Ben Hankamer leads a group focused on examining a selection of proteins with potential application for bio-hydrogen production. His research as a biochemist and protein chemist at the Institute of Molecular Bioscience, University of Queensland in Brisbane, involves the Solar Bio-Hydrogen Project, single particle analysis, and 2D crystallisation of membrane proteins for electron crystallography. As part of this work, he has been exploring the use of a green algal system that uses solar energy to split water into hydrogen and oxygen with potential for large-scale production of hydrogen as well as water purification. He gained a PhD in structural studies on Photosystem II from the Imperial College of Science Technology and Medicine in London. His research as a biochemist and protein chemist particularly covers producing hydrogen fuel from water.

Solar-powered H₂ production from H₂O using engineered green algal cells
by Dr Ben Hankamer

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I would like to thank the Academy for giving me the opportunity to speak today. I also realise that I am between the audience and lunch, so I will try and make this brief.

I want to present to you the work we are carrying out through a consortium of researchers and industry. We have called this project the Solar Bio-H₂ Project, and it is aimed at using engineered green algal cells for hydrogen production from water.

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The underlying principle is the process of photosynthesis. You shine sunlight on algae, for example, and they use this process to split water, essentially into protons, electrons and oxygen. However, under anaerobic conditions many algae have developed a survival mechanism whereby they generate hydrogen instead of oxygen. It is this process that we are using.

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In the long term, what we would also like to do - is to try and couple this to water desalination. So if you use marine algae, for example, what you can do is to split the water into essentially the hydrogen and oxygen that comprises water, and on combustion through fuel cells you get fresh water back.

I realise this project focussed towards the long term, but this is also a symposium on long-term future directions.


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What we have tried to do, because it is an important problem and a difficult problem, is to establish a consortium of a wide range of researchers. So we have work being done, firstly, in the areas of molecular biology and the genetic screening of algae that are directed at identifying their ability to split water and generate hydrogen, to optimise the available biochemistry.


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This slide illustrates how we are working on the structural biology of the various complexes involved in catalysing these processes.


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We also have Professor Posten, in Germany, who has been involved in one of the largest bioreactor designs available for algae. Shown here is a two million litre bioreactor facility, with glass tubes which are filled with algae.

So we are really trying to cover all aspects.


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When we started this project in 2002, we really didn’t want to enter the project without having done some kind of feasibility study and analysis of whether this was something that was worthwhile doing. So in a sense, we have done some work that runs parallel to the types of work presented by George Crabtree this morning.

For example, we recently completed a study that looked at fossil fuel supply and climate change.

We also carried out a study on the underlying biochemistry of the process. Some questions that were specifically asked were: is this biochemical approach feasible?
What kind of efficiencies might be achievable? and, would the efficiencies that one can achieve, be economically interesting, if they could be achieved?

Finally, with the kind help of Thiess, we put together an industrial feasibility study on the whole process, whereby we looked at 10 parallel research streams to develop the biochemistry, engineering, molecular biology, and whole set of components that make up such a process. We then looked at the evaluation of the process and whether it could be economically feasible if the targets that we identified were achieved. The conclusion from this process was positive.


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I now just want to show you some of the highlights of this research.


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On this slide I have placed some of the various headlines that people can see about global warming and fuel supply issues. In relation to these topics, I will now take you through some of the more important results (as I see them), as well as the main constraints. The reason we did this research is that we wanted to get a sense of the time frame. For example, can we achieve this goal by 2050, or does it have to happen before this time;  and what are the constraints?

One interesting headline, from the World Wildlife Fund, came out just a couple of days ago and this made the suggestion that ‘mitigating climate change is affordable and achievable’. The estimation was that it would cost approximately $250 per person to achieve a 40 per cent reduction in CO2 emissions by 2030. I think that this is very encouraging. Indeed, I think often one is put off by all the difficulties, but in fact when one looks at all the costs of climate change, one has to ask whether this is an investment that is worth making. Clearly, this suggests that it is.


