A GRADIENT IN BENTHIC INTERTIDAL ALGAL ASSEMBLAGES ALONG THE SOUTHERN CALIFORNIA COAST1

Based on an analysis floristic data, a gradient appears to exist in the composition of intertidal algae along the 450 km of southern California coastline immediately south of Point Conception. Reciprocal averaging ordination of the algal flora at 51 sites in this area suggests that the gradient is not strictly latitudinal. Variation from a latitudinal gradient occurs ca. 60-80 km south of Point Conception. An ordination of frequently occurring species indicated that compositional changes are substantial when sites at the extreme north and south are compared, but that the changes are gradual and continuous. Possible explanations for the gradient are discussed, and these include: sea temperature, upwelling, sand movement, human disturbance (i.e., pollution, trampling), and wave action.     

ADAPTATION OF STYROFOAM SUBSTRATE TO BENTHIC ALGAL PRODUCTIVITY STUDIES IN LAKE TAHOE,CALIFORNIA-NEVADA1

A routine sampling technique has been developed using artificial styrofoam substrate to estimate benthic algal productivity in the littoral zone of lakes. Estimation of maximum carbon fixed in Lake Tahoe ranged from 11.1 mg C·m^?2· day^?1 at 0.5 m to 17.1 mg C·m^?2· day^?1 at 1.0 m. Estimates were made for communities composed of both diatom and green algal populations in water between 0.5 and 3.0 m. Maximum productivity occurred between 1–2 m. The technique developed can give comparable estimates of productivity if adequate replication is undertaken to decrease problems associated with periphytic heterogeneity.     

NUTRIENT LIMITATION OF BENTHIC ALGAE IN LAKE MICHIGAN: THE ROLE OF SILICA1

In the Laurentian Great Lakes, phytoplankton growth and biomass are secondarily limited by silica (Si), as a result of phosphorus (P) enrichment. Even modest levels of P enrichment can induce secondary Silimitation, which, in turn, promotes a shift from the native diatom phytoplankton flora to chlorophyte and cyanobacteria species. However, very little is known about the nutritional status of benthic populations and their response to nutrient enrichment. Two experiments were performed in the littoral zone of Lake Michigan where nutrients were delivered to in situ benthic algal (episammic and epilithic) assemblages using nutrient‐diffusing substrata. In order to test the hypothesis that benthic algae in Lake Michigan are Si limited, a 2 × 3 factorial experiment was used to deliver all combinations of Si, N, and P to resident assemblages growing on artificial substrata composed of natural (Si rich) versus calcium carbonate (Si poor) sand. A second experiment utilized a serial enrichment to evaluate the role of Si in mediating changes in taxonomic composition. These findings indicate that benthic algae in Lake Michigan exhibit signs of secondary Si limitation, and that their response to enrichment is similar to the phytoplankton. Moreover, natural sand substrata may provide a source of Si to resident benthic algae.     

QUANTITATIVE DIFFERENCES BETWEEN BENTHIC ALGAL COMMUNITIES ALONG A DEPTH GRADIENT IN LAKE MICHIGAN1,2

This study analyzes the impact of conditions associated with depth upon benthic algal communities in Lake Michigan. Diatom abundance was greater at 9.1 and 14.6 m depths than 6.5, 22.6 and 27.4 m. Shallow (6.5 m), mid-depth (9.1 and 14.6 m) and deep (22.6 and 27.4 m) zones were distinguishable on the basis of community composition, structure and abundance. Dominance of benthic species, high diversity and low abundance in shallow communities probably resulted from substantial substrate disturbance by wave action in this productive zone. Dominance of benthic species, high diversity and high abundance characterized mid-depth communities where less wave disturbance enabled algal accumulation. Preponderance of living planktonic taxa, low diversity and low abundance delineated deep communities where planktonic algae accumulated and low light levels reduced growth of benthic species.     

