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Backyard bacteria rout a stubborn toxin

In deep volcanic Idaho rock, engineers track how native soil bacteria clean up the groundwater

Kent Sorenson stands in the southeastern Idaho desert where he and his team at INEEL found that microorganisms efficiently clean up the groundwater.

Engineering and Microbiology — In a portion of fractured basalt more than 200 feet below the surface of the Idaho National Engineering and Environmental Laboratory lies a highly concentrated sludge of the heavy liquid toxin trichloroethene (TCE). INEEL engineers are determined to rid the rock of the toxic solvent which, over more than 30 years, has gradually leached into the groundwater of the Snake River aquifer. Cleaning up a persistent contaminant like TCE usually means going to the expense and the trouble of bringing the polluted groundwater to the surface. Fortunately, however, the underground rock is also home to a crowd of resourceful soil bacteria.

In 1998, environmental engineer Kent Sorenson and a team of scientists at INEEL recruited native microorganisms to break down the recalcitrant TCE. The experiment worked even better than planned, and the Laboratory has now turned large-scale cleanup over to the bacteria. A summary of the experiment will be published this month in the Battelle Bioremediation Symposium Proceedings. Engineers estimate that the microbial cleanup of both the concentrated sludge and its plume of contaminated groundwater is saving the Laboratory $15 million over 30 years.

Naturally, the cleanup crew and the government are happy with the successful (and cost-cutting) results. But the scientists are just more intrigued. When the Idaho bacteria degraded the toxin more efficiently and thoroughly than expected, the scientists could not really say why. The process, in-situ bioremediation, is now widely used as a cleanup strategy, but "it's like a black box," said Lisa Alvarez-Cohen, a professor of engineering at UC Berkeley who collaborates with Sorenson. Nobody knows the exact combination of microorganisms that do the work.

Sorenson and his collaborators are now taking the experiment further to unearth the details of the complex underground microbial communities.

Underneath the surface

TCE is one of the most common and intractable groundwater contaminants in the United States. It is widely used as a solvent to clean greasy metal tools and to remove paint and fabric stains. Breathing a small amount may cause a headache. Drinking a large amount could cause irreversible liver damage or even death. The difficulty of removing TCE once it gets into the groundwater is that it degrades very slowly. It will persist within an aquifer for years, gradually dissolving into the flowing groundwater, and spreading farther and farther away from the source.

"Solvent plumes often grow to miles long," said Sorenson. And although the amount of pollutant that actually dissolves in the water is often very small, that amount is still above the EPA's strict drinking water standard of five parts per billion, or about one drop of TCE per four full bathtubs of water. "You can have concentrations above five parts per billion for centuries," Sorenson said.

The solvent originally entered the aquifer through a well, into which the Laboratory injected waste, including TCE, tritium and other chlorinated solvents, from the mid-1950s to 1972.

In 1995, the government pledged to restore the groundwater to drinking water standards. Traditionally, cleanup would mean pumping the groundwater up from the aquifer, removing the TCE, and then returning the clean water to the ground. Yet "pump-and-treat" is slow and expensive, and in the end, the engineers still must decide what to do with the toxic TCE they have removed.

A natural remedy

Sorenson and his co-workers found a far better alternative to pump-and-treat: in-situ bioremediation, a process that encourages microbes already in the underground rock to break down the toxin. Bioremediation has become increasingly common as a cleanup method. It is relatively inexpensive, fast and good for the environment. Plus, said Sorenson, "the bugs that do it are pretty ubiquitous. People have found them in uncontaminated areas."

To decontaminate, the microorganisms first need a high-energy food called an electron donor. Sorenson decided to use lactate, a molecule that the heart commonly uses for fuel. With the energy from lactate, soil microorganisms can break down TCE by successively stripping it of chlorine molecules. They turn TCE, a three-chlorine compound, into a two-chlorine compound called dichloroethene (DCE), a one-chlorine compound called vinyl chloride (VC), and finally, into ethene, a completely nontoxic and nonchlorinated molecule found naturally in plants.

One of the potential hitches in TCE cleanup, and what has happened at other sites, is that the microbes won't take TCE all the way to ethene—they may only take it as far as DCE, a compound still considered toxic to the soil. It isn't always a smooth, continuous cascade down to ethene.

