FEMS Microbiology Ecology 101 (! 992) 26 ! -270
© 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
261
FEMSEC 00411
Kinetics of
CH 4
oxidation in oxic soils exposed to ambient air or
high CH 4 mixing ratios
Martin B e n d e r ~ and R a l f C o n r a d
b
a Fakultiit fiir Biologie, Unicersitiit Konstanz, Konstanz, FRG, and b May.~Tar.ck-lnstitut fur Terrestrische Mikrobiologie, Marburg,
FRG
Received 21 April 1992
Revision received 7 July 1992
Accepted l0 July 1992
Key words: Methane oxidation; Oxic soil; Vmax; Km; Threshold; Methanotrophic bacteria; Most probable
number
1. SUMMARY
The kinetic parameters of C H 4 oxidation ( K m,
Vma~, apparent threshold = The) were measured
using different oxic soils (cultivated cambisol, forest 1uvisol, meadow cambisol, paddy soil) both in
a fresh state and after 3 weeks preincubation
under high CH 4 mixing ratios (20%). The preincubation resulted in an increase of the most probable number of methanotrophic bacteria. In fresh
soils, CH 4 oxidation followed Michaelis-Menten
kinetics with a low K m (30-51 nM CH4), low
Vma~ (0.7-3.6 nmol CH4 h - l g - l d w soil), and low
Tha (0.2-2.7 ppmv CH4). In preincubated softs,
C H 4 oxidation exhibited biphasic kinetics in
which two different CH a saturation curves were
apparently superimposed on each other. Eadie-
Correspondence to: R. Conrad, Max-Planck-lnstitut fiir Terrestrische Mikrobiologie, Karl-von-Frisch-Stras~e, D-3550
Marburg, FRG.
Hofstee plots of the data showed two activities
with different kinetic parameters: a high-affinity
activity with low K m (13-470 nM CH4), low Vma~
(2.1-150.0 nmol CH 4 h - J g - t d w ) and low Th a
(0.3-4.1 ppmv CH 4) being similar to the kinetic
parameters in fresh soils; and a !ow-affinit~j activity with high K m (1740-27900 nM CH4), high
Vma~ (270--3690 nmol CH 4 h - l g - l d w ) and high
Th a (11-45 ppmv CH 4) being similar to the kinetic parameters known from methanotrophic
bacteria. The low-affinity activity was also observed in a soil over a deep natural gas source
which was permanently exposed to high CH 4
mixing ratios ( > 5% CH4). Bacteria cu!turable as
methanotrophs are probably responsible: for the
low-affinity activity which is typical f o r t h e soils
exposed to high CH4 mixing ratios. However, :the
bacteria responsible f o r the high-affinity activity
are still unknown. This activity is typical for the:
soils exposed to only ambient CH4 mixing ratios~
Both high- and low-affinity activities were inhibited by autoclaving and by acetylene.
i:
262
2. INTRODUCTION
Methane is a radiatively active trace gas which
increased by about 1% per year over the last
decades [1-3]. This increase is probably due to
increased CH 4 emission rates by sources like
combustion of organic compounds, paddy fields,
cattle breeding, landfills, termite nests and wetlands [4-6], In the global CH 4 cycle, CH 4 is
consumed by chemical and biological processes.
About 90% of the sinks for atmospheric C H 4 a r e
due to chemical oxidation in the troposphere [5].
B i o l o g i c a l C H 4 oxidation in oxic soils seems to
play only a minor role (up to 20%) in the global
budget [5,7].
However, biological CH 4 oxidation plays an
important role in controlling the CH 4 emissions
at anoxic-oxic interfaces in soils and sediments
[8-12]. In these environments, high concentrations of C H 4 a r e supplied from anoxic parts of
the soil or deeper sediment layers, and are oxidized in the aerated soil regions or the shallow
layer of oxic surface sediment. The oxidation
efficiency may reach > 90% of the C H 4 produced [10-12].
In principle, two types of oxic soils can be
distinguished: (1) soils which are at least temporarily exposed to high CH 4 mixing ratios, and
(2) soils which are exclusively exposed to low
atmospheric CH 4 mixing ratios, i.e. to mixing
ratios typically lower than 1.7 ppmv, equivalent to
< 2.5 nM in the soil water. (CH 4 mixing ratio =
volume C H 4 per volume air; 1 ppmv C H 4 = 1 /~l
C H 4 l-lair). The first type of soil is represented
for example by tundra soils, landfill cover soils,
soils over natural gas reservoirs, or soils preincubated under high CH 4 mixing ratios. The CH 4
oxidation kinetics in these types of soil have been
studied previously [13,14]. The second types of
CH4-oxidizing soils act as a sink for atmospheric
C H 4 [15-23]. Only little is known about the kinetics of the microbial C H 4 oxidation in this
second type of soil. It is also unknown whether
the CH4-oxidizing microbial populations in the
two different soil types are the same.
Possible candidates for C H 4 oxidation in soils
are methanotrophic bacteria which can grow on
C H 4 a s sole energy source, and ammoniumoxidizing nitrifiers which can co-oxidize CH 4 [24].
The question whether CH4-oxidizing bacteria are
able to grow on the C H 4 present in ambient air is
connected with the kinetic properties of these
bacteria. The half saturation constants (Kin),
maximum oxidation rates (Vm~) and threshold
mixing ratios (Th) of C H 4 oxidation are characteristic parameters which determine the ability of
bacteria to grow on atmospheric CH 4 [25]. Conrad [25] concluded that ambient CH 4 mixing ratios are probably too low and the K m values of
the methanotrophs are too high to allow for
growth.
We studied the differences in the kinetic parameters ( K i n , l/max, Th) of C H 4 oxidation and in
the cell numbers of methanotrophic bacteria in
soils which were exposed to ambient mixing ratios
of CH 4 (fresh soils) and in the same soils exposed
to high CH 4 mixing ratios (preincubated soils). In
addition, a forest soil which was 'naturally prein-
Table 1
Characteristics of the soils
Soil
CC
MC
FL
PS
pH (H ~O)
WHC a (%)
Organic C (%)
Total N (%)
C/N
Loamyclay
8.0
57
4.2
0.16
15
Sandysilty loam
7.5
75
8.0
0.42
11
Sandyclayloam
5.0
66
5.7
0.10
33
Sandyclayloam
6.8
56
3.9
0.19
12
a:WHC = water holdingcapacity(g H20/100 g dw soil).
i
:
i
NG
4.7
263
cubated' by exposure to high CH 4 mixing ratios
from a sub-surface natural gas source was investigated.
3. M A T E R I A L A N D M E T H O D S
leis [32]. The microtiter plates were incubated
under 20% C H 4 in air at 25°C in the dark for 3
weeks and then tested for bacterial growth. Controls were incubated under CH4-free air and
showed no growth in the dilutions which were
positive under 20% CH 4.
3.1. Soils
3.3. Kinetic experiments
Soil samples were taken from the A h horizons
(10 cm deep) of 5 different sites: cultivated cambisol (CC), forest luvisol (FL), and meadow cambisol (MC) were sampled near Konstanz (Germany), and paddy soil (PS) was sampled in Verceili
(Italy). The soils have been described before
[9,26,27]. A forest soi! (NG) percolated with high
C H 4 mixing ratios from a sub-surface natural gas
source was sampled in Kleinteil (Obwalden,
Switzerland). C H 4 was emanating at this site into
the atmosphere in quantities sufficient for ignition (i.e. > 5% CH4)o The soils were characterized by standard protocols [28]. The main soil
characteristics are summarized in Table 1. After
sampling, the soils were passed through a sieve
( < 2 mm mesh) and stored in fresh state in
polyethylene bottles at I°C in the dark. Only the
PS soil was stored as air-dried soil lumps at room
temperature [29]. The experiments with fresh soil
(without pretreatment) were done within 2 days
after sampling. The experiments with preincubated soils were done after incubation of the soil
under 20% c a 4 in air for 2 - 3 weeks in the dark
at 25°C.
Fresh or preincubated soil (100 g; 35-50%
WHC) was filled in glass flasks (1.2 1), flushed
with air, d o s e d with silicone septa and pressur 7
ized to 1.05 bar (105 kPa). The water content was
determined gravimetrically before and after the
experiment, which typically lasted not longer then
24 h. Controls were done by autoclaving the soil
samples for 1 h at 120°C or adding 1% acetylene
(C2H 2) into the headspace. C2H 2 is an inhibitor
of C H 4 oxidizing bacteria [24]. CH 4 was added to
give the desired initial mixing ratio, and the flasks
were incubated in duplicate at 25°C in the dark~
Gas samples (1 ml) were repeatedly taken over
time with a gastight syringe and analyzed in a gas
chromatograph with flame ionisation detector [9].
All data were averages of duplicate m e a s u r e ments which usually deviated < 15% from each
other. Rates of CH 4 oxidation were proportional
to the amount of soil up to 300 g indicating that
gas transfer between gas phase and soil phase
was not rate-limiting. At the end of a kinetic
experiment, the CH4 mixing ratio was adjusted to
the initial CH 4 mixing ratio again, and the C H 4
oxidation rate was measured a :second time t o
ensure that the activity had not changed during
the course of the experiment.
Up to 20 oxidation rates (V) were measured at
increasing initial CH 4 mixing ratios (m) in air to
determine the kinetic parameter~ ( K m, Vm~)using Eadie-Hofstee plots [33]. The data were also
analyzed for cooperativity using Hill plots [33].
The threshold mixing ratios of CH 4 in the d i f f e r '
ent oxidation experiments were determined i n
two ways: The Th m value (measured threshold)
was determined by following the C H 4 mixing
ratio from ambient air values (1.7-Z0 ppmv)down
to the lowest C H 4 value which then remained
constant for more than 3 days, T h e Tha value
(approximated threshold) was calculated by e x trapolating the linear part of the Michae!is~i
3.2. Numbers of methanotrophic bacteria
The numbers of methanotrophic bacteria were
determined by the most probable number (MPN)
technique. A modified ammonium mineral salts
(AMS) medium [30] was used for incubation and
extraction of the methanotrophic bacteria. The
medium contained per liter distilled water (pH
6.9): 10 mmol NH4CI, 0.4 tzmol MgSO 4 - 7H20, 4
mmol K 2 H P O 4, 0 . 1 / ~ m o l CaCI 2, 1 ml trace
element solution (SL-10) [31]. Fresh soil (5 g) was
suspended in 15 ml AMS and shaken for 12 h at
4°C in the dark~ This suspension served as inoculum f o r determination of the numbers of methanotrophic bacteria. The MPN was determined in
microtiter plates using 2-fold dilutions in 8 paral-
•
i¸i:•~i
264
2,5
CH4[ppmv] "
CH4 [ppmv] x 1000 in air
250
with acetylene
2C - -
0
O
D
autoclaved
(~
[2
200
D
D
150
1,51
100
0,5
S0
control
o
Thin
~ ~
A
.................................................................................
-r
i
"r
~
~.~ ~................
..................
25
fi0
75
100
125
150
Fig. 1. Progress curve of
in fresh soil (MC)
under ambient air with ([]) and without (©) acetylene (1%).
The measured threshold (Th,n) was obtained from the final,
constant C H 4 mixing ratio.
Menten saturation curve (first-order range of CH 4
oxidation) to zero oxidation rate where V = 0 [34].
The Th~ value represents the lowest CH 4 mixing
ratio at which the oxidation of CH 4 I U I I U W U U
first-order-kinetics. The CH 4 concentrations in
soil water were calculated by using the Bunsen
Coefficient for C H 4 at 25°C (0.033 [35_t) and the
molar gas volume of an ideal gas at 25°C (24.46 1).
" - "
i
100
175
Time[ h ]
CH 4 oxidation
.....
"
4. RESULTS
4.1. Microbial C H 4 oxidation in soil
The C H 4 oxidation activity in the tested soils
was abolished by autoclaving or by treatment with
C2H 2. The inhibition was observed for CH 4 oxidation at ambient (Fig. 1) and at high CH 4 mixing ratios (Fig. 2). Incubation of soil in presence
of 20% CH 4 in air resulted in increasing oxidation rates of CH 4 after about 100 h (Fig. 2) and
simultaneously in increased numbers of methanotrophie bacteria (Table 2).
r
200
Time [ h i
300
400
500
Fig. 2. Increasing oxidation of CH 4 during incubation of fresh
soil (MC) under 20% CH 4 (<3), and control with autoclaved
soil ( [] ).
relatively low K~f and l/maxt values (Table 3). The
suffix 'f' symbolizes 'fresh soil'. The Kmf values
of the three soils varied between 22 and 37 ppmv
CH4 in the headspace, equivalent to 30-51 nM
CH4 in the aqueous phase. The Vmaxfvalues
ranged between 0.7 and 3.b nmoi h - : g - : d w (Table 3). The measured thresholds (Thmf) were very
low (about 0.02 ppmv) and close to the detection
limit of our analytical system ([34]; data not
shown). The approximated thresholds (Thaf) were
10-140 fold higher than the measured Thmf values (Table 3).
I V [nmollh
0,8
Vmaxf
/
0
| d.w.]
,
6
~
0,4
0,2
4.2. Fresh soil
CH 4 oxidation in fresh soils (CC, MC, FL)
showed a typical Michaelis-Menten saturation
curve (Fig. 3). The Eadie-Hofstee plot of a typical
kinetic is shown in Fig. 4. Fresh soils showed
Kmf
0
0
20
40
60
80
100
t
i
~
i
i
120
140
160
180
200
CH4lppmv]in air
Fig. 3. Substrate saturation curve of CH 4 oxidation in fresh
soil (MC).
265
V [nmol/h
Table 2
Numbers of methanotrophic bacteria (-+ SE%) in various soils
assayed in fresh state (exposed to ambient CH 4) and after
preincuhation under 20% CH 4
g d.w.]
0'8~Vmaxf
°"I
0,006
0,01
0,016
0.0 '~
v/e [I/h g d.,,.]
Soils
Numbers of methanotrophs ( ± SE%)
(cells g- I dw)
Fresh
Preincubaled
CC
MC
FL
PS
NG a
3.6×106±14
3.6× 10s -+ 17
2.4 × 105 +_34
4.2× I06 ± 13
10 x 106-+ 14
3.3× 10° _+21
6.4 x 105 ± 20
2.3× 1 0 7 ± 19
2.1 X 107 + 16
The numbers were significantly different in the two treatments (t-test; p < 0.05).
a Forest soil over a natural gas source in Switzerland.
Fig. 4. Eadie-Hofstee diagram of the C H 4 oxidation kinetics
in fresh soil (FL).
W h e n a i r - d r y p a d d y soil ( P S ) w i t h 1.4% H 2 0
was remoistened to 36% WHC no CH 4 oxidation
activity c o u l d b e m e a s u r e d , a l t h o u g h t h e d r y soil
contained significant numbers of culturable
m e t h a n o t r o p h s ( T a b l e 2). E v e n p r o l o n g e d i n c u b a t i o n (12 w e e k s ) o f t h e r e m o i s t e n e d P S u n d e r
air d i d n o t r e s u l t i n d e t e c t a b l e C H 4 o x i d a t i o n .
T h e C H 4 o x i d a t i o n activity o n l y d e v e l o p e d a f t e r
p r e i n c u b a t i o n u n d e r 2 0 % C H 4.
4.3. Preincubated soil
T h e p r e i n c u b a t e d soils ( C C , M C , FL, P S ) w h i c h
were enriched with methanotrophic bacteria (Tab l e 2) s h o w e d s a t u r a t i o n c u r v e s w h i c h w e r e dif-
Table 3
Kinetic parameters (Kra, Vmax, Th a) of C H 4 oxidation in fresh and preincubated oxic soils
Soil
CC
MC
FL
PS
NG c
Parameter
a
Vmax
(nmol h - tg- t d.w.)
Fresh so:;
Preincubated soil (1)
(2)
0.7_+ 7
15 ± 3
270 _+11
0.9_+ 5
2.1 ± 6
410.0±18
3.6± 8
4!.0± 6
450.0+ 8
0a
150.0_+ 16
3690.0± 7
44500.0_+ 12
(nM)
Fresh soil
Preincubated soil(!)
(2)
50.6± 13
91 +22
1740.0 ± 34
49.9± 7
12.6_+36
4560.0 ± 33
29.7± 35
470.0_+21
27900.0 ± 24
34.0+_24
8000.0 ± 31
t00000.0_+21
Th a b
(ppmv)
Fresh soil
Preincubated soil (1)
(2)
0.2± 13
2.3± 5
42.0± 5
0.2_+ I
4.1± 15
11.3+ i8
2.7± 16
1.3_+ 2
12.8± 10
0.3±21
45.3_+ 14
Km a
a Km' Vmaxand ± SE (%) were determined from linear regression of Eadie-Hofstee diagrams.
b The approximated thresholds +_SE (%) (Th a) were determined by extrapolation of the linear part of Michaelis-Menten
saturation curves (first-order oxidation rate) to zero C H 4 oxidation using linear regression.
¢ Naturally CH4-preincubated soil over a deep gg~ssource in Switzerland.
a Air dried paddy soil remoistened to ,'36% WHC
266
ferent from those of the fresh soils (Fig. 5A,B).
Hill plots resulted in a constant coefficient of
h = 1 indicating that the shape of the saturation
curve was not due to concentration-dependent
changes in cooperativity of the CH4-oxidizing enzyme systems. It rather appeared that the complete saturation curve was composed of two different saturation curves, which were superimposed on each other. The Eadie-Hofstee plot
revealed two different sets of kinetic parameters
which could be clearly discerned (Fig. 6). One
V [nraol/h g d.w.]
.500 ~
400
300
200
/-Km2
100
0
400
•
i i
0
V [ n m o l / h g d.w.]
Vlnax2
Vmax2
i
)
0,ol 0,0z o,oa o,04 0.05 o.06 0,07 0,08 o.09 0,1
v/c [l/h e d.w.l
+
Fig. 6. Eadie-Hofstee diagram of the CH 4 oxidation kinetics
in preincubated soil (FL).
300
-
200-
,oo-÷ I
/t
30
4O
CH4 [ppmv] x t000 i n a i r
1o
V [ n m o l C H 4 / h g d.w.]
9 "~
fresh
8 • "4"- preineubated
j j ~ , - r
/ / -
/
/
vm~
3
2
...............
40
1"ta~l
7haf
60
Vmaxt
B
80
100
CH4 [ppmv] i n a i r
Fig. 5. Substrate saturation curve of CH 4 oxidation in fresh (.)
and CH4-preincubated ( + ) soil (MC). (A) Whole range of
CH 4 mixing ratios. (B) Magnification of diagram (A) for low
CH 4 mixing ratios. The suffixes 'f', '1', and '2' symbolize
'fresh
soil', 'high-affinity activity', and 'low-affinity activity', respectively.
saturation curve was characteristic for the lower
CH a mixing ratios (up to 200 ppmv) and showed
low values for K m, Vma~ and Th a. This activity is
symbolized by the suffix '1'. This saturation curve
was apparently due to a 'high-affinity' activity.
The second curve was characteristic for CH 4 mixing ratios > 200 ppmv and showed relatively high
values for K m, Vm~, and Th a. This activity is
symbolized by the suffix '2'. This saturation curve
was apparently due to a 'low-affinity' activity.
The high-affinity Kml values ranged between
9.3 and 348 ppmv in air (13-470 nM in soil
water), whereas the low-affinity Kin2 values
ranged between 1290 and 20 680 ppmv in air
(1740-27900 nM in soil water) being 19- to 362fold higher than the Kml values. The values of
the high-affinity Kml in the preincubated soils
were similar to the Kmf values measured in the
fresh soils (Table 3).
The high-affinity Vm~I values ranged between
2 and 150 nmol h - l g - l d w ' whereas the low-affinity Vma~2 values varied betweeen 270 and 3690
nmol h - l g - l d w being 11- to 195-fold higher than
the //maxI values. The Vm~xl values in preincubated soils were generally higher than the Vma~
values in the fresh soils (Table 3).
The forest soil (NG) which was naturally
preincubated from a natural gas source below
showed high values for Kmr (100000 n M ) a n d
Vma~ (44500 nmol h - l g - l d w ) . These values resembled the low-affinity Kmz and Vma~ values of
267
the other soils (Table 3). The kinetics were not
sufficierJtly resolved at the low CH 4 mixing ratios
to detect a high-affinity activity.
The approximated threshold (Th a) of the
high-affinity and low-affinity activities were also
different (Fig. 5B). Tha2 values were 2.8- to 151fold higher than Tha~ values (Table 3). All Th~
values were significantly higher than the measured thresholds (Thin; [34] data not shown)which
did not differ from the Th~ values determined in
the fresh soils. Even the NG soil showed Thm
values as low as 20 ppbv.
5. DISCUSSION
Exposure of soils to percentage levels of CH 4
over 3 weeks resulted in CH 4 oxidation kinetics
that were more complex than those of fresh soils
which never had been in contact with CH 4 mixing
ratios higher than ambient (about 1.7 pprnv). At
least two different C H 4 oxidation activities could
be identified in CH 4- preincubated soils displaying biphasic saturation curves. The first actMty
with high-affinity for C H 4 w a s comparable with
the activity in fresh soil, adapted to low, ambient
C H 4 mixing ratios. This activity displayed low
values for Kin, Vmax and Th a. A second activity
with low-affinity for C H 4 appeared only after
preincubation at increased C H 4 mixing ratios,
and displayed high values for K m, Vmax and Tha.
Both, the low-affinity and the high-affinity activities were apparently due to microorganisms, as
they were abolished by autoclaving. The inhibition by acetylene indicates that the microorganisms were probably methanotrophic or nitrifying
bacteria [24].
The bacteria responsible for the low-affinity
activity which arose during preincubation with
h i g h C H 4 mixing ratios seemed to be the methanotrophic bacteria which are usually counted and
isolated from soils using standard techniques
[30,36]. This conculsion is supported by our observation that the number of the culturable
methanotrophs increased in parallel with the induction of the low-affinity activity for CH 4 oxidation in soils. It is furthermore supported by the
similarity of our Kin2 values with those observed
in pure culture studies [14,24,37] and those determined in soils which were exposed to high mixing
ratios of CH 4 [13,14]. The naturally preincubated
NG soil (Obwalden, Switzerland)also showed
high numbers of methanotrophs and high Km~,
Vmax values comparable to those known from
literature or determined as low-affinity values in
preincubated soils.
Earlier kinetic experiments by other investiga,
tots [13,14] did not detect the simultaneous presence of a high-affinity activity, probably since
C H 4 oxidation kinetics were not resolved at sufficiently low CH4 mixing ratios. The CH 4 oxidation activity with high affinity displayed K m values that were much lower than any value which
has so far been reported in pure cultures of
methanotrophs or CH4-oxidizing nitrifiers
[24,37-40]. This high-affinity activity may be due
either to bacteria which have so far not been
isolated and characterized, or to an activity of the l
known methanotrophs which has so far escaped
discovery. However, since preliminary experi.
ments with a methanotrophic enrichment culture
from the forest soil showed only a high K m value
(ca. 25 ttM CH4), we assume that the high-affinity activity is due to unknown microorganisms.
The high-aff'mity activity was present in fresh as
well as in preincubated soils. However, the Vr,a~!
values observed in the preincubated soils w e r e
significantly higher than the Vma~ values in fresh
soils indicating an induction of the high-affinity
activity by either increasing the unknown microbial population or by increasing the specific CH 4
oxidation activity of the individual bacteria after
exposure to high C H 4 mixing ratios.
Both soil preparations (fresh and preincu,
bated) exhibited CH 4 oxidation activity below
ambient mixing ratios of CH 4 (about 1.7 ppmv)
and thus were able to act as sinks for atmospheric
CH 4. They showed measurable thresholds (Th m)
which were significantly lower (near the detection
limit of our analytical system) than the approximated thresholds (Th a) [34]. Even a forest SOft
over a deep natural gas source (NG soil) which
was adapted to high CH 4 mixing ratios: over a
long period, displayed Th m values similar to those
in the other fresh soils. Apparently, soils did not i
loose their ability to oxidize ambient CH~ mixing:
i
i
•
•
/
•
iill i i!i
i
i
ii
-
•
il
•
ii
i
i ~:::•
!
!':
!: !i.;i:~i ¸
, i! !ili
268
ratios even when they were exposed to percentage levels of CH 4 for a long time. However,
remoistened air-dried paddy soil did not oxidize
ambient CH 4 unless it was preincubated under
high CH 4 concentrations.
The quantitative relation of the measured rates
of C H 4 oxidation (Vmax) tO the numbers of
methanotrophic bacteria in soil is problematic,
since the MPN technique probably underestimates the actual bacterial numbers of the
methanotrophs, since some types of methanotrophs may not be able to grow under the cultivation conditions used. On the other hand, we
cannot exclude that the MPN estimates also include resting or inactive states of methanotrophs.
Therefore, we t~sed only the data from CH4-preincubated soils in which the methanotrophs
should be in a relatively active stage. Using the
MPNs of methanotrophs and the Vma~ values
determined in the kinetic experiments, we calculated bacterial CH 4 oxidation rates between 30
and 21 200 fmol h-tcell-~ in the different preincubated soils. These values are considerably
higher than those known from pure culture studies ranging between 3 to 438 nmol min-~mg -1
cells which is equivalent to 0.1 to 15.5 fmol
h-~cell -~ using a dry weight of 0.5 pg r~er bacterial cell [24,37,41]. The difference of cellular activities between CH4-preincubated soil samples
and pure cultures is so high ( < 103) that unrealistically high numbers of methanotrophs must be
present in the soil unless we postulate that the
methanotrophic populations have a higher cellular activity in soil than indicated by the pure
culture studies.
In fresh soil, the determined MPN of culturable methanotrophs did probably not represent
the microbial population which was actually oxidizing CH 4. A large number of the counted
methanotrophs were obviously resting stages, like
exospores or cysts. This is indicated by the observation that remoistened paddy soil, which did not
show any CH 4 oxidation activity at ambient CH 4
mixing ratios, displayed numbers of methanotrophs which were comparable to those of the
other soils which did show C H 4 oxidation activity
at ambient CH 4 mixing ratios. Efforts to determine the numbers of the CH4-oxidizing bacteria
with low-affinity activity were not successful. An
MPN assay which was incubated under 10 ppmv
CH 4 showed no difference to the control which
was incubated under CHa-free air.
The bacteria responsible for the high-affinity
activity of C H 4 oxidation so far remain unknown.
Their low K m may be sufficient to allow growth
even on the low C H 4 mixing ratios in ambient air
[25]. It would be interesting to isolate and study
these methanotrophic microorganisms.
ACKNOWLEDGEMENTS
We thank Dr. Bodmer, Sulzer KG, Switzerland, for showing us the soil site over the deep
natural gas source. This work was financially supported by the Fonds der Chemischen Industrie.
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