 My name is Arvind Raman. I'm the associate dean for the faculty in the College of Engineering. And today's celebratory event is co-organized. It actually brings together two different series. We have the Celebration of Faculty Careers series in the College of Engineering and the Celebration of Distinguished Professors in the College of Agriculture. We're combining them for today's event. The Celebration of Faculty Careers series in the College of Engineering really started as an outcome of the last strategic plan and the Faculty of 2020. And it invites faculty full professors in rank for seven years or more as an opportunity to present highlights and achievements of their career. And today it is my distinct honor to invite Dr. Karen Plott, the interim dean of College of Agriculture to introduce our distinguished speaker today. Sorry about my voice. I'm kind of fighting a cold. It's a great opportunity to introduce Mike Lottish. He got his degree in 1977 in chemical engineering and then got a position in the department of ag and biological engineering. And as most of you know, that department reports to both College of Engineering and the College of Agriculture, which is why we get to celebrate together today and really gets to the cross-disciplinary nature of some of the things that ABE has done for years and that we want to continue to do in the future. Mike does have an extremely distinguished record. He's going to talk to you a little bit about biorefining, science, technology, and applications. I'll let him talk about that. But I wanted to mention that he is a fellow of the American Institute of Medical and Biological Engineering and a member of the National Academy of Engineering. He received the Marvin Johnson Award in Biomedical Technology from the American Chemical Society. And he was named one of the 100 engineers of the modern airify AICNG, which is the American Institute of Chemical Engineers. Yes, I did look it up. He also received the Charles Scott Award and he's a fellow of ACAAAS, the National Academy of Engineering and AICNG. He really is a distinguished faculty and has done so many different things in his career to really promote many of you in the room, many of those in the College of Engineering and the College of Agriculture, as well as really move LORI, which is the Laboratory of Renewable Resources Engineering, he started this before this area was popular and really championed this area of study. And so with that, I'll turn the floor over to Mike. So anyhow, it's my pleasure to address everyone and it's really nice to have all our friends come and spend some time from their afternoons to listen about biorefining, which is what the Laboratory of Renewable Resources Engineering has been doing for some time, and which is an area of my research. What I want to look at is the science, the technology and the applications of what we call biorefining. So what biorefining is, it's a sustainable processing and this includes renewable resources into fuels, into chemicals, other types of bioproducts, then also it's bioenergy, which we biofuels, broadly defined, power. Think about, for instance, recovery of energy from renewers or other types of waste and then also heat. And just got some of the updates and it turns out there are 14 biorefineries, corn ethanol plants operational right now in the state of Indiana. I didn't realize and there's 201 in the US. But basically one bushel of corn, assuming the price is 320, it's a little bit higher right now. It's half the weight. We'll give approximately five dollars and 20 cents of value added products. And that includes ethanol, fiber, protein and now thanks to work such as being done by Abbey Engelberth, other types of small volume, very high value molecules that can be added to foods, nutraceuticals. Cellulose conversions being introduced, another statistic I was surprised to learn that 1% of the ethanol production now in the US is from cellulose And so, again, we're talking Ali about whispers and what's coming next. This is it. And it's coming very quickly. It's using our technology developed and patented here at Purdue, which is now off-patent. But thanks to Abhijit, there'll be Act 2, right? We have some other patents required to practice our technology. The GHG reduction, you'll see this number over and over again. GHG means greenhouse gas reduction. This is the reduction of a product over what would be required in terms of energy and inputs if it were derived from petroleum, a non-renewable source. So GHG reduction of 45%, which has now been qualified for the corn ethanol industry, means a 45% reduction based on fuel ethanol over what it would take to make an equivalent amount of energy from gasoline. But anyhow, here's some statistics. 15.8 billion gallons were produced in 2017. And surprisingly, this ethanol, some of it was exported, I think, to the tune of 400 million gallons in 2017. There was 71,000 direct jobs, 285,000 indirect jobs. I'm not sure what that means, but the 71,000 basically would be operators of corn plants by my estimate, about 12,000 people. And all of those that feed the inputs, deliver the products, et cetera. There's about $45 billion added to the GDP, about $10 billion or so in tax revenue. And again, this includes the workers that work in these plants. There's 201 plants. When I first entered the field, it was over here. And then I just kept growing and growing. When we first introduced the use of corn to dry ethanol was somewhere in this range, kept growing and growing. When we first discovered the use of liquid hot water as a pretreatment, it was here. Still not growing. And then things took off. Somewhere in here, I started a company with Bowen Engineering. Bob Bowen and Brian Stader called Celsus, which is acquired by Moscoma. And then the Mortgage Crisis hit. But still, things went forward. And so now, this is all corn ethanol, except for 1%. So now, last year, it was about 15.7 billion gallons of ethanol, fuel ethanol produced from corn. So biorefining has common denominators. It's not just fuel ethanol. It includes many other types of products. The major current products are