GROWTH, PRIMARY PRODUCTIVITY, AND NITROGEN FIXATION POTENTIAL OF NODULARIA SPP. (CYANOPHYCEAE) IN WATER FROM A SUBTROPICAL ESTUARY IN THE UNITED STATES

Nodularia is a halotolerant, filamentous, dinitrogen-fixing cyanobacterium that forms massive blooms in some coastal oceans, estuaries, and saline lakes worldwide. Although the genus is globally distributed, its blooms are sporadic and appear to be confined to certain water bodies. Blooms are frequently associated with phosphorus enrichment; therefore Nodularia may benefit from increased anthropogenic nutrient loading to coastal waters. We studied the potential for Nodularia to grow in the nitrogen-limited Neuse River Estuary (North Carolina, U.S.A.) with laboratory growth experiments in Neuse River Estuary water and by examining physico-chemical data from the estuary. Analysis of nutrients (nitrogen and phosphorus), salinity, and temperature data from the Neuse River Estuary between 1994 and 1998 revealed that suitable conditions for Nodularia prevailed during the summer of each of these years for time spans ranging from 1.5 to 5 months. Growth of two laboratory strains in Neuse River Estuary water was as fast or slightly slower than in artificial growth medium, as long as the culture inoculum had phosphorus reserves. Phosphorus addition did not stimulate growth of already phosphorus-sufficient inocula. Phosphorus starvation of the inoculum before the experiment decreased growth rates in the estuarine water unless additional phosphorus was supplied. Although phosphorus addition had a stimulatory effect on dinitrogen fixation and productivity, the effect differed for the two Nodularia strains. Results suggest that growth of Nodularia in North Carolinian estuaries is possible, and that such growth would be phosphorus-limited at times. Phosphorus availability may determine the times and locations for potential establishment of Nodularia in this and similar estuarine ecosystems.     

Direct evidence for production of microcystins by Anabaena strains from the Baltic Sea

Anabaena is a filamentous, N(2)-fixing, and morphologically diverse genus of cyanobacteria found in freshwater and brackish water environments worldwide. It contributes to the formation of toxic blooms in freshwater bodies through the production of a range of hepatotoxins or neurotoxins. In the Baltic Sea, Anabaena spp. form late summer blooms, together with Nodularia spumigena and Aphanizomenon flos-aquae. It has been long suspected that Baltic Sea Anabaena may produce microcystins. The presence of microcystins has been reported for the coastal regions of the Baltic proper, and a recent report also indicated the presence of the toxin in the open Gulf of Finland. However, at present there is no direct evidence linking Baltic Sea Anabaena spp. to microcystin production. Here we report on the isolation of microcystin-producing strains of the genus Anabaena in the open Gulf of Finland. The dominant microcystin variants produced by these strains included the highly toxic MCYST-LR as well as [d-Asp(3)]MCYST-LR, [d-Asp(3)]MCYST-HtyR, MCYST-HtyR, [d-Asp(3),Dha(7)]MCYST-HtyR, and [Dha(7)]MCYST-HtyR variants. Toxic strains were isolated from the coastal Gulf of Finland as well as from the easternmost open-sea sampling station, where there were lower salinities than at other stations. This result suggests that lower salinity may favor microcystin-producing Anabaena strains. Furthermore, we sequenced 16S rRNA genes and found evidence for pronounced genetic heterogeneity of the microcystin-producing Anabaena strains. Future studies should take into account the potential presence of microcystin-producing Anabaena sp. in the Gulf of Finland.     

Metabolites produced by cyanobacteria belonging to several species of the familyNostocaceae

This paper provides a comprehensive overview of metabolites, including lipids and lipid-like compounds, nitrogen metabolites, oligopeptides and amino acid derivatives, produced by cyanobacteria of the genera Anabaenopsis, Aphanizomenon, Aulosira, Cylindrospermopsis, Cylindrospermum, Nodularia, and Richelia of the family Nostocaceae.     

