- by Nancy Shute

Craig Criddle, a soft-spoken environmental engineer at Stanford University, is on a mission to purge the Oak Ridge site of the deadly contaminants. His weapons are microorganisms that, it was recently discovered, chomp on uranium like it was candy, rendering it more stable and less apt to leach into the water. “This is the most complicated site I've ever worked at,” says Criddle, who has worked on major experimental bioremediation projects. At Oak Ridge, the soil is not very permeable, and the geology is unusual, with saprolite lying at a 30-degree angle. High levels of nitrate contamination interfere with uranium reduction. Criddle is using two rows of wells to inject ethanol into the ground, as food for microbes that will convert the uranium into an insoluble form that won't leach into groundwater. An above-ground, fluidized-bed reactor is being tested to transform the nitrate into benign nitrogen. “It remains to be seen if it will work,” Criddle says. “In the next 9 months I hope we can prove it.” Making it work is what counts. “My goal is the actual cleanup,” he says. “That's where I get the buzz. People in bioremediation really want to do something for the environment.”

The Oak Ridge project is part of the Natural and Accelerated Bioremediation Research (NABIR) program, a $20 million effort by the Department of Energy (DOE) to fund research on bioremediation of radionuclide and metal contamination at DOE sites around the country. “We have made a lot of progress in the past five years,” says Anna Palmisano, director of the program. “But it's a tough problem.” Dealing with metals has been the toughest problem yet. “People just threw up their hands,” Palmisano says. “Unless you're an alchemist, you can't convert one metal to another.”

Cleaning up hazardous wastes is a huge task—the U.S. Geological Survey estimates that cleaning up existing environmental contamination in the United States could cost as much as $1 trillion. Much of the cost of traditional cleanup technologies comes from digging up and carting away contaminated soils. Criddle and other researchers around the country are trying to devise new methods of bioremediation—using living organisms to reduce or eliminate environmental hazards by immobilizing the contaminant, or by transforming it into a benign chemical. One of bioremediation's biggest benefits is that it treats the contamination in place, avoiding the transport costs of traditional cleanup methods. Bioremediation also typically disturbs the site less than traditional methods, reducing stress on the environment. And bioremediators are increasingly relying on the power of natural microbial processes already at work at the site. That can drive down the costs of a cleanup considerably.

But bioremediation, which has been used in various forms since the 1980s, has not always delivered as much as it has promised. And bioremediators still know too little about how microorganisms function, and how they interact with their environment and each other, to use them to the fullest. Many researchers say the power of this invisible army is only beginning to be realized. New research suggests that discredited techniques such as bioaugmentation, in which proprietary brews of bugs are injected into a site, may actually work when researchers have a deeper understanding of how the microbes function.

More importantly, bioremediation is gaining new energy from advances in a field of science most would consider quite remote from engineering—microbiology. In the last 10 years, researchers studying bacteria and other simple organisms have made extraordinary discoveries, including “extremophiles” that can live in hot-water vents miles below the ocean's surface, or locked in Antarctic ice. At the same time, huge advances in the tools available to microbiologists—including quick, inexpensive gene sequencing—have made it easier to identify and understand these invisible, cryptic organisms and harness them to good ends. “It's a way to learn about the novelty of the microbial world,” says James Tiedje, a pioneer of bioremediation who is a professor of microbial ecology at Michigan State University and president of the American Society for Microbiology. “It's the extreme end of life.”

Criddle is an environmental engineer, an offshoot of civil engineering that traditionally has focused on cleaning up water. That's an important bit of background, because the big problems in hazardous waste come when contaminants migrate into ground water, poisoning drinking water, lakes and streams, and the natural environment. Bioremediation is really an attempt to clean up nature's plumbing system, once we've gunked it up.