ethanol, biodiesel, and to a smaller extent, fuels that are qualified to be used in aviation, sometimes known as aviation biofuels. I'm sure you've heard about Virgin Atlantic that used the first biofuel. And what they did is they added 5% of a very highly purified biodiesel into their jet fuel. And the reason this is being driven by the industry is because of the regulations in Europe that call for reductions in carbon. And one way to get this is for the airlines to voluntarily use this aviation biofuels. The other way would be to have Europe regulated for them. And if I incorrect on that, what happened was they decided to have internal goals in order to reduce the amount of carbon emitted by blending in renewable fuels into their aviation biofuels. Another product is amino acids. And there's also enzymes used in detergents. More valuable enzymes used as biocatalysts. Some moderate-volume chemicals are currently being produced, but many are still in the pipeline. And there's many small-volume high-value chemicals being produced as well, which are also in various commercialization stages. Now, there was two researchers in the area. I've worked with both of them. One is Joe Bozell from University of Tennessee. He used to be at DUA. The other is Gene Peterson, who I worked with very closely when I was at Moscoma and previously. And they came up with criteria of how you would define a bioproduct that would be suitable for renewables and have some commercial chances of success. And there were nine criteria, and you can look it up to what the criteria were in this journal article. But basically, it has to come from a renewable source. It has to be sustainable. And it has to have a documented use and documented properties. And there's nine criteria, basically. And some of those that have met these criteria include glycerol, which is a co-product, for example, of biodiesel production. And with hydrogenolysis, and hydrogen is an expensive-added chemical that or component that is required. But nonetheless, it's still economic to make ethylene glycol, propane diol, two, three PDOs used by Dupont to make serona carpets, for example. And then sorbitol, which is a smaller use. 500,000 tons sounds like a lot, but it is not. But it's a smaller use as a sweetener. And so really, the bioproducts that require hydrogen will probably evolve as biosources of hydrogen become available. Renewable sources of hydrogen become available. But there's a huge number of companies that are in this space already producing chemicals and bioproducts from renewable resources. Many of these companies are using bioprocessing, either enzymes or fermentations. And again, this is from a report by the National Academy of Sciences, which discusses the use of bi... They call it industrial biology in order to produce these molecules. The companies on the right and the products on the right, the green arrow, are currently commercial, most in small quantities. The products on the left are not yet commercial. But you can see names such as Geanomatica, the vice president of research is a lorry alum, for example, making two, three butane diol, which was first pioneered by George Sao in 1986. So it takes a while for these things to develop, okay? Or you have Ivonic, which is producing various amino acids as is Aginomoto and for that matter, ADM. And these are used for animal feed, lysine, for certain human products, but again, from renewables. And so you have all sorts of products. And again, you can look this up. DSM is a partner of Poet. Poet is one of the few companies remaining that is looking at cellulose conversion up in Iowa, okay? And they have a fairly large effort in order to try to make commercial success. There are about six major fundamental unit operations that define a biorefinery. The first step, still not solved, is feedstock preparation. The second is pretreatment, and those two have to be considered together. Although we're working with Karl Wasgren in mechanical engineering and some of our other colleagues in materials engineering, Nate Mosier, and hopefully soon the DOE will fund a project to look at how to liquefy this material and make it much easier to transport, more on that later. The third step is hydrolysis to get sugars. The fourth step then is fermentation to convert the sugars into ethanol, but also many other types of useful products. For example, working with our colleagues in USDA, Bruce Dean, he's able to take the glucose we get from renewable resources and ferment it into oil, which can then be recovered as a low quality oil but used for use either in foods or as a fuel. The fifth step is separations. The separations are required to purify the ethanol, remove the water, recover products that are present in smaller quantities, and basically add value. The reason it's called a biorefinery is not just ethanol or fuel that is a product. It's all these other co-products that add a lot of value that are very important to the overall economics. The solids that are left over, mainly lignin at this point, are then burned and the CO2 that is generated goes back into the environment, but since the source of the sugars and the source of the lignin, which is burned, it's like a, we used to call it a young coal, incorrect language, no. But basically, it's a soft coal, if you will. And there's so much energy in the lignin that at least when I was working at Moscoma, the greenhouse gas reduction was certified as being greater than 100%. The reason being that when we used wood, wood was not fertilized, the lignin that was left over in fact had sufficient energy to run the plant and there was so much energy, it became an economic issue because you had to develop some sort of way of taking that energy short of burying it, which by the way doesn't count, as far as greenhouse gas reduction necessarily. And so that meant generation of electricity, so you'd have to bring in transmission lines in parts of the country that had biomass, but no infrastructure. So these are all the sorts of things that you don't think about when you're developing technology that become very important. So a suitable feedstock, we