Biomedical Engineering Reference
In-Depth Information
Just as the examination of soil borings and groundwater hydrogeology augment historical
document evaluation in determining the appropriateness of traditional treatment technologies
such as pump-and-treat, the use of molecular biological tools (MBTs) now permit a more
accurate assessment of potential bioremediation options.
Even more so than groundwater or soil sampling for chemical analyses, MBT use can
suffer from careless planning, sloppy field sampling protocols and misinformed interpretation.
The great power of MBTs is countered by their extreme sensitivity, which is capable of
compounding any errors in sample collection and rendering the acquired data useless or
(even worse) nonrepresentative of site conditions. Coupled with their relatively high cost
when compared with traditional chemical analyses, MBTs often are neglected as useful and
cost-effective tools for site characterization and remedial design. This chapter provides a
practical introduction to the science behind several MBTs, as well as their value to remedial
practitioners for site characterization, remedial
technology screening and performance
monitoring.
MBTs measure specific biomarkers (e.g., deoxyribonucleic acid [DNA] or ribonucleic acid
[RNA] sequences, peptides, proteins or lipids) that are indicators of the ability to degrade
specific contaminants. A comprehensive summary of MBT techniques, applications, issues,
questions and associated research needs was presented in a SERDP/ESTCP workshop report
(Stroo et al.,
2006
). Among all MBTs, application of nucleic acid-based tools is the most
advanced, and specific tests for the presence and abundance of key dechlorinating (i.e.,
Dhc
bacteria) as well as petroleum degrading bacteria are commercially available. Peptide, protein
and lipid biomarkers also are useful for monitoring target bacterial populations; however,
the techniques targeting these biomarkers are not as specific as nucleic acid-based tools for
site assessment and bioremediation monitoring. Hence, the current focus is on nucleic
acid-based tools.
Used in conjunction with contaminant and geochemical data, nucleic acid-based MBTs
can be utilized to: (1) confirm the presence of naturally occurring bacterial populations capable
of biodegradation; (2) identify the need for bioaugmentation at a site; and (3) monitor
the performance of a bioremediation treatment. Quantitative real-time polymerase chain
reaction (qPCR) techniques have been most widely used to quantify key bacteria and the
functional genes (e.g.,
tceA
,
vcrA
and
bvc
A) responsible for reductive dechlorination processes
(Figure
6.1
). Other nucleic acid-based techniques (e.g., fluorescent
in situ
hybridization [FISH],
denaturing gradient gel electrophoresis [DGGE] and terminal restriction fragment length
polymorphism [T-RFLP]) have been developed, but their use for site assessment and bioreme-
diation monitoring is currently limited. Additional techniques such as reverse-transcriptase
qPCR (RT-qPCR) of RNA, qPCR of messenger ribonucleic acid (mRNA) and compound
specific isotope analysis (CSIA) have considerable promise but require further research for
field-scale application (Stroo et al.,
2006
).
This chapter discusses monitoring strategies and sampling procedures for ground-
water sampling, as well as data interpretation for qPCR, FISH and community profiling
techniques. These procedures are currently applied to
Dhc
biomarker analysis in support of
bioremediation at chlorinated solvent sites, but the protocols are universal and should be
applicable for monitoring other groundwater bacterial populations of interest. The sampling
process is generally the greatest source of variability in the use of MBTs (Stroo et al.,
2006
;
Lebr
´
n et al.,
2011a
; SERDP and ESTCP,
2005
). Although not standardized like sampling
procedures for chemical analysis, the procedure described herein is common in the reme-
diation field and can be considered a current best practice (Lebr
´
n et al.,
2011b
; Ritalahti
et al.,
2010
).
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