9UNP1255SD_Caryn_Wadler_WordPress

- Welcome everyone to another edition of Wednesday Nite @ The Lab. My name is Garret Suen, I'm an assistant professor the Department of Bacteriology here at UW-Madison and it is my absolute pleasure to introduce Caryn Wadler. Caryn was born in Vernon Hills, Illinois where she grew up and she did her undergraduate work at the University of Illinois Urbana-Champaign. She also did her master's work there as well, with her adviser, John Cronan. Before she came here to the University of Wisconsin, where she joined the Microbial Doctoral Training Program, she originally joined the lab of Dr. Jorge Escalante, but Jorge left for greener pastures, maybe, and she ended up joining my lab, here I'm very fortunate to have been able to recruit her and it gives me great pleasure to introduce her because she just recently defended her PhD, in fact, on July 20th, and so tonight, she is going to be telling us a little bit about the work that she did for her thesis. So, the title of her talk is "The Dirt Beneath Our Feet": How a Wisconsin Soil Bacterium Is Advancing Biofuel's Technologies. Dr. Caryn Wadler. 

(audience applause)
- Thanks Garret, for that introduction, and thanks to Tom and everyone for giving me a chance to speak here at Wednesday Nite @ The Lab. Indeed, Garret has mentioned the title of my talk, and so with that, I'm just gonna get right to it. So, if we wanna talk about biofuel technologies, we first have to talk about energy. Energy use in the world has been going up through the delightful advent of smart phones, internet, and all sorts of other processies that require energy. And you'll notice by this graph of 2010, energy use per country, the United States uses a large amount of energy, especially relative to the rest of the world. Now this energy is consumed in four basic sectors. The industrial sector, which makes a lot of the products that we use. The transportation sector, which makes products and people get back and forth. Residential sector, which is all of your homes, and the commercial sector, which is the businesses that we visit. 

This energy comes from a variety of different sources, most notably, petroleum, but also natural gas, coal, nuclear, and what I'm going to be talking about today, renewable energy, of which biofuels is only a small subset.

Now, this is a very complex graph of how all of those different sources of energy relate to the different sectors that use them. And the one that I want you to focus on is the renewable energy down in the bottom left in green, and the transportation sector in the upper right, and you'll notice a little green number four by transportation, which means four percent of the energy used by transportation sector comes from renewable energy in the form of biofuels. Now, why am I pointing this out? Back in 2006, the DOE issued the Billion Ton Challenge. They want to reduce the transportation sector's fossil fuel use by 30 percent, or one third by 2030. And they want to do this by increasing the production of biofuels. So, what that means, is they want the little green number four there, the percent of biofuels feeding the transportation energy, to rise up to 34 percent, and the 93 percent, coming from petroleum, to drop by a third to 63 percent. So, biofuels are, as it was a surprise to me, actually not really a very new technology. Biofuels have been around since the 1100's, when they first distilled ethanol from wine, or from grapes. 

And then since then, they've used ethanol for lamp oils and cooking in the 1600's, and in the 1700's, farmers were turning crop waste, which is how we get our biofuels nowadays, into lamp oils and other stove fuels for cooking. In the 1800's, with the invention of the combustion engine, they immediately started seeing whether they could put ethanol in that, and in the 1920's, adding ethanol to gasoline was actually a very large part of the commercial enterprise. And you can see that picture above is 1933 in Nebraska, where they have corn alcohol gasoline, which has a 10 percent mixture of ethanol with gasoline, which is the same mixture that we use now when we put fuel in our cars. Now, since then, with the Great Depression, the cost of petroleum became cheaper to put into cars to ethanol, and there's been kind of a back and forth interplay between the economics of biofuels and the affordability and availability of petroleum. Until recently, the rising prices of oil, and the environmental concerns of what happens when we burn it, have led to renewed interest in biofuels and we again, actually have commercial ethanol production plants active in the U.S. So, one of the things that is different about biofuels nowadays back from biofuels in the 1920's is the way that we make them. And biofuels are generated by bacteria nowadays, so we harvest the leftover material after we grow our crops, and they go through this initial processing, which is really just grinding up all of this plant matter into kind of fine particles. These particles are then treated by enzymes to break them down so that bacteria can ferment the sugars from these broken down plants into the biofuels and ethanol that we're all familiar with. So, when we look at how much biofuels and ethanol we make now, and the fact that we want to increase this by such a huge fold, we have to look at the process and think about what areas we can improve on, and how we can make biofuels more affordable, more available. And the two steps that are currently the most economically and physically difficult are this treatment by enzymes and the following bacterial fermentation. Now, the reason this is so difficult, is because plant matter is actually not a very easy substrate to break down, either by enzymes or by bacteria. 

