Anabaena Fertilissima Research Articles

Extracellular antibacterial substances in Anabaena fertilissima CCC597 kill bacteria by triggering oxidative radicals and destroying membrane integrity

An axenic culture of a cyanobacterium in the spent medium produced hexane-extracta- ble compound(s) that antagonized growth of several Gram+ve and –ve bacteria, including a few potential pathogens. Phylogenetic investigations classified the strain to be Anabaena fertilissima strain CCC597. Using Escherichia coli MTCC443 as a test organism, we have shown that ROS (O 2; H 2O 2) production and outer and inner membrane (OM: IM) permeabilization were induced upon such treatments. Consequently, leakage of proteins and cytosolic acidification processes were initi- ated. Suppression of cytoplasmic membrane-bound respiratory O 2consumption was most likely the physiological aberration that killed the bacteria. Several antioxidant enzymes viz. superoxide dis- mutase, catalase, and peroxidases showed concomitant increase in the enzymatic activities and band intensities in the corresponding substrate gels. Notwithstanding, the counteraction mechanism(s) was not preventive, and sufficient oxidative radicals still generated to manifest lipid peroxidation. Chemical analysis of the hexane-extract of A. fertilissima culture filtrates revealed presence of a number of long chain unsaturated fatty acids, including cis-13,16-docosadienoic acid, with proven antibacterial properties.

Extraction, purification and characterisation of Phycocyanin from Anabaena fertilissima PUPCCC 410.5: as a natural and food grade stable pigment

Diverse phycocyanin (PC) extraction and purification methods for cyanobacteria have been reported in literature. A particular method is not applicable to all cyanobacteria and the best method seems to be genus/species specific. Here, we report PC extraction and purification method for the filamentous cyanobacterium Anabaena fertilissima, a hyperproducer of phycobiliproteins. PC was also characterised in terms of unit assembly and stability. PC was extracted by freeze-thawing in distilled water, concentrated by ammonium sulphate fractionation and purified by size exclusion chromatography. The purification process resulted in 5.85-fold increase in PC purity reaching to 3.28. SDS-PAGE of purified PC demonstrated two bands of 19 and 21 kDa. Based on analysis of non-denaturing PAGE, the native PC from this organism is suggested to be a hexamer (αβ)6 of 240 kDa. Lyophilised PC in powder form was more stable compared to PC in the solution. Half-life of dry PC is 110 days when stored at 4 °C in dark. Storage of PC in light at elevated temperature decreased its half life. When added to milk products, PC was stable up to 9 days when stored at 4 °C. Aqueous solution of PC also exhibited significant free radical scavenging activity. The PC from this organism has potential applications in food as a natural pigment and conservative due to its antioxidant properties.

Proteomic analysis of the salt-adapted and directly salt-(NaCl and NaCl+Na2SO4 mixture) stressed cyanobacterium Anabaena fertilissima

Salinity is a serious threat to agriculture productivity. Beneficial microbes could be simple and low-cost biological methods to mitigate the salt toxicity. The present study describes proteome dynamics and salt stress tolerance of the salt-primed (pre-exposed to salt) and directly salt-stressed cyanobacterium Anabaena fertilissima. The difference in the proteome of differently salt-treated and the control cells was the abundance of proteins. Out of 130 proteins resolved in control, 51.8% remained constant, 25.5% were upregulated, and 22.7% protein spots were downregulated in salt-adapted cells (exposed to 500 mM NaCl). However, in cells exposed to 250 mM NaCl, percentage of constant, upregulated, and downregulated proteins was 56.8, 16, and 27.2, whereas in the cells exposed to equimolar NaCl+Na2SO4 mixture, these values were 41.8, 29.4, and 28.7, respectively. This indicated that an altered protein expression occurred in large number of protein species under different salt-stress regimes. Four proteins showing significant and reproducible changes under salt stress showed close homology with photosystem I reaction center subunit XII, epoxyqueuosine reductase, response regulator protein VraR, and molybdopterin biosynthesis protein. The kinetic analysis revealed that salt treatment increased the abundance of all the four identified proteins. Despite the exposure to higher NaCl concentration (500 mM), salt-adapted cells showed minimal hyper accumulation of these four proteins, followed by NaCl and NaCl+Na2SO4 mixture. Lesser accumulation of these identified proteins in salt mixture stressed cells than that of NaCl stressed cells revealed that the availability of sulfur during salt-stress relieved the salt-toxicity, while Cl− increased it.