just described it, is ag residues. We have a lot of corn residue in this part of the country in Brazil, and thanks to Dr. Shamanus, who's in the back, we've had very, very good program in relations with Brazil, Professor Cristian Farinas, also Abadino, and in Brazil they have two large cellulose ethanol plants and a lot of impetus to develop some of these new areas in cellulose conversion. In wood is a very important resource, particularly in the upper Midwest, particularly mixed hardwoods, and then there's purposely grown energy crops. Some are being developed here in the US. For instance, at the University of Illinois, Steve Long made the case that he has a new plant that can fact sequester more CO2 than other types of plants, I'm not sure what the science is, I'm not an expert in it. But anyhow, Miss Kansas has often been said, but it's a tuber, and so as a consequence, you have to plant the roots or the plant, and once you're done, you have to tear it up if you ever want to use the crops for farmland. On the other hand, switchgrass, which is being developed here at Purdue and some other universities, is attractive, but again, it has to be grown in quantities, harvested, stored, transported, et cetera, so that's economic. In Africa it's yet to be determined, but despite this, there's a lot of biomass available. In fact, in the US alone, this is from what's called the Billion Ton Update Reports. These are surveys by the US DOE, together with USDA, looking at how much cellulosic material can be sustainably supplied to these plants should the demand arise. And these are online, there was a report in 2011, another in 2016. But long story short, there's a lot out there right now, about 140 million dry tons of cornstover alone. If you add all these up, it's between 400 and 800 million tons per year on a sustainable basis. The problem is it's very distributed, and the plants that would utilize this material can only afford to ship material within a 50 mile radius. So this limits the size of the plants, and the unit capital that goes into these plants can be rather large, because you don't have the, in most cases, you don't have the ability to greatly expand the production, because the feedstock has to be shipped, and that's an issue. So ag residue is around here, and what we've worked with is our hardwood and corn. There's two parts of corn, there's the leaves, of course the kernels from the, removed from the coven, and the stalk in the rye. And as far as what Poet is doing very interestingly is they are recommending the farmers harvest the part of the corn that is above this height, because below it is where all the phosphate goes. And if you remove it at this height, increase your cellulose yield, you also have to replace the phosphate in the field. So there are costs of getting agricultural biomass as well. So is corn stover available without getting into the debates about indirect land use? Basically the answer is yes. This is an Iowa corn stover trial that's been carried out for over four years by Poet. Poet is a large ethanol company. They run 20 or 21 ethanol production facilities in the Midwest, including Indiana. And so before baling, this is what the field likes using equipment that's been developed by John Deere and several others, this is what it looks like afterward and notice they have left enough stover on the ground to prevent erosion. Also notice that the stalks are left standing in order to maximize the amount of fertilizer value that particularly minerals that go back into the ground. They're now testing the storage of these. I calculate each bail will produce about 50 gallons of ethanol, which is not a lot. So you need a lot of corn stover to run one of these facilities. The problem has been, at least in the DuPont facility, is they at least catch fire. If they're too wet, the fungus starts to grow, it heats up the bail and they actually combust. And so they're still learning how to store these materials. Unlike wood, hardwood, you can just store it for a long time. But the feedstock is available, but the problem is it's lignocellulosic, it's not starch. Corn kernel probably about 80% of it is fermentable or more. In the case of wood or ag residue, it's only about 60% or so and 32% is lignan, which has to be dealt with. Lignan also protects the structure. Corn stover, wood, sugarcane, big ass have similar total compositions, but very different components, very different structures of the lignan. Lignan itself is underutilized, burning it would seem a waste after you recovered it. Economically, it works out, but there's many other uses it could be put into. And at the same time, lignan in fact will inhibit hydrolysis and fermentation and protect the biomass from being broken down. So in order to make these plants go, you have to do pretreatment and also have cost-effective enzymes. If you put enough enzyme in, these work. But you can hardly afford to use $5 worth of enzyme to make a product that's worth a dollar and 20 cents, which is what much the literature actually does these days. So there's a lot of ways to decrease the cost, but it requires a knowledge, a deep knowledge of some of the science that impacts how these materials are broken down. And inhibitors and inhibition is a key. So this is a figure from one of Nate Mosier's papers, we all co-authors on it, and say what, Nate, 5,500 times. Everybody likes a cartoon, right? Plus the science that's in the paper, of course, yeah. And so what happens during pretreatment is you have hemicellulose and cellulose. Cellulose is a black line, it's very crystalline. Hemicellulose is a polymer of five carbon sugars, that's the green line, and lignan is a polyphenolic, that's the purple line, and they're structured in the cell wall in order to protect the biomass, the plant from attack by microorganisms or funguses that have these enzymes that try to attack these plants by drilling a hole in the side and then infecting the plant. And so the plant has a lot of protective mechanisms. During pretreatment, what happens is a lignan is melted, it's disrupted, some of the hemicellulose is dissolved away and the structure