Biology Project 1
Biology Project 1: The Contribution of Eukaryotic Diversity to Deep Subsurface Microbial Ecosystems
Cynthia Anderson, BHSU Associate Professor of Biology
The diversity of Eukaryotic microbes in subsurface terrestrial ecosystems remains underexplored. While the likelihood of finding Protista and Fungi in the pristine rock strata at any significant depth diminishes due to spatial and nutrient constraints, it is not entirely unreasonable to expect that some endogenous Eukaryotes live and perhaps thrive in the seemingly inhospitable subsurface environment where nutrients are limiting and temperatures increase with depth. Some species of fungi are capable of surviving in extremely nutrient poor rock environments. For example, dematiatious meristematic fungi have been isolated from rock surfaces (granite, limestone, and calcite), and from stone monuments. It is well documented that fungi play an important role in geomicrobiological transformations, ranging from facilitating syntrophic interactions to aiding rock decay and diagenesis. While prevalent in terrestrial environment on and slightly below the earth’s surface, these interactions of fungi within the geologic environment of the deep subsurface remain to be explored. Even if endogenous fungi are found to be lacking in the deep subsurface, the role of exogenous fungi in rock decay and diagenesis, as well as biofouling of research infrastructure in the subsurface needs to be understood, especially in light of the establishment of subsurface research infrastructure.
There is no doubt that fungi have been introduced to the subsurface areas of SURF through anthropogenic activities ranging from the mining activities of the past 125 years, to the more recent flooding and dewatering of the mine (past 6 years), to the re-ventilation of the drifts and subsequent construction of the subsurface research infrastructure. Preliminary culture independent environmental genomic data on the Eukaryotic diversity found in a water sample, and a biofilm sample taken from the 4850L indicate the presence of several Eukaryote taxa representing both Protista and Fungi (Fig 3).
The constant influx of ventilation air introduces microbial propagules that can become established on rock surfaces, especially near slow flowing water seeps, and anthropogenic activity during infrastructure construction such as the production of pollutants from fossil fuel run equipment, introduction of wood and other construction materials have introduced organic and inorganic nutrients upon which some of the microbes can feed. Thus it could be hypothesized that the drift walls at the 4850L throughout SURF will continue to harbor a diverse microbial community that includes eukaryotes. The drift wall communities will consist of both aerobic and facultative anaerobic Prokaryotic and Eukaryotic microorganisms some living independently, but most forming complex syntrophic and symbiotic relationships. The prevalence of endemic Eukaryote taxa in SURF is unknown. Furthermore, the influence of exogenous Fungi on the endemic microflora, the level of syntrophy among members of the subsurface microbial communities, the role of endogenous and exogenous Eukaryotic organisms in biogeochemical processes such as rock weathering, and the extent of biogeochemical interactions within the miles of subsurface drifts throughout the former Homestake mine areas are completely unknown.
Example Student Project: The pilot data depicted above was generated from a rDNA amplicon clone library. While informative, the Chao diversity indices estimated that only 45% and 48% of the diversity of the water and biofilm samples was captured. Next Generation sequencing technologies would vastly improve the ability to capture the entire diversity present. Furthermore, comparisons of air, open water, water from deep drill holes, water emanating from drill holes and biofilms would provide us with a much more thorough view of the eukaryotic diversity in this isolated ecosystem and lead to hypotheses about colonization of water and biofilms, and the organismal relationships occurring in the systems. It would also help us to identify those eukaryotic microbes that may actually be endemic to the system and not simply introduced. Water emanating from subsurface drill holes, biofilms, and air from various areas of SURF over varying seasons will be sampled. Culture independent DNA analyses will be employed to explore the eukaryotic microbial diversity. Students will PCR amplify the intergenic spacer region of the rDNA, ITS1-5.8S-ITS2, and construct amplicon libraries for NextGen sequencing and will use bioinformatics data analysis tools to estimate the eukaryotic diversity throughout several sampling areas. Additionally, students will visually survey various substrates throughout SURF, choosing interesting biofilms and instances of colonization to sample in order to attempt to culture fungi that may be colonizing the biofilms and other substrates observed. By collaborating with students working in projects 6 – 9, a deeper understanding of the possible roles of eukaryotic microbes will allow us to further our understanding of their roles in biogeochemical processes as well as their roles in syntrophic relationships in a complex ecosystem.