 Good afternoon. My name is Ellen Mantis. I'm the Director of the Chemical Sciences Roundtable at the National Academies of Sciences, Engineering, and Medicine. For those of you that don't know anything about the Roundtable, the Roundtable provides a neutral forum to advance understanding of issues of importance to the chemical sciences. And we look to promote exchange of information among all the different sectors, government, industry, academic, and other. Normally, our activities are workshops that we have during the year, but this year for 2020, we are excited to launch a series of webinars on emerging topics. And this is the first of our series. And today's topic is inorganic biohybrids. The format for today will be two presentations, one by Dr. Catherine Brown from the National Renewable Energy Laboratory and the other by Daniel Nezera from Harvard University. Their biographical information is on our website, so you can read more about them there. After the presentations, we will have a moderated question-answer session with the speakers. Mark Jones, who is a member of our Chemical Sciences Roundtable, will moderate that discussion. And to ask a question of the speakers, you simply submit your question via the chat feature. To do so, click on the chat icon at the bottom of the Zoom screen and type in your message, either to everyone or to Mark Jones. And Mark will ask questions on behalf of the audience. So with that, I'd like to turn it over to Dr. Brown, who will lead off the presentation. Good morning, everyone. Sorry, getting the technical details set. So hopefully everyone can see a full screen version of my slides now. Good morning. I'm Dr. Catherine Brown. I'm a researcher in the Redox Biochemistry. Kate, sorry for interrupting. Yes. We see your presentation view. Sorry. It's the correct view. No. No. Is that the correct view? Yes. Thank you, Kate. Thank you. Sorry, everyone. So I'm Dr. Catherine Brown. I'm a research scientist at the National Renewable Energy Lab in the Redox biochemistry group. And I'm excited to tell you today about our work and the work of other colleagues in semi-artificial photosynthesis. And really, the work that we do is trying to use biohybrids to as model systems for understanding the fundamental processes of photo catalysis and photosynthesis. So we are really focused on basic science of how these processes work. And just to start off, I'd like to talk a little bit about what we mean when we say artificial photosynthesis, because in the most general sense, we mean systems that use water oxidation to generate high energy electrons. But that can encompass an enormous amount of variety in terms of the kind of devices and science that's going on. There's just three examples of many. So in the top left is a system where you have water oxidation and fuel generation, in this case hydrogen production, occurring at separated electrodes that are then wired together. At the bottom, this is sort of a classic artificial synthesis take. It's a scheme that was first designed or proposed by Art Nozick in the 70s, where it's really reproducing the energy scheme that you see in photosynthesis of this offset between the water oxidation catalyst and the fuel producing catalyst where they have these mesh energy schemes, which allows you to up convert the energy and produce high energy electrons. And then over on the right, we have Professor Nozara's work where he's taking this concept of splitting water and wiring it up to biology to create this artificial photosynthesis that's really connected to the biology that already exists. And obviously I'm not going to talk a lot about that because he's going to cover it later. So really the point I'd like you to take home from this is that there's this enormous diversity in the concept of artificial photosynthesis. And if you Google this in Google Scholar, you'll get 350,000 results. So there's an enormous diversity in what we call artificial photosynthesis and the kind of science that's going on. Given that diversity, I'd like to take a step back and look at the natural system one more time to give grounding and how we in our labs see photosynthesis and see the processes that we can learn from in the system. So I'm sure most of you are familiar with how photosynthesis works, but I'm going to go through it very briefly. So we have light absorption at photosystem 2 that generates a highly oxidizing hole that allows you to drive water oxidation. And those electrons are then processed through the membrane through the plasticinone pool to put the cytochrome B6F to sign into PS1. And at PS1 you have absorption of another photon, which creates a high energy electron that then gets transferred to ferrodoxin and onto ferrodoxin NADP reductase to produce these reduced electron sources for the cell in the form of NADPH and then reduce ferrodoxin. The other aspect of photosynthesis that I'd really like to point out here is that in addition to this moving of electrons, you also have the generation of this proton motive force, which is linked to ATPase and is another way that the cell uses photosynthesis to maximize the amount of energy it's getting out of this process. So in addition to moving electrons and creating these high energy reduced products, you're also creating this proton motive force, which gives you the cell an additional source of energy. And what this really means to me is that photosynthesis is more than just water splitting. It's actually a finely tuned system for moving charges and creating energy for the cell on a higher level than just water splitting. So if we look at photosynthesis from a different perspective, and we think about how this charge transfer is happening, you can see that both photosystem one and photosystem two are designed to move charges to create long lived charge separated states. And if we look at the work that's been done summarizing the time scales of these, you can see that each charge transfer step is extremely fast. So within a millisecond, in both cases, you get charge separated states that have extremely long lifetimes and allow the cell to do a lot of chemistry. And the other thing that this system creates is extremely high quantum yield for the system. So photosystem one, for example, has a quantum near 100%. So that means almost all of the electrons that are absorbed by photosystem one result in a reduced product. And that's a system that artificial systems have yet to match photosynthesis, particularly photosystem one is extremely efficient. And that's an element that we can really use photosynthesis photosynthesis to learn about how to generate artificial systems that can begin to match this. And in our group and in our colleagues groups, the way to do this is with bio hybrids, or the way to study this is with bio hybrids. So there's one more element that a photosynthesis that I want to talk about before I get into the specifics of our work and colleagues works. And that's that photosynthesis in terms of this on the cellular level is about more than just photosystem one and photosystem two and the way they move these charges. There's this enormous diversity of chemistry is that happened downstream of photosynthesis. This links in with Professor Sarah's work as well, in that we're really interested just as much in all of this rich diversity of downstream chemistry that you get from out of photosynthesis, as we are in the charge transfer within the system as well. So, by creating these three energy sources NADPH reduced ferrodoxin and ATP the cell can then do an enormous diversity of chemistry so you have reductive chemistry and dehydrogenases oxidor reductases, and so on. You have all of the biosynthesis that goes on within the cell is linked to these energy carriers nitrogen and sulfur assimilation and then redox metabolism carbon metabolism all rely on these energy carriers produced by photosynthesis. And really the point that I want to bring out here is that in artificial systems often you're talking about linking water oxidation to a single fuel product. Hydrogen or an alcohol, some fuel product that we're going to use within our energy infrastructure, but the cellular photosynthesis creates energy carriers that can do an enormous range of chemistry. And that provides a flexibility that would be really great to be able to reproduce an artificial systems. And that brings me again to bio hybrid systems. The first very briefly I'm going to talk about some of the work, looking at how you can link up PS one to catalysis to make bio hybrids and study PS one. So this is just two examples there are others, but the gold back group that Penn State they were able to wire photosystem one to artificial to catalyst so they've in here they're showing a nano crystal, but you've also had. They've also been able to link this to enzymes and to catalysts and do some really fundamental work looking at the how fast electrons can move through photosynthesis and what kind of catalysis you can drive by directly linking that. And then a group at Argonne National Laboratory did a similar work but instead of using a molecular