Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions

Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years. Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls). U-Th disequilibrium dating indicates that these halite layers were deposited at 9600 and 97 000 years before present (ybp) respectively. Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-specific 16S rDNA primers or placed into haloarchaea culture medium. Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloarchaea-containing prepared crystals (positive controls), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (negative controls) resulted in no growth. Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobacterium salinarum. DNA-DNA hybridization experiments confirmed that BBH 001 was closely related to H. salinarum (81-75% hybridization) and its ascription to this haloarchaea species. The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites.