Methanotrophy potential versus methane supply by pore water diffusion in peatlands

Low a ﬃ nity methanotrophic bacteria consume a signiﬁcant quantity of methane in wet-land soils in the vicinity of plant roots and at the oxic-anoxic interface. Estimates of the e ﬃ ciency of methanotrophy in peat soils vary widely in part because of di ﬀ erences in approaches employed to quantify methane cycling. High resolution proﬁles of dis-5 solved methane abundance measured during the summer of 2003 were used to quantify rates of upward methane ﬂux in four peatlands situated in Wales, UK. Aerobic incubations of peat from a minerotrophic and an ombrogenous mire were used to determine depth distributions of kinetic parameters associated with methane oxidation


Introduction
Alpha and gamma Proteobacteria belonging, respectively, to the Methylocystaceae and Methylococcaceae families are ubiquitous at oxic-anoxic interfaces in the Earth system where oxygen (O 2 ) is present and methane (CH 4 ) is transported in large quantities under the influence of concentration gradients or ebullition.These microorganisms, also known as Type I (gamma) and Type II (alpha) methanotrophs, serve as an efficient filter, removing CH 4 that otherwise would enter the troposphere.Collectively low affinity methanotrophs in such environments annually consume a quantity of CH 4 well in excess of the ∼600 Tg that does enter the Earth's atmosphere from biological and geological sources (Mikaloff Fletcher et al., 2004).
The anoxic soils of natural wetlands are one of the main perennial sources of CH 4 flux that help to maintain a low but significant quantity of this chemically and radiatively active organic gas in the Earth's highly oxidizing atmosphere.More than three decades of study of methanotrophs in wetlands and peatlands have yielded significant insights into their phylogeny, distribution, kinetics, and preferred growth conditions (e.g., Segers, 1998;Gutknecht, 2006;Chen et al., 2008).Methanotroph populations in the rhizosphere and with depth in peat soils have been mapped using molecular biology techniques, including PCR amplification of DNA extracts and hybridisation with specific phylogenetic 16S rRNA and functional gene primers (e.g., Krumholz et al., 1995;Mc-Donald et al., 1996, 1999;Ritchie et al., 1997;Calhoun and King, 1998;Edwards et al., 1998;Dedysh, 2002;Dedysh et al., 2001Dedysh et al., , 2003;;Wartiainen et al., 2003;Miller et al., 2004), quantification of membrane phospholipid fatty acids (PLFAs) (Krumholz et al., 1995;Sundh et al., 1995Sundh et al., , 1997)), and more recently stable isotope probing techniques involving 13 C-labelling of PLFAs and nucleic acids (Morris et al., 2002;McDonald et al., 2005;Kreuzer-Martin, 2007;Chen et al., 2008).Both Type I and II methanotrophs occur in wetland soils, occupying oxic zones immediately adjacent to plant roots (King, 1994(King, , 1996;;Schipper and Reddy, 1996;Calhoun and King, 1998;van der Nat and Middelburg, 1998;Popp et al., 2000) and shallow zones within peat soils to which at-Introduction

