Advancing Microbial Electrochemistry: Biophysical Characterisation of the Electron-Transfer Interactome in S. oneidensis MR-1

Lead Research Organisation: University of Leeds
Department Name: School of Biomedical Sciences


Reports concerning dwindling reserves of fossil fuels and concerns over fuel security are frequent news headlines. The rising costs of fuel are a daily reminder of the challenges faced by a global society with ever increasing energy demands. In this context it is perhaps surprising that the energy supplies available to us, namely, sunlight, winds, waves and bio-organic produce, remain largely untapped resources. A significant amount of our energy demand is currently needed to process our waste water and sewage. The latter is done by bacteria, which consume and thereby remove organic waste materials from our sewage and convert it to clean water and carbon dioxide. The bacteria extract energy out of this process in the same way we extract energy out of the food we eat. However, in a very intriguing process some bacteria excrete electricity as a result of their digestive processes. This bio-electricity can be harvested by interfacing these bacteria with electrodes. Recently it has been discovered that by applying an appropriate voltage to the electrode the flow of electricity can also be reversed. In this way electrons are pushed into the bacteria and they can drive the activities of enzymes to make otherwise costly, petroleum derived chemicals and fuels from low-value, abundant molecules such as carbon dioxide.

The examples above illustrate how bacterial electrochemistry offers multiple routes to support a sustainable future. However, full realisation of these promising technologies requires electricity to flow optimally across a network of proteins in the bacteria and be delivered to the appropriate enzyme(s). In much the same way at home, electricity should be directed to the TV in front of you rather than one in another room, or next door when you wish to watch a film. Well understood circuitry, switches and fuse boxes ensure the desired flow of electricity occurs in a safe manner from the power station to your TV. Here we aim to elucidate the bacterial equivalents of the electrical grid that surrounds us. We will establish how the flow of electricity is regulated by the network of proteins within a bacterium called Shewanella oneidensis that is the 'lab rat' for developing bacterial electrochemistry.

Technical Summary

Shewanella oneidensis MR-1 (MR-1) possesses a remarkably versatile respiratory system. Key to this versatility is the inner-membrane menaquinol dehydrogenase, CymA, that represents a hub for electron transfer between catabolic enzymes and terminal reductases. However, very little is known about the factors that determine electron exchange between CymA and the network of periplasmic and outer-membrane proteins with which it interacts. Here we propose to address this situation through recent advances in the applicants' laboratories. Specifically, we propose to use novel supported membrane technology with quartz-crystal microbalance and electrochemical measurements complemented by contemporary biochemistry with the aims of a) mapping out the periplasmic and outer-membrane proteins that interact with CymA, and, b) quantifying the factors that regulate protein-protein interaction and electron flux across this network. This will allow us to test our hypothesis that respiratory electron transfer by MR-1 may be regulated by the presence of terminal electron acceptors and predominantly controlled at the metabolic level through reconfiguration of the network of electron-transport proteins around CymA.
Key advances will include a) an understanding of the biophysical basis for respiratory flexibility in MR-1, and b) insight into the determinants of catalytic bias in CymA which belongs to a phylogenetically widespread family of quinol-dehydrogenases. Thus, we envisage our results will inform the understanding of electron flux and respiratory versatility in a number of bacteria. In addition, our results will define conditions where electron exchange between CymA and the outer-membrane electron transfer conduit MtrCAB is optimised. Thus, we envisage our results will underpin advances in microbial biotechnologies that exploit electrode-MtrCAB electron transfer such as microbial fuel cells and electrosynthesis for which MR-1 is a model organism.

Planned Impact

Impact summary
The aim of this project is two-fold: (a) to use biochemical, biophysical and (bio)nanotechnological approaches to study Shewanella oneindensis MR-1 (MR-1) to optimise the exploitation of MR-1 and related microbes in biotechnology and (b) development of techniques in membrane biology. This work will impact the global society (timescale > 10 year) and research sectors in alternative energy sources (timescale > 3 year) and drug development (timescale > 2 years). Finally, public-engagement events organised during the life-time of the project will have a direct impact to the local community, while also the training of PDRAs and closely-involved research staff and PhD students will impact the scientific community.

