Isolation and Characterization of Manganese Oxidizing Bacteria
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Isolation and Characterization of Manganese Oxidizing Bacteria
Intern: GrahamMentors: Dr. Roberto Anitori & Professor Brad Tebo
Tebo,1995
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2Manganese 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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3Significance of Mn Oxidation
• 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
Tebo, 1995
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4Project 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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5Peptide Capture Method
Peptide
BiotinBead
Streptavidin
• 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
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6Peptide 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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7Peptide Capture #2
• Fresh specific peptide• Also used synthetic oxides
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9Project 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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10Bacteria Cultured from Columbia River Water
• Mn-oxidizing cultures from Columbia River Plume water
• Isolates being purified by subculturing
• Several strains showing oxidation (below)
Original Cultures from Columbia River Water
LBB Positive
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1116S rDNA Analysis
Agarose Gel Electrophoresis• 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
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12Results 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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13Project 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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14Halomonas LOB-5
• Isolated from Loihi Seamount
• Lithoautotrophic, microaerophillic, also grows heterotrophically Photos
Courtesy of Rick Davis
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15LOB-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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16LOB-5 Growth Conditions (cont.)
Solid Fe Oxidation Medium
High Oxygen, Low Fe(II)
Low/ No Oxygen,High Fe(II)
Uniform Mn(II) Conc.
High Oxygen
Low/ No Oxygen
Headspace with air
Solid Mn Oxidation Medium
Semi-solid Fe Oxidation Medium
nZVI plug
Abiotic Fe Oxides
Headspace with air
High Oxygen, Low Fe
Low/ No Oxygen,High Fe
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17Halomonas 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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18Conclusions
• 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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19Future 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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20Acknowledgements
• ASE– Mattie Courtright
• CMOP– Professor Brad Tebo: Mentor– Dr. Roberto Anitori: Mentor– Dr. Antonio Baptista– Karen Wegner– Elizabeth Woody– Tebo and Haygood Labs