The Geochemical and Spatial Argument for Microbial Life Surviving Into Early Diagenesis in the Appalachian Basin

The Geochemical and Spatial Argument for Microbial Life Surviving into Early Diagenesis in the Appalachian Basin

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Edwin R. Buchwalter

Graduate Program in Earth Sciences

The Ohio State University

2016

Master's Examination Committee:

Professor David Cole (Advisor)

Professor Matthew Saltzman

Professor Michael Wilkins

Copyrighted by

Edwin Robert Buchwalter

2016

Abstract

While life is known to exist in the subsurface environment, specific limitations to microbial populations inhabiting deep subsurface habitats are assumed and include organic substrate and terminal electron acceptor availability, temperature and space to live. Microbial populations have been found in environments where they are least expected, notably 3 km deep in granite as well as in the boreholes and near-borehole environment of oil and gas wells where they often cause problems for oil and gas operators. While an anthropogenic source is assumed for microbes in and near the borehole of oil and gas wells in the Utica-Point Pleasant system due to the high temperatures the rock has undergone, the question remains whether microbial populations could have survived in less mature rock to the West of contemporary oil and gas operations. As a component of an NSF funded study “Microbial Biodiversity and

Functionality of Deep Shale and it’s Interfaces” this research attempts to answer whether microbes could have survived to the present day in pores as well as questions relating to biological limitations and whether these are present in the Utica-Point Pleasant. Looking at sulfur, organic carbon and potential micro-lithologies within the Utica-Point Pleasant organic-rich mudstone may yield a better understanding of how the diagenesis of a marine mud affects anaerobic microbial populations established in these muds. Utilizing a variety of petrophysical, geochemical and high resolution imaging techniques this research has identified micro-lithologies within the Utica-Point Pleasant system that ii likely provided safe harbor for anaerobic microbes until the habitat was either sterilized due to temperature or in-filled with minerals, sealing off these habitats. Further, these micro-lithologies may respond to hydraulic fracturing chemicals and processes and become inhabitable for anthropogenically introduced microbial populations.

iii

Acknowledgments

The author would like to acknowledge the assistance of several people and labs within the Ohio State School of Earth Sciences. First my advisor, Dr. Dave Cole for supporting me throughout this endeavor and providing guidance when needed and the freedom to follow my ideas. Julie Sheets and Sue Welch were always available with their expertise, their invaluable instrument training, and willingness to listen to my ideas. Sincere thanks to Mostafa Fayek and Ryan Sharpe at the University of Manitoba for their rapid turnaround on Sulfur isotopes. For his efforts in obtaining µXCT data a big thank you to

Alex Swift is due, your help is always appreciated. I am grateful for the support provided by the National Science Foundation’s grant DEB-1342701, as I quite literally couldn’t have done this without them. To the faculty of the School of Earth Sciences, your passion for our shared field of study shows through in every single class that I have taken. Thanks

Derek for helping with the sulfur isotopes of this study, and for all the lunches we spent talking politics. Last but not least, Kathryn Johnson, while others see the fruits of my labor, they do not see the sacrifices and are mostly unaffected by late nights at the lab. I am grateful for your support and love.

Vita

June 2002 ...... Diploma, Buckeye Valley High School

March 2010 ...... Associate of Arts, The Ohio State University

December 2013 ...... B.S. Geological Science, The Ohio State

University

2013 to present ...... Graduate Research Associate, School of

Earth Sciences, The Ohio State University

Publications

Buchwalter, E., Swift, A. M., Sheets, J. M., Cole, D. R., Prisk, T., Anovitz, L. L. &

Chipera, D. (2015). Mapping of Microbial Habitats in Organic-Rich Shale.

Unconventional Resources Technology Conference (URTEC: 2174226 http://archives.datapages.com/data/urtec/2015/2174226.htm).

Fields of Study

Major Field: Earth Sciences

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita ...... v

List of Tables ...... ix

List of Figures ...... x

Introduction ...... 1

Relevance of Black Shales ...... 1

What is a Black Shale...... 3

Biological Requirements ...... 6

Major Limitations to Biology ...... 6

Geologic Setting...... 10

Depositional Environment...... 10

Utica-Point Pleasant Lithologic Description ...... 14

Methods...... 16

Sample Selection ...... 16

MICP ...... 17

SEM ...... 18

Sulfur Isotopes...... 19

Micro-XCT ...... 19

Matrix Assisted Laser Desorption Ionization (MALDI) ...... 20

Sample Suite ...... 22

Pore Assessment ...... 31

SEM ...... 31

MICP ...... 40

Geochemical Assessment...... 50

Origin of Pyrite and the Role of Microbes ...... 50

Rapid Assessment of Mobile Organic Matter ...... 61

Understanding the Spatial Context of Organic Matter and Pyrite ...... 64

Conclusions ...... 70

Future Research ...... 75

References ...... 77

Appendix A: SEM images ...... 82

Appendix B: MICP data...... 86

Appendix C: SIMS sulfur isotopes ...... 89

vii

Appendix D: MALDI spectra ...... 91

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List of Tables

Table 1. Sample suite with applied techniques...... 23

Table 2. MICP sample details with maturity, mineralogy and TOC ...... 41

Table 3. Summary results of SIMS measurements...... 54

Table 4. SIMS data for SW 3955, Ro 0.59...... 89

Table 5. SIMS data for MU 4471, Ro 0.78...... 89

Table 6. SIMS data for JP 10930, Ro 2.49...... 90

List of Figures

Figure 1. Paleogeographic map of the Middle Ordovician, Black line represents the transect of the sample suite (Modified from Colorado Plateau Geosystems http://deeptimemaps.com)...... 11

Figure 2. Paleogeographic map of the Late Ordovician, Black line represents the transect of the sample suite (Modified from Colorado Plateau Geosystems http://deeptimemaps.com)...... 12

Figure 3 Litho-facies map showing the Point Pleasant sub-basin with the surrounding carbonate platforms (map from Patchen et al., 2006)...... 13

Figure 4 Ternary diagram illustrating the different mineralogies present in the sample suite...... 22

Figure 5. SEM image of SW 2958 matrix, note dolomite rhombohedrons (Do) carbonates

(CaCO3) and Phosphates. Interspersed white Euhedrons are pyrite...... 25

Figure 6. SEM image of SW 3055 matrix, note elongate organic objects...... 26

Figure 7 SEM of JP 10855 matrix...... 28

Figure 8. SEM of JP 10930 matrix, with elongate organic stringers and carbonate fossil.

...... 29

Figure 9. SEM of OM-hosted porosity in the high maturity JP 10930 sample (Ro 2.49) 32

Figure 10. SEM image showing non-porous OM, indicated by the yellow arrows...... 33 x

Figure 11. A. Linear clusters of pyrite framboids in JP 10930 (scale bar 200 µm). B.

Framboid cluster observed in a fracture in JP 10930 (scale bar 10 µm). C. Pyrite framboids aligned with organic stringer in JP 10930 (scale bar 100 µm). Bedding parellel to scale bar in A,C, perpendicular in B...... 34

Figure 12. Two in-filled fossil pore environments that have a pair of minerals that could be produced via microbial reduction of sulfate...... 36

Figure 13. Various SEM images showing ovoid objects within a carbonate cement on the underside of a brachiopod. (JP 10930) ...... 37

Figure 14. Pyrite framboids and euhedrons occupy the underside of a brachiopod hash bed in carbonate cement (JP 10930)...... 39

Figure 15. Results of the equilibration experiment on KB 8440 ...... 42

Figure 16. MICP results for carbonate dominated samples, legend in order from least to most mature...... 45

Figure 17. MICP results for clay-dominated samples...... 46

Figure 18. Graphs of porosity accessed through pore-throats smaller than 50 µm vs maturity, total organic carbon, carbonate content, and clay content...... 47

Figure 19. A.) Large euhedron replacing carbonate, small framboids scattered throughout.

B.) Heavily pyritized OM, framboids and very tiny cubic pyrite crystals. C.) Pyrite inside pelycopod fossil. D.) Pyrite framboid cluster in matrix. E.) Non-framboidal pyrite in OM.

F.) Weathered pyrite among bi-pyramidal pyrite crystals...... 50

Figure 20. Temperature-dependent isotopic fraction factors for various sulfur species

(calculated from Ohmoto et al., 1982)...... 52

Figure 21 Open versus closed system fractionation of sulfate, at 250⁰ C starting with a

δ34S of 30‰ (Calculated with data from Ohmoto 1982)...... 53

Figure 22. Many pyrite framboids in a small habitat within a fossiliferous zone. MU .... 56

Figure 23. Cluster of pyrite framboids occupying a pentagonally shaped carbonate, likely a crinoid stem or other fossil. MU 4471 ...... 57

Figure 24. Multiple SIMS measurements on euhedrons and framboids (JP 10930, 2.49

Ro)...... 57

Figure 25. Heavy δ34S pyrite euhedrons near a fossiliferous zone in the lowest maturity sample, SW 3055 ...... 59

Figure 26. Matrix Assisted Laser Desorption Ionization Mass Spectrometry spectra for four samples, two low maturity (SW 2958 and SW 3055) and two high maturity (JP

10855 and JP 10930) samples...... 61

Figure 27. Solvent extractions with Dichloromethane and 30⁰C. The two left-hand samples are low maturity (SW 2958 and SW 3055) and right-hand samples are high maturity (JP 10855 and JP 10930). Note difference in color...... 63

Figure 28. High density objects are white in this grayscale µXCT image. Yellow 3D objects have framboid-like appearance, suggesting white objects are pyrite...... 64

Figure 29. A.) µXCT image of JP 10930, 3D pyrite grains and framboids, 2D OM stringer. B.) 3D pyrite turned off, perpendicular to bedding µXCT 2D image. C and D.)

OM stringer in two different bedding parallel orientations...... 66

Figure 30. µXCT image of JP 10930, showing several large OM stringers (OM is red) and dispersed pyrite (Pyrite is yellow)...... 67

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Figure 31. Connecting reflected light, µXCT and SEM across multiple techniques and length scales. (Image taken from 2015 URTEC presentation by Buchwalter et al. 2015) 68

Figure 32. SEM image of JP 10855 with a fossiliferous zone that is nearly completely filled with pyrite...... 73

Figure 33. Cross-sections of ovoid objects observed outside of matrix and adjacent to carbonate cement. Similar objects are observed in the carbonate cement...... 82

Figure 34. SEM image of poorly crystalline carbone cement in the underside of a brachiopod...... 83

Figure 35. Low maturity OM stringers in MG 5778, note smooth well-defined edges. .. 84

Figure 36. OM-stringer in JP 10930, high maturity sample. Note less well-defined edges.