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This slide contains information that you have seen a couple of times this morning. So I am not going to dwell on it, but the point is to show the types of atmospheric CO₂ levels that we have had over the last 400,000 years.


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Here you see what has been happening over the last 150 years or so. We are currently at about 370 or 380 parts per million (ppm) CO₂ in the atmosphere, and the evidence that I am going to show you in a minute suggests that the safe limit is about 450 ppm CO₂. Again the reason this is important, is that it dictates the pace at which research developments and implementation need to move.


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So what I did, together with my postdocs and my group, was to trawl through the literature that we could find on this subject, and just try to extract some of the highlights.

At the moment, people are suggesting that we have about 33 per cent coral reef damage, and that by 450 ppm we can expect severe coral reef damage. Then at levels of 550 ppm we can expect a level of about 24 per cent plant and animal extinction and there will also be a number of other effects. By 650 ppm we are looking at something like an extinction rate of 35 per cent of plants and animals. Now I would just point to the fact that these are articles in Science and Nature, and that there are a whole heap of other articles that have come out in the last couple of years, which also suggest that these targets should be taken seriously. Thus when you look at these targets, you really come to the conclusion that perhaps the most responsible thing to do is to stay below a level of about 450 ppm.

So the two constraints affecting the management of this issue involve  oil supply and climate change, as we have heard this morning.


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I haven’t really come to a firm conclusion on oil resources. Respected scientists such as Ken Deffeyes, in Princeton, have predicted a peak of oil production at around 2000–2005; other people in the industry suggest peaking at around 2007–2038 or on average, about 2020. If you talk to some people in the mining industry, they say, “Oh, we’ve got heaps of coal and oil et cetera, and we can extend production for a long time beyond that with the development of new technologies such as injecting steam into oilwells to try and get thick oils out.” Of course, the question that arises is: At what point it is worth doing these things? - taking into account the cost of doing this, and  the energy requirements.

However, I think on balance that one can say that prices will go up with demand, and demand is expected to double by about 2025.

I would also say that although the development of new technologies will be driven by the issues of fuel supply and cost of fuels, the most limiting factor - to my mind, is climate change. When you look at the models developed by Hoffert, which were also presented earlier by George Crabtree, what you see is that if you take a business-as-usual model where you have about 2 to 3 per cent economic growth per year (which is what we would like to keep, roughly) and 1 per cent increase in energy intensity, energy requirements go up from about 13 terawatt in 2000 to 46 terawatt by the end of the century.

If we want to stay below the level of 450 ppm, which I would suggest is the environmentally responsible thing to do, then this will require a clean energy level of 11 terawatt years. This is almost the entire global energy amount that we are currently using at the moment, to be installed by 2025. I found this particular study to be important because it puts a kind of timeframe on the speed at which things should be directed. I also think that it is interesting to observe that a country such as Sweden has now decided that by 2020 they want to move to an ‘oil-free economy’. Certainly, they have clearly made a political decision that this goal is something they want to achieve.


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The next stage of the process that we carried out was to look at what clean energy supplies and renewable resources we could use to generate hydrogen. This diagram shows that solar is by far the largest clean energy source, compared to all the other energy sources that we have available. At the bottom of the diagram I have shown the world energy demand, at 13 terawatt years, as compared to 178,000 terawatt of solar energy that we receive annually.

We get 13,000 times more energy from the sun than we use, on an annual basis. So in this project, we are attempting to capture solar energy and convert it to hydrogen.


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Another interesting point about this technology is the fact that all other clean energy technologies are really focused on generating electricity whereas we are focused on using this for fuel production. The importance of this point is illustrated by the fact that only 33 per cent of our energy is currently used in the form of electricity, with 67 per cent used as fuels.

The process of producing fuel at the moment consists of three stages, although the second one shown here, in blue, is not absolutely necessary and ultimately would be something that we would probably like to avoid.

