MICROBIAL MONITORING DURING BIOAUGMENTATION WITH DEHALOCOCCOIDES - Bioaugmentation for Groundwater Remediation - page 182

Table 6.1. Dehalococcoides qPCR Analysis Rules of Thumb
Dhc 16S rRNA (gene copies per L) Interpretation

10 3 or lower Suboptimal Dhc to sustain dechlorination rates

10 4 -10 6

May sustain appreciable dechlorination rates

10 7 or greater Usually associated with high rates of dechlorination

and ethene production

There is some uncertainty regarding the numbers of Dhc that are needed for acceptable
rates of dechlorination, or that would indicate complete biodegradation to ethene is occurring.
However, some rules of thumb have been developed for interpreting Dhc results (Lebr ´ n et al.,
2011a , b ). In general, Dhc concentrations less than 10 3 per L indicate suboptimal concentrations
of Dhc cells in groundwater, and such levels may not be capable of sustaining complete
dechlorination (van der Zaan et al., 2010 ; Lebr ´ n et al., 2011a ). Concentrations of 10 4 -10 6 per
L may provide acceptable dechlorination rates, while concentrations greater than 10 6 per L
often achieve high rates suitable for effective bioremediation (van der Zaan et al., 2010 ;
Lu et al., 2006 , 2009 ) and are considered the threshold concentration. These rules of thumb
for Dhc data interpretation also are shown in Table 6.1 .
As noted above, the limitations of qPCR must be considered when analyzing data obtained
from environmental samples. For example, even when sufficient numbers of Dhc cells are
present, complete reductive dechlorination may not occur, because the Dhc group contains a
diverse number of species and strains, with some capable of transforming TCE through to
ethene, and others capable of transformation only to DCE or VC. Research studies discovered a
correlation between particular genes (named “dehalogenases”) and the varied ability to trans-
form these compounds. One example is the vcrA gene mentioned in the introduction, which is
responsible for the final dehalogenation step reducing VC to ethene, and is not found in the
genomes of all known Dhc . Although qPCR analysis of the Dhc 16S rRNA gene may indicate
greater than 10 8 cells per L (a quantity likely capable of sustained and vigorous reductive
dechlorination), if a sufficient quantity of these cells are Dhc species lacking the vcrA gene, VC
will accumulate. Thus, analysis using qPCR for determining the concentration of the vcrA gene
(and/or other VC reductase genes) may be required.

6.5.5 Conclusion

The development of qPCR as a viable molecular biological tool for environmental analyses
provides a tremendous opportunity for collecting data in support of bioaugmentation design,
implementation and performance monitoring. However, as with chemical data obtained from
the environment, careful planning and a consistent approach are critical to ensure an accurate
assessment of site conditions is made. This is especially true given the nature of qPCR, where
sampling variability may result in reported concentrations orders of magnitude different from
actual concentrations.

6.6 FLUORESCENT IN SITU HYBRIDIZATION

6.6.1 Introduction

Fluorescent in situ hybridization (FISH) is a molecular technique used to detect genes
within microbes of interest in environmental samples, including Dhc (Madigan et al., 2003 ;
Yang and Zeyer, 2003 ). Through the use of fluorescently-labeled molecular probes, microbial