Mediator-Dependent Transfer of Cathodic Electrons into Shewanella oneidensis · ·...
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Mediator-Dependent Transfer of Cathodic Electrons into Shewanella oneidensis Holly L. Sewell 1 , Svenja T. Lohner 1 , Ann Lesnefsky 1 , Alfred M. Spormann 1,2 1 Department of Civil and Environmental Engineering, 2 Department of Chemical Engineering, Stanford University, Stanford, CA, 94305 Funding for this project is provided by the Global Climate and Energy Program (GCEP) at Stanford University. Acknowledgements 2) Biological production of hydrogen on the cathode Introduction There is a high demand for developing novel energy technologies that decrease net CO 2 release and reduce dependence on fossil fuel hydrocarbons as primary energy sources for production of liquid (transportation) fuels. Generating fuels via microbial electrosynthesis is one promising alternative to fossil fuels that makes use of unique autotrophic prokaryotic organisms capable of consuming cathodic electrons and reducing CO 2 to produce useful compounds such as biofuels and their precursors. Although recent data successfully demonstrate this concept Model system for mediated electron transfer Utilizing a wide range of electron acceptors, cathodic electron transfer into Shewanella oneidensis MR-1 can be monitored via the metabolic reduction of fumarate to succinate: concept, the mechanism of cathodic electron transfer is still highly speculative and has been shown to present a bottleneck for bioelectro- synthetic processes. Med ox Med red succinate Cathode fumarate e- In this project we investigate different modes of external electron transfer between the cathode and microbial cells. Mediator compounds play a major role in these processes as they can be continuously re-reduced by the cathode to shuttle electrons to redox active membrane-bound proteins of the cell Shewanella oneidensis MR-1 represents an excellent model organism for extracellular electron uptake as it is known for its versatile electron accepting capabilities. It utilizes a complex electron transfer network consisting of various tetraheme and decaheme cytochromes that extend electron transport from the cytoplasmic membrane through the periplasm and to the outer membrane. Previous studies have shown that S. oneidensis MR-1 is electrochemically active and capable of synthesizing endogenous mediator compounds. The genetic tractability of S. oneidensis enables genetic modifications of its electron transfer networks to pinpoint the entry point of cathodic electrons into the cell. Experimental Setup For the microbial electro-synthesis experiments, a dual-chamber electrochemical reactor was employed in which the anode and cathode compartments are separated by a cation exchange membrane. The three-electrode setup allows for precise control of the cathode at a specified potential, tunable according to the mediator being used. Specific Research Questions 1. Can mediators be used to transfer cathodic electrons into the metabolic pathways of S. oneidensis MR-1? 2. Can biofuels or biofuel precursors be produced using mediator-dependent cathodic electron transfer? Fumarate + 2 e - + 2 H + Succinate References Rabaey K., Rozendal R.A. (2010) Microbial Electrosynthesis – Revisiting the Electrical Route for Microbial Production. Nature Reviews Microbiology, Vol. 8, 706-716. Ross D.E. et al.. (2011). Towards Electrosynthesis in Shewanella: Energetics of Reversing the Mtr Pathway for Reductive Metabolism. PLOS one, 6(2): e16649. doi:10.1371/journal.pone.0016649 Meshulam-Simon G.. et al.. (2007). Hydrogen metabolism in Shewanella oneidensis MR-1 . App. & Env. Microbio., vol. 73 no. 4 1153-1165 [H 2 ] ppb In the absence of fumarate as electron acceptor, electrons from reduced methyl viologen are transferred to H + to form H 2 as end product. Formate is also observed under these conditions. In the absence of methyl viologen and/or applied potential, no hydrogen was observed. Abiotic hydrogen production cannot account for the total amount of hydrogen observed. 0 20 40 60 80 100 120 140 160 0 2 4 6 8 10 12 + (-500mV) vs SHE + 5mM MV + fumarate + (-500mV) vs SHE + 5mM MV Cell suspensions of S. oneidensis MR-1 were placed in an electrochemical reactor with electrochemically reduced methyl viologen (5 mM) provided as the sole electron donor and fumarate provided as an electron acceptor. The formation of succinate from fumarate and the transferal of electrons from the cathode to the system were recorded and monitored by HPLC and potentiostat during the course of the experiment. Current is transferred when the electron acceptor fumarate is present. Fumarate reduction to succinate is dependent upon the presence of reduced methyl viologen. I (mA) Using methyl viologen as mediator, fumarate is completely reduced to succinate. 