Voltammetric Technique for Rapid Voltammetric Technique for Rapid Screening of Microbial Iron(III) Screening of Microbial Iron(III) Reduction by Reduction by Shewanella oneidensis Shewanella oneidensis strain MR- strain MR- 1 1 Morris E. Jones , Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert EAS GSS 2006 NSF – Biogeosciences Program Photo by Ken Nealson * Dept of Biology
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Morris E. Jones , Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert
Voltammetric Technique for Rapid Screening of Microbial Iron(III) Reduction by Shewanella oneidensis strain MR-1. Morris E. Jones , Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert EAS GSS 2006 NSF – Biogeosciences Program. * Dept of Biology. Photo by Ken Nealson. - PowerPoint PPT Presentation
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Voltammetric Technique for Rapid Screening of Voltammetric Technique for Rapid Screening of Microbial Iron(III) Reduction by Microbial Iron(III) Reduction by Shewanella oneidensisShewanella oneidensis
strain MR-1strain MR-1
Morris E. Jones, Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert
EAS GSS 2006
NSF – Biogeosciences Program
Photo by Ken Nealson* Dept of Biology
Why study iron reducing bacteria (FeRB)?
• Carbon cycling – anaerobic
iron vs sulfate
• Bioremediation– radionuclides
• Mineral stability– iron surface chemistry
• Metal corrosion– petroleum pipeline
• One of the first respiratory processes on earth
Dissimilatory microbial iron reduction
• At circumneutral pH Fe3+ mostly found as a solid (Stumm and Morgan 1996)
• Two common oxidation states, Fe3+ and Fe2+
• Fe2+ rapidly oxidized in the presence of oxygen (Millero, et.al. 1987)
• The iron reductase has not been found (DiChristina, et.al. 2005)
• How are FeRB able to use FeOx as a Terminal Electron Acceptor
• Four hypotheses for microbial iron reduction– Direct contact (Meyers and Meyers 1993)
– Nanowires (Lovley, et.al. 2005)
– Electron shuttles (Lovely et.al. 1996)
– Ligand promoted dissolution (Nevin et.al. 2002)
Potential iron reduction pathways
Dichristina, 2005
Lovley, 2005
What we see with voltammetry
• Square wave voltammetry– Scan potential– Measure current
• Under anaerobic conditions, with FeOx as TEA, Org-Fe(III) is produced
– Environment– Laboratory
• Conventional screening techniques only detect Fe(II) reduction product
Pt Counter
Hg-Au Working
Ag/AgCl Reference
100 m diameter Au wire
Single colony MR1, 40mM FeOx, 24 hrs
0.000.10
0.200.300.400.50
0.600.700.80
0.901.00
-1.80-1.30-0.80-0.30
E - [V]
I - [u
A]
w/o iron
w/ iron
0.0 -0.5 -1.0 -1.5
0
20
40
60
80
100
120
140
160
I -
[nA
]
E - [V]
Org-Fe(III)
Fe(II)
Org-Fe(III)
Fe(II)
FeS
Satilla River sediment core
Arnold, 1988
Research plan
• Use voltammetry as a screening tool for iron reduction activity
• Create mutants from wild type S.oneidensis• Random vs targeted mutants• Screen for iron reducing activity
– Org-Fe(III)– Fe(II)– Possible intermediates
• Locate genes• Identify proteins
Mutation and Complementation
X
X
Random single nucleotide
mutation
Wild-type S. oneidensis MR-1
Voltammetric screening for iron reduction deficiency
Clone Bank
Fe(III)
Fe(III)
Fe(III)
EMS
ethane methyl sulfonate
Mobilize wild-type gene clone bank
into mutants
Voltammetric screening for iron reduction activity
Voltammetric screening array
• Eight electrodes• 13.5 min per row• 3 hrs per tray• Anaerobic