...... 85

Figure 37. Example of the prepared sample loaded into a penetrometer (A) and on weigh paper showing sawn surfaces (B)...... 86

Figure 38. MICP data for SW 2958 (Clay-rich) and SW 3055 (Carbonate-rich)...... 87

Figure 39. MICP data for RH 6528 (Clay-rich) and RH 6644 (Carbonate-rich)...... 87

Figure 40. MICP data for KB 8334 (Clay-rich) and KB 8440 (Carbonate-rich)...... 88

Figure 41. MICP data for JP 10855 (Clay-rich) and JP 10930 (Carbonate-rich)...... 88

Figure 42. MALDI spectra of Sinapinic Acid and Dichloromethane...... 92

Figure 43. MALDI spectra of Alpha-Cyano-40Hydroxycinnamic Acid (CHCA) and

Dicholoromethane (DCM solvent)...... 93

Figure 44. MALDI Spectra for SW 2958 prepared with CHCA...... 94

Figure 45. MALDI spectra for SW 3055 prepared with CHCA...... 94

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Figure 46 MALDI Spectra for JP 10855 prepared with CHCA...... 95

Figure 47. MALDI spectra for JP 10930 prepared with CHCA...... 95

Figure 48. MALDI spectra for sinapinic acid (SA)...... 96

Figure 49. MALDI spectra for SW 2958 prepared with SA ...... 97

Figure 50. MALDI spectra for SW 3055 prepared with SA...... 97

Figure 51. MALDI spectra for JP 10855 prepared with SA...... 98

Figure 52. MALDI spectra for JP 10930 prepared with SA...... 98

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Introduction

Relevance of Black Shales

The modern economically successful extraction of hydrocarbons from black shales, classically viewed as a hydrocarbon source rock, has been a process taking the better part of half a century to come to fruition. While often viewed as a modern technical achievement, commercial hydrocarbon production from black shales has a long history dating to a gas well drilled in 1821 to provide gas lighting in the town of Fredonia, New

York (Peebles, 1980). While the scale of production of these early gas-shales was limited, the ability to produce gas from shales was well-known by the early 1900’s from the

Devonian shales of the Appalachian Basin (Peebles 1980, Curtis 2002).

The key to production of black shales in the United States is that a fracture network is critical to access economically viable natural gas. The Department of Energy took shale- gas seriously when it invested in the Eastern Gas Shale Project, beginning in 1976 and carrying through to 1994 (D.O.E.’s Unconventional Gas Research Programs, 2007). This project focused on the Devonian age shales in the Eastern part of the United States and highlighted the importance of shales as an economic resource, funding a flurry of research over the life of the program (D.O.E.’s Unconventional Gas Research Programs,

2007).The project ended before horizontal drilling and hydraulic fracturing technique development enabled these black shales to be commercially viable , however the body of 1 knowledge gained during this project laid the groundwork for the shale-gas boom that came a decade later (King et al., 1993).

Hydrocarbon-bearing black shales are once again in the limelight as these account for

48% of 2016’s natural gas production in the United States according to the Energy

Information Administration. Unlike traditional gas or oil wells, shale-gas systems like the

Ordovician age Utica-Point Pleasant and the Devonian Marcellus span the oil, condensate and gas maturity windows, allowing for operators with large acreage that spans these windows to drill wells that are high in natural gas liquids such as ethane, butane and other

C1-C8 hydrocarbons allowing for greater diversity in products from a single well

(Blackstock et. al., 1968). There is little doubt that as the nation’s energy requirements increase so too will shale gas exploration and production as this has been the driver of cheap electrical energy in the United States, driving the cost per thousand cubic ft.

(MMCF) of natural gas down from a high of $7.97/MMCF in 2008 to a low of

$1.63/MMCF in March 2016 according the Independent Energy Agency website. Though popular media (e.g. movies such as Gasland, Fracknation) and environmental interest groups look upon shale gas negatively due to the potentially negative impacts of hydraulic fracturing, it is important to note that as electric power transitions from coal to natural gas, the United States carbon output dropped to pre-2000 levels, in large part due to the lower CO2 output of natural gas power plants (Carey 2012, Lueken et al., 2016).

These are serious resources that have changed the energy industry and will continue to do so for the foreseeable future.

An invaluable by-product of the production of these important resources has been the unique access vertical and horizontal drilling provides for studying the physical, chemical and biological properties and processes of hydrocarbon-producing formations. One of the most poorly understood areas of study within the subsurface is the depth window in which biogeochemical processes participate in the diagenesis of sediment to rock. While much of the Utica and Point Pleasant formations have been subjected to temperatures in excess of those considered survivable for thermophilic microbes, many microbial metabolic processes leave mineralogical signatures behind. As such the focus of this research is looking for mineralogical and textural signs of past microbial inhabitation, petrophysical characterization of the rock to identify zones where nutrient-flux would be high enough to support life and looking ahead to the future of Utica and Point Pleasant formations to where anthropogenic microbes could survive if introduced through hydraulic fracturing.

What is a Black Shale?

From a purely geologic standpoint, sedimentary rocks are characterized by component minerals, color, grain-size, induration, and the presence or absence of laminae a (e.g. a rose-colored massive sandstone). Thus when the term black shale is used, the kind of rock comes to mind is a finely laminated very dark colored rock that is composed of clay sized grains, often aluminosilicate minerals with detrital quartz. When samples of the

Point Pleasant or other “black shales” are closely examined, this description falls short on nearly every account due to the complexity of the rock fabric and mineral assemblage.

The Point Pleasant is first and foremost composed primarily of carbonate material, either individual grains or fossils, rather than clay and is well indurated, so much so that is more akin to a black limestone. There are micro-lithologic changes that transition from a carbonate-rich matrix to a fossiliferous packstone. From a mineralogical perspective it is more proper to refer to the Point Pleasant as a carbonate mudstone as opposed to a shale.

A mudstone is defined as a fine-grained siliciclastic rock consisting primarily of clay and/or fine quartz grains (Boggs, 2012). The term black shale refers to not only the color of a shale, but the organic-rich character of the rock which should at a minimum contain at least 1% total organic content (TOC) by weight though some black shales have TOC in excess of 10% (Tourtelot, 1970, Arthur et al., 1994). All of the samples analyzed in this study have a TOC greater than 1% and as such will be referred to as an organic matter

(OM)-rich mudstone.

OM-rich mudstones often concentrate trace metals and ions due to the high surface area and charge of the OM and aluminosilicate platelets sweeping these metals and ions out of the water column during settling. This enrichment can produce metallic ore deposits of commercial value within OM-rich mudstones, in some cases enough to warrant mining of uranium and silver (Tourtelot, 1970, Arthur et al., 1994). Though many controversies exist as to the origin of black shales and whether they are a product of primary production and rapid burial or slow burial in a restricted, anoxic basin, what has become quite clear is that there is truly no direct analog present in today’s oceans (Tourtelot, 1979, Arthur et al., 1994, Wignall, 1994). The often quoted modern analog for shale deposition is the aptly named Black Sea, which is a restricted basin environment that produces black shale

4 like sapropels; however this is a basin that is more than 2,000m deep and is a silled-basin, creating euxinic bottom-water conditions (Wignall, 1994). When applying this analog to an expansive shale unit such as the Utica, it falls especially short as the Utica is interpreted to have been deposited in 20-50 meters of water casting doubt that this could represent a stratified water column in a restricted basin and trace-metal evidence suggests poorly oxygenated bottom-water conditions (Schumacher et al., 2013, Foley 2016).

Biological Requirements

Major Limitations to Biology

In order to identify zones within the Utica-Point Pleasant formation where microbial habitats could exist, an understanding of the biological limitations of microbes is paramount. A generalized set of requirements for subsurface life can be laid out which represent obstacles that even intense adaptation would likely be unable to overcome.

While many aspects of black shales or mudstones are different, the environment is almost certainly an anaerobic one with abundant sulfur, iron, organic matter and high salinity.

This environment would exclude more energetic aerobic phototrophs, allowing less energetic thermophilic and halophilic chemotrophic archaea and bacteria to become the dominant lifeforms. While these less energetic metabolisms (possibly show figure of energy/metabolic chart) would find the chemistry of a black shale inviting, the tiny pores and pore-throats found in most black shales are too small to allow microbes to reside there. Biogenic hydrocarbon degradation in deep subsurface reservoir and source rocks suggests microbes can survive in these environments and that biogenic activities contribute to the creation of methane from n-alkanes and other heavy oils (Frederickson et al., 2001; Head et al., 2003). Keeping these thoughts in mind, a loose set of requirements can be laid out that govern whether a subsurface microbial population can survive in or near a black shale environment. First, the availability of a carbon-based substrate from which electrons can be stripped and donated to a terminal electron 6 acceptor in an energetically favorable reaction through which the microbe can sustain itself. Secondly, temperatures less than 80ᵒ C are required to allow the slower microbial metabolisms to continuously replenish Adenosine Triphosphate (ATP) which degrades in a process called amino acid racemization in less than 30 minutes or less as temperatures exceed 80ᵒ C (Adams et al., 2006, Berhardt et al., 1984). Lastly organisms need sufficiently large pore or fracture-space that is reasonably permeable so that nutrients can continuously replenish nutrients for microbial metabolisms.

Failure to meet these requirements is known to prevent life from surviving in any environment, and even with intense adaptation, these are unlikely to be overcome

(Frederickson et al., 2001). The likelihood of microbes being pervasive within a black shale is somewhat low, however there are zones within a black shale such as the Utica-

Point Pleasant that would contribute to the habitability of microbes. Further, production of H2S gas and flow impedance from biofilms and slimes suggest that modern microbial populations inhabit the Utica-Point Pleasant formation, creating operating challenges for producers of oil and gas in eastern Ohio, Pennsylvania and West Virginia (Duncan,

2009). While their presence is detected and observed in cell count studies of produced water, their origin remains unclear (Daly et. al, 2016). Arguments can be made that these populations are introduced anthropogenically, as terrestrial microbes are not removed from water and other fluids prior to hydraulic fracturing of the Utica-Point Pleasant (Daly et. al, 2016). To compound the issue any biomass present at depth would be difficult to identify due to the amount of biomass being delivered downhole with the hydraulic fracturing slurry.

