Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

Michigan Technological University

With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples of the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities.

Metagenomic applications in environmental monitoring and bioremediation.

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by , , , , and  species, while the 5°C consortia were dominated by several species of the genera , , and  Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills.  and , known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of  was observed. At 25°C, , , and  were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly,  was greatly stimulated by the addition of oil at 5°C. While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms.

Deep marine oil spills like the Deepwater Horizon (DWH) in the Gulf of Mexico have the potential to drastically impact marine systems. Crude oil contamination in marine systems remains a concern, especially for countries around the Mediterranean Sea with off shore oil production. The goal of this study was to investigate the response of indigenous microbial communities to crude oil in the deep Eastern Mediterranean Sea (E. Med.) water column and to minimize potential bias associated with storage and shifts in microbial community structure from sample storage. 16S rRNA amplicon sequencing was combined with GeoChip metagenomic analysis to monitor the microbial community changes to the crude oil and dispersant in on-ship microcosms set up immediately after water collection. After 3 days of incubation at 14 °C, the microbial communities from two different water depths: 824 m and 1210 m became dominated by well-known oil degrading bacteria. The archaeal population and the overall microbial community diversity drastically decreased. Similarly, GeoChip metagenomic analysis revealed a tremendous enrichment of genes related to oil biodegradation, which was consistent with the results from the DWH oil spill. These results highlight a rapid microbial adaption to oil contamination in the deep E. Med., and indicate strong oil biodegradation potential.

Rapid Response of Eastern Mediterranean Deep Sea Microbial Communities to Oil.

Oil biodegradation as a weathering process has been extensively investigated over the years, especially after the Deepwater Horizon blowout. In this study, we performed microcosm experiments at 5 °C with chemically dispersed oil in non-amended seawater. We link biodegradation processes with microbial community and metagenome dynamics and explain the succession based on substrate specialization. Reconstructed genomes and 16S rRNA gene analysis revealed that Bermanella and Zhongshania were the main contributors to initial n-alkane breakdown, while subsequent abundances of Colwellia and microorganisms closely related to Porticoccaceae were involved in secondary n‑alkane breakdown and beta‑oxidation. Cycloclasticus, Porticoccaceae and Spongiiabcteraceae were associated with degradation of mono- and poly-cyclic aromatics. Successional pattern of genes coding for hydrocarbon degrading enzymes at metagenome level, and reconstructed genomic content, revealed a high differentiation of bacteria involved in hydrocarbon biodegradation. A cooperation among oil degrading microorganisms is thus needed for the complete substrate transformation.

Microbial community and metagenome dynamics during biodegradation of dispersed oil reveals potential key-players in cold Norwegian seawater.

The environmental impacts of hydraulic fracturing, particularly those of surface spills in aquatic ecosystems, are not fully understood. The goals of this study were to (1) understand the effect of previous exposure to hydraulic fracturing fluids on aquatic microbial community structure and (2) examine the impacts exposure has on biodegradation potential of the biocide glutaraldehyde. Microcosms were constructed from hydraulic fracturing-impacted and nonhydraulic fracturing-impacted streamwater within the Marcellus shale region in Pennsylvania. Microcosms were amended with glutaraldehyde and incubated aerobically for 56 days. Microbial community adaptation to glutaraldehyde was monitored using 16S rRNA gene amplicon sequencing and quantification by qPCR. Abiotic and biotic glutaraldehyde degradation was measured using ultra-performance liquid chromatography--high resolution mass spectrometry and total organic carbon. It was found that nonhydraulic fracturing-impacted microcosms biodegraded glutaraldehyde faster than the hydraulic fracturing-impacted microcosms, showing a decrease in degradation potential after exposure to hydraulic fracturing activity. Hydraulic fracturing-impacted microcosms showed higher richness after glutaraldehyde exposure compared to unimpacted streams, indicating an increased tolerance to glutaraldehyde in hydraulic fracturing impacted streams. Beta diversity and differential abundance analysis of sequence count data showed different bacterial enrichment for hydraulic fracturing-impacted and nonhydraulic fracturing-impacted microcosms after glutaraldehyde addition. These findings demonstrated a lasting effect on microbial community structure and glutaraldehyde degradation potential in streams impacted by hydraulic fracturing operations.