Physiological responses to salt stress of salt-adapted and directly salt (NaCl and NaCl+Na2SO4 mixture)-stressed cyanobacterium Anabaena fertilissima.

Soil salinity in nature is generally mixed type; however, most of the studies on salt toxicity are performed with NaCl and little is known about sulfur type of salinity (Na2SO4). Present study discerns the physiologic mechanisms responsible for salt tolerance in salt-adapted Anabaena fertilissima, and responses of directly stressed parent cells to NaCl and NaCl+Na2SO4 mixture. NaCl at 500mM was lethal to the cyanobacterium, whereas salt-adapted cells grew luxuriantly. Salinity impaired gross photosynthesis, electron transport activities, and respiration in parent cells, but not in the salt-adapted cells, except a marginal increase in PSI activity. Despite higher Na+ concentration in the salt mixture, equimolar NaCl appeared more inhibitive to growth. Sucrose and trehalose content and antioxidant activities were maximal in 250mM NaCl-treated cells, followed by salt mixture and was almost identical in salt-adapted (exposed to 500mm NaCl) and control cells, except a marginal increase in ascorbate peroxidase activity and an additional fourth superoxide dismutase isoform. Catalase isoform of 63kDa was induced only in salt-stressed cells. Salinity increased the uptake of intracellular Na+ and Ca2+ and leakage of K+ in parent cells, while cation level in salt-adapted cells was comparable to control. Though there was differential increase in intracellular Ca2+ under different salt treatments, ratio of Ca2+/Na+ remained the same. It is inferred that stepwise increment in the salt concentration enabled the cyanobacterium to undergo priming effect and acquire robust and efficient defense system involving the least energy.

Effects of Cyanobacterial Inoculation on the Growth and Yield of Triticum aestivum L. var. Deva K9117

The exponential growth in human population is become a serious concern in terms of their nutritional requirements. For the solution of this problem several methods and technologies have been adopted like development of high yielding varieties with improved agricultural practices. These varieties require more chemical fertilizers for the maximum production but excessive utilization of these chemical fertilizers may cause the deficiencies and infertility in the agricultural soils. So there is need to replace these chemical fertilizers with biological fertilizers due to their environmental sustainability. The utilization of cyanobacteria as bio-fertilizers are an eco-friendly, easily manageable and self-regenerating process which improve the nutrient status as well as health of soil. In addition to biological nitrogen fixation they also produce several growth promoting substances. During the present investigations we have adopted an experimental research approach to examine the effects of cyanobacteria (inoculation of live Anabaena fertilissima C.B. Rao and Nostoc linckia Bornet ex Bornet and Flahault isolates collected from river Ganga at Kanpur, U.P, India) as bio-fertilizer in Triticum aestivum L. var. Deva K9117 on the basis of average height of plants, weight of grains and number of grains/spike. The observations showed significant changes/improvements in growth and productivity of Triticum aestivum L. var. Deva K9117. The inoculation of Anabaena fertilissima C.B. Rao (100 ml) increased the 19.81% grains/spike, 9.86% weight of grains and 7.91% height of plants while inoculation of Nostoc linckia Bornet ex Bornet and Flahault (100 ml) increased the 19.36% grains/spike, 7.27% weight of grains and 5.64% height of plants over the control. The most considerable finding of the study was the mixture of Anabaena fertilissima C.B. Rao+Nostoc linckia Bornet ex Bornet and Flahault (60:40) increased 24.32% grains/spike, 15.09% weight of grains and 10.09% height of plants over the control.

