Long-term CRISPR locus dynamics and stable host-virus co-existence in subsurface fractured shales.

Viruses are the most ubiquitous biological entities on Earth. Even so, elucidating the impact of viruses on microbial communities and associated ecosystem processes often requires identification of unambiguous host-virus linkages-an undeniable challenge in many ecosystems. Subsurface fractured shales present a unique opportunity to first make these strong linkages via spacers in CRISPR-Cas arrays and subsequently reveal complex long-term host-virus dynamics. Here, we sampled two replicated sets of fractured shale wells for nearly 800 days, resulting in 78 metagenomes from temporal sampling of six wells in the Denver-Julesburg Basin (Colorado, USA). At the community level, there was strong evidence for CRISPR-Cas defense systems being used through time and likely in response to viral interactions. Within our host genomes, represented by 202 unique MAGs, we also saw that CRISPR-Cas systems were widely encoded. Together, spacers from host CRISPR loci facilitated 2,110 CRISPR-based viral linkages across 90 host MAGs spanning 25 phyla. We observed less redundancy in host-viral linkages and fewer spacers associated with hosts from the older, more established wells, possibly reflecting enrichment of more beneficial spacers through time. Leveraging temporal patterns of host-virus linkages across differing well ages, we report how host-virus co-existence dynamics develop and converge through time, possibly reflecting selection for viruses that can evade host CRISPR-Cas systems. Together, our findings shed light on the complexities of host-virus interactions as well as long-term dynamics of CRISPR-Cas defense among diverse microbial populations.

Dissolved organic matter within oil and gas associated wastewaters from U.S. unconventional petroleum plays: Comparisons and consequences for disposal and reuse.

Wastewater generated during petroleum extraction (produced water) may contain high concentrations of dissolved organics due to their intimate association with organic-rich source rocks, expelled petroleum, and organic additives to fluids used for hydraulic fracturing of unconventional (e.g., shale) reservoirs. Dissolved organic matter (DOM) within produced water represents a challenge for treatment prior to beneficial reuse. High salinities characteristic of produced water, often 10× greater than seawater, coupled to the complex DOM ensemble create analytical obstacles with typical methods. Excitation-emission matrix spectroscopy (EEMS) can rapidly characterize the fluorescent component of DOM with little impact from matrix effects. We applied EEMS to evaluate DOM composition in 18 produced water samples from six North American unconventional petroleum plays. Represented reservoirs include the Eagle Ford Shale (Gulf Coast Basin), Wolfcamp/Cline Shales (Permian Basin), Marcellus Shale and Utica/Point Pleasant (Appalachian Basin), Niobrara Chalk (Denver-Julesburg Basin), and the Bakken Formation (Williston Basin). Results indicate that the relative chromophoric DOM composition in unconventional produced water may distinguish different lithologies, thermal maturity of resource types (e.g., heavy oil vs. dry gas), and fracturing fluid compositions, but is generally insensitive to salinity and DOM concentration. These results are discussed with perspective toward DOM influence on geochemical processes and the potential for targeted organic compound treatment for the reuse of produced water.

In Situ Enhancement and Isotopic Labeling of Biogenic Coalbed Methane.

Subsurface microbial (biogenic) methane production is an important part of the global carbon cycle that has resulted in natural gas accumulations in many coal beds worldwide. Laboratory studies suggest that complex carbon-containing nutrients (e.g., yeast or algae extract) can stimulate methane production, yet the effectiveness of these nutrients within coal beds is unknown. Here, we use downhole monitoring methods in combination with deuterated water (D2O) and a 200-liter injection of 0.1% yeast extract (YE) to stimulate and isotopically label newly generated methane. A total dissolved gas pressure sensor enabled real-time gas measurements (641 days preinjection and for 478 days postinjection). Downhole samples, collected with subsurface environmental samplers, indicate that methane increased 132% above preinjection levels based on isotopic labeling from D2O, 108% based on pressure readings, and 183% based on methane measurements 266 days postinjection. Demonstrating that YE enhances biogenic coalbed methane production in situ using multiple novel measurement methods has immediate implications for other field-scale biogenic methane investigations, including in situ methods to detect and track microbial activities related to the methanogenic turnover of recalcitrant carbon in the subsurface.

Utica Shale Play Oil and Gas Brines: Geochemistry and Factors Influencing Wastewater Management.

