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Isolation and
Characterization of
Manganese Oxidizing
Bacteria
Tebo,1995
Intern: Graham
Mentors: Dr. Roberto Anitori
& Professor Brad Tebo
Manganese Oxidation
• Mn occurs in 1 of 3
oxidation states
– Mn(II)
– Mn(III)
– Mn(IV)
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• Abiotic vs. biological Mn
oxidation
• In bacteria: Enzymes
are responsible for Mn
oxidation (outermembrane proteins)
– Bacteria become
encrusted in oxides
Tebo et. al., 2004
Significance of Mn Oxidation
Tebo, 1995
• Mn(III) and Mn(IV) are
strong oxidizers, have
high sorption capacities
• Key role in other
biogeochemical cycles
(i.e. Fe, S, C)
• Control distribution of
trace and contaminant
elements
• Useful in
bioremediation
processes
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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
Bead
Streptavidin
Biotin
Peptide
5
• 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
Peptide Capture #1
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• Using synthetic Mn oxides rather than
Columbia River water samples
Specific
Peptide
Expected
Result
Random
Peptides
No Peptides
+
(for MnOx)
-
-
Observed LBB
Result
+
+
+
Observed
Phase Contrast
Result
+
+
+
• Specific peptide may have been old and/or
faulty
Peptide Capture #2
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• Fresh specific peptide
• Also used synthetic oxides
Mn Oxides Remaining on “Chain”
Following Each Wash
1
0.9
0.8
0.7
LBB absorption 0.6
at 618nm
0.5
Specific
0.4
Random
0.3
No Peptide
0.2
0.1
0
1
2
3
4
5
6
7
Wash Number
8
9
10
11
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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
9
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
Agarose Gel Electrophoresis
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• 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
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
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Photos
Courtesy of
Rick Davis
LOB-5 Growth Conditions
Medium
Incubation T (°C)
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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
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Microaerobic
Manganese Oxidation
Semi-solid X tube
22
Microaerobic
Iron Oxidation
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(II)
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High
Oxygen,
Low Fe
High
Oxygen
Abiotic Fe
Oxides
Uniform
Mn(II)
Conc.
Low/ No
Oxygen,
High Fe(II)
Low/ No
Oxygen,
High Fe
nZVI plug
Low/ No
Oxygen
Halomonas LOB-5: Current Results
Growth
Condition
Aim
Mn
Fe
Incubation
Oxidation? Oxidation?
Time
Aerobic K Plates
Mn
Oxidation
No
Aerobic X Plates
Mn
Oxidation
No
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
5 weeks
5 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
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