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Isolation and Characterization of Manganese Oxidizing Bacteria

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). Abiotic vs. biological Mn oxidation

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Isolation and Characterization of Manganese Oxidizing Bacteria

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  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 • 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

  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

  6. Peptide Capture #1 • Using synthetic Mn oxides rather than Columbia River water samples • Specific peptide may have been old and/or faulty

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

  8. 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

  10. 16S 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

  11. 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

  12. 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

  13. Halomonas LOB-5 • Isolated from Loihi Seamount • Lithoautotrophic, microaerophillic, also grows heterotrophically Photos Courtesy of Rick Davis

  14. LOB-5 Growth Conditions

  15. 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

  16. Halomonas LOB-5: Current Results

  17. 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

  18. 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)

  19. 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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