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Tables Figures Full Screen / Esc Printer-friendly Version Interactive Discussion mospheric O 2 is able to diffuse under edaphic conditions and vegetation groundcover specific to particular types of wetlands (Krumholz et al., 1995;McDonald et al., 1996;Watson et al., 1997;Edwards et al., 1998;Beckman and Lloyd, 2001;Megonigal and Schlesinger, 2002).The tolerance of methanotrophs to anoxia appears to vary (Roslev and King, 1996).
Greatly diminished levels of methanotrophic activity have been reported in post-anoxia incubations of rhizome material (King, 1994) while sediment and peat from other wetlands upon return to O 2 -rich conditions have shown CH 4 oxidation capacities ranging from moderately attenuated (e.g., King, 1990) to rapid and vigorous (Whalen and Reeburgh, 2000).Methane supply is most commonly cited as the factor limiting methanotrophy in peat soils (Boon and Lee, 1997;Megonigal and Schlesinger, 2002;Berestovskaya et al., 2005;Basiliko et al., 2007 ) although O 2 availability also may restrict rates of CH 4 uptake (King, 1990(King, , 1994(King, , 1996;;Mikkel ä et al., 1995;Beckman and Lloyd, 2001).Differences in the limiting factors between peatlands likely results from a combination of soil properties affecting gas exchange and heat transfer, the abundance and types of flora present, and water table depth, all of which impact the potential for CH 4 oxidation and production (Kettunen et al., 1996;Kettunen, 2003).
Water table level is a particularly critical parameter because it controls the thickness of the unsaturated zone, which enhances the capacity for methanotrophy, but also can eliminate a key zone for CH 4 production at shallow depths in the vicinity of the rhizosphere where methanogens benefit from higher temperatures and an abundant supply of labile substrates from root exudation (Roulet et al., 1993;Sundh et al., 1994;Kettunen et al., 1999;Str öm et al., 2005).Despite the presence of methanotrophy in this zone, CH 4 flux from wetlands is significantly enhanced by gas exchange with the atmosphere through the aerenchyma of vascular flora (Shannon et al., 1996;Joabsson et al., 1999;Joabsson and Christensen, 2001;Oquist and Svensson, 2002;Str öm and Christensen, 2007).In the absence of high temporal resolution measurements of CH 4 flux capable of detecting sporadic ebullition events (Baird et al., 2004;Tokida et al., 2007a, b), estimates of CH 4 emission from wetlands will be dominated by passive or Attempts to quantify the efficiency of methanotrophy in peat soils have yielded a wide range of estimates of CH 4 consumption, in part, because of different methods employed and the limitations associated with specific approaches as discussed by Pearce and Clymo (2001).Le Mer and Roger (2001) concluded from a survey of literature that ∼60 to 90% of CH 4 produced in wetland soils is oxidized by methanotrophs in the rhizosphere or shallow subsurface horizons; however, other estimates suggest a range of proportions, including 20-40% in general for natural wetlands (Whalen, 2005), 15 to 76% of potential diffusive CH 4 flux seasonally and ∼43% annually of CH 4 entering the oxic zone of a freshwater marsh (Roslev and King, 1996), ∼22% for conversion of CH 4 to CO 2 during transport through 10 cm of acrotelm Sphagnum-rich peat (Pearce and Clymo, 2001), complete consumption within 20 cm of the water table in an undrained peatland (Roulet et al., 1993), 65±24% of CH 4 entering the rhizosphere of Sagittaria lancifolia estimated by CH 3 F inhibition and 79±20% by mass balance (Schipper and Reddy, 1996), 34.7±20.3%and 16.1±7.9%in the rhizosphere, respectively, of bulrush and reed wetlands (van der Nat and Middelburg, 1998), 55% of upward diffusing CH 4 in an Alaskan boreal peatland (Whalen and Reeburgh, 2000), 52±10% and 81±9% in two tidal freshwater wetland forests (Megonigal and Schlesinger, 2002), 0 to 34% rhizosphere oxidation of CH 4 in a Carex fen determined using 13 C mass balance (Popp et al., 1999), and 58 to 92% or <20% in the same peatland depending upon whether CH 4 consumption was quantified by subtracting in situ methane emission rates from CH 4 production rates measured in the laboratory or in situ use of the CH 3 F inhibitor technique (Popp et al., 2000).Much of the variability in estimates of CH 4 oxidation efficiency appears to stem from differences in methodology.As noted by Popp et al. (2000), CH 4 production rates determined in vitro likely lead to an overestimation of CH 4 supply in calculate the supply of CH 4 into the methanotrophic zone at the sites.The gas concentration profiles enabled determination of complete attenuation of CH 4 flux by pore water diffusion when the abundance of dissolved CH 4 was ∼0 µmol l −1 (i.e., below detection limits) within the saturated zone.We compared estimated rates of CH 4 transport by pore water diffusion to total quantites of CH 4 emitted to the atmosphere.We also used aerobic incubations of peat amended with CH 4 to assess differences in CH 4 uptake kinetics with depth and between two of the peatlands (a raised bog and an intermediate fen).In situ CH 4 concentration data and the determined µ m (maximum rates of CH 4 oxidation) and K s (half saturation concentrations) values were then employed to estimate the capacity for CH 4 consumption in relation to the supply of CH 4 by pore water diffusion.Finally, we also investigated relationships between cumulative rainfall in the period preceding pore water sampling and the distribution of CH 4 with depth in the peatland soils to determine whether the timing of sampling impacted our results.

Peatland descriptions
The locations of the four peatlands investigated in Wales, UK are shown in Fig. 1 and further details for the sites are provided in Table 1.Crymlyn Bog and Gors Lywd both receive water input from surrounding uplands via overland and subsurface flow and thus have slightly more alkaline pore water than Blaen Fign and Cors Caron, which are ombrogeneous bogs.Sphagnum spp.were common at all sites; however, predictably the abundance and diversity of vascular flora were highest at the minerotrophic peatlands Crymlyn Bog and Gors Lywd.At each peatland, two adjacent stations (∼1 m apart) were chosen for installation of pore water equilibrators and ground collars to support flux chambers.At Crymlyn Bog and Blaen Fign the ground collars enclosed significantly different proportions of bryophytes and vascular flora with Sphagnum moss dominating at station 1 and sedge species at station 2.  the lid was kept open during sampling to prevent subambient pressures from forming while air samples were collected.Each chamber contained a small battery operated fan to mix the headspace.Air samples for CH 4 flux measurements were collected at 0 (chamber open), 5, 15 and 30 min in 60 ml polypropylene syringes fitted with gas-tight valves.Independent flux determinations were conducted in triplicate for each station during each sampling trip.