CHARACTERIZATION OF PSYCHROPHILIC OSCILLATORIANS (CYANOBACTERIA) FROM ANTARCTIC MELTWATER PONDS

Oscillatorian cyanobacteria dominate benthic microbial mat communities in many polar freshwater ecosystems. Capable of growth at low temperatures, all benthic polar oscillatorians characterized to date are psychrotolerant (growth optima > 15° C) as opposed to psychrophilic (growth optima ≤ 15° C). Here, psychrophilic oscillatorians isolated from meltwater ponds on Antarctica's McMurdo Ice Shelf are described. Growth and photosynthetic rates were investigated at multiple temperatures, and compared with those of a psychrotolerant isolate from the same region. Two isolates showed a growth maximum at 8° C, with rates of 0.12 and 0.08 doublings·d ^{? 1}, respectively. Neither displayed detectable growth at 24° C. The psychrotolerant isolate showed almost imperceptible growth at 4° C and a rate of 0.9 doublings·d ^{? 1} at its optimal temperature of ～23° C. In both photosynthesis versus irradiance and photosynthesis versus temperature experiments, exponentially growing cultures were acclimated for 14 days at 3, 8, 12, 20, and 24° C under saturating light intensity, and [¹⁴C] photoincorporation rates were measured. Psychrophilic isolates acclimated at 8° C showed greatest photosynthetic rates; those acclimated at 3° C were capable of active photosynthesis, but photoincorporation was not detected in cells acclimated at 20 and 24° C, because these isolates were not viable after 14 days at those temperatures. The psychrotolerant isolate, conversely, displayed maximum photosynthetic rates at 24° C, though photoincorporation was actively occurring at 3° C. Within acclimation temperature treatments, short‐term photosynthetic rates increased with increasing incubation temperature for both psychrophilic and psychrotolerant isolates. These results indicate the importance of temperature acclimation before assays when determining optimal physiological temperatures. All isolates displayed photosynthetic saturation at low light levels (<128 μmol·m ^{? 2}·s ^{? 1}) but were not photoinhibited at the highest light treatment (233 μmol·m ^{? 2}·s ^{? 1}). Field studies examining the impact of temperature on photosynthetic responses of intact benthic mats, under natural solar irradiance, showed the mat communities to be actively photosynthesizing from 2 to 20° C, with maximum photoincorporation at 20° C, as well as capable of a rapid response to an increase in temperature. The rarity of psychrophilic cyanobacteria, relative to psychrotolerant strains, may be due to their extremely slow growth rates and inability to take advantage of occasional excursions to higher temperatures. We suggest an evolutionary scenario in which psychrophilic strains, or their most recent common ancestor, lost the ability to grow at higher temperatures while maintaining a broad tolerance for fluctuations in other physical and chemical parameters that define shallow meltwater Antarctic ecosystems.     

GROWTH AND ORGANIC OSMOLYTES OF GEOGRAPHICALLY DIFFERENT ISOLATES OF MICROCOLEUS CHTHONOPLASTES (CYANOBACTERIA) FROM BENTHIC MICROBIAL MATS:RESPONSE TO SALINITY CHANGE1

The comparative growth and osmotic acclimation often culture strains of the marine benthic cyanobacterium Microcoleus chthonoplastes Thuret isolated from microbial mats in Germany, Spain, Egypt, the United States, Mexico, Chile, and Australia were investigated in salinities ranging from freshwater to twice seawater. All isolates showed a broad growth versus salinity response consistent with the dominance of this species in intertidal and hypersaline microbial communities. Growth optima, salinity preferences, and maximum growth rates differed for each isolate and could be related to the habitat from which they were isolated. This is most obvious when comparing strains from brackish habitats with those from a hypersaline lake. While the former isolates exhibited sharply pronounced growth optima under hyposaline conditions, cultures from the hypersaline environment grew best in salinity more than double seawater. The major low-molecular weight organic compounds present in all M. chthonoplastes strains were the carbohydrates glycosylglycerol and trehalose. This was proven by using ¹³C-nuclear magnetic resonance spectroscopy. Glycosylglycerol was synthesized and accumulated with increasing salinities, indicating its role as an osmolyte. In contrast, trehalose was present in relatively high concentrations under hyposaline conditions only. Differences in the patterns of growth versus salinity, as well as in those of osmotic acclimation among the M. chthonoplastes isolates, point to the development of different physiological ecotypes within the species.     

