Shigeo Tsujimura

Simultaneous production of homoanatoxin-a, anatoxin-a, and a new non-toxic 4-hydroxyhomoanatoxin-a by the cyanobacterium Raphidiopsis mediterranea Skuja

A neurotoxin, homoanatoxin-a, was identified from a toxic strain of the cyanobacterium Raphidiopsis mediterranea Skuja (strain LBRI 48) isolated from Lake Biwa, Japan, as the major toxin component (0.57% of dry cell-weight). This cyanobacterium produced anatoxin-a and a new homoanatoxin-a derivative as minor components (0.04 and 0.06%, respectively). The structure of a new compound was assigned based on the spectral data as 4-hydroxyhomoanatoxin-a, which was not toxic to mice up to 2.0 mg/kg by intraperitoneal injection. The isolation of minor components was accomplished by improved extraction and separation procedures: (1) extraction with methanol-water (4:1) from dried cells, (2) adsorption of aqueous residue on ODS column (or cartridge) after evaporation of methanol, (3) cleaning up of the column by successive elution with water and 50% methanol/water, (4) elution of a toxic fraction by 20% methanol/water containing 0.1% TFA and (5) HPLC (ODS) purification with methanol/water containing 0.05% TFA. The procedures were effective in removing impurities and concentrating alkaloidal neurotoxins. It should be noted that this is the first report demonstrating the simultaneous production of anatoxin-a and homoanatoxin-a by the same strain of cyanobacterium.

Seasonal variations of Microcystis populations in sediments of Lake Biwa, Japan

Seasonal variations of colony numbers of Microcystis aeruginosa(Kütz.) Kütz. and M. wesenbergii(Komárek) Komárek in N. V. Kondrat. in sediments of Lake Biwa were investigated over a period of 1 year. At two stations located in the shallow South Basin of Lake Biwa (ca. 4 m water depth), the colony number of Microcystisfluctuated seasonally. The number had a tendency to gradually decrease from winter to early summer, while it increased through mid-summer and autumn. Since the Microcystispopulation in sediment was rather small, intensive growth and accumulation in the water column should be important for the formation of Microcystisblooms in Lake Biwa. Microcystiscolonies in the sediment samples after June were observed to be floating in a counting chamber under a microscope. The observation suggests that the recruitment of Microcystis colonies into the water column mostly occurs in early summer. The number of Microcystiscolonies in the deep North Basin of Lake Biwa (70 – 90 m water depth) was larger than in the South Basin. Because the seasonal variation of colony numbers was not observed in the North Basin, and Microcystiscells do not have gas vesicles, these colonies will not return into the water column. The colonies isolated from the sediment of the North Basin were able to grow in cultured conditions, in the same way as those from the sediment of the South Basin. Therefore, Microcystiscolonies may survive for a long time under stable conditions of low temperature (ca. 8 °C) and darkness, in the sediment of the deep North Basin, accumulating gradually each year.

Transport and accumulation of bloom-forming cyanobacteria in a large, mid-latitude lake: the gyre-Microcystis hypothesis

Abstract Toxic cyanobacterial blooms have occurred in the near-shore waters of the North Basin of Lake Biwa, Japan, since 1994, and have been attributed to deterioration of water quality in the enriched littoral zone of the lake. From 1997 onwards, the bloom-forming cyanobacteria have been observed with increasing frequency in the deep offshore waters of the North Basin. In the present study, we examined the mechanisms responsible for these bloom populations in the main body of the lake. Specifically, we addressed the hypothesis that buoyant, nutrient-replete colonies of cyanobacteria are generated inshore, are advected offshore by large-scale horizontal transport processes, and subsequently accumulate in the downwelling center of large surface gyres that characterize the overall circulation pattern in the epilimnion of the North Basin. Diel variations of Microcystis biomass at the center and the edge of the Lake Biwa gyre were monitored at 6-h intervals on August 23–24, 2000, and the horizontal distribution of buoyant Microcystis was determined on October 6. The hydrodynamic structure of the first gyre was determined over the preceding 2 days by an on-board Acoustic Doppler Current Profiler (ADCP). The gyre was characterized by a counterclockwise horizontal current that could potentially advect material large distances offshore, a downwelling current near the center of the gyre, and an upwelling current at the edge of the gyre, caused by the radial pressure gradients. The biomass of Microcystis near the water surface was greater at the center than at the edge of the gyre, and the biomass at 5 m depth at the edge of the gyre was greater than that at the water surface or at the thermocline near the edge of the gyre. The results are consistent with the gyre-Microcystis hypothesis, and show the potential for accumulation of large concentrations of cyanobacteria in deep offshore lake environments that are normally considered unsuitable for cyanobacterial blooms.

