Astrobiology: The Search for Life on Earth and

Beyond

Susan Pfiffner and Kim DavisUniversity of Tennessee

Jane Skinner, Amy Arnold, John Smithand a host of contributors

The Study of the Living Universe

Planet X

MultidisciplinaryCross-cutting ResearchState-of-the–art Science Beyond...

The Deep Underground Science and Engineering

Laboratory (DUSEL): An Opportunity for Multidisciplinary Deep Subsurface

Geomicrobiology Research

A National Science Foundation initiative to establish a national underground laboratory for research in physics, Earth and environmental sciences, civil and Mining engineering, and the biosciences.

What is DUSEL?

“The science cuts across disciplines and Directorates(ENG, GEO, and MPS) and provides opportunitiesfor transformational breakthroughs and to educatethe scientists & engineers of the 21st century”Michael S. Turner, Assist, Dir. for MPS (NSF)

NSF Solicitations

• Solicitation 3: Planning grant to develop full proposal including suite of initial experiments, MREFC and Presidential Budget for 2008.

• Solicitation 1: Planning grant to define science and infrastructure requirements for science modules will result in a glossy publication and a NSF report (December 2005).

• Solicitation 2: Site development grants has been award to Henderson, CO and Homestake, SD (July 2005).

Earth’s Subsurface Microbial Ecology

• The biosphere extends deep into the subsurface• Limited by geothermal gradient and nutrient flux• Biomass generally low relative to the surface• Distribution is very patchy and heterogeneous• Rates of community metabolism are very low• Volumetrically largest

part of the biosphere

Todd Stevens

Subsurface Microbiology 1986-present• Drilling and tracer technologies• Extended known biosphere

greater than 3 km• Revealed biomass &

biodiversity• Isolates in culture collections• Linked microbial activity with

geological interfaces• Slow rates of subsurface

microbial activity• Indications of autotrophic

ecosystems

CHRONOLOGY OF SUBSURFACE MICROBIAL INVESTIGATIONS

SALT MINES

OIL&GAS FIELDS

DEEP-SEA VENTS

SUBSURFACE

POLAR-ENDOLITHS

1930 1950 1970 1990

1 9 8 7 1 9 9 0 1 9 9 5

HanfordINEEL

Taylorsville Bas inCerro Negro

Piceance Bas in

Marine Sediment s

Coas t al Plain

Oys t e r

Nevada Test Sit e

MolSt ripa Äspö

WIPP

Savannah R.

Life in the slow lane!

2.0 Ga

2.9 Ga2.7 Ga

2.0 Ga 300 Ma

2.3 Ga

Transvaaldolomiticsediments

10 kmbls

Basement 3.4 Ga

120oC

9-15oC/km25oC/km 20oC/km

Introduction

• The terrestrial and oceanic deep subsurface biosphere is spatially expansive and is a large component of the global biomass.

• The structure, distribution and function of subsurface microbial communities remain poorly understood due to the difficulty of access

• The ultradeep gold mines of the South African Witwatersrand provide an excellent opportunity for direct exploration of the deep subsurface biosphere

• Why do we want to look at extreme environments in the deep subsurface?– To gain a better understanding of the

types of microbial communities existing in the subsurface• Abundance and Diversity

• Activities and the geobiochemical processes that support them

• Potential for in-situ or ex-situ processes

How do we look for microorganisms?• Direct microscopic counts (acridine orange)

– Estimate of total microbial abundance• Cultivation methods - bacteria, fungi, and protozoa

– Plate counts and most probable number– Selective and differential media

• Activity assays - metabolic and respiratory capabilities– Utilization and formation of metabolic end-products– Radiolabeled electron donors or acceptors– Utilization of methane and hydrogen– Enzyme screening

How do we look for microorganisms?• Analytical Chemistry

– Quantifying cellular macromolecules• Phospholipid fatty acids and DNA

– Petrology, chemical and isotopic analyses

• Statistical Analyses– Correlations– Artificial neural networks– Modeling software

Major Questions in Geoscience Fundamental Energy Exchange Processes at Depth• How do the coupled Hydro-Thermal-Mechanical-

Chemical-Biological (HTMCB) processes in fractured rock masses vary as function of the physical and time scales involved?

