Advancing Biotechnologies for Fuel Generation: Exploiting Transmembrane Cytochromes for Solar Energy Conversion

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 so many of the renewable energy supplies available to us, namely, sunlight, winds and waves, remain largely untapped resources. This is mainly due to the challenges that exist in converting these energy forms into fuels from which energy can be released 'on demand' when we wish to play computer games, drive a car and so on. However, during plant photosynthesis fuels are made naturally from the energy in sunlight. Light absorption by the green chlorophyll pigments generates an energised electron that is directed, along chains of metal centres, to catalysts that make sugars. These sugars fuel us, and all animals, when their energy is released following digestion of a meal. However, using farmed plants to produce biofuels is controversial as agriculture is also required to feed the world. As a consequence, and inspired by natural processes, we propose to build a system for artificial photosynthesis. In essence, we wish to place tiny solar-panels on microbes in order to harness sunlight to drive the production of hydrogen - a fuel from which the technologies to release energy on demand are well-advanced. We will use dyes and semi-conductor particles as mechanically and chemically robust materials to capture the energy in sunlight and generate energised electrons. We will couple these particles to biology's version of conducting wires. These wires are made from heme proteins that span membranes that provide Nature's solution to compartmentalising water-filled chambers (i.e., the inside of the bacterium). The heme-wires are produced naturally by 'rock-breathing' microorganisms and after these wires have transferred the energised electrons across the membrane they will drive enzyme catalysis to produce hydrogen Our novel bio-mimetic photocatalysts will establish new principles for the design of homogeneous photocatalysts with spatially segregated sites for fuel-evolution and the supply of electrons that is needed to sustain this process. We imagine that our photocatalysts will proove versatile and that with slight modification they will be able to harness solar energy for the manufacture of drugs and fine chemicals.

Technical Summary

Most artificial homogeneous photosynthetic systems suffer a major drawback: a short-lived charge separated state, which is due to the failure to spatially decouple the reductive and the oxidative sites required to sustain charge separation. Inspired by membrane bilayers as Nature's solution to spatially decouple reduction and photoexcitation/oxidation, we propose to utilise synthetic biology to develop a novel approach that adopts the principles of natural photosynthesis; light harvesting, charge separation and catalysis. Our photocatalysts will exploit the outer-membrane spanning, cytochrome-based electron-transfer conduits produced naturally by Shewanella oneidensis MR-1. We will establish methods to attach photosensitisers, e.g., dye-sensitised TiO2 and CdS nanoparticles, to the external cytochrome of this conduit in a manner that allows for a rapid charge separation across the membrane creating a long-lived charge separated state. We will develop methods to deliver electrons from the internal face of the conduit to redox catalysts. As a proof-of-principle, the conduit will be coupled to hydrogen-evolving catalysts that will include a [NiFeSe]-hydrogenase, a synthetic cobaloxime catalyst that evolves hydrogen in pH neutral conditions, and colloidal platinum, well-known for its hydrogen evolving properties. Voltammetric and spectroscopic methods together with quantification of hydrogen evolution by gas-chromatography will define the solar conversion efficiencies, electron transfer rate and catalytic properties of these systems. Conditions will then be established to combine systems with the desired properties as hybrid photocatalysts in the bilayers of liposomes and also in S. oneidensis MR-1. Two methods will be employed to deliver the electrons required to sustain hydrogen evolution, sacrificial electron donors such as triethanolamine and electrodes. The latter is explored as it offers opportunities for simultaneous production of electricity.

Planned Impact

Societal impact

The aim of this project is to use biotechnological, biophysical, (bio)nanotechnological and synthetic biological approaches to study and exploit Shewenella sp. and Shewanella proteins. In particular, we aim to exploit Shewenalla sp. and their respiratory proteins to harvest solar energy and produce carbon-neutral fuels such as hydrogen. A renewable energy cycle is recognized as a top national strategic priority in the UK (UK White Paper on Energy). In the last 18 months, several incidents have demonstrated the fragility of the global energy supply: the sharp rise in oil prices following the outbreak of conflicts and civil wars in the Middle-East and the ecological and humanitarian threat of a nuclear meltdown in Fukushima, Japan. The search for alternative energy sources is therefore of major importance to THE GLOBAL SOCIETY. A solution to this problem has to be sought by combining a multitude of 'alternative' energy sources; this research will contribute to this progress.

