Biohybrids for Solar Chemicals and Fuels: Whole-cell Photocatalysis by Non-photosynthetic Organisms.

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

Abstract

From news headlines to social media streams, we are constantly reminded of the need to help secure a healthy planet for future generations. The abundance of solar panels in urban and rural landscapes immediately illustrates how, nationally and as individuals, we are embracing technology to do this. Solar panels absorb sunlight and convert its energy to electricity. This helps to mitigate against climate change by lowering our use of fossil carbon reserves and harnessing sunlight is very sensible; in just two hours the sun provides Earth with sufficient energy to meet its present annual energy demand. However, solar panels are not without problems since they are constructed with toxic materials and suffer from intermittent output, which may not match patterns of energy demand. Developing more sustainable routes to harness the energy of sunlight is a global challenge. To help meet this challenge, and inspired by photosynthesis in green plants, we aim to combine the best of natural and synthetic approaches to solar energy conversion for the sustainable production of chemicals including fuels.

Photosynthesis is Nature's way of harnessing solar energy. Using abundant and non-toxic elements assembled as environmentally benign proteins, green plants absorb sunlight to drive the synthesis of fats and sugars. Amazingly, the only side-product of this reaction is the oxygen we need to breath. Fats and sugars provide fuels that we, like plants, can use for energy whether it is day or night, sunny or cloudy. Natural photosynthesis is a self-sustaining, renewable model for solar energy conversion but it is not without bottlenecks. The light-harvesting systems absorb only a small fraction of the solar spectrum and are easily damaged - during the day they are usually replaced every 30 minutes. By contrast, synthetic light-harvesting materials like those in solar panels, are more robust than natural photosystems. In addition 'rainbow absorbers' can harness much more of the solar spectrum.

Our research will combine robust, synthetic light-harvesting materials with non-photosynthetic bacteria in a powerful, sustainable solution to delivering complex chemical transformations. We aim to develop systems producing fuels, for example, ethanol (from a major underutilised by-product of biodiesel production), hydrogen (from water) and formate (from carbon dioxide a greenhouse gas). Importantly our biohybrids will simultaneously produce two different fuels but no side products so they are true mimics of natural photosynthesis in their chemical efficiency.

How will we do this? We will use detailed knowledge of the structure of a protein conducting electrons from the inside to the outside of Shewanella bacteria. This knowledge allows us to engineer the external surface of the Shewanella bacteria for selective labelling with light-harvesting electrocatalysts. Then, powered by the energy of sunlight, electrons will move between enzyme catalysts inside the bacteria and the synthetic catalyst outside the bacteria in order to couple the production of one fuel inside the bacterium and a different fuel outside the bacterium. Our approach allows us to use enzymes to deliver complex transformations without expensive purification that can result in fragile systems. By using bacteria there is also the possibility that the performance of our systems will benefit from natural processes of enzyme self-repair and regeneration.

Technical Summary

We propose to tap into sunlight, an underutilised source of clean power, and address the direct exchange of electrons between bacterial cells and inorganic photocatalysts for the biophotocatalytic production of solar chemicals including fuels. Current state-of-the-art technology, referred to as "bionic leaf", relies on the transfer of energy via intermediates such as hydrogen. By contrast, recent results from the applicants indicate that cytochromes purified from the extracellular respiratory machinery of Shewanella oneidensis MR-1 (MR-1) enable direct exchange of solar energy with synthetic photosensitisers. The MR-1 extracellular respiratory machinery exchanges energy/electrons across the bacterial outer membrane. To this end, we propose a novel synthetic biology approach in which bespoke photocatalysts are directly coupled to the extracellular cytochromes of MR-1 in vivo. Modular biohybrid assemblies will be produced that use intracellular redox transformations to sustain light-driven extracellular catalysis thereby closing the redox loop and enabling self-sustaining production of solar fuels. Two proof-of-principle light-driven reactions are proposed: (a) MR-1 catalysed reductions, such as protons to hydrogen or carbon dioxide to formate, coupled to inorganic photo-oxidation of industrially-relevant alcohols to aldehydes, and (b) MR-1 catalysed oxidation of glycerol (a major underutilised by-product of biodiesel production) to ethanol coupled to the inorganic photo-reduction of carbon dioxide to formate.

