Biomedical Engineering Reference
In-Depth Information
9.5.1 Trace Metals
As noted previously, the Cu:PDTC complex is the agent of transformation, therefore trace
Cu is required for CT transformation. Induction of CT transformation also requires iron
limitation. This is because synthesis of PDTC is under
fur
control (Sepulveda-Torres et al.,
2002
). The
fur
regulator is activated at soluble Fe levels of about 1 micromolar (
m
M) or less.
These low levels can be achieved by adjusting the pH of the growth medium to around 8 (Criddle
et al.,
1990
; Tatara et al.,
1993
), to cause precipitation of Fe(III) hydroxide, or by adding iron
chelating agents (Lewis and Crawford,
1995
). To date, field efforts involving bioaugmentation
with strain KC have relied upon adjusting the pH to around 8 prior to strain KC introduction.
Adjustment of pH to 8 triggers
fur
genes and also confers an ecological advantage for strain
KC. Adjusting the pH requires careful attention to the potential for calcium carbonate precipi-
tation, given that many groundwaters are close to saturated with respect to calcite.
9.5.2 Cell and CT Concentration
Before PDTC was identified as the secreted agent responsible for CT transformation,
Tatara et al. (
1993
) found that the initial rate of reaction was first-order with respect to cell
concentration and first-order with respect to CT concentration. They described the initial rate of
degradation using a second-order rate expression:
k
0
CX, where C is CT concentra-
tion, X is biomass concentration, and k
0
is a pseudo second-order rate coefficient. Initial values
for k
0
are high, in the range of 4 liters/milligram protein/day (L/mg protein/d).
Dybas et al. (
1995a
) traced CT-degradation activity to the supernatant of strain KC
culture medium, but in the absence of actively respiring cells, both the rates and extent of
CT transformation were limited. These and other experiments led to the conclusion that
transformation of CT involved a cell-free component, later identified as PDTC, as well as one
or more cell-associated components. In the absence of cells, the high CT transformation rates
initially observed were not sustained, and the extent of CT transformation was limited. By
contrast, in the presence of living cells, a period of rapid CT transformation was followed by
a period of sustained, but slower CT transformation. The slower sustained rate of transfor-
mation enabled a greater extent of transformation, permitting removal of CT at levels of up
to 5 mg/L.
The combined results of Tatara et al. (
1993
) and Dybas et al. (
1995a
) suggest that CT
transformation is initially limited by the concentration of the reduced PDTC-copper complex,
and subsequently becomes limited by the rate of synthesis and/or regeneration of the reduced
PDTC-Cu complex. Values for k
0
used in transport models reflect the slower rates of transfor-
mation of the slower transformation period, and are in the range of 0.04-0.19 L/mg protein/d
(Phanikumar et al.,
2002a
,
b
; Vidal-Gavilan,
2000
).
To determine whether specific cell types are required for regeneration of the transforma-
tion activity, Tatara et al. (
1995
) combined partially purified cell-free supernatants from strain
KC, now known to contain PDTC, with cells that were incapable of CT transformation or that
transform it slowly. Table
9.1
summarizes the first-order rate coefficient (k
00
) and the half-lives
obtained. Very rapid CT transformation was obtained when culture supernatant was combined
with other cultures, including: other
Pseudomonads
(
P. stutzeri
strains other than KC,
P. fluorescens
); another gram-negative organism (
Escherichia coli
K-12); a gram-positive
organism (
Bacillus subtilus
); a consortium (SC-1) derived from CT-contaminated groundwater
at Schoolcraft, Michigan; a consortium (HC-14) derived from CT-contaminated groundwater at
Hanford, Washington; and yeast (
Saccharomyces cerevisiae
). The results clearly indicated that
specific cell types are not required for regeneration of the CT transformation activity.
dC/dt
ΒΌ
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