Modulation of fungicidal potential of <Emphasis Type="Italic">Anabaena</Emphasis> strains by light and temperature

The regulation of fungicidal and hydrolytic enzyme activity was investigated in a set of cyanobacterial strains belonging to the genus Anabaena (Anabaena laxa RPAN8, Anabaena iyengarii RPAN9, Anabaena variabilis RPAN59 and Anabaena oscillarioides RPAN69), with A. variabilis RPAN16 serving as negative control. Time course studies undertaken with cultures incubated under different light and temperature conditions revealed enhancement in growth and fungicidal activity under continuous light (CL) and light dark (LD, 16:8) conditions and temperature of 30 °C and 40 °C. A significant increase of 3–18 % in chitosanase activity was recorded in all the 4-week-old cultures under CL condition and at 40 °C. Endoglucanase activity of RPAN8 and 9 was twofolds higher than the other strains under all light/dark conditions and temperature in the 4-week-old cultures, while continuous dark (CD) enhanced CMCase activity in RPAN69. This study provided useful information regarding the most suitable conditions of light and temperature for maximizing hydrolytic enzyme activity and fungicidal activity, as a prelude to their effective use as biocontrol agents.     

Melafen action on the growth and physiological parameters of phototrophic and heterotrophic microorganisms

Melafen, a synthetic plant growth regulator, stimulates the growth of periodic cultures of cyanobacteria Anacystis nidulans and Anabaena variabilis and green microalgae Chlorella vulgaris by 30–80%, when applied within the concentration range of 10⁻⁸–10⁻⁶ M. If the melafen concentration lies out of this range, it is either ineffective or inhibits the growth of these microorganisms. The growth and development of green microalgae Dunaliella maritima are stimulated, and the cell growth of heterotrophic bacteria Pseudomonas diminuta is inhibited in a wide range of the increased melafen concentrations. This agent also stimulates the growth of dialyzed phototrophic cultures and the formation of heterocysts and vegetative cells in the filaments of Anabaena variabilis, increases the redox potential of a cultivation medium, and reduces a carbohydrate excretion from the cells of phototrophic microorganisms. It is supposed that melafen activates the processes of photosynthesis and atmospheric nitrogen fixation.     

Action of hydroxyurea on Anabaena variabilis

Summary Low concentrations of hydroxyurea stimulated the growth of the blue-green alga Anabaena variabilis that had been pretreated with sublethal concentrations of chloramphenicol or of certain nucleic acid base analogues. When supplemented to the culture medium, hydroxyurea also counteracted the growth inhibitory effect of chloramphenicol on this organism. In contrast, when A. variabilis cells grown in the presence of hydroxyurea were subsequently treated with chloramphenicol, they were found to have become highly susceptible to the growth inhibitory effects of chloramphenicol. The growth of hydroxyurea pretreated cells in basal medium was attended by a lag that was shorter than that of untreated controls; on the other hand, when hydroxyurea pretreated cells were inoculated into chloramphenicol-supplemented medium, they exhibited a longer lag than that shown by untreated cells in chloramphenicol.The results obtained are discussed in terms of the probable effects of hydroxyurea and chloramphenicol on certain enzyme systems.     

Molybdenum isotope fractionation by cyanobacterial assimilation during nitrate utilization and N₂ fixation

We measured the δ⁹⁸Mo of cells and media from molybdenum (Mo) assimilation experiments with the freshwater cyanobacterium Anabaena variabilis, grown with nitrate as a nitrogen (N) source or fixing atmospheric N₂. This organism uses a Mo‐based nitrate reductase during nitrate utilization and a Mo‐based dinitrogenase during N₂ fixation under culture conditions here. We also demonstrate that it has a high‐affinity Mo uptake system (ModABC) similar to other cyanobacteria, including marine N₂‐fixing strains. Anabaena variabilis preferentially assimilated light isotopes of Mo in all experiments, resulting in fractionations of ?0.2‰ to ?1.0‰ ± 0.2‰ between cells and media (ε_{cells–media}), extending the range of biological Mo fractionations previously reported. The fractionations were internally consistent within experiments, but varied with the N source utilized and for different growth phases sampled. During growth on nitrate, A. variabilis consistently produced fractionations of ?0.3 ± 0.1‰ (mean ± standard deviation between experiments). When fixing N₂, A. variabilis produced fractionations of ?0.9 ± 0.1‰ during exponential growth, and ?0.5 ± 0.1‰ during stationary phase. This pattern is inconsistent with a simple kinetic isotope effect associated with Mo transport, because Mo is likely transported through the ModABC uptake system under all conditions studied. We present a reaction network model for Mo isotope fractionation that demonstrates how Mo transport and storage, coordination changes during enzymatic incorporation, and the distribution of Mo inside the cell could all contribute to the total biological fractionations. Additionally, we discuss the potential importance of biologically incorporated Mo to organic matter‐bound Mo in marine sediments.     

