Enhanced dechlorination of carbon tetrachloride and chloroform in the presence of elemental iron and Methanosarcina barkeri, Methanosarcina thermophila, or Methanosaeta concillii

Article abstract of DOI:10.1021/es970785h

Previous experiments in our laboratory have demonstrated that the rate and extent of carbon tetrachloride (CT) and chloroform (CF) dechlorination were enhanced when a methanogenic enrichment culture and iron (Fe⁰) were incubated together. Batch experiments with three pure cultures of methanogens, Methanosarcina barkeri, Methanosarcina thermophila, and Methanosaeta concillii were performed to determine how this enhanced transformation occurred. When hydrogen (H₂) was added as an electron donor, degradation of CT for all organisms and CF for M. thermophila was more rapid. H₂ was produced from the oxidation of iron, which therefore served as an H₂ source for the organisms, enhancing the transformation of CT and CF. Experiments with M. thermophila and M. concillii, which could not grow on H₂-CO₂ under the conditions tested, showed that H₂ could serve as an electron donor for dechlorination of CT and CF with these organisms as well. Experiments with supernatants from M. thermophila grown with and without iron indicated the presence of an excreted biomolecule active in the enhanced transformation of CT and CF. Previous experiments in our laboratory have demonstrated that the rate and extent of carbon tetrachloride (CT) and chloroform (CF) dechlorination were enhanced when a methanogenic enrichment culture and iron (Fe⁰) were incubated together. Batch experiments with three pure cultures of methanogens, Methanosarcina barkeri, Methanosarcina thermophila, and Methanosaeta concillii were performed to determine how this enhanced transformation occurred. When hydrogen (H₂) was added as an electron donor, degradation of CT for all organisms and CF for M. thermophila was more rapid. H₂ was produced from the oxidation of iron, which therefore served as an H₂ source for the organisms, enhancing the transformation of CT and CF. Experiments with M. thermophila and M. concillii, which could not grow on H₂-CO₂ under the conditions tested, showed that H₂ could serve as an electron donor for dechlorination of CT and CF with these organisms as well. Experiments with supernatants from M. thermophila grown with and without iron indicated the presence of an excreted biomolecule active in the enhanced transformation of CT and CF.

Full text of DOI:10.1021/es970785h

Environ. Sci. Technol. 1998, 32, 1438-1443
(
5, 6). Electrons are made available through the production
Enhanced Dechlorination of Carbon
Tetrachloride and Chloroform in the
Presence of Elemental Iron and
Methanosarcina barkeri,
Methanosarcina thermophila, or
Methanosaeta concillii
0
2+
of hydrogen gas (H
2
) from the corrosion of Fe to Fe
.
0
+
4
Fe + 8H f 4Fe2+ + 4H2
(3)
(4)
4
H + CO f CH + 2H O
2 2 4 2
Methanogens, which under anaerobic conditions mediate
the hydrogenolysis and hydrolytic reduction of halogenated
aliphatics (7), appear to grow solely on CO
2
and the H
2
0
produced from the corrosion of Fe (5, 6).
A combined system of elemental iron plus methanogens
should provide a low-maintenance support for an anaerobic
bacterial population in a treatment system, exploiting the
dechlorinating abilities of both the biological and abiotic
agents present. Preliminary studies have shown that, when
iron is combined with a mixed population of methanogens,
enhanced degradation (both in rate and extent) of carbon
tetrachloride (CT) and chloroform (CF) (1, 2) occurred. This
enhanced dechlorination was much greater than expected
by simply adding the effects of biodegradation by resting
cells with iron-mediated degradation.
P . J . N O V A K , * L . D A N I E L S , A N D
G . F . P A R K I N
The Department of Civil and Environmental Engineering,
Engineering Building, The University of Iowa,
Iowa City, Iowa 52242
Previous experiments in our laboratory have demonstrated
that the rate and extent of carbon tetrachloride (CT) and
chloroform (CF) dechlorination were enhanced when a
From these experiments (1, 2), the question arose, how
does this enhanced degradation take place? The research
0
methanogenic enrichment culture and iron (Fe ) were
presented here addressed this question. Objectives included
incubated together. Batch experiments with three pure
cultures of methanogens, Methanosarcina barkeri, Metha-
nosarcina thermophila, and Methanosaeta concillii were
performed to determine how this enhanced transformation
occurred. When hydrogen (H2) was added as an electron
donor, degradation of CT for all organisms and CF for M.
thermophila was more rapid. H2 was produced from the
oxidation of iron, which therefore served as an H2 source
for the organisms, enhancing the transformation of CT
and CF. Experiments with M. thermophila and M. concillii,
which could not grow on H2-CO2 under the conditions
tested, showed that H2 could serve as an electron donor for
dechlorination of CT and CF with these organisms as well.
