 As he just said, we actually do chemistry and biology side by side, although in two different labs, unfortunately now at Rutgers, but nonetheless we do both the chemistry and the biology. We are completely agnostic as to how we choose to do energy conversion, whether it's a photo-electrochemical cell that we make, or whether it is a biological system that is made by nature and modified to become a transgenic reaction center. So the hope would be that we can actually tell you who is blowing smoke when it comes to the question of what's going to have a real impact on the world, right? When are we playing the toys and when are we talking about real catalytic systems that can have impact? I want to start off by pointing out that this is a collaboration. By the way, this is my first, actually we haven't finished our first year, we will I guess in January or so. So this is a progress report and it's great to be back in the GSEP family. I was here probably about 15 years ago or so. I see Richard over there. It's Sally, the new director. Franklin Orr was the director at that time. I was a reviewer at that point and so I had no idea this was going to become an even closer relationship where I'm benefiting directly. So thank you. So as Mateo mentioned, we're over at Rutgers now. We're at the Waxman Institute of Microbiology, Selman Waxman in Nobel Prize in that area. So we have the resources of an entire laboratory and multiple collaborators to do anything we want at almost 500 to 1000 liter scale at least. And then over in chemistry with a huge consortium of material scientists and chemists working together on materials. So it's been literally paradise. For GSEP, we partnered with a former collaborator and visitor in my lab, Jun Cheng at Jejong University who has over 110 bioreactors each one the size of a football field. So if you want to test anything at scale, we can do it. And more recently, a collaboration with Kristal Benning's lab at Michigan State who had agreed to give us some of their mutant strains. And this is morphed into a much more close relationship with mutagenesis going on in the two labs together. Sometimes it's useful to look at pictures like this and you can get a sense of what is the scale of the problem that we're trying to tackle. 300 billion tons of carbon dioxide collectively into the atmosphere. So when we start thinking about microbes and land plants and photoelectric chemical cells, we have to factor into this. Are we investing our money in the solutions that are going to work? Or are we just playing with toys? I'm not going to answer that question today, but it has to play into GSEP decisions for sure. Here's a slide I prepared in 2007 or so as part of a, you know, give me money strategy through DOE, I believe at that time. And it depicted the corn crop because they were anticipating developing corn ethanol, of course. That took off big time with the Renewable Fuels Act in 2007. And that mandated the cap of corn grain ethanol at about 15 billion gallons per year so that we wouldn't take too much for food. We quickly within two years capped that and we've been pegged at 50 billion per year gallons of ethanol. And it hasn't budged up. So it's clearly an artificial number that is dictated solely by the subsidy that's provided to the ethanol producers of 51 cents. The numbers you're looking at land area in 2011, about 25% of the land that was used for corn ethanol, but really a total of 84 million acres for all of our needs for corn, be it locally for food, for animals and for overseas and so forth. So that turns out to be 4.4% of the U.S. land area right there in 2011. That generates our 15 billion gallons of ethanol. Oddly enough, they actually have to take some of the non-dedicated corn ethanol or corn grain from other applications to make up this 15 billion. So it's about 41% of all corn grain goes into corn ethanol. It's an absolutely huge number. And where we're going, 2026, it would be predicted these are conservative estimates by the people who are most likely to give you the lowest numbers, about 11% to 90% land area mass for cellulosic ethanol. That's to provide only about 15% of the transportation fuel and light vehicles, right? Only 15% ethanol is capped. Can you imagine that this planet, if we use agriculture for all of our bioproduct needs and fuel needs, it would be unlivable, literally unlivable. So this notion of using agriculture based upon photosynthesis to provide commodity chemicals and biofuels is an absurd concept. And it's absurd because we have a photosynthetic efficiency which is capped at about 1% best case in greenhouses and so forth. It's typically well below that. So photosynthesis is abysmally inefficient. If I went to just up the street up to your investors, Silicon Valley, right? I said, I got a great process for whatever, formate or some hydrocarbon. But my plant that makes it is less than 1% efficiency. They would