Pore water methane
Collection methods and pore water CH 4 data for Blaen Fign were reported previously in Bowes and Hornibrook (2006).The dissolved CH 4 abundances for Crymlyn Bog, Gors Lywd and Cors Caron are reported here for the first time.Briefly, the collection technique employed membrane-exchange equilibrators constructed of PVC that were installed ∼15 cm from each ground collar.The equilibrators enabled sampling of pore water gases at closely spaced depth intervals (2 cm resolution) for measurement of dissolved CH 4 abundance.The design of Hesslein (1976) was modified to permit input and removal of de-ionised, de-gassed water after ground installation through 3-mm OD Tygon ® tubes connected to 1×25×0.5 cm (H×W×D) troughs that were sealed with a gas and ion permeable membrane filter (0.2 µm pore size; HT-200, Pall Life Sciences).

Peat cores
Peat samples for porosity measurements and CH 4 oxidation kinetic experiments were obtained from monoliths (100 cm 2 cross-sectional area × 120 cm length) collected using a Wardenaar ® peat corer (Eijelkamp, Netherlands).The peat was sectioned in the field into 1 dm 3 blocks, sealed in gas tight bags and then packed in ice for transport to the laboratory.Introduction

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Tables Figures Printer-friendly Version Interactive Discussion

Soil temperature and water table level
Soil temperature was measured using an Omega Model HH-41 handheld thermistor thermometer and a thermistor probe.The latter consisted of a nylon-coated type-K thermocouple encased within a 5-cm long brass tube that had a wall thickness of 0.15 mm.The lead wire of the thermocouple was passed through a 2-m long stainless steel tube enabling the protected thermocouple tip to be inserted to specific depths in the peat soil.A nylon plug was used to isolate thermally the thermocouple tip from the stainless steel tube.
The ambient water-table level at each peatland was measured relative to the ground surface in a 10×10 cm hole that had been cut during a previous visit using the Wardenaar ® corer.

Methane concentration analysis
Methane concentrations in air samples collected for determining flux rates were analyzed using a Carlo Erba HRGC5300 gas chromatograph (GC) equipped with gassampling valve (1 cm 3 sample loop), Porapak ® QS packed column (3 mm×4 m), and flame ionization detector (FID).The carrier gas was helium at 35 ml min −1 , and FID support gases were hydrogen at 30 ml min −1 and zero air at 400 ml min −1 .Samples were injected through 1 cm 3 cartridges packed with magnesium perchlorate to remove H 2 O.The relative precision of CH 4 analysis in air samples typically was better than ±2% based on replicate injections of BOC Specialty Gases alpha-gravimetric standards and actual samples.Flux rates were determined from the slope of linear regression equations fitted to the change in chamber CH 4 concentration versus time.Rates were corrected for the areal coverage and volume of the chambers, and are expressed in units of mg m −2 d −1 .Methane was stripped from pore water into a headspace of helium using the method of McAullife (1971).The resulting gas samples were analyzed on the Carlo Erba Introduction

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Tables Figures Printer-friendly Version Interactive Discussion HRGC5300 gas chromatograph (GC) under the same conditions used for analysis of CH 4 in flux samples.Pore water concentrations of CH 4 were corrected for differences in peat porosity and are expressed in units of µmol CH 4 l −1 .

Methane oxidation kinetics
Peat monoliths obtained from Cors Caron and Crymlyn Bog were subsampled in 5 cm slices (∼0.5 dm 3 ) centred on five depths (5, 12.5, 20, 27.5 and 35 cm).The material was slurried in a 1:1 ratio with autoclaved de-ionised water.Slurry from each depth was incubated in triplicate at 15 • C in crimp-top 35 ml Wheaton ® serum vials containing a headspace of CH 4 in zero air corresponding to initial dissolved CH 4 concentrations (S 0 ) of ∼10, 25, 50, 100, 250 and 500 µM.An additional slurry sample for each depth was incubated in singular as a blank containing a headspace of air only to confirm the absence of CH 4 production.Within two hours of loading the vial headspace, the actual value of S 0 in each vial was determined by GC-FID analysis of CH 4 in the headspace and Henry's Law.The rate of CH 4 oxidation was determined subsequently from the decrease in headspace concentration of CH 4 from time 0 (initial) to 24, 48 and 72 h.
Gas samples were extracted using a 50 µl Hamilton ® glass syringe fitted with a sidehole needle and gas-tight valve.Methane abundance was analyzed in triplicate using a Perkin Elmer ® Clarus 500 gas chromatograph fitted with an Elite ® PLOT Q mega-bore column (30 m×0.53 mm diameter) and FID.The carrier gas was helium at 45 ml min and FID support gases were hydrogen at 35 ml min −1 and zero air at 450 ml min −1 .The CH 4 oxidation rates determined independently in triplicate for each of the six S 0 values (i.e., 18 rate measurements per depth) were used to determine the maximum specific rate of CH 4 uptake (µ m ) and half saturation concentrations (K s ) for each depth interval in the two peatlands.Oxygen presumably was not a limiting factor in our experiments given that the incubations were conducted in zero air and hence the single Monod Introduction