Experiments with supernatants from M. thermophila
grown with and without iron indicated the presence of an
excreted biomolecule active in the enhanced transforma-
tion of CT and CF.
0
(
a) the determination of whether H
2
from oxidizing Fe could
serve as an electron donor for biologically mediated dechlo-
rination and (b) whether in the combined system a biological
factor was excreted into the media that was active in the
dechlorination of CT and CF. The model contaminants CT
and CF were used with pure cultures of methanogens
(
Methanosarcina barkeri 227, Methanosarcina thermophila,
and Methanosaeta concillii) for the experiments presented.
Experimental Section
Chem icals. High-pressure liquid chromatography (HPLC)-
grade CT, CF, and dichloromethane (DCM) were obtained
from Sigma. HPLC-grade methanol (MeOH) was used as a
substrate (Fisher). A single bottle of elemental iron powder
(
Aldrich Chemical Co.) was used in all of the experiments.
The iron powder (99.9% pure, 10 µm) was unwashed and
2
had a specific surface area of 2.02 m / g. Methane gas (CH
-CO (80:20 vol/ vol), and N -CO (80:20 vol/ vol) were
obtained from Air Products.
4
),
H
2
2
2
2
Organism and Culture Conditions. Studies were per-
formed with pure cultures of either M. thermophila (DSM
1825; Deutsche Sammlung von Mikroorganismen, Braun-
schweig, Germany), M. barkeri 227 (DSM 1538), or M. concillii
(DSM 3671). These three cultures were chosen for their varied
Introduction
Halogenated aliphatics have been in widespread use for
several decades in industrial applications such as degreasing,
cleaning, and extraction. Many of these compounds are listed
as priority pollutants by the United States Environmental
Protection Agency and are known or suspected human
carcinogens, mutagens, or toxins (3).
abilities to utilize H
readily on H -CO with no acclimation period, M. thermo-
phila will only grow poorly on H -CO after a long lag period
(about 9 days) (8, 9), and M. concillii is unable to grow on
. All three cultures were grown according to the
2
2
-CO for growth. M. barkeri will grow
2
2
2
2
0
Elemental iron (Fe ) has been shown to facilitate the
abiotic hydrogenolysis of chlorinated aliphatics (4):
H
2
-CO
2
directions provided by the DSM.
0
2+
-
Methanogens were cultured in 140-mL bottles (Supelco),
with thick butyl rubber stoppers (Bellco) or in a 1-L Pyrex
bottle fitted with a constructed stopper. The constructed
stoppers consisted of the following. A 20-mL headspace
autosampler vial (Kimble) with the bottom of the vial removed
was sealed with a thick butyl rubber stopper (Bellco). This
vial was fit into a large butyl rubber stopper with the center
removed, and the entire unit was then fit into a drilled hole
in the plastic media bottle cap. In all cases, freshly prepared
media was added to the bottles; the bottles were then sealed,
autoclaved, and allowed to cool. Sterile techniques were
Fe f Fe + 2e
(1)
(2)
H+ + 2e + CCl f CHCl + Cl
-
-
4
3
0
Several elemental metals, including Fe , can support the
growth of methanogenic bacteria as the sole electron donor
*
Corresponding author present address: Institute of Technology,
University of Minnesota, 122 Civil Engineering Building, 500 Pillsbury
Dr. SE, Minneapolis, MN 55455-0220; phone: (612)626-9846; fax:
(
612)626-7750; e-mail: novak010@tc.umn.edu.
1
4 3 8
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 10, 1998
S0013-936X(97)00785-2 CCC: $15.00

1998 Am erican Chem ical Society
Published on Web 04/11/1998

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
9
1 4 3 9

-
1
TABLE 1. First-Order Rate Coefficients for CT and CF Degradation by M. barkeri, M. thermophila, and M. concillii (day )
M. barkeri
M. thermophila
M. concillii
treatment
(
parent com pd)
CT
CF
CT
CF
CT
cells-only
2.13 ( 0.30
0.39 ( 0.14
0.64 ( 0.74
NAa
0.76 ( 0.43
0.21 ( 0.13
0.16 ( 0.05
8.31 ( 0.33
14.0 ( 0.92
11.9 ( 3.62
18.6 ( 1.64
10.3 ( 0.62
0.27 ( 0.25
7.45 ( 1.58
11.1 ( 1.74
11.7 ( 3.76
16.2 ( 0.47
0.31 ( 0.03
0.13 ( 0.01
NAa
Cells-plus-H2
MeOH-fed cells
iron-plus-cells
iron-only
5.38 ( 0.43
0.44 ( 0.03
0.31 ( 0.07
8.25 ( 2.06
10.0 ( 1.61
0.20 ( 0.05
a
b
NA
9.84 ( 1.09
4.74 ( 0.15
0.29 ( 0.01
m edium control
a
Not applicable. b Acetate fed.
column. The column had a 1.8 µM DB-VRX stationary phase
Supelco). Nitrogen was the carrier gas. The oven temper-
(
ature was held constant at 35 °C for a 10-min run length.
Detection limits were approximately 0.412 µM.
Methane and H
00 µL were directly injected into a gas chromatograph
Hewlett-Packard 5890) with a thermal conductivity detector
GC-TCD) for CH and H measurement. A 6-ft stainless
2
Measurem ent. Headspace samples of
1
(
(
4
2
steel-packed column with Haysep Q packing was used.
Nitrogen was the carrier gas. The oven temperature was
held at 120 °C for a run length of 2.5 min. Detection limits
were approximately 10 µL/ reactor for each gas.
VSS Measurem ent. The methodology was followed as
outlined in Standard Methods (Method 2540 G; 10).
Results and Discussion
FIGURE 1. CT transformation profiles and subsequent CF formation
and transformation profiles in experiments with M. thermophila
and added CT. Error bars represent ( one standard deviation.
Symbols are 0, cells-only CT; 9, cells-only CF; 4, iron-plus-cells
CT; 2, iron-plus-cells CF; O, iron-only CT; b, iron-only CF. The
Direct comparisons of the CT and CF first-order transforma-
tion coefficients for the three different species should be
viewed with caution. However, relationships between treat-
ments, product distribution, and transformation patterns
can be compared. In addition, because these experiments
were conducted at the optimal growth conditions for each
organism, the rates of transformation are very high and should
not be extrapolated to field applications. Replicate experi-
ments were performed and, when statistically compared with
a two-way analysis of variance (ANOVA) test with interaction,
were found to behave in the same manner as those
experiments reported in the paper. These results are not
included for brevity.
2
cells-plus-H , MeOH-fed cells, and medium control treatments have
been deleted for clarity.
thermophila, the iron-only system produced a nearly sto-
ichiometric amount of CF from CT; this CF underwent slow
subsequent transformation. In all treatments containing
organisms, less CF was formed (ranging from 20 to 50% of
initial CT concentration). CF was very quickly transformed
2
in the treatments containing cells-plus-H and iron-plus-
cells (Figure 1). In treatments of cells-only and MeOH-fed
cells, less CF was formed, and it was subsequently trans-
formed more slowly.
In experiments where CF was added, very little trans-
formation occurred in the iron-only and medium systems
In the experiments described here, M. thermophila was
not exposed to H
containing iron or an H
2
-CO
2
until introduced to the bottles
2
headspace (approximately 1 h before
CT or CF addition). Because experiments with M. thermo-
phila generally lasted less than 2 days, no acclimation to
(
Table 1), while systems containing M. thermophila trans-
growth on H
2
2
-CO was expected. Indeed, methane and
formed CF readily. In the systems where all the CF was
transformed (the systems containing organisms), approxi-
mately 50% (on a molar basis) was transformed to DCM;
Figure 2 shows DCM production by M. thermophila. Cells-
only treatments produced DCM but were unable to degrade
it (Figure 2). However, in biological systems containing added
biomass measurements showed no growth in the M. ther-
mophila and M. concillii treatments containing iron or an
H
2
headspace.
H
2
was produced in all iron-containing
and H
treatments with time (data not shown). The CH
4
2
profiles showed the expected trends for each treatment based
on the catabolic abilities of these three organisms (data not
shown).