Distribution of nitrogen-fixing cyanobacteria (Nostocaceae) during rice cultivation in fertilized and unfertilized paddy fields

This study describes the development of nitrogen-fixing cyanobacteria (Nostocaceae) during different stages of rice seedlings plantation in fertilized and unfertilized paddy fields in north Bihar, India. A heterogeneous population of cyanobacteria was randomly harvested around day 20, 40 and 60 of rice seedlings plantation, and the diversity was analyzed. Thirtytwo species (7 genera) were identified from unfertilized fields, of which Anabaena was represented by 12 species, Anabaenopsis and Aulosira by 3 each, Cylindrospermum by 4, Nostoc by 8 and Aphanizomenon and Nodularia by 1 species each. However, fertilized fields contained only 25 species (7 genera), of which 8 each belonged to Nostoc and Anabaena , 3 to Cylindrospermum , 2 to each Anabaenopsis and Aulosira and 1 to each Aphanizomenon and Nodularia . Although Nostoc and Anabaena were dominant in both fertilized and unfertilized paddy fields, a marked decrease in nitrogen-fixing cyanobacteria was recorded from fertilized fields. In both treatments, the diversity of nitrogen-fixing cyanobacteria was at a maximum around day 60 after seedling plantation. The current study concludes that there is a negative effect of nitrogenous fertilizers on the development of heterocystous cyanobacteria in rice fields. It is proposed that early appearing efficient nitrogen-fixers should be used as nitrogen fertilizers in the management for better establishment and exploitation of heterocystous cyanobacteria for sustainable agricultural practices.     

Growth, Nitrogen Fixation, and Nodularin Production by Two Baltic Sea Cyanobacteria

In late summer, nitrogen-fixing cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae form blooms in the open Baltic Sea. N. spumigena has caused several animal poisonings, but Baltic A. flos-aquae is not known to be toxic. In this laboratory study, performed with batch cultures, the influences of environmental conditions on the biomass and nitrogen fixation rate of N. spumigena and A. flos-aquae were compared and the toxin (nodularin) concentration produced by N. spumigena was measured. Several differences in the biomasses and nitrogen fixation rates of N. spumigena and A. flos-aquae were observed. A. flos-aquae preferred lower irradiances, salinities, and temperatures than N. spumigena. The biomass of both species increased with high phosphate concentrations and with accompanying bacteria and decreased with unnaturally high inorganic nitrogen concentrations. Nodularin concentrations in cells and growth media, as well as nitrogen fixation rates, were generally highest under the conditions that promoted growth. Intracellular nodularin concentrations increased with high temperature, high irradiance, and high phosphate concentration and decreased with low and high salinities and high inorganic nitrogen concentrations. Nodularin concentrations in growth media increased with incubation time, indicating that intracellular nodularin was released when cells lysed. The different responses of A. flos-aquae and N. spumigena to changes in salinity, irradiance, and temperature may explain the different spatial and temporal distribution of these species in the Baltic Sea. According to the results, toxic N. spumigena blooms may be expected in late summer in areas of the Baltic Sea with high phosphorus concentrations and moderate salinity.     

Salinity tolerances of 62 strains of Pflesteria and Pfiesteria-like heterotrophic flagellates (Dinophyceae)

The salinity tolerance of 62 strains of Pfiesteria and Pfiesteria‐like heterotrophic dinoflagellates was measured. All strains were acclimated at 12 psu for at least 1 year before experimentation. Strains isolated from the Chesapeake Bay and Neuse River systems tolerated lower salinities than strains isolated from the Wilmington River system (P< 0.005). Swimming cells were still observed after 5 days at 0.5 psu for one strain, and at 1 psu for most other Chesapeake Bay and Neuse River strains. Swimming cells for the Wilmington River were still observed after 5 days at 3–5 psu, but no swimming cells were observed at ≤ 2 psu. With regard to the upper salinity tolerance, the Wilmington River strains tolerated higher salinities than the Chesapeake Bay and Neuse River systems (P< 0.005). Most Wilmington River strains were swimming after 5 days at salinities ≥ 50 psu, whereas the Chesapeake Bay and Neuse River system strains rarely had swimming cells at salinities exceeding 35–45 psu. For all three water systems and for both lower and higher salinities, cells apparently encysted in many instances. However, when salinities were returned to 12 psu, swimming cells often re‐appeared. Statistically significant geographic differences in salinity tolerance suggest a geographic adaptation has occurred and that salinity tolerance is under genetic control. The results also suggest there is diversity among the strains.     

