Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria): Temperature Characteristics and Potential Use in Biophotolysis

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10⁴ ergs cm⁻² s⁻¹. Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of M. laminosus NZ-86-m produced hydrogen at comparable rates to Anabaena cylindrica. The conversion efficiency of light to hydrogen energy at maximum rates of hydrogen production was 2.7%.     

Effect of Temperature on Blue-Green Algae (Cyanobacteria) in Lake Mendota

The temperature optimum for photosynthesis of natural populations of blue-green algae (cyanobacteria) from Lake Mendota was determined during the period of June to November 1976. In the spring, when temperatures ranged from 0 to 20°C, there were insignificant amounts of blue-green algae in the lake (less than 1% of the biomass). During the summer and fall, when the dominant phytoplankton was blue-green algae, the optimum temperature for photosynthesis was usually between 20 and 30°C, whereas the environmental temperatures during this period ranged from 24°C in August to 12°C in November. In general, the optimum temperature for photosynthesis was higher than the environmental temperature. More importantly, significant photosynthesis also occurred at low temperature in these samples, which suggests that the low temperature alone is not responsible for the absence of blue-green algae in Lake Mendota during the spring. Temperature optima for growth and photosynthesis of laboratory cultures of the three dominant blue-green algae in Lake Mendota were determined. The responses of the two parameters to changes in temperature were similar; thus, photosynthesis appears to be a valid index of growth. However, there was little photosynthesis by laboratory cultures at low temperatures, in contrast to the natural samples. Evidence for an interaction between temperature and low light intensities in their effect on photosynthesis of natural samples is presented.     

Observations of Toxic Blue-Green Algae (Cyanobacteria) in Some Scandinavian Lakes

Blooms of blue-green algae from 51 eutrophic Scandinavian lakes were investigated during the period 1978–1984, to ascertain the occurrence of toxinogenic species. Toxicity assays were performed by intraperitoneal injection of suspensions of freeze-dried algal material in mice. Toxin-producing blue-green algae were found in 30 lakes. They belonged to 11 different species of the six genera Anabaena, Aphanizomenon, Gomphosphaeria, Microcystis, Nodularia and Oscillatoria. The presence of toxinogenic strains of blue-green algae seemed quite constant in several of the localities studied. In some lakes, more than one toxic species were found to develop simultaneously. The level of toxicity showed large variation (MLD100, 6 to > 2500 mg/kg), but clinical and pathological changes were quite uniform. The results indicate that water-blooms of toxin-producing blue-green algae, in the geographical area in question, are regionally widespread. In some localities, blooms of blue-green algae are apparently always toxic. Several aspects of the toxic blue-green algae problem are discussed.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N(2)-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N(2)-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with P(i), and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N₂-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with ³²P_i, and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Lytic Organisms and Photooxidative Effects: Influence on Blue-Green Algae (Cyanobacteria) in Lake Mendota, Wisconsin

The effects of exposure to high light intensities on blue-green algal (cyanobacterial) populations were examined in Lake Mendota, Wis. The algal populations were shown to be susceptible to inhibition of photosynthetic activity and pigment bleaching as a result of exposure. These effects generally influence only a small percentage of the lake population and thus are probably not important in causing major declines in chlorophyll a. Lytic organisms were shown to increase in numbers in the lake in response to the seasonal development of blue-green algae, reaching values of greater than 1,000 plaque-forming units per ml in midsummer. Both bacteria and protozoa were observed in plaque zones, but it could not be determined whether these lytic organisms had a major role in algal biomass declines.     

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Summary: This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N₂ fixation and/or H₂ formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H₂ as a source of combustible energy. To enhance the rates of H₂ production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H₂ formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.     

Fixation of Elemental Nitrogen by Marine Blue-green Algae

Three blue-green algae, Calothrix scopulorum, Nostoc entophytum,and Oscillatoria brevis, isolated from the upper littoral andsupralittoral fringe of the sea-shore were obtained in pureculture and tested for fixation of elemental nitrogen. Appreciablefixation by Calothrix and Nostoc was detected, a proportionof the total nitrogen fixed being liberated into the culturemedium. There was no evidence of fixation by Oscillatoria. Thisappears to be the first evidence that blue-green algae isolatedin pure culture from marine habitats fix nitrogen.     

The Presence of Cellulose in Heterocyst Envelopes of Blue-Green Algae and its Role in Relation to Nitrogen Fixation

The present study gives evidence for the presence of cellulose in the heterocyst envelope of blue-green algae by means of electron microscopy, cellulase treatments and specific staining and demonstrates the role of this cellulose for the protection of the heterocyst nitrogenase during acetylene reduction. Experiments with lysozyme and cellulase suggest that nitrogen fixation in heterocystous blue-green algae under aerobic conditions is functionally effective only when an intimate relationship exists between vegetative cells and heterocysts and both cell types have intact wall structures.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Effect of Rice Plants on Nitrogenase Activity of Flooded Soils

In samples of flooded soil containing blue-green algae (cyanobacteria), the presence of rice plants did not influence the nitrogenase activity of the algae. Nitrogenase activity of heterotrophic bacteria was enhanced by the presence of rice plants, but this activity was not affected by changes in plant density. The rate of nitrogen fixation in the rhizosphere, however, varied significantly among the 16 rice varieties tested. A simple method was devised to test the nitrogen-fixing activity in the root zone of rice varieties, and data were obtained showing marked differences in the activities of the 16 varieties. In tests of two varieties with dissimilar rates of nitrogen fixation in their rhizospheres, the variety which had the greater root weight and lesser shoot weight and which supported greater methane formation had the greater nitrogenase activity.     

