Soil Biology & Biochemistry 54 (2012) 36e47
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Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
pH controls over anaerobic carbon mineralization, the efficiency of methane
production, and methanogenic pathways in peatlands across an
ombrotrophiceminerotrophic gradient
Rongzhong Ye a, Qusheng Jin b, Brendan Bohannan a, Jason K. Keller c, Steven A. McAllister a,
Scott D. Bridgham a, *
a
b
c
University of Oregon, Institute of Ecology and Evolution, 5289 University of Oregon, Eugene, OR 97403, USA
University of Oregon, Department of Geological Sciences, Eugene, OR 97403, USA
Chapman University, School of Earth and Environmental Sciences, Orange, CA 92866, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 December 2011
Received in revised form
15 May 2012
Accepted 16 May 2012
Available online 5 June 2012
Methane (CH4) production varies greatly among different types of peatlands along an ombrotrophice
minerotrophic hydrogeomorphic gradient. pH is thought to be a dominant control over observed
differences in CH4 production across sites, and previous pH manipulation experiments have verified the
inhibitory effect of low pH on CH4 production. In this experiment, we asked (i) if the major effect of low
pH is direct inhibition of one or both pathways of methanogenesis and/or inhibition of ‘upstream’
fermentation that provides substrates for methanogens, and (ii) to what extent is pH sufficient to explain
differences in CH4 production relative to other factors that co-vary across the gradient. To address these
questions, we adjusted the pH of peat slurries from 6 peatlands to 4 levels (3.5, 4.5, 5.5, and 6.5) that
reflected their range of native pH, maintained these pH levels over a 43-day anaerobic laboratory
incubation, and measured a suite of responses within the anaerobic carbon cycle. Higher pH caused
a significant increase in CO2 production in all sites. Regardless of site, time, and pH level, the reduction of
inorganic electron acceptors contributed to <12% of total CO2 production. Higher pH caused acetate
pooling by Day 7, but this effect was greater in the more ombrotrophic sites and lasted throughout the
incubation, whereas acetate was almost completely consumed as a substrate for acetoclastic methanogenesis by Day 43 in the minerotrophic sites. Higher pH also enhanced CH4 production and this process
was up to 436% more sensitive to changes in pH than CO2 production. However, across all sites and pH
levels, CH4 production accounted for <25% of the total gaseous C production. Fermentation appeared to
be the main pathway for anaerobic C mineralization. Our results indicate that low pH inhibits CH4
production through direct inhibition of both methanogenesis pathways and indirectly through its effects
on fermentation, but the direct effects are stronger. The inability of acetoclastic methanogenesis to fully
compensate for acetate pooling in ombrotrophic peats at higher pH suggests that CH4 production is
inhibited by some factor(s) in addition to pH in these sites. We examine a variety of other potential
inhibitory mechanisms and postulate that humic substances may provide an important inhibitory effect
over CH4 production in ombrotrophic peatlands.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
pH
Anaerobic carbon mineralization
Acetate pooling
Efficiency of methane production
Methane pathways
Ombrotrophiceminerotrophic gradient
Peatlands
1. Introduction
Peatlands cover less than 3% of the earth’s land surface, yet they
contain 513 Pg soil carbon (C) (Maltby and Immirzi, 1993), or about
22% of the world’s soil C pool to 3m depth (Jobbágy and Jackson,
* Corresponding author. Tel.: þ1 541 346 1466.
E-mail addresses: [email protected] (R.
(S.D. Bridgham).
Ye),
[email protected]
0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2012.05.015
2000). Wetlands are responsible for between 20 and 40% of
global methane (CH4) emissions, with an important but poorly
defined contribution from northern peatlands (Denman et al.,
2007). The formation and maintenance of peatlands requires
a water table close to the surface (Belyea and Baird, 2006), and thus
a substantial portion of the soil profile normally undergoes anaerobic mineralization, which produces carbon dioxide (CO2) and CH4
as end products. Given that CH4 has approximately 25 times the
global warming potential of CO2 (Forster et al., 2007), it is essential
to understand the fundamental controls over the efficiency and
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
pathways of CH4 production during anaerobic C mineralization in
peatlands.
During anaerobic C mineralization, organic polymers are
initially hydrolyzed into monomers, followed by the fermentation
of monomers to short-chain fatty acids (including acetate), simple
alcohols, dihydrogen (H2), and CO2. These fermentation products
are subsequently oxidized coupled with the reduction of several
inorganic terminal electron acceptors (TEAs), such as nitrate (NO
3 ),
manganese (Mn(IV)), ferric iron (Fe(III)), and sulfate (SO2
4 )
(Megonigal et al., 2004). Humic substances have recently been
identified as important organic TEAs in natural environments, and
may be particularly important in peatland soils (Lovley et al., 1996;
Heitmann et al., 2007; Keller et al., 2009; Lipson et al., 2010). In
general, microbes will preferentially utilize these TEAs, producing
CO2 as a byproduct, before CH4 production becomes important,
resulting in a high ratio of CO2:CH4 produced during anaerobic
respiration. After these more favorable TEAs have been depleted,
methanogenesis dominates as the terminal step of anaerobic C
mineralization. In freshwater wetlands, CH4 production mostly
occurs via acetate disproportionation (acetoclastic methanogenesis) and CO2 reduction by H2 (hydrogenotrophic methanogenesis). Acetoclastic methanogenesis generally accounts for
two-thirds of CH4 production resulting in an overall 1:1 ratio of
CO2:CH4 production under methanogenic conditions (Conrad,
1999).
Different types of peatlands within a landscape are typically
defined by a hydrogeomorphic gradient ranging from
precipitation-fed (ombrotrophic) bogs to groundwater-fed (minerotrophic) fens. The source of incoming water and associated
nutrients and cations explains a range of ecosystem characteristics
(Vitt et al., 1995; Bridgham et al., 1996, 1998). Ombrotrophic bogs
are typically highly acidic (pH 4). Their acidity is primarily
generated from organic acids and secondarily from sequestration of
cations in peat, and their pH reflects the buffering characteristics of
organic acids because by definition all mineral inputs are only
atmospheric (Clymo, 1987; Urban et al., 1987). In contrast, minerotrophic fens receive inputs of water and minerals from groundwater and/or overland runoff, which provide substantially more
mineral-derived alkalinity and higher pHs (Bridgham et al., 1999).
With increasing pH, fens can be further subdivided into acidic fens,
intermediate fens, and rich fens (Sjörs, 1950). In addition to soil pH,
base cation concentrations, soil nitrogen and phosphorus availability, vegetation community structure, and soil carbon quality
vary dramatically along with this gradient.