of the cellulose is opened up. So the enzymes, which are fairly large, relatively speaking, can access and hydrolyze away the cellulose and hemicellulose fractions. Now this gives you an idea what it looks like. This is untreated material. Bars are about four or five microns in size. This is what the plant cell wall structure looks like. The plant cell walls are very large, 100 microns, excuse me, 100 microns are larger. After pretreatment, this is an intermediate severity, probably about 180 degrees centigrade in liquid hot water for 15 minutes. You start to disrupt the structure and you see these balls. After even higher severity, I think this one is about 210 degrees C for 15 minutes. You can see these very large, finely identified balls are about one to two microns in diameter. These balls are lignan. They're very good at absorbing enzymes. They would break down the surrounding cellulose. So you need pretreatment to get high yield, but to get the high yield after you pretreat it, you may need a lot of enzyme because this lignan structure will absorb the bio catalyst. And so the economics don't work out very well. So why liquid hot water? Well, it's been 20 years or more. It's hard to believe how time flies. But anyhow, what we found out is if you cook hardwood or corn stover in water at high pressure, 200 degrees C or so, what'll happen is you'll open up the structure but minimize hydrolysis, break down of the cellulose into soluble components. However, if you add a little bit of acid, which is the methodology still used by many, or has been used by many companies, what'll happen is you'll break down the cellulose, make it active, then hydrolyze the glucose and then immediately go to degradation products because of the reaction kinetics. Degradation products are aldehyde that are very, very inhibitory of the fermentation. So what we found is a sweet spot, a temperature range and a pressure where we added no chemicals, cook the cellulosic material in water. We were able to get high conversion by stopping the reaction here or even here. So we separated the physical change, breaking open the structure from the chemical change, making glucose and degradation products. By separating these two steps, it was patented in 1998 or so or 1997, we found that we could use water as a liquid phase and that the materials in biomass, such as corn stover and wood, had the necessary compositions to self-buffer to the desired pH range. Second of all, the temperatures were in a reasonable range as far as chemical processing goes. The run times also were in a pretty reasonable range and we could do this at solids loadings anywhere from 10 to 30%. And if you do the pretreatment, this is one example that at this time we're using a lot of enzyme, you can see the difference between no pretreatment and cooking it liquid hot water. The conversion really jumps, it gets into a range where it's economically attractive. The problem is the yield was there, but the amount of enzyme used was just way too much. This is across the board for many other types of pretreatment as well. Since liquid hot water is now the methodology of preference and growing very rapidly, matter of fact, I think the Anderson's up in Delphi, you just saw Rod the other day, we're thinking of using it. It was invented here, patented here. Okay, but now off patent and I think as the industry develops, you'll see more and more of this. But the problem is you still have to deal with the inhibitors. So the way we did this is we did a series of pretreatment, lots and lots of work and many, many students worked on this. So the initial lab experiments were to fill up a tube with, looks something like this. Leave about 30% freeboard open volume because water expands as you heat it. I guess one of our students found that out. Fortunately, it was in the sand bath and sand went all over the tape. It was all according to OSHA standards. Oh, I'm being taped here, okay. Too late now, right? Okay, so basically you fill it up, you cap it and then you drop it into a sand bath. A sand bath is something that can heat up to two or 300 degrees C very easily. Then after 15, 20 minutes, you drop it into a bucket, literally a five gallon bucket of water. It cools back to 100 degrees C or less and the reaction stops. We then scaled that up after we showed it would work. We worked together with Bowen Engineering and we started a company in 2006 called Celsis and the co-founders were Bob Bowen, Brian Stader and myself and we had a number of angel investors from Indianapolis who contributed. And so Brian was able to very rapidly raise funding to build a pilot unit. Pre-treatment occurred in this tube. This is a steam generator. This is the order in which the cellulosic material was fed and we actually ran this on the agronomy farm, on South 231 out there. And Nate was there at the time as well and Tommy Creek who's now working for the Indiana Department of Environmental Management, I believe. And we got that to work. So in those days, things move quickly. That's when the curve was starting to go up. So within 14 months, Celsis was acquired by Moscoma. This is after Credit Suisse and three other large venture companies came to Lafayette, Indiana, believe this or not. We met Stu Benning's office. We decided to work with Moscoma because they had the wherewithal to couple the other technologies needed, including fermentation, processing, qualification of greenhouse gas reduction, et cetera. So I became their CTO. That was one of the requirements of the deal and Purdue in fact owned part of Celsis and then part of Moscoma. So I got at Moscoma, I started commuting to Boston every week, but thanks to a wonderful group here at Purdue we were able to keep the lab going and so forth. So I got there in November, by January we had a warehouse, by August it was built out, November it was ready to start up. So when industry decides they wanna do something it's very intense and very fast. The cost of this pilot plant was quite significant but at that point then four years or five years more of work ensued in order to prove the process. Long story short we were able to