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Applied and Environmental Microbiology, October 1999, p. 4537-4542, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Enhanced Biotransformation of Carbon Tetrachloride
by Acetobacterium woodii upon Addition of Hydroxocobalamin
and Fructose
Syed A.
Hashsham1,* and
David L.
Freedman2
Center for Microbial Ecology and Department
of Civil and Environmental Engineering, Michigan State University,
E. Lansing, Michigan 48824,1 and
Department of Environmental Engineering and Science, Clemson
University, Clemson, South Carolina 296312
Received 2 April 1999/Accepted 19 July 1999
 |
ABSTRACT |
The objective of this study was to evaluate the effect of
hydroxocobalamin (OH-Cbl) on transformation of high concentrations of
carbon tetrachloride (CT) by Acetobacterium woodii (ATCC
29683). Complete transformation of 470 µM (72 mg/liter [aqueous])
CT was achieved by A. woodii within 2.5 days, when 10 µM
OH-Cbl was added along with 25.2 mM fructose. This was approximately 30 times faster than A. woodii cultures (live or autoclaved)
and medium that did not receive OH-Cbl and 5 times faster than those
controls that did receive OH-Cbl, but either live A. woodii
or fructose was missing. CT transformation in treatments with only
OH-Cbl was indicative of the important contribution of nonenzymatic
reactions. Besides increasing the rate of CT transformation, addition
of fructose and OH-Cbl to live cultures increased the percentage of
[14C]CT transformed to 14CO2 (up
to 31%) and 14C-labeled soluble materials (principally
L-lactate and acetate), while decreasing the percentage of
CT reduced to chloroform and abiotically transformed to carbon
disulfide. 14CS2 represented more than 35% of
the [14C]CT in the presence of reduced medium and OH-Cbl.
Conversion of CT to CO was a predominant pathway in formation of
CO2 in the presence of live cells and added fructose and
OH-Cbl. These results indicate that the rate and distribution of
products during cometabolic transformation of CT by A. woodii can be improved by the addition of fructose and OH-Cbl.
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INTRODUCTION |
Metallocoenzymes like corrinoids,
cytochrome P-450, and iron(II) porphyrins are known to play a major
role in biotransformation and detoxification of carbon tetrachloride
(CT) (22). The homoacetogen Acetobacterium woodii
(1) is one of many organisms that transform CT at relatively
high rates due in part to its use of the acetyl coenzyme A (CoA)
pathway and correspondingly high levels of corrinoids (25).
CT is transformed by fructose-grown A. woodii to mainly chloroform (CF), dichloromethane (DCM), and several soluble
nonchlorinated products (9, 10, 24). Autoclaved A. woodii cultures also transform CT at an appreciable rate, perhaps
indicating the heat stability of the enzymes involved. CT is also
transformed by Shewanella alga BrY, an organism lacking the
acetyl-CoA pathway (28). BrY reduces vitamin
B12, which subsequently transforms CT primarily to carbon
monoxide (CO).
These and other studies suggest that the role of microorganisms in
dechlorinating CT may be limited to reduction of corrinoids or other
cofactors, which can be accomplished equally well with chemical
reducing agents (5, 17). However, previous results with a
mixed culture grown on DCM and amended with cyanocobalamin indicates
that an active microbial population may play a bigger role in CT
transformation than simply providing reducing power (2, 14).
Addition of cyanocobalamin significantly increases the rate of CT
transformation and decreases CF accumulation, in comparison to
autoclaved culture or sulfide-reduced basal medium supplemented with
B12. Avoiding CF accumulation is a major concern with in
situ bioremediation of CT.
The objective of this study was to determine the role of live A. woodii cells in CT transformation when provided with fructose and
hydroxocobalamin (OH-Cbl). Based on results from a methanogenic enrichment culture (14), we hypothesized that a pure culture of A. woodii supplemented with fructose and OH-Cbl would
transform CT faster than autoclaved cells with the same amount of
OH-Cbl and faster than OH-Cbl present in sulfide-reduced basal medium. In addition, we expected the presence of live cells supplemented with
fructose and OH-Cbl to shift the product distribution from CT away from
CF and DCM and towards CO and CO2. The results indicate that A. woodii recycles supplemental corrinoid to its
reduced form, increases the rate of CT transformation to CO versus
carbon disulfide (CS2), and drives metabolism of CO to
CO2 and fermentation products.
