INTRODUCTION_NN1 
The_AT0 intricacies_NN2 of_PRF the_AT0 deep_AJ0 subsurface_NN1 have_VHB triggered_VVN many_DT0 investigations_NN2 into_PRP the_AT0 microbial_AJ0 habitability_NN1 sustaining_VVG itself_PNX in_PRP the_AT0 rock-hosted_AJ0 subsurface_NN1 ._SENT The_AT0 chemical_AJ0 drivers_NN2 that_CJT govern_VVB the_AT0 transition_NN1 from_PRP terrestrial_AJ0 surface_NN1 to_PRP subsurface_NN1 life_NN1 are_VBB not_XX0 fully_AV0 understood_VVN ,_PUN especially_AV0 in_PRP near-subsurface_AJ0 zones_NN2 where_AVQ the_AT0 system_NN1 may_VM0 behave_VVI as_PRP a_AT0 hybrid_NN1 between_PRP surface_NN1 and_CJC subsurface_NN1 processes_NN2 ._SENT Field_NN1 sites_NN2 spanning_VVG terrestrial_AJ0 ,_PUN lacustrine_AJ0 ,_PUN hot_AJ0 springs_NN2 ,_PUN and_CJC marine_AJ0 environments_NN2 have_VHB gained_VVN traction_NN1 for_PRP molecular_AJ0 biological_AJ0 investigation_NN1 due_AJ0 to_PRP unique_AJ0 ,_PUN extreme_AJ0 conditions_NN2 mimicking_VVG potential_AJ0 environmental_AJ0 settings_NN2 of_PRF early_AJ0 life_NN1 on_PRP Earth_NN1 as_CJS well_AV0 as_CJS analogs_NN2 to_PRP those_DT0 on_PRP other_AJ0 planetary_AJ0 systems_NN2 ._SENT While_CJS marine_AJ0 systems_NN2 have_VHB unveiled_VVN the_AT0 extent_NN1 of_PRF microbial_AJ0 subsurface_NN1 habitability_NN1 in_PRP oceanic_AJ0 settings_NN2 ,_PUN research_NN1 aimed_VVN at_PRP understanding_VVG accessible_AJ0 terrestrial_AJ0 environments_NN2 has_VHZ gained_VVN considerable_AJ0 interest_NN1 because_CJS of_PRF more_DT0 uniquely_AV0 limiting_AJ0 constraints_NN2 (_PUL low_AJ0 nutrient_NN1 access_NN1 ,_PUN low_AJ0 O2_AJ0 availability_NN1 ,_PUN and_CJC elevated_AJ0 temperatures_NN2 )_PUR toward_PRP microbiological_AJ0 habitability_NN1 ._SENT 
The_AT0 large_AJ0 biodiversity_NN1 of_PRF microbial_AJ0 life_NN1 in_PRP the_AT0 deep_AJ0 biosphere_NN1 remains_VVZ to_TO0 be_VBI explored_VVN through_PRP terrestrial_AJ0 subsurface_NN1 investigations_NN2 ._SENT Terrestrial_AJ0 sites_NN2 can_VM0 offer_VVI further_AV0 geologic_AJ0 and_CJC hydrologic_AJ0 variability_NN1 than_CJS marine_AJ0 environments_NN2 due_AJ0 to_PRP the_AT0 presence_NN1 of_PRF unconstrained_AJ0 fluid_NN1 sourcing_NN1 and_CJC lithology_NN1 in_PRP surface_NN1 soils_NN2 and_CJC bedrock_NN1 ._SENT Even_AV0 more_AV0 ,_PUN water-rock_AJ0 interactions_NN2 demonstrated_VVN in_PRP terrestrial_AJ0 subsurface_NN1 environments_NN2 distinguish_VVB the_AT0 chemical_AJ0 disparity_NN1 of_PRF chemolithotrophic_AJ0 processes_NN2 that_CJT sustain_VVB ecosystems_NN2 of_PRF low_AJ0 biomass_NN1 utilizing_VVG restricted_AJ0 sources_NN2 of_PRF energy_NN1 ._SENT The_AT0 contemporary_AJ0 outlook_NN1 of_PRF an_AT0 "_PUQ extreme_AJ0 "_PUQ environment_NN1 encompasses_VVZ some_DT0 variation_NN1 of_PRF a_AT0 chemical_AJ0 or_CJC hydrological_AJ0 endpoint_NN1 for_PRP microorganisms_NN2 to_TO0 exist_VVI primarily_AV0 due_AJ0 to_PRP physiological_AJ0 constraints_NN2 ._SENT However_AV0 ,_PUN new_AJ0 insights_NN2 into_PRP shallow_AJ0 and_CJC deep_AJ0 subsurface_NN1 research_NN1 may_VM0 not_XX0 