Microorganisms that can withstand high pressure within an environment are termed piezophiles. These organisms are considered extremophiles and inhabit the deep marine or terrestrial subsurface. Because these microorganisms are not easily accessed and require expensive sampling methods and laboratory instruments, advancements in this field have been limited compared to other extremophiles. This review summarizes the current knowledge on piezophiles, notably the cellular and physiological adaptations that such microorganisms possess to withstand and grow in high-pressure environments. Based on existing studies, organisms from both the deep marine and terrestrial subsurface show similar adaptations to high pressure, including increased motility, an increase of unsaturated bonds within the cell membrane lipids, upregulation of heat shock proteins, and differential gene-regulation systems. Notably, more adaptations have been identified within the deep marine subsurface organisms due to the relative paucity of studies performed on deep terrestrial subsurface environments. Nevertheless, similar adaptations have been found within piezophiles from both systems, and therefore the microbial biogeography concepts used to assess microbial dispersal and explore if similar organisms can be found throughout deep terrestrial environments are also briefly discussed.

Linear and crosslinked polymers are commonly used in the oil and gas industry. Guar-derived polymers have been extensively utilized in hydraulic fracturing processes, and recently polyacrylamide and cellulose-based polymers have also found utility. As these polymers are used during various phases of the hydraulic fracturing process, they can accumulate at formation fracture faces, resulting in undesired filter cakes that impede oil and gas recovery. Although acids and chemical oxidizers are often added in the fracturing fluids to degrade or ‘break’ polymer filter cakes, the constant use of these chemicals can be hazardous and can result in formation damage and corrosion of infrastructure. Alternately, the use of enzymes is an attractive and environmentally friendly technology that can be used to treat polymer accumulations. While guar-linkage-specific enzyme breakers isolated from bacteria have been shown to successfully cleave guar-based polymers and decrease their molecular weight and viscosity at reservoir conditions, new enzymes that target a broader range of polymers currently used in hydraulic fracturing operations still require research and development for effective application. This review article describes the current state-of-knowledge on the mechanisms and enzymes involved in biodegradation of guar gum, polyacrylamide (and hydrolyzed polyacrylamide), and carboxymethyl cellulose polymers. In addition, advantages and challenges in the development and application of enzyme breaker technologies are discussed.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.

The widespread use of hydrocarbon-based fuels has led to the contamination of many natural environments due to accidental spills or leaks. While anaerobic microorganisms indigenous to many fuel-contaminated groundwater sites can play a role in site remediation (e.g., monitored natural attenuation, MNA) via hydrocarbon biodegradation, multiple lines of evidence in support of such bioremediation are required. In this study, we investigated two fuel-contaminated groundwater sites for their potential to be managed by MNA. Microbial community composition, biogeochemical indicators, fumarate addition metabolites, and genes diagnostic of both alkane and alkyl-monoaromatic hydrocarbon activation were assessed. Fumarate addition metabolites and catabolic genes were detected for both classes of hydrocarbon biodegradation at both sites, providing strong evidence for in situ anaerobic hydrocarbon biodegradation. However, relevant metabolites and genes did not consistently co-occur within all groundwater samples. Using newly designed mixtures of quantitative polymerase chain reaction (qPCR) primers to target diverse assA and bssA genes, we measured assA gene abundances ranging from 10⁵–10⁸ copies/L, and bssA gene abundances ranging from 10⁵–10¹⁰ copies/L at the sites. Overall, this study demonstrates the value of investigating fuel-contaminated sites using both metabolites and genes diagnostic of anaerobic hydrocarbon biodegradation for different classes of hydrocarbons to help assess field sites for management by MNA.

