Microbial communities in fluid inclusions and long-term survival in halite

Fluid inclusions in modern and ancient buried halite from Death Valley and Saline Valley, California, USA, contain an ecosystem of “salt-loving” (halophilic) prokaryotes and eukaryotes, some of which are alive. Prokaryotes may survive inside fluid inclusions for tens of thousands of years using carbon and other metabolites supplied by the trapped microbial community, most notably the single-celled alga Dunaliella, an important primary producer in hypersaline systems. Deeper understanding of the long-term survival of prokaryotes in fluid inclusions will complement studies that further explore microbial life on Earth and elsewhere in the solar system, where materials that potentially harbor microorganisms are millions and even billions of years old. INTRODUCTION Microbes are known to exist in subsurface habitats, such as sub-seafloor sediments and continental and oceanic crust, to depths of up to ~3 km (Parkes et al., 2000; Kerr, 2002; Lin et al., 2006; Onstott et al., 2006). Prokaryotes (single-celled organisms lacking a nucleus and other membrane-bound specialized structures) in these subsurface environments live in water within sediment pores and rock fractures. Most are heterotrophic and depend upon preexisting organic matter around them for metabolism, but some are autotrophic and can use non-photosynthetically derived energy sources (Lin et al., 2006). Other prokaryotes that live in Earth’s subsurface under such so-called “extreme” conditions have been found in ice as old as 120 ka from Antarctica, Greenland, and mountain glaciers, and in permafrost, perhaps as old as 8 Ma (Christner et al., 2000; Miteva et al., 2004, 2005; Bidle et al., 2007; Johnson et al., 2007). Collectively, these discoveries have extended the realm of the biosphere into Earth’s crust and have given hope for finding life beneath the surface of other planets, moons, asteroids, and comets of our solar system where present surface conditions are inhospitable. The world’s “oldest living organisms” come from another subsurface setting, buried salt deposits. Over the past 50 years, a series of papers have claimed long-term survival of prokaryotes (Bacteria and Archaea) in these deposits, in some cases for >250 m.y. (Reiser and Tasch, 1960; Dombrowski, 1963; Norton and Grant, 1988; Grant et al., 1998; Stan-Lotter et al., 1999; McGenity et al., 2000; Vreeland et al., 2000, 2007; Radax et al., 2001; Mormile et al., 2003; Schubert et al., 2010a). Prokaryotes in ancient salt deposits also apparently survived in water, but in this case were confined to brine-filled “fluid inclusions” in the halite itself, isolated from surrounding poreand fracturefilling waters. Reports of extreme microbe longevity in salt are controversial. The well-known Permian bacterium from the Waste Isolation Pilot Plant (WIPP) site, Salado Formation, New Mexico, USA (Vreeland et al., 2000), for example, comes from a brine inclusion within a large, diagenetically formed halite crystal. That brine inclusion could have been trapped after the Permian during burial cementation and recrystallization processes (Hazen and Roedder, 2001). Later study of those fluid inclusions, however, shows that they most likely contain evaporated Figure 1. Map of Death Valley and Saline Valley, California, USA, with locations of cores DV93-1 and SV-4A; modified from Schubert et al. (2009a).