reproduce using microorganisms that Lee Linde had developed at Dartmouth and also some other organisms at the company at that time was their chief technology officer had been able to obtain from other companies. So we showed that we could in fact directly ferment wood into ethanol and that the laboratory results the guideline and the pilot plant results, I think this is for 200 gallons on your left in fact worked out. And so we're getting about 40 grams per liter of ethanol which doesn't sound like a lot but for this technology is the minimum you need in order to be able to recover the ethanol economically. And we scaled it up to 1,000 gallons. It worked a little bit better. We finally got to 5,000 gallons and by the time I left Moscoma which was in 2013 I stepped down as CTO. By the way Karen I was only gonna do it for a year but lasts a little bit longer and Jay said well you might wanna build in a little bit of extra time there. So I did. So we got to 70 grams per liter and what this process does it takes wood, no chemicals added, cooks it in liquid hot water and adds a yeast that's been genetically modified to ferment both glucose and xylose to ethanol and then also makes cellulose enzyme to break down the wood. And it was not Nancy's, that was a different yeast. And so as a consequence that looked very attractive and what we learned along the way technically is that materials handling the front end was a limitation. The pre-treatment of step alone was 18% of the total capital cost. I just finished writing a report on my experience at Moscoma which had been funded by DOE and the plant itself, large scale plant itself the capital cost alone were about $120 million for 20 million gallon per year capacity. And then the installation is about double that. So you figure 240, this is for the first plant. And one huge issue was getting this material to this state. Once you get here you can ferment it and pump it, hydrolyze it, recover it, distill it. What's interesting here is this material, this is after pre-treatment, this material have all the same solids content, 22%. So again, thanks to the team effort in Moury, thanks to some shrewd observations by Eduardo Shimanus and Christine Christian Ferenas and others. Myself prior to that, we found that some enzymes for reasons we didn't understand at that time enabled this material to become a fluid that's pumpable. It's a shear thinning fluid, it's still viscous but it's pumpable. And it changes the capital investment required immensely. The reason is you're trying to take a solid material, introduce it into a chamber that's 360 PSI or so, 200 degrees centigrade and hold it for 15 minutes and then pump it and get it back out. You have augers, you have locks and everything, all these mechanical things that can go wrong. So this is by the way 22% weight solids. If you wet a piece of paper with water, that's 10% by weight solids. That's actually double the dilution. And so this is a major technology. There's two ways to go, one is using enzymes, the other is using malacic acid which is an enzyme emetic that Nate discovered. Again, it's off patent though, right? But don't worry, we'll get back to it. So basically then we said, oh okay, if we can get the stuff to pump and we hopefully will start a project, Carl, soon they set the date for the review March 23rd, by the way. So basically the other thing that happens is when you pretreat the material, the ligand in itself gives off inhibitors. And so this is again a GC and I think it may aspect identify what they were. And all these inhibitors turns out are quite effective in reducing the activity of cellulose enzymes. So if you take this mixture of phenols and you have two milligrams of phenol for milligram cellulose protein which is very low concentration. Recognize we're trying to get conversions that occur where we use less than three or four milligrams of protein per gram of biomass. So this is going to be in the same range as the phenols that are given off by that gram of biomass. And so what you can see the activity itself drops precipitously. You lose half the activity. So what's the answer? Somebody said, well, they should be soluble, let's wash them out. By the way, I'm making a long story short but anyhow, what these phenols did is they stopped the activity of the enzyme which converted the soluble cellulose fractions into glucose. Then the cellobiosidimer would build up and that would in turn inhibit the CVH. This part here turned out to be my PhD thesis. It's really strange how things work. 1977 and it turns out we thought this was a major inhibition. Well, this turns out is not. It's maybe a hundred times less than the phenols but the CVH is very strongly inhibited by cellobios. As soon as that builds up, the reaction stops for all practical intents and purposes. So what happened is one of our colleagues at NREL, McMillan, Jim McMillan, said, okay, let's wash out the phenols from the corn stover, add back the enzyme and lo and behold, yeah, you get a much higher conversion, everything else being equal. So we said, okay, let's wash it, we're home free and realize all the while I'm at Moscoma and when you have investors and we had a lot of investors and quite a bit of funding to build this thing, you know, you get calls like, I think the CEO gets a call. How's it going, Bruce? Oh, okay, four o'clock in the afternoon. Well, what else has happened, you know? Maybe I'm exaggerating a little bit, but basically a lot of pressure to get this thing running. So we said, okay, we should be in good shape. Well, it turned out, remember, we're aiming at two and a half FPU or less. That's our economic level of enzyme we can use. Well, this is what two and a half gave us with pre-treated material that's been washed. This is where you start to get white here and you know, you're spending somebody else's money so they're not real happy with it. Okay, well, it turned out that, well, possibly go up to 40 FPU, we're home free. No. So at that point, we were trying to figure out what's going on and this has worked from Jake Koko and young me, Kim, who's now teaching at University of Wisconsin