1

Isolation and Characterization of Manganese Oxidizing Bacteria

Intern: GrahamMentors: Dr. Roberto Anitori & Professor Brad Tebo

Tebo,1995

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

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

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

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

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

+ + +

7Peptide Capture #2

• Fresh specific peptide• Also used synthetic oxides

8

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

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

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

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

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

14Halomonas LOB-5

• Isolated from Loihi Seamount

• Lithoautotrophic, microaerophillic, also grows heterotrophically Photos

Courtesy of Rick Davis

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

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

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

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

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)

20Acknowledgements

• ASE– Mattie Courtright

• CMOP– Professor Brad Tebo: Mentor– Dr. Roberto Anitori: Mentor– Dr. Antonio Baptista– Karen Wegner– Elizabeth Woody– Tebo and Haygood Labs