At the INEEL Site, however, the TCE was taken straight to ethene. Even in the first month, the microbes started to consume 300 gallons of lactate within two days after an injection. After pumping batches of lactate into the soil every week for eight months, the scientists found more ethene in the groundwater than any of its chlorinated precursors.

The researchers weren't surprised that it worked. They just didn't know it would work quite so well. Within 24 months, the TCE concentration at the injection well itself dropped from 240 times the drinking water standard to below a detectable level.

One exceptional result contributed to the impressive success. Near the end of the initial 12 months of the experiment, Sorenson noticed a rather odd phenomenon. Once the engineers stopped adding lactate, TCE degradation sped up. Sorenson reasoned that since lactate itself breaks down in the soil into acetate and propionate, some of the microbes were using these breakdown products as food—or electron donors—and using them more efficiently than the original lactate. The microbial community apparently shifted once the lactate was gone, and became more efficient in the presence of lactate offspring than the original lactate.

The engineers found they didn't need to—and in fact shouldn't—send lactate into the soil as frequently. Since less frequent injections led to faster cleanup, it decreased the cost overall. Sorenson wants to understand why the microbes work so much better after the lactate is gone and which microbes do it. He thinks that understanding will lead to even greater cost savings.

"When you have questions in the field, you take them back to the lab to get answers. You thus create a process for continuous improvement," he said. He would like to set up an optimizing system, where glitches, puzzling results and interesting data from the field are brought back into the laboratory for investigation. The laboratory answers are applied in the field again, and the cycle repeats itself in a feedback loop.

So back in the laboratory, an Idaho State University graduate student, funded by INEEL's Laboratory Directed Research and Development fund, is trying to decipher which combination of microorganisms work most efficiently.

Back at the lab

Tamzen Wood says it is hard to recreate an underground environment above ground. In the field, the microorganisms she studies live a football field's length below the surface in something of a microbe-Mecca. TCE was only one of many waste products injected into the aquifer, and it is therefore "an extraordinarily rich area," said Wood. Scores of microbes grow and feed on the waste. The active microorganisms used up all of the oxygen in the vicinity long ago and created a strongly anaerobic system. Wood is careful, when she brings the field sample to the laboratory, to never expose it to the oxygen that the microbial communities have learned to live without.

It's important for Wood that oxygen not contaminate the sample, because only in an anaerobic environment do the microbes degrade TCE. "They're basically using TCE like we use oxygen," said Wood. In essence, in the absence of oxygen, the bacteria that use oxygen give way to a population that uses whatever's available (TCE, for example) as a replacement.

Wood is letting the community grow in a cooled, oxygen-free flask, and then will divide it into five smaller ones. She will add different types of food—different electron donors at different intervals—to each flask. After letting them grow for a time, she'll gather the results. She's looking for two things: which bacteria grow and what byproducts they produce.

She's pretty sure that whatever she finds, it will be not just one species, but rather a combination of microorganisms that do the work. Sorenson said that, in general, it takes more than one type of microbe to break down TCE--one species will take TCE only so far, and then hand it off to another to finish the job. Even bacteria not directly involved in TCE degradation change the environment to make it more or less favorable for the TCE work. The separate populations wax and wane with their food sources to create shifting boom-and-bust communities.

As Wood explained, "You've got bugs in there that are helping your bugs and bugs in there that are hurting your bugs and that is what I am trying to tease apart in my project."

Wood collaborates with Anna-Louise Reysenbach's laboratory at Portland State University to identify the microbial species. She does so by isolating and identifying the DNA. She's first trying to catalog all the species that live there (somewhere between 18 and 34—perhaps even more). Then she compares her master list to what she finds in each experimental flask. That way, she knows which combination of microbes is breaking down the greatest amount of TCE fastest.

Combined, the scientists hope to understand the natural underground population booms and what causes them. That revelation, Sorenson firmly believes, will ultimately translate to more cost-effective, efficient TCE bioremediation. The idea is to understand the process well enough that they can encourage the right microbes to grow, at the right time, to fully decompose one of the most obstinate groundwater contaminants.

Kent Sorenson is now working at an environmental engineering firm (North Wind Environmental, Inc.) that subcontracts to the Laboratory. The lead researcher for the TCE bioremediation research at INEEL is Dave Cummings.



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