Microorganisms that grow under extreme conditions are being studied at ORNL because of their potential to make useful materials, trap uranium contaminants, and produce hydrogen for energy.

Amazing Microbes

Tommy Phelps is a microbe hunter who will go to extremes to find exotic bacteria. In the late 1980s the microbiologist, then at the University of Tennessee at Knoxville (UTK), picked through buffalo dung in Yellowstone National Park to find a microorganism that produced an enzyme that had special characteristics sought by the client, Eastman Kodak. Three years later, the enzyme was being used in laundry detergent manufactured in Europe.

In 1993, as a research staff member in ORNL's Environmental Sciences Division (ESD), he set up a lab by a Texaco oil-and-gas exploration rig near Fredericksburg, Virginia. Studying samples extracted by Texaco workers from the Taylorsville Triassic Rift Basin, he discovered novel bacteria in a mixed culture. Incredibly, these subsurface microbes had been geologically isolated for some 100 million to 140 million years. They had lived at a depth of 2800 meters (9100 feet) at a temperature of 75°C (167°F). Later Phelps showed that a strain of these bacteria produced fine-grained particles of magnetic material that might have industrial uses.

These bacteria are called extremophiles because they love extreme environments. Extremophiles, microorganisms that grow under extreme conditions, thrive in environments we would avoid, such as boiling or ice-cold water, concentrated brine, household ammonia, or vinegar. The bacteria from Virginia are thermophiles because they love heat, just like their cousins who flourish in hot springs.

Other extremophiles prefer environments that are very cold (psychrophiles), high in salt (halophiles), very acidic (acidophiles), or high in alkalinity or pH (alkalinophiles). Their habitats may be Alpine glaciers, cold polar seas, deep-sea sediments, highly saline lakes, carbonate springs, or soda lakes. Extremophiles are of great interest to ORNL researchers trying to find better ways to make useful materials, prevent uranium pollutants from leaving Department of Energy sites, or produce hydrogen for energy.

A Gold Mine for Extremophiles

Tommy Phelps (left) and his wife, microbiologist Susan Pfiffner, are suited up for their two-mile descent into a South African gold mine to hunt for bacteria with useful talents.

In his search for other extremophiles that might prove useful, Phelps went to extremes again in 1998 as part of an expedition by a number of scientists to South Africa's East Driefontein gold mine. (This expedition is described in the cover story of the July 1999 issue of Discover magazine). He joined his wife Susan Pfiffner, a UTK microbiologist, and a team of American scientists in braving temperatures between 32°C (90°F) and 49°C (120°F) for 4 hours a day in a gold mine shaft 2.3 kilometers (2 miles) deep. The participating organizations in this daring venture, which was funded by the National Science Foundation, included ORNL, UTK, Princeton University, Cornell University, and two other DOE labs — Pacific Northwest National Laboratory and Idaho Engineering and Environmental Laboratory. The microbe hunters took samples of bacteria from brown stains on the seam of the gold mine. Thermophiles were found in "carbon leaders," fractures in quartzite that allowed the flow of seawater from which gold and organic matter precipitated.

Phelps (right) and geologist T.C. Onstott of Princeton University take samples of bacteria from the South African gold mine.

"My wife and I brought back 70 cultures of extremophiles called acetogens," Phelps says. "We found evidence of fermenting microbes that feed on hydrogen and carbon dioxide, reduce sulfates, and produce acetic acid."

Bacteria That Make Magnetite

The samples collected in Virginia in 1993 contained micron-sized bacteria and nanometer (nm)-sized particles of magnetic iron in what Phelps calls "their bug poop." Phelps brought the samples back to ORNL. He and his ESD colleagues Chuanlun Zhang and Jizhong Zhou later purified the samples, isolated a strain of bacteria they named TOR-39, and showed that these anaerobic (non-oxygen-breathing), rod-shaped extremophiles can convert iron hydroxide to magnetic iron, or magnetite.

"These bacteria respire iron," Phelps says, while sliding a horseshoe magnet along a test tube to coax the microbes' black residue inside to rise up the wall, showing it is magnetic. "Just as humans get rid of electrons by forming carbon dioxide and water from their food, these bacteria dump electrons on nearby electron-accepting metals, such as iron."

In the process, they reduce iron hydroxide [Fe(OH)3] to magnetic iron oxide (Fe3O4). Zhang learned in 1996 that TOR-39 bacteria can also ferment glucose and other carbohydrates when grown at temperatures from 50°C (122°F) to 70°C (158°F). Phelps says that these bacteria, when properly fed, form carbonates from carbon dioxide.