Riverside, right? So what happened is we ran into what we later found out and called the pre-treatment conundrum. And remember, the purple lines are lignin. The light spaces are cellulose and hemicellulose. The circles, if there's any biologists in the room, Karen, you'll have to forgive me. The circles are supposed to be enzymes, okay? Not to scale. So what happens is if you have no pre-treatment, you get very little conversion because everything's protected. If you have moderate pre-treatment, you get a little bit better conversion at moderate enzyme loadings but things are still protected. If you in fact get very severe pre-treatment, that's a pre-treatment level with a severity of about 11, you get a great conversion if you use enough enzyme but the problem is you also expose the lignin and the function of the lignin is to absorb the enzymes that will impact the cell walls. So the better you got at pre-treatment, the more enzyme you needed, in which case it's not economic. So how did we unravel this just in a few steps? Well, first of all, cellulose enzyme is not a single enzyme, it's a component, three major components, cellobiahedralase, endoglucanase, beta-glucosidase. One attacks randomly, sort of chips open, the cellulosic structure. The second one de-plimerizes it two at a time. The third one takes that dimer and converts it into glucose. Well, the story doesn't stop there and then matter of fact, the last year one of our really top-notch students in Brazil did a secretome analysis on aniger and t-reci, which are the two fungal organisms that are the major source of the enzymes. I mean, enzyme components you think they found in proteins, 200 of them. This is in nature. Well, for the ones that are produced through molecular biology at large scale by commercial vendors, there's only 10 to 20. But the real problem is the hydrophobicity changes there's different types of components. Molecular weight changes. So that means you have to have different porosities to get these enzymes to get in. Okay. The protein families are different a little bit as well. And so in addition to the PI, the isoelectric point changes and when you're at the isoelectric point, the protein precipitates. So the preferred condition for hydrolysis is pH 48. So this was quite an interesting, that's euphemism, may you live an interesting time, right? But quite an interesting development. So what we did is we took lignin and prepared lignin, put it in a microfiber tube with buffer and enzyme and then very systematically measured how much enzyme absorbed onto the lignin. The way the lignin was prepared is through liquid hot water pretreatment using lots of enzyme, hydrolyze the way the cellulosic fraction and then clean off the proteins that may be absorbed onto the lignin using enzymes called proteases. Well, it turns out we had something else going against us. The higher the severity, the lignin that was left over would absorb more enzyme. Right? So this is your partition coefficient, higher is worse for us, more absorption. And so as the severity went up, the absorption would go up. So that's the problem. And the other thing we found out, some cellulases absorb onto lignin irreversibly. They'll never come off, no matter what conditions you use. So what do you do? Or what has been done in literature? You titrate the lignocellulose with enzyme to the point where it's not economic. If you look at across the board, there's probably thousands of papers like this where the whole idea is just to get it to work. But at this point, for commercial application, you have to get it to work economically. So the way we did this is we took cellulase to absorb, we add a second enzyme called a protease. It hydrolyzes proteins, got the cellulase to desorb. And what we found is the initial pre-treated solids would have a small amount of protein in the background anyhow. But even after we went through all these steps to remove the protein, we found out that the lignin itself had three or four times the amount of protein that would be naturally present, and that protein is enzyme. And by the way, a lot of this credit goes to Jay Kongco, Young Lee Kim, insights from Nate Mosier, and Warren Shimanez, and many of our students. And so what we then found is the worst news. The enzyme that it irreversibly absorbs is inhibited and precipitated even by phenolics is called beta-glucosidase. And you'll recall that is the last enzyme in the step that goes from cellobios to glucose. As soon as that shuts down, everything shuts down. And here's the activity remaining. Here's where we start, right? Now at this point in time, Eduardo made an interesting discovery that the preferred industrial microorganism, terecii, had a beta-glucosidase that's very, very sensitive to inhibition. The second industrial microorganism known as Aniger has a beta-glucosidase, which is not sensitive to inhibition. So what we did is we bought two commercial preps, one from Novo, okay, and one from the other company. Well, our graduates work for Novo, okay. So, and mixed them together. One was Aniger from Novo, the other was terecii, mixed them together, and lo and behold, inhibition suddenly gets a lot less. That's the spesime, C-P, Aniger mixture. And to further prove it, and there's a lot of other background work that was carried out, is we ran protein gels. These are gels where we separate the proteins by molecular weight, and then we look for the activity that remains. So it turns out, when you had lignin, the free protein, the supernate, these bands, three molecular weights disappear, were really decreased over what was originally present, and that was due to the absorption of beta-glucosidases. So then what we did is we diluted the enzyme. We used bovine serum albumin, which is not economic. And lo and behold, everything else being equal, no pretreatment, no conversion. No BSA added, 40% conversion. Add BSA on top of that, and suddenly the conversion doubles. So what's happening? What's happening? It's like at Elyse ass, how you add the protein, the protein absorbs onto the lignin, preferentially keeps the cellulase from getting on there and boom, the