Methane oxidation potential of the arctic wetland soils of a taiga-tundra ecotone in northeastern Siberia

Arctic wetlands are significant sources of atmospheric methane and the observed accelerated climate changes in the arctic could cause the change in methane dynamics, where methane oxidation would be the key process to control methane emission from wetlands. In this study we determined the potential methane oxidation rate of the wetland 5 soils of a taiga-tundra transition zone in northeastern Siberia. Peat soil samples were collected in summer from depressions covered with tussocks of sedges and Sphagnum spp. and from mounds vegetated with moss and larch trees. A bottle incubation experiment demonstrated that the soil samples collected from depressions in the mossand sedge-dominated zones exhibited active methane oxidation with no time lag. The 10 potential methane oxidation rates at 15 oC ranged from 94 to 496 nmol h-1 g-1 dw. Methane oxidation was observed over the depths studied (0-40 cm) including the water-saturated anoxic layers. The maximum methane oxidation rate was recorded in the layer above the water-saturated layer: the surface (0-2cm) layer in the sedge-dominated zone and in the middle (4-6 cm) layer in the moss-dominated zone. The methane oxidation rate was 15 temperature-dependent, and the threshold temperature of methane oxidation was estimated to be −4 to −11 oC, which suggested methane oxidation at subzero temperatures. Soil samples collected from the frozen layer of Sphagnum peat also showed immediate methane consumption when incubated at 15 oC. The present results suggest that the methane oxidizing bacteria in the wetland soils keep their potential activities even under 20 anoxic and frozen conditions and immediately utilize methane when the conditions become favorable. On the other hand, the inhibitor of methane oxidation did not affect the methane flux from the sedge and moss zones in situ, which indicated the minor role of plant-associated methane oxidation. 2 Biogeosciences Discuss., https://doi.org/10.5194/bg-2019-98 Manuscript under review for journal Biogeosciences Discussion started: 1 April 2019 c © Author(s) 2019. CC BY 4.0 License.


Abstract.
Arctic wetlands are significant sources of atmospheric methane and the observed accelerated climate changes in the arctic could cause the change in methane dynamics, where methane oxidation would be the key process to control methane emission from wetlands. In this study we determined the potential methane oxidation rate of the wetland 5 soils of a taiga-tundra transition zone in northeastern Siberia. Peat soil samples were collected in summer from depressions covered with tussocks of sedges and Sphagnum spp. and from mounds vegetated with moss and larch trees. A bottle incubation experiment demonstrated that the soil samples collected from depressions in the mossand sedge-dominated zones exhibited active methane oxidation with no time lag. The 10 potential methane oxidation rates at 15 ºC ranged from 94 to 496 nmol h -1 g -1 dw. Methane oxidation was observed over the depths studied (0-40 cm) including the water-saturated anoxic layers. The maximum methane oxidation rate was recorded in the layer above the water-saturated layer: the surface (0-2cm) layer in the sedge-dominated zone and in the middle (4-6 cm) layer in the moss-dominated zone. The methane oxidation rate was 15 temperature-dependent, and the threshold temperature of methane oxidation was estimated to be −4 to −11 ºC, which suggested methane oxidation at subzero temperatures.
Soil samples collected from the frozen layer of Sphagnum peat also showed immediate methane consumption when incubated at 15 ºC. The present results suggest that the methane oxidizing bacteria in the wetland soils keep their potential activities even under 20 anoxic and frozen conditions and immediately utilize methane when the conditions become favorable. On the other hand, the inhibitor of methane oxidation did not affect the methane flux from the sedge and moss zones in situ, which indicated the minor role of plant-associated methane oxidation.