wire to connect the catalysts to photosynthesis that photosystem one. They absorbed platinum nanoparticle and they've since continued this work using other catalysts as well to drive light driven catalysis. And this is really allowed some fundamental work on looking at the spectroscopy within PS one and understanding how charges move between those energies. So that's some primers on some simple ways you can use natural systems in bio hybrids. Now I'm going to move into the work in our lab, which is much more focused on this side of things looking at the downstream chemistry that can happen. Using the high energy electrons and to do that we've replaced photosystem one with an artificial system to generate high energy electrons. And I'm going to start off with quite a bit on our work on hydrogenases and then later in the talk I'll tell you a little bit more about some of the other work that we're doing. So the system that we use is semiconductor nanoparticle enzyme bio. And what this is is linking the charge transfer and charge generation light capture properties of nanocrystals to redox enzymes. So as in photosystem one and photosystem two, when you excite a semiconductor nanoparticle you you promote an electron to the conduction band from the valence band and you then have this high energy electron which can be transferred out of the nanoparticle. To species and solution. And in the case of our work, those species are enzymes so we can transfer this electron into an enzyme. And these enzymes have their own internal charge transfer networks, and that allows us to drive catalysis with light, and then the hole that remains behind we use a chemical donor to regenerate regenerate the system and allows us to do this again. So the first question you might ask is how are we forming these bio hybrids and getting stable structures and we do that by mimicking the biological electron so shown here is the hydrogenase that we're working with and it's typical cellular redox partner which is ferred oxen. And these are electrostatic maps of the enzyme and in red is negatively charged and in blue is positively charged and you can see there's this match between the negatively charged ferred oxen and the positive patch on the enzyme which allows ferred oxen to deliver electrons into these iron sulfur clusters which can drive catalysis. So to mimic this, we use negatively charged nanomaterials, which have liggins on the surface which create a highly high density negative charge, which then absorbs to these positive patches in the same orientation that ferred oxen binds and allows us access to these iron sulfur clusters and promotes electron transfer that results in catalysis. And we've done a number of biochemical studies looking at how these nanoparticles can block ferred oxen binding to confirm that in fact we're binding at this site and so if you bind a nanoparticle, if you add nanoparticles to a solution of enzyme and then try and drive the catalysis with ferred oxen, you don't get any ferred oxen driven catalysis so we're fairly certain that we are mimicking this biological electron transfer step and binding the nanoparticle at that site of biological electron injection. And then this is just a cartoon of how we envision the whole system so we have a nanoparticle bound to the enzyme and you get electron transfer through all of these iron sulfur clusters to the active site which then drives catalysis. So the next question you might ask is does it actually work can we actually drive catalysis with light and the answer is conveniently yes. So here is a grass chromatogram of the product formation so this is measuring hydrogen in the headspace of a reaction vessel. So we have a small uptake as the machine is sampling the headspace and then we'll turn the light on you can see we get this linear production of hydrogen. You turn the light off again, it goes flat and then you get hydrogen production again. So we can in fact drive hydrogen production fairly efficiently using these bio hybrids. So the question then becomes alright, so we can make this work what are we going to do with this and the question I get asked frequently is, you know, could we turn these into devices and generally the answer is probably not because one thing to note is that these hydrogenases are not simple to make it's quite a labor intensive process. And they're also quite oxygen sensitive. So what we really envision these bio hybrids for is they are wonderful tools for understanding the fundamental mechanisms of photo catalysis, electron transfer and enzyme mechanism that really allow us to ask some interesting and fundamental questions about what's going on. And I'm going to give you some examples of the kind of work that we've been doing. So first, we've been able to use these systems in conjunction with our collaborators at University of Colorado. Gordana Dukovic's group to look at quantifying the electron transfer kinetics. The efficiency of these systems is going to depend in large part on how well we can get the electron from the nanoparticle into the enzyme. And we can study this using ultra fast transient absorption spectroscopy so I'm not I'm not going to go over the details of this but essentially when you excited an electron into this conduction band. It can either be transferred to the enzyme or it can recombine and reform the ground state of the system. And we can look at the kinetics of those different processes how much recombination occurs how much electron transfer occurs by looking at the nano scale nanosecond scale time resolved spectra of the nanoparticles. And what's shown here is the that those kinetics as we increase the amount of enzyme presence so you can see as we increase the enzyme, the decay of this curve gets faster and faster meaning the electron is being transferred faster and faster. And Gordana Dukovic's group has done an enormous amount of modeling to understand this data on a really detailed way. And our collaboration has generated a lot of really interesting information about the overall efficiency of photo catalysis and how these internal processes of recombination and electron transfer can drive the efficiency of photo catalysis and the overall product formation. And this provides useful information for how to engineer artificial systems and the kind of catalysis you're going to want kind of catalyst you're going to want an artificial system and what properties of a light absorber you want in order to maximize this electron transfer versus this recombination because these competing reactions are present in any system artificial or natural. And we've been able to tease out some fundamental insights into how to engineer these systems to optimize catalytic rates. So another advantage of these bio hybrids is they give us some unique tools to play with the system and drive catalysis. And one of those is the ability to modulate the size of the nanoparticle to change the potential of the electron so the potential of the conduction band is dependent on the size of the nanoparticle and as you get larger, the energy of the electron goes down. And why is this interesting for us. Well, biology, in general, operates within a very narrow potential window. Essentially, you have the potential of pherodoxin, you have the potential of NADPH, you have the potential of NADH and there's some, there's a little bit of wiggle room but the vast majority of enzymes operate within this very narrow potential window. And that is in stark contrast to a large number of synthetic catalysts which require much higher driving forces because they don't have the advantage of the enzyme 3D structure to reduce the energy of intermediates and flatten out the potential landscape. And what bio hybrids can allow us to do is ask some fundamental questions about how those that enzymes operate within that narrow potential window. And this work, as an example, we were able to use four different sizes of nanomaterials with four different potentials, which, if you think about the Marcus electron potential calculations where the rate of electron transfer is going to depend on the potential difference between the donor and the acceptor, the distance and the coupling constant, you expect the rate of electron transfer to decrease as you decrease the potential of the electron. And what we found in the case of our bio hybrids is that in fact all four sizes and all four potentials had essentially the same electron transfer rate. And this is because of the tuning than the enzyme. So we were actually able to make some novel insights into how the enzyme flattens that potential and allows it to take electrons of different potentials and transfer them into the enzyme at essentially the same rate. And that provides again some design principles for how you would want to design an enzyme to allow you to use lower energy electrons. This also has the advantage of, if you look at the spectrum of these nanoparticles larger nanoparticles absorb much more of the visible spectrum which this covers roughly 300 to 800 nanometers. So if you can tune catalysts to allow you to have