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Soil Biology & Biochemistry 32 (2000) 977±988
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Speci®city of chloroform, 2-bromoethanesulfonate and
¯uoroacetate to inhibit methanogenesis and other anaerobic
processes in anoxic rice ®eld soil
Amnat Chidthaisong, Ralf Conrad*
Max-Planck-Institut fuÈr terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany
Accepted 6 December 1999

Abstract
Chloroform (CHCl3), 2-bromoethanesulfonate (BES) and ¯uoroacetate have frequently been used as methanogenic inhibitors
in rice ®eld soil and in other environments, but their e€ects on other microbial processes have not received sucient attention.
Therefore, we comparatively determined the e€ects of CHCl3, BES and ¯uoroacetate on di€erent microbial processes in rice
®eld soil slurry upon incubation under anoxic conditions: on the reduction of the electron acceptors nitrate, ferric iron, sulfate;
on the production of CH4 and CO2; on the temporal change of the electron donors H2, acetate and propionate; and on the
turnover of [2-14 C]acetate during the early reduction phase (day 7), and during the later methanogenic phase (day 30). The
results demonstrate: (1) ¯uoroacetate inhibited acetate consumption by all microorganisms, (2) BES generally inhibited CH4
production, and (3) CHCl3 not only inhibited methanogenesis, but partially also acetate-dependent sulfate reduction, and
perhaps H2-dependent homoacetogenesis. The speci®city of the di€erent inhibitors resulted in characteristic patterns of the
temporal change of electron donors and acceptors and of CH4. The pattern of propionate change was consistent with

production by fermenting bacteria and consumption by sulfate reducers either using sulfate or methanogens as electron acceptor.
Sulfate reducers were also found to be important for acetate consumption during the early phase of soil anoxia. Later on,
however, methanogenic acetate consumption was much more pronounced. The application of inhibitors with di€erent speci®city
was helpful for elucidating the ¯ow of carbon and electrons during degradation of organic matter in anoxic rice ®eld soil. 7 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Methane production; Acetate turnover; Sulfate reduction; Iron reduction; Nitrate reduction; Hydrogen; Propionate

1. Introduction
Metabolic inhibitors can be very useful to study microbial processes in the environment (Oremland and
Capone, 1988). Many chemical substances have been
applied to study the relative importance of methanogenic and other processes for carbon mineralization
both in bacterial cultures and in the environment. For
example,
2-bromoethane
sulfonate
(BES,

* Corresponding author. Tel.: +49-6421-178-801/800; fax: +496421-178-809.
E-mail address: conrad@mailer.uni-marburg.de (R. Conrad).

BrCH2 CH2 SO3ÿ † is often used to speci®cally inhibit
CH4 formation by methanogenic archaea (Oremland
and Polcin, 1982; Zinder et al., 1984; Alperin and Reeburgh, 1985; Nozoe, 1997; Nollet and Demeyer, 1997).
BES is a structural analog of coenzyme M which is
found in all methanogens but not in other Bacteria or
Archaea (Balch and Wolfe, 1979). Thus, it is regarded
as a ``speci®c'' inhibitor for methanogens. Another
compound which has been widely used to inhibit
methanogenic activity is chloroform (CHCl3) (Lovley
and Klug, 1982; Jones and Simon, 1985; Thebrath et
al., 1992; Achtnich et al., 1995a; Chin and Conrad,
1995; DeGraaf et al., 1996). CHCl3 is known to block
the function of corrinoid enzymes and to inhibit

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
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A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

methyl-coenzyme M reductase (Oremland and Capone,
1988). A further inhibitor, ¯uoroacetate (FCH2COOÿ),
has widely been used to block acetate metabolism.
Fluoroacetate is converted to ¯uorocitrate which then
inhibits the activity of aconitase in the tricarboxylic
cycle and thus, blocks acetate metabolism (Lehninger,
1975). Interestingly, it also inhibits acetoclastic methanogenesis, although the tricarboxylic acid cycle is not
involved. The mechanism of inhibition is unknown.
Mutants of Escherichia coli defective in acetate kinase
and phosphotransacetylase are resistant to ¯uoroacetate (LeVine et al., 1980) indicating that ¯uoroacetate
is activated similarly as acetate before it exerts its
adverse e€ect. Despite the unknown inhibition mechanism ¯uoroacetate has been applied to distinguish
acetoclastic from hydrogenotrophic methanogenesis or
to study the relative importance of acetate as the key
intermediate in organic carbon mineralization (Mountfort et al., 1980; Alperin and Reeburgh, 1985; DeGraaf
et al., 1996; Schulz and Conrad, 1996).
Recent investigation has shown that CHCl3 can inhibit not only the activity of methanogenic archaea but
also the activity of homoacetogenic bacteria and of
acetate-consuming sulfate-reducing bacteria (Scholten

et al., 2000). Sulfate reducers are only inhibited if they
degrade acetate via the acetyl-CoA pathway but not
via the tricarboxylic acid cycle. It has been reported
that Desulfotomaculum sp., which possess the acetylCoA pathway, and other acetate-utilizing sulfate reducers are present in rice ®eld soil (Wind and Conrad,
1995; Wind et al., 1999). Therefore, we hypothesized
that CHCl3 addition might have a di€erent e€ect on
the activity of microorganisms present in anoxic rice
®eld soil than addition of BES or ¯uoroacetate and
thus, may help to elucidate the ¯ow of carbon and
electrons during methanogenic degradation of organic
matter. We did incubation experiments with Italian
rice ®eld soil and studied the e€ect of the typical
methanogenic inhibitors BES, CHCl3 and ¯uoroacetate
on the reduction of nitrate, ferric iron, sulfate and the
formation of CH4, on the accumulation of metabolic
intermediates (H2, acetate, propionate), and on the
turnover of [2-14 C]acetate.

2. Materials and methods
2.1. Soil sample and slurry incubation

Rice ®eld soil was taken in 1993 from the experimental ®elds of the Italian Rice Research Institute at
Vercelli, in the valley of the river Po. Detailed site
descriptions and soil characteristics were already given
in a previous study (Holzapfel-Pschorn et al., 1986).
The soil was mechanically crushed and sieved (99% and the
speci®c activity of 57 mCi mmolÿ1, American Radiolabeled Chemical).
Soil incubation was terminated by adding 1 ml 7 N
H2SO4. The increase in the concentration of CO2 and
14
CO2 upon acidi®cation was used to correct for dissolved radioactive and non-radioactive CO2 by assuming that the CO2 in the gas and aqueous phases was in
equilibrium throughout the experiment. Typically,
slurry acidi®cation resulted in the release of 14 CO2
from dissolved radioactive bicarbonate and carbonate
to the gas phase, but had no signi®cant e€ect on the
recovery of 14 CH4 : When used below, the term 14 CO2

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

is assumed to represent total 14 CO2 (CO2 + bicarbonate + carbonate).
Since acetate uptake at day 7 was very slow, we

determined the acetate transformation rate constant,
i.e. the formation of 14 CO2 plus 14 CH4 , instead of the
acetate uptake rate constant, as described by Phelps
(1991). The transformation rate constant was shown to
be similar to the uptake rate constant and thus, can be
used alternatively to estimate the turnover of acetate
in anoxic soil and sediment (Phelps, 1991). At day 30,
uptake of acetate was rapid enough, so that the acetate
uptake rate constant was estimated from a semilogarithmic plot of residual [2-14 C]acetate against the incubation time (Phelps and Zeikus, 1984). The actual
turnover rate was obtained by multiplying the transformation rate constant or uptake rate constant with
the pool size (pore water pool) of acetate. The respiratory index (RI) was used to compare the carbon ¯ow
toward CH4 and CO2; RI ˆ f…14 CO2 †=…14 CO2 ‡ 14 CH4 †g:
The rate of CH4 production from acetate was calculated from: acetate turnover rate  (1ÿRI), assuming
that CH4 is produced only from acetate and CO2/H2.
2.3. Analytical techniques
CH4, CO2, and H2 concentrations were measured by
the gas chromatography (Conrad et al., 1989). Radioactivity of gaseous products was measured by gas proportional counting (Conrad et al., 1989). Nitrate,
nitrite, and sulfate ions were determined by HPLC

Fig. 1. E€ects of methanogenic inhibitors on the temporal change of

H2 partial pressure in anoxically incubated rice ®eld soil; mean2SD
of duplicates.

979

equipped with conductivity plus UV detector (Bak et
al., 1991). Ferrous iron was determined by a recently
developed HPLC system (Schnell et al., 1998). Organic
acids and their radioactivity were determined by
HPLC with the outlet connected to a radioactive scintillation monitor (KrumboÈck and Conrad, 1991).

3. Results
3.1. Accumulation of H2
H2 is usually found to accumulate during the ®rst
1±2 days of soil incubation in this Italian rice ®eld soil
(Chidthaisong et al., 1999). In the present study, H2
accumulation reached the maximum after one day of
soil incubation regardless of the presence of inhibitors
(Fig. 1). In all treatments, H2 concentrations decreased
rapidly to below 2.5 Pa at day 7, then increased again.

After day 25, the highest H2 concentration was
observed in the bottles with BES (6±10 Pa), whereas
the CHCl3 treatment showed concentrations similarly
as in the control (2.5±5 Pa) (Fig. 1). With ¯uoroacetate, H2 remained at a low concentration until about
day 25, after which it increased to the same level as in
the control (Fig. 1).
3.2. Reduction of nitrate, ferric iron and sulfate
Nitrate is usually reduced within a day of soil ¯ooding, thus the examination of the e€ect of inhibitors on
its reduction was performed on the ®rst day of soil incubation (0±8 h). For this purpose, all inhibitors were
added soon after the gas phase was exchanged with
pure N2 (time = 0 h). The temporal decrease of
nitrate is shown in Fig. 2. Reduction of nitrate in the
control progressed steadily during the ®rst six hours
and then accelerated. Interestingly, addition of BES
decreased the extractability of nitrate from the pore
water (Fig. 2A) and resulted in a lag of nitrite production (Fig. 2B). However, the other inhibitors
showed no signi®cant e€ect on nitrate decrease and
nitrite production. The rates of nitrate reduction calculated from the decrease of nitrate during the time of
0±8 h are given in Table 1. None of the inhibitors signi®cantly altered the rate of nitrate reduction ( p =
0.05) in comparison to the control.

The production of Fe(II) in the presence and
absence of inhibitors is shown in Fig. 3. Reduction of
Fe(III) as seen from the rapid increase of Fe(II) started
immediately when the soil was incubated under anoxic
conditions. Addition of inhibitors had no signi®cant
e€ect on this process. The rates of Fe(II) production
calculated between 0 and 10 days of incubation ranged
between 15 and 22 mmol gÿ1 dÿ1 (Table 1).
In contrast to nitrate and Fe(III) reduction, sulfate

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A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

Fig. 2. Reduction of (A) nitrate and production of (B) nitrite in rice ®eld soil incubated under anoxic conditions in the presence of di€erent inhibitors; mean2SD of duplicates.

reduction was signi®cantly a€ected by addition of the
inhibitors (Fig. 4). The initial sulfate concentration
was similar (ca. 2 mM). In the control, sulfate was
reduced without lag phase and reduction was complete

at about day 15. Addition of BES delayed the initiation of sulfate reduction by 5 days, but thereafter
sulfate reduction progressed even more rapidly than in
the control so that the reduction of sulfate in the presence of BES was also complete at day 15. However,
when CHCl3 was added sulfate concentration did not
decrease for about 10 days and then proceeded only
slowly, so that the residual concentration of sulfate
was still above 1 mM at the end of the experiment.
Apparently, CHCl3 had a pronounced inhibitory e€ect
on sulfate reduction. In the bottles with ¯uoroacetate,
on the other hand, sulfate reduction was only transiently inhibited. Sulfate reduction in these bottles was
inhibited before day 17, but then progressed with a
similar rate as the control and was complete at day 23

(Fig. 4). The rates of sulfate reduction calculated
during the phase of fastest decrease of sulfate concentrations (4±15 d in control; 6±14 d with BES; 8±32 d
with CHCl3; 8±23 d with ¯uoroacetate) were signi®cantly lower in the presence of CHCl3 than in the control, but were not signi®cantly di€erent in the other
treatments (Table 1).
3.3. Accumulation of acetate and propionate
Intermediate accumulation of acetate to millimolar
concentrations is a general characteristic of this soil

(Chidthaisong et al., 1999). In the present study, up to
2.5 mM acetate transiently accumulated in the control
with a maximum on day 10 (Fig. 5A). Acetate concentration subsequently decreased and stayed around 100
mM thereafter. In the presence of inhibitors, acetate
accumulated continuously but to di€erent extent. With
BES, a lag phase of about 5 days, similarly as in the

Table 1
E€ect of methanogenic inhibitors on the rates of nitrate, ferric iron and sulfate reduction procesess in rice ®eld soil (mean2SD of duplicates;
Treatments

Nitrate reduction (mmol gÿ1 dayÿ1)

Ferric iron reduction (mmol gÿ1 dayÿ1)

Sulfate reduction (nmol gÿ1 dayÿ1)

Control
BES
Chloroform
Fluoroacetate

2.820.6
3.120.5
2.821.0
2.420.9

20.423.5
17.123.1
14.923.9
21.921.7

163.3220.5
188.4251.3
45.424.3a
126.0236.6

a

indicates signi®cant di€erence at p = 0.01.

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

Fig. 3. Production of ferrous iron in rice ®eld soil incubated under
anoxic conditions in the presence of di€erent inhibitors.

control, was observed, but then acetate concentrations
increased further and reached about 12 mM at the end
of the experiment. With CHCl3, the pattern of acetate
accumulation was similar to that with BES, but accumulation started somewhat earlier and acetate accu-

981

mulated to higher concentrations (ca. 20 mM)
(Fig. 5A). With ¯uoroacetate, accumulation of acetate
started without lag phase and reached the highest ®nal
concentration (ca. 25 mM). The rates of acetate accumulation (between days 10 and 32) in the presence
of BES, CHCl3 and ¯uoroacetate were about 400, 740
and 740 nmol gÿ1 dÿ1, respectively.
Propionate accumulation was also found in all treatments. In the control, transient accumulation of propionate reached its maximum at about day 12, slightly
delayed compared to acetate accumulation (Fig. 5B),
and disappeared rapidly thereafter. With BES, propionate transiently accumulated during day 7±12, became
undetectable shortly afterwards, but then started to accumulate continuously beginning day 15 until the end
of incubation, when >200 mM of propionate had
accumulated. With CHCl3, propionate reached a transient maximum between days 5 and 12 and then
decreased below the detection limit similarly as in the
control. With ¯uoroacetate, propionate showed a similar pattern as with BES, but with the transient maximum reached between days 10 and 20 and start of
continuous accumulation beginning on day 22
(Fig. 5B). Accumulation of other intermediates (e.g.,
butyrate, valerate, caproate etc.) was not observed.
3.4. Production of CH4 and CO2
Production of CH4 accelerated around day 10
(Fig. 6A), i.e. about the same time as acetate started
to decrease from its transient maximum (Fig. 5A).
After day 15, when acetate was decreased to a low
steady state concentration, CH4 production transiently
slowed down, but then resumed the former rate after
day 25. The rate of CH4 production in the control
averaged between day 10 and 40 was about 410 nmol
gÿ1 dÿ1, after day 25 it was about 600 nmol gÿ1 dÿ1.
Production of CH4 was completely inhibited by addition of BES or CHCl3. In the presence of ¯uoroacetate, CH4 production was also inhibited but resumed
after day 25 (Fig. 6A). Thus, inhibition of methanogenesis by ¯uoroacetate was not complete. There was
no signi®cant di€erence in CO2 production during
days 0±17 (Fig. 6B). Later on, however, addition of
both CHCl3 and ¯uoroacetate partially inhibited CO2
production, whereas addition of BES had no e€ect.
3.5. Turnover of [2-14 C]acetate

Fig. 4. Reduction of sulfate in rice ®eld soil incubated under anoxic
conditions in the presence of di€erent inhibitors; mean 2 SD of
duplicates.

Turnover of [2-14 C]acetate was measured after 7 and
30 days of soil incubation by measuring the formation
of 14 CH4 and 14 CO2 : On day 7, Fe(III) reduction was
just about to ®nish while sulfate reduction was still
going on. On day 30, all inorganic electron acceptors
had been reduced and methanogenesis was the only
active reduction process remaining. Results of a pre-

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A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

Fig. 5. Change of the concentrations of (A) acetate and (B) propionate in rice ®eld soil incubated under anoxic conditions in the presence of
di€erent inhibitors; mean2SD of duplicates.

vious study have shown that during the phase of reduction of endogenous electron acceptors (day 0±15)
acetate turnover rate constants are much lower than
during the methanogenic phase (Chidthaisong et al.,
1999). Indeed, conversion of [2-14 C]acetate at day 7
was slow in all treatments. The uptake rate constant of

acetate in control, BES, CHCl3 were 0.028, 0.017 and
0.012 hÿ1, respectively (Table 2), equivalent to turnover times of 40, 59 and 80 h. With ¯uoroacetate [214
C]acetate uptake was not detectable (Table 2). BES
and CHCl3 inhibited acetate uptake by 40% and 60%,
respectively.

Fig. 6. Accumulation of (A) CH4 and (B) CO2 in rice ®eld soil incubated under anoxic conditions in the presence of di€erent inhibitors; mean2
SD of duplicates.

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

983

Table 2
Uptake of [2-14 C]acetate and its recovery as 14 CH4 and 14 CO2 at di€erent days of soil incubation. Uptake of acetate at day 7 was determined
from the accumulation of 14 CO2 between 100 and 7200 min after its addition. At day 30, uptake of acetate was estimated from the decrease in
[2-14 C]acetate. Recovery of 14 CH4 and 14 CO2 is given as the maximum value found during the time course of the experiment (7200 min)
Treatments

Uptake rate constant (hÿ1)

Maximum recovery (%) as
CO2

Addition at day 7
Control
BES
Chloroform
Fluoroacetate
Addition at day 30
Control
BES
Chloroform
Fluoroacetate

0.028
0.017
0.012
0
0.23320.006
0.00420.001
0.00720.002
0.00720.002

CH4

0.03
0.06
0.08
0
8.4
14.3
7.5
3.4

0.01
0
0
0

78
86
65
99

90.4
0
0
0

99
104
100
99

All of the inhibitors applied completely inhibited
CH4 formation from [2-14 C]acetate (Fig. 7A). In the
control, production of 14 CH4 was observed about 1 d
after addition of [2-14 C]acetate. However, small
amounts of 14 CO2 were produced earlier, ca. 2 h after
addition of [2-14 C]acetate. The ®nal RI of the control
was 0.6. Addition of BES and CHCl3 did not inhibit
14
CO2 production in day-7 soil. Fluoroacetate completely inhibited both 14 CH4 and 14 CO2 production from
[2-14 C]acetate.
In contrast to the results obtained with day-7 soil,
[2-14 C]acetate was rapidly converted to 14 CH4 in day30 soil. In the control, it was exclusively converted to
CH4 (RI = 0.08) (Fig. 8A). Production of some 14 CO2
14

Fig. 7. Conversion of [2-14 C]acetate to (A) 14 CH4 and (B)
conditions for 7 days; mean2SD of duplicates.

Total radioactive recovery (% of initially added acetate)

14

was only observed after 1 d (Fig. 8B). Addition of
BES, CHCl3 or ¯uoroacetate completely inhibited
14
CH4 production. The turnover rate constants of acetate in di€erent treatments were 0.223, 0.004, 0.007 and
0.007 hÿ1 in control, BES, CHCl3 and ¯uoroacetate,
respectively (Table 2). Addition of these inhibitors also
resulted in an increase of the acetate pool size
(Fig. 8C). The amount of acetate accumulated in the
presence of BES, CHCl3 and ¯uoroacetate was similar
indicating that acetotrophic methanogenesis was inhibited to a similar extent.
In the control, the acetate pool stayed constant at 68
2 10.3 nmol gÿ1 (Fig. 8C). The turnover rate of acetate in the control was calculated to 365 nmol gÿ1

CO2 in the presence of di€erent inhibitors using rice ®eld soil incubated under anoxic

984

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

dayÿ1. With a RI of 0.08, this rate is equivalent to a
rate of acetotrophic CH4 production of 336 nmol gÿ1
dÿ1 or 56±82% of the total CH4 production of 410±
600 nmol gÿ1 dÿ1. Since the pool sizes of acetate in
other treatments were not constant during the time
course of the experiment, reasonable acetate turnover
rates could not be estimated.
An interesting observation was that accumulation of
H2 was only caused by the addition of BES and even
more by CHCl3, while ¯uoroacetate did not a€ect the
H2 concentration (Fig. 8D).

4. Discussion

4.1. Selectivity of methanogenic inhibitors
The results of our experiments have con®rmed that
CHCl3, BES and ¯uoroacetate function as methanogenic inhibitors. However, they also demonstrate that
the selectivity of these inhibitors is di€erent and that
anaerobic processes other than methanogenesis are
also inhibited to di€erent extents. The data are consist-

Fig. 8. Conversion of [2-14 C]acetate to (A) 14 CH4 and (B) 14 CO2 , and change of (A) the acetate concentrations and (B) the H2 partial pressure in
the presence of di€erent inhibitors using rice ®eld soil incubated under anoxic conditions for 30 days; mean2SD of duplicates.

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

ent with the following speci®city of the di€erent inhibitors:
Fluoroacetate generally inhibited acetate consumption as seen from the fact that the turnover of [214
C]acetate was completely inhibited at day 7 (Fig. 7)
as well as at day 30 (Fig. 8A, B) and that acetate accumulated right from the beginning of anoxic incubation
(Fig. 5). Since only acetate but not H2 accumulated
upon addition of ¯uoroacetate (Fig. 8D), H2 consumption by methanogens apparently was not a€ected.
BES generally and completely inhibited the production of CH4 (Fig. 6). BES resulted in the accumulation of both acetate and H2 which could no longer
be consumed by the methanogens (Fig. 8C, D). Conversion of [2-14 C]acetate to 14 CH4 was completely
inhibited by BES both in 7-day and 30-day old soil
(Figs. 7A and 8A). Reduction of nitrate and Fe(III)
was not inhibited by BES, and reduction of sulfate
was only marginally inhibited (Figs. 3 and 4). In longterm experiments (Fig. 5), BES resulted in accumulation of acetate as soon as Fe(III) and sulfate were
reduced and thus could no longer serve as electron
acceptors. After 10 d, however, the rate of acetate accumulation decreased. Possibly, acetate producing processes were partially inhibited by BES.
CHCl3 also inhibited the production of CH4 from
both H2/CO2 and acetate (Figs. 6±8). In addition,
however, sulfate reduction was also inhibited, at least
partially (Fig. 4). On the other hand, reduction of
nitrate and Fe(III) was not a€ected by CHCl3 (Figs. 2
and 3). It should be noted that inhibition was achieved
by application of low concentrations (100 mM) of
CHCl3 which should not cause changes in the soil microbial community. This is in contrast to the so-called
chloroform fumigation which applies high CHCl3 concentrations (CHCl3 vapor) to kill a large part of the
soil microbial populations for subsequent extraction
and determination of the microbial biomass carbon
(Shibahara and Inubushi, 1995). Soil fumigation with
chloroform preferentially kills Gram-negative bacteria
and thus may result in a shift towards a microbial
community dominated by Gram-positive bacteria
(Zelles et al., 1997).
If the inhibitors indeed were as speci®c as outlined
above, the patterns of changing metabolites observed
in the experiments with the di€erent inhibitors might
be used to interpret organic matter degradation under
anoxic conditions with respect to the contribution of
di€erent microbial groups.
4.2. Reduction of nitrate and iron
Since nitrate reducers and iron reducers were active
right from the beginning of incubation, but were not
inhibited by ¯uoroacetate (Figs. 2 and 3), they all
must have been able to use other electron donors than

985

acetate, e.g. H2 which was available at relatively high
partial pressure (Fig. 1) or organic compounds such as
glucose (Chidthaisong et al., 1999) or propionate. The
rate of acetate accumulation in the presence of ¯uoroacetate (about 740 nmol gÿ1 dÿ1) was too low to stoichiometrically (8 Fe/acetate) account for the rate of
Fe(II) formation (about 22,000 nmol gÿ1 dÿ1). Propionate concentrations were low during the phase of
nitrate and iron reduction (Fig. 5). Propionate started
to accumulate at day 5±10 when Fe(III) was largely
depleted. Active nitrate and iron reduction either consumed propionate or prevented a substantial production of propionate by fermenting bacteria, as
nitrate reducers and iron reducers have probably an
energetic advantage against fermenting bacteria when
using common precursor substrates, such as sugars.
4.3. Reduction of sulfate
Sulfate reduction was inhibited by ¯uoroacetate for
15 days, then the inhibition was released and sulfate
was depleted until day 25 (Fig. 4). Apparently, sulfate
reducers were dependent on acetate metabolism until
day 25. The interpretation that acetate was important
for sulfate reduction is in agreement with the inhibition pattern of CHCl3. CHCl3 completely inhibited
sulfate reduction until day 10 and then allowed only a
rather small rate. However, the inhibition patterns by
¯uoroacetate and CHCl3 were complex. Thus, in 7-day
old soil [2-14 C]acetate was converted to 14 CO2 both in
the control and in soil treated with either BES or
CHCl3 (Fig. 7). However, this reaction was completely
inhibited by ¯uoroacetate. We have to assume that the
conversion of acetate in the presence of CHCl3 was
catalyzed by sulfate reducers, since the 7 days old soil
contained only sucient sulfate, but not Fe(III) and
nitrate. Therefore, only part of the acetate-dependent
sulfate reducers can have been inhibited by CHCl3,
presumably those using the acetyl-CoA pathway
(Scholten et al., 2000). Consequently, acetate in 7 days
old soil would have been degraded by a population of
sulfate reducers using the tricarboxylic acid cycle as
degradation pathway. This population would have
been resistant to CHCl3 but sensitive to ¯uoroacetate.
Although accumulation of acetate occurred at a similar
rate in the presence of either ¯uoroacetate or CHCl3
(Fig. 5A), this observation is not necessarily a contradiction to the radiotracer experiments (Fig. 7). The
two experiments were not strictly comparable, since
they were done on di€erent time scales, and tested for
the net e€ect on production minus consumption of
acetate or on only acetate consumption, respectively.
The question remains why ¯uoroacetate eventually
allowed rapid depletion of sulfate after day 15. Possibly, sulfate reducers switched from acetate to propionate utilization. In the presence of ¯uoroacetate,

986

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

propionate started to accumulate at day 10 when
Fe(III) was depleted and decreased during days 15 and
25 when sulfate reduction was active (Fig. 5). Thereafter, it continuously accumulated. A similar pattern
was observed with BES. In the presence of CHCl3, on
the other hand, sulfate was available until the end of
incubation and indeed, propionate only transiently
accumulated after Fe(III) was depleted and then was
consumed and stayed below the detection limit (Fig. 5).
The pattern of propionate accumulation both in the
absence and presence of inhibitors is explained when
assuming that sulfate reducers were involved in propionate degradation as long as sulfate was available.
We also have to assume that sulfate reducers consumed H2, since the H2-partial pressures were low as
long as sulfate was available (Figs. 1 and 4). This conclusion is in agreement with earlier observations which
have found H2-dependent sulfate reducers to be very
active (Achtnich et al., 1995a, 1995b). Sulfate reducers
may have started to consume H2 when Fe(III) was
depleted after day 5±10 and competition for H2 by
iron reducers was released. Iron reducers should outcompete sulfate reducers on H2 for thermodynamic
reasons (Lovley and Goodwin, 1988). Neither ¯uoroacetate nor CHCl3 should have inhibited H2-dependent
sulfate reduction. Indeed, there was a slight decrease
of sulfate after day 10 (Fig. 4) in the presence of either
inhibitor. In the presence of CHCl3, H2 concentrations
stayed at a low level until the end of incubation
(Fig. 1) which is explained by the extended activity of
sulfate reducers due to available sulfate (Fig. 4). However, the di€erent rates of sulfate reduction in the presence of ¯uoroacetate versus CHCl3 after day 15
remain unexplained. If the sulfate reducers would then
have consumed propionate or H2 as suggested above,
rates of sulfate reduction should be the same in the
presence of ¯uoroacetate or CHCl3.
We assume that sulfate reducers were also involved
in propionate degradation when sulfate was not available, then acting as fermenting bacteria that degrade
propionate in syntrophy with methanogens (Krylova et
al., 1997; Krylova and Conrad, 2000). It is known that
propionate is syntrophically degraded in association
with methanogens (Schink, 1997). Under this condition, methanogens play a crucial role in maintaining
a low partial pressure of H2, and thus allow exergonic
propionate degradation. When H2-dependent methanogenesis was inhibited by BES, accumulation of H2
probably inhibited propionate degradation and
resulted in its accumulation as observed in the present
study. Indeed, the ®nal H2 concentrations were higher
in the presence than in the absence of BES (Fig. 1).
However, H2 did not accumulate since the lack of propionate degradation abolished the further supply of
H2. Krylova et al. (1997) have shown that H2-dependent CH4 production is driven by propionate degra-

dation. With CHCl3, propionate did not accumulate
since sulfate was still present and thus allowed the oxidation of propionate by sulfate reduction. With ¯uoroacetate, propionate did accumulate, since H2dependent methanogenesis had not yet started and H2dependent sulfate reduction was not possible either,
since sulfate was already depleted. Hence, propionate
consumption was not possible.
Our results are consistent with recent experiments
(Chidthaisong and Conrad, 2000) showing that sulfate
reducers are involved in the acetate turnover in anoxic
rice ®eld soil. By contrast, earlier observations found
that acetate-dependent sulfate reduction was not important and that spores of acetotrophic sulfate reducers were only present in very low numbers and
germinated only at a later time (Achtnich et al., 1995b;
Wind and Conrad, 1995). The discrepancy between
these results may be due to the usage of di€erent
batches of soil which allowed the establishment of
di€erent communities of sulfate reducers. Obviously,
more research is required to elucidate the role of sulfate reduction in the degradation of organic matter in
anoxic rice ®eld soil.
4.4. Methanogenesis
Consistent with earlier results (Krylova et al., 1997;
Yao and Conrad, 1999) CH4 started to accumulate as
soon as Fe(III) and sulfate were depleted. BES and
CHCl3 inhibited CH4 production completely, but in
the presence of ¯uoroacetate CH4 started to accumulate after day 25 when sulfate was depleted and H2
became available. Apparently, ¯uoroacetate only
inhibited acetoclastic but not H2-dependent methanogenesis. The rate of acetate turnover explained about
56±82% of the rate of CH4 production in the uninhibited control being in agreement with earlier observations (SchuÈtz et al., 1989; Rothfuss and Conrad,
1993).
Using 30 days old methanogenic soil, CHCl3
resulted in a much higher accumulation of H2 than
BES (Fig. 8D). We assume that some H2 consumption
was due to homoacetogenic bacteria which were only
inhibited by CHCl3 but not by BES. CHCl3 inhibited
homoacetogenic bacteria utilizing H2 or methanol in
pure culture (Scholten et al., 2000).

5. Conclusion
The results of our study in rice ®eld soil are in good
agreement with the previous studies (Scholten et al.,
2000; DeGraaf et al., 1996) that CHCl3 inhibits acetate
consumption especially by sulfate reducers and methanogens. By using di€erent inhibitors we are able to
demonstrate that sulfate reduction plays a special role

A. Chidthaisong, R. Conrad / Soil Biology & Biochemistry 32 (2000) 977±988

in propionate and acetate metabolism in rice ®eld soil.
Furthermore, it seems that the sulfate reducers are the
most sensitive population responding to the addition
of inhibitors such as CHCl3 and ¯uoroacetate. As a
result, when CHCl3 is used to study microbial processes, such as methanogenesis in environmental
samples, special care should be taken to the side e€ects
of the inhibitors used. Our experiments showed the
combined application of ¯uoroacetate, CHCl3 and
BES, which have a di€erent speci®city, may help to
elucidate the processes involved in electron and carbon
¯ow during the degradation of organic matter in
anoxic environments.

Acknowledgements
We thank S. Ratering for advice and help during
the determination of iron and J.C.M. Scholten for critically reading the manuscript. Amnat Chidthaisong
was supported by a fellowship of the Alexander-vonHumboldt foundation. We also thank the Fonds der
Chemischen Industrie for ®nancial support.

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