INVOLVEMENT OF MICROCYSTINS AND COLONY SIZE IN THE BENTHIC RECRUITMENT OF THE CYANOBACTERIUM MICROCYSTIS (CYANOPHYCEAE)1

The benthic recruitment of Microcystis was simulated in vitro in order to characterize the colonies of Microcystis recruited and to study the impact of intracellular and extracellular microcystins (MCs), and the influence of colony size on the recruitment process. We observed recruitment dynamics consisting of a lag phase followed by a peak and then a return to low recruitment rates, mainly controlled by passive resuspension throughout the experiment, and by physiological processes during the recruitment peak. Ninety‐seven percent of the Microcystis colonies recruited were <160 μm in maximum length, and their cells contained much greater amounts of MCs (0.26 ± 0.14 pg eq microcystin leucine‐arginine variant [MC‐LR] · cell^?1) than those in benthic colonies (0.021 ± 0.004 pg eq MC‐LR · cell^?1). The MC content of recruited Microcystis varied significantly over time and was not related to changes in the proportion of potentially toxic genotypes, determined using real‐time PCR. On the other hand, the changes in MC content in the potentially toxic Microcystis recruited were closely and negatively correlated with recruitment dynamics; the lowest MC contents corresponded to high recruitment rates, and the highest MC contents corresponded to low recruitment rates. Thus, depending on temperature and light conditions, these variations are thought to result from the selection of various subpopulations from among the smallest and the most toxic of the initial benthic population. Adding purified MC‐LR to experimental treatments led to a decreased recruitment of Microcystis and more specifically of mcyB genotypes.     

BIOGEOGRAPHY OF STREAM MACROALGAE IN NORTH AMERICA1

In a survey of 1000 20-m long stream segments in North America from 73°N to 10° N, 259 infrageneric taxa were identified, composed of 35% Chlorophyta, 24% Cyanophyta, 21% Chrysophyta, 20% Rhodophyta, and one species of Phaeophyta. The most common morphological forms were mats (42%), gelatinous colonies (23%), and gelatinous filaments (13%); the majority of taxa were vegetative (78%). The frequency of reproductive states varied among the biomes examined, with the highest values observed as follows: vegetative (85%) in the tundra, sexual (35%) in the deciduous forest, and asexual (21%) in the tropical rainforest. In terms of total species, the tundra had the lowest number (54), the boreal forest had the greatest number (100), and other well-sampled biomes had 84 to 87. The Chlorophyta had the greatest species numbers in all biomes, and the relative contribution of this division did not vary significantly throughout the continent. With regard to similarity of species composition, the two closest associations were the boreal forest with the western coniferous forest and the eastern hemlock–hardwood forest with the deciduous forest. Species numbers per stream segment ranged from 0 to 11 (X?= 3.1), cover ranged from 0 to 100% (X?= 15%), and there was no significant difference in these values among the biomes. The cyanophyte Phormidium retzii (C. Ag.) Gom. was the most widespread species (in 172 segments and all biomes). The majority of species were found in moderate current velocities (X?= 40 cm X? s^?1), neutral to slightly alkaline pH (X?= 7.5), and moderately low ion waters (X?= 261 μS · cm^?1). On both tropical and Arctic islands, there was no significant increase in total species or species numbers per stream segment with increasing surface area.     