Introduction
Methane is a greenhouse gas produced in natural and anthropogenic anaerobic environments as the terminal product of organic decomposition. Arctic wetlands, where large amounts of organic carbon are stored (Tarnocai et al., 2009;Hugelius et al., 2014), 5 are one of the largest sources of atmospheric methane (Kirschke et al., 2013;Intergovernmental_Panel_on_Climate_Change, 2014). Methane emission from the Arctic wetlands could be increased by the climate changes that include increasing temperatures, changing precipitation patterns, and permafrost thaw (Olefeldt et al., 2013;Schuur et al., 2015;Treat et al., 2015). 10 Methane emission from the wetlands to the atmosphere is the results of the balance between methane production and consumption. The potential methane oxidation rate is typically one order of magnitude higher than that of methane production (Segers, 1998).
The oxic-anoxic interfaces such as the surface of the wetland soils and the rhizosphere of aerenchymatous plants are often characterized by the active methane oxidation thus 15 playing a key role in controlling methane flux from the wetlands (Zhuang et al., 2004;Preuss et al., 2013).
The methane oxidation in arctic wetland soils has been reported repeatedly. In many cases the potential methane oxidation was determined by the incubation experiment in which the collected samples incubated under high concentrations of methane, and its 20 controlling factors have been studied (Wagner et al., 2003(Wagner et al., , 2005Liebner and Wagner, 2007;Knoblauch et al., 2008;Christiansen et al., 2015). The potential methane oxidation rate differs spatially and temporally under the influence of different environmental conditions. Wagner et al. (2003) reported that methane oxidation in polygon depression in the Lena Delta, Siberia, was higher than in polygon rim with the increasing rate and expanding active depth with time in summer. The methane oxidation rate at the in situ temperature (0.4-7.5°C) ranged 1.9−7.0 nmol h -1 g -1 except for the boundary to the frozen ground where no methane oxidation was recorded (Wagner et al., 2003(Wagner et al., , 2005. Much higher potential methane oxidation rates were recorded in permafrost-affected soils of 5 Northeast Siberia with 45-87 nmol h -1 g -1 for mineral soils and 835 nmol h -1 g -1 for organic soil at the in situ temperature (5 °C) (Knoblauch et al., 2008); 8-32% of the maximum oxidation rate was observed at 0 °C. Thus, water level, soil depth, and temperature are the major factors that affect the methanotrophic activity in the artic wetlands.
Aerenchymatous plants provide a niche for methane oxidizing bacteria in the 10 rhizosphere where oxygen and methane are both available in wetlands (Frenzel, 2000).
Moss has a symbiotic association with methanotrophs: methanotrophs use oxygen supplied from moss to oxidize methane and moss utilizes CO2 produced by methanotrophs for photosynthesis (Raghoebarsing et al., 2005;Kip et al., 2010Kip et al., , 2011Larmola et al., 2010;Liebner et al., 2011). Plant-associated methane oxidation in wetland 15 soils has been studied by comparing the methane flux under the conditions with and without the specific inhibitor of methane oxidation (Frenzel and Bosse, 1996;Frenzel and Rudolph, 1998;Kruger et al., 2001). However, the role of plant-associated methane oxidation in methane dynamics has been poorly studied in the arctic wetlands (Liebner et al., 2011;Nielsen et al., 2017). 20 Most studies to determine the potential methane oxidation rates of the arctic wetlands have been done by in vitro incubations; the target samples were transferred from the study sites to the laboratory and the methane oxidation activity was measured after some time of storage, which may affect the enzymatic activities of soils depending on the type 4 Biogeosciences Discuss., https://doi.org /10.5194/bg-2019-98 Manuscript under review for journal Biogeosciences Discussion started: 1 April 2019 c Author(s) 2019. CC BY 4.0 License. (Burns et al., 2013). Also, it is not clear if the measured methane oxidation represents the actual potential of the collected samples or the methanotrophic activity was induced by incubation since the temporal change in methane concentration in the system is poorly documented in the incubation experiments. In this study, we measured the potential methane oxidation of the wetland soils in the northeastern Siberia immediately (< 24 h)  Collected samples were stored at 4 °C and subjected to measurement of potential methane oxidation within a day.

Vertical profiling of dissolved oxygen in soil
The vertical profile of dissolved oxygen in peat soil of the sedge and moss wetlands was measured by inserting a D.O. meter (HI 2040-01, Hanna Instruments, RI, U.S.A.) into small wells (diameter: ca. 1.5 cm) that were made by drilling the peat with a wodden stick 15 a few days prior to measurement.

Methane oxidation potential of soil samples
Samples were homogenized by cutting into pieces (< 5 mm) with scissors and mixing, Methane concentration in the headspace was monitored using a photoacoustic field gas monitor (Innova 1412, LumaSense Technologies, Ballerup, Demmark). The methane oxidation rate was calculated from the linear regression of the methane concentration decreasing with time. The methane oxidation rate for the initial two days of incubation 5 (ca. 50 hrs) was calculated and the threshhold temperature for methane oxidation was estimated from the linear regression between methane oxidation rates and incubation temperatures. The methane oxidation rate was expressed per dry weight of samples that was obtained by drying at 80 °C for 48 hrs. To examine the effect of nutrients and black carbon on methane oxidation, the potential atomospheric depositions, the samples (10 g) 10 were applied with 1 ml of inorganic solution (10 μM NH4NO3, 250 μM NaCl, 40 μM CaCl2, 20 μM MgSO4, 10 μM KCl) and/or 1 ml of 100 μg l -1 charcoal powder of oak (Quercus L., <47 μm). 15 Methane oxidation associated with the wetland plants was estimated using CH2F2, the specific inhibitor of methane oxidation, in 2014 and 2015 according to Kruger et al.  10 Differences in methane oxidation rate between treatments were tested with a one-way ANOVA and the effect of CH2F2 on methane emission was assessed by Wilcoxon's test using SPSS for Windows Ver. 22.0.