necessitate_VVI harsh_AJ0 geochemical_AJ0 gradients_NN2 or_CJC temperatures_NN2 ._SENT Instead_AV0 ,_PUN restricted_AJ0 access_NN1 to_PRP nutrients_NN2 ,_PUN vital_AJ0 for_PRP microbial_AJ0 activity_NN1 ,_PUN makes_VVZ terrestrial_AJ0 subsurface_NN1 studies_NN2 of_PRF interest_NN1 to_TO0 investigate_VVI how_AVQ moderately_AV0 warm_AJ0 and_CJC /_PUN or_CJC acidic_AJ0 environments_NN2 can_VM0 still_AV0 be_VBI uniquely_AV0 constraining_AJ0 to_PRP microbiota_NN1 ._SENT 
In_PRP order_NN1 to_TO0 study_VVI subsurface_NN1 microbiology_NN1 ,_PUN many_DT0 continental_AJ0 deep_AJ0 subsurface_NN1 observatories_NN2 have_VHB been_VBN established_VVN ._SENT Reaching_VVG depths_NN2 of_PRF up_AVP to_PRP ∼2.4_CRD km_NN0 ,_PUN the_AT0 Sanford_NP0 Underground_NN1 Research_NN1 Laboratory_NN1 in_PRP South_NP0 Dakota_NP0 has_VHZ investigated_VVN the_AT0 geochemistry_NN1 and_CJC DNA_NN1 sequences_NN2 of_PRF up_AVP to_PRP 10_CRD ,_PUN 000-year-old_AJ0 groundwater_NN1 to_TO0 understand_VVI the_AT0 energetics_NN1 of_PRF chemolithotrophic_AJ0 organisms_NN2 ._SENT These_DT0 subsurface_NN1 laboratories_NN2 extend_VVB across_PRP multiple_AJ0 continents_NN2 and_CJC countries_NN2 ,_PUN including_PRP the_AT0 United_NP0 States_NP0 ,_PUN Finland_NP0 ,_PUN France_NP0 ,_PUN Japan_NP0 ,_PUN and_CJC the_AT0 United_NP0 Kingdom_NP0 ._SENT Another_DT0 exists_VVZ in_PRP Canada_NP0 at_PRP the_AT0 Kidd_NP0 Creek_NN1 Observatory_NN1 ,_PUN where_AVQ fracture_NN1 waters_NN2 at_PRP 2.4_CRD km_NN0 in_PRP depth_NN1 were_VBD acquired_VVN in_PRP order_NN1 to_TO0 cultivate_VVI microorganisms_NN2 from_PRP the_AT0 fluids_NN2 and_CJC determine_VVB the_AT0 metabolic_AJ0 energetic_AJ0 pathways_NN2 from_PRP parallel_AJ0 geochemical_AJ0 analysis_NN1 ._SENT Onstott_NP0 et_NP0 al._NP0 have_VHB established_VVN the_AT0 presence_NN1 of_PRF a_AT0 microbial_AJ0 community_NN1 in_PRP a_AT0 gold_NN1 mine_NN1 in_PRP the_AT0 deep_AJ0 subsurface_NN1 from_PRP fluids_NN2 obtained_VVN up_AVP to_PRP 3_CRD km_NN0 below_PRP the_AT0 surface_NN1 ._SENT These_DT0 fluids_NN2 proved_VVD microbiota_NN1 indigenous_AJ0 to_PRP the_AT0 host_NN1 rock_NN1 environment_NN1 and_CJC further_AV0 demonstrate_VVB the_AT0 potential_NN1 for_PRP life_NN1 driven_VVN by_PRP equilibration_NN1 of_PRF water-rock_AJ0 interactions_NN2 ._SENT Microorganisms_NN2 that_CJT exist_VVB in_PRP the_AT0 secluded_AJ0 hematite_NN1 iron_NN1 formation_NN1 of_PRF the_AT0 Soudan_NN1 Underground_NN1 Mine_NN1 State_NN1 Park_NN1 were_VBD examined_VVN with_PRP metagenomic_AJ0 analyses_NN2 to_TO0 determine_VVI genes_NN2 representing_VVG diverse_AJ0 metabolic_AJ0 strategies_NN2 from_PRP a_AT0 low_AJ0 biomass_NN1 system_NN1 to_TO0 overcome_VVI nutrient_NN1 limitations_NN2 ._SENT 