从塔北隆起奥陶纪钙藻化石探讨奥陶纪的古环境

塔里木盆地塔北隆起轮南46井、英买1、2井奥陶纪石灰岩内含有大量的钙藻化石、蓝细菌以及疑难微体化石。这些钻井岩芯均在深达5000-6200m的地下深部取得。钙藻主要为绿藻类的Dasyporella,Ver‘miporella,Moniliporella以及?Plexa;红藻类的管孔藻类Solenoporaceans;钙化蓝细菌则有Girvanella,Botomaella,?Subtifloria等;疑难微体化石有Bevocastria,Nuia,Rothpletzella。这些钙藻生活于热带或亚热带正常盐度的浅海水内,其水深不到20m。世界各地的奥陶纪Vermiporella均位于古赤道的两侧,这表明它们是在气候炎热、温暖海水中生活的一类海洋藻类。Girvanella以藻灰结核和内碎屑最为常见,某些球粒可能代表Girvanella破碎后形成的单管或小棒。塔里木盆地钙藻植物群相似于哈萨克斯坦、波罗的海周围地区以及北美同时代植物群,这表明这些钙藻和蓝细菌化石具有遍布于全球的性质。塔北隆起早、中奥陶世沉积属于典型的碳酸盐岩台地相沉积。到晚奥陶世时,碳酸盐岩沉积被浅水陆棚沉积所取代,以陆源碎屑岩为主,夹少量的碳酸盐岩。     

山西省辛安泉泉域三种泉溪大型藻类植物附生藻类的研究

对三种泉溪大型藻类(脆弱刚毛藻, 弧形串珠藻和普生轮藻)藻体上附生藻类的种类、分布及环境因子与寄主本身对其的影响进行了研究。2004 年7 月至2005 年4 月在山西省辛安泉泉域采集三种大型藻类植物(每个季节采集一次)。研究结果表明, 三种大型藻类藻体上的附生藻类种类数不同, 普生轮藻上的附生藻类有49 种, 脆弱刚毛藻上37 种, 弧形串珠藻上6 种, 其中硅藻门种类数最多。这说明寄主本身结构的复杂程度在一定意义上会影响附生藻类的种类数及多样性。就季节变化而言, 三种大型藻类藻体上的附生藻类也表现出了明显的季节性,大致趋势为: 春季>秋季>夏季>冬季, 同时, 灰关联分析结果也表明, 环境因子在一定程度上对附生藻类也有影响, 最关键的影响因子是流速、水深和电导。这说明影响大型藻类植物藻体上附生藻类的影响因素是多方面的, 不仅有寄主本身, 同时流速、水深和电导这三种环境因子也起到了很关键的作用。       

SEASONAL FLUCTUATIONS IN TISSUE NITROGEN FOR FIVE SPECIES OF PERENNIAL MACROALGAE IN RHODE ISLAND SOUND1

Tissue nitrogen was assessed monthly for 16 months in five species of perennial macroalgae representing three phyla at one location in Rhode Island Sound. The species showed a remarkable similarity in their pattern of seasonal fluctuation in both nitrate and total nitrogen. The period of greatest accumulation (January through March) coincided with the period of highest concentration of inorganic nitrogen in the water, and for most of these algae it was also the time of-least growth. Conversely, the period of lowest tissue nitrogen (50% of the winter value, May through July) coincided with the period of lowest inorganic nitrogen in the water and highest algal growth. The greatest accumulation of nitrate was found in Laminaria saccharina (L.) Lamour. (80 μmol·g dry wt.^?1), four times as much as that measured simultaneously in the other species and 560 times the ambient concentration. By April the concentration of internal nitrate had dropped to nearly undetectable levels, but in August it began to accumulate again—a pattern that was repeated in Chondrus crispus Stackh. In Ascophyllum nodosum (L.) Le Jolis, Fucus vesiculosus L. and Codium fragile subsp. tomentosoides (Van Goor) Silva, the period of negligible internal nitrate level extended from March to December. The greatest concentration of total tissue nitrogen was measured in C. crispus (4.8% dry wt.), double the maximum in L. saccharina (2.3% dry wt.).     

