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Preservative biocides are designed to control microbial growth and biogenic souring in the downhole environment. We report the prevention of biogenic souring by 4,4-dimethyloxazolidine (DMO, a preservative biocide) and glutaraldehyde as compared to that afforded by tributyl tetradecyl phosphonium chloride (TTPC, a cationic surface-active biocide), in a first-of-its kind suite of High Pressure, High Temperature (HPHT) Bioreactors that simulate hydraulically fractured shale reservoirs. The design of these new bioreactors, which recreate the downhole environment (temperatures, pressures, formation solids, and frac additives) in a controlled laboratory environment, enables the evaluation of biocides under field-relevant conditions. The bioreactors receiving either no biocide treatment or treatment with a high concentration of TTPC (50 ppm active ingredient) rapidly soured within the first two weeks of shut-in, and all surpassed the maximum detectable level of H2S (343 ppm) after the addition of live microbes to the reactors. Conversely, a higher loading of DMO (150 pppm active ingredient) maintained H2S concentrations below the minimum dectable level (5 ppm) for six weeks, and held H2S concentrations to 10.3 +/- 5.2 ppm after fifteen weeks of shut-in and two post shut-in microbial rechallenges. In a second study, a lower concentration of DMO (50 ppm active ingredient) maintained H2S concentrations below the minimum detectable level through the addition of live microbes after three weeks, and H2S concentrations only registered above 10 ppm upon a second addition of live microbes after five weeks. In this same study (which was performed at moderate temperatures), a 50 ppm (active ingredient) treatment of glutaraldehyde also maintained H2S concentrations below the minimum detectable level through the addition of live microbes after three weeks, and H2S concentrations registered 15.0 +/- 9.7 ppm H2S after four weeks. Similar time scales of protection are observed for each treatment condition through the enumeration of microbes present in each reactor. The differentiation in antimicrobial activity (and specifically, prevention of biogenic souring) afforded by DMO and glutaraldehyde suggests that such nonionic, preservative biocides are a superior choice for maintaining control over problematic microorganisms as compared to surface-active biocides like TTPC at the concentrations tested. The significant duration of efficacy provided by DMO and glutaraldehyde in this first-of-its-kind suite of simulated reservoirs demonstrates that comprehensive preservation and prevention of biogenic souring from completion through to production is feasible. Such comprehensive, prolonged protection is especially relevant for extended shut-ins or drilled but uncompleted wells (DUCS) such as those experienced during the COVID-19 pandemic. The environment simulated within the bioreactors demonstrates that the compatibility afforded by a preservative biocide offers downhole protection that cationic, surface-active biocides do not.

MIC is characterized by the acceleration of corrosion rates in the presence of microorganisms and has been traditionally associated with microbial metabolic processes that result in the production of acid and the reduction of sulfate to hydrogen sulfide. For this reason, topside corrosion monitoring programs typically include efforts to detect both sulfate-reducing bacteria (SRB) and acid-producing bacteria (APB). However, the advent of bioinformatics in the oil and gas industry now allows for more comprehensive microbiological field monitoring programs that may be able to detect other microorganisms that threaten asset integrity. In this chapter, a case study will be presented that illustrates the use of the bug bottle, qPCR, and next-generation sequencing (NGS) techniques to monitor the corrosion risk of a topside oilfield system. This case study highlights how three monitoring techniques can be used not only to identify potential problem areas within a system but also to diagnose the underlying cause of the corrosion so that it can be addressed.