These_DT0 established_AJ0 observatories_NN2 in_PRP the_AT0 deep_AJ0 subsurface_NN1 offer_NN1 novel_NN1 investigations_NN2 into_PRP niche_NN1 environmental_AJ0 conditions_NN2 ._SENT However_AV0 ,_PUN the_AT0 scope_NN1 of_PRF which_DTQ microorganisms_NN2 survive_VVB in_PRP shallow_AJ0 subsurface_NN1 environments_NN2 from_PRP young_AJ0 meteoric_AJ0 groundwater_NN1 infiltration_NN1 from_PRP the_AT0 surface_NN1 versus_PRP chemical_AJ0 energy_NN1 sources_NN2 originating_VVG in_PRP local_AJ0 settings_NN2 remains_VVZ insufficiently_AV0 answered_VVN ._SENT Studies_NN2 have_VHB utilized_VVN deep_AJ0 fracture_NN1 fluids_NN2 or_CJC ancient_AJ0 aquifers_NN2 to_TO0 constrain_VVI life_NN1 in_PRP the_AT0 subsurface_NN1 ,_PUN yet_AV0 only_AV0 limited_AJ0 research_NN1 has_VHZ considered_VVN the_AT0 influence_NN1 of_PRF meteoric_AJ0 water_NN1 penetration_NN1 into_PRP the_AT0 near_AJ0 subsurface_NN1 ._SENT The_AT0 role_NN1 of_PRF young_AJ0 meteoric_AJ0 groundwater_NN1 infiltration_NN1 as_PRP a_AT0 transportation_NN1 mechanism_NN1 for_PRP nutrients_NN2 introduces_VVZ questions_NN2 regarding_PRP the_AT0 greater_AJC potential_NN1 for_PRP life_NN1 ,_PUN the_AT0 connectivity_NN1 of_PRF the_AT0 surface_NN1 to_PRP the_AT0 subsurface_NN1 ,_PUN and_CJC the_AT0 relation_NN1 of_PRF surface_NN1 processes_NN2 to_PRP subsurface_NN1 microorganisms_NN2 ._SENT 
An_AT0 additional_AJ0 consideration_NN1 for_PRP deep_AJ0 observatories_NN2 is_VBZ the_AT0 cost_NN1 and_CJC accessibility_NN1 of_PRF boreholes_NN2 for_PRP sampling_NN1 and_CJC inquiry_NN1 ._SENT Due_AJ0 to_PRP expenses_NN2 from_PRP boreholes_NN2 and_CJC gaining_VVG access_NN1 to_PRP subsurface_NN1 environments_NN2 ,_PUN numerous_AJ0 questions_NN2 remain_VVB unanswered_AJ0 from_PRP both_DT0 marine_AJ0 and_CJC terrestrial_AJ0 subsurface_NN1 ecosystem_NN1 exploration_NN1 ._SENT Therefore_AV0 ,_PUN easier_AJC access_NN1 into_PRP the_AT0 subsurface_NN1 is_VBZ advantageous_AJ0 not_XX0 only_AV0 to_TO0 establish_VVI consistent_AJ0 research_NN1 regarding_PRP the_AT0 microbial_AJ0 communities_NN2 existing_AJ0 in_PRP the_AT0 subsurface_NN1 but_CJC also_AV0 to_TO0 monitor_VVI geochemical_AJ0 variables_NN2 influencing_VVG subsurface_NN1 habitability_NN1 with_PRP greater_AJC control_NN1 over_PRP experimental_AJ0 design_NN1 and_CJC statistical_AJ0 power_NN1 ._SENT 
This_DT0 paper_NN1 discusses_VVZ the_AT0 advantages_NN2 of_PRF studying_VVG subsurface_NN1 microbiology_NN1 at_PRP the_AT0 Edgar_NP0 Experimental_AJ0 Mine_NN1 ,_PUN an_AT0 easily_AV0 accessible_AJ0 portal_NN1 to_PRP the_AT0 subsurface_NN1 with_PRP ample_AJ0 opportunities_NN2 to_TO0 unveil_VVI and_CJC experiment_VVI with_PRP the_AT0 subsurface_NN1 biosphere_NN1 ._SENT Once_CJS an_AT0 active_AJ0 economic_AJ0 mining_NN1 site_NN1 ,_PUN it_PNP was_VBD repurposed_AJ0 for_PRP teaching_VVG demonstrations_NN2 by_PRP Colorado_NP0 School_NN1 of_PRF Mines_NN2 for_PRP mining_NN1 engineering_NN1 practices_NN2 ._SENT Four_CRD boreholes_NN2 drilled_VVN from_PRP previous_AJ0 mining_NN1 teaching_NN1 practices_NN2 are_VBB the_AT0 focus_NN1 of_PRF this_DT0 study_NN1 ,_PUN located_VVN centimeters_NN2 to_PRP meters_NN2 apart_AV0 along_PRP the_AT0 same_DT0 wall_NN1 of_PRF our_DPS field_NN1 site_NN1 ._SENT Meteoric_AJ0 groundwater_NN1 infiltrating_VVG the_AT0 fractures_NN2 of_PRF the_AT0 mine_NN1 