Phycobiliprotein production by a novel cold desert cyanobacterium Nodularia sphaerocarpa PUPCCC 420.1

The present study focuses on finding a good source of phycobiliproteins (PBP). We report a new cyanobacterium Nodularia sphaerocarpa PUPCCC 420.1 as a good producer of PBP. The organism produced 445.6 μg PBP mg−1 dry biomass. The growth and PBP production of organism were optimized by varying pH of growth medium, nitrogen sources, light quality and sugars. The optimized conditions for PBP production were as follows: pH 8.0, 5 mmol L−1 KNO3, 10 mmol L−1 NaNO2, 0.5% sucrose and green light. The PBP production under these conditions ranged from 486 to 676.3 μg mg−1 dry biomass. The PBP were more stable when stored in alkaline pH at 4 °C under dark. As per survey of literature, except for Anabaena fertilissima, the amount of PBP is significantly higher than the amount of PBP produced by other cyanobacteria. Thus, this organism is a good candidate for the PBP production at commercial level.

Biphasic ROS accumulation and programmed cell death in a cyanobacterium exposed to salinity (NaCl and Na2SO4)

High salinity increases antioxidative activities in plants; however, their significance for overall plant salt tolerance remains to be established. This work provided in vivo evidence of salinity induced biphasic reactive oxygen species (ROS) accumulation evoking oxidative stress in the cyanobacterium Anabaena fertilissima. First, a transient increase in ROS (intense and short-lived) was observed within 5min of salt exposure, which peaked within 15min and reached basal level by 2h. This was followed by a second relatively long-lived and low magnitude ROS accumulation that started at 4h of salt stress, attained its maximal at 6h, followed by a gradual decline but did not attain the basal level by the end of experimentation (12h). Phase I ROS accumulation timing corresponded to the reaction of cyanobacterial cells to the salt stress, while altered photosynthetic and respiratory parameters corresponded with the phase II ROS generation. Relatively lower magnitude of ROS generation during phase II may be attributed to the rapid activation of robust antioxidative systems in cyanobacteria. Consequently, ROS generation lead to the activation of programmed cell death (PCD) undergoing various apoptotic stages such as externalization of phosphatidylserine, DNA laddering and loss of plasma membrane integrity. A. fertilissima exposed to salt in the presence of SO4¯ was relatively better equipped to deal with salt stress.

Biodegradation Capability and Enzymatic Variation of Potentially Hazardous Polycyclic Aromatic Hydrocarbons—Anthracene and Pyrene by Anabaena fertilissima

This study encompasses the biodegradation capacity of Anabaena fertilissima to model PAH (Polycyclic Aromatic Hydrocarbon) compounds namely Anthracene (ANT) and Pyrene (PYR) at different LC50 concentrations viz., 5.0 mg/L and 3.0 mg/L, respectively, on growth in terms of Chlorophyll-a and protein. Depletion in chlorophyll-a and protein content was registered with rise in PAHs concentration while the inhibition of nitrogen fixing enzymes such as nitrate reductase and glutamine synthetase activity was also concentration dependent and showed more sensitivity for high molecular weight aromatic compound PYR as compared to ANT. GC/MS analysis explained the degradation of ANT by 46% and PYR by 33%, at 5.0 mg/L and 3.0 mg/L, respectively. Moreover, the common degraded product for ANT was 2, 4-Dimethyl-1-heptene and for PYR it was 2, 3, 4-Trimethylhexane. Results indicate that PYR was more stable and recalcitrant compared to ANT. This study suggests that Anabaena fertilissima could be used for bioremediation of ANT and PYR pollution in surface waters and terrestrial environments.

Hyperproduction of phycobiliproteins by the cyanobacterium Anabaena fertilissima PUPCCC 410.5 under optimized culture conditions