We report the design, operation and biogenic souring data from a first-of-its kind suite of High Pressure, High Temperature (HPHT) Bioreactors for hydraulically fractured shale reservoirs. These bioreactors vet the ability of microbial control technologies, such as biocides, to prevent the onset of microbial contamination and reservoir souring at larger experimental volumes and higher pressures and temperatures than have been previously possible outside of field trials. The bioreactors were charged with proppant, crushed Permian shale, and sterile simulated fracturing fluids (SSFF). Subsets of bioreactors were charged with SSFF dosed with either no biocide, tributyl tetradecyl phosphonium chloride (TTPC, a cationic surface-active biocide), or 4,4-dimethyloxazolidine (DMO, a preservative biocide). The bioreactors were shut in under 1,000-2,500 psi and elevated temperatures for up to fifteen weeks; hydrogen sulfide (H2S) and microbial counts were measured approximately once per week, and additional microbes were introduced after weeks three and five. Across two separate studies, the bioreactors containing no biocide soured within the first week of shut-in and H2S concentrations increased rapidly beyond the maximum detectable level (343 ppm) within the first three to six weeks of shut-in. In the first study, the bioreactors treated with TTPC soured within two weeks of shut-in (prior to the first addition of fresh microbes), and H2S concentrations increased rapidly to nearly 200 ppm H2S within the first six weeks of shut-in and beyond the maximum detectable level after fifteen weeks of shut-in. The bioreactors containing DMO did not sour during either study until at least the first addition of fresh microbes, and higher levels of the preservative biocide continued to prevent the biogenic formation of H2S even during and after the addition of fresh microbes. Microbial counts correlate with the H2S readings across all bioreactor treatments. The differentiation in antimicrobial activity afforded by the different types of biocide treatments validates the use of these simulated laboratory reservoirs as a biocide selection tool. This first-of-its-kind suite of HPHT Bioreactors for hydraulic fracturing provides the most advanced biocide selection tool developed for the hydraulic fracturing industry to date. The bioreactors will guide completions and stimulation engineers in biocide program optimization under reservoir-relevant conditions prior to beginning lengthy and expensive field trials.

Drilling and completion operations introduce microbial contaminants into the reservoir via numerous pathways including drilling muds and hydraulic fracturing fluids. Uncontrolled microbial growth in the reservoir can lead to reduced flow and production rate of hydrocarbons due to accumulation of biofilm and plugging. Microbial contaminants can also lead to microbially influenced corrosion (MIC) of topside and downhole production equipment, causing undesirable impacts to capital spending and operational efficiency. As a mitigation strategy, biocides are often added to hydraulic fracturing fluids to control microorganisms which cause these operational issues. Oilfield service laboratories often receive water samples from the field to provide recommendations on the most optimum biocide use and concentration. The standard industry method (NACE TM-0194) used for measuring biocide performance is based on a time-kill test in which water collected from the field is spiked with bacteria. Biocides are added at desired concentrations and samples are withdrawn to determine log reductions. Bacterial levels are often determined by ATP so results can be collected within minutes. A wide number of biocides are available for oil and gas operations. They differ primarily by mechanism of action, speed of kill, and interaction with other components of fracturing fluids. As the NACE method relies on rapid activity, misleading test results are often observed with preservative biocides which are intended for slow-release / long-term activity. Preservatives are utilized for extended protection of the well and reservoir after drilling and/or completion operations, not for the rapid reduction of microbes in a water sample. Therefore, as current methods are intended for rapid-kill biocides, novel methods are required to demonstrate the efficacy of preservatives. Methods were designed primarily to demonstrate efficacy of preservative treatment for waters used in hydraulic fracturing. Using culture-based methods, biocides were added to water samples that were incubated at elevated temperatures and challenged periodically. Glutaraldehyde was an effective preservative at 55 °C, providing antimicrobial efficacy for >7 days. 4,4-dimethyloxazolidine (DMO) was effective for 4 weeks or more, with improved efficacy at elevated temperature. The methods described in this work can differentiate rapid-kill from preservative biocides, be performed at a service company laboratory, and provide results within an "actionable" period (approximately 7-10 days) of time.

About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes (MAGs). Utilizing six other previously sequenced isolate genomes and MAGs, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs.

The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative Halanaerobium species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause “clumping” of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems.

The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative Halanaerobium species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause “clumping” of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems., Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. Halanaerobium is believed to be inadvertently introduced into this environment during the drilling and fracturing process and must therefore tolerate large changes in pressure, temperature, and salinity. Here, we used a Halanaerobium strain isolated from a natural gas well in the Utica Point Pleasant formation to investigate metabolic and physiological responses to growth under high-pressure subsurface conditions. Laboratory incubations confirmed the ability of Halanaerobium congolense strain WG8 to grow under pressures representative of deep shale formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological shifts were identified, including higher abundances of proteins associated with the production of extracellular polymeric substances. Confocal laser scanning microscopy indicated that extracellular polymeric substance (EPS) production was associated with greater cell aggregation when biomass was cultured at high pressure. Changes in Halanaerobium central carbon metabolism under the same conditions were inferred from nuclear magnetic resonance (NMR) and gas chromatography measurements, revealing large per-cell increases in production of ethanol, acetate, and propanol and cessation of hydrogen production. These metabolic shifts were associated with carbon flux through 1,2-propanediol in response to slower fluxes of carbon through stage 3 of glycolysis. Together, these results reveal the potential for bioclogging and corrosion (via organic acid fermentation products) associated with persistent Halanaerobium growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes. IMPORTANCE The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative Halanaerobium species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause “clumping” of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems.

