DCO Project Summary

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Project Title
Evaluation of Sample Collection and DNA Extraction Procedures for Characterizing Bacterial Diversity in Ultrabasic Groundwaters ‐ CoDL Option 1
Start DateEnd Date
2014-07-01 2014-12-31
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Over the past several years, a number of studies have used culture-independent molecular sequencing surveys to describe the microbial diversity of groundwaters hosted in continental bedrock. Water-rock reactions in many of these systems, especially those associated with the process of serpentinization, lead to H2  and CH4 enriched fluids at ultrabasic pH (10-12). The diversity of microbial communities in these systems is commonly extremely low, consisting of only a few major taxa. Clostridia and related species are predominant inhabitants of serpentinizing subsurface environments. Also, Candidatus ‘Desulforudis audaxviator’ is a persistent member of microbial populations in a multi-year study of a well at Cabeço de Vide (CVA) in Portugal. Furthermore, bacterial taxa related to the order Clostridiales have been detected in numerous subsurface environments, and many of these are related to spore-forming species. Additionally, recent studies have shown that spores originating in warm subsurface environments are easily dispersed, and persist in subsurface environments. This proposal is spurred by our observations of discrepancies between the taxonomic composition of microbial communities at CVA which seem to be dictated by whether cells were collected by filtration on 0.2 μm filters and extracted using gentle (chemical) cell lysis, versus collection on 0.1 μm filters and extracted using physical lysis (bead beating). The 0.1 μm samples had a high percentage (5-24%) of Desulforudis sequences, whereas the 0.2 μm samples did not.  Furthermore, it is known that cell sizes in energy- and nutrient-poor environments can lead to the formation of “dwarf cells” which can pass through 0.2 μm filters. Hydrogenophaga-like organisms, which are common inhabitants of serpentinizing subsurface environments are known to form such miniaturized cells. Our objective is to use samples of ultramafic groundwaters collected in parallel on 0.1 and 0.2 μm filters from the CROMO site in northern California, USA and the CVA site to test the hypotheses that (a) microbial populations <0.2 μm in size contribute to the natural microbial diversity of ultrabasic groundwaters, and (b) a significant portion of the microbial diversity described using physical lysis methods is derived from recalcitrant bacterial endospores. We will compare the taxonomic compositions of community DNA extract by gentle (chemical) and rigorous (physical) lysis approaches. These studies will be conducted in concert with the quantification of spore abundances using differential centrifugation and light microscopy, and through analysis of glass fiber filters and determination of the spore protein dipicolinic acid. We propose to have the purified DNA samples sequenced for bacterial 16S RNA via CoDL Option 1.

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Reporting Year 2014 Click to expand

  • RY2014-1 - submitted on Aug 31, 2014

    Update Details:

    Samples will soon arrive at the MBL for DNA sequencing.


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