Speaker: Tim McDermott, Thermal Biology Institute, Montana State University

Title: Geomicrobiology of Acid Sulfate Chloride Springs

Date: April 2004

Back to Science Talks in Yellowstone

Tim McDermott: About six I think. Somewhere around six.

John Varley: Yeah, six. They went out and generated a bunch of money to support it among the scientific advisory council of TBI and so I’m exposed to that on a fairly regular basis and they are doing a lot of great work up there. In no small part due to him (gesturing at Tim McDermott). So he’s going to talk to us today about some of the acid springs and they’re fascinating and he’ll convince you of that fact, momentarily.

Tim McDermott: I hope so. Well, thanks John. And I want to thank the folks who originally invited me down. I never miss an opportunity or try very hard not to miss an opportunity to come down to the park and work in this capacity. One of my jobs is education outreach, clearly. What better opportunity to disseminate the latest and greatest, in terms of what we are finding in these springs, than to speak with people like you because, the ripple effect is huge. Of all the millions of people who come to the park every year, the number of people that you interact with, regardless of how small, results in a very large dissemination factor. So I always jump at the opportunity to do this. Also, let me point out, that I do it in part as a debt to John because when I write grants to the National Science Foundation, which is one of the primary funders of my research, John is always ready to supply these really very well crafted letters of support of my research. To have a total package that is going to be rated highly and acquire funding, you have to have these kinds of letters of support. John has done a great job in that regard and so I view it as a symbiosis, if you will, because anything that John can do to help us get money, and then I come back down and provide as much education and outreach as I can. And everyone benefits.

As the title says, Geomicrobiology of Acid Sulfate Chloride Springs. What I’ll be talking about today is focused almost like a laser beam on Norris Geyser Basin, because that is where I do most of my work. You’ve seen springs like this off the boardwalk at Norris Geyser Basin; the yellows, the greens, the browns, etc. I’ll show you a few more examples of that and try to illustrate or explain to some degree what all is involved.

Before we get there though, I thought it would be worthwhile spending a few minutes going through some microbiology 101, if you will. In a place like Norris Geyser Basin, you hear a lot about bacteria for example, but in addition to bacteria you have eukaryotic organisms as well. Bacteria or prokaryotes are very simple in structure. They are cellular. They have membrane structure. They have some DNA in the cell. It’s not in the nucleus like over here (pointing to eukaryote image). In a eukaryotic cell you have organelles, a much more complex structure. You have a nucleus that contains the DNA. So you can see just by the comparison visually that the eukaryotic cell is much more advanced, much more sophisticated than the prokaryotic cell.

If you find yourself on the boardwalk, and you choose to go to issues of size, that’s what this slide is all about. The top depicts what a virus is in terms of relative size to a prokaryotic cell or bacterium. And this whole large yellow object is about the size of a yeast or a eukaryotic algae. So you might compare a virus such as comparing a golf ball to a beach ball in comparison to a eukaryotic cell. Or a golf ball to a basketball if you are comparing a virus to a bacterium. That gives you some idea of relative size differences.

In terms of the microbes we are talking about in Yellowstone, these thermophiles, as the name implies, are thermal-loving. These organisms are not just hanging by their fingernails, OK. They like it here. If you take them out of the high temperature environment, they either stop growing or they just die altogether. So they not only live here. They thrive here. The upper limit for bacteria is about 113 degrees Centigrade (235 degrees F.) although a recent report in Science by Derek Levi’s Lab, suggests it could be even higher than that. Fungi, the upper limit is about 55 degrees Centigrade , and plants about 48 degrees Centigrade (118 degrees F.). There are many examples of all three categories in a place like Yellowstone. I’m sure you’ve seen many of them as well.

Many of these thermal environments, in particular Norris, in addition to having high temperature have other extremes like ph and toxic metals. I’ll show you a few examples of those in a minute.

These thermophiles are actually part of a big family tree, depicted here with the hypothetical origin of evolution being right about there at the fork between the bacteria, the archaea, and the eukarya. The eukarya are the higher organisms, if you will. The plants, fungi, animals. Of course humans, we’re at that branch with the animals. You can see however how humans, elk, wolves, grizzly bears, etc., the high profile animals in the park, are represented by one single branch on a very, very large tree. These thermophiles can be found in many different places in the tree, however there are certain locations that are … you primarily find them down towards the root of the tree. For many evolutionary scientists, that suggests that early earth was a high temperature environment and that these were the most early evolving organisms, the hyperthermophiles here. The hyperthermophiles branching early within the archaea.

Just to give you some idea of what this acidity business is all about. Neutral ph is pure water, ph 7. As you get increasing acidity, examples include volcanic soils like here at Yellowstone. Lemon juice is a good example of a very low ph, highly acidic type of material. On the other end of the spectrum, like at Mammoth, you have pH’s near neutral and alkaline, ph 8.5 range. There are very few places in the park that (inaudible) this region although, John, I think there are a couple of places around ph 11. So that is why Yellowstone is such an attractive place for people like me to work because you can come up with almost any combination of temperature and ph that you are after. In terms of finding microbes that thrive in these various locations and various extreme environments, again it is like a candy store in that regard.

Now, there are some places in the park that are very toxic. John has heard me say this, and it is not very far from the truth when I say that there are some places, like this location in Norris Geyser Basin, that if it wasn’t in a national park it would be an EPA superfund site. Here are some examples of the heavy metals that we find in this location. Those numbers that are shown in parenthesis are from a garden soil in Bozeman. It gives you an idea of just how different we are talking about here. Here’s lead, .94 (this is in milligrams per liter, soluable) whereas in the garden soil it is less than .05. aluminum 429, Zinc 5.9, manganese, copper. Check out the mercury (75-300 mg/kg). When my colleague at the University of Minnesota first ran the analysis on the Mercury, it broke his machine, it was so high. As a point of reference you might find .2 or .3 nanograms of mercury per kilogram of soil in a farm field or your back yard.