cellulase is going back to hydrolyze the cellulase. Yeah. Well Charlie, it took a long time to figure that one out, but we should have had you on the team. Yeah. Yeah. By the way, Dr. Babs is a very famous physician, and works in the Biomedical Engineering Department. And you did teach, IU has a med school here, did you know that? And I believe they don't have a sign anymore, do they? They do? I know it's south of you, but Chris says the sign wasn't there anymore. Somebody take it down, anyhow. So basically, here's another way to describe it is we ran different yields, and if when we add the BSA to all these other conditions, gave a little bit of boost to the BSA. Again, everything else being equal gave a higher yield. Now, if we use avacel, avacel by the way, is a pure cellulose that's used in the excipient. You ever take those little aspirin, you know? Well, what the active ingredient is compressed in avacel, right? It's what you swallow. Well, we use it to measure hydrolysis. It's a lignin-free cellulose, and what happens is, except for pH 5.5, which is away from the optimum for the enzyme, the avacel hydrolysis is not really affected by BSA. But if you have lignocellulose at the equivalent conditions and equivalent cellulose concentration, you have a huge effect. So then what we did is we did an experiment. We took cellulase, and we decreased the cellulase content per total protein added by diluting it with BSA. And so as we got the cellulase, milligram cellulase per milligram total protein decreased, the conversion went from 20% to over 80%. Less enzyme, much higher conversion. And the reason again is, and by the way, you wouldn't use BSA because that's almost as expensive as cellulase, but it was to prove a point. The BSA absorbs onto the lignin. The cellulase does not. So you need a lot less to get the job done. So then we really worked with Rick Milans here. All my friends are here today, yeah, and Vlad as well. I'm not gonna talk about our BIC. I didn't have time. They wouldn't give me till 7.30, you know? So basically with Rick and Clint Chappell, and this is what's so wonderful about Purdue is across the disciplinary environment, the cooperation between agriculture and engineering. And so what Clint and his group did, particularly Bonnewitz was a lead student. I think he now works at Dal Agra, right? So basically they were studying how to redirect lignin synthesis. Long story short, people have been trying to redirect lignin synthesis for years, and when they were successful in redirecting the synthesis and changing the composition, the plants would get sick. And this is what they would look like when you tried to grow them. So what Clint and his team found out is that if they redirect the synthesis, shut down something called mediator complex, what'll happen is a lignin amount will stay roughly the same, but it will now be mainly H-lignin. It turns out H-lignin doesn't have anything on here to sort of enzymes. That's not why they did it, but it's one of the tests we ran. So what we found out for those plants with H-lignin, you got really good hydrolysis, even without pretreatment. That's up here 60%. If you pretreat it, you might get 85%. Again, the reason for this is is the cellulase is not absorbing on the lignin, this time because the composition is different. So pretreatment and enzymes are key, really, really key. They make the substrate susceptible, they decrease the enzyme, they keep the costs in line, they may form fermentation inhibitors, but the science really led to strategies for managing the inhibition, and the engineering was able to get the job done and show how to do it practically. And that effect is we reduced, and this research is at Purdue, we have reduced the cost of enzyme by use of enzyme by 5X. So that means that the costs of cellulose conversion are now within range of being practical. Since then, we've made some other interesting discoveries, including the fact that you've now converted cellulose pericarp without pretreatment. Again, you start to see some of the connections here, and hopefully we'll be talking to the Andersons and some other companies about this. So the conclusions really are what I've covered before. The surface area is important. If you can mask the lignin or add protein that prevents absorption, you're in a great place to be. As a matter of fact, our colleague in Brazil, Christian Ferenas, in fact, added soybean meal, which is one-tenth the cost of BSA, got the same effect, small amounts. So future research, what are we gonna do in Laury? Well, future research really, somebody asked me what I did, and I tried to think about it. We build things, we're engineers, so we like to build things as a team. But what ties everything together is proteins that interfaces. What do proteins do at high concentrations when they connect with themselves, when they absorb on a solid surface? For instance, if you're doing bioseparations or chromatography, it's on a solid surface, okay? And so what do they do? So it's proteins that interfaces, and so this led to another project, and this is where Purdue's such a great place. And Carl's here, really a leading figure in the US, if I'm gonna say this, Carl, I know you'll be embarrassed, but he's one of the key modelers of understanding how solid materials interact so you can get them to flow mathematically. And so thanks to that, we're working together with Idaho National Lab, David Thompson, who's a Purdue alum from Laury. And one of your students, Tyler, all right. And so basically, we're assuming DOE approves, we're gonna now look at the modeling and figure out how to get these slurries before pretreatment so we can reduce the capital costs. Remember, $250 million reduced the capital costs by 20%. That's a lot of money. Why do I know these numbers? I was CTO at Moscoma for six years, and we just published approved report and you look at upline DOE, which gives all the costs. Okay, so basically it'll reduce the price of the ethanol. Also, the other thing we're working on very exciting is injectable biologics thanks to Charest-Garamella and Ken Sannell, they negotiated, and many