laugh and sort of go somewhere else with their money. So that's the task is making photosynthesis efficient. And so here's one of the partial ways out is to build a algal bioreactor. Algal photosynthesis is more efficient than land-based photosynthesis. You would provide nutrients, sea water, hopefully, low-cost CO2 from fossil fuel plants. You need to get typically organisms that can tolerate 15% CO2 in the flue gas. And that's a challenge right there. Is this going forward? All right. Typically yields 20 to 40 grams per meter, per square meter per day, what you're looking at. So in an optically thin bioreactor, so it says if you want to have an impact on that 300 billion tons, you need to cover the earth multiple times over to achieve that. So there are some advantages to the algal system. And certainly we work on this largely to understand the process of light energy conversion. So that we can use those tricks to make better man-made total electrochemical systems. So we're going to be making oil this way. The organisms that are doing this are algae of one type or another. Nanochloropsis was chosen for our GSEP project because of a significant toolbox for modification of this organism. We know that wild strains won't do it. We certainly still look for wild strains that are metabolic outliers. We're looking for the Usain Bolt of algae, literally, or the hummingbirds amongst the class of birds. Of course, we're looking for the metabolic outliers. And we have certainly found some that are far better than nanochloropsis, but no genetic tools for them at this stage. So what we learned in nanochloropsis might be transferable to something that is a Usain Bolt, let's say, of algae, we hope. Lipid yields in nano are around a quarter of the biomass yield. You can get it up to about almost 50% by nitrogen deprivation from the growth medium. Another aspect here of its benefit here is indicated in the theoretical lipid yield here. These are numbers from scientists who are completely disconnected with algal culturing and mass scale. So these numbers are polyana-based, pie in the sky, estimates based upon a one-liter bioreactor. 10,000 grams per, what's the number here? I can't even read it myself. Does he have it? The area, 10,000 kilograms per hectare per year. So knock that down by a factor of 10 for the normalization and then probably another factor of 10 for the challenge of growing outside. So the numbers are out there from estimates. Nitrogen deprivation, after you grow up the cells, removal of nitrogen from the medium gives rise to lipid accumulation in many cells. You can visibly see these. Anyone who's read most of the algal literature, you see pictures like this by every other article. But you rarely see the predictions of what it does at large scale or actual experiments at large scale. So that's the big challenge. So why nano? Nano has a very high diacylglyceride acyltransferase set of genes. If you look at the number of gene copies amongst the heteroconts, which include these are the diatoms of which nanocleropsis is a member, but it doesn't have a silicate frustal. It has the largest number of DGAD genes of any phototrope known. And apparently because of evolution, it evolved late in the photosynthetic life from the combination of a heterotrope with green and red algal chloroplast or algae to make chloroplast. And so it has all of those in its genome. So very versatile at least on the end stage of lipid biosynthesis. So that was another reason for choosing it. So hype, lipid productivity, complete sequence genome for a couple of the strains here from the lab. The Benning lab did one of those genome sequences. And so we've been collaborating with them on modification of this alga. There's also targeted mutagenesis based upon transformation of this described by two different groups. First a group here, Chris Nyoges group on a homologous transformation and then more recently a CRISPR-Cas9 observation that you can transform this. So it looks as though you'll be able to make any mutation that you want. A big problem with algae and particularly with nano is that it has a carbon concentrating mechanism, which means that it concentrates CO2 in the cell. And looking at the genome you can see that it has two carbonic anhydrase genes indicative of classical carbonic anhydrases. It has two bicarbonate transporter genes. So it looks as though it's well set up to bring in CO2 and bicarbonate and concentrate it in the cell. So there's consequences for this if you're growing algae at 15% CO2, which is shown here. The biomass concentration in grams per liter here in air, if you supplement up to 2%, many phototropes, land plants grow better of course, algae do too. You go up to 5%, 15%, you knock it way down, way down. So you have to figure out a way of either eliminating the expression of this set of genes in here or other ways in which you can make it more tolerant to the CO2 