So plants, such as switchgrass, which is a biofuels feed stock, is made up of plant cells, and these plant cells have a matrix of lignin, hemicelluloses, and cellulose. Now the cellulose is what we actually want to break down into ethanol, but you can see it's that yellow core under the blue hemicellulose and lignin, it's very difficult to get to. You have to break through all of those layers before you can even access the cellulose. And then the cellulose itself is in these really tight, rigid sheets, which are difficult for enzymes to act on. And then, each of those little cellulose fibers is then a chain of the glucose cellobiose and other sugars that is actually what the bacteria act on after enzyme treatment to make cellulosic ethanol. And this works by using cellulases, which are the enzymes that break down cellulose, into these small sugars that the bacteria can then use and ferment and make cellulosic ethanol. So, when we think about that two-step process of breaking down the cellulose into sugars and then fermenting, some of the ways that we can make this easier and more affordable, is to understand how bacteria degrades cellulose. Is there a way that they can degrade just maybe switchgrass, this big plant, instead of having to go through all this enzymatic pre-treatment. So, I'm not the only one who thinks about things like this. The university started a freshman interest group, which is a special class focused on, in this case, studying biofuels. 

It's called Agronomy 375 last year. And the course took advantage of some of the things we have on our campus, we have actually several campus facilities interested in biofuels, Wisconsin Energy Institute and the Great Lakes Bio-research Energy Center, and they worked in collaboration with this freshman class, to let them do visits to the labs, go out into the fields, see the technology that's available. Many different professors came in and lectured about how their labs are doing these different things. Professors from each of these institutes talked about how the institutes as a whole are working towards biofuels. And on top of all of this lecturing that they were given, they were allowed to go into the lab and do experiments to see what scientists who are actually working on biofuels might be doing and how that biofuel research is actually carried out. And so to do this, they wanted to isolate a bacteria that could degrade cellulose. So, they went out and looked for an environment where they might find bacteria that degrades cellulose, and one of the most prevalent environments is the dirt. Our soil is where all the plant matter falls after it decays, tree leaves and all those other things, and the soil's actually a very large repository of the cellulose degrading bacteria and fungi. And what you can see in this picture here, down at the bottom there's a cellulose fiber covered with little dots, and those little dots are cellulose degrading bacteria and then there's the kind of smooth long things, those are fungi that are breaking open the plant tissue so that the bacteria can get to that cellulose. So they went outside the University of Madison campus on to Howard Temin path, right about where that little pink pointer dot is, and they grabbed a scoopful of soil and they took it back to the lab to see if they could find the cellulose degrading bacteria. 

So obviously there would not be much of a story if they hadn't, so the girl there pipetting is Madeline Hughan, and she helped isolate this bacteria which I'm calling A375-1, for Agronomy 375, first isolate. And so they take the soil into lab, they pipette it into a bunch of different media, and they're looking for bacteria that degrades cellulose and you can see on the right, there's two different tubes. The one with the yellowness has bacteria in it, and the one on the right doesn't, and each of them have this kind of white filter paper, I don't know how easy it is to see there, but it's a long white strip, and that's pure cellulose that they have put in the tube to feed these bacteria and as it grows, it will digest it. And you can see in the tube with the bacteria, you can't see very much of that filter paper cuz they've just been going through it. So now that they've done the very hard and time-consuming work of isolating a pure bacteria from that culture that had all the different bacteria and fungi in it, the question became, what have they found? What is this organism? So when you look at mammals, there's a lot that you can determine just by physically looking at it and seeing what it is. So looking at this image here, you can tell down at the bottom that there's a brown bear and a polar bear. We know that those are bears and they're kind of closely related, but they're definitely two different bears. When you look at bacteria, you can get an idea of what they are based on their shape, some of them are kind of the spherical cocci shape, or a rod bacillus shape or a spiral shape, and there are other tests that you can do to try to figure out what they are. 