leak_NN1 out_AVP of_PRF the_AT0 boreholes_NN2 ,_PUN offering_VVG the_AT0 opportunity_NN1 to_TO0 study_VVI the_AT0 subsurface_NN1 microbiology_NN1 of_PRF meteoric_AJ0 water_NN1 influenced_VVN by_PRP young_AJ0 fluid-rock_AJ0 equilibration_NN1 (_PUL Fig._NN1 1_CRD )_PUR ._SENT Additionally_AV0 ,_PUN the_AT0 periodic_AJ0 (_PUL ∼2_CRD weeks_NN2 )_PUR to_PRP seasonal_AJ0 recharge_NN1 of_PRF water_NN1 makes_VVZ it_PNP possible_AJ0 to_TO0 study_VVI temporal_AJ0 influences_NN2 through_PRP incubation_NN1 and_CJC recharge_NN1 periods_NN2 ._SENT Here_AV0 ,_PUN we_PNP apply_VVB genomic_AJ0 ,_PUN geochemical_AJ0 ,_PUN and_CJC isotopic_AJ0 approaches_NN2 to_TO0 better_AV0 define_VVI the_AT0 variable_AJ0 microbial_AJ0 composition_NN1 of_PRF borehole_NN1 fluids_NN2 and_CJC their_DPS relationship_NN1 to_PRP surface_NN1 influences_NN2 from_PRP meteoric_AJ0 penetration_NN1 into_PRP the_AT0 subsurface_NN1 ._SENT 
RESULTS_NN2 
Borehole_NN1 geochemistry_NN1 ._SENT 
Over_PRP the_AT0 4-year_AJ0 sample_NN1 collection_NN1 period_NN1 ,_PUN the_AT0 subsurface_NN1 boreholes_NN2 remained_VVD relatively_AV0 uniform_AJ0 in_PRP their_DPS recorded_AJ0 analyte_NN1 and_CJC physical_AJ0 chemistry_NN1 measurements_NN2 ._SENT While_CJS boreholes_NN2 minorly_AV0 fluctuate_VVB in_PRP analyte_NN1 (_PUL major_AJ0 anion_NN1 and_CJC cation_NN1 )_PUR concentration_NN1 ,_PUN no_AT0 discrete_AJ0 patterns_NN2 were_VBD observed_VVN ._SENT Despite_PRP a_AT0 lack_NN1 of_PRF change_NN1 ,_PUN each_DT0 borehole_NN1 hosts_VVZ nuanced_AJ0 geochemical_AJ0 profiles_NN2 ,_PUN providing_VVG niche_NN1 microenvironments_NN2 for_PRP microbial_AJ0 communities_NN2 ._SENT Fluid_NN1 recharge_NN1 in_PRP the_AT0 boreholes_NN2 was_VBD unpredictable_AJ0 regarding_PRP volume_NN1 and_CJC temporal_AJ0 repetition_NN1 ._SENT Borehole_NN1 2_CRD was_VBD able_AJ0 to_TO0 consistently_AV0 fill_VVI with_PRP greater_AJC than_CJS 1_CRD liter_NN1 of_PRF water_NN1 each_DT0 sampling_NN1 period_NN1 ,_PUN whereas_CJS all_DT0 other_AJ0 boreholes_NN2 required_VVN months_NN2 or_CJC years_NN2 before_PRP refilling_VVG with_PRP fluid_NN1 (_PUL Fig._NN1 1_CRD )_PUR ._SENT The_AT0 remaining_AJ0 boreholes_NN2 did_VDD not_XX0 refill_VVI with_PRP predictable_AJ0 volumes_NN2 of_PRF water_NN1 at_PRP each_DT0 sampling_NN1 period_NN1 ,_PUN further_AV0 emphasizing_VVG a_AT0 complex_AJ0 fracture_NN1 pathway_NN1 for_PRP fluid_NN1 transport_NN1 ._SENT Physical_AJ0 parameters_NN2 and_CJC chemical_AJ0 composition_NN1 remained_VVD relatively_AV0 homogenous_AJ0 among_PRP boreholes_NN2 ._SENT Water_NN1 temperature_NN1 ranges_NN2 varied_VVN seasonally_AV0 ,_PUN generally_AV0 fluctuating_AJ0 between_PRP 10.6_CRD and_CJC 13.5_CRD °_NN0 C_ZZ0 and_CJC reaching_VVG 17.6_CRD °_NN0 C_ZZ0 in_PRP summer_NN1 ._SENT Dissolved_AJ0 oxygen_NN1 varied_VVD most_AV0 strongly_AV0 from_PRP 1.97_CRD to_PRP 7.7_CRD mg_NN0 /_PUN liter_NN1 ,_PUN while_CJS pH_NN1 was_VBD generally_AV0 uniform_AJ0 within_PRP individual_AJ0 boreholes_NN2 but_CJC varied_AJ0 among_PRP all_DT0 samples_NN2 ranging_VVG from_PRP pH_NN1 5.30_CRD to_PRP 7.16_CRD ._SENT 
