Nondestructive and three-dimensional visualization by identifying elements using synchrotron radiation microscale X-ray CT reveals microbial and cavity distributions in anaerobic granular sludge.

We developed a nondestructive three-dimensional microbial visualization method utilizing synchrotron radiation X-ray microscale computed tomography to better understand the relationship between microorganisms and their surrounding habitats. The method was tested and optimized using a mixture of axenic Escherichia coli and Comamonas testosteroni. The osmium-thiocarbohydrazide-osmium method was used to stain all the microbial cells, and gold in situ hybridization was used to detect specific phylogenetic microbial groups. The stained samples were embedded in epoxy resin for microtomographic analysis. Differences in X-ray absorbances were calculated by subtracting the pre-L3-edge images from the post-L3-edge images to visualize the osmium and gold signals. Although we successfully detected cells stained with osmium, those labeled with gold were not detected, probably because of the insufficient density of gold atoms in the microbial cells. We then applied the developed technique to anaerobic granules and visualized the distribution of microbial cells and extracellular polymeric substances. Empty spaces were highlighted to determine the cavity distribution in granules. Numerous independent cavities of different sizes were identified in the granules. The developed method can be applied to various environmental samples for deeper insights into microbial life in their habitats. IMPORTANCE: Microorganisms inhabit diverse environments and often form biofilms. One factor that affects their community structure is the surrounding physical environment. The arrangement of residential space within the formed biofilm plays a crucial role in the supply and transportation of substances, as well as the discharge of metabolites. Conventional approaches, such as scanning electron microscopy and confocal laser scanning microscopy combined with fluorescence in situ hybridization, have limitations as they provide information primarily from the biofilm surface and cross-sections. In this study, we developed a method for detecting microorganisms in biofilms using synchrotron radiation X-ray microscale computer tomography. The developed method allows nondestructive three-dimensional observation of biofilms at a single-cell resolution (voxel size of approximately 200 nm), facilitating an understanding of the relationship between microorganisms and their physical habitats.

Microbial anabolic and catabolic utilization of hydrocarbons in deep subseafloor sediments of Guaymas Basin.

Guaymas Basin, located in the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nano-scale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Expedition 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin.

Single-cell analysis reveals an active and heterotrophic microbiome in the Guaymas Basin deep subsurface with significant inorganic carbon fixation by heterotrophs.

The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce carbon sequestration in the subsurface, depending on their metabolic lifestyle. However, the activity of subsurface microbes is rarely measured. Here, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3-75 m below the seafloor, 0-14°C). We observed that a large majority of cells were active (83%-100%), although the rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in microbial activity decreased with increasing sediment depth and age. Using a dual-isotope labeling approach, we determined that all active cells analyzed were heterotrophic, deriving the majority of their cellular carbon from organic sources. However, we also detected inorganic carbon assimilation in these heterotrophic cells, likely via processes such as anaplerosis, and determined that inorganic carbon contributes at least 5% of the total biomass carbon in heterotrophs in this community. Our results demonstrate that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by not only assimilating organic carbon but also fixing inorganic carbon. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface. IMPORTANCE: The global subsurface is the largest reservoir of microbial life on the planet yet remains poorly characterized. The activity of life in this realm has implications for long-term elemental cycling, particularly of carbon, as well as how life survives in extreme environments. Here, we recovered cells from the deep subsurface of the Guaymas Basin and investigated the level and distribution of microbial activity, the physicochemical drivers of activity, and the relative significance of organic versus inorganic carbon to subsurface biomass. Using a sensitive single-cell assay, we found that the majority of cells are active, that activity is likely driven by the availability of energy, and that although heterotrophy is the dominant metabolism, both organic and inorganic carbon are used to generate biomass. Using a new approach, we quantified inorganic carbon assimilation by heterotrophs and highlighted the importance of this often-overlooked mode of carbon assimilation in the subsurface and beyond.

Metagenomic profiles of archaea and bacteria within thermal and geochemical gradients of the Guaymas Basin deep subsurface.

