Spring 2009


In search of hot springs to sample, scientists hike along Yellowstone's White Creek, which is fed entirely by the dozens of springs that dot its path. Kelly Gorham

On the ice boardwalk above Black Pool, Tom Schoenfeld is working as fast as his numbing fingers will allow. He lowers a hose into the steaming pool, one of dozens of hot springs in the West Thumb portion of Yellowstone National Park, and then slides the other end into a keg-sized plastic jug. Shuffling along the slick wood planks, he begins piecing together the rest of the water filtration system that he developed for Lucigen Corporation, a Madison-area biotech company where he is vice president of enzyme discovery. The system, which concentrates the bacteria and viruses living within the spring, takes about 30 minutes to set up and an additional two hours to produce a couple of liters of teeming liquid. And though Schoenfeld arrived here at the break of dawn on this freezing September morning, he barely has enough time to get his work done before the tourists arrive. Tourists always delay things.

Despite its name, Black Pool is gemstone blue and perfectly clear. Its scalding waters produce a thick steam that rises from the pool and floats across the boardwalk, enveloping Schoenfeld in a fine mist. Water droplets soak his clothes and frost his eyelashes. As he reaches to switch on the generator that powers his equipment, he hesitates. There’s water all over it-and on the electrical cords leading to the pumps.

A thick mat of colorful microbes carpets the outflow channel of Yellowstone’s Octopus Spring, where water is so scalding that only a few microbes survive. Kelly Gorham

“Wear your gloves when you turn that on, man,” says David Mead, Lucigen’s president, who has accompanied Schoenfeld on seven visits to Yellowstone’s hot springs. As much as they can, the two look out for each other in the field and so far so good. Neither has been electrocuted, and they hope to keep it that way.

Electric shock, however, is only one of many perils they face collecting biological samples from Yellowstone’s springs. Once, a herd of unruly bison flushed the researchers from the edge of a backcountry pool, forcing them to wait several hours before they could return to collect Schoenfeld’s equipment. Another time, hiking through a dense forest, Mead tripped and narrowly missed impaling himself on the jagged branch of a downed tree. But above all else, they worry about the hot springs themselves. According to the book Death in Yellowstone, at least 19 people have died after falling or jumping unwittingly into the park’s pools, which are heated by an underground volcano. And as the book relates in sometimes gruesome detail, no matter how quickly a person scrambles out, falling into near-boiling water is a death sentence.

But for Schoenfeld and Mead, these risks are worth taking. That’s because they believe there are million-dollar microbes living in the park’s pools-bugs that, if found and studied, could unlock the doors to major medical breakthroughs and biotechnological advances. And if history is any guide, the bugs are almost certainly there, surviving and thriving in conditions that would kill almost any other form of life.

All Schoenfeld and Mead have to do is find one of them.

Welcome to the adventurous world of extreme microbiology. Like hundreds of other research scientists, Mead and Schoenfeld travel to Yellowstone to seek out extremeophiles-microorganisms that thrive in extremes of temperature and pressure and other inhospitable environments. These bacteria and viruses survive not only in hot springs but in metal-contaminated soils, pools of acid and lakes so salty that crystals bejewel the shoreline. The researchers who seek them out are partly motivated by curiosity, but also by the realization that extreme forms of life, like high-endurance athletes, have some extraordinary abilities. They harbor powerful proteins-known as enzymes-that enable them to make the most of their surroundings, efficiently turning otherwise inaccessible materials into the food and energy needed to sustain life.

“The bottom line,” explains UW-Madison microbial geologist Eric Roden, who teaches an undergraduate course on extremophiles, “is that extremophiles can do things that other organisms can not.”

Scientists first stumbled onto these rare organisms just a half century ago, when microbial ecologist Thomas Brock, then a professor at Indiana University, found a type of bacteria living in Yellowstone’s Mushroom Spring. Brock, now an emeritus professor of bacteriology at UW-Madison, had been searching for an ecosystem hot enough to support only a few forms of life. On a field trip to Yellowstone in 1964, he began examining the spring’s outflow channel. Starting at the cool end of the channel, where a lush, colorful mat of organisms covered the streambed, he worked his way up to hotter and hotter sections.

Becky Hochstein, a former Lucigen staffer now at Montana State University, scoops water from one of Yellowstone’s 10,000 hot springs in a search for heat-loving life. Kelly Gorham

“When I got up close (to the spring), I started seeing this stuff,” says Brock, who joined the UW-Madison faculty in 1970. “It didn’t have any pigments. It didn’t have any chlorophyll or anything like that, but it looked like it was alive.”

And it was. Brock was able to grow and study this “stuff” in the lab, and in this way discovered the first extremophile, a heat-loving bacterium he named Thermus aquaticus, which is capable of growing at temperatures up to 80 degrees Celsius, not far below the boiling point of water. (Later, it was discovered that T. aquaticus lives in most residential hot water heaters, a harmless squatter.)