fast electron transfer even with low energy, low potential electrons can actually use more of the solar spectrum which will give you more efficient artificial systems. So we've also been able to use these systems to investigate the mechanism of enzyme active sites. So again, this is our cartoon of a nanoparticle enzyme biohybrid. And this is a rendering of the active site of hydrogen is and this active site serves as a model for hydrogen catalysts because the this enzyme can catalyze hydrogen production very quickly and very efficiently. But there's still quite a few mechanistic questions about how this reaction works and how the enzyme modulates the intermediates. And one of the things that bio hybrids has allowed us to do is have time resolved spectroscopy of the active site changes. So this plot here is a three dimensional plot of time and wavelength showing the changes in the active site and I'm not going to get into the details of the infrared spectroscopy and what signal goes with what in the active site. But what I want you to notice that we're seeing real time changes when we turn on the light, and we're able to actually track the kinetics of individual species of this active site. The other advantage of these bio hybrids is in contrast to how you typically do these kind of experiments of studying the redox state of an enzyme. Usually you'll take the system and you will poison the reduced state or poison in the oxidized state, chemically. So that's you can't watch it change. But what we can also do with bio hybrids is because this electron transfer is not relying on diffusion, we can actually lower the temperature and still be able to inject electrons in. And that means we can slow down these catalytic processes and watch catalysis, which can still go on at low temperatures if you can deliver an electron, and actually tease out a few more details by being able to do them at low temperatures which is a unique capacity of the bio hybrids that we're able to do. So I've told you very brief overview of a lot of the work that we've done with hydrogenases. Now I'd like to switch gears and talk a little bit about some of the work we've done, looking at NADPH and linking it to some of the reductives chemistry that So what we were able to do is take Faridox and NADP reduxase, which has again been shown on all of those photosynthesis pictures as the final step in electron transfer and replace Faridoxin with this nanoparticle to drive electron transfer into the Flavin active site and reduce NAD plus to NADPH. So here you can see this is a fluorescence signal of NADPH, it's accumulating, you can see we can accumulate, the longer we illuminate the more NADPH we can accumulate. And we were then able to take this system and put it into a solution with an alcohol dehydrogenase and actually generate a fuel product, in this case isobutenol from isobuteraldehyde recycling NADPH and we were able to show that not only do we produce NADPH from illumination but that we were actually able to drive this cycle repeatedly and and regenerate NADPH have it used again and then regenerate it. And then showing that you can couple biohybrids to biofuels production situations and really mimic some of the downstream properties of photosynthesis and advantages of photosynthesis. So finally, I want to finish up telling you about our work with nitrogen reduction. So we've, we spent a great deal of time and effort along with our collaborator. To understand the enzyme nanoparticle hydrogenase system and really get some mechanistic insights. So our next challenge was to say, what would happen if we tried to use the same kind of model system in a much more challenging multi electron reaction and in this case, we focused on nitrogen production. So industrial nitrogen fixation is a really essential product process. There have been estimates that half the people alive on earth today are the result of are able to be alive because of the Haber-Bosch process, which takes nitrogen in the air and reduces it to ammonia which is then used in fertilizer. So this is a very energy intensive process. It has to be done at very high temperatures and pressures in order to crack that very stable nitrogen triple bond, and it's been estimated that it uses up to 1% of global nitrogen. So I said how it's estimated that half the people are able to be alive because of the Haber-Bosch process. So what about the other half that comes from biological nitrogen fixation. So if you've ever heard that certain plants can enrich nitrogen in soils, if they're planted after other crops, it's not actually those plants, it's the bacteria in their root nodules, which are able to do biological nitrogen fixation and produce ammonia naturally in the ground. And in contrast to the Haber-Bosch process, that biological nitrogen fixation can happen at ambient conditions, meaning room temperature, room pressure, no need to concentrate the nitrogen and put it at high pressure. But the cost of this is that it's got a very high biological energy demand. So in addition to the need for eight electrons to crack an N2 molecule, you also need 16 ATP molecules. So this is for a cell, this is an enormously energy intensive process. And these ATP are essential for driving electron transfer into the nitrogenase that actually does the chemistry of reducing nitrogen to a molecule. So our question was could we make a biohybrid that replaces all of this energy intensive side of things with a photocatalytic light driven process. And the short answer is we were successful in doing this. We were able to couple a nano rod to nitrogenase and drive nitrogen reduction. We were able to achieve about 70% of the maximum rate for this protein, this Mothi protein. And essentially, one essential point is that we were able to show that this chemistry was actually happening at the iron molybdenum active site of nitrogenase by showing that the classic inhibitors that are known to stop this process and the enzyme still inhibited the process. So we were able to show that this was actually occurring at the active site of the nitrogen. So just briefly, I'd like to show you some of the ongoing work we're doing in our lab. So one essential element of nitrogen, nitrogenase activity is that in addition to producing ammonia, you also produce hydrogen and it's actually essential to the mechanism of the enzyme. But the amount of hydrogen that you produce depends on the reaction conditions, and you can under certain conditions you can get more hydrogen and less ammonia produced which reduces the efficiency. So obviously this is very important for understanding our photocatalytic system and we're not currently, we're still building a picture of how that works. We've been able to do some work showing that the light intensity that we use, i.e. the number of photons hitting the system within a given amount of time actually affects the nitrogen, the amount of ammonia and the amount of hydrogen made. And we measure this using N15 labeled gas so that we're getting very accurate measures of the amount of ammonia being made. And we have some results showing that as you increase the intensity of the light, you get more ammonia and less hydrogen, meaning that you're more efficiently driving electrons into ammonia production versus hydrogen production, which is increasing the efficiency of the system overall. And then we're also doing work on reaction mechanism. So you can see this is quite a complicated proposed mechanism. There's a lot of individual steps. Using these biohybrids to look at the accumulation of these intermediates as each electron transfer occurs. And just briefly, we've been able to show spectroscopic evidence of moving through these different states and you can see changes in the spectrum, new peaks coming in, the main peaks are getting shorter. And we're able to make some mechanistic insights into what's happening in this reaction mechanism as we shine light on the system and move it through these different states. So I'd just like to finish up by giving you a brief overview of some of our colleagues and the amazing work that they're doing in biohybrids. So Brian Dyer's group at Emory University, they use time-resolved infrared spectroscopy and they're actually using a mediated system where they can reduce hydrogenase using a mediator generated at the nanoparticle. And they're able to make mechanistic insights and they've built these very beautiful maps of how hydrogenase moves through different states using biohybrids. So the Ryzen group at Cambridge, they work also with nanoparticle biohybrids and they've been able to link these nanoparticles to CO2 reducing enzymes and make a format from CO2 and study some of the processes involved. Our collaborator, Gerdana Dukovic, has this very recent in the last couple of weeks published this lovely paper where they were showing carbon-carbon bond formation. You're able to drive carbon-carbon bond formation from CO2 using a nanoparticle enzyme hybrid. And then finally, a little bit different, more like the first set of work I showed using photosystem 2. Several groups have been