Biotechnological potential of filamentous cyanobacterial strains of the area of Vojvodina

Abstract

<p>S obzirom&nbsp; da su cijanobakterije (modrozelene alge) identifikovane kao jedna od<br />najperspektivnijih grupa organizama za izolaciju novih i biolo&scaron;ki aktivnih prirodnih<br />produkata, cilj ove teze bio&nbsp; je utvrđivanje biotehnolo&scaron;kog potencijala&nbsp; autohtonih<br />filamentoznih sojeva&nbsp; cijanobakterija izolovanih sa područja Vojvodine koji pripadaju<br />azotofiksirajućim rodovima&nbsp;<em> Nostoc</em>&nbsp; i&nbsp; <em>Anabaena</em>&nbsp; i neazotofiksirajućem rodu&nbsp;&nbsp;&nbsp; <em>Spirulina</em>. Biotehnolo&scaron;ki potencijal testiranih sojeva je određen&nbsp; u smislu produkcije biomase, fikobiliproteinskih pigmenata, masnih kiselina, fenolnih jedinjenja, antioksidanata, antibakterijskih i antikancerogenih agenasa. Dobijeni rezultati su pokazali&nbsp; da su produkcija biomase i sadržaj fikobiliproteinskih pigmenata kod svih testiranih sojeva zavisili od primenjenih uslova kultivacije, pri&nbsp; čemu je&nbsp; kod sojeva roda&nbsp; <em>Spirulina</em> produkcija biomase bila jače stimulisana primenom kontinualnog osvetljenja, a kod azotofiksirajućih sojeva rodova&nbsp;<em> Nostoc&nbsp;</em> i&nbsp;<em> Anabaena</em>&nbsp; organskim izvorima ugljenika (glicerolom i glukozom). Kao soj sa najvećim potencijalom za&nbsp; proizvodnju biomase izdvaja se soj&nbsp; <em>Spirulina&nbsp; S1</em>, a za produkciju fikobiliproteina sojevi&nbsp; <em>Spirulina</em>&nbsp; S1,&nbsp;<em> Nostoc</em> 2S1,&nbsp; <em>Anabaena</em>&nbsp; Č2 i&nbsp; <em>Spirulina</em>&nbsp; S2.&nbsp; Određivanjem sadržaja masnih kiselina GC-FID metodom utvrđeno je da su&nbsp; kod svih sojeva&nbsp; najzastupljenije bile palmitinska, palmitoleinska, oleinska i linolna kiselina, pri čemu su&nbsp; sojevi roda <em>Spirulina</em> produkovali i&nbsp; &gamma;-linolensku kiselinu, dok su svi&nbsp; sojevi rodova&nbsp; <em>Nostoc&nbsp;</em> i&nbsp;<em> Anabaena&nbsp;</em> produkovali&nbsp; -linoleinsku kiselinu.&nbsp; Najzastupljenije fenolne komponente testiranih etanolnih ekstrakata određene HPLC-MS/MS metodom&nbsp; bile su hinska kiselina i katehin, pri čemu je najveći sadržaj fenolnih jedinjenja registrovan kod soja&nbsp; <em>Nostoc</em>&nbsp; 2S7B. Hemijskom karakterizacijom ekstrakata kod testiranih sojeva takođe je utvrđen značaj azotnih uslova kultivacije&nbsp; u cilju povećanja produkcije&nbsp; fenolnih jedinjenja,&nbsp; kao i&nbsp; -linoleinske kiseline. Poređenjem rezultata antioksidantne aktivnosti u kori&scaron;ćenim testovima&nbsp; DPPH&nbsp; i FRAP, kao sojevi sa najvećim&nbsp; antioksidantnim potencijalom izdvajaju se&nbsp; <em>Spirulina</em>&nbsp; S1 i<em> Spirulina</em>&nbsp; S2. Antibakterijska aktivnost metanolnih ekstrakata&nbsp; registrovana je kod sojeva<em> Nostoc</em> 2S7B, <em>Nostoc</em> 2S1, <em>Anabaena</em> Č2, <em>Anabaena</em> Č5, <em>Spirulina</em> S1 i&nbsp;<em> Spirulina</em> S2, koji su ispoljili efekat na Gram-pozitivne i Gram-negativne bakterije, pri čemu su&nbsp; sojevi <em>Anabaena</em>&nbsp; Č2,<em> Nostoc&nbsp;</em> 2S7B&nbsp; i&nbsp; <em>Nostoc</em>&nbsp; 2S1&nbsp; delovali na najvi&scaron;e bakterijskih sojeva.&nbsp; Kod svih testiranih sojeva je primenom MTT testa uočena antikancerogena tj. citotoksična aktivnost dimetil sulfoksidnih (DMSO) ekstrakata prema HepG2 ćelijskoj liniji, među kojima su najveću aktivnost&nbsp; ispoljili sojevi&nbsp;<em> Nostoc</em>&nbsp; LC1B i&nbsp; <em>Nostoc&nbsp; </em>2S7B.&nbsp; Primenom bioeseja&nbsp; <em>Artemia salina, Daphnia magna</em>&nbsp; i&nbsp;&nbsp;<em> Danio rerio&nbsp; </em>konstatovan je mali broj sojeva koji su ispoljili toksičnost na test organizme, dok na ćelijsku liniju&nbsp; RTL-W1&nbsp; testirani sojevi nisu ispoljili citotoksičnost&nbsp;<em> in vitro</em>, &scaron;to sa aspekta potencijalne biotehnolo&scaron;ke primene sojeva ima veliki značaj. Kao najtoksičniji izdvojili su se sojevi<em>&nbsp; Nostoc&nbsp; </em>LC1B&nbsp; i <em>Nostoc </em>S8 koji su ispoljili&nbsp; toksičnost u sva tri bioeseja. Ispitivanjem toksičnosti<em> in vitro</em> u enzimskim esejima konstatovano je da je manji broj sojeva inhibirao aktivnost enzimaprotein fosfataze 1 (PP1) u odnosu na aktivnost enzima&nbsp; acetilholinesteraze&nbsp; (AChE). Primenom Analitičkog hijerarhijskog procesa u grupnom kontekstu, najveću težinu su dobili kriterijumi antikancerogena ativnost, produkcija biomase i sadržaj fikocijanina, navedenim redom. Konačno, u vi&scaron;ekriterijumskom kontekstu najbolje rangiran soj je<br /><em>Spirulina</em> S1, na drugom mestu je soj <em>Spirulina</em> S2, dok je na trećem soj&nbsp;<em> Nostoc&nbsp; </em>LC1B.</p>