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Tables Figures Printer-friendly Version Interactive Discussion expression was used to describe methanotroph consumption of CH 4 in the vials: where µ is the rate of methanotrophy (µmol l −1 h −1 ), µ m is the maximum specific rate of CH 4 uptake (µmol l −1 h −1 ), [CH 4 ] is the concentration of CH 4 (µmol l −1 ) (i.e., S 0 values) and K s is the concentration of CH 4 (µmol l −1 ) required to attain half the maximum rate of CH 4 uptake.Equation ( 1) was fitted to the CH 4 oxidation rate and S 0 data using nonlinear regression software (Prism v4.0, GraphPad Software, San Diego, CA, USA).

Diffusion rates for CH 4 in pore water
The rate of upward CH 4 diffusion in pore water at each peatland was determined using Fick's 1st Law: where J is the flux rate (µmol cm 2 s −1 ), D s is the temperature and porosity corrected diffusion coefficient for CH 4 in water (cm 2 s −1 ) and d [CH 4 ]/dz is the CH 4 concentration gradient (µmol cm −3 cm −1 ) with depth (cm) in peat soils.Fick's 1st law was used because the amount of time required to sample an equilibrator profile (∼1 h) is small and hence the measured gradients can be treated as being effectively steady state.A value of D was calculated for each CH 4 profile based upon the mean soil temperature measured in situ across the depth interval for which pore water CH 4 concentration data were linearly regressed to determine d [CH 4 ]/dz.Values of D were porosity corrected using Eq. ( 4) from Lerman (1979): where φ is porosity (unitless).For each pore water data set, an average value of φ was calculated from in situ porosity measurements collected across the d [CH 4 ]/dz depth interval.

Daily precipitation and timing of sampling
The timing of sample collection at Crymlyn Bog, Cors Caron, Blaen Fign and Gors Lywd is shown in Pore water CH 4 concentration profiles at all sites exhibited a similar shape although the size of the zone beneath the water table in which dissolved CH 4 abundance was below the detection limit of our analysis method varied widely between peatlands and sampling months at individual sites.The potential impact of the magnitude and timing of rainfall events on the size of the zone where [CH 4 ]<0 µmol l −1 will be explored further in Sect.5.1.

CH 4 oxidation kinetics
Maximum potential rates of CH 4 oxidation (µ m ) and half saturation concentrations (K s ) determined from incubations of slurried peat are presented in Table 2.The methanotrophy rate and S 0 data were fitted twice with Eq. (1): once using all data (S 0 =10 to 500 µmol l −1 ) and a second time excluding the S 0 =250 and 500 µmol l −1 measurements (i.e., S 0 =10 to 100 µmol l −1 ).The µ m and K s values determined using all data are anomalous, in particular, the K s values which exceed all half saturation constants reported to date for low affinity methanotropy by 1 to 2 orders of magnitude.The µ m values are similarly high with values from the two samples in the depth interval 10 to 22.5 cm at Cors Caron being ∼10 times greater than any maximum potential rates for CH 4 oxidation in freshwater environments reported to date.These anomalous values appear to result from the disproportionate effects of high CH 4 oxidation rates determined from the small number of incubations having S 0 values >100 µmol l and consequently, Eq. ( 1) was fitted to the data a second time excluding CH 4 oxidation rates from the two highest values of S 0 (250 and 500 µmol l −1 ).The resulting µ m and K s values are consistent with kinetic parameters typically associated for low affinity methanotrophy in aerobic environments.The half saturation concentrations are still amongst the highest reported to date; however, they are similar to published values of K s for peat soils, which tend to be large relative to other methanotrophic environments (Segers, 1998).
Notably the S 0 =10 to 100 µmol l −1 set of depth profiles of µ m and K s values do not show maxima at depths near the lower limit of water table fluctuations (which are present in the µ m and K s values from analysis of the complete data set).Instead µ m values decrease steadily with increasing depth.The large standard errors associated with the K s parameter preclude any broad generalisation about trends with depth of the half saturation constant in soils at either site.(Table 3).The potential for methanotrophy in the 3 cm thick zone was estimated by integrating rates of CH 4 oxidation calculated by substituting values of µ m and K s , and in situ dissolved CH 4 concentrations into Eq.(1).A peat interval of 3 cm downward from the point [CH 4 ] 0 was chosen because (i) depths above the point [CH 4 ] 0 yield methanotrophy rates (µ) equal to zero using Eq. ( 1), (ii) 3 cm was the minimum depth reported by Beckmann and Lloyd (2001) for penetration of O 2 by diffusion into a Scottish peat soil, and (iii) our aim was to provide a conservative estimate of CH 4 oxidation potentials based upon the kinetic parameters determined in laboratory incubations.For example, the values of potential capacity for CH 4 uptake noted in Table 3 (mg CH 4 m −2 d −1 ) are ∼3 orders of magnitude smaller than integrated oxidation rates reported by Sundh et al. (1994) for boreal peatlands in Sweden that were based upon a 0 to 60 cm depth interval (3.0 to 22.1 g CH 4 m −2 d −1 ).Integration over large depth intervals is accurate when a double Monod expression incorporating availability of O 2 can be employed; however, we did not measure either in situ concentrations of pore water O 2 or kinetic parameters associated with O 2 consumption, hence we opted for the conservative approach of applying the determined µ m and K s values to a small depth interval in which O 2 was likely to be available.
The integrated methanotrophy potential rates were scaled to an area of 1 m 2 to facilitate comparison with pore water CH 4 diffusive fluxes and directly measured rates of CH 4 emission to the atmosphere.The latter also are shown in Table 3 and have been reported previously in Bowes and Hornibrook (2006) and Hornibrook and Bowes (2007).The CH 4 fluxes to the atmosphere are due only to steady-state diffusion processes (i.e., pore water or plant-mediated transport).Chamber measurements that exhibited erratic pulses (i.e., ebullition) were excluded from the flux analysis because it could not be determined conclusively whether the events were natural or induced during sample collection.
In the minerotrophic peatlands (Crymlyn Bog and Gors Lywd), CH 4 emission rates to the atmosphere typically exceeded maximum rates of CH 4 transport by pore water diffusion by one to two orders of magnitude, in particular, during summer months (Ta-Introduction