One type of bacteria (PBI) studied at ORNL produces both magnetic iron oxide and calcium carbonate (first test tube at right) when exposed to carbon dioxide. Under controlled conditions (second test tube) the bacteria produce a nonmagnetic ferritic oxide. In the third test tube, the bacteria produce magnetic iron oxide only when not exposed to carbon dioxide. Stirring-bar magnets are placed between the test tubes.

Phelps is working with Bob Lauf of ORNL's Metals and Ceramics Division to stir up industrial interest in the magnetic particles produced by the TOR-39 bacteria (for which a patent application has been filed). Lauf has found that if the biologically formed magnetite is doped with cobalt, nickel, or palladium, it becomes more magnetic. Phelps and his colleagues have shown that the TOR-39 bacteria efficiently produce 10-NM to 300-NM of magnetic particles in a temperature range of 45 to 70°C. They also found that magnetite particles of a desired size can be obtained by harvesting the bacteria at the proper time.

"We believe that these particles could be used to make superfine magnetic coatings that may be needed by the computer industry for faster magnetic disks and by companies that need faster motors for drills and other uses," Phelps says.

The discovery that microscopic creatures of the deep may produce magnetite by respiration is of great interest to scientists. It suggests that bacteria may have played a role in the evolution of the atmosphere and of creatures that breathe (like us). It suggests further that bacteria may be partly responsible for the banded iron formations that provide Earth's magnetic field. Because biologically formed magnetite is similar to extraterrestrial magnetite in the Martian meteorite ALH84001, some scientists see this as evidence supporting the existence of past biological activity on Mars. But future space probes may have to dig deep to find positive proof.

In fact, an unmanned NASA space probe planned for 2013-2020 will drill 100 meters beneath the surface of Mars to obtain samples that may contain bacteria. Tommy Phelps has been named to this project's science team, so once again he is going to extremes as he joins the scientific search for extraterrestrial life.

Can Microbes Sequester Carbon?

One of DOE's interests is to find better ways to capture carbon from power plant emissions and the atmosphere and store it securely. The goal is to prevent atmospheric carbon dioxide levels from climbing enough to cause potentially devastating climate change. To achieve such carbon sequestration, Phelps believes that extremophiles such as TOR-39 bacteria could play a role.

"We have shown that, when exposed to carbon dioxide dissolved in water in a test tube medium, our TOR-39 strain precipitates carbonates faster than other microbes," Phelps says. "We propose using our bacteria to coat fly ash and waste coal dust from coal power plants with carbonates as a way of sequestering carbon. Then instead of hauling this material to the landfill, it could be sold as road fill."

The micrograph at left shows carbonate-coated fly ash as a result of bacterial action. Coal fly ash is shown at right.

A possible microbial route to removing carbon from power plant emissions might be to lace a pond next to a coal-fired power plant with the TOR-39 bacteria. Carbon dioxide could be captured from the stack emissions and injected into the pond. The bacteria could use the dissolved waste gas to produce iron carbonate or calcium carbonate, which would settle into the pond's bottom sediments as a mineral.

Phelps and his colleagues have done experiments that show that PB-1 bacteria from a marine environment can produce iron carbonate a week after being exposed to carbon dioxide. But further studies showed that TOR-39 bacteria, when fed sugar and exposed to carbon dioxide, can produce iron carbonate in less than a week. Phelps thinks it is possible to make the TOR-39 bacteria generate the material within one to three days.

This carbonate-containing "bioherm" conglomerate was found in halophilic algae in the Great Salt Lake in Utah.

"Many people think that biologically sequestered carbon in rocks such as calcium carbonate conglomerates is formed only in the presence of light and photosynthesis," Phelps says, showing a carbonate-containing "bioherm" conglomerate that he found in halophilic algae in the Great Salt Lake in Utah.

"However, there is evidence from methane gas hydrates deep in the ocean that carbonate-containing conglomerates can be formed without photosynthesis. It is possible that extremophiles may serve as nucleation catalysts for the formation of minerals. If this is so, we might be able to identify or design bacteria that could form conglomerates much faster and better for carbon sequestration."

Bacteria That Prevent Uranium from Straying

At the Oak Ridge Y-12 Plant and other DOE sites, waste uranium in groundwater, streams, and ponds may be in the oxidized state. That's a potential problem. Such uranium is soluble and mobile, increasing the risk that it might remain in water that flows off site. Because DOE wants to confine the uranium to its sites, it is interested in exploiting bacteria to change the chemical state of the uranium. The goal is to make the uranium stick to soil, rocks, and stream sediments to ensure that it stays on site.

