then used to inoculate the media with the appropriate
organism. Organism growth was monitored via methane
concentration in the bottles. Periodically, the samples were
checked under a microscope (Zeiss) to assess culture purity.
All cultures were grown near their optimal temperatures (35
capped. Vials were spun at 10 000 rpm (5700g) for 15 min
in a centrifuge outside of the glovebag.
Once spun, supernatant exchange took place. One iron-
plus-cells treatment (iron-stay) and one cells-only treatment
(cells-stay) were shaken to resuspend the biomass and iron
and were placed back into their original 38-mL bottles.
Alternatively, the pellets from one iron-plus-cells treatment
and one cells-only treatment were placed back into their
original 38-mL bottles. The supernatants from the cells-
only systems were placed in the bottles containing the iron-
plus-cells pellets (iron-swap), and the iron-plus-cells su-
pernatants were placed in the bottles containing the cells-
only pellets (cells-swap). The supernatant from the final
cells-only treatment was decanted into sterile, empty 38-mL
bottles (supernatant control). After the 38-mL bottles were
filled with the appropriate supernatants and pellets, the
bottles were capped, and the experiment was started with
the amendment of CT (5-10 µM). The reactors were
incubated statically on their sides for the duration of the
experiment.
Data Analysis. Because all of the various treatments did
not contain organisms or iron, it was thought that the best
way to compare transformation rates between treatments
was first-order rate coefficients. Each system was expected
to be heterogeneous; however, a complicated transformation
rate model would not facilitate a better comparison between
treatments. Because the different treatments were handled
in as similar a manner as possible, a first-order rate coefficient
calculation provides the most suitable means by which
comparisons could be made. First-order rate coefficients
were calculated as follows. Degradation of the contaminant
°C for M. barkeri and M. concillii and 50 °C for M. thermophila)
and incubated quiescently on their sides.
Experim ental Procedure. Triplicate reactors were used
for each treatment in all experiments. CH
4
and H
2
were
monitored over time to quantify growth and H
2
production
and/ or utilization. Biomass was measured [as mg/ L volatile
suspended solids (VSS)] before and after each experiment to
monitor organism growth. All experiments were performed
in 38-mL crimp-top bottles sealed with a thick butyl rubber
stopper lined with Teflon. Experiments were performed at
5
0, 35, or 35 °C for M. thermophila, M. barkeri, and M. concillii,
respectively.
Saturated aqueous stock solutions were prepared by
equilibrating neat CT and CF (3 mL each) with NanoPure
water. The solutions were shaken vigorously for several hours
and then allowed to settle for several days at 20 °C before
use. Primary standards for calibration were prepared gravi-
metrically with neat CT, CF, and DCM in methanol. Cali-
bration standards were prepared by adding specific quantities
of primary standards to 38-mL bottles containing 25 mL of
NanoPure water.
Dechlorination Experim ents. Reactors for these studies
were prepared and incubated in the following manner. The
bottles were filled with iron plus N
2
-CO
2
, or only H
2
-CO
2
or N -CO The bottles were then capped, sealed, and
2
2
.
autoclaved. Once the bottles had cooled, they were filled
with either 25 mL of medium or 25 mL of medium and cells
in the stationary phase. Approximately 1 h after filling, CT
or CF (approximately 5-10 µM, total concentration) was
added to the bottles to begin the experiment. Bottles were
incubated quiescently on their sides.
(
C) is represented by
dC
dt
) -k′C
where k′ is the first-order rate coefficient and t is time. This
expression can be integrated, yielding the equation
The following treatments, with an N
2
2
-CO headspace
unless otherwise stated, were used (in triplicate) for the
dechlorination experiments: a medium control consisting
of medium only; a cells-only control consisting of resting
cells and medium; an iron-only control consisting of iron
C
C0
-ln
) k′(t - t0)
and medium; a cells-plus-H
2
system consisting of resting
headspace; and an iron-
where C
zero (t ). When -ln (C/ C
initial slope is equal to k′.
0
is the initial contaminant concentration at time
cells and medium with a H -CO
2
2
0
0
) is plotted against time, t, the
plus-cells system consisting of iron, resting cells, and medium.
For experiments with M. thermophila, there was also a MeOH-
fed system consisting of cells, medium, and added MeOH
The figures show average concentration versus time
profiles for the triplicate reactors for each treatment. Error
bars in the figures represent the standard deviation between
the concentrations in each of the triplicate reactors for a
given treatment. For each treatment, the rate of transfor-
mation and standard deviation (given in the tables) represent
initial rates of CT or CF transformation averaged over
triplicate reactors. The standard deviation was calculated
from the individual rates of transformation in each of the
triplicate reactors. For data that contained an initial lag
period, rates of transformation are based on the transforma-
tion rate after the lag.
(
100 mM). For M. concillii, there was an acetate-fed system
containing excess acetate and no resting cell system. For
experiments with M. concillii, growth of the cells took
approximately 3 months. The added acetate (83 mM) was
never completely used. At the end of the experiment,
approximately 8-16 mM (or 480-960 mg/ L) acetate re-
mained; therefore, true resting cell treatments could not be
investigated without risking general loss of cell viability.
CT, CF, and DCM were monitored by gas chromatography
(
GC) over time, as were H
2 4
and CH . Experiments with M.
thermophila were of a 24-30 h duration, experiments were
CT, CF, and DCM Measurem ent. Headspace samples
1
-4 days in length for M. barkeri, and experiments were 5
(
100 µL) were taken with a gastight locking syringe (Precision
days in length for M. concillii.
Sampling Corp., Baton Rouge, LA). These were directly
injected into a gas chromatograph (Hewlett-Packard 5890
series II) equipped with an electron capture detector (GC-
ECD) for CT and CF measurement. A fused silica capillary
column (J&W) with a DB-5 stationary phase was used with
nitrogen as the carrier gas. The oven temperature was held
at 35 °C for a run length of 2.5 min. Method detection limits
were 0.00034 and 0.025 µM for CT and CF, respectively, as
determined by the method outlined in Standard Methods
(Method 1030 E; 10). DCM was measured by direct head-
space injection into a GC-ECD equipped with a 60 m capillary
Supernatant Exchange Experim ents. The reactors for
these experiments were prepared as follows. Thirty-eight-
milliliter bottles contained iron plus N
CO . After autoclaving, the bottles were filled with 25 mL of
cell suspension entering the exponential growth phase (as
monitored by CH production). For each experiment, there
2
-CO
2 2
or only N -
2
4
were two iron-plus-cells treatments and three cells-only
treatments (with each in triplicate). Once cells had reached
the stationary phase (approximately 3 days), the reactors
were decanted into centrifuge vials in the glovebag and
VOL. 32, NO. 10, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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