The Utica and Marcellus Shale Plays in the Appalachian Basin are the fourth and first largest natural gas producing plays in the United States, respectively. Hydrocarbon production generates large volumes of brine ("produced water") that must be disposed of, treated, or reused. Though Marcellus brines have been studied extensively, there are few studies from the Utica Shale Play. This study presents new brine chemical analyses from 16 Utica Shale Play wells in Ohio and Pennsylvania. Results from Na-Cl-Br systematics and stable and radiogenic isotopes suggest that the Utica Shale Play brines are likely residual pore water concentrated beyond halite saturation during the formation of the Ordovician Beekmantown evaporative sequence. The narrow range of chemistry for the Utica Shale Play produced waters (e.g., total dissolved solids = 214-283 g/L) over both time and space implies a consistent composition for disposal and reuse planning. The amount of salt produced annually from the Utica Shale Play is equivalent to 3.4% of the annual U.S. halite production. Utica Shale Play brines have radium activities 580 times the EPA maximum contaminant level and are supersaturated with respect to barite, indicating the potential for surface and aqueous radium hazards if not properly disposed of.

Repetitive Sampling and Control Threshold Improve 16S rRNA Gene Sequencing Results From Produced Waters Associated With Hydraulically Fractured Shale.

Sequencing microbial DNA from deep subsurface environments is complicated by a number of issues ranging from contamination to non-reproducible results. Many samples obtained from these environments - which are of great interest due to the potential to stimulate microbial methane generation - contain low biomass. Therefore, samples from these environments are difficult to study as sequencing results can be easily impacted by contamination. In this case, the low amount of sample biomass may be effectively swamped by the contaminating DNA and generate misleading results. Additionally, performing field work in these environments can be difficult, as researchers generally have limited access to and time on site. Therefore, optimizing a sampling plan to produce the best results while collecting the greatest number of samples over a short period of time is ideal. This study aimed to recommend an adequate sampling plan for field researchers obtaining microbial biomass for 16S rRNA gene sequencing, applicable specifically to low biomass oil and gas-producing environments. Forty-nine different samples were collected by filtering specific volumes of produced water from a hydraulically fractured well producing from the Niobrara Shale. Water was collected in two different sampling events 24 h apart. Four to five samples were collected from 11 specific volumes. These samples along with eight different blanks were submitted for analysis. DNA was extracted from each sample, and quantitative polymerase chain reaction (qPCR) and 16S rRNA Illumina MiSeq gene sequencing were performed to determine relative concentrations of biomass and microbial community composition, respectively. The qPCR results varied across sampled volumes, while no discernible trend correlated contamination to volume of water filtered. This suggests that collecting a larger volume of sample may not result in larger biomass concentrations or better representation of a sampled environment. Researchers could prioritize collecting many low volume samples over few high-volume samples. Our results suggest that there also may be variability in the concentration of microbial communities present in produced waters over short (i.e., hours) time scales, which warrants further investigation. Submission of multiple blanks is also vital to determining how contamination or low biomass effects may influence a sample set collected from an unknown environment.

Environmental Drivers of Differences in Microbial Community Structure in Crude Oil Reservoirs across a Methanogenic Gradient.

Stimulating in situ microbial communities in oil reservoirs to produce natural gas is a potentially viable strategy for recovering additional fossil fuel resources following traditional recovery operations. Little is known about what geochemical parameters drive microbial population dynamics in biodegraded, methanogenic oil reservoirs. We investigated if microbial community structure was significantly impacted by the extent of crude oil biodegradation, extent of biogenic methane production, and formation water chemistry. Twenty-two oil production wells from north central Louisiana, USA, were sampled for analysis of microbial community structure and fluid geochemistry. Archaea were the dominant microbial community in the majority of the wells sampled. Methanogens, including hydrogenotrophic and methylotrophic organisms, were numerically dominant in every well, accounting for, on average, over 98% of the total Archaea present. The dominant Bacteria groups were Pseudomonas, Acinetobacter, Enterobacteriaceae, and Clostridiales, which have also been identified in other microbially-altered oil reservoirs. Comparing microbial community structure to fluid (gas, water, and oil) geochemistry revealed that the relative extent of biodegradation, salinity, and spatial location were the major drivers of microbial diversity. Archaeal relative abundance was independent of the extent of methanogenesis, but closely correlated to the extent of crude oil biodegradation; therefore, microbial community structure is likely not a good sole predictor of methanogenic activity, but may predict the extent of crude oil biodegradation. However, when the shallow, highly biodegraded, low salinity wells were excluded from the statistical analysis, no environmental parameters could explain the differences in microbial community structure. This suggests that the microbial community structure of the 5 shallow, up-dip wells was different than the 17 deeper, down-dip wells. Also, the 17 down-dip wells had statistically similar microbial communities despite significant changes in environmental parameters between oil fields. Together, this implies that no single microbial population is a reliable indicator of a reservoir's ability to degrade crude oil to methane, and that geochemistry may be a more important indicator for selecting a reservoir suitable for microbial enhancement of natural gas generation.