Outdoor evaluation of herbicide resistant strains of Anabaena variabilis as biofertilizer for rice plants

Application of diazotrophic cyanobacteria, Anabaena variabilis, as biofertilizer for rice cultivation has a beneficial effect on crop productivity and maintenance of soil fertility. However, periodic applications of herbicides used to obtain high crop productivity are not only detrimental to weeds but to biofertilizer strains of cyanobacteria also. Therefore, research was undertaken to isolate four herbicide resistant strains (Arozin-R, Alachlor-R, Butachlor-R and 2,4-D-R) and a multiple herbicide resistant strain (MHR) of natural isolates of A. variabilis exhibiting resistance against these common rice field herbicides. The outdoor survivability of mutant strains and the productivity of rice crop (IR-36) were evaluated by inoculating the wild type and herbicide resistant mutant strains of A. variabilis in the presence and absence of recommended field dosages of test herbicides. No difference in survival and biofertilizer potentials of the herbicide resistant strains was observed in herbicide treated or in untreated conditions. Highest survivability (87%) was exhibited by MHR relative to other mutants. Highest growth and grain yield (76%) were recorded in plants treated with MHR as compared to uninoculated control rice plants. In conclusion, the mutant strains of A. variabilis had stable resistance to herbicides under outdoor conditions in flooded soils. Not only did the herbicide resistance strains increase growth of rice relative to the uninoculated pots, they were more beneficial for rice growth than the wild type strain. Responsible Editor: Richard W. Bell.     

Treatment of mixed domestic–industrial wastewater using cyanobacteria

Alexandria Sanitary Drainage Company (ASDCO), Alexandria, Egypt has two primary treatment plants, the eastern and the western wastewater treatment plants (EWTP and WWTP) that receive mixed domestic–industrial influents and discharge into L. Mariut. The lake is subjected therefore to severe levels of pollution and dominated by members of cyanobacteria that can cope with the high pollution load in the lake water. Isolation and utilization of the locally generated cyanobacterial biomass for remediation processes of highly toxic pollutants offers a very efficient and cheap tool for governmental or private industrial activities in Alexandria and will generate a source of revenue in Egyptian localities. The main objective of the present study was to investigate the biodegradation and biosorption capacity of some potential cyanobacterial species dominating the lake ecosystem toward organic and inorganic contaminants polluting the primary-treated effluents of the EWTP and WWTP. The primary effluents were subjected to biological treatment using three axenic cyanobacterial strains (Anabaena oryzae, Anabaena variabilis and Tolypothrix ceytonica) as batch system for 7 days. Removal efficiencies (RE) of the different contaminants were evaluated and compared. Results confirmed the high efficiencies of the investigated species for the removal of the target contaminants which were species and contaminant-dependent. BOD₅ and COD recorded 89.29 and 73.68% as maximum RE(s) achieved by Anabaena variabilis and Anabaena oryzae, respectively. The highest RE of the TSS recorded 64.37% achieved by Tolypothrix ceytonica, while 38.84% was recorded as the highest TSD RE achieved by Anabaena variabilis. Tolypothrix ceytonica also exhibited the highest RE for FOG recorded 93.75%. Concerning the contaminant metals, Tolypothrix ceytonica showed the highest biosorption capacity where 86.12 and 94.63% RE were achieved for Zn and Cu, respectively. In conclusion, results of the present study confirmed the advantageous potential of using the tested cyanobacterial species for the treatment of contaminated wastewater. Results also clearly showed the quality improvement of the discharged wastewater which in turn will eliminate or at least minimize the expected deterioration of the receiving environment.     

Surface lectin binding toAnabaena variabilis and to cultured and freshly isolatedAnabaena azollae

Five fluorescein isothiocyanate (FITC)-labeled lectins and Calcofluor white ST were tested for their binding abilities to vegetative cells and heterocysts of culturedAnabaena variabilis (AVA) and toA. azollae from four species ofAzolla; toAnabaena azollae freshly isolated fromAzolla pinnata (AP),A. caroliniana (AC),A. mexicana (AX), andA. filiculoides (AF); and to cultured akinetes ofAnabaena variabilis and four isolates ofA. azollae. Heterocysts of cultured cells of threeAnabaena isolates (APC, ACC, AXC) were most intensively surface-stained with soybean agglutinin fromGlycine max (SBA)-FITC; those of AX and AC were dimly stained with wheat germ agglutinin fromTriticum vulgaris (WGA); and only heterocysts of AX were dimly stained withDolichos biflorus agglutinin (DBA). Akinetes of cultured cells stained only with ConA. Vegetative cells and heterocysts of all four fresh isolates stained with Jack Beam aglutinin fromCanavalia ensiformis (ConA). None of the cell types were stained with either peanut agglutinin fromArachis hypogea (PNA) or Calcofluor.     

