Higher temperature sensitivity of forest soil methane oxidation in colder climates

Introduction

Methane (CH₄) is a highly potent greenhouse gas that contributes disproportionately to Earth’s ongoing warming^1,2, and has increased rapidly from 0.7 ppm to over 1.9 ppm since the industrial revolution³. This increase poses a significant challenge to the world’s goal of restricting global temperature rise to 1.5–2 °C over pre-industrial temperatures⁴. However, forest soils can provide an important terrestrial CH₄ sink, owing primarily to the activity of methanotrophs⁵, and this sink is expected to strengthen in the face of climate warming^6,7,8. As a result, the warming-induced CH₄ sink changes may mitigate the growth in atmospheric CH₄ concentration caused by human activities and climate change, triggering a negative feedback that may slow down the rate of global warming⁹. However, the magnitude of this forest soil CH₄ sink in response to warming remains uncertain¹⁰, in part because it is not well understood how the temperature sensitivity of forest soil microbial CH₄ oxidation (i.e., CH₄ uptake by soil methanotrophs) varies over a broad geographical scale^{11,12,13,14,15,16}.

Soil methanotrophs show very different responses in CH₄ oxidation capacities with increasing temperature depending on the climates and soil properties in which they are embedded^17,18. It is clear that soil CH₄ oxidation is temperature-sensitive and variable across forest ecosystems^11,12,18, as a result of variation in climate^19,20, soil abiotic factors^21,22,23, and the microbial community properties^24,25. For instance, previous studies have shown that increased mean annual temperature reduces the abundance of methanotrophs²⁶ and inhibits the activity of enzymes involved in CH₄ oxidation²⁷ that would lead to reduced CH₄ uptake²⁰. Moreover, sandy soils have been demonstrated to be generally more efficient at oxidizing CH₄ than silty soils, likely due to their better ability to diffuse gases^28,29. Along with soil texture, soil nitrogen is a crucial factor in controlling CH₄ oxidation since it is an essential nutrient element for the growth of methanotrophs^30,31. How these biotic and abiotic factors interact to impact the temperature sensitivity of forest soil CH₄ oxidation, however, remains largely unidentified and thus is not explicitly represented in process-based models^10,32. In addition, the current modeling efforts generally use a fixed temperature sensitivity parameter and do not incorporate spatial variation in temperature sensitivity of CH₄ oxidation, which will lead to large uncertainty in global forest soil CH₄ sink estimation^9,33,34. By evaluating the spatial variation and ecological drivers of the temperature sensitivity of forest soil CH₄ oxidation, we can begin to gain more accurate predictions of the magnitude and direction of the response of soil CH₄ sink to warming. This would allow us to improve processed-based models to quantify the relative importance of soil in the global CH₄ cycle and its potential ability to reduce atmospheric CH₄ under future global change scenarios.

To better understand the temperature sensitivity of forest soil microbial CH₄ oxidation at a large scale, we evaluated the temperature response curves of soil microbial CH₄ oxidation using soils sampled from 84 sites along a ~4000 km north-south transect in eastern China. This transect included high levels of variation in both mean annual temperature and mean annual precipitation (Supplementary Data 1) across many climate zones. Our main objectives are to answer the following questions: (i) How does the temperature sensitivity of forest soil microbial CH₄ oxidation change at large geographic scales? (ii) What are the important factors of temperature sensitivity in forest soil microbial CH₄ oxidation?

Results

Across the latitudinal gradient, we observed a high degree of variation in temperature sensitivity values, reflecting the increase in soil microbial CH₄ oxidation rate per degree of warming (Fig. 1a). These sensitivity values ranged from 0.03 to 0.77 μg CH₄ g⁻¹ soil d⁻¹ °C^–1 (Fig. 1b and Supplementary Data 1), and increased along a latitudinal gradient from tropical to cold-temperate forests (Supplementary Table 1). Similar patterns were observed in the temperature sensitivity of soil microbial CH₄ oxidation across different temperature ranges (Supplementary Fig. 1).

**Fig. 1: The temperature sensitivity of forest soil microbial CH₄ oxidation across the 84 sites in eastern China.**

We found that the temperature sensitivity of soil microbial CH₄ oxidation was significantly influenced by variations in climate factors, soil resources (e.g., total carbon, total nitrogen, and available nitrogen content), bacterial community composition, and methanotroph traits, while soil properties (i.e., pH and clay content) had no notable effect (Fig. 2). Variance decomposition analysis revealed that climate factors accounted for 44.45% of the variation in the temperature sensitivity, surpassing the individual contributions of soil resources, soil properties, bacterial community composition, and methanotroph traits (Fig. 3a; R² = 0.52). The key role of climate factors in driving the temperature sensitivity of soil microbial CH₄ oxidation was further confirmed by independent random forest analyzes (Fig. 3b), which identified mean annual temperature as the primary factor determining the observed spatial patterns of temperature sensitivity. Specifically, the temperature sensitivity of soil microbial CH₄ oxidation exhibited a significant negative correlation with mean annual temperature (Fig. 4).

Fig. 2: The relationship between the temperature sensitivity of soil microbial CH₄ oxidation and climate factors, soil properties, soil resources, bacterial community composition, and methanotroph traits.

**Fig. 3: Relative importance of biotic and abiotic variables in determining the temperature sensitivity of forest soil microbial CH₄ oxidation.**

**Fig. 4: The relationship between the temperature sensitivity of forest soil microbial CH₄ oxidation and mean annual temperature across 84 forest sites.**

Structural equation model (SEM) was used to further assess the direct and indirect effects of climate factors, soil resources (including total carbon, total nitrogen, and ammonium nitrogen content), soil properties (i.e., pH and clay content), bacterial community composition, and methanotroph traits on the temperature sensitivity of soil microbial CH₄ oxidation (Fig. 5). The SEM revealed that climate factors and the relative abundance of type II methanotrophs had direct effects on the temperature sensitivity of soil microbial CH₄ oxidation (Fig. 5). Additionally, soil resources and the relative abundance of Verrucomicrobiota had indirect effects on temperature sensitivity of soil microbial CH₄ oxidation, mediated through changes in the relative abundance of type II methanotrophs (Fig. 5). Furthermore, climate factors indirectly influenced the shifts in the relative abundances of both type II methanotrophs and Verrucomicrobiota by altering soil resources (Fig. 5).

**Fig. 5: Structural equation model (SEM) examining the multivariate effects on the temperature sensitivity of forest soil microbial CH₄ oxidation.**

Discussion

Although forest soils play an important role in the global CH₄ sink⁵, the magnitude of this effect can be strongly but variably influenced by climate warming. Our findings show that colder regions had the highest CH₄ oxidation potential, as well as the greatest increase in forest soil CH₄ oxidation capacity in response to warming (Fig. 4 and Supplementary Table 1). As a consequence, we expect forest soils in colder regions to become more effective at oxidizing CH₄ as the global climate warms, especially since these regions account for more than half of the world’s forested area³⁵. Given that global warming tends to be most pronounced at high latitudes³⁶, these colder regions will play a vital role in mitigating the rise in atmospheric CH₄ concentrations by enhancing microbial-driven CH₄ oxidation capacity in a warming climate. Furthermore, it is important to note that the warming-induced enhancement of soil CH₄ oxidation capacity may lead to a potential increase in CO_₂ emissions from the soil, with significant implications for the global greenhouse gas budget^37,38.

Unlike other soil carbon cycling processes, CH₄ uptake in forest soils is driven by methanotrophs, which primarily rely on CH₄ as their carbon and energy source^39,40,41,42. Previous studies have shown a strong relationship between soil CH₄ oxidation rates and the abundance of key methanotroph functional groups^25,40,43. Similarly, our finding indicates that the temperature sensitivity of soil microbial CH₄ oxidation is significantly influenced by the predominant methanotroph functional group, with sensitivity increasing in relation to the relative abundance of type II methanotrophs (Supplementary Figs. 2 and 3). These results suggest that variations in type II methanotrophs abundance may significantly impact methanotroph-mediated CH₄ oxidation under warming, thus affecting the capacity of soil CH₄ sinks. However, current terrestrial ecosystem models do not account for the role of dominant methanotroph functional groups in CH₄ oxidation and its temperature sensitivity^9,21,32. Our findings emphasize the importance of incorporating variations in type II methanotrophs abundance into biogeochemical models to improve estimates of global forest CH₄ sinks under climate change.