As part of a larger National Science Foundation funded study “Microbial Biodiversity and Functionality of Deep Shale and its Interfaces” with Prof. Paula Mouser as PI, this study will look at the Utica-Point Pleasant formation to determine whether or not microbes could survive in the formation in the present-day. The primary question involves whether there are micro-lithologies within the Utica-Point Pleasant that could meet or exceed the basic requirements for life (e.g. pore-space, carbon substrates and sufficiently cool temperatures).

We will address this question through the examination of the mineralogy, porosity and organic content of the individual micro-lithologies observed in the Utica and Pt Pleasant formations. While these formations as a whole is likely a poor habitat for microbes if only due to a lack of pore-space, the primary goal of this research is to identify zones within the Utica-Pt. Pleasant formations that meet the basic criteria to support microbial life. Further, while the initial source of nutrients in the Utica-Point Pleasant is seawater, what clues do the mineralogy provide for redox conditions within these micro-lithologies and can electron donors and acceptors be identified within these zones? Mineralogy and texture may yield clues as to whether pores may once have been open, allowing for the interpretation of timeframes where the formation may have been more favorable to microbes at some point in the past.

The Utica and Point Pleasant formations today have a high total organic content

(TOC>2%) along with various electron accepting iron and sulfur species known to support microbial metabolisms, and a current formation temperature mostly below 80ᵒC to the west and approaching 100o C on the eastern edge of Ohio. The geothermal gradient

8 in eastern Ohio is 22-25o C/km suggesting that samples 3-4 km deep will be cool enough to allow thermophilic bacteria to survive in most of the Utica-Point Pleasant formations

(Nathenson et al., 1988). The challenge from a microbiological perspective becomes the availability of sufficient pore-space in the Utica-Point Pleasant. Mudstones in general have porosity that is too small for known bacteria to survive, as the smallest confirmed microbes in starvation/desiccated forms are on the order of 2-300 nm, larger than most pores in a mudstone (Luef et al., 2015; Velimirov, 2001). The Utica and Point Pleasant formations, however, are not just an organic-rich mudstone as portions of the Point

Pleasant are more similar to a limestone than a shale or mudstone, and it is within these limestone like layers that original porosity may have been much higher. A feature of the

Utica-Point Pleasant that shows promise for larger pores are the thick banded packstones, often made up of imperfectly stacked brachiopod fossils. These bands will be prioritized in the search for signs that microbial life existed, as these would have likely been open pores during clay de-watering.

Geologic Setting

Depositional Environment

Understanding the deposition and stratigraphic relationships of the Utica Shale and Point

Pleasant formation and the conditions under which they formed is critical in identifying the early microbial inhabitants of the formation. During the Middle Ordovician large portions of the Laurentian continent (the landmass for much of the present day U.S. and

Canada) is inundated under shallow and expansive epicontinental seas, home to expansive, highly productive tropical carbonate platforms (Figure 1).

Figure 1. Paleogeographic map of the Middle Ordovician, Black line represents the transect of the sample suite (Modified from Colorado Plateau Geosystems http://deeptimemaps.com).

These aerobic environments would have a different microbiota than that of the Point

Pleasant sub-basin, utilizing much of the available carbon substrates prior to establishing anaerobic conditions. Off the Southern coast the Iapetus ocean basin is closing, eventually forcing a collision between the Taconic volcanic island arc and the Laurentian continent (Van Der Pluijm, 2004). The Black River limestone and the subsequent

Lexington and Trenton limestones were deposited during this quiescent low-stand period, however the presence of regionally correlative potassium-rich bentonites are indicative of powerful volcanic eruptions (Kolata et al., 1996). The presence of these bentonites in the carbonate platforms may indicate the commencement of tectonic activities that later formed the Point Pleasant sub-basin.

Figure 2. Paleogeographic map of the Late Ordovician, Black line represents the transect of the sample suite (Modified from Colorado Plateau Geosystems http://deeptimemaps.com).

By the late Ordovician, tectonic and sea-level changes began to disrupt the continued deposition of the Lexington and Trenton carbonate platforms, giving way to OM-rich mudstone deposition. The existence of these high-productivity shelf carbonates suggests shallow, tropical and oxygenated marine conditions, calculated to be somewhere near 20 degrees south latitude as shown in Figure 2 (Schumacher et al., 2013, Patchen et al.,

2006).

Figure 3 Litho-facies map showing the Point Pleasant sub-basin with the surrounding carbonate platforms (map from Patchen et al., 2006).

Surrounded by high productivity carbonate shelves on three sides as depicted in Figure 3 and underlain by a thin portion of the Trenton limestone, the basin seems to have been experiencing local subsidence and/or sea-level rise greater than that experienced by the surrounding carbonate platforms. This could have been exacerbated by several pre- existing basement tectonic features, such as the Rome Trough and East Continental Rift

Basin that has shaped much of the subsequent and antecedent sediment packages

(Patchen et al., 2006). It was not until the Taconic Orogeny was in full swing that the

Lexington and Trenton carbonate platforms were also drowned and the Utica shale was deposited over top much of the Appalachian Basin, marking the transition to high-stand sea-level conditions (Van Der Pluijm, 2004, Patchen et al., 2006). The sequence of rocks

13 from the carbonate platforms through the overlying Utica shale represent a transgressive sequence as the Appalachian basin is subsiding in response to the Taconic orogeny.

Utica-Point Pleasant Lithologic Description

The Utica shale formation underlies much of Ohio and Pennsylvania with parts extending into Kentucky, West Virginia and Western New York where it is correlative with the

Upper Indian Castle formation (Hickman et al., 2015). First described as the Utica Shale by Ebenezer Emmons in his 1842 Geology of New York: Part II, the formation was correlated by Professor Edward Orton as a member of the Cincinnati group (Logan et al.,

1922). The Utica shale is fine grained organic rich dark brown to black mudstone varying in thickness from 0 to 340 feet. The Utica is over 95% shale with some limestone dominated beds with bedding ranging from laminated to thick in the shale intervals and laminated to thin in the limestone intervals (Schumacher 2013). Though the Utica is not considered fossiliferous, the unit is biostratigraphically identified by its pelagic swimming faunal fossil assemblage primarily consisting of graptolites (Schumacher

2013).

The Point Pleasant formation is known outside of Ohio as the Clay’s Ferry formation to the south into Kentucky, and as the Lower Indian Castle formation to the north into western New York (Hickman et al., 2015). The Point Pleasant formation was first described by Edward Orton in 1873 based on an outcrop near the city of Point Pleasant,

Ohio along the Ohio River (Logan et al., 1922). At the time Orton attributed these limestone-rich beds to the upper portion of the Trenton Limestone, however they are now

14 recognized as the lowest member of the Cincinnati group. The Point Pleasant formation’s lower contact with the Lexington and Trenton limestone is gradational, becoming more argillaceous before transitioning to interbedded fine grained organic rich mudstone and carbonate facies below the Utica. The Point Pleasant’s thickness varies from 50 to 120 feet and is roughly 60% limestone on average (Schumacher 2013). This gives the Point

Pleasant formation a marly appearance as the limestone interbeds range from regular to irregular, creating a contrasting black shale and gray limestone appearance in core and leading various workers to interpret the unit as transitional, representing a change from a shallow sea to deeper water (Schumacher et al., 2013, Patchen et al., 2006). This is the specific target formation for much of today’s horizontally drilled and hydraulically fractured wells due to high organic richness and the brittle, fractural nature of the carbonate rich shale.

Methods

Sample Selection

Utilizing the various imaging and analysis techniques of the Subsurface Energy Materials

Characterization and Analysis Laboratory (SEMCAL) at The Ohio State University

School of Earth Sciences, areas of interest are defined as locations within the sample suite where mineralogical associations suggest that intense local reduction has occurred in the presence of OM, as well as areas where OM appears to be relatively unaltered as suggested by a lack of sulfides. As part of the identification process, optical light microscopy was used to locate areas within fossiliferous zones that contain differing sulfide morphologies when compared with the matrix. A commonly cited indicator of microbial activity is the creation of spherical clusters of well-ordered small pyrite euhedrons, which have been imaged in various studies and attributed to microbial activity

(e.g., Berner, 1970, Berner, 1980). While many workers believe the correlation between framboidal pyrite and microbial reduction of sulfate to be clearly illustrated in modern sediments, there are others that attribute this pyrite morphology to other diagenetic processes (Butler et al., 2000). While certain sulfide morphologies can be attributed to different stages in diagenesis with large euhedral crystals forming later as well as carbonate replacement by pyrite, the origin of framboidal pyrite is still an area of debate.

MICP

To understand pore communication and fluid flow, Mercury Injection Capillary Pressure

(MICP) analysis of the sample suite is required. Mercury is a non-wetting fluid that must be forced into available pores, unlike water which readily invades pores due to capillary pressure created through its low wetting angle. Using a step-pressurized hydraulic bath, the MICP analysis calculates pore-throat diameters using a modified Washburn equation, equating pressure required to allow for mercury intrusion to pore throat diameter

(Ethington, 1990; Kemball, 1946; Webb 2001). Equivalently sized samples were analyzed that are carbonate-rich and carbonate poor, from low and high maturity samples to further extrapolate the effect of maturation on porosity and permeability. Once selected, samples were dried in a vacuum oven at 50ᵒ C for a minimum of 24 hours to ensure humidity is baked off. Upon assembly of the penetrometer, the sample was placed under a vacuum of <50µmHG for no less than 30 minutes prior to filling the penetrometer with mercury. Upon completion of a low pressure run up to 28.8 psi (2 bar) the samples are switched to the high pressure port and pressurized step-wise to 60,000 psi. While the matrix pore network likely restricts fluid flow, it is possible the fossiliferous zones offer greater connected porosity and larger pore throats and pores due to the resistance to compression of fossil material versus the clay constituents.

Fossiliferous layers exhibiting greater connected porosity and larger pores may provide inhabitable settings for introduced microbes or allow indigenous microbes to survive diagenesis. While often sample damage is a concern when using MICP, the durable nature of the clays found in the Utica-Point Pleasant (illite, chlorite) and low content of

17 other compressible mineral phases suggest little if any grain compression would occur to add noise to the signal of mercury intruding into open pore space. MICP sample analyses were performed in-house at the Ohio State School of Earth Sciences in the SEMCAL research laboratory, using a Micromeritics Autopore IV®.

