Browsing by Author "Wilkins, Michael J., advisor"
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Item Open Access Metagenomic insights into microbial colonization & persistence in subsurface fractured shales(Colorado State University. Libraries, 2023) Amundson, Kaela K., author; Wilkins, Michael J., advisor; Wrighton, Kelly C., committee member; Borch, Thomas, committee member; Ross, Matthew, committee memberMicroorganisms are pervasive yet important components of hydraulically fractured shale systems. Subsurface shales harbor oil & gas and require unconventional techniques, such as hydraulic fracturing, to access these trapped hydrocarbons. Shale microbiomes are of crucial importance as they can directly impact the recovery of oil & gas and associated infrastructure. The overarching theme of this dissertation was to characterize the metabolisms and key traits that underpin the colonization and persistence of fractured shale microbiomes using a multi-omic approach to better understand the microbial impact on this important ecosystem. In Chapter 1, I first discuss the importance of subsurface shales as an important energy reserve, summarize what is known about microorganisms in these ecosystems, and highlight the strength of using a metagenomic approach to studying shale microbiomes. Subsurface shales are heterogeneous – varying in their mineral content, temperature, and other physiochemical conditions. The microbial communities that persist can have substantial impact on the fractured shale ecosystem and contribute to common challenges in hydrocarbon recovery such as corrosion, souring, and bioclogging. The literature review presented here highlights the need to study the functional potential of shale microbiomes as most studies have mostly focused solely on taxonomic composition of persisting microbial communities, and a vast majority of these studies have focused on samples from wells in the Appalachian Basin. However, functional potential of shale microbiomes across a variety of physiochemical conditions must be considered in order to gain an understanding of the role of microorganisms and what possible influences they may have on hydraulically fractured shales systems. Here, I highlight the need (1) to study the whole community at a functional scale and (2) apply a metagenomic approach to a variety of less characterized shale basins to gain a holistic understanding of shale microbiomes and the effects they may have on the broader ecosystem. In Chapter 2, I apply this metagenomic approach to study the persisting shale microbiomes of three fractured shale wells in the Anadarko Basin – a western shale basin characterized by elevated temperature and salinity. No studies using metagenomics had been applied to shale basins in the western United States prior to this research. We sampled five wells in the Anadarko basin over a timeseries >500 days and preformed NMR metabolomics and metagenomic sequencing to uncover the dominant metabolisms, community composition, and other functional traits of the Anadarko shale microbiome. This system was dominated by a fermentative microbial community and a less-abundant sub-community of inferred sulfate reducing microorganisms. Using paired NMR metabolomics and metagenomics, I demonstrated how many fermentative microorganisms have the potential to degrade common complex polymers, such as guar gum, and have potential to produce organic acids that may serve as electron donors for sulfate and thiosulfate reducing microorganisms. Thus, in this study I provided a framework for how carbon may move through the closed fractured shale ecosystem to sustain the microbial community. Finally, I investigated viral presence and diversity across all thirty-six metagenomes and found that inputs were large sources of viral diversity, but that only an extremely small proportion of viruses recovered from produced fluids were genetically similar to viruses previously reported from fractured shales. I observed that a majority of the dominant and persisting genomes encoded a CRISPR-Cas viral defense system, likely in response to the viral community. This highlights viral defense as another key trait for persisting microorganisms, as viruses are the only predators to bacteria and archaea in fractured shale ecosystems. Overall, this study expanded our knowledge of sulfate and thiosulfate reducing microorganisms in fractured shales, demonstrated the potential for common chemical inputs such as guar gum to be utilized by shale microbiomes, and highlighted how other key traits, such as CRISPR-Cas viral defense systems, may be a crucial trait for persisting shale microbiomes. Building on results from viral analyses in Chapter 2, in Chapter 3 I next sought to investigate the temporal dynamics between hosts and viruses to better understand the role of microbial defense against viruses in fractured shale ecosystems. To do this, I sampled six shale wells in the Denver-Julesburg Basin for >800 days, performed metagenomic sequencing, and identified host (bacterial & archaeal) and viral genomes from this data. I observed evidence of ongoing host defense to viral predation at both the community and genome-level through quantifying spacers from CRISPR arrays in metagenomic reads and MAGs. Through these analyses leveraging timeseries sampling and age differences between the shale wells, I provided evidence that suggested migration toward CRISPR arrays that may be more efficient at protecting the microbial host against a wider suite of viruses. Finally, I observed a temporal increase in host-viral co-existence in the closed, fractured shale ecosystem – suggesting the CRISPR defense does