Initial Initial Geochemical Geochemical and and Microbiological Microbiological CharacterizationCharacterizationof Henderson Fluidsof Henderson Fluids

• How does knowledge of the site-specific chemistry at Henderson enhance our ability to identify subsurface microbial

organisms (phylogentically and functionally)

-- and their direct dependency upon fluid-rock interaction?

BIOSCIENCE SAMPLING TEAMBIOSCIENCE SAMPLING TEAM

Templeton: Henderson DUSEL Capstone Workshop May 4-7th

Tom Kieft (New Mexico Tech)for

Alexis Templeton (University of Colorado – Boulder)

Fluid Chemistry: 7025 level

Warm, ~40C; pH 5.9 to 6.2

Dissolved O2: 0.3 to 3.8 mg/L

High DIC (~30 mM)

High Mn2+, Fe2+ (1 to 20 mM)

High SO42- (4 to 48 mM)

Trace metals: Zn > Ni > Mo

Low (but detectable) organics (50 M)

Abundant nitrogen species:includes NO3

-, NO2- , N2O, NH4

+

Flowing boreholes variably mixed with oxygen

O 2 concentrations increase

as borehole flow-rate decreases

From D1 D3

O2 levels drop 1000x within 1 hour of packer-insertion

D1

D2

D3

Establishing the basic chemistry, nutrient levels and cell numbers Establishing the basic chemistry, nutrient levels and cell numbers in deeply-sourced, hot fluids:in deeply-sourced, hot fluids:

Natural Fracture Surface: Degassing CONatural Fracture Surface: Degassing CO22

Can also detect NCan also detect N22O (6-18 microM), only low CHO (6-18 microM), only low CH44 (sub-microM), and H (sub-microM), and H22 not measured yet. not measured yet.

CO2 Movie:

High CO2 important for autotrophic growth

Fe-oxidesFe-oxides

SulfatesSulfates

FeFe2+2+ rapidly oxidizes and oxide and sulfate minerals precipitate rapidly oxidizes and oxide and sulfate minerals precipitate

New minerals hard to structurally identify – relatively amorphous

What’s the disequilibrium between the fluids and the surface? Oxic-Anoxic Interface

Adjacent to the Fracture: Adjacent to the Fracture: Abundant Mn(IV)-oxides form black mineral coatings on the tunnel walls.Abundant Mn(IV)-oxides form black mineral coatings on the tunnel walls.

LBB-testShows Mn(IV)In minerals;

Mn(II) in fluids

First assessement: First assessement: Microbial Diversity in the Ancient FluidsMicrobial Diversity in the Ancient Fluids

10%

14%

29%

14%

3%

3%

17%

10%

Fungus; AscomycotaGreen Non-Sulfur or ChlorofexiDelta-ProteobacteriumBeta-ProteobacteriumAlpha-ProteobacteriumSpirocheteActinobacteriumFirmicutes; Clostridium

Data from John Spear, CSM

Surprising abundance ofEukaryotic fungal sequences..

Second assessment: Second assessment: Microbial Diversity in fluids of varying chemistryMicrobial Diversity in fluids of varying chemistry

(samples only separated by meters….)(samples only separated by meters….)

More dilute fluids

(7025-D1), pre-packer:

Dominated by uncultured beta-Proteobacteria (e.g. Japanese Gold

Mine): 80%

Fluids with high Fe, Mn, sulfate, NH4+, N2O (7025-D4) post-packer:

Abundant delta-Proteobacteria (SRB)

Abundant Nitrospira

Remarkable diversity (~27 other groups)

•See John Spear talk on Friday…

Thermophilic Metal-oxidizing bacteria?Thermophilic Metal-oxidizing bacteria?

Microbes more abundant in the Fe-oxide matsMicrobes more abundant in the Fe-oxide mats

Rapid growth on Mn-media at 50°C using D4 mineral mat! Rapid growth on Mn-media at 50°C using D4 mineral mat! First 3 isolates being sequencedFirst 3 isolates being sequenced

Water and Mat samples:

Fe-oxidizing bacteria Mn-oxidizing bacteria

Establish enrichments:

*site-specific Henderson medium*

with and without organicsnear-neutral pHstabilized on mineral surfacesculture at 40°C-55°C

Growth onFe-media alsosuccessful…

DNA-labeled cells

Fe-oxide particles

Dissolved Oxygen now below detection

Now target anaerobic enrichments:Isolates for biochemical and proteomic studies

Anaerobic Fe-oxidizers

Mn-reducersFe-reducers

Sulfate-reducers

In-situ biofilms recently

extracted for analysis

Summary to-dateSummary to-date• Yes, microorganisms are present in the fluids, at low cell numbers.

• The fluid chemistry at Henderson is unique due to water-rock interaction, which has implications for the likely modes of metabolism and enzymatic activities.

– CO2 and sulfate concentrations are very high– N and C nutrient levels are above detection limits, but not P– Millimolar concentrations of Mn, Fe and Zn

• Fluid chemistry will vary significantly depending upon location inside vs. outside mineralized rock.

• The microbial diversity is high and includes unexpected fungal sequences

• There exists unexpected potential for a subsurface microbial N-cycleNH4

+, NO2-, NO3

- and N2O all abundant (naturally or not?)

• Microbial organisms are more abundant at the oxic-anoxic interface, where the Fe-oxide mineral mats are forming.

• Initial culturing efforts targeting Fe and Mn-oxidizing bacteria appear to be successful: sequenced isolates anticipated in the near-term, to be compared to clone libraries.

• Noble gas & stable-isotope geochemistry will constrain the age & source of the fluids.– Preliminary 14C data shows 26,000 years