Phosphorus-limited growth dynamics in two Baltic Sea cyanobacteria, Nodularia sp. and Aphanizomenon sp

Rates of carbon (C) specific growth and nitrogen (N2) fixation were monitored in cultures of Baltic Sea Nodularia and Aphanizomenon exposed to gradual limitation by inorganic phosphorus (P). Both cyanobacteria responded by decreased cellular P content followed by lowered rates of growth and N2 fixation. C-specific growth and cellular N content changed faster in Aphanizomenon both when inorganic P was lowered as well as during reintroduction of P. Aphanizomenon also showed a more rapid increase in N-specific N2 fixation associated with increased C-specific growth. When ambient concentrations of inorganic P declined, both cyanobacteria displayed higher rates of alkaline phosphatase (APase) activity. Lower substrate half-saturation constants (KM) and higher Vmax : KM ratio of the APase enzyme associated with Nodularia suggest a higher affinity for dissolved organic P (DOP) substrate than Aphanizomenon. Aphanizomenon, which appears more sensitive to changes in ambient dissolved inorganic P, may be adapted to environments with elevated concentrations of P or repeated intrusions of nutrient-rich water. Nodularia on the other hand, with its higher tolerance to increased P starvation may have an ecological advantage in stratified surface waters of the Baltic Sea during periods of low P availability.     

Enumeration, isolation, and characterization of n(2)-fixing bacteria from seawater

Marine pelagic N(2)-fixing bacteria have not, in general, been identified or quantified, since low or negligible rates of N(2) fixation have been recorded for seawater when blue-green algae (cyanobacteria) are absent. In the study reported here, marine N(2)-fixing bacteria were found in all samples of seawater collected and were analyzed by using a most-probable-number (MPN) method. Two different media were used which allowed growth of microaerophiles, as well as that of aerobes and facultative anaerobes. MPN values obtained for N(2)-fixing bacteria ranged from 0.4 to 1 x 10 per liter for water collected off the coast of Puerto Rico and from 2 to 5.5 x 10 per liter for Chesapeake Bay water. Over 100 strains of N(2)-fixing bacteria were isolated from the MPN tubes and classified, yielding four major groups of NaCl-requiring bacteria based on biochemical characteristics. Results of differential filtration studies indicate that N(2)-fixing bacteria may be associated with phytoplankton. In addition, when N(2)-fixing bacteria were inoculated into unfiltered seawater and incubated in situ, nitrogenase activity could be detected within 1 h. However, no nitrogenase activity was detected in uninoculated seawater or when bacteria were incubated in 0.2-mum-filtered (phytoplankton-free) seawater. The ability of these isolates to fix N(2) at ambient conditions in seawater and the large variety of N(2)-fixing bacteria isolated and identified lead to the conclusion that N(2) fixation in the ocean may occur to a greater degree than previously believed.     

低温对氯化钠胁迫下蓝藻固氮活性的影响

低温加剧氯化钠对蓝藻固氮的抑制,营养液中氯化钠浓度增高时,抑制程度更甚.能源受限(暗处理和加抑制剂时的光合受抑,N_2和Ar的厌氧下呼吸代谢受阻)和氧下固氮酶受到伤害时,低温处理使氯化钠对蓝藻固氮的抑制进一步加剧.在能源和还原剂供应,合成固氨酶蛋白的物质基础(如CO_2和N_2的加合).光合作用正常进行的条件得到改善和保证,以及供应CO_2、外源蔗糖和氮氧加合时,低温加剧氯化钠对蓝藻固氮的抑制程度明显变小.     

Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland

The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena . Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena . Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland.     

