Microbial Characterization and Diversity

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Microbial Characterization and Diversity
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The Study of the Living Universe
Planet X
Multidisciplinary Cross-cutting
Research State-of-theart Science
Beyond...
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Why study extreme environments in the deep
subsurface?
Gain a better understanding of the types of
microbial communities existing in the subsurface

• Abundance and diversity
• Activities and the biogeochemical processes that
  support them
• Potential for in-situ or ex-situ processes
• Test tools for extraterrestrial exploration

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Outline

• Part 1 Characterizing a microbial isolate
• Part 2 characterizing a microbial community

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Part 1 Characterizing an isolate

• What does it look like?
• Rods
• Cocci
• Spiral
• Other?

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Morphology

• Eukaryotes vs prokaryotes
• Presence of organelles
• Membrane-enveloped nucleosome
• Prokaryotes
• Cell morphology
• Gram reaction
• Motility
• Survival and storage structures

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Prokaryotic and Eukaryotic Cells Compared with
Organelles
Prokaryotic cell Eukaryotic cell Eukaryotic organelles (chloroplast and mitochondria)
DNA Circular Linear Circular
Ribosome 70S 80S 70S
Growth Binary fission Mitosis Binary Fission
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Some Characteristics of Bacteria, Archaea, and
Eukarya
Bacteria Archaea Eukarya
Representative organism Methanosarcina E. coli Ameoba
Cell type Prokaryotic Prokaryotic Eukaryotic
Cell wall contains peptidoglycan contains no peptidoglycan, varying composition contains carbohydrates, varying composition
Cell membrane composed of Straight carbon chains attached to glycerol by ester linkage Branched carbon chains attached to glycerol by ether linkage Straight carbon chains attached to glycerol by ester linkage
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How does the isolate make a living?

• Energy source?
• Chemical
• Organic C sugars, amino acids, fatty acids, etc.
• Inorganic H2, H2S, Fe(II), etc.
• Light
• Carbon source?
• Organic C -- heterotrophs
• Inorganic C CO2, -- autotrophs

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How does a cell make energy?

• Energy flows is conserved
• Matter is cycled

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How does a cell make energy?

• Energy flows is conserved
• Matter is cycled
• electron flow
• electron donors
• electron acceptors

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Electron Donors
nitrate, sulfate, carbon dioxide
Anaerobic respiration
Organic e-donor
oxygen
Aerobic respiration
Fermentation
endogenous organic electron acceptor
oxygen, sulfate, nitrate
Inorganic e-donor
Chemolithotrophy
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Metabolism

• Sources of energy and carbon for cells
• Autotrophs (primarily CO2)
• Photoautotrophs (Light and CO2)
• Chemoautotrophs (Inorganic compounds and CO2)
• Heterotrophs
• Energy catabolism of organic compounds
• Anabolism (synthesis) from imported organic
  compounds

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Selective and differential growth media
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Whats the terminal electron acceptor?

• O2
• NO3-
• Fe(III)
• SO42-
• CO2

Fe(III) reduction to Fe(II) by a Thermus isolate
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Why is temperature important? http//helios.bto.ed
.ac.uk/bto/microbes/thermo.htm Psychrophiles
(cold-loving) can grow at 0C, and some even as
low as -10C their upper limit is often about
25C. Flavobacterium species Mesophiles grow in
the moderate temperature range, from about 20C
(or lower) to 45C. Escherichia coli
Thermophiles are heat-loving, with an optimum
growth temperature of 50C or more, a maximum of
up to 70C or more, and a minimum of about 20C.
Bacillus sterothermophilus Hyperthermophiles
have an optimum above 75C and thus can grow at
the highest temperatures tolerated by any
organism. Thermocuccus celer An extreme example
is the genus Pyrodictium, found on geothermally
heated areas of the seabed. It has a temperature
minimum of 82C, optimum of 105C and growth
maximum of 110 C.
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Why is pH important? Acidophile organisms that
grow at low pH values (generally between 1 and
5) Neutralphile organisms that grow at
nearneutral pH Alkaphile organisms that grow
optimally at pH values above 9, often between 10
and 12, but cannot grow or grow only slowly at
the near-neutral pH.
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S. African Au Mine Bacterial Isolates
S. African Au Mine Bacterial Isolates
Metal-reducing Thermus scotoductus
Geobacillus thermoleovorans
Alkaliphilus
transvaalensis
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Culture-Independent Microbial Characterization

