Cyanobacterial Diversity of Phototrophic Biofilms in Serbian Thermomineral Springs: An Integrated Morphological, Ecological, and Molecular Study

Abstract

Thermomineral springs provide stable thermal and geochemical regimes that promote the development of dense phototrophic biofilms dominated by cyanobacteria. Phototrophic communities were investigated across eight thermomineral springs in Serbia (six in eastern and two in central Serbia) using an integrated morphological, ecological, and molecular approach. Biofilms were sampled in 2024 from contrasting microhabitats (submerged, overflow, and splash/aerosol-exposed zones) and biomass was assessed through chlorophyll a (Chl a) quantification. Cyanobacterial taxa were identified via light microscopy using standard taxonomic keys and simple trichal morphotypes represented the highest diversity; Leptolyngbya showed the broadest distribution, while heterocytous taxa (Calothrix) were rare. A total of 73 cyanobacterial taxa were recorded across the eight investigated springs, with richness ranging from 1 to 33 taxa per locality; the highest richness was observed in Brestovac (33 taxa), followed by Šarbanovac (28) and Nikoličevo (22). Molecular characterization (16S rRNA) of nine cultured isolates revealed multiple filamentous cyanobacterial lineages, including Elainella (Oculatellales) and Wilmottia-related clades, while several isolates showed “Leptolyngbya-like” BLASTn affinities but phylogenetically separated clustering. Overall, the results indicate that Serbian thermomineral springs host diverse cyanobacteria-dominated biofilms shaped by local physical and chemical gradients and microhabitat structure and represent promising reservoirs of thermotolerant strains for further biotechnological use.