ELSEVIER
FEMS Microbiology
Methanogenesis
Ecology 18 (1995)2 15-224
and methanotrophy
Lee R. Krumholz
within a Sphagnum peatland
a***‘,Julie L. Hollenback a-2,Sanford J. Roskes a*3,
David B. Ringelberg b
’ Department of Cil,il and Environmental Engineering. Massachusetts Institute of Technology, Bldg. 48-423, Cambridge, MA 0213Y. USA
b Ceruerfor
Environmental
Biotechnolog!.
Unkersity
of Tennessee. Knoxville, TN 37932, USA
Received IO May 1995: revised 21 July 1995; accepted 23 July 1995
Abstract
Methane production and consumption activities were examined in a Massachusetts peatland. Peat from depths of 5-35
cm incubated under anaerobic conditions, produced an average of 2 nmol CH, g- ’ hK’ with highest rates for peat fractions
between 25-30 cm depth. Extracted microbial nucleic acids showed the strongest relative hybridization with a 16s rRNA
oligonucleotide probe specific for Archaea with samples from the 25-30 cm depth. In aerobic laboratory incubations, the
peat consumed methane with a maximum velocity of 67 nmol CH, g-’ h-’ and a K, of 1.6 FM. Methane consumption
activity was concentrated 4-9 cm below the peat surface, which corresponds to the aerobic, partially decomposed region in
this peatland. Phospholipid fatty acid analysis of peat fractions demonstrated an abundance of methanotrophic
bacteria
within the region of methane consumption activity. Increases in temperature up to 30°C produced an increase in methane
consumption rates for shallow samples, but not for samples taken from depths greater than 9 cm. Nitrogen fixation
experiments were carried out using 15Nz uptake in order to avoid problems associated with inhibition of methanotrophy.
These experiments demonstrated that methane in peat samples did not stimulate nitrogen fixation activity, nor could activity
be correlated with the presence of methanotrophic bacteria in peat fractions.
Ke~~~ord.stMethanogenesis: Methanotrophy; Sphagnum; Peatland; Nitrogen fixation; Phospholipid
1. Introduction
Northern peatlands are characterized by high but
variable water tables, low nutrient levels, and low
= Corresponding
author. Tel: + 1 (405) 325-0437: Fax: + 1
(405) 325-7541: E-mail: [email protected]
’ Present address: Department of Botany and Microbiology,
University of Oklahoma, Norman, OK 73019. USA.
’ Present address: Department of Bacteriology,
University of
Wisconsin, Madison. WI 53706, USA.
’ Present address: Ecology and Environment, Washington, DC.
USA.
Ol68-6496/95/$09.50
0 1995 Federation
SSDI 0 ‘68.6496(95)0006
‘-5
of European
Microbiological
fatty acid; Wetland
pH (around 4.0). Under these conditions decomposition occurs slowly, causing thick deposits of peat to
accumulate. The water table is often below the surface of the peat mat leaving an unsaturated zone of
varying depth. Anaerobic bacteria inhabit the saturated zone [ 1] and release carbon as methane. As this
methane diffuses and bubbles toward the atmosphere, some of it is consumed [2,3] probably by
oxidation to carbon dioxide by methanotrophic
bacteria in the unsaturated
regions. Total peatland
methane emissions are therefore dependent on the
activities of both methanogenic
and methanotrophic
bacteria.
Societies.
All rights reserved
216
L.R. Kntmhol:
e? al. / FEMS Microbiology
The emission of methane is especially important
because methane is radiatively twenty times as active, mol for mol, in the atmosphere as carbon
dioxide [4]. Existing data on peatland methane emissions are highly variable, with values ranging from
0.7 to 866 mg CH, mm2 day-’ [5-71. Previously
reported values of methane consumption in peat bogs
are also highly variable, ranging from 0.6- 18.7 pmol
(1 peat)-’
h-’ [3,8]. It is difficult to separate net
methane emissions into gross production and internal
consumption.
