Deep-branching acetogens in serpentinized subsurface fluids of Oman.

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman.

Serpentinization can generate highly reduced fluids replete with hydrogen (H2) and methane (CH4), potent reductants capable of driving microbial methanogenesis and methanotrophy, respectively. However, CH4 in serpentinized waters is thought to be primarily abiogenic, raising key questions about the relative importance of methanogens and methanotrophs in the production and consumption of CH4 in these systems. Herein, we apply molecular approaches to examine the functional capability and activity of microbial CH4 cycling in serpentinization-impacted subsurface waters intersecting multiple rock and water types within the Samail Ophiolite of Oman. Abundant 16S rRNA genes and transcripts affiliated with the methanogenic genus Methanobacterium were recovered from the most alkaline (pH, >10), H2- and CH4-rich subsurface waters. Additionally, 16S rRNA genes and transcripts associated with the aerobic methanotrophic genus Methylococcus were detected in wells that spanned varied fluid geochemistry. Metagenomic sequencing yielded genes encoding homologs of proteins involved in the hydrogenotrophic pathway of microbial CH4 production and in microbial CH4 oxidation. Transcripts of several key genes encoding methanogenesis/methanotrophy enzymes were identified, predominantly in communities from the most hyperalkaline waters. These results indicate active methanogenic and methanotrophic populations in waters with hyperalkaline pH in the Samail Ophiolite, thereby supporting a role for biological CH4 cycling in aquifers that undergo low-temperature serpentinization.IMPORTANCE Serpentinization of ultramafic rock can generate conditions favorable for microbial methane (CH4) cycling, including the abiotic production of hydrogen (H2) and possibly CH4 Systems of low-temperature serpentinization are geobiological targets due to their potential to harbor microbial life and ubiquity throughout Earth's history. Biomass in fracture waters collected from the Samail Ophiolite of Oman, a system undergoing modern serpentinization, yielded DNA and RNA signatures indicative of active microbial methanogenesis and methanotrophy. Intriguingly, transcripts for proteins involved in methanogenesis were most abundant in the most highly reacted waters that have hyperalkaline pH and elevated concentrations of H2 and CH4 These findings suggest active biological methane cycling in serpentinite-hosted aquifers, even under extreme conditions of high pH and carbon limitation. These observations underscore the potential for microbial activity to influence the isotopic composition of CH4 in these systems, which is information that could help in identifying biosignatures of microbial activity on other planets.

An untargeted exometabolomics approach to characterize dissolved organic matter in groundwater of the Samail Ophiolite.

The process of serpentinization supports life on Earth and gives rise to the habitability of other worlds in our Solar System. While numerous studies have provided clues to the survival strategies of microbial communities in serpentinizing environments on the modern Earth, characterizing microbial activity in such environments remains challenging due to low biomass and extreme conditions. Here, we used an untargeted metabolomics approach to characterize dissolved organic matter in groundwater in the Samail Ophiolite, the largest and best characterized example of actively serpentinizing uplifted ocean crust and mantle. We found that dissolved organic matter composition is strongly correlated with both fluid type and microbial community composition, and that the fluids that were most influenced by serpentinization contained the greatest number of unique compounds, none of which could be identified using the current metabolite databases. Using metabolomics in conjunction with metagenomic data, we detected numerous products and intermediates of microbial metabolic processes and identified potential biosignatures of microbial activity, including pigments, porphyrins, quinones, fatty acids, and metabolites involved in methanogenesis. Metabolomics techniques like the ones used in this study may be used to further our understanding of life in serpentinizing environments, and aid in the identification of biosignatures that can be used to search for life in serpentinizing systems on other worlds.

Intact polar lipidome and membrane adaptations of microbial communities inhabiting serpentinite-hosted fluids.

The generation of hydrogen and reduced carbon compounds during serpentinization provides sustained energy for microorganisms on Earth, and possibly on other extraterrestrial bodies (e.g., Mars, icy satellites). However, the geochemical conditions that arise from water-rock reaction also challenge the known limits of microbial physiology, such as hyperalkaline pH, limited electron acceptors and inorganic carbon. Because cell membranes act as a primary barrier between a cell and its environment, lipids are a vital component in microbial acclimation to challenging physicochemical conditions. To probe the diversity of cell membrane lipids produced in serpentinizing settings and identify membrane adaptations to this environment, we conducted the first comprehensive intact polar lipid (IPL) biomarker survey of microbial communities inhabiting the subsurface at a terrestrial site of serpentinization. We used an expansive, custom environmental lipid database that expands the application of targeted and untargeted lipodomics in the study of microbial and biogeochemical processes. IPLs extracted from serpentinite-hosted fluid communities were comprised of >90% isoprenoidal and non-isoprenoidal diether glycolipids likely produced by archaeal methanogens and sulfate-reducing bacteria. Phospholipids only constituted ~1% of the intact polar lipidome. In addition to abundant diether glycolipids, betaine and trimethylated-ornithine aminolipids and glycosphingolipids were also detected, indicating pervasive membrane modifications in response to phosphate limitation. The carbon oxidation state of IPL backbones was positively correlated with the reduction potential of fluids, which may signify an energy conservation strategy for lipid synthesis. Together, these data suggest microorganisms inhabiting serpentinites possess a unique combination of membrane adaptations that allow for their survival in polyextreme environments. The persistence of IPLs in fluids beyond the presence of their source organisms, as indicated by 16S rRNA genes and transcripts, is promising for the detection of extinct life in serpentinizing settings through lipid biomarker signatures. These data contribute new insights into the complexity of lipid structures generated in actively serpentinizing environments and provide valuable context to aid in the reconstruction of past microbial activity from fossil lipid records of terrestrial serpentinites and the search for biosignatures elsewhere in our solar system.