able to link photosystem 2s to catalysts to drive catalysis. And then at Aria National Laboratory, Lisa Utsig's group actually was able to take their earlier work on just PS1 and place it within the context of the entire thylakoid membrane and drive catalysis linked to water splitting within the thylakoid membrane. And finally, I'd just like to thank the people who contributed to this work, the Redox Biochemistry Group at NREL. Paul King is our group leader and the PI of these projects. David Kara, along with myself, are co-PIs on these projects. Mike was a scientist with our group who left us to join the Peace Corps and he did a lot of the IR studies that we shared and then Bryant Chickas currently a postdoc intergroup working on the nitrogens. We also have a number of wonderful collaborators, Gordana Dukovic's work with our group almost from the start on all of our hydrogenase work. The Nitrogenase Group is a large group, including our group, the Dukovic Group and Lance Seafelt at Utah State and John Peters at Washington State. And then we also collaborate with Alex Gao at Carnegie Mellon on spectroscopy, and then we have a number of wonderful collaborators at NREL who have worked on these projects at various points. And then, most importantly, I'd like to say none of this work would have happened without the support of the DOE Office of Science, Basic Energy Sciences. The hydrogenase work was supported by Photosynthetic Systems and Solar Photochemistry and our Nitrogenase work is supported by Physical Biosciences and Solar Photochemistry together. And none of this would be possible without their support. And thank you all for attending this lecture. Thank you so much. We're going to move on to Dr. Nassara. Just a reminder to our audience to please remember to type in your questions in the chat feature so that we can have a vibrant discussion at the close of Dr. Nassara's presentation. So I'll turn it over to you now, Dr. Nassara. Hello. Hi, you're on. Yeah, I said, could you see my presentation? Oh, no, we cannot. Okay, presentation. Just wait one second. Sure. I guess. So, first it's fine to present it. It's on the desktop. Oh, there it is. Wait one sec. A little problem. Would you like me to pull up your slides from my end? Get back to the... Maybe. Do you like get back to the program? Okay, I got it. Can you see it? We can. Yes. Thank you. So I'll talk about artificial photosynthesis sort of the way we just brought up. And what I'm going to show is using just sunlight, air, and water. You can almost make anything. And actually greatly exceed photosynthesis. I'm going to talk about photosynthesis more in a systems engineering way. And that is, and this is what I think was Kate did a really great job explaining this, but my field forgets this, that when you do photosynthesis, the plant only uses sunlight to split water to hydrogen and oxygen. It then takes, and that means the energy of the sunlight is in the rearrangements of water to basically make fuels. Oxygen and hydrogen and nature is form of hydrogens, NADPH. It then takes that hydrogen and in the dark, the so-called Calvin Benson or dark cycle combines it with carbon dioxide to make biomass. And so what I want you to concentrate on, at least for the talk that I'm giving, is sunlight is used to split water. The biomass, think about whether it's polymers or wood or sugar, you name it. That's not energy storage. That's just simply a form of hydrogen storage. That's how nature said, I can't deal with H2 as a gas. And so what I'll do is store hydrogen with carbon dioxide in the form of biomass. And so water splitting is energy storing, biomass is hydrogen storing. The reason for that simple thermodynamics, water splitting is 1.23 volts uphill. So that's how you store the energy of sunlight. Once I have the hydrogen, and I'm showing five products here, I could show thousands, literally millions of products. And CO2 plus hydrogen, and in this instance, I've split my hydrogen up. So this is three moles of hydrogen in the first reaction, 4, 9, 12, 15 moles of hydrogen. Once I have hydrogen combined with carbon dioxide, it's thermonutrile or downhill. So we took that lesson from photosynthesis and said, we're going to do a two-step process. First, we'll figure out how to use sunlight to split water to hydrogen and oxygen. Then in the dark, we'll figure out some way to then take the hydrogen with CO2 and make biomass. And that's basically the inorganic biological hybrid approach. The inorganic materials chemistry catalysis, that's on the light side, the one I'm about to talk to. And then the biology, that then needs to get interfaced with the biology, which then will run the dark reaction, which is H2 fixation with CO2 to make biomass. I'm not going to go into, this we published, these are well-known catalysts, and I published them eight years ago. The last four years actually really spent a lot of time thinking about mechanisms. But the bottom line is we invented, this is just one of these catalysts. It's a cobalt phosphate catalyst. It's inorganic. When it gets, it gets charged on it. So initially we just plugged it into the wall and took electricity out of the wall. It splits water to oxygen. It leaves four protons or left behind. And then we made two catalysts. These are two shown here, a nickel, molybdenum zinc alloy or a cobalt phosphorus alloy that could then take the electrons and protons and make hydrogen. There's two important things about this catalyst. One of these catalysts, that's one is there's no sun. So I got to figure out how to get sunlight into them. But another thing which we spent a lot of time doing, these are the first self-healing catalysts, meaning as long as these catalysts are operating, they never die. And we, with DOE support, have figured out a lot about how to generate self-healing catalysts. But they never die as long as they're doing catalysts. And the bottom line is they self-assemble. And the energy for self-assembly is less than the energy for catalysis. So as long as they have enough energy to do catalysis, they always have enough energy to self-assemble. So if the catalyst degrades, it has enough energy to reform itself. And a simpler way to say this is as long as these catalysts are operating, a self-healing catalyst has a turnover number of an infinity. It can never die. Why did we spend so much time making self-healing catalyst? One is we can use literally any water source. So we can run this out of pure water, which is how most water-splitting catalysts operate. We can run it out of a puddle on the ground. We can run it out of the Charles River. We can run it out of seawater. We can run it out of urine. And a lot of this science was dedicated to getting energy to the poor. And if I'm going to do something for the poor, I can't say I need 18 million to own pure water or concentrated base to do water-splitting. So that was important to us. But for the purposes of this talk, when you're in neutral water, it's easy to stabilize materials. And so remember, I don't have any light in the picture yet, so I'm going to have to interface these catalysts to light absorbing materials. And then, if I'm in neutral water and buffered waters, that allows me to interface the biology. So the key to this whole front end was self-healing catalysis to interface the materials. Just here's the first sort of path we took. And this is using a triple-junction silicon cell, that inner wafer of silicon. There's three layers. That's engineered. We took amorphous silicon. That absorbs at 400 nanometers. And then we introduced germanium into the strained amorphous silicon lattice. That changes absorption spectrum. The more germanium you dope into amorphous silicon, the red ship that you can go. So we dope enough germanium to get that middle spectrum shown here. And then we dope even more germanium in and get the bottom spectrum. And we engineered the germanium such that that three wafer system absorbs top, middle, bottom. And that spectrum absorbs the total visible absorption spectrum of solar light. You then have to protect the silicon. And we did that with ITO. We initially now use FTO. You put the catalyst on top, the water splitting oxygen catalyst. You put the hydrogen catalyst on the bottom. When sunlight goes into the system, you get one unit of charge separation. Just like in photosynthesis, you need four units of charge. When you charge up the top catalyst with four holes, it makes oxygen. When you charge up the bottom catalyst with four electrons, it makes hydrogen from the leftover protons from the water splitting above. So the key here, so this is called a very junction. It was the first very junction. It's totally wireless. It's only coatings. The beauty of this is it's modular. You can replace the inner absorber with any absorber you would like. You can use different catalysts on top and bottom. So it's a modular design. There's a lot more work to be done in this area for people who are interested. You could use organic photovoltaics. You could use perovskites. That's something I want to do with NREL. You can use different