SEM

Scanning electron microscopy (SEM) analysis allows for detailed inspection of sulfide, organic and carbonate relationships in a two dimensional plane once areas of interest have been determined from reflected light microscopy. for the SEM provided images at the scales in which microbes operate, thus providing insights into potential habitats available within the rock. The FEI Quanta Field Emission Gun 250® SEM used for these images is located at the Ohio State School of Earth Sciences in the SEMCAL research group. Sample preparation for the SEM was performed using a step-wise polishing process, on a Buehler Iso-Met® polisher. Samples chosen for SEM work were representative of the background clay-carbonate matrix and carbonate dominated fossiliferous zones to compare and contrast these two dominant lithologies. Samples were then cut using a slow-speed saw in order to mount these to SEM stubs for further smoothing. After affixing the samples to SEM stubs the samples were polished for 20 minutes at 15 µm, 40 minutes at 6µm and 1µm, followed by at least 40 minutes at 0.5µm though most samples required an additional 20-40 minutes to remove scratches caused by previous steps. All polishing was performed in an ethylene glycol bath which was drained, rinsed and refilled every 20 minutes. To obtain the required highest resolution,

18 select samples were ion-milled with a Leica RES 102® to produce surfaces that are as close to perfectly flat as possible. This ensures that electrons are able to fully interact with the sample surface and reflect toward the electron detectors as efficiently and accurately as possible.

Sulfur Isotopes

While conventional separation and gas source mass spectrometry would provide useable results, the very small scale (<30µm) of pyrite framboids and the importance of their orientation with respect to surrounding mineralogy and pores makes this type of destructive method unfavorable. Secondary ion mass spectrometry (ion microprobe) was used for in-situ measurement of the 34S/32Swithout damaging the pyrite’s surrounding host habitat or disrupting the context of the pyrite grains. This portion of research has been outsourced to Prof. Mostafa Fayek and his team of experts working at the

University of Manitoba who have a Cameca IMS 7F® ion microprobe.

Micro-XCT

Although micro X-ray computed tomography (µXCT) has insufficient resolution to identify porous organic matter compared to non-porous organic matter, it does distinguish between mineral matrix grains and organic-filled pores. Encasing core in low vacuum epoxy and using a 1/8” drill bit allows for careful extraction of shale cores to use in medical grade micro XCT scanners. A custom 1 mm pure aluminum pre-filter is used to attenuate the high energy x-rays being emitted from the x-ray source, allowing for better

19 quality imaging of the dense shale, lessening beam hardening artifacts (Bazalova et al.,

2007, Klotz et al., 1990, Ketcham et al., 2001). The end-product for µXCT is a three dimensional view of the internal fabric of the rock, allowing for the interpretations of mineral and OM associations without the damage associated with polishing and grinding of a thin section. This three dimensional imagery can be utilized along with simple assumptions of pore geometry and pore volume to create fluid migration simulations. The work performed using MICP and SEM allow for reasonably accurate characterizations of pore geometry and pore volume, and the µXCT data complement these data by providing three-dimensional microbial habitability map. Three-dimensional images of these samples have been obtained on the 13 BMD Beamline at the Advanced Photon Source,

GeoSoilEnviroCARS facility, as well as the Hart Biomedical Imaging Laboratory in the

Department of Biomedical Engineering at The Ohio State University.

Matrix Assisted Laser Desorption Ionization (MALDI)

With the Utica-Point Pleasant mudstone, there are two different habits for OM, one termed intergranular in spaces between mineral grains, and one that is much larger, termed OM stringers. A short term solvent extraction should preferentially dissolve the intergranular OM due to the high surface area to volume ratio. The intergranular OM is important as this is where the OM-hosted porosity is often seen developing, suggesting it is more reactive than the larger OM objects. Utilizing a crush and dissolve method, samples from the clay-dominated matrix zone and from fossiliferous carbonate- dominated zones were ground for interrogation using MALDI. Samples are crushed to a

20 fine powder before adding 5 mL of dichloromethane to extract the more labile organic components. After a period of 48 hours 1 µL of solvent was added to 1 µL of a non- reactive matrix that responds to a 635 nm wavelength laser, ionizing whole organic matter carbon chains and sending these through a wide-field mass spectrometer. Three different matrices were used (α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxy benzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid ) as these are optimized for different organic molecules, so each sample was prepared with all three matrices in triple replicate in hopes of capturing the full spectrum of solubilized organic molecules. This research was carried out at the OSU Chemistry and BioChemistry Mass Spectroscopy lab (CBC) on a Bruker Microflex® MALDI-TOF.

Sample Suite

Figure 4 Ternary diagram illustrating the different mineralogies present in the sample suite.

The sample suite to be interrogated in this study was donated by the Chesapeake Energy

Corporation, and from appearance and identified depth intervals includes a deeper sample that can be described as an organic and carbonate-rich black mudstone, and a shallower organic and clay-rich dark grey mudstone (Figure 4). Given the depths and counties that the wells were located in, we conclude these represent the Point Pleasant and lowermost

Utica mudstones, respectively. The samples are less than 100’ in depth apart, and as such are likely to be in close proximity to the target environment of a hydraulically fractured production well and are a suitable sample suite for identifying microbial habitats that could accessed and inoculated through hydraulic fracturing. The samples span a west to east transect across Ohio and into Pennsylvania, and vary from as shallow as 3,000’ in the west to more than 10,900’ in the East, representing maturities of 0.59 to 2.49 Ro, respectively. Geochemical analysis of these rocks for maturity, TOC, and mineralogy were performed prior to obtaining the sample suite from Steve Chipera of the Chesapeake

Energy Corporation, and as such are treated as bulk samples, as minute variations with the individual rock samples is not only likely but visible to the naked eye (e.g. fossil bands, carbonate banding and variations in color).

Table 1. Sample suite with applied techniques.

Technique SW SW MU RH RH KB KB JP JP 2958 3055 4471 6528 6644 8334 8440 10855 10930 MICP X X X X X X X X SEM X X X X X X Δ34S X X X µXCT X X MALDI X X X X

In order to focus on how these rocks may inhibit or promote microbial inhabitation, end- members with respect to mineralogy and maturity were studied with the full suite of techniques as shown in Table 1. Samples for detailed study were chosen on the basis of

23 the lowest and highest maturity (depth) examples of the Utica and Pt. Pleasant. Samples between these maturities were often used for other experiments to identify trends or fill in the gaps of data to better understand the diagenetic history of the Utica-Point Pleasant system. As these samples represent varying depths and maturities from across the eastern half of Ohio and into western Pennsylvania, samples that showed markedly different features such as fossiliferous bands and or excess pyrite content as compared to the end- member samples were explored further using appropriate techniques. Discussed in detail below are end-members of the sample suite, providing a look at some key differences that the samples display at opposite ends of the maturity spectrum.

SW 2958

Figure 5. SEM image of SW 2958 matrix, note dolomite rhombohedrons (Do) carbonates (CaCO3) and Phosphates. Interspersed white Euhedrons are pyrite.

Figure 5 shows an example SEM from the shallowest and lowest maturity sample within the sample suite, with a maturity of 0.59 Ro and is a clay dominated sample according to bulk mineralogy obtained with the sample suite. Given the depth of this sample and color, this sample is believed to be from the lower Utica formation with high OM content. The carbonates observed in this sample are often dolomitized and euhedral, making up a surprisingly large portion of the matrix as shown in the SEM image above. The carbonates are highly dolomitized (as compared to similar samples in the eastern portion 25 of the basin) suggesting interaction with a magnesium-rich fluid, either due to evaporation or the thermal alteration of magnesium-rich aluminosillicates at deeper portions of the basin with subsequent magnesium-rich fluid transported up-section.

SW 3055

Figure 6. SEM image of SW 3055 matrix, note elongate organic objects.

At a depth of 3055’ in central Ohio (Figure 6), this sample underlies SW 2958 and displays the characteristic carbonate and organic-rich matrix of the Point Pleasant. Darker

26 in color than the shallower sample this rock has bands of fossil material that appears more or less intact, suggesting short transport of fossils. The relatively low abundance of clays in the matrix of the sample suggest this is more akin to a black carbonate, which is a major reason why the brittle nature of this rock is attractive to oil and gas operators.

Despite a lower clay content, there is a clear background layering suggesting quiescent but not completely still waters, and the abundance of algal-like OM stringers suggests a shallower depositional environment or rafted OM. These stringers appear to be un- reactive in higher maturity rock, or perhaps these are the source for the intergranular OM that is clay-clogged and porous in higher maturity samples.

JP 10855

Figure 7 SEM of JP 10855 matrix.

The matrix of this high maturity (2.49 Ro) sample shows more clays and silica grains, a noticeable decrease in dolomite and some large OM stringers (Figure 7). As this is most likely from the overlying Utica, the mineralogy and high OM content are not surprising features of this rock.

JP 10930

Figure 8. SEM of JP 10930 matrix, with elongate organic stringers and carbonate fossil.

A significant amount of effort was devoted to this sample, which exhibits a high maturity

(2.49 Ro) with a high carbonate content, again more akin to a black limestone (Figure 8).

Fossil beds are thin (<1 cm) and composed of brachiopod body fossils exclusively, suggesting a periodicity to bottom-water environments as these are adjacent to organic- rich black muds. These small brachiopod fossils are mostly intact, suggesting little to no transport after death and thus the bottom-water conditions were stressed while not purely anoxic or euxinic while. OM in this sample is similar to that in the shallowest sample,

29 with the exception of the appearance of the larger OM objects looking quite “ragged” having been subjected to rather high temperatures during metagenesis. There is an increase in the quantity of pyrite is observed in this sample, these are the bright flecks of cubic or framboidal habit.

Pore Assessment

SEM

Observations in the SEM show various different pore types due to the complexity that exists within the OM-rich Point Pleasant mudstone, especially with respect to how fluids may migrate through the rock. What those pathways are composed of and how the mineralogy and maturity may govern fluid migration is a critical component to understanding the survivability of microbes in this environment. Three possible modes of fluid transport can be observed within the Utica-Point Pleasant depending on mineralogical composition and maturity.

Figure 9. SEM of OM-hosted porosity in the high maturity JP 10930 sample (Ro 2.49)

The first is clearly visible in Figure (9) where porous organic matter (highlighted by yellow arrows) occupies the space between carbonate, silica and aluminosilicate minerals. This porous network of organic matter develops as the more volatile organic compounds crack into natural gas, and becomes an increasingly important fluid flow component as maturity increases. JP 10930 has a Ro of 2.49 and exhibits a great deal of porosity contained within the organic matter, hereon referred to as OM-hosted porosity.