While this discovery inspired some of Brock’s academic peers to start studying extremophiles right away, the part of the story that galvanized commercial bioprospectors came two decades later, when Kary Mullis of Cetus Corporation requested a sample of T. aquaticus, among a number of other thermophiles, from a microorganism distribution facility. Mullis was searching for heat-tolerant enzymes that could expedite a common, but cumbersome, DNA analysis procedure called the polymerase chain reaction, or PCR. By chance, the enzyme from T. aquaticus worked wonderfully. It’s hard to overestimate the impact that this enzyme, known as Taq polymerase, has had on science; it lies at the heart of genetic testing, as well as the forensic technique known as DNA fingerprinting used to solve crimes and determine paternity. In 1991, Cetus sold the pertinent patents to Hoffman-La Roche for $300 million, and since then, this technology is believed to have generated more than $2 billion in royalties.

The commercial success of Taq polymerase helped spark a wave of entrepreneurial interest in extremeophiles. Researchers began to plumb remote environments that had previously been assumed too harsh to support life-places such as permafrost soils, deep-sea vents and the acidic channels flowing from contaminated mines. The microorganisms turned up through these efforts comprise a motley and interesting group. Some munch on dynamite. Others are able, after exposure to huge doses of radiation, to fix their DNA in just a few hours. Another group thrives in lakes as acidic as battery acid.

“I just think they are cool from a biology point of view,” says Charles Kaspar, a professor of bacteriology at CALS who has researched extremeophiles. While his main research program centers around understanding the bacterial pathogen E. coli, Kaspar has indulged an interest in acid-tolerant organisms, studying a microbe isolated by a former UW colleague from an abandoned mine in California. He hopes to travel to Costa Rica to scour an acidic lake on top of a volcano for similar bacteria.

But extremeophiles have major implications for the biotechnology, medical and manufacturing industries as well. Their enzymes are used to facilitate certain large-scale reactions, such as extracting metals from composite rock and converting chemicals into new forms. Some of Kaspar’s work, for instance, led to the engineering of an acid-tolerant strain of yeast designed to improve ethanol production. But extreme life is at play in more mundane processes, as well. The protein-busting enzymes at work in some laundry detergents trace their roots to thermophilic bacteria.

“It makes sense to go to natural organisms for biologically active molecules,” explains Jo Handelsman, a UW-Madison professor of bacteriology, “because these molecules have evolved, in many cases, over billions of years of natural selection…to perform (a certain) role in nature.”

David Mead teeters over a boardwalk as he checks the temperature and acidity of a pool, which tells him whether it may be a good source for biomass-degrading extremeophiles. Nicole Miller

Bioprospecting is one of the major thrusts of the Great Lakes Bioenergy Research Center, the U.S. Department of Energy-funded research effort charged with developing non-food sources of biofuels that is housed in the UW-Madison bacteriology department. The GLBRC helped fund Lucigen’s expedition to Yellowstone in the hopes that Mead and Schoenfeld might uncover naturally existing microbes that are exceptionally good at breaking down wood and plant material. By searching in hot-water environments, the researchers hope to find bugs that not only can degrade biomass but are already well adapted to the high temperatures used in the early stages of biomass conversion.

But finding the desired bugs is an inexact science. While Mead and Schoenfeld have worked out some rough guidelines about which pools to sample, they really have no idea what they will scoop up from any given pool. Using the jury-rigged system he designed, Schoen-feld probes the park’s hottest pools, searching for viral enzymes for DNA analysis applications. As Schoenfeld monitors his system’s progress, Mead meanders around to find nearby springs, measuring each one’s temperature and acidity. When he sees a pool with a few bits of decayed grasses or twigs, he gets especially intrigued. Such pools seem likely to harbor bugs that survive by degrading plant material. When the conditions seem right, he dons a heat-proof glove and submerges a liter-sized plastic bottle to collect a sample for analysis back at the lab.

Mead has been lucky before. While working as a staff scientist in a UW-Madison chemistry lab, he helped develop a technology known as TA cloning that became a multi-million dollar product for Invitrogen, the California biotech company that licensed the approach. This success, as well as subsequent research experiences, inspired him to start Lucigen in his basement. “But you know what they say about lightning striking twice,” he laughs.

In 2000, Mead hired his first employee, Schoenfeld, who proposed the idea of bioprospecting among Yellowstone’s hot springs for viral enzymes. “When I first started thinking about this type of research,” says Schoenfeld, “I pulled out some review papers that said people were detecting (viruses in) ocean water and lake water, but nobody had even thought about hot spring water.” Before joining Lucigen, he’d already proposed this idea at two other Madison-area biotech companies and been shot down. Scooping up a super-bug containing a super-enzyme not only relies on luck, but some tricky lab work. Before it can be identified, the gene that encodes that super-enzyme must survive the sample preparation process, which often involves chopping all of the sample’s genetic material into more manageably sized pieces. Finally, should the enzyme be found, there remains the monumental task of developing it into a useful product that people will buy.

“Until you come up with a product to sell, (the discovery of an enzyme) doesn’t really matter,” says Schoenfeld. “And you need to make it user-friendly so the customer can just open up a kit and make it work.”