Two-sided, paired t-tests over the first-order transforma-
tion coefficients were determined between various treatments
at the 95% confidence level. The results from these tests
were used to establish significance; this significance is
mentioned in the text.
Dechlorination Experim ents. First-order rate coefficients
for CT and CF transformation by M. thermophila, M. barkeri,
and M. concillii are listed in Table 1 for each of the treatments
studied. Transformation rate coefficients were calculated
as described when either CT or CF was added. Coefficients
were not calculated for daughter products.
iron, H
degraded.
M. barkeri. In experiments with M. barkeri, CT was
2
, or MeOH, DCM was formed and subsequently
transformed in the treatments containing iron-plus-cells at
a rate significantly faster than in all other treatments (Table
1, Figure 4). The cells-plus-H treatments also transformed
2
CT at a significantly faster rate than the cells-only treatment.
Accumulation of CF followed a pattern similar in some ways
to that observed for M. thermophila, with approximately
stoichiometric CF formation in the iron-only system and
only a transient CF accumulation in the iron-plus-cells
reactors (Figure 3). However, unlike M. thermophila, reactors
with cells-plus-H and cells-only accumulated CF at levels
2
M. thermophila. CT transformation profiles and con-
comitant CF formation and transformation profiles are shown
in Figure 1 for M. thermophila. In experiments with M.
similar to the iron-only reactors. Subsequent degradation
of CF was very slow in all of the treatments except the iron-
plus-cells reactors.
1
4 4 0
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 10, 1998

FIGURE 4. CT transformation profiles and subsequent CF formation
and transformation profiles in experiments with M. concillii and
added CT. Error bars represent ( one standard deviation. Symbols
are 0, acetate-fed cells CT; 9, acetate-fed cells CF; 4, cells-plus-
H CT; 2, cells-plus-H CF; O, iron-only CT; b, iron-only CF. The
2 2
iron-plus-cells and medium control treatments have been deleted
for clarity.
FIGURE 2. DCM formation and subsequent transformation profiles
in experiments with M. thermophila and added CF. Error bars
represent ( one standard deviation. Symbols are: 0, cells-only;
2
2
, cells-plus-H ; 4, iron-plus-cells; 9, MeOH-fed cells; O, iron-
only treatments. No DCM was formed in the medium control
treatments; therefore, these points are not shown.
transformation was quite fast; therefore, the rate of CT
transformation in the iron-plus-cells system was dominated
by the abiotic reaction, and no enhanced degradation was
observed when organisms were added. Transformation
profiles for CT and subsequent formation and transformation
profiles for CF are given in Figure 4.
Discussion of Dechlorination Experim ents. An H
2
headspace with cells enhanced CT and CF degradation by
M. barkeri and M. thermophila as compared to cells-only
systems. An H headspace with cells also enhanced CT and
2
CF degradation by M. concillii as compared to the acetate-
fed cells system. For M. barkeri, when one adds the rate
2
coefficient for CT transformation by cells-plus-H to the iron-
only transformation coefficient, it equals that of the iron-
plus-cells systems. For M. thermophila, the CT transfor-
mation coefficient for cells-only plus iron-only approximates
that of the iron-plus-cells systems. However, when one adds
the CF transformation coefficients for the cells-plus-H
systems to that for the iron-only systems, it is a better
approximation of the combined system than the cells-only
2
FIGURE 3. CT transformation profiles and subsequent CF formation
and transformation profiles in experiments with M. barkeri and
added CT. Error bars represent ( one standard deviation. Symbols
are 0, cells-only CT; 9, cells-only CF; 4 iron-plus-cells CT; 2,
iron-plus-cells CF; O, iron-only CT; b, iron-only CF. The cells-plus-
2
plus the iron-only systems. These results show that H was
a key factor in the enhanced degradation of CT and CF for
both organisms, but not the only factor of importance in the
transformation of these contaminants by M. thermophila.
H
2
and medium control treatments have been deleted for clarity.
