Effect of Cyanobacterial Combinations on Peanut Yield

The characteristics of two cyanobacterial strains, Anabaena oryzae and Nostoc muscorum, were studied in order to use them as biofertilizers in a field experiment conducted in the two winter seasons of 2021 and 2022 at the Ismailia Agricultural Research Center Station to study the effect of both strains on peanut plant in sandy soil. Cyanobacterial strains were used individually by coating seed, soil drench, and foliar applications, as well as mixed applications of two strains in various ways. Both cyanobacterial strains morphological examination revealed that they both have heterocysts, nitrogen, phosphorus, and potassium in their culture filtrate, and they were able to produce chlorophyll a and phosphatase enzymes. The results of an agricultural experiment showed that using Nostoc muscorum and Anabaena oryzae separately had a positive effect on peanut plants in a variety of applications, but combining both of these applications with 75% nitrogen increased the growth traits, nutrient contents, and soil biological activities in both peanut plants and their rhizosphere soil. The soil drench treatment with A. oryzae and Nostoc muscorum plus 75% nitrogen produced the highest growth results and peanut yields in a single application. The A. oryzae Soil Drench Application (S) + N. muscorum Foliar Application (F) with 75% N reported the best outcomes in mixed treatments. However, compared to single applications, all blended applications displayed better growth and yield characteristics. The results of the study suggest that employing cyanobacteria in a mixed application will enhance its advantages over a single use.

Impact of Cyanobacterial Inoculant on Growth and Productivity of Peanut Plants in Sandy Soil

During the summer growing seasons of 2021 and 2022, two field experiments were conducted at Ismailia Agricultural Research Center Station(Latitude 30? 35? 41.901? N and Longitude 32? 16? 45. 843?E) to study the effect of cyanobacterial inoculation (Anabaena oryzae (A. oryzae) and Nostoc mascarum (N. mascarum)) on peanut yield, quality and certain soil biological activities under various nitrogen fertilization conditions and three types of applications, thefirst treatment was carried out as coating seeds with powder of individual of each cyanobacterial strain and the before planted, second treatment seeds were drenched withsuspension of eachcyanobacterial strain individually andthe last treatment was by foliar doses after 15, 45 and 60 days from seeds planting. Results showed that applying cyanobacteria inoculation to peanut plants generally enhanced peanut plant growth, leading to significantly higher yields of peanut and grains than uninoculated treatments. Treatment of N. mascarum + 75% N recorded the highest peanut yield and plant characteristics followed by N. mascarum+ 75% N in soil drench application compared to other tested treatments and types of applications. Cyanobacteria enhanced the amount of N, P, Kand Ca in peanut plants overall. By increasing the total chlorophyl, carotenoids, dehydrogenase, urease activities and nutrients in the peanut rhizosphere, cyanobacteria inoculation had a favorable impact on soil fertility. In general, cyanobacteria inoculation with 75% nitrogen amounts can benefit under peanut growth in sandy soil conditions.

Antibacterial Activity Of Desiccated Cyanobacterium Anabaena Sp. Isolated From Terracotta Monuments Of Bishnupur, West Bengal

Anabaena sp. are the dominant cyanobacterial species on terracotta monuments of Bishnupur which exposed to high solar radiation, ultraviolet and in a desiccated condition in most part of the year. In the present study three Anabaena species were isolated from crust samples and its antibacterial activities were evaluated against pathogenic bacteria Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli. We observed good antibacterial activity in ethyl acetate and ethanol extract of Anabaena sp. (VBCCA 052002) which having highest antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, Staphylococcus aureus and E. coli respectively. We have validated the antibacterial assay by using resazurin based antimicrobial assay in microtiter plates and calculated the MIC value of ethyl acetate extract of Anabaena sp. (VBCCA 052002) which is found to be 100 µg/ml against Staphylococcus aureus and 150 µg/ml against Salmonella typhimurium.

Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Influence of light intensity, temperature and CO2 concentration on growth and lipids in green algae and cyanobacteria.