How do these organisms deal with these toxins? I chose arsenic as an example because arsenic is prevalent throughout Yellowstone. Particularly we find it in more acidic ph regions, like Norris Geyser Basin. So what happens is arsenate, shown here as As5, is a chemical analog for phosphate. It can be taken up in the phosphate transport system in the membrane of this bacterium. Once it gets inside the cell, however, the cell detects it and it goes into its arsenic detoxification mode and it will reduce arsenate to arsenite. Once it is in the form of arsenite, which by the way is even more toxic to the cell than arsenate, it can readily dispose of this arsenite through an active transport process. There are transporters in the membrane that will shoot it back out of the cell. So the strategy of the cell is to just get it out of the cell. Just get it out. You can imaging opening a window in your house and just throwing out the dirt. OK, that’s the same thing. If the arsenite remains in the cell, it will kill the cell. But they have adaptive mechanism for actively exporting these toxins. They do the same thing for tetracycline resistance. You hear about antibiotic resistant microorganisms popping up at hospitals, etc. Well, there are genes that code for these antibiotic resistances. In the case of tetracycline, one of the main mechanisms of resistance is an export protein. It takes the tetracycline that gets into the cell and actively transports it back out faster than it can come in the cell.

In reviewing some of the organisms that we find in Yellowstone, again Norris being the primary example here. Staying in the same step-wise grading, of size that I showed you in the previous slide, bacteria, etc, here are some new thermophilic viruses that my colleague Mark Young has discovered in Yellowstone. This one is particularly interesting because it has these little turrets on the surface. The genomes that code for these viruses are so different that when they first started sequencing on these genomes, and they started doing blast searches, that is, you sequence the DNA, and then you take that sequence and you compare it with that which is known in major databases, public databases, and lets say if you extracted some DNA from the garden soil in your yard and sequenced it, you would probably have about a 50% chance of identifying the encoded proteins. Because the databases are so large. You know, 500,000 or so accessions, or even more. It’s growing daily.

When they first did these blast searches with the genomic DNA of these strange new viruses, there were zero hits. Nothing. Suggesting that these life forms are so different that they don’t resemble anything else known to man or known to the databases at least. That is why viruses have been so exciting, so fun to study. Other microbes you find in places like Norris, organisms like sulfolobus and thermoplasma, these organisms again are examples of the hyperthermophiles. They really like the super-high temperatures. They can live on things like hydrogen sulfide as an energy source. They oxidize it to sulfuric acid which contributes to the extreme acidity of their environment.

Moving up the size scale, you have the cyanidia. Here’s Lemonade Creek. All of this green color is due to the chlorophyll of the cyanidium and gualdiaria. This is a microscopic image. These are eukaryotic algae thought to be evolutionarily ancient. My lab is working in conjunction with Dick Casenoles to try to find out more about their ecology. Some, as it turns out, are very, very resistant to things like arsenic, like sulfide, like zinc, heavy metals etc., that you often find in these acid environments. And I showed you an example a few minutes ago.

Here’s an example of a plant that I mentioned in previous slides, Dichanthelium is the genus name. My colleague Rich Stout works on this particular plant. Notice the probe that is in the crown of the plant is registering 50.8. I founded instances of live, viable plants where the temperature probe registered 63 degrees Centigrade. Now they are not necessarily growing at these extreme temperatures but they are tolerant of these high temperatures. So when you see these types of plants, to my eye it is almost like crabgrass in my yard, but it is an interesting plant and oftentimes this will be the only show in town, in these thermal areas, because it can tolerate these high temperatures.

Picture yourself on a boardwalk in Norris Geyser Basin. In fact I took these pictures right off the boardwalk at Norris. You’ve seen these kind of color formations before. You’ve got the brown, the green, more brown. Whenever you seen these color gradations, typically it means you have different organisms setting up shop. You have thermal niches, they are niches along thermal gradients, or chemical gradients, which I’ll show you an example of in a minute. An organism finds that this particular area are best suited for their needs and that’s where they will establish.

Here’s a zagagonium, another algae, black, you see right along the boardwalk. Typically you don’t associate this with high temperatures. You always find it close to some kind of sulfide source, however. I don’t know of anyone who has spent a lot of time studying this particular organism even though it is very prevalent in places like Norris.

Here is a place where you have two different streams coming together. This one has a very bright green color. About two years ago I would have said this one was probably higher temperature because we have a lot of cyanidia there and they are proliferating anywhere from 45 to 53 degrees Centigrade whereas here, the temperature is probably lower, yadayadayada. Now we’re finding that some of our pure culture isolates, their clothial content, is much heavier, much more intense, than some other pure culture isolates, so we know, for example, that the numbers of cyanidia here (pointing at one color in the photo) could be as intense as here (pointing to another color in the photo). They could both be cyanidia but it just could be a different strain. The question would be then, why do we have a different strain of cyanidia here, or gualdiaria for that matter, than here. We don’t know that. We are trying to get at that with some of our studies.

Just another example, this time you see a lot more brown. More often than not, and in fact at this time I would say that all of this brown color is due to microbial activity. In fact almost, AHHH, I’m biased because I’m a microbiologist, but so much of this color is due to not so much abiotic chemical reactions occurring but the microbes that are reacting with the chemical constituents of the water. This water, if you measure it, you will see that it has a very high content of ferrous iron, Fe+2. These microbes are making a living off oxidizing the iron, the ferric iron, which is not soluable, precipitates and forms these mats. I’ll have a better example of that in a minute.

