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Research Magazine > ARCHIVE > Spring 92 > Article

Life at the Boiling Point
by Judy Purdy

It's probably been there all along, but nobody ever thought to look for life at the rims of erupting undersea volcanoes. Until lately.

Now scientists are probing the bacteria that thrive in boiling water -- exotic organisms that weren't even imagined until the past decade.

The research may lead to breakthroughs in biotechnology. And it is literally changing the way scientists look at life itself.

"These are completely new organisms. They're a new breed of life forms," said biochemist Mike Adams. "They promise to revolutionize ideas on the origin and chemistry of life and applied industrial biology."

In his University of Georgia laboratory, Adams is deciphering how these new-found bacteria can survive in the pressure-cooker environment of undersea volcanoes and boiling geysers like those in Yellowstone National Park.

Supported by competitive grants from the U.S. Department of Energy, the National Science Foundation, the National Institutes of Health, the Office of Naval Research and others, Adams analyzes the structures of the molecules inside "hyperthermophilic" organisms to find out how they can withstand such intense heat and pressure without disintegrating. And he's made remarkable discoveries about their enzymes, the proteins found in all living organisms that carry out and control life's essential chemical reactions.

"There are a number of organic molecules that you and I use -- that all life forms use -- and we know they fall apart in a matter of seconds at temperatures near the boiling point. For example, proteins, including enzymes, are usually inactivated when you boil them," Adams said. "To me, it seemed we needed to answer a very fundamental question: What makes the molecules in hyperthermophilic bacteria stable at 212oF and above?"

But before Adams could seek the answer, he first had to get his hands on the bacteria. And that was no easy task considering where they live.

Collecting 'Hyperthermophiles'

High-temperature bacteria must be snatched from caldrons near the deep sea vents of active volcanoes on the ocean floor. Scientists collect the rare samples using robotic arms attached to cramped underwater craft called deep sea submersibles.

"Quite a number of these organisms have now been discovered in deep sea hydrothermal vents, many of which are found around two miles down on the ocean floor," Adams said.

Only in the past decade have scientists detected the new organisms, mainly because "no one had looked before then," Adams said. "It takes a certain amount of technology just to be able to send humans two to three miles down into the ocean to collect samples. There are tremendous pressures almost 300 times atmospheric pressure at those depths."

The underwater craft must come close enough to the volcanic vents to collect samples of the bacteria, but not so close that superheated water will endanger the crew. Dr. John Baross, a University of Washington microbiologist who has participated in a number of deep sea collecting trips, said the deep-sea submersible Alvin is constructed of titanium and has acrylic windows to protect the craft and crew from the intense heat and pressure.

Adams' bacteria samples are supplied by scientists from the United States and Germany, who share their deep-sea finds.

Adams often works with bacteria before their discovery has been published in research journals. Some are so recently discovered they don't even have scientific names -- just reference numbers assigned to them as they are collected.

These heat-loving organisms thrive in an environment that would have captured the imagination of Jules Verne. As ocean water seeps into the earth's crust surrounding an underwater volcano, it is heated to temperatures up to 750oF. This intensely hot water, which remains liquid far above the normal boiling point of 212oF because of the incredible pressure at that depth, is thrust upward violently.

Emanating as spectacular jets at the ocean floor, the superheated water mixes with the nearly freezing water (only 36oF) at the bottom of the sea.

The result is a clash of forces that occurs nowhere else.

" All these dissolved minerals precipitate out of the water, and that's when you get the `black smoker effect.' It's where the water has cooled down near the normal boiling point (212°F) that you find these hyperthermophilic bacteria," Adams said.

Hyperthermophilic bacteria are not isolated directly from the high temperature water, but from "chimney" material, debris that breaks off the black smoker walls, said Dr. Holger Jannasch, a microbiologist at the Woods Hole Oceanographic Institution who studies the ecology and physiology of hyperthermophilic life forms.

