 Hello and welcome. I'd like to tell you today about some of the hydrolysis-determining substrate characteristics of liquid hot water pretreated hardwood. I've worked together with my colleagues on this area, and we think we found some very interesting results that perhaps you'd like to hear about. So, pretreatment opens up the structure upon enzyme hydrolysis. I think this is pretty well known. And if you look at our cartoons, this is one from Mosier et al. a few years ago. You can see the lignite, the solid line, the cellulose, the black line, the green is the hemicellulose. When we carry out pretreatment, we open up the structure so that the enzyme, the red circles and the light gray, can access the cellulose and hydrolyze it, forming glucose, a soluble product in the process. So this is the way it's supposed to work, and in fact it does. However, it's very important to have low enzyme litigants so that the process itself is cost-effective. What interferes with this is something else. If you look at the product, which would be glucose or cellulobiotis, that product will inhibit the enzymes. It's only for glucose. It inhibits the enzymes. The other thing that's given off when we do the pretreatment is phenols. And these phenols also, their soluble, they also interfere with enzyme hydrolysis because they will, in particular, inhibit, as shown by Jimannus and all, will inhibit this final step and literally precipitate beta-glucosidase in some cases, which will stop the reaction. So the question is, if we wash away these phenols that we saw in the inhibitors, we should be in pretty good shape. However, that's not the way it works now. So what we found is, even if we wash the materials as we go through different enzyme levels, 40, 10, 5, and 2.5, decreasing enzyme levels, and we plot these enzyme levels as a function of the severity of pretreatment, the higher the severity, the more the pretreatment. We find at the lowest enzyme level, the conversion is 5%, even it for the most severe pretreatment. If we add a lot of enzyme, however, we can get up to 70%, and what this tells us is that the pretreatment's working, but something is interfering with the efficient action of the enzyme. So we looked at this further, and here's what we found. If you look at adding BSA-Volan-Sex around human to the enzyme and what it does to hydrolysis, it's very, very interesting. First of all, if we have no pretreatment, no conversion, if we have pretreatment, exact same conditions, and we increase time, we will get up to 30 or 35% conversion, and this is for about 5 FPU per gram lukin or about 8 milligrams of protein per gram lukin, 3.5 milligrams of grams per little solace, okay? Now, if we dilute the enzyme with BSA, we add BSA to the reaction mixture, everything else is the same, but the conversion increases to 90%. So you might ask, how can this happen? Well, we went and investigated further, and here's what we found. We did another experiment. So this time we added BSA, but the cellulase loading was only 1.8 FPU per gram lukin or about 1.3 per gram pretreated solace. And so then, as we decrease the activity, in other words, the milligrams, cellulase per milligram total protein, as we decrease it, you'll notice the conversion yield actually goes up, and the reason for this is it's called, we call it the pretreatment commander. As we pretreat the cellulase material, it opens up not only the cellulase structure, so the enzyme can get added, but also the lignin. And the way this works is if you look at these scanning electron micrographs, the untreated material is very smooth. The pretreated material, on the other hand, shows lignin globules, and this was identified by Mike Kimmel a number of years ago, also published in biotank BioLange. And these lignin globules have a very high surface area, and they will absorb protein, any protein, in particular cellulase protein. So what we did is we came up with an explanation and a graphic to try and explain how this conundrum, in fact, is implemented. This first square represents cellulose and lignin structure. It's a graphic, of course. The purple represents lignin, the light's colored squares represent the cellulose, the circles, the enzyme. This is before pretreatment. We do get some hydrolysis. We likely also have some exposure to lignin, which will absorb a small amount of the enzyme, and this is shown as a flattened circle here. As we increase the severity of pretreatment, what you'll see as you look at the graphic under moderate, you have more lignin that is exposed, also more cellulose, and, again, the hydrolysis increases, but also the amount of enzyme absorbed on the lignin, again shown as flattened circles. At the most severe pretreatment, the cellulose is exposed, the lignin itself still absorbs enzymes, the flattened circle, and if we add enough enzyme, we'll get a high yield because the cellulose is now exposed. So what we did is we added BSA to the mixture, got it to absorb a lignin first, and that then enabled the cellulose enzyme to, in fact, be directed toward the pretreatment cellulose to give us high yield at low enzyme loading. So then that effect was to decrease the enzyme loading by a factor of 5 or 10 at the same conditions. And so in summary, what these papers show is that low cost production processes will define cellulose ethanol, that's known and has been described by others, and that lignin drive inhibitors on the next target, I think, to reduce some costs. We're not going to use BSA in a process, we use BSA in our research because the properties are known, what their weight is defined, and we know what sorts of interactions we would expect. But this gives us some information on what to do next, and also there's further background in this very interesting area and related papers that have recently been published by the Technology Bioengineering. I hope this was of interest to you, and I hope this short video abstract will, in fact, motivate you perhaps to read some of the papers that we have put out. Thank you very much, and I look forward to talking to you next time.