Effects of the environmental variables such as light intensity (µmol photons m-2s-1), temperature (°C) and CO2 concentration (ppm) on chlorophyll, total soluble proteins and lipids were studied in selected microalgal strains from Chlorophyceae (Chlamydomonas sp., Scenedesmus sp., Chlorella sp., Kirchneriella sp.) and cyanobacteria (Nostoc sp.1, Anabaena sp., Nostoc sp. 2, Cylindrospermum sp.). Cultures were grown under controlled conditions at the National Phytotron Facility, Indian Agricultural Research Institute (IARI), New Delhi. Our results showed that chlorophyll concentration enhanced with increased CO2. Chlorella exhibited the highest chlorophyll at 850 ppm CO2 and 28°C; for Chlamydomonas it was at 78 µmol photons m-2s-1 light intensity. In Cylindrospermum, total soluble proteins decreased with enhanced CO2, and were highest at 18°C. In Anabaena, a light intensity of 65 µmol photons m-2s-1 was best for maximum total soluble proteins. In Chlorella, CO2 @ 850 ppm was most suited for maximum lipid accumulation. In Kirchneriella, increase in temperature, from 18°C up to 37°C, increased total lipids; the highest was at 28°C. In Chlamydomonas, the light intensity of 78 µmol photons m-2s-1 was optimum for lipid accumulation and the maximum total lipids was 30.8 (% dry wt.).

COMPARATIVE GROWTH AND BIOCHEMICAL COMPOSITION OF FOUR STRAINS OF Nostoc AND Anabaena (CYANOBACTERIA, NOSTOCALES) IN RELATION TO SODIUM NITRATE / Comparación del crecimiento y Composición Bioquímica de cuatro cepas de Nostoc y Anabaena (Cyanobacteria, Nostocales) en relación con el nitrato de sodio

Nitrogen concentration is an essential parameter in cyanobacterial cultures to produce enriched biomass with biotechnological purposes. Growth and biochemical composition of Nostoc LAUN0015, Nostoc UAM206, Anabaena sp.1 and Anabaena sp.2 were compared at 0, 4.25, 8.5 and 17 mM NaNO3. Cultures under laboratory conditions were maintained for 30 days at a volume of 500 mL. Anabaena sp.1 yielded the highest value of dry mass of 0.26 ± 2.49 mg mL-1 at 8.5 mM NaNO3. For chlorophyll, phycocyanin and phycoerythrin, maximum values were achieved at 17 mM NaNO3 with 18.09 ± 1.74, 102.90 ± 6.73 and 53.47 ± 2.40 μg mL-1, respectively. Nostoc LAUN0015 produced its maximum value of protein 644.86 ± 19.77 μg mL-1, and 890 mg mL-1 of carbohydrates in the absence of nitrogen. This comparative study shows that the most efficient strain for the production of protein, carbohydrates and lipids in diazotrophic conditions corresponded to Nostoc LAUN0015. However, Anabaena sp.1 and Anabaena sp.2 required high nitrogen concentrations to achieve higher values of metabolites, comparing with Nostoc strains. Nitrogen dependence for the production of pigments and high protein production in strains of Anabaena and in diazotrophic conditions for Nostoc was demonstrated. Nostoc can be cultured under nitrogen deficiency and Anabaena in sufficiency, for biomass production enriched with proteins and carbohydrates.

La concentración de nitrógeno constituye un parámetro esencial en cultivos de cianobacterias para la producción de biomasa enriquecida con fines biotecnológicos. Se comparó el crecimiento y composición bioquímica de las cepas Nostoc LAUN0015, Nostoc UAM206, Anabaena sp.1 y Anabaena sp.2 a 0, 4,25; 8,5 y 17 mM NaNO3. Los cultivos en condiciones de laboratorio fueron mantenidos durante 30 días a un volumen de 500 mL. En masa seca, Anabaena sp.1 obtuvo el mayor valor, con 2,49 ± 0,26 mg mL-1 a 8,5 mM NaNO3. Para clorofila, ficocianina y ficoeritrina, los máximos se alcanzaron a 17 mM NaNO3 en Anabaena sp.1, con 18,09 ± 1,74; 102,90 ± 6,73 y 53,47 ± 2,40 μg mL-1, respectivamente. Nostoc LAUN0015 produjo su máximo valor de proteínas de 644,86 ±19,77μg mL-1, y alrededor de 890 μg mL-1 de carbohidratos en ausencia de nitrógeno. El estudio comparativo indica que la cepa más eficiente para la producción de proteínas, carbohidratos y lípidos, en condiciones diazotróficas, correspondió a Nostoc LAUN0015. En cambio, las cepas de Anabaena sp.1 y sp.2 requieren de elevadas concentraciones de nitrógeno para alcanzar los mayores valores de metabolitos, respecto a las cepas de Nostoc. Se demuestra la dependencia de nitrógeno para la producción de los pigmentos y la alta producción proteica en las cepas de Anabaena y en condiciones diazotróficas para Nostoc. Esta última puede ser cultivada bajo una deficiencia de nitrógeno y Anabaena con suficiencia para la producción masiva de biomasa enriquecida con proteínas y carbohidratos.