Collecting the bacteria is replete with danger. Even before the scientists can confront the pressure and heat of two-mile-deep vents in the ocean floor, skin divers must guide the deep-sea submersible from the surface, through shark-infested waters, to depths where the craft can maneuver on its own.

Even exploring shallow waters can pose a risk. On a recent expedition off the Polynesian coast, Karl Stetter, the German biochemist who discovered the hyperthermophilic life only a decade ago, approached an underwater volcano about 130 feet deep just as it began to erupt. Luckily, the researchers were not close enough to be in danger, and after the eruption, they were able to collect viable hyperthermophilic organisms from the cooled-down volcano and from floating volcanic debris up to several miles from the eruption.

The deep-sea collecting trips require careful planning, said Jannasch, who participated in his first deep-sea submersible dive to the ocean floor more than 20 years ago aboard the Alvin.

"It takes two years to prepare for an exhaustive two-week program of isolating hyperthermophilic bacteria," he said. "Our last Alvin cruise was in March 1991 and we started planning it in 1989. We took 18 scientists from many countries on that cruise."

The planning and preparation time are well worth the effort for Jannasch as well as researchers like Adams. Almost all hyperthermophilic organisms discovered by Woods Hole scientists have been collected near deep-sea vents.

Incubating Life at 212°

That bacteria can live in environments where water would normally boil is amazing enough. But imagine what it's like to grow them in a laboratory.

That's the challenge Adams faced before he could begin his study of the bacteria's heat-resistant enzymes: He had to replicate the organisms' natural environment in order to grow them.

"When we first started growing these bacteria, only limited information was available on them, and it was obtained by the group that had isolated them. We had to figure out how to grow them to optimize the amount of live material so that we could purify some of their enzymes," he said.

Most of the recently discovered hyperthermophilic bacteria are marine organisms whose native habitats are as many as three miles below sea level. The high salt concentrations and tremendous pressures found at those depths are difficult to emulate in a lab setting.

Until this year, Adams had no fermenter that could incubate life at the boiling point. Conventional fermenters are designed to incubate bacteria at temperatures around 90° to 120° F. Some will heat up to 185°F, which still doesn't provide an optimal climate for hyperthermophilic bacteria. And even then it's difficult to control the temperatures inside the fermenters.

"These bacteria grow naturally in very hot places, and very little is known about their growth requirements," Adams said. "The first thing we do is try to optimize growth in small-scale cultures by adding different chemical components, including various metals and other nutrients, to increase growth at high temperatures. Because these organisms grow in exotic places, we have no hard and fast rules as to what might stimulate their growth."

Most hyperthermophilic bacteria also need sulfur as an energy source -- which creates additional problems because of the chemical's corrosive properties.

"These organisms grow in a sulfur-rich environment. Most of them actually derive their energy by converting elemental sulfur to hydrogen sulfide -- the compound that is the smell of rotting eggs," Adams said. "Hydrogen sulfide is toxic and very corrosive. It actually eats away at the stainless steel lining of a conventional fermenter."

The problem of producing large quantities of the bacteria were alleviated this spring with the installation of a new 150-gallon fermenter on campus. The fermenter -- specially designed for Adams' research -- uses a corrosion-resistant enamel lining to withstand the effects of sulfur compounds and high salt concentrations. Better still, it can be heated in a controlled manner to 300° F.

That will allow Adams and his team of graduate students and post-doctoral fellows to grow a wider variety of hyperthermophiles and learn more about what makes them tick.

In his lab in the University's recently built Life Sciences Building, Adams has been mass producing the limited number of hyperthermophilic bacteria able to grow without sulfur in the department of biochemistry's 150-gallon stainless steel fermenters. These yield roughly a pound of bacteria at a time, which is enough to extract the various biological molecules he needs for his work.

