Soil Biology & Biochemistry 32 (2000) 1581±1590

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Evaluation of potential inhibitors of methanogenesis and methane

oxidation in a land®ll cover soil
A.S.K. Chan a, T.B. Parkin b,*
a
Department of Microbiology, Iowa State University, Ames, IA 50011, USA
b
USDA, Agricultural Research Service, National Soil Tilth Laboratory, 2150 Pammel Drive, Ames, IA 50011-4420, USA
Accepted 1 March 2000

Abstract

Biological methane (CH4) production is an anaerobic process, while CH4 consumption occurs predominantly under aerobic
conditions; however, both processes can occur simultaneously in soil. Thus, ®eld measurements of CH4 ¯ux re¯ect the net result
of both consumption and production reactions. Speci®c inhibitors of either CH4 consumption or production processes o€er the
opportunity for assessing the rates of these two processes independently. The objective of this work was to identify potential
gaseous inhibitors of either CH4 oxidation or methanogenesis, and to evaluate the e€ect of inhibitor concentration on these two
processes. For these studies, sieved cover soil from a municipal land®ll was treated with a variety of compounds including
acetylene (C2H2), ethylene (C2H4), ethane (C2H6), methyl chloride (CH3Cl) and methyl ¯uoride (CH3F). Each experiment
consisted of six di€erent treatments which included sterile soil, untreated soil and soil with test compound concentrations of
0.00%, 0.01%, 0.1% and 1%. Incubations were conducted under both aerobic and anaerobic conditions. Several compounds
completely inhibited CH4 oxidation while not signi®cantly in¯uencing CH4 production; including C2H2 at 0.001%, C2H4 at
0.1%, and CH3F at 0.1%. One compound (CH3Cl) was unique; in that a concentration of 0.1% inhibited methanogenesis by
88.9% but CH4 oxidation was not signi®cantly a€ected. We recommend the use of C2H2 or C2H4 for inhibition of CH4
oxidation, and CH3Cl for inhibition of methanogenesis. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Methanogenesis; Methane oxidation; Methanotrophy; Inhibitors; Land®ll

1. Introduction soils, when conditions are predominately aerobic

Recent concern about global warming has intensi®ed
interest in the role of terrestrial systems in controlling
atmospheric CH4 levels. Terrestrial systems can func-
tion as net sources or sinks for atmospheric CH4.
Methane ¯ux measured at the soil/atmosphere inter-
face is the net e€ect of two processes Ð CH4 oxidation
and methanogenesis (Knowles, 1993). A negative CH4
¯ux, i.e., consumption of CH4 by soil, occurs when the
magnitude of the CH4 uptake process is larger than
methanogenesis. This is commonly observed in arable
* Corresponding author. Tel.: +1-515-294-6888; fax: +1-515-264-
8125.
E-mail address: parkin@nstl.gov (T.B. Parkin).
(Schutz et al., 1990; Mosier et al., 1991; Bronson and
Mosier, 1993; Hansen et al., 1993). On the other hand
a positive CH4 ¯ux indicates net CH4 production, and
is observed when the magnitude of the methanogenic
process is larger than CH4 uptake. This is the case in
rice paddies and wetlands (¯ooded or water saturated
areas) which are predominately anaerobic (Schutz et
al., 1990; Lauren and Duxbury, 1993). Both soil and
wetland systems often support a mixture of anaerobic
and aerobic sites, and it is under these conditions that
the use of inhibitors are of value in distinguishing CH4
consumption and production activities.
Inhibitors have been applied to a variety of ecosys-
tems to quantify the role of CH4 oxidizers in mitigat-
ing atmospheric CH4 increases. Boeckx and Van
Cleemput (1997), using CH3F in ®eld chambers, deter-

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 1 - 7
1582 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590

mined that CH4 oxidation was responsible for con- been performed with land®ll cover soil. Land®lls rep-
sumption of 34±67% of the CH4 produced in a wet- resent major contributors to atmospheric CH4 (Topp
land system. Using CH3F in investigations of CH4 and Pattey, 1977) and the cover soils of land®lls have
cycling in the rhizosphere of Pontederia cordata, Sagit- been reported to have high CH4 oxidation activity
taria lancifolia and Typha latifolia, Lombardi et al. (Jones and Nedwell, 1993; Bogner, 1997; Borjesson
(1997) determined that CH4 oxidation consumed and Svensson, 1997). Keeping in view the importance
22.9% of the CH4 produced in ®eld experiments and of land®lls to the global CH4 budget, along with the
64.9% of the CH4 produced in greenhouse exper- lack of information about inhibitor ecacy for land®ll
iments. King (1996) used C2H2 as an inhibitor of CH4 cover soils, our investigations were designed to evalu-
oxidation and determined that 1±58% of the potential ate ®ve gaseous compounds over a wide concentration
CH4 produced was consumed by CH4 oxidation as- range for their inhibitory e€ects on methanogenesis
sociated with burweed (Sparganium eurycarpum ) roots. and CH4 oxidation associated with a land®ll cover
Data from Oremland and Culbertson (1992a) show soil. This study includes compounds which have been
that CH4 oxidation in a seasonally exposed sandbar previously investigated (C2H2, C2H4, C2H6 and
accounted for 75±96% of the total CH4 production. CH3F), as well as an evaluation of the inhibitory
By utilizing the CH3F technique on plants (S. lancifo- e€ects of methyl chloride (CH3Cl) and low concen-
lia ), Schipper and Reddy (1996) determined that 65% trations of C2H4 and C2H6, which have not been pre-
of the total CH4 produced was oxidized. viously determined.
Several gaseous compounds have been examined as
potential inhibitors of CH4 oxidation and methanogen-
esis. Ethylene was observed to inhibit methanogenesis 2. Materials and methods
in marine sediments at concentrations of 20% and
5%, but not at 1.25%; however, C2H6 had no e€ect 2.1. Soil properties
on CH4 production (Oremland and Taylor, 1975). The
e€ects of C2H4 or C2H6 on CH4 oxidation have not The soil used in our experiments was topsoil col-
been evaluated. Historically, C2H2 has been used as an lected from a capped city land®ll located on Dayton
inhibitor of both CH4 oxidation and methanogenesis, Road, Ames, Iowa. Initial testing indicated that this
depending upon the concentration. Inhibition of soil had high CH4 oxidizing activity under aerobic
methanogenesis has been reported at C2H2 concen- conditions and high methanogenic activity under an-
trations of 0.5% (King, 1996) and at 1.25% (Oremland aerobic conditions.
and Taylor, 1975), while CH4 oxidation is much more Nitrate (+nitrite) (NOÿ ÿ
3 , NO2 ) and ammonium
+
sensitive to C2H2 and inhibition has been reported to (NH4 ) were determined by colorimetric analyses of 2
occur at a C2H2 concentration as low as 0.003% Molar KCl soil extracts (KCl:soil, 4:1) on a Lachat
(Watanabe et al., 1995). autoanalyzer (Lachat Instruments, Mequon, WI) fol-
Methyl ¯uoride has a similar di€erential e€ect on lowing the procedure described by Keeney and Nelson
CH4 oxidation and production. Oremland and Cul- (1982). The pH was determined on 1:1 (dH2O:soil)
bertson (1992b) recommended that in soil, a CH3F extracts with a standard glass electrode as described in
concentration of 0.4% would selectively block CH4 the study by McLean (1982). Soil water content was
oxidation without a€ecting methanogenesis. Schipper determined gravimetrically after overnight drying at
and Reddy (1996) found no signi®cant e€ect of 1% 1058C (Gardner, 1982). Microbial biomass carbon
CH3F on methanogenesis, but CH4 oxidation was measurements were determined by fumigation-extrac-
completely inhibited in soil slurries. Also, Epp and
Chanton (1993) observed that while CH4 oxidation in
the rhizosphere of aquatic macrophytes was inhibited Table 1
by 1.5% of CH3F, methanogenesis was not signi®- Properties of land®ll soil used in inhibitor evaluation studiesa
cantly a€ected. However, there have been reports of
methanogenesis inhibition at lower CH3F concen- pH 6.71 (0.79)
trations. Frenzel and Bosse (1996) observed that CH4 NOÿ 3 (mg N g
ÿ1
dry soil) 3.89 (1.34)
NH+ 4 (mg N g
ÿ1
dry soil) 0
production in soil slurries was reduced by 75% in the Moisture (%) 54.32 (0.82)
presence of 0.1% CH3F and 90% inhibition of metha- Microbial biomass (mg C gÿ1 dry soil) 595.8 (12.63)
nogenesis occurred at 1% CH3F. Also, >99% inhi- Soluble C (mg C gÿ1 dry soil) 31.12 (7.09)
bition of methanogenesis has been observed at CH3F Total N (%) 0.15 (3.94)
concentrations as low as 0.01% in anaerobic sediments Total C (%) 1.92 (1.04)
Soil texture 57% sand 25% silt 18% clay
(King, 1996).
Whereas inhibitors have been evaluated in materials a
Values in brackets are percentage coecients of variation of
collected from a wide variety of habitats, none have three measurements.
 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590
tion (Rice et al., 1996). Fifty grams of 5 mm sieved
soil was extracted with 100 ml of 0.5 Molar potassium
sulfate (K2SO4). Soluble organic carbon was measured
on a Dohrmann DC-180 carbon analyzer (Rosemount
Analytical Services, Santa Clara, CA). Biomass carbon
was calculated using the correction factor (K = 0.33)
of Sparling and West (1988). Total N and C were
determined by combustion on a Carlo Erba Carbon/
Nitrogen Analyzer (Fisons Instruments, Rodano,
Milano, Italy). Soil texture analyses (sand, silt and
clay) were performed by Midwest Laboratories
(Omaha, NE). General properties of this soil are listed
in Table 1.
2.2. Laboratory experiment
For these tests 20 g portions of sieved (0.5 cm mesh)
soil were incubated in 250 ml erlenmeyer ¯asks. Flasks
were sealed with a rubber stopper in which a glass
tube accommodating a butyl rubber septum (20 mm)
Fig. 1. E€ect of headspace methyl ¯uoride concentration on methane
consumption (A) and methane production (B). Each line and symbol
type represent di€erent methyl ¯uoride concentrations from 0% to
10%.
1583
(Bellco Glass, Vineland, NJ, USA) was mounted.
Compounds were tested at concentrations of 0%,
0.001%, 0.01%, 0.1%, 1%, and in some cases 10% (v/
v). Autoclaved soil was included as a sterile control.
Each treatment was replicated three times. The e€ects
of these compounds on CH4 consumption and CH4
production were evaluated in two di€erent incubation
regimes. Aerobic conditions (headspace of approxi-
mately 20% O2) with added CH4 (initial headspace
concentration of 1±3%) were used to evaluate com-
pound in¯uences on CH4 oxidation, while anaerobic
conditions (He atmosphere) with a 10% H2 and 10%
CO2 headspace were established to evaluate compound
e€ects on methanogenesis. Incubations were performed
in the dark at room temperature (218C) and spanned
up to 30 h for the evaluation of CH4 consumption and
up to 7 days for the evaluation of CH4 production.
2.3. Gas chromatography
Methane and the initial test compound concen-
trations were measured by injecting 0.2 ml of gas
sample obtained from the headspace of each ¯ask into
a Tracor 540 gas chromatograph (Tracor Instruments
Austin, Austin, TX) equipped with a ¯ame ionization
detector running at 2008C, a Porapak Q column (All-
tech Associates, Deer®eld, IL, USA) and He carrier
gas ¯owing at the rate of 30 ml minÿ1. Certi®ed stan-
dards of CH4, C2H2, C2H4, and C2H6 were obtained
from Scott Speciality Gases, Troy, MI, USA. Methyl
chloride and CH3F were obtained from Matheson Gas
Products, Montgomeryville, PA.
2.4. Rate determinations
An example of the response of these processes to
increasing concentrations of CH3F is shown in Fig. 1.
Methane consumption appeared to be a ®rst order
process (Fig. 1(A)), but increased concentrations of
CH3F reduced CH4 consumption. Rate coecients for
CH4 consumption were estimated by applying linear
regression to the natural log of the concentration ver-
sus time data (slope = ÿk, Paul and Clark, 1996).
Methane production rate increased with time but was
not well described by ®rst order kinetics (Fig. 1(B)).
The impact of inhibitors on this process was evaluated
by computing the maximum rate of CH4 production.
This analysis was done by ®tting the CH4 concen-
tration versus time data with a curve and evaluating
the ®rst derivative of the ®t equation using the Table
Curve software package (SPSS, Chicago, IL).
2.5. Statistical analysis
Results for each inhibitor tested are presented in
terms of a mean and a con®dence limit. Statistical
1584 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590

di€erences between treatments and the control were 3. Results

determined by Tukey's test (Sigma Stat software pack-
age, SPSS, Chicago, IL). E€ects within test concen-
trations can be determined by observation of the
overlap of 97.5% con®dence limits (Birnbaum, 1961;
Natrella, 1963; Parkin, 1993).
Methane oxidation was inhibited by all of the test
compounds, but inhibitor e€ectiveness di€ered (Fig. 2).
Acetylene had a strong inhibitory e€ect on CH4 oxi-
dation over the entire concentration range (Fig. 2(A)).

Fig. 2. E€ects of di€erent concentrations of inhibitors on methane oxidation ®rst order rate constant. Error bars are the 97.5% con®dence limits
of the mean of three replicates. In cases where only two replications are available, the distance between the error bars are actual ranges of the in-
dividual rate constants. Asterisks indicate that the treatment is statistically signi®cant (P < 0.05) from the control (no inhibitor). Ten percent con-
centrations of acetylene, ethylene and ethane were not tested.
 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590 1585

At C2H2 concentration of 0.001% the ®rst order rate
constant dropped 4.2  10ÿ2 minÿ1, corresponding to
an average of 93.5% inhibition and at higher C2H2
concentrations CH4 oxidation was 100% inhibited.
Methane oxidation was less sensitive to C2H4
(Fig. 2(B)). At 0.001% C2H4, CH4 oxidation was unaf-
fected, but partial inhibition (33%) was observed at
0.01% C2H4, with near complete inhibition (96%)
occurring at concentrations of 1%. Neither C2H6 nor
CH3Cl (Fig. 2(C) and (D)) were e€ective inhibitors of
CH4 oxidation at concentrations less than 0.1%; how-
ever, at a concentration of 1%, a 53% and 78% inhi-
bition was observed for C2H6 and CH3Cl, respectively.
Methyl chloride was completely inhibitory at a concen-
tration of 10%. Increased concentrations of CH3F
progressively inhibited CH4 oxidation (Fig. 2(E)), and

Fig. 3. E€ects of di€erent concentrations of inhibitors on methane production maximum rate. Error bars are the 97.5% con®dence limits of the
mean of three replicates. In cases where only two replications are available, the distance between the error bars are actual ranges of the individual
rates. Asterisks indicate that the treatment is statistically signi®cant …P < 0:05† from the control (no inhibitor). Ten percent concentrations of
acetylene, ethylene and ethane were not tested.
1586 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590

concentrations at and exceeding 0.1% complete inhi-
bition was observed.
The e€ects of the test compounds on methanogen-
esis are presented in Fig. 3. Acetylene only slightly
inhibited CH4 production at 0.001%, but with increas-
ing concentration greater inhibition was observed
(Fig. 3(A)). Ethylene was ine€ective at inhibiting CH4
production at low concentrations, but at 1% signi®-
cant inhibition was observed. In contrast, C2H6
showed essentially no e€ect on CH4 production ac-
tivity (Fig. 3(C)). Methyl chloride, on the other hand,
was completely inhibitory at concentrations of 1% and
10%, and partial inhibition (89%) was observed at
0.1% (Fig. 3(D)). Methyl ¯uoride exhibited slight inhi-
bition (39%) at 1% and inhibition was nearly com-
plete (93%) at 10% (Fig. 3(E)).

Fig. 4. E€ects of di€erent concentrations of inhibitors on methane oxidation and methane production activity. Solid bars indicate the mean
methane oxidation activity. Open bars indicate the mean methanogenic activity. Error bars are the 97.5% con®dence limits (n = 3). In cases
where only two replications were available, error bars indicate the range. Asterisks indicate that the treatment is statistically signi®cant (P <
0.05) from the control (no inhibitor). Ten percent concentrations of acetylene, ethylene and ethane were not tested.
 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590
A comparison of the e€ects of the inhibitors on
both processes is presented in Fig. 4, where CH4 con-
sumption and production activity are expressed as a
percentage of the respective rates with no inhibitor
added. Acetylene, at a concentration of 0.001% did
not signi®cantly reduce CH4 production activity, but
did inhibit CH4 oxidation (Fig. 4(A)). Methane oxi-
dation at 0.001% C2H2 was reduced to 6.6% of the
control. At higher C2H2 concentrations, both CH4 oxi-
dation and methanogenesis were signi®cantly lower
than the control (no inhibitor). There was no signi®-
cant e€ect of C2H4 on CH4 production at concen-
trations less than 1%, but 89% inhibition of
methanogenesis was observed at 1% C2H4 (Fig. 4(B)).
Methane oxidation was more sensitive to C2H4 than
methanogenesis and a C2H4 concentration of 0.1%
and greater resulted in 90% inhibition. Methanogen-
esis was una€ected by C2H6 over the range of concen-
trations tested (Fig. 4(C)). Methane oxidation was
only partially inhibited (35% and greater) by C2H6 at
higher concentrations (>0.1%).
The pattern of inhibition produced by CH3Cl was
unlike those of the other inhibitors tested, in that
methanogenesis was more sensitive than CH4 oxidation
(Fig. 1(D)). Methanogenesis was impacted by CH3Cl
at concentrations exceeding 0.01%, and at a CH3Cl
concentration of 0.1% methanogenesis was inhibited
by 89%. Methane oxidation was not signi®cantly
a€ected by CH3Cl less than 0.1%, but concentrations
of CH3Cl greater than 1% did inhibit CH4 oxidation.
Methanogenesis was una€ected by CH3F concen-
trations at and below 0.1% (Fig. 4(E)). Partial inhi-
bition of methanogenesis (38.8%) was observed at 1%
CH3F and 92.6% inhibition at 10% CH3F. Methane
oxidation was partially inhibited (39.5%) by CH3F at
0.01% and completely inhibited by CH3F concen-
trations >0.1%.
4. Discussion
Inhibitors present potentially powerful tools in the
study of microbial process; however, they must be
used judiciously due to potential nonspeci®c or unin-
tended e€ects. As pointed out by Oremland and
Capone (1988), `` . . .employing inhibitors in ecological/
biogeochemical studies must be tempered by a knowl-
edge of their limitations.'' This caution is underscored
by the variability in the literature concerning inhibitory
e€ects of C2H2 and CH3F on CH4 oxidation and pro-
duction in the wide range of systems they have been
applied.
Generally, there is consistency in reports regarding
the inhibitory concentrations of CH3F required to
inhibit CH4 oxidation. Data from Oremland and Cul-
bertson (1992b) indicate complete inhibition of CH4
1587
oxidation by soil at CH3F concentrations of 0.3% and
1.7% over a 150 h period. Decreasing CH3F concen-
trations which resulted in a decrease in the time inhi-
bition was sustained, with CH3F concentrations of
0.16 and 0.06% resulting in nearly complete inhibition
of CH4 oxidation for 100 and 30 h, respectively. In
short term incubations (130 h) of S. eurycarpum
roots, King (1996) observed nearly complete inhibition
of CH4 oxidation (86±96%) over a CH3F concen-
tration range of 0.01±1.0%. Our aerobic incubations
of land®ll topsoil yielded results similar to these past
studies. Over a 30 h incubation, we observed a 39%
inhibition of CH4 oxidation at a CH3F concentration
of 0.01%, and complete inhibition at 0.1% CH3F.
The e€ects of CH3F on methanogenesis are less con-
sistent. Oremland and Culbertson (1992a) reported a
36% inhibition of methanogenesis in anoxic salt marsh
sediments incubated with 1.25% CH3F, but observed
no inhibition of methanogenesis at 0.4% CH3F in an-
aerobic incubations of composted paper waste. Epp
and Chanton (1993) found that in the rhizosphere of
aquatic macrophytes, CH3F concentration of 1.5%
was sucient to inhibit CH4 oxidation, but did not
a€ect methanogenesis. However, King (1996) observed
that methanogenesis was a€ected by much lower
CH3F concentrations. He reported that methane pro-
duction in anaerobic incubations of peat material was
inhibited by 89% and 96% at CH3F concentrations of
0.01% and 0.1%, respectively and suggested that a
possible reason for the higher sensitivity of methano-
genesis to CH3F may be due to di€erences in sediment
sources (marine vs. freshwater). Recently, Frenzel and
Bosse (1996) observed that methanogenesis in di€erent
systems exhibited a di€erential sensitivity to CH3F. In
the rhizosphere of cat-tail (T. latifolia ), these workers
found that methanogenesis was una€ected by CH3F
up to concentrations of 1.0%. Methane production in
a hypersaline microbial mat was also found to be
insensitive to CH3F concentrations up to 4%, but in
anoxic rice ®eld soil low CH3F concentrations (0.1%)
inhibited methanogenesis by 75%. The di€erential
e€ects of CH3F on methanogenesis were thought to be
related to the characteristics of the methanogenic
populations, with acetoclastic methanogens showing a
greater sensitivity to CH3F than organisms using
methylamine or formate (Frenzel and Bosse, 1996).
Evidence supporting this e€ect is provided by Janssen
and Frenzel (1997) who observed that the growth of
acetoclastic methanogens was inhibited by lower CH3F
concentrations than growth of methanogens using H2
or formate. Our results are consistent with past obser-
vations in that, in anaerobic incubations of soil with
added H2 and CO2, methanogenesis was not impacted
by CH3F concentrations of <0.1%, but at high CH3F
levels (>1.0% ) CH4 production was inhibited.
Unlike CH3F, there is more agreement in the litera-
1588 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590
ture concerning the inhibitory e€ects of C2H2 on CH4
oxidation and production. Early work indicated that
C2H2 at high concentrations (>1.25%) strongly inhi-
bits methanogenesis (Oremland and Taylor, 1975;
Sprott et al., 1982). Recently, CH4 production has also
been reported to be sensitive to much lower C2H2 con-
centrations. King (1996) observed that 0.01% C2H2
inhibited methanogenesis associated with peat by
>60%. Also, data of Watanabe et al. (1997) indicate
that in a 7-day incubation of soil amended with glu-
cose, C2H2 at a concentration of 0.001% only had a
slight e€ect on methanogenesis, but that 0.01% C2H2
resulted in 60% inhibition. Our observations of 80%
inhibition of methanogenesis at a 0.01% C2H2, but no
signi®cant e€ect at 0.001% C2H2 are consistent with
these recent studies.
Acetylene also inhibits CH4 oxidation, but most past
studies only have examined C2H2 concentrations
exceeding 0.1% (Yavitt et al., 1990; Bender and Con-
rad, 1995; McDonald et al., 1996; Miller et al., 1998).
For C2H2 to be useful in distinguishing between gross
and net rates of CH4 production, it must inhibit CH4
oxidation at a concentration of <0.01% because
methanogenesis is also sensitive to 0.01% C2H2. King
(1996) reported an 89% inhibition of CH4 oxidation
associated with excised S. eurycarpum roots at 0.01%
C2H2. An evaluation of lower C2H2 concentrations on
CH4 oxidation have only been presented by Wanta-
nabe et al. (1995). In laboratory incubations of rice
®eld soils, these investigators observed 90% inhibition
of CH4 oxidation at a C2H2 concentration of 0.003%.
The e€ects of C2H4 on CH4 cycling reactions are
largely unknown. Oremland and Taylor (1975) found
nearly complete inhibition of methanogenesis at 5%
C2H4, but there have been no reports of C2H4 e€ects
on CH4 oxidation. Our results not only con®rm that
high C2H4 concentrations inhibit methanogenesis, but
also that CH4 oxidation is nearly completely inhibited
(90%) at C2H4 concentrations exceeding 0.1%. Despite
the fact that C2H4 is produced in soils by microorgan-
ism (Arshad and Frankenberger, 1989, 1990) it is
doubtful that this process signi®cantly impacts CH4
oxidation in nature. In anaerobic incubations of forest
soils Sexstone and Mains (1990) measured C2H4 pro-
duction rates of 205±71.8 nM C2H4±C kg soilÿ1 hÿ1.
Assuming a bulk density of 1 g cmÿ3 and an air ®lled
porosity of 50%, we calculated that these rates of pro-
duction would result in soil atmosphere C2H4 concen-
trations of only 0.0005% and 0.00017% over an 1 h
period (assuming no loss through di€usion). Based on
our results these concentrations are too low to impact
either CH4 consumption or production.
There is little information on the other compounds
we examined. Our observations that methanogenesis is
not signi®cantly a€ected by C2H6 at concentrations up
to 1% are similar to results of Oremland and Taylor
Table 2
Recommended compound concentrations to selectively inhibit either
methanogenesis or methane oxidation
Compound Concentration % % Average inhibition
(v/v)
CH4 production CH4 oxidation
Acetylene 0.001 8.8 93.5
Ethylene 0.1 17.7 90
Methyl chloride 0.1 88.9 11.2
Methyl ¯uoride 0.1 7.7 100
(1975), who reported no inhibitory e€ect of C2H6 on
CH4 production at concentrations of 1.25% and 20%
using marine sediments. Methyl chloride, as far as we
know, has never been used to study its e€ects on CH4
oxidation or methanogenesis; however, our results in-
dicate that CH3Cl is distinctly di€erent than the other
compounds of our study, in that methanogenesis was
more sensitive than CH4 oxidation. Methanogenesis
was signi®cantly inhibited (89%) by 0.1% CH3Cl, but
CH4 oxidation was una€ected by concentrations
<0.1%.
Conclusions of our study are summarized as rec-
ommendations of concentrations of compounds which
may be useful for distinguishing between CH4 oxi-
dation and CH4 production (Table 2). Acetylene at a
concentration of 0.001% inhibited CH4 oxidation by
93.5% but did not signi®cantly impact CH4 pro-
duction. Ethylene at a higher concentration (0.1%)
had a similar e€ect. Methyl chloride is unique, in that
at a concentration of 0.1% methanogenesis was inhib-
ited by 89%, but CH4 oxidation was not signi®cantly
a€ected. Finally, CH3F at a concentration of 0.1%
showed complete inhibition of CH4 oxidation but did
not signi®cantly in¯uence CH4 production. Of the
compounds we investigated, C2H2, C2H4 and CH3Cl
show the greatest application for use in CH4 cycling
studies. Despite its widespread use, CH3F should be
used with caution, because of the reported di€erential
response of methanogenic bacteria to CH3F (Frenzel
and Bosse, 1996; Janssen and Frenzel, 1997).
Finally, it is realized that extrapolation of our
results to other soils may be tenuous; however, for
many of the compounds tested, similar ecacious con-
centrations have been reported over a wide range of
habitats, including mineral and organic soils, sediments
and plant systems. Also, the land®ll soil used in our
study had the highest CH4 oxidation and production
activity of any soil we have examined, and we specu-
late that the inhibitor concentrations we report (with
the exception of CH3F) may be at least as e€ective in
other soils. However, a prudent course of action would
be to evaluate the precise inhibitor concentration
 A.S.K. Chan, T.B. Parkin / Soil Biology & Biochemistry 32 (2000) 1581±1590
required to achieve the desired degree of inhibition in
each speci®c system of interest.
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