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

Lead Research Organisation: University of East Anglia
Department Name: Chemistry

Abstract

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.

Publications

10 25 50
 
Description Lipid bilayers are used in biology to define cells and their compartments, provide barriers against toxins and allow for energy conservation through ATP generation by respiration and photosynthesis. Stacked lipid bilayers are a common principle as they afford opportunities to greatly increase membrane protein concentration, and achieve spatial organisation. We have developed a method for step-by-step assembly of multi-layered lipid membranes through alternate exposures of an appropriate surface to positively charged polymer and negatively charged lipid membranes. Redox active enzymes can be incorporated throughout these stacked bilayer assemblies and their electrocatalytic activity driven by the potential applied to an underlying electrode. Electron exchange between the electrode and membrane confined enzymes is facilitated by very rapid diffusion of electron carrying quinones and quinols that are contained in the lipid-bilayers. The resulting assemblies are similar to the interconnected photosynthetic thylakoid membranes or the folded lamella of mitochondria such that they have potential to mimic energy production or biosensing for biotechnology or fundamental research. In addition we have purified an important electron transfer enzyme from the inner membrane of the bacterium Shewanella oneidensis and established that this exists as a dimer, rather than monomer. This has implications for the mechanism of catalysis by this enzyme during anaerobic respiration.
Exploitation Route The development of a method producing stacked lipid bilayers provides a resource to gain fundamental insight into electron transfer at/across membrane bilayers. This method can be used by others aiming to study membrane associated proteins and enzymes typically found in such environments. It can also be used for bioelectrochemical synthesis when enzymes such as hydrogenase are hosted in these bilayers and driven by the voltage applied to an underlying electrode. Knowledge of oligomeric state of CymA from Shewanella oneidensis will aid researchers aiming to use electron transfer between the external and internal environments of this bacterium for chemical synthesis by microbial electrosynthesis and electricity production by microbial fuel cells.
Sectors Chemicals,Energy,Manufacturing, including Industrial Biotechology

 
Description Through public engagement activities, workshops and talks, the public are increasingly well informed of climate friendly approaches to producing chemicals and electricity using bacteria. This is contributing to changes in domestic culture and practice that impact positively on environmental sustainability, protection and impact. Through discussions with representatives of Anglian Water our research has contributed to the knowledge base of a major utility provider.
First Year Of Impact 2017
Impact Types Societal

 
Description Workshop on Protein Electrochemistry BISON
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact New skills and approaches to research were displayed by the participants after completing the course.
 
Description Biohybrids for Solar Chemicals and Fuels: Whole-cell Photocatalysis by Non-photosynthetic Organisms.
Amount £1,220,000 (GBP)
Funding ID BB/S002499/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description Royal Society Leverhulme Trust Senior Research Fellowship
Amount £54,000 (GBP)
Organisation The Royal Society 
Department Royal Society Leverhulme Trust Senior Research Fellowship
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2016
 
Description Solid state electronic conduction in multiheme cytochromes 
Organisation Weizmann Institute of Science
Country Israel 
Sector Academic/University 
PI Contribution Multiheme cytochrome designed to have single cysteine adjacent to terminus of heme wire at defined site on surface was designed and purified.
Collaborator Contribution Measurements of solid state conductance by the aforementioned multiheme cytochrome were performed.
Impact Manuscript describing key findings has been submitted for peer-review. Multi-disciplinary: physics, biophysics, biochemistry, theoretical chemistry.
Start Year 2017
 
Description Anglian Water Discussion 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Technology Director for Anglian Water visited UEA and spoke with research leaders on a number of projects identified as having potential interest to their business. The use of bacteria in 'sludge' to produce electricity was a novel concept to the Director, follow up e-mails passed on relevant technical reports and literature.
Year(s) Of Engagement Activity 2017
 