One of the most common ones is a Gram stain, where you look at the membranes of the cells and you can stain them, and based on the membranes, the cells will either stain pink or purple. You can do other microscope work to see if they have a characteristic such as making spores, and spores are those big white circles in the center of the black bacteria. Or you can just look microscoply with like a scanning electron microscope to see everything that's there. So, looking at these pictures, you would say, okay, there's definitely a lot of different things going on. These look like there's obviously pink and purple cells in this Gram stain, there's different shapes of these spores, and there's different like cocci and rod shapes on the right, it looks like each of these have a bunch of different bacteria in them. But that's actually not the case. This image on the left is all the bacteria in there are actually bacillus cereus, it has the special phenotype that as cells age, it changes the way that they behave with this Gram stain. Sometimes they start out purple and then as they age they kind of turn pink, cuz the membrane changes as the cells grow older. These spores are all from the same bacillus, and again, it's an aged phenotype and also based on how long the cells have been in different conditions, they form these spores, so the ones on the left are kind of happy and growing, and the ones in the middle are in a difficult condition and have formed these tough spores. And the one on the right is actually a bunch of different bacteria on a grain of sand. 

Those are different shapes, there was no tricking in that one. So given that these phenotypical tests, these tests that we look at to see what their behavior is, can give different results, even for the same bacteria, it makes it very difficult for us just to look at these organisms and tell what they are. So how do we figure it out? Well, the one thing that is harder for the bacteria to disguise from us, is their DNA. So it order to figure out what these bacteria are, we take the DNA from their cells and we sequence sections of it, and we've gotten to the point with bacteria where we mostly just look at one or two different gene sequences, and can compare that to a library that we have of all the other DNA sequences from other bacteria that have been published, and try to get an idea of what it is, based on how closely its sequence resembles other sequence bacteria that we have in our collection. We then take all of these sequences and we can make something called a Tree of Life. So this is kind of a graph that shows how closely related different organisms are based on how similar their DNA sequences are. And so you can see over on the right, there's animals and they're all kind of closely related, fungi, and then there's this whole section on the left in blue of bacteria. So we took the bacteria, A375 that was isolated by this class, sequenced its DNA, and found that it is a Gram-positive organism. And further than that, we could tell that it was actually an organism from the genus Cellulomonas. 

And you can see right down there, that the DNA sequence is different enough from these other Cellulomonas to form a discreet branch on this tree, but still similarly related enough to be within the same, we call it a clade, but the same clustering of bacteria. So given this, we renamed this bacteria Cellulomonas wisconsinensis. So, and this is where I came in because I actually study Cellulomonas in my lab and the class came to me and said we found this organism, we think it's a Cellulomonas, what do you think? So, I'm going to talk a little bit here about why I study the genus Cellulomonas. The genus Cellulomonas has been established since 1923, its in the phylum Actinobacteria, along with other organisms that you might've heard of like Streptomyces. And it's comprised of short Gram-positive rods that are known to degrade cellulose. These bacteria are difficult to characterize because kind of like Bacillus cereus, which I showed you earlier, its Gram stain can be variable, so sometimes when you look at it, you think it's Gram-negative or Gram-positive, and it also behaves differently in several other of those tests. And so we have to rely on the DNA for that. But, as of now, there are 25 type species of Cellulomonas that have been isolated from soils all over the world. There's several American soils, different sorts of farmlands, compost heaps, municipal dumps, rotting logs, all sorts of places that you would expect to find lots of cellulose, and environments that are very similar to where these students found Cellulomonas wisconsinensis. 