Previous studies of microbial communities in subseafloor sediments reported that microbial abundance and diversity decrease with sediment depth and age, and microbes dominating at depth tend to be a subset of the local seafloor community. However, the existence of geographically widespread, subsurface-adapted specialists is also possible. Here, we use metagenomic and metatranscriptomic analyses of the hydrothermally heated, sediment layers of Guaymas Basin (Gulf of California, Mexico) to examine the distribution and activity patterns of bacteria and archaea along thermal, geochemical and cell count gradients. We find that the composition and distribution of metagenome-assembled genomes (MAGs), dominated by numerous lineages of Chloroflexota and Thermoproteota, correlate with biogeochemical parameters as long as temperatures remain moderate, but downcore increasing temperatures beyond ca. 45 ºC override other factors. Consistently, MAG size and diversity decrease with increasing temperature, indicating a downcore winnowing of the subsurface biosphere. By contrast, specific archaeal MAGs within the Thermoproteota and Hadarchaeota increase in relative abundance and in recruitment of transcriptome reads towards deeper, hotter sediments, marking the transition towards a specialized deep, hot biosphere.

Cultivation of Piezotolerant and Piezophilic Hyperthermophiles with a Newly Developed Constant High Pressure and Temperature Culturing and Monitoring System.

The Earth's microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules. The main characteristics of our system are (1) a chamber made of gold with gold-etched lid parts that suppress the majority of non-biological reactions, (2) the exceptional containment of dissolved gas, even small molecules, such as hydrogen, and (3) the sampling capacity of intra-chamber liquid without depressurization and the isobaric transfer of a culture to inoculate new medium. We initially confirmed the retention of dissolved hydrogen in the incubation container at 82°C and 20| |MPa for 9 days. Cultivation tests with an obligate hyperthermophilic piezophile (Pyrococcus yayanosii), hydrogenotrophic hyperthermophile (Archaeoglobus profundus), and heterotrophic hyperthermophile (Pyrococcus horikoshii) were successful based on growth monitoring and chemical ana-lyses. During HTP cultivation, we observed a difference in the duration of the lag phase of P. horikoshii, which indicated the potential effect of a pressure change on the physiology of piezophiles. The present results suggest the importance of a cultivation system designed and developed explicitly for HTP conditions with the capacity for sampling without depressurization of the entire system.

Microbial gene expression in Guaymas Basin subsurface sediments responds to hydrothermal stress and energy limitation.

Analyses of gene expression of subsurface bacteria and archaea provide insights into their physiological adaptations to in situ subsurface conditions. We examined patterns of expressed genes in hydrothermally heated subseafloor sediments with distinct geochemical and thermal regimes in Guaymas Basin, Gulf of California, Mexico. RNA recovery and cell counts declined with sediment depth, however, we obtained metatranscriptomes from eight sites at depths spanning between 0.8 and 101.9 m below seafloor. We describe the metabolic potential of sediment microorganisms, and discuss expressed genes involved in tRNA, mRNA, and rRNA modifications that enable physiological flexibility of bacteria and archaea in the hydrothermal subsurface. Microbial taxa in hydrothermally influenced settings like Guaymas Basin may particularly depend on these catalytic RNA functions since they modulate the activity of cells under elevated temperatures and steep geochemical gradients. Expressed genes for DNA repair, protein maintenance and circadian rhythm were also identified. The concerted interaction of many of these genes may be crucial for microorganisms to survive and to thrive in the Guaymas Basin subsurface biosphere.

Uniquely low stable iron isotopic signatures in deep marine sediments caused by Rayleigh distillation.

Dissimilatory iron reduction (DIR) is suggested to be one of the earliest forms of microbial respiration. It plays an important role in the biogeochemical cycling of iron in modern and ancient sediments. Since microbial iron cycling is typically accompanied by iron isotope fractionation, stable iron isotopes are used as tracer for biological activity. Here we present iron isotope data for dissolved and sequentially extracted sedimentary iron pools from deep and hot subseafloor sediments retrieved in the Nankai Trough off Japan. Dissolved iron (Fe(II)aq) is isotopically light throughout the ferruginous sediment interval but some samples have exceptionally light isotope values. Such light values have never been reported in natural marine environments and cannot be solely attributed to DIR. We show that the light isotope values are best explained by a Rayleigh distillation model where Fe(II)aq is continuously removed from the pore water by adsorption onto iron (oxyhydr)oxide surfaces. While the microbially mediated Fe(II)aq release has ceased due to an increase in temperature beyond the threshold of mesophilic microorganisms, the abiotic adsorptive Fe(II)aq removal continued, leading to uniquely light isotope values. These findings have important implications for the interpretation of dissolved iron isotope data especially in deep subseafloor sediments.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Terpene Synthase Gene Amplicons from Subseafloor Sediment.