catalysts. But the key here is the solar piece of photosynthesis we can greatly exceed. So this version of using the cobalt catalyst with the nickel catalyst, Cassandra, who's now working at DASF, she achieved solar to hydrogen efficiencies of 12.8%. So that's a true efficiency. We take energy of sunlight in. We look and see how much hydrogen we made. We know how much energy is in the hydrogen we made. And you just divide one by the other and you get a true efficiency. And that's 12.8%. So what that means is on the light side of the reaction, which I now have darkened out, we have a very high solar to hydrogen energy conversion efficiency. We then said, how do we take that hydrogen, combine it with carbon dioxide? And we went back to photosynthesis. And this is what you just heard. You've heard the whole photosynthetic chain. So photosystem 2 splits water to oxygen. There's electron transfer relays in between that send the electrons over to PS1. And then there are catalysts there to make hydrogen. And like I said, nature stores hydrogen as NADPH. What I just told you is I can replace that entire part of the photosynthetic membrane. I just get rid of it because I can now use sunlight to split water to make hydrogen at very high efficiencies using sunlight. What I can't get rid of, however, is how do I take the hydrogen and then somehow get it to combine with CO2? So there's a large field trying to do this chemically. We said we'll hijack biology and now interface the inorganic water splitting to a biological organism. And where I need to enter then is that NADPH. So I need to convert my hydrogen to NADPH. Once I have NADPH, I can drive an ATPase. This is John Walker's Nobel Prize. And once I have NADPH and ATP, I can drive CO2 fixation. And so that's here. We'll take hydrogen. Somehow I need to get it into a bio organism that breeds in carbon dioxide and then I'll hijack biology. So here's a schematic. I'm going to take photosystem one and two in the membrane, get rid of it because I have water splitting with very junctions. But now how do I get hydrogen into an organism? And the easy way to do that is to use a hydrogenase. And so hydrogenase enzymes, you just heard about them, they take H2 to two protons and two electrons. Once I have two protons and two electrons, I can backend it to an NADPH reductase. Once I have that, I can basically follow the biological pathway with ATP synthase and NADPH and ATP. I can run carbon fixing cycles. So that's what we did. We took bacteria, ralstonia, eutropha. You can overexpress if you would like. Hydrogenetically, you can put hydrogenases in the membrane. And once you do that, it enables you to wire inorganic chemistry to biology. So we did that. There's different ways you can do it. You can use the artificial leaf directly. You can use a photovoltaic and then wire up your catalyst. They make the hydrogen and the bugs eat the hydrogen directly. The important point here is the only food source for this bacteria is hydrogen. Because of the way they're designed now, they can't eat anything else. So if they don't have hydrogen, they go dormant and die. If they have hydrogen, they can live. And just like any bioorganism, they can two go to four or four go to eighth. And you can go into an exponential growth cycle. So you take a pinch of bugs, these ralstonia, eutropha. You load them up with their hydrogenases and then you start feeding them hydrogen and they grow and you grow biomass. And it's all in the form of lipids. Most of the bug is mostly lipid and you can grow a lot of biomass. One of the students who did this is China Liu and I believe he's actually participating in this seminar series. So the other thing China did is he said, once I'm at acetyl coenzyme A, I can take the ralstonia eutropha and you can add different genes. And these genes deliver different enzymes in the cell. And just like you heard, the enzymes can carry out chemistry. So we put these four genes in the cell. One makes a ketoviolase, one makes a transferase, a decarboxylase and a dehydrogenase. And we chose those four genes because they do this chemistry. So a ketoviolase cuts the acetyl coenzyme carbon bond and makes a carbon-carbon bond to transferase, hydrolyzes it, the decarboxylase, decarboxylase CO2, and then you hydrogenate. And so if you have this ralstonia eutropha with its hydrogenases and then these four genes, it makes isopropanol. You can do different biosynthetic pathways. And we did. And you can make, that's the C3 I just told you about. You can make C4 and C5s. We've made, this isn't published yet. I can make C8s. So I can make gasoline. If I start with propanol coenzyme A, there's three carbons. I add two carbons at a time. You can make C11s and 13s. You can make diesels. So you can engineer. And this is just using the tools of synthetic biology. I can engineer different biosynthetic pathways. I want to come back here. And this is important and talk about efficiency because the literature is a bit of a mess. There's lots of papers and science in nature. People taking nanowires and putting bugs on them. And there's a bunch of biohybrid stuff. And they talk about quantum yields and faradaic efficiency. And they'll say it's 90 or 100%. And then you see the paper in nature and science. But that's not the whole story. And the reason I say that is that's not energy, a quantum yield or a faradaic efficiency. So faradaic efficiencies is electrons into product made. That's not a true energy efficiency. If my electrons go in at 20 volts, which is most of these papers because they're keeping constant current, they let the voltage swing and a typical potential stack to maintain voltage from 20 or 30 volts. So putting electrons in, which is C, faradaic efficiency. And I'm doing that at 30 volts versus 1.23, which is thermodynamic efficiency. Then I take a hit of a factor of 30 in overall energy efficiency. So these are true energy efficiencies I'm talking about. We look to see how much hydrogen we made at a voltage. And that gives you an energy for that hydrogen and electrical efficiency because we take the energy of the electrons to make hydrogen and then we divide it by the amount of material we made times the heat of formation. So that's energy of product over energy of electrons that made hydrogen. And in there is the solubility of hydrogen and everything. It's electrons to hydrogen. I know how much energy is there. And then I say how much energy did I make in my product? That gives an electrical efficiency. If I use a 20% PV, I take the electrical efficiency times 0.2 and it gives me a true energy efficiency. So one thing this field needs to start doing is talking about true efficiencies, not just faradaic efficiency or quantum yield. If you do that with the system I just told you for growing in biomass, we hit 10.8% with a 20% typical PV driver. So that is well over natural photosynthesis. The best crops grow at 1%. So the biomass we're growing, these bugs are growing at 10.8%. You can make isopropanol at 8%. We're making C5s at 5% efficiency. So that's sunlight in to product made. This purple bar I want you to concentrate on now. This is a different type of biomass. That's polyhydroxybutyrate. So this biomass is in liquid. The red and blue bars are in a fuel. That's the energy we're counting. The polyhydroxybutyrate is a biopolymer that's stored inside the cell. And so like I told you now, we've converted hydrogen to a solid biopolymer inside the cell. So that's the first part. This is an organism that fixes carbon and you can artificially greatly exceed natural photosynthesis. In my opinion now, nobody should be growing crops for biomass. They should be doing this. And then you can take your biomass and meet up with your favorite chem engineer and turn out into a biofuel. And now you can put a factor of 10 in front of all your calculations because most calculations use 1% soy or switchgrass. But as you heard in the last talk, another big hydrogen fixing need is nitrogen to make food. So what we did here is, and this is the trick of polyhydroxybutyrate, which I'm indicating with these white balls of plastic inside the organism. Now what we did is we took a carbon and nitrogen fixing bacteria. And so by doing that, I can take water splitting, fix it with CO2 to make a biopolymer, PHB. And then the organism has nitrogenases in it. The nitrogenase can get its hydrogen from PHB like I told you biomass is for hydrogen. It can then also degrade the PHB to get energy, ATP. And I can drive a nitrogenase cycle. So this organism now is a different organism. It's a xanthobacteria. It's carbon and nitrogen fixing. And it's a three-step process. Split water to make hydrogen. Have blood like I just showed you take CO2 plus hydrogen and make PHB, solid biopolymer. And then you draw on the PHB to drive nitrogen fixation to make ammonia. And that is important because as you heard, nitrogenases to fix nitrogen, it's extremely energy-intensive. So most natural organisms down-regulate nitrogen fixation because it's so exhausting to the cell. But with this trick, I made my cells super energetic. They don't