Microbial colonization and persistence in deep fractured shales is guided by metabolic exchanges and viral predation

Abstract

Background: Microbial colonization of subsurface shales following hydraulic fracturing offers the opportunity to study coupled biotic and abiotic factors that impact microbial persistence in engineered deep subsurface ecosystems. Shale formations underly much of the continental USA and display geographically distinct gradients in temperature and salinity. Complementing studies performed in eastern USA shales that contain brine-like fluids, here we coupled metagenomic and metabolomic approaches to develop the first genome-level insights into ecosystem colonization and microbial community interactions in a lower-salinity, but high-temperature western USA shale formation.

Results: We collected materials used during the hydraulic fracturing process (i.e., chemicals, drill muds) paired with temporal sampling of water produced from three different hydraulically fractured wells in the STACK (Sooner Trend Anadarko Basin, Canadian and Kingfisher) shale play in OK, USA. Relative to other shale formations, our metagenomic and metabolomic analyses revealed an expanded taxonomic and metabolic diversity of microorganisms that colonize and persist in fractured shales. Importantly, temporal sampling across all three hydraulic fracturing wells traced the degradation of complex polymers from the hydraulic fracturing process to the production and consumption of organic acids that support sulfate- and thiosulfate-reducing bacteria. Furthermore, we identified 5587 viral genomes and linked many of these to the dominant, colonizing microorganisms, demonstrating the key role that viral predation plays in community dynamics within this closed, engineered system. Lastly, top-side audit sampling of different source materials enabled genome-resolved source tracking, revealing the likely sources of many key colonizing and persisting taxa in these ecosystems.

Conclusions: These findings highlight the importance of resource utilization and resistance to viral predation as key traits that enable specific microbial taxa to persist across fractured shale ecosystems. We also demonstrate the importance of materials used in the hydraulic fracturing process as both a source of persisting shale microorganisms and organic substrates that likely aid in sustaining the microbial community. Moreover, we showed that different physicochemical conditions (i.e., salinity, temperature) can influence the composition and functional potential of persisting microbial communities in shale ecosystems. Together, these results expand our knowledge of microbial life in deep subsurface shales and have important ramifications for management and treatment of microbial biomass in hydraulically fractured wells. Video Abstract.

Keywords: Metabolomics; Metagenomics; Shale; Subsurface; Thermotoga; Viruses.

Fig. 1

Sampling design for input and produced fluid samples with metagenomic sequencing from the STACK shale play. Input samples obtained during the development of the well (A) are color coded to match the produced fluid timeseries for each well (B) in which they are associated with. Drill muds were collected from a nearby, drilling operation and are thus not colored to match the three STACK wells

Fig. 2

Temporal dynamics of the 24 MAGs representing the dominant and persisting taxa (> 5% relative abundance at in at least one sample) in the three distinct STACK shale play wells (STACK-14, STACK-16, STACK-17). Relative abundances were calculated from the metagenomic read recruitment to MAGs as described in the methods. The relative abundance of each MAG is indicated by the width of its respective band in the alluvial plot at each timepoint, with the most abundant MAG on top and least abundant on the bottom and colored by respective taxonomy. Completeness estimates for each MAGs are listed following MAG taxonomy, and unique identifiers for each MAG are listed in parentheses. Trends in alpha diversity through time are shown above each plot for each well

Fig. 3

Carbon flow in the STACK shale play. From left to right, complex polymers may be degraded by inferred fermentative microorganisms and converted to organic acids, which could be utilized by sulfate- and thiosulfate-reducing microorganisms. Color of each MAG oval corresponds to taxonomic classification, and the size of each circle within each MAG indicates max. % relative abundance for the STACK 16, 17, & 14 wells, respectively. Completeness estimates for each MAG are listed after the MAG taxonomy in the key. All graphs depicting organic acid concentrations are in μM measurements. Solid lines between inferred fermenters (far left) and organic acids indicate genomic potential and statistical prediction to that metabolite via sPLS (VIP>2), while dashed lines only indicate genomic potential. All other solid lines between MAGs and complex polymers, and between organic acids and sulfate reducers, indicate genomic potential for degradation or uptake, respectively.

Fig. 4

Key MAGs encoding taxa inferred to be involved in sulfide generation within the STACK shale play ecosystem. Colored circles indicated the respective MAG contained genomic evidence of the gene in the specified pathway to transform tetrathionate/thiosulfate/sulfate to sulfide. Completeness estimates for each MAG are provided after taxonomy in the key

Fig. 5

Viral-host dynamics in the STACK shale play. A Visual representation of each of the 24 STACK MAGs “relevance” and viral connections. Relevance is evaluated by the number of samples where a MAG is present, and the maximum relative abundance that each MAG reaches (considering any given sample). Each MAG is depicted as a colored circle, with a solid line indicating the presence of CRISPR-Cas viral defense system and dashed the absence of one. Small, connected circles represent the viral linkages, and the dashed gray line connecting virus-to-virus indicates an identical spacer sequence (but likely not an identical virus). B Evaluation of viral and host dynamics where linkages could be made. Relative abundances of hosts and the summed relative abundance of their linked viruses are plotted for each timepoint that the host is present, revealing that the most abundant viruses are associated with the most abundant microbial hosts

Fig. 6

16S rRNA gene relative abundances of Thermotogae across US shale formations and their respective salinities, as reported in this study (STACK, DJ Basin) and previously by others (Utica, Marcellus, Bakken, Three Forks)