In this announcement, we present the set of putative terpene synthase (TS) gene fragments detected in a subseafloor sediment sample collected off Shimokita Peninsula, Japan. This data set contains sequences with 72 to 100% identity to TS from actinobacteria and cyanobacteria.

Simple In-liquid Staining of Microbial Cells for Flow Cytometry Quantification of the Microbial Population in Marine Subseafloor Sediments.

Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM). We initially examined the efficiency of microbial cell staining at various dye/sediment ratios (volume ratio of SYBR-I/sediment [vSYBR/vSed]). Non-cell particles in sediment strongly and preferentially adsorbed SYBR-I dye, resulting in the unsuccessful staining of microbial cells when an insufficient ratio (<1.63 vSYBR/vSed) of SYBR-I dye was present per volume of sediment. SYBR-I dye at an abundance of 10 vSYBR/vSed successfully and stably stained microbial cells in green fluorescence, while the fluorescent color of non-cell particles red-shifted to yellow-orange with the overaccumulation of SYBR-I dye. A low vSYBR/vSed ratio was quickly recognized by a colorless supernatant after centrifugation. At the appropriate vSYBR/vSed ratio, FCM-measured cell concentrations in subseafloor sediments were consistently similar to microscopy counts (>106 cells cm-3). Samples with low cell abundance (<105 cells cm-3) still require cell separation. This modified staining allows us to efficiently process and perform the microbial cell counting of sediment samples to a depth of a few hundred meters below the seafloor with a higher throughput and capability to scale up than procedures employing microscopy-based observations.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Temperature limits to deep subseafloor life in the Nankai Trough subduction zone.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

Global diversity of microbial communities in marine sediment.

Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species-area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 103 to 6.10 × 105 and 3.28 × 104 to 2.46 × 106 amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth's global biosphere.

Aerobic microbial life persists in oxic marine sediment as old as 101.5 million years.

Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

EDTA-FISH: A Simple and Effective Approach to Reduce Non-specific Adsorption of Probes in Fluorescence in situ Hybridization (FISH) for Environmental Samples.

Fluorescence in situ hybridization (FISH) is a widely used molecular technique in microbial ecology. However, the non-specific adsorption of fluorescent probes and resulting high intensity of background signals from mineral particles hampers the specific detection of microbial cells in grain-rich environmental samples, such as subseafloor sediments. We herein demonstrated that a new buffer composition containing EDTA efficiently reduced the adsorption of probes without compromising the properties of the FISH-based probing of microbes. The inclusion of a high concentration of EDTA in the buffer in our protocol provides a simple and effective approach for reducing the background in FISH for environmental samples.

Deep microbial proliferation at the basalt interface in 33.5-104 million-year-old oceanic crust.

The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 1010 cells/cm3. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.

Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

Origin of Short-Chain Organic Acids in Serpentinite Mud Volcanoes of the Mariana Convergent Margin.

