Progress on Using Bacteria to Clean Aquifers

IN Lewis Semprini's basement laboratory, tall glass columns covered with aluminum foil and filled with sand rest on top of a workbench. The sand comes from a nearby underground water source, or aquifer, that is contaminated with hazardous organic chemicals. But thanks to a growing colony of microorganisms, the sand inside the column gets a little cleaner every day. Fed on a mixture of oxygen and methane gas, the bacteria are working day and night to transform the chlorinated solvents into safe compounds like carbon dioxide and water.

``What we have done to date is work with bacteria in the subsurface, bacteria that are already there, and change the environmental conditions to enhance the transformation process,'' explains Dr. Semprini, a research scientist in the civil engineering department at Stanford University here.

Feeding the bacteria in the laboratory is easy: Just bubble the gases through the glass columns. Last year, a Stanford research group headed by Dr. Paul V. Roberts demonstrated that the bacteria can also be fed in their native environment. During a three-year experiment, the bacteria broke down as much as 85 to 95 percent of some hazardous compounds tested, according to the group's final report.

Bioremediation, the process of harnessing bacteria to break down hazardous, man-made chemicals, ``has the potential to clean up many contaminated aquifers,'' says Semprini, the project's manager. For years, bioremediation has taken the back seat to other methods of cleaning up contaminants, but the technique is quickly gaining in popularity.

``We've been trying to make it a trendy topic for a long time,'' says Fran Kremer, a senior environmental engineer at the United States Environmental Protection Agency's office of research and development. This month, the EPA will sponsor a three-day conference on the its biosystems technology development program.

Since most environmental engineers have not been trained in bioremediation, says Dr. Kremer, informing professionals about the techniques and convincing them to consider biological methods when planning a cleanup is a big part of her job. Since biological methods can't be used at every site, ``treatability studies need to be conducted up front,'' she says.

A prospective site must be surveyed to make sure that there are bacteria in the ground that can break down the chemicals that are present: Hence the glass columns in Semprini's basement.

``When we started our field program, we got core samples'' from a contaminated aquifer near the Moffett Naval Air Station, a half-hour drive from San Francisco, explains Semprini. ``We tried to take them as aseptically as possible: You try to get the bugs that are [underground], not the bugs from the surface.''

Back in the laboratory, the scientists wrapped the columns with aluminum foil to keep the soil in the dark, helping to simulate the underground environment. Then they exposed them to the mixture of methane and oxygen to see if the particular bacteria present could break down the hazardous organic compounds.

The choice of gases was not accidental, says Dunja Grbic-Galic, an associate professor at Stanford working with the group.

Since the mid-1980s, scientists have known that chlorinated industrial solvents like trichloroethene and vinyl chloride could be broken down by microbes called methanotrophs - bacteria that feed on methane.

``The chlorinated compounds which we wanted to degrade are not used by bacteria for growth or energy,'' explains Dr. Grbic-Galic, whose field is molecular biology. But the enzyme the bacteria use to break down methane will ``fortuitously attack'' the chlorinated compounds. ``[The enzyme] doesn't have a very narrow substrate specificity: It can degrade not only its substrate for which it is made but other things [as well].''

The methanotrophs transform the chlorinated solvents into very reactive chemicals called epoxides, says Grbic-Garlic. ``Once the epoxide is formed, and once it breaks down by itself, the product can be used by heterotrophs,'' another kind of bacteria that is also found in the aquifer. ``That means the compound will be completely degraded.''

After the group verified the presence of the required bacteria, they set up an automated research station on top of the aquifer. Water was pumped out of the ground at an ``extraction well,'' run through a bubbler that filled it with methane and oxygen, and then pumped back into the ground at the ``injection well,'' says Semprini. Three smaller wells pumped tiny amounts of water out of the ground for analysis, using laboratory equipment that the group specially modified to work unattended. A personal computer ran the entire experiment 24 hours a day.

THE system bubbled the methane into the water for four hours, then oxygen for eight. Alternating the gases, explains Semprini, kept ``the methanotrophs [from] growing up and clogging the injection well.'' It also had the side effect of distributing the gases evenly through the test zone, which made the bacteria grow more uniformly.

The results of the experiment were encouraging: The bacteria started transforming the trichloroethene within eight days; after the first year, 20 percent of the chemical was gone, says the group's report. Another chemical, vinyl chloride, was completely degraded in less than two days. One of the most impressive results, says Semprini, is that the depletion of organic compounds in the water almost perfectly matched the group's mathematical predictions.

``I think it's excellent work,'' says John Wilson, who pioneered the use of methanotrophic bacteria in the early 1980s. ``It is becoming a model for doing precise performance evaluations at the field scale.''

Simply running the extraction pump might cause the concentration of trichloroethylene (TCE) to decrease because of dilution, says Dr. Wilson, a senior research microbiologist at the EPA's Kerr environmental research laboratory in Ada, Okla. Concentrations can also be reduced by the absorption of the TCE on the solids in the aquifer. ``What is unique about their work is that they clearly separate all of the possible influences that attenuate the concentration of the contaminants concerned.

``TCE is one of the most common and economically important contaminants of drinking water in the industrial world,'' continues Wilson. ``The most common remedy is called pump-and-treat, where water is pumped from the aquifer and treated at the surface. Stanford's process, and the design model, can be used to shorten the time required for pump-and-treat by adding biological degradation, maybe by as much as one half, and that reduces the cost of the cleanup proportionally.''

The Stanford group is now surveying a large Superfund site in Michigan to see if they can try the approach there. As with the experimental site, the first step is to find out if bacteria in the ground can break down the particular toxic chemicals present.

``Sites are very different,'' says Grbic-Galic, who is still studying the precise reactions that the bacteria use to break down the chemicals. ``Just because one process works at one site does not mean that it will work at others.''

Although it might be possible to pump down bacteria if they are not present, ``I think it is easier if the bugs are already there to convince a regulatory agency [that the procedure is safe],'' says Semprini. And if the bacteria are already present, he adds, then they will be distributed throughout the site.

Underground biological treatment is a particularly attractive way to clean up contaminated aquifers, says Semprini. ``With the appropriate microbial process, you can completely degrade the contaminants to nontoxic end products: You're getting rid of the problem,'' he says. Some pump-and-treat systems simply remove the contaminants from the aquifer and either release them into the air or deposit them on charcoal filters, which must then be disposed of.

Another problem with pump-and-treat techniques, he adds, is that the organic compounds tend to stick to the sand that make up the aquifer, making the process less efficient as time goes on. ``What happens with the bacteria is that they are actually growing attached to the solids'' right where the contamination is most likely to be, Semprini says with a smile.

But the approach isn't a cure-all for contaminated ground water, he quickly adds. For example, the methanotrophs broke down some contaminants that were in the aquifer but left others untouched. And even with a large-scale bioremediation system it might still take years to clean a large aquifer.

In the future, says the EPA's Kremer, bacteria might be genetically tailored to eat specific organic compounds. ``We have some major research programs on-going within the agency. The main obstacle is the regulatory issue and [not understanding] a lot of the ecological impacts for utilizing genetically engineered microorganisms in the environment.''