This OM-hosted porosity can be quite extensive, and may contribute significantly to permeability of this rock due to the connected nature of the intergranular OM. Lower maturity samples contain similar intergranular organic matter, however it lacks porosity despite having similar local mineralogy (Figure 10).

Figure 10. SEM image showing non-porous OM, indicated by the yellow arrows.

Another fluid migration feature seen in the Utica-Point Pleasant were fractures, though the majority of these appear to be de-lamination fractures caused by the removal of lithostatic stress from the rock after coring and sub-sampling. Many of these fractures follow bedding planes, an expected morphology for fractures caused by the removal of overburden stress. Another sign that fractures were present in the subsurface environment are “healed” fractures containing carbonate or other minerals that were deposited after the fracture opened, eventually sealing it. While observed in other black shales, this type of fracture was not observed frequently in the Utica-Point Pleasant samples studied herein.

Some of these fractures, however appear in or near anomalous pyrite framboid zones, something that may give a clue to fluid flow through that zone. As shown in Figure 11 several examples of pyrite framboids occurring in or near these delamination fractures, often in concentrations many times greater than framboid concentrations in the mudstone

33 matrix and approaching the framboid density seen in fossiliferous zones. This degree of pyrite framboid creation may signal that local conditions were different with respect to bedding parallel fluid flow, and that a hydrogen sulfide-rich fluid was able to migrate along planes of weakness in the rock, forming these framboids and later providing a nucleation point for the delamination fractures.

Figure 11. A. Linear clusters of pyrite framboids in JP 10930 (scale bar 200 µm). B. Framboid cluster observed in a fracture in JP 10930 (scale bar 10 µm). C. Pyrite framboids aligned with organic stringer in JP 10930 (scale bar 100 µm). Bedding parellel to scale bar in A,C, perpendicular in B.

An alternate mechanism to explain why these fractures occur where they do may be simply due to the local stress field caused by the creation of the pyrite framboids, as these minerals would be more brittle than the surrounding rock, and when overburden is removed the pyrite framboids prevent the local rock from decompressing uniformly, causing a fracture. The concentration of pyrite framboids and their bedding parallel orientation suggests that their origin differs from the euhedral pyrite crystals found 34 dispersed throughout the matrix. Given the similarity of these pyrite framboids to those framboids found in the underside of brachiopods, where early permeability would have been higher simply due to greater pore-volume, similar controls may be guiding the creation of these bedding-parellel framboid clusters.

Minerals that are formed after the deposition of rock are the result of diagenesis and are considered secondary, and in mudstones diagenetic minerals are often carbonates like dolomite and calcite, as well as pyrite. A poorly understood part of the diagenetic process is the extent to which microbial processes have an impact on the development of these minerals. Certain morphologies of pyrite have been attributed to the biological reduction of sulfate in pore-waters, and a by-product of this metabolism is CO2 as shown in

Equation 1.

Equation 1. Oxidation of carbon substrates through sulfate reduction.

푦푖푒푙푑푠 2− 2− 2퐻2퐶푂 + 푆푂4 → 2퐶푂2(푎푞) + 2퐻2푂 + 푆

This equation, however simplified, provides some insight into the minerals that are likely produced during microbial reduction of sulfate. In an environment with abundant reduced iron pyrite is formed readily as a result of the interaction between reduced Sulfur and

Iron. Further in an environment that is acidic, reduced sulfur becomes a proton sink creating hydrogen sulfide, preserving this sulfur in the pore-fluids. The aqueous phase carbon dioxide if produced within an alkaline environment will precipitate carbonate cements. Given this information, special attention was paid to samples that contained

35 abundant pyrite and carbonate in order to identify any link between these two minerals that may be the result of microbes. As shown in Figure 12 within fossiliferous bands a strong positive correlation between pyrite framboids and carbonates. This is the sort of environment where micro-lithologic changes create aberrant features in the Point Pleasant mudstone, in this case an originally large pore environment adjacent to OM and nutrient- rich mudstone. Another feature of these zones are the very large (~50 to 100 µm) pyrite euhedrons, which are believed to have formed later stage as they are often seen replacing carbonate crystals and fossils.

Figure 12. Two in-filled fossil pore environments that have a pair of minerals that could be produced via microbial reduction of sulfate.

Figure 13. Various SEM images showing ovoid objects within a carbonate cement on the underside of a brachiopod. (JP 10930)

One of the most intriguing discoveries of this investigation involves small ovoid three- dimensional objects that have a very distinct morphology. These objects appear to be individual crystals that have replaced an organic ovoid-shaped object with a central sphere-like structure. In some images, there appears to be two of these objects closely linked, very suggestive of cell-splitting. These objects are shown in Figure 13, and are

37 located in a place where post-depositional microbial survival would be easily attainable due to the abundant pore space inside of a brachiopod shell. Such a region could have originally been host to abundant open pore-space with abundant nutrient flux from the adjacent OM-rich mudstone.

These objects have a long axis measurement of approximately 1 µm and a short axis of .3

µm. The organic shape of these objects is very suggestive of an origin closely related to biology, and not attributable to a rolled clay or other single mineral as these are clearly clusters of individual minerals that have coalesced or originated on an object. Two samples within the sample suite have objects displaying this morphology, suggesting that this is not an isolated event, and an exhaustive study of current published work has not yielded a reasonable alternative explanation for these objects. Given their location within what would have been open pore-space prior to cementation by carbonate, within a nutrient rich fossil bed adjacent to organic-rich black shales, it is possible that these are fossilized microbes, or that the crystals comprising these objects are nucleated around the remains of microbes. Prior to the discovery of these ovoid objects it was hypothesized that these interfaces between shelly limestone-like beds and adjacent organic rich mudstones would provide a comfortable habitat prior to being filled in. with the discovery of these features suggestive microbial shape provide further evidence of the existence of habitats available for microbes to inhabit early in the diagenetic cycle of an organic and fossil rich black mudstone.

It is possible that these objects are not what they appear to be, however, a suitable explanation for their organic forms has not been found, lending credence to the validity to the hypothesis that microlithologic changes such as fossil hash beds within an OM-rich black mudstone would likely by the last place in which a microbe may find safe harbor prior to being sterilized from the environment due to amino acid racemization. Heavily pyritized undersides in brachiopods strongly suggest a microbial influence, such as this example housed within a brachiopod shell, shown in Figure 14.

Figure 14. Pyrite framboids and euhedrons occupy the underside of a brachiopod hash bed in carbonate cement (JP 10930).

It is within these micro-lithologic changes that during the early stage of diagenesis, prior to carbonate cement in-filling that one may suspect microbes would find a hospitable habitat. The poorly crystalline nature of these carbonate cements suggest impurities (clay is clearly visible on the right side of Figure 14) and almost certainly came after deposition as they are filling the underside of more durable carbonate fossils. Without this cement these habitats would likely have harbored life and the microbe-sized mineral assemblages imaged in Figure 13 suggest that there may have been microbial life in these habitats after deposition and that they may have contributed the aqueous CO2 that later crystallized into the cement.

MICP

Due to the fossiliferous zones being mineralized and cemented with carbonate (as seen in the SEM images of the previous section), it is likely that the fossiliferous zones in the present-day formation are barriers to fluid-flow as opposed to the conduits they likely were prior to mineralization. In lieu of this for analyzing pores and pore-throats active today a decision was made to target zones that represent the matrix of the mudstone in an attempt to analyze the contribution to porosity from mineral interfaces and OM-hosted porosity. For MICP, eight samples from across central Ohio to western Pennsylvania were selected for analysis, ranging in maturity from a low of 0.59 Ro to 2.49 Ro. Table 2 below lays out the relevant bulk mineralogy and geochemical data for the eight samples for analysis with MICP.

Table 2. MICP sample details with maturity, mineralogy and TOC

Sample identifier SW SW RH RH KB KB JP JP 2958 3055 6528 6964 8334 8440 10855 10930 Maturity (Ro) 0.59 0.59 1.06 1.06 1.62 1.62 2.49 2.49 Carbonate (%) 20.6 48.8 16.3 57.2 14.3 49.3 15.3 52.3 Clay (%) 44.9 24.8 54.0 20.2 54.5 21.5 54.3 21.8 TOC (wt%) 4.5 3.2 1.9 3.5 1.9 3.6 1.4 4.1

These samples were then cut into 3-4 rectangular shaped blocks roughly 1.5 g each in order to ensure thickness remained less than 500 microns to allow greater access to the pore systems present in the rock. Using samples cut on a saw as opposed to being crushed and/or selected as chips, reduces the surface area presented to the mercury. Mercury is a non-wetting fluid and requires pressure to come into contact with the sample. The more rough this surface is, the more pressure is required for the mercury to fully contact the sample before it can begin probing the pore-systems contained within the rock. This process of getting the mercury into contact with the sample can create a large signal, indeed if a sample is sufficiently “rough” this early noise can often amount to a greater signal than the mercury intruding into the true pore-system contained there-in. Through careful sample preparation this noise can be reduced, which is why this technique was used for this study.

Another critical component is selecting a sample that is sufficiently representative of the rock matrix, which required a visual inspection to identify macro-fossiliferous material that may be present in the blocks. While fractures and fossils may provide conduits of

41 enhanced permeability and porosity, by excluding these known high-permeability features a better understanding of the whole-rock permeability can be obtained. For an example of the prepared sample prior to MICP analysis, see images of sample and penetrometer in Appendix B as well as comparison of clay and carbonate dominated samples. While the pressures remain the same, early on looking at the extrusion curve

(where pressures are reduced in a step-wise fashion as well) it seemed that mercury continued to intrude into the sample throughout the extrusion curve, suggesting that the amount of time the sample was pressurized for was insufficient to fully investigate the pore-system of the rock.