Ion_NN1 chromatography_NN1 (_PUL IC_NP0 )_PUR and_CJC inductively_AV0 coupled_VVN plasma_NN1 atomic_AJ0 emission_NN1 spectroscopy_NN1 (_PUL ICP-AES_NP0 )_PUR results_NN2 of_PRF borehole_NN1 fluids_NN2 demonstrated_VVD weakly_AV0 variable_AJ0 subsurface_NN1 fluid_NN1 chemistry_NN1 ._SENT Concentrations_NN2 of_PRF anions_NN2 (_PUL putative_AJ0 electron_NN1 acceptors_NN2 for_PRP microbial_AJ0 growth_NN1 as_CJS well_AV0 as_CJS phosphorous_AJ0 sourcing_NN1 )_PUR tend_VVB to_TO0 be_VBI very_AV0 low_AJ0 or_CJC undetected_AJ0 ,_PUN allowing_VVG for_PRP selective_AJ0 substrate_NN1 availability_NN1 of_PRF microbial_AJ0 communities_NN2 ._SENT Fluoride_NN1 concentrations_NN2 across_PRP all_DT0 boreholes_NN2 ranged_VVD from_PRP below_AV0 detection_NN1 or_CJC from_PRP 0.15_CRD to_PRP 0.33_CRD ppm_NN0 ._SENT Nitrite_NN1 concentrations_NN2 were_VBD undetected_AJ0 except_CJS for_PRP three_CRD samples_NN2 that_CJT recorded_VVD values_NN2 of_PRF 0.19_CRD ,_PUN 0.27_CRD ,_PUN and_CJC 0.37_CRD ppm_NN0 ._SENT Nitrate_NN1 remained_VVD undetected_AJ0 or_CJC reported_AJ0 values_NN2 ranging_VVG from_PRP 0.45_CRD to_PRP 1.27_CRD ppm_NN0 ._SENT Chloride_NN1 concentrations_NN2 varied_VVN unexpectedly_AV0 within_PRP individual_AJ0 boreholes_NN2 with_PRP concentrations_NN2 between_PRP 1.59_CRD and_CJC 22.08_CRD ppm_NN0 but_CJC spiked_AJ0 as_CJS high_AV0 as_CJS 474.85_CRD ppm_NN0 ._SENT Sulfate_NN1 was_VBD the_AT0 most_AV0 concentrated_AJ0 analyte_NN1 among_PRP all_DT0 boreholes_NN2 and_CJC consistently_AV0 remained_VVD high_AJ0 (_PUL usually_AV0 greater_AJC than_CJS 1_CRD ,_PUN 000_CRD ppm_NN0 )_PUR ._SENT Analytes_NN2 chosen_VVN from_PRP IC_NP0 and_CJC ICP_NP0 analysis_NN1 were_VBD selected_VVN to_TO0 demonstrate_VVI greatest_AJS variability_NN1 among_PRP the_AT0 borehole_NN1 fluids_NN2 (_PUL Table_NN1 S1_CRD in_PRP the_AT0 supplemental_AJ0 material_NN1 )_PUR ._SENT These_DT0 analytes_NN2 were_VBD evaluated_VVN with_PRP a_AT0 principal-component_AJ0 analysis_NN1 (_PUL PCA_NP0 )_PUR to_TO0 observe_VVI the_AT0 relationship_NN1 of_PRF geochemical_AJ0 measurements_NN2 collected_VVN from_PRP the_AT0 individual_AJ0 boreholes_NN2 (_PUL Fig._NN1 2_CRD )_PUR ._SENT Of_PRF interest_NN1 ,_PUN principal_AJ0 component_NN1 2_CRD appears_VVZ to_TO0 suggest_VVI a_AT0 strong_AJ0 delineation_NN1 driven_VVN by_PRP metals_NN2 concentrations_NN2 ._SENT In_PRP particular_AJ0 ,_PUN manganese_NN1 and_CJC zinc_NN1 were_VBD the_AT0 highest_AJS notable_AJ0 metal_NN1 concentrations_NN2 with_PRP maximum_AJ0 values_NN2 of_PRF 27.15_CRD mg_NN0 /_PUN liter_NN1 and_CJC 18.46_CRD mg_NN0 /_PUN liter_NN1 ,_PUN respectively_AV0 ._SENT There_EX0 is_VBZ a_AT0 linear_AJ0 relationship_NN1 between_PRP the_AT0 dissolved_AJ0 manganese_NN1 and_CJC zinc_NN1 concentrations_NN2 in_PRP the_AT0 borehole_NN1 fluids_NN2 (_PUL Fig._NN1 3_CRD )_PUR ._SENT This_DT0 highlights_VVZ the_AT0 distinction_NN1 between_PRP the_AT0 metal-rich_AJ0 fluids_NN2 found_VVN