Tinfen Yan

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Molecular Characterization of Microbial Community
Structure in Contaminated Groundwater
Tinfen Yan Xandui Liu Kitt E. Bagwell Susan L.
Carroll Terry C. Hazen Jizhong Zhou Melinda E.
Clark Judy D. Wall Zhili He Qiang He Liyou Wu
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Field Research Center at Oak Ridge, TN Bear Creek
Valley at the Y-12 Security Complex
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NABIR-Field Research Center
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FRC Groundwater Geochemistry
Well pH nitratea uraniumb
nickelc aluminumc sulfatec (mM) (µM)
(µM) (mM) (mM) FW-300 6.1 0.02 ND
0.85 0.01 0.06 FW-005 3.9 6.27
27.0 84.3 1.74 0.15 FW-010
3.5 713 0.71 322 41.5 2.24 FW-015
3.4 173 32.4 147 22.9
1.02 TPB-16 6.3 0.48 4.62 ND 0.01
8.03 FW-003 6.0 17.1 0.04 0.26 0.02
0.17 a nitrate was determined via ion
chromatography b uranium was determined via
ICP-mass spectroscopy C nickel and aluminum were
determined via ICP
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Bacterial Isolates from FW-005
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FW-005 16S rDNA 175 ppm NO3, 6 ppm U pH 3.9
Acidovorax
Caulobacter
Rhodoferax
Delftia
Mesorhizobium
Acinetobacter
Zoogloea
Stenotrophomonas
Ralstonia
Pseudomonas
Azoarcus
Nitrospira
Cytophaga
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FW-300 16S rDNA Background, pH 6.0
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TPB-16- 30 ppm NO3, 1 ppm U, pH 6.3 16S rRNA gene
clonal library
Nitrospira
Chlorobium
Acidobacterium
Corynebacterium
Polyangium
Cytophagales
Hyphomicrobium
Ferribacter
Methylobacter
Azoarcus
Pseudomonas
Herbaspirillum
Ralstonia/Achrombacter
Zoogloea
Achromatium
Acidovorax
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Diversity based on SSU 16S rDNA clonal library
and partial sequences
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Relative abundances of bacterial genera based on
SSU rDNA clonal libraries from FRC groundwater
high nitrate pH 6.0
high nitrate pH 4.0
high nitrate pH 4.0
high nitrate pH 4.0
low nitrate pH 6.0
background
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Sequence libraries from FRC groundwater along
contaminant plume
a Fields et al. (in review) b Yan et al., 2003
c Yan et al., (in preparation) d Bagwell et
al., (in review)
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Groundwater Based Upon SSU rDNA
0.8
FW-300
0.4
FW-003
FW-015
0
FW-010
FW-015
-0.4
TPB-16
-0.8
-0.2
0
0.2
0.4
0.6
0.8
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PCA1
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? When the geochemical PC1 value was compared to
the diversity index (1/D) estimated from the SSU
rRNA gene sequences, a power function correlation
was observed (r0.85).
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• Conclusions
• PCA analyses was sufficient for simple
  separation of sites by geochemistry
• ? A sole geochemical parameter could not account
  for overall geochemical variability
• Different combinations of chemical variables
  may be necessary to explain spatial differences
  in the microbiology within and along the
  contaminant plume
• Correlations between groundwater chemistry and
  the recovery and diversity of different
  functional gene sequences gave very different
  results.
• The data reduction with PCA did consider
  phylogenetically distinct OTUs, but the analysis
  did not consider the relatedness between the
  distinct OTUs.
• Future work is underway to compare distance
  matrices of all sequences between sites in order
  to capture the degree of similarity within the
  genomics sequences of the sampled community.

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GS-acetate-Fe(III) enrichment. Clostridium
sphenoides (98) Clostridium bifermentans (98)
Sporomusa (lt90) Anaerovibrio (lt90)
GS-lactate-Fe(III) enrichment. Uncultivated clone
(95) Anaerovibrio glycerini (89) Selenomonas
lacticifex (88) Clostridum sphenoides (98)
GM-lactate-Fe(III) enrichment. Anaerovibrio
glycerini (91) Clostridium algidixylanolyticum
(99) Anaerovibrio/Sporomusa spp. (87)
GM-lactate/ethanol nitrate enrichment.
Uncultivated clone (99) Citrobacter sedlakii
(97) Azoarcus eutropha (96) Ochrobactrum
intermedium (93)
GM-lactate/ethanol sulfate enrichment.
Desulfosporosinus Blif (88) Desulfosporosinus
orientis (96) Clostridium chromoreductans
(98) uncultivated clones (87)
GM-lactate/ethanol-Fe(III) enrichment (10-5).
Uncultivated Anaeromyxobacter (94) Anaeromyxobac
ter dehalogenans (94)
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GM-sulfate with lactate and ethanol
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GM-Fe(III) with lactate and ethanol
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• Conclusions
• ? Different electron donors and acceptors
  resulted in different bacterial communities
• ? Novel and unique iron-, sulfate-, and
  U-reducing microorganisms are present at the FRC
  and represent communities that contain novel and
  unique genomic sequences
• Sulfate- and iron-reducing enrichments were
  dominated by low GC Gram-positive bacteria
• The highest dilution iron-reducing enrichment
  was predominated by Anaeromyxobacter spp.

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• The microarrays performed well with logarithmic
  and stationary phase cells grown in LS
• Several putative molecular chaperone proteins
  were up-regulated in stationary phase
• An ORF with a predicted flocculin domain was
  up-regulated in stationary phase
• A hypothetical protein that has low homology
  with a proteophosphoglycan from Leischmania was
  up-expressed in stationary phase

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As cells entered stationary phase growth, a
transient increase in total carbohydrate levels
was observed for cells grown in both LS and LS4D
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Use Klebsiella pneumoniae to test methods for the
extraction of extracellular carbohydrate ? NaOH
wash ? 2 EDTA wash ? NaCl/Formaldehyde ?
Ultracentrifugatin ? Zwittergent 3-14
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• D. vulgaris cultures increased glycogen levels
  as the cells entered stationary phase growth

• Future work includes the whole-genome
  expression analysis over time of batch cultures
  and determine possible roles for glycogen in
  survival and stress response in D. vulgaris

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