Potencijal sekundarnih metabolita cijanobakterija kao biomarkera u paleoklimatskoj rekonstrukciji

Abstract

U doktorskoj disertaciji je rađena analiza produkcije i procena stabilnosti sekundarnih metabolita cijanobakterija. U analizi pigmenata cijanobakterija kao biomarkera u paleoklimatskoj rekonstrukciji poslužio je nov model analize-AMMI model, koji pruža značajan doprinos odabiru odgovarajućih biomarkera u paleoklimatiskoj rekonstrukciji. Analizom produkcije MOSA i MOMA 15 vodenih i zemljišnih cijanobakterija NSCCC sposobnost produkcije MOSA je uočena kod 8, a MOMA kod svih sojeva. Kultivacija u različitim pH vrednostima podloge i različitim temperaturnim uslovima nije pokazala znatan uticaj na produkciju MOSA. Produkcija MOMA je bila izraženija u baznoj sredini (pH 9.0) i na višim temperaturama (30-35 °C). UV svetlost se pokazala kao najznačajniji faktor i inicirala je najveću produkciju MOSA i MOMA kod svih sojeva. Veća koncentracija azota u podlozi nije uticala na povećanje produkcije MOSA kod većine analiziranih sojeva, dok je znatno uticala na povećanje produkcije MOMA kod svih analiziranih sojeva. Analizom produkcije pigmenata 19 lesnih cijanobakterija NSCCC prisustvo fikobilina i MOSA je uočeno u svim analiziranim kulturama, dok sposobnost produkcije MOMA nije uočena u dve kulture lesnih cijanobakterija NSCCC. Tokom posmatranog vremenskog perioda uočena je razgradnja ukupnih fikobilina u kontrolnim uslovima rasta, dok za isti vremenski period nije došlo do degradacije MOSA i MOMA u kontrolnim uslovima. Procenom stabilnosti MOSA i MOMA delovanjem abiotičkih faktora (različitih pH i temperaturnih vrednosti podloge, različitog vremena izlaganja UV svetlosti) utvrđeno je da su MOSA i MOMA pokazali izraženu stabilnost na testirane abiotičke faktore. Procenom stabilnosti pigmenata nakon delovanja biotičkih faktora primetna je intenzivnija razgradnja ukupnih fikobilina posmatrano u zavisnosti od vremena, do postizanja potpune degradacije u pojednim kulturama lesnih cijanobakterija, dok je uočeno da su MOSA i MOMA pokazali stabilnu strukturu i u testu biodegradabilnosti nije došlo do njihove degradacije. Analizom prisustva MOSA i MOMA u lesnom sedimentu i biološkim lesnim pokoricama njihovo prisustvo je uočeno u svim analiziranim uzorcima. Takođe, korišćenjem LC-MS(/MS) metode utvrđeno je prisustvo scitonemina u 10 terestričnih kultura NSCCC. Analizom toksičnosti i produkcije mikrocistina, cilindrospermopsina i saksitoksina lesnih cijanobakterija NSCCC dobijeni su negativni rezultati. Razvojem novih metoda za detekciju cijanotoksina u terestričnim ekosistemima potrebno je proveriti dobijene rezultate. Procenom stabilnosti mikrocistina referentnog soja Microcystis aeruginosa PCC 7806, uočena je njegova izrazita stabilnost tokom vremenskog perioda od 96 h u kontrolnim uslovima i delovanjem tri bakterijska soja. Shodno dobijenim rezultatima, UV zaštitni pigmenti su mnogo podesniji za paleoklimatsku rekonstrukciju od fikobilina s obzirom da molekuli MOSA i MOMA imaju postojaniju strukturu i da nisu degradirani tokom posmatranih stresnih uslova. Usled nemogućnosti detekcije cijanotoksina lesnih cijanobakterija, i pored izražene stabilnosti mikrocistina referentnog soja, cijanotoksini se ne mogu smatrati biomarkerima cijanobakterija u geološkim istraživanjima. Postavljanje BLOCDUST teorije i otkriće stabilnosti MOSA i MOMA i njihove upotrebe kao pouzdanih biomarkera u paleoklimatskoj rekonstrukciji predstavlja osnovu za mnoga buduća istraživanja od neprocenjivog naučnog značaja, pogotovo u paleoklimatskoj rekonstrukciji lesa. Predloženi scenario se može smatrati osnovom u paleoklimatskoj rekonstrukciji.

