of the Problem
1,1,1-Trichloroethane (1,1,1-TCA) is a solvent that
was first sold commercially in the 1950’s as
a safer alternative to other degreasing products such
as Tricholoethelyne (TCE). Laboratory studies later
showed that 1,1,1-TCA had toxic effects on laboratory
animals – it damaged the animals nervous systems
and livers and caused delays in fetal development.
To protect human health, the USEPA set a limit of
0.2 parts of 1,1,1-TCA per million parts of drinking
water (0.2 ppm) (ATSDR, 1996).
1,1,1-TCA moves easily through soils and has become
a prevalent groundwater contaminant. It has been found
at about half of the sites identified for cleanup
by the federal Superfund Program and was among the
most common volatile organic compounds encountered
in a national study of contaminants in ambient groundwater
In addition, 1,1,1-TCA is a concern because it breaks
down into two additional contaminants, 1,1-Dichloroethane
(1,1-DCA) and 1,1-Dichloroethene (1,1-DCE). 1,1-DCE
is more toxic than 1,1,1-TCA itself -- the USEPA has
set a limit of 0.007 parts of 1,1-dichloroethene per
million parts of drinking water (0.007 ppm). (ATSDR,
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.
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.
Click on highlighted words and definitions and illustrations
will pop up.
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
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).
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
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
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
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.
For More Information
Contact Dr. Lewis Semprini
or Dr. Mark Dolan
or read more in the following references:
Kim, Y., Arp, D.J., Semprini, L., 2002, Kinetic and Inhibition
Studies for the Aerobic Cometabolism of 1,1,1-Trichloroethane,
s,s-Dichloroethylene, and 1,1-Dichloroethane by a Butane-grown
Mixed Culture cometabolism of 1,1,1-Trichloroethane, 1,1-Dichloroethylene,
and 1,1-Dichloroethane by a Butane-grown Mixed Culture butane-grown
mixed culture, Biotechnology and Bioengineering, v. 80,
Kim, Y., Arp, D.J., Semprini, L., 2000, Chlorinated Solvent
Cometabolism by Butane-Grown Mixed Culture, Journal of Environmental
Engineering, v. 126, no. 1, p.934-942.
Kim, Y., Arp, D.J., 2002, A Combined Method for Determining
Inhibition Type, Kinetic Parameters, and Inhibition Coefficients
for Aerobic Cometabolism of 1,1,1-Trichloroethane by a Butane-grown
Mixed Culture, Biotechnology and Bioengineering, v. 77,
ATSDR, 1995, Public Health Statement for 1,1-Dichloroethene,
ATSDR, 1996, Public Health Statement for 1,1,1-Trichloroethane,
Squillace, P.J., Scott, J.C., Moran, M.J., Nolan, B.T.,
and Kolpin, D.W., 2002, VOCs, pesticides, nitrate, and their
mixtures in groundwater used for drinking water in the United
States: Environmental Science & Technology, v. 36, no.
9, p. 1923-1930.(report
--pdf version). Supplemental
material (MS Excel format).