Given that inorganic TEAs are often at very low concentrations
in peatlands (Vile et al., 2003a; Keller and Bridgham, 2007), it might
be expected that methanogenesis dominates anaerobic C mineralization in these systems (resulting in a 1:1 ratio of CO2:CH4). In
contrast to this expectation, observed CO2:CH4 ratios in peats are
typically much higher and can vary by several orders of magnitude
among different types of peatlands, suggesting distinctive pathways and controls of anaerobic C mineralization (Bridgham et al.,
1998; Segers, 1998; Vile et al., 2003b; Yavitt and Seidman-Zager,
2006; Keller and Bridgham, 2007; Galand et al., 2010). The
predominance of CO2 production in peatlands has been attributed
to either humic substances acting as TEAs (Yavitt and SeidmanZager, 2006; Keller and Bridgham, 2007) or the buildup of
fermentation products (Vile et al., 2003a; Galand et al., 2010).
However, a number of other factors that affect anaerobic C cycling
co-vary along the ombrotrophiceminerotrophic gradient and could
influence the production rates of CO2 and CH4 and the efficiency of
CH4 production (i.e., the CO2:CH4 ratio), including C quality,
nutrient status, and soil pH (Segers, 1998; Blodau, 2002). Furthermore, methanogenic pathways and archaeal community composition differ dramatically along this gradient (Horn et al., 2003;
37
Dettling et al., 2007; Keller and Bridgham, 2007; Kotsyurbenko
et al., 2007). Thus, the relative importance of various factors
controlling observed variations in anaerobic C mineralization in
different peatland types remains elusive.
Of the many potential controlling factors, pH is known to pose
fundamental physiological restrictions on soil microbial communities and therefore impact C mineralization (Goodwin and Zeikus,
1987a). Low rates of CH4 production and high CO2:CH4 ratios are
commonly observed in anaerobic incubations of ombrotrophic
peatland soils (Bridgham et al., 1998; Avery et al., 2002; Galand
et al., 2005; Dettling et al., 2007; Keller and Bridgham, 2007).
Manipulations of soil pH demonstrate that excess acidity contributes to the low CH4 production in these peatlands (Williams and
Crawford, 1984; Dunfield et al., 1993; Kotsyurbenko et al., 2004).
There are a number of potential mechanism by which pH
could regulate anaerobic carbon mineralization and CH4 production, but the importance of these mechanisms across the
ombrotrophiceminerotrophic gradient has not been well studied.
Valentine et al. (1994) suggested that low CH4 production in more
acidic conditions was a result of substrate limitation caused by pH
suppressions of fermentation. Addition of fermentative products,
such as acetate and CO2/H2, has been shown to stimulate CH4
productions in a bog (Bräuer et al., 2004) and an intermediate fen
(Wüst et al., 2009). However, other studies have found an accumulation of acetate in ombrotrophic peatlands and suggested that
low pH causes a disconnect between acetogenesis and acetoclastic
methanogenesis (Shannon and White, 1996; Duddleston et al.,
2002; Keller and Bridgham, 2007). Hydrogenotrophic methanogenesis often dominates in ombrotrophic peatlands whereas
acetoclastic methanogenesis dominates in more minerotrophic
conditions (Duddleston et al., 2002; Galand et al., 2005; Dettling
et al., 2007). Again, manipulative pH experiments suggest that
acidity is a primary control over the relative importance of the
two methanogenesis pathways (Kotsyurbenko et al., 2007). Yavitt
and Seidman-Zager (2006) postulated that soil pH was the
main factor that determines the dominance of acetoclastic versus
hydrogenotrophic methanogenesis in peatlands.
Thus, while it is well established that low pH has a negative
effect on CH4 production, its effects co-vary with many other factors
along the ombrotrophiceminerotrophic gradient in peatlands and
the specific mechanisms for the pH inhibition are unclear.
Accordingly, we asked the following questions in this study: Do
ombrotrophic peatlands have low CH4 production rates solely
because of their low pHs or are other factors also important? Is the
inhibitory effect of low pH due to its direct effect on methanogens
or due to an “upstream” effect on fermentation and production
of H2 and acetate? Does low pH inhibit one methanogenesis
pathway more than another? In the present study, we manipulated
soil pH and analyzed anaerobic C mineralization in six peatlands
with different native pHs (ranging from 3.7 to 6.0) along an
ombrotrophiceminerotrophic gradient. To our knowledge, no other
study has performed a manipulative pH experiment in such a broad
range of peatlands. Our objectives were to determine to what
extent that differences in soil pH among these peatlands control (i)
acetate accumulation, (ii) CH4 and CO2 production rates, (iii) the
efficiency of CH4 production vs. CO2 production, and (iv) the
dominant methanogenic pathway.
2. Materials and methods
2.1. Site description
We sampled six peatlands in the Upper Peninsula of Michigan,
USA that spanned the ombrotrophiceminerotrophic gradient based
upon dominant vegetation and soil pH. Five of these peatlands are
38
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
located on the property of the University of Notre Dame Environmental Research Center and the sixth site is approximately 100 km
east in Crystal Falls, Michigan, USA. Based upon our extensive
previous research in the peatlands of this region and a survey of
more than 20 potential peatlands in 2008 (unpublished data), sites
were chosen to be representative of the region and to incorporate
differences in CH4 production and CO2:CH4 ratios.
Bog 1 (N46 60 600 , W88 160 2500 ) is an ombrotrophic bog with well
developed hummocks and minimal hollows and lawn area. Vegetation is dominated by >90% cover of Sphagnum spp. mosses with
stunted (<1-m height) ericaceous shrubs such as leatherleaf
(Chamaedaphne calyculata (L.) Moench), small cranberry (Vaccinium
oxycoccos L.), and bog Labrador tea (Rhododendron groenlandicum
Oeder), and scattered low-stature black spruce (Picea mariana
(Mill.) Britton, Sterns & Poggen). Peat depth is w380 cm, and the pH
of the surface peat was 3.7 at the time of sampling. The water table
averaged 27 cm during the growing season (all water-table
depths are reported from hollow surfaces). Bog 2 (N46130 5700,
W89 340700 ) has similar vegetation to bog 1 except that it has much
less hummock development. It has a peat depth of w490 cm, and
a pH of 4.1. It had an average water-table depth of 16 cm. The
acidic fen (N46120 4800 , W89 300 200 ) has a Sphagnum spp. lawn with
little cover from other species in the area of sampling. Peat depth is
w500 cm, pH was 4.1, and average water-table depth was 10 cm.
The intermediate fen (N461501700, W89 320 2000 ) also has >90%
cover of Sphagnum spp., the same ericaceous shrubs as the bog
sites, the deciduous shrubs speckled alder (Alnus incana (L.)
Moench, ssp. rugosa (Du Roi) Clausen) and purple chokeberry
(Photinia floribunda (Lindl.) Robertson & Phipps), and a variety of
graminoid and forb species. Peat depth is w340 cm, soil pH was 4.5,
and the average water-table depth was 17 cm. The rich fen site
(N46130 2700, W89 290 5300 ) is dominated by upright sedge (Carex
stricta Lam.) tussocks with leatherleaf also present on the tussocks.
