AbstractA comparative study of bioactive compounds in two marine cyanobacteria species, Oscillatoria acutissima and Oscillatoria simplicissima, grown in F/2 media was performed by estimation of many of bioactive compounds after 12th days, The greatest level of Phenolics and tannins was observed in Oscillatoria acutissima. Methanolic extracts of two species of cyanobacteria demonstrated diverse antimicrobial activity toward various human pathogenic microbial species which were associated with their bioactive components. Two species of cyanobacteria were observed to be the most active one in relation to all examined pathogenic microorganisms. The methanolic extracts of the algae were chemically distinguished by gas chromatography mass spectroscopy “GC–MS”. Based on the results obtained, the two species of marine cyanobacteria can be a renewable provider of beneficial bioactive complexes for health and nutritional products.IntroductionCyanobacteria are gram-negative, photosynthetic prokaryotic organisms that have the ability to grow in a wide range of aquatic and terrestrial habitats1. Their structural diversity and capacity to produce a wide range of compounds such as pigments, vitamins, and enzymes make them pioneer species in most of the ecosystems. Additionally, cyanobacteria are known to be sources of a wide range of natural products, some of which are toxins, but have potential applications in the pharmaceutical industry2,3,4. Nostoc genus is a morphologically diverse and large group of phototrophic cyanobacteria that are located in various environments. The presence of both primary and secondary metabolites with varied bioactive properties in plants and cyanobacteria underscores their importance to phytopharmaceutical uses5. Cyanobacteria have antioxidant, anticancer and antiviral properties and find applications in a wide range of industries, such as agriculture, industry, medicine, biotechnology, and pharmaceuticals6. They also exhibit antimicrobial effects capable of inhibiting or destroying pathogenic microorganisms. These antimicrobial agents work by modifying or disrupting cytoplasmic membrane, disabling enzymes, and preventing the synthesis of proteins7. The antimicrobial effect differs according to the algal species and the kind of solvent used8. Moreover, cyanobacteria synthesize natural compounds that enhance their survival under diverse environmental stresses. These natural metabolites have been applied in disease management for decades, and cyanobacteria continue to offer promising sources for developing novel drugs against previously incurable diseases9. Recent progress in biotechnology has focused on enhancing the synthesis of valuable compounds in cyanobacteria for use across diverse industrial sectors10. Cyanobacteria are known to generate a range of bioactive metabolites, such as those with antitumor properties11, toxic compounds12, and enzyme inhibitors13. These metabolites serve multiple biological roles, including defense against predators, chemosensory signaling, and protection from light-induced damage. Because of their adaptive responses to antimicrobial agents, such bioactive substances have potential applications in nutraceuticals, pharmaceuticals, and industrial biotechnology, particularly in the development of cosmeceuticals14. This adaptive capacity underscores the importance of ongoing investigations into novel antimicrobial agents15. The current study focuses on analyzing the phytochemical composition of two cyanobacterial strains gathered from the Gulf of Aqaba along the Red Sea coast of Alexandria, Egypt, with the goal of identifying their chemical constituents for potential industrial and other practical uses.Materials and methodsAlgal IsolationThe study’s algae species were gathered from the Gulf of Aqaba on Alexandria’s Red Sea coast. The F/2 medium was used to culture the samples16. After that, the medium was autoclaved for 30 min at 120 °C. The culture was incubated at pH 8, 30 ± 1 °C, and 3000 lx of light intensity. The ideal conditions were maintained for the algae. Based on the available literature, the isolated strain was recognized morphologically17.The marine cyanobacterial strains used in this study were isolated from environmental water samples. Monocultures of single species were successfully separated and established using the standard capillary micropipette washing technique under an optical microscope, followed by successive streak-plating onto F/2 agar plates to ensure unialgal purity before scale-up.The study’s algae species were gathered from the coastal waters of Alexandria, Egypt. The two marine cyanobacteria species (Oscillatoria acutissima and Oscillatoria simplicissima) were cultured separately in 500 mL glass Erlenmeyer flasks containing a working volume of 300 mL of sterile F/2 medium. The medium was prepared using natural seawater filtered through 0.45 μm membranes and adjusted to a baseline marine salinity of 35‰. The medium was autoclaved for 30 min at 120 °C. Culturing was conducted in a controlled environmental incubator at a temperature of 25 ± 2 °C, under a light intensity of 3000 lx with an operating pH of 8. A strict 16:8 h light: dark diurnal photoperiod cycle was maintained. To ensure continuous gas exchange and prevent biomass sedimentation, the cultures were continuously agitated by bubbling with sterile-filtered air using an automated aeration system. Based on the available literature, the isolated strains were recognized morphologically.Measurements of algal growthAs