Impacts of Glutaraldehyde on Microbial Community Structure and Degradation Potential in Streams Impacted by Hydraulic Fracturing.

Microbial adaptation to extreme conditions takes many forms, including specialized metabolism which may be crucial to survival in adverse conditions. Here, we analyze the diversity and environmental importance of systems allowing microbial carbon monoxide (CO) metabolism. CO is a toxic gas that can poison most organisms because of its tight binding to metalloproteins. Microbial CO uptake was first noted by Kluyver and Schnellen in 1947, and since then many microbes using CO via oxidation have emerged. Many strains use molecular oxygen as the electron acceptor for aerobic oxidation of CO using Mo-containing CO oxidoreductase enzymes named CO dehydrogenase. Anaerobic carboxydotrophs oxidize CO using CooS enzymes that contain Ni/Fe catalytic centers and are unrelated to CO dehydrogenase. Though rare on Earth in free form, CO is an important intermediate compound in anaerobic carbon cycling, as it can be coupled to acetogenesis, methanogenesis, hydrogenogenesis, and metal reduction. Many microbial species-both bacteria and archaea-have been shown to use CO to conserve energy or fix cell carbon or both. Microbial CO formation is also very common. Carboxydotrophs thus glean energy and fix carbon from a "metabolic leftover" that is not consumed by, and is toxic to, most microorganisms. Surprisingly, many species are able to thrive under culture headspaces sometimes exceeding 1 atmosphere of CO. It appears that carboxydotrophs are adapted to provide a metabolic "currency exchange" system in microbial communities in which CO arising either abiotically or biogenically is converted to CO  and H  that feed major metabolic pathways for energy conservation or carbon fixation. Solventogenic CO metabolism has been exploited to construct very large gas fermentation plants converting CO-rich industrial flue emissions into biofuels and chemical feedstocks, creating renewable energy while mitigating global warming. The use of thermostable CO dehydrogenase enzymes to construct sensitive CO gas sensors is also in progress.

Life on the fringe: microbial adaptation to growth on carbon monoxide.

Many freshwater environments experience dramatic seasonal changes with some systems remaining ice-covered for most of the winter. Freshwater systems are also highly sensitive to environmental change. However, little is known about changes in microbial abundance and community composition during lake ice formation and times of persistent ice cover. The goal of this study is to characterize temporal dynamics of microbial communities during ice formation and persistent ice cover. Samples were collected in triplicate, five days per week from surface water in the Keweenaw Waterway between November and April. Environmental conditions along with microbial abundance and microbial community composition was determined. Distinct community composition was found between ice-free and ice-covered time periods with significantly different community composition between months. The microbial community underwent dramatic shifts in microbial abundance and diversity during the transitions into and out of ice cover. The richness of the microbial community increased during times of ice cover. Relatives of microbes involved in nitrogen cycling bloomed during times of ice cover as sequences related to known nitrifying taxa were significantly enriched during ice cover. These results help to elucidate how microbial abundance and diversity change over drastic seasonal transitions and how ice cover may affect microbial abundance and diversity.

Microbial Community Dynamics During Lake Ice Freezing.

Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA. Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.

Surface Water Microbial Community Response to the Biocide 2,2-Dibromo-3-Nitrilopropionamide, Used in Unconventional Oil and Gas Extraction.

In the past, ballast water has been a key vector in the ship-mediated dispersal of invasive species. Here, we evaluate the potential for port microorganisms to enter and colonize the hull and bilge water of ships. Due to the small size and ubiquitous nature of bacteria, they also have the potential to be spread through hull fouling and bilge water discharge. The goal of this study was to identify the extent to which the boat microbial community is shaped by the microbial community in the port water where the boat spends most of its time. Here, we compared the microbial communities of the hull and bilge compartments of 20 boats to those of the port water in 20 different ports in five regions around the world. We found that there was a significant difference in microbial diversity between boat and port microbial communities. Despite these differences, we found that  were present at high abundances in the bilge water of most vessels. Due to the limited light in the bilge, the presence of  suggests that port microorganisms can enter the bilge. Using source-tracking software, we found that, on average, 40% of the bilge and 52% of the hull microbial communities were derived from water. These findings suggest that the bilge of a vessel contains a diverse microbial community that is influenced by the port microbial community and has the potential to serve as an underappreciated vector for dispersal of life. Invasive species have been a worldwide problem for many years. However, the potential for microorganisms to become invasive is relatively underexplored. As the tools to study bacterial communities become more affordable, we are able to perform large-scale studies and examine bacterial communities in higher resolution than was previously practical. This study looked at the potential for bacteria to colonize both boat surfaces and bilge water. We describe the bacterial communities on boats in 20 shipping ports in five regions around the world, describing how these microorganisms were similar to microorganisms found in port water. This suggests that the water influences the bacterial community of a boat and that microorganisms living on a boat could be moved from place to place when the boat travels.

