Insights into aerobic microbial community in the Kamniška Bistrica river sediment with culture-dependent and molecular biology approach | Vpogled v aerobno mikrobno združbo rečnega sedimenta Kamniške Bistrice s klasičnim gojitvenim in molekularno biološkim pristopom

anglais. River systems are exposed to anthropogenic disturbances, including chemical pollution and eutrophication. They may affect a structure and diversity of microbial communities in river sediment. The aim of the study was to describe diversity and structure of a bacterial community in the Kamniška Bistrica river sediment using culture-dependent and molecular methods. Sediment samples were collected from three sampling stations along the Kamniška Bistrica river: upstream (Kamniška Bistrica), midstream (Vir) and downstream (Bišče), and physico-chemical analysis was conducted on-site. Isolates were recovered from three different agar plate media (LB, NB in R2A) and micro- as well as macromorphologically described. DNA was extracted from pure cultures and directly from sediment samples, and 16S rRNA gene segments were PCR amplified using universal bacterial primers. The PCR products were subjected to denaturing gradient gel electrophoresis (DGGE). The results showed that the DGGE was an appropriate method for monitoring the changes in the community structure of the river sediment and allows to determine the impact of environmental factors on bacterial diversity. The comparative analysis of the DGGE profiles from isolates and sediment samples showed that the DGGE banding patterns revealed different bacterial community structures for all three sampling stations, while the DGGE profiles of isolates did not represent dominant bacterial populations of the sediment. On the bases of the DGGE profiles, 25 isolates were chosen and their 16S rRNA genes were amplified and sequenced. All partial nucleotide sequences of 16S rRNA genes were preleminary taxonomically identified by using BLAST. The majority of sequenced isolates belonged to bacteria normally present in aquatic environments.

slovène. Rečni sistemi so največkrat izpostavljeni antropogenim vplivom, ki vključujejo kemično onesnaženost in evtrofikacijo. Vse to povzroča spremembe tudi v strukturi in pestrosti mikrobnih združb v rečnem sedimentu. Namen študije je bil z gojitvenimi in molekularnimi metodami proučiti pestrost in strukturo mikrobne združbe sedimenta Kamniške Bistrice. Vzorce sedimenta smo odvzeli na treh mestih vzdolž toka reke: izvir (Kamniška Bistrica), srednji del (Vir) in spodnji del (Bišče) ter na terenu opravili osnovne fizikalno-kemijske analize. Čiste kulture smo iz sedimenta osamili in gojili na treh vrstah agarskih gojišč (LB, NB in R2A) in jih mikro- in makromorfološko opisali. Iz čistih bakterijskih kultur in iz vzorcev sedimenta smo izolirali tudi genomsko DNAK. S širokospecifičnimi bakterijskimi začetnimi ologonukleotidi smo v reakciji PCR pomnožili ustrezne dele genov za 16S rRNA in jih analizirali z metodo gelske elektroforeze v gradientu denaturanta (DGGE). Ugotovili smo, da je metoda DGGE primerna za proučevanje pestrosti celokupne mikrobne združbe rečnega sedimenta in omogoča ugotavljanje vplivov dejavnikov okolja na raznolikost mikrobne združbe. Profili analize DGGE mikrobnih združb so se med mesti vzorčenja razlikovali, medtem ko profili čistih kultur po pričakovanju niso predstavljali številčno pomembnejših bakterijskih populacij sedimenta. Na podlagi profilov DGGE smo izbrali 25 izolatov in za direktno sekvenciranje pripravili njihove pomnožke. Pridobljene sekvence dela gena za 16S rRNA smo z orodjem BLAST preliminarno taksonomsko uvrstili med skupine bakterij, ki so običajno prisotne v vodnih okoljih.