Figure 15. Results of the equilibration experiment on KB 8440

In an effort to understand what an appropriate equilibration time is, a simple experiment was performed to determine whether 10, 100, or 1000 seconds was a sufficient amount of time to allow mercury to infiltrate the pore-system. The resulting intrusion/extrusion data are shown in Figure 15. With each increase in the equilibration time, more mercury intruded into the sample’s pore-system, and more mercury remained in the pore-system after releasing the pressure from the sample suggesting much of this mercury was trapped inside of the pore-system, in a manner often described as “ink bottle theory” (Geisch,

2007). After commencing pressure drawdown, the 10 second sample displays a large positive hump in the extrusion curve, suggesting mercury is continuing to intrude into the sample despite the pressure decreasing until roughly 10,000 psi. This suggests that though the pore-system accessible above 10,000 psi is accessed during intrusion, it continues to fill with mercury after pressure release, indicating a10 second equilibration time is insufficient. This results in an incomplete interrogation of the pore-system accessed at pressures greater than 10,000 psi. Moving to a 100 second equilibration time, the positive hump in the extrusion curve is smaller and true extrusion commences at roughly 30,000 psi, indicating this equilibration time was still insufficient to fully investigate the pore-system. At 1000 seconds the positive hump after pressure drawdown had decreased significantly, suggesting that mercury was able to fully intrude the pore- system at the limit of interrogation for this instrument. This experiment shows that an increase in equilibration time decreases the size of the positive hump in the extrusion curve, suggesting that nearly all accessible porosity was filled with mercury at 1000 seconds.

Another concern with MICP is sample compaction/mineral distortion (damage), and that perhaps longer equilibration times may allow for greater distortion of the sample fabric.

While a convincing argument could be made that long term stresses on an object may cause plastic deformation, in the case of a material largely composed of brittle mineral grains, the strain caused by a given amount of stress is likely to be a brittle failure response, which is instantaneous. Thus the additional pore-throats investigated by the preceding experiment with equilibration times are unlikely to be sourced from material failure as failure due to pressure should be instantaneous, and the resulting intrusion graphs should be similar regardless of equilibration time. The observed behavior in these data more closely resembles what we should see in a rock that maintains its fabric, forcing the mercury to travel through the same pore-system as oil, gas or water would have to move in the subsurface.

Due to the high surface area of shales, mercury porosimetry “measures” a good deal of porosity at low pressures, a process that SEMCAL refers to as “shrink-wrap”. This initial porosity has nothing to do with the porosity within a shale, and is attributed to mercury being pressed against a high surface area rock. This results in an artificial bi-modal pore structure as illustrated well in Figure 15 by the plateau in the data between 100 and 1000 psi. As such the data representing the internal porosity of the rock is measured at pressures greater than 1000 psi, and is best illustrated on a graph with the Y-axis representing the percentage of porosity accessed, and the X-axis representing the pore- throat diameter through which this porosity is accessed. Illustrating these data in this manner provides a useful interpretation of what the dominant pore-throat size is, and an

44 effective comparison of samples based on their true porosity rather than a measure of the roughness of their surfaces.

Figure 16. MICP results for carbonate dominated samples, legend in order from least to most mature.

The first four samples to be discussed represent the deeper, carbonate-dominated mudstone interpreted to be the Point-Pleasant. Figure 16 shows that dominant pore- throats for the Point Pleasant mudstone range between 10 and 5 nm, with the largest pore- throat observed in the highest maturity sample at 10nm. This result is compelling as the

OM produces more and more gas, the porosity housed within the OM would have to store more gas creating larger pores and pore-throats. There is also a visible trend that with

45 greater maturity, the dominant pore-throats open up from 5 nm to 10 nm as shown in

Figure 16. The relationship between maturity and porosity within the carbonate- dominated Point Pleasant is correlative, with the highest maturity sample having the greatest differential porosity at the largest pore-throat of 10 nm. These results align with the SEM observations made earlier, that with increasing maturity the role that OM-hosted porosity plays in fluid-flow increases.

Figure 17. MICP results for clay-dominated samples.

In the clay-dominated samples (Figure 17) the clay has a negative effect on total porosity

(note the scale on the Y-axis) and the pore-throat sizes have become smaller. The limit of MICP is 4 nm, and three of the four samples show an obvious up-tick at this pore- 46 throat size, indicating the dominant pore-throat is smaller than the resolution of the porosimeter. Though the correlation between maturity and higher porosity is tenuous in these clay-dominated samples, it is present suggesting that it has some effect on total porosity. These data suggest that in general maturity has an impact on porosity in both sample sets.

Figure 18. Graphs of porosity accessed through pore-throats smaller than 50 µm vs maturity, total organic carbon, carbonate content, and clay content.

When plotting the net porosity accessed through pore-throats smaller than 50 µm against bulk mineralogy and geochemistry data, some other controls emerge. In the carbonate- dominated sample set, the two best correlative controls on net porosity are related to organic matter, with the clearest control being the total organic carbon (TOC) with an R2 value of 0.9915, followed by maturity with an R2 value of 0.9339. With abysmally low correlative values with respect to mineralogy (R2 0.0897 for carbonate and 0.2847 for clay) the sample set at first suggests that mineralogy has little to do with affecting porosity. When looking at the clay-dominated samples, the opposite correlations are true with a high positive correlation between clay content and porosity (R2 = 0.977) and a strong negative correlation between carbonate content and porosity (R2 = 0.9891). TOC also has a positive correlation with porosity (R2 = 0.9319) suggesting that the pore network within the OM is playing a role in increasing measured porosity. However the problem here is that when taken as a whole, the samples themselves separate into a high porosity carbonate dominated sample set and a low porosity clay-dominated sample set.

This is problematic at first, considering TOC and maturity govern the porosity in the carbonate sample set and seem to have little correlation with mineral content, yet as a whole the sample set is more porous with larger pore throats. This can be attributed to the fact that there is more clay within the mudstone matrix restricting access to the pore- system hosted within the OM in samples, and forcing mercury to access this system through the tiny pore-throats in the clay. The carbonate dominated sample, having less clay restricting access to the pore-system allows mercury to travel through the larger

48 pore-throats contained in the OM, allowing the dominant signal to come from the pore- throats of the OM-hosted porosity. Through the same OM-hosted pore system exists in both sample sets, the doorways to access these pore-systems is controlled by how much clay is inter-mixed in the matrix with the OM.

Geochemical Assessment

Origin of Pyrite and the Role of Microbes

Figure 19. A.) Large euhedron replacing carbonate, small framboids scattered throughout. B.) Heavily pyritized OM, framboids and very tiny cubic pyrite crystals. C.) Pyrite inside pelycopod fossil. D.) Pyrite framboid cluster in matrix. E.) Non-framboidal pyrite in OM. F.) Weathered pyrite among bi-pyramidal pyrite crystals.

The origin of pyrite has and will remain a topic of research in the marine mudstone environment. Pyrite habits differ in the Utica-Point Pleasant mudstone depending on the

50 associated mineral assemblage, and examples of the dominant habits are shown in Figure

19. These pyrite crystals theoretically have different origins discussed at length by other authors in the isotope geochemistry field (Ohmoto et al., 1996, Berner et al., 1980, Ohfuji et al., 2005). Pyrite framboids are generally considered to be developed early on through biological reduction of pore-water sulfate (Figure 19 B and D), while the larger pyrite euhedrons replacing other minerals such as carbonate are considered to be late stage diagenetic products (Figure 19 A and F). OM stringers often contain both pyrite framboids and small dodecahedral and cubic pyrite crystals, or strictly dodecahedrons.

One way of ascertaining the relationship of pyrite and biology is by looking at the isotopic signature of sulfur in pyrite. Biological processes naturally fractionate 32S and

34S through the metabolic activities of sulfate-reducing microbes, resulting in waste products enriched in the lighter isotope, in some cases by as much as 40‰ with an organic electron donor (Kohn et al., 1998, Chambers et al., 1979). Certain morphologies of pyrite have been attributed to microbial reduction of sulfate, specifically small spherical clusters of pyrite called framboids (Berner, 1980). These small clusters of pyrite as well as pyrite dodecahedrons are often smaller than 20 µm in diameter, the diameter of the ion beam used in Secondary Ion Mass Spectroscopy at the University of Manitoba,

Canada where samples were sent for analysis. Large pyrite euhedrons and several clusters of pyrite framboids are >20 µm in diameter allowing for δ34S measurement.

Figure 20. Temperature-dependent isotopic fraction factors for various sulfur species (calculated from Ohmoto et al., 1982).

As shown in Figure 20 the fractionation factor between sulfate and hydrogen sulfide is many times greater at lower temperatures than at high temperatures. Many ore-deposit sulfides have positive isotopic values near that of the parent sulfate due to the low fractionation as temperatures approach 1000⁰ C (Ohmoto et al., 1982). For a system like the Utica-Point Pleasant, where maximum temperatures are likely to have been less than

250⁰ C across much of the basin, the left-hand portion of Figure 20 can be ignored for the

time being. This leaves a minimum thermal fractionation factor of ~25‰, allowing for late stage thermodynamically driven sulfate reduction to achieve positive δ34S values as thermogenic sulfate reduction exhausts available sulfate in a closed system, depicted in

Figure 21.

Figure 21 Open versus closed system fractionation of sulfate, at 250⁰ C starting with a δ34S of 30‰ (Calculated with data from Ohmoto 1982).

With the introduction of microbial reduction of sulfate, early fractionation of sulfate is driven by the availability of electron donors and the extent to which microbes can survive 53 in the sediment column. In a less permeable rock such as a mudstone the system can be considered closed with respect to equilibrium conditions, and the degree to which sulfate is processed is likely to be variable depending on how long microbes can act on the closed system in a given habitat prior to running out of electron donating carbon substrates or pore-space. This depletes the 32Sin the sulfate pool prior to the onset of thermogenic sulfate reduction, allowing for heavier δ34S than that produced by thermogenic sulfate reduction alone due to starting with a heavier δ34S pool of sulfate than seawater values.

Table 3. Summary results of SIMS measurements.

Sample Maturity 0.59 Ro 0.78 Ro 2.49 Ro Number of Framboids 0 4 2 sampled Framboid average δ34S N/A -12.63‰ -3.05‰

Framboid standard N/A ±5.42 ±19.46 deviation Number of Euhedrons 12 6 12 sampled Average Euhedral δ34S +13.9‰ +9.18‰ +11.76‰

Euhedral standard ±1.72 ±.95 ±1.91 deviation

Pyrite framboids measured in this sample suite have values ranging from -8 to -19‰

δ34S, lighter than the euhedrons measured by a significant margin (Table 3). Assuming a seawater sulfate during the Ordovician to be approximately 28‰ δ34S these framboids 54 have fractionated by -41.5 ± 5.1‰ agreeing nicely with the stated fractionation factor of -

45 ± 20‰ (Ohmoto et al., 1990). This is an expected result as pyrite framboids are commonly attributed to microbial processing of pore-water SO4 in seafloor sediments after anoxic conditions are established. The framboid to framboid variability within each sample suggests a variable amount of microbial sulfate reduction occurred. In other words in a closed system an early formed framboid will most likely have the lightest

δ34S, and in environments where microbes persisted longer (i.e. in a large habitat such as a fossil with large pores) the sulfate pool is more processed resulting in heavier framboids. An example of an “isotopically heavy” cluster of framboids is shown in

Figure 22, with a signal that is likely the combination of the small and large framboids, as the inset SEM image shows the darker interaction area of the ion microprobe. With as many samples as possible, SEM images of the sample spots were taken to insure that the signal was coming from the intended target. Especially with pyrite framboids being smaller than the interaction area of the ion microprobe a mixed signal from another sulfide in the area would have a significant impact on the signal.

