Phylogenetic and functional analysis of microbial communities

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Phylogenetic and functional analysis of microbial
communities
Eoin Brodie Lawrence Berkeley National
Lab Berkeley, CA, USA ELBrodie_at_lbl.gov
vimss.lbl.gov greengenes.lbl.gov www-esd.
lbl.gov/CEB/index.html
http//www-esd.lbl.gov/ERT/hanford100h
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Non-invasive geophysical monitoring
Injection well
Monitoring well
Estimated hydraulic conductivity Showing zonation
of permeability
302 days after HRC injection
3 days after HRC injection
2 days after HRC injection
Pre-HRC injection
40
40
Depth (ft)
Depth (ft)
50
50
Change in conductivity
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Geochemistry Injection well
Sulfate
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Geochemistry Monitoring well
---- 45 ft ---- ---- 56.5 ft ----
Nitrate
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Geochemistry Iron reduction
Upgradient control well
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Bacterial biomass - Direct counts
Bacterial biomass enriched rapidly by 2 orders of
magnitude remained elevated over one year later
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PhyloChip

• PhyloChip 500,000 probes (300k target 16S)

Amplicon pool fragmented, biotin labeled
16S rRNA gene is amplified from genomic extract
or 16S rRNA molecules are used directly
16S rRNA gene used as biomarker due to large
database and availability of universal primers.
Biotin labeled 16S hybridizes to its complement
sequence on the array surface.
PhyloChip is scanned, fluorescence data analyzed
and probe sets with gt90 probes positive are
considered present
PhyloChip stained and washed using automatic
fluidics station
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Data mining Bidirectional clustering
I Injection well M Monitoring well
Highest intensity
Lowest intensity
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Functional groups Iron reduction
Fe(II) can abiotically reduce Cr(VI) to Cr(III)
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Functional groups Sulfate reduction
Hybridization intensity
Days post HRC injection
Days post HRC injection
H2S can abiotically reduce Cr(VI) to Cr(III)
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Functional groups Methanogenesis
Hybridization intensity
Days post HRC injection
Days post HRC injection
Presence of methanogens indicates strongly
reducing conditions
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Functional microarray analysis
April 17th 2006
Mean signal to noise ratio
Nitrate, Sulfate, Iron reduction. Methanogenesis,
Methane oxidation, Sulfur oxidation. Many
chromium tolerance/reduction genes.
Joe Zhou, Joy Van Nostrand - Oklahoma
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Molecular summary

• A single application of HRC maintained strongly
  reducing (methanogenic) conditions in a chromate
  contaminated aquifer for at least 20 months
• Phylogenetic arrays demonstrate continued
  enrichment of iron, sulfate, nitrate reducers,
  methanogens, methane oxidizers, sulfur oxidizers
• Preliminary Functional array data shows
  enrichment of nitrate reducers, chromate
  reducers, iron reducers, sulfate reducers, sulfur
  oxidizers, methanogens, methane oxidizers
• Combined molecular analyses indicates chromate
  reduction is potentially mediated by both direct
  (chromate reductase) and indirect (Fe2, H2S)
  microbial interaction

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Initial Culture-based Results from Hanford
sediments
Romy Chakraborty, Richard Phan, Terry C Hazen
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Isolating microorganisms
Sediment collected
Lactate
Enrichments set up
Pure culture of bacterium
Electron acceptor
Isolated colonies developed by agar shake tubes
method
Periodic transfers and microscopic counts
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• 2 isolates - sulfate reducers obtained from the
  blue depth-55.5
• Closest to Desulfosporosinus orientis and
  Desulfosporosinus meridiei species.
• Both are big curved bacteria, sometimes appear
  straight. known to have spores.
• Both utilize sulfate and thiosulfate as
  alternative electron acceptors. not nitrate
• Both utilized lactate and pyruvate as electron
  donors.
• Both grow best at 370C.
• Direct Enzymatic Chromate utilization not tested
  yet.

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• One isolate obtained from the yellow depth-45
• Its closest relative is Xanthobacter flavus
• Organism ferments lactate.
• Does not reduce sulfate.
• Not known if it can reduce Chromium.

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Currently, more enrichments are being processed
for isolation from the blue and the black depths.
Isolates will be obtained in a week.
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From the sediments collected in July 06
Organism type Media E-acceptor Result Sediment depth
Sulfate reducer LS4D Sulfate 47.2-47.7
Sulfate reducer Fresh water Sulfate 47.2-47.7
Nitrate reducer Fresh water Nitrate 39-39.5 47.2-47.7
Iron reducer Fresh water Ferric-NTA 39-39.5 47.2-47.7
Chromium reducer Fresh water Chromium -
Enrichments are in agar,Isolates will be obtained
7-10 days.