not entirely protect against viral predation. Chapter 4 ultimately leverages the approaches, insights, and data gained from Chapters 2 and 3 to study shale microbiomes at a cross-basin, geographic scale. Here, I collected samples from many collaborators who have previously worked in shale systems, performed metagenomic sequencing, and processed all samples in a standardized pipeline to build a comprehensive genomic shale database. In total, this database contains 978 unique MAGs and >7 million unique genes recovered from 209 metagenomic samples obtained from 36 fractured shale wells spanning eleven shale basins from North America, China, and the United Kingdom. In this chapter I analyze the functional potential of shale microbiomes at a genome-resolved level to better understand the geographic distribution of microbial metabolisms and other key traits that likely contribute to colonization and persistence of microorganisms. Here I also leveraged bioinformatic tools to build a custom annotation summary toolkit to process and analyze the large amount of sequencing data for traits of interest. The complete absence of a taxonomic core microbiome across shale basins illustrated in this chapter underscores the necessity of a genome-resolved and functional approach to studying shale microbiomes. Results from analyzing shale microbiomes at this scale could ultimately help to inform microbial management of fractured shale systems. The final chapter of this dissertation (Chapter 5) summarizes the key findings of my research into fractured shale microbiomes, and the mechanisms that may promote microbial colonization, persistence, and survival in these relatively harsh and economically relevant ecosystems. In this chapter I conclude this work by discussing future directions and lingering knowledge gaps for studying fractured shale microbiomes, as well as implications of these findings for other subsurface engineered ecosystems. Ultimately, this body of work contributes a substantial amount of new and informative insights into the functional potentials of persisting shale microbiomes across broad geographic scales.Item Open Access Tracking the impact of wildfire on the soil microbiome across temporal scales(Colorado State University. Libraries, 2024) Nelson, Amelia Rose, author; Wilkins, Michael J., advisor; Hall, Ed, committee member; Borch, Thomas, committee member; Rhoades, Charles, committee member; Wrighton, Kelly, committee memberAs climate change progresses, the western United States is experiencing shifting wildfire behavior to more frequent and severe wildfires. Wildfires reduce soil microbial biomass and alter the soil microbiome community composition, selecting for "pyrophilous" microbial taxa with encoded traits that enable them to persist during wildfire or thrive in the soil thereafter. The soil microbiome is a key player in ecosystem carbon (C) cycling through the mediation of soil organic matter decomposition and stabilization. In addition to post-fire shifts in the soil microbiome, wildfire decreases soil C pools through combustion and alters C quality via fire-induced transformations to aromatic pyrogenic C (PyC). The intricate interplay between wildfire-induced alterations to soil microbiome composition and function, and subsequent ecosystem C cycling, remains poorly understood across different temporal and spatial scales. Leveraging multi-omics data alongside soil chemistry information (e.g., mass spectrometry) can offer insights into how shifting wildfire behavior may influence microbially mediated C cycling in forest ecosystems across the western US. To address this knowledge gap, I developed an extensive multi-omic dataset from burned Colorado subalpine coniferous forest soils collected over time (spanning 1 to 60 years following burning) and disturbance severity (low and high fire severity). This dataset includes 108 metagenomes and 12 metatranscriptomes, resulting in 1651 metagenome-assembled genomes (MAGs) that represent many of the dominant putative pyrophilous taxa previously identified in compositional studies. This dissertation presents the key findings derived from this comprehensive dataset, with the primary goal of addressing how wildfire impacts the soil microbiome with a focus on microbial interactions with soil C. Chapter 1 serves as a comprehensive literature review, providing an overview of prior research relevant to the research presented thereafter. It underscores the timely relevance of this dissertation research by examining how wildfire behavior is shifting globally with climate change and anthropogenic forcing. Given the critical role of forest ecosystems as significant global C sinks, understanding the repercussions of wildfires on ecosystem biogeochemistry is imperative. I broadly summarize previous research regarding severe wildfire impacts to soils and the soil microbiome and focus on existing knowledge gaps regarding the function of the post-wildfire soil microbiome across differing burn severities and time since fire. In Chapter 2, I characterize how burn severity impacts the soil microbiome one year post-fire in Colorado (CO) subalpine coniferous forests using soil samples collected in July 2019 from within the 2018 Ryan and Badger Creek fire burn scars that represent a burn severity gradient (control, low, moderate, and high severity burned soils). I used a suite of tools to understand both the impacts to soil chemistry and the soil microbiome, including Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to characterize dissolved soil organic matter, 16S rRNA gene and ITS amplicon sequencing for soil microbiome composition, and coupled metagenomics