ESD researchers Tony Palumbo and Jizhong Zhou are studying bacteria that reduce uranium by adding an electron to each uranium atom, making it insoluble. As a result, the uranium should precipitate out of the water, drop into the sediments, and remain within the confines of the DOE site. The work is supported by the National Accelerated Bioremediation (NABIR) Program of Doe's Office of Biological and Environmental Research.

Palumbo is working on a project with a group at the Georgia Institute of Technology to help meet Doe's goal. One way to make uranium precipitate out of the water is to let it combine naturally with the phosphorus already in the water. The problem is that most phosphorus is unavailable for attachment to uranium because it is tied up with soil in and around the stream. One solution is to add to the soil an organic compound containing phosphorus that can move into the water. Palumbo and the Georgia Tech team genetically engineered a strain of bacteria that chops the organic compound from phosphorus in a high oxygen environment.

Enrichments from site samples are used to isolate bacteria that digest different forms of organic phosphorous. Bacterial growth is obvious in media containing, from left, triethyphosphate (TEP) and glucose-6 phosphate (G6P) in comparison with the control, which has no added soil sample (right). The bacteria growing in the enrichments are used to obtain pure cultures. The resulting cultures from the TEP media are the first to be documented as users of TEP.

"We genetically engineered the Pseudomonas bacteria to rapidly metabolize organic phosphorus," Palumbo says. "We isolated six to twelve bacterial strains that use triethylphosphate, or TEP, which is a very mobile organic form. We are identifying the genes responsible for the organism's ability to break up the TEP. We do this by disrupting different sets of genes in various strains. We find which mutants do not metabolize TEP and then figure out which genes are disrupted in each mutant. The normal forms of those genes are linked to TEP metabolism."

After they isolated two genes responsible for TEP metabolism, they identified the enzyme produced by these genes. Called phosphatase, this enzyme breaks down TEP, freeing phosphorus to combine with uranium in contaminated waters.

Indicator media are used to detect the activity of the enzyme phosphatase, which breaks down organic compounds containing phosphorus. The control plate (left) contains organic phosphorus and a dye, which appears bright green. The plate at right contains several strains of Pseudomonas bacteria that have been growing on the organic phosphorus. The strains are genetically engineered to produce large amounts of the enzyme. As a result, the bright green areas are disappearing and, in some cases, the dye is concentrated in the phosphorus-metabolizing bacteria (the green streaks).

DOE is interested in an extremophile that is known to be able to make uranium insoluble. It is Shewanella putrefaciens MR-1 bacterium. Shewenella is intriguing because it not only reduces metals (which may make it ideal for treating contaminated waters) but it also produces hydrogen and magnetite.

An intensive effort will be made by DOE in FY 2000 to completely sequence this bacterium to identify the genes that can change uranium's chemical state. The genes from this microbial genome might be used to design microorganisms for advanced bioremediation of Doe's contaminated streams.

Extremozymes and Hydrogen Production

Hydrogen may be the fuel of the future once hydrogen fuel cells for propelling cars are perfected. Combined with oxygen in a fuel cell, hydrogen provides electricity and a little heat. Its only waste product is water. The hydrogen car will be clean because it will not discharge nitrogen oxides and carbon dioxide.

But how can hydrogen be obtained cheaply? One approach may be to use special enzymes to (1) transform cellulose into glucose sugar, and (2) convert the glucose product and its byproduct, gluconic acid, into hydrogen. Sources of cellulose are old newspapers, grass clippings, and other waste products of renewable resources.

In 1996 Jonathan Woodward and his associates in ORNL's Chemical Technology Division (CTD) reported an important advance. They learned how to produce a molecule of hydrogen from a molecule of glucose using two enzymes, both of which are "extremozymes." An extremozyme is an enzyme produced by an extremophile.

Because the glucose-to-hydrogen process is more efficient if run at a higher temperature, it makes sense to replace some standard enzymes with extremozymes. The reason: Standard enzymes will stop working when exposed to higher temperatures unless special, costly measures are taken to protect these proteins. Some extremozymes might eliminate the need for protective steps, increasing efficiency and reducing costs. In addition, extremozymes may be more stable and react faster than their "mesophilic" counterparts that prefer benign conditions.

In October 1999 CTD researchers Woodward, Mark Orr, and Elias Greenbaum and CTD student Kimberley Cordray reported that the CTD group had produced 11.6 hydrogen molecules for every glucose molecule in the substrate. The researchers achieved 97% of the maximum stoichiometric yield possible — 12 hydrogen molecules for each glucose molecule. This is the highest yield of hydrogen ever obtained from glucose by a biological process.