Global Comparison of the Bacterial Communities of Bilge Water, Boat Surfaces, and External Port Water.

Biocides used in unconventional oil and gas (UOG) practices, such as hydraulic fracturing, control microbial growth. Unwanted microbial growth can cause gas souring, pipeline clogging, and microbial-induced corrosion of equipment and transportation pipes. However, optimizing biocide use has not been a priority. Moreover, biocide efficacy has been questioned because microbial surveys show an active microbial community in hydraulic fracturing produced and flowback water. Hydraulic fracturing produced and flowback water increases risks to surface aquifers and rivers/lakes near the UOG operations compared with conventional oil and gas operations. While some biocides and biocide degradation products have been highlighted as chemicals of concern because of their toxicity to humans and the environment, the selective antimicrobial pressure they cause has not been considered seriously. This perspective article aims to promote research to determine if antimicrobial pressure in these systems is cause for concern. UOG practices could potentially create antimicrobial resistance hotspots under-appreciated in the literature, practice, and regulation arena, hotspots that should not be ignored. The article is distinctive in discussing antimicrobial resistance risks associated with UOG biocides from a , not a chemical toxicology, perspective. We outline potential risks and highlight important knowledge gaps that need to be addressed to properly incorporate antimicrobial resistance emergence and selection into UOG environmental and health risk assessments.

Unconventional Oil and Gas Energy Systems: An Unidentified Hotspot of Antimicrobial Resistance?

We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to characterize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats (, , , and ) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the overwhelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography. Microbes are ubiquitous throughout the world and are highly diverse. Characterizing the extent of variation in the microbial diversity across large geographic spatial scales is a challenge yet can reveal a lot about what biogeography can tell us about microbial populations and their behavior. Machine learning approaches have been used mostly to examine the human microbiome and, to some extent, microbial communities from the environment. Here, we display how supervised machine learning approaches can be useful to understand microbial biodiversity and biogeography using microbes from globally distributed shipping ports. Our findings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system.

Biogeographic Patterns in Members of Globally Distributed and Dominant Taxa Found in Port Microbial Communities.

Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Phylogeny and diversity of alkane-degrading enzyme gene variants in the laurentian great lakes and western atlantic.

Advances in nucleic acid sequencing technology have enabled expansion of our ability to profile microbial diversity. These large datasets of taxonomic and functional diversity are key to better understanding microbial ecology. Machine learning has proven to be a useful approach for analyzing microbial community data and making predictions about outcomes including human and environmental health. Machine learning applied to microbial community profiles has been used to predict disease states in human health, environmental quality and presence of contamination in the environment, and as trace evidence in forensics. Machine learning has appeal as a powerful tool that can provide deep insights into microbial communities and identify patterns in microbial community data. However, often machine learning models can be used as black boxes to predict a specific outcome, with little understanding of how the models arrived at predictions. Complex machine learning algorithms often may value higher accuracy and performance at the sacrifice of interpretability. In order to leverage machine learning into more translational research related to the microbiome and strengthen our ability to extract meaningful biological information, it is important for models to be interpretable. Here we review current trends in machine learning applications in microbial ecology as well as some of the important challenges and opportunities for more broad application of machine learning to understanding microbial communities.

Stephen M. Techtmann

Department of Biological Sciences

Michigan Technological University

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Stephen M. Techtmann

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

Michigan Technological University

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Department of Biological Sciences