Before the C. antiqua bloom, some diatoms were dominant, with a chlorophyll a (Chla) concentration ranging between 2 and 8 μg litre−1 at the surface. The maximum Chla concentration reached 27 μg litre−1 at the surface during the bloom, this being due to C. antiqua. Violaxanthin, which is a constituent carotenoid of C. antiqua, varied in line with cell concentration of the species. The relative abundance of C. antiqua was estimated as its Chla contribution to total Chla; the Chla contributions of Raphidophyceae, Bacillariophyceae and Dinophyceae were calculated from pigment ratios obtained by multiple regression analysis of pigment concentrations. The relative abundance of C. antiqua at the surface was calculated to be 0–16% before the bloom and 83–100% during the bloom.