• Extract all DNA
• Amplify rRNA genes (rDNA) using polymerase chain
  reaction (PCR) and universal Archaeal 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

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Phylogenetic analysis using small subunit rRNA
sequences (16S and 18S rRNA gene
sequences). Where does your isolate Belong on
the Tree of Life?
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Part 2 Characterizing the entire microbial
community

• Challenges
• Fewer than 1 of microbes in natural environments
  can be grown in culture.
• Wed like to know what theyre doing in situ.

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Membrane lipids
Enrichments, genes, enzymes
Sampling
Community Structure
Function
Subsurface Microbial Biogeochemical Cycling
Isolates Archives
16S rDNA
Environment
Dissolved Gases, Cosmogenic Stable Isotopes
Microscopy Mineral Geochemistry
Aqueous Geochemistry
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What sources of energy are available to the
community?

• Organic C
• Reduced Inorganic compounds
• H2
• H2S
• NH3
• Fe(II)

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What is the dominant terminal electron accepting
process?Quantify available e- acceptors and
reduced products

• Electron acceptors
• O2
• NO3-
• Fe(III)
• Mn(IV)
• SO42-
• CO2

• Reduced products
• H2O
• N2, NH3
• Fe(II)
• Mn(II)
• H2S
• CH4

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Changes in aquifer chemistry along a flowpath
oxygen is depleted, sequential depletion of other
oxidants
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Phelps, et.al. 1994. Microbial Ecol. 28 335-350
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Physical and Chemical Parameters for Fracture
Waters in South African Gold Mines Results
being published in the Geomicrobiology Journal
For summary of the South African
research http//geoweb.princeton.edu/people/onstot
t/research.html
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Weeping Borehole Characterization
Open to mine air Development of metalliferous and
microbially rich biofilms
SRBs grown at 60oC
16S rDNA DSR Desulfotomaculum geothermicum
ASM 2006 press release , Desulforudis
audaxviator bold traveler
34S of sulfate sulfide indicate 90 conversion
FeS drip with 108 cells/g
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Abundance vs. chemical energy flux
Cells/ml or Cells/g
1.E01
1.E02
1.E03
1.E04
1.E05
1.E06
1.E07
1.E08
0.0E00
Be16
Savannah River
1.0E03
Flow Cytometry
2.0E03
Depth (m)
MS 151
MP 104
TauTona 100
3.0E03
PLFA
4.0E03
5.0E03
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What are the rates of in situ activity?

• Requires geochemical modeling approaches to
  estimate subsurface rates

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Life in the slow lane!
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Death-O-Meter figure is from Oak Ridge National
Laboratory, ORNL 98-759/lmh
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Down-Hole Probe
52 mm
On board Computer transformers
Circulating Pump
Cintered Filter Array
Microcantilever array
360o rotation
CCD w/ White LED's UV laser fluorescence
Retractable Sampling Syringe
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Did the surface residing microbes move
underground? or Did subsurface residing
microbes move to the surface?
Permafrost
Brine and gas
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Summary Community characterization requires an
array of techniques for

• Abundance (biomass)
• Community structure
• Diversity
• In situ metabolic activities
• In situ geochemical processes

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pictures of Lake Vostok bacteria http//www.hero.a
c.uk/resources/5957.jpg
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Extraterrestrial Extremes

• Europa

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3. How does the interplay between biology and
geology shape the subsurface?

• Microbially mediated mineral dissolution
• Microbially mediated mineral precipitation

e.g., magnetite formation
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Reference Brocks Biology of Microorganisms 10th
Edition Machael Madigan, John Martinko, and Jack
Parker Prentice Hall 2003