With the new fermenter, Adams said he will be able to "mass culture" under optimal conditions any of the hyperthermophilic organisms currently known. However, he still has to use some tricks to grow them. Since most of the organisms die rapidly in the air, they must be grown at high temperatures in the absence of oxygen. To grow large batches of bacteria in the new equipment, he must experiment in his lab with different growing techniques for each of the new organism. Once he figures out their growth requirements, he must produce enough to "inoculate" the new 150-gallon fermenter.

"When we're satisfied we've done whatever we can, we move from small scale to the large fermenters where we can grow enough bacteria to study their enzymes," he said.

Building Blocks of Life

Understanding their enzymes is one of the keys to understanding hyperthermophilic life.

All living organisms contain proteins, the building blocks of cells. Enzymes are proteins that carry out chemical reactions necessary to life such as converting sugar, or sulfur-containing compounds in the case of some hyperthermophilic bacteria, into energy.

But enzymes produced by conventional organisms, and the protein material that comprises them, are very sensitive to high temperatures. Consider how an egg white -- almost pure protein -- instantly turns solid when dropped into boiling water. The high temperature destroys the protein structure, and when the proteins fall apart and precipitate out, they no longer function.

So what's different about the proteins and enzymes in hyperthermophilic bacteria? Do they withstand high temperatures because of basic differences in their proteins or do they use the same proteins and just put them together differently?

The answer is both.

Adams has subjected temperature-stable enzymes from hyperthermophiles to all kinds of experiments to solve the mystery. Each test he and his colleagues perform reveals a different clue to the structure and function of heat-stable enzymes.

Studies, including chemical analyses, Nuclear Magnetic Resonance (NMR) spectroscopy and crystallography, have revealed that hyperthermophilic enzymes and proteins are made with the same building blocks as conventional proteins and enzymes. The hyperthermophilic proteins simply have more chemical bonds inside of them. The result is a "superglue" that maintains the structure of protein even at high temperatures.

Among the enzymes Adams has isolated and identified from hyperthermophiles is one also found in a large number of conventional bacteria that grow at "normal" temperatures. Called hydrogenase, this enzyme helps bacteria convert hydrogen gas to a useable energy source within the cell. Unlike conventional forms of hydrogenase, the enzyme Adams isolated from hyperthermophilic bacteria is not destroyed at high temperatures.

Adams also was the first to isolate and purify a tungsten-containing enzyme from hyperthermophilic bacteria. Metals such as iron, copper and manganese are common ingredients of enzymes, but tungsten is very rare. In fact, before Adams' discovery only one other tungsten-containing enzyme had ever been isolated from a living organism, and that was done by UGA biochemist Dr. Lars G. Ljungdahl and his post-doctoral associate, Dr. Jan R. Andreesen, in 1971.

Adams first noticed the bright red tungsten-containing enzyme while purifying hydrogenase from Pyrococcus, a round-shaped bacterium whose scientific name means "fireball." His recent research has shown the presence of similar tungsten-containing enzymes in all five genera that he has examined.

The tungsten protein helps release waste products from the cell. Both it and the hydrogenase have potential biotechnological uses because they can catalyze high-temperature reactions important in manufacturing chemically-based products.

Applications for Industry

Because enzymes from hyperthermophilic organisms remain stable at high temperatures, they retain their catalytic effectiveness-- even at temperatures exceeding the boiling point. And that's good news for the chemical industry.

"Industrial chemists are frequently intrigued by the specificity and activity of enzymes in contrast to many synthetic catalysts," Adams said. "But with the discovery of hyperthermophiles, we can start to think of using enzymes to catalyze reactions that preiously we wouldn't have since no enzymes were known that were active at 250o Fahrenheit.

Until now, manufacturers could only dream of using enzymes to speed up production at temperatures above the normal boiling point. The hyperthermophilic enzymes may offer good news for many industries.

·The sugar industry could use them to convert corn starch to something sweeter-- high fructose corn syrup. The syrup is widely used in the beverage industry and in a variety of processed foods. The stability of hyperthermophilic enzymes at higher temperatures could mean a more efficient, less expensive way to make the sweetener.