Effects of amino acids on methionine-sulfoximine-induced heterocyst formation in Anabaena

Heterocyst development in ammonia-grown cultures of Anabaena variabilis and Anabaena 7120 was fully induced by the amino-acid analog methionine sulfoximine (MSO) at concentrations of 0.5–1.0 M. Glutamine, glutamate, aspartate, and alanine at 0.5 mM blocked the induction of heterocysts by MSO in A. variabilis. With Anabaena 7120, glutamine and glutamate were fully effective and alanine partially effective in preventing MSO-induced heterocyst formation. In MSO-treated algae, glutamine synthetase activity was reduced to less than 15% of control values within 4–6 h. Inactivation of the enzyme was prevented by all four amino acids tested.     

Promising microbial consortia for producing biofertilizers for rice fields

Two cyanobacterial cultures from rice paddies of Kyzylorda Provence, Kazakhstan were isolated and characterized: Anabaena variabilis and Nostoc calsicola. Based on these cultures, new consortia of cyanobacteria, microalgae and Azotobacter were developed: ZOB-1 (Anabaena variabilis, Chlorella vulgaris, and Azotobacter sp.) and ZOB-2 (Nostoc calsicola, Chlorella vulgaris, and Azotobacter sp.). High growth rate and photosynthetic activity of microalgae were observed in these consortia. The active consortium ZOB-1 was selected, which improved germination and growth of rice plants. ZOB-1 was recommended as a biostimulator and biofertilizer for crops.     

Studies on sulphur-selenium antagonism in blue-green algae

Summary Anacystis nidulans and Anabaena variabilis contain sufficient sulphur reserves to enable them to perform only one round of growth cycle in the non-sulphur growth medium. Sulphate, sulphite, l-methionine and d-methionine, each can act as a suitable sulphur source, but they differ in respect of their growth promoting action; sulphate uptake seems to be a light driven phenomenon and the sulphate metabolizing enzymes are inducible in nature. Methionine appears to act as a repressor of sulphate-metabolizing enzymes.     

Effect of Mineral Phosphate Solubilization on Biological Nitrogen Fixation by Diazotrophic Cyanobacteria

The ability of two diazotrophic cyanobacteria Westiellopsis prolifica and Anabaena variabilis were examined to solubilize extracellular insoluble tricalcium phosphate (TCP) and Mussorie rock phosphate (MRP). The two strains exhibited a differential response to insoluble forms of phosphorus used. W. prolifica showed better growth in presence of MRP while A. variabilis proliferated better in presence of TCP. Biological nitrogen fixation measured in terms of acetylene reduction (AR) activity showed significant variation among the concentrations of TCP or MRP and time of incubation. W. prolifica and A. variabilis showed maximum AR activity on 14 and 21 days of incubation respectively. In general AR activity in presence of MRP was always less than that in presence of TCP at all concentrations. Among the two cyanobacteria A. variabilis was best in terms of P-solubilization and nitrogen fixation and TCP (20 mg P l⁻¹) was the best source of insoluble P rather than MRP or K₂HPO₄.     

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE-GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.     

Destruction of Pigments and Ultrastructural Changes in Cyanobacteria during Photodamage

Sequential stages of pigment degradation and ultrastructural changes were examined in cyanobacteria Anabaena variabilis ATCC 29413, Synechococcus sp. PCC 6301 (Anacystis nidulans) and S. elongatus B-267 during irradiation of cell suspensions with high-intensity light. Early manifestations of photooxidative destruction were evident as profound changes in ultrastructure of thylakoids; in A. variabilis these changes appeared even before bleaching of pigments. Concomitant to these alterations, the cytoplasmic matrix turned homogenous and the nucleoid was subject to degradation, while ultrastructural changes of cytoplasmic membrane and cell walls became evident in some species. In A. variabilis these changes were related to a subsequent autolysis of cells. Synechococcus strains demonstrated comparatively high resistance to irradiation. The experimental data were compared with previously described behavior of the same species of cyanobacteria cultured under photooxidative conditions. This comparison revealed principal similarity and species-specific features in the destructive changes of thylakoids and other cell components of cyanobacterial cells.     