This high stoichiometric yield of hydrogen from glucose was attained through an "oxidative pentose phosphate cycle" using 11 enzymes. In this cycle, glucose is oxidized completely to the compound NADPH and carbon dioxide. In the presence of hydrogenase, hydrogen is released.

This hydrogenase is produced by the extremophile Pyrococcus furiosus, a strain of bacteria from a deep-sea hydrothermal vent. It works most efficiently at a temperature of 85°C. This hydrogenase is also one of only two enzymes known to accept electrons from NADPH to produce hydrogen.

"We ran our hydrogen-producing reaction at 30°C because the yeast enzymes we used are inactivated above 45°C," Woodward says. "Thus, the activity of the hydrogenase was ten times lower than it would have been at 85°C. We could probably produce hydrogen much more efficiently from glucose if all eleven enzymes were isolated from thermophiles."

Woodward, CTD staff researcher Barbara Evans, and CTD students Cordray, Robert Emonston, Maria Blanco-Rivera, and Susan Mattingly showed that hydrogen could be obtained enzymatically from other biomass substrates. These substrates were lactose, sucrose, xylan, starch, and steam-exploded aspen wood.

Woodward, Evans, Gerard Bunick of the Life Sciences Division, and ESD's Jizhong Zhou and Tony Palumbo have been studying extremozymes obtained from extremophiles of the ORNL subsurface culture collections, the Methanococcus jannaschii genome clones available from the American Type Culture Collection, and biotechnology companies. Some enzymes are produced at ORNL by cloning purchased DNA sequences and inserting them into E. coli bacteria, which churn out the desired protein.

"We have identified, isolated, purified, and characterized extremozymes that might be useful for bioprocessing and energy production," Evans says. 'To produce hydrogen fuel from cellulose, we must first break the cellulose down to its component sugar — glucose. We use the enzyme cellulase to hydrolyze the cellulose — that is, add water to the bonds connecting the glucose molecules that make up the cellulose polymer."

Converting the cellulose cheaply and efficiently to glucose continues to bog down the two-step process of producing hydrogen from cellulose. "The bottleneck in the cellulose-to-glucose process is the cellulase enzyme," says Woodward, who has long been experimenting with the fungal enzyme to increase its efficiency in breaking down cellulose into glucose. "We need a stable enzyme that works faster to catalyze production of glucose from cellulose. Right now it takes two days to produce glucose from cellulose. Our goal is to make cellulase work ten times faster to obtain glucose in 30 minutes to an hour."

These atomic force microscopy images show, at top, control cotton fibers (cellulose) incubated in a buffered solution without enzyme and, at bottom, cotton fibers after treatment for 6 hours with the cellulase CBH 1 from the fungus Trichoderma reesei. The image shows that the crystalline structure of the cotton fibers is disrupted by the cellulase enzyme.

If a reaction is run at higher temperatures, shouldn't thermophilic cellulases digest cellulose faster and be more stable than "mesophilic" cellulases, which works at moderate temperatures?

"That's not necessarily so," Evans says. "Mesophilic cellulases can break down cellulose in 30 minutes if you add enough enzyme to the cellulose. But the enzyme is too expensive to make this approach economically feasible.

"We need a combination of cellulase enzymes that works effectively in very small concentrations. It must speed up the slow step of the process, disrupting the cellulose fibers and making them accessible so they can be digested into glucose. Even thermophilic enzymes appear to be slow in carrying out this initial step."

Evans is studying a variety of cellulases, including thermophilic ones, to determine their properties, such as stability and reaction speed, in the digestion of cellulose. She will be studying the interaction of various cellulases during the digestion process. Bunick uses X-ray diffraction to determine the structure of extremozymes, which could help explain why some are stable under extreme conditions.

Hugh O'Neill, a postdoctoral scientist in Woodward's group, is studying hydrogen-producing bacteria that prefer different temperature ranges to see which ones are the most efficient. He is looking at how efficient each microbe is at producing hydrogen from different substrates. He is studying psychrophiles (4-33°C), mesophiles (20-45°C), and thermophiles (45-70°C). He will then isolate the hydrogenase enzyme from the most efficient hydrogen producers.

If a psychrophilic hydrogenase is found, it could be useful for producing hydrogen for fuel cells in cold places, from Antarctica to outer space. The U.S. Defense Applied Research Projects Agency is funding this project because of its interest in producing hydrogen enzymatically for military fuel cells, using substrates from the environment, such as sucrose in tree sap. Such fuel cells could be used to power biosensors needed to detect biological and chemical warfare agents.

At ORNL, the X-10 files on extremophiles are expanding.

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