One of the other interesting things about the genus Cellulomonas is that two of its members Cellulomonas uda and fermentons, are the only reported bacteria capable of degrading cellulose and generating ethanol both aerobically and anaerobically. So, why this is a big deal is because the ways that bacteria degrade cellulose under conditions that are aerobic with oxygen and anaerobic, without oxygen, are very very different. Aerobic bacteria use this non-complex cullulase system, which means that they excrete their cellulases, their cellulose degrading enzymes, outside of the cell, and then they move around to pick up all the sugars that happen when the cellulases break down cellulose. Anaerobically, the cells have what's called a cellulosome, which attaches the cells and the cellulases to the cellulose so it can bring up the sugars that are made when cellulose is broken down immediately. And the...oops. The organisms that use the aerobic and the anaerobic system are both very different in terms of what you see and the way they degrade cellulose. So how can there be these two very different systems? The reason that there are two very different systems comes down to how the bacteria function. So bacteria growing aerobically, with oxygen, go through glycolysis and this TCA cycle, which is a metabolic process which generates them 38 ATP. 

And ATP are the energy unit of the cell. That's what bacteria use to do everything that they need to do. And so that 38 ATP gives them a lot of energy to make cellulases and to move around to pick up the break-down products from cellulose. Anaerobically, the bacteria don't have oxygen and they use what's known as a mixed acid fermentation, which generates only 2 ATP. So that's much fewer ATP than under aerobic conditions, which means they don't have energy to make many cellulases and the cellulases that they make, they have to keep closely tethered to the cell and they have to be closely tethered to the break-down products so they can get those as quickly as possible and make those 2 ATP to keep functioning. So, the questions that I wanted to ask with my research about the Cellulomonas is whether all Cellulomonas are capable of degrading cellulose under both aerobic and anaerobic conditions. And how do they do so?

So, going back to the tree that we had shown, the two organisms that are known to degrade cellulose aerobically and anaerobically, Cellumonas uda and fermentons, are actually very spacially separate on this tree, which suggests that if those two organisms can do this, then maybe all of them can. So to study this, I took Cellulomonas wisconsinensis, and 19 of these other Cellulomonas that I got from strain collections all around the world, I got them to send me their Cellulomonas so that I could test whether or not they can make ethanol and degrade cellulose aerobically and anaerobically. So, the way that this would work was that bacteria are going to excrete cellulases, the cellulases will break down the cellulose into sugars that they can then use for bacterial metabolism. 

And I can then look at the end products of bacterial metabolism using a machine that can measure these metabolites, and I looked for the amount of ethanol, acetate, formate, lactate, and succinate that these bacteria made after they grew. So, just to show you again, I actually tested their ability to do this on several different sources. Switchgrass, the full complete plant stalk, cellulose, the crystalline fibers, and then cellobiose, which is the broken-down sugar that they can take up directly. And you can see here that under aerobic and anaerobic conditions, they produced different amounts of these different metabolites. They did all produce ethanol, which is the dark blue color that you can see at the top of those graphs. But under anaerobic conditions, they made many more fermentation products than under aerobic conditions, and that in general, they made more of these metabolites on cellobiose than either cellulose or switchgrass. And this made sense from what we knew about metabolism in that aerobically, the cells are not going to make very many metabolites because they're going to be shuttling the energy sources through and making ATP, whereas anaerobically, they're gonna be making more of these metabolites. When I went and then looked at all of the Cellulomonas, I saw similar trends. Anaerobically, there were more products produced than aerobically, and there were more products produced on cellobiose, the bottom row, than either cellulose or switchgrass. And again, this makes sense, because cellulose and switchgrass take more energy to break down, they're more difficult substrates, and so they have to spend energy making the enzymes to break that down, instead of making these metabolic products. 

So now that I knew that these bacteria were undergoing these different metabolisms, I wanted to know how many cellulases they were making to do this, and how active they were. And there are a couple of different assays that I used to do this. The first, looking at the number of cellulases, I again looked at the media after cells had grown, and I looked at what they had produced. And I can run this media, the spent media without cells on a gel, and then look at the ability of the cellulases that now get trapped in this gel, to degrade cellulose. And I used a special cellulose called carboxymethylcellulose, that dyes red with this congo red dye, and then everywhere that a cellulase was able to break down that cellulose, it also meant that it couldn't get dyed, so all of those yellow spots there are where cellulases have broken down the cellulose that was trapped in this gel with them. You can then count up all of those yellow spots, and in this case I have Cellulomonas wisconsinensis shown here, again growing on those same substrates cellobiose, cellulose and switchgrass, and count up then how many cellulases it was able to produce. And you can see here, the trend that aerobically, more cellulases were produced than anaerobically, and on cellulose and switchgrass, there were more cellulases produced than on cellobiose. And again, that holds true with the trend that cellulose and switchgrass need to be broken down by the cellulases, so it's going to want to be producing more of them. This trend was again consistent among all of the Cellulomonas, and you can see more cellulases were produced on cellulose and switchgrass than cellobiose, and more cellulases produced aerobically than anaerobically. I used a similar assay to look at the activity of these cellulases, but instead of running that sample on a gel to see how many were there, I kept them all together in the tube and looked at their ability to break down this cellulose into breakdown products over time. 