and bicarbonate that's going in the cell. So here's the overall strategy. We're going to start with nanocoropsis oceanica. This is an open strain which has no patent leans on it. Anything that we make will be free to the public. We're working with Michigan State. First they gave us 400 of their random mutants that they created. We were going to screen for high CO2. We've had to throw those mutants away and made a thousand more because the techniques for making them improved in the interim. So we went back to them and worked on improving that and both they and also now using the better method for random mutagenesis. And then targeted mutagenesis for high lipid-producing strains. We searched the literature for what are the kinetic roadblocks or limitations in the biosynthesis of triacylglycerides. So that would presumably get us winter strains, both random and targeted hopefully. And then we would unleash the arsenal of modern molecular biology and chemistry and characterization of chemical, the metabolite profiling, the physiological characterization and the mutant localization as well. That would then bring us some winter strains. A very small number over to Zhejiang University with our partner Jun Cheng where they would begin to scale up from small bioreactors to greenhouses. And then all the way up to these football field trials. So we're not at Zhejiang yet, but that's in the future. Forward random mutagenesis. So here's the strategy that we're using is to introduce a plasmid into the nanocoropsis electrophoretically and do that randomly and then screen for phenotype. The screening that we're doing is on a fluorescence activated cell sorting instrument that can distinguish between high lipid yield using a stain or high chlorophyll by fluorescence. And you pull those winners out that way, grow them up and identify which ones you want to propagate further. And then so they're grown on antibiotic resistance colonies. This plasmid would have a marker for an antibiotic resistance cassette there. You can then also screen for high CO2 tolerance in a reactor where you get up to 15% without killing your students hopefully. That's non-trivial actually. It turns out the health and safety people are very, very conscientious about this, which is good. So further characterization once they pass through those three screens would tell us what are the winners. Here's an example of metabolic engineering for targeted mutagenesis. So I presume you all remember undergraduate work where you studied photosynthesis, which has light reactions that produces both NADPH or reductant and ATP. Those are used in the Calvin cycle to fix carbon dioxide, which makes C3 and C2 intermediates from that. And the C2 intermediates asked hill coins I may feed directly into. Excuse me, what does the yellow light mean? Should I pay attention to that yellow light? Okay, it's changing colors over here. Okay, I didn't think there was a problem. So we would be going into the glycolytic pathway of those products that go into acetyl coins I may. That's the entry point into the tricarboxylic acid cycle, which goes on to bake both energy, but also proteins, right? Or precursors to protein. So we know that nitrogen deprivation elimination of protein biosynthesis elevates lipid production. So we know that we're stopping the TCA cycle by nitrogen deprivation. So the strategy we chose was to knock out citrate synthase here. And that was targeted in a nonphotosynthetic organism and shown to produce higher lipid content. In fact, that along with lipases, knockout, and still yet another organism was shown to increase tag production, triacylglycerides. And both the diacylglyceride and phosphatidic acyltransferase enzymes involved in lipid production from fatty acids were shown in independent organisms to actually increase lipid production. And then in a nonphotosynthetic organism, the glycerol pathway to make glycerol, remember the backbone of lipids is glycerol, overexpression of that in a nonphototrope was shown to increase that. So we targeted these five, three for upregulation, two for knockout, and GSEP liked that idea, they funded us. And very quickly we learned that our person who was giving us the random mutants was already doing P-DAT and D-GAT. So rather than copy that, we said, well, let's do the other ones and then we can stack those mutants together in a single organism and see if we can really kick the lipid production up. So that's what has happened with Michigan State doing P-DAT and D-GAT. We're doing G3-PDH, glycerol-3-phosphate dehydrogenase, lipase and citrate synthase. I'll just show you one of the results here, which is the overexpression of the glycerol-3-phosphate dehydrogenase, which is this gene here, so in a plasmid it has a bidirectional promoter with that that can both express, overexpress the G3-PDH as well as bleomycin. And bleomycin confers resistance to an antibiotic, zeosin, so you can