others, they negotiated a research project with Eli Lilly to find a way of injecting monoclonals. There's 300 of them in the pipeline right now at very high concentrations so they can be used to treat cancer and other immune dysfunctions while avoiding the need, in many cases anyhow, Charlie, for intravenous administration, which takes four to six hours once a week. Not real pleasant. So we just started that project. We're searching for low-crossed lignin blocking molecules and thanks to Karen. Thank you very much. We're able to, and EVPR and Bernie, thank you. We're able to buy a process LC and pack columns with biomass and now what we're doing is looking at things that prevent enzymes from absorbing onto the biomass, i.e. the lignin. And so we're screening those, obviously, soybean oil. We're trying to find some very, very low-cost molecules that we could add to get the job done. On the protein? Yes. Take cheaper molecules, we'll find them at the same site. No, ultimately you would try to do some molecular biology or Kevin, Christopher, right? And change the hydrophobic regions or the charged region. I probably, his amination has something to do with it as well so that they don't bind at all to the surfaces that are presented. And the way we can study this is thanks to Clint. He can make lignins, at will now, that have different properties. So we can understand better what we're looking for. We're also working with rapid-pathogen injection with LAD and Young Chen and others. And again, it's proteins and interfaces because in this case, the proteins get into a hollow fiber membrane, they get into the pores, they precipitate and plug everything up. We've been able to overcome this by using enzymes suggested by Eduardo. And so as a consequence, we're able to rapidly filter and recover high-concentrated living pathogens so they can be probed. And we're coupling this with optical methods of various types through the BIC grant. And DITRA, which just came last week, DARPA, et cetera, are very interested in this for other reasons. So what we do is we take a very large, sloppy sample, 225 mils, concentrate down to half a mil, recover the organisms, put it in a microfuge tube, spin it down, give it to LAD, and he says, aha, I wouldn't touch this stuff, right? Or it's okay, yeah. And then we have enzyme-assistant fractionation to corn kernels. This was funded initially by the Indiana corn and now is being pursued with industry. And this is where we're using cellulose enzymes to avoid the use of chemicals to fractionate corn kernels into identifiable components, corn, starch, germ, pericarp, and protein. And last but not least, with Nate and others, nano-cellulose healing properties, again with Eduardo. We had a student now that is able to grow nano-cellulose from glucose. So you hydrolyze the cellulose to a fermentation broth, you then ferment it into nano-cellulose, and you get these sheets and you can see your fingers through it. That's how you know it's nano, it's clear. So all this takes a lot of money, so I just listed them there. And this is part about, I guess, two thirds of our research group. And as Nate and I were looking at this, we were a little bit sad because two thirds of these people have graduated. So we have a new group. We try to get our students through in four years from BS to PhD. And all of them have some very successful careers. And last but not least, I just wanted to point out that George Sal, who's the founder of Lorie, Lorie will celebrate its 40th year in May 18th and 19th. We have something over five, according to Joel 580, but let's say over 500 alumni, we're gonna try to bring a number of these back and George will also come. And so I think we have a real debt of gratitude to George Sal because he was a visionary and saw this coming 40 years ago. Was that? Thank you very much. I wanna say, Mike, you are a visionary. Because you are working on the premier problems of the 21st century, and you started doing it before anybody thought it was cool in the 20th century. And I applaud you. Well, thank you, Charlie. Thank you very much. I also wanna say as far as I ask about the licking and blocking problem because I think there might be an opportunity to leverage some knowledge here on campus of people who have worked with drug receptor binding and a drug development group. Because it very well may be that the cellulase is binding by just this very specific site on the lignin and you could flood that with cheap low-cost blocking molecules just like a classical competitive antagonist in pharmacology. Or you could identify the domain that is responsible for absorption, produce it in quantity using Kevin's CRISPR without a genetically modified organism, right? Not yet, okay. Right, but where we're extrapolating that to and I'm really getting back into it, and in fact, my intent is to study it very deeply is the delivery of highly concentrated monoclonal antibodies. Same problems. We're looking at 200 or 300 milligrams per mil. This is supported through the Lilly research challenge. And so we're already making progress but it's very interesting how some of the very fundamental biology and biochemistry and engineering is very similar to what we saw with the lignin. It's a different system obviously and different enzymes but you're exactly right, Charlie. If you look at the fundamentals of science, science can tell you what's going on. The engineering tells you how to make it work. Yes. Well, since we're here also for your reason to celebrate your success in the research I'd like also to add a couple words. I admire how brave you are. I mean, so Mike doesn't have any psychological barrier to cross boundaries between very different fields. Like I'm doing none of the phonics and Mike would approach me and say, hey, what none of the phonics could do for this pathogen detection. And he pretty much challenged us, inspired us, motivated us and I truly admire this. I mean, I believe that real success comes for people who don't have this problem. You're in interdisciplinary areas and you're clearly one of those heroes. Thank you. Well, thank you, Vlad. Thank you very much.