The relationship between the vegetation types and CH4 oxidation
The methane concentration in the headspace of the bottle with peat samples from wetlands methane oxidation of any samples. We also tested 10 times higher concentrations of nutrients and charcoal powder but found no influence on methane oxidation of the all samples (data not shown). 5 The time course incubation experiment using the surface (0-10 cm) wetland peat samples showed that methane was oxidized with no induction period (Figs. 3A & B). The highest activity of methane oxidation was recorded at the middle (4-6 cm) layer and the top (0-2 cm) layer of moss and sedge peat samples, respectively. Immediate methane oxidation of 10 the moss peat sample was even observed in the frozen layer (30-40 cm) with the lower rate than the upper layers (Fig. 3C). Peat samples from the sedge up to 20 cm in depth also showed active methane oxidation, while a mineral soil collected from the top of the frozen layer did not exhibit any methane oxidation. The calculated methane oxidation rate ranged from 54 to 496 nmol h -1 g -1 (Table 1). In contrast to the high potential of methane 15 oxidation through the active layer, the in situ concentration of dissolved oxygen in the pore water of the wetlands was very low and undetectable below 10 cm with one exception in the sedge wetland (Fig. 4). at 0 °C did not differ from that at 5 °C (Fig. 4C). The threshold temperature for methane oxidation was estimated to be −13 and −9 °C for the moss and sedge peat samples, respectively, when the simple linear regression model was applied.

vegetations
The methane emission rate for the first measurement ranged from 2.4 to 1800 μmol h -1 m -2 ; the mound showed the lowest rate (Fig. 5). The methane emission rate in the second measurement ranged from 0.19 to 1840 μmol h -1 m -2 and did not differ from that of the 10 initial measurement even when the second mesurement was conducted after the treatiment with the inhibitor.

15
The wetland soils in the depression area of the taiga-tundra transition zone in the northeast Siberia exhibited the active methane oxidation in the incubation experiment. The potential rates estimated in this study were at the higher end of those previously reported for other Arctic regions including Siberia (Wagner et al., 2003(Wagner et al., , 2005Liebner and Wagner, 2007;Knoblauch et al., 2008;Christiansen et al., 2015). The highest rate was recorded at the 20 subsuface (4-6 cm) and surface (0-2 cm) of the moss and sedge dominated wetlands, respectively; these depths corresponded to the water level of the study site, where the maximum methane oxidation rate has been often reported in other wetlands (e.g., (Vecherskaya et al., 1993;Sundh et al., 1995;Whalen and Reeburgh, 2000). On the other hand, the mound soil in the same area showed no methane oxidation. The results show the spatial heterogeneity of the potential methane oxidation of the soils in the arctic at inter-and intra-regional scales. We tested our hypothesis that methane oxidation of organic soils may be constrained by limited amounts of mineral nutrients including nitrogen, but application of the mineral salts and charcoal that are supposed to be  Roslev and King (1996) reported that peat samples from the freshwater marsh maintain 30% of the initial methane oxidation capacity after 32 days 20 of anoxic incubation and methanotrophs from anoxic peat initiated aerobic methane oxidation within 1-7 hours after oxygen addition. The subzero temperatures would not also be motal for methanotrophs to keep their potential activity as estimated by the temperature dependence of methanotrophic activity (Fig. 4C), which is same as the microbial respiration on added carbon at temperatures as low as -15 ºC in the Canadian high Arctic soil (Steven et al., 2007). The immediate methane oxidaiton upon thaw is also reported for the frozen permafrost soil from a black-spruce forest in Alaska (Mackelprang et al., 2011). In this study, the methane oxidation potential over the soil depth was estimated at 15 ºC, but the deeper sample could have the higher activity at the lower 5 optimum temperature (Liebner and Wagner, 2007).
The methane emission rate in the moss-and sedge-dominated wetlands observed in this study ranging from 7.36 to 1,840 μmol m -2 h -1 was mostly comparable to or more than that reported in the previous studies (Cao et al., 1998;Kutzbach et al., 2004;Petrescu et al., 2008). Addition of the inhibitor of methane oxidation did not affect the methane

2011)
. The no effect of the inhibitor added in the headspace on the methane flux from the moss-dominated wetlands suggests that moss-associated methanotrophs may not use oxygen diffused from the atmosphere but use oxygen released from moss by photosynthesis (Raghoebarsing et al., 2005). Moss-associated methanotrophs may keep their activity even after the moss is dead and accumulated in the deeper layer where the 5 conditions are not favorable for methane oxidation due to the anoxia (King, 1996).
In conclusion, the wetland soils of the taiga-tundra ecotone in the northeastern Siberia keep the high methane oxidation potential even under the unfavorable conditions. Though the plant associated methane emission may be influenced by methane oxidation, the oxic-