Recent studies have inferred methane
flux from measured methane gradients at various
depths in the field [2], or have incubated peat aerobically and anaerobically in the laboratory to estimate
potential for methane consumption
and production
[3,9]. Based on the field results at the present study
site [2], maximum methane consumption
occurs at
6-12 cm depth. Maximum methane production usually occurs in the upper sections of peat mats just
below the oxycline [IO]. Oxycline depth varies depending on the relative depth of the water table in
the peat with oxygen often disappearing just below
the water table level.
Although the above studies have documented
zonation in the distribution of methanogenesis
and
methane consumption,
they have not demonstrated
the presence of microorganisms
capable of carrying
out these processes, nor have they drawn a clear
boundary between the methane-oxidizing
and the
methane-producing
layers of the peat bog. A number
of studies have demonstrated
kinetic controls on
methane consumption within wetlands [ 12- 151. Factors such as light [ 16,171, oxygen, pH, temperature
and relative moisture content all have a significant
influence on the kinetics of methane consumption
within wetlands. In forest soils, the major limitation
on methane consumption
may be the rate of mass
transfer of methane to the microorganisms [ 141. Each
of these controlling factors varies significantly
with
the depth of the active microbial community,
with
increasing influences of oxygen and temperature limitation as the depth of the microorganisms
increases
within the peat mat.
In this study, we examined depth profiles of
methane-consuming
and methane-producing
bacteria
from peat samples obtained at Thoreau’s Bog in
Concord, Massachusetts. After establishing methane
consumption
in intact samples, we determined the
Ecolo,qy 18 CIYY51 215-224
location of methane consumption
activity within a
core. We also examined the effects of oxygen concentration and temperature on the consumption rate.
A molecular approach was then used to localize the
responsible
organisms.
We obtained phospholipid
fatty acid (PLFA) profiles of the upper peat fractions
in order to confirm the presence of methanotrophic
bacteria as well as to determine the major microbial
components of the community. We also attempted to
quantify
specific organisms
with oligonucleotide
probes specific for 16s rRNA. This part of the
project used group-specific
oligonucleotide
probes
for the Archaea in order to quantify methanogenic
bacteria.
Within the context of gaining a greater understanding of the role of methanotrophic bacteria within
the peat mat, we examined peat profiles in order to
determine where nitrogen fixation was occurring.
and in particular whether the methanotrophic
bacteria contributed significantly to nitrogen fixation. Previous studies have reported nitrogen fixation to be
catalyzed by heterotrophic bacteria in aerobic peat
[ 181, or by cyanobacteria
associated with wetland
plants [ 19.201. The assay for nitrogen fixation used
in the above studies involves the use of acetylene, a
known methanotrophic inhibitor [21]. Since Group 2
methanotrophs
and Methylococcus
capsulatus possess nitrogenase [21,22] and nitrogen fixation is felt
to be an important process in peatlands [23], there
exists the possibility of an important role for methanotrophs as nitrogen-fixing
organisms
in peatland
ecology.
2. Materials
and methods
2.1. Site characterization
Peat samples were taken from Thoreau’s Bog in
Concord, Massachusetts (47”27’43” N, 7 lo1 9’42” W).
This acidic bog covers 0.4 hectare in the Merrimack
River Watershed. The vegetation is composed primarily of Sphagnum sp. and small shrubs (see [24]
for a description of flora). The water table is 7-14
cm below the peat surface. Precipitation provides the
only hydrologic input to the bog, and evaporation
accounts for 80% of hydrologic output [24]. The
remaining output is due to groundwater flow and
L.R. Krumhok et al. / FEMS Microbiology Ecology 18 (1995) 215-224
intermittent
stream flow from the bog [25]. The
biogeochemistry
of this bog has been characterized
in detail by Hemond and colleagues [2,24,25].