And you can look at the amount of cellulases, the amount of cellulase activity as a measure of how much of this CMC got broken down over time. So when I did this, I saw that a large percent of CMC was degraded on cellulose and switchgrass because of high cellulolytic activity and less on cellobiose, and again, more activity aerobically than anaerobically. This was consistent with the number of cellulases that I had seen, the more cellulases that were present, the more activity that was seen in this assay. This trend held true again for all of the Cellulomonas, in that there was more activity aerobically than anaerobically and more activity on the complex substrates that produced more cellulases with cellulose and switchgrass.

So, what I have going on here is very different behavior of these two cells under aerobic and anaerobic conditions. Aerobically, they're using a respiration metabolism that produces lots of ATP, which allows them to make many cellulases that have high activity and get excreted into the outside of the cells. Anaerobically, there are few or no excreted cellulases, which leads to lower cellulolytic activity.

So, to answer my question, are all Cellulomonas capable of degrading cellulose under both aerobic and anaerobic conditions? The answer to that was yes. 20 Cellulomonas that I tested were able to degrade cellulose aerobically and anaerobically, and they were able to produce ethanol as a byproduct of that metabolism. 

So then I wanted to ask, what cellulolytic strategy are they using to do this metabolism? So given these two very different behaviors where there were many excreted cellulases versus few excreted cellulases, I came up with the hypothesis that aerobically, these bacteria are using this non-complex aerobic cellulose degrading metabolism, where they excrete their cellulases, and then move around and pick up the break-down products. And then anaerobically, I hypothesized that they were doing something more similar to the anaerobic metabolism where they somehow attach to the cellulose. Now this would be a kind of novel thing, because not only are no bacteria able to degrade cellulose aerobically and anaerobically, that have been published other than the Cellulomonas, no bacteria has been shown to have two different strategies for doing this. So to go about figuring out if this is what these cells are actually doing, I started using Cellulomonas gilvus as a model organism. It's one of the three species of Cellulomonas that has a complete sequence genome, which is a very useful tool in trying to understand what bacteria are doing. Because you can see all of the genes that are encoded and all of the potential proteins that they might make. Of these three sequenced Cellulomonas, Cellulomonas gilvus has the smallest genome, while still having comparable amounts of enzymes that are predicted to be involved in breaking down cellulose, which suggests it might be the Cellulomonas with the most minimal system for degrading cellulose, and therefore, the most basic one for understanding. So, going back to this model, it seems that if I believe it's excreting cellulases under aerobic conditions, and tethering itself under anaerobic conditions, the easiest way to test if this is what's actually going on, is to look at the cellulases that are being excreted free from the cells and the cellulases that are remaining attached to the cells. So I used a cellulase activity assay like I have described before, and I separated out by centrifuging the bacterial cells, and that's that hard yellow pellet on the bottom, and the media outside of the cells and looked to see how many, how much cellulitic activity was present in each of these fractions. 

So for cells growing aerobically on cellulose, they had high numbers of excreted cellulases and anaerobically, they had low numbers of excreted cellulases, and this is consistent with what I had seen before. But when I looked at the cellulase activity on themselves, I saw low or no cellulase activity under either aerobic or anaerobic conditions. So, this made me pause and take a step back. What's going on here? We know they're degrading cellulose, there's cellulose breakdown products, how are they able to degrade cellulose if I can't find any cellulases? Very frustrating question. So I had to take a step back and say, okay, if they're degrading cellulose under aerobic and anaerobic conditions, in very different ways, what's happening within the cell and the cellular metabolism has to be a big global change, and there are ways that we can look at global changes in bacteria. And it gets back to something called the Central Dogma. So the Central Dogma says that DNA in the cell is gonna be transcribed into messenger RNA, which is then gonna be translated into proteins, and that will lead to a change into bacterial behavior. So, any bacterial behavioral change, such as differences in cellulase production, can be categorically seen in changes in protein levels, mRNA levels, and then eventually back to which DNA sections are being turned on or off to cause those changes. 