Planned Impact

Societal Impact:

Renewable energy is recognised as a top national strategic priority (UK White Paper on Energy). Several incidents have demonstrated the fragility of the global energy supply such as 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 GLOBAL importance. The Paris Agreement in 2015 has set out a global strategy to minimise the impact of climate change by reducing greenhouse gas emissions. A solution to this problem has to be sought by combining a multitude of complementary 'alternative' energy sources; this research will contribute to this progress. Specifically, we aim to use synthetic biology, protein engineering, (bio)nanotechnology and chemistry to develop bacterial biohybrids that harvest solar energy for novel and innovative approaches to produce value added chemicals and fuels. For example, we will explore MR-1 oxidation of glycerol to ethanol and CO2 coupled to external light-driven inorganic reduction of CO2 to formate. This carbon-neutral reaction photocatalysis would add considerable value to glycerol, a low-commodity chemical, through the production of two fuels. In the US, approximately 1 kg of crude glycerol is produced for every 10 kg of biodiesel which equates to the production of approximately 0.8 x 10^9 kg of glycerol in 2016 .

Technological Impact:

The state-of-the-art in solar-driven microbial catalytic systems, 'bionic leaf' technology, first produces hydrogen and, in a second step, uses this hydrogen as an energy vector to drive downstream bioproduction of higher value compounds, e.g. reduction of CO2 to fusel alcohols by Ralstonia. This proposal presents fundamental research that aims to advance the state-of-the-art by directly coupling the photocatalyst and microbial catalytic systems. We envisage that successful completion of this project will demonstrate proof-of-principle for a disruptive technology contributing to the future design of hybrid bacterial-inorganic (photo-)catalytic systems for chemical conversions including those requiring NAD(P)+/NADPH recycling.

Conjugation between inorganic materials and biomacromolecules has wide-ranging relevance to technology, including bioenergy (as proposed here), health technology, e.g. drug delivery and the development of novel probes for cellular localisation and trafficking. We envisage that our research will also be of immediate impact in the development of emerging technologies for electrically interfacing living systems and abiotic materials. In this area, our research could impact the development of artificial vision and light-dependent sensing/signalling pathways.

Impact through Collaborations and Training:

This project will consolidate the recently formed partnership between Butt, Clarke (UEA), Jeuken (Leeds) and Reisner (Cambridge). The project will extend the partnership to Gralnick (USA), who will bring expertise in engineering Shewanella for targeted biotransformations, and the Molecular Foundry (USA), who will bring expertise for the analysis of protein-nanoparticle conjugates. Within this project we will also provide top-quality cross-disciplinary training for three BBSRC PDRAs and a research technician, to provide expertise in the development of alternative energy biotechnologies, an area of critical scientific, technological and economic importance for the future.

Publications

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Butt J (2023) Protein film electrochemistry in Nature Reviews Methods Primers

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Piper SEH (2022) Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors. in Angewandte Chemie (International ed. in English)

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Van Wonderen JH (2021) Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme. in Proceedings of the National Academy of Sciences of the United States of America

 
Description This work is inspired by photosynthesis. It aims to combine the best synthetic absorbers of sunlight with biology's catalysts to provide more sustainable routes to chemical synthesis. Molecular dyes are more robust than natural absorbers of sunlight and we have succeeded in attaching one such dye to a protein creating a biohybrid photoenzyme. When exposed to light, the purified photoenzyme accepts energised electrons from the dye. We have i) demonstrated that our purified photoenzyme is a robust light-driven reducer of azo-dyes, major contaminants of waste-water from the textile industry and printing, and ii) engineered our photoenzyme to stabilise the photoenergised electron and are now assessing the impact on catalytic performance. Furthermore we have developed a method allowing our photoenzyme to associate with the surface of bacterial cells. The association forms a complex allowing electron transfer across the bacterial outer membrane. Ongoing experiments aim to establish whether bacteria carrying the photoenzyme support light-driven electron exchange between redox partners inside and outside the bacteria.
Exploitation Route We have incorporated outcomes of our research through public engagement. An interactive display was delivered at the Royal Norfolk Show and Norwich Science Festival. Due to the pandemic, planned face-to-face activities with local schools were cancelled. We have used Instagram and Twitter to publicise activities. A presentation to the Oxford Energy Society shared research from this grant with PhD students and post-docs from a wide range of different disciplines.