It has w6.4 m of peat, a pH of 5.9, and consistently about 30 cm of
standing water. The cedar swamp site (N46140 4100, W89 320 3800 )
has an overstory of northern white cedar (Thuja occidentalis L.) and
speckled alder and a mixed understory of mosses, forbs, and
unvegetated ground. Peat depth is w290 cm, pH was 6.0, and the
water-table depth averaged 6 cm. Three of the sites (bog 2, the
acidic fen, and the rich fen) bordered lakes, reflecting a common
situation of peat in-filling of lakes in this region, but observations
and differences in chemistry between the lakes and porewater
suggest no significant exchange of surface or groundwater between
the lakes and bog 2 and the acidic fen. In contrast, the rich fen
receives continuous surface water flow from the adjacent lake,
giving it a minerotrophic status.
2.2. Sample preparation
Four soil cores were randomly collected in hollows from each
site with PVC tubes (10-cm diameter) to a depth of 15 cm below the
water table in August 2009. The samples were taken at a relatively
dry period, so the water-table level was 34 cm at bog 1, 23 cm at
bog 2, 15 cm at the acidic fen, 23 cm at the intermediate
fen, þ35 cm at the rich fen, and 16 at the cedar swamp. Upon
extraction, cores were intermediately capped after filling the cores
with porewater to prevent oxidation of the soil. Soil cores and
water samples were transported on ice to our laboratory at the
University of Oregon and frozen at 20 C until use.
In the laboratory, peat cores were processed in a glove box filled
with 98% N2 and 2% H2 (Coy Laboratory Products Inc., Grass Lake,
Michigan, USA). Each core was homogenized following the removal
of large roots, woody material, and green vegetation. Moisture
content was measured on a subsample that was dried at 60 C for 3
days. Rubbed fiber content and the von Post index were determined
on a subsample according to Parent and Caron (2006) and Clymo
(1983), respectively, to estimate the degree of decomposition of
the peats. Lower rubbed fiber content generally indicates a greater
degree of decomposition and hence a lower carbon quality,
although the plant material from which the peat is derived is also
important (Bridgham et al., 2001). In contrast, a low humification
value of the von Post index suggests a lower degree of decomposition. Four additional subsamples were prepared from each core
for the pH manipulation experiment, with individual cores serving
as the replicate unit. Approximately 120 g of field-moist peat was
transferred to a 440 mL Mason jar, slurried with 240 mL of degassed
and deionized water, and adjusted to a pH of 3.5, 4.5, 5.5, or 6.5 with
either 10 N HCl or 10 N NaOH. We adjusted the pH daily on the first
7 days of incubation and once every 2 or 3 days thereafter in the
glove box since pH changes in most of the peat slurries were less
than 0.2 pH units after 7 days. After pH adjustment, peat slurries
were capped and bubbled vigorously with oxygen- free N2 gas for
10e15 min, followed by incubation at 17 C in the dark, which was
the average field temperature when the soil cores were extracted.
2.3. Analyses of porewater chemistry
On the 2nd, 7th, 15th, and 43rd day of incubation, 20 mL of
water was collected from each slurry sample in the glove box, followed by additions of equal amounts of degassed and deionized
water. The dilution effect of these additions was corrected for when
calculating the concentrations of dissolved constituents at different
sampling events. Water samples were centrifuged at 5000 rpm for
5 min and filtered with Whatman GF/F glass-fiber filter paper,
followed by quantification of reduced iron (Fe (II)) as described by
Gibbs (1976). An aliquot of the water sample was frozen at 20 C
for additional chemical analyses. Concentrations of SO2
4 , NO3 , and
)
were
determined
with
a
Dionex
DX500
ion
chromanitrite (NO
2
tography system equipped with an Ionpac AS11 column, an AES
suppressor, and an ED50 electrochemical detector (Dionex Corporation, Bannockburn, Illinois, USA). The detection limits for SO2
4 ,
NO
3 , and NO2 were 4, 16, and 4 mM, respectively. Acetate concentrations were measured with a Dionex DX500 ion chromatography
system equipped with a HC-75 (Hþ) column (Hamilton Company
USA, Reno, Nevada, USA) and a Dionex AD20 absorbance detector,
with a detection limit of 11 mM.
2.4. Analyses of methanogenic pathways and CO2, CH4, and acetate
production
Immediately after collection of water samples and pH adjustment, approximately 10 g of slurried peat was removed from each
Mason jar and transferred into a 40 mL I-CHEM vial (Thermo Fisher
Scientific Inc., Rockwood, Tennessee, USA) and subsequently
bubbled vigorously with oxygen-free N2 gas for 5e10 min. To
estimate potential rates of hydrogenotrophic methanogenesis,
0.1 mL of 3.5 mCi mL1 NaH14CO3 was added to each of the slurries,
followed by gently shaking and incubation at 17 C in the dark. After
incubation for 2 days, slurries were shaken to release trapped gas
bubbles. Headspace gases were then analyzed for CO2 and CH4 by
gas chromatography using a flame ionization detector equipped
with a methanizer (SRI Instruments, Torrance, California, USA).
14
CO2 and 14CH4 were determined simultaneously with an in-line
radioactive gas detector (LabLogic Systems Inc., Brandon, Florida,
USA). Total CO2, CH4, 14CO2, and 14CH4 production were calculated
from both gas and liquid phases, adjusting for solubility, temperature, and pH (Stumm and Morgan, 1995). Rates of respiration and
methanogenesis were calculated from total CO2 and CH4 production during the 2-day incubation and expressed per gram dry
mass peat. Methane production efficiency was defined as CH4
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
39
Table 1
Selected properties of native peat samples across an ombrotrophiceminerotrophic gradient (mean 1 standard error, n 4).
Site
pH
Bog 1
Bog 2
Acidic fen
Intermediate fen
Rich fen
Cedar swamp
3.7
4.1
4.1
4.5
5.9
6.0
0.0
0.0
0.0
0.0
0.0
0.1
von Post index
Rubbed fiber volume content (%)
H3
H3
H3/H4
H4/H5
H5
H5
38
38
35
13
15
11
10
10
9
4
5
3
Fe(II) (mM)
23.3
3.9
1.5
0.9
1.2
0.36
9.3
1.9
0.5
0.4
0.5
0.2
SO2
4 (mM)
18.7
14.9
20.8
25.3
6.0
12.2
Acetate (mM)
6.8
1.8
9.6
8.3
3.6
6.0
BD
20.4
61.4
60.3
94.9
75.6
7.1
29.7
9.0
18.7
21.2
Note: BD, below detection limit; both NO
3 and NO2 were not detected.
production/(CO2 production þ CH4 production) and expressed as
a percentage.