STEROL PROFILES IN 18 MACROALGAE OF THE PORTUGUESE COAST1

The sterol profiles of dominant macroalgae occurring in the western Portuguese coast were evaluated. An analytical procedure, involving alkaline hydrolysis and extraction followed by separation by reversed‐phase HPLC–diode array detection (HPLC–DAD), was optimized for the study of their sterols composition. The validated methodology is short in analysis time (as the compounds are determined in <20 min), sensitive, reproducible, and accurate. It was then successfully applied to the determination of campesterol, cholesterol, desmosterol, ergosterol, fucosterol, stigmasterol, and β‐sitosterol in 18 species (three Chlorophyta, five Rhodophyta, and 10 Phaeophyta). The profiles obtained for the several macroalgal species were considerably different. C29 sterols were predominant in Phaeophyta and Chlorophyta (71%–95% of total sterol content), while in Rhodophyta cholesterol content is significantly higher (34%–87%). Among the studied species, Asparagopsis armata Harv. contained the lowest sterol amount (555 mg · kg^?1 dry weight), and Cystoseira tamariscifolia (Huds.) Papenf. the highest one (6,502 mg · kg^?1 dry weight). Data obtained may be helpful in identifying suitable marine sources of sterols, with potential applications in the food and pharmaceutical industries.     

RELEASE OF CARBOHYDRATES AND PROTEINS BY A SUBTROPICAL STRAIN OF RAPHIDIOPSIS BROOKII (CYANOBACTERIA) ABLE TO PRODUCE SAXITOXIN AT THREE NITRATE CONCENTRATIONS1

Raphidiopsis brookii P. J. Hill (cyanobacteria) was isolated from a small subtropical eutrophic pond (Biguá Pond) located in the grounds of Rio Grande University in the extreme south of Brazil, following a toxic bloom of this species. Growth, saxitoxin production, and release of carbohydrates and protein were monitored at three sodium nitrate concentrations (500, 1,000, and 1,500 μM), from inoculation up to the stationary growth phase. Growth was monitored by determining the biovolume, chl content, and trichome count. Growth was better described in terms of biovolume and chl measurements, because trichome fragmentation was observed to increase at the stationary growth phase. Carbohydrates and proteins were released in small amounts during most of the experiment, with a significant increase during the stationary phase. Extracellular polysaccharides were essentially composed of glucose, galactose, N‐acetyl‐glucosamine, mannose, xylose, rhamnose, arabinose, and fucose. The relative proportions of these units showed no significant variation during growth. Small quantities of extracellular free carbohydrates were also detected, and only fucose was released in significant amounts at the lowest nitrate concentration (500 μM). R. brookii produced both saxitoxin and dc‐saxitoxin, the former at four times the rate of the latter. This was the first study demonstrating saxitoxin production and the release of both carbohydrate and protein by R. brookii.     

AN INVESTIGATION OF THE VERTICAL DISTRIBUTION OF SELECTED BENTHIC MARINE ALGAE WITH A TIDE–SIMULATING APPARATUS1

The growth yields of 8 species of red and 4 species of green algae, collected from various vertical levels on the western Atlantic (New Jersey) shore, were investigated in a range of emersion-submersion regimes in the laboratory. The sinusoidal tide-simulating apparatus consisted of a plexiglass frame connected by a nylon thread to a rotating wheel, powered by an electric motor geared to make one revolution every 12 h. Inocula of settled spores on glass cover-glasses were placed at 6 levels on the frame and alternately raised out of, and lowered into, a seawater tank every 6 h with consequent subjection to 0, 18, 37, 58, 79, and 100% submergence regimes per tidal cycle. There was little correlation between the vertical position of a species on the shore in nature and its ability to grow as the percentage submergence per tidal cycle increased. All species tested grew best when continuously submerged, even those that are usually restricted to elevated positions on the shore in nature. The majority grew well in all regimes in which there was some submergence per tidal cycle, except for one sublittoral species (Callithamnion byssoides) whose growth decreased sharply as the percentage exposure to the air was increased.     