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Tables Figures Printer-friendly Version Interactive Discussion ble 3).Fluxes of CH 4 to the atmosphere were much smaller from the ombrogenous peatlands (Blaen Fign and Cors Caron) with the exception of the sedge-rich plot (station 2) at Blaen Fign, consistent with the well known ability of many aquatic vascular flora to mediated gas transport via aerenchymatous tissue.

5.1
The influence of precipitation events on pore water CH 4 profiles Rates of both aerobic and anaerobic microbial processes in peat soils can be impacted by rainfall events through the introduction of electron acceptors such as O 2 , SO 2− 4 and NO − 3 (Dise and Verry, 2001;Gauci et al., 2002Gauci et al., , 2004)).Concentrations of microbial substrates in shallow peat layers, including dissolved gases (e.g., CH 4 ), also may be influenced through dilution which may affect rates of processes such as methanogenesis and methanotrophy (Kettunen et al., 1996).Thus the timing of CH 4 flux measurements or sampling of pore water CH 4 concentrations should be considered when possible in relation to short-term precipitation records.
The distance between the water table level and depth where [CH 4 ]=0 µmol l −1 (i.e., [CH 4 ] 0 ) differed greatly between the four peatland sites and sampling periods at individual sites (Figs. 3 to 6).The potential influence of precipation input on this parameter was explored by comparing the depth to [CH 4 ] 0 in the saturated zone with rainfall amounts on (i) the day of sampling, and (ii) the periods 1, 3, 5 and 7 days before sampling of pore water.Significant correlations existed with cumulative rainfall during the period 3 days prior to pore water sampling (Fig. 7 and Table 4) but not the amount of rainfall over shorter or longer periods before sample collection (data not shown; r 2 values typically <0.40).A few of the weaker correlations in Fig. 7 (e.g., Cors Caron, stations 1 and 2) result from single data points heavily skewing the linear regression analysis because of the small size of the data sets (i.e., typically n=4).Regression lines which receives significant moisture input from groundwater as well as precipitation.
For the other three peatlands, including Gors Lywd which is positioned at the head of a small catchment, the slopes of the regression equations are positive.The analysis in Table 4 and Fig. 7 suggests that in the absence of significant rainfall events, the depth of [CH 4 ] 0 is not as variable as implied in Figs. 3 to 6.The large range of values for this parameter likely reflect differences in recent input of precipitation rather than microbiological driven changes in methane production and consumption.The "normal" depth of [CH 4 ] 0 appears to vary between individual peatlands as indicated by differences in the y-intercepts of the regression equations in Table 4 (e.g., ∼10 cm for Gors Lywd versus ∼5 cm for Blaen Fign).
Noteworthy in Fig. 7 are the infilled data points for Cors Caron and Crymlyn Bog that lie largely at x-values of ∼0 mm (i.e., when little or no rainfall occurred prior to the sampling period).The infilled points (5 in total) represent times when the concentration of dissolved CH 4 at the water table level exceeded 0 µmol l −1 and CH 4 transport was occurring across the subsurface air-water interface.The correlations in Table 4 will be unimportant during periods of low rainfall and at those times CH 4 most likely is able to diffuse across the water table surface because heterotrophic activity in the unsaturated zone has depleting O 2 from pore spaces.