At the beginning of the twentieth century, the nation's fast-growing cities had serious public-health problems due to the lack of clean drinking water and municipal sewage systems. By World War II, thanks to a huge effort to build municipal water and sewer systems, safe drinking water was the norm throughout the United States. Environmental engineering came into its own as a result of those efforts. As manufacturing boomed and the population grew, environmental engineers found themselves faced with new challenges, including air pollution, hazardous waste, and radioactive contamination. “We've got a lot of cleanup to do, from years of doing things the wrong way,” says Bill Anderson, executive director of the American Academy of Environmental Engineering. In 1980, Congress passed CERCLA, the “Superfund” law, mandating cleanup of the nation's hazardous waste sites, but progress has been slow and costly.

Since the 1960s, scientists have known that some microbes can degrade petroleum by using oxygen. In the early 1980s, researchers studying how toxic wastes interact with the environment came to realize that microorganisms living underground could radically effect what happened to toxic substances seeping into groundwater. This might not seem like a huge revelation to anyone who has owned a home with a septic tank. Those simple systems use drain fields to disperse household sewage into the soil, where microbes neutralize nitrates and other contaminants. But researchers soon found that subsurface bacteria could degrade far-more-toxic substances: crude oil, chlorinated solvents, gasoline, creosote, herbicides, and pesticides. Within the past decade, researchers also have proven that other microbes can degrade pollutants without using oxygen at all. These anaerobes instead typically borrow electrons from subsurface iron for their metabolic processes. In some cases, the metabolic processes draw organic carbon or electrons from contaminants such as petroleum hydrocarbons; in other cases, a contaminant like trichloroethene (TCE) may take on electrons. Environmental engineers, geologists, and microbiologists started exploring how these anaerobic cleanup specialists could be induced to take on society's most intractable messes.

TEAM EFFORT

One of the charms of bioremediation is the broadly multidisciplinary nature of the science required. Clearly, an understanding of chemical processes is a boon. And since these processes are happening underground, in soil and in aquifers, an understanding of geology and hydrology doesn't hurt, either. And since the essential tools are living organisms, a background in microbiology is also useful. Because the organisms often need special nutrients delivered underground, and the progress of the cleanup needs to be monitored, engineering capabilities would be useful, too. Thus people working in the field come from a host of backgrounds and almost always have an understanding of the other disciplines involved.

“The business is changing from purely engineering to the realization that you need to have more people with a microbiology background,” says Derek Lovley, a microbiologist who is principal investigator for the Geobacter project at the University of Massachusetts-Amherst. Lovley has pioneered research on using geobacters, naturally occurring microbes found in soil, to clean up uranium—the same type of process that Criddle and his colleagues are experimenting with at the Oak Ridge test site. “It uses metal the same way we use oxygen,” Lovley says. “If there's any uranium around, it transfers electrons from it. It changes the uranium into an insoluble form.”

For the past two summers, Lovley's group has been testing geobacter's ability to eat uranium at a uranium mill tailings site outside Rifle, Colo. Todd Anderson, an environmental engineer and a postdoc at UMass, is supervising the efforts to “feed” the geobacter with acetate, a mild acid. Feeding bacteria lodged in mud underground is no small task, and generally involves designing a system of wells and pumps to deliver the nutrients deep into the soil. The Rifle site lacks electricity, so Anderson set up a system with a 560-gallon tank of acetate and bromide, pressurized to 1.5 psi with bottled nitrogen. The acetate is fed into 20 injection wells set 6 inches apart, in a “fence.” Wells downstream monitor levels of nitrogen, bromide, uranium, and iron. Last summer's run neutralized 70 percent of the uranium in groundwater in 5 weeks. “Within 9 days we started to see uranium concentrations decrease. And we saw an incredible bloom of geobacters.”

But in last year's field test, the uranium levels didn't stay down. Anderson speculates that as iron at the site was depleted by the growing population of geobacters, other microbes gobbled up the acetate and stalled the process. This summer, he's returning to the site to experiment with increasing the acetate. Indeed, delivering the food to microbes in the soil is one of the biggest challenges in bioremediation. “Our challenge is to maintain the stimulation of iron reducers in the subsurface,” he says.