Growth and survival potentials of immobilized diazotrophic cyanobacterial isolates exposed to common ricefield herbicides

Summary The effect of graded concentrations of four common ricefield herbicides (Arozin, Butachlor, Alachlor, 2,4-D) on diazotrophic growth, macromolecular contents, heterocyst frequency and tolerance potentials of Ca-alginate immobilized diazotrophic cyanobacterial isolates Nostoc punctiforme, N. calcicola, Anabaena variabilis, Gloeocapsasp., Aphanocapsa sp. and laboratory strain N. muscorum ISU (Anabaena ATCC 27893) was studied and compared with free-living cultures. Cyanobacterial isolates showed progressive inhibition of growth with increasing dosage of herbicides in both free and immobilized states. There were significant differences in the relative toxicity of the four herbicides. Arozin proved to be more growth toxic in comparison to Alachlor, Butachlor and 2,4-D. Growth performance of the immobilized cyanobacterial isolates under herbicide stress showed a similar diazotrophic growth pattern to free cells with no difference in lethal and sub-lethal dosages. However, at lethal concentrations of herbicides, the immobilized cells exhibited prolonged survivability of 14–16 days as compared to their free-living counterparts (8–12 days). The decline in growth, macromolecular contents and heterocyst frequency was found to be similar in both the states in graded dosages of herbicides. Of the test organisms, A. variabilis showed maximum natural tolerance towards all the four herbicides tested. Evidently immobilization by Ca-alginate seems to provide protection to the diazotrophic cyanobacterial inoculants to a certain extent against the growth-toxic action of herbicides.     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

Chromate (VI) uptake by and interactions with cyanobacteria

Summary The short-term accumulation of chromate by the cyanobacteriaAnabaena variabilis andSynechococcus PCC 6301 has been described as consisting of a rapid and relatively low level of biosorption of chromate to the cell walls; no energy-dependent uptake was detected. This biosorption was dependent on chromate concentration and could be described by a Freundlich adsorption isotherm for both cyanobacterial species studied. Decreasing the external pH increased the chromate accumulation by both species. Over a longer time period with growth it was shown thatA. variabilis was capable of reducing chromate (VI) to chromium (III) and then accumulating the chromium (III).Synechococcus PCC 6301 showed no further interaction with chromate concentrations over the same time period after the initial biosorption.     

Melafen action on the growth of cyanobacteria and green microalgae cultured in stress conditions

Melafen stimulating effect on cell growth of cyanobacteria Synechococcus sp. PCC 6301 cultures amounted to 30–45% at 1000 lx illumination. The melafen effect decreased when cell cultures were exposed at the illumination of the saturation range (4000 lx). Growth rate and biomass increase of Anabaena variabilis, as well as the observed melafen stimulating effect, were higher on nitrogen-free medium compared to a nitrogen-containing one by 20–25%. We conclude that melafen activates photosynthetic processes and, probably, stimulates fixation of the atmospheric nitrogen in the cells. Opposite to the stimulating effect of melafen, ions of the heavy metal Cd²⁺ inhibited both biomass increase and the average number of the cells in the cyanobacteria A. variabilis colonies. The melafen added to the medium together with the Cd²⁺ ions decreased their negative effect. The other heavy metal ions, Cu²⁺, inhibited the growth of the cyanobacteria Synechococcus sp. PCC 6301 and green microalgae Chlorella vulgaris but had a stimulation effect on carbohydrate excretion by the cell cultures. Again, the melafen decreased the toxic effect of Cu²⁺ in this case. We suppose that melafen has an antistress activity at heavy metal ions presence and reduces their toxic effect on growth of phototrophic microorganisms.     

Note on the survival of algal resting cells during long-term maintenance in darkness and minimum lake bottom temperature. Comparison of Anabaena (cyanobacteria) and Haematococcus (volvocales)

Resting cell formation in algae and cyanobacteria is one of the strategies developed for withstanding adverse environmental conditions. This note informs about the different capacities of such resting cells to germinate after long-term dark preservation at 4 °C in Anabaena variabilis (Cyanobacteria, Cyanophyta) and Haematococcus lacustris (Chlorophyta, Volvocales).     

Electron donation to nitrogenase in heterocysts of cyanobacteria

Various electron donors were found to stimulate C₂H₂ reduction (N₂ fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H₂, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C₂H₂ reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C₂H₂ reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C₂H₂ reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C₂H₂ reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C₂H₂ reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday