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Methanogenic Archaea in Marcellus Shale: A Possible Mechanism for Enhanced Gas Recovery in Unconventional Shale Resources Yael Tarlovsky Tucker, James Kotcon, and Thomas Mroz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00765 • Publication Date (Web): 29 Apr 2015 Downloaded from http://pubs.acs.org on May 5, 2015
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Environmental Science & Technology
Methanogenic Archaea in Marcellus Shale: A Possible Mechanism for Enhanced Gas Recovery in Unconventional Shale Resources Yael T. Tucker*, 1, 2, James Kotcon2, and Thomas Mroz1 1
National Energy Technology Laboratory, United States Department of Energy, Morgantown, WV, USA
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Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, USA
*National Energy Technology Laboratory, United States Department of Energy, 3610 Collins Ferry Rd., PO Box 880, Morgantown, WV, 26505 USA; ph. (304) 285-1398;
[email protected] 1
ABSTRACT
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Marcellus Shale occurs at depths of 1.5 to 2.5 km (5,000 to 8,000 ft.) where most geologists generally assume that
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thermogenic processes are the only source of natural gas. However, methanogens in produced fluids and isotopic
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signatures of biogenic methane in this deep shale have recently been discovered. This study explores whether those
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methanogens are indigenous to the shale or are introduced during drilling and hydraulic fracturing. DNA was
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extracted from Marcellus Shale core samples, pre-injected fluids and produced fluids and was analyzed using Miseq
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sequencing of 16s rRNA genes. Methanogens present in shale cores were similar to methanogens in produced fluids.
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No methanogens were detected in injected fluids suggesting that this is an unlikely source and that they may be
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native to the shale itself. Bench-top methane production tests of shale core and produced fluids suggest that these
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organisms are alive and active under simulated reservoir conditions. Growth conditions designed to simulate the
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hydrofracture processes indicated somewhat increased methane production; however, fluids alone produced
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relatively little methane. Together, these results suggest that some biogenic methane may be produced in these wells
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and that hydrofracture fluids currently used to stimulate gas recovery could stimulate methanogens and their rate of
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producing methane.
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INTRODUCTION
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With continued development of second- and third-world economies and continued population growth,
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worldwide energy demands have become more difficult to fulfill. Currently, the United States uses more than 96
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Quadrillion British Thermal Units (BTUs) of energy per year, only 8 % of which comes from renewable sources.1
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The majority of that energy is provided by coal, petroleum, and natural gas. One of the newest and most
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controversial frontiers in energy production is the natural gas in the Marcellus Shale of the Appalachian Basin. That
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gas is believed to have the capacity of ensuring at least 100 years of additional reliable energy and can be acquired
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domestically, so it is of special interest. Although other sources of natural gas were used in the past, it is within
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recent years that technology has been used advantageously to extract natural gas trapped in the deep, low-
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permeability shale rock. However, due to its continuing development, both challenges and benefits are still arising. 2
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Although it has been generally accepted that methane gas from Marcellus Shale is completely thermogenic,
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recent studies using isotope measurements have found evidence of potentially biogenic gases present in these shale
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wells.3 Such a finding may be of great importance because biogenic methane is generated by micro-organisms at a
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much more rapid rate than methane generated by inorganic, thermogenic processes, which require geologic time
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periods at current reservoir temperatures.4 Therefore, biogenic sources would mean faster regeneration of natural gas
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in these shales and potential for secondary gas recovery. Such recovery efforts can be seen in coal today because
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coal has been shown to contain methanogens that are able to produce significant amounts of biogenic methane.5, 6
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Similarly, much work has also been done to engineer methanogens for gas recovery from waste coal materials.7-9
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Economic production of biogenic methane has also been found in shales. For example, in Michigan, where the
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Devonian-age Antrim Shale was discovered to contain large numbers of Methanohalophilus, the shale naturally
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produces enough biogenic methane for economic recovery.10 Although this shale is younger and shallower (0.05).
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All of the produced water Barnett Shale samples indicated homology to uncultured Methanosarcina. This suggests
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that methanogens are present in the Barnett well and may not originate from the injected hydrofracture water.