Anderson “got hooked on metal reducers” while working at the U.S. Geological Survey. He credits his undergraduate degree in chemistry, and his longtime interest in microbiology, with preparing him for bioremediation. At the same time, his engineering skills are invaluable, especially in a department where colleagues spend all day sitting in front of computers analyzing genetic data. “I'm the ‘applied' guy,” he says, laughing.

Lovley's lab is also exploring how the same metabolic process that reduces uranium could be used to generate electricity. In the laboratory, geobacter microbes generate tiny amounts of electricity as they take electrons from iron. Much of Lovley's work is basic research, funded by an $8.9 million Department of Energy grant in the Genomes to Life program, which is funding basic research that could solve problems in energy production, environmental cleanup, and carbon recycling.

Despite these recent successes, all bioremediators, in the field and in the laboratory, stress that they have a lot more to learn about what's happening underground, and how those processes could be improved. “A lot of people think they're bioremediating the metals,” says Terry Hazen, head of the Center for Environmental Biotechnology at the Lawrence Berkeley National Laboratory. “You're biotransforming them. They're still there. They're just not as toxic and mobile.” This raises the unsettling possibility that uranium and other dangerous metals could “biotransform” back into their more lethal state. But, Hazen adds, biotransformation might be the only option for contamination by metals and radionuclides deep within the earth—some more than 1,000 feet deep. Hazen, who holds a Ph.D. in microbial ecology and has worked on the bioremediation of more than 50 sites, is now working on a test project at the Hanford federal laboratory in Washington state that aims to biotransform a deeply buried plume of chromium, a common contaminant at industrial sites (and the villain in the film Erin Brockovich) into a less toxic state. “It requires us to have a much better understanding of the biology and chemistry that's going on there,” Hazen says. “If you understand the biology and chemistry you can model it better, understand the process.”

Hazen's lab is part of a consortium—which includes the University of Washington, the University of California-Berkeley, private industry, and the Oak Ridge National Lab—that has received $33.6 million from the Department of Energy's From Genomes to Life program in order to study the basic processes that drive bioremediation. Hazen is investigating how anaerobic microbes handle oxygen stress. “It looks like they've got some really neat responses to overcome that stress.”

However, most hazardous waste sites don't have much room for experimentation—the clients are typically private companies, or state and federal agencies who want the job done the first time. “Right now it's very empirical,” Lovley says. “We know Geobacter likes acetate, so we dump in some acetate and see how it works.” Only now, he says, are researchers able to do the kind of laboratory research that lets them start to understand the science behind these processes.

Researchers and entrepreneurs have long experimented with growing bugs in the laboratory and injecting them into a bioremediation site. They've also dreamed of creating superbugs designed to attack specific contaminants. Indeed, the first patent given on a life form was for a petroleum-degrading microbe. But such “bioaugmentation,” even with naturally occurring microbes, has proved a disappointment. When the lab-bred organisms are distributed at a hazardous waste site, they curl up and die. But new research, published in the July 3 issue of Nature, suggests for the first time that lab-bred bugs can help. The research team, led by Frank Loeffler, a microbiologist and assistant professor of environmental engineering at Georgia Tech, isolated a naturally occurring bacterium, Dehalococcoides strain BAV1, and tested its abilities to clean up tetracholorethene (PCE) and trichloroethene (TCE), two solvents commonly used in dry cleaning and for degreasing metal parts. Previous efforts to bioremediate PCE and TCE worked only partway, leaving toxic intermediate substances such a vinyl chloride, which causes cancer.

Loeffler and his crew tested BAV1 at the Bachman Road residential area in Oscoda, Mich., where PCE from a dry-cleaning operation has contaminated drinking water wells and seeped into nearby Lake Huron. BAV1 bacteria were already growing at the site but in small numbers. So the researchers used traditional bioremediation techniques at one part of the site, adding lactate and nutrients to a 20-foot-deep test plot. In another section, they injected nutrients and high numbers of BAV1. The BAV1 solution degraded the PCE to harmless ethene within six weeks; the usual biostimulation method took three months longer. “Bioaugmentation had a relatively poor reputation,” Loeffler says. “In cases targeting petroleum candidates, it didn't help any more than less-expensive strategies. Now, we have a good example of bioaugmentation at work.”