Here’s an example of a low temperature system off the boardwalk, the zagagonium chlorella, probably. I’ve not done the analysis. This is a clover situation. The cyanidium that I mentioned, the gualdiaria that I mentioned, those kinds of algae, their niche is acid ph and higher temperatures, 45 to 53 C. These chlorella can’t withstand those higher temperatures so you will see them associated with this lower temperature zagagonium.

These three springs I have worked a lot with Bill Inskeep, a chemist at MSU. These aren’t the official names but names that we or post-docs have applied to them; Dragon Spring, Beowulf Spring, and Succession Spring. Here’s some chemical analysis data and some physical factors or features that measured pretty constantly at these three different springs. It gives you an idea of the kinds of things that are going on. Temperatures can range from the low to mid 60s up to low to mid 80s, such as here at Succession. Succession, up here in the right panel, is typically right at 80 degrees Centigrade. The ph is remarkably stable; 3, 3.1. The arsenic content, typically is arsenite, ranges anywhere from 35 micromolar to close to, in some cases 100 micromolar. There are very high concentrations of sulfide, ferrous iron, CO2, and hydrogen gas. A lot less hydrogen but our data is beginning to tell us that there are a lot of microbes that potentially are making a living using hydrogen as an energy source. I’ll speak to more of that in a minute. Arsenite, hydrogen sulfide, ferrous iron, and hydrogen are all energy sources for these organisms. They all thrive on these inorganic constituents as an energy source. They oxidize them. In the case of sulfide they oxidize it to elemental sulfur or sulfuric acid. In the case of ferrous iron, they oxidize it to ferric iron, giving it the orange-red-brown mats. CO2 is a source of carbon. They take carbon dioxide and convert it into a glucose molecule or some structural carbohydrate. So everything they need to make a living is right there in the spring. They have the energy source in terms of sulfide, ferrous iron, or hydrogen or arsenite and they have plenty of carbon in the form of CO2. These are super-saturated conditions. Much of the chemistry we are measuring here, I’m showing you, is right at the source. It changes with distance from the source.

These are some up-close images to show you how these different microbial communities develop along these transition points, from yellow, to brown, to green, that I spoke of earlier. Here in the yellow zone you have these rhomboid crystals that are elementus sulfur and you have filamentous bacteria. In the brown zone you have more filamentous bacteria but they are encrusted within this iron that they are oxidizing and which gives you the brown color. And then when you get into the green zone you have these cyanidia, these eukayrotic algae, and of course you have the very large, circular morphologies. The analytical SEM work has verified what you see visibly, that is you have a very prominent iron peak coming from the brown zone and a very prominent elemental sulfur peak associated with the yellow zone.

What we are finding out is very important and is very pertinent to understanding and passing on and conveying this information in terms of colors and gradients and band patterns. There are many gradients operating in these springs contribute to the color formation. We’ve got temperature gradients. For example in this particular graph of Dragon Spring the temperature is shown on the right here. It starts out at about 63 (degrees Centigrade) and then with distance from the source, as you might imagine, it cools down rapidly or fairly rapidly. Last December when we were out there at Dragon air temperature was minus 25 (F) in the morning and that temperature had a significant impact on the water temperature, especially out here farther downstream. When the springs are only a couple of centimeters deep and we have very cold temperatures like that it will impact on the water temperature and impact on the microbes that are living there.

Hydrogen sulfide, as I pointed out in the previous slide, is a potential energy source for some microbes but it can also be toxic. We’re finding there’s a distribution pattern, we think at this point, of different microbes that have set up shop at different distances from the source. So we find, for example, that the sulfide concentration declines rapidly as you get further and further from the source until when you are 4 to 5 meters form the source you drop down to very, very low sulfide concentrations. That corresponds to the end of the yellow zone. So we’re seeing this hydrogen sulfide is gassing off but also it is being converted to elemental sulfur.

Our most recent evidence suggests that a very significant part of that sulfide oxidation is microbial. I’m not going to go on record as saying that the microbes are responsible for all of that oxidation, or a majority of the oxidation, but right now the evidence suggests that they are responsible or participating a lot more than we ever gave them credit for. I’m sure Kirk Nordstrom, for example, might have….It would make for an interesting conversation with Kirk. We’ve converted him on the arsenic issue already though so maybe sulfide won’t be so difficult.

Hydrogen is also utilized by many microbes, not just thermophiles. The source water concentrations are down in nano-molar levels. It’s not very high but when you consider flux issues, it is. It is constantly coming out, these concentrations, then you can begin to understand how it can be important to some organisms. So we have these different gradients of hydrogen that also gasses off; the hydrogen sulfide, the temperature. Some of these things are toxic to some organisms. Some have preferences of higher temperatures or higher sulfide concentrations, or at least tolerances of higher sulfide concentrations. So you can start to see how these temperature and chemical gradients start establishing niches or allowing niches to be established by different organisms. The gradients can occur perpendicular to the steam flow as well as in a vertical direction. One point here I want to make is that when we measure arsenite oxidation to arsenate…We’ll just use it as an example of microbial activity. I don’t want to get into why these organisms are doing it necessarily but we see that it is not happening until the sulfide concentration drops very close to zero. One of our first questions was “What’s up with that?” Is it due to the fact that there are no arsenite oxidizers along the channel here? It’s only out here that we start seeing aresenite oxidation because that’s where they’re at? Maybe they’re sensitive to sulfide. Maybe they’re sensitive to temperature. Any of these kinds of gradient issues.

