Comparative Genomics and Physiological Investigation of a New Arthrospira/Limnospira Strain O9.13F Isolated from an Alkaline, Winter Freezing, Siberian Lake.

Cyanobacteria from the genus Arthrospira/Limnospira are considered haloalkalotolerant organisms with optimal growth temperatures around 35 °C. They are most abundant in soda lakes in tropical and subtropical regions. Here, we report the comprehensive genome-based characterisation and physiological investigation of the new strain O9.13F that was isolated in a temperate climate zone from the winter freezing Solenoye Lake in Western Siberia. Based on genomic analyses, the Siberian strain belongs to the Arthrospira/Limnospira genus. The described strain O9.13F showed the highest relative growth index upon cultivation at 20 °C, lower than the temperature 35 °C reported as optimal for the Arthrospira/Limnospira strains. We assessed the composition of fatty acids, proteins and photosynthetic pigments in the biomass of strain O9.13F grown at different temperatures, showing its potential suitability for cultivation in a temperate climate zone. We observed a decrease of gamma-linolenic acid favouring palmitic acid in the case of strain O9.13F compared to tropical strains. Comparative genomics showed no unique genes had been found for the Siberian strain related to its tolerance to low temperatures. In addition, this strain does not possess a different set of genes associated with the salinity stress response from those typically found in tropical strains. We confirmed the absence of plasmids and functional prophage sequences. The genome consists of a 4.94 Mbp with a GC% of 44.47% and 5355 encoded proteins. The Arthrospira/Limnospira strain O9.13F presented in this work is the first representative of a new clade III based on the 16S rRNA gene, for which a genomic sequence is available in public databases (PKGD00000000).

Spirulina ameliorates aspirin-induced gastric ulcer in albino mice by alleviating oxidative stress and inflammation.

Aspirin is a commonly prescribed non steroidal anti-inflammatory drug, but its prolonged use injures the gastric mucosa. The present study was carried out to evaluate the ameliorative effect of spirulina against aspirin-induced gastric ulcer in albino mice. Gastric ulcer was induced by oral administration of aspirin (500 mg/kg bw). Spirulina (250 and 500 mg/kg bw) was given orally for 3 days after the induction of gastric ulcer. Spirulina ameliorated aspirin-induced gastric ulcer by improving the gross morphology, histology and mucous layer of gastric tissue, augmenting the endogenous enzymatic and non-enzymatic antioxidants (reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase) and the cytoprotective marker (COX-1), and by alleviating tissue levels of the lipid peroxidation marker (malondialdehyde) and inflammatory mediators (TNF-α, COX-2 and NO). In conclusion, spirulina has a therapeutic potential in aspirin-induced gastric injury by alleviating oxidative stress and inflammation.

Characterization of phototrophic microorganisms and description of new cyanobacteria isolated from the saline-alkaline crater-lake Dziani Dzaha (Mayotte, Indian Ocean).

The saline-alkaline crater-lake Dziani Dzaha (Mayotte, Indian Ocean) is dominated by the bloom-forming cyanobacterium Arthrospira. However, the rest of the phototrophic community remains underexplored because of their minute dimension or lower biomass. To characterize the phototrophic microorganisms living in this ecosystem considered as a modern analog of Precambrian environments, several strains were isolated from the water column and stromatolites and analyzed using the polyphasic approach. Based on morphological, ultrastructural and molecular (16S rRNA gene, 18S rRNA gene, 16S-23S internal transcribed spacer (ITS) region and cpcBA-IGS locus) methods, seven filamentous cyanobacteria and the prasinophyte Picocystis salinarum were identified. Two new genera and four new cyanobacteria species belonging to the orders Oscillatoriales (Desertifilum dzianense sp. nov.) and Synechococcales (Sodalinema komarekii gen. nov., sp. nov., Sodaleptolyngbya stromatolitii gen. nov., sp. nov. and Haloleptolyngbya elongata sp. nov.) were described. This approach also allowed to identify Arthrospira fusiformis with exclusively straight trichomes instead of the spirally coiled form commonly observed in the genus. This study evidenced the importance of using the polyphasic approach to solve the complex taxonomy of cyanobacteria and to study algal assemblages from unexplored ecosystems.

Development of coaxial alginate-PCL nanofibrous dressing for controlled release of Spirulina extract.

Spirulina has widely been highlighted as a source of bioactive material that can be impregnated into dressing materials. The aim of this study was to widen the application fields of Spirulina extract-containing nanofiber, which has been suggested as an attractive dressing material in several previous studies. The bioactivity release pattern, water absorbance, and mechanical strength must be controllable. Spirulina extract was physically impregnated inside a nanofiber without significant chemical bonding to polycaprolactone or alginate polymers. This led to an initial burst and continual release of bioactive molecules from the nanofiber. By altering the concentration of Spirulina extract, mechanical strength and water absorbance were controllable. In addition, the dressing patch showed no cytotoxicity towards human epithelial cells, not causing skin-irritation. This indicates that the coaxially fabricated patch is a controllable dressing material that can be customized to have a specific mechanical strength, water absorbance, and bioactive release pattern, making it suitable for wide applications.