Eco-Friendly Approach for Treating Dairy Effluent and Lipid Estimation Using Microalgae.

Aim: In this study, an attempt has been made to examine the utility of these species in treating dairy wastewater. Bioreactor was studied using immobilized Chlorella vulgaris and Anabaena ambigua to treat dairy effluent. Study Design: The entire study including the treatment and filtration was conducted in Centre for biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad, between November 2014 to February 2015. Methodology: The treatment of dairy effluent consists of two stages; the first stage includes dairy effluent treatment using immobilised Chlorella vulgaris and Anabeana ambigua, while the second stage involves sand bed and coal bed filtration. Results: Chlorella vulgaris reported a high lipid content of 12% when compared with Anabaena ambigua 5% after the cultivation period. Whereas the protein content of Anabaena ambigua (40%) was higher than Chlorella vulgaris (28%) when compared with after harvesting. Whilst ammonium nitrate was completely removed by bead treatment it was 96% reduction when treated with Chlorella vulgaris. A 98% removal of phosphates was achieved on an average after algal bead treatment for both species in both modes of operation. Moreover, a significant decrease in BOD and COD was achieved by this method. Conclusion: It can be concluded that, the cultivation of algae in dairy effluent results the combined advantages of treating the dairy effluent and also producing algal biomass, which can further use of food additives for aquatic culture, animal and human feed, energies such as biogas and fuels and bio-fertiliser.

Effects of a cyanobacterial extract containing-anatoxin-a(s) on the cardiac rhythm of Leurolestes circunvagans

This work presents the effects of an anatoxin-a(s)-containing extract on a cockroach semi-isolated heart preparation and the results supporting the extract’s biological activity on acetylcholinesterase (purified from ell). The presence of the toxin in cyanobacterial strains Anabaena spiroides (ITEP-024, ITEP-025 and ITEP-026) isolated from the Tapacurá reservoir in Pernambuco, Brazil, was confirmed by means of liquid chromatography coupled to an ion-trap mass spectrometer. The anticholinesterase activity was assessed biochemically by the Ellman test and was confirmed by measuring the cockroach’s heart rate. The concentration of the extract containing the tested anatoxin-a(s) (antx-a(s)) (10, 16 and 100 μg.μL-1) inhibited the eel acetylcholinesterase (AChE) by more than 90%. The cockroach cardiac frequency increased by a maximum of about 20% within 29 min after the addition of 2.5x10³ μg of extract containing antxa (s).g-1 bw (n=9, p<0.05). Our results strongly indicate that antx-a(s) is capable of exerting biological effects on cockroach, indicating that more research might be conducted to determine its role in the environment, especially on insects.

Alterations in proteins and amino acids of the Nile cyanobacteria Pseudanabaena limnetica and Anabaena wisconsinense in response to industrial wastewater pollution

The effect of industrial wastewater on the Nile cyanobacteria Pseudanabaena limnetica and Anabaena wisconsinense was investigated. The data showed that P. limnetica was more sensitive to pollution than A. wisconsinense. The treatments with different levels of wastewater exerted pronounced reductions in protein and amino acids content. SDS-PAGE analysis revealed that the cyanobacteria grown in the industrial wastewater showed induction in the synthesis of certain polypeptides and repression of others. The treatments of P. limnetica with wastewater stimulated the appearance of six protein bands with molecular masses of 28, 30, 31, 32, 58 and 97 kDa. The same treatments caused the disappearance of 20, 38 and 56 kDa. The structural protein pattern of the treated A. wisconsinense showed appearance of 16, 30, 170 and 230 kDa and disappearance of 56 kDa. The treatment of the two investigated cyanobacteria with different levels of wastewater stimulated the biosyntheses of different amino acids and inhibited others.