It turns out the sulfide in the water actually inhibits the enzymes involved in the arsenite oxidation. We are doing some follow up experiments this summer to ask, “Well, what happens if we take sediment samples here and suspend them in water from downstream where the sulfide concentration has dropped to zero, can we demonstrate aresenite oxidation?” These are the kinds of things that scientists get involved in and sometimes they are nitpicky but never-the-less, it helps us understand and model the microbial activities going on here and how the chemical environment impacts on these microbes.

Along the borders of the yellow zone we see these long streamer structures and so we have taken a look at which organisms are involved in the formation of these structures. As it turns out… let me back up… The most recent evidence, only about 10 days old, is telling us that the organisms in the middle of this yellow zone are not the same organisms that are on the periphery of the yellow zone. It gives you yet another example of how gradients matter and how the microbial populations change significantly only by a distance of a few centimeters.

These filament structures are composed of more rhomboid sulfur structures. We also find these very perfectly formed spheres that are elemental sulfur. We don’t know, for example, if there are microbes inside these spheres that are a nucleation source for the sulfide oxidation that forms this elemental sulfur and therefore we have this nice sphere. We have some transmission electron microscopy of it showing that we do have microbes within these spherical structures. A lot more work remains to be done to try to connect these kind of images (pointing) conclusively with these kind of images (pointing) and understand who is doing what with what. But we think we have a pretty good start.

Here are some reactions that I alluded to earlier that are going on in these springs. The hydrogen is being combined with oxygen to form water. Sulfide is getting oxidized into elemental sulfur and can be oxidized further on to sulfate, contributing to the acidity. A couple of reactions that, we don’t know how prevalent they are in Yellowstone, not as prevalent as the first two or this last one (pointing), were ferrous iron Fe2+ is being oxidized to Fe3+, which is insoluble. These are the reactions that are contributing to the microbiology and therefore the colors that you guys are seeing in these springs.

I’m not going to try to convert anybody into a biochemist or microbial physiologist but I thought if we devote just one slide to try to explain how a microbe makes a living by oxidizing these chemical constituents, it might make it easier to comprehend, understand, how they are making a living and why they are existing here. So I chose iron oxidation because in a place like Beowulf you have these very prominent iron crusts. So what happens is, these bacteria, in their membranes, they have enzymes like this Rusticyanin for example that will oxidize the ferrous iron (Fe2+) to ferric iron (Fe3+). They take the electrons that they liberate from the ferrous iron and they pass it down from protein to protein – This they refer to as electron transport. – and in the process they expel these protons, shown here as hydrogen. When these protons come back through a certain channel, know as ATPaise in the membrane it creates ATP. ATP is the common currency in biochemistry. If you’re not making ATP, you’re dying or you’re dead. That goes for us too, not just microbes. ATP is essential. It drives all sorts of bio-synthetic reactions in the cell. So the cell has to have some way of making ATP and this is one of the ways they do it, in particular for these organisms that live in this low ph environments. Protons are always trying to get back into the cell and the iron oxidation is one mechanism that allows them to do that. The same thing happens with hydrogen oxidation, hydrogen sulfide oxidation. I didn’t to the trouble of showing more slides to show the same effect, the same phenomenon.

This is basically what they are doing. They have to maintain that membrane because if they don’t have a membrane to keep things separate, … in fact you might view this as poles on a battery, plus on the outside, negative on the inside. So you have this energy potential across the membrane that allows the cell to do work and sometimes that work involves bringing in phosphate, bringing in certain other nutrients from the environment into the cell that the cell needs. That’s how the cell makes a living, how it generates energy to do all the different functions. These organisms that live in these low ph environments have a different membrane structure that is more resistant to low ph. That’s one of the reasons that a bacterium from your garden would not live in these low ph environments because its membrane couldn’t tolerate this low ph acidity. If the membranes structural integrity is not maintained, the cell dies. It’s as simple as that. They have to maintain that charge difference from the outside to the inside.

(Lights go out in the room.)

Now, I thought it would be interesting … and John has seen this demonstration … many of you, I am guessing, have not. You can all see this luminescent culture. (It is barely visible on the video.) This is a luminescent bacterium, it is not a thermophile at all, but what I am trying to demonstrate here is the speed at which enzymes in a bacterium, or any microbe, or for that matter any organism including us, the speed at which enzymes work.

If we now take a look at this biochemical pathway, again this is not a biochemistry class, but the emphasis of this slide is to show you that all of these kinds of things have to happen before luminescence happens. You have some redux reactions going on up here (pointing), you have some oxygen being introduced in the pathway at this stage (pointing) and then finally you have this release of a photon of light.

So what I am now going to show you is the same luminescent culture except now I have it in this long tube. (Tim takes a moment to try to block the light from the projector.) So, same luminescent culture except it is not glowing. Look what happens when I invert the tube and the oxygen bubble that is at the top of the tube goes through the culture. (Again the luminescent activity is barely visible on the video. The tube lights up quickly from bottom to top.) Remember now, as the air bubble goes through the culture, the oxygen has got to diffuse in the medium and then be taken up by the bacterium. And so, all of this happens, just like that (snapping his fingers).

So all of those reactions you just saw in the pathway, all happened in a millisecond. That’s the time scale in which enzymes operate and that’s the time scale in which microbes also operate. As it turns out, those kinds of enzymatic capabilities are one of the main reasons that these organisms are interesting to many people, in particular the bio-tech industry. I’ll get to that in just a minute.