CH 4 oxidation kinetics
The µ m values determined for different depth intervals at Crymlyn Bog and Cors Caron (Table 3; S 0 =10 to 100 µmol l −1 values) lie within the range of potential methane oxidation rates (0.1 to 100 µmol m −3 s −1 ) compiled by Segers (1998) for different types of environments that host low affinity methanotrophic activity.Conversion of units in Table 3 for comparison yield µ values of 0.5 to 1.1 and 2.9 to 5.9 µmol m −3 s −1 , respectively, for Crymlyn Bog and Cors Caron.Half saturation constants for Cors Caron also are higher than values for Crymlyn Bog.It is unclear why methanotrophs in the raised bog environment should have a lower affinity for substrate (i.e., higher K s ) and molecular bi-Introduction

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Tables Figures Printer-friendly Version Interactive Discussion ology data are unavailable to determine whether differences existed in methanotrophic communities at the two peatlands.Although the two peatlands differ in the composition and pH of their soil solution, we cannot speculate about potential relationships between the parameters µ m and K s , and factors such as pH because slurries were diluted 1:1 with deionized water.The buffering capacity of peat from the two sites would have differed considerably (i.e., rainfed versus groundwater influenced).However, K s values cited by Segers (1997) for peat soil ranged from 1 to 45 µmol l −1 , encompassing the values determined for Crymlyn Bog. Watson et al. (1997) reported a K s of 57.9 µmol l −1 for CH 4 oxidation in acidic peat from Ellergower Moss, comparable to the range of half saturation constants determined with depth for Cors Caron, which also is a raised bog (i.e., 42.6 to 68.1 µmol l −1 ; Table 3).The ranges of µ m and K s values in Table 3 are noteworthy also because of the difficulties such variability presents in efforts to model CH 4 dynamics in peatland soils.For example, one of the more rigorous process-based models for estimating CH 4 flux from peatlands (Walter and Heimann, 2000) employs the assumption that the parameters µ m and K s for methanotrophy are constant with depth and in different types of wetlands, assigning values of 20 µmol l −1 h −1 and 5 µmol l −1 , respectively.The mean µ m values for Crymlyn Bog and Cor Caron suggest that maximum rates of CH 4 oxidation may differ between minerotrophic and ombrogenous mires and in both cases appear to decrease gradually with depth.As noted previously, half saturation concentrations also may be higher in acidic rainfed peatlands (e.g., Table 3 and Watson et al., 1997).Availability of kinetic parameters describing CH 4 oxidation in peatlands is too limited at present to attempt to develop generalised relationships describing µ m and K s in different types of peatlands and spatially and temporally within individual sites.profiles collected during the summer of 2003.Rates of CH 4 emission to the atmosphere from Blaen Fign and Cors Caron were the same order of magnitude as pore water CH 4 diffusion rates; however, it is unlikely that CH 4 transport by this mode contributed to atmospheric flux.The stable carbon isotope compositions (δ 13 C values) of CH 4 in pore water and surface flux have been used previously to demonstrate that emission of CH 4 to the atmosphere at all four peatlands occurs predominately via plantmediated transport (Bowes and Hornibrook, 2006;Hornibrook and Bowes, 2007).For example, CH 4 emitted at a higher rate from sedge-rich station 2 at Blaen Fign has δ 13 C values that are statistically indistinguishable from CH 4 emissions from Sphagnum-rich station 1 (Bowes and Hornibrook, 2006).The δ 13 C composition of CH 4 emissions from both plots are 13 C-depleted by ∼15 to 20‰ relative to pore water CH 4 , which eliminates the possibility that the small quantities of CH 4 emitted from station 1 is residual CH 4 that has survived transit across the unsaturated zone (Happell et al., 1994;Popp et al., 1999).Similarly, CH 4 emissions from Cors Caron, Crymlyn Bog and Gors Lywd also are 13 C-depleted relative to the pore water CH 4 pool (Hornibrook and Bowes, 2007).
These conclusions about transport processes based upon stable isotope data are consistent with the observation reported here that low affinity methanotrophs in the 3 cm thick zone where CH 4 first appears in the pore water pool (i.e., immediately below the depth [CH 4 ] 0 ) have a capacity for CH 4 consumption that significantly exceeds the upward CH 4 supply via pore water diffusion (Table 3).While low affinity methanotrophs appear to consume the bulk of CH 4 transported along concentration gradients in pore water, they do not provide a robust barrier to CH 4 flux from peatlands because of the prevalence of CH 4 movement through vascular flora which bypasses the methanotrophy filter.During June to August, microbial CH 4 oxidation rates ranged from 0.8 to 40.7% of total CH 4 flux to the atmosphere in Crymlyn Bog and Gors Lywd; however, the majority of values were <10%.In the same months, the percentages were higher at sedge-poor plots at the ombrogeneous mires (Blaen Fign, ∼9.surficial emissions (Hornibrook and Bowes, 2007).Consequently, in the absence of bacterial CH 4 oxidation the CH 4 flux rate from minerotrophic peatlands would not be significantly greater in absolute terms but the increase would be proportionally much larger in ombrogenous bogs.The steady state flux rates of >100 mg CH 4 m −2 d −1 commonly observed from wetland soils (e.g., Whalen, 2005) would be difficult to achieve if pore water diffusion alone was the dominant transport mechanism of CH 4 .The bulk of CH 4 emitted from peatlands typically occurs via vascular flora and possibly ebullition, although data for the latter transport process remain sparse (Baird et al., 2004).

