This project focuses on The Cedars Peridotite, a site in Northern California where active serpentinization occurs, resulting in spring waters so extreme that no current paradigms of microbial metabolism are compatible with life existing there. The intellectual driver of the work thus revolves around the understanding of how microbes manage to eke out a living in this extreme inorganic world of ultra-basic (pH ~ 12) water that has: 1) low levels of organic carbon for heterotrophic growth; 2) (at this pH) virtually no dissolved CO2 or bicarbonate for autotrophic carbon uptake; 3) an Eh that is routinely lower than -550 mV; 4) no obvious abundant electron acceptors; and, 5) low levels of Na+. The absence of H+ and the low concentration of Na+ suggest that the microbes present in this environment are equipped with an energy metabolism that is potentially unlike anything previously described. Thus, identifying and characterizing the resident microbes and the metabolic approaches they utilize may allow us to form an understanding for just how microbes (individuals and/or syntrophic groups) can exploit an environment that appears, at first glance, to be impossibly inhospitable. Investigators will study the microbiology of several different high pH pools, with the following goals in the two year project: 1) the identification of the dominant microbial species by 16S rDNA characterization and metagenomic sequencing; 2) the characterization of the environmental and organismal gene content by metagenomics and genome assembly; 3) the determination of which of these genes are expressed and/or active by metatranscriptomics, and, 4) the geochemical characterization of the sites, so that the biological data can be placed in a geochemical context. This approach is a necessary first step towards hypothesis generation in terms of testing which strains grow in this harsh ecosystem, and how they do it (i.e., which metabolic mechanisms and pathways are utilized). It also relates to the question of whether it is the gene content or the species content (or both) that truly defines a successful ecosystem. This project should lead to the identification of the types of genes required for survival and growth in this harsh environment: e.g., cytochromes, ATPases, hydrogenases, cation pumps, etc. Preliminary results have revealed a number of dominant species and very low diversity. Results of 16S rRNA gene sequence analyses suggest that these microbes are distinct from any previously seen microbes, perhaps not surprising, given the metabolic challenges in these sites. The results of this work will also provide insight into the adaptation and evolution of microbial life on present-day Earth, and given that these ultra-basic, anaerobic environments are close analogs to the early earth conditions prior to photosynthetic activity, the results may have significance regarding the adaptation and evolution of life on the early Earth. Investigators thus expect these results to significantly contribute to a greater understanding not only of extremophiles (the data may provide a milestone in terms of understanding a new type of alkaline microbial community), but to models of early earthly life. The project, which will include both field and laboratory work, will be carried out by a postdoctoral fellow, a graduate student, and several undergraduates.