Karakterizacija zemljišnih cijanobakterijskih sojeva izolovanih iz šumskih ekosistema planinskih područja Republike Srbije / Characterization of soil cyanobacterial strains isolated from forest ecosystems of mountain areas of the Republic of Serbia

As one of the oldest groups of photoautotrophic microorganisms, cyanobacteria represent widespread prokaryotes with diverse metabolic strategies in order to survive and adapt to different environmental conditions. For this reason, cyanobacteria are significant as producers of various biologically active metabolites. However, most of the studies are mainly related to cyanobacteria of aquatic ecosystems. The subject of the research of this dissertation is to determine the diversity of autochthonous cyanobacteria of forest ecosystems and to characterize selected terrestrial cyanobacterial representatives of mountainous areas of Serbia isolated during three year monitoring by determining their basic ecophysiological, biochemical and genetic characteristics. The results obtained in this dissertation have show the diversity of soil cyanobacteria of forest habitats of the investigated mountain areas as well as their metabolic diversity and potential to produce various bioactive compounds. Based on taxonomically important features, 20 cyanobacterial strains have been identified to belong to the following genera: Nostoc, Anabaena, Tolypothrix, Calothrix, Cylindrospermum, Lyngbya, Oscillatoria, Phormidium. Identification of the isolated cyanobacteria using the molecular marker 16S rRNA in most cases (90%) confirmed the preliminary identification of genera based on morphological criteria. In terms of biomass production, the obtained results showed that the production of biomass in the selected tested cyanobacterial strains depended on the applied cultivation conditions. It was found that nitrogen, glucose and sucrose acted towards the stimulation of biomass production in a large number of strains. The largest biomass production was detected in strain Calothrix M2 in the presence of nitrogen in the medium. In the presence of glucose and sucrose in the medium the highest increase in biomass production was recorded in cyanobacterial strain Nostoc T18. Also, the content of phycobiliproteins has been increased in most of the tested strains in the presence of glucose and sucrose in the medium. Examination of the carbohydrate content (glucose, fructose and xylose) showed that all three monosaccharides were present in all strains and that each strain had a specific carbohydrate profile whereby the content of monosaccharides in all tested strains declined in the following order: glucose ˃ fructose ˃ xylose. Among the tested strains, three strains namely Nostoc M1, Phormidium T11 and Calothrix M2 showed the exceptional ability to produce hexose and pentose. The antibacterial activity of intracellular extracts was recorded in 16 tested cyanobacterial strains and depended on the combination of cyanobacterial-bacterial strain and the type of applied extract. Compared to hexane extracts, methanol extracts showed greater efficiency, indicating on the nature of bioactive compounds with antibacterial activity. The most effective were 75% MeOH extracts of cyanobacterial strains Oscillatoria M2, Calothrix M2, Lyngbya T7 and Cylindrospermum K1 which exhibited antibacterial activity on 4 tested bacteria. Results of the analysis of the fatty acid composition showed that the fatty acid content of tested cyanobacterial strains varied depending on the strain. The most significant constituents of the tested cyanobacterial strains were 18 and 16 carbonic fatty acids such as linoleic acid, α-linoleic. The highest content of linoleic acid was detected in two strains, Phormidium T11 and Tolypothrix K11, indicating that these strains can be potentially significant sources of essential fatty acids. Results of antioxidant tests showed that all tested strains had antiradical activity. In the case of DPPH assay, ethanolic extracts of Calothrix M2 exhibited the most effective ability to scavenge DPPH ·radical while in the case of the FRAP method, ethanolic extract of Cylindrospermum K1 had the greatest reduction power. According to data obtained from chemical analysis of the phenolic composition of the analyzed cyanobacterial strains, a total of 21 phenol compounds were identified and quantified. The phenolic composition varied depending on the strain, and the most frequently detected phenolic compounds were luteolin-7-O-glucoside, baicalin and kaempferol. The strain with the most significant ability to produce phenolic compounds was Phormidium M1, in which the presence of 11 phenolic compounds was identified. The results of the toxicity of intracellular extracts obtained in A. salina, D. magna and D. rerio biotests, showed that a total of 40% of the tested strains exhibited a toxic effect. The most toxic strain was Nostoc T7 due to the fact that it showed toxicity in all three tests. In the case of A. salina biotest, the most potent strains were Nostoc T7, Oscillatoria M2, Oscillatoria T18 and Nostoc K15. Cyanobacterial strains that exhibited the most prominent activity in D. magna biotest were Tolypothrix K15, Nostoc T7 and Calothrix M2. In biotest with zebrafish embryos, the strain with the most significant teratogenic effect was Cylindrospermum K1. Regarding the effect of cyanobacterial extract on gene expression in model organism D. rerio, cyanobacterial strain Cylindrospermum K1 exhibited the ability to modulate biological processes such as circadian rhythm as well as the ability to produce compounds with an estrogenic effect. The results of the toxicogenetic analysis of the tested cyanobacterial strains have shown that the genes mcyB and mcyE involved in the production of cyanotoxin microcystins have not been detected in any of the tested strains. The absence of two genes from the mcy gene cluster indicates that other compounds are responsible for the observed toxic effect in applied biotests. The obtained results point out on the importance of the study of soil cyanobacteria, since the obtained results have indicated that the tested strains possess a large metabolic diversity and potential to produce various bioactive compounds.