This study was targeted to find new sources of phycobiliproteins (PBP) as these are important as natural colours, antioxidants and fluorescent markers. Here we report Anabaena fertilissima PUPCCC 410.5 as a good producer of PBP. Control cultures of the cyanobacterium produced 383μg PBP mg−1 dry biomass on day 8. The pH of the medium, supplementation of nitrogen source, sugar and illumination of cultures with different colours of light were optimized for PBP production. Optimized individual parameters were: pH of the medium 7.5; supplementation of 2mmol nitrite L−1 or 0.5% sucrose; and illumination of cultures with blue light. Total PBP production under these conditions ranged between 627 and 696μgmg−1 dry biomass, resulting in nearly 1.6 fold increase, with a significant increase in phycocyanin content. Incubation of 2mmol nitrite L−1 supplemented cultures in blue light resulted in nearly 4.5 fold increase in phycoerythrin compared to the control cultures. The PBP/phycocyanin/phycoerythrin production under the optimized conditions by Anabaena fertilissima PUPCCC 410.5 is significantly higher than the PBP production by the other cyanobacterial strains reported in literature. Thus this cyanobacterial strain is a promising candidate for PBP production at commercial level.

Consequences of Environmentally Hazardous Polycyclic Aromatic Hydrocarbon- Anthracene Treatment on Cyanobacteria

The study was aimed to determine the chronic toxicity of Polynuclear aromatic hydrocarbon – Anthracene in response to pigments and metabolic study on three different cyanobacterial species such as Synechocystis sp., Anabaena fertilissima, and Nostoc muscorum. Test organisms were treated at different doses and encountered LC50/Mean Lethal Concentration (at which 50% lethality/ growth reduction occur) separately at 7.0 ppm for Synechocystis sp, 5.0 ppm for Anabaena fertilissima and 1.5 ppm for Nostoc muscorum. The influence of anthracene on pigments, metabolites and enzymes was carried out. The test doses caused concentration dependent and decreased pigments like carotenoids and phycobilliproteins. Depletion of carbohydrate by 65 to 80% and proteins by 58 to 78% was encountered with rise in Anthracene concentrations after 16th day exposure in case of Synechocystis sp however, phenols were found to raise by 26 to 37% with increased anthracene concentrations. Similar trend also observed in other two tested blue green algae. Thus the Synechocystis sp.is more tolerant to anthracene treatments as compare to Anabaena fertillissima but Nostoc muscorum showed highest sensitivity to anthracene.Int J Appl Sci Biotechnol, Vol 3(3): 381-386

Chronic Response of Three Different Cyanobacterial Species on Growth, Pigment, and Metabolic Variations to the High Molecular Weight Polycyclic Aromatic Hydrocarbon – Pyrene

A study was carried out to investigate the chronic response of three different cyanobacterial species namely Anabaena fertilissima, Synechocystis sp., and Nostoc muscorum to Pyrene, a high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). Organisms were treated based upon LC50 (lethal concentration at which 50% reduction in growth) concentrations, i.e., 3.0 ppm for Anabaena fertilissima, 3.0 ppm for Synechocystis sp., and 0.5 ppm for Nostoc muscorum as well as finalized one lower and one higher dose to LC50. The influence of pyrene on growth, pigments, release of metabolites, such as carbohydrates, protein, amino acid, and phenols, was carried out. The test doses caused a concentration dependent decrease in carotenoids and phycobilliproteins and showed more sensitivity for pyrene. Depletion of carbohydrate by 47–98% and proteins by 39–81% was encountered with rise in pyrene concentrations after 16th day of exposure in case of high tolerant strain Synechocystis sp. However, phenols were found to rise by 64–93% with increased pyrene concentrations on the contrary, amino acids were reported to decline by 59–93%. The order for the sensitivity of the test species is as follows: Nostoc muscorum > Anabaena fertillissima > Synechocystis sp.

Chronic Toxicity Of High Molecular Weight Polynuclear Aromatic Hydrocarbon- Pyrene On Freshwater Cyanobacterium Anabaena Fertlissima Rao