Essentially, you use the photosystem II complex to split water and generate protons, electrons and oxygen. Using the normal processes of photosynthesis you then store the protons and electrons by combining them with carbon dioxide, through the processes of photosynthesis and CO₂ fixation. This process produces something like starch which is a complex carbohydrate which is insoluble in water and is used by plants as a way to store excess glucose. Now ultimately you can then extract the protons and electrons from this starch and recombine them through a hydrogenase (enzyme) to generate hydrogen. This occurs under an anaerobic phase while the oxygen in the reaction is released under an aerobic phase.


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The two stages are depicted here. Now, I am not going to go through all these details, but here you see the photosystem II complex in the photosynthetic electron transport chain. It carries out the water-splitting reaction, producing atmospheric oxygen, and protons, and the electrons then get passed down the electron transport chain. During this process, the protons and the electrons ultimately become recombined to form starch. Under anaerobic conditions (shown in the lower panel) you can feed these electrons down the electron transport chain to a hydrogenase enzyme, and the protons can also be fed upward to generate hydrogen.


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Now I will cover some of the advantages of the system. One is the potential for targeting the chemical fuel cell market. Then there is the fact that, of course, plants assemble themselves, and so you don’t have to put in the considerable energy required to develop expensive solar capture systems that one would otherwise have to make in other ways. Furthermore, these systems absorb CO₂ in the process, which is a beneficial factor (especially in terms of the aforementioned climate targets).

These systems could also be coupled to water purification through the use of marine algae, and Australia of course has a plentiful supply of seawater. The other consideration that I think is an issue, particularly for Australia, is that if we examine the biofuels available (and while I am all in favour of them), there is the critical question of water supply in competing with crops. Fresh water is required to water all those crops, in order to produce those biofuels. By comparison, an advantage of systems that employ algae is that enclosed bioreactors can be used and these can be placed on non-arable land.

So the possibilities, as least as indicated by our feasibility study, are that one could, in the long term, achieve economic processes in this way, if one could achieve the associated scientific targets.

The current limitations that we really face at present  are biological efficiency and engineering challenges.


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I now want to show you some electron micrographs that were produced by my colleague Alasdair McDowall. Here you see here a cell of Chlamydomonas, which is a freshwater alga that we are using as our study system. This is the chloroplast, which carries out the reactions involved in generating hydrogen and oxygen from water. Pictured here is also the pyrenoid, which fixes carbon dioxide and generates starch, and out to the side you can see the starch molecules. The mitochondria carry out the respiration and normal processes.


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The structural biology, which is something that I am particularly interested in, is highlighted here.

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In this figure I have zoomed into a part of the cell where you have the thylakoid membranes and the photosystem II complexes sitting within this part of the structure. Using various processes of structure determination, we can now solve the structure of these proteins and try to analyse how they catalyse these reactions.


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So what we have done is to carry out random mutagenesis to try to develop a high-hydrogen producing mutant such as ‘Stm6’. We have also been working on identifying the biochemical pathways that drive hydrogen production.


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Those pathways are shown here. Again I am not going to go into all the details.


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What should also be highlighted is the fact that the hydrogenase itself is oxygen sensitive. This is the reason why this process is carried out under anaerobic conditions.


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So the first advantage of this mutant type of algae is that it has a modified cyclic electron transport process. Normally the electrons get passed from photosystem II through a linear electron transport chain and onto the hydrogenase - HydA. However, there is a competition with cyclic electron transport, and this is knocked out in this mutant - so now all the electrons go towards hydrogen production. We have also down-regulated photosystem II, and as a result we get less oxygen being produced, as shown here.


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Now in the case of the mutant alga, we have shown that there is also a much larger supply of starch, which means that we have an excellent store of protons and electrons. If you like, this can be fed into the hydrogenase enzyme. In the electron micrographs shown here, you can see the white densities that are starch, and which are shown in our high-hydrogen producing mutant. So the mutant has much higher levels of starch than the wild type.


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We have also generated an up-regulated alternative oxidase which absorbs oxygen, and as a result, maintains lower cellular oxygen concentrations. This results in an improved functionality of the hydrogenase itself. So here you have a trace level of oxygen for the mutant and much higher oxygen concentrations for the wild type.


