Summary of the Problem
One of the goals of environmental
biotechnology is to manage microbial communities that
provide service to society. Services include the removal
of contaminants from groundwater, sediment or soil
and the protection of environmental and human health.
(PCE) and trichloroethene (TCE) are two of the
most hazardous and widespread groundwater contaminants
in the United States. A group of microbes called Dehalococcoides
is able to detoxify these compounds. Cleanup practitioners
have successfully promoted degradation of PCE and
TCE at field sites by introducing microbial enrichment
cultures containing Dehalococcoides organisms.
Modern molecular biology tools provide a way to study
and monitor the ecology of beneficial microbial communities
such as Dehalococcoides. Techniques such
as polymerase chain reaction (PCR) and fluorescence
in situ hybridization (FISH) provide a way to identify
and enumerate microbial populations and monitor their
activity in the environment with a high degree of
spatial and temporal resolution. This information
can then be used to create conditions that promote
the microbes’ performance of a desirable service
like the detoxification of contaminants.
WRHSRC researchers Alfred Spormann and Sebastian
Behrens are exploring ways that PCR and FISH can be
used to monitor and optimize the dechlorinating activity
Applying Molecular Techniques
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.
Click on highlighted words for illustrations and links.
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.
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
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
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
Alfred Spormann, or refer to the following:
Amann, R., Fuchs, B.M., Behrens, S., 2001,
The identification of microorganisms by fluorescence in
situ hybridisation, Environmental Biotechnology, 12, pp
Behrens, S., J. McMurdie, G. Meshulam, A.
Spormann. 2005, Evaluation of a CARD-FISH Protocol for the
Quantification of Dehalococcoides sp. in Soil. 2005 American
Society of Microbiology General Meeting.
Muller, J.A., Rosner, B.M., von Abendroth,
G., Meshulam-Simon, G., McCarty, P.L. and Spormann, A.M.,
2004, Molecular Identification of the Catabolic Vinyl Chloride
Reductase from Dehalococcoides sp. Strain VS and Its Environmental
Distribution, Applied and Environmental Microbiology, 70(8),
Semprini, L., M. Azizian, A. Sabalowsky,
M. Dolan, P. Ruiz-Hass, J. Ingle, S. Behrens, A. Spormann.
2005. A Continuous Flow Column Study of Anaerobic PCE Transformation
with the Evanite Culture and Hanford Aquifer Solids. Joint
International Symposia for Subsurface Microbiology (ISSM
2005) and Environmental Biogeochemistry (ISEB XVII), Wyoming,
August 14-19, 2005.