A new academic partnership & training of new leaders in the energy sector

This project will establish a new academic partnership between Butt, Clarke, Richardson at Univ. East Anglia, Jeuken at Univ. Leeds and Reisner at Univ. Cambridge. The strong ties through this BBSRC project will allow us to form a nucleus around which future networks and collaborations will be built. Within this project we will also provide top-quality cross-disciplinary training for three BBSRC PDRAs, to provide expertise in the development of alternative energy biotechnologies, an area of critical scientific, technological and economic importance for the future.

Contribution to technology of alternative energy sources

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 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 or hydrogen. Research into the electron transport of Shewanella will increase our understanding of their capabilities in microbial fuel cells. At this stage, these are basic research aims, with academic beneficiaries. However, after successful completion of this project, we propose that our work will contribute to the future design of such microbial fuel cells, in particular where future work aims to genetically or synthetically modify the microbes to enhance electron transfer rates to the anode (i.e., increase electrical current). Furthermore, this BBSRC proposal explores a novel and innovative approach in which the natural electron transfer pathway is reversed. Instead of generating electricity by respiring hydrogen or a carbon source, we propose to use solar energy to produce hydrogen. Although the overall concept of this proposal is the harvesting of solar energy and the storage of energy (in the form of hydrogen), the fact that electricity can be used by microbes to make 'higher-energy' organic molecules, including hydrogen and a variety of hydrocarbons, is of major economic value. Microbes as catalysts are ideal as they are relatively cheap to make and maintain (i.e., they grow and regenerate). This makes them ideal catalyst to synthesise organic molecules, such as formate, using electricity and CO2.

Publications

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Ainsworth EV (2016) Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO. in Chembiochem : a European journal of chemical biology

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Hutton GA (2016) Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis with Redox Enzymes. in Journal of the American Chemical Society

 
Title Life, Light and Insight: Photographic Exhibition 
Description Researchers photographed in lab and one image chosen to represent research in the group was exhibited as part of an exhibition profiling Norwich scientists and their research activities. Exhibition was held in Blickling Hall, National Trust property just outside of Norwich. 
Type Of Art Image 
Year Produced 2014 
Impact discussion in public audience of technologies relating to solar H2 production and sustainable energy production in general. 
 
Description We have demonstrated a cell-based strategy to harness nature's intrinsic catalytic diversity for light-driven chemical synthesis in a non-photosynthetic microorganism. A water-soluble, membrane permeable mediator shuttles photoenergized electrons from abiotic photosensitisers to enzymes catalyzing H2-evolution and C=C or C=O bond hydrogenation. The predominant reaction is selected by the experimental conditions and the microorganism shown to act as a multi-faceted catalyst with a repertoire defined by its intrinsic, or genetically enhanced, complement of enzymes. This approach to light-driven chemical synthesis provides a versatile route to accessing a diverse but selectable product range because the mediator used has a well-established ability to deliver electrons to enzymes in numerous microorganisms. Furthermore our method has no requirement for protein purification, for which the associated costs and frequent loss of enzyme activity represent major challenges in delivering scalable, cost-efficient technologies exploiting the catalytic power offered by these remarkable catalysts.
We have demonstrated that water-compatible photosensitisers, both soluble dyes and nanoparticles, can deliver photoenergised electrons to purified extracellular cytochromes and membrane spanning porin:cytochrome complexes from Shewanella oneidensis. We have not yet been able to use these photosensitisers for spontaneous and direct, i.e. non-mediated, light-driven catalysis by enzymes of Shewanella oneidensis and this raises the question of how the extracellular matrix impacts on the ability of water-compatible photosensitises to access cytochromes presented on the external surface of these bacteria. In response to these findings we a) developed a modular approach to assembling functional outermembrane spanning electron transfer conduits in Shewanella oneidensis that we envisage that this will allow bespoke assembly of conduits functionalised extracellular photosensitisers to enable light-driven electron exchange between the intracellular and extracellular environments, and, b) quantified the catalytic properties of >10 Shewanella species with a view to subsequent exploration of their ability to support spontaneous, non-mediated light-driven catalysis.
Protein film electrochemistry has revealed new properties of the extracellular cytochrome MtrC. This protein served as a conduit for electron transfer between an electrode and redox enzymes with the MtrC redox activity regulating the direction of catalysis in a diode-like manner. MtrC adsorbed on mesoporous indium tin oxide electrodes catalysed H2O2 reduction at low overpotential at rates outperforming horse radish peroxidase.
Light-driven electron transfer across liposome bilayers was supported by porin:cytochrome complexes with water-soluble photosensitizers. However, photocatalysis using these reaction chambers could not be demonstrated and our research suggested that this was most likely to be because we were unable to achieve efficient encapsulation of our chosen electrocatalysts.
Exploitation Route Our results link fundamental science to a global challenge in studies that we believe to be of interest to a wide audience working in the fields of biocatalysis/catalysis, solar chemicals, microbial synthesis and photochemistry. The development of scalable and cost-efficient routes to light-driven chemical synthesis represents a major challenge in moving towards a sustainable society. Coupling the light-harvesting capabilities of abiotic photosensitisers with enzymes as electrocatalysts represents an attractive way to meet this goal. Abiotic photosensitisers can improve on Photosystem I and II as light-harvesting components through increased absorbance across the solar spectrum, effective charge separation and longevity. Importantly, abiotic photosensitizers also offer the prospect of enabling photocatalysis in non-photosynthetic organisms. We have demonstrated this possibility and described a cell-based strategy to harness nature's intrinsic catalytic diversity for light-driven chemical synthesis in a non-photosynthetic microorganism. A water-souble, cell-permeable mediator shuttles photoenergized electrons from abiotic photosensitisers to enzymes catalyzing H2-evolution and C=C or C=O bond hydrogenation. The predominant reaction is selected by the experimental conditions and the microorganism shown to act as a multi-faceted catalyst with a repertoire defined by its intrinsic, or genetically enhanced, complement of enzymes.