1) Uptake of cathodic electrons into S. oneidensis While fumarate is present, nearly 100% of the electrons donated by the cathode end up in succinate. 0 0.5 1 1.5 2 2.5 0 2 4 6 8 10 cathodic electrons consumed electrons recovered in product mmol of electrons Time (hours) 0 2 4 6 8 10 12 -12 -10 -8 -6 -4 -2 0 0 2 4 6 8 10 12 current [fumarate] [succinate] Time (hours) Concentration (mM) 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 0 2 4 6 8 10 [fumarate] (mM) [Hydrogen] (ppb) [H 2 ] ppb Time (hours) Concentration (mM) It is also observed that hydrogen is produced when fumarate is depleted as an electron acceptor. This indicates a possible electron switch in which electrons will be directed to the production of succinate or hydrogen based on a certain hierarchy of the electron acceptors available. Conclusions and Future Work These results indicate that cathodic electrons can successfully be transferred into S. oneidensis MR-1 by means of methyl viologen to form fumarate and biofuel precursors, hydrogen and formate. We hypothesize that these electrons enter through the metal reduction (Mtr) pathway for fumarate reduction. To test this hypothesis, genetic analyses are being performed to determine the molecular Cathode MtrC Fcc MtrB MtrA CymA CymA MQH2 MQ fumarate succinate Outer Membrane Periplasmic Space Cytoplasmic Membrane 2H+ H2 Medox Medred 2e< ? H 2 ase = Mtr pathway = knockout e < Results pathway(s) of how mediator-delivered electrons enter cellular electron transferring networks. Additionally, other mediators are being examined and, in preliminary studies, S. oneidensis also seems to be able to take up electrons from these compounds.
Transcript of Mediator-Dependent Transfer of Cathodic Electrons into Shewanella oneidensis · ·...
Mediator-Dependent Transfer of Cathodic Electrons into Shewanella oneidensis Holly L. Sewell1, Svenja T. Lohner1, Ann Lesnefsky1, Alfred M. Spormann1,2
1Department of Civil and Environmental Engineering, 2Department of Chemical Engineering, Stanford University, Stanford, CA, 94305
Funding for this project is provided by the Global Climate and Energy Program (GCEP) at Stanford University.
Acknowledgements
2) Biological production of hydrogen on the cathode
Introduction There is a high demand for developing novel energy technologies that decrease net CO2 release and reduce dependence on fossil fuel hydrocarbons as primary energy sources for production of liquid (transportation) fuels. Generating fuels via microbial electrosynthesis is one promising alternative to fossil fuels that makes use of unique autotrophic prokaryotic organisms capable of consuming cathodic electrons and reducing CO2 to produce useful compounds such as biofuels and their precursors. Although recent data successfully demonstrate this concept
Model system for mediated electron transfer
Utilizing a wide range of electron acceptors, cathodic electron transfer into Shewanella oneidensis MR-1 can be monitored via the metabolic reduction of fumarate to succinate:
concept, the mechanism of cathodic electron transfer is still highly speculative and has been shown to present a bottleneck for bioelectro-synthetic processes.
Medox
Medred succinate C
atho
de fumarate
e- In this project we investigate different modes of external electron transfer between the cathode and microbial cells. Mediator compounds play a major role in these processes as they can be continuously re-reduced by the cathodeelectrons from the cathode to cathode to shuttle electrons to redox active membrane-bound proteins of the cell Shewanella oneidensis MR-1 represents an excellent model organism for extracellular electron uptake as it is known for its versatile electron accepting capabilities. It utilizes a complex electron transfer network consisting of various tetraheme and decaheme cytochromes that extend electron transport from the cytoplasmic membrane through the periplasm and to the outer membrane. Previous studies have shown that S. oneidensis MR-1 is electrochemically active and capable of synthesizing endogenous mediator compounds. The genetic tractability of S. oneidensis enables genetic modifications of its electron transfer networks to pinpoint the entry point of cathodic electrons into the cell.