Facultative diazotrophy increases Cylindrospermopsis raciborskii competitiveness under fluctuating nitrogen availability

Relative fitness of three bloom-forming and potentially toxic cyanobacteria from the subtropical St. John's River, Florida was investigated under a range of nutrient conditions, during a bloom dominated by Cylindrospermopsis raciborskii. Nitrogen (N) was the primary nutrient limiting phytoplankton primary productivity and biomass. Phytoplankton biomass was also enhanced by phosphorus (P) added either alone or jointly with N, suggesting different components of the phytoplankton experienced distinct nutrient limitations. Based on quantitative PCR, the diazotrophic cyanobacteria Anabaena sp. and C.?raciborskii were responsible for the primary production response to P additions, while the nondiazotrophic Microcystis aeruginosa appeared to benefit from N released from the diazotrophs. Cylindrospermopsis raciborskii maintained high net growth rates under diazotrophic and nondiazotrophic conditions, while Anabaena sp. growth was significantly reduced under DIN enrichment. C.?raciborskii appears to be a generalist with regard to N source, a lifestyle traditionally not considered a viable ecological strategy among diazotrophs. Using facultative diazotrophy, C.?raciborskii gains a growth advantage under fluctuating DIN conditions, such as systems that are under the influence of anthropogenic N loading events. The described niche differentiation may be a key factor explaining the recent global expansion of C.?raciborskii.     

Nitrogen fixation by marine cyanobacteria

Discrepancies between estimates of oceanic N(2) fixation and nitrogen (N) losses through denitrification have focused research on identifying N(2)-fixing cyanobacteria and quantifying cyanobacterial N(2) fixation. Previously unrecognized cultivated and uncultivated unicellular cyanobacteria have been discovered that are widely distributed, and some have very unusual properties. Uncultivated unicellular N(2)-fixing cyanobacteria (UCYN-A) lack major metabolic pathways including the tricarboxylic acid cycle and oxygen-evolving photosystem II. Genomes of the oceanic N(2)-fixing cyanobacteria are highly conserved at the DNA level, and genetic diversity is maintained by genome rearrangements. The major cyanobacterial groups have different physiological and ecological constraints that result in highly variable geographic distributions, with implications for the marine N-cycle budget.     

Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta

Tolerance to salinity stress in higher plants correlates to levels of antioxidant enzymes and/or substrates. Do hyperosmotic and hypoosmotic stress induce antioxidant responses in salt tolerant algae, and if so, are these responses the same for both excess and minimal salinity? To answer these questions, cultures of the marine alga Dunaliella tertiolecta (Chlorophyta) were grown in seven salinities covering a 60-fold range from 0.05 to 3.0 mol/L NaCl. Long-term effects of salinity on growth and antioxidant parameters were determined. Growth rates were reduced at the salinity extremes (0.05 mol/L NaCl and 3 mol/L NaCl) indicating the cultures were stressed. The levels of six antioxidant enzymes and three antioxidant substrates were quantified at these growth salinities. Compared to growth at optimum salinities (i.e. 0.2-0.5 mol/L NaCl), high salinities produced a 260% increase in monodehydroascorbate reductase, a doubling of ascorbate peroxidase activity and a three-fold increase in the rate of dark respiration. Cells acclimated to low growth salinities (hyposaline stress, i.e. < 0.2 mol/L NaCl) showed major increases in glutathione and alpha-tocopherol coupled with decreases in Fv/Fm ratios and in total and reduced ascorbate compared to moderate and high external salinities. Cell volumes remained unchanged, except at the lowest salinity where they doubled. Catalase, superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities were not altered by extreme salinities. The involvement of oxidative stress at both salinity extremes is implied by the alterations in antioxidant enzymes and substrates, but the specific changes are very different between hypo and hypersaline stresses.     

十四属蓝藻液泡的检查和分离

采用压片法,渗透冲击法和原生质球法,对14个属的不同蓝藻进行了液泡存在的检查和分离试验,每种蓝藻都检查到液泡,无一例外。其中鱼腥藻、念珠藻、项圈藻、席藻、简孢藻等都能形成液泡化原生质球,原生质球低渗破裂释放液泡,液泡得率极高,不同种的液泡都耐低渗。因此,本研究证明液泡在蓝藻中的存在是普遍的。     

