Diversity of cultured bacteria from the perennial ice block of Scarisoara Ice Cave, Romania

Cave ice ecosystems represent a poorly investigated glacial environment. Diversity of cave ice bacteria and their distribution in perennial ice deposits of this underground glacial habitat could constitute a proxy for microbial response to climatic and environmental changes. Scarisoara Ice Cave (Romania) hosts one of the oldest and largest cave ice blocks worldwide. Here we report on cultured microbial diversity of recent, 400, and 900 years-old perennial ice from this cave, representing the first characterization of a chronological distribution of cave-ice bacteria. Total cell density measured by SYBR Green I epifluorescence microscopy varied in the 2.4 x 10(4) - 2.9 x 10(5) cells mL(-1) range. The abundance of cultured bacteria (5 x 10(2) - 8 x 10(4) CFU mL(-1)) representing 0.3-52% of the total cell number decreased exponentially with the ice age, and was higher in organic rich ice sediments. Cultivation at 4 degrees C and 15 degrees C using BIOLOG EcoPlates revealed a higher functional diversity of cold-active bacteria, dependent on the age, sediment content and physicochemical properties of the ice. The composition dissimilarity of ice microbiota across the ice block was confirmed by growth parameter variations when cultivated in different liquid media at low and high temperatures. PCR-DGGE and sequencing of bacterial 16S rRNA gene fragments from the cultured ice samples led to the identification of 77 bacterial amplicons belonging to Gammaproteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, showing variation in distribution across the ice layers. Several identified OTUs were homologous to those identified in other glacial and karst environments and showed partial conservation across the ice block. Moreover, our survey provided a glimpse on the cave-ice hosted bacteria as putative biomarkers for past climate and environmental changes.