Skills training of the UEA PDRA, technician and PI our developing through regular discussions with the Leeds and Cambridge based PDRAs and PIs.
Sectors Education

Environment

Manufacturing

including Industrial Biotechology

 
Description Our findings have underpinned public engagement activities which have informed the general public about our research and led to a change in their thinking about biotechnology and the opportunities for using bacteria for sustainable production of electricity and chemicals including fuels. Our findings have also allowed us to secure further BBSRC funding in partnership with Johnson Matthey to explore our photosensitised-cytochromes as photocatalysts for recovery of valuable metals from 'waste streams'.
First Year Of Impact 2022
Sector Education,Manufacturing, including Industrial Biotechology
Impact Types Societal

Economic

 
Description BETTER BY DESIGN: CHEMICALLY DIVERSE BIOMOLECULAR WIRES
Amount £188,675 (GBP)
Funding ID RPG-2020-085 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Creating and comprehending the circuitry of life: precise biomolecular design of multi-centre redox enzymes for a synthetic metabolism
Amount £4,900,000 (GBP)
Funding ID BB/W003449/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2022 
End 11/2027
 
Description EPSRC iCASE Studentship 'Tuneable bioelectronics - Manipulation of bacterial proteins towards biodegradable, inducible, designer electronics with bespoke functionality'
Amount £42,000 (GBP)
Funding ID Voucher Award 240066 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2024 
End 09/2028
 
Description Engineering Biology Hub for environmental processing and recovery of metals; from contaminated land to industrial biotechnology in a circular economy
Amount £14,881,000 (GBP)
Funding ID BB/Y008456/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2024 
End 02/2029
 
Description Norwich Biosciences BBSRC DTP PhD studentship
Amount £12,000,000 (GBP)
Organisation John Innes Centre 
Sector Academic/University
Country United Kingdom
Start 09/2020 
End 09/2024
 
Description Tuning extracellular cytochromes for enhanced metal recovery and nanoparticle formation
Amount £290,637 (GBP)
Funding ID BB/X011453/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2023 
End 01/2025
 
Title Liquid-Chromatography Mass Spectrometry Describes Post-Translational Modification of Shewanella Outer Membrane Proteins 
Description LC-MS data for MtrCAB complexes from Shewanella oneidensis MR-1 and Shewanella baltica OS-185.LC-MS data for a soluble form of MtrC from Shewanella oneidensis MR-1 carrying a C-terminal Strep II tag. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://figshare.com/articles/dataset/Liquid-Chromatography_Mass_Spectrometry_Describes_Post-Transla...
 
Title Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme using pump-probe spectroscopy. 
Description Data from spectroscopic, electrochemical, voltammetric and computational studies as presented in van Wonderen et al 'Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme'. Data presented in the Main and Supporting Information Appendix are included. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
URL https://figshare.com/articles/dataset/van_Wonderen_et_al_PNAS_2021_Data_Sets_xlsx/16621714
 
Title Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme using pump-probe spectroscopy. van Wonderen et al 
Description Spectroscopic, electrochemical and voltammetric data desribing properties of photosensitized MtrC proteins. The data are presented as figures in van Wonderen et al 'Nanosecond heme-to-heme electron transfer rates in a spectrally unique His/Met-ligated heme', the manuscript and supporting information appendix. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
URL https://figshare.com/articles/dataset/Nanosecond_heme-to-heme_electron_transfer_rates_in_a_multiheme...
 
Title Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors Piper et al 
Description Data from spectroscopy and gas chromatography with corresponding plots of analyte concentration with time. Data from dynamic light scattering and mass spectrometry (LC-MS) 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
URL https://figshare.com/articles/dataset/Photocatalytic_Removal_of_the_Greenhouse_Gas_Nitrous_Oxide_by_...
 
Title Research data supporting "Size-dependent activity of carbon dots for photocatalytic H2 generation in combination with a molecular Ni cocatalyst" 
Description Data for publication, including UV-vis data, H2 evolution data, IR spectra, and PL spectra. The data set is organised by Figures and each folder contains a specific readme.txt file that provides detailed explanations. Publication abstract: Carbon dots (CDs) are low-cost light-absorbers in photocatalytic multicomponent systems, but their wide size distribution has hampered rational design and the identification of the factors that lead to their best performance. To address this challenge, we report herein the novel use of gel filtration size exclusion chromatography to separate amorphous, graphitic, and graphitic N-doped CDs depending on their lateral size to study the effect of their size on photocatalytic H2 evolution with a DuBois type Ni cocatalyst. Transmission electron microscopy and dynamic light scattering confirm size-dependent separation, while UV-vis and fluorescence spectroscopy of the more monodisperse fractions show a distinct response which computational modelling attributed to a complex interplay between CD size and optical properties. A size-dependent effect on the photocatalytic H2 evolution performance of the CDs in combination with a molecular Ni cocatalyst is demonstrated with a maximum activity at approximately 2-3 nm CD diameter. Overall, size separation leads to a two-fold increase in the specific photocatalytic activity for H2 evolution using the monodisperse CDs compared to the as synthesized polydisperse samples, highlighting the size-dependent effect on photocatalytic activity towards H2 evolution. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://www.repository.cam.ac.uk/handle/1810/357252
 