When CF was added to M. barkeri as the parent com-
M. barkeri can use H
increased biomass should degrade CT and CF at a faster rate
if H was present. Also, H would be expected to serve as an
electron donor for dechlorination because M. barkeri contains
hydrogenase, which would utilize the reducing equivalents
2
-CO
2
for growth and through
pound, very slow transformation of CF was observed in all
of the treatments (Table 1). M. barkeri was sensitive to higher
concentrations of CF. In the experiments where CT was
added, less CF was formed than when CF was added directly
2
2
(
2.5 versus 5.5 µM, respectively). When less CF was present,
2 2
from H to reduce CT or CF. In other studies, H has also
it was transformed over two times faster than when the higher
concentration of CF was added to the organisms. M.
thermophila did not exhibit such sensitivity.
When CF was added at 5.5 µM, the transformation rate
coefficient for CF was not significantly different in any of the
treatments. DCM formed and increased in concentration
over the experimental period in the cells-plus-H and iron-
2
plus-cells treatments. No DCM was formed in the other
treatments (data not shown).
been implicated as the electron donor for dechlorination of
PCE (11).
Although H
2
2
-CO cannot be used as a growth substrate
for M. thermophila under the conditions studied here,
enhanced degradation of CT and CF occurred in the presence
of an H
with the observation that even though M. thermophila does
not grow on H -CO , it does exhibit a low level of hydrogenase
activity (8, 12). This demonstrates that H can be used as an
electron donor for dechlorination when the cells were unable
to utilize H -CO for growth. This has important implications
2
headspace with this organism. This was consistent
2
2
2
M. concillii. In experiments with M. concillii, transfor-
mation of CT occurred very slowly in the systems without
iron (Table 1 and Figure 4). CT did degrade significantly
2
2
for bioremediation since field conditions (such as pH,
micronutrients, and temperature) are rarely ideal for organ-
2
faster in the cells-plus-H reactors than in the acetate-fed
reactors, but the difference was slight. However, unlike the
results with M. thermophila and M. barkeri, CT transforma-
tion in the iron-plus-cells system was not faster than in the
iron-only system. The rate of CT transformation by M.
concillii was very slow, and the rate of iron-mediated
ism growth. It can be inferred from these results that if H
2
is present (via the corrosion of iron) and methanogens, having
active hydrogenase, are also present, the organisms will not
need to be able to grow in order to effectively use the H
an electron donor for dechlorination.
2
as
VOL. 32, NO. 10, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1 4 4 1

TABLE 2. First-Order Rate Coefficients for CT Degradation by
M. barkeri and M. thermophila in the Supernatant
-
1
Exchange Experiments (day )
treatmenta
M. barkeri
M. thermophila
cells-stay
cells-swap
iron-swap
iron-stay
0.87 ( 0.16
1.18 ( 0.24
5.47 ( 0.58
5.43 ( 1.14
0.31 ( 0.28
10.8 ( 0.06
15.7 ( 0.22
15.6 ( 0.60
34.7 ( 3.20
16.2 ( 0.19
supernatant control
a
Cells-stay contains pellet and supernatant not exposed to iron;
cells-sway contains pellet not exposed to iron and exposed supernatant;
iron-swap contains pellet exposed to iron and supernatant not exposed;
iron-stay contains pellet and supernatant exposed to iron. The
supernatant control is supernatant only from the system not exposed
to iron.
2
For experiments with M. concillii, H was also able to
serve as an electron donor for dechlorination at very low
levels when it was unable to serve as an electron donor for
growth. M. concillii is a relatively poorly studied organism;
it is only able to utilize acetate for growth and is therefore
FIGURE 5. CF profiles in the supernatant exchange experiments
with M. thermophila and added CT. Error bars represent ( one
standard deviation. Symbols are 4, cells-stay; 9, cells-swap; 2,
iron-swap; b, supernatant control treatments; O, iron-stay treat-
ments. Treatment names are as defined in the Experimental Section.
not thought to contain hydrogenase. It is unclear how H
2
would be utilized as an electron donor for dechlorination
without the presence of hydrogenase. It is possible that these
organisms do contain such enzymes, but at a low enough
level that they are unable to contribute to organism growth.
In early characterization studies of this organism, a hydro-
genase assay showed very slight activity (13). It is not clear
whether this was an artifact or did in fact represent a very
small quantity of this enzyme in the culture. In an in situ
situation where H is cathodically produced, organisms
2
similar to M. concillii, present at a distance from the iron,
could still contribute to the enhanced dechlorination by
utilizing this H for dechlorination.