So I chose to look at mRNA levels using something called RNA sequencing or RNA-seq. So to do this, we look at, we take the total mRNA from cells that are grown in these different conditions. And then we use cDNA library to translate it back and get an idea of exactly how many copies of these mRNAs are present under each of these different conditions. And then we can compare the levels of each of these different mRNAs for every gene in the cell under aerobic and anaerobic conditions with cellulos and cellobiose, and then by comparing what is there and isn't there under these different conditions, we can get an idea of everything that's going on within these cells, in terms of what happens when we change their carbon source or oxygen availability. So, looking at every single gene in a whole cell is very daunting so I broke this down into the major components necessary to degrade cellulose. And those are motility, chemotaxis, adherence, and cellulases. So motility is the movement of the cells and they use something called a flagella, which is a large apparatus that is anchored into the membrane of the cell and it uses ATPs to make it rotate, and then it moves around based on the direction that this flagella rotates. And you can see it's actually a very complex structure with many components, it's difficult for the cells to make. So I'm gonna be showing that in further pictures by the little cell with the flagella. Chemotaxis is the bacterial sensory network. 

So you see here a picture of a bacteria with receptors and the receptors bind these little blue signal molecules. So bacteria don't have quite as many senses as you or I do and this is how they interact with their environment. They have different receptors for different molecules and they can sense those molecules and move towards them or away from them and it's kind of then you can see the little line related to, it tells the flagella which way to go.

So there it is, with some little molecules there for sensing. And then adherence. So adherence is mostly used by the anaerobic cellulose degrading bacteria and I have a picture here of clostridium that we saw previously, and you can see it attaching to the cellulose fiber. And it's actually the very tight attachment mediated by the cellulosome. And I'm gonna show it kind of like that in the future. And then we have the cellulases, which I have talked about a lot in this brief time, and those are the enzymes that they use to degrade cellulose. So, these are the four components I'm gonna be looking at for cellulose degradation, and I went and looked at the mRNA that had been taken from cells grown aerobically and anaerobically, and compared what was different when they were growing on cellulose versus cellobiose. 

And there were many genes that were different, but there were also many that overlapped. So in the things that were different were mRNAs involved with metabolism, so like I had shown before, the things necessary for the TCA cycle, glycolysis, as compared to mixed acid fermentation. Regulators, those are proteins involved with which things are on or which things are off, and small hypothetical proteins which are predicted to be involved with other signaling, and those would be the sorts of things that receptors might be targeting. However, all of the things involved with the cellulose degradation machinery that I knew of, cellulases, other enzymes involved in cellulose degradation, flagellar components, chemotaxis, all of those were looking exactly the same under both aerobic and anaerobic conditions. They were both turned on or turned off to very similar levels. So, how does this explain my different bacterial behavior of aerobically, they're excreting lots of cellulases, and anaerobically, they're not, but this mRNA says those cellulases are there, but I haven't been able to detect them. So what I thought at this point was maybe whatever is happening is more subtle than I imagined originally. Maybe it's not a large global change, but a smaller change. So I decided to break down each of these components and look at them in depth with both this RNA-seq data that I had, and some specific assays to get at what was happening with these cells. So, my hypothesis I started out with was that aerobically, cells are motile and they move towards breakdown products such as cellobiose. 

