# Presentation: 424711

## The Subsurface Biosphere

*Tom Kieft*
*New Mexico Inst. of Mining and Technology*
*T. C. Onstott*
*Princeton University*

## CHRONOLOGY OF SUBSURFACE     MICROBIAL  INVESTIGATIONS

## Major accomplishments & findings:

**Tracer technologies**
**Extended known limits of biosphere to > 3 km depth**
**Established biomass & biodiversity**
**Isolates in Culture Collections**
**Correlated microbial activity with **
**   ****geological interfaces**
**Rates of subsurface microbial activity**
**Long-term survival of subsurface microbes**

## sulfate

- DOC, H2 & NH3

- Contact metamorphic source

- Fluid inclusions

- Mantle source

- Deep crustal meteoric circulation

- Deep crustal meteoric circulation

- Ventersdorp dyke

- Pilansberg dyke

- Vertical fluid transport along fault

- CH4
- filled
- fracture porosity

- DOC

- Nitrate

- NH3?

- DIC

- N2

- NATURAL FLUID & SUBSTRATE MIGRATION

- Permeable
- Sedimentary Layers

- DOC, H2 & NH3

- CH4

- AUTOTROPHY

- HETEROTROPHY

- ENERGY

## LIFE IN THE EXTREMELY SLOW LANE

## Slide 6

## Community Structure

**Subsurface**
**Microbial**
**Biogeochemical**
**Cycling**

_**Community Structure**_

_**Function**_

_**Aseptic Sampling**_
_**w/ Tracers**_

_**Environment**_

**Build a team!**

- Sherwood Lollar-Univ. Toronto

- DeFlaun-Envirogen Inc.

- Takai-JAMSTEC

- Pedersen-Univ. Goteborg

- Balkwill-Florida State

- Pfiffner&White-Univ. Tenn.

- Southam-Northern Arizona

- Omar&Hoek-Univ. Penn.

- Boone-Portland State

- Pratt-Indiana Univ.

- Stahl-Univ. Wash.
- Baker-Univ. Wisconsin

- Stute-LDEO

- Suflita-Univ. Oklahoma

- Phelps-ORNL

- Fredrickson&Brockman**-**PNNL

- Wilson-Turgis Tech.
- Alexander-Univ. Wits.
- Spoelstra-Driefontein

## Community Structure

**Subsurface**
**Microbial**
**Biogeochemical**
**Cycling**

**Community Structure**

**Function**

**Sampling**

**Aqueous**
**Geochemistry**

**Dissolved Gases**
** ****& Stable Isotopes**

**Microscopy**
**& Mineral **
**Geochemistry**

**Environment**

## 9-15oC/km

- 2.0 Ga

- 2.9 Ga

- 2.7 Ga

- 2.0 Ga

- 300 Ma

- 2.3 Ga

***Transvaal***
***dolomitic***
***sediments***

- 10 kmbls

***Basement 3.4 Ga***

- 9-15oC/km

- 25oC/km

- 20oC/km

- 120oC

## MANTLE CH4 & Hydrocarbons

- MANTLE CH4 & Hydrocarbons

**H****2**

**H****2**

**CH****4**

**CH****4**

**CH****4**

- ENERGY SOURCES AND CARBON SUBSTRATES

**CO****2**

- E5

## Witwatersrand Gases

***CH******4****** (30-80%)***
***C******2******+ (3-4%) ***
***H******2****** (up to 30%)***
***He (up to 10%)***
***balance N******2***
***some NH******3******?***

**Similar to Canadian and Fennoscandian Shield mines**

## Sampling

*Rocks from freshly mined face*
*Cores*
*Fissure water from flowing boreholes*
*biofilms*

## Direct extraction, PCR and sequencing of 16SrDNA from Au- and U-rich carbon leader yielded Thermus and 

**Direct extraction, PCR and sequencing of 16SrDNA from**** ****Au- and**** ****U-rich carbon leader yielded Thermus and **
**cyanobacteria? **
**sequences**

**Phosphorimage**

**Carbon Leader**

## How can you collect uncontaminated ore samples?

** ****SAMPLES CHISELED FROM FRESHLY BLASTED FACE**
** ****LARGE BLOCKS COLLECTED FROM FRESHLY BLASTED RUBBLE**
** ****SIDE WALL CORING INTO INACTIVE STOPE FACE**

