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July 2000
Genetic method has promise for assessing
environmental cleanup
WEST LAFAYETTE, Ind. – Engineers are using genetics to develop a simple, quick method for assessing the progress of environmental cleanup efforts at sites contaminated with petroleum-based pollutants like gasoline and diesel fuel.
The technique works by screening soil for genes that reveal the presence of an enzyme produced by pollution-busting bacteria. If the enzyme is detected, that means bacteria probably are cleaning the soil. Information about the bacteria's presence and concentration might then be used to assess the progress of efforts to remove toxins from the contaminated soil.
"It's like a direct biochemical method to take the attendance of the bacterial organisms in the soil," says Loring Nies, an associate professor of civil engineering at Purdue University who specializes in bioremediation, or using microorganisms to clean up environmental contamination.
The method could be used to test soil at contaminated sites within a few hours. Conventional methods require that soil samples be taken to a laboratory, where bacteria are cultured on a growth medium. But that can take days and is not always effective because some microorganisms will not grow under laboratory conditions, Nies says.
The technique being developed at Purdue works by detecting the enzyme catechol 2,3-dioxygenase, which several types of bacteria produce when soil is contaminated by petroleum-based toxins such as benzene, toluene and xylenes. Engineers first extract DNA from contaminated soil samples and then search for the specific genes that reveal the enzyme's presence. Detecting the enzyme is made possible because of a genetic tool called a "primer," which is a small piece of DNA that matches the gene sequence indicating the enzyme's presence.
"When you extract DNA from soil you can get DNA from a billion different microorganisms, and we only want to know about this one little piece," says Nies, who is developing the technique with Cindy Nakatsu, a Purdue associate professor of agronomy. The primer was specially designed to detect only catechol 2,3-dioxygenase, which neutralizes the petroleum-based contaminants. After the primer finds and attaches to a matching piece of DNA, thousands of copies are produced through a well-known method known as polymerase chain reaction, which enables scientists to make billions of copies of a single strand of DNA.
"Our detection technique requires several thousand or even millions of pieces of DNA before we can see them," Nies says.
Catechol 2,3-dioxygenase is present in several types of bacteria that degrade environmental pollutants. The toxins typically get into soil through leaking storage tanks. They are absorbed by the bacteria and broken down by enzymes into less toxic compounds, such as methyl catechol, which are further degraded into harmless chemicals by catechol 2,3-dioxygenase. While this is not the only enzyme used by microorganisms to neutralize petroleum-based toxins, it is one of the key biochemical "pathways" through which the contaminants are degraded.
"Of all the genes that we know about and all the pathways that we know about, this is a very important one and it is probably the dominant one," says Nies, who sees the technique as a potential new way to monitor the progress of cleanup efforts at contaminated sites.
"Suppose you took a soil sample at a site that had petroleum contamination and you didn't detect this gene, and then you took steps to enhance biodegradation, such as adding fertilizer or oxygen, or something else to improve the conditions for microorganisms. Then you could go back in a month and take another soil sample. If you detected the gene the second time, you would have some measure of how well the steps you took to improve biodegradation worked. You have some assessment tool to determine whether adding oxygen had any effect."
In some ways, the method is better than simply measuring the changing concentration of soil contamination.
"It's a very direct measurement," Nies says. "Measuring the changes in concentration of the contaminant is very important, but then you still don't know why the contaminant has decreased. For example, it could have disappeared because it flowed away in the ground water. So, in parallel with chemical analysis, we can do some microbial assessment."
A research paper about the method appeared in February in the journal Applied and Environmental Microbiology, published by the American Society for Microbiology. The paper was written by graduate student Matthew B. Mesarch, Nakatsu and Nies.
The technique has been shown to work in the laboratory, and researchers are now testing it in the field at actual contamination sites.
Sources: Loring Nies, (765) 494-8327, nies@ecn.purdue.edu
Cindy Nakatsu, (765) 496-2997, cnakatsu@purdue.edu
Writer: Emil Venere, (765) 494-4709, evenere@purdue.edu
Purdue News Service: (765) 494-2096; purduenews@purdue.edu
NOTE TO JOURNALISTS: A copy of the research paper referred to in this news release is available from Emil Venere, (765) 494-4709.
ABSTRACT
Development of Catechol 2,3-Dioxygenase-Specific Primers
for Monitoring Bioremediation by
Competitive Quantitative PCR
Matthew B. Mesarch, Cindy H. Nakatsu and Loring Nies
Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 102 to 103 gene copies, which was lowered to 100 to 101 genes by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegradation genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.
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