A limited study on diurnal temperature fluctuations was carried out. During a typical sunny day in
August temperatures reached 32°C by early afternoon at the surface but decline rapidly to 22°C
during the night. The water temperature during August typically reaches about 18°C and fluctuates only
slightly (< 1 Co) during the day. Peat material in the
unsaturated zone is partially insulated depending on
depth, and shows an increase of between 2 and 9°C
through the day, cooling off in late afternoon (data
not shown).
2.1.1. Sample collection
Samples were collected using a stainless steel
cylinder, 12.5 cm internal diameter with a band saw
blade welded to the bottom. This sharpened edge
was twisted into peat with slight downward pressure
to minimize compression, and cores of up to 40 cm
in length were routinely obtained. For methane production experiments, a plastic cap was placed over
the bottom of the corer in order to minimize contact
with the atmosphere. Intact cores were transported to
the laboratory. For the other experiments, cores were
separated into fractions in the field, and subsamples
from each fraction were transported in polypropylene
tubes to the laboratory. Samples for rRNA analysis
were stored at -20°C.
2.2. Nucleic acid and phospholipid
sis
fatty acid analy-
For the isolation of nucleic acids, bacteria were
first isolated from peat samples using a modified
protocol from Holben et al. [26]. Peat samples were
blended with homogenizing
solution (0.5 g 1-l
KH,PO,, 0.4 g 1-l MgC12. 6Hz0, 0.4 g ll’ NaCl,
0.4 g I-’ NH,Cl, 0.05 g Il’ CaCl, .2H,O, and 100
g 1-l acid-washed
polyvinylpolypyrrolidone)
and
then centrifuged at 1000 X g to remove soil debris.
Remaining
bacterial
cells were centrifuged
out,
washed with 2% sodium hexametaphosphate,
and
resuspended in 50 mM sodium acetate buffer (pH
5.1). The suspended cells were then mechanically
disrupted with glass beads as described by Stahl et
al. [27], after addition of 2.5% Pmercaptoethanol
to
217
the bead mixture as an RNase inhibitor. The resulting nucleic acid preparation had an A26,,,280 ratio
greater than 1.5. Blotting and hybridization
also
followed the protocol of Stahl et al. [27]. The sequences used for the oligonucleotide
probes were
described by Stab1 and Amann [28] (archaeal and
eubacterial specific probes). Modifications
included
addition of 1% heat-denatured
salmon sperm DNA
and 0.05% sodium dodecyl sulfate to the hybridization buffer, as well as use of 2 X lo6 cpm of labeled
oligonucleotide
per blot rather than 2 X lo5 cpm. In
addition, optimal wash temperatures
were determined experimentally
for each probe using DNA
isolated from pure control cultures. Quantities of
nucleic acids blotted (per dot) and wash temperatures
for each probe were: archaeal 2 pug, 70°C; and
eubacterial I pg, 63°C. Membranes were wrapped in
plastic wrap after washing and exposed to film with
an intensifying
screen at - 20°C for 19-22 h. The
response was calculated on a computing densitometer. Relative hybridization
signal was calculated as
signal obtained from the group-specific
probe divided by signal from the eubacterial specific probe.
The eubacterial probe was used as a correction factor
instead of a universal probe to avoid including plant
nucleic acids. Eubacterial:universal
hybridization ratios varied from 0.39 to 4.5.
For the measurement of phospholipid fatty acids
(PLFA), peat samples collected on two dates were
fractionated into a O-2.5 cm and a 5-7.5 cm fraction. These samples were then lyophilized. One gram
aliquots were extracted and analyzed for PLFA content following the protocols described in Guckert et
al. [29].
2.3. Methane production
Cores were placed in an anaerobic glovebox with
a N,-H, (1% HZ) atmosphere. Samples of 1.5-2.0 g
fresh weight from each depth were placed in 120-ml
serum bottles, that were capped with butyl rubber
serum stoppers and crimped before being removed
from the glovebox. Control bottles incubated without
peat samples demonstrated that these stoppers efficiently contained methane with undetectable losses
over the course of the experiments. The samples, wet
but not saturated, were incubated at 10 or 22°C.