Conclusions
The depth below ambient water table levels at which dissolved methane is depleted to a concentration of ∼0 µmol l −1 by methanotrophic activity varied widely between peatlands and temporally within individual peatlands.Short-term precipitation events appeared to increase the depth to [CH 4 ] 0 without necessarily disturbing dissolved CH 4 profiles.In the absence of recent rainfall input, the depth of [CH 4 ] 0 below the water table level ranged from ∼5 to 10 cm, although the size of the interval diminished to 0 (i.e., CH 4 present at the water table surface) during prolonged periods without precipitation input.
The capacity for methanotrophy in peatland soils from both minerotrophic and ombrogenous peatlands typically was greater than the available supply of upward diffusing CH 4 .Kinetic parameters (µ m and K s ) describing the response of methanotroph populations to substrate (i.e., CH 4 ) concentrations are not constant with depth as assumed in some process models and both parameters were larger in the ombrogenous versus minerotrophic peatlands.Low affinity methanotrophic activity effectively consumes the majority of upward diffusing CH 4 in peatland soil (in most cases 100%); however, in the absence of bacterial CH 4 oxidation the flux rate from minerotrophic peatlands would not be significantly greater.Maximum rates of CH 4 flux by pore water diffusion were at most 10 to 20 mg m  niques, FEMS Microbiol. Ecol., 21, 197-211, 1996. McDonald, I. R., Upton, M., Hall, G., Pickup, R. W., Edwards, C., Saunders, J. R., Ritchie, D. A., and 4.
peat soils, contributing to the calculation of anomalously high proportions of CH 4 removal by methanotroph activity.We measured detailed (cm scale resolution) in situ profiles of dissolved CH 4 concentration at four different peatlands situated in Wales, UK during the summer of 2003 to

3. 2
Methane fluxCollection methods and CH 4 flux data for all sites were reported previously inBowes and Hornibrook (2006) andHornibrook and Bowes (2007).Briefly, flux chambers and ground collars were constructed of polyvinyl chloride (PVC) and had a combined volume of either 11 or 15 litres.The chambers were sealed onto the collars using large neoprene rubber o-rings coated with silicon grease and then covered with opaque lids also fitted with greased o-rings.Air samples were collected via a 4-m length of 3-mm OD Tygon ® tube installed in the lid of each chamber.A second identical tube fitted in Final values of J are expressed in mg CH 4 m −2 d −1 to facilitate comparison with CH 4 fluxes to the atmosphere measured using static chambers.The temperature dependency of D S was based upon polynomial regression of diffusion coefficients for CH 4 in water in range 0 to 35 • C (83rd Edition of the Handbook of Physics and Chemistry) which yielded the relationship: D = 8.889 × 10 −11 T 3 − 1.714 × 10 −9 T 2 + 3.721 × 10 −7 T + 8.771 × 10 −6 Fig. 2 in relation to total daily precipitation measured at UK Meteorological Office Stations (MOSs) situated near the peatlands.Swansea Victoria Park (Fig. 2a) and Swyddffynnon (Fig. 2b) MOSs are located immediately adjacent to Crymlyn Bog and Cors Caron, respectively, providing accurate daily precipitation records for each site.There are no active MOSs in close proximity to either Blaen Fign or Gors Lywd because of their remote locations in the Elan Valley.Consequently daily precipitation records from the Cwmystwyth and Llangurig MOSs, which geographically bracket the peatland sites, have been used (Fig. 2c and d) 4.2 Pore water CH 4 Pore water profiles of dissolved CH 4 measured in soils at the four peatlands during the summer of 2003 are shown in Figs. 3 to 6. Also shown in each figure panel is the ambient water −1 .Such concentrations of CH 4 are very rare in situ at the oxic-anoxic interface in peatlands