(Some preliminary results of this study were presented at the 96th
Annual Meeting of the American Society for Microbiology, New Orleans,
La., 19 to 23 May 1996.)
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MATERIALS AND METHODS |
Chemicals.
CT, CF, and DCM (all 
99.9%) and acetate,
lactate, fumarate, and isobutyrate were obtained from Aldrich Chemical
Co.; CS2 was from EM Industries, Inc. OH-Cbl (98%) and
cyanocobalamin (99%) were purchased from Sigma Chemical Co.
[14C]CT was obtained from Dupont NEN Products and diluted
to 1.9 × 107 dpm/ml with distilled deionized water
(0.54 mM CT). The radiochemical purity of this [14C]CT
stock solution was evaluated to be 99.6% ± 0.11% (± 1 standard deviation for duplicate bottles) by using the procedures described under "Analysis of 14C products." EcoScint (Baker
Diagnostics, Inc.) was used as liquid scintillation cocktail for all
14C radioactivity measurements.
Growth conditions.
A. woodii (ATCC 29683) was grown on
fructose in a basal medium described by Freedman and Gossett
(11), modified as follows: the Fe2+
concentration was reduced to 1.2 mg/liter, and 10 ml of a vitamin solution per liter was added (27). Fructose was added as a
sterile stock solution (2.8 or 25.2 mM).
A. woodii was grown in 160-ml serum bottles that were
modified by connecting a 1-cm inside-diameter test tube at a right
angle to the side of the bottles near the base, resulting in a final bottle volume of 173 ml. These modified serum bottles resemble culture
flasks with a side arm (e.g., Bellco Biotechnology or Ace Glass),
making it possible to monitor growth of A. woodii by optical
density at 620 nm (Bausch and Lomb Spectronic 20 spectrophotometer). A
correlation was developed between optical density and dry weight. Cells
were harvested by centrifugation and resuspended in the basal medium.
The suspension was filtered through a glass fiber filter (Whatman, 21 mm), dried at 105°C overnight, and weighed. An optical density of 0.1 using a 1-cm light path corresponded to 45 mg of cells per liter.
Basal medium was distributed to serum bottles in an anaerobic glove box
(Coy Laboratory products, Inc.). The headspace of
the bottles was then
purged with an N
2-CO
2 gas mixture (70%/30%
[vol/vol]) to remove hydrogen and equilibrate the bicarbonate
in the
basal medium with CO
2 (resulting in a pH of 7.0). The serum
bottles were sealed with slotted gray butyl rubber septa, covered
with
aluminum foil, and incubated at 35°C on a gyratory shaker
table, with
the liquid in contact with the
septum.
Volatile organic product analyses.
CT, CF, DCM,
CH4, and CS2 were measured by gas
chromatographic analysis (Perkin-Elmer model 9000) of 0.5-ml samples
taken from the 65-ml headspace of the serum bottles. A Carbopack 1% SP-1000 column was used along with a flame ionization detector, as
previously described (11). Although flame ionization is not the most suitable detector for measurement of CS2, a linear
response was obtained for concentrations less than 50 µmol per
bottle. Detection limits (nanomoles per bottle) were 60 for
CH4, 20 for DCM, 100 for CS2, 20 for CF, and 4 for CT. CO was analyzed isothermally (90°C) on a gas chromatograph
(GOW-MAC Instruments) equipped with a thermal conductivity detector
(150°C), a 3.2-mm by 2.44-m stainless steel 80/100 mesh, Molecular
Sieve 5A column (GOW-MAC Instruments), and helium as the carrier gas
(30 ml/min). The detection limit for CO was approximately 1 µmol per bottle.
All of the CT transformation products reported in this study are in
micromoles per bottle. Aqueous phase concentrations of
the volatile
compounds can be obtained by using their Henry's
constant
(mol-m
3 gas concentration/mol-m
3 aqueous
concentration) at 35°C, as reported previously (
14),
and
the liquid and headspace volumes in the serum bottles. The
percentages
of each compound found in the aqueous phase (108 ml)
were 4.8% for
CH
4, 3.4% for CO, 93% for DCM, 56% for CS
2,
88%
for CF, and 47% for
CT.
CT transformation studies.
Experiments were initiated by
adding 2 ml of a log-phase culture of A. woodii growing on
fructose to 106 ml of basal medium in serum bottles. Initially 2.8 mM
fructose was added, to grow the culture to a dry cell mass of 12.2 ± 0.44 mg/bottle. During this period, the bottles were not shaken.
After growth was complete (i.e., no further increase in optical
density) by day 3, 11 treatments were set up, using duplicate serum
bottles for each treatment. CT (100 µmol per bottle), additional
fructose (25.2 mM), and OH-Cbl (108 nmol per bottle) was then added to
appropriate bottles, which were then monitored for optical density and
CT transformation products. Treatments 1 to 7 consisted of
sulfide-reduced basal medium (2.1 mM sulfide) with combinations of live
or autoclaved A. woodii, OH-Cbl, and fructose. Water
controls (treatment 8) consisted of 160-ml serum bottles with 100 ml of
autoclaved deionized water and 100 µmol of CT, to evaluate losses
through the septum and measure the radiochemical purity of the
[14C]CT. Treatments 9 through 11 were included to check
growth of A. woodii on fructose and/or OH-Cbl.
Analysis of 14C products.
[14C]CT
was added (approximately 1.9 × 106 dpm) along with
unlabeled CT to bottles representing treatments 1 to 8 (see Table 1). Treatment 1 (live A. woodii culture plus [14C]CT plus OH-Cbl plus
fructose) included two identical sets; one analyzed for 14C
transformation products after 2.5 days and the other after 13 days
(along with all of the other treatments) prior to analysis of
14C products.
The amount of
14C activity associated with volatile
products was determined by gas chromatographic separation and
combustion
tube analysis of headspace samples (
11). The
efficiency of product
recovery measured as trapped CO
2
after passing through the combustion
tube was 95% ± 2.7% for CT, CF,
and DCM and 98% ± 0.01% for methane.
14CO
2
was determined by stripping acidified samples with N
2 and
trapping in NaOH, as previously described (
11).
Analysis of
14C nonvolatile products was carried out by
acidifying a 20-ml portion of the liquid sample with HCl and sparging
with N
2 for 30 min.
14C not purged at acid pH
corresponded to nonstrippable residue
(NSR).
14C-labeled
NSR retained by a 0.22-µm-pore-size filter (Whatman,
Inc.) was
presumed to be cell-associated.
14C-labeled NSR filtrate
(i.e., soluble NSR) was either concentrated
50- to 100-fold in a vacuum
centrifuge before high-performance
liquid chromatography (HPLC)
analysis or injected directly onto
the HPLC (10 to 250 µl).
Separation was achieved on a 300-mm-diameter
HPX-87H ion exclusion
column (Bio-Rad Laboratories) connected
to a UV-visible light
absorbance detector (model 486; Millipore
Corp.) set at 210 nm. Three
different mobile phase conditions
(0.013 N
H
2SO
4, 0.7 ml/min, 30°C; 0.013 N
H
2SO
4 with 5% acetonitrile,
0.5 ml/min,
27°C; and 0.007 N H
2SO
4 with 10.8%
acetonitrile, 0.5
ml/min, 27°C) were used in order to confirm the
identity of the
compounds by shifts in retention times. The presence of
acetate
and butyrate was also confirmed by gas chromatographic analysis
(Hewlett Packard 5890A series II) with a 4-mm inside diameter
and
183-cm-long glass column (GP 10% SP-1200-1%
H
3PO
4 on 80/100
Chromosorb W AW; Supelco,
Inc.).
To quantify which of the peaks from the HPX-87H column contained
14C activity, fractions were collected in 0.5- to 1-min
intervals,
mixed with 15 ml of scintillation cocktail, and counted on a
liquid
scintillation counter. The identity of
14C labeled
L-lactate was also confirmed by its reactivity with
lactate
dehydrogenase. The average percent recovery during the
HPLC analysis
[
(
14C in all fractions)/
14C injected] was
89% ± 5.0%.