The abundance and functional characteristics of soil microbial groups are influenced by soil resource availability and interactions with other microbial groups⁴⁴. As expected, our results showed that soil resources directly promote the relative abundance of type II methanotrophs (Fig. 5). This is likely due to the nitrogen requirements of methanotrophs, which typically need a 1:4 nitrogen-to-carbon ratio^25,45,46,47. Additionally, soil resources indirectly affect the type II methanotrophs by influencing the abundance of the predominant bacterial phylum, Verrucomicrobiota (Fig. 5). Given the critical role of type II methanotrophs in regulating the temperature sensitivity of soil microbial CH₄ oxidation, soil resources are key in shaping this sensitivity. Moreover, climate has been proven to influence soil weathering, leading to significant variability in soil resources on a large-scale^48,49. These findings highlight the significance of the interactive effects of climate, soil resources, and key microbial groups in regulating the temperature sensitivity of soil microbial CH₄ oxidation, suggesting that considering these interactions is essential for robust prediction of future global forest soil CH₄ uptake under warming.

While our study provides a consistent way to examine the temperature sensitivity of forest soil microbial CH₄ oxidation across a broad geographic scale, some uncertainties remain. The temperature sensitivity values we reported (Supplementary Data 1) are higher than those found in previous studies^12,16,18. One possibility is that CH₄ oxidation rates in our study were measured under conditions of ample CH₄ substrate and optimal moisture, which may have mitigated the limiting effects of environmental factors at elevated temperatures, thus increasing temperature sensitivity^22,50. Additionally, the temperature sensitivity of soil microbial CH₄ oxidation was assessed within a range of 15–25 °C, reflecting the average growing season temperatures at the sites, which ranged from 10.04 to 24.19 °C (Supplementary Data 1). However, this range may not fully capture in situ responses of forest soil CH₄ oxidation to ambient temperature changes⁵¹, especially in regions where temperatures are low during the non-growing season. To better understand how microbial CH₄ oxidation responds to climate warming, future research should focus on capturing seasonal variations in the temperature sensitivity through in situ observations.

Overall, our research reveals the broad-scale spatial variation and key drivers of forest soil CH₄ oxidation in response to warming. We found that the temperature sensitivity of soil microbial CH₄ oxidation was highest in colder regions, suggesting a greater potential for the CH₄ sink in these areas under global warming. Additionally, our results emphasize the critical roles of soil resources and type II methanotrophs in shaping the spatial patterns of temperature sensitivity in soil microbial CH₄ oxidation. Our findings provide a previously unrecognized perspective that improves our understanding of forest soil CH₄ oxidation and its response to warming in the face of variable climate and edaphic factors, thereby helping to refine and validate biogeochemical models that predict the global CH₄ sink under warming.

Methods

Study area and soil sampling

Between June and August 2023, we collected 336 topsoil (0–10 cm) samples from 84 forest sites across eastern China along a ~4000 km south–north transect. This transect spanned a wide range of latitudes (18.7°–53.4° N) and longitudes (100.3°–134.7° E) and covered five climatic zones (cold-temperate, mid-temperate, warm-temperate, subtropical and tropical zones). The mean annual temperature of these sites ranges from −4.6 to 24.19 °C, and the mean annual precipitation varies from 337 to 1884 mm. Detailed information about the sites is provided in Supplementary Data 1.

At each forest sampling site, we randomly established four 10 × 10 m sampling plots separated by more than 100 m from each other. We took six soil core samples (0–10 cm) following a zigzag path through each plot and then mixed them to form a composite sample. We then sieved the samples through a 2 mm mesh and divided them into three subsamples. One subsample was stored at 4 °C for soil incubation, one was stored at −20 °C for microbial DNA extraction, and the third was air-dried to constant weight and processed for measurements of soil properties. The soil samples were kept at 4 °C for no longer than 4 weeks before the incubation experiment began. Previous studies have shown that storing soil samples at 4 °C for up to 7 weeks does not significantly affect microbial activity or its temperature sensitivity^52,53. Therefore, the storage conditions used in this study are unlikely to have a major impact on the subsequent analyzes of microbial CH₄ oxidation rates^52,54.

