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Scarisoara Ice Cave (Romania) hosts one of world's largest and oldest underground glacier. While no studies were carried out on the existence of microorganisms in this cave's ice block, our interest is to investigate the presence of microorganisms and their chronological distribution in the cave's subterranean ice in relationship with past climatic changes. Samples were collected from ice layers of different age (from present to similar to 900 cal. yrs. BP), and the diversity of embedded microbial communities was assessed by classical cultivation and molecular techniques. The microorganisms from icesediments were cultivated at 4 degrees C and 15 degrees C, in the presence and absence of light. Epifluorescence microscopy analysis indicates the presence of autotrophic prokaryotes and eukaryotes in sunlightexposed ice and water samples. Total DNA was isolated from each ice sample and the bacterial and eukaryotic SSU-rRNA genes were amplified by PCR. The chemical composition and organic content of both deeply buried (> 10 m inside the ice block) and surface (supra- glacial pond water) habitats were analyzed in relation to their age and organic composition. This study is the first to report on the presence of both prokaryotic and eukaryotic microorganisms in the subterranean ice block of Scarisoara Ice Cave, thriving in both organic-rich ice and clear ice layers. Phototrophic prokaryotes and eukaryotes were identified in sun-exposed recent ice. The composition of cold-adapted ice embedded microbiota varied with the habitat age and organic content, as resulting from dissimilarities in growth curve profiles at two different temperatures. The presence of bacteria and eukaryotes in all the analyzed samples was asserted by PCR amplification of SSU-rRNA gene fragments. These findings can be further used to reconstruct changes in the microbial diversity over the past approximately 5000 years, in correlation with climatic and environmental changes recorded by the ice block.

Searching for cold-adapted microorganisms in the underground glacier of Scarisoara Ice Cave, Romania