Hydraulically fractured shale reservoirs have facilitated studies of unexplored niches in the continental deep biosphere. In high-salinity North American shale systems, members of the genus Halanaerobium seem to be ubiquitous. Polymers like guar gum used as gelling agents in hydraulic fracturing fluids are known to be fermentable substrates, but metabolic pathways encoding these processes have not been characterized. To explore this, produced water samples from the Permian Basin were incubated both at 30°C to simulate above-ground storage conditions and at 60°C to simulate subsurface reservoir conditions. Guar metabolism coincided with Halanaerobium growing only at 30°C, revealing genes for polymer biodegradation through the mixed-acid fermentation pathway in different metagenome-assembled genomes (MAGs). Whereas thiosulfate reduction to sulfide is often invoked to explain the dominance of Halanaerobium in these settings, genes for thiosulfate metabolism were lacking in Halanaerobium genomes with high estimated completeness. Sulfide production was observed in 60°C incubations, with corresponding enrichment of Desulfohalobium and Desulfovibrionaceae that possess complete pathways for coupling mannose and acetate oxidation to sulfate reduction. These findings outline how production of fermentation intermediates (mannose and acetate) by Halanaerobium in topside settings can result in reservoir souring when these metabolites are introduced into the subsurface through produced water reuse. IMPORTANCE: Hydraulically fractured shale oil reservoirs are ideal for studying extremophiles such as thermohalophiles. During hydraulic fracturing, reservoir production water is stored in surface ponds prior to reuse. Microorganisms in these systems therefore need to withstand various environmental changes such as the swing between warm downhole oil reservoir temperatures and cooler surface conditions. While most studies on hydraulically fractured oil reservoirs mimic the environmental conditions found in oil wells, this study follows this water cycle during fracking and the associated microbial metabolic potential during topside-produced water storage and subsurface oil reservoir conditions. Of particular interest are members of the genus Halanaerobium that have been reported to reduce thiosulfate contributing to souring of oil reservoirs. Here, we show that some Halanaerobium strains were unable to grow at hotter temperatures reflective of oil reservoir conditions and lack genes for thiosulfate reduction, despite the proposed importance of this metabolism in other studies. Rather, it is likely that these organisms metabolize complex organics in fracking fluids at lower temperatures, thereby generating substrates that support reservoir souring by thermophilic sulfate-reducing bacteria at higher temperatures. In this way, Halanaerobium promotes souring indirectly by feeding sulfate-reducing microorganisms fermentation products (e.g., acetate and hydrogen) rather than via direct sulfidogenesis via thiosulfate reduction. Therefore, the novelty of this research is not within the detection of known oil reservoir colonizing bacteria but rather in the relationship between bacteria and the indirect involvement of Halanaerobium , promoting souring throughout the produced water reuse cycle.

Nitrate addition for mitigating sulfide production in oil field systems has been studied in laboratory settings and in some subsurface oil reservoirs. To promote water recycling and reuse associated with oil reservoirs produced by hydraulic fracturing, high-salinity produced waters are temporarily stored in surface ponds prior to subsequent reinjection into the subsurface. In this study, nitrate was added directly to a storage pond to prevent sulfide accumulation. DNA sequencing of pond water over a 4-week period revealed a decrease in the proportion of sulfate-reducing microorganisms following nitrate application. Sulfate levels remained stable during this period, whereas nitrate and nitrite fluctuated in the days following the nitrate addition. Metagenome-assembled genomes (MAGs) reconstructed from the pond water microbiome highlighted different organisms with genes for organoheterotrophic and lithoheterotrophic nitrate reduction, whereas genes associated with sulfide production via sulfate or thiosulfate reduction were barely detected. Within those MAGs, genes for acetate metabolism were observed, consistent with acetate decreasing substantially in the pond water in the presence of nitrate. After nitrate was consumed an increase in relative abundance of putative autotrophic microorganisms was observed (e.g. Arhodomonas, Guyparkeria, and Psychroflexus), corresponding to a drop in total inorganic carbon measurements in the storage pond. This trial offers an overview on microbial processes taking place in storage pond environments in response to nitrate addition.