Headspace samples were taken periodically, begin-
ning after 2 h and continuing for I 10 h. Headspace
methane concentrations
were determined
using a
Shimadzu W-80
gas chromatograph
fitted with a
l/8 inch X 6 foot stainless steel column packed
with 80/100 Carbosieve S (Supelco) and a flame
ionization detector.
2.4. Methane
consumption
Subsections of peat cores were divided into 1.52.0 g samples, placed in 120-m] serum bottles and
sealed. Dilutions of methane in air were injected to
desired initial concentration.
Aqueous methane concentrations
were assumed
to be 3.64% of the
headspace concentration
based on the Henry’s Law
constant for methane of 661. In experiments where
oxygen
concentration
was varied, bottles were
flushed with N, prior to addition of pure O1 to the
desired concentration.
Methane concentrations
were
determined at IO-20 h intervals for 48 h as described above. Rates were calculated by plotting the
methane concentration
over time and dividing the
slope of the line (determined by regression analysis)
by the wet weight of the peat.
In order to determine the maxima1 rate of methane
consumption and the half-saturation constant, results
from a series of rate determination
experiments
(seven methane concentrations
ranging from 15 nM
to 15 PM) were plotted and the curve was fitted
using the equation: Rate = V,,, . CH, Concentration/( K, + CH, Concentration),
where a best fit
was determined for V,,, and K, using the Sigma
Plot for PC application.
additions. After 48 h incubation at 22°C. all samples
were removed from the bottles and dried at 70°C for
20 h.
An additional field experiment was carried out in
order to localize nitrogen fixation within the peat
mat. Three liter acid bottles were prepared with the
bottom removed. The coring device was used to
create an opening in the peat mat and the bottle was
inserted to a depth of 20 cm. A silicone rubber
septum was put on each bottle top and 15N2 was
injected into each bottle to a final concentration
of
either 0.5% or 2.0% (calculated 6 15N enrichment of
headspace is 1728 and 691 1 respectively). A plastic
canopy was set up to partially shade the bottles and
they were incubated for 24 h. After this time period.
the bottles were removed, and the peat was removed.
The core was cut into fractions and a part of each
fraction was dried for later analysis.
The samples were ground with a mortar and
pestle and screened through a 0.5 mm mesh. 6 15N
analysis was carried out at the Boston University
Stable Isotope Laboratory. The samples were combusted at 1000°C in a Hereus C-N analyzer. A
Finnigan C-T box cryogenically
separated the products and the N? was then introduced into a Finnigan
Delta-S isotope ratio mass spectrometer. Calculation
of nitrogen fixed over the course of the experiment
was based on calculations
[30] to determine the
percent nitrogen in the sample derived from atmospheric N2 over a given time period. ~Mol nitrogen
fixed/g dry weight/h was determined with the following equation: (%nitrogen in sample derived from
atmosphere
X total N in sample)/(dry
weight X
time).
2.5. Nitrogen fixation
Core sub-sections
were obtained as described
above and 4.0 g samples were placed in 120-ml
serum bottles with air. Pure 15N2 was added to 0.1%
by volume to each treatment (natural abundance is
0.3663% so the calculated
6 “N enrichment
of
headspace is 344). A control set of bottles was also
included in which no 15N2 was added. In order to
test for the significance of methanotrophic
and phototrophic bacteria in nitrogen fixation, one set of
bottles had an addition of 2.7 pmol methane and one
set was incubated under 200 PEinsteins
mm’ of
light. The last set was incubated in the dark with no
3. Results
3. I. Localization
of bacteria
Peat sub-samples
from several cores extending
well below the water level indicated that methane
consumption
capabilities
were present in the top
9-l 1 cm of peat, but in general, absent below 11 cm
depth (Fig. I). This corresponds roughly to the layer
of unsaturated
peat material, where oxygen and
methane mix. Maximum methane consumption usually occurred between 4-6 cm. Relative variations in
L.R. Krumholr
et al. / FEMS Microbiology
Ecology
18 (19951 215-224
219
,100
7
_c
7
UJ
'ij 80
E
c
5
60
s
"a
E
40
ii
z
0
E
20
cm
2
0
0
2
10
12
14
0
10
20
30
II
Average Depth (cm)
Fig. I. Methane oxidation rates at different depths of the peat mat,
repeated four times with different cores. Incubation occurred for
48 h at 24°C under air with an aqueous concentration
of 3.0 /.LM
methane. Peat samples were collected in August 1991.