Isolation and characterization of high protein and phycocyanin producing mutants of Arthrospira platensis.

Cyanobacteria are known to exhibit their efficiency in producing high concentrations of compounds of commercial value. Arthrospira is one such cyanobacterium which is considered as important source of protein (65%) and other nutrients. In present study, chemical mutagenesis using N-methyl-N-Nitro-nitrosoguanidine (NTG), a proven potent mutagen for cyanobacteria was used to bring stable and desirable alteration in Arthrospira platensis ARM 730. Three morphological mutants (G-1, G-2, and SF) were selected and characterized. The G-1 and G-2 were helical, more bluish in pigmentation than the wild type strain where G-1 also showed enlarged cell size. The SF mutant was an altered straight-filament having maximum biomass. Among three mutants, higher protein and phycocyanin contents were observed in G-1 and G-2 mutants whereas chlorophyll was less in these mutants as compared to wild type strain indicating change in the pigment ratio. Carotenoid content was higher in SF mutant as compared to wild type and other mutants. Variation in total sugar content was not observed in comparison to wild type strain. The analysis of amino acid spectrum of all the mutants and wild type showed significant increase in proline content. Overall, it is revealed from the results that G-1 and G-2 mutants showed higher biomass, phycocyanin, and protein contents in comparison to wild type which indicated their great potential to be used in food and pharmaceutical industries.

Potential application of microalga Spirulina platensis as a protein source.

The high protein level of various microalgal species is one of the main reasons to consider them an unconventional source of this compound. Spirulina platensis stands out for being one of the richest protein sources of microbial origin (460-630 g kg-1 , dry matter basis), having similar protein levels when compared to meat and soybeans. The use of S. platensis in food can bring benefits to human health owing to its chemical composition, since it has high levels of vitamins, minerals, phenolics, essential fatty acids, amino acids and pigments. Furthermore, the development of new protein sources to supply the shortage of this nutrient is an urgent need, and protein from S. platensis plays an important role in this scenario. In this sense, extraction processes that allow maximum protein yield and total utilization of biomass is an urgent need, and ultrasonic waves have proven to be an effective extraction technique. The number of scientific papers related to protein fraction from S. platensis is still limited; thus further studies on its functional and technological properties are needed. © 2016 Society of Chemical Industry.

[Phylogenetic comparison between Spirulina and Arthrospira based on 16S rRNA and rpoC1 gene].

OBJECTIVE: Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. METHODS: We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. RESULTS: The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-35 1, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. CONCLUSION: Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

Fouling characterization and control for harvesting microalgae Arthrospira (Spirulina) maxima using a submerged, disc-type ultrafiltration membrane.

This study characterized the fouling of a novel circular-disc ultrafiltration membrane in a submerged bioreactor system to harvest Arthrospira maxima cells. Flux-stepping study showed that the value of critical flux was below the smallest flux tested at 28.8lm(-2)h(-1), and that the membrane was to operate above the critical flux to sustain the necessary rate of cell concentration. The membrane with similar pore size but greater pore density experienced not only lesser degree of total resistance, but also possessed smaller fraction of irreversible resistance. Membrane fouling was mainly attributed to fragmented cells rather than to soluble or extracellular polymeric substances. Furthermore, flux recovery studies demonstrated that membrane relaxation and surface cleaning could partially recover fluxes for both low (6gl(-1)) and high (40gl(-1)) cell densities, whereas backwashing could fully recover fluxes. Calculation of energy consumption and cell harvesting productivity also favoured membrane filtration with backwashing.

Divergence in three newly identified Arthrospira species from Mexico.

Arthrospira (Spirulina) is a microalgae that has a unique set of biological characteristics which are very useful for a broad range of applications. Based on its worldwide requirements, this investigation was conducted to collect, isolate and identify the local Arthrospira strains in the central and western part of Mexico. We have successfully collected, isolated and identified (morphologically as well as molecularly) three Arthrospira strains from different regions in Mexico. Morphological studies were conducted by analyzing the size and shape of the helix, the spiral pattern, cell length and width with the help of light microscopy and for molecular analysis, the 16S rRNA and internally transcribed spacer (ITS, 16S-23 rRNA) gene partial sequence were used followed by phylogenetic analysis. The three species were completely different in their filament size and width whereas their ITS sequences were the same in size and more than 87 % similar in nucleotide sequence. The resulted morphological and phylogenetic analysis concluded that the three stains were identified as Arthrospira platensis. Inspite of their morphological variations and differences they were grouped genetically into one cluster along with the A. platensis of reported strains of Gene Bank database (NCBI). One of the isolated strains NPS-0, is probably the biggest Arthrospira strains ever reported and can be suitable for industrial scale biomass and protein production.