Rates of hydrogenotrophic methanogenesis (CH4,hyd) were
calculated as described by Keller and Bridgham (2007) with slight
modifications: CH4,hyd ¼ a[SCO2]a/Atg, where a is the recovered
activity of 14CH4, SCO2 is the available CO2 pool in the liquid and
gaseous phases, a (¼1.12) is the 14C:12C isotope fractionation factor
for hydrogenotrophic methanogenesis, A is the activity of available
H14CO
3 in the liquid and gaseous phases, t is the incubation time,
and g is the dry mass of peat in the slurry. The size of the CO2 pool in
the peat slurry was minimal initially because of the flushing of the
headspace and reached its maximum at the end of the incubation as
a result of anaerobic respiration, so using SCO2 at the end of the
incubation (as done in Keller and Bridgham, 2007) overestimates
CH4,hyd. We therefore assumed that the increase of CO2 production
during the 2-day incubation was linear and used the mid-point value
of SCO2 to estimate CH4,hyd. Similarly, we assumed that the
consumption of H14CO
3 was linear, and A was calculated from the
activity of H14CO
3 at the mid-point of the incubation. We have
extensively compared both analytical approaches in a number of
experiments and found the “traditional” approach used by Keller and
Bridgham (2007) and others (e.g., Avery et al., 1999) can substantially
overestimate hydrogenotrophic methanogenesis, even to the extent
that it is sometimes greater than total methanogenesis. In the
current experiment, hydrogenotrophic methanogenesis rates were
always lower using the current method, but generally acetoclastic
methanogenesis dominated with both analytical approaches (see
below) and provided similar statistical results.
Rates of acetoclastic methanogenesis were calculated by subtracting hydrogenotrophic production from total CH4 production.
In preliminary experiments, we have found that acetoclastic
methanogenesis rates are underestimated with 14C-acetate, as
reported by Avery et al. (1999). Net acetate production/consumption was calculated as the increase/decrease in acetate concentrations in each slurry between two consecutive time points.
2.5. Statistical analyses
Results were analyzed using restricted maximum likelihood in
the MIXED procedure of SAS 9.1 (SAS Institute) (Littell et al., 2006).
The fixed effects were pH, peat type (site), time, and their interactions with time as a repeated variable. Significant differences
between individual pH levels, sites, and time intervals were
analyzed with Tukey’s test at a ¼ 0.05. All data were tested for
normality and log-transformed if the transform resulted in significant improvements in overall distribution.
3. Results
3.1. Physicochemical analyses
Sites had a wide range of in situ pH (3.7e6.0) that represents the
range typically found along the ombrotrophiceminerotrophic
gradient in this region (Table 1). Initial rubbed fiber volume was
much higher in the ombrotrophic peats than in the minerotrophic
peats (Table 1), indicating the more decomposed nature of the
latter (i.e. less availability of labile C) and the woody origin of the
sapric peat from the cedar swamp. The rubbed fiber values were
relatively low, especially in the ombrotrophic sites, because of the
low water-table at the time that the samples were taken and,
hence, the great depth at which the samples were taken. The von
Post humification score and porewater acetate concentrations
generally increased along the ombrotrophiceminerotrophic
gradient (Table 1). Nitrite and NO
3 were not detected in any of
the water samples during the course of experiment. Initial SO2
4
concentrations were 25 mM but were below detection in all
samples on the 2nd day of incubation and thereafter (Table 1).
Fe(II) concentrations increased as pH decreased, with a large
increase in concentration during the first week at pH 3.5 and 4.5
(Table 2, Fig. 1). At pH 3.5 and 4.5, samples from the more minerotrophic sites (intermediate fen, rich fen, and cedar swamp)
consistently had higher Fe(II) concentrations than those from other
sites (Fig. 1).
Acetate concentrations were also significantly influenced by pH
adjustment, with complicated interactions among pH, site, and
incubation time (Table 2). On Day 2, pH had little effect on acetate
concentrations in any of the samples (Fig. 2a). Acetate concentrations increased dramatically between Day 2 and Day 7 in most
samples within each pH level, especially those from the more
ombrotrophic sites (Fig. 2a, b). Acetate concentrations were
Table 2
Effect of pH, peat type, and incubation time on selected variables of peat samples.
Source
DF
F-values
Fe(II)
Acetate
CO2
CH4
CH4/(CO2 þ CH4)
Acetoclastic CH4
Hydrogenotrophic CH4
pH
Peat
Time
pH*Peat
pH*Time
Peat*Time
pH*Peat*Time
3
5
3
15
9
15
45
529.8**
27.5**
21.6**
14.9**
4.9**
2.8**
1.1
13.8**
77.4**
182.4**
4.0**
4.4**
32.7**
3.1**
99.5**
60.5**
117.0**
2.1**
1.9
1.7*
1.2
134.8**
96.5**
26.4**
2.9**
3.1**
4.1**
0.7
41.1**
100.9**
112.8**
2.4**
1.5
3.8**
0.8
96.8**
66.5**
31.4**
3.5**
1.7
2.6**
0.8
91.4**
62.4**
7.32**
1.85*
3.2**
4.9**
1.7
Note: *, p < 0.05; **, p < 0.01.
40
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
Fig. 1. Fe(II) concentrations in peat samples across an ombrotrophiceminerotrophic
gradient (from left to right) adjusted to various pH levels. Bars indicate mean 1
standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
typically greater at higher pH after Day 2 in peat from the three
more ombrotrophic sites (bog 1, bog 2, and acidic fen) and in
intermediate fen peat on Days 7 and 15, but pH had little effect on
acetate concentrations in peat from the two most minerotrophic
sites (rich fen and cedar swamp), which remained low throughout
the experiment. Acetate concentrations also decreased gradually
from the Day 7 to Day 43 at pHs > 4.5 in rich fen and cedar swamp
peat and from Day 15 to Day 43 in intermediate fen peat
(Fig. 2bed).
3.2. CO2 production
There was a positive effect of increasing pH on CO2 production
rates across the peatland gradient, although that effect varied
Fig. 2. Acetate concentrations in peat samples across an ombrotrophiceminerotrophic
gradient (from left to right) adjusted to various pH levels. Bars indicate mean 1
standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
somewhat by peat type (Table 2). By Day 2 an increase in pH from
3.5 to 6.5 increased the rates of CO2 production by 607% in bog 1,
332% in bog 2, 175% in the rich fen, and 208% in the cedar swamp
(Fig. 3a). These pH effects were maintained throughout the experiment, although increases in peat from individual sites were not
always significant (Fig. 3). In general, CO2 production rates
decreased from Day 2 to Day 7 and remained relatively stable
thereafter.