CLEAN GENES

One of the most exciting advances in bioremediation comes from gene sequencing—the newfound ability to quickly and inexpensively catalogue, and then analyze, the genes in key cleanup microbes. Last October, the Institute for Genomic Research in Rockville, Md., sequenced the genome of Shewanella oneidensis, an unusually versatile microbe used to bioremediate both heavy metals and chlrorinated solvents. Shewanella's genome turns out to have an unusually high number of cytochromes, which are enzymes associated with electron transport—the engine behind the microbe's bioremediating power. Many other labs around the country are sequencing and studying the genes of bioremediating microbes. For instance, Lovley's lab is sequencing the genomes of geobacter microbes gathered at the Rifle, Colo., site. “We'll understand which genes get expressed, and under what conditions,” Lovley says. “No one's ever done that before. Environmental microbiology's still operating in the dark to a large extent.” The group has already discovered that the microbe has the ability to swim and seek out different metals. Jim Tiedje is excited about his discovery that some bioremediating organisms have 7 to 12 different genes that could be active in these processes. “They're not related to anything else in the DNA databases,” Tiedje says. “It raises the question of what these genes are for. They're not identical, so they must have diversified over time.” And several companies are developing real-time polymerase chain reaction (PCR) tests, which identify DNA from organisms, to quickly detect what microbes are in the field. Loeffler's group used these tests to quickly detect which microbes were working at the Bachman Road site.

Even as bioremediators face the challenges of using bugs to fight radioactivity and toxic metals, they see an even bigger challenge, and opportunity, ahead: endocrine disruptors. These chemicals, which are byproducts of common industrial and agricultural chemicals, are increasingly suspected of causing health problems in humans and wildlife, including sterility, impaired development, birth defects, and metabolic disorders. The effects may be caused by extremely small amounts of contamination—parts per trillion. They have been detected in groundwater in various parts of the United States. Some European countries have started banning the use of endocrine-disrupting chemicals. Hazen says: “The only way to get at those is by bioremediation.”

 

Nancy Shute is a freelance writer based in the Washington, D.C., area. She can be reached at nshute@asee.org.

-By Thomas K. Grose

The successful use of bioremediation technologies to clean hazardous waste sites is a tribute to the field's multidisciplinary approach. When bioremediation works, it's because specialists with myriad scientific backgrounds have pooled their skills. But the pooling of experts from diverse fields requires those involved to have an understanding of what their colleagues do.

Frank Loeffler, a microbiologist and assistant professor of environmental engineering at Georgia Tech, says it is not enough to assemble a team if the participants can't communicate with one another. And, he admits, that's not always easily achieved because scientists speak in the language of their own discipline, which can leave outsiders—no matter how smart they are—bewildered. So it helps to have a working knowledge of the other sciences but something well short of expertise. That's why Georgia Tech recently introduced a required class for its undergraduate environmental engineering students: Engineering and the Environment. It covers hydrology, atmosphere, and biosystems, and is taught by three different professors (Loeffler handles the biosystems duties). “It's a quick, broad overview,” he says.

But students at other schools may have to pick up extra knowledge on an ad hoc basis. Craig Criddle, an associate professor of civil and environmental engineering at Stanford University, recommends that environmental engineering students who want to become bioremediators take classes outside their own curriculum to get a taste of the other sciences they'll be dealing with. He suggests courses in hydrology, geochemistry, and environmental microbiology. Most large schools offer these courses, Criddle says, but they may call them different things. For instance, hydrology may be called flow and porous media, while geochemistry might be referred to as aquatic chemistry. When it comes to joining a bioremediation team, a little bit of knowledge can be a good thing.