While Mead was undaunted by the challenge, he says it was tough finding funding for Lucigen’s field expeditions. Even when they finally succeeded, winning a Small Business Innovation Research grant from the federal government, the reviewers didn’t hesitate to make their reservations known. “Reviewers don’t usually fund fishing expeditions,” says Schoenfeld. “But in this case they said, ‘Sure, it’s a fishing expedition, but if it works, it would be worth it.'”

Schoenfeld applied for a research permit from Yellowstone, which approves between 30 and 50 such permits for microbial research each year. Early on, they met with some luck. Just as their first grant was running out, they discovered a new type of DNA polymerase-an enzyme similar to the famous Taq polymerase, but with some promising differences-in one of the springs along the park’s Firehole River. They spent five years developing it into a basic PCR kit for DNA analysis. Now, capitalizing on this enzyme’s unique properties, Schoenfeld is in the process of developing a 30-minute diagnostic test that can be used to detect a number of viral and microbial infections, including HIV and tuberculosis. It would require no equipment, and if he can get it to work, Schoenfeld is optimistic that he can make it precise enough to recognize one flu strain from another.

Taking the heat: The steaming waters of a bubbling mud pot in Yellowstone National Park are no match for extremophilic microbes, which have a remarkable ability to survive tough conditions. Microbiologists are interested in what else bugs like these can do.

But in many ways, bioenergy is an even bigger gamble. Mead and Lucigen scientist Phil Brumm began building a library of enzymes for the industry several years ago, but they did so recognizing that the industry they hope to sell to does not yet exist. To date, no industrial process for the conversion of plant material to biofuels has proved cost-effective, and research on new methods of bioconversion remains in its infancy. Even as Mead fills his bottles with potentially promising bacteria, he does so with the knowledge that it may take years of lab work before he can say what he has-and whether it can play a meaningful role in making plant-based ethanol a commercial reality.

And that’s one of the hard realities of bioprospecting. Although the possibilities are enticing, the work involves a level of delayed gratification. On their visits to Yellowstone, Mead and Schoenfeld stick to their daily routines, performing the repeated tasks of setting up equipment, sampling and packing up with cold efficiency. The only inspirational moments come from the setting-the vast western sky, the steaming landscape, the glimpses of eagles and elk. Otherwise, the hours are filled with hiking and waiting-and hoping that the next bottle will pull up the microbial Moby Dick.

The trips usually span three or four days, but they can still feel incessantly long. The hours getting into and out of the park. The hikes laden with 40-pound packs of equipment. The nights spent at the kitchen sink, re-filtering samples to separate bacteria from viruses. Then an early bedtime so they can rise and repeat the whole thing the next day.

By the end of their seventh trip to Yellowstone, Schoenfeld will have collected five samples of concentrated viruses, and Mead will have filled more than 20 plastic bottles with bacteria-laced spring water, covering nearly 10 miles of trail in the process. From this catch, they’ll continue the search for Lucigen’s first million-dollar enzyme, a goal that-as long as it’s still ahead of them-will lead them to hot springs sites year after year. They don’t plan to stop until they find what they are looking for. It’s business, of course, but also something more.

“It’s like hunting or fishing in a way,” explains Schoenfeld. “The same brain chemical that makes people fish makes us go back for more (enzymes). You want to get the big one.”

SIDEBAR — Some like it hot: 4 microbes that thrive in extreme conditions

Thermus aquaticus

Where it lives: In near-boiling water, from hot springs to hot water heaters.

How it does it: T. aquaticus has adjusted the composition of its cell wall so it doesn’t melt at high temperatures. It also contains proteins and enzymes that function best in the heat.

Notable achievement: The key enzyme used in some types of genetic testing-Taq polymerase-comes from this bug.

Ferroplasma acidiphilum
Where it lives: In the highly acidic drainage pools of abandoned mines.

How it does it: By constantly pumping protons out of its extracellular space to keep its internal pH levels close to neutral.

Notable achievement: F. acidiphilum uses the iron in pyrite (fool’s gold) as an energy source and produces sulfuric acid as a waste product.

Deinococcus radiodurans
Where it lives: About anywhere-deserts, acid lakes, frozen tundras and even sites of extreme radiation.

How it does it: D. radiodurans has an extraordinary ability to quickly fix its DNA. During lean times it hunkers down and waits for things to get better, and then when the time comes it quickly fixes its DNA and reproduces itself.

Notable achievement: Researchers are exploring whether its DNA reassembly mechanisms can be used to piece together fragments of DNA recovered at crime scenes.

Pseudomonas putida
Where it lives: In soils contaminated with solvents like tolulene and naphthalene. It also munches on polystyrene foam, a substance that was previously believed to be non-biodegradable.

How it does it: P. putida‘s diverse metabolism allows it to be a rather indiscriminate eater. In a pinch, it is able to generate energy by breaking down nasty organic pollutants, detoxifying them in the process.

Notable achievement: The first organism to be patented, this bug is a potent bioremediator, used to clean up toxic soils.

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