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Alternatively, it is possible that methanogenic Archaea are at concentrations that are too low for detection in these
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Barnett Shale pre-injection fluids.
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Five Marcellus Shale wells were sampled for produced fluids; however, pre-injection samples were only
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available for one well (MB). Produced water samples were taken from the separator or the storage tank or both
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(Table 1). The pre-injection water from well MB contained no sequences that could be identified with methanogens
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and a few sequences that identified with uncultured Archaea (and therefore may belong to non-methanogenic
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species). However, produced water from the storage tank on the same well (MB), on day 60 of production, did
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contain a 0.02% propostion of methanogenic organisms. The presence of organisms found in the flowback fluids is
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significant (p ≤ 0.05), using a Poisson test of significance when compared to the pre-injection fluids. These
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organisms included those with homologies to the genus Methanococcoides and other uncultured species from the
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order Methanosarcinales. Organisms in Methanococcoides are characterized as methanogens that consume
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methylated compounds.35 The water from the holding tanks of the other wells from the same region, (M1, M2, M3)
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all contained DNA with homologies to methanogenic Archaea. Wells M1 and M2 were also sampled later in
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production but only well M2 contained sequences with homology to methanogenic Archaea in the later samples.
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Samples of separator water from both M1 and M2 were also analyzed to consider possible contamination from the
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holding tank or negative selection due to the oxygenated conditions during storage. The initial sample taken from
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the M1 well separator on day 661 of production contained a much enriched methanogenic culture (17%); however,
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other samples taken from this separator at later times did not contain sequences from methanogenic Archaea. It is
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unclear why this sample was so much more enriched than the others at just one point in time. Notably, this sample
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also contained a biofilm producer (Halanaerobium prevalens) in a high percentage, so it is possible that a biofilm
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dislodged from the well and was carried into the separator. This may indicate why a small percentage of
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methanogens are usually seen in produced fluids, as methanogens may normally be immobile in their biofilms.36
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DNA isolated from two different depths (7860 and 7872 feet) of Marcellus Shale core samples from
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Greene County, PA (Core 1, 2 respectively) indicated the presence of methanogens in the population (0.01-0.47%).
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These values were not statistically different from those in the produced fluids. Nearly all of these methanogenic
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sequences identified with >97% homology to uncultured Methanosarcina, much like the produced water samples
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above. Evidence of methanogens in produced fluids was paired with evidence from core samples to examine any
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overlap in methanogenic species. Despite the non-sterile nature of these samples, the cores have never come in
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contact with hydrofracturing fluids; therefore, contamination from injection water would not explain an overlap in
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organisms between core and produced water. Presence of methanogens in both sets of samples suggests that the
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methanogens seen in produced fluids may have come from the shale, and that there may be a native community of
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these organisms in the shale before drilling began. The fact that these organisms are not similar enough to currently
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isolated organisms in these conserved regions also supports this hypothesis, since these organisms would have had
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much time while in isolation to differentiate from methanogens at the Earth’s surface.
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A matched pairs test indicated that the number of sequences from methanogenic Archaea was highly
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correlated with the number of sequences from certain non-methanogenic organisms, with r values of 0.99, 0.95,
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0.96, and 0.95 for Halanaerobium, Anaerophaga, Clostridium and Fastidosipila, respectively (NCBI sample
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numbers SAMN03273679-SAMN03273731). These genera all include extremophiles that could have sourced
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alongside these methanogens. Diversity analysis illustrated both differences and similarities between the organisms
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present in samples of pre-injection water, storage tank water, separator water, and cores. Although the absence of
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organism detection in any sample may have been because their abundance was below detectable levels, overlaps
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between all types of organisms found in storage tank water, separator water, and cores but absent in pre-injection
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water provides information on which organisms may be in (or perhaps native to) the shale.
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Metagenomic Assay. To eliminate possible primer bias, an enriched separator sample (from well M1) indicating
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17% methanogenic composition, as identified by the DNA sequencing, was analyzed using metagenomic
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techniques. Although the percentages are different, metagenomic analysis identified sequences that are homologous
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to proteins or 16S rRNA regions of several methanogenic Archaea (Figure 1). Despite its relatively low (97% homology)
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