We aim to increase our understanding of the bioenergetics of MR-1 and their respiratory proteins, which is urgently required to optimise the use of microbes to harvest energy and produce fuels using microbial electrochemistry, which includes microbial fuel cells and microbial electrosynthesis. Of particular interest for the studies proposed here are the multi-heme proteins in Shewanella which mediate electron transfer to the outside of the cell or to inorganic substrates. Shewanella bacteria serve as an important model system for mediator-less microbial fuel cells that run on waste carbon sources (such as in waste water) to produce electricity. This is a new area with much potential in the future. We propose that our work will contribute to the future design of such microbial electrochemistry, in particular where future work aims to genetically or synthetically modifying the microbes to enhance electron transfer rates to the electrodes (i.e., increase electrical current).

The study proposed here includes the further development of so-called solid-supported membranes. Recent pilot data has shown the latter tool to be very useful in the study of protein-protein interactions in cases where one or two of the proteins are membrane proteins. Currently, membrane biology is an extremely active and important research area. For instance, although only ~20% of the human genome, membrane proteins represent approximately 50% of today's drug targets. Having tools available that can screen protein-protein interactions of membrane proteins (this includes pharmaceutically important antibody mimetics) will be of high importance to the wider scientific community, including the industrial pharmaceutical sectors.

Societal impact
The search for alternative energy sources is of major importance to THE GLOBAL SOCIETY where conventional energy sources, based on burning of earth-stored carbon sources, are thought to be responsible for global warming. Furthermore, the extraction of these carbon sources themselves is becoming increasingly more expensive and, combined with the higher demand for energy world-wide, this has led to rapid increases in energy prices. A solution to this problem has to be sought by combining a multitude of 'alternative' energy sources; this research will contribute to this progress.
Timescale of likely impact: >10 years

The technology development of solid-supported membranes will also impact on the pharmaceutical industry, where improvements in drug development and screening of antibody mimetics will be of benefit to the THE GLOBAL SOCIETY on the longer timescale.
Timescale of likely impact: >10 years

As part of this grant we plan to contribute to a variety of events aimed to engage the public in alternative energies (see Pathways for Impact for more details). These events will contribute to the public understanding of communities in or near Leeds and Norwich.
Timescale of likely impact: During the lifetime of the project.

Commercial/industrial impact
Both the alternative energy industry sector and the biopharmaceutical sector may benefit from this research, as explained above.
Timescale of possible impact: >2-3 years after starting the project.


10 25 50
Description Multilayer lipid membranes form many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae) and sequential processing (golgi cisternae). In this project, we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly-l-lysine (PLL) as an electrostatic polymer linker. This system can be used to study the processes that occur in this biological multimembrane systems.
Subsequently, we discoveredthat the multilayer lipid membrane can also be tailored such that the lipid membranes in each layers are 'connected' to each other via so-called stacks. The later is similar to the way thylakoid discs are connected to each other in chloroplast. The interconnectiveness made it possible to create electrocatalytic systems that could find application in fuel cell technology. The principle was tested using two membrane enzymes, the oxygen reducing, ubiquinol oxidase from E. coli and the hydrogen oxidizing, [NiFe] hydrogenase from R. eutropha.
We are currently exploiting this system to test electron transfer reaction from the inner to outer membrane to study the molecular mechanisms underlying respiration in Gram negative bacteria.
Exploitation Route The multibilayer lipid systems could be used by the academic community to study structural and functional features biological systems, e.g., , such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae) and sequential processing (golgi cisternae).
The multibilayer lipid systems could be used in biotechnological electrocatalytic systems with membrane enzymes (for instance for fuel cell technology).
Sectors Energy