Localized Tetrazolium Reduction in Relation to N(2) Fixation, CO(2) Fixation, and H(2) Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N(2) fixation (acetylene reduction), CO(2) fixation, and H(2) utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, CO(2) fixation and H(2) utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N(2)-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Ecological and Biogeochemical Interactions Constrain Planktonic Nitrogen Fixation in Estuaries

Many types of ecosystems have little or no N₂ fixation even when nitrogen (N) is strongly limiting to primary production. Estuaries generally fit this pattern. In contrast to lakes, where blooms of N₂-fixing cyanobacteria are often sufficient to alleviate N deficits relative to phosphorus (P) availability, planktonic N₂ fixation is unimportant in most N-limited estuaries. Heterocystic cyanobacteria capable of N₂ fixation are seldom observed in estuaries where the salinity exceeds 8–10 ppt, and blooms have never been reported in such estuaries in North America. However, we provided conditions in estuarine mesocosms (salinity over 27 ppt) that allowed heterocystic cyanobacteria to grow and fix N₂ when zooplankton populations were kept low. Grazing by macrozooplankton at population densities encountered in estuaries strongly suppressed cyanobacterial populations and N₂ fixation. The cyanobacteria grew more slowly than observed in fresh waters, at least in part due to the inhibitory effect of sulfate (SO₄ ²⁻), and this slow rate of growth increased their vulnerability to grazing. We conclude that interactions between physiological (bottom–up) factors that slow the growth rate of cyanobacteria and ecological (top–down) factors such as grazing are likely to be important regulators excluding planktonic N₂ fixation from most Temperate Zone estuaries. Received 26 April 2002; Accepted 12 July 2002.     

Temporal and spatial distribution of planktic cyanobacteria in Lake Kastoria, Greece, a shallow, urban lake

The temporal and spatial distribution of planktic cyanobacteria and some environmental parameters were studied in the shallow, urban Lake Kastoria, Greece from June 1996 to June 1997. Water temperature varied from 6–27 °C, pH from 7.5–8.9 and dissolved O₂ concentration from 0.7–12 mg m^-3 10^-3. The mean annual Chl a concentration was 83 mg Chl a m^-3 indicative of the eutrophic-hypertrophic state of the lake. Cyanobacterial biomass ranged from 11–238 g FW m^-3, constituting about 90% of the total phytoplankton biomass throughout the year. Cyanobacterial biomass was non-uniformly distributed both vertically and horizontally from August to November 1996 and resulted mainly from the distribution of Microcystis. Seven cyanobacterial taxa were reported for the first time in Lake Kastoria. Six taxa were dominant: Microcystis aeruginosa, M. flos-aquae, M. novacekii. Limnothrix redekei, Anabaena sp. and Cylindrospermopsis raciborskii. The dominant cyanobacterial taxa can be grouped on the basis of their distribution patterns (1) Microcystis species: maximum biomass occurring at pH > 8, temperature 12–17 °C, depth < 0.2 m; (2) Anabaena sp. and Cylindrospermopsis raciborskii : maximum biomass at temperatures 23–26 °C; (3) Limnothrix redekei : maximum biomass at temperatures 6–27 °C. Usually, non-uniform, vertical distributions of cyanobacterial biomass were associated with the formation of temperature, pH and O₂ gradients. L. redekei was considered to be a key lake organism since it contributed up to 59% of the cyanobacterial biomass. Interestingly, three of the dominant cyanobacteria Microcystis aeruginosa, Anabaena sp. and Cylindrospermopsis raciborskii belong to genera that include toxin-producing species.     

海洋固氮生物多样性及其对海洋生产力的氮、碳贡献

固氮生物及其共生体在自然生态系统的氮素供给和生产力的持续发展中有重要作用。是贫营养盐海域和海洋生态系统中新生产力的主要贡献者。就海洋固氮生物研究的历史和现状,以及在世界不同海域和海洋生态环境条件下,海洋固氮生物的物种多样性和在海洋生态环境中的生存方式、共生互作关系多样化,作较全面的概述。综合分析了海洋固氮生物的固氮能力（活性）、在光合固碳中的作用和对初级生产力的贡献。介绍了固氮生物在海洋生物链和海洋不同生态系统和生物群落中的作用及其对环境的影响的最新研究概况。