Preferred temporal-spatial parameters, physical maturation, and sex are related to vertical and braking forces in adolescent long-distance runners.

For adults, increasing cadence reduces ground reaction forces, but a lower preferred cadence does not predispose adults to experience higher ground reaction forces. Pubertal growth and motor control changes influence running mechanics, but it is unknown if preferred cadence or step length are associated with ground reaction forces for pre-adolescent and adolescent runners. Pre-adolescent and adolescent runners underwent an overground running analysis at a self-selected speed. Mixed model multiple linear regressions investigated the associations of preferred cadence, step length, physical maturation, and sex on ground reaction forces, while accounting for running speed and leg length. Running with a lower preferred cadence or longer preferred step length was associated with larger peak braking and vertical forces (p ≤ .01), being less physically mature was associated with larger vertical impact peak force and vertical loading rate (p ≤ .01), and being a male was associated with larger loading rates (p ≤ .01). A lower preferred cadence or longer preferred step length were associated with higher braking and vertical forces and being less physically mature or a male were associated with higher loading rates. An intervention to increase cadence/decrease step length could be considered if ground reaction forces are a concern for an adolescent runner.

Subsurface biogeochemical cycling of nitrogen in the actively serpentinizing Samail Ophiolite, Oman.

Nitrogen (N) is an essential element for life. N compounds such as ammonium ( NH 4 + ) may act as electron donors, while nitrate ( NO 3 - ) and nitrite ( NO 2 - ) may serve as electron acceptors to support energy metabolism. However, little is known regarding the availability and forms of N in subsurface ecosystems, particularly in serpentinite-hosted settings where hydrogen (H2) generated through water-rock reactions promotes habitable conditions for microbial life. Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg2+- HCO 3 - type fluids comprised mostly of NO 3 - , and Ca2+-OH- fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as NO 3 - . High concentrations of NO 3 - (>100 µM) with a relict meteoric oxygen isotopic composition (δ18O ~ 22‰, Δ17O ~ 6‰) were observed in shallow aquifer fluids, indicative of NO 3 - sourced from atmospheric deposition (rainwater NO 3 - : δ18O of 53.7‰, Δ17O of 16.8‰) mixed with NO 3 - produced in situ through nitrification (estimated endmember δ18O and Δ17O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH3 (>100 µM) with little NO 3 - detectable. We interpret that NH3 in hyperalkaline fluids is a product of NO 3 - reduction. The proportionality of the O and N isotope fractionation (18ε / 15ε) measured in Samail Ophiolite NO 3 - was close to unity (18ε / 15ε ~ 1), which is consistent with dissimilatory NO 3 - reduction with a membrane-bound reductase (NarG); however, abiotic reduction processes may also be occurring. The presence of genes commonly involved in N reduction processes (narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of NO 3 - . Production of NH 4 + as the end-product of NO 3 - reduction via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.

Analysis of limb kinetic asymmetry during a drop vertical jump in adolescents post anterior cruciate ligament reconstruction.

BACKGROUND: Limb asymmetry after ACL reconstruction is often cited as a risk factor for ACL reinjury. We assessed ground reaction forces on each limb during a drop vertical jump, and compared kinetic symmetry between limbs in adolescents post-ACL reconstruction versus healthy controls. METHODS: Forty-four participants who underwent an ACL reconstruction (16 male/28 female, mean age 16.1 ± 1.5, mean 7.3 ± 0.9 months post-ACL reconstruction) and 34 controls (20 male/14 female, mean age 14.9 ± 1.1) completed a drop vertical jump captured on a Vicon system and Bertec force plates. Kinetic variables were calculated individually for each limb. Inter-limb asymmetry was calculated as an index between each limb (involved versus uninvolved for the ACL reconstruction group, and left versus right for controls), and was compared between groups using independent t-tests. FINDINGS: Asymmetry was significantly more pronounced in the ACL reconstruction group versus the controls for peak contact ground reaction force (11.6% vs 4.4%, p = 0.009), eccentric impulse (8.8% vs 3.8%, p = 0.009), eccentric mean force (8.0% vs 3.4%, p = 0.006), concentric peak ground reaction force (4.1% vs 0.8%, p = 0.003), concentric impulse (5.1% vs 1.1%, p = 0.001), and peak landing ground reaction force (12.7% vs 1.7%, p < 0.001). INTERPRETATION: Limb kinetic asymmetry during a drop vertical jump was more pronounced in adolescents post-ACL reconstruction versus controls for both eccentric- and concentric-phase variables, which may indicate the use of compensatory strategies to offload the post-operative limb. Targeted interventions to produce more symmetric loading and unloading during jumping tasks should be developed, tested, and monitored to determine the impact on rehabilitation programs, return-to-sport evaluations, and injury prevention outcomes.

Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation.

Metagenome assembled genomes (MAGs) and single amplified genomes (SAGs) affiliated with two distinct Methanobacterium lineages were recovered from subsurface fracture waters of the Samail Ophiolite, Sultanate of Oman. Lineage Type I was abundant in waters with circumneutral pH, whereas lineage Type II was abundant in hydrogen rich, hyperalkaline waters. Type I encoded proteins to couple hydrogen oxidation to CO2 reduction, typical of hydrogenotrophic methanogens. Surprisingly, Type II, which branched from the Type I lineage, lacked homologs of two key oxidative [NiFe]-hydrogenases. These functions were presumably replaced by formate dehydrogenases that oxidize formate to yield reductant and cytoplasmic CO2 via a pathway that was unique among characterized Methanobacteria, allowing cells to overcome CO2/oxidant limitation in high pH waters. This prediction was supported by microcosm-based radiotracer experiments that showed significant biological methane generation from formate, but not bicarbonate, in waters where the Type II lineage was detected in highest relative abundance. Phylogenetic analyses and variability in gene content suggested that recent and ongoing diversification of the Type II lineage was enabled by gene transfer, loss, and transposition. These data indicate that selection imposed by CO2/oxidant availability drove recent methanogen diversification into hyperalkaline waters that are heavily impacted by serpentinization.

Physiological adaptations to serpentinization in the Samail Ophiolite, Oman.

Hydration of ultramafic rock during the geologic process of serpentinization can generate reduced substrates that microorganisms may use to fuel their carbon and energy metabolisms. However, serpentinizing environments also place multiple constraints on microbial life by generating highly reduced hyperalkaline waters that are limited in dissolved inorganic carbon. To better understand how microbial life persists under these conditions, we performed geochemical measurements on waters from a serpentinizing environment and subjected planktonic microbial cells to metagenomic and physiological analyses. Metabolic potential inferred from metagenomes correlated with fluid type, and genes involved in anaerobic metabolisms were enriched in hyperalkaline waters. The abundance of planktonic cells and their rates of utilization of select single-carbon compounds were lower in hyperalkaline waters than alkaline waters. However, the ratios of substrate assimilation to dissimilation were higher in hyperalkaline waters than alkaline waters, which may represent adaptation to minimize energetic and physiologic stress imposed by highly reducing, carbon-limited conditions. Consistent with this hypothesis, estimated genome sizes and average oxidation states of carbon in inferred proteomes were lower in hyperalkaline waters than in alkaline waters. These data suggest that microorganisms inhabiting serpentinized waters exhibit a unique suite of physiological adaptations that allow for their persistence under these polyextremophilic conditions.

Microscale Biosignatures and Abiotic Mineral Authigenesis in Little Hot Creek, California.

Hot spring environments can create physical and chemical gradients favorable for unique microbial life. They can also include authigenic mineral precipitates that may preserve signs of biological activity on Earth and possibly other planets. The abiogenic or biogenic origins of such precipitates can be difficult to discern, therefore a better understanding of mineral formation processes is critical for the accurate interpretation of biosignatures from hot springs. Little Hot Creek (LHC) is a hot spring complex located in the Long Valley Caldera, California, that contains mineral precipitates composed of a carbonate base (largely submerged) topped by amorphous silica (largely emergent). The precipitates occur in close association with microbial mats and biofilms. Geological, geochemical, and microbiological data are consistent with mineral formation via degassing and evaporation rather than direct microbial involvement. However, the microfabric of the silica portion is stromatolitic in nature (i.e., wavy and finely laminated), suggesting that abiogenic mineralization has the potential to preserve textural biosignatures. Although geochemical and petrographic evidence suggests the calcite base was precipitated via abiogenic processes, endolithic microbial communities modified the structure of the calcite crystals, producing a textural biosignature. Our results reveal that even when mineral precipitation is largely abiogenic, the potential to preserve biosignatures in hot spring settings is high. The features found in the LHC structures may provide insight into the biogenicity of ancient Earth and extraterrestrial rocks.