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 eects on other microbial processes have not received sucient attention. Therefore, we comparatively determined the eects of CHCl3, BES and ¯uoroacetate on dierent 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 [2-14C]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 dierent 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 dierent speci®city was helpful for elucidating the ¯ow of carbon and electrons during degradation of organic matter in anoxic rice ®eld soil.72000 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 mi-crobial processes in the environment (Oremland and Capone, 1988). Many chemical substances have been applied to study the relative importance of methano-genic and other processes for carbon mineralization both in bacterial cultures and in the environment. For example, 2-bromoethane sulfonate (BES,
BrCH2CH2SO3ÿ is often used to speci®cally inhibit
CH4 formation by methanogenic archaea (Oremland
and Polcin, 1982; Zinder et al., 1984; Alperin and Ree-burgh, 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). CHCl3is known to block
the function of corrinoid enzymes and to inhibit
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* Corresponding author. Tel.: 6421-178-801/800; fax: +49-6421-178-809.
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 metha-nogenesis, 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 ¯uoroace-tate (LeVine et al., 1980) indicating that ¯uoroace¯uoroace-tate is activated similarly as acetate before it exerts its adverse eect. Despite the unknown inhibition mech-anism ¯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 (Mount-fort et al., 1980; Alperin and Reeburgh, 1985; DeGraaf et al., 1996; Schulz and Conrad, 1996).
Recent investigation has shown that CHCl3can
inhi-bit 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 acetyl-CoA pathway, and other acetate-utilizing sulfate redu-cers are present in rice ®eld soil (Wind and Conrad, 1995; Wind et al., 1999). Therefore, we hypothesized that CHCl3 addition might have a dierent eect 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 eect of the typical methanogenic inhibitors BES, CHCl3and ¯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 [2-14C]acetate.
2. Materials and methods
2.1. Soil sample and slurry incubation
Rice ®eld soil was taken in 1993 from the exper-imental ®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 (<0.5 mm). Soil slurry was prepared in a 120 ml serum
bot-tle by adding 28 ml of distilled sterilized water to 28 g of air-dried soil giving a ®nal volume of the soil slurry of 32.7 ml. The bottles were closed with black rubber stoppers and crimped with aluminum caps. The gas-eous head space was exchanged with N2 and the
bot-tles were incubated at 308C without shaking to avoid destruction of microbial consortia (Dannenberg et al., 1997). All incubations were performed in duplicate.
2.2. Inhibition and tracer experiments
Inhibitors were added at dierent times during the course of the experiment. The ®nal concentrations of inhibitors were: 100 mM CHCl3 (99±99.4% purity,
Merck KGaA, Germany), 10 mM BES (sodium salt, 97% purity, Fluka AG, Germany) and 1 mM ¯uoroa-cetate (sodium salt, 95% purity, Fluka AG). These concentrations have been shown to completely inhibit methanogenesis or acetate metabolism in both pure culture of microorganisms and in environmental samples (Oremland and Capone, 1988; Schulz and Conrad, 1996; DeGraaf et al., 1996). All inhibitors were dissolved in sterilized water, injected into the bot-tles and mixed into the soil slurry by vigorous shaking. In one experiment, the inhibitors were added at the beginning of soil incubation, soon after the head space was exchanged with N2. At given time intervals, gas
samples (1 ml) were taken from the head space after vigorously shaking the bottles by hand and then ana-lyzed for CH4, CO2 and H2. Liquid samples (1 ml)
were taken and centrifuged at 14,000 g for 5 min. The supernatant was membrane-®ltered (0.2 mm; poly-tetra¯uoroethylene; Sartorius, GoÈttingen, Germany) and stored frozen (ÿ208C) until analysis of dissolved compounds. Liquid samples (0.5 ml) were also extracted with 4.5 ml 1 N HCl for analysis of ferrous iron.
In the other experiments, the inhibitors were added at day 7 of soil incubation when methanogenesis became active and at day 30 when methanogenesis was at steady state. Eight hours after addition of the in-hibitors, the soil slurries were used to measure the con-version of [2-14C]acetate to14CO
2 and14CH4 by adding
ca. 2 mCi of [2-14C]acetate (purity of >99% and the
speci®c activity of 57 mCi mmolÿ1, American Radio-labeled Chemical).
Soil incubation was terminated by adding 1 ml 7 N H2SO4. The increase in the concentration of CO2 and
14CO
2 upon acidi®cation was used to correct for
dis-solved radioactive and non-radioactive CO2by
assum-ing that the CO2in the gas and aqueous phases was in
equilibrium throughout the experiment. Typically, slurry acidi®cation resulted in the release of 14CO2
is assumed to represent total 14CO2 (CO2+
bicarbon-ate + carbonbicarbon-ate).
Since acetate uptake at day 7 was very slow, we determined the acetate transformation rate constant, i.e. the formation of 14CO2 plus 14CH4, 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 semiloga-rithmic plot of residual [2-14C]acetate against the
incu-bation time (Phelps and Zeikus, 1984). The actual turnover rate was obtained by multiplying the trans-formation rate constant or uptake rate constant with the pool size (pore water pool) of acetate. The respirat-ory index (RI) was used to compare the carbon ¯ow toward CH4 and CO2; RI f 14CO2= 14CO214CH4g:
The rate of CH4 production from acetate was
calcu-lated from: acetate turnover rate(1ÿRI), assuming that CH4is produced only from acetate and CO2/H2.
2.3. Analytical techniques
CH4, CO2, and H2concentrations were measured by
the gas chromatography (Conrad et al., 1989). Radio-activity of gaseous products was measured by gas pro-portional counting (Conrad et al., 1989). Nitrate, nitrite, and sulfate ions were determined by HPLC
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 scin-tillation 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, H2concentrations 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 ¯uoroace-tate, 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 ¯ood-ing, thus the examination of the eect of inhibitors on its reduction was performed on the ®rst day of soil in-cubation (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 pro-duction (Fig. 2B). However, the other inhibitors showed no signi®cant eect on nitrate decrease and nitrite production. The rates of nitrate reduction calcu-lated from the decrease of nitrate during the time of 0±8 h are given in Table 1. None of the inhibitors sig-ni®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 eect on this process. The rates of Fe(II) production calculated between 0 and 10 days of incubation ranged between 15 and 22mmol gÿ1dÿ1(Table 1).
In contrast to nitrate and Fe(III) reduction, sulfate
reduction was signi®cantly aected 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 in-itiation 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 pre-sence 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, CHCl3had a pronounced inhibitory eect
on sulfate reduction. In the bottles with ¯uoroacetate, on the other hand, sulfate reduction was only transi-ently 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 concen-trations (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 CHCl3than in the
con-trol, but were not signi®cantly dierent 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 concen-tration subsequently decreased and stayed around 100
mM thereafter. In the presence of inhibitors, acetate accumulated continuously but to dierent extent. With BES, a lag phase of about 5 days, similarly as in the
Fig. 2. Reduction of (A) nitrate and production of (B) nitrite in rice ®eld soil incubated under anoxic conditions in the presence of dierent in-hibitors; mean2SD of duplicates.
Table 1
Eect 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 gÿ1dayÿ1) Ferric iron reduction (mmol gÿ1dayÿ1) Sulfate reduction (nmol gÿ1dayÿ1)
Control 2.820.6 20.423.5 163.3220.5
BES 3.120.5 17.123.1 188.4251.3
Chloroform 2.821.0 14.923.9 45.424.3a
Fluoroacetate 2.420.9 21.921.7 126.0236.6
a
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 ac-cumulation started somewhat earlier and acetate
accu-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 ac-cumulation (between days 10 and 32) in the presence of BES, CHCl3 and ¯uoroacetate were about 400, 740
and 740 nmol gÿ1dÿ1, respectively.
Propionate accumulation was also found in all treat-ments. In the control, transient accumulation of pro-pionate reached its maximum at about day 12, slightly delayed compared to acetate accumulation (Fig. 5B), and disappeared rapidly thereafter. With BES, propio-nate transiently accumulated during day 7±12, became undetectable shortly afterwards, but then started to ac-cumulate continuously beginning day 15 until the end of incubation, when >200 mM of propionate had accumulated. With CHCl3, propionate reached a
tran-sient maximum between days 5 and 12 and then decreased below the detection limit similarly as in the control. With ¯uoroacetate, propionate showed a simi-lar pattern as with BES, but with the transient maxi-mum 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 CH4and 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 gÿ1dÿ1. Production of CH4 was completely inhibited by
ad-dition of BES or CHCl3. In the presence of
¯uoroace-tate, CH4 production was also inhibited but resumed
after day 25 (Fig. 6A). Thus, inhibition of methano-genesis by ¯uoroacetate was not complete. There was no signi®cant dierence 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 eect.
3.5. Turnover of [2-14C]acetate
Turnover of [2-14C]acetate was measured after 7 and
30 days of soil incubation by measuring the formation of 14CH4 and 14CO2: 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-Fig. 3. Production of ferrous iron in rice ®eld soil incubated under anoxic conditions in the presence of dierent inhibitors.
vious study have shown that during the phase of re-duction 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 [2-14C]acetate at day 7
was slow in all treatments. The uptake rate constant of
acetate in control, BES, CHCl3were 0.028, 0.017 and
0.012 hÿ1, respectively (Table 2), equivalent to turn-over times of 40, 59 and 80 h. With ¯uoroacetate
[2-14C]acetate uptake was not detectable (Table 2). BES
and CHCl3inhibited acetate uptake by 40% and 60%,
respectively.
Fig. 5. Change of the concentrations of (A) acetate and (B) propionate in rice ®eld soil incubated under anoxic conditions in the presence of dierent inhibitors; mean2SD of duplicates.
All of the inhibitors applied completely inhibited
14CH
4 formation from [2-14C]acetate (Fig. 7A). In the
control, production of 14CH
4 was observed about 1 d
after addition of [2-14C]acetate. However, small
amounts of14CO2 were produced earlier, ca. 2 h after
addition of [2-14C]acetate. The ®nal RI of the control
was 0.6. Addition of BES and CHCl3 did not inhibit
14CO
2 production in day-7 soil. Fluoroacetate
comple-tely inhibited both 14CH4 and 14CO2 production from
[2-14C]acetate.
In contrast to the results obtained with day-7 soil, [2-14C]acetate was rapidly converted to 14CH
4 in
day-30 soil. In the control, it was exclusively converted to CH4(RI = 0.08) (Fig. 8A). Production of some14CO2
was only observed after 1 d (Fig. 8B). Addition of BES, CHCl3 or ¯uoroacetate completely inhibited
14CH
4 production. The turnover rate constants of
acet-ate in dierent 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 inhib-ited to a similar extent.
In the control, the acetate pool stayed constant at 68
210.3 nmol gÿ1(Fig. 8C). The turnover rate of acet-ate in the control was calculacet-ated to 365 nmol gÿ1
Table 2
Uptake of [2-14C]acetate and its recovery as14CH4and 14CO2 at dierent days of soil incubation. Uptake of acetate at day 7 was determined from the accumulation of14CO2between 100 and 7200 min after its addition. At day 30, uptake of acetate was estimated from the decrease in [2-14C]acetate. Recovery of14CH4and14CO2is given as the maximum value found during the time course of the experiment (7200 min)
Treatments Uptake rate constant (hÿ1) Maximum recovery (%) as Total radioactive recovery (% of initially added acetate)
CO2 CH4
Addition at day 7
Control 0.028 0.03 0.01 78
BES 0.017 0.06 0 86
Chloroform 0.012 0.08 0 65
Fluoroacetate 0 0 0 99
Addition at day 30
Control 0.23320.006 8.4 90.4 99
BES 0.00420.001 14.3 0 104
Chloroform 0.00720.002 7.5 0 100
Fluoroacetate 0.00720.002 3.4 0 99
Fig. 7. Conversion of [2-14C]acetate to (A)14CH
4and (B)14CO2in the presence of dierent inhibitors using rice ®eld soil incubated under anoxic
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 aect the
H2concentration (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
methano-genic inhibitors. However, they also demonstrate that the selectivity of these inhibitors is dierent and that anaerobic processes other than methanogenesis are also inhibited to dierent extents. The data are
consist-Fig. 8. Conversion of [2-14C]acetate to (A)14CH
4and (B)14CO2, and change of (A) the acetate concentrations and (B) the H2partial pressure in
ent with the following speci®city of the dierent inhibi-tors:
Fluoroacetate generally inhibited acetate consump-tion as seen from the fact that the turnover of
[2-14C]acetate was completely inhibited at day 7 (Fig. 7)
as well as at day 30 (Fig. 8A, B) and that acetate accu-mulated right from the beginning of anoxic incubation (Fig. 5). Since only acetate but not H2 accumulated
upon addition of ¯uoroacetate (Fig. 8D), H2
consump-tion by methanogens apparently was not aected. BES generally and completely inhibited the pro-duction of CH4 (Fig. 6). BES resulted in the
accumu-lation of both acetate and H2 which could no longer
be consumed by the methanogens (Fig. 8C, D). Con-version of [2-14C]acetate to 14CH4 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 long-term experiments (Fig. 5), BES resulted in accumu-lation 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 ac-cumulation decreased. Possibly, acetate producing pro-cesses 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 aected by CHCl3(Figs. 2
and 3). It should be noted that inhibition was achieved by application of low concentrations (100 mM) of CHCl3which should not cause changes in the soil
mi-crobial community. This is in contrast to the so-called chloroform fumigation which applies high CHCl3
con-centrations (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 dierent inhibitors might be used to interpret organic matter degradation under anoxic conditions with respect to the contribution of dierent 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
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 ¯uoroa-cetate (about 740 nmol gÿ1 dÿ1) was too low to stoi-chiometrically (8 Fe/acetate) account for the rate of Fe(II) formation (about 22,000 nmol gÿ1dÿ1). Propio-nate 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 con-sumed propionate or prevented a substantial pro-duction 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 inhi-bition 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 CHCl3were complex. Thus, in 7-day
old soil [2-14C]acetate was converted to 14CO
2 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 sucient 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 contra-diction to the radiotracer experiments (Fig. 7). The two experiments were not strictly comparable, since they were done on dierent time scales, and tested for the net eect on production minus consumption of acetate or on only acetate consumption, respectively.
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). There-after, 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 propio-nate degradation as long as sulfate was available.
We also have to assume that sulfate reducers con-sumed H2, since the H2-partial pressures were low as
long as sulfate was available (Figs. 1 and 4). This con-clusion 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 out-compete sulfate reducers on H2 for thermodynamic
reasons (Lovley and Goodwin, 1988). Neither ¯uoroa-cetate nor CHCl3should 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, H2concentrations
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). How-ever, the dierent rates of sulfate reduction in the pre-sence 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 avail-able, 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 con-dition, methanogens play a crucial role in maintaining a low partial pressure of H2, and thus allow exergonic
propionate degradation. When H2-dependent
methano-genesis 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 H2concentrations were higher
in the presence than in the absence of BES (Fig. 1). However, H2did not accumulate since the lack of
pro-pionate degradation abolished the further supply of H2. Krylova et al. (1997) have shown that H2
-depen-dent CH4 production is driven by propionate
degra-dation. With CHCl3, propionate did not accumulate
since sulfate was still present and thus allowed the oxi-dation of propionate by sulfate reduction. With ¯uor-oacetate, propionate did accumulate, since H2
-dependent methanogenesis had not yet started and H2
-dependent 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 im-portant and that spores of acetotrophic sulfate redu-cers 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 dierent batches of soil which allowed the establishment of dierent communities of sulfate reducers. Obviously, more research is required to elucidate the role of sul-fate 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
accumu-late after day 25 when sulfate was depleted and H2
became available. Apparently, ¯uoroacetate only inhibited acetoclastic but not H2-dependent
methano-genesis. The rate of acetate turnover explained about 56±82% of the rate of CH4production in the
uninhib-ited control being in agreement with earlier obser-vations (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 H2consumption
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 CHCl3inhibits acetate
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
pro-cesses, such as methanogenesis in environmental samples, special care should be taken to the side eects of the inhibitors used. Our experiments showed the combined application of ¯uoroacetate, CHCl3 and
BES, which have a dierent 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 cri-tically reading the manuscript. Amnat Chidthaisong was supported by a fellowship of the Alexander-von-Humboldt foundation. We also thank the Fonds der Chemischen Industrie for ®nancial support.
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