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I am presenting this slide to show you some of the biochemistry that we have conducted here. These are the typical types of flasks that we use for small scale experiments. We can collect the hydrogen gas through a tube and measure the volume and composition of the gas present. We also have oxygen sensors and pH sensors; and we can inject various chemicals to modify the process through the side ports.

In some work, we have also taken the hydrogen directly off the top of these samples and used it to run a fuel cell powered car, without any other purification. (I can’t vouch for the quality of the fuel cell after we have done this a few times, but it does work!)


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One of the other points about what we have done, is that because we have modified the electron transport chain (and all the electrons move in a linear process), we have now up-regulated the rate at which electrons are donated to the enzyme - hydrogenase.

So we thought that perhaps the arrival of the protons from one side of the membrane to the other might be a limiting factor. Thus we included an uncoupler in the process which allows protons to move across the thylakoid membrane from the lumen to the stroma. By doing this in our mutant, we indeed obtained about a ninefold increase in hydrogen production over the wild type.


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In other developments, we have shown that in the long term, over say two weeks or so, we can obtain about five times more hydrogen production, but we think that with modification we can improve this even further. I would perhaps estimate that the numbers you probably want are around this level and that, at the moment, under lab conditions we are at 2 per cent efficiency at an illumination of 20W/m2. The challenge is going to be to run these systems at about 7 to 10 per cent efficiency to make it much more economically viable at external light levels.


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In my lab we also examine structural biology whereby we take pictures of molecules using an electron microscope, and this is done in much the same way as when a person goes into a hospital and a head scan is produced. You take pictures of the head from different angles, and then you can build a three-dimensional model of the brain. So in a similar way, we can do the same thing with molecules, by taking pictures of molecules at all sorts of different orientations and then building 3D reconstructions of the proteins. In this manner we can start to understand the organisation of the proteins in terms of their larger macromolecular assemblies in the cell, and how they drive the catalytic processes.


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Using this type of work while I was at Imperial College and before I moved to the University of Queensland, we used electron crystallography to solve the structure of the higher plant photosystem II core dimer. We also used the single particle approaches, which I have just shown you, to develop the outlines of much larger complexes. So these methods enable you to start building up a picture of how all these things fit together.

In its current form, the whole project is part of a much larger ‘Visible Cell Program’ which we have running at the University of Queensland. We are now using this project based on the Chlamydomonas cell to try to dock these types of macromolecular assembly structures back into cellular volumes of the types that I showed earlier.


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In summary, what I have shown you is that in the mutant we have comparable levels of hydrogenase, much higher levels of starch, lower cellular oxygen concentrations, and we have also blocked cyclic electron transport.

So in this system we now have a much more efficient electron delivery to the hydrogenase. We also have increased the level of hydrogenase activity due to the reduced amount of oxygen present in the cell. Thus overall, we have generated a greater amount of hydrogen.

We have also been able to extend hydrogen production times, due to the fact that we now have these high-starch mutants which are able to store a lot of the protons and electrons in the form of starch.


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So we completed our fuel cell and climate change study and have looked at the various bottlenecks in the process. We have also done the initial feasibility study with Thiess. Furthermore, we have characterised the high-hydrogen producing mutants and published these findings. So at present, the situation to my knowledge is that compared with leading labs like those of NREL, stm6 is one of the highest-hydrogen producing mutants (at least under controlled conditions). (They have some wonderful new developments in terms of the technology which we are hoping to couple together with our research.) Indeed, we have put this into a National Phase Entry now for Australia, the US, Japan and Europe, and we have also shown that we can power a small-scale fuel cell powered car. We have at least achieved this once or twice, although we haven’t yet trialled it more than on these occasions. So to proceed with these objectives, we have been trying to establish linkages with industry.


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Now I just want to leave you with an idea of some of the various designs that people are using when scaling-up bioreactors. At the top left of this slide you see an example of one of these systems by Clemens Posten (in Germany) where algae are cultured in flat plates. You shine light on them, absorb the light and collect the hydrogen.

