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Thermodynamic calculations provide valuable insights into the reactions that drive the profound fluid transformations during serpentinization, where surface fluids are transformed into some of the most reduced and alkaline fluids on Earth. However, environmental observations usually deviate from thermodynamic predictions, especially those occurring at low temperatures where equilibrium is slowly reached. In this work, we analyzed 138 low‐temperature (<40°C) fluids from the Samail ophiolite in Oman to test thermodynamic predictions with environmental observations. Four fluid types were identified through this work. (i) Type 1 circumneutral (pH 7–9) fluids result from fluid interactions with serpentinized rocks common in the shallow subsurface. (ii) Fluids with pH ranging from 9 to 11 and low Si concentrations are products of intermediate stages of serpentinization. (iii) Type 2 hyperalkaline (pH > 11) fluids approach equilibrium with diopside, and with serpentine and brucite actively forming during advanced stages of serpentinization. Lastly, (iv) most fluids sampled in this work deviate from predicted equilibrium compositions and depict various degrees of mixing between Type 1 and 2 fluids. Mixed fluids fall within the same pH range but have considerably higher dissolved Si than intermediate‐type fluids. Hyperalkaline fluids exhibit variable degrees of mixing despite maintaining pH > 11, implying strong buffering capacity of serpentinization‐generated fluids. Overall, this work demonstrates that predicted and measured compositions of serpentinization‐derived fluids can be reconciled using a combination of equilibrium and fluid‐transport simulations. This work substantiates these calculations as useful tools in exploring serpentinization reactions in continents and perhaps in other low‐temperature environments on Earth and beyond.

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A geochemical gradient established by mixing between reduced, hyperalkaline (pH > 11), H2‐rich fluids generated through the process of serpentinization and surrounding surface water (pH ∼ 8) in the Samail Ophiolite of Oman provides an opportunity to characterize the geochemical and biological factors that influence the distribution of H2 oxidizing chemotrophs, hydrogenotrophs. In this study, 16S rRNA gene amplicon sequencing was implemented to characterize hydrogenotrophs in sediments underlying surface expressed serpentinized fluids in Oman. Hydrogenotroph phylotype distribution was evaluated as functions of chemical energy supplies for their given metabolic redox reactions. Through this approach, it was discovered that hydrogenotrophic taxa are likely constrained to sediments with overlying fluids that have <∼60 μm O2, including microorganisms of the genus, Hydrogenophaga. Sulfate reducers of the family, Thermodesulfovibrionaceae, likely require >∼10 μm SO4−2 for survival. In sediments with fluids having >∼10 μm SO4−2, sulfate reducers likely outcompete microorganisms of the methanogen genus, Methanobacterium, for H2. Additionally, differences in distribution between Thermodesulfovibrionaceae and Methanobacterium may be driven by the availability of electron acceptors and the redox reaction that is most energy yielding in the fluid. Taken together, observations from the Oman geochemical gradient result in a hydrogenotroph niche model that can be used to evaluate global distribution patterns of hydrogenotrophs in continental serpentinized fluids. On a global scale, based on previous studies, Methanobacterium is constrained to fluids that have <∼10 μm SO4−2.