Description Hydrogenase biomembrane-modified anodes for hydrogen-oxygen fuel cells.
Amount £10,657 (GBP)
Funding ID BB/S009736/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 02/2019
Description Interaction between lipid bilayers and polylysine for multilayer lipid surfaces
Amount £800 (GBP)
Funding ID TG 31-05-15 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 08/2015 
End 08/2015
Description Paul OShea, Nottingham 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We applied to a travel grant from the Directed Assembly Network (EPSRC) for the Post Doctoral Research Fellow on this project (Dr. George Heath) to visit the lab of Prof. Paul O'Shea at the University of Nottingham. This travel grant was successful. Dr. George Heath then prepared samples at the University of Leeds, which were taken to Nottingham to characterize. In Nottingham, Dr. George Heath characterised the effect of poly-Lysine on charge distribution of lipid membranes (liposomes). This data formed part of larger amount of data that ultimately led to the publicaiton in Biomacromolecules in 2016.
Collaborator Contribution The partner, the group of Paul' Oshea in Nottingham, contributed by sharing his experimental setup and expertise that enabled us to characterise the influence of poly-lysine on liposomes.
Impact This work led (in part) to a publication in Biomacromolecules in 2016 (DOI: 10.1021/acs.biomac.5b01434)
Start Year 2015
Description University of East Anglia 
Organisation University of East Anglia
Country United Kingdom 
Sector Academic/University 
PI Contribution This award was a collaboration between us and the University of East Anglia. All details for the collaboration and research are described in detail in the research proposal.
Collaborator Contribution This award was a collaboration between us and the University of East Anglia. All details for the collaboration and research are described in detail in the research proposal.
Impact This partnership/collaborations has been funded by multiple BBSRC proposals. All outputs and outcomes are described in detail under the respective BBSRC awards.
Start Year 2009
Description AchiBio, the Superposition 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact The Superposition is a network and space for artists, makers and scientists to collaborate. Within the superposition there are many smaller 'work groups', including BioLeeds and ArchiBio. Within ArchiBio, through an ongoing series of workshops, we are comparing architectural and biological methods and structures. The ultimate aim is create a series of exhibits and organise workshops for the general public as part of "ASMbly", (A)rt, (S)cience, (M)ake labs, which is the Superposition's annual big event. So far, the Superposition, has run 2 editions of a week long pop up art science lab with the aim of generating new artworks and prototypes in a fast, hotbed environment of cross disciplinary collaboration. These have taken place in a disused city centre spaces.
Year(s) Of Engagement Activity 2015,2016,2017
Description Biotechnology in solar power 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Event organised by SciBar Nottingham.
Year(s) Of Engagement Activity 2016
Description Discovery Zone (Par of Science Week at the University of Leeds) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact It engaged school children into science in general and aims to excite them about science and science education. It also aims to raise awareness that scientist are 'normal' people, with exciting jobs.

The impact is not directly measurable, as the impact is raised awareness of and excitement towards science. However, the organisers of the discovery zone received many letters from school thanking us for a wonderful day and one of the school kids was interviewed at BBC Radio 2 about her experiences.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016,2017
Description Public Engagement Event on Bioenergy, connected to a symposium "Membrane Proteins From A to Z: A commemoration of the life and work of Prof. Stephen Baldwin" 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 60 Students and their teachers participated in an event delivered by a team of our early career researchers in which they enjoyed a tour of the research facilities and a poster session on membrane related research in Leeds. At the poster session the students discussed our research with our early career researchers.

The feedback from the school clearly indicated that a major impact was to stimulate thinking and to create enthusiasm for science:

"Our students thoroughly enjoyed the chance to talk to scientists (great poster displays!), see the labs, and listen to Sir John's thought-provoking and inspiring talk. I think they felt rather special as he talked to them as the next generation with responsibilities".
Dr Kirsty Bryant Allerton High School

"It is easy to think about the highlig
Year(s) Of Engagement Activity 2015