down-regulate nitrogen production. And so the bottom line is after they fix water, they make the polyhydroxybutyrate. They draw on that and run nitrogen fixation. We prove it's all the nitrogen's coming from air. We use N15 like you saw in the last talk, and we see all the ammonia's N15 label. But the really incredible thing here is these bugs per cell are having a turnover number of nitrogen of three times 10 to the ninth because they're so energy-rich, they don't down-regulate nitrogen fixation. That's a lot of ammonia per cell. Remember, my cells don't need sunlight anymore. I preloaded them with sunlight in the form of PHB. I can put the bugs in the ground. They begin drawing on the PHB. They breathe in nitrogen and I can grow crops. So here what I'm showing you over here on the left with no bio-fertilizer. This is just out of fertile soil. We get radishes. But then I can put my bugs in the ground and I can get radishes. And I'm getting increased crop yields of 150% because I now have a natural renewable fertilization method. The story's a little better than I'm telling you because the soil I grew these radishes, which are 1.5 times bigger than those, I didn't grow it out of fertile soil. The gray bars, I inoculated the soil, took all the nutrients out, and then put the bugs in the ground because what the bugs are doing are they're renewably re-energizing the soil with carbon and nitrogen. One thing I didn't tell you about is if you put them against the natural waste stream, they'll also fix phosphorus in terms of polyphosphate. So now I have a way to take phosphorus out of waste stream, take carbon and nitrogen out of air, I'll driven by sunlight and be new for five soils. To end, I'm going to show you our latest crop and I don't do chemistry anymore. I guess I become a farmer. So what I did here is this is a 400-acre farm that grows lettuce and corn, and I took bugs that were energized with PHB. They're a lean. I've only energized these bugs at 5% to 8%. We now are up to 25% so I can get rid of... Let me just do this experiment. I took synthetic fertilizer. It's called UAN, urea ammonium nitrate. For growing corn, typical corn per acre, you use 130 pounds of nitrogen per acre. So nitrogen per pound of UAN. I replace half of the fertilizer with my bugs and I get the same crop yields. And so that's important because, remember that means I'm not fixing nitrogen with methane, that's how you do Harbor Bosch. So I save that CO2. And after the bug degrades the PHB, it takes the hydrogen out of the polymer. It leaves carbon in the ground. So first, I'm carbon neutral. I'm using hydrogen, not from methane. I'm saving that CO2. And I'm carbon negative because the PHB, which, remember, is being fixed at 10 times natural photosynthesis, meaning 10 times the rate of natural photosynthesis I'm putting in a cell. After the cell degrades the PHB, it leaves the carbon in the ground and it sequesters it. So this is true data from my farm trial. For that corn crop trial, I saved, by replacing half the fertilizer, I saved 109,000 pounds of carbon dioxide from going into the air. And after I degrade the PHB, I knew how much PHB is in the cells I put in the ground, I end up sequester. I pull or suck 16,000 pounds of carbon into the ground. So that's for a 400-acre farm trial for growing corn. It would use 26,000 pounds of nitrogen, I'd say. And that's what the carbon footprint is. So the important point here is if you start combining fast-growing agriculture the way I'm talking about using a biohybrid approach with water splitting, which is how you get the solar light into the system, you can be carbon neutral in fuels and you're actually carbon negative. And this is a way to do massive carbon fixation, I believe. And you can grow crops with beneficial yields. We're very happy about this because this means I can do both fissure tropes and harborbosh in a distributed way using only sunlight, air, and any water source. And that's how I believe the future needs to be, especially for the poor parts of the world. To end, I just want to tell you this approach is totally general. Synthetic biology is growing by leaps and bounds. So not only can you make fertilizer in fuels, you can make polymers, plastics, you can make drugs, you can make starches. There are seven out of the 10 drugs on the market are biological now. I've been invited by the CEOs of three top pharma companies. They are now realizing this is a way they can renewably make drugs by interfacing solar water splitting via hydrogenases into their organisms. So this guy went to Harvard for only one year and I heard he was stuck on Mars. So when you're going to Mars, you have sunlight, you have urine, you have CO2. You could literally start thinking about making things this way. And actually NASA is interested in this. So that's it. To end this, I have to tell you, I never fit into any program. So none of this was federally funded. It was done, and I know a lot of gratitude to my friends, Tom Steyer and Kat Taylor. All this research was funded privately by them. And I hope now this method gains traction in the federal agencies will get interested in it. So with that, I'd like to thank you for your attention. I'm done, Jessica. Thank you. Thank you, Dan. This is Mark. This is Mark Jones. I'll step in. How are you guys? Everybody can hear me? Good. Okay. So we have had some questions come in. Please continue to type questions in the chat window if you have questions for Catherine or for Dan. And I'll try to get started here. I'll start with Catherine. There was a, you mentioned the quantum yield near 100% in photosystem one and things like 70% of maximum biological yield. There are a couple of questions coming in about the, what the efficiencies mean here. When you define that, can you give a little more color to what that, what those efficiencies mean? Sure. So when we talk about quantum yield, what we usually mean is for every photon that's absorbed, how much product are you getting out? So in the case of hydrogen, for example, every molecule of hydrogen that we produce requires two electrons. So that's two photons. So if you were getting 50 molecules of hydrogen for every 100 electrons absorbed, you would have 100% quantum efficiency. In reality, you're limited by the electron transfer rate, the recombination rate of the nanoparticles. So that's generally what I mean by quantum yield. To take, move up a level and talk about some of the efficiency of photosynthesis. That's actually a topic of ongoing debate that happens at NREL a lot because we have, you know, a great diversity of folks. So we have electrochemists and physical chemists who will tell you that biology is extremely inefficient because photosynthesis is usually 1% to 3% in some engineered crops. You get about 7% efficiency, meaning every, for every photon absorbed, you're only getting 7% out of electrons. But what I think, what Professor Nisera's work shows, and I think if you look at it from a different perspective, biology is inefficient because it's not interested in giving us what we want. It's interested in staying alive and growing. And so you have to look at quantum efficiency from a slightly different perspective, which I think Professor Nisera's talk showed very nicely that you can get around some of those challenges. But when you're talking about efficiency on a biological scale, you need to think about the fact that these plants and animals are looking to stay alive and reproduce and grow, not give us what we want. So efficiency depends on what perspective you're looking at. Yeah, could I jump in? Yeah, the DOE organized the conference, a little workshop actually eight years ago now, but it was run by Bob Blankenship. And he has physical chemists like me and Art Nozikin and lots of biologists and scientists and just for the people listening in, the paper was published in Science in 2011. Its volume number was 332 and page 805. And that paper is a consensus paper on photosynthetic efficiencies leading to crops. And that's exactly what Kate said. Biology didn't know it was supposed to grow up and be oil wells for us. So for two billion years of evolution, it had to go in a different direction. But if you want to find out what really is happening in an efficiency for both C3 and C4 products, that paper is pretty definitive and people put a lot of time in it. And I think there's consensus in the community that that paper is accurate and that's what people use for plant growing efficiencies. And if people who are on the line want to click, Jake Eston has just typed that link into the chat window so that you can click on Dan's work and get to Dan's work immediately. That's not my work. It's a group of work. It's that paper. But I do want to say you can get around it just like was mentioned by Catherine and biology didn't know what was supposed to solve our world's problems by growing plants two billion years ago. So do you have a couple of questions coming in? I'll start with you, Dan. Where do you draw the line? What are the efficiencies that are needed to make biohybrid's actually industrially