How E. coli produces hydrogen

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

Prokaryotes are the simplest living organisms on planet Earth. They include the single-celled bacteria and their cousins the archaea, which are the closest surviving examples of the earliest life-forms that ever existed. Many of these organisms can grow without oxygen, and instead utilise other chemicals from the environment to generate energy for life. Sometimes the chemicals used are unusual and bacteria can use one of the simplest molecules in the Universe to gain energy for growth; hydrogen. Moreover, a range of microorganisms, from photosynthetic algae to strictly anaerobic bacteria, can actually produce hydrogen as a by-product. For example, in the absence of oxygen some bacteria, such as the gut-dwelling Escherichia coli, grow by a process known as fermentation. This initially results in formic acid being produced, which is ultimately used by the cell to generate hydrogen gas. This process requires the action of a complicated enzyme called formate hydrogenlyase (FHL), which comprises at least seven different proteins together with iron, sulphur, nickel and molybdenum atoms. The activity of E. coli FHL was first described as long ago as 1932 by Marjory Stephenson, the first female Fellow of the Royal Society. In the years that have followed, no scientist has been able to isolate FHL in order to study it more closely. In this research, we now describe an innovative new approach that has allowed the purification of FHL for the first time. The overall aim of this project is to understand how FHL works at the molecular level, and modify this activity so it will be suitable for industrial applications. Biological approaches to hydrogen production (so-called 'biohydrogen') are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. Hydrogen has the highest energy per weight of any fuel, and its use (particularly in a fuel cell) is clean and efficient. At present 99% of hydrogen is produced by reforming fossil fuels and 1% comes from electrolysis, with most being used as a feedstock by the chemical industry. Most importantly, biohydrogen offers the prospect of FULLY RENEWABLE hydrogen, freed from any dependence on fossil fuel, and the scope for taping into this resource is enormous. The biochemistry of hydrogen production depends upon normally oxygen-sensitive enzymes known as hydrogenases. FHL contains a hydrogenase (the so-called 'Hyd-3' enzyme) that is responsible for all of the hydrogen produced by E. coli. The active site of Hyd-3 contains nickel, iron, carbon monoxide and cyanide molecules (which can be studied using the advanced spectroscopy available in Oxford), and is thus termed a [NiFe]hydrogenase. Indeed, we and others have proposed that the active sites of such hydrogenases are as active in hydrogen chemistry as platinum catalysts - an expensive and limited resource. Hyd-3 is rapidly inactivated by oxygen, and this may be a reason why its isolation has proven problematic for so long. Our recent studies of [NiFe]hydrogenases, together with that of others, has identified an important subset of enzymes that can function in air (so-called 'oxygen-tolerant hydrogenases'). These enzymes hold the key to technological developments of biohydrogen and we now have fresh insight into the molecular mechanism of their oxygen tolerance. Another important aim of this project, therefore, is to use this new knowledge to engineer oxygen tolerance into FHL. The Oxford and Dundee groups are superbly complementary. Dundee has expertise in studying the molecular cell biology of hydrogenases in E. coli, and Oxford has pioneered biophysical methods for studying hydrogenases, most notably protein film electrochemistry (PFE) and spectroscopy. PFE is the most powerful of all techniques for studying the properties of hydrogenases and has been instrumental in understanding the mechanistic details of their chemistry.

Technical Summary

Escherichia coli is a bacterium with a flexible metabolism and renowned genetic tractability that has established it as an important 'model', or 'chassis', organism for biotechnologists interested in genetically modifying metabolism, or even designing completely synthetic activities. E. coli can perform a 'mixed-acid fermentation' in which glucose is metabolised to ethanol and various organic acids, including formate. This formate is further disproportionated to carbon dioxide and hydrogen by the formate hydrogenlyase (FHL) complex. The scope for tapping into this activity is enormous since it offers the possibility of fully renewable biohydrogen, freed from any dependence on fossil fuel. Although the genetics and physiology of FHL are understood, the instability and oxygen-lability of this important enzyme have meant that it has never been isolated in an intact or active form. In this proposal, we describe an innovative genetic solution to this problem and demonstrate the isolation of FHL for the first time. Our isolation of FHL is a major breakthrough in the fields of bioenergetics, membrane biology, and bioenergy research. An intensive, fully complementary, and cost-effective program of studies is thus planned to address the molecular basis of biohydrogen production by E. coli. FHL is a membrane-bound multi-enzyme complex comprising a formate dehydrogenase and a [NiFe]hydrogenase. The Dundee/Oxford collaboration offers superbly complementary approaches to the characterisation of FHL. The Dundee group has internationally-recognised expertise in cell and molecular biology of E. coli hydrogenases, and in the biochemistry of bacterial membrane proteins. The Oxford group have pioneered protein film electrochemistry for studying hydrogenases, which has now identified the molecular basis of the oxygen-tolerance mechanism utilised by some [NiFe]hydrogenases, and are world-leaders in unravelling the mechanisms of complex metalloenzymes using advanced spectroscopy.

