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The discovery that most of the prokaryotic diversity and biomass on Earth resides in the deep subsurface, calls for an improved definition of habitability, which should consider the existence of dark biospheres in other planets and moons of the Solar System and beyond. The discovery of “interior liquid water worlds” on some ice moons with waterless surfaces has piqued wide astrobiological interest, but the sporadic mentions of the possibility of life in the deep subsurface of rocky planets in recent habitability reviews calls for a methodical effort to develop sufficient knowledge, both scientific and technological, to include the dark biospheres in our habitability assessments. In this review we analyze recent developments and the methodologies employed to characterize Earth’s continental hard rock deep subsurface to both prepare the future exploration of the putative dark biosphere of Mars and to highlight its importance when evaluating planetary habitability.

The subsurface is one of the last remaining 'uncharted territories' of Earth and is now accepted as a biosphere in its own right, at least as critical to Earth systems as the surface biosphere. The terrestrial deep biosphere is connected through a thin veneer of Earth's crust to the surface biosphere, and many subsurface biosphere ecosystems are impacted by surface topography, climate, and near surface groundwater movement and represent a transition zone (at least ephemerally). Delving below this transition zone, we can examine how microbial metabolic functions define a deep terrestrial subsurface. This review provides a survey of the most recent advances in discovering the functional and genomic diversity of the terrestrial subsurface biosphere, how microbes interact with minerals and obtain energy and carbon in the subsurface, and considers adaptations to the presented environmental extremes. We highlight the deepest subsurface studies in deep mines, deep laboratories, and boreholes in crystalline and altered host rock lithologies, with a focus on advances in understanding ecosystem functions in a holistic manner. [ABSTRACT FROM AUTHOR]

The subsurface is one of the last remaining ‘uncharted territories’ of Earth and is now accepted as a biosphere in its own right, at least as critical to Earth systems as the surface biosphere. The terrestrial deep biosphere is connected through a thin veneer of Earth’s crust to the surface biosphere, and many subsurface biosphere ecosystems are impacted by surface topography, climate, and near surface groundwater movement and represent a transition zone (at least ephemerally). Delving below this transition zone, we can examine how microbial metabolic functions define a deep terrestrial subsurface. This review provides a survey of the most recent advances in discovering the functional and genomic diversity of the terrestrial subsurface biosphere, how microbes interact with minerals and obtain energy and carbon in the subsurface, and considers adaptations to the presented environmental extremes. We highlight the deepest subsurface studies in deep mines, deep laboratories, and boreholes in crystalline and altered host rock lithologies, with a focus on advances in understanding ecosystem functions in a holistic manner.

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system.

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system. [ABSTRACT FROM AUTHOR]

Although information from devoted geomicrobiological drilling studies is limited, it is clear that the results obtained so far call for a systematic exploration of the deep continental subsurface, similar to what has been accomplished in recent years by the Ocean Drilling Initiatives. In addition to devoted drillings from the surface, much of the continental subsurface data has been obtained using different subterranean "windows," each with their correspondent limitations. In general, the number and diversity of microorganisms decrease with depth, and the abundance of Bacteria is superior to Archaea.Within Bacteria, the most commonly detected phyla correspond to Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Within Archaea, methanogens are recurrently detected in most analyzed subsurface samples. One of the most controversial topics in the study of subsurface environments is whether the available energy source is endogenous or partly dependent on products photosynthetically generated in the subsurface. More information, at better depth resolution, is needed to build up the catalog of deep subsurface microbiota and the biologically available electron acceptors and donors. [ABSTRACT FROM AUTHOR]

Relatively few studies have described the microbial populations present in ultra-deep hadal environments, largely as a result of difficulties associated with sampling. Here we report Illumina-tag V6 16S rRNA sequence-based analyses of the free-living and particle-associated microbial communities recovered from locations within two of the deepest hadal sites on Earth, the Challenger Deep (10,918 meters below surface-mbs) and the Sirena Deep (10,667 mbs) within the Mariana Trench, as well as one control site (Ulithi Atoll, 761 mbs). Seawater samples were collected using an autonomous lander positioned ~1m above the seafloor. The bacterial populations within the Mariana Trench bottom water samples were dissimilar to other deep-sea microbial communities, though with overlap with those of diffuse flow hydrothermal vents and deep-subsurface locations. Distinct particle-associated and free-living bacterial communities were found to exist. The hadal bacterial populations were also markedly different from one another, indicating the likelihood of different chemical conditions at the two sites. In contrast to the bacteria, the hadal archaeal communities were more similar to other less deep datasets and to each other due to an abundance of cosmopolitan deep-sea taxa. The hadal communities were enriched in thirty four bacterial and four archaeal operational taxonomic units (OTUs) including members of the Gammaproteobacteria, Epsilonproteobacteria, Marinimicrobia, Cyanobacteria, Deltaproteobacteria, Gemmatimonadetes, Atribacteria, Spirochaetes, and Euryarchaeota. Sequences matching cultivated piezophiles were notably enriched in the Challenger Deep, especially within the particle-associated fraction, and were found in higher abundances than in other hadal studies, where they were either far less prevalent or missing. Our results indicate the importance of heterotrophy, sulfur-cycling, and methane and hydrogen utilization within the bottom waters of the deeper regions of the Mariana Trench, and highlight novel community features of these extreme habitats.