Isolation and identification of homoanatoxin-a from a toxic strain of the cyanobacterium Raphidiopsis mediterranea Skuja isolated from Lake Biwa, Japan

Abstract A neurotoxin, homoanatoxin-a, was isolated and characterized from a strain of the cyanobacterium Raphidiopsis mediterranea (from the Lake Biwa Research Institute, designated as LBRI 48) isolated from Lake Biwa, Japan. The toxicity of the lyophilized cells was estimated at 330 mg kg−1 by intraperitoneal injection in mice. A toxic substance was extracted with a methanol–water mixture (4:1) and separated by an octadecylsilanated silica gel (ODS) cartridge, followed by high-performance liquid chromatography purification (ODS, 17% methanol–water containing 0.05% trifluoroacetic acid). Signs of toxicity observed following lethal doses of the cell extract and the toxin were intense body paralysis, convulsions, gasping, dyspnea, and death in 5–10 min. The toxin was identified, based on its ultraviolet absorption spectrum (maximum at 230 nm), fast atom bombardment mass [(M + H)+ at m/z 180], and 1H-nuclear magnetic resonance spectral data, as homoanatoxin-a, which has previously been identified only from Oscillatoria formosa strain NIVA-CYA 92. This is the first report demonstrating that R. mediterranea produces cyanotoxins.

Effect of temperature on growth of the cyanobacterium Aphanizomenon flos‐aquae in Lake Biwa and Lake Yogo

The water bloom‐forming cyanobacterium Aphanizomenon flos‐aquae Ralfs ex Bornet et Flahault (Nos‐tocales, Cyanophyceae) appeared in Lake Biwa and Lake Yogo in 1999 for the first time. The morphological characteristics were described using natural samples. In contrast to the other water bloom‐forming cyanobacteria such as Microcystis and Anabaena in Lake Biwa and Lake Yogo, the small summer population of A. flos‐aquae is apt to grow in winter, suggesting the low temperature preference or tolerance of this species. In order to clarify the effect of temperature on the growth, culture experiments were conducted using an axenic strain isolated from Lake Biwa. The strain could grow at above 8°C with an optimum temperature ranging from 23 to 29°C, and survived even at 5°C for at least 25days under low light conditions. Although these results confirmed the ability of the bloom formation during late autumn and winter, it is still unclear why the Aphanizomenon bloom occurred at temperatures of ca 10°C in December and not immediately after the disappearance of Microcystis and/or Anabaena bloom during autumn.

Seasonal succession of phytoplankton in Lake Yogo over 2 years : effect of artificial manipulation

We studied the seasonal variation in concentrations of nutrients and phytoplankton in Lake Yogo for 2 years, from May 2000 to May 2002, in order to clarify the seasonal succession of phytoplankton and the effect of various manipulations on it. It was revealed that in spite of the installation of aeration systems and the pumping of mesotrophic water from Lake Biwa during the summer season, the trophic state of Lake Yogo overall has not improved during the past few decades. However, the pumping of water from Lake Biwa did affect the concentrations of nutrients and the periods of cyanobacterial bloom during the summer. The pumping period was different in each year, and the cyanobacterial bloom occurred during the period without pumping in both years. The aeration destratification was not strong enough to prevent cyanobacterial blooms. Cyanobacteria and Bacillariophyceae contributed most to the phytoplankton biomass in both years. Aphanizomenon, Anabaena, and Microcystis were the main genera among cyanobacteria. The bloom of Aphanizomenon or Anabaena occurred early in the summer, and was then replaced by Microcystis. Aphanizomenon was almost always present, and often formed bloom even in winter. The seasonal succession of Bacillariophyceae was almost the same in both years and was well categorized: winter-growing species such as Aulacoseira pusilla (F. Meister) Tuji et Houki and species of Thalassiosiraceae, spring-growing species such as Asterionella formosa Hassall, Fragilaria crotonensis Kitton, and Synedra cf. acus, and fall-growing species such as Aulacoseira ambigua (Grunow) Simonsen, and Aulacoseira granulata (Ehrenb.) Simonsen.

Effect of cyanobacterial blooms on thermal stratification

Abstract Enclosure experiments were performed at Akanoi Bay, Lake Biwa, in 1995 to determine whether the blooms of cyanobacterial algae changed thermal stratification in the lake. We used four rectangular enclosures, each 10 m × 10 m, with a volume of 200 m3, which were open to the sediments. Two enclosures, A and B, were mixed artificially by aquatic pumps from 1000 to 1400 every day, and the other two enclosures, C and D, were controls with no mixing. The experiment was conducted during late summer from August 3 to September 27. Chlorophyll a concentrations were highest in enclosure D, followed by enclosure C, both of which were controls without mixing. Enclosure A had lower concentrations than enclosures C and D, and enclosure B had the lowest concentrations. No large cyanobacterial algae blooms of Anabaena sp. and Microcystis sp. were seen in the mixed enclosures A and B. In enclosures C and D, blooms of Anabaena sp. occurred in the middle of August, and Microcystis sp. later became dominant in enclosure D at the end of August. In enclosure D, the water temperature changed over the diel cycle before August 17, with thermal stratification during the day and complete mixing at night. After August 17, as Anabaena sp. and Microcystis sp. became dominant, the temperature at the bottom of the enclosure did not change clearly over the 24-h cycle. The APE (available potential energy) density (a measure of water column stability) in the enclosures increased by almost 100% when the biovolume of Anabaena sp. + Microcystis sp. exceeded 20 mm3 l−1. These results indicate that blooms of Anabaena sp. and Microcystis sp. can increase the available potential energy in the water column and create more stable stratification for their growth.