screen by that root. And if you want, you can also see it light up because you put in a luciferase gene as well. You can see the colony is lighting up because it has incorporated the luciferase gene. You can do the PCR on the plasmid and demonstrate. Here's six different insertial mutants in which the one kilobase gene fragment for this plasmid was inserted, so we know it's in there both by two different markers. So the hope would be then that we'll move on to physiological characterization. Is it a winner? Does it do anything? Which of these mutants, because it's random insertion, are the better overexpressors and don't knock out other functions? So that's the strategy where we stand on that. Here's some growth attributes of nanocoropsis here in the laboratory, whether it's on shakers or with bubbling. One of the first, and we're typically growing at 22 and low light intensity, 35 micro Einstein, 22 degrees and 35 micro Einstein. So that's a low light flux, and you can ask the question, well, what's the temperature dependence of that? That's in progress at that large scale, but also in our physiological studies, which I'll get to shortly. So here's one of the first things you find is that you better aerate this. It doesn't like to be shaken, it likes to be aerated, so literally that's an important feature here. So one is going to have to do a lot of mixing if one uses this in open cultures. If you add glucose, and this is the doubling time in days, so they're all in this 10 to 20 hour doubling, I'm sorry, that should be hours, 20 hours doubling time in days of growth, sorry about that. But if you add a chemical reductant like glucose, a native chemical reductant, you can see that the doubling time gets quite a bit longer. So what this tells us is that if cells lice and it spills out glucose and any other reduced carbohydrate, it is going to suppress the growth of the remaining part of the culture. So you better take good care of your culture because any reduced carbon around ends up reducing the plasticinone pool for the experts and means it shuts, turns off photosystem 2. So some practical things are learned by that. Here's the first selection of the random mutants. Again, these are selected based upon growth will tolerate hygromycin. Here's the wild type strain. It's dead on three levels of hygromycin. Here's a mutant strain. You can see it's growing. So we would take in principle our up to 10,000 random selected mutants. We're only at the 96 well level right now. We've got a thousand mutants and we took our first plate and found some winners. We selected them out based upon chlorophyll content. We then grew them up at larger scale. As depicted here, you can see wild type on the end and you can see there are some differences here in growth rate as far as chlorophyll is this B1 happens to be quite a bit higher. If you look at the doubling time here, and here we got it right. It's in hours between 20 and 40. So some are slower than others relative to wild type. But here's one that's growing better. This G2 organism, a faster doubling time. If you now look at those same mutants, again you can measure the moles of oxygen per cell per flash. So this is a classic experiment of Bessel Koch and Pierre Joliot from 50 plus years ago where you use a single turnover xenon flash and you measure the yield of oxygen and it oscillates with period 4 which is the signature of the manganese cluster that splits the water. So in clearly just taking equal aliquots of these you see quite a difference in the yield of oxygen here. If you divide the oxygen yield by what is called FV which is the chlorophyll fluorescence quantum yield for photosystem 2. You're saying this is how many oxygens can I make for every charge separation event that photosystem 2 does. So here's wild type and you can see we have two winners one of which is this B1 which is growing great. You can see the chlorophyll is also a positive indicator that this is great and this is saying it's because probably it's making a whole lot more energy conversion in this phototrope as is another one down here as well. So this would be one of the more advanced levels of characterization. So we're on our way. The next step, at least when we get the real winners the final winners will be to take these to Géjean University where they will grow them up in their laboratory first at increasing scales demonstrate that they can validate our results move it over into a greenhouse at somewhat larger scale and then ultimately out to the open ponds where they have about 100,000 square meters of surface area where they could explore if necessary. So that's where we stand. We have a thousand mutants created by electrophoretic random insertion into their linear DNA constructs designed for screening of antibiotic selection in site localization. We have a citrate synthase knocked down targeted mutant already. We're trying to redirect carbon into