per the procedure outlined by18. measuring the growth of algae using chlorophyll a. Centrifugation was used for 15 min at 5000 rpm in order to harvest. By comparing the absorbance at 663 and 645 nm in a 1 cm quartz cell to a blank of 80% hydrous acetone using a spectrophotometer, the pigment concentration in the filtered extract was calculated employing the subsequent formula:Chlorophyll a = 12.7. E663-2.69. E645.Preparation of the algal extractsIn F/2 media, three microalgae were cultivated under aeration. In order to extract antimicrobial compounds, microalgae pellets were collected for development during the stationary stage, the culture was subjected to centrifugation, and the pellets were subjected to hot air drying (60 °C) until they reached a consistent weight. Each of the three microalgae’s half-gram dry biomass was extracted using ten millilitres of hexane, chloroform, ethanol, and methanol. According to19, every extract was kept at -4° C.“In F/2 media, the microalgae were cultivated under continuous aeration. In order to extract antimicrobial compounds, microalgae pellets were collected during the stationary stage via centrifugation at 4000 rpm for 15 minutes. The collected algal pellets were washed with distilled water and subjected to hot air drying (60°C) until they reached a constant weight. For extraction, a half-gram of each microalga’s dry biomass was extracted using ten millilitres of absolute analytical-grade methanol under continuous shaking at 150 rpm for 48 hours at room temperature in the dark. The mixture was filtered using Whatman No. 1 filter paper, and the solvent was completely evaporated under reduced pressure using a rotary evaporator at 40°C to yield the crude methanolic extract. According to19, every extract was kept at -4°C.”Phytochemical characteristicsExtraction of secondary metabolitesDried samples, 10 g, were placed within Soxhlet equipment and obtained with 100 ml methanol for 8 h, subsequently the filtrate (crude extracts) was gathered.Formulation of methanolic algae extractsApproximately 1 g of every dried algal biomass sample was individually blended in methanol and subjected to sonication for 20 min employing an ultrasonic micro tip probe (400 W, ULTRASONIC Get 750). The mixtures were subsequently subjected to centrifugation for 10 min at 4500 rpm. The resulting supernatants were gathered independently, while the remaining pellets were subjected to extraction again two times following the same procedure. All collected supernatants were combined and refrigerated for subsequent analysis. Some portions of the supernatants underwent evaporation until dry at 40 °C employing a rotary evaporator, and the resulting dried samples were transferred into marked sterile vials and preserved at − 20 °C in a deep freezer for antimicrobial evaluation20.Qualitative analysisPhenolics, phytosterols, triterpenes, saponins, tannins, flavonoids, anthraquinones, coumarins and cardiac glycosides were detected in the algal methanolic fractions based on standard methods21.Antimicrobial activity assayThe bioactivity of the methanolic crude algal fractions was investigated toward four gram negative bacteria: Vibrio cholerae, Pseudomonas aeruginosa, Escherichia coli, and Aeromonashydrophila, two gram positive bacteria “Enterococcus faecalisand Staphylococcus aureus” and one fungal species “Candida albicans” employing disc diffusion procedure of Kirby-Bauer procedure22. Employing cotton swabs, bacterial cultures containing “150 CFU/ml” were uniformly added to nutrient agar plates. Around 0.75 × 106 fungal spores/ml were aseptically swabbed Czapex-Dox plates. Sterilized discs (6 mm) from Whatman No. 1 filter paper were impregnated with either extract (10 mg/ml) and dried entirely in sterile circumstances subsequently were maintained at 37 °C for 24 h for bacteria and at 28 °C for 48-72 h for fungal forms, correspondingly. DMSO served as a negative control, while the positive control was ciprofloxacin (10 mg/ml). The diameter of clear zone plus the diameter paper disc (mm) was used to calculate antibacterial activity. The average of triplicate analyses is used for all estimated antibacterial outcomes.GC-ISO Mass analysis of methanolic algal extractA GC-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) was employed to analyze bioactive complexes present in various methanolic preparations. The analysis was carried out applying a TG–5MS capillary column (30 m × 0.25 mm × 0.25 μm film thickness). The temperature of the oven was initially set at 55 °C, subsequently elevated at a pace of 5 °C/min to 250 °C, sustained for 2 min, and finally rose to 300 °C at 25 °C/min. The injector temperature was set at 270 °C. The carrier gas was helium at a steady flow rate of 1 mL/min. A solvent delay of 4 min was applied, and 1 µL of each diluted sample was automatically introduced in split mode employing the AS3000 Autosampler integrated with GC. In full scan mode, electron ionization (EI) mass spectra were obtained at 70 eV across the m/z interval of 50–650. Compound identification was executed by contrasting retention intervals and mass spectra with entries in the NIST14 and WILEY 09 mass spectral libraries23.Statistical analysisAll experimental treatments and chemical assays were conducted in independent triplicates ($n = 3$). Data are presented as mean ± standard deviation (SD). The findings for diverse biochemical variables were interpreted by one-way ANOVA followed by Duncan’s multiple range test to compare differences between means ($P