Javna obrana doktorskog rada studentice doktorskog studija BIOFIZIKA IVE STOJAN,

SAŽETAK:

U ovom su radu tijekom jednogodišnjeg istraživanja određeni sastav i dinamika mikrobne zajednice na prostorno-sezonalnoj skali područja srednjeg Jadrana s naglaskom na aerobne anoksigene fototrofe (AAF). Za određivanje strukture ukupne mikrobne zajednice na finijim taksonomskim razinama korištena je kombinacija metabarkodiranja 16S rRNA gena te kvantitativnog CARD-FISH pristupa. Metabarkodiranjem pufM gena i IC-FISH metodom određen je sastav zajednice AAF. Kako bi se otkrili obrasci unutar zajednica i protumačili u odnosu na najznačajnije okolišne čimbenike, provedeni su algoritmi umjetnih neuronskih mreža. Pripadnici klada SAR11 prevladavali su u raznolikim okolišnim uvjetima. Uočen je opći obrazac povećanja ukupne mikrobne raznolikosti na račun brojnosti u oligotrofnim uvjetima, posebice povećanjem saliniteta. Primijećena je značajna sezonalnost u prosječnim apsolutnim AAF brojnostima (maksimumi u proljeće: 2,136 ± 0,081 x 10⁴, minimumi ljeti: 0,86 x 10⁴ stanica mL^-1), IC-FISH grupama (klad Roseobacter prevalentan u jesen, razredi Alpha- i Gammaproteobacteria ljeti) i pufM podatcima (razlike u mjerama raznolikosti). Nisu uočene razlike u prostornom ni vertikalnom profilu. Zajednica je bila heterogenog sastava s najvećim prosječnim relativnim udjelom klada Roseobacter (37,66%), a slijedili su pripadnici Gammaproteobacteria (35,25%) i općih Alphaproteobacteria (31,15%). Otkrivena je prevalencija roda Luminiphilus na svim postajama, mjesecima i dubinama. Obrnuti odnos AAF brojnosti i mjera raznolikosti primijećen je ljeti, ali nije uočen za ostala godišnja doba. Visoka raznolikost zajednice bila je neovisna o temperaturi, salinitetu i trofičkom statusu. Dodatno je izvršena detaljna usporedba pet metoda izolacije DNA i izravnog PCR-a na uzorcima „mock“ i morske zajednice kao temeljnog koraka u metabarkodiranju. Nijedna metoda nije bila najbliža teoretski očekivanom sastavu „mock“ zajednice. Dokazano je da se standardni protokol temeljen na toksičnom fenolu može zamijeniti kraćim i sigurnijim alternativama temeljenima na taloženju izopropanolom i etanolom, dok su rezultati izravnog PCR-a pokazali prednosti smanjenog vremena rukovanja uzorcima.

 

Title:  MICROBIAL COMMUNITY ECOLOGY WITH AN EMPHASIS ON AEROBIC ANOXYGENIC PHOTOTROPHS IN THE CENTRAL ADRIATIC SEA

 

ABSTRACT:

In this thesis, composition and dynamics of the microbial community of the central Adriatic were determined during a one year study on a spatio-temporal scale, with an emphasis on aerobic anoxygenic phototrophs (AAPs). A combination of 16S rRNA metabarcoding and quantitative CARD-FISH approach was used to determine the structure of the total microbial community at finer taxonomic scale. The composition of the AAP community was estimated by pufM gene metabarcoding and IR-FISH method. In order to detect and interpret patterns within microbial communities in relation to the most significant environmental factors, artificial neural network algorithms were implemented. Members of SAR11 clade prevailed in diverse environmental conditions. A general pattern was recorded of total microbial diversity increase at the expense of abundance in oligotrophic conditions, especially with increased salinity. A significant seasonality was detected in average absolute AAP counts (maxima in spring: 2.136 ± 0.081 x 10⁴ cells mL^-1, minima in summer: 0.86 x 10⁴ cells mL^-1), IR-FISH groups (Roseobacter clade predominant in autumn, classes Alpha- and Gammaproteobacteria in summer) and pufM dataset (differences in diversity measures). No differences were observed in the spatial nor vertical profiles. The community had a heterogeneous composition with the highest average relative proportion of the Roseobacter clade (37.66%), followed by members of Gammaproteobacteria (35.25%) and Alphaproteobacteria (31.15%). The prevalence of genus Luminiphilus was revealed at all stations, months and depths. An inverse relationship between AAP abundance and diversity was observed in summer, but not for other seasons. High AAP diversity was independent of temperature, salinity and trophic status of the environment. Additionally, a detailed comparison of five DNA isolation protocols and direct PCR was performed on mock and marine community samples as a fundamental step in metabarcoding. No method was closest to the theoretically expected mock community composition. It has been demonstrated that conventional and toxic phenol-based protocol can be replaced by its shorter and safer alternatives based on isopropanol and ethanol precipitation, while direct PCR results have shown advantages of reduced sample manipulation time.