lipid biosynthesis under that test. I mentioned the glyceraldehyde 3-phosphate dehydrogenate over expression strain. We'll be looking at that shortly. We have a stacked mutant with the citrate synthase and the G3-DPH over expression in a single strain that we'll be testing shortly, we hope. Energy conversion yields and kinetic choke points during PS2 turnover have been determined. So we've done a complete physiological characterization of two strains, Gatatana and Oceanica thus far. And soon we'll be going over to China, we hope, with some winners. Future directions would be to build further random mutant libraries, you want to get up to 10,000 and looking for higher lipids, higher growth rates, and tolerance to CO2. We're going to then certainly identify the winners as far as where they are, which genes they're targeting and so have a knowledge base growing. And then selected random and targeted mutants so with high lipid and high growth rates would be further characterized as to what is their photosynthetic capability. Are they really over doing photosynthesis, which is what we think is limiting things overall, the light reactions. So with that, I am going to thank the people who did all the work, as I mentioned, we're in the Waxman Institute of Microbiology but also in the chemistry department. And at Waxman, Gennady Ananyev is chief of the physiological characterization energy conversion. And then we have two graduate students, Yuan Zhang and Hua Wu, who are doing targeted mutagenesis and growth physiological characterization and Yun Bing Ma is doing the random mutagenesis work. We have a couple of great undergraduates here in red working with them. And I don't want to forget our collaborators, Jun Cheng at Zhejiang University. We're going to be putting him to big work very soon, we hope. And certainly Kristoff Benning for his very generous offer to provide both mutants but also then co-training and stacked mutants will work together with him and his student, Eric Paulinar. So thank you very much. Thank you, Charles, for the great presentation. Questions from the audience. As far as getting them out into the open ponds, is there any thought that's going into keeping them competitive with regular algae strains that would get introduced once it's in an open environment? No, we're thinking very monoculture, as scientists are, so I think you're asking would it be beneficial to have a polyculture that might be more stable, robust, and so forth. I can show you samples from Yellowstone National Park that were gathered in 2009. The only thing we've added to them is deionized water, and there is green and lush as possible. They have everything they need in there. They have the nitrogen fixers, they have the sulfate reducers, they're happy. So you're right, we've got to go that way. But I think we have to do the hard science first. One more question, please. Is there a microphone? Right, I think the question might have been a little different. At least my question is, how do you keep the wild types from poisoning the systems? Certainly a good question. The title of the talk really is different than what I presented, all about metabolic scaling and so forth. And indeed, what you typically find is that the wild types are not necessarily the fastest growers, but they're more robust and tolerant to stress. And so that will be the big challenge. Can we break the well-known scaling relation that says if you make more energy, you make more osmotic pressure inside the cell, you make it more susceptible to lysis. Will we be able to solve that? I don't know. Good question. Very last one. Thank you, Professor, for a great presentation. I'm from China, so I'm interested to know because you're cooperating with Jersey University. So when you cooperate with them, do you need to collaborate or report to any government departments? And the second question is, how do you solve the intellectual property rights ownership issue? Fortunately, I don't have to deal with the Chinese government. Jun Cheng does. And I'm sure there is significant work in reporting that. I was here in May, and we went up to the Yantai Bioreactor Facility in Shandong province. The engineers were very gracious and eager to show us all the facilities. They are eager to collaborate and so forth. The reason for choosing Oceanica as the strain is that there's no patent lien on that. So any of the development that we do, we're openly sharing, I was Jay-Jong University. I have no interest whatsoever in getting patent rights for Rutgers. It's all about solving this big problem. And they were willing to donate at no cost. It doesn't cost GCEP anything to use these bioreactors. And you can imagine the significant investment that that takes. So I don't really have good answers to your question other than, in my particular case, I'm not allowing that to be a roadblock in our collaboration. Great. Let's thank Charles one more time.