ULTRASTRUCTURE OF UNICELLULAR N2 FIXING CYANOBACTERIA FROM THE TROPICAL NORTH ATLANTIC AND SUBTROPICAL NORTH PACIFIC OCEANS1

Nitrogen fixing unicellular marine cyanobacteria may have a major role in the global biogeochemistry of N; nevertheless, little is known about their phylogeny and morphology. We isolated N₂ fixing unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific Oceans and examined ultrastructural dynamics during dark:light cycles when grown in incubators. The isolate from the subtropical North Pacific was larger and showed a size variation from 3 to 7 μm but had similar morphology and cell division‐plane characteristics as the isolate from the North Atlantic (2.5 μm). Nitrogen fixation only occurred during the dark phase, and ultrastructural analysis demonstrated changes in the appearance and quantity of large carbohydrate‐like granules present in the cells. To verify the composition of these carbohydrate‐like granules, staining with periodic acid, thioacetic acid, and silver was carried out, and a positive reaction was obvious in all cells. The cells from the Atlantic seemed to empty their polysaccharide granules during the night, whereas those from the Pacific showed a decrease in the number of their granules. Our work suggests that phylogenetically related strains of unicellular N₂ fixing cyanobacteria from different oceans showed similar carbohydrate‐like granules that could be used to fuel N₂ fixation during darkness.     

SALINITY AND NUTRIENT LIMITATIONS ON GROWTH OF BENTHIC ALGAE FROM TWO ALKALINE SALT LAKES OF THE WESTERN GREAT BASIN (USA)1

Enrichment cultures of littoral benthic algae from Mono Lake, California, and Abert Lake, Oregon, were grown under conditions of varied salinity and nutrient content. Field-collected inocula were composed mainly of diatoms and filamentous blue-green and green algae. The yield of long-term cultures (30 days) showed tolerance over a broad salinity range (50–150 g·L^?1) for Mono Lake-derived algae. Algae from Abert Lake had a lower range of tolerance (25–100 g·L^?1) Organic content and pigment concentrations of algae from both lakes were also reduced above the tolerated salinity level. Within the range of salinity tolerance for Mono Lake algae, initial growth rates and organic content were reduced by increased salinity. The effects of macro- and micronutrient enrichment on algal growth in Mono Lake water were also tested. Only nitrogen enrichment (either as ammonium or nitrate) stimulated algal growth. Although the benthic algae cultured here had wide optima for salinity tolerance, the rates of growth and storage were limited by increased salinity within the optimum range. Although the lakes compared had similar species composition, the range and limits of tolerance of the algae were related to salinity of the lake of origin.     

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O₂ concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters N_m, N_d, and α were derived. In both strains, nitrogenase activity (N_tot = N_m + N_d) was the highest at 39°C–42°C and at 0% O₂. The ratio N_tot/N_d was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (N_d). N_tot/N_d increased with decreasing O₂ concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (N_m). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O₂ concentration and temperature as the latter may dynamically alter the flux of O₂ into the heterocyst.     

INTERACTIONS BETWEEN NITRATE UPTAKE AND NITROGEN FIXATION IN CONTINUOUS CULTURES OF THE MARINE DIAZOTROPH TRICHODESMIUM (CYANOBACTERIA)1

Diazotrophic cyanobacteria can take up combined nitrogen (nitrate, ammonium, amino acids, dissolved organic nitrogen) from solution, but the interaction between N₂ fixation and uptake of combined nitrogen is not well understood. We studied the effects of combined nitrogen ) additions on N₂ fixation rates in the cyanobacterium Trichodesmium erythraeum (IMS‐101) maintained in continuous culture in an N‐free medium (YBCII) and a 12:12‐h light:dark cycle. We measured acetylene reduction rates, nutrient concentrations, and biomass throughout the 12 h of illumination after the addition of nitrate (0.5–20 μM) at the start of the light period. Compared with unamended controls, Trichodesmium showed strong inhibition of acetylene reduction (up to 70%) in the presence of , with apparent saturation of the inhibition effect at an initial concentration of approximately 10 μM. The inhibition of acetylene reduction persisted through much of the light period as concentration in the culture vessel decreased. Recovery of N₂ fixation was observed late in the light period in cultures amended with low concentrations of (<5 μM) when ambient concentrations had decreased to 0.3–0.4 μM in the culture vessel. Nitrate uptake accounted for as much as 86% of total N uptake and, at the higher treatment concentrations, more than made up for the observed decrease in N₂ fixation rates. We conclude that Trichodesmium can obtain significant quantities of N through uptake of nitrate and does so in preference to N₂ fixation when sufficient is available.     

INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.