The recovery efficiency during the
14C analyses was defined
as the total disintegrations per minute recovered in all components
(CT + CF + DCM + CS
2 CO + CO
2 + pyruvate +
L-lactate + acetate
+ isobutyrate + unidentified soluble NSR + cell
associated fraction)
divided by the total disintegrations per minute
present at the
time of analysis. The total disintegrations per minute
present
in a bottle at the time of
14C analysis was the sum
of 0.5-ml headspace sample disintegrations
per minute and 100-µl
aqueous phase sample disintegrations per
minute measured by direct
injection into scintillation cocktail.
For bottles with low
CS
2 levels (i.e.,
22%), the recovery based
on
14C remaining was 96% ± 3.7%; for bottles with high
CS
2 levels,
the recovery was 80.8% ± 1.2%. All
14C transformation products are expressed as percent of
disintegrations
per minute initially injected without correcting for
losses through
the septum during the incubation period. CT
transformation rates
are obtained by dividing the difference of initial
and final aqueous
CT concentrations by the number of days it took for
the transformation,
adjusted for losses in the water
controls.
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RESULTS AND DISCUSSION |
Rate enhancement due to OH-Cbl addition.
The focus of this
research was to determine if the rate of CT biotransformation by
A. woodii can be enhanced by biochemical amendments,
especially at high CT concentrations. OH-Cbl was selected for this
purpose because cobalamins are one of the main coenzymes in the
acetyl-CoA pathway implicated in the cometabolic transformation of CT
by A. woodii (10), and they are also known to
transform CT in abiotic systems (18).
Results presented in Fig.
1a demonstrate
that adding OH-Cbl and fructose to
A. woodii caused a
30-fold increase in the rate
of CT biotransformation with respect to
controls that did not
receive additional OH-Cbl (26 mg/liter/day for
treatment 1 versus
0.83 mg/liter/day for treatment 5). When fructose or
A. woodii cells were omitted or
A. woodii cells
were killed (treatments
2, 3, and 4), the addition of OH-Cbl still
increased the rate
fivefold, indicating the importance of abiotic
sulfide-mediated
CT transformations. The difference in rate between the
treatment
that contained
A. woodii with fructose and OH-Cbl
and all others
that received only OH-Cbl indicates that
A. woodii cells needed
fructose to take advantage of the added OH-Cbl
for CT transformation.
The rate of CT transformation by
A. woodii receiving only fructose
was similar to previously reported
rates (0.8 to 1.7 mg/liter/day)
by Stromeyer et al. (
24) and
Egli et al. (
10). Biomass concentrations
estimated from
protein and optical density data in all three studies
were comparable
(300 to 500 mg/liter). The amount of OH-Cbl (1
µmol/bottle) added in
this study was only 1% of the initial CT,
but it substantially
increased the total amount of corrinoids
present. Based on a corrinoid
content of 0.52 nmol/mg of dry cell
mass (
7) and a cell mass
of 40 mg/bottle, the corrinoid contributed
by
A. woodii
cells was only 2% of the added OH-Cbl.
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FIG. 1.
Transformation of CT (a), cell growth (b), and
accumulation of chloroform (c), CO (d), and CS2 (e) by
A. woodii and basal medium under various treatment
conditions. 1, live A. woodii with CT, OH-Cbl, and fructose
( ); 2, live A. woodii with CT and OH-Cbl but no fructose
( ); 3, autoclaved A. woodii with CT and OH-Cbl ( ); 4, autoclaved medium with CT and OH-Cbl ( ); 5, live A. woodii with CT and fructose but no OH-Cbl ( ); 6, autoclaved
A. woodii with CT but no OH-Cbl ( ); 7, autoclaved medium
with CT but no OH-Cbl ( ); 8, autoclaved water with CT but no OH-Cbl
(×); 9, A. woodii seed control with fructose but no CT and
no OH-Cbl (+); 10, A. woodii seed control with OH-Cbl and
fructose but no CT ( ); and 11, A. woodii seed control
with OH-Cbl but no fructose and no CT ( ). Vertical bars represent 1 standard deviation for duplicate bottles.
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When OH-Cbl was replaced with an equimolar amount of cyanocobalamin in
the live culture with fructose, there was only a marginal
enhancement
in the rate of CT transformation over live cells alone
(data not
shown), perhaps due to the toxicity of cyanide to
A. woodii
(
20). Cyanocobalamin was successful in improving the
rate of
CT and CF transformation, as reported previously in a
DCM-grown
enrichment culture (
2,
14) as well as in a mixed
culture
(
15).
Since biomass concentration typically has a significant effect on the
rate of CT transformation, growth was also monitored
in all treatments
involving
A. woodii. The presence of CT inhibited
growth of
A. woodii (Fig.
1b). This was expected, since the aqueous
CT
concentration used was much higher than in most previous studies
(72 versus <2 mg/liter). However, once CT was consumed and most
of the
accumulated CF was also transformed, (treatment 1; Fig.
1a and c),
A. woodii grew to a cell density that was slightly
higher
than the maximum cell density for the controls with no
CT. The absence
of growth with fructose and OH-Cbl until nearly
all of the CF was
consumed (day 8, Fig.
1c) may be indicative
of the activity of
A. woodii towards toxicity reduction. There
was no indication of
OH-Cbl being used as a carbon source for
growth in the presence or
absence of fructose during the period
of this study (Fig.
1b,
treatments 9, 10, and
11).
Effect of OH-Cbl addition on volatile product distribution.
The distribution of CT transformation products is significantly
influenced by experimental conditions, including the type of reducing
environment, organisms present, and concentration of CT and coenzymes
(5, 12, 16, 17, 26). The major intermediates observed during
CT transformation are shown in Fig. 2.
Egli et al. (10) presented a similar diagram based on
studies with A. woodii, although it did not include CO or
CS2. CO formation from CT is known to be catalyzed by
corrinoids under reduced conditions. Further biotransformation of CO
yields nonhazardous CO2 and organic acids. CS2
is produced from CT mainly under sulfide-mediated reducing conditions.
Although it is a neurotoxin (13), its presence in drinking
water is not currently regulated. CF is the most common undesirable
transformation product of CT under various anaerobic conditions. DCM
may also accumulate to some extent, via reduction of CF. Strategies
that minimize accumulation of CF, DCM, and CS2 are of
interest for application of in situ bioremediation.
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FIG. 2.
Pathways for biotransformation of CT by A. woodii in sulfide-reduced basal medium. Solid arrows represent
major processes observed in this study; dashed arrows represent
pathways reported elsewhere, but were minor in this study. Further
reduction of DCM to chloromethane is possible, but occurs at very slow
rates. Numbers in parentheses represent oxidation states.
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|
Addition of OH-Cbl promoted accumulation of CO (Fig.
1d, treatment 1).
Without OH-Cbl, there was no detectable CO (below 1
µmol/bottle in
treatments 5, 6, and 7). The increase in CO beyond
day 8 corresponded
to an increase in cells in bottles with
A. woodii, OH-Cbl,
and fructose (Fig.
1b), following depletion of
the accumulated CF (Fig.
1c). Addition of OH-Cbl also promoted
the accumulation of
CS
2 (Fig.
1e). In the bottles with live culture
plus OH-Cbl
and fructose (treatment 1), the CS
2 peaked after 2.5
days
and remained nearly constant thereafter, since all of the
CT was
consumed. CS
2 formation from CT has been observed in other
microbial studies carried out in sulfide-reduced media (
6,
14). However, neither Stromeyer et al. (
24) nor Egli
et al.
(
10) reported any CS
2 formation in their
studies with
A. woodii in a sulfide-reduced
medium.
OH-Cbl addition also had a significant effect on accumulation of CF. In
other studies with
A. woodii (
9,
10,
24) and
several anaerobic pure cultures (
4,
19) without supplemental
cobalamins, CF and DCM were among the major metabolites that
accumulated
during CT transformation. In this study also, accumulation
of
CF occurred in all of the treatments that received CT, but to
a
lower extent when OH-Cbl and fructose were also present with
A. woodii.
The fastest rate of CT transformation and the highest level of CO
accumulation occurred with the fructose and OH-Cbl amended
live
A. woodii (treatment 1), with a correspondingly lower level
of CS
2 (compared to treatments 2, 3, and 4) and CF
accumulation.
Thus, active metabolism of an electron donor by
A. woodii in the
presence of supplemental OH-Cbl shifted the
transformation of
CT in favor of CO and away from CS
2 and
CF. The combination of
live cells, metabolism of the electron donor,
and OH-Cbl was necessary
to affect this shift. CT transformation still
occurred at a high
rate with sulfide-reduced OH-Cbl, but in the absence
of fructose
and active cells, CS
2 became the predominant
product.
