DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 66

2.10 DEHALOCOCCOIDES BIOMARKERS
Dhc -containing consortia can be mass-produced (Vainberg et al., 2009 ) and have been used
successfully at numerous sites to initiate dechlorination or increase dechlorination rates and
achieve desired end points (Ellis et al., 2000 ; Lendvay et al., 2003 ; Major et al., 2002 ).
Prognostic site assessment and diagnostic bioremediation monitoring tools are desirable to
detect and quantify total Dhc and individual Dhc strains with specific dechlorination activities
of interest. Various laboratories utilize primer sets that target slightly different regions of the
Dhc 16S rRNA gene (Table 2.7 ).
Typically, DNA is extracted from a groundwater sample (Ritalahti et al., 2010a ) and Dhc
biomarker genes are either detected with qualitative PCR methods (Hendrickson et al., 2002 ;
L¨ffler et al., 2000 ) or quantified using qPCR. Robust qPCR protocols that either use TaqMan
or SYBR-Green detection chemistries have been published (Behrens et al., 2008 ; Holmes et al.,
2006 ; Mackay, 2004 ; Ritalahti et al., 2006 ; Hatt and L¨ffler, 2012 ). TaqMan detection relies on
fluorescently-labeled, linear hybridization probes and the application of probes carrying differ-
ent fluorophores allows quantification of up to four targets in multiplex format (Mackay,
2007 ). The 16S rRNA gene serves as an excellent biomarker to detect and quantify Dhc in
laboratory and environmental samples, although this analysis has its limitations. As more
environments are explored and new sequences are added to the GenBank database, the
available information expands the diversity and reveals the unexpected breadth of this bacterial
group. As a result, some tests designed to specifically assay for Dhc later have been found to
target a broader range of organisms. For example, several primer pairs designed to be Dhc -
specific also amplify the 16S rRNA genes of 1,2,3-trichloropropane-dechlorinating Dehalogen-
imonas lykanthroporepellens strains, which are distant Dhc relatives (Figure 2.3 ) (Yan et al.,
2008 ). In other words, the lack of knowledge of the diversity of Dhc and related bacteria can
result in false positives and lead to erroneous conclusions, possibly limiting the value of the
analysis.
Another drawback of 16S rRNA gene targeted analyses is the similarity of 16S rRNA gene
sequences among Dhc strains that exhibit different dechlorination activities. For example, some
members of the Pinellas group share identical 16S rRNA genes but the strains use different
chlorinated compounds as electron acceptors (Table 2.4 ) indicating that the resolution of the
16S rRNA gene is insufficient to infer dechlorination activity. Thus, the 16S rRNA gene
analysis provides information about the presence of Dhc and related dechlorinators but the
analysis falls short of providing insights into specific dechlorination activities (L¨ffler and
Edwards, 2006 ). Despite this shortcoming, quantitative monitoring of Dhc 16S rRNA gene
provides useful information, especially when the data are correlated with contaminant trans-
formation over time (i.e., temporal assessment of the same monitoring wells).
To overcome the limitation of the 16S rRNA gene analysis, genes that correlate directly
with dechlorination activity are being sought. Specific function has been assigned to few Dhc
RDase genes (Table 2.6 ) and a major task is to elucidate the substrate range of each functional
RDase represented on the Dhc genomes. Knowledge of the full spectrum of haloorganic
substrates is pivotal for designing a comprehensive suite of molecular tools for monitoring
abundance and expression of individual RDase genes and predicting dechlorination activity.
Figure 2.7 shows RDase genes that have been implicated in the reductive dechlorination of
chlorinated ethenes. The genes tceA , vcrA and bvcA have served as useful markers to track
individual Dhc strains relevant for chlorinated ethene detoxification (Behrens et al., 2008 ;
Holmes et al., 2006 ; Ritalahti et al., 2006 ; Scheutz et al., 2008 ).
Although the utility of these targets for monitoring chlorinated ethene reductive dechlori-
nation has been demonstrated, it is clear that only a small fraction of all RDase genes