Cloning and High Expression Anabaena Class-II Fructose-1, 6-bisphosphate Aldolase Gene in Escherichia coli / 中国生物工程杂志

The complete genomes of more cyanobacterial strains have been completed for sequence, and genetic engineering of cyanobacteria has evolved in the post-genome era. Since Kaneko and colleagues had completed the sequence for the complete genome of Anabaena sp. PCC 7120 in 2001, functions of some genes in this genome, including fructose-1,6-bisphosphate aldolase (FBA) gene, have been predicated using the method of bioinformatics. However, little information is available regarding whether this gene can encode FBA and its product characteristics of related enzyme. Here, to explore this information, the predicted II-FBA gene-encoding region in Cyanobase database was cloned by PCR method and then ligated into pET-32a to generate the expression vector, pET-FBA-II. The results of SDS-PAGE indicated that the expression level of the expected target polypeptide was approximate 23.4 percent as compared to total protein and the molecular weight is about 40 kDa as compared to the protein molecular marker. The results of enzyme activity analysis showed that the activity of II-FBA was ~11.8 U per mg protein and owned a standard activity of II-FBA. To sum up, the results not only prove the functional prediction of this II-FBA gene from the Cyanobase database, but also provide the important conditions for further studying its physiological and biochemical characteristics and functions of the gene expression product.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

The activity of specific glycosidases during the degradation of the extracellular polysaccharide (EPS) produced by Anabaena spiroides was determined using MUF-substrates (MUF-monosaccharides). Polysaccharide degradation was found to occur in a two-phase process. The first consisted of high enzymatic activity that consumed 41% of the EPS at a relatively high rate, while the second consumed the remaining polysaccharide (59%) at a slower rate. A transition phase from the higher to the slower degradation rates was marked by a replacement of bacterial populations from coccoid to bacillus cells. During the degradation process, the bacterial biomass increased with the decrease of EPS, as revealed by bacterial cell counts. The enzymatic activity detected through the substrates MUF-alpha-D- and MUF-beta-D-glucoside was higher than that detected by other substrates tested. The remaining glycosides were MUF-alpha-L-rhamnopyranoside, MUF-beta-D-galactoside, MUF-alpha-D-mannopyranoside, MUF-beta-D-fucoside, MUF-beta-D-mannopyranoside, MUF-alpha-L-arabinopyranoside, and MUF-beta-L-fucoside. The fluorescence emitted by each MUF-substrate was proportional to the concentration of the corresponding monosaccharide in A. spiroides EPS. This demonstrates the susceptibility of EPS produced by A. spiroides to enzymatic attack by bacterial populations.

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41% do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59%). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-alfa-D- e 4-MUF-beta-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-alfa-L-ramnopiranosídeo, MUF-beta-D-galactosídeo, MUF-alfa-D-manopiranosídeo, MUF-beta-D-fucosídeo, MUF-beta-D-manopiranosídeo, MUF-alfa-L-arabinopiranosídeo, e MUF-beta-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability in Anabaena sp. strain PCC 7120.

When deprived of combined nitrogen, aerobically-grown filaments of Anabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus in Anabaena.

Control of sporulation in the filamentous cyanobacterium A nabaena torulosa.

In the cyanobacterium Anabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculated A. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.

Canavanine-lnduced inhibition of growth and heterocyst differentiation in Anabaena doliolum and isolation of a canavanine-resistant mutant.

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen–fixing alga Anabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μΜ. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. Α mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.

Extracellular polypeptides of Anabaena L-31 : Evidence for their role in regulation of heterocyst formation.

Extracellular polypeptides released by both N2-grown [peptide I] and NO3-grown [peptide II] Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N2-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differenttiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, that Anabaena L-31 synthesises "inducer" or " inhibitor " peptides which could possibly control pattern formation.