The pipelines and vast infrastructure required for the production and transport of oil and gas are largely constructed of carbon steel. This material is highly susceptible to damage and failure as a result of direct or indirect Microbially Influenced Corrosion (MIC). One approach to reduce and/or remediate MIC is the application of microbicides to affected assets such as storage tanks, pipelines, heat exchangers and pumps. The objective of this paper is to generate efficacy data for biocidal formulations against corrosion-associated biofilms grown under anoxic and flowing conditions, which is difficult to obtain due to the distinct nature of biofilms and growth conditions. This data can be more representative when mimicking petroleum and water transporting pipelines. Thus, to facilitate the testing and comparison of products that successfully attenuate MIC under field-relevant conditions, a method that allows consistent corrosion measurements to be made, for corroding biofilms grown on steel surfaces under flowing and anoxic conditions, has been developed. This method utilizes microorganisms that were enriched on carbon steel from a North Sea sediment sample that, when grown in recirculating flow circuits, can generate corrosion rates of up to 65 mpy after four weeks of growth. The samples taken for the enrichment of corroding cultures came from a low tide anoxic sediment sample (black sand). In this presentation and paper, this method and its application to the benchmarking of field-relevant biocidal chemistries in MIC control experiments are described. Attention will be given to the reproducibility of the method as well. Distinct differences can be observed in biocidal performance against biofilms developed on metal surfaces.

The deep terrestrial biosphere harbours a substantial fraction of Earth's biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus-host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) spacers elucidated lineage-specific virus-host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.

This study examined the responses of two termite species, the Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar), to three types of wood decay fungi: a brown rot fungus, Gloeophyllum trabeum (Persoon: Fries) Murrill; a white rot fungus, Phanerochaete chrysosporium Burdsall; and a litter rot fungus, Marasmiellus troyanus (Murrill) Singer. We also examined the responses of termites to these three types of fungi grown on different substrates. For all three fungal species, both termite species showed a strong preference for fungus-infected sawdust over uninfected sawdust. In choice tests, both termite species preferred sawdust infected with either M. troyanus or P. chrysosporium over G. trabeum. However, termites did not show any preference for fungus-infected potato dextrose agar over uninfected potato dextrose agar. Tunneling activity of C. formosanus was greater in sand treated with methanol extracts of fungus-infected sawdust than in sand treated with extracts of uninfected sawdust. Because chemicals in the fungal extracts caused termites to tunnel further into treated sand than untreated sand, these chemicals could potentially be used to direct termite foraging toward bait stations in the field.

Benzo[a]pyrene (B[a]P) is a carcinogenic polyaromatic hydrocarbon that enters the environment as an incomplete combustion production of fossil fuels. Several species of filamentous fungi are capable of biotransforming and/or mineralizing B[a]P in liquid cultures, however there has been less success in soil habitats. In this study, the litter rot fungus Marasmiellus troyanus was encapsulated in alginate and delivered to B[a]P-spiked soil microcosms (100 μg B[a]P/g soil) for 1, 2 and 6 weeks, with and without a fertilizer solution. After 2 weeks, 32.5% of B[a]P was recovered from soil microcosms treated with M. troyanus compared to 55–70% for controls. After 6 weeks, controls demonstrated an average percent recovery of B[a]P of 54% while M. troyanus-inoculated samples gave an average percent recovery of 11%. Similar bioaugmentation of contaminated habitats with appropriately formulated fungi has potential for practical bioremediation in soil environments. Journal of Industrial Microbiology & Biotechnology (2000) 25, 116–119.

