Subsurface Microbial Carbon Cycling: Rates and Processes

orRecovery and Characterization

of a Deep Microbial Ecosystem

Brian J. Mailloux

Barnard College

For the

Sloan Deep Carbon Workshop

May 16, 2008

Talk overview

• Background

• Sampling Requirements

• Use of Carbon isotopes

State of Knowledge

• Examining depths to 120°C

• Lower cell numbers at greater depth

• Lower diversity at greater depths

• Slow

• Hard to sample

Can we use carbon isotopes to understand rates and turnover times and in the future link to diversity?

State of Knowledge

Low Diversity from a 2.825 km deep fault (Lin et al.,)

0.0

1.0

2.0

3.0

4.0

102 103 104 105 106 107 108Cells/ml or Cells/g

De

pth

(k

m)

10

Pfiffner et al. 2006

Requirements of Subsurface SamplingConstraints

• CLEAN

• Molecular sample constraints?

• Sample Size-How large a sample do we need?

• Location-Where and how can we sample?

Requirements of Subsurface SamplingMolecular Constraints

• PCR– Nanograms of DNA

• Metagenomes– 10’s to 100’s of micrograms of DNA– Amounts can be lower with whole

genome amplification

• Isotopes– 100’s of micrograms of DNA

• PLFA’s generally have smaller sample sizes than DNA

Kno

wle

dge

DNA

Requirements of Subsurface SamplingSample Size

• 1011 cells. (0.25 mg of DNA)

• ROCK– 103 cells/g therefore need 108 grams!!

• WATER– 103 cells/ml therefore need 105 liters (10,000L)

• At 1 gpm≈2 days

• If you have flowing water you can get good samples!

Requirements of Subsurface SamplingLocation

• Cores– Access to novel locations– Expensive and size limited

• Wells– Access to novel locations– Deep wells can be hard to sample

• Mines– Access to the subsurface– Locations limited– Can get clean samples– Can go back repeatedly and run experiments

Carbon Isotopes of DNA

• Bangladesh Example

• How it could be used in the deep subsurface

• 12C=99%, 13C=1%, 14C=1ppt but t1/2=5730 yr

• Microarrays

Analyzing 14C of DNA Bangladesh ExampleAtmospheric derived 14C

• Sampled ~2000 liters from a 180’ deep well.

• Extracted DNA ~150μg (Not trivial!)

• 14C DOC ~5700 yr bp

• 14C DIC ~6240 yr bp

• 14C DNA ~300 yr bp

Small, Young, Labile Pool of Organic Carbon!

E. Reichert, Senior Thesis

How can we use Carbon Isotopes to Understand Subsurface Growth

Rates?

14C is generated in situthrough decay of U and Th.

14C in DIC, Hydrocarbons, CH4…..

14C in Microbes (DNA)

Steady-stateProduction=Decay.

Steady-stateProduction=Decay.

No ProductionOnly Decay after incorporation!

Imagining an Experiment

• Collect 14C and 13C of DNA, DIC, DOC and compound specific electron donors

• 14C of DNA should be “older” with a more negative Δ14C

• The Δ14C offset should be directly related to the turnover rate (“age”) of the microbes.

• Can then directly get to turnover times in the deep subsurface.

• Can then use a 14C microarray in a subsurface Beta Cage to relate specific genes to Δ14C

Goals-Need to Link

• Isotopes

• Geochemistry

• Genomics/Proteomics

• With good subsurface access

ConclusionsACKNOWLEDGEMENTS

T.C. Onstott and collaborators within his lab including: Dylan Chivian, Eric J. Alm, Eoin L. Brodie, David E. Culley, Thomas Gihring, Alla Lapidus, Li-Hung Lin, Steve Lowry, Duane P. Moser, Paul Richardson, Gordon Southam, Greg Wanger, Lisa M. Pratt, Adam P. Arkin, Terry C. Hazen, Fred J. Brockman, Duane Moser

Columbia University- Greg Freyer, Martin Stute, Lex van Geen, Elizabeth Reichert

LLNL-Bruce Buccholz