The aim of this work was to determine the consequences of Polynuclear aromatic hydrocarbon – Pyrene in response to growth, pigments and metabolic study on Anabaena fertilissima Rao. Test organisms were treated at different doses and encountered LC50 (Lethal concentration at which 50% growth reduction occur) concentration separately at 1.5 mg/l, 3.0 mg/l and 6.0 mg/l respectively for Anabaena fertilissima Rao. The influence of Pyrene on growth, pigments, release of metabolites such as carbohydrates, protein, amino acid, phenols was carried out. The test doses caused a concentration dependent decrease in pigments like carotenoids and phycobilliproteins and showed more sensitivity to pyrene. Depletion of carbohydrate by 13% to 81% and proteins by 47% to 93% was encountered with rise in pyrene concentrations after 16th day of exposure. However, phenols were found to rise by 27% to 50% with increased pyrene concentrations on the contrary, amino acids were reported to decline by 79% to 92%. This study therefore suggests high molecular weight pyrene that decreases in metabolite content and enzyme activity can be used as a signal of PAHs toxicity in cyanobacteria. International Journal of Environment, Volume-2, Issue-1, Sep-Nov 2013, Pages 175-183 DOI: http://dx.doi.org/10.3126/ije.v2i1.9220

Physiological evidence indicates microcystin-LR to be a part of quantitative chemical defense system

The functional role of microcystins (MC) is poorly understood. Here, we investigated the effect of pure MC-LR recovered from the freshwater planktonic cyanobacterium Nostoc sp. BHU001 on five closely related cyanobacteria (Nostoc muscorum, Nostoc commune, Anabaena fertilissima, Anabaena doliolum, and Cylinderospermum majus) isolated from different habitats as well as on the producer itself (Nostoc sp. BHU001). MC-LR was found to be a general growth inhibitor active at nanomolar range (25–100 μg L−1). It inhibited the growth of all cyanobacterial strains in a concentration-dependent manner, except the producer. A. fertilissima was the most sensitive species. MC-LR affected vital metabolic processes such as photosynthesis, respiration, and nitrogen fixation. Nitrogenase activity showed maximum sensitivity, followed by respiration, photosynthesis, and general growth. The photosynthetic electron transport activity was maximally inhibited at PSI, followed by whole chain and PSII activities. Thus, MC-LR is active at multiple sites causing energy constraint to the vital metabolic processes of the target organisms. However, its requirement at high concentration, which is environmentally irrelevant, and lack of quantitative information on the extracellular release of MC-LR suggest that MC-LR has no allelopathic function and could be a part of a quantitative chemical defense system.

Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima

An isolate of the cyanobacterium Anabaena from paddy fields was cultured and identified as Anabaena fertilissima based on morphometric features and 16S rRNA gene sequence matching. Cell extracts prepared using bead beater hydrolyzed casein. The caseinolytic protease with native molecular mass of 49 kDa was purified using ammonium sulfate fractionation, hydrophobic, affinity and ion-exchange chromatography, and gel filtration. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified protease was resolved in 17-kDa homologue of microcompartment protein and 27-kDa fragment of unknown protein. The enzyme in native state was digested with gelatin and fibrin in substrate gels producing bands corresponding to ca. 49 kDa. Moreover, a plasmin-specific substrate d-Val-Leu-Lys p-nitroanilide was also hydrolyzed with apparent K m = 0.18 mM and V max = 4.9 × 10−7 M s−1; while Ca2+ stimulated, phenylmethanesulfonyl fluoride, leupeptin, and chelators completely abolished the amidolytic activity. The enzyme exhibited pH and temperature stability over a wide range. Upon incubation with fibrinogen, the Aα- and Bβ-chains preferentially cleaved, though the products thus resolved on SDS-PAGE moved at masses different from those of thrombin- and plasmin hydrolysates, and unlike thrombin, cross-linking of fibrinopeptides was not observed. In the plate assays, fibrinolysis was revealed at comparable strengths to that of plasmin, and the dissolute so obtained upon SDS-PAGE lacked bands corresponding to γ-dimer. Consequently, the degraded D-Dimer peptides appeared. The cyanobacterial protease displayed several unique properties not found in microbial and snake venom fibrinolytic enzymes.