We report the isolation, characterization, and development of a robust genetic system for a halophilic, Fe(II)-oxidizing bacterium isolated from a vertical borehole originating 714 m below the surface located in the Soudan Iron Mine in northern Minnesota, USA. Sequence analysis of the 16S rRNA gene places the isolate in the genus Marinobacter of the Gammaproteobacteria. The genome of the isolate was sequenced using a combination of short- and long-read technologies resulting in 2 contigs representing a 4.4 Mbp genome. Using genomic information, we used a suicide vector for targeted deletion of specific flagellin genes, resulting in a motility-deficient mutant. The motility mutant was successfully complemented by expression of the deleted genes in trans. Random mutagenesis using a transposon was also achieved. Capable of heterotrophic growth, this isolate represents a microaerophilic Fe(II)-oxidizing species for which a system for both directed and random mutagenesis has been established. Analysis of 16S rDNA suggests Marinobacter represents a major taxon in the mine, and genetic interrogation of this genus may offer insight into the structure of deep subsurface communities as well as an additional tool for analyzing nutrient and element cycling in the subsurface ecosystem.

Relatively few studies have described the microbial populations present in ultra-deep hadal environments, largely as a result of difficulties associated with sampling. Here we report Illumina-tag V6 16S rRNA sequence-based analyses of the free-living and particle-associated microbial communities recovered from locations within two of the deepest hadal sites on Earth, the Challenger Deep (10,918 meters below surface-mbs) and the Sirena Deep (10,667 mbs) within the Mariana Trench, as well as one control site (Ulithi Atoll, 761 mbs). Seawater samples were collected using an autonomous lander positioned ~1m above the seafloor. The bacterial populations within the Mariana Trench bottom water samples were dissimilar to other deep-sea microbial communities, though with overlap with those of diffuse flow hydrothermal vents and deep-subsurface locations. Distinct particle-associated and free-living bacterial communities were found to exist. The hadal bacterial populations were also markedly different from one another, indicating the likelihood of different chemical conditions at the two sites. In contrast to the bacteria, the hadal archaeal communities were more similar to other less deep datasets and to each other due to an abundance of cosmopolitan deep-sea taxa. The hadal communities were enriched in 34 bacterial and 4 archaeal operational taxonomic units (OTUs) including members of the Gammaproteobacteria, Epsilonproteobacteria, Marinimicrobia, Cyanobacteria, Deltaproteobacteria, Gemmatimonadetes, Atribacteria, Spirochaetes, and Euryarchaeota. Sequences matching cultivated piezophiles were notably enriched in the Challenger Deep, especially within the particle-associated fraction, and were found in higher abundances than in other hadal studies, where they were either far less prevalent or missing. Our results indicate the importance of heterotrophy, sulfur-cycling, and methane and hydrogen utilization within the bottom waters of the deeper regions of the Mariana Trench, and highlight novel community features of these extreme habitats. [ABSTRACT FROM AUTHOR]

We report the isolation, characterization, and development of a robust genetic system for a halophilic, Fe(II)-oxidizing bacterium isolated from a vertical borehole originating 714 m below the surface located in the Soudan Iron Mine in northern Minnesota, USA. Sequence analysis of the 16S rRNA gene places the isolate in the genus Marinobacter of the Gammaproteobacteria. The genome of the isolate was sequenced using a combination of short- and long-read technologies resulting in two contigs representing a 4.4 Mbp genome. Using genomic information, we used a suicide vector for targeted deletion of specific flagellin genes, resulting in a motility-deficient mutant. The motility mutant was successfully complemented by expression of the deleted genes in trans. Random mutagenesis using a transposon was also achieved. Capable of heterotrophic growth, this isolate represents a microaerophilic Fe(II)-oxidizing species for which a system for both directed and random mutagenesis has been established. Analysis of 16S rDNA suggests Marinobacter represents a major taxon in the mine, and genetic interrogation of this genus may offer insight into the structure of deep subsurface communities as well as an additional tool for analyzing nutrient and element cycling in the subsurface ecosystem. [ABSTRACT FROM AUTHOR]