and metatranscriptomics to identify shifts in soil microbial functional potential. The combination of these tools allowed me to characterize the entire soil microbiome, including bacteria, fungi, and viruses. From metagenomic sequencing, I recovered 637 MAGs, 1982 unique DNA and RNA viral populations, and 2 fungal genomes from low and high severity burned soil samples. I broadly found that Actinobacteria dominated the fraction of enriched and active bacterial taxa within high severity surficial soils and exhibited traits (e.g., heat resistance, fast growth, expression of genes for degrading aromatic PyC) that enabled them to survive the soil heating and thrive after the disturbance. Ectomycorrhizal fungi (EMF), key symbionts of coniferous trees and other plant taxa, were depleted in severely burned soils. Lastly, there were abundant viruses targeting dominant Actinobacteria MAGs that likely played important roles in assembly of the post-wildfire soil microbiome and serve as top-down controls of C cycling within the system. Overall, this study served as a holistic and comprehensive snapshot of the post-wildfire soil microbiome at one point in time and laid the foundation for forming hypotheses and guiding the subsequent studies. Building upon the groundwork laid in Chapter 2, Chapter 3 broadly evaluates the relative importance of putative pyrophilous traits identified between one year and 11 years following wildfire. Additionally, I explored the applicability of other proposed conceptual life history strategy frameworks (e.g., Y-A-S framework) in defining post-wildfire soil microbial dynamics. I utilized a series of soil samples collected from a chronosequence of CO wildfire burn scars representing 1, 3, 5, and 11 years following low- and high-severity wildfire. Using genome-resolved metagenomic approaches and combining this newly generated MAG catalog with the MAGs reconstructed from sequencing in Chapter 1 resulted in a total of 825 bacterial MAGs. Again, this dataset was coupled to various soil chemistry datasets, microbial biomass measurements (via PLFA), and marker gene sequencing data. I found that the potential for fast growth was an important bacterial trait driving dominance in the post-wildfire soil microbiome for up to approximately 11 years post-fire. Moreover, I observed that MAGs investing in traits aimed at acquiring diverse resources from the external environment often dominated severely burned soils, aligning with the 'A' strategy outlined in the Y-A-S framework. These insights suggest that microbial trait profiles play a pivotal role in shaping post-wildfire soil microbial successional dynamics. Furthermore, the study marks a significant step towards unraveling how trait-based frameworks can offer valuable insights into post-disturbance microbial ecology. In Chapter 4, the focus shifts to investigating one of the most extreme scenarios that can occur in a terrestrial ecosystem with severe wildfire: a burning-induced aboveground vegetation shift. Pile burning is a common fuel reduction or site preparation practice wherein logging residue is burned on the forest floor and, because of the high soil temperatures often reached during pile burning, can serve as a surrogate for studying impacts to soil caused by severe wildfire. Following clear-cut harvesting, pile burning can lead to the creation of persistence openings dominated by herbaceous plants within successfully regenerating conifer forest. In this study, a paired 60-year chronosequence of burn scar openings and surrounding forests that regenerated after clear-cut harvesting provided a unique opportunity to study soil microbiome changes associated with two distinct ecosystem development trajectories (i.e., burning-induced aboveground vegetation shift, regenerating coniferous forest). The primary objective was to identify whether the belowground soil microbiome exhibited resilience to a disturbance-induced aboveground vegetation shift. I collected soils from the aforementioned chronosequence and interrogated soil microbiome composition (via marker gene sequencing), functional potential (via metagenomics), and function (via laboratory incubations). There were compositional shifts in the soil microbiome that mirrored the ongoing aboveground vegetation shifts, with short-term changes to microbial community composition and C cycling functionality closely resembling a post-wildfire soil microbiome (e.g., PyC degradation). However, over the six-decade chronosequence the soil microbiome composition and function both displayed resilience, converging with that of the surrounding regenerating forest. This final research chapter extended the findings from the previous studies by exploring the longevity of wildfire impact to the soil microbiome in the extreme case of a burning-induced aboveground vegetation shift. The final chapter (Chapter 5) summarizes the key findings of this doctoral research and discusses potential research implications and applications along with future research directions and remaining knowledge gaps. In summary, the aims of this dissertation research were to identify how burn severity influences the soil microbiome composition and function one year post-fire (Chapter 2), assess the longevity of these impacts and the applicability of conceptual traits-based frameworks to the post-fire soil microbiome (Chapter 3), and evaluate the resilience of the belowground soil microbiome to a burning-induced multidecadal aboveground vegetation shift (Chapter 4). This research significantly advances our understanding of the impacts of wildfires on crucial forest ecosystems, with a specific emphasis on ecosystem C cycling.