4. 4
Rates of CH 4 flux and consumption A summary of rates of internal and external CH 4 fluxes (all sites) and subsurface methanotrophy potentials (Crymlyn Bog and Cors Caron only) are presented in Table3.Rates of upward CH 4 flux into the methanotrophic zone were determined according to the method described in Sect.3.8 and then scaled to a cross-sectional area of 1 m 2 .The majority of CH 4 fluxes by pore water diffusion had a magnitude <10 mg m−2 d −1 .The concentration of dissolved CH 4 at the water table surface was >0 µmol l −1 in only 5 of the 33 pore water CH 4 profiles (Figs.3c, e, g, 6c and f), suggesting that diffusion of CH 4 across the air-water interface contributes little to atmospheric emissions of CH 4 at these sites.Methane escaping from the water surface within the peat profile must still transit pore spaces and methanotroph populations in the unsaturated zone before reaching the atmosphere.In all cases the rate of upward CH 4 flux was less than the capacity for CH 4 oxidation determined in a 3 cm thick zone immediately below the depth at which [CH 4 ]=0 µmol l have a negative slope only for Crymlyn Bog, the most minerotrophic of the peatlands

5. 3
Methane supply, demand and net fluxThe amount of upward CH 4 transport in all four peatlands via pore water diffusion typically was <10 mg m −2 d −1 and exceeded this value in only 4 of the 33 pore water CH 4 3 to 53.4%; Cors Caron, 11.0 to 21.1%), but the difference in proportions is unimportant because as indicated by δ 13 C data little or none of the diffusion transported CH 4 contributed to

2
at Crymlyn Bog and Blaen Fign.b Rates of internal CH 4 flux into the zone of methanotrophy based upon Fick's 1st law (Eq.2) and linear regression analysis of pore water CH 4 data shown in Figs. 2 to 5. c Potential rate of CH 4 oxidation in a 3 cm thick zone below the depth at which [CH 4 ]=0 µmol l −1 defined by the yintercept of linear regression analysis of pore water CH 4 concentration data in Figs. 2 to 5. The total potential rate of CH 4 oxidation in the 3 cm thick zone is based upon actual CH 4 concentrations measured in peat soils and the depth distribution of µ m and K s parameters determined experimentally for Crymlyn Bog and Cors Caron (

Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .
Fig. 3. Pore water profiles of dissolved CH 4 measured at Crymlyn Bog during the summer of 2003 from pore water equilibrators E1 and E2.Watertable level in each panel is indicated by a dotted horizontal line.The dashed line through shallow CH 4 values trending to zero concentration is a regression line fitted to the data to determine the gradient d [CH 4 ]/dz, which was used to calculate rates of CH 4 flux into the methanotrophic zone.
[CH 4]/dz was obtained to calculate rates of upward CH 4 diffusion and the depth at which[CH 4]=0 µmol l −1 (i.e., the y-intercept denoted as [CH 4 ] 0 ).The gray horizontal bar delineates a 3 cm thick zone immediately beneath depth[CH 4] 0 in which potential rates of CH 4 oxidation were calculated based upon experimentally determined kinetic parameters (see Sect. 4.3) and in situ dissolved CH 4 concentrations.Where gaps existed in pore water[CH 4] data, missing values were interpolated between adjacent CH 4 concentrations, including when necessary the point[CH 4 table level at the time of sampling of the membrane equilibrators.The dashed line through the dissolved CH 4 data is a linear regression curve from which Introduction Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia, Appl.Environ.Microb., 64, 1099-1105, 1998.Chen, Y., Dumont, M. G., McNamara, N. P., Chamberlain, P. M., Bodrossy, L., Stralis-Pavese, N., and Murrell, J. C.: Diversity of the active methanotrophic community in acidic peatlands as assessed by mRNA and SIP-PLFA analyses,Environ.Microbiol., 10, 446-459, 2008.Introduction Hall, G. H., Pickup, R. W., and Murrell, J. C.: Methane oxidation potential and preliminary analysis of methanotrophs in blanket bog peat using molecular ecology tech- −2 d −1 , which in minerotrophic mires represents typically <10% of

Table 2 .
Maximum CH 4 oxidation rates (µ m ) and half saturation constants (K s

Table 3 .
Internal and external methane fluxes and subsurface oxidation potentials.

Table 2
Locations of peatland study sites and Met Office MIDAS Land Surface Observation Stations (MOS = Met Office Station) in Wales, UK.Details of the four peatlands are provided in Table 1, including map coordinates.Introduction ). d Total diffusive CH 4 flux to the atmosphere measured using closed dynamic chambers and reported previously in Introduction