How Do Prokaryotes Survive In Fluid Inclusions In Halite for 30,000 Years?

Prokaryotes (Archaea and Bacteria) and algal cells (Dunaliella) occur in primary fluid inclusions in 10,000-100,000 year old halite crystals from a 90-m long salt core from Death Valley, California. Microbial cells in fluid inclusions were identified (1) in situ using transmitted and UV microscopy, and, (2) under SEM from surface sterilized and filtered samples. Probable prokaryote cells can be distinguished from mineral microparticles using criteria including morphology, size distribution, and birefringence. Ancient prokaryote cells are miniaturized cocci (<1 µm) and rare rods (<2 µm long), which supports interpretations that the microorganisms are indigenous to the halite and that starvation-survival may be the response of some microbes to long-term trapping in fluid inclusions. Microorganisms occur almost exclusively in fluid inclusions in halite formed in a perennial saline lake 10,000-35,000 years ago, which suggests trapping and preservation of microorganisms in fluid inclusions is influenced by the original environment in which the halite precipitated. Culturing experiments were designed to test the viability of ancient prokaryote cells in fluid inclusions. Surface sterilization and dissolution of 891 halite crystals from the Death Valley core produced growth of six halophilic Archaea (in media containing glycerol) and two halotolerant Bacteria, from perennial saline lake halite, 22,000-31,000 years old. Samples yielding positive cultures all contained abundant Dunaliella and prokayotes in fluid inclusions, observed in situ. If algae and bacteria are trapped in the same fluid inclusions, glycerol released from Dunaliella (used to maintain osmotic equilibrium with their saline environment), may provide an energy source for prokaryotes to survive in fluid inclusions for 10,000-100,000 years.