Figure 22. Many pyrite framboids in a small habitat within a fossiliferous zone. MU

While some framboids exist as individual isolated spheres, targeting them with the ion beam proved to be a challenge. In order to best illustrate the framboid δ34S variability within each sample, another group of framboids is shown in Figure 23. This cluster of framboids are occupying the space within what is likely the remnants of a crinoid stem or other fossil, note the pentagonal symmetry and a size of ~50 µm. The signal coming from this aggregate of framboids is the lightest of the sample set, yet appears to be in an environment where microbes may have persisted for some time. Both aggregates of framboids are near fossiliferous zones, and likely had some fluid migration supplying sulfate from the surrounding sediment prior to mineral in-filling.

Figure 23. Cluster of pyrite framboids occupying a pentagonally shaped carbonate, likely a crinoid stem or other fossil. MU 4471

Figure 24. Multiple SIMS measurements on euhedrons and framboids (JP 10930, 2.49 Ro).

Figure 24 best illustrates the challenges of measuring the δ34S of pyrite framboids, as the signal from the framboids (yellow arrow) is washed out by the adjacent large grain of pyrite in the upper third of the beam interaction area. Also illustrated well in Figure 24 is the lower variability in euhedral pyrite grains, giving some confidence into the thermogenic origins of the parent hydrogen sulfide. Euhedral pyrite grains studied in this project yielded low standard deviations grain to grain within a sample, and 2 of the three samples followed a trend that suggested increased temperature brought an increase in the

δ34S of pyrite euhedrons. The least mature sample, however did not follow this trend and had an average δ34S of 13.9‰, making these pyrite euhedrons the heaviest measured in this sample set. There were 12 pyrite euhedrons analyzed with a standard deviation of

1.7‰ which is reasonably low suggesting that the pyrite euhedrons, and the sulfate pool from which they came were heavier than the equivalent higher maturity samples. A clue to the reason behind this heavier δ34S in the lowest maturity sample may come from the high dolomitization observed in the SEM. It may be possible that the hydrogen sulfide responsible for forming these pyrite grains came from a hotter source or a more evolved pore-fluid and migrated into the formation along fossiliferous bands, examples of which are shown in Figure 25. Another possibility is that the fossiliferous bands within this sample provided habitats for microbes to survive longer than the other two samples, extensively reducing sulfate over a longer period of time and enriching the pore-fluids in heavier sulfate. However thermogenic fractionation of sulfate at 100⁰ C results in a fractionation factor of almost 45‰, compared to 25‰ at 250⁰ C, requiring the sulfate pool from which the pyrite’s hydrogen sulfide evolved to be more than 20‰ heavier than

Figure 25. Heavy δ34S pyrite euhedrons near a fossiliferous zone in the lowest maturity sample, SW 3055

the sulfate pool in JP 10930. This would require a significant amount of microbial reduction of sulfate to have taken place in this sample prior to the onset of thermogenic sulfate reduction.

These data indicate that pyrite framboids do have a microbial origin, and that euhedral pyrite grains replacing carbonate are a later stage of pyrite formation, indicating less fractionation between the parent sulfate pool and the daughter hydrogen sulfide. These results seem reasonable, with varying degrees of microbial sulfate reduction producing greater deviation in the pyrite framboid δ34S and the assumed later stage pyrite having a positive δ34S signature that has a low standard deviation. The unexpected result was that instead of δ34S following thermal maturity with the lowest maturity sample having the

59 lightest δ34S and the highest maturity the heaviest, the lowest maturity sample has the heaviest δ34S. For the full SIMS data set see Appendix C.

Rapid Assessment of Mobile Organic Matter

Figure 26. Matrix Assisted Laser Desorption Ionization Mass Spectrometry spectra for four samples, two low maturity (SW 2958 and SW 3055) and two high maturity (JP 10855 and JP 10930) samples.

Organic matter characterization often involves the thermal destruction of organic molecules in a pyrolysis process, analyzing the hydrocarbons produced through artificial cooking of crushed rock samples. As an alternative, MALDI TOF is a soft ionization

61 technology, resulting in carrying whole organic molecules into the mass spectrometer.

Initial results (Figure 26) indicate relative abundance of small organic molecules (mass

>900 daltons) decrease with increasing maturity, however most of the signal peaks are present in both high and low maturity samples. These results were unexpected, as it was believed that the short time-frame would be difficult to extract larger organic molecules from the crushed sediment. Clearly larger organic molecules were extracted, with peaks centered at ~250, ~400 and 640 daltons suggesting some amino acids may have been extracted as these are often seen with a mass of 400 daltons. Blank MALDI samples spots were prepared for each of the three matrices, Sinapinic Acid (SA), Alpha-Cyano-4-

Hrdroxycinnamic Acid (CHCA), and 2,5-Dihydroxybenzoic Acid (DHB), and each of the four samples had 3 spots prepared with each matrix. Though the intensity varies graph to graph due to the nature of MALDI, in general, intensity is equivalent to the number of molecules measured, the two lower maturity samples have orders of magnitude greater intensity than the more mature samples. This makes sense, as the lower maturity samples showed a significant color change during the solvent extraction process, as shown in

Figure 27.

The most promising results were obtained using SA and CHCA, developing a method for future use of MALDI with respect to OM characterization in OM-rich mudstones.

Dicholormethane proved to be an effective solvent for extracting organic molecules and amino acids from shale without the need to acidify the sample to remove carbonate and aluminosilicate minerals as has been done previously. For raw MALDI spectra of matrices and individual samples prepared with SA and CHCA, see Appendix D

Understanding the Spatial Context of Organic Matter and Pyrite

Figure 28. High density objects are white in this grayscale µXCT image. Yellow 3D objects have framboid-like appearance, suggesting white objects are pyrite.

Originally µXCT was utilized in with the expectations of visualizing OM in three dimensions, and simulate how fluids may migrate through the OM-hosted porosity. A periphery goal was to understand the relationship between pyrite and OM, and establish

64 any relationship the two may have. With µXCT, high density objects are easily observed, as seen in Figure 28, with framboid-like objects with diameters of roughly 20 µm clearly composed of small (~2-4 µm) cubic crystals. Large OM objects can also be seen, however, a common issue with µXCT is that the scanners are built to scan low-density objects, and introducing x-rays to a wider range of densities decreases the ability to record subtle differences in material density (Ketcham et al., 2001). This limits the scanner from discerning fine-grained intergranular matter which hosts porosity, limiting the usefulness of this technique in mapping porous networks. While quantifying a porous network may be beyond the resolution of the scanners used at the Advanced Photon

Source, identifying large organic objects (such as the stringers imaged with SEM ) can be accomplished fairly well by adjusting the grayscale values down to highlight OM and filtering out objects with volumes less than 50 µm. The closer the object size is to the resolution of the machine, the resulting data becomes difficult to separate from instrument noise, forcing the prioritization of targets at an appropriate scale to instrument resolution. Looking at a mudstone as if it were a simple 3 phase system, you can loosely group the major rock components into OM and associated clays (1g/cc) carbonate, phosphate, silica and aluminosillicates (~2.65-2.8 g/cc) and pyrite or other metallic sulfides (5g/cc). Fossil matter can often be identified by the uniform x-ray attenuation creating a recognizable three dimensional object, especially if the fossil is near lower density objects such as OM or clay.

Figure 29. A.) µXCT image of JP 10930, 3D pyrite grains and framboids, 2D OM stringer. B.) 3D pyrite turned off, perpendicular to bedding µXCT 2D image. C and D.) OM stringer in two different bedding parallel orientations.

These three phases are resolvable utilizing µXCT and reveal important qualitative information, such as the spatial distribution of OM and pyrite as shown in Figure 29.

Utilizing three dimensional micro-tomography allows a qualitative analysis of the quantity and distribution of the end-member components of an organic-rich mudstone, such as sulfides and organic-matter. While the resolution of the scanner could not pick up

66 on the subtle changes between porous and non-porous OM, it shows the relationship between OM stringers and high-density objects, such as those shown in Figure 30.

Yellow objects are created by thresholding grayscale values in excess of those of carbonate, while red objects are the result of a porosity analysis wizard that separates objects with grayscale values lower than a user-set value and filters out objects smaller than a user-set value, in this case objects smaller than 25µm.

Figure 30. µXCT image of JP 10930, showing several large OM stringers (OM is red) and dispersed pyrite (Pyrite is yellow).

The challenge with µXCT is that it requires significant amount of user input to determine what grayscale value corresponds to density. In order to use this technique to its fullest, ground-truthing minerals to grayscale is critical in a complex mudstone such as the Utica-

Point Pleasant. Even after collecting reflected-light, SEM and µXCT data and locating the same pyrite cluster within them, a reasonable method for decreasing noise and extending the detection limit to identify intergranular OM proved a difficult task to accomplish. In Figure 31 a comparison of techniques are shown to illustrate what is reasonably discernible in µXCT. The 20µm euhedral pyrite grain is easily distinguishable as well as the somewhat smaller phosphate grain above it, however the mass of pyrite framboids are indistinguishable as individual framboids. Intergranular OM is significantly smaller than the pyrite framboids and indistinguishable in the µXCT and reflected light images.

Figure 31. Connecting reflected light, µXCT and SEM across multiple techniques and length scales. (Image taken from 2015 URTEC presentation by Buchwalter et al. 2015)

Though the data was less useful for understanding the pore-system within the OM, the 3 dimensional nature of the OM stringers would have been easily overlooked, and µXCT provides an excellent method for viewing individual objects down to approximately 25

µm.

Conclusions

Based on the results of this study, some conclusions as to the habitability of the Utica-

Point Pleasant can be drawn. Microbial populations left their mark on pyrite framboids through sulfate cycling, with higher variability and lighter δ34S values than thermogenic sulfate reduction produced in pyrite euhedrons measured with SIMS. Clusters of framboids within fossiliferous zones suggest these were areas where either microbes survived for a longer period of time or more microbes were processing sulfate within these zones, and one of these contained a framboid aggregate with a -8.7 ‰ δ34S, indicating that either the pool of sulfate was anomalously higher prior to microbial reduction of sulfate, or that microbial reduction of sulfate in that location was longer lived and pushed the δ34S of the remaining sulfate higher.

