Applying Molecular Techniques to Bioremediation

Molecular biology tools that allow organisms to be identified and quantified on the basis of their genetic composition present an exciting opportunity for bioremediation. Researchers at the WRHSRC are developing applications for two of these techniques – polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Led by Alfred Spormann and Sebastian Behrens at Stanford University, the team’s focus is on bioremediation of PCE and TCE and the use of molecular techniques to monitor the abundance and activity of Dehalococcoides, a group of bacteria that can couple the detoxification of these compounds to growth through a process called metabolic halorespiration.

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PCR

PCR is a widely applicable technique that can be used to confirm the presence of a species by targeting and replicating a genetic sequence unique to the organism of interest. Standard PCR is used commercially to identify the presence of Dehalococcoides in environmental samples and establish the potential for dechlorination to take place. Real-time PCR is a modification of the basic PCR technique that allows quantification of the initial target sequence concentration by using a variety of different chemistries. The quantification principle is based on changes of the optical properties of fluorescent dyes that accumulate in proportion to the number of copies of the targeted gene sequence.

For the general identification of Dehalococcoides organisms, unique signature sequences of the 16S ribosomal RNA (16S rRNA) gene are targeted. However, a critical short coming of this technique has been a lack of primers that distinguish between strains of the Dehalococcoides bacteria. Sub-species identification is necessary because only some strains can carry out the complete reaction sequence that transforms PCE to the harmless substance ethene (Figure 1). Other strains initiate dechlorination, but cannot transform the highly toxic, intermediate product, vinyl chloride.

To solve the strain identification problem, the WRHSRC team has focused on genetic information in Dehalococcoides organisms that encodes for enzymes called reductive dehalogenases. These enzymes catalyze the dechlorination reactions of specific chlorinated hydrocarbons, like PCE or TCE. Different strains of Dehalococcoides differ in their genetic composition of these enzymes. For example, only some strains produce a VC reductase - an enzyme that catalyzes the transformation of vinyl chloride to ethene. The WRHSRC researchers at Stanford have developed primers that target genes coding for VC reductases and other reductive dehalogenases. Their goal is to use the primers in real-time PCR assays to identify and quantify different strains of Dehalococcoides in the environment.

The same set of primers can also be used to quantify how the activity of reductive dehalogenase genes changes in response to environmental conditions. For this type of analysis, mRNA is targeted in real-time PCR assays instead of genomic DNA. mRNA is the messenger that triggers production of a particular protein, therefore its presence is a measure of gene expression and indicates that a particular gene has been “turned on”. Analysis of mRNA levels of reductive dehalogenases under different environmental conditions will lead to a better understanding of how Dehalococcoides organisms are likely to function in contaminated environments.

FISH

The team is also exploring the use of another molecular technique called FISH. FISH is a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation. The technique combines the precision of nucleic acid hybridization with the visual information of microscopy (Figure 2). In theory, each ribosome within a targeted bacterial cell is stained by one fluorescently-labeled probe molecule during the hybridization procedure. This permits the abundance of the targeted microorganisms to be estimated. However, the majority of environmental bacteria are small, slowly growing or starving, and contain low amounts of ribosomes (e.g. Dehalococcoides). This means that fluorescence intensities are frequently below detection limits or they can be overwhelmed by high levels of background fluorescence.

These limitations can be overcome by the use of horseradish peroxidase (HRP) labeled oligonucleotide probes and tyramide signal amplification (TSA), also known as catalyzed reporter deposition (CARD). CARD is based on the deposition of a large number of fluorochrome-labeled tyramine molecules by peroxidase enzyme activity. This technique leads to greatly enhanced signal intensity and detection rates by introducing numerous fluorescent molecules at the probe hybridization site inside each cell. The WRHSRC team at Stanford is exploring the applicability of CARD-FISH protocol for the identification and quantification of Dehalococcoides organisms in environmental samples.

Column Experiments

The WRHSRC team has applied PCR and CARD-FISH to monitor and evaluate the performance of Dehalococcoides organisms in continuous flow columns with PCE or TCE dechlorinating conditions. They chose this experimental setting because the columns mimic some of the complexities of a field setting -- the columns are packed with aquifer solids and the flow makes the setting dynamic. Trials involve adding the contaminants, an electron donor, and different bacterial cultures containing Dehalococcoides organisms and observe changes in microbial community composition, detoxification activity and product formation.

The team samples the microbial community by withdrawing material through special ports located along the 30-cm length of the column. Comparisons of the microbial community structure with data about dechlorination rates and the expression of reductive dehalogenase genes gives the team insight into the complex microbiology of bioremediation.