Our approach to light-driven chemical synthesis provides a versatile route to accessing a diverse but selectable product range because the mediator has a well-established ability to deliver electrons to enzymes in numerous microorganisms. Furthermore our method has no requirement for protein purification, for which the associated costs and frequent loss of enzyme activity represent major challenges in delivering scalable, cost-efficient technologies exploiting the catalytic power offered by these remarkable catalysts.
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 USC Tubulation of Proteoliposomes 
Organisation University of Southern California
Country United States 
Sector Academic/University 
PI Contribution Intellectual contribution, skills in (proteo-)liposome preparation and protein handling, purified proteins, training of research students and post doctoral researchers.
Collaborator Contribution Intellectual contribution, access to and experience with microscopies (white light, fluoresence, atomic force, transmission electron and cryo-electron) and measurement of the electrical conductivity of bacterial nanowires spanning interdigitated electrodes, training of research student and project supervisor in aforementioned methods.
Impact studies ongoing, too early for outputs
Start Year 2016
 
Description A-level demonstration 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact much interest in spectroscopy and forensic protein chemistry arose

A-level students noted more interest in science and protein chemistry to forensic sciences
Year(s) Of Engagement Activity 2013
 
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 BBC Radio Norfolk interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Broadcast Interview to highlight advances in understanding structures that conduct electrons through proteins. Impact was raised profile of our research and University.
Year(s) Of Engagement Activity 2014
 
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 Microbe Munch: A power for good 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Positive impressions formed of our research from those who read the magazine (most readers are unknown and scattered geographically so immediate feedback is difficult to gain'.

none known
Year(s) Of Engagement Activity 2013
 
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 On-line Media Coverage - Artificial Photosynthesis & MtrC photoanode 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interviews given by team members contributed to content of on line article featuring information on research achieved by BBSRC grant.
Year(s) Of Engagement Activity 2016
URL https://howwegettonext.com/sugar-batteries-and-fake-leaves-will-light-up-your-world-ef04dace9e83#.tl...
 
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 Press release; grant launch 
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 several on-line and print articles covered this news nationally and internationally

not known
Year(s) Of Engagement Activity 2013
 
Description Public Lecture (Forum, Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Approximately 40 people attended the lecture which was followed by a lively Q&A/discussion afterwards.

none I am aware of
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 RSC sponsored Science Investigation Day 
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 practicals sparked questions and discussion

pupils asked to learn more about my research at UEA
Year(s) Of Engagement Activity 2012,2013,2014
 
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 Spectroscopy in a Suitcase 
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 greater understanding of spectroscopy and practical 'hands on' experience.

discussion about a range of spectroscopic methods and their applications arose
Year(s) Of Engagement Activity 2012,2013,2014
 
Description Spectroscopy in a Suitcase 
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 greater understanding of spectroscopy and practical 'hands on' experience.

discussion about a range of spectroscopic methods and their applications arose
Year(s) Of Engagement Activity 2012,2013,2014
 
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
 
Description demo of light driven redox activity 
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 lots of discussion of artificial photosynthesis

interest in A-level science studies from GCSE students.
Year(s) Of Engagement Activity 2014,2017
 
Description guest speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact talk triggered discussion of how passionate science communicators/mentors influence the career choices of others particularly pre-PhD study

lots of discussion about my science and 'career' as model pathway
Year(s) Of Engagement Activity 2012