Low cellular P-quota and poor metabolic adaptations of the freshwater cyanobacterium Anabaena fertilissima Rao during Pi-limitation

Anabaena fertilissima is a filamentous freshwater N(2)-fixing cyanobacterium, isolated from a paddy field. Growth of the cyanobacterium was limited by the non-availability of inorganic phosphate (Pi) in the growth medium and was found to be directly related to the cellular P quota, which declined rapidly in Pi-deficient cells. To overcome Pi-deficiency, cells induced both cell-bound and cell-free alkaline phosphatase activities (APase). The activity of cell-bound APase was rapid and 5-6 times higher than that of the cell-free APase activity. Native gel electrophoresis revealed the presence of two APase activity bands for both the cell bound and cell-free APase (Mr ≈42 and 34 kDa). For Pi-deficient cells, APase activity was inversely related to cellular P-quota. In A. fertilissima phosphate uptake was facilitated by single high-affinity phosphate transporter (K ( s ), 4.54 μM; V(max), 4.84 μmol mg protein(-1) min(-1)). Pi-deficiency severely reduced the photosynthetic rate, respiration rate and nitrate uptake, as well as the activities of nitrate reductase, nitrite reductase and nitrogenase enzymes. In photosynthesis, PSII activity was maximally inhibited, followed by PSI and whole chain activities. Transcript levels of five key glycolytic enzymes showed the poor adaptability of the cyanobacterium to switch its metabolic activity to PPi-dependent enzyme variants, which has rather constant cellular concentrations.

Chronic response of Anabaena fertilissima Rao, C.B. on growth, metabolites and enzymatic activities by chlorophenoxy herbicide

Study was carried out to investigate the chronic response of cyanobacteria, Anabaena fertilissima to chlorophenoxy herbicide 2,4-dichlorophynoxyacetic Acid (2,4-D) ethyl ester at different concentrations 15, 30 and 60 ppm. The influence of 2,4-D on growth (pigments), release of metabolites such as carbohydrates, protein, amino acid, phenols and nitrate reductase and glutamine synthetase activities was analyzed. The test concentrations caused a concentration-dependent decrease in pigments. Depletion in carbohydrate and protein content was registered with rise in herbicide concentrations. However, phenols were found to rise with increased herbicide concentrations but amino acids were reported to decline. The inhibition of nitrate reductase and glutamine synthetase activity was also concentration-dependent and showed more sensitivity for substituted phenoxy herbicide. This study therefore suggests that decrease in metabolite content and enzyme activity can be used as a signal of herbicide toxicity in cyanobacteria.

Changes in the phycobiliproteins during spore (akinete) differentiation in a cyanobacterium, Anabaena fertilissima

Spore (akinete) differentiation in Anabaena fertilissima in accompanied by changes in the composition of allophycocyanin, phycocyanin and phycoerythrin. Phycoerythrin in transformed from C-type in vegetative cells to R-type in spores. The apparent disappearance/reduction in the amount of allophycocyanin/phycocyanin in spores is mainly due to the loss of chromophores (bilins) rather than due to the loss of the apoproteins.

Effects of temperature pre-treatment, desiccation and aging on the viability of spores of halophilic blue-green algae

The effects of temperature, desiccation and aging on the viability of spores of Sambhar salt lake blue-green algae, Anabaena fertilissima and Anabaenopsis arnoldii, were studied. Spores of A. arnoldii were found to be more susceptible to temperature variation, desiccation and storage than spores of A. fertilissima. Pre-treatment of spores with higher temperatures, 37° and 47°C, stimulated germination in A. fertilissima. In a sporulated filament, spores which developed first were generally bigger and more resistant to adverse environmental conditions than spores formed later. The differential loss of viability in spores of a filament may be due to certain intrinsic differences in the physiological/ biochemical properties of the spores.

Some Observations Related to Red — Far Red Antagonism in Germination of Spores of the Cyanobacterium Anabaena fertilissima

Effects of intermittent short duration exposures of red (650 nm) and/or far red (725-750 nm) illuminations on spore germination were studied in Anabaena fertilissima . Spore germination was induced by red light while far red irradiation inhibited it. In reversal experiments, induction of germination by red light was annulled when the spores were subsequently exposed to far red light whereas inhibition due to far red illumination was overcome when spores were subjected to consequent red light treatment. Such antagonistic effects of red - far red irradiations provide evidence for the operation of photoinduction-photoreversal phenomenon mediated by a pigment which seems to be functionally similar to phytochrome of higher plants.