Biomedical Engineering Reference
In-Depth Information
The mutual effects of binary mixtures of TCE, VC, DCA and
cis-
DCE on the kinetics of
degradation by JS666 were investigated (F. Liu, E. Wood, and J. M. Gossett, unpublished).
Although the presence of TCE, VC or DCA reduced the degradation rate of
cis-
DCE,
both
cis-
DCE and the cocontaminant can be degraded to completion. Thus, JS666 may be
able to completely degrade mixtures of chlorinated solvents in the field. However, it is
unknown how the presence of the non-
cis-
DCE substrate might have affected growth because
these were short-term, batch experiments designed in such a manner that biomass would remain
constant during the assays. Observation of approximately stoichiometric chloride release from
VC and TCE degradation indicates their mineralization by JS666 (F. Liu and J. M. Gossett,
unpublished).
With
cis-
DCE at 40
m
M (ca. 4 mg/L), presence of VC or TCE over a wide range of
concentrations (up to 10
M) caused maximum
cis-
DCE-degradation rates to decrease by as
much as half. There was no evidence that one compound was being preferentially degraded,
since the fraction of compounds remaining was essentially the same for each of the binary
mixtures. Degradation of VC was enhanced by the presence of
cis-
DCE, while TCE degrada-
tion was unchanged. Preliminary results suggest that TCE and VC may be degraded by
different enzymes or pathways than
cis-
DCE. The effect of the presence of DCA on
cis-
DCE degradation differs from that of VC or TCE.
cis-
DCE and DCA degradation rates are
inversely correlated with each others' concentrations - i.e., the highest concentration of DCA
caused the slowest
cis-
DCE degradation, and vice-versa (F. Liu and J. M. Gossett, unpublished).
Preliminary results thus suggest that DCA competitively inhibits
cis-
DCE degradation in JS666,
which indicates that they may be transformed by the same enzyme or pathway. Future studies
of the degradation of mixtures of contaminants in JS666 can be expected to provide insight into
the mechanisms of degradation.
m
7.2.5 Development of a Molecular Probe for Process Monitoring
The ability to track the presence of the bioaugmented organism in the subsurface is
important for monitoring the progress of
in situ
bioaugmentation. A quantitative polymerase
chain reaction PCR (qPCR) assay was developed for the specific detection of JS666 in soil
(Giddings et al.,
2010
a). The qPCR assay targeting the JS666 isocitrate lyase gene is specific,
accurate and reproducible in soil (Giddings et al.,
2010
a). The molecular probe was used in a
field bioaugmentation study with JS666 (Section
7.4.4
). One objective of the field study was to
combine the qPCR assay with microcosm studies to determine whether the presence of JS666
can be correlated with degradation activity.
There are a number of different techniques available to quantify microorganisms in soil,
including culture-based techniques (e.g., most probable number or selective plating), fluores-
cent
in situ
hybridization (FISH) and qPCR. Techniques based on cultivation are tedious and
cannot be used to quantify the vast majority of microbes that have not yet been cultured.
Techniques such as FISH (Amann et al.,
1995
), do not require cultivation, but can be difficult to
apply to soil or to cultures with low cell concentrations. qPCR is quickly becoming the method
of choice for quantifying microbes in mixed communities because of the ability to detect
extremely low concentrations of cells without the need for cultivation (Sharma et al.,
2007
;
Smith et al.,
2006
). qPCR has been applied to the quantification of genes or microbes associated
with the biodegradation of chlorinated ethenes (Cupples,
2008
; He et al.,
2003
; Lendvay et al.,
2003
; Smits et al.,
2004
) and ethanes (Van Raemdonck et al.,
2006
).
A qPCR assay for JS666 was developed using strain-specific primers (Giddings et al.,
2010
a) and SYBR Green reagent (Van Raemdonck et al.,
2006
). Primers were designed to target
the chromosomal gene that encodes isocitrate lyase, because the 16S ribosomal gene in JS666
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