in_PRP boreholes_NN2 2_CRD and_CJC 3_CRD and_CJC more_DT0 dilute_AJ0 fluids_NN2 demonstrated_VVN in_PRP boreholes_NN2 6_CRD and_CJC 8._CRD Additionally_AV0 ,_PUN this_DT0 relationship_NN1 between_PRP the_AT0 boreholes_NN2 shows_VVZ the_AT0 consistency_NN1 of_PRF borehole_NN1 fluid_NN1 chemistry_NN1 despite_PRP sampling_NN1 period_NN1 and_CJC how_AVQ this_DT0 can_VM0 serve_VVI as_PRP an_AT0 indicator_NN1 for_PRP variation_NN1 in_PRP microbial_AJ0 community_NN1 composition_NN1 ._SENT 
The_AT0 δ18O_NN1 and_CJC δ2H_NN1 signatures_NN2 for_PRP borehole_NN1 fluids_NN2 were_VBD compared_VVN to_PRP the_AT0 global_AJ0 meteoric_AJ0 water_NN1 line_NN1 (_PUL GMWL_NP0 )_PUR along_AVP with_PRP samples_NN2 from_PRP other_AJ0 subsurface_NN1 systems_NN2 or_CJC observatories_NN2 (_PUL Fig._NN1 4_CRD )_PUR ._SENT This_DT0 trendline_NN1 serves_VVZ as_PRP an_AT0 investigative_AJ0 tool_NN1 for_PRP understanding_NN1 if_CJS the_AT0 subsurface_NN1 fluids_NN2 have_VHB undergone_VVN some_DT0 extent_NN1 of_PRF low-temperature_AJ0 water-rock_AJ0 interactions_NN2 or_CJC remain_VVB isotopically_AV0 conserved_AJ0 to_TO0 reflect_VVI meteoric_AJ0 recharge_NN1 from_PRP the_AT0 surface_NN1 ._SENT Samples_NN2 are_VBB plotted_VVN above_PRP the_AT0 GMWL_NN1 with_PRP slightly_AV0 enriched_AJ0 δ2H_NN1 values_NN2 ._SENT Edgar_NP0 Mine_PNP is_VBZ located_VVN at_PRP greater_AJC elevation_NN1 and_CJC drier_AJC climate_NN1 ,_PUN which_DTQ often_AV0 causes_VVZ enriched_AJ0 δ2H_NN1 values_NN2 of_PRF meteoric_AJ0 water_NN1 as_PRP a_AT0 result_NN1 from_PRP the_AT0 rainout_NN1 effect_NN1 ._SENT 
Microbial_AJ0 community_NN1 analysis_NN1 ._SENT 
16S_CRD rRNA_NN1 gene_NN1 sequence_NN1 analyses_NN2 were_VBD performed_VVN on_PRP filtered_AJ0 water_NN1 to_TO0 determine_VVI the_AT0 microbial_AJ0 community_NN1 composition_NN1 within_PRP each_DT0 borehole_NN1 ._SENT Several_DT0 samples_NN2 from_PRP each_DT0 borehole_NN1 were_VBD taken_VVN to_TO0 contrast_VVI composition_NN1 over_PRP time_NN1 ._SENT The_AT0 fluid_NN1 samples_NN2 were_VBD dominated_VVN by_PRP Bacteria_NN2 (_PUL 98.5_CRD %_NN0 )_PUR with_PRP only_AV0 a_AT0 small_AJ0 presence_NN1 of_PRF Archaea_NN1 (_PUL 1.5_CRD %_NN0 )_PUR belonging_VVG predominantly_AV0 to_PRP samples_NN2 from_PRP borehole_NN1 2._CRD All_DT0 boreholes_NN2 were_VBD collectively_AV0 dominated_VVN by_PRP the_AT0 phylum_NN1 Proteobacteria_NN1 (_PUL 39.2_CRD %_NN0 )_PUR and_CJC also_AV0 contained_VVN sequences_NN2 for_PRP divisions_NN2 of_PRF Firmicutes_NP0 (_PUL 16.1_CRD %_NN0 )_PUR and_CJC Bacteroidota_NP0 (_PUL 13.0_CRD %_NN0 )_PUR known_VVN to_TO0 represent_VVI a_AT0 diverse_AJ0 group_NN1 of_PRF microorganisms_NN2 ._SENT 