Fig. 2. Production of methane by peat from 5 cm depth
ments. The samples were collected in November 1991 and
bated in anoxic serum bottles at 10°C (0)
and 22°C
Methane concentrations
were determined every 20-30 h for
120 h.
methane consumption
rates, as determined in four
independent cores collected on a single day, tend to
be larger at the extremes of depth and rates were
much more uniform within the 4 to 9 cm range (Fig.
1). The rates were linear initially but decreased as
methane levels decreased. Initial rates averaged 47
nmol g- ’ h- ’ between 4-9 cm. Two of four cores
exhibited no methane consumption
in the top 4 cm
of the core.
We observed methane production activity in samples at depths as shallow as 5-10 cm and as deep as
30-35 cm. averaging 2 nmol gg ’ hh ’ (Fig. 2).
Methane production activity outside this depth range
was not detected in our studies.
The depth range of methanogenic bacteria within
peat cores was examined
using group specific
oligonucleotide
probes. Cores collected in May and
July of 1992 were sectioned and screened for the
presence of microorganisms
whose rRNA was capable of hybridizing with these group-specific probes.
The archaeal probe showed strong hybridization
at
depths of 1.25, 15, and 20 cm in May, while the
sample from July had a clear peak around 25-30 cm
below the peat surface (Fig. 3). This is consistent
with our methane production results, which showed
production from 5-35 cm and a peak at 27.5 cm.
The difference is most likely due to seasonal variation.
PLFA analysis was performed on core fractions in
order to provide evidence
of the existence
of
methanotrophic and other bacteria within the unsaturated zone of the peat column. The data provide
evidence for the existence of a significant population
of microorganisms
within both the surficial layer as
well as the actively methanotrophic layer at 5 to 7.5
cm depth (Table I). These populations appear to be
composed of significant numbers of Gram-positive
organisms
and/or
Gram-negative
obligate anaerobes, Gram-negatives
in general, Actinomycetes,
increincu(a ).
I IO-
0.6-
I
10
8.
20
I.
30
40
Average Depth (cm)
Fig. 3. Hybridization
of oligonucleotide
probe to nucleic acid
preparations of peat samples from different depths. Samples were
collected in May (0) and July (A) of 1992. Values plotted are
the ratios of the densitometer signal obtained with the Archaeal
probe to the signal obtained with the eubacterial probe.
220
L.R. Krumhok
et al./ FEMS Microbiology
anaerobes and in the deeper layer, methanotrophs
[29,3 1,321. Because about twice as much PLFA per g
was recovered from the surficial layer, and the mol%
of most microbial fatty acids were equivalent, we
can say that microbial abundance is greater in the
surficial layer of peat. However, several groups of
organisms increased in numbers in the lower layers
(5-7.5 cm). These include both methanotrophs and
obligate anaerobes. Certain groups also appeared to
increase in numbers in the deeper layer during the
August sampling in comparison to the June sampling
as indicated by the differences in mol% on the two
dates. The greatest percentage of methanotrophic
PLFA was observed in the September sample.