Experimental Setup
For the microbial electro-synthesis exper iments , a dua l -chamber e l e c t r o c h e m i c a l r e a c t o r w a s employed in which the anode and cathode compartments are separated by a cation exchange membrane. The three-electrode setup allows for precise control of the cathode at a specified potential, tunable according to the mediator being used.
Specific Research Questions
1. Can mediators be used to transfer cathodic electrons into the metabolic pathways of S. oneidensis MR-1? 2. Can biofuels or biofuel precursors be produced using mediator-dependent cathodic electron transfer?
Fumarate + 2 e- + 2 H+ Succinate
References Rabaey K., Rozendal R.A. (2010) Microbial Electrosynthesis – Revisiting the Electrical Route for Microbial
Production. Nature Reviews Microbiology, Vol. 8, 706-716.
Ross D.E. et al.. (2011). Towards Electrosynthesis in Shewanella: Energetics of Reversing the Mtr Pathway
for Reductive Metabolism. PLOS one, 6(2): e16649. doi:10.1371/journal.pone.0016649
Meshulam-Simon G.. et al.. (2007). Hydrogen metabolism in Shewanella oneidensis MR-1 . App. & Env.
Microbio., vol. 73 no. 4 1153-1165 [H
2] p
pb
Time (hours)
In the absence of fumarate as electron acceptor, electrons from reduced methy l v io logen a re transferred to H+ to form H2 as end product. Formate is also observed under these conditions. In the absence of methyl viologen and/or applied potential, no hydrogen was observed. Abiotic hydrogen production cannot account for the total amount of hydrogen observed. 0
20
40
60
80
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120
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0 2 4 6 8 10 12
+ (-500mV) vs SHE + 5mM MV + fumarate
+ (-500mV) vs SHE + 5mM MV
Cell suspensions of S. oneidensis MR-1 were placed in an electrochemical reactor with electrochemically reduced methyl viologen (5 mM) provided as the sole electron donor and fumarate provided as an electron acceptor. The formation of succinate from fumarate and the transferal of electrons from the cathode to the system were recorded and monitored by HPLC and potentiostat during the course of the experiment.
Current is transferred when the electron acceptor fumarate is present. Fumarate reduction t o s u c c i n a t e i s dependent upon the presence of reduced methyl viologen.
I (m
A)
Using methyl v io logen as mediator, fumarate is completely reduced to succinate.
1) Uptake of cathodic electrons into S. oneidensis
While fumarate is present, nearly 100% of the electrons donated by the cathode end up in succinate. 0
0.5
1
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0 2 4 6 8 10
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cathodic electrons consumed
electrons recovered in product m
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of e
lect
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-4
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0 2 4 6 8 10 12
current [fumarate] [succinate]
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[fumarate] (mM) [Hydrogen] (ppb)
[H2 ] ppb
Time (hours)
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ratio
n (m
M)
It is also observed that hydrogen is produced w h e n f u m a r a t e i s depleted as an electron acceptor. This indicates a poss ib le e lect ron switch in which electrons will be directed to the production of succinate or hydrogen based on a certain hierarchy of the e l e c t r o n a c c e p t o r s available.
Conclusions and Future Work
These results indicate that c a t h o d i c e l e c t r o n s c a n successfully be transferred into S. oneidensis MR-1 by means of methyl viologen to form f u m a r a t e a n d b i o f u e l precursors, hydrogen and formate. We hypothesize that these electrons enter through the metal reduction (Mtr) pathway for fumarate reduction.
To test this hypothesis, genetic analyses are being performed to determine the molecular pathway(s)
Cathode
MtrC
Fcc
MtrB MtrA CymA
CymA%
MQH2%MQ%
fumarate% succinate%
Outer%Membrane%Periplasmic%Space%
Cytoplasmic%Membrane%
2H+% H2%
Medox
Medred
2e<%?%
H2ase =%Mtr%pathway%
=%knockout%
e<%
Results
pathway(s) of how mediator-delivered electrons enter cellular electron transferring networks. Additionally, other mediators are being examined and, in preliminary studies, S. oneidensis also seems to be able to take up electrons from these compounds.
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