Ordination_NN1 via_PRP principal-coordinate_AJ0 analysis_NN1 (_PUL PCoA_NP0 )_PUR with_PRP a_AT0 weighted_AJ0 UniFrac_NN1 distance_NN1 matrix_NN1 was_VBD performed_VVN to_TO0 determine_VVI statistical_AJ0 similarity_NN1 of_PRF microbial_AJ0 communities_NN2 from_PRP each_DT0 borehole_NN1 (_PUL Fig._NN1 5_CRD )_PUR ._SENT Ordination_NN1 suggested_VVD strong_AJ0 clustering_NN1 as_PRP a_AT0 result_NN1 of_PRF grouping_NN1 by_PRP borehole_NN1 location_NN1 ._SENT Additional_AJ0 soil_NN1 samples_NN2 were_VBD collected_VVN from_PRP the_AT0 surface_NN1 of_PRF the_AT0 mine_NN1 to_TO0 confirm_VVI the_AT0 distinctive_AJ0 compositions_NN2 of_PRF borehole_NN1 microbiota_NN1 ._SENT An_AT0 Adonis_NP0 test_NN1 was_VBD run_VVN on_PRP the_AT0 dissimilarity_NN1 matrix_NN1 to_TO0 verify_VVI sampling_NN1 source_NN1 as_PRP distinct_AJ0 and_CJC significant_AJ0 (_PUL R2_CRD =_NP0 0.44_CRD ,_PUN P_ZZ0 =_NN1 0.001_CRD )_PUR ._SENT Weighted_AJ0 UniFrac_NP0 distance_NN1 matrices_NN2 were_VBD also_AV0 tested_VVN by_PRP Adonis_NP0 for_PRP the_AT0 number_NN1 of_PRF days_NN2 since_PRP the_AT0 initial_AJ0 extraction_NN1 of_PRF fluid_NN1 from_PRP the_AT0 boreholes_NN2 ,_PUN the_AT0 number_NN1 of_PRF days_NN2 between_PRP sampling_NN1 events_NN2 ,_PUN and_CJC the_AT0 season_NN1 in_PRP which_DTQ samples_NN2 were_VBD collected_VVN ._SENT Results_NN2 demonstrated_VVD the_AT0 number_NN1 of_PRF days_NN2 since_PRP the_AT0 initial_AJ0 extraction_NN1 period_NN1 as_CJS significant_AJ0 (_PUL R2_CRD =_NP0 0.38_CRD ,_PUN P_ZZ0 =_NN1 0.001_CRD )_PUR as_CJS well_AV0 as_CJS seasonality_NN1 (_PUL R2_CRD =_NP0 0.36_CRD ,_PUN P_ZZ0 =_NN1 0.001_CRD )_PUR and_CJC the_AT0 number_NN1 of_PRF days_NN2 between_PRP sampling_NN1 events_NN2 as_CJS modestly_AV0 significant_AJ0 (_PUL R2_CRD =_NP0 0.073_CRD ,_PUN P_ZZ0 =_NN1 0.047_CRD )_PUR ._SENT These_DT0 values_NN2 suggest_VVB that_CJT each_DT0 borehole_NN1 is_VBZ host_NN1 to_PRP unique_AJ0 microbial_AJ0 community_NN1 compositions_NN2 despite_PRP their_DPS relatively_AV0 close_AJ0 proximity_NN1 and_CJC similarity_NN1 of_PRF host_NN1 rock_NN1 ._SENT The_AT0 inherent_AJ0 covariance_NN1 in_PRP our_DPS study_NN1 between_PRP days_NN2 since_PRP initial_AJ0 extraction_NN1 and_CJC seasonality_NN1 suggests_VVZ that_CJT further_AJC work_NN1 is_VBZ needed_VVN to_TO0 decipher_VVI these_DT0 two_CRD signals_NN2 ._SENT Nonetheless_AV0 ,_PUN a_AT0 clear_AJ0 shift_NN1 in_PRP community_NN1 composition_NN1 occurs_VVZ over_PRP time_NN1 ,_PUN predominantly_AV0 in_PRP boreholes_NN2 2_CRD and_CJC 3._CRD However_AV0 ,_PUN the_AT0 source_NN1 of_PRF this_DT0 change_NN1 requires_VVZ additional_AJ0 sampling_NN1 to_TO0 account_VVI for_PRP unsampled_AJ0 seasons_NN2 and_CJC how_AVQ microbial_AJ0 diversity_NN1 may_VM0 change_VVI with_PRP even_AV0 more_DT0 time_NN1 allowed_VVD to_TO0 pass_VVI within_PRP the_AT0 boreholes_NN2 ._SENT 
To_TO0 further_AV0 investigate_VVI the_AT0 nuances_NN2 in_PRP microbial_AJ0 community_NN1 composition_NN1 and_CJC shifts_NN2 occurring_VVG over_PRP the_AT0 course_NN1 of_PRF the_AT0 experiment_NN1 ,_PUN differential_AJ0 abundance_NN1 tests_NN2 revealed_VVD the_AT0 top_AJ0 15_CRD most_DT0 abundant_AJ0 genera_NN2 in_PRP the_AT0 boreholes_NN2 and_CJC soil_NN1 samples_NN2 (_PUL Fig._NN1 6_CRD and_CJC 7_CRD )_PUR ._SENT Boreholes_NN2 2_CRD and_CJC 3_CRD appear_VVB to_TO0 host_VVI similar_AJ0 microbial_AJ0 