## Microbiological, petrological, chemical and 

**Microbiological, petrological, chemical and **
**isotopic analyses of fracture fluid**

## MINING IMPACT

** ****induced fluid migration **
** ****biological contamination**
** ****oxidation interface **

## Service, Dolomite and Fissure water: pe vs. pH

**Service, Dolomite and Fissure water: pe vs. pH**

## >1 Myr.

- Stute @  LDGO

## Dolomite

- Acid Mine Water

- Hi Sulfate
- Fissure Water

- Microbial sulfate reduction

- Pratt @  IU

- Sulfide Ore

- Low Sulfate
- Fissure Water

## Environment

**Subsurface**
**Microbial**
**Biogeochemical**
**Cycling**

**Sampling**

**Microscopy**
**& Mineral **
**Geochemistry**

**Aqueous**
**Geochemistry**

**Environment**

**Dissolved Gases, Cosmogenic **
** ****& Stable Isotopes**

**PLFA-cell membrane**

**Isolates &**
**Archives**

**Function**

**16SrDNA**

**Community Structure**

**Enrichments**
**genes, & enzymes**

## West Driefontein

- Crenarchaeota

- Kieft @ NMT

## Acid Mine & Service Water 16S rDNA Sequences

- W6

- Beatrix, W. Drie & Kloof Fissure Water Sequences

- Takai @  JAMSTEC

## 0.10

- Nitrospira marina

- clone WCHB1-02

- Thermus thermophilus

- Deinococcus radiodurans

- Fervidobacterium islandicum

- Petrotoga mobilis

- Coprothermobacter proteolyticus

- Coprothermobacter platensis

- Hydrogenobacter subterranea

- Aquifex pyrophilus

- Thermodesulfobacterium hveragerdicum

- clone OPB45

- Methanothermus fervidus

- .10

- 041

- 036

- Anaerobaculum thermoterrenum Group

- Fibrobacter and Acidobacterium

**Low G+C Gram-positive Bacteria**

- Nitrospina Subdivision

- Flexistipes sinusarabici Assemblage

- Fusobacteria and Relatives

- Gamma Proteobacteria

- Beta Proteobacteria

- Alpha Proteobacteria

- Flexibacter-Cytophaga-Bacteroides

- Delta-Proteobacteria

- High-G+C Gram-positive Bacteria

- Cyanobacteria and Chloroplasts

- Planctomyces and Relatives

- Environmental Clone OPB2 Group

- Prosthecobacter Group

- Environmental Clone WCHB1-31 Group

- Leptospirillum-Nitrospira Group

- Green Non-Sulfur Bacteria & Relatives

- Thermotogales

- Coprothermobacter proteolyticus Group

- Thermophilic Oxygen Reducers

- Environmental Clone OPB45 Group

- Archaea (outgroup)

- Green Sulfur Bacteria

- Spirochetes

- Environmental Clone OPB80 Group

- Desulfotomaculum halophilum

- Desulfotomaculum ruminis

- Desulfotomaculum luciae

- Desulfotomaculum thermosapovorans

- unnamed organism AJ012599

- Sporotomaculum hydroxybenzoicum

**12/98 Clone Group 1 (18 clones**

**2/99 Clone Group 1 (27 clones**

**2/99 Clone Group 2 (2 clones)**

- Anaerovibrio burkinabensis

- Sporomusa str.  DR1/8

- 112

- Desulfitobacterium frappieri

- Thermoanaerobacter thermohydrosulfuricus

- Thermoanaerobacter kivui

- 094

- Bacillus thermocatenulatus

- Thermoanaerobacter kivui

- East Driefontein
- Bacteria

- Balkwill @  FSU

## Complete 16S rDNA Sequence Analyses

- Moser @  PU&PNNL; Takai @ JAMSTEC

## Pyrococcus  in mine fissure water?   - a biological tracer of upwelling geothermal water?

*Fissure water characteristics:*
*Anoxic*
*60 C*

*Pyrococcus abysii*
*Found at deep sea hydrothermal vents*
*Anaerobic*
*Growth at 67-102 C*
*Optimum: 95 C*