They don't adhere to celllulose, and they excrete their cellulases outside of the cells. Anaerobically, cells also are motile towards this cellulose breakdown products, but with the goal of attaching to this cellulose fiber, so that they can use their attached enzymes to break it down and bring those products into the cells. So, starting off with motility and chemotaxis, I went back to my RNA sequencing data, and I noticed that there were some differences here. Based on oxygen availability, flagellar motor genes, the genes that make the flagella turn, were not as, not turned on as much under anaerobic conditions as aerobic conditions. Which made sense, cells are predicted to be more motile aerobically than anaerobically because they have more energy to do so. And the flagellar membrane components, the things that made the length of the flagellar, were different in terms of on cellulose or cellobiose. But again, some of the flagellar genes were regulated by both conditions. So, to determine how these bacteria were able to respond to cellobiose, I did what's called a chemotaxis capillary assay. So you take a little well of your bacteria, and you take a little capillary with either media or with cellobiose, which the cells should want to swim towards, and you dip it in your wells, and you pull it out, and you count how many bacteria actually swam up your little capillary towards the energy source. And then you can count those bacteria, and what you can see is that aerobically on cellobiose, the number of cells that swam towards them were very high, although at higher concentrations of cellobiose, as it went towards the one up at the very top, they got less motile. 

And this makes sense, because once you find your food source, you don't wanna swim away from it. So the more food sources present, the more they would slow down. If you use just cellulose, you don't see this sort of effect. You see kind of a random, maybe sometimes they went in there, maybe not. It's not a strict response. And then anaerobically, cells were much less motile than aerobically, which makes sense given the energy constraint, but you did still see that trend where, when the cellobiose concentration got higher, they would stop moving. So this supported my hypothesis that the cells would be motile toward cellobiose, a breakdown product of cellulose, that they would want to eat. So what about adherence to cellulose? Aerobically, my hypothesis is that the cells will not attach to cellulose, anaerobically, I hypothesized that they would. So the RNA sequencing data had nothing to indicate whether or not they would be attaching, but all of the normal attachment mechanisms like a cellulosome and those proteins, were not present. 

So I went directly to another one of my phenotypic assays. And to do this, you take a culture of bacteria that's growing and you add in some cellulose. And then you wait for an hour for it to settle, and if the bacteria combined to the cellulose, then they're gonna settle with it, they're gonna, cellulose is gonna kind of pull them out of the media, and if they can't, then they'll just keep swimming around like the culture on the left where they're kind of everywhere. And then you can take a sample from the top of the tube with cellulose and without cellulose and you can compare how many bacteria are there to see what percent of your cells actually attached to that cellulose. And when I did this, I saw something really surprising, which is that a percentage of cells attach to cellulose under both aerobic and anaerobic conditions. Now normally, this attachment is affected by proteins and the proteins will help it bind to the cellulose, like with the cellulosome. So I tried a bunch of different treatments to see if I could change how this binding was working, and I tried making the temperature 32 degrees, which is a more favorable temperature for these proteins to work, and then I tried boiling and treating with proteinase k, both of which would degrade proteins and make it so any proteins involved couldn't bind these cells. And I saw no affect of any of these treatments, which suggested that these are attaching to cells, to cellulose, but not in a protein mediated way. So what's going on? So, I contacted some friends over at Pacific Northwest National Lab, and they performed scanning electron microscopy for me of Cellulomonas gilvus growing aerobically and anaerobically on cellulose so that I could see what was going on with these cells. 

And you can see in these images that there is cellulose attachment by these bacteria under both aerobic and anaerobic conditions, but it's a mixed population. There are also some that aren't attached, and that more cells attach as you go towards stationary phase, so as the cells have more time with the cellulose, they're going to move towards it and attach. But, the other thing that I noticed in the anaerobic stationary column there, was that the cells started looking a little bit different than they were under these other images. They got fatter and fuzzier and more compact, and other people in the literature had seen this sort of affect with cellulose attaching bacteria, and they said it was something called a glycocalyx. So a glycocalyx is a coat of polysaccharides, so sugars, that the cells excrete outside of their membrane that can help it stick to different growth substrates and it's so sticky and dense that sometimes cellulases can get stuck in there too as they're being excreted. So I was wondering if this is what my cells were doing and I ripped off this extra polysaccharide coat, and it turns out that there was actually a glycocalyx there, and the composition, what this was made out of, was the same as that other organism that uses a glycocalyx to attach to cellulose. So I got really excited about this and I went back and said how does it do it, how does it do it? And I found the pathway that it uses to make all of these precursors for the glycocalyx, and I then went back to my RNA-seq data with this new information that there were these other genes that might be involved, and saw that this one there that makes the largest precursor for 65 percent of that glycocalyx, was indeed upregulated on cellulose growing anaerobically.

