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 In bacteria: Enzymes are responsible for Mn oxidation (outer-membrane proteins) Bacteria become encrusted in oxides Tebo et. al., 2004

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

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

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

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

Peptide Capture #2 Fresh specific peptide Also used synthetic oxides

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

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

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

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

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

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

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

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

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

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

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)

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