Microbial Diversity in Drain Hole Waters of the Henderson Mine

John R. SpearJason Sahl

Division of Environmental Science and EngineeringColorado School of Mines, Golden, CO

jspear@mines.edu

$125 x 106

$125 x 106

Amount spent in the U.S. on marshmallows in 2005

What Is Life?

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Euryarchaeota

Crenarchaeota

A “Map” of LifeEuryarchaeota

Crenarchaeota

N.R. Pace, Science, 1997

Molecular MethodEnv. Sample

Bulk DNA/RNA

Extraction

CommunityrRNA/rDNA

rRNA/rDNAClones

PCR

Cloning

rRNA/rDNA Sequences

Sequencing

PhylogeneticTrees

Databases

7150’ DH-3 Data

10%

14%

29%14%

3%

3%

17%

10%

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

Fungus

Sulfate Reducing Bacteria

7025’ DH-1 Data

Fungus

Sulfate Reducing Bacteria

73%

1%4%5%

16%

1%

B-Proteobacteria

Nitrosomonas

Acidobacteria

Henderson Ungrouped#3Bacteroidetes

Fungi

7025’ DH-1-Post Packer Data

Fungus

Sulfate Reducing Bacteria

6%4%

1%

60%

14%

12%

1%1%

1%Henderson Ungrouped#1Henderson Ungrouped#3B-Proteobacteria

Chloroflexi

Chlorbi

Firmicutes

Nitrospira

OS-K

NBO1

7025’ DH-4-Post Packer Data

Fungus

Sulfate Reducing Bacteria

3% 1%5%

7%

1%

39%

44%

Henderson Ungrouped#1Gamma-Proteo

Delta-Proteo

Acidobacteria

Chloroflexi

Chlorobi

Crenarchaeota

DH Chemistry7025-D1 7025-P1 7025-D4 7025-P4

O2 (mg/L) 0.26 0.14 3.8 0.04Temp (oC) 38 40 35 35

pH 5.9 5.8 6.3 6DIC (mM) 29 16 3.2 20DOC (mM) 0.05 0.03 0.05 0.03

Total N (mM) 0.05 0.05 0.17 0.17SO4

2- (mM) 4.3 4 10 9.8NO3

- (mM) 0.03 0.017 0.086 0.047NO2

- (mM) 0.05 0.027 0.048 0.046

HM

Conclusions

Potential for 3 new groups (phyla) of Bacteria!!Something is happening with N!!Community shift pre- verses packed- drill

holes.Surprising diversity for a subsurface.Low abundance of both Archaea and Eucarya!Publish?

Thanks To:

My Start-up money….Team Bio--Tom K., Jeff M., Alexis T.,

Betsy S., Jeff W., Kevin M.HUSEP FolksHenderson Mine and Chip deWolfe

“If it wasn’t for the optimist,the pessimist would never know….

how happy he wasn’t.”

Henderson, New Drill Core

Plan, to drill a core into the HUSEP facility area

For some portion of that:– Take a segment for biological consideration

• Sterile? BEADS + Tracer• Core collection--on site• Core sample sections to Socorro, CSM, CU

– Continue drilling to depth

Henderson Mine, 2 Nov 05

Molecular MethodEnv. SampleContact Slide

Bulk DNA/RNA

Extraction

CommunityrRNA/rDNA

rRNA/rDNAClones

PCR

Cloning

= Method = Result

rRNA/rDNASequences / Databases

Nucleic AcidProbes

Sequencing

ProbeDesign

Whole-CellHybridization

(FISH)

PhylogeneticTrees

Nucleic Acid

Hybridization

GGCC

TACG

CCGG TACGGGCC ATGC

Polymerase Chain Reaction

CCGG TACG

GGCC ATGC

GGCC ATGC

CCGG TACG

GGCC ATGC

TACG

CCGG TACGGGCC

GGCC ATGC

TACG

CCGG TACG

GGCC

DH-2, DH-3 + ???? To Do….

Water

Water

= 1.5’ PVC Pipe Inserted with Glass and/or Fe Growth Slides

Anoxic H2Oout

DH-1 Water Chemistry + CulturesSpecies Concentration Si4+ 0.05 mMCa2+ 8.7 mMMg2+ 1.3 mMAl3+ 4.8 mMNa+ 7.9 mMSr2+ 0.04 mMK+ 1.0 mMFeT 1.5 mMMn2+ 21 mMF- 12 mMCl- 0.15 mMNO2- Not detectedNO3- 1.0 mMBr- Not detectedPO43- Not detectedSO42- 48 mM

DH-1 at 7150’, pH 5.8

Culture efforts:Swanner & Templeton, CU:

DH-1 3 (Fe + Mn)DH-3 3 (Mn)DH-4 2 (Fe + Mn)