competitive with fossil fuels or for fertilizer? All right. So the fossil fuel thing, there is nothing any human being is ever going to do that's going to displace fossil fuels when you can poke a hole in the ground, take them out of the ground. That's over 90 years of infrastructure that's all paid off. You will never, ever, ever be competitive unless you do carbon pricing. So anybody who thinks they're going to be competitive commercially making fuels versus putting holes in the ground in a paid off infrastructure with no price on carbon, you will have all failed futures. It's just no way to be competitive. So that's why I didn't try to commercialize the carbon piece. Someday, if you price carbon, this isn't, you can make fuels this way, but you're going to need incentives with carbon pricing. On the fertilizer piece, it's already competitive. This is what I'm talking about. It's already getting commercialized and there are countries all over the world now interested in this. It's an interest, I know there's some USDA people on the line. This is an interesting fertilizer because by definition it's non-chemical. I never put nitrogen in the ground. I put some carbon in the ground. I put some phosphorus in the ground. But from a nitrogen point of view, this is a non-nitrogen fertilizer when it goes in the ground. It starts making nitrogen from the air. So this is kind of an interesting approach to think about fertilization. The cost targets are already competitive. I'll just leave it at that. And again, sticking with you for a moment, you only replaced half in the data that you showed. Can you explain the reason why you didn't can't completely replace with your end-year organism? Yeah. So one is I told you I made a lean bug. It was only 8% loaded with PHB. When it finally runs out of its own internal energy supply, it starts down regulating because it says now I'm going to get exhausted and die. If we go to 25%, which we can do now, I can go all the way down to a replacement of 90%. I still need 10% because the bugs need to get kick-started. And when you first, this gets into growing crops, like I told you, I'm slowly becoming a farmer and learning all this, you need immediate nitrogen in the form of nitrate. So we need a kick-start with a little bit of nitrate. But when they're super fat, I can replace up to 90% of the chemical fertilizer. That's important because EPA is starting in their regulations in California right now looking for, is there a way to replace 50% and finally, that's on the books, I believe, some legislations in the work because of harmful algal blooms. And we're already at 50. I think we can top out at 90 with a kick-start of nitrate. At the end of the day, I can probably get rid of the nitrate because I can formulate some nitrate-fixing bacteria that make nitrate quickly and eat the ammonia from my bug and make nitrate. But I should say this is a big step down the road in terms of EPA initiatives of getting rid of harmful algal blooms. But it literally comes down to how much loaded they are with their energy before they begin down-regulating. Okay, thank you. So returning to Catherine for a moment. If we are talking about the efficiencies, which you've spoken about in your review paper, what is currently the reasonable upper bound that you think these systems can get to and what's limiting you, what's your wish list for what you could figure out that you don't know today? All right, well, so for our systems, what we found is that a major limitation is actually the recombination rate of the nanomaterial. So that's, and again, this provides a nice contrast to photosynthesis because both PS1 and PS2, they move those charges very rapidly away from the central chromophore so that they can't recombine, which makes the system much more efficient. In the biohybrids that we work with, you have an inherent recombination rate within that nanoparticle, and that competes with electron transfer into the enzymes. And so the longer you can make that excited state last by engineering nanomaterials, the more chance, essentially, it's a probability argument. The longer it hangs around, the more chance you have to transfer it out into the enzyme. So that's one of the major limitations for biohybrid systems, the kind of biohybrids that we work with. And again, we're really focused on those fundamental principles of how to engineer the systems to be more efficient. So really it's that rapid charge separation and creating long-lived states that's the key. Based on the questions that are coming in, I think that Dan is onto something talking about food. Seems everybody is thinking with their stomach a little bit here. So I'm going to return to you, Dan. A little bit of confusion around exactly how you use your, you've created an organism that you photo illuminate so that it stores PHBs or PHAs in its cellular structure, and then you introduce that into the soil. Is that correct? Yeah, people always get confused by this. That's why I said it's three steps. So first I need sunlight and I do water splitting. And that's just like I told you at the beginning with photosynthesis. I make hydrogen. So step one, I have sunlight. Step two, I don't need, once I make hydrogen, I can turn sunlight off because I've stored the sunlight in the rearranged bonds of water. Now in the dark, the bug takes the hydrogen, which came from sunlight. That's where I stored the energy thermodynamically. In a breathing carbon dioxide, it makes a solid fuel. So it makes PHB. I've stored the sunlight in the biopolymer. And I can do that in the dark. I only need sunlight for water splitting. Then I do carbon fixation in the dark, polyhydroxybutyrate. Once the bug has polyhydroxybutyrate, it still has all the energy. I still don't need sunlight. I can then put the bug in the ground where the sun doesn't shine, but it's been loaded up by a solar energy supply and it draws on that solar energy supply, PHB, to do nitrogen fixation. Because again, people forget, nitrogen plus hydrogen and ATP, that's thermonutrile basically. It's a little bit downhill. I only need the energy to drive the nitrogenase. And now it's from the PHB. So you only need sunlight for water splitting. And then we played the trick of storing the sunlight with carbon and then telling the bug, draw on that stored hydrogen and internal energy supply and do nitrogenase. And that's why they can go in the ground and they don't need sunlight anymore. That also means that they don't then regenerate the PHBs. That's correct. That's why I'm calling it a true fertilizer. When it runs out of PHB, the bugs start down regulating and they don't make any more ammonia. And so you have to put more bugs in the ground like you would with a fertilizer. This is an organic bio fertilizer. Load it up with PHB so they can start generating it. Once they're in the ground, matter of fact, a lot of them die. When they finally run out of their PHB, this organism dies. There's a kill switch. Okay. So a question kind of for both of you. In the first presentation, we had a bunch of things that were nano rods with like cadmium and other things. You're now engineering an organism. Can you speak broadly about the safety of these bio and organic systems as we are talking about releasing them or getting them out of the lab? Katherine, can we start with you? Yeah, so I mean, I think there's ongoing work with carbon-based quantum dots and non-toxic materials that can be used. The Reisner group, for example, uses TIO2, which is not going to be toxic. So you're not necessarily wedded to toxic materials. The cadmium-based systems that we use, they're really excellent for laboratory scale, fundamental question, basic science research. We're really asking questions about how these things work without designs in mind. But I think there are a lot of work in the material science realm making new materials that you could use that wouldn't be toxic in similar systems. But again, the work that we do, it's really not focused on making design level or device level systems. It's really focused on fundamental understanding. And that requires, I think, a different approach than the kind of work that Professor Nussero talks about, which is much more focused on end use. And I think they're just two different approaches that require slightly different focus and different choices of material. So Dan, same question to you. What about the safety aspects? Okay, so making fuels, the bugs aren't released just like anything. You can think of like a fermenter. They're eating hydrogen, so on splitting water, there's a photovoltaic. Let's do this design. You can have a lot of different reactor designs. But I can have a photovoltaic. The leads went to two electrodes. Actually, we run these in PVC piping and we just run the bugs over, around in a loop, and they grow inside a reactor. So none of those are released into the environment. For the food to make my future life easier, and probably the EPA and USDA, we didn't want to use a GMO. So what we did is, first we did synthetic biology, and there's three genes. It's called the 5A, B, and C