Date of Graduation


Document Type


Degree Type



Davis College of Agriculture, Natural Resources and Design



Committee Chair

Jianbo Yao

Committee Co-Chair

James Kotcon

Committee Member

Joseph Morton

Committee Member

Tom Mroz

Committee Member

Joginder Nath


The gas-productive part of the Marcellus Shale occurs in the Appalachian basin at depths of 1.5 to 2.5 km (5,000 to 8,000 ft.), where most geologists generally assume that thermogenic processes occurring over geologic time periods are the only source of natural gas. This is because these sediments are believed to be sterile due to conditions these sediments have endured in the past, which are beyond those that most organisms are currently known to withstand. Recently, Marcellus shale drilling processes have allowed for the study of the microbiology of these sediments by analysis of microorganisms carried in "produced" waters that emerge to the surface over time after injection. Studies of geological and chemical processes and how they may impact the environment are numerous, but little has been done to characterize microbiological interactions. Many microorganisms have been identified in these samples, and composition in the produced fluids is known to change over time. These changes generally have been explained as a natural selection of the injected organisms, but growth of microbes originating from the subsurface environment provides an alternative explanation. Consequently, investigations were conducted to determine the possible sources of microorganisms and methanogens in flowback fluids. DNA extracts from pre-injection and produced fluid samples were compared to those from Marcellus core samples using Next Generation Sequencing of the barcoding region of the 16S rRNA gene. Identified organisms in the produced fluids were then compared using SourceTracker and principal components analysis. SourceTracker analysis indicated that a majority of the microorganisms found in the waters returning to the surface were more likely to have come from communities seen in shale cores than those seen in pre-injected fluids. Principal components analysis supported this as microbial communities in core samples grouped closer to those in produced fluids than in pre-injection fluids, suggesting that the deep subsurface Marcellus shale may contain native organisms. Microbes indigenous to the shale would be among the deepest living organisms ever found, possibly deposited during the original sedimentation, or transported in during a more recent water influx event.;Methanogens produce methane at a faster rate than thermogenic processes. Therefore, a second study was conducted to examine methanogens specifically. To determine whether methanogens are indigenous to the shale itself, or are introduced as contaminants during drilling and hydraulic fracturing, results from DNA extractions in the initial study were analyzed with special focus on Archaeal sequences, most specifically, DNA of known methanogens,. Absence of methanogens in injected fluids suggests that these organisms are unlikely to have been introduced with these fluids and therefore may be native to the shale itself. Bench-top growth analyses measuring methane production in these samples suggested that organisms are not only present, but are potentially alive and active in simulated shale conditions without the need for external microbial or chemical sources. Growth conditions designed to simulate conditions in shale after the hydrofracture processes indicated somewhat increased methane production compared to those seen in shale alone. Fluids alone produced little methane, supporting the conclusion that the shale is an essential element for methanogenesis. Together, these results suggest that some biogenic methane may be produced in these wells and that the introduction of hydrofracture fluids currently used to stimulate gas recovery could affect methanogens and methane production rates. Further experimentation could yield ways to increase biogenic methane production in the Marcellus Shale, providing more natural gas and reducing the number of wells drilled. These two studies indicate that microbes, possibly native to that environment, are present and further analyses may offer key information on their role in natural gas production in shale. Further experimentation may be useful to modify current well management techniques and increase biogenic methane production in these shales.