BIOAUGMENTATION WITH DEHALOCOCCOIDES: A DECISION GUIDE - Bioaugmentation for Groundwater Remediation - page 122

4.7.1 Laboratory Diagnostic Tests

Laboratory testing is by nature conducted at relatively small scales under controlled
conditions so information can be gathered with greater precision and lower costs than in the
field. However, there can be laboratory artifacts and difficulties in extrapolating to field scale.
The laboratory diagnostic tests useful for bioaugmentation decisions include molecular
biological analyses, compound specific isotope analyses and laboratory microcosms.
The advantages, limitations and interpretations of the most commonly used diagnostic analyses
are summarized in Table 4.1 .

4.7.1.1 Molecular Biological Tools
The least costly and quickest option for additional site testing is to perform specific
molecular diagnostic analyses, which are collectively described as molecular biological tools
(MBTs). These analyses have been applied to several environmental problems in recent years
(SERDP and ESTCP, 2005 ). These types of molecular analyses are discussed in greater detail in
Chapter 6 , so they are described only briefly below.
MBTs target key biomarkers (e.g., specific nucleic acid sequences, proteins or lipids) that
provide information about organisms and processes important for site characterization and
remediation. These methods have great potential to improve environmental characterization and
remediation (Lovley, 2003 ; Koenigsberg et al., 2005 ) and in particular they have the potential to
determine whether bioaugmentation will be needed or beneficial at a site (Ritalahti et al., 2005 ).
The method that has proven most useful to date is the quantitative polymerase chain
reaction (qPCR), especially as the range of genes analyzed is broadened and the technique is
extended to messenger ribonucleic acid (mRNA) (Stroo et al., 2006 ). A method that may help in
future bioaugmentation decisions is fluorescence in situ hybridization (FISH), which allows
direct visualization of cells with specific gene sequences of interest (Yang and Zeyer, 2003 ).
Dehalococcoides Biomarkers . The difficulties in isolating and studying Dhc by conventional

microbiological methods have spurred the effort to develop meaningful MBTs for these
important microorganisms (Cupples, 2007 ). MBTs can measure specific microbial capabilities
within a site and may be useful to quantify the current degradation potential and to identify
environmental conditions that are limiting the current potential.
The qPCR method can quantify several key gene sequences important in reductive dechlo-
rination of chlorinated ethenes (Figure 4.2 ) including:
1. 16S rRNA characteristic of Dhc (L¨ffler et al., 2000 ; Fennel et al., 2001 ; Hendrickson
et al., 2002 ),
2. pceA , a sequence from gene (PCE reductive dehalogenase) capable of dechlorinating
PCE to form TCE,
3. tceA , a sequence from gene (TCE reductive dehalogenase) capable of dechlorinating
TCE and other chlorinated aliphatic hydrocarbons (Magnuson et al., 2000 ),
4. vcrA , a gene sequence from the first VC reductase gene identified (M¨ller et al., 2004 ),
and
5. bvcA , a separate sequence from a VC reductase found in a different Dhc strain that
also reduces VC to ethene (Krajmalnik-Brown et al., 2004 ).
To date, the most useful of these biomarkers has been the 16S rRNA probe, which is
now routinely used to quantify Dhc in a sample by qPCR. This analysis has made it possible to