·Stain-removing enzyme cleaners are nothing new for the detergent industries, but the new heat-stable enzymes may erase stubborn protein-derived stains, such as blood, at much hotter temperatures.

Dr. Robert Kelly and colleagues at Johns Hopkins University study the protein-digesting and starch-degrading qualities of hyperthermophile-derived enzymes. Their research has shown these enzymes are active at temperatures around 285°F.

·The high-temperature enzymes also may penetrate the petroleum industry. Adams and coworkers are investigating enzyme potential for removing hydrogen sulfide and other waste sulfur compounds from coal and other petroleum products that could lead to cleaner fuels and less environmentally harmful petroleum waste products.

In addition, "hydrogen is an extremely important intermediate in the chemical industry," Adams said, because petroleum refining involves several hydrogenation reactions. "Hydrogenase could be used extensively in chemical processing for large-scale chemical productions and small-scale specialized chemicals".

"It's also an ideal fuel because it's clean burning," he said. "When burned, hydrogen gas produces water instead of greenhouse-type gases such as carbon dioxide or carbon monoxide."

Treatments to process other industrial waste products, to mine precious metals from minerals and to convert complex carbohydrates and lignin-type substances produced in the pulp and paper industry into useable materials are also potential applications of the new heat-stable enzymes.

The enzymes may also offer potential biotechnology for the pharmaceutical industry.

One enzyme newly available from two different high-temperature bacteria, already has been adopted commercially. It's used to synthesize DNA for certain kinds of biological research, Adams said.

Hyperthermophilic enzymes also are yielding a less tangible, but perhaps more important product: a better understanding of how enzymes work. Enzyme usually react so fast that it's difficult to capture or study intermediate products. To slow the reaction rate, scientists have to cool enzymes to temperatures below freezing in "antifreeze" solutions. But hyperthermophilic enzymes react slowly if at all, at room temperature, which is almost two hundred Fahrenheit degrees colder than the temperatures at which they function; therefore, they can be studied conveniently at or near the normal room temperature.

The Origin of Life

The growing body of knowledge about hyperthermophilic bacteria is causing scientists to question the universally accepted "tree of life." These most recently discovered hyperthermophilic organisms may be, in a sense, the "oldest" on earth; scientists now consider them the closest living relatives to the original forms of life on the planet.

They are better adapted to survive in climates that prevailed when the earth was a seething ball of hot liquids and erupting volcanoes. The early atmosphere, devoid of oxygen, was perfect for hyperthermophilic bacteria, the majority of which cannot grow in the presence of oxygen.

"It now appears that the deep-sea hydrothermal vent environments are akin to those under which life on earth first arose," Adams said. "So we should examine the hyperthermophiles in terms of links to ancient life on earth."

Life at high temperatures was the early norm, but as the earth cooled, life forms that could exist at cooler temperatures fluorished.

Scientists are finding the recent biochemical information uncovered by Adams and others supports the new theory. The biochemistry of the high-temperature bacteria suggests "that these organisms or something like them were among the first on ancient earth," said Baross. "Part of the lure of studying these organisms is that we may be looking at analogues of some of the first life forms on this planet."

And Adams' recent discovery of the presence of tungsten-based proteins in every genus of high-temperature bacteria he has examined so far poses new possibilities. Perhaps the synthesis of proteins with tungsten, which is found in sufficient supply in the effluent of deep-sea vents, was an ancestral process common among many early organisms that has been lost over time. In fact, the majority of conventional life forms, including man, have some enzymes that contain molybdenum, an element chemically related to tungsten. Perhaps as the early earth cooled and life forms evolved that eventually gave rise to present-day organisms, the early dependence of life upon tungsten was replaced by molybdenum.

"It now seems clear that conventional life forms didn't give rise to these hyperthermophilic bacteria; rather all conventional life forms evolved from high temperature organisms," Adams said. "In other words, you and I are adaptations to much colder (below boiling point) environments."

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