3.3. CH4 production
Methane production rate also increased as pH increased, but
this effect varied greatly with site and time (Table 2, Fig. 4). On Day
2, the CH4 production rate was up to 12 times smaller at a pH of 3.5
compared to higher pHs in peat from the intermediate fen and rich
fen, with no pH effect in peat from the other sites (Fig. 4a). On Days
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
41
Fig. 4. Rates of total methanogenesis in response to pH adjustment in peat samples
across an ombrotrophiceminerotrophic gradient (from left to right). Bars indicate
mean 1 standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
Fig. 3. Rates of anaerobic CO2 production in response to pH adjustment in peat
samples across an ombrotrophiceminerotrophic gradient (from left to right). Bars
indicate mean 1 standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
7, 15, and 43, lower pH typically had a strong inhibitory effect on
CH4 production rates in all sites, with CH4 production rates often
several orders of magnitude greater at a pH of 6.5 compared to a pH
of 3.5 (Fig. 4bed). At pH > 3.5, the rates of CH4 production generally
increased from Day 2 to Day 15 (Fig. 4a, b). One of the bog sites (bog
2) was an outlier with very small CH4 production rate overall and
no significant pH effect.
Methane production efficiency (CH4/(CO2 þ CH4)) was significantly affected by pH, which was dependent on site but not time of
incubation (Table 2). Averaged across time, increasing pH from 3.5
to 6.5 enhanced CH4 production efficiency by 159%e334% (Fig. 5).
Bog 2 was again an outlier with very low CH4 production efficiency
and no effect of pH. Within each pH level, the rich fen generally had
the highest CH4 production efficiency, followed by the intermediate
fen, cedar swamp, acidic fen, bog 1, and bog 2 (Fig. 5). Interestingly,
CH4 production efficiency was consistently highest at pH 5.5
regardless of the site, although it was not always statistically
different from pH 4.5 and 6.5 (Fig. 5).
On Day 2, rates of acetoclastic CH4 production were 3e12
times smaller at pH 3.5 than at higher pHs in the three most
minerotrophic peats (intermediate fen, rich fen, and cedar
swamp), but there was no effect in peat from the other sites
(Fig. 6a). Acetoclastic methanogenesis was inhibited by low pH in
peat from all sites on the last three sampling dates, except that peat
from bog 2 had low overall rates and no significant pH effect until
Day 43. On Day 15, the rates of acetoclastic methanogenesis in peat
from the intermediate fen and rich fen were much higher than the
three most ombrotrophic peats (bog 1, bog 2, and acidic fen), which
was also observed on Day 43 (Fig. 6c, d). Rates of acetoclastic
methanogenesis in most samples increased gradually from Day 2 to
Day 15, especially in peat from the intermediate fen, rich fen, and
cedar swamp at pH > 4.5 (Fig. 6aec). However, the rates decreased
after Day 15 except in peat from the intermediate fen.
Low pH generally inhibited hydrogenotrophic methanogenesis
in peat from all sites throughout the incubation (Fig. 7). The pH
effect was not significant in acidic fen peat on Day 2 and in peat
from bog 2 and the intermediate fen on Day 7 (Fig. 7a, b). There
were large changes in rates through time but this varied by site.
42
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
Fig. 5. pH effects on the efficiency of CH4 production relative to total gaseous C
(CO2 þ CH4) production in peat samples across an ombrotrophiceminerotrophic
gradient (from left to right). Bars indicate mean 1 standard error.
The contribution of acetoclastic methanogenesis to total CH4
production in peat from bog 1 generally decreased as pH increased
from 3.5 to 6.5, though the difference was not always significant
(Fig. 8). Similar pH effects were also observed in bog 2 on Day 43
and in the acidic fen on Day 15, but not in the three most minerotrophic sites (intermediate fen, rich fen, and cedar swamp) (Fig. 8).
However, regardless of pH, acetoclastic methanogenesis dominated
the total CH4 production (>54%) in all the peats, especially after Day
2 (>66%).
4. Discussion
In the present study, we addressed the questions of whether
the differences in pH in peatlands across an ombrotrophice
minerotrophic gradient are sufficient to explain the large variation in the efficiency of CH4 production and what are the underlying mechanisms of this pH effect in the context of the entire
anaerobic C cycle. As with any manipulative biogeochemical laboratory study, extrapolation of rates to in situ conditions should be
done with extreme caution, and even more so in an experiment
where something as fundamental as pH is changed with its myriad
effects on abiotic and biotic processes. However, like many other
manipulative studies, our experiment provides insights to understanding mechanisms that cannot be achieved with a comparative
field approach. Overall our results indicate that, as we expected,
low pH is an important control over anaerobic C mineralization and
CH4 production in peatlands, but other factors are of at least equal
importance in explaining low rates of CH4 production and low CH4
production efficiency in ombrotrophic peatlands. Based upon the
current experiment and ancillary experiments, we examine other
factors likely to inhibit CH4 production.
4.1. CO2 production
Our results suggest strong pH limitation on anaerobic C
mineralization as increasing pH from 3.5 to 6.5 greatly stimulated
rates of CO2 production in all sites across the peatland gradient,
but particularly in bog 1 and bog 2 on Day 2 (Fig. 3). The two bogs
had the lowest native pH along with high rubbed fiber contents
and low humification scores (i.e. high C quality) (Table 1). When
potential pH limitation in these bog soils was removed, more C
substrates were made available to microbial decomposers through
Fig. 6. Rates of acetoclastic methanogenesis in response to pH adjustment in peat
samples across an ombrotrophiceminerotrophic gradient (from left to right). Bars
indicate mean 1 standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
increased fermentation, as evidenced by greater CO2 and acetate
production.
We did not quantify denitrification or sulfate reduction rates,
but we assume that they had minimal contribution to anaerobic C
mineralization given that neither NO
3 nor NO2 were detected in
concentrations
were
below
the detection
our samples and SO2
4
limit after 24 h. Low rates of these processes are common in many
northern peatlands (Blodau et al., 2002; Duddleston et al., 2002;
Vile et al., 2003a), although high rates of sulfate reduction have
been observed in some peatlands during short-term incubations
(Wieder et al., 1990; Vile et al., 2003b).
Iron reduction is also generally assumed to be unimportant in
northern peatlands due to low availability of Fe(III) (Blodau
et al., 2002). Although we did not measure the concentrations of
Fe(III) directly in the present study, our results clearly indicated
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
Fig. 7. Rates of hydrogenotrophic methanogenesis in response to pH adjustment in
peat samples across an ombrotrophiceminerotrophic gradient (from left to right). Bars
indicate mean 1 standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
substantial accumulation of Fe(II) at pH 4.5, especially in
more minerotrophic peats (Fig. 1). More minerotrophic peatlands
generally have much higher peat mineral content (Bridgham et al.,
2001; Gorham and Janssens, 2005), but more ombrotrophic peatlands have higher porewater iron concentrations because iron is
more soluble at low pH (Vitt et al., 1995). We demonstrated the
interaction of these factors in this study, with maximal porewater
Fe2þ concentrations in minerotrophic peats at low pH. Using the
increase in Fe(II) concentration over time and the ratio of 4 mol
Fe(III) reduced to 1 mol CO2 mineralized to estimate the carbon
equivalents of Fe reduction (Megonigal et al., 2004), our results
demonstrate that Fe reduction contributed <12% of total CO2
production at pH 3.5 and <3% at pH 4.5. In a separate experiment
we measured Fe reduction rates by the difference in oxalateextractable Fe(II) under in situ conditions using peat from the
same sites and sampling event as the current experiment. We
found that Fe reduction accounted for <8% of anaerobic CO2
production and was much lower in most samples (data not shown).