Estimation of soil algal biomass in salinized irrigation land: a comparison of culture dilution and chlorophyll a extraction methods

Two representative methods for quantitative estimation of soil algae, the culture dilution method and chlorophyll a extraction, were compared using soil samples collected from irrigation land in the flood plain of the River Ili, Kazakhstan, where the distribution of soil algae had been studied in the previous year. The estimate by the culture dilution method was almost the same as in the previous year, except for one site, which was enclosed by shrubs of sakusaul, Haloxylon aphyllum (Minkw.) Iljin. The important role of wind in transport of airborne algal cells was pointed out. There was a significant correlation (p<0.001) between the logarithm of the number of colonies by the culture dilution method andthe logarithm of the concentration of chlorophyll a, when data from all samples were analyzed. However, no significant correlation was observed with the data of cropland sites alone. Furthermore, the seasonal variations of both values at each site did not necessarily agree with each other. One reason for the inconsistency may have been the over estimation of chlorophyll a caused by inclusion of litter from vascular plants. Other reasons may relate to differences between the methodologies. The density of soil algae estimated by culture dilution reflects the algal biomass in a certain previous period of time. Therefore, it is suggested that the method is suitable for spatial, but not for seasonal studies.

Effect of nutrient availability on the C, N, and P elemental ratios in the cyanobacterium Microcystis aeruginosa

To clarify whether nutrients limit the growth of Microcystis aeruginosa (Kütz.) Kütz during the growing season in Lake Yogo, we examined the cellular ratios of carbon (C), nitrogen (N), and phosphorus (P) in the populations of M. aeruginosa from August to December 2001. We also measured cellular C, N, and P ratios of M. aeruginosa under batch culture conditions. The cellular levels of N and P of M. aeruginosa in natural population changed more than twofold. The atomic N: C ratio of natural populations of Microcystis fluctuated from 0.11 to 0.26. The atomic P: C ratio fluctuated from 0.0080 to 0.024. The N: C, P: C, and N: P ratios of exponentially growing M. aeruginosa in N-and P-rich medium were 0.19, 0.013, and 15 on average. The growth of M. aeruginosa was suppressed below the N: C ratio of 0.13 under the N-free condition and below the P: C ratio of 0.0026 in the P-free condition. In the natural population, the N: C ratio was low on August 1-2 (0.11) and the P: C ratio was low (less than 0.011) until September. The Microcystis population on August 1-2 was N limited, judging from the results of the culture experiment. In other periods, the population seemed to be supplied with a sufficient amount of N. Although the P: C ratio was low (approximately 0.01) during August and September, it was several times larger than the value of the reduction of growth rate that occurred in culture. P limitation did not occur during the study period. N became more of a limiting factor than P for the formation of blooms of Microcystis. No blooms were observed in August and September, in spite of the increase of cellular levels of N. The formation of Microcystis blooms in Lake Yogo seems to be affected by artificial manipulations such as pumping from Lake Biwa and outflow.

Fatty acid composition as an indicator of physiological condition of the cyanobacterium Microcystis aeruginosa

Abstract The present study examined the fatty acid composition of Microcystis aeruginosa grown in a batch culture and that of Microcystis-dominated plankton collected in an experimental enclosure in a shallow, eutrophic embayment of Lake Biwa (Akanoi Bay). In pure culture, we detected 16 : 0, 18 : 2ω6, 18 : 3ω3, 18 : 3ω6, and 18 : 4ω3 acids as major fatty acids of M. aeruginosa, with trace amounts of C20 polyunsaturated fatty acids. In both pure culture and the field enclosure, the ratio of total fatty acid weight to dry weight decreased with decreasing availability of dissolved inorganic nitrogen. The ω3/ω6 ratios of C18 polyunsaturated fatty acids [(18 : 3ω3 + 18 : 4ω3)/(18 : 2ω6 + 18 : 3ω6)] varied greatly (range, 2–5) in response to the changes in physical and chemical conditions for Microcystis growth. Most notably, the ω3/ω6 fatty acid ratios were significantly positively correlated with the growth rate of cells in a batch culture. We suggest that the fatty acid composition is a useful indicator of the physiological state of Microcystis in freshwater lakes.