Planned Impact

In recent years, biological approaches to energy production (so-called 'bioenergy') are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. Hydrogen gas is among the most exciting of the current options for future energy needs. As the immediate product of energizing water by photolysis (sunlight) or renewable-powered electrolysis, or via biological routes (photosynthetic algae or 'dark' fermentation of waste materials), hydrogen is potentially the 'greenest' and most renewable of fuels. Currently, hydrogen is predominantly used as a feedstock in the chemical industry - but it is increasingly being used as a fuel. Hydrogen as a fuel is being championed by advanced countries, particularly through the governments of the USA, Australia, Germany and Sweden. Although the drawbacks of hydrogen are frequently aired (e.g. low energy density, storage difficulties, primitive supply and distribution infrastructure) these issues cannot be allowed to hold back its research and development, particularly in an academic context. 'Needs must' and hydrogen (including biohydrogen) will eventually become a dominant part of human lives and economies. Presently, 99% of hydrogen is produced by reforming fossil fuels. This approach is unsustainable and alternative, fully renewable, solutions must be found. This research project focuses on the mechanism of hydrogen production by Escherichia coli - the model organism of choice for biotechnologists the world over. Multinational energy companies, as well as innovative SMEs, will be greatly interested in the breakthroughs offered by this research. Industrialists who specialise in bioprocessing will also benefit from this work since we will be exploring methodology for the isolation of multi-enzyme, and membrane-bound, protein complexes. Biomedical companies will also be interested in this study of bacterial hydrogen metabolism, since hydrogenases are key virulence factors in pathogens related to E. coli - most notably Salmonella. Researchers interested in the emerging field of 'synthetic biology' should also be interested in this work. The principles of synthetic biology, some of which are applied here, can help overcome many hurdles in basic biological science research. This is the way forward for molecular biology. The staff trained on this project will be of immediate great interest to the industrial biotechnology and biomedical sectors. The project constantly applies electrochemistry to addressing biological questions, and this is a key modern technique at the interfaces of chemistry, physics and biology. Experts in this area will be in high demand, especially for companies interested in developing biosensors and other hybrid devices. Workers skilled in modern molecular biology, as well as membrane protein biochemistry, will also be in immediate demand. The Universities of Dundee and Oxford work hard to provide generic skills training in a broad spectrum of areas so that scientists are ready and able to contribute to the UK economy in whatever future career they so choose. Scientists at both institutions are exposed to a wide range of subjects, and the latest cutting-edge technology, so cannot fail to acquire both a strong work-ethic and broad understanding of the sciences. Overall, this project will benefit the nation's wealth as we move to a low carbon economy over the next 10-20 years. It will improve the nation's health over the same timescale as the use of hydrogen as a fuel increases (zero toxic or environmentally harmful emissions). It at will also place UK science at the cutting edge as the leading player in global biohydrogen research and development.

Publications

10 25 50
 
Description We have discovered, contrary to legend, that it is possible to purify and characterise the Formate HydrogenLyase (FHL) complex - a large bacterial enzyme. FHL can drive H2 production from formic acid and is therefore potentially one of the most important enzymes in biotechnology. We have published our purification and characterisation of this enzyme. We have also published the first mutagenic analysis of this enzyme and used modern synthetic biology approaches to engineer stability into this normally fragile enzyme.
Exploitation Route This enzyme could be used to understand the mechanism of H2 production by proteins - this will allow further harnessing of nickel-dependent hydrogenases for biofuel production. Understanding the reverse reaction (linking H2 uptake to CO2 fixation) will also interest climate scientists. We have secured NIBB funding to collaborate with industry on these ideas.


The University has an effective Research and Innovation Services Team that will be consulted with as appropriate.
Sectors Chemicals,Energy,Environment

 
Description This project is completed. Key papers on formate hydrogenlyase, which can also act as a hydrogen-dependent CO2 reductase, have been published. This has allowed public engagement activities to be designed in the bioenergy area and has attracted interest from industry, for example Ingenza LtD.
First Year Of Impact 2014
Sector Energy,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description Bioeconomy Accelerator
Amount £191,394 (GBP)
Organisation Zero Waste Scotland 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2018
 
Description C1net BBSRC NIBB
Amount £49,981 (GBP)
Funding ID POC-8-Sargent-C1net 
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 06/2016
 
Description Magnificent Microbes 2014 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a two-day event designed by academics and staged at Dundee Science Centre. Several interactive stalls were set up and these were used to stimulate discussions with the visiting public. The first day involved visits from selected primary schools from the East of Scotland. The second day was open to the public.

School children and their teachers were surveyed before and after the event on their knowledge and understanding of microbiology. There was a notable shift afterwards.
Year(s) Of Engagement Activity 2014
URL http://www.lifesci.dundee.ac.uk/impact/schools-outreach/media/magnificent-microbes-2014
 
Description Magnificent Microbes 2016 
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 This was a two-day event designed by academics and staged at Dundee Science Centre. Several interactive stalls were set up and these were used to stimulate discussions with the visiting public. The first day involved visits from selected primary schools from the East of Scotland. The second day was open to the general public. The media were invited and many interviews took place.
Year(s) Of Engagement Activity 2016
 
Description Magnificent Microbes 2018 
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 This was a two-day event designed by academics and staged at Dundee Science Centre. Several interactive stalls were set up and these were used to stimulate discussions with the visiting public. The first day involved visits from selected primary schools from the East of Scotland. The second day was open to the general public. The media were invited and many interviews took place.
Year(s) Of Engagement Activity 2018
URL https://www.dundee.ac.uk/news/2018/university-scientists-aim-to-inspire-with-celebration-of-life-in-...
 
Description Magnificent Microbes, Dundee Science Centre, 2016 
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 A two day event aimed at introducing the general public to the types of microbiology research that is happening at Dundee University. The first day - Friday 11 March 2016 - involved selected schools visiting interactive stalls. The local media (TV, radio, print media) were also present. The second day - Saturday 12 March 2016 - opened up the event to all comers. Hundreds of visitors enjoyed the event.
Year(s) Of Engagement Activity 2016
URL http://www.lifesci.dundee.ac.uk/impact/schools-outreach/media/magnificent-microbes-2016