In another version, by Mike Borowitzka, who is working in WA, he has actually coiled tubing around large cylinders and then the algae are pumped through the tubing. In this way you can significantly increase the surface area to volume ratio. The inset shows the green tubes filled with algae.

The third bioreactor, located at the right of this slide, is just slightly larger than the two million litre bioreactor facility that I showed you earlier. I actually think that bioreactors might look quite different from this in the future, for a number of different reasons that I don’t have time to go into, but I think it gives you an idea that a lot of the problems that we are facing in developing these technologies are beginning to be addressed.


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I would like to finish by acknowledging the contribution of many of my collaborators, particularly who are highlighted on this slide in green: Olaf Kruse, with whom I started up the project; my postdocs and my group; Alasdair McDowall, who generated the structures of the algal cells that I showed you; Peer Schenk at UQ; and Clemens Posten at University of Karlsruhe. I also acknowledge our industry partners and granting agencies.

Thank you very much.

Discussion

Cyril Appleby – I am a retired biologist who has grown any amount of non-photosynthetic and photosynthetic micro-organisms in my time. There is this terrible mess of getting rid of the spent materials. I think I heard you proposing that starch could be extracted and used, but what about the rest?

Ben Hankamer – In this case, no - the starch isn’t extracted. You have the algae and you grow them under aerobic conditions, and that allows you to build up the starch supply within the cell. You then put the whole culture under anaerobic conditions, and the starch is converted to hydrogen within the cell. At the end of the process you have a depleted biomass, compared with what you had at the start.

However, I take your point seriously with regard to the disposal of biomass. There are now gasification units that can deal with biomass gasification, and so one would deal with both the disposal of the waste and the extraction of additional hydrogen from biomass.

Oliver Mayo – To follow up on that point then: so you take the spent algae and you put them in an anaerobic bacterial fermenter. Is that it?

Ben Hankamer – No, I am sorry, I must not have made that clear. The algae themselves drive the hydrogen production, using the process of photosynthesis. You have two phases. One is where you have the splitting of water, and you generate the protons and electrons. The reason the starch is an important intermediate is that it is the proton-electron store. If you then transfer your algae into anaerobic conditions, those protons and electrons can be extracted out of the starch and, using this process of photosynthesis to then drive hydrogen production.

Oliver Mayo (cont.) – I understand that part of it. But your algae are going to keep reproducing and you are going to have more algae, and these algae will be present at different stages. Can you recycle them, or what?

Ben Hankamer – You can recycle them.

Oliver Mayo (cont.) – So you don’t, in fact, have a waste stream of algae under this system?

Ben Hankamer – A typical cycle for us in our lab, for example, runs at the aerobic stage for two days, and then in the anaerobic mode for two weeks or something like that, although this can be shortened, depending on the production efficiencies that you want. The NREL teams in the US have shown that you can recycle these and do it for about six months. I don’t know if you can do it for longer; I think they are probably stopped at this stage.

Les Field – I think you would agree with me that we have had four excellent talks in this session, ranging from hydrogen production by various means to hydrogen storage, and all the different ways that we can envisage hydrogen being utilised. Could you please join with me, firstly in thanking Ben for his talk, but also in thanking the rest of the speakers in this session.

Symposium program

Other speakers

Dr John Wright
Setting the scene: What is the hydrogen economy?

Dr George Crabtree
The two hydrogen economies

Professor Cameron Kepert
Hydrogen storage in nanoporous materials

Dr Sukhvinder Badwal
Fuel cells

Professor Andrew Dicks
Advanced nanomaterials for fuel cells

Dr Evan Gray
Hydrogen storage: status and prospects

Dr Catherine Grégoire Padró
Production of hydrogen

Professor David Trimm
Catalysis and syngas for the production of hydrogen

Dr Wes Stein
Making hydrogen from the Sun

Professor Harry Watson
Hydrogen car prospects