As research on the biodegradation of benzo[a]pyrene (B[a]P) shifts from laboratory to field based studies, a need has arisen for reproducible, economical, and accurate methods for extraction and quantification of B[a]P. This study describes a method for the analysis of B[a]P using batch extraction techniques with quantification by reversed phase high performance thin layer chromatography (HPTLC) and densitometry. A loam soil was fortified with B[a]P at a level of 100 mg B[a]P kg−1 soil. Several solvents were evaluated as potential soil extraction systems including: acetone, acetonitrile, ethyl acetate, methanol, and methylene chloride:acetone (1:1). Reverse phase thin layer chromatography of B[a]P was performed on hydrocarbon impregnated, C18 reverse phase plates using methanol:acetonitrile (1:1) as the developing solvent. The Rf for B[a]P in this system was found to be 0.52. Recovery of B[a]P from soil ranged from 11% with methanol to 84% with ethyl acetate.

Some 17 strains of filamentous fungi, encompassing 13 different species, were tested for their ability to decolorize the polymeric dye R-478. Decolorization was observed with both living and dead mycelia of the three Aspergillus species tested, indicating bioadsorption, not biodegradation. With mycelia of other strains tested, the most decolorization was obtained with Marasmiellus troyanus, Pleurotus sapidus, and Pleurotus ostreatus; with extracellular filtrates, the most decolorization was observed with Laetiporus sulphureus. Parallel experiments incubating benzo[a]pyrene (B[a]P) with mycelia and filtrates showed that six of the species removed over 40% of B[a]P in comparison with HgCl2-killed controls. The highest B[a]P removal by mycelia was shown by M. troyanus (95.0%); the highest level by extracellular filtrates was shown by Hericium erinaceous (44.8%). With the exception of A. ochraceous, no products of B[a]P metabolism were detected for any of the species tested. For most species, the disappearance of B[a]P was correlated with the ability to decolorize poly R-478 ( r = 0.78 for mycelia; r = 0.74 for culture fluids). M. troyanus gave rise to more disappearance than decolorization. The removal of B[a]P by M. troyanus and Phanerochaete chrysosporium was compared over 30 days: M. troyanus gave significantly better removal in a biphasic pattern.

Averufin is a polyhydroxyanthraquinone first isolated from Aspergillus versicolor (Vuill.) Tiraboschi (Pusey and Roberts, 1963; Holker et al., 1966). This anthraquinone inhibits ATP synthesis in mitochondria by exerting an extremely potent uncoupling effect and a significant depression on mitochondrial respiration (Kawai et al., 1984). Averufin is an intermediate in aflatoxin biosynthesis and has been extensively studied in a blocked mutant of A. parasiticus Speare (Donkersloot et al., 1972; Lin et al., 1973) and has been useful in elucidating the origin of the difuran ring in the polyketide pathway leading to sterigmatocystin and subsequently aflatoxin production (Townsend et al., 1982). Because of its bright orange color, averufin is also a useful marker in parasexual studies of aflatoxigenic fungi (Bennett and Papa, 1988). Aflatoxins are highly toxic secondary metabolites produced by certain strains of A. parasiticus and A. flavus Link (Betina, 1989). Genetic studies of aflatoxin biosynthesis have been hampered by the absence of a sexual (teleomorphic) stage in either aflatoxigenic species. Transformation systems have recently been developed for both species (Woloshuk et al., 1989; Skory et al., 1990) and will facilitate future molecular analyses. Nevertheless, it will require years of research to characterize A. flavus or A. parasiticus to the level of A. nidulans, which has well developed classical and molecular genetic systems (Smith and Pateman, 1977; Timberlake, 1980). A recent paper reports that sterigmatocystin, a late intermediate in aflatoxin biosynthesis, is produced in significant amounts by the "Glas

Mycelia from the mushroom Marasmiellus troyanus were grown in the presence of radiolabeled benzo[a]pyrene in liquid culture. After 15 days, 8.1% of the label from M. troyanus cultures was recovered in CO2 as compared to 1.1% for Phanerochaete chrysosporium and 0.2% for Aspergillus niger. M. troyanus efficiently transformed B[a]P into water soluble metabolites with 64% of the label recovered in the water soluble fraction as compared to 11.7% for P. chrysosporium and 4.1% for A. niger. Glucuronic acid and sulfate conjugates of B[a]P were identified from the aqueous fraction of cultures of M. troyanus, after 17 days.