Biotehnološki potencijal mikroalgi izoliranih iz rijeke Gacke i Jadranskoga mora- karakterizacija i optimizacija uzgojnih uvjeta

doctoral thesis

• Title (english): Biotechnological potential of microalgae isolated from river Gacka and Adriatic Sea- characterization and optimization of growth conditions
• Author: Marina Grubišić
• Mentor: Mirela Ivančić Šantek (mentor)
• Committee member: Blaženka Kos (predsjednik povjerenstva)
• Committee member: Ivana Radojčić Redovniković (član povjerenstva)
• Committee member: Marin Roje (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2022-09-23, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 62 - Engineering. Technology in general

Abstract

U ovom radu je izolirano i identificirano pet mikroalgi (Nitzschia sp. S5, Nanofrustulum shiloi D1, Tetraselsmis sp. C6, Tetraselmis sp. Z3, Picochlorum sp. D3) i jedna cijanobakterija (Euhalothece sp. C1) iz Jadranskog mora, te tri mikroalge iz rijeke Gacke (Chlorella vulgaris S2, Clorococcum sp. S1 i Tetradesmus obliquus S3). Provedena je karakterizacija izoliranih vrsta s obzirom na kinetiku rasta, makromolekulski sastav, antimikrobno i antioksidacijsko djelovanje. Mikroalga Picochlorum sp. D3 istaknula se najvećom brzinom rasta, dok je najveći prirast biomase zabilježen kod mikroalge Tetraselmis sp. Z3 (0.48± 0.11 g Lˉ¹) i Tetraselmis sp. C6 (0.45 ± 0.04 g Lˉ¹). Proteini su bili najzastupljenija makromolekula u svim mikroalgama (32.83–57.94%, g gˉ¹). Lipidi morskih mikroalgi bili su bogati na višestruko nezasićenim masnim kiselinama (eikosapentaenoična, dokosaheksaenoična, alfa- linolenska), dok su karotenoidi bili najzastupljeniji pigmenti. Metanolni ekstrakti svih istraženih mikroalgi pokazali su dobru antioksidativnu aktivnost, te su snažno inhibirali rast bakterija Escherichia coli, Salmonella typhimurium i Staphylococcus aureus. Provedena je optimizacija sastava hranjive podloge i uvjeta uzgoja odabranih mikroalgi. Šaržnim uzgojem s pritokom supstrata Nitzschia sp. S5 u hranjivoj podlozi optimiranog sastava [Si]: [N-NO3]:[PO43-] =6.67:23.33:1 [mM:mM:mM] postignuta je šest puta veća koncentracija biomase u odnosu na šaržni uzgoj u izvornoj f/2 podlozi. Šaržnim uzgojem s pritokom supstrata u barbotirajućoj koloni postignuta je najveća koncentracija biomase Picochlorum sp. D3 od 1.68 g Lˉ¹. Prihranom kulture natrijevim bikarbonatom potaknuto je nakupljanje lipida u biomasi čiji je udjel udvostručen. Učinkovita samoflokulacija Picochlorum sp. D3 (>98 %) postignuta je pri pH>11, te flokulacija (>97%) primjenom 1100 mg Lˉ¹ željezovog klorida pri pH=9 i 25 mg Lˉ¹ hitozana pri pH=8. Uspješno je provedena i bioflokulacija s plijesni Mortierella isabelina pri optimalnim uvjetima (mikroalga: plijesan=1:50 g g-1, pH=2 i T=30°C) s učinkovitošću od 88.47%. Najveća učinkovitost adsorpcije stanica mikroalge C. vulgaris S2 na hife plijesni postignuta je s Aspergillus niger. Pogodan izvor ugljika za uzgoj slatkovodnih mikroalgi u miksotrofnim i heterotrofnim uvjetima bila je glukoza, galaktoza, melasa i octena kiselina. Uzgoj mikroalge C. vulgaris S2 uz veće molarne omjere izvora ugljika i dušika (C:N=10, 30, 50, 75 mol molˉ¹) potaknuli su nakupljanje lipida tijekom šaržnog uzgoja i uzgoja s pritokom supstrata. Heterotrofni uvjeti uzgoja pokazali su se pogodnijim za rast biomase slatkovodnih mikroalgi u odnosu na miksotrofne uvjete. Dvostupanjskim šaržnim uzgojem C. vulgaris S2 postignuta je visoka koncentracija biomase od 19.31 g Lˉ¹ i produktivnost procesa od 0.075 g Lˉ¹.