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.     

Reversible and Irreversible Inactivation of Photosynthesis in Relation to the Lipid Phases of Membranes in the Blue-Green Algae (Cyanobacteria) Anacystis nidulans and Anabaena variabilis

The Arrhenius plots of photosynthetic oxygen evolution in theblue-green algae Anacystis nidulans and Anabaena variabiliswere composed of two straight lines with break points whichwere very close to temperatures for the onset of phase separationof the thylakoid membranes. Irreversible inactivation of photosynthesisbegan to appear at the same temperature as the onset of phaseseparation of the cytoplasmic membranes in A. nidulans. Electrolytesbegan to leak from the cytoplasm into the outer medium, indicatingthat the permeability of the cytoplasmic membranes increasedwhen they entered the phase separation state. In A. variabilis,in which the cytoplasmic membranes had remained in the liquidcrystalline state above 0?C, no irreversible damage to photosynthesisnor leakage of electrolytes was observed between 0 and 20?C.These findings suggest that photosynthesis of the blue-greenalgae is reversibly suppressed when only the thylakoid membranesare in the phase separation state, and irreversibly inactivatedwhen the cytoplasmic membranes are in the phase separation state. (Received April 9, 1984; Accepted June 19, 1984)     

Carbon Concentration Mechanism(s) in Unicellular Green Algae and Cyanobacteria

Unicellular green algae and cyanobacteria have mechanism(s) to actively concentrate dissolved inorganic carbon (DIC) into the cells, only if they are grown with air levels of CO₂. The DIC concentration mechanisms are environmental adaptations to actively transport and accumulate inorganic carbon into the chloroplasts of green algae or into the carboxysomes of cyanobacteria. The current working model of cyanobacterial carbon concentration mechanism consists of at least two basic components: an active C_i transport system and a Rubisco-rich polyhedral carboxysome. In case of unicellular green algae, the working model for DIC concentration mechanism includes several isoforms of carbonic anhydrase (CA), and ATPase driven active bicarbonate transporters at the plasmalemma and at the inner chloroplast envelopes. In the past twenty years, significant progress has been made in isolating and characterizing the isoforms of carbonic anhydrase. However, active transporters are yet to be characterized. This mini-review summarizes the current status of research on DIC-pumps including its significance and possible application to increase the productivity of plants of economic importance.     

Effect of Indoleacetic Acid on Growth and Dinitrogen Fixation in Cyanobacteria

The effect of IAA on growth, dinitrogen fixation, and heterocystsfrequency of Anabaena PCC 7119 and Nodularia sp. have been investigated.Concentrations of IAA ranging from 10^–10 to 10^–4M did not change the growth of Anabaena PCC 7119. Concentrationshigher than 10^–4 M were inhibitory. Similar results werefound in Nodularia sp. although in this case the inhibitoryeffect appeared with 10^–5M of IAA. Neither the nitrogenaseactivity nor the heterocysts frequency were enhanced by IAAtreatment. (Received June 17, 1986; Accepted January 22, 1987)     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Studies on Nitrogen Fixation by Blue-green Algae: II. NITROGEN FIXATION BY MASTIGOCLADUS LAMINOSUS COHN

Mastigocladus laminosus Cohn, a blue-green alga of hot springs,has been obtained in pure bacteria-free culture and has beenshown to be capable of fixing elementary nitrogen. This appearsto be the first instance in which this capacity has been demonstratedin a blue-green alga belonging to a family other than the Nostocaceae.     

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.     

Nitrogen Fixation (Acetylene Reduction) by Epiphytes of Freshwater Macrophytes

The involvement of epiphytic microorganisms in nitrogen fixation was investigated in a shallow freshwater pond near Ithaca, N.Y. The acetylene reduction technique was used to follow diel and seasonal cycles of nitrogen fixation by epiphytes of Myriophyllum spicatum. Acetylene-reducing activity was maximal between noon and 6 p.m., but substantial levels of activity relative to daytime rates continued through the night. Experiments with the seasonal course of activity showed a gradual decline during the autumn months and no activity in January or February. Activity commenced in May, with an abrupt increase to levels between 0.45 and 0.95 nmol of ethylene formed per mg (dry weight) of plant per h. Through most of the summer months, mean rates of acetylene reduction remained between 0.15 and 0.60 nmol/mg (dry weight) per h. It was calculated from diel and seasonal cycles that, in the pond areas studied, epiphytes were capable of adding from 7.5 to 12.5 μg of N per mg of plant per year to the pond. This amount is significant relative to the total amount of nitrogen incorporated into the plant. Blue-green algae (cyanobacteria), particularly Gloeotrichia, appeared to bear prime responsibility for nitrogen fixation, but photosynthetic bacteria of the genus Rhodopseudomonas were isolated from M. spicatum and shown to support high rates of acetylene reduction.