genes. And we use those genes to have the bug fixed carbon to make polyhydroxybutyrate. We then learned how the organism was doing that. The microbiology, so when I tell you, by the way, if I do the synthetic biology, I can fix over 90% by their weight with polyhydroxybutyrate. So when you look at the cell, it just looks like a big white blob, almost. But for the bugs I'm putting in the ground, I just did these field trials after we figured out how the bug was fixing the carbon dioxide to make polyhydroxybutyrate. We were able to grow them naturally under certain microbiology growth conditions, and that's why we get up, the ones I'm telling you, we get up to 25% now. There's no genetic engineering. It's using this strange bug we have, this xanthobacteria, and then developing the microbiology, which we learned from the synthetic biology, and they do everything naturally, so there's no genetic engineering for those bugs that are in the ground. Also for you, Dan, you are somewhat famous for the artificial leaf. You're now dealing with microbes. Is there any potential that these same ideas could be used in something like a higher organism, a leafy plant, or is that out of the realm of even your thoughts? It's out of the realm of my thoughts because coming in the front, I'm all about energy efficiency. Coming in the door, whenever I get tied to biology, they need to live. It's like Kate was saying, that whole step down procedure that leads to the high quantum efficiency, they're moving electrons down a potential ramp. So they're quote-unquote losing energy, but she's exactly right. They aren't losing energy. Every time the electron steps down, it's putting energy elsewhere and reproduce. My hydrogen production is dead. I don't need to work, so I can collect all that energy. So I don't ever want to go into a natural organism because coming out of the blocks, I'm going to be limited by energy efficiency. And by using the hybrid approach, I've blown way by that limitation. So I haven't had a thought about trying to go into a natural organism yet. Now, you can use natural organisms that do other things, and then you just play my trick of using solar water splitting and making ATP and NADPH the way I told you, and then use those bio organisms. So there's lots of possibilities that way. But I don't want to rely on natural photosynthetic energy efficiencies when they're stuck at 1% and just coming out of the blocks on the 10 or 11%. And I can go higher. If I make hydrogen at a higher efficiency, I'm going to even have higher efficiency of photosynthesis. So you're somewhat using the same trick that cyanobacteria used to be able to live at night by... But many of those excrete extracellular. You're keeping all your material intracellular. Is there a reason you couldn't also do extracellular such that you could then, through the growing season, add food, if you would, back onto the soil or even have microbial degradation give you the food that you need? Yeah, so look, I mean, if we're going to go into advance, like in the future, why grow crops? I'll just have the bacteria to make start. Okay. That gets back to the Martian movie. Like why grow a potato? I'll just have them emit the starch directly. And I'm serious about that. That's something we're looking at in our lab now, just making sugars and starches directly and just skip all growing in the ground. But you are right. There's a big problem with cyanobacteria, though. And this gets down to also microalgae. For energy efficiency, every organism needs to catch photons. And so that's why when you get reactor design or designed at algae reactors, it gets complicated because if you don't absorb photons, you're wasting solar energy. And this approach gets around that. Like I showed you, you can engineer the inorganic part to do all the light absorption. So you have much simpler reactor designs which translates into lower cost up front. But you are right. I could choose some other organism and have them excrete stuff. And like I said, the ones we're concentrating on now are sugars and starch directly. Okay. Again, another question is just to come in. War for you. Can you use the term self-healing? And I've read several of your papers and it always kind of causes me to scratch my head on your artificial leaf. Can you talk about the self-healing nature of the water-splitting catalyst? Yeah. I know these papers are pretty complicated. I'm going to just explain it simply. There's a paper I published in PNAS. Again, a little complicated on the physical chemistry of self-healing. But this is how I'll explain it. If I have cobalt in solution, cobalt 2+, and I have to put a potential on it to cobalt 3+, and then in the presence of phosphate, the catalyst forms spontaneously. It turns out for this catalyst, I have to put 0.9 volts on an electrode, 900 millivolts, to oxidize the cobalt 2 to 3. And then the cobalt dissembles and spontaneously forms. To run the water-splitting reaction by the catalyst, I put 1.4 volts on the catalyst. The thermodynamic potential is 1.2. So when the catalyst is splitting water, I have 1.4 volts there. If some of the catalyst degrades, it gets reduced by biological stuff in the Charles River. It reduces the cobalt down to cobalt 2 and it can start to fall apart. But as soon as I go to cobalt 2 stage, I only need 0.9 volts to get it back up to 0.3. I'm applying 1.4 to keep the catalysis going. So as soon as cobalt 2 gets reduced, before it can dissolve and go away, it gets re-oxidized back up to cobalt 3 and therefore the catalyst never dies. And we've shown it. We can take the catalyst film and just run it. We actually did very careful experiments. We used cobalt 57, radioactive and we ran the catalyst for like three weeks. And after three weeks of continual operation, we can only detect I think 0.001% cobalt in solution by radioactive tracing. And so the concept of self-healing, the papers are very complicated because we had to figure out all the mechanisms. But the simple line is you get a self-healing, a self-assembling catalyst that assembles at a potential less than the potential you need to do catalysis. And if you do everything right, like what those papers explain, then the catalyst never dies. It's always fixing itself and it's turnover numbers infinite as long as it's obvious. Well, let's hope that helped the other listeners as much as it did me. We're about two minutes away from our appointed hour here. So we're kind of like last words for both of you. I'll let you go first Dan and then I'll give Catherine really the last word. Any thoughts that you want to leave us with with respect to this field? Well, one is that people should start looking at that hybrid biology and organic systems give rise to very fast biomass and photosynthesis, way past natural photosynthesis. That's number one. And then the bigger issue is, because I'm always, you know, I'm an energy person and I worry about carbon. And if you then put fast growing biomass by playing this trick, interfacing energy science with agriculture and it's fast, fast growing biomass, you can do a lot of damage in the good way to the carbon budget. And that's a very good way to start sucking carbon out of the air. And I hope people will start. I should probably write a little review or perspective on this. I never do write those. But fast biomass can lead to significant carbon sequestration. Okay. Catherine, the floor is yours for your last words. I mean, I would just like to return to I think the point that I hope was made and as part of my talk was, you know, really just that natural photosynthesis results in this enormous diversity of chemistry and I mean, that's part of what Dan's done is tap into that diversity and take advantage of it. But that, you know, oftentimes in an artificial system, it's focused on one fuel product and that's for very good reason. But if we're going to use natural photosynthesis as a model, should really think about the flexibility that it provides and the way in which biology has managed to make energy carriers that can drive so many diverse chemistries, many of which are relevant to both our energy infrastructure and then also, you know, biomedical the biomedical fields, for example, and really that flexibility and that diversity and that richness of chemistry that's available to us is really something I think where we can think deeply about how to drive the field forward and use biology as an inspiration and understand it's how it manages these processes to make so many different products possible. Well, thank you very much. Thank you both for a very interesting seminar and for the very uplifting thoughts there at the end. So it's always good to have something to be, to look forward to, right? So thank you all. Jessica, are you taking it back or Alan? Yes, I just want to add my thanks to Dr. Brown and Dr. Nassara for very interesting presentations and great discussion. And I want to thank the audience for tuning in and just tune in May 11th when we have our next webinar in the series on opioid sensing. And I thank everyone again and have a good afternoon. Bye.