MICROBIAL MONITORING DURING BIOAUGMENTATION WITH DEHALOCOCCOIDES - Bioaugmentation for Groundwater Remediation - page 181

Even when DNA extraction occurs from a representative environmental source and
contains no inhibitory compounds, qPCR requires a solid understanding of the distribution
and diversity of any genes being measured. Poorly designed or selected primers may result in
qPCR-determined gene concentrations not reflective of actual environmental conditions.
Appropriate selection of target genes must reflect whether they are conserved, variable, or a
combination of both. Measuring a broadly distributed target gene may result in cellular
quantification errors resulting from differing gene copies being present in differing microbes.
Careful primer development, target gene selection and use of standard curve quantification
methods can overcome this limitation. Commercially available and well validated primers and
probes can be readily attained, specifically for analysis of Dhc -related genes.
PCR bias is a commonly reported limitation of these techniques (Reysenbach et al., 1992 )
and refers to the preferential amplification of one target sequence over another. For example,
in a groundwater sample containing equal concentrations of different species of bacteria, PCR
may preferentially amplify the 16S rRNA gene of one species, leading to an overestimate of
the relative abundance of that species. These preferential amplifications may be due to the
nucleotide content, secondary structure or even degradation of the target sequences. The effect
of these biases may be reduced by performing replicate PCR analysis on multiple DNA extracts
and pooling the replicates. Practical solutions to PCR bias under high biomass and high copy-
number amplifications also are described elsewhere (Polz and Cavanaugh, 1998 ).
Finally, it also must be remembered that the presence of a given gene does not guarantee
activity of that gene, but instead informs of its potential activity. In response to stimuli, DNA is
transcribed to messenger RNA that is then translated to a protein (enzyme). Many environ-
mental influences may result in inhibition or cessation of messenger RNA synthesis, so that
even though a gene is present in a microbial genome, it may not be transcribed or translated.
In these instances, the use of other molecular techniques capable of measuring actual activity
may be required. RT-qPCR is one such technique, which was developed to measure gene
expression. Assays of messenger RNA for Dhc activity should be interpreted with caution,
as transcript abundance may not correlate with activity (Fletcher et al., 2011 ).
The use of 16S rRNA genes as qPCR target genes carries with it an explicit assumption that
all members of the target population exhibit the same potential metabolic activity. As discussed
in the general example below, this is not always the case and care must be taken when choosing
the appropriate target gene of interest. Quantification of functional gene targets, in addition to
rRNA genes, can help to provide a more complete description for the metabolic capabilities of a
microbial community.
6.5.4 Dhc Analysis
Bioaugmentation of aquifers impacted by chlorinated ethenes is now commonly accepted
by regulatory agencies and practiced by remediation experts. Bioaugmentation cultures con-
taining Dhc species with the ability to respire chlorinated ethenes are commercially available.
Using qPCR, remedial design engineers may assess the need (or lack thereof) for augmentation
of native microbial populations with Dhc . Typically, groundwater samples are collected for
Dhc analysis, followed by extraction and purification of microbial DNA. The extracted DNA
undergoes qPCR analysis using primers specific for the 16S rRNA gene of the genus Dhc .
The genomes of all known Dhc species (like many microbes) contain a single copy of this 16S
rRNA gene that encodes for 16S rRNA, and ultimately relates to a protein subunit involved in
biosynthetic reactions. Thus, one copy of this gene corresponds with a single Dhc cell and the
greater number of this gene present in a sample, the higher the likelihood reductive dechlorina-
tion will occur.