Influence of carbon-dioxide on the growth of Spirulina sp. (MCRC-A0003) isolated from Muttukadu backwaters, South India.

Growth of Spirulina sp. (MCRC-A0003), a cyanobacterium, was evaluated under different concentrations of carbon-dioxide (CO2) (4-50 %) in a closed glass photobioreactor. Although significant CO2 utilization by the cyanobacterial strain was observed up to 50 % concentration, complete utilization was observed only at 4, 10 and 20 % concentrations on 3rd, 6th and 8th day respectively. However, considerable reduction was witnessed in reactors containing 30-50 % CO2 only between 6th and 9th day. A corresponding increase in the biomass and primary metabolites like chlorophyll-a, carbohydrate and protein were observed. Biomass productivity of Spirulina in reactors sparged with 4, 10 and 20 % CO2 were 13.7, 43 and 44 % more than that in control reactor without CO2. While CO2 increased the levels of primary metabolites in the cyanobacterial cells, it was quite prominent in 10 % CO2 concentration with the chlorophyll-a, carbohydrate and protein contents were 64, 183 and 626 mg g(-1) respectively. While 10 and 6.6 % increase were noticed in chlorophyll-a and protein, 17 % increase in carbohydrate levels was observed in Spirulina cells, which could be attributed to the conversion of CO2 to carbohydrate by the cyanobacterium.

Exploration of the gasification of Spirulina algae in supercritical water.

This study presents non-catalytic gasification of Spirulina algae in supercritical water using a plug flow reactor and a mechanism for feeding solid carbon streams into high pressure (>25 MPa) environments. A 2(III)(3-1) factorial experimental design explored the effect of concentration, temperature, and residence time on gasification reactions. A positive displacement pump fed algae slurries into the reactor at a temperature range of 550-600°C, and residence times between 4 and 9s. The results indicate that algae gasify efficiently in supercritical water, highlighting the potential for a high throughput process. Additional experiments determined Arrhenius parameters of Spirulina algae. This study also presents a model of the gasification reaction using the estimated activation energy (108 kJ/mol) and other Arrhenius parameters at plug flow conditions. The maximum rate of gasification under the conditions studied of 53 g/Ls is much higher than previously reported.

Aeration effect on Spirulina platensis growth and ã-linolenic acid production

The influence of aeration on algal growth and gamma-linolenic acid (GLA) production in a bubble column photobioreactor was investigated. Studies were performed in a 20-L reactor at different aeration rates (0.2-2.5 vvm). Static, continuous, and periodic operation of air resulted in 41.9%, 88.4%, and 108% air saturation of dissolved oxygen, for which the corresponding values of GLA were 2.3, 6.5, and 7.5 mg·g-1 dry cell weight, respectively. An increase in the aeration rate from 0.2 to 2.5 vvm enhanced both the specific growth rate and GLA content under periodic sparging in the bicarbonate medium. With a 6-fold increase in the aeration rate, the GLA content of the alga increased by 69.64% (5.6-9.5 mg· g-1 dry cell weight). In addition, the total fatty acid (TFA) content in dry biomass increased from 2.22% to 4.41%, whereas the algae maintained a constant GLA to TFA ratio within the aeration rate tested. The dependence of GLA production on the aeration rate was explained by interrelating the GLA production rate with the specific growth rate using the Luedeking and Piret mixed growth model.

Molecular classification of commercial Spirulina strains and identification of their sulfolipid biosynthesis genes.

Cyanobacterial strains of the genus Spirulina have recently been identified as an excellent source of sulfolipids, some of which possess anti-HIV properties. Thus, to investigate the distribution of sufolipid biosynthesis pathways in Spirulina, a genetic screening/phylogentic study was performed. Five different strains of Spirulina [Spirulina (Jiangmen), Spirulina sp., S. platensis, S. maxima, and Spirulina seawater] sourced from different locations were initially classified via 16S rDNA sequencing, and then screened for the presence of the sulfolipid biosynthesis genes sqdB and sqdX via a PCR. To assess the suitability of these strains for human consumption and safe therapeutic use, the strains were also screened for the presence of genes encoding nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs), which are often associated with toxin pathways in cyanobacteria. The results of the 16S rDNA analysis and phylogenetic study indicated that Spirulina sp. is closely related to Halospirulina, whereas the other four Spirulina strains are closely related to Arthrospira. Homologs of sqdB and sqdX were identified in Spirulina (Jiangmen), Spirulina sp., S. platensis, and the Spirulina seawater. None of the Spirulina strains screened in this study tested positive for NRPS or PKS genes, suggesting that these strains do not produce NRP or PK toxins.