## Enrichments:

**Fe(III), Mn(IV), NO****3****-****, S****o****, SO****4****2- ****reducers, **
**S****o**** & Mn(II) oxidizers, methanogens, acetogens, methanotrophs, and fermenters. **
** ****Samples inoculated aerobically/**
**    ****anaerobically, +/-NaCl, +/- H****2**** & MPN’s.**
** ****Incubate up to 6 months at RT or 60****o****C.**
** ****Transfer, isolate & characterize growth**
**    ****vs. T, pH, salinity, radiation.**
** ****16Sr DNA & archive. **

## S. African Au Mine Bacterial Isolates

*Thermus multireducens*

*Auriminutor *
*transvaalensis*

*Bacillus *
*thermoaureus*

- Vesicles

- PHB’s

## Thermus multireducens – isolated from a West Rand Au mine

*Reduces a variety of electron acceptors:*
*O**2**, nitrate, Fe(III), Mn(IV), S**0**, Co(III), Cr(VI), U(VI), Tc(VII)*
*Thermophilic: optimum temp. = 65**0**C*
*Relatively radiation resistant*
*D**37** = 2000 Gy*

## Auriminutor transvaalensis

*Extreme alkalophile: optimum pH 11,       range 8-12*
*Anaerobic, heterotrophic, fermentative metabolism with proteinaceous complex organic substrates as sole sources of carbon and energy*
* *
*yeast extract, peptone, and casein*
*no growth on organic acids, alcohols etc.*
*elemental sulfur, thiosulfate or fumarate added as accessory electron acceptors improved growth*
*growth with thiosulfate induced black precipitates-presumably iron sulfide*

- Takai @  PNNL

## New genus in low G+C gram positives

*New genus in **low G+C gram positives*

***Auriminitutor transvaalensis***

- Takai @  PNNL

## Bacillus thermoaureus

*Thermophilic: 55-70**o**C*
*Microaerophilic growth with nitrate reduction to nitrite; anaerobic growth with nitrate reduction to ammonia*
*no growth w/other anaerobic electron acceptors*
*Tolerant of a wide range of PAHs and straight chain hydrocarbons but not as growth substrates*
*Produces thermostable lipases and esterases*
*Spore former*
*Forms PHBs under low nitrogen, low oxygen conditions*

## Phylogeny-new species?

*Phylogeny-new species?*

*BACILLUS THERMOAUREUS*

## Biofilms form from weeping fractures

** ****Biofilm samples had high lipid biomass: 1300 to 6600 pmol/g (3x10****7**** - 2x10****8 ****cells/g).**
** ****PLFA profiles indicate a diverse microbial community. **
** ****Biofilm lipid profiles contain a higher relative proportion of free fatty acids and unidentified fatty acids as compared to fissure water or carbon leader samples.**
** ****Sulfate reduction in enrichment cultures under saline and non-saline conditions and at 60****o****C. **

*Biofilms form from weeping fractures*

- Pfiffner @  UT

## Sulfate Reduction @ 3.2 kmbls., 37°C

**SRB’s grown **
**at 60****o****C**

**FeS  drip with**
**10****8**** cells/g**

**16S rDNA & DSR **
*Desulfofotomaculum*
*geothermicum*

******34****S of sulfate &**
**sulfide indicate **
**90% conversion**

**Sulfate Reduction @ 3.2 kmbls., 37°C**

## Growth Conditions in situ- E5-46-BH1 borehole isolation experiment

*Terminally branched saturates and branched monoenoic fatty acids increased over time.*
*These fatty acids are indicative for **sulfate- reducing bacteria** .*
*Monoenoic fatty acids decreased over time in later three samples (containing >1pmol PLFA/ml total biomass).*

- Pfiffner @  UT

## Sulfite reductase gene expressed

- Columbia R. Basalt

- E5 46 Bh1

- West Driefontein

- Archaea?

- Stahl @  UW

## Results to date:

**  ****Four new bacterial species and many new 16S rDNA sequences.**
** ****Hyperthermophilic(?) Archaea may **
**   ****reflect deep circulating groundwater (>1 Ma).**
** ****Radiolysis is source of H****2**** at West **
**   ****Rand**

## Further questions:

**Are the hyperthermophiles really**
**   ****hyperthermophiles?**
** ****Does autotrophy support**
**   ****subsurface biosphere?**
** ****What is the source of NH****3****?**
** ****What are the in situ microbial**
**   ****growth rates?**