Since Mn(IV) concentrations in peat soils are much less than Fe(III)
43
Fig. 8. pH effects on the percentage of acetoclastic CH4 production relative to total CH4
production in peat samples across an ombrotrophiceminerotrophic gradient (from left
to right). Bars indicate mean 1 standard error. a, Day 2; b, Day 7, c, Day 15; d, Day 43.
(Gorham and Janssens, 2005), we assumed that Mn reduction was
negligible in the present study.
Acetoclastic methanogenesis produces CO2, whereas hydrogenotrophic methanogenesis consumes CO2. In the current experiment there was a net production of CO2 by these two processes
because acetoclastic methanogenesis was the dominant pathway
(see below). We used the ratio of 1 mol acetate oxidized to 1 mol
CO2 generated during acetoclastic methanogenesis and the ratio of
1 mol CO2 reduced to 1 mol of CH4 produced during hydrogenotrophic methanogenesis to calculate the CO2 balance during
the CH4 production (Conrad, 1999). The net production of CO2
during methanogenesis explained only 1e25% of the CO2
production.
In sum, methanogenesis and the reduction of inorganic TEAs can
only explain a very small proportion of CO2 production, especially
at higher pH where Fe reduction was unimportant and respiration
rates were highest. Large amounts of CO2 production that cannot be
44
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
explained by the consumption of inorganic TEAs has been repeatedly demonstrated in peatlands (Duddleston et al., 2002; Vile et al.,
2003a and b; Yavitt and Seidman-Zager, 2006; Keller and
Bridgham, 2007). Galand et al. (2010) found that anaerobic CO2
production in a minerotrophic fen occurred primarily via acetogenesis, whereas it was primarily through an unknown pathway in
an oligotrophic fen and a bog. Keller and Bridgham (2007) examined anaerobic C pathways seasonally in a bog, intermediate fen,
and rich fen in northern Michigan, where 29e85% of the CO2
production was through an unknown pathway, with a somewhat
higher percentage in the rich fen. In the current study, we found
that an unexplained pathway contributed 60e99% of CO2 production, with lower percentages in the three most minerotrophic sites
(intermediate fen, rich fen, and cedar swamp) at pH 4.5.
Some have suggested that the unexplained CO2 production in
peatlands is due to humic substances acting as organic electron
acceptors (Segers, 1998; Yavitt and Seidman-Zager, 2006;
Heitmann et al., 2007; Keller and Bridgham, 2007). Humic
substances may act indirectly as electron shuttles for Fe and sulfate
reduction or act directly as electron acceptors (Heitmann et al.,
2007), but since Fe and sulfate reduction rates were likely
minimal in this experiment, the direct effect as an electron
acceptor is most pertinent for this experiment. Reduction of humic
substances is more thermodynamically favorable than methanogenesis (Cervantes et al., 2000) and both concentrations of dissolved and solid humic substances are typically very high in
peatlands (Thurman, 1985). Studies have also demonstrated clear
inhibitory effects of humic substances on methanogenesis either
by substrate competition or by direct toxicity to methanogens
(Cervantes et al., 2000; Keller et al., 2009; Minderlein and Blodau,
2010). Humic substances are important electron acceptors and
inhibit methanogenesis in peats from our study sites (unpublished
data). Nonetheless, the peats utilized in the current experiment
were collected and processed under anaerobic conditions and the
humic substances may have already been completely reduced. It is
therefore doubtful that the effect of humic substances is substantial
enough to explain the remaining CO2 production and low CH4
production observed in the current experiment.
Many fermentative processes produce CO2 (Reddy and DeLaune,
2008), and it has been suggested that the unexplained CO2
production may be explained by the buildup of fermentative low
molecular weight alcohols and acids (Duddleston et al., 2002; Vile
et al., 2003a and b; Galand et al., 2010). In the present study, we
observed significant acetate pooling along with increasing CO2
production (Figs. 2 and 3). It is likely that fermentation is the ultimate explanation for the unexplained CO2 production in the
current experiment.
The consistently higher CO2 and acetate production with an
increase in pH in all sites (Figs. 2 and 3) indicates a fundamental pH
limitation of fermenters and rates of fermentation (Goodwin and
Zeikus, 1987b; Valentine et al., 1994; Bergman et al., 1999).
Increased pH also greatly increases the solubility of organic matter
in wetland soils (Clark et al., 2005; Grybos et al., 2009), which may
increase the availability of labile C to microbes and hence enhance
CO2 production.
4.2. Acetate dynamics
Porewater acetate concentrations vary greatly over time in
peatlands (Shannon and White, 1996; Keller and Bridgham, 2007),
reflecting the temporal dynamics of production and consumption
mechanisms. These concentrations were initially low in all samples,
and particularly so in the bog peats (Table 1). This is potentially
because the samples were collected in mid-summer when the
water table was relatively far from the surface, providing an
extended aerobic zone that would promote the aerobic degradation
of acetate, while also reducing the anaerobic zone for acetate
production and accumulation.
It is generally thought that under anaerobic condition acetate is
mostly derived from incomplete oxidation of organic C in heterotrophic respiration with TEAs and fermentation reactions (Reddy
and DeLaune, 2008). As discussed previously, heterotrophic respiration with TEAs likely played a minor role in C mineralization in
the present study and hence was not a significant source of acetate
production. Acetate can also be produced through the process of
homoacetogenesis (i.e., acetate formation from CO2 and H2), but
thermodynamics suggests that hydrogenotrophic methanogens
should outcompete homoacetogens for their common substrate, H2
(Zinder and Anguish, 1992), except possibly at low temperature
when H2 is sufficient (Hoehler et al., 1999; Kotsyurbenko et al.,
2001). In the present study, we did not measure H2 concentrations, but we did determine rates of homoacetogenesis in samples
near their native pHs by quantifying the incorporation of 14CO
3 into
acetate (unpublished data). In contrast to accepted theory, homoacetogenesis was an important (if not dominant) pathway of
acetate production in these peats at least their native pHs. Thus,
fermentation reactions were likely the dominant source of acetate
in our experiment.
Acetate rarely accumulates in most anaerobic environments
because of its high turnover rates (King et al., 1983; Kotsyurbenko
et al., 2004). However, accumulation of acetate has been documented repeatedly in acidic bogs (Shannon and White, 1996; Avery
et al., 1999; Duddleston et al., 2002; Keller and Bridgham, 2007;
Hines et al., 2008). Much greater acetate pooling is often observed
in ombrotrophic peatlands than in minerotrophic peatlands (Keller
and Bridgham, 2007; Hines et al., 2008), although Galand et al.