This PhD thesis analyzed the production and stability of cyanobacterial secondary metabolites. The results describe the effects of pH, temperature and light source combined with the effects of medium nitrogen content on the production of the MOSA and MOMA compounds of aquatic and soil cyanobacterial strains through AMMI model. The application of the AMMI model represents a significant contribution to the selection of appropriate biomarkers in the paleoclimatic reconstructions because it reveals the increased production of certain secondary metabolites in certain environmental conditions. MOSA compounds were observed in 8 out of 15 soil and aquatic cyanobacterial strains, while MOMA compounds were found in all 15 strains. Results show that exposure to UV light induced a higher synthesis of both pigments. The production of the MOSA compounds was clearly increased by UV irradiation and other treatments did not show a significant impact on its production. The production of MOMA compounds was increased by several stress factors including pH (pH 9.0), temperature (30-35 °C), nitrogen content and UV irradiation. A higher concentration of nitrogen in the medium did not influence the increase in the production of MOSA compounds in most of the analyzed strains, while it significantly influenced the increase in the production of MOMA in all analyzed strains. By analyzing the production of pigments in 19 loess cultures, phycobilins and MOSA were present in all examined loess isolates, while presence of MOMA was not detected in two samples from China. In control conditions, it was observed degradation of phycobilins depending on time, but MOSA and MOMA showed a stable structure. Stability assessment of the MOSA and MOMA by the treatment with abiotic factors (different pH and temperature values of the medium, different time of exposure to UV light) revealed their pronounced stability on tested abiotic factors. Considering unstable structure of phycobilins in the presence of bacterial strains, phycobilins cannot be considered as biomarkers in loess studies. Detected results indicate that MOSA and MOMA have stable core structures resistant to bacterial strains, which makes them potentially good biomarkers for paleoclimatic reconstruction. Moreover, the presence of MOSA and MOMA compounds was confirmed in loess sediment samples and BLC. Also, the LC-MS(/MS) method revealed the presence of scytonemin in 10 terrestrial cyanobacterial cultures. Testing of the toxicity of loess cyanobacterial cultures and their ability to produce microcystins, cylindrospermopsin and saxitoxins, negative results were obtained. The development of new methods for detection of cyanotoxins in terrestrial ecosystems is necessary in order to revise obtained results. By assessing the stability of microcystins of the reference strain of Microcystis aeruginosa PCC 7806, its pronounced stability was observed over a 96 h in control conditions and in the presence of three bacterial strains. Due to MOSA and MOMA narrow environment and organism specificity, as well as its structural stability, these metabolites are designated with a strong potential to be used as a cyanobacterial biomarker in paleoclimatic research. Due to the inability to detect cyanotoxins in loess cyanobacteria, despite the pronounced stability of the microcystin of the reference strain, cyanotoxins cannot be considered as adequate biomarkers of cyanobacteria in geological research. The production of the MOSA and MOMA compounds across examined stress conditions, and further, their presence in loess samples and BLCs indicate the potential of these compounds to be regarded as biomarkers in paleoclimatic research of lacustrine/marine and loess sediments. Setting up the BLOCDUST theory and determining stability of MOSA and MOMA compounds and their aplication as a convinient biomarkers of cyanobacteria in paleoclimatic reconstruction provides the foundation for many future research of invaluable scientific significance, especially in the paleoclimatic reconstruction of loess. The proposed scenario can be considered as one of the basic model for paleoclimatic reconstruction.