BIOAUGMENTATION FOR GROUNDWATER REMEDIATION: AN OVERVIEW - Bioaugmentation for Groundwater Remediation - page 17

1.4.4 Monitoring Effectiveness

Once the bioaugmentation treatment is in place, it is necessary to monitor the presence of
the inoculum and/or the degradation of the pollutant. Pollutant levels are primarily monitored
to ensure the objective of the treatment - namely pollutant removal. It also would be ideal to
monitor for the accumulation of toxic metabolites. Inoculum levels are monitored to ensure
that the bacteria are alive and active and to be able to correlate pollutant reductions with
microbial activity. Loss of inoculum would signal a need for reinoculation or use of a different
inoculum. Ideally, once the treatment is complete, the inoculated strains should cease to be an
active part of the system, and tracking the inoculum would verify this. There are several
methods available for tracking the inoculum and pollutant degradation, including using

microbiology, molecular biology or physicochemical techniques (Table 1.3 ).
Conventional microbiological techniques like plating and most probable number (MPN)
counts take samples from the site of interest and then grow the organisms in the sample on
defined media. In the case of plating, dilutions of the sample are spread onto agar plates with
some kind of selective agent (usually the target compound) to isolate the degrading species and
confirm their degradation activity. With MPN, the samples are diluted until the activity of
interest can no longer be detected in liquid media.
Recent innovations include fluorescence in situ hybridization (FISH), which uses fluores-
cent probes that bind to a gene of interest (either phylogenetic or catabolic) so that organisms
containing the target gene can be observed directly (Yang and Zeyer, 2003 ). Successful
identification of the gene is observed using a fluorescent microscope or flow cytometry.
If genetically-modified bacteria were to be used in the field, monitoring their presence and
activity could be facilitated by incorporating a reporter gene - like the luc gene encoding firefly
luciferase or the gfp gene encoding green fluorescent protein - downstream of the catabolic
genes (Jansson et al., 2000 ).
Modern molecular methods avoid the pitfalls of culturing bacteria and can be especially
useful with consortia or uncultured organisms because they use genetic material extracted
directly from the medium. Molecular methods often revolve around the polymerase chain
reaction (PCR) technique to monitor nucleic acid sequences - particularly the 16S ribosomal
ribonucleic acid (rRNA) sequences - from the microbes of interest (Gentry et al., 2004 ).
The benefit of PCR is that it amplifies a quantitatively small amount of target sample to a
level where it can be detected either on gels or with fluorescent markers. PCR can be used to
detect the presence of the gene, while real-time quantitative PCR (qPCR) can be used
to quantify gene levels in a system (Van Raemdonck et al., 2006 ). Reverse-transcriptase PCR
(RT-PCR) reflects what genes are being expressed, and involves extraction of messenger RNA
(mRNA), reverse transcription of that RNA to DNA and amplification of the gene of interest.
RT-qPCR combines the reverse transcription step with a quantitative PCR. Analysis of mRNA
is currently considered a semi-quantitative method because it often is unstable. However, the
presence of detectable mRNA demonstrates that the gene of interest is being expressed, and the
results can indicate activity levels, particularly in comparison to other samples (ESTCP, 2005 ).
If there are numerous genes or strains to be monitored, a microarray of the target genes can
detect thousands of sequences (associated with those genes/strains) simultaneously (Johnson
et al., 2008 ). Microarray analysis is performed by first labeling the sample genetic material,
usually with fluorescent tags or radioactivity, and then hybridizing the sample with the micro-
array chip onto which the target genes have been affixed. The chip is then washed to remove the
non-hybridized sample and read using the appropriate technology, like a fluorescence scanner.
These and other molecular methods of monitoring bioaugmentation have been reviewed more
thoroughly elsewhere (Saleh-Lakha et al., 2005 ), and are reviewed in Chapter 6 of this volume.