2
Section. For M. thermophila, there was a significant dif-
ference between the cells-swap and the cells-stay treatments
(Table 2), with the cells-swap treatment transforming CT at
a faster rate. The supernatant control also transformed CT
very rapidly (Table 2). However, in experiments with M.
barkeri, no statistical difference was seen between the cells-
swap and cells-stay treatments (Table 2). There was also no
significant difference between the iron-swap and iron-stay
treatments (Table 2). In addition, no appreciable transfor-
mation of CT was observed in the M. barkeri supernatant
control.
The transformation patterns of CF (when CT was added)
or DCM (when CF was added) indicated that the presence
of H gas increased the rate at which CF and DCM were
2
subsequently degraded. Again, this indicates that the
enhanced degradation of CT, CF, and DCM observed when
elemental iron and methanogenic bacteria were present
together was due, at least in part, to the H gas produced
2
from corroding iron serving as an electron donor for
dechlorination.
In experiments with M. thermophila, the CF formation
and subsequent degradation profile was very different for
the cell systems with or without exchanged supernatants
(Figure 5). A buildup of CF in the cells-stay reactors to about
50% of the initially added CT occurred, followed by slow
degradation. In the cells-swap reactors, less CF was formed
and was quickly transformed. In the iron-plus-cells systems
with or without exchanged supernatants about 50% of the
initially added CT was transformed to CF, which was quickly
degraded. The rate of CF transformation appeared to be
about the same in the three groups of reactors that contained
iron or contained supernatant exchanged from the iron-plus-
cells systems. CF was also formed in the supernatant control
treatments, which was slowly transformed.
In experiments with M. barkeri, the CF profile in the cells-
swap and cells-stay treatments was approximately the same
(Figure 6). In both systems CF was formed from CT, but was
not further transformed. Likewise, the CF profile in the iron-
stay treatment was mimicked in the iron-swap treatment,
with CF forming but failing to undergo further dechlorination.
Nearly stoichiometric quantities of CF were produced from
CT in the iron-containing systems. Little CF was formed in
the supernatant control, and no further transformation of
the CF was observed.
The degradation of CT by M. barkeri was not affected by
exchanging the supernatants between the cells-only and iron-
plus-cells treatments. This indicates that no extracellular
compound was active in these systems. However, when the
same experiments were run with M. thermophila, there was
an enhanced degradation of CT and CF in the cells-only
systems containing supernatant from the iron-plus-cells
systems. There was no visual evidence of iron carryover in
the exchanged supernatants. The enhanced transformation
could therefore be due to an extracellular factor released
from the organisms, which was active in CT and CF
2
In a mixed anaerobic culture, H would be expected to
serve as both an electron donor for growth as well as an
electron donor for dechlorination. One would expect to find
organisms that would fall into each of the categories
investigated in this research: cells that grow readily on H
CO , cells that will grow poorly on H -CO after an acclima-
tion period, and cells that are completely unable to utilize
-CO for growth. The results presented here demonstrate
2
-
2
2
2
H
2
2
that each of these culture types will be able to use H
electron donor for dechlorination.
2
as an
Research by Weathers and Parkin (1) and Weathers et al.
2) corroborates that H was responsible for the enhanced
(
2
transformation of CT and CF in a mixed anaerobic culture.
Dechlorination occurred at a much faster rate when organ-
isms were in the presence of iron rather than under an N
CO headspace.
Supernatant Exchange Experim ents. Supernatant ex-
2
-
2
change experiments were performed to determine whether
an excreted biomolecule was responsible for all or a portion
of the enhanced CT and CF transformation. The superna-
tants from treatments where organisms were grown in the
presence of iron were exchanged with those where organisms
were grown in media only. First-order transformation
coefficients were calculated for CT for each treatment with
M. barkeri and M. thermophila and are presented in Table
2
. Treatment names are as described in the Experimental
1
4 4 2
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 10, 1998

dechlorination (as for M. thermophila or M. concillii). Iron
was thereby able to enhance the CT (and to some extent CF)
dechlorination rate by serving as an electron donor for
dechlorination. Finally, an excreted biomolecule from M.
thermophila was implicated in the enhanced transformation
of both CT and CF when grown in the presence and absence
of elemental iron. No such biomolecule was detected from
M. barkeri. Further studies were conducted to study this
biomolecule and will be discussed in an additional paper
(
16).
Acknowledgments
Funding for this research was provided by the Great Plains
Rocky Mountain Hazardous Substance Research Center, the
National Institute of Health, and The University of Iowa
Center for Biocatalysis and Bioprocessing. Dr. N. Belay and
Dr. L. Weathers helped with the early stages of the research.
Dr. J. G. Ferry provided initial samples of M. thermophila.
C. Just assisted with instrumentation, and Dr. K. Nagaranthal
provided the constructed stoppers.
FIGURE 6. CF profiles in the supernatant exchange experiments
with M. barkeri and added CT. Error bars represent ( one standard
deviation. Symbols are 4, cells-stay; 9, cells-swap; 2, iron-swap;
b, supernatant control treatments; O, iron-stay treatments. Treatment
names are as defined in the Experimental Section.
Literature Cited
(
1) Weathers, L. J.; Parkin, G. F. Enhanced Transformation of
Chlorinated Methanes Under Methanogenic Conditions Using
Elemental Iron as the Ultimate Electron Donor. Abstract for the
Third International Symposium on In Situ and On-Site Biore-
mediation, San Diego, CA, 1995.
transformation. The rapid transformation observed in the
supernatant control indicates that the difference between
the cells-only and cells-plus-iron supernatants could be that
growth in the presence of iron results in the production of
a greater quantity of a factor always released by the cells or
the release of an additional factor.
(
(
(
2) Weathers, L. J.; Parkin, G. F.; Alvarez, P. J. Environ. Sci. Technol.
1
997, 31, 880-885.
3) National Institute of Environmental Health Sciences (NIEHS).
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Technol. 1996, 30, 2634-2640.
Evidence for similar extracellular factors can be found in
the literature. Dybas and co-workers (14) have found that
a Pseudomonas species will degrade CT completely to CO
2
(5) Daniels, L.; Negash, B.; Rajagopal, B. S.; Weimer, P. J. Science
987, 237, 509-511.
1
and a nonvolatile fraction under denitrifying conditions. This
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(
6) Lorowitz, W. H.; Nagle, D. P., Jr.; Tanner, R. S. Environ. Sci.
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nogen, Methanobacterium bryantii BKYH, was exposed to
high concentrations of copper, the organism responded by
excreting 4-fold increased levels of three extracellular pro-
teins. These proteins were thought to protect the organism
from toxicity and allow it to grow in an environment where
it would otherwise be unable to live. M. thermophila may
respond in a manner similar to Fe by excreting a protein
that also happens to be active in the dechlorination of CT
and CF.
Experiments conducted to assess the cause of enhanced
CT and CF transformation when methanogens were in the
presence of elemental iron allowed the following conclusions
(8) Zinder, S. H.; Mah, R. A. Appl. Environ. Microbiol. 1979, 38,
996-1008.
(
9) Mukhopadhyay, B.; Purwantini, E.; de Macario, E. C.; Daniels,
L. Curr. Microbiol. 1991, 23, 165-173.
(
10) APHA. Standard Methods for the Examination of Water and
Wastewater, 17th ed.; American Public Health Association:
Washington, DC, 1989.
(11) DiStefano, T. D.; Gossett, J. M.; Zinder, S. H. Appl. Environ.
Microbiol. 1992, 58, 3622-3629.
(
12) Zinder, S. H.; Anguish, T. Appl. Environ. Microbiol. 1992, 58,
323-3329.
13) Patel, G. B. NRC, Ottawa, Canada, personal communication,
997.
3
0
(
1
(14) Dybas, M. J.; Tatara, G. M.; Criddle, C. S. Appl. Environ. Microbiol.
1995, 61, 758-762.
15) Kim, B.-K.; Pihl, T. D.; Reeve, J. N.; Daniels, L. J. Bacteriol. 1995,
(
1
77, 7178-7185.
(
16) Novak, P. J.; Daniels, L.; Parkin, G. F. Submitted to Environ. Sci.
2
to be drawn. First, providing H as an electron donor resulted
Technol
in faster degradation of CT for all organisms and CF for M.
thermophila; therefore, the enhanced dechlorination when
organisms were incubated with iron was likely due to iron
Received for review September 4, 1997. Revised manuscript
received February 20, 1998. Accepted February 24, 1998.
providing a source of H
2
. Organisms did not need to be able
to act as an electron donor for
to grow on H -CO for H
2
2
2
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