Ecology 18 (19951215-224
Table 2
Results of a nitrogen fixation experiment
Nitrogen fixation was determined by the
course of a 48 h incubation. Samples
variety of conditions in order to simulate
with samples from peat.
uptake of “N during the
were incubated under a
their in situ effects
Depth cm
Nitrogen fixation ( pmol N (g dry weight)Dark
Dark plus CH, a
Illuminated
o-4
4-8
8-12
12-16
16-20
0.205
0.160
0.083
0.123
0.076
0.192
0.167
0.133
0.074
0.082
0.166
0.183
0.128
0.091
0.097
’ h- ’ )
b
a Methane (2.5 prnol) was added to each of the 120-ml serum
bottles.
’ Bottles were illuminated continuously for 48 h.
3.2. Nitrogen jixation
Nitrogen fixation experiments (Table 2) indicated
that nitrogen fixing organisms were present throughout the peat core. The ability to fix “N2 was greater
in the upper layers of the core, with undetectable
influences of either light or methane. Typical 6 15N
enrichments in peat samples varied from 2.2 to 7.2
with a background
of 0.5. The field incubations
showed that nitrogen fixation could take place in the
top (O-4 cm) section of the core at a rate of approximately 0.5 pmol nitrogen fixed hh’ gg ’ dry weight.
No detectable nitrogen fixation activity was observed
in other fractions down to a depth of 20 cm.
Table I
Representative
phospholipid
PLFA
3.3. Parameters
at Thoreau’s
Bog during the summer of 1992
PLFA mol8
group
5-7.5
O-2.5 cm
il5:O
a15:O
lOme16:O
lOme18:O
16: I w7c
16:l w7t
18:lw7c
lS:lw7t
16: I w8c
18:lwSc
i17:Iw7c
Total nmol PLFA/g
Gram-positive
or Gram-negative
Actinomycete
Gram-negative
Group I methanotroph
Group II methanotroph
Anaerobe or Streptomyces
methane consumption
Methane consumption rates increased relative to
methane concentration (Fig. 4) with a K, for methane
of I .6 PM and with a V,,, of 67 nmol g- ’ hh ’ at
25°C. Experiments
to determine the influence of
temperature on methane consumption showed significant activity occurring at temperatures as low as
10°C (Fig. 5). No significant activity was observed at
4°C. Temperature influence is dependent on original
depth from which peat samples are taken: methane
consumption
rates peak at temperatures which de-
fatty acid levels from peat samples collected
Representative
influencing
obligate anaerobe
cm
6/22/92
9/28/92
6/22/92
9/28/92
3.23
2.05
1.70
0.07
2.42
0.28
5.89
0.22
0.00
0.00
0.46
2.73
1.13
0.68
0.09
2.4 I
0.30
6.64
0.27
0.00
0.00
0.45
3.78
1.64
2.49
0.44
3.24
0.64
7.28
0.35
0.23
0.00
0.93
6.06
3.51
2.59
0.33
4.24
I .46
12.81
2.20
3.05
5.90
2.44
304
521
169
216
221
L.R. Krumholz et al./ FEMS Microbiology Ecology 18 (1995) 215-224
concentrations
concentration
between 1 and
of 0.3 PM.
21%
and
methane
4. Discussion
0
initial hethane Cckentratiod&M)
Fig. 4. Methane oxidation rates at varying initial methane concentrations. Incubation occurred at 24°C. Samples were collected in
July 1991.
crease with increasing
original
depth. Resident
methanotrophs in deeper areas are presumably acclimated to lower temperatures.
Methane production
was also influenced
by temperature,
with a large
increase in the rate of production as the temperature
increased from 10 to 22°C (Fig. 2).
Oxygen concentration
is likely to have a significant influence on the rate and extent of methane
consumption.
We observed methane consumption
rates of 6 to 8 nmol g - ’ hh ’ at headspace oxygen
0
10
20
30
Temperature (OC)
Fig. 5. Methane oxidation rates at different incubation temperatures. Incubation occurred under air with methane at 3.0 PM
aqueous concentration.
Peat fractions were collected from depths
of O-4 CO), 4-6.5 (A). 6.5-9 (+). and 9-11.5 (0) cm in July,
1991.
