Field Studies of Cometabolism

Most bioremediation technologies make use of microbes that feed on contaminants and convert them into harmless end-products. WRHSRC environmental engineers Dr. Lewis Semprini, Dr. Mark Dolan and Dr. Perry McCarty are studying an alternative approach. They are leading a field study on cometabolism – a process where microbes do not consume contaminants directly, but instead live on an alternate food source and fortuitously create conditions that degrade contaminants.

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The study focuses on the cleanup of 1,1,1-Trichloroethane (1,1,1-TCA) a toxic solvent that is found at nearly half the sites identified for cleanup by the federal Superfund Program (ATSDR, 1996). In experimental trials at Moffett Federal Airfield in California, the researchers have been able to use cometabolism to lower the concentration of 1,1,1-TCA in test wells by 70 to 80%.

The experiments utilize a microbial culture that grows on butane. Dr. Dolan isolated the culture from an enriched culture obtained from Hanford Nuclear Reservation during the 1990s (Kim et al, 2000). Years of laboratory studies showed that the culture could not only transform 1,1,1-TCA but also a variety of other chlorinated aliphatic hydrocarbons (CAHs) (e.g. Kim et al, 2002).

The isolated culture seemed particularly promising for cleanup of 1,1,1-TCA. In laboratory studies it could degrade both the contaminant itself and its breakdown products, 1-1 Dichloroethane (1,1-DCA) and 1,1 Dichloroethene (1,1-DCE) (Figure 1). 1,1-DCE is of particular concern; it is extremely toxic and very low concentrations make drinking water unsafe (see box at left).

The success of the culture in the lab prompted the Moffett Airfield study: would cometabolism of 1,1,1-TCA, 1,1,-DCA, and 1,1-DCE work in the field? The study is funded by the Strategic Environmental Research and Development Program (SERDP).

Field Trials

The Moffett Airfield study site consists of two “test legs” of wells six meters deep extending over a seven meter length of a confined aquifer. Wells at one end are used for injection and wells at the other end for extraction; this pumping pattern sets up two parallel but isolated flow fields (Figure 2). One of these “test legs” is used as the experimental system where researchers inject the contaminants, the study culture, and supplements such as butane, oxygen, or hydrogen peroxide dissolved in groundwater. The other leg becomes the control (stimulation of native microorganisms) where they inject only the contaminant and supplements. Observation wells allow samples to be taken over the length of the flow fields.

In three years of trials, the WRHSRC researchers have experimented with adding different combinations of the culture, contaminants, and supplements over different time intervals. For example, Figure 3 shows a trial during the second year where they added 1,1,1-TCA on day 10, and the experimental culture, butane, and oxygen on day 24. The graph shows that the concentration of 1,1,1-TCA dropped dramatically after the addition of the culture. By day 40, its concentration in the S3 monitoring well was about 70% below the injection concentration. In contrast, the researchers did not observe a decrease in 1,1,1-TCA concentrations in the control leg.

Unfortunately, the graph also shows that the concentration of 1,1,1-TCA in the S3 monitoring well gradually began to increase and after 70 days approximately 50% removal was achieved. For some reason, the effectiveness of the added culture declined with time. Semprini and Dolan aren’t certain why, but it’s a result they encountered repeatedly in different trials. “Perhaps indigenous microorganisms, which utilize butane but do not cometabolize 1,1,1-TCA, begin to out compete the injected culture,” says Semprini.

Molecular Studies

Dr. Dolan is leading molecular studies that may help to answer this question. His research uses DNA techniques to compare the microbial communities in different test wells and at different time intervals during the experiments. He hopes his studies will show how the composition of the microbial community changes through time and as butane or other supplements are added.

One molecular technique he uses is called Terminal Restriction Fragment Length Polymorphism (T-RFLP). T-RFLP creates a “community fingerprint” that gives a qualitative picture of the types of organisms present in the culture. T-RFLP involves extracting a sample of the mixed culture's DNA and amplifying a universal DNA sequence using polymerase chain reaction (PCR). The amplified sequences are then labeled on one end with a fluorescent tag and cut into fragments using a restriction enzyme. Because the DNA comes from different organisms with different base-pair sequences, the restriction enzymes will cut the DNA into fragments of different lengths. Each length is characteristic of a particular organism and the relative number of fragments with that length is a qualitative indication of the abundance of that organism. For example, Dr. Dolan nicknamed one of the organisms in the Hanford culture the “183 base-pair organism” because its signature in T-RFLP was a DNA fragment with a 183 base-pair length.

Dr. Dolan has used other microbial techniques to isolate and identify specific organisms within the experimental cultures. The more he learns about the individual organisms and the dynamics of the microbial communities, the better he will be able to create conditions that will maximize their growth and their ability to transform contaminants.

The Moffett Airfield study demonstrates that 1,1,1-TCA, 1,1,-DCA, and 1,1-DCE can be removed from groundwater by adding a cometabolizing culture and its food source. The next step will be to learn more about the microbial community that makes up the culture and about ways to prolong its effectiveness in the field.