That leads me to the next part of the talk which is, who cares who lives in these extreme environments. Well, NASA’s exobiology program does, for one. They are interested in finding out more and more about life in extreme environments on Earth so it helps them understand what is possible out there beyond Earth. The picture on the left just shows a scientist who has found a meteorite in a place in Antarctica where there is so much snow and ice that when you come across a rock that is miles away from the nearest mountain, you’ve got to ask, how did it get there? Typically it’s a meteorite. It’s a great place to find meteorites. At one time there was a big brew-ha-ha about microscopic evidence suggesting that some of these meteorites had microbial fossils. Well, a lot of people spent a lot of time looking at those meteorites and concluded that , well, maybe it’s not such a worthwhile hypothesis to continue chasing. There is other evidence. A place like Mars could have supported life at one time because of all the work that is going on in places like Yellowstone, Antarctica, the Gobi Desert, all these different places, we found one common denominator that is always necessary is liquid water. If you have liquid water, a microbe can eak out an existence, it can eak out a living almost on anything or nothing for that matter. Things like temperature extremes or ph extremes are really irrelevant because we find organisms that are capable of tolerating all of these different kinds of extremes.

Here is an image from a satellite shot over the surface of Mars. You can see from the scale here, I don’t have a scale bar here, but this is in hundreds of kilometers. This is a picture of one location on Mars where there was massive movement of some kind of liquid. You can’t tell what kind of liquid but certainly you can see it is erosion type geology suggesting that there was some kind of liquid that moved across the surface of Mars. So there is always the issue, If we can’t find life on Mars now, can we find evidence that life once existed in a place like Mars? That’s what the rovers on Mars are trying to do right now. It’s research in places like Yellowstone that helps the engineers and scientists at NASA understand the kinds of experiments that they want to try to program into these probes. The kinds of experiments they want to conduct so that if they can find the smoking-gun evidence that life once existed on Mars, they would be able to determine it with some authority.

What is the connection with a place like Yellowstone between NASA’s interest in life in extreme environments? Here is Jupiter with a couple of its moons, Europa and Io. It turns out there has been volcanic activity on Io. Here are a couple of shots. So that is the connection with a place like Yellowstone. They are interested in high-temperature environments. What does it take for a microbe to make a living? What are the chemicals signatures or bio-chemical signatures that we should be looking for to try to obtain evidence that life does exist in a place like Io.

Moving on to another area of interest, that is the bio-tech industry, they are also very interested as John and Christie can attest, in terms of the number of permits that they’re working with every year. I don’t know how many permit applications are coming from the bio-technology industry but it is my impression that there are still several. Here’s some of the reasons why bio-tech is interested in these extremophiles.

Extremophiles, like thermophiles that have adapted to high temperatures, like Yellowstone hot springs, are of interest because there are a lot of applications of heat-stable enzymes, one I’ll mention in a minute, that I think you are all aware of, is taq polymerase. These microbes are the natural place to look for these high temperature stable enzymes. They have no way of insulating themselves from the environment so whatever temperature it is outside is the same temperature on the inside of the cell. So these enzymes are performing life-sustaining functions and therefore they are active, they’re stable, and therefore these organisms are a great place to look for enzymes that are stable at these high temperatures.

There are some industrial processes that can use bio-catalysts or enzymes that are stable under these high-temperature conditions. Enzymes are cheaper than organic synthesis and organic chemists will tell you that he, or she, can synthesize anything, almost, in their laboratory, and that’s true but many times it is very expensive. If you have an enzyme that can convert ‘A’ to ‘B’ it’s much, much cheaper however the enzyme has to be stable to the application conditions and sometimes they’re exothermic and they generate a lot of heat. So that is where again, these extreme enzymes from these extreme organisms come into play.

Who has heard of PCR? So I’m preaching to the choir here. The Polymerase Chain Reaction enables scientists to amplify DNA. There’s a heat cycling step here in one part of the Polymerase Chain Reaction so that is where you have to have this heat stable enzyme this taq polymerase that was purified from Thermos aquaticus in Yellowstone. Do I need to say anything more about…I admit, I forgot about the crowd I was talking to… If anyone should know taq polymerase and the Polymerase Chain Reaction, it is you folks. Do I need to say anything more about that? I can expand a little bit if you like.

Basically in a Polymerase Chain Reaction you take a DNA template and you apply heat. That will denature the double-strand DNA into two single strands. You add primers that will kneel at certain locations within the DNA and then you use an enzyme, a DNA polymerase, shown as a blue dot, that will then amplify or replicate the DNA starting from that point of primary kneeling. You then apply heat again to re-denature the DNA into two single strands and you start the whole process over again. In the initial invention of PCR they did not have a heat stable enzyme so they had to keep adding enzyme after each heat denaturation step. But with the discovery of taq polymerase, that no longer was required.

And so, again many of you are aware of this information. In fact, this picture up here, I obtained from John (Varley) and Kevin Schneider. But taq polymerase is from Thermos aquaticus, discovered, or isolated from Yellowstone National Park. It’s been used in a variety of application: criminal identification, medical diagnoses, managing Yellowstone’s natural resources, medical diagnosis prior to PCR, and this is an example of how PCR impacts a society in a very positive way. Everyone is aware, for example, of the internet, I mean we use it everyday, and so yes, it has impacted society, but so has PCR. Prior to PCR, prior to the discovery of taq polymerase, if a physician suspected that that you had a genetic disease, it would be very difficult or impossible for that physician to make the correct diagnosis, in anything short of maybe a year or so, in terms of extracting the DNA, sequencing the DNA, and determining, yes, the sequence has changed therefore it looks like a mutated gene, that’s why you no longer have a functional enzyme, therefore you have the disease.

Now, the physician could have the answer within 48 hours due to PCR. Last century was a century of physics; this century is a century of biology largely because of polymerase chain reaction –tracing back to Thermos aquaticus.