Combining methane consumption and production
assays with a molecular analysis facilitated both
localization
of methanotrophic
and methanogenic
bacteria and a determination
of the environmental
factors which influence them. All cores examined
had high methane consumption rates 4-9 cm below
the peat surface. In Thoreau’s Bog, the region is
comprised of partially decomposed peat and is generally unsaturated. The presence of methanotrophic
bacteria here, as determined by activity profiles and
by PLFA analysis agrees with previous reports from
Thoreau’s Bog [2], from several Appalachian mountain peatland ecosystems
[3,8] and from several
Swedish peatlands [9]. The results of the PLFA
analysis clearly demonstrate the presence of methanatrophic organisms in the lower layer of peat (5-7.5
cm depth).
There is considerable variation in the potential for
methane consumption within the top few centimeters
of peat: some cores showed no activity between O-4
cm while others showed levels as high as 70 nmol
g -’ h-r. This is due to spatial variability within the
bog, with some areas having a thinner unsaturated
layer or a more accessible methane source. The drop
in consumption
rates around 9-l 1 cm, just at the
water table, is likely caused by insufficient oxygen
levels, as the supply of oxygen to the methanotrophs
is limited by diffusion through water and the decrease in the peat porosity overlying the water table
[2]. In addition, the PLFA results suggest that both
Group I and Group 2 methanotrophs may be important in methane oxidation.
The K, value for methane uptake (1.6 PM) is
slightly lower than previously reported values. K,
values reported for methane consumption
by soils
and sediments range from 2.5 for a landfill cover soil
[ 151 and 2.2 to 3.7 for a Danish Wetland [ 131 to 8.3
to 10.7 for Lake Washington sediments [33]. Yavitt
et al. [3] reported K, = 3.7 to 42.0 pmol I_ , peat
from intact peat cores of a West Virginia bog. Our
slightly lower result may be due to a lessening of the
mass transfer limitations which are known to influ-
ence methane consumption
under certain conditions
[ 141. Our experiments
were carried out with very
small (1-2
g) samples with a relatively
large
headspace. In these experiments individual peat fibers
were typically exposed to gaseous methane, with
only a thin layer of water on their surface. In contrast, the majority of experimental analyses of sedimentary methane consumption
(e.g. [3,8,13]) use
slurries where the water barrier is much thicker,
resulting in both a longer period of equilibration and
a greater possibility for mass transfer limitation.
The maximal rate of methane consumption (V,,,
= 67 nmol g-’ hh’ ) is similar to those determined
in previous studies. Yavitt et al. [3] noted a V,,, of
11.9 nmol crnm3 hh’ for homogenized peat slurries
at Big Bog Run, although they noted that slurries had
three to eight times lower rates than did intact samples. Methane consumption rates reported for a number of Appalachian peatlands range from 0 to 18.7
PM hh’ ( pmol 1-I peat h- I : [8]). Since I cm’ of
fresh peat from the upper unsaturated zone has an
approximate mass of 0.15 g. the V,,, from Thoreau’s
Bog peat was about 10.0 nmol cmm3 hh ’ . Fechner
and Hemond [2] reported an average in situ consumption rate of 115 nmol cm-’ hh ‘, which when
corrected for the 10 cm depth interval over which
methane consumption occurs gives an in situ methane
consumption
rate of 11.5 nmol cm-j
h- ’ for
Thoreau’s Bog in late summer. This would allow
approximately 75% of the methane they found entering the unsaturated zone to be removed. This rate is
significantly higher than our laboratory values when
we take into account the fact that gaseous methane
concentrations
in the bog are typically 220-9000
nmol ll’ which roughly corresponds to a concentration of 8-330 nM in the aqueous phase of the
unsaturated peat. With a K, of 1.6 FM. the organisms are therefore operating well below their V,,,.
Moore and Knowles [7] have reported a significant influence of the water table depth on methane
emissions from peatlands. The higher the water table
is relative to the surface of the mat, the less methane
oxidation occurs. Although we have not monitored
the flux in the depth of the water table over the
course of our experiments, this type of fluctuation
may explain some of the variability associated with
our results.
A direct influence of temperature on methane
consumption
has been reported in several recent
studies [8,14.15]. Two of these studies [ 14,151 have
shown maximal rates of methane consumption
to
occur at approximately
30 to 40°C. In contrast, the
optimal rates for methane consumption observed in
our studies ranged from 25 to 30°C and varied with
the depth of the peat. The variable effects of temperature on different depths were noted by Williams and
Crawford [I]. although they examined only methane
production, and their samples were from a broader
range of depths. Variations in temperature effects
among samples from different
depths, both in
methane production and oxidation, may be explained
by the in situ temperatures that microorganisms
normally experience. The microbial population near the
surface is subject to large (13°C) daily changes
during the summer and may be better suited to take
advantage of them while bacteria in deeper fractions
are subject to much lower temperatures and smaller
daily changes.
The methanogen population appears to decrease
below 30-35 cm depth. probably reflecting a decrease in available hydrogen and acetate below that
level. Methane production was constant from 2- 1 10
h. making it unlikely that release of previously absorbed methane contributed to the observed rate.
The PLFA profiles showed two significant differences between the top (O-2.5 cm) and the deeper
methane consuming fraction. A significant increase
in the level of methanotrophic bacterial fatty acids as
well as several other increases were observed in the
lower fraction including
1O-methyl saturated fatty
acids and i 17: 1w7c. These latter increases could
have resulted from either an increase in the level of
sulfate reducers. of which Desulfobacter
sp. is
known to contain both 1Ome 16:O and i 17: 1 w7c fatty
acids [31], or an increase in the levels of certain
actinomycetes [32].
Chapman and Hemond [23] have reported that
nitrogen fixation is a major source of nitrogen to
Thoreau’s Bog, based on experiments
using the
acetylene
reduction
assay. We feel that since
methanotrophic
bacteria within peatlands have the
energy available and therefore the potential to be
important peatland nitrogen fixing organisms, the
possibility of their involvement in peatland nitrogen
fixation needed to be explored. Previous studies
looking at peatland nitrogen fixation almost all used
L.R. Krurnhok
et al. / FEMS Microhiolq~
acetylene
[ l8-20,23,34],
a known
inhibitor
of
methanotrophic bacteria [2 I]. The” Nz nitrogen fixation assay requires the assumption that isotopic fractionation during the experiment is insignificant. The
magnitude of isotopic fractionation
associated with
nitrogen cycling is known to be small and is therefore inconsequential
in studies with labeled nitrogen
[30]. Our results, showing no significant influence of
either methane or light on “N, fixation, provide a
strong indication that neither methane oxidizing bacteria nor actively phototrophic bacteria are the dominant nitrogen
fixing organisms
in aerobic peat.
Waughman [34] has reached similar conclusions regarding the influence of light. It has been shown that
ambient concentrations
of oxygen will inhibit nitrogen fixation by one strain of Methylococcus capsulatl4s
in pure culture [22]. However all other nitrogen
fixing strains examined in that paper exhibited activity at ambient oxygen concentrations.
Our field experiments designed to localize in situ nitrogen fixation demonstrated that, under the conditions of the
experiment,
nitrogen fixation took place predominantly in the upper-most layer of the peat cores.
These latter data provided additional evidence that
methanotrophic
nitrogen fixation was not the most
important nitrogen fixation process in the bog.
Acknowledgements
Special thanks to David White at the Center for
Environmental
Biotechnology,
University
of Tennessee, for help with the PLFA analysis. We thank
Harold Hemond for helpful comments,
and Lisa
Snider for technical assistance. The Isotope ratio
analysis was carried out by Robert Michener at the
Boston University Stable Isotope Laboratory. This
work was supported by the Massachusetts Institute
of Technology Undergraduate
Research Opportunities Program.
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