First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria.

Anatoxin-a is a potent neurotoxin produced by several species of cyanobacteria. This alkaloid may cause fatal intoxication to exposed organisms and this has raised concerns over the increasing popularity of food supplements containing cyanobacteria. These are being marketed with alleged health properties for animal and human consumption. These supplements most commonly contain the genera Spirulina (Arthrospira) and Aphanizomenon and their consumption represent a potential route for anatoxin-a exposure in cases where adequate quality control is not undertaken. In this work, several dietary supplements containing cyanobacteria from different commercial suppliers were evaluated for the presence of anatoxin-a by high performance liquid chromatography with fluorescence detection. Additionally, the presence of the previously derivatized anatoxin-a was confirmed by using Gas chromatography-mass spectrometry. A total of 39 samples were analysed in the study. Results showed that three of the samples (7.7%) contained anatoxin-a, at concentrations ranging from 2.50 to 33 microg g(-1). Quality control of cyanobacterial food supplements is required to avoid potential health effects in humans and animals.

Cultivation of Arthrospira (spirulina) platensis in desalinator wastewater and salinated synthetic medium: protein content and amino-acid profile

Arthrospira (Spirulina) platensis was cultivated in laboratory under controlled conditions (30ºC, photoperiod of 12 hours light/dark provided by fluorescent lamps at a light intensity of 140 µmol photons.m-2.s-1 and constant bubbling air) in three different culture media: (1) Paoletti medium (control), (2) Paoletti supplemented with 1 g.L-1 NaCl (salinated water) and (3) Paoletti medium prepared with desalinator wastewater. The effects of these treatments on growth, protein content and amino acid profile were measured. Maximum cell concentrations observed in Paoletti medium, Paoletti supplemented with salinated water or with desalinator wastewater were 2.587, 3.545 and 4.954 g.L-1, respectively. Biomass in medium 3 presented the highest protein content (56.17 percent), while biomass in medium 2 presented 48.59 percent protein. All essential amino acids, except lysine and tryptophan, were found in concentrations higher than those requiried by FAO.

Arthrospira (Spirulina) platensis foi cultivada em laboratório sob condições controladas (30ºC, intensidade luminosa de 140 µmol fótons.m-2.s-1, 12 horas claro/escuro e insuflação constante de ar atmosférico), em três meios de cultivo: (1) meio de Paoletti (controle), (2) meio de Paoletti suplementado com 1,0 g.L-1 de NaCl (água salinizada) e (3) meio de Paoletti preparado com rejeito de dessalinizador. Foi verificado o efeito destes tratamentos no crescimento, teor de proteínas e aminoácidos. As concentrações celulares máximas obtidas foram de 2,587; 3,545 e 4,954 g.L-1 no meio controle, meio de Paoletti suplementado com água salinizada ou com rejeito de dessalinizador, respectivamente. Com relação às concentrações protéicas, estas foram maiores na biomassa cultivada no meio 3, com 56,17 por cento, enquanto que a biomassa cultivada no meio 2 apresentou 48,59 por cento. A maioria dos aminoácidos essenciais encontrou-se acima dos limites requeridos pela FAO, com exceção apenas de lisina e triptofano.

Isolation and functional characterization of Spirulina D6D gene promoter: role of a putative GntR transcription factor in transcriptional regulation of D6D gene expression.

Delta6-Desaturase (D6D) is a key enzyme that catalyzes the synthesis of gamma-linolenic acid (GLA), an essential polyunsaturated fatty acid. We report here the isolation and first functional characterization of the D6D gene promoter from Spirulina platensis C1. Functional analysis of this isolated promoter showed that the Spirulina promoter was functional in Escherichia coli. Site-specific mutation studies demonstrated that the -10 sequence (TATAAT), located at -33bp relative to the translation start site, was essential for D6D promoter function. Temperature responsive deletion analysis studies identified the minimal core promoter within the region -51 to +1, which was sufficient for basal D6D promoter activity, and several cold-shock responsive cis-acting elements with activating and repressing activity. Electrophoretic mobility shift assay and LC-MS/MS studies demonstrated that an 'AT-rich Inverted Repeat' (-192 to -164) served as a target-binding site for a transcriptional regulator (probably a member of the GntR family) from Spirulina. Western blot analysis studies revealed that the DNA-binding transcriptional regulator underwent phosphorylation after a temperature downshift and possibly associated with transcriptional regulation of D6D gene expression. Taken together, our results suggest complex regulation of D6D gene expression in Spirulina.