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.
We have investigated geomicrobiology of ultra-basic, low salinity springs associated with a serpentinizing peridotite body known as The Cedars, near Cazadero, California. The long-term goal of this research is to understand how the diversity of the microbial community and its members’ specific ecophysiologies (i.e. gene regulation, biochemistry, and physiology) intersect and interact with the ultra-basic geochemical environment of the site, allowing microbial growth in an environment where, according to present day paradigms of metabolism, no growth should occur. The objectives of this project focused on elucidating the geochemistry of the site and the nature of the microbial communities present in low salinity, ultra-basic and highly reducing springs. The extremely high pH, low Eh and the near absence of electron acceptors, sodium, potassium and carbon dioxide make The Cedars springs some of the harshest environments on Earth. These springs represent environments where no described microbes should be able to grow, although, as will be shown, microbial life has been detected in the springs. First, we have conducted the geochemical characterization of ultra-basic (pH 11–12) reducing (-656 to -585 mV) groundwater springs discharging from serpentinized peridotite. The spring waters investigated were thought to be of meteoric origin, but geochemical modeling suggested that there were two sources of groundwater, a shallow source with sufficient contact with The Cedars’ peridotite body to be altered geochemically by serpentinization, and a deeper groundwater source that not only flows through the peridotite body but is also in contact with the marine sediments of the Franciscan Subduction Complex (FSC) below the peridotite body. We thus proposed that the groundwater discharging from lower elevations (GPS1 and CS1) reflect the geochemistry of the deeper groundwater in contact with FSC, while groundwaters discharging from springs at higher elevations (NS1 and BSC) were a mixture of the shallow peridotite-only groundwater and the deeper groundwater that has been in contact with the FSC. All of the springs had very low (< 1 µM) levels of several important elements and/or electron acceptors (e.g. nitrate/ammonium, sulfate, and phosphate) required for (microbial) growth, which is not uncommon at sites of continental serpentinization. Gases rich in N2, H2, and CH4were exsolving from the springs. The stable carbon isotope signatures of the various springs suggested that the methane was of mixed (i.e., microbial and geological) origin . Aromatic and alkane compounds detected in the spring water were originated in the deeper FSC groundwater. Second, to understand the taxonomic nature and temporal stability of the microbial communities at the site, we conducted multiyear, culture-independent geomicrobiological study of three springs at The Cedars that differ with respect to the nature of the groundwater feeding them. Within each spring, both geochemical properties and microbial diversity in all three domains of life remained stable over a 3-y period, with multiple samples each year. Between the three springs, however, the microbial communities showed considerable differences that were strongly correlated with the source of the serpentinizing groundwater. In the spring fed solely by deep groundwater, phylum Chloroflexi, class Clostridia, and candidate division OD1 were the major taxa with one phylotype in Euryarchaeota. Less-abundant phylotypes include several minor members from other candidate divisions and one phylotype that was an outlier of candidate division OP3. In the springs fed by the mixture of deep and shallow groundwater, organisms close to the Hydrogenophaga within Betaproteobacteriadominated and coexisted with the deep groundwater community members. The shallow groundwater community thus appears to be similar to those described in other terrestrial serpentinizing sites, whereas the deep community is distinctly different from any other previously described terrestrial serpentinizing community. For further understandings of the microbial characteristics in this environment, we have conducted metagenomic and metatranscriptomic analyses. The metagenomic analyses were successfully conducted and resulted in the assembly of 30 of high-coverage genomes of microbes inhabiting in these environments. The genomic features suggested that the microbes there are unusual in terms of both genome size and metabolic genes content when compared to the known microbes. In addition, we have isolated three strains that are the dominant members in all of the continental serpentinizing sites on Earth. The detailed physiological, genomic and transcriptomic studies revealed the unique features of the strains. The research project contributed to a broader social impact in various aspects: 1) providing interdisciplinary post-doctoral training; 2) facilitating undergraduate research opportunities and extended STEM education through summer and school-year internship programs at JCVI; 3) resulting in reporting by the local newspaper, 4) resulting in press-releases about the paper published in PNAS from JCVI, 5) resulting in a BBC documentary featuring the Cedars (in preparation), 6) providing public outreach through JCVI participation in regional and national science festivals promoting STEM learning, and, 7) disseminating results in high-impact peer-reviewed journals, national and international conferences, and classroom lectures.