So, going back to my hypothesis, what actually was happening is that there's this mixed population of cells under both aerobic and anaerobic conditions that are both attached to the cellulose, and not attached to the cellulose. Now this is also something very novel for cellulose degrading bacteria, they usually only do one thing, they either attach, or they don't attach. 

However, this kind of gave me a little bit of insight into why my assays previously looking for cellulases that were attached to cells and not attached to cells, was failing. And that's because if cells are attached to cellulose, their cellulases are going to be binding that cellulose and not free to be using the other substrates that I was giving it in my assays. So armed with this knowledge, I went back to my hypothesis that cells that were free swimming, aerobically, were excreting cellulases, anaerobically, were tethering cellulases, and I performed that same assay, but with a very important change. And that change was I first separated my cells, the same way that I had done in that attachment assay, and I looked at the cells that were free swimming, versus attached to cellulose, and I subjected them to a freeze-thaw to break them away from the cellulose that they were binding to. So then I would have these two different populations, and I did this assay where I looked at the degrading of CMC over time, and what I saw was that the free swimming cells that were not attached to cellulose had more activity of excreted cellulases as compared to cell attached cellulases, both aerobically and anaerobically, and the attached cells, the ones that were attached to cellulose, had more cell associated cellulase activity both aerobically and anaerobically. So what this looked like was that there was cell associated cellulases both aerobically and anaerobically and that the ones that were attached to the cells, the cellulose, had more attached cellulases and the cells that were not attached to cellulose were excreting more cellulases. So I looked at all of these different components and I put them together to make a model for how Cellulomonas gilvus was degrading cellulose. And this model is that cells start out motile, excreting cellulases, which break down cellulose and create a bunch of cellobiose, the sugar that the cells want to eat. The cells then follow this gradient back towards the cellulose fiber where they can attach and continue using their attached cellulases to degrade the cellulose.

And this is a hybrid version then of that aerobic and anaerobic model where aerobically they're excreting cellulases and anaerobically they're attached, in that it does both.

So, the conclusions that I had for my study were that 20 of the Cellulomonas species degraded cellulose and switchgrass, which is a very difficult thing to degrade under both aerobic and anaerobic conditions, and that this likely is a defining characteristic of the genus. And that in Cellulomonas gilvus, degradation of cellulose under these conditions is accomplished using components of both the canonical aerobic and anaerobic stategies, creating a novel cellulose degrading strategy, which is a hybrid of the two. So, this is all very good, but how does it relate to biofuels? So, back when I was talking about biofuel generation, the two most difficult cost expensive steps were this treatment by enzymes and bacterial fermentation. By having a bacteria that can go from the initial processing, just ground up switchgrass directly to biofuels, makes this a one step process, which could be slightly more effective, and by looking at how different bacteria can degrade cellulose, we can potentially find more effective enzymes or better ways to engineer other bacteria to degrade cellulose and make biofuels for us. So future directions for this project are looking at the genome of all these different Cellulomonas and comparing them to see whether Cellulomonas gilvus is indeed a model for all of them, or whether it's unique within the genus, and then using RNA sequencing to determine what's different between the Cellulomonas that are free swimming versus attached, and which other genes and proteins are necessary for Cellulomonas gilvus to attach to cellulose, because if we can find those, we could also engineer those into other bacteria to help them attach as well. And with that, I'd like to acknowledge Garret Suen, my mentor and adviser, Andrew Steinberger, our freshman, well, a student in my lab who's done a lot of work on this project as well. Melissa, who I got the lab from, our collaborators at PNNL who helped us with SEM, JGI, who helped with RNA sequencing, Paul Weimer and Chris Odt at the USDA helped with all of my metabolite analysis, and of course the Agronomy 375 FIG, John Greenler was in charge of it, Gina Lewin was the TA, who helped Maggie, Isaiah, and Madeline isolate Cellulomonas wisconsinensis for us. And our funding sources, the U.S. Department of Energy, the JGI and Oscar Allen predoctoral fellowship from the Department of Bacteriology. And with that, thank you for your attention and I'd be happy to answer any questions that you might have. 

(audience applause)