<p>Cyanobacteria (blue-green algae) have been identified as one&nbsp; of the most promising groups of organisms for the isolation of new and biologically active natural products, therefore, the aim of this thesis was to determine the biotechnological potential of autochthonous filamentous cyanobacterial strains isolated from&nbsp; Vojvodina region,&nbsp; which belong to the N₂-fixing genera&nbsp; <em>Nostoc</em>&nbsp; and&nbsp; Anabaena&nbsp; and non-N₂-fixing genus&nbsp; <em>Spirulina</em>. Biotechnological potential of tested strains was determined using the production of biomass, phycobiliprotein pigments, fatty acids, phenolic co mpounds, antioxidants, antibacterial and anticancer agents. The obtained results showed that the production of biomass and phycobiliprotein pigments, in all tested strains, depended on the cultivationconditions, whereas biomass production was strongly stimulated by continuous light in<em> Spirulina</em>&nbsp; strains, and by organic carbon sources (glycerol and glucose) in N₂-fixingstrains. The highest potential for biomass production was shown in&nbsp;<em> Spirulina</em>&nbsp; S1 strain.On the other hand, the highest potential&nbsp; for the production of phycobiliproteins wasshown in strains&nbsp; <em>Spirulina</em>&nbsp; S1,&nbsp; <em>Nostoc&nbsp;</em> 2S1,&nbsp; <em>Anabaena</em> C2 and&nbsp; <em>Spirulina</em>&nbsp; S2. By determination of the content of fatty acids using GC-FID method it was found that in allthe tested strains the most common fatty acids were palmit ic, palmitoleic, oleic andlinoleic acid, whereby the strains of the genus&nbsp; <em>Spirulina</em> produced &gamma;-linolenic acid as well,while all strains of the<em> Nostoc&nbsp;</em> and <em>Anabaena&nbsp;</em> genera produced y-linolenic acid. The most frequent phenolic compounds of tested strains determined by using the HPLC-MS/MSmethod were quinic acid and catechin, with the highest content of phenolic compounds registered in Nostoc&nbsp; strain 2S7B. By chemical characterization of the extracts in the tested strains it was also stated a significance of&nbsp; the nitrogen cultivation conditions in order toincrease the production of phenolic compounds, as well as&nbsp; y-linolenic acid. Comparing the results of the antioxidant activity in the DPPH and FRAP tests, it&nbsp; was shown that strains&nbsp; <em>Spirulina</em>&nbsp; S1 and&nbsp; <em>Spirulina&nbsp;</em> S2 had the highest antioxidant potential. The antibacterial activity of the intracellular methanolic extracts was registered in strains <em>Nostoc&nbsp;</em> 2S7B,&nbsp; <em>Nostoc&nbsp;</em> 2S1,&nbsp; <em>Anabaena</em>&nbsp; C2,&nbsp; <em>Anabaena</em>&nbsp; C5,&nbsp; Spirulina&nbsp; S1 and&nbsp; Spirulina&nbsp; S2, that&nbsp; inhibited the growth of&nbsp; Gram-positive and Gram&nbsp; -negative bacteria. Using MTT test, anti-cancer ie. cytotoxic activity of&nbsp; dimethyl sulfoxide&nbsp; (DMSO) extracts to the HepG2 cell line was detected in all tested strains, however, the highest activity was exhibited in strains <em>Nostoc</em>&nbsp; LC1B and<em> Nostoc</em> 2S7B . In bioassays <em>Artemia salina,&nbsp; Daphnia magna</em>&nbsp; and <em>Danio rerio</em>&nbsp; a small number of strains exhibited toxicity to the test organisms, while in case of cell line RTL-W1 tested strains did not show&nbsp; in vitro&nbsp; cytotoxicity, which is of great importance from the aspect of the potential biotechnological application of thestrains.&nbsp;<em> Nostoc</em>&nbsp; LC1B and&nbsp; <em>Nostoc</em>&nbsp; S8 strains induced toxicity in all three bioassays, and therefore considered as the most toxic strains. By testing&nbsp; in vitro&nbsp; toxicity in enzyme assays, it was found that few strains inhibited the activity of&nbsp; protein phosphatase (PP1) enzyme in relation to&nbsp; acetylcholinesterase&nbsp; enzyme&nbsp; (AChE) activity. Using the Analytical hierarchical process in the group context, the highest weight was given to the criteria of anticancer&nbsp; activity, biomass production, and the phycocyanin content, respectively. Finally, in the multi-criteria context, the best-ranked strain is&nbsp;<em> Spirulina</em>&nbsp; S1,&nbsp; <em>Spirulina&nbsp;</em> strain S2 is on the second place, while <em>Nostoc</em> strain LC1B is the third one.</p>