Diversity of cultivable bacteria involved in the formation of macroscopic microbial colonies (Cave silver) on the walls of a cave in Slovenia

Abstract

Karstic caves often support white, yellow, grey or pink microbial colonies that are termed ‘cave silver’ by speleologists. Using various sample pre-treatments and culture media, a wide variety of bacteria associated with these colonies were recovered from a cave in Slovenia, Pajsarjeva jama. Decreasing the inoculum size resulted in significant increases in viable counts, while pre-treatments had the opposite effect with the exception of microwave irradiation. While all growth media yielded viable counts, the maximal counts were observed on a low-nutrient TWA medium. Based on the 16S rRNA gene sequence of OTU representatives, the majority of the 80 isolates examined belonged to Streptomyces (25%), Micrococcus (16%) and Rhodococcus (10%) Other abundant groups were Pseudomonas (9%), Agrobacterium (8%), Lysobacter (6%) and Paenibacillus (5%), while members of genera Microbacterium, Agrococcus, Arthrobacter, Bacillus, Kocuria, Oerskovia, Sphingomonas, Aerococcus, and Bosea represented a minor portion of cultivable diversity encountered. Members of Streptomyces and Agrobacterium were common to all samples. Although these microorganisms readily form colonies under laboratory conditions, they were unrelated to abundant environmental phylotypes recovered from same samples in a previous study. However, the comparative 16S rRNA analysis showed that microorganisms highly related to the ones obtained in this study were cultivated from other subterranean environments indicating that they might represent true microbial cave dwellers.