Serpentinitic systems are potential habitats for microbial life due to frequently high concentrations of microbial energy substrates, such as hydrogen (H2), methane (CH4), and short-chain organic acids (SCOAs). Yet, many serpentinitic systems are also physiologically challenging environments due to highly alkaline conditions (pH > 10) and elevated temperatures (>80°C). To elucidate the possibility of microbial life in deep serpentinitic crustal environments, International Ocean Discovery Program (IODP) Expedition 366 drilled into the Yinazao, Fantangisña, and Asùt Tesoru serpentinite mud volcanoes on the Mariana Forearc. These mud volcanoes differ in temperature (80, 150, 250°C, respectively) of the underlying subducting slab, and in the porewater pH (11.0, 11.2, 12.5, respectively) of the serpentinite mud. Increases in formate and acetate concentrations across the three mud volcanoes, which are positively correlated with temperature in the subducting slab and coincide with strong increases in H2 concentrations, indicate a serpentinization-related origin. Thermodynamic calculations suggest that formate is produced by equilibrium reactions with dissolved inorganic carbon (DIC) + H2, and that equilibration continues during fluid ascent at temperatures below 80°C. By contrast, the mechanism(s) of acetate production are not clear. Besides formate, acetate, and H2 data, we present concentrations of other SCOAs, methane, carbon monoxide, and sulfate, δ13C-data on bulk carbon pools, and microbial cell counts. Even though calculations indicate a wide range of microbial catabolic reactions to be thermodynamically favorable, concentration profiles of potential energy substrates, and very low cell numbers suggest that microbial life is scarce or absent. We discuss the potential roles of temperature, pH, pressure, and dispersal in limiting the occurrence of microbial life in deep serpentinitic environments.

An Improved Method for Extracting Viruses From Sediment: Detection of Far More Viruses in the Subseafloor Than Previously Reported.

Viruses are the most abundant biological entities on Earth and perform essential ecological functions in aquatic environments by mediating biogeochemical cycling and lateral gene transfer. Cellular life as well as viruses have been found in deep subseafloor sediment. However, the study of deep sediment viruses has been hampered by the complexities involved in efficiently extracting viruses from a sediment matrix. Here, we developed a new method for the extraction of viruses from sediment based on density separation using a Nycodenz density step gradient. The density separation method resulted in up to 2 orders of magnitude greater recovery of viruses from diverse subseafloor sediments compared to conventional methods. The density separation method also showed more consistent performance between samples of different sediment lithology, whereas conventional virus extraction methods were highly inconsistent. Using this new method, we show that previously published virus counts have underestimated viral abundances by up to 2 orders of magnitude. These improvements suggest that the carbon contained within viral biomass in the subseafloor environment may potentially be revised upward to 0.8-3.7 Gt from current estimates of 0.2 Gt. The vastly improved recovery of viruses indicate that viruses represent a far larger pool of organic carbon in subseafloor environments than previously estimated.

Microbial Metabolism and Community Dynamics in Hydraulic Fracturing Fluids Recovered From Deep Hydrocarbon-Rich Shale.

Hydraulic fracturing is a prominent method of natural gas production that uses injected, high-pressure fluids to fracture low permeability, hydrocarbon rich strata such as shale. Upon completion of a well, the fluid returns to the surface (produced water) and contains natural gas, subsurface constituents, and microorganisms (Barbot et al., 2013; Daly et al., 2016). While the microbial community of the produced fluids has been studied in multiple gas wells, the activity of these microorganisms and their relation to biogeochemical activity is not well understood. In this experiment, we supplemented produced fluid with 13C-labeled carbon sources (glucose, acetate, bicarbonate, methanol, or methane), and 15N-labeled ammonium chloride in order to isotopically trace microbial activity over multiple day in anoxic incubations. Nanoscale secondary ion mass spectrometry (NanoSIMS) was used to generate isotopic images of 13C and 15N incorporation in individual cells, while isotope ratio monitoring-gas chromatography-mass spectrometry (IRM-GC-MS) was used to measure 13CO2, and 13CH4 as metabolic byproducts. Glucose, acetate, and methanol were all assimilated by microorganisms under anoxic conditions. 13CO2 production was only observed with glucose as a substrate indicating that catabolic activity was limited to this condition. The microbial communities observed at 0, 19, and 32 days of incubation did not vary between different carbon sources, were low in diversity, and composed primarily of the class Clostridia. The primary genera detected in the incubations, Halanaerobium and Fusibacter, are known to be adapted to harsh physical and chemical conditions consistent with those that occur in the hydrofracturing environment. This study provides evidence that microorganisms in produced fluid are revivable in laboratory incubations and retained the ability to metabolize added carbon and nitrogen substrates.