Probing Metabolic Activity of Deep Subseafloor Life with NanoSIMS

There are very few natural environments where life is absent in the Earth's surface biosphere. However, uninhabitable region is expected to be exist in the deep subsurface biosphere, of which extent and constraining factor(s) have still remained largly unknown. Scientific ocean drilling have revealed that microbial communities in sediments are generally phylogenetically distinct from known spieces isolated from the Earth's surface biosphere, and hence metabolic functions of the deep subseafloor life remain unknown. In addition, activity of subseafloor microbial cells are thought to be extraordinally slow, as indicated by limited supply of neutrient and energy substrates. To understand the limits of the Earth's subseafloor biosphere and metabolic functions of microbial populations, detection and quantification of the deeply buried microbial cells in geological habitats are fundamentary important. Using newly developed cell separation techniques as well as an discriminative cell detection system, the current quantification limit of sedimentary microbial cells approaches to 102 cells/cm3. These techniques allow not only to assess very small microbial population close to the subsurface biotic fringe, but also to separate and sort the target cells using flow cytometric cell sorter. Once the deep subseafloor microbial cells are detached from mineral grains and sorted, it opens new windows to subsequent molecular ecological and element/isotopic analyses. With a combined use of nano-scale secondary ion masspectrometry (NanoSIMS) and stable isotope-probing techniques, it is possible to detect and measure activity of substrate incorporation into biomass, even for extremely slow metabolic processes such as uncharacteriszed deep subseafloor life. For example, it was evidenced by NanoSIMS that at least over 80% of microbial cells at ~200 meters-deep, 460,000-year-old sedimentary habitat are indeed live, which substrate incooporation was found to be low (10-15 gC/cell/day) even under the lab incubation condition. Also microbial activity in ultraoligotrophic biosphere samples such as the South Pacific Gyre (i.e., IODP Expeditions 329) will be shown. Our results demonstrates metabolic potential of microbes that have been survived for geological timescale in extremely starved condition.