Abstract (english)

Five microalgae (Nitzschia sp. S5, Nanofrustulum shiloi D1, Tetraselsmis sp. C6, Tetraselmis sp. Z3, Picochlorum sp. D3) and one cyanobacterium (Euhalothece sp. C1) from the Adriatic Sea as well as three microalgae from the Gacka River (Chlorella vulgaris S2, Clorococcum sp. S1 and Tetradesmus obliquus S3) were isolated and identified. Microalgae were characterized regarding growth kinetics, macromolecular composition, and antimicrobial and antioxidant activity. Microalga Picochlorum sp. D3 showed the highest growth rate, while the highest biomass concentrations were achieved with microalgae Tetraselmis sp. Z3 (0.48± 0.11 g Lˉ¹) and Tetraselmis sp. C6 (0.45± 0.04 g Lˉ¹). Proteins were the most abundant macromolecule in all characterized microalgae (32.83–57.94%, g gˉ¹). Marine microalgae lipids were rich in polyunsaturated fatty acids (eicosapentaenoic, docosahexaenoic, alpha-linolenic), while carotenoids were the most abundant pigments. Methanolic extracts of all investigated microalgae showed good antioxidant activity and strongly inhibited the growth of Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The nutrient concentration in the growth medium and the growth conditions were optimized for selected microalgae. In fed-batch cultivation of Nitzschia sp. S5 in the optimized growth medium with a nutrient molar ratio of [Si]: [N-NO3]:[PO43-] of 6.67:23.33:1 [mM:mM:mM] biomass concentration was six times higher compared to batch cultivation using an original f/2 medium. The highest biomass concentration of Picochlorum sp D3 was achieved in fed-batch cultivation in a bubble column (1.68 g Lˉ¹). Supplementing the culture with sodium bicarbonate stimulated lipid accumulation resulting in doubled lipid content. Effective self-flocculation (>98 %) of Picochlorum sp. D3 was achieved at pH>11, and flocculation (>97%) using 1100 mg L-1 ferric chloride at pH=9 and 25 mg Lˉ¹chitosan at pH=8. Bioflocculation with the fungi Mortierella isabelina was successfully performed under optimized conditions (microalgae: fungus=1:50 g gˉ¹, pH=2 and T=30°C) with an efficiency of 88.47%. The highest adsorption efficiency of microalgae C. vulgaris S2 cells was achieved with Aspergillus niger. Suitable carbon sources for cultivating freshwater microalgae under mixotrophic and heterotrophic conditions weres glucose, galactose, molasses, and acetic acid. Cultivation of the microalgae C. vulgaris S2 with higher molar ratios of carbon and nitrogen (C:N=10, 30, 50, 75 mol molˉ¹) promoted the lipid accumulation during batch and fed-batch cultivation. Heterotrophic cultivation conditions were more suitable for the biomass growth of freshwater microalgae than mixotrophic conditions. The two-stage batch cultivation of C. vulgaris S2 resulted in a high biomass concentration of 19.31 g Lˉ¹ and productivity of 0.075 g Lˉ¹.

• Keywords: mikroalge; morske mikroalge; Jadransko more; biotehnološki potencijal; fotobioreaktor; optimizacija uzgoja; barbotirajuća kolona; dijatomeje; flokulacija; bioflokulacija
• Keywords (english): microalgae; marine microalgae; Adriatic Sea; biotechnological potential; photobioreactor; cultivation optimization; bubble column; diatoms; flocculation; biofloculation
• Language: croatian
• URN:NBN: urn:nbn:hr:159:627687
• Promotion: 2022
• Study programme: Title: Postgraduate PhD Study in Biotechnology and Bioprocess Engineering; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti (doktor/doktorica znanosti)
• Type of resource: Text
• Extent: 303 str ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-11-08 09:42:48