(2010) observed the opposite trend. These studies have ascribed
this phenomenon to a variety of factors that co-vary along the
ombrotrophiceminerotrophic gradient, including plant community composition, microbial community composition, and the low
nutrient and low pH characteristics of ombrotrophic peats.
Acetate accumulated to very high concentrations throughout
the incubation in the two bog peats and the acidic fen peat, especially at higher pHs (Fig. 2), indicating much greater acetate
production relative to consumption by acetoclastic methanogenesis
and other potential pathways, such as TEA reduction. The higher
rates of acetate production in the more ombrotrophic sites at
higher pH likely resulted from the less decomposed nature of the
ombrotrophic peats (i.e., more labile C) and hence greater rates of
fermentation when the native pH limitation of these peats was
ameliorated. In the intermediate fen peat, there was similar acetate
accumulation through Day 15, but there was substantial acetate
consumption by Day 43 at pHs 5.5 and 6.5, likely because of high
rates of acetoclastic methanogenesis at these pHs during this
period (Fig. 6). The rich fen and cedar swamp peats had a much
lower degree of acetate pooling, and almost all of the acetate was
consumed at pH > 3.5 by the end of the incubation. These minerotrophic peat types likely had lower rates of acetate production
than the more ombrotrophic peats because their rates of acetoclastic methanogenesis were not substantially greater, although
other pathways of acetate consumption cannot be completely discounted. Low rates of acetate production in the rich fen and cedar
swamp samples are likely attributable to their more decomposed
nature of the peats (i.e., less labile C for fermentation) inferred from
their higher humification scores and lower rubbed fiber contents
(Table 1).
We demonstrate that the acetate pooling sometimes observed
in ombrotrophic peats is not due solely to their naturally low pH,
because we found even greater acetate accumulation in these sites
at higher pH. We surmise that acetate pooling in these sites is due
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
to their low rates of acetoclastic methanogenesis, reflecting their
low native pH and other potential inhibitory factors, including
humic-like substance derived from Sphagnum spp. mosses, as
discussed below.
4.3. Methanogenesis
Methanogens exist in peat soils across a wide range of pH
(Dunfield et al., 1993; Horn et al., 2003; Kotsyurbenko et al., 2004,
2007). Dunfield et al. (1993) found that optimal pH values for
methanogenesis were about 2 pH units higher than the native pH in
acidic peats and only 0e1 unit higher in near neutral peats. Our pH
manipulation experiment revealed that raising a native pH of 3.7
(bog 1) by 2e3 pH units resulted in up to 697% increase in CH4
production, while reducing a native pH of 6.0 (cedar swamp) by
2e3 pH units suppressed CH4 production potentials by up to 2105%
(Fig. 4), indicating strong pH limitation on methanogenesis across
the ombrotrophiceminerotrophic gradient, which agrees with
other manipulative studies (Williams and Crawford, 1984; Dunfield
et al., 1993; Valentine et al., 1994; Bräuer et al., 2004). Studies of
pure cultures of methanogens also demonstrate pH effects (Sizova
et al., 2003; Bräuer et al., 2006).
Our results demonstrate that increasing pH increased the efficiency of CH4 production across the gradient, with a maximum
percentage consistently at a pH of 5.5 (Fig. 5). Increasing pH
increased fermentative activity and generally the availability of
acetate (and presumably H2), and this increase in methanogenic
substrates is likely partially responsible for the increased efficiency
of CH4 production. Regardless of the pH, CH4 production contributed <25% of total gaseous C production (CO2 þ CH4) (Fig. 5), in
contrast to the optimal ratio of efficiency of 50% in a purely
methanogenic system (Zinder, 1993). A lower CH4 efficiency in
ombrotrophic versus minerotrophic peats at their native pHs
has been repeatedly demonstrated in previous studies (e.g.,
Bridgham et al., 1998; Yavitt and Seidman-Zager, 2006; Keller and
Bridgham, 2007). Even after experimentally increasing the pH in
ombrotrophic peats, CH4 efficiency was generally much lower than
in more minerotrophic peats with the same pH despite relatively
minor differences in CO2 production and increased acetate accumulation. This suggests that while pH is important, differences in
CH4 production across the ombrotrophiceminerotrophic gradient
are controlled by other factors in addition to soil pH.
Peat derived from different plant sources has very different C
quality and CH4 production rates (Yavitt et al., 1987; Bridgham et al.,
1998), but the pooling of acetate and the dominance of acetoclastic
methanogenesis in the ombrotrophic sites strongly suggest that the
low CH4 efficiency in these sites cannot be explained predominantly by C quality. However, the role of acetate as a substrate
for acetoclastic methanogenesis is complicated by its role as an
organic acid at low pH (pK ¼ 4.7) and its consequent inhibitory
effect on acetoclastic methanogens (Russell, 1991; Bridgham and
Richardson, 1992). Bräuer et al. (2004) suggested that acetate can
be utilized by acetoclastic methanogens when concentrations are
less than 5e10 mM. In the present study, none of the peat samples
had acetate concentrations >2 mM, suggesting acetate protonation
was unlikely to cause significant inhibitory impacts.
A number of additional physiochemical variables vary along
the ombrotrophiceminerotrophic gradient and could play a role
in regulating CH4 dynamics in peatlands. For example, methanogenesis may be limited in ombrotrophic peats by trace metal
limitation (Basiliko and Yavitt, 2001), but we have found no such
limitation in peat from our sites (unpublished data). Nutrient
availability also differs across this gradient, but previous work
suggests that nutrient limitation does not fully explain differences in
anaerobic carbon cycling in peatlands (e.g., Keller et al., 2005, 2006).
45
An alternative explanation for low CH4 production potential in
more ombrotrophic peats is that Sphagnum mosses have high
concentrations of humic-like substances (Williams et al., 1998) that
can be quite inhibitory to microbes (Aerts et al., 1999; Minderlein
and Blodau, 2010). It is possible that methanogens are particularly sensitive to these compounds. Hines et al. (2008) found low
CH4 efficiency in peatlands dominated by Sphagnum spp. mosses
and that CH4 efficiency increased with an increasing proportion of
vascular plant cover. Our results are consistent with the hypothesis
that increasing Sphagnum spp. cover decreases CH4 efficiency: plant
cover was dominated by Sphagnum spp. in the two bogs and acidic
fen, was somewhat less dominant in the intermediate fen, was
absent in the rich fen because of the consistently high water table,
and was a subdominant ground layer in the cedar swamp. In
a subsequent experiment we added the humic-substance analog,
anthraquinone-2,6-disulfonate (AQDS), to peat from the bog 1 and
the rich fen, and it was highly inhibitory of methanogenesis in bog 1
but acted as an organic TEA in the rich fen (unpublished data).