Description transient absorbance at RAL 
Organisation Research Complex at Harwell
Country United Kingdom 
Sector Public 
PI Contribution Initiated the collaboration, provided the samples for measurements, assisted with data collection, performed the data analysis and interpretation.
Collaborator Contribution instrument development necessary to perform the desired measurements (time resolved absorbance pico- to micro-second resolution)
Impact Multidisciplinary collaboration: biochemistry, physics, computation. One paper published from the collaboration (van Wonderen et al J. Am Chem Soc 2019 DOI: 10.1021/jacs.9b06858) Data collection and analysis for a second publication is complete. Manuscript writing will start soon.
Start Year 2017
 
Description Institution for Engineering and Technology (IET) Savoy Place, London (Feb 2023) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Lecture followed by Q&A with members of IET London. These were delivered/led by myself in collaboration with Prof. Yannis Ieoropoulos (Univ. Southampton). The Q&A activity revealed great interest in our research and its potential applications. It was also clear that the audience were hearing about microbial fuel cells and approaches to green electronics for the first time - it was clear that many of the audience left with a renewed appreciation of biotechnology for electricity production from materials often considered as waste.
Year(s) Of Engagement Activity 2023
 
Description Interactive Display on our research as part of the 'Norwich Science Festival' at the Royal Norfolk Show June 2019 
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 Interactive displays on energy transfer, energy for life, electroactive bacteria as biobatteries, proteins that form molecular wires, new approaches to artificial photosynthesis.
All displays were available for use by the general public. Questions raised by, and discussion with, the visitors were wide-ranging and allowed us to describe our research in great detail and in the context of contributions to novel biotechnology for energy and chemicals.
Year(s) Of Engagement Activity 2019
 
Description Norwich Science Festival 
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 >200 members of the general public attended our interactive display as part of the Norwich Science Festival in the city centre location. Participants helped create a biofilm of novel bacteria, learnt how synthetic biology can be used to design bacteria with new, desirable properties for sustainable production of electricity, medicines and toiletries. The activity sparked much discussion and insightful questions.
Year(s) Of Engagement Activity 2021
 
Description Pint of Science Festival - Norwich Virtual Event 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The group created an Instagram site and posted pictures to illustrate features of our research. The site was launched and the postings made live to as part of a week of Pint of Science reponse to the pandemic. We have subsequently attracted an undergraduate from UEA's Natural Science programme to help us develop the site as part of their Science Communications project.
Year(s) Of Engagement Activity 2020
URL https://www.instagram.com/juleab_lab/
 
Description Solar Chemicals Webinar - Using Sunlight to Build Molecules 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Webinar hosted by Royal Society of Chemistry. A panel comprised of Theme Leads from EPSRC-funded UK Solar Chemicals Network discussed talking points raised by pre-recorded interview with experts from research and commercial labs.
Year(s) Of Engagement Activity 2023
URL https://www.rsc.org/events/detail/77684/solar-chemicals-using-sunlight-to-build-molecules
 
Description SynBio 4 Schools Teacher Resource Pack 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Developed by OpenPlant scientists from across the University of Cambridge, Norwich Research Park and beyond, a free to use resource, SynBio 4 Schools, aims to inspire and educate the next generation of biological engineers. With a focus on plant science the materials and activities provided within the resource cover under-represented topics in synthetic biology, while complementing the national curriculum and existing teaching resources. The SynBio 4 Schools booklet, which includes a full introduction to the resource, step by step activity instructions and supplementary information, is also accompanied by a teacher briefing document to help teachers and schools, identify activities relevant to each stage of the curriculum. This research project contributed an introduction to the biomolecular protein nanowires that allow electricity production by bacteria growing on root exudates. SynBio 4 Schools was officially launched by video conference in Feb 2022 to an international audience. Materials were developed from 2019 and the planned in person launch was delayed and finally postponed due to COVID-19.
Year(s) Of Engagement Activity 2019,2020,2022
URL https://www.openplant.org/synbio-4-schools
 
Description Talk for Oxford Energy Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Attendees joined a virtual seminar hosted by the Oxford Energy Society. The presentation sparked questions and discussion on a range of topics indicating engagement with participants from a range of different disciplines.
Year(s) Of Engagement Activity 2021
 
Description Work experience for 2 college students 
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 Two students from different schools joined the research of our group for 2 weeks. They shadowed researchers doing various types of experiments. They learnt about research in the molecular biosciences and synthetic biology and how this can inspire improved biotechnology.
Year(s) Of Engagement Activity 2019