communities_NN2 with_PRP subtle_AJ0 changes_NN2 in_PRP taxa_NN2 present_NN1 and_CJC changes_NN2 over_PRP the_AT0 course_NN1 of_PRF the_AT0 sampling_NN1 period_NN1 ._SENT The_AT0 boreholes_NN2 share_VVB a_AT0 large_AJ0 relative_AJ0 abundance_NN1 of_PRF Desulfosporosinus_NP0 ,_PUN a_AT0 potential_AJ0 sulfate-reducing_NN1 bacteria_NN2 ,_PUN and_CJC Sulfurifustis_NP0 ,_PUN a_AT0 potential_AJ0 sulfur-oxidizing_NN1 bacteria_NN2 ,_PUN from_PRP each_DT0 sampling_NN1 occurrence_NN1 ._SENT Boreholes_NN2 2_CRD and_CJC 3_CRD exhibit_VVB the_AT0 same_DT0 relationship_NN1 where_AVQ an_AT0 increased_AJ0 relative_AJ0 abundance_NN1 of_PRF Desulfosporosinus_NN1 leads_VVZ to_PRP a_AT0 dramatic_AJ0 decrease_NN1 in_PRP the_AT0 relative_AJ0 abundance_NN1 of_PRF other_AJ0 microbial_AJ0 members_NN2 within_PRP the_AT0 top_AJ0 15_CRD most_DT0 abundant_AJ0 genera_NN2 of_PRF fluid_NN1 samples_NN2 from_PRP boreholes_NN2 2_CRD and_CJC 3._CRD This_DT0 stark_AJ0 contrast_NN1 in_PRP microbial_AJ0 composition_NN1 is_VBZ demonstrated_VVN from_PRP the_AT0 last_ORD two_CRD sampling_NN1 periods_NN2 ,_PUN after_PRP 948_CRD days_NN2 have_VHB passed_VVN into_PRP the_AT0 experiment_NN1 (_PUL Fig._NN1 6_CRD )_PUR ._SENT In_PRP borehole_NN1 2_CRD ,_PUN this_DT0 shift_NN1 appears_VVZ to_TO0 drastically_AV0 change_VVI the_AT0 microbial_AJ0 composition_NN1 where_AVQ the_AT0 relative_AJ0 abundance_NN1 of_PRF Desulfosporosinus_NN1 continues_VVZ to_TO0 increase_VVI ,_PUN and_CJC there_EX0 is_VBZ little_DT0 relative_AJ0 abundance_NN1 represented_VVN from_PRP additional_AJ0 microorganisms_NN2 ._SENT In_PRP contrast_NN1 ,_PUN borehole_NN1 3_CRD displays_VVZ a_AT0 shift_NN1 in_PRP composition_NN1 at_PRP this_DT0 same_DT0 sampling_NN1 instance_NN1 ,_PUN but_CJC Desulfosporosinus_NP0 decreases_VVZ in_PRP relative_AJ0 abundance_NN1 while_CJS other_AJ0 taxa_NN2 increase_NN1 (_PUL Fig._NN1 6_CRD )_PUR ._SENT Additionally_AV0 ,_PUN this_DT0 shift_NN1 does_VDZ not_XX0 last_VVI through_PRP the_AT0 final_AJ0 sampling_NN1 period_NN1 ,_PUN and_CJC Desulfosporosinus_NP0 increases_VVZ in_PRP relative_AJ0 abundance_NN1 similar_AJ0 to_PRP earlier_AJC fluid_NN1 samples_NN2 ._SENT The_AT0 other_AJ0 taxa_NN2 present_AJ0 in_PRP the_AT0 borehole_NN1 fluid_NN1 samples_NN2 are_VBB indicators_NN2 of_PRF metal-rich_AJ0 environments_NN2 ,_PUN including_PRP genera_NN2 such_DT0 as_PRP Candidatus_NP0 Nitrotoga_NP0 and_CJC Chelatococcus_NP0 ._SENT 
Comparatively_AV0 ,_PUN the_AT0 microbial_AJ0 community_NN1 composition_NN1 of_PRF boreholes_NN2 6_CRD and_CJC 8_CRD appear_VVB to_TO0 be_VBI fixed_VVN without_PRP any_DT0 distinctive_AJ0 influence_NN1 as_CJS observed_VVN in_PRP boreholes_NN2 2_CRD and_CJC 3._CRD However_AV0 ,_PUN the_AT0 genera_NN2 observed_VVN in_PRP boreholes_NN2 6_CRD and_CJC 8_CRD juxtapose_VVB the_AT0 microbial_AJ0 communities_NN2 observed_VVN in_PRP boreholes_NN2 2_CRD and_CJC 3_CRD ,_PUN where_AVQ the_AT0 microorganisms_NN2 represented_VVN do_VDB not_XX0 indicate_VVI metal-rich_AJ0 environments_NN2 ._SENT 