These findings together are suggestive that methanogenesis in
ombrotrophic peatlands is strongly inhibited by humic-like
substances that may be derived from Sphagnum spp. mosses.
Our experiment demonstrates that low pH limits methanogenesis rates and CH4 efficiency, but it is also clear that other factors
are important in limiting methanogen activity in all peatlands,
and particularly in ombrotrophic peatlands. Despite substantial
research to date on this topic, the mechanisms limiting methanogenesis in peatlands remain elusive; however the possibility that
organic substances directly inhibit methanogenesis deserves
considerably more attention.
4.4. Methanogenic pathways
In freshwater habitats, CH4 is theoretically produced from
acetate and H2/CO2 at a ratio of 2:1 (Conrad, 1999). While CH4
production in most natural anaerobic habitats follows this ratio
relatively closely, peatlands are often an exception. More minerotrophic surface peats typically are dominated by acetoclastic
methanogenesis, whereas more ombrotrophic peats, deep peat,
and far northern peats are often dominated by hydrogenotrophic
methanogenesis (Chasar et al., 2000; Keller and Bridgham, 2007;
Galand et al., 2010). It has been suggested that low pH is the main
factor that determines the dominance of hydrogenotrophic methanogenesis in ombrotrophic peats (Horn et al., 2003; Yavitt and
Seidman-Zager, 2006; Kotsyurbenko et al., 2007).
Our results clearly demonstrate that higher pH stimulated the
rates of both hydrogenotrophic and acetoclastic CH4 production in
samples across the peatland gradient (Figs. 6 and 7); however pH
did not have substantial effects on the relative importance of the
two pathways (Fig. 8). Acetoclastic methanogenesis generally
accounted for greater than the expected 66% of CH4 production in
all sites after Day 2 and hydrogenotrophic methanogenesis never
dominated, even in the two bog sites at the lower pH. These results
are in contrast to those of Kotsyurbenko et al. (2007), who found
a shift from acetoclastic to hydrogenotrophic methanogenesis in
a peat with a moderately acidic native pH (4.8) as pH decreased
from 6.0 to 3.8 at temperatures 15 C. They also isolated an
acidophilic hydrogenotrophic methanogen of the genus Methanobacterium that was postulated to be primarily responsible for
the hydrogenotrophic CH4 production at low pH in their system.
Methanobacterium was the dominant methanogen genus in all of
our sites in a May 2010 sampling, but bog 1 also had the highest
proportion of Methanosarcina, a genus that is putatively acetoclastic
(unpublished data).
It is possible that in our experiment bubbling the slurries at the
beginning of the CH4 pathway measurements reduced CO2 and H2
46
R. Ye et al. / Soil Biology & Biochemistry 54 (2012) 36e47
levels enough to limit hydrogenotrophic methanogenesis. But we
have found in parallel experiments that CO2 quickly accumulates in
the headspace and would only be potentially limiting for a brief
period, whereas H2 concentrations are always low even after
extended incubations indicating rapid coupling of production and
consumption. These H2 dynamics are typical of other peatland
studies (Horn et al., 2003; Yavitt and Seidman-Zager, 2006).
As discussed above, in an ancillary experiment we found
substantial rates of homoacetogenesis in peats near their native
pHs (unpublished data). Homoacetogenesis may affect the relative
dominance of methanogenic pathways, because acetate is the
substrate for acetoclastic methanogenesis and homoacetogens
potentially compete for H2 with hydrogenotrophic methanogens.
Thus, the low contribution of hydrogenotrophic methanogenesis to
CH4 production may have been at least partially due to high rates of
homoacetogenesis.
It has also been suggested that hydrogenotrophic methanogenesis is more dominant under situations of low C quality
(Hornibrook et al., 1997). Our results contradict this hypothesis as
hydrogenotrophic methanogenesis was greatest in the bog sites at
higher pH (Fig. 8), where we also observed the highest CO2 production and acetate accumulation (Figs. 2 and 3). Overall, heterotrophic
activity (CO2 production þ acetate production) was uncorrelated
with % acetoclastic methanogenesis (r ¼ 0.03, p ¼ 0.61).
Overall, our results suggest that pH is not a major factor in the
relative importance of the two methanogenesis pathways in peatlands. Given the contradictory results of our study and others in the
literature, we suggest that further experiments are necessary to
examine the interaction of pH, C availability, homoacetogenesis,
and temperature on the relative contribution of the CH4 pathways
in a broad range of peat types.
5. Conclusions
pH adjustment significantly altered many facets of anaerobic C
mineralization in six peat types with a broad range of native pH, but
not always in expected ways. Both CO2 and acetate production were
greater at higher pH in all sites in the early stage of experiment,
demonstrating a general pH control over fermentation in peatlands
across the ombrotrophiceminerotrophic gradient. Acetoclastic CH4
production accounted for <25% and the reduction of various TEAs
contributed to <12% of total gaseous C production, indicating
that fermentation was the major pathway for anaerobic C mineralization across the different peat types and pHs. Increased pH
promoted significant acetate accumulation in ombrotrophic
peats, mainly because of enhanced acetogenesis, whereas acetate
production and consumption were tightly coupled in minerotrophic peats. Our results suggest that the acetate accumulation
often observed in ombrotrophic peatlands is not mainly driven by
their low pH.
More C flow was directed to methanogenesis as pH increased to
a pH of 5.5. Methane production via the hydrogenotrophic pathway
was not greater at lower pHs, suggesting that pH alone is not the
reason that this pathway is often so important in ombrotrophic
peatlands under field conditions. Stimulation of both hydrogenotrophic and acetoclastic methanogenesis at higher pH may
have been a direct pH effect or due to an increase in substrate
availability, but the large acetate pooling in the ombrotrophic peats
at higher pH indicates that acetoclastic methanogens were not able
to adjust to their environmental conditions as rapidly as acetogenic
bacteria over the extended incubation period. Similar disequilibrium between acetate production and consumption may explain
the temporary acetate pooling often observed in situ. The relatively
low CH4 efficiency in the more ombrotrophic peats across pHs
suggests that pH alone does not explain the low CH4 production in
these types of peatlands. Overall, this study suggests that pH is
a primary factor controlling fermentative activity in peatlands. pH
is also an important control over rates of CH4 production, but other
factors are likely of equal or greater importance in explaining the
low CH4 production typically observed in ombrotrophic peatlands.
Acknowledgments
We thank N.M. Eisenhut and A. Harvey for their help during the
experiment, James Ziemer and Yvonne Ziemer for access to their
private field site, and the University of Notre Dame Environmental
Research Center for access to field sites and laboratory facilities.
This work was supported by NSF grant DEB-0816575. A. Harvey
was supported by the University of Oregon Summer Program
for Undergraduate Research. Comments from two anonymous
reviewers greatly improved this manuscript.
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