Here is some more information that I obtained from Kevin and John. These are some of the applications of some of the research emanating from the park. Many of these I couldn’t elaborate on for a couple of reasons, one is that either I don’t know enough about them, or two, they are proprietary information so we are not going to find out any more about them. But suffice to say that there are a large number of applications here that you know, you think about it, if someone the boardwalk were to ask you, “Well, how does it matter? Why does it matter?” Then you can start rattling off applications: used tire bioremediation; food processing enzymes; acid mine reclamation. The enzymes that are being advertised to be in laundry detergent agents are thermal-stable enzymes. Think about it, you are using hot water and you are also using alkaline conditions, so any enzymes, including in the laundry detergent that’s going to remove the blood or grass stains, proteases for example, they are all going to be thermal-stable enzymes, and they all are going to be stable at high pH conditions, which are prevailing in your washing machine. And they all derive from these extreme organisms.

Now here’s another example that did not come from Yellowstone, but I wanted to through it in because this is yet another example of how microbes have changed things significantly in a very positive way for society. Some scientists at Sandoz at one time it w as common practice at one time that whenever they went on vacation, for example, they always take plant or soil samples and bring it back to the lab and they would isolate organisms that generate antibiotics. In this particular case the scientist was touring Norway with his wife and his wife wanted to take a picture of a fjord and so they stopped the car, and while she was taking the photograph, he was sampling soil in the road ditch. Well, eventually the isolated a fungus that generated an antibiotic known as Cyclosporin A. Now in terms of impact, has had as big or probably even more impact than PCR. It could be argued either way, nevertheless, let me give you some more information—you can then make your own mind up. This particular antibiotic has antifungal activity; its active against malaria; it has anti-inflammatory effects for people who suffer from auto-immune diseases; it does have the potential for controlling the HIV virus; and this last category is selective immunosuppressant. As it turns out the cyclosporine A is used in organ transplant surgery; it suppresses your immune system. You remember when surgeons initiated organ transplants? The biggest problem was rejection. Remember? They had to spend lots of time coming up with the perfect match. Well, Cyclosporin A suppresses your immune system. Transplant patients have to be on Cyclosporin A or some derivative of this drug for life to continue suppressing their immune system, otherwise they’ll go through organ rejection. But this is an example of a metabolite, and antibiotic synthesized by a microorganism t hat has a profound positive impact on man. It also had a profound impact on somebody’s wallet, too.

Ok, here are some more examples of application of microbes, in this case, thermophiles in Yellowstone. I colleague of mine, Joan Henson at MSU, she discovered a fungus that lives in the roots of this (inaudible) that I mentioned a few minutes ago. She found that this fungus, by mechanisms that are not quite understood, bestows her tolerance on plants. These seedlings are watermelon seedlings, ok, grown to 38, 40, 46 and 50 degrees centigrade. The plants on the left were not inoculated with the fungus; the plants on the right were inoculated with this thermophilic fungus. It gives you an example of – the potential application her is, you can imagine agriculture. By using this fungus as a seed born inoculant—that kind of technology, by the way, is very common, so it’s not like you’d have to create a whole new set of technology to transfer this fungus to another environment. But if this fungus could be used as a seed born inoculant to enhance thermal tolerance to wheat, for example, then you might be able to expand the growing range of wheat throughout the world. One example.

Keith Cooksey, in collaboration with some scientists at Ohio University and Oakridge Laboratories, isolated some thermophilic cyanobacteria and used it in a reactor design which is geared towards scrubbing of CO2 rich gases coming from a coal-fired power plant. So the idea here is to pass this hot flue gas through this reactor and these panels you have these cyanobacteria (phototropes) that would use the CO2 to convert it into cell biomass. Because they are thermophiles they are not worried about the high-temperature application and so they reduce the CO2 content in the flue gas so the exhaust is now something more tolerable in terms of CO2 emissions.

I am beginning to wonder if we need to go through bioprospecting Yellowstone, you guys are probably very familiar with that. Should I go through some of this?

Varley: Probably most of the people have not been exposed to this.

McDermott: Have not?

Varley: Yes.

McDermott: Ok. Most of this information, again, I got from Kevin Schneider and John’s office. As you also remember Diversa corporation in San Diego, Calif., at one point several years ago entered into a contract with Yellowstone and the National Park Service, and this contract was to allow them to take samples, to bioprospect in Yellowstone. As I recall some of the facts, and John jump in at anytime that I misspeak, it was almost immediately attacked by certain groups that were very much against this kind of activity. Well, this original agreement Diversa and Yellowstone had the following traits: Diversa could not sale park resources; there are no exclusive rights granted; there are no third-party transfers; and Yellowstone would retain the right to audit Diversa’s books.

As I said, this agreement was immediately attacked. The Edmunds Institute, the International Center for Technology Assessment, the Alliance for the Wild Rockies and some private citizens joined together in a lawsuit to try to null this contract. The plaintiffs alleged that the Yellowstone-Diversa CRADA or cooperative research and development agreement was illegal. There are many laws on the books that ultimately the courts ruled the CRADA was proper and did not constitute a consumptive use of Yellowstone or any other National Park for that matter and therefore was consistent with the National Park Service mission.

Now, there are several different laws on the books, bills laws, that the courts used to make this ruling. There’s the Organic Act that created the National Park Service in 1916 and defined one of the missions as “to conserve the scenery and the natural and historic objects and the wild life therein and to provide for the enjoyment of the same in such a manner and by such means, as well will them unimpaired for the enjoyment of future generations.”

Now, this is where John’s and Christy’s job comes in because they need to ensure that the kind of work we are doing is not consumptive and we a re not damaging these thermal features. The main thing to keep in mind here is the kind of sampling we do is not by backhoe or front-end loader as you might imagine with a mining operation. And perhaps that was some of the thought that went into the objections of this kind of activity. We are talking about thimblefuls of soul or just a few mills of water, sometimes a little bit more than that but on a scale basis, w e are talking about very, very small-scale samples. As it turns out from our experience at a place like Dragon Spring, we take a sample of that yellow zone, for example, the spring will regenerate that yellow material in just a few days if not in a week or so. For example, last summer I was out there with the Yellowstone Association Institute group and I came up to Dragon Spring and the yellow zone was just completely wiped out, gone, just a very faint yellow deposit at the bottom of the stream sediments. I was shocked because I had never seen it like that before. I didn’t quite know how to react accept that, well there you go, you got it. It’s something we hadn’t seen before, but it happens I suppose. Well, I came back two weeks later with a different group and that yellow zone had completely restored itself. So the rejuvenating power of these springs is significant because it is almost like a chemostat where the nutrient flow is constant and you are never going to get rid of all these microorganisms. Not that we are trying and that our sampling activity would take it to that extreme, but that is an example of a natural phenomenon, whatever caused it, literally wiped out the spring and yet it rejuvenated itself, rebuilt itself.

But the point here, this slide, is the kinds of sampling activity we take part in is as unobtrusive as possible. The Federal Technology Transfer Act allows federal laboratories to enter into benefits-sharing agreements such as that between Diversa and Yellowstone. The non-federal parties provide funds, personnel, services, facilities, equipment and other resources, that Diversa has subsequently done for Yellowstone. And by way of doing some genetic marker analysis of the Yellowstone bears and wolves. This federal technology act authorizes CRADAs and benefits-sharing. These kinds of CRADAs had already been in effect and operating with other federal agencies like NIH and DOE.

Then there’s the National Parks Omnibus Management Act: “The Secretary of the Interior may enter into negotiations with the research community and private industry for the equitable, efficient benefits-sharing arrangements.” Which is, as I understand it, exactly what the Diversa agreement was all about.

So I am left with the situation of describing or summarizing the kinds of things we’re doing. We don’t, in terms of biotechnology and applications at MSU, we don’t necessarily seek to develop research along the lines of biotechnology or commercialization. There too many companies like Diversa that do it much better than we do. They have a lot more resources to throw at it than we do, and that’s what their focus is. Every once in awhile we’ll come across an application like those that I described a few minutes ago about the phototropes that are cleaning up the exhaust gases of coal-filed power plants; the fungus that provides enhanced thermo-tolerance of plants—those kinds of applications. If you are aware of the literature, sometimes you stumble into these observations and you say, “Aha, ok, so this may have an application.” And so we pursue it to that extent. But we don’t set out in our research to try to do technology per se.

With every talk you have your acknowledgements, and for me, the lab personnel here have been very important to the success of my research; funding from these funding sources; and permitting and help from John Varley and Christy Hendrix. This is my 14-foot self-bailer hitting House Rock a couple years ago on the Gallatin. So, there must be someone else I must acknowledge because that was an interesting flow through that water.

Thanks for your patience. I would be happy to try to answer any questions at this point.

Q (John Varley): Tim, what do you know about the membrane that survives a pH of zero?

A: I personally don’t know anything about that particular membrane. When you get into these acid environments, they are so selective that your community diversity drops off significantly. There aren't very many organisms that have the capability of synthesizing a membrane that can tolerate these kinds of pH extremes.

Q: So if we think of an analogy... It’s so hard for me to comprehend an organism or any tissue surviving.

A: So here’s what it might feel like. I don’t know what kind of analogy you are looking for, but if you have a cut on your finger, for example, and you happen to drop a drop of lemon juice on it, it will sting like the dickens. That’s what acidity feels like. I don’t know if that’s what it feels like for a microorganism. I don’t think they have sensors and feelings, well, they do have sensors, I can’t say that, they do—they are always sensing their environment, but I can’t honestly come up with a good analogy to explain other than acid in a cut, how low pH might seem to any other organism. Is that what you were going for there?

Q: Well, ahhhh, if you stick your hands in there, you’ve got a rubber glove….

A: Yes, yes, always.

Q: So, then can we assume that without a rubber glove of some kind…. a rubber glove is inert, it’s not a tissue; that’s where I am stuck.

A: Well, the membrane’s structure is such that the hyrdrophobic portions help repel water. Everything inside the cell is kept separate, it’s a separate compartment from the outside. The cell can adjust the components and the structural components of the membrane relative to different environmental conditions. We find that in (inaudible) ameciaphylic? organisms, that is, non-extreme organisms. You change the phosphate concentration in the medium and you’ll see that the phosphate levels in membranes of microbes will drop off but they will compensate with some other kind of chemical structure—anything they can do to maintain that membrane integrity. These membranes can be viewed like out skin. I mean the skin separates our body from the environment. I guess our skin is similar to a membrane in the function of separating inside from outside. We don’t generate energy across the skin, like the microbes use the membrane for energy generation, but maintaining that separate compartmentalism between inside and outside is no less essential.

Q: It’s some tough skin.

A: It is. It is.

Q: Tim, can you kind of elaborate for us how researchers are able to come in and make these discoveries like tac polymerase without bioharvesting per se. They don’t have to collect tons and tons of microbes to use.

