Microbial Manganese Oxidizers and Reducers in the Columbia River

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Microbial Manganese Oxidizers and Reducers in
the Columbia River

• By Kira Kranzler
• Frontline Mentor Suzanna Brauer
• Senior Mentor Brad Tebo

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Significance

• Estuaries
• Transition Zone
• Nutrient Rich
• Protected from Ocean
• High Biodiversity
• Microbial Populations
• 60-90 Earths Biomass
• Geochemical Cycling
• Carbon Fixation
• Manganese Utilizers
• Largest Mn Source to CA Current
• Carbon Fixation
• DOM from POM

• Massive algal bloom off the coast of Vancouver
  Island BC.

http//www.cosmosmagazine.com/node/387
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Bacterial Mn Cycle Links to C cycle

• Mn Reducers Can Release DOM from POM.

Mn2 1/2O2 H2O MnO2 2H
?G? -70.9 kJ/mole
Courtesy of Greg Dick
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The Calvin Cycle(Carbon Fixation)
http//www.msu.edu/smithe44/calvin_cycle_process.
htm
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The Problem

• Showing a concrete link between the RuBisCO gene
  and Manganese oxidation.
• RuBisCO gene not shown to function in SI85-9A1.
• SI85-9A1s RuBisCO gene HAS been shown to work in
  E. coli.
• -Caspi, R., Haygood, M. G. Tebo, B. M. (1996).
  Unusual ribulose-1,5-bisphosphate
  carboxylase/oxygenase genes from a marine
  manganese-oxidizing bacterium. Microbiology-Uk
  142, 2549-2559.

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Objectives

• To better understand the Mn utilizing population
  in the Lower Columbia River Estuary.
• Grow and culture individual species of Mn
  utilizing microbes.
• Determine size of culturable Mn oxidizing and
  reducing populations in the estuary.
• Determine potential for C fixation by the Mn
  oxidizing population, using RuBisCO gene.

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Sample Collection on the Lower Columbia River
Estuary
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Study Sites
June 14th Cruise 2007
July 17th Cruise 2007
Astoria
Astoria
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The Estuary Turbidity Maximum

• As a salt wedge from the ocean tide moves
  upriver, water near the front of the wedge can
  well upwards and sink back down, scouring the
  bottom and forcing nutrients and microbes
• from the river bottom
• up into the higher
• water levels.

Fresh Water
ETM
Salt Water
http//www.hpl.umces.edu/jpierson/images/etmlogo.
JPG
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Microbial growth from the June 14th Columbia
River Cruise.
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Mn (III)-PPi Glycerol Plates Show Positive for
Manganese Oxidizers
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Colony Growth Results from Columbia River Water
Samples Plated on Varying C sources
We plated samples from casts 3, 8/9 9 and
obtained similar results from all three casts
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• The oxidation of Mn(II) to Mn(IV) passes through
  a transient Mn(III) intermediate
• Mn(II) Mn(III) MnO2
• -Trouwborst, R. E., Clement, B. G., Tebo, B. M.,
  Glazer, B. T. Luther, G. W. (2006). Soluble
  Mn(III) in suboxic zones. Science 313, 1955-1957.
• -Webb, S. M., Dick, G. J., Bargar, J. R. Tebo,
  B. M. (2005). Evidence for the presence of
  Mn(III) intermediates in the bacterial oxidation
  of Mn(II). Proceedings of the National Academy of
  Sciences of the United States of America 102,
  5558-5563.

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Manganese Reduction
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Courtesy of Nick Hagerty
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96 Well MPN Plates
25 Seawater, Top is INT, Bottom is LBB
-Top 4 rows on each plate are duplicate Glycerol
Formate Ascorbate-Hepes media. -The bottom 4
rows on each plate are duplicate Succinate media.
-Columns from left to right are serial
dilutions to 10-9 (top) and 10-10 (bottom), then
negative and positive controls.
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Amplification of RuBisCO gene from Lower Columbia
River Estuary water sample
RuBisCO Gene
- control
Controls
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Conclusion

• Mn levels in samples parallel ETM events.
• Mn oxidizers make up a significant percentage of
  the Lower Columbia River Estuary culturable
  microbial population that uses succinate or
  glycerol.
• Amount of DNA extracted in the ETM event
  significantly greater than outside the ETM event.
• Some Mn oxidizers are capable of forming Mn
  oxides starting from Mn(III).

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The Next Steps

• Process MPNs from July cruise.
• Colony PCR and sequencing of isolated Mn
  oxidizing colonies and compare to community
  analyses.
• Clone library of 16S ribosomal gene.
• Data analysis with BLAST.

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Special Thanks To

• Suzanna Brauer
• Brad Tebo
• Antonio Baptista
• Andrew Han
• Craig Anderson
• Dan Murphy
• Holly Simon
• Lydie Herfort
• Rick Davis
• Michael Wilkin and the Crew of the Forerunner
• And everyone else in my lab!

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References

• Caspi, R., Haygood, M. G. Tebo, B. M. (1996).
  Unusual ribulose-1,5-bisphosphate
  carboxylase/oxygenase genes from a marine
  manganese-oxidizing bacterium. Microbiology-Uk
  142, 2549-2559.
• Pichard, S. L., Campbell, L. Paul, J. H.
  (1997). Diversity of the ribulose bisphosphate
  carboxylase/oxygenase form I gene (rbcL) in
  natural phytoplankton communities. Applied and
  Environmental Microbiology 63, 3600-3606.
• Selesi, D., Schmid, M. Hartmann, A. (2005).
  Diversity of green-like and red-like
  ribulose-1,5-bisphosphate carboxylase/oxygenase
  large-subunit genes (cbbL) in differently managed
  agricultural soils. Applied and Environmental
  Microbiology 71, 175-184.
• Tebo, B. M., Johnson, H. A., McCarthy, J. K.
  Templeton, A. S. (2005). Geomicrobiology of
  manganese(II) oxidation. Trends in Microbiology
  13, 421-428.
• Tebo, B. M., Bargar, J. R., Clement, B. G., Dick,
  G. J., Murray, K. J., Parker, D., Verity, R.
  Webb, S. M. (2004). Biogenic manganese oxides
  Properties and mechanisms of formation. Annual
  Review of Earth and Planetary Sciences 32,
  287-328.
• Trouwborst, R. E., Clement, B. G., Tebo, B. M.,
  Glazer, B. T. Luther, G. W. (2006). Soluble
  Mn(III) in suboxic zones. Science 313, 1955-1957.
• Webb, S. M., Dick, G. J., Bargar, J. R. Tebo,
  B. M. (2005). Evidence for the presence of
  Mn(III) intermediates in the bacterial oxidation
  of Mn(II). Proceedings of the National Academy of
  Sciences of the United States of America 102,
  5558-5563
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