Incubation experiments

The incubation experiment aimed to assess the intrinsic temperature sensitivity of soil microbial CH₄ oxidation, which is defined as the change in CH₄ oxidation rate with temperature under otherwise constant conditions⁵⁵. For each site, 10 g of dry-weight fresh soil, with four replicates, was weighed into 140 mL incubation bottles. To control for potential variations in soil moisture across different sites, we adjusted the soil moisture to 60% of the water-holding capacity using sterile deionized water, which is optimal for microbial activity and gas diffusion⁵⁶. To activate microorganisms and minimize possible disturbances, we performed a 3-day pre-incubation at 20 °C, a period sufficient to restore soil microbial CH₄ oxidation activity (Supplementary Fig. 4). Since the growing season is typically the most critical period for soil CH₄ oxidation⁵¹, with average growing season temperatures at the sampling sites ranging from 10.04 °C to 24.19 °C (Supplementary Data 1), the subsequent incubation experiments were conducted at three temperatures—15 °C, 20 °C, and 25 °C—to capture the temperature response of microbial CH₄ oxidation during the active period of methanotrophs in forest soils.

During the incubation, each bottle was sealed with a butyl rubber stopper, and pure CH₄ was injected to approach a concentration of ~200 ppm. This concentration was chosen because it is high enough to enable the maximum potential CH₄ oxidation rate across different soils, as lower, atmospheric-like CH₄ concentrations might limit the activity of soil methanotrophs (Supplementary Fig. 5). Next, a 5 mL gas sample was collected using a syringe, and 5 mL CH₄-free air was immediately injected into the bottle to avoid the air pressure changes in the incubation bottles. The sealing times for the bottles at 15 °C, 20 °C, and 25 °C were ~3, 2, and 2 h, respectively. After the sealing period, another 5 mL gas sample was collected from the headspace. The CH₄ concentrations in the gas samples were measured using gas chromatography (Agilent 7890 A, Agilent Technologies Inc., Santa Clara, California, USA). The potential soil CH₄ oxidation rate was then calculated as follows:

$$P=frac{{dc}}{{dt}}times frac{{Vh}}{{Ws}}times frac{{MW}}{{MV}}times frac{{Ts}}{T}$$

(1)

where P indicates the potential soil CH₄ oxidation rate (μg CH₄ g^–1 soil d^–1), dc/dt is the rate of CH₄ concentration change. Vh indicates incubation bottle headspace volume (m³), and Ws indicates soil dry-weight (g). MW and MV indicate molar mass of CH₄ (16 g mol^–1) and gas molar volume under standard air pressure (22.4 L mol^–1), respectively. Ts and T represent standard temperature (273.15 K) and incubation temperature (K), respectively.

Model choice for soil methane oxidation rate and its temperature sensitivity

The response of biological processes to temperature change can often be fitted using different functional forms⁵⁷. In this study, we evaluated the performance of three widely used models—the linear, exponential, and Arrhenius equations—to describe the relationship between soil microbial CH₄ oxidation rate and temperature. Our analysis revealed that the linear equation provided the best fit for the majority of sampling sites, outperforming both the exponential and Arrhenius models (Supplementary Table 2). Therefore, we determined the temperature sensitivity of microbial CH₄ oxidation as the slope of the linear relationship between soil CH₄ oxidation rate and temperature change (Supplementary Fig. 6), as follows:

$$P=a , * , T+b$$

(2)

where P indicates the potential CH₄ oxidation rate (μg CH₄ g^–1 soil d^–1), T indicates incubation temperature (°C), and a and b are fitting parameters.

Given that climate warming exhibits marked non-uniformity across the globe, the intrinsic temperature sensitivity is thus defined here as the change rate of P with respect to temperature while all other variables are held constant⁵⁸. Mathematically, this corresponds to the partial derivative of P with respect to temperature, ∂P/∂T, meaning the absolute change of P for a given unit change in temperature^58,59. In this study, the parameter a value represents the temperature sensitivity of soil microbial CH₄ oxidation and allows direct comparisons of the absolute change in soil CH₄ oxidation rate for a 1 °C temperature increase over a large geographic scale. Indeed, the temperature sensitivity of biological processes expressed as partial derivatives has been widely used^60,61.

Furthermore, we calculated the Q₁₀ value, which reflects the relative increase in microbial metabolic rate with a 10 °C rise in temperature⁶², for soil microbial CH₄ oxidation rate at both lower (between 15 and 20 °C) and higher (between 20 and 25 °C) temperature ranges. The Q₁₀ value was calculated using the following equation:

$${Q}_{10}={left(frac{R({T}_{2})}{R({T}_{1})}right)}^{frac{10}{({T}_{2}-{T}_{1})}}$$

(3)

where R(T₁) and R(T₂) are soil CH₄ oxidation rates in temperatures T₁ and T₂, respectively.

Climate data and soil analysis

Climate data, including mean annual temperature, mean annual precipitation, annual maximum temperature, and annual temperature variation (defined as the difference between the annual maximum and minimum temperatures), were extracted from the Worldclim dataset for the period 1970–2000⁶³.

Soil water content was determined by drying samples at 105 °C for 24 h. The maximum water-holding capacity of the soil was measured using the funnel-filter paper-drainage method⁶⁴. In this process, deionized water was used to pre-saturate the filter paper in the funnel. Then, 10 g of air-dried soil was placed into the wet filter paper funnel, and the soil was saturated by wetting the outer rim of the exposed filter paper. Once the soil appeared glistening and a small amount of water was visible on the surface, the funnel was covered with cling film that had small holes to minimize evaporation. After 6 h, the drained soil was weighed and then oven-dried to determine its dry weight.

Total carbon (TC) was measured using a TOC analyzer (Multi N/C 3100, Germany), while total nitrogen (TN) was quantified using the Kjeldahl method⁶⁵. The contents of ammonium (NH₄⁺) and nitrate (NO₃⁻) in the soil were determined in 2 M KCl extracts (with a soil-to-extract ratio of 1:4 by weight) using a flow injection analyzer (AutoAnalyzer 3 SEAL, Bran and Luebbe). Soil pH was measured using a 1:2.5 soil-to-water suspension and a pH meter. Soil texture was analyzed using a particle size analyzer (BT-9300ST, China).

DNA extraction, PCR amplification, and high-throughput sequencing

Soil total DNA was extracted from 300 mg of composite frozen soil sample from each site using the ALFA-SEQ Magnetic Soil DNA Kit (Findrop Biosafety echtnology (Guangzhou) Co. Ltd) according to the manufacturer’s instructions and was stored at −80 °C for later use. The pmoA gene, which encodes the critical enzyme methane monooxygenase (particulate MMO), is commonly employed to identify methanotroph communities. We determined the copy numbers of the pmoA gene using quantitative PCR (qPCR) with the primer pairs pmof1/pmor (Supplementary Table 3). We determined the bacterial community and methanotroph community using high-throughput sequencing analysis of the 16S rRNA gene with primers 515F/806R and the pmoA gene with primers pmof1/pmor (Supplementary Table 3). This pmoA primer has proven useful for amplifying both cultured and uncultured methanotrophs (Supplementary Table 4)^66,67. Samples with clear, bright main bands were selected for further experiments. The sequencing library was prepared and sequenced on an Illumina NovaSeq 6000 platform at Guangdong Magigene Biotechnology Co., Ltd, Guangzhou, China. This allowed for a comprehensive analysis of the microbial communities present in the soil samples.

The QIIME2 was used to process the raw sequences⁶⁸. DADA2, integrated within QIIME2, was employed to cluster the sequences into amplicon sequence variants (ASVs) after filtering out adaptor sequences, low-quality reads, ambiguous nucleotides, and barcodes. Taxonomic assignment of the 16S rRNA and pmoA sequences was performed using the Ribosomal Database Project (RDP) classifier, referencing the SILