Biomedical Engineering Reference
In-Depth Information
hydroxylase gene decreased by several orders of magnitude after 33 days of monitoring with
FISH, yielding lower numbers than
in situ
PCR. The authors indicated that in the rRNA-
targeted FISH, the fluorescence intensity was dependent on the copy number of the rRNA in
an individual cell, which is not the case for
in situ
PCR.
R. eutropha
probably did not have
ribosomal content high enough for detection by FISH because it was not synthesizing protein or
growing actively under the oligotrophic conditions of the aquifer. The study provides a good
example of how molecular methods can be applied to monitor the survival of bioaugmented
microorganisms.
8.4.2 Bioaugmentation Approach II
Approach II uses an effective constitutive strain that can transform TCE or other CAHs
while being maintained
in situ
on a non-inhibiting growth substrate. A mutant strain of
Burkholderia cepacia
G4 (PR1
301
) that constitutively expresses TMO was developed for this
purpose, and tested in microcosms (Munakata-Marr et al.,
1996
,
1997
) and in the field
(Bourquin et al.,
1997
; McCarty et al.,
1998b
).
8.4.2.1 Bioaugmentation with a Constitutive
Burkholderia cepacia
G4:
Microcosm Tests
Munakata-Marr et al. (
1996
) evaluated TCE cometabolism in small laboratory columns
packed with aquifer material from the Moffett Test Facility. The columns were bioaugmented
with a wild type strain of
Burkholderia (Pseudomonas) cepacia
G4 and a nonrecombinant
mutant of G4 (PR1
301
) that is capable of constitutive degradation of TCE in the absence of
toluene or phenol. The bacterial strain used in the bioaugmentation was the wild type strain
Burkholderia (Pseudomonas) cepacia
G4 that was isolated by Nelson et al. (
1986
). Strain G4
cometabolizes TCE using the
o
TOM enzyme, and is induced by either phenol or toluene. Two
mutants of G4 also were evaluated:
B. cepacia
PR1
23
(Shields et al.,
1995
) and PR1
301
developed
by Munakata-Marr et al. (
1996
). Both mutants express
o
TOM constitutively when grown on
substrates such as lactate.
The columns were periodically exchanged with groundwater amended with TCE (250
m
g/L),
DO (31 mg/L) and either lactate (15 mg/L) or phenol (6.5 mg/L). Two types of tests were
performed: (1) a high density single bioaugmentation (1-11 mg of cells) to sterile and nonsterile
aquifer columns; and (2) low density semicontinuous bioaugmentation (70
m
g of cells were
added with each exchange).
In the high density tests, dissolved oxygen was completely consumed in all the phenol- and
lactate-fed microcosms when higher primary substrate concentrations were used. Even when
the substrate concentrations were lowered, TCE degradation was limited. The high density
bioaugmentation with PR1
301
did not successfully degrade TCE.
In the low density tests, the microcosm that was fed G4 with no substrate achieved the same
degree of TCE removal as the microcosm fed only phenol and not bioaugmented. This result
indicated the successful bioaugmentation of an induced culture for its transformation potential
alone (Approach I). The addition of PR1
301
culture when grown on lactate, but fed phenol, was
effective in transforming TCE. The authors indicated that this result has practical implications
for field scale bioaugmentation, in that large quantities of cells for bioaugmentation can be
grown on a non-toxic compound, such as lactate, and then induced on concentrations of phenol
as low as 1 mg/L. Lactate-grown G4 and PR1
301
, when fed phenol, transformed TCE. Bioaug-
mentation with cultures known to degrade TCE more than doubled the extent of TCE
degradation compared with microcosms fed phenol only and that stimulated indigenous strains.
Search WWH ::
Custom Search