Among the various observations made with the SEM, two stand out with respect to ancient microbial habitats. The first is the observation that where framboidal pyrite has accumulated, so too has a sugary carbonate cement, which is important as aqueous phase

CO2 is also a byproduct of the microbial reduction of sulfate allowing for the possibility that these cements are biogenic in origin as well as the pyrite. The second and most intriguing observation are the 1 µm ovoid objects found within the underside of a brachiopod fossil in the sugary carbonate cement. Their size and organic shape are highly suggestive of a biological origin, and being found within a zone that also harbors pyrite 70 framboids and poorly crystalline carbonate cement further hinting at the possibility that these are fossilized microbes by identifying a metabolic pathway that fits the mineralogy.

Lastly the location of these objects in a fossiliferous zone adjacent to OM-rich mudstones provides a diffusive pathway for organic substrates and terminal electron acceptors to support a microbial population in what can be termed a “deep shale interface”. Though very small these fossiliferous zones fit the description of a fossiliferous limestone, adjacent to OM-rich mudstones and prior to mineralization by carbonate cements these zones would have had significantly more porosity than the adjacent mudstones.

MICP data illustrates the importance of mineralogy and OM-hosted porosity with respect to fluid flow through the matrix of the Utica-Point Pleasant. The shallower clay-rich samples are much tighter with respect to dominant pore-throat, and the greatest control on porosity is mineralogical, followed by total organic content. In the clay-rich samples, clay platelets act as a barrier to accessing the porosity hosted within the organic matter, and have a positive correlation between clay content and net porosity. The deeper, carbonate- rich samples show little correlation between mineralogy and porosity, however the data indicate total organic carbon and maturity are the biggest controls on porosity, followed by a negative relationship between clay content and net porosity, whereas clay content increases, the clay platelets act as barriers to the larger pore-throats contained within the

OM-hosted porosity.

These observations combined would suggest that for a period of time between deposition and mineralization, these fossiliferous zones would have provided a habitat rich in carbon substrates and with access to pore-fluids being expelled from the adjacent mudstones.

These fossiliferous zones today appear to be tightly sealed by these carbonate cements, and are now barriers to fluid flow at least prior to anthropogenic exploitation of the formations oil and gas reserves. These carbonate layers, being more brittle than the surrounding mudstones readily fracture, once again becoming critical to fluid flow in the

Utica-Point Pleasant. It is likely that within these freshly fractured zones that anthropogenically introduced microbial populations may find fracture space and food sources that allow for re-inhabitation.

Along with microbes, terminal electron acceptors are also transported downhole into the formation due to this area’s coal mining history. Water for hydraulic fracturing is sourced locally from surface waters, many of which have elevated sulfate and heavy metal contents due to coal mine drainage. This combination of stagnant ponds and lakes combined with run-off from coal mines allows anaerobic bacteria to establish an environment in the lakes and ponds. When this water is used in hydraulic fracturing, it provides an initial environment for the microbes to inoculate the subsurface environment.

Other investigators have found that not only are surficial microbes introduced into the hydraulically fractured shale system, the system establishes an anaerobic bacterial ecosystem that is diverse at first, waning to one composed almost entirely of

Halanaerobium, a producer of hydrogen through fermentation of sugars (Cluff et al.,

2014).

Figure 32. SEM image of JP 10855 with a fossiliferous zone that is nearly completely filled with pyrite.

Environments similar to fossiliferous zone shown in Figure 32 would have provided the most accessible environment for a microbial population, with abundant nutrients being expelled with pore-water from the adjacent mudstones during compaction. This steady in- flux of carbon substrate and terminal electron acceptors once supplied a microbial

73 population with an adequate habitat in these fossiliferous zones. Once fossiliferous zones like this are accessed with hydraulic fracturing and become inoculated, they will likely again provide a habitat.

Future Research

Further research on the topic of subsurface microbial habitats will continue through the

NSF dimensions study. Prior to the completion of this document, I reached out to our collaborators at the University of Manitoba to look further at Sulfur isotopes, and found them to be booked through early next year. In lieu of this, several additional samples from both the overlying Utica and Point Pleasant have been identified for further SIMS measurements. These samples will help establish the trends observed during this research, and will focus more on the framboid clusters that are present in the formation in hopes of bolstering the number of data points for framboids.

A different approach, and one that could be performed in-house at SEMCAL is measurement of the carbonate cement δ13C that has in-filled the fossiliferous layers.

Should this carbonate cement prove to be of biologic origin, this would support the hypothesis that the last habitat for a microbe in the Point Pleasant is in these fossiliferous zones. A challenge with this measurement will be obtaining a sufficiently small drill bit to remove the carbonate cement from the underside of the overlying brachiopods. This proved to be a challenge with performing this analysis during my tenure at the

University.

While µXCT lacks the resolution to image porosity in OM, dual beam focused ion beam milling (FIB) has the capability to reveal OM-hosted porosity, and to image the small 75 ovoid-shaped objects found in carbonate cement in the fossiliferous zones. The challenge here was locating a sample spot in the SEM that had a sufficiently interesting series of elements to warrant the cost of dual beam FIB. JP 73 certainly still holds promise, the habitat hosting the ovoid microbe-like objects was destroyed through polishing in an attempt to obtain higher quality SEM images.

Organic matter characterization is another potential research area to expand into in an attempt to identify the source of the two different types of organic matter observed in the

SEM, the intergranular more mobile OM versus the large (>100µm) OM stringers. One avenue of research that could be pursued involves Infrared Raman probing, which can analyze the ratio of hydrogen to carbon within the OM in question. This could be done at

The Ohio State University in the Chemistry department, however sample selection challenges exist. Large OM objects will be easy to find and measure in the instrument, however the smaller intergranular OM is too tiny to see with the reflected light microscope. Another technique available to us at The Ohio State University is Matrix

Assisted Laser Desorption and Ionization Mass Spectrometry. While initial results presented here indicate this is a worthwhile pursuit, lessons learned from the initial experiment have yet to be implemented into a second experiment.

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Appendix A: SEM images

Figure 33. Cross-sections of ovoid objects observed outside of matrix and adjacent to carbonate cement. Similar objects are observed in the carbonate cement.

Figure 34. SEM image of poorly crystalline carbone cement in the underside of a brachiopod.

Figure 35. Low maturity OM stringers in MG 5778, note smooth well-defined edges.

Figure 36. OM-stringer in JP 10930, high maturity sample. Note less well-defined edges.

Appendix B: MICP data

Figure 37. Example of the prepared sample loaded into a penetrometer (A) and on weigh paper showing sawn surfaces (B).

Figure 38. MICP data for SW 2958 (Clay-rich) and SW 3055 (Carbonate-rich).

Figure 39. MICP data for RH 6528 (Clay-rich) and RH 6644 (Carbonate-rich).

Figure 40. MICP data for KB 8334 (Clay-rich) and KB 8440 (Carbonate-rich).

Figure 41. MICP data for JP 10855 (Clay-rich) and JP 10930 (Carbonate-rich).

Appendix C: SIMS Sulfur Isotopes

Sample Sample spot name Pyrite Morphology Δ34S SW 35 Ches-SW35-10-01 Euhedron 12.8 Ches-SW35-10-02 Euhedron 12.0 Ches-SW35-7-01 Euhedron 14.3 Ches-SW35-6-01 Euhedron 17.1 Ches-SW35-8-01 Euhedron 16.3 Ches-SW35-4-01 Euhedron 13.9 Ches-SW35-3-01 Euhedron 14.5 Ches-SW35-3-02 Euhedron 14.4 Ches-SW35-5-01 Euhedron 13.6 Ches-SW35-5-02 Euhedron 13.0 Ches-SW35-1-01 Euhedron 13.7 Ches-SW35-2-01 Euhedron 10.7 Table 4. SIMS data for SW 3955, Ro 0.59.

Sample Sample spot name Pyrite Morphology Δ34S MU 61 Ches-MU61-04-01 Framboid -18.9

Ches-MU61-07-01 Framboid -15.3

Ches-MU61-02-01 Framboid -8.8

Ches-MU61-03-01 Framboid -7.5

Ches-MU61-10-02 Euhedron 8.1

Ches-MU61-10-01 Euhedron 8.7

Ches-MU61-08-01 Euhedron 8.9

Ches-MU61-09-01 Euhedron 9.1

Ches-MU61-01-01 Euhedron 9.3

Ches-MU61-06-01 Euhedron 10.9 Table 5. SIMS data for MU 4471, Ro 0.78. 89

Sample Sample spot name Pyrite morphology Δ34S JP 73 Ches-4-1 Framboid -16.8 Ches-3-1 Euhedron 7.8 Ches-6-3 Euhedron 9.4 Ches-8-1 Euhedron 9.8 Ches-6-1 Euhedron 10.6 Ches-7-2 Framboid 10.7 Ches-7-1 Euhedron 11.3 Ches-2-2 Euhedron 11.4 Ches-6-4 Euhedron 11.6 Ches-0-2 Euhedron 11.8 Ches-2-1 Euhedron 11.9 Ches-7-3 Euhedron 12.3 Ches-1-1 Euhedron 12.9 Ches-1-2 Euhedron 13.0 Table 6. SIMS data for JP 10930, Ro 2.49.

Appendix D: MALDI Spectra

Figure 42. MALDI spectra of Sinapinic Acid and Dichloromethane.

Figure 43. MALDI spectra of Alpha-Cyano-40Hydroxycinnamic Acid (CHCA) and Dicholoromethane (DCM solvent).

Figure 44. MALDI Spectra for SW 2958 prepared with CHCA.

Figure 45. MALDI spectra for SW 3055 prepared with CHCA.

Figure 46 MALDI Spectra for JP 10855 prepared with CHCA.

Figure 47. MALDI spectra for JP 10930 prepared with CHCA.

Figure 48. MALDI spectra for sinapinic acid (SA).

Figure 49. MALDI spectra for SW 2958 prepared with SA

Figure 50. MALDI spectra for SW 3055 prepared with SA. 97

Figure 51. MALDI spectra for JP 10855 prepared with SA.

Figure 52. MALDI spectra for JP 10930 prepared with SA.