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Discriminating Native Subsurface Microbes from Drilling Mud Contaminants at
Depths of 1.0-2.2 KM in the Illinois Basin Using 454 Pyrotag Sequencing
Yiran Dong1,2,3, Isaac Cann3,4,6, Roderick Mackie3,4, Nathan Price3,5, Theodore M. Flynn2,
Robert A. Sanford2, Philip Miller2, Nicolas Chia3, Charu Gupta Kumar3, Pan-Jun Kim3,
Mayandi Sivaguru5, Bruce Fouke*1,2,3,6
* corresponding author email: fouke@illinois.edu
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Energy Bioscience Institute, University of Illinois at Urbana-Champaign
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Department of Geology, University of Illinois at Urbana-Champaign
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Institute for Genomic Biology, University of Illinois at Urbana-Champaign
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Department of Animal Science, University of Illinois at Urbana-Champaign
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Chemical and Biomolecular Engineering Department at University of Illinois,
Urbana-Champaign
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Department of Microbiology, University of Illinois at Urbana-Champaign
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Knowledge of composition, structure and activity of microbial communities in deep
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subsurface is of fundamental importance for practical management of groundwater
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resources, hydrocarbon extraction and carbon sequestration. However, technical
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challenges of collecting deep subsurface samples free of contamination have left the
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identity and function of these populations a mystery. To overcome this issue, the newly
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developed Schlumberger Quicksilver MDT probe (MDT) and conventional drill stem test
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(DST) were deployed to collect formation water from different horizons of a 2.2 km deep
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well in the Illinois Basin. IL. Microbial communities of these samples were compared
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with that of the drilling fluid to differentiate indigenous species from contamination.
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Five water samples contaminated with drilling mud were collected at 1 km depth using
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the DST method, and 3 formation water samples were obtained using the Quicksilver
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MDT probe at the horizons ranging from 1.8 to 2.2 km. Geochemistry and microbial
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communities inhabiting in these sampling horizons were evaluated with a suite of
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geochemical, microscopic, and molecular phylogenetic analyses (e.g., T-RFLP, clone
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libraries and 454 pyrosequencing). In addition to distinct geochemical conditions
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observed, significantly different microbial communities among horizons were also
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suggested by molecular analyses. Pyrosequencing results indicated the MDT samples to
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be much less similar to the drilling mud (< 2 % shared) than the mud-contaminated DST
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samples (11 – 27% of sequences shared). Moreover, comparison of the microbial
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communities from which potential contaminating populations had been removed
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indicated heterogeneous distribution of indigenous microbial composition in the Illinois
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Basin, even within the same stratigraphic geological formation.
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Our work suggests that pyrosequencing is a sensitive tool to determine indigenous
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subsurface bacterial communities. The diversity of indigenous microbial communities
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inhabiting in different horizons may result from the geochemical gradients (e.g. total
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dissolved salts (46 – 158 g/L), pH (5.9 – 8) or T (28.7 – 52.1 ˚C)) in the formations.
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