The ocean as regional enabler A Pacific Northwest success story

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Isolation and Characterization of Manganese
Oxidizing Bacteria
Tebo,1995
Intern Graham Mentors Dr. Roberto Anitori
Professor Brad Tebo
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Manganese Oxidation

• Mn occurs in 1 of 3 oxidation states
• Mn(II)
• Mn(III)
• Mn(IV)

• Abiotic vs. biological Mn oxidation
• In bacteria Enzymes are responsible for Mn
  oxidation (outer-membrane proteins)
• Bacteria become encrusted in oxides

Tebo et. al., 2004
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Significance of Mn Oxidation

• Key role in other biogeochemical cycles (i.e. Fe,
  S, C)
• Control distribution of trace and contaminant
  elements
• Useful in bioremediation processes

Tebo, 1995

• Mn(III) and Mn(IV) are strong oxidizers, have
  high sorption capacities

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Project Goals

• Optimize the Peptide Capture method for isolating
  Mn-oxidizing bacteria from the Columbia River
  Estuary
• Isolate Mn-oxidizing bacteria from Columbia River
  Estuary and identify with 16S ribosomal RNA gene
  analysis (16S rDNA)
• Confirm Mn and Fe oxidation in Halomonas LOB-5

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Peptide Capture Method
Biotin

• Bacteria encrusted in Mn oxides are held to the
  side of the tube while other suspended particles
  are removed
• Presence of Mn oxides in samples determined with
  LBB colorimetric assay and phase contrast

Bead
Peptide
Streptavidin
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Peptide Capture 1

• Using synthetic Mn oxides rather than Columbia
  River water samples
• Specific peptide may have been old and/or faulty

Specific Peptide Random Peptides No Peptides
Expected Result (for MnOx) - -
Observed LBB Result
Observed Phase Contrast Result
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Peptide Capture 2

• Fresh specific peptide
• Also used synthetic oxides

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Project Goals

• Optimize the Peptide Capture method for isolating
  Mn-oxidizing bacteria from the Columbia River
  Estuary
• Isolate Mn-oxidizing bacteria from Columbia River
  Estuary and identify with 16S ribosomal RNA gene
  analysis (16S rDNA)
• Confirm Mn and Fe oxidation in Halomonas LOB-5

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Bacteria Cultured from Columbia River Water

• Mn-oxidizing cultures from Columbia River Plume
  water
• Isolates being purified by subculturing
• Several strains showing oxidation (below)

LBB Positive
Original Cultures from Columbia River Water
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16S rDNA Analysis

• Colony PCR
• At least one sample from each of 4 colony types
• One sample of bacteria cultured from filter used
  on Plume water
• LOB-5
• Analyze PCR for positives (band)
• Obtain DNA sequence
• BLAST database search to identify microbes

• Agarose Gel Electrophoresis

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Results of Sequence Analysis

• One Mn oxidizing Pseudoalteromonas species
• Pseudoalteromonas found to oxidize Mn in places
  like the Black Sea
• 6 cultures of Mn oxidizers appear to be bacteria
  from the Rheinheimera genus
• Most likely only one species present in isolates
• No Rheinheimera species have previously been
  observed oxidizing Mn
• Raises questions about purity of LOB-5 culture

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Project Goals

• Optimize the Peptide Capture method for isolating
  Mn-oxidizing bacteria from the Columbia River
  Estuary
• Isolate Mn-oxidizing bacteria from Columbia River
  Estuary and identify with 16S ribosomal RNA gene
  analysis (16S rDNA)
• Confirm Mn and Fe oxidation in Halomonas LOB-5

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Halomonas LOB-5

• Isolated from Loihi Seamount
• Lithoautotrophic, microaerophillic, also grows
  heterotrophically

Photos Courtesy of Rick Davis
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LOB-5 Growth Conditions
Medium Incubation T (C) Oxygen Content Aim
K plate 10, 30 Aerobic Manganese Oxidation
X plate 10, 30 Aerobic Manganese Oxidation
Solid X tube 10, 22 Microaerobic Manganese Oxidation
Solid X tube 10, 22, 30 Microaerobic Iron Oxidation
Semi-solid X tube 22 Microaerobic Manganese Oxidation
Semi-solid X tube 22 Microaerobic Iron Oxidation
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LOB-5 Growth Conditions (cont.)
Solid Fe Oxidation Medium
Semi-solid Fe Oxidation Medium
Solid Mn Oxidation Medium
Headspace with air
Headspace with air
High Oxygen, Low Fe
High Oxygen
High Oxygen, Low Fe(II)
Abiotic Fe Oxides
Uniform Mn(II) Conc.
Low/ No Oxygen, High Fe
Low/ No Oxygen, High Fe(II)
nZVI plug
Low/ No Oxygen
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Halomonas LOB-5 Current Results
Growth Condition Aim Mn Oxidation? Fe Oxidation? Incubation Time
Aerobic K Plates Mn Oxidation No   5 weeks
Aerobic X Plates Mn Oxidation No   5 weeks
Microaerobic Solid Tubes Mn Oxidation Yes   4 weeks
Microaerobic Semi-solid Tubes Mn Oxidation No   2 weeks
Microaerobic Solid Tubes Fe Oxidation   No 4 weeks
Microaerobic Semi-solid Tubes Fe Oxidation   No 2 weeks
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Conclusions

• The current Peptide Capture method is somewhat
  useful for capturing Mn oxides
• However, the specific peptide has not proven
  any more specific than a random mix of peptides
• Identified novel manganese oxidizing species from
  the Reinheimera genus
• Mn oxidation by LOB-5 has been confirmed in
  cultures in solid media with low oxygen
  concentrations

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Future work

• Continue isolation and purification of cultures
  isolated from Columbia River Plume
• Monitor for Fe- and Mn-oxidation by LOB-5 in
  semi-solid gradients and remaining solid
  gradients
• Set up more gradients (e.g. without acetate)

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Acknowledgements

• ASE
• Mattie Courtright
• CMOP
• Professor Brad Tebo Mentor
• Dr. Roberto Anitori Mentor
• Dr. Antonio Baptista
• Karen Wegner
• Elizabeth Woody
• Tebo and Haygood Labs