A: Good point. So Diversa, for example, when they take a sample of soil, sediment or water, they’ll extract all of the DNA out of that sample, and they’ll clone it into these plasmids, which they then stick into ecoli, a lab rat. They’ll use ecoli as a host vector to amplify, to continue growing and making this DNA that they extracted and cloned into these plasmids. These plasmids are smaller DNA elements that are self-replicating and replicate separately from the genome—the main chromosome of the organism. and so, they can go in and take one sampling, clone all the DNA into these plasmids and then they freeze it away and it’s there forever., unless there freezer breaks down, they lose what’s ever in the freezer. But they always have back-ups, okay. I don’t want to be facetious, but the point is…is that kinda where you are going with this? But the sampling is a one-time thing and in academia we often have to go back and resample to verify that ok, what we observed this one time is exactly what’s going on, ok, it’s just not a one-time thing. As it turns out, these springs like Dragon, like Beowulf, are very stable. You have temperature fluctuations and so you will see some wobble in chemistry, but it is pretty remarkable how stable they are. So we don’t have to go in and resample over, and over, and over again unless we have to acquire some observations that are a new phenomenon, say one day we set up some experiments to measure hydrogen sulfide oxidation, and the next day we might set up experiments to hydrogen oxidation kinds of things. We try to double up on the samples, and, indeed, when we take samples back to the lab, I’ll keep them in my incubator for yonks? I’ll just keep them and keep adding moisture to them with the idea that I may be able to use them for some application at some point, rather than through them away. We do try to extend the usefulness of each sample, and the Diversa example, one sampling time is all they need to acquire all of the DNA that they need.

Many of the people working in the park focus on (inaudible) So we amplify the DNA, that gene, that portion of the gene, we sequence them to find out who’s home, because each sequence is different for each organism. What we are finding is that there are different sequences down the middle of the yellow zone than there are on the edge of the yellow zone. The information available to us in the literature, the closest relatives of these organisms here and here suggest that there are many organisms in the middle of the mat involved in hydrogen and hydrogen sulfide oxidation, whereas the organisms on the edge of the mat seem to be, at least based on the similarity of characteristics of the organisms, are more involved in sulfur reduction. Now, is that due to tolerance of sulfide concentrations? Maybe these organisms on the edge of the yellow zone aren’t tolerant of sulfide. As I mentioned, sulfide can be toxic, or is toxic to many organisms. Hank was just telling me that he’s monitoring sulfide concentrations in a place like Yellowstone because sulfide can be toxic. Wasn’t it several months ago that some bison were found dead because of sulfide, or CO2 poisoning? Which one was it?

So this is an example of how these gases can be toxic to organisms, and there’s no difference with respect to microbes. So we don’t know if their location is due to intolerance of sulfide for example…sorry, the location of the organisms on the edge of the mat is due to their intolerance to sulfur, or it could be since the temperature is cooler there that’s where they are happiest. You can almost calibrate your thermometer to where you find the cyanidia, it will almost be, bang, 47 degrees, 48 degrees. It’s consistent, very reliable in that regard. It’s difficult to necessarily apply function to location because so many organisms are capable of, for example, using hydrogen as an energy source. But because they can’t handle the high temperatures, you won’t find them closer to the spring source, but rather you will find them farther down stream, even though they would to use the hydrogen as an energy source. It’s a combination of overlaying chemistry and temperature gradients that influence where these organisms are going to set up shop.

Q: And are they all bacteria?

A: Yes, they are all bacteria in that yellow zone. We don’t any eukaryotic organisms. It’s when you get in the cooler regions that the green bands we saw along the edge of a place like Dragon, that’s where you have the cyanidium.

Q: Well, wouldn’t it seem then (inaudible)?

A: Yes and no. It’s how we view it. If you view it in terms of spring flow, you might say, well they are arranging themselves relative to and parallel wit the spring flow, but likely, more than anything they are arranging themselves relative to temperature in that case. Remember that one slide I showed had the red arrows going parallel with the spring flow, perpindicular…

Q: (inaudible)

A: Well, when you get farther down stream, the water is slowing down, the spring current is slowing down considerably, and so things are almost like a shallow pond at a place like Dragon, as opposed to a constant stream. It’s just a question of where you are at in location with these gradients, temperature or chemical gradients. And the gradients can go that way, this way, this way, actually, in all dimensions. You have to be a little bit careful about where you are sampling it so you can with some certainty determine that, OK, the temperature is cooling from here to here to here and you are setting a micro transact if you will and taking your samples along that temperature gradient, keeping in mind that you may also be sampling on top of a sulfide gradient. But if you keep track of your stream flow, you might be able to isolate a sulfide concentration to a certain concentration running this way and sample this way across a temperature gradient a s the water cools closer and closer to the edge. So you have to be just a little bit careful how you do your sampling so that you become more aware of what might be causing chemical signature differences…sorry, the molecular signature differences that were are encountering in a place like the middle part of the yellow zone versus the edge.

Q: So pool depth and flow rate and maybe even the substrate all would effect…..

A: Pool depth, knowing water depth but also the sediment depth. Some other relatively unrelated research at MSU, biofilm research, they have shown using oxygen electrodes, micro electrodes that if you have a 100 micron thick biofilm of psuedomonstreigarosa it’s an anaerobic organism being grown aerobically. As you pass that electrode down through that 100-micron thick biofilm you find that after the first 20 microns it goes completely anaerobic because all of the microbes are consuming all of the oxygen faster than the oxygen can diffuse into the biofilm. If you were to grow microbes on an agar plate, ecoli for example, in the center of that colony it would largely be anaerobic, again because oxygen consumption exceeds oxygen diffusion. And so, the spring sediments in a place like Dragon we fully expect to find…..I’m trying to think of some of the names of the guys who are coming this summer, we just found out, they specialize in micro electrode work…Meco Cool is coming. We hope to be able to measure sulfide, oxygen concentrations as you move farther and farther into the sediments. It’s a perfect example of yet one more gradient that they are all sort of mixed in on top of each other. But spring depth, water depth does matter. Oxygen solubility is lower at higher temperatures and so if you have a large depth like some of the deeper springs in the Mud Volcano area, the ability of oxygen to diffuse into the sediments at the bottom of these springs is pretty limited. So the oxygen concentrations there I would assume would be close to zero.

Any more questions?

John Varley: Thanks again, Tim.

McDermott: Oh, thank you for having me. I appreciate it.

Audience applause.