Description Interactive Display at Latitude Festival, Suffolk, 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact >1100 people visited our interactive display over 3 days in the Kids Zone at Latitude festival. Through experiments, games and virtual reality our visitors learnt about different types of energy, interconversion of types of energy,microbial fuel cells, electricity producing bacteria and proteins that conduct electricity across cell walls. Our engaging activities sparked lively discussion among our visitors and it was clear that they all (kids, parents and grandparents) left us with a great excitement for science.
Year(s) Of Engagement Activity 2018
URL https://www.sawtrust.org/news/latitude-festival-2018/
 
Description Interactive Display at Norwich Science Festival October 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Forum, in the centre of Norwich, was the location for our interactive display during a day of the Norwich Science Festival. Through experiments, games and virtual reality our visitors learnt about different types of bacteria, learnt about microbial fuel cells and electrically conductive proteins. There were > 1000 visitors and informal feedback showed they had learnt something new about sustainable technology, physics and biology from their visit.
Year(s) Of Engagement Activity 2018
URL https://norwichsciencefestival.co.uk/
 
Description Norwich Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Demonstrations of microbial fuel cells accompanied by virtual reality headsets that allowed a 'journey' through the bacteria to see the enzymes key to operation of the fuel cell. Much discussion and questions arising from this.
Year(s) Of Engagement Activity 2016,2017
 
Description PTI Subject Day - Photosynthesis 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 15 teachers attended subject specific training day organised by the Princes Teaching Institute. Alongside events designed to re-invigorate teaching of photosynthesis at A(S)-level I delivered a talk on research at forefront of artificial photosynthesis. There was lively debate following the talk and subsequent feedback indicated the teachers had greatly enjoyed learning about how research is moving forward in extending the principles of plant based photosynthesis to systems that may deliver sustainable strategies to chemicals and fuel production.
Year(s) Of Engagement Activity 2015
 
Description Press Release MHC review 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact not known
Year(s) Of Engagement Activity 2014
 
Description Public Talk 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact An informal presentation on 'biobatteries' and extracellular electron transfer at pub in Norwich City Centre. Attended by approximately 30 members of public including school teachers and electrical engineers.
Year(s) Of Engagement Activity 2017
URL https://www.meetup.com/Norwich-Biomakers/events/243884032/?_cookie-check=XxyoJwuy8pUP8Sx_
 
Description Royal Norfolk Show 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Over 2 days many representatives from industry/business in addition to general public and school children visited our display and engaged in discussion about our research, its possible technological impact and the underlying scientific principles.
Year(s) Of Engagement Activity 2016,2017
 
Description Royal Norfolk Show 2017 Education Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Prior to the event Local Schools supplied soil from their grounds. This was loaded into Microbial Fuel Cells and their performance (power output and longevity) was reported at the Royal Norfolk Show alongside virtual reality experience of extracellular electron transfer process for H2 production and a short talk covering key principles of biological H2 production.
Year(s) Of Engagement Activity 2017
 
Description School visit (Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 30 high school pupils were present for a talk followed by Q&A. Many questions and increase in pupils considering career in science was reported.
Year(s) Of Engagement Activity 2017
 
Description School visit (Norwich) with experiments 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact science workshops for key stage 1 (5 - 7 yr)
Year(s) Of Engagement Activity 2016,2017
 
Description Talk at IET meeting Shrewsbury March 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation at a meeting of The Institution of Engineering and Technology in Shrewsbury. The significance of bio-electricity to ATP production was explained, electricity production by bacteria was explained and microbial fuel cells demonstrated and discussed. Our related, new research into (semi-)artificial photosynthesis was introduced. Discussion after the talk was lively and touched on wide-ranging topics.
Year(s) Of Engagement Activity 2019
 
Description Talk at Norwich Science Festival October 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A talk on electrically conductive proteins and electricity producing bacteria. There were lots of questions and discussion, during and after the talk. From those it was clear that the topics sparked lots of interest in the physics of biology and new approaches to eco-friendly electricity production.
Year(s) Of Engagement Activity 2018
URL https://norwichsciencefestival.co.uk/
 
Description UEA Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact interactive demonstrations run throughout the day prompted ongoing discussions with visitors about our research.

many people noted the research was fascinating and they were not aware of it previously
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014