Product distribution based on [14C]CT.
Use of
[14C]CT made it possible to determine the distribution of
soluble products and CO2 originating from CT and to confirm the distribution of volatile products obtained by gas chromatographic analysis of headspace samples (Fig. 1). Table 1 shows the distribution of products from 14C[CT] for each of the treatment
conditions. The amount of 14C lost during incubation was
proportional to the total amount of CT remaining, as indicated by CT
losses from the water controls (treatment 8). The maximum percentage of
14CO2 we observed when fructose and OH-Cbl were
added to live A. woodii cells (31%) was significantly
higher than those reported in other pure culture studies, including
A. woodii grown on fructose (9, 10, 24). Although
oxidation of 14CO appears to be the principal mode of
14CO2 formation in the presence of live cells,
small amounts of 14CO2 (<9%) did form with
autoclaved cells and with basal medium alone (treatments 6 and 7);
somewhat higher levels formed in the presence of supplemental OH-Cbl
(treatments 3 and 4). How CT is converted to CO2 in reduced
medium without cells is not well established. One possibility is
hydrolysis of CS2 (17), which is favored at
alkaline pH. With the OH-Cbl and fructose-supplemented active cultures,
oxidation of CF may have contributed to CO2 formation (2, 10). However, the extent of CF accumulation was small relative to direct conversion of CT to CO. It should be noted that
although CO was detected in treatment 1 by gas chromatographic analysis
on day 13 (Fig. 1d), virtually none of it was 14C labeled
at this point. The decrease in 14CO between days 2.5 and 13 in treatment 1 was accompanied by increases in
14CO2 and 14C-labeled
L-lactate, acetate, ethanol, and isobutyrate. This is consistent with involvement of the acetyl-CoA pathway in CT
transformation as proposed by Egli et al. (10); i.e.,
oxidation of CO to CO2 and formation of fermentation
products. A. woodii typically produces 3 mol of acetate per
mol of fructose under nonlimiting conditions, although formation of
other compounds has been observed under stressed conditions
(3). In this study, the stress was most likely due to CT and
CF. Vitamin B12 and carbon monoxide dehydrogenase, two key
enzymes in the acetyl-CoA pathway, are adversely affected by CT
(8). This effect may have been partially mitigated by the
addition of supplemental OH-Cbl.
The results of this study are in agreement with previous experiments
that observed enhanced CT transformation by a methanogenic
enrichment
culture supplemented with cyanocobalamin (
14). When
provided
with fructose and supplemental OH-Cbl,
A. woodii plays
a
bigger role in CT transformation than simply supplying coenzymes
that
carry out nonenzymatic transformations. Metabolically active
cells are
needed to realize the highest transformation rates and
most favorable
product distribution. The mechanism by which this
occurs needs further
investigation. For example, it is not yet
known if the added OH-Cbl
acts extracellularly, (as shown with
vitamin B
12 and
Shewanella alga BrY [
28]), intracellularly,
or by some combination of the
two.
Supplemental addition of cobalamin along with an electron donor offers
the prospect of accelerating the rate of in situ anaerobic
transformation of CT in contaminated groundwater, as well as minimizing
formation of hazardous metabolites like CF. The relatively high
cost of
cobalamins may be more than offset by a reduction in the
time required
for remediation, as well as minimization of hazardous
products. One
negative aspect of this approach is the formation
of CS
2 in
the presence of sulfides, although it can be removed
under aerobic
conditions, if necessary (
23). Adsorption of cobalamin
to
aquifer material has also been found to be low, with a retardation
factor of 2.1 in sand (
15). This is in the same range as for
CT (
21), suggesting that distribution of cobalamin along
with
an electron donor throughout a contaminated area is a feasible
process.
| |
ACKNOWLEDGMENTS |
This research was supported in part by a grant from the Oak Ridge
Institute for Science and Education, U.S. Department of Energy, and by
the U.S. Army Construction Engineering Research Laboratory.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Civil and Environmental Engineering, A126 Research
Complex
Engineering, Michigan State University, East Lansing, MI
48824. Phone: (517) 355-8241. Fax: (517) 355-0250. E-mail:
hashsham{at}pilot.msu.edu.
| |
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Applied and Environmental Microbiology, October 1999, p. 4537-4542, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