• Relative influences of these processes on – groundwater flow; contaminant transport; ore forming processes

– long-term isolation of hazardous and toxic wastes, carbon sequestration and hydrocarbon storage underground

– energetic slip on faults and fractures; stability of undergroundexcavations

Scientific Case for DUSEL: Access

•Drive•Drill•Shaft

Scientific Case for DUSEL: Isolation

• Longtime sequestered• Old water• Old sediments/rocks• Multiple interfaces• Varied T, P, lithology,

stress

Scientific Case for DUSEL: Scale

10-3 m10-9 m

103 m

Scale1 m10-6 m

Major Questions in Geomicrobiology1. How deeply does life extend into the Earth?

What are the lower limits of life in the biosphere? What is the temperature barrier, the influence of pressure, the interplay of energy restrictions with the above?

0.0E+00

1.0E+03

2.0E+03

3.0E+03

4.0E+03

5.0E+03

1.E+01 1.E+03 1.E+05 1.E+07 1.E+09

Cells/ml or Cells/g

De

pth

(m

)

Field sampling

REU Site:Biogeochemical Educational Experience -South Africa

Weeping BoreholeWeeping BoreholeOpen to mine airOpen to mine airDevelopment of Development of metalliferousmetalliferous and and microbiallymicrobially rich rich biofilmsbiofilms

16S rDNA & DSR Desulfotomaculumgeothermicum

SRB’s grown at 60oC

34S of sulfate &sulfide indicate 90% conversion

FeS drip with108 cells/g

Sampling Fracture Waters

• CH4 (30-80%)• C2+ (3-4%) • H2 (up to 30%)• He (up to 10%)• balance N2

• some NH3?

SubsurfaceSubsurfaceMicrobialMicrobial

BiogeochemicalBiogeochemicalCyclingCycling

SamplingSampling

MicroscopyMicroscopy& Mineral & Mineral

GeochemistryGeochemistryAqueousAqueous

GeochemistryGeochemistry

EnvironmentEnvironmentDissolved Gases, Dissolved Gases,

Cosmogenic Cosmogenic & Stable Isotopes& Stable Isotopes

PLFAPLFA--cell membranecell membrane

Isolates &Isolates &ArchivesArchives

FunctionFunction

16SrDNA16SrDNA

Community StructureCommunity Structure EnrichmentsEnrichmentsgenes, & genes, & enzymesenzymes

Before

After

S. African Au Mine Bacterial Isolates

Vesicles

PHB’s

Thermusscotoductus

Geobacillus thermoleovoransAlkaliphilus

transvaalensis

Culture-Independent Microbial Characterization

• Extract all DNA• Amplify rRNA genes (rDNA)

using polymerase chain reaction (PCR) and universalArchaeal or Bacterial primers

• Clone genes into Escherichia coli• Screen clones by restriction

fragment length polymorphism (rflp) analysis

• Sequence rDNA of unique clones• Compare sequences to databases• Construct phylogenetic trees

Phylogenetic studies

fimicutes and proteobacteria

Dec-98

Feb-99

Nov- 2001

Nov-2002

Isolate DR504

Clone 24

Clone 24

Clone 24

Clone 24

DR546BH1 T-RFLP 16S DNA extracted and amplified

Isolate DR 520

Isolate DR 520

from: Gihring, Moser, et al., in prep

Major Questions in Geomicrobiology

2. What fuels the deep biosphere? – Do deep microbial ecosystems exist that are

dependent upon geochemically generated energy sources ("geogas": H2, CH4, etc.) and independent from photosynthesis. How do such systems function, their members interact to sustain the livelihood?

3. Does the interplay between biology and geology shape the subsurface? Role of microbes in Hydro-Thermal-Mechanical-Chemical-Biological (HTMCB)

Earthlab Report 2003

Major Questions in Geomicrobiology

Drilling forDrilling forsubsurfacesubsurfacelife on Mars?life on Mars?

5. Did life on the earth's surface come from underground?– Does the deep subsurface

harbor primitive life processes today?

– Has the subsurface acted as refuge during extinctions.

– What "signs of subsurface life" should we search for on Mars?

Major Questions in Geomicrobiology

4. What are subsurface genomes telling us?

– Microbes may have been isolated from the surface gene pool for very long periods of time. How different are they from microbes on the surface?

– How do they evolve with very low population density, extremely low metabolism rate and high longevity?

* SASFiG-1

SASFiG-2

SASFiG-3SASFiG-4

SASFiG-5

SASFiG-6

SASFiG-7SASFiG-9

SASFiG-8

*

*SASFiG-9 (isolated)

Detected within a water-bearing dyke/fracture at 3.2 Km depth.

strictly anaerobic; iron-reducer

optimal growth temperature = 60 oC

virgin rock temp = ~ 45 oC

Novel Bacterial lineages unique to the SA deep-subsurface:South Africa Subsurface Firmicutes Groups (SASFiG)

1 µm

Novel and unusual microbes and sequences, deep branching indicative of ancestral linkages, less evolved sequences (early life?), biomed and biotech applications

image courtesy of Gordon Southam

Major Questions in Geomicrobiology

6. Is there life as we don't know it?– Does unique biochemistry, e.g. non-nucleic acid

based, and molecular signatures exist in isolated subsurface niches?

– Is the subsurface a reservoir for unexpected and biotechnologically useful enzymes?

Exciting Science and Engineering• Compelling questions: • National resource for research in physics, life sciences,

chemistry, geosciences and engineering• Multidisciplinary

– Not just a juxtaposition for political convenience– Overlap of questions: between fields and between

fundamental and applied – Multidisciplinary approaches: e.g. geo-micro-biologists– New Synergies

• Instrumentation of the rock prior to construction of physics cavities

• Low radioactivity methods, instrumentation, data acquisition

Exciting Science and Engineering• Marvelous education and outreach

opportunity– Training of a new generation of multidiciplinary

scientists and engineers

– Exciting the imagination of K-12 students (Bio+Earth+Physics+Astronomy)

– Involvement of local population (Native Americans, Southern Appalachia)

Education and Public Outreach

Discover Underground!

A national resource for research in physics, life sciences, chemistry, geosciences and engineering

Undergraduate students extract DNA from underground rocks to search for life.

Young children in hard hats quiz an engineer about where that underground water comes from and where it goes.

High schools students deliver a detector component they built for testing.

Summary •Research to be conducted at DUSEL will address fundamental questions in physics, biology, earth sciences and engineering. The results will help us to address wide ranging practical issues of societal interest, including:

• Development of advanced detectors for use in medicine, seismology and for national security

• Development of improved techniques to search for fresh water and to clean up contaminated aquifers

• Development of new techniques to explore for and extract valuable resources needed by modern societies, such as copper, gold, silver, oil, coal, etc.

• Identification and testing of novel bioengineering techniques that lead to development of new drugs

•DUSEL is unique: located in the U.S., dedicated, controlled access, isolated environment, multiple scales, many disciplines, education and outreach

AcknowledgementNational Science Foundation

S-1 planning grantwith special thanks

Bernard Sadoulet, Hamish Robertson, Eugene Beier, Charles Fairhurst, Tullis Onstott, James Tiedje, and

S-1 working group leaders

ReferencesEarthLab 2003, A Subterranean Laboratory and

Observatory to Study Microbial Life, Fluid Flow, and Rock Deformation, Geosciences Professional Services, Inc.

NeSS02 Final Report, International Workshop on Neutrino and Subterranean Science, September 19–21, 2002, Washington D.C.

Proc. Intern’l Symp. ROCKSTORE 77; 80 (Stockholm)

Spatial scale, x,y,z

Depth, z -> ∆σ; ∆Τ

Time, t

Astrobiology Astrobiology Adventures into extreme environments continue to yield exciting developments in geomicrobiology of the deep subsurface on Earth and beyond.

http://www.indiana.edu/~deeplife/