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

2. 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

3. 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

4. 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

5. 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
Procedural flow chart

6. 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

7. Peptide Capture #2
Fresh specific peptide
Also used synthetic oxides

9. 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

10. 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

11. 16S rDNA Analysis Colony PCR Agarose Gel Electrophoresis
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

12. 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

13. 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

14. Halomonas LOB-5 Isolated from Loihi Seamount
Lithoautotrophic, microaerophillic, also grows heterotrophically
Photos Courtesy of Rick Davis
Overall comments:
* An excellent start. Now the presentation needs polishing up, and it needs to be done in a way that provides more explanations to the audience. Remember that many of them will be non-scientists.
needs some images to make it more appealing (make sure you include the source of the image with the picture)
Slide titles - font size is usually bigger than text size in main part of slide

15. LOB-5 Growth Conditions
Medium
Incubation T (°C)
Oxygen Content
Aim
K plate
10, 30
Aerobic
Manganese Oxidation
X plate
Solid X tube
10, 22
Microaerobic
10, 22, 30
Iron Oxidation
Semi-solid X tube 22

16. LOB-5 Growth Conditions (cont.)
Solid Fe Oxidation Medium
Solid Mn Oxidation Medium
Semi-solid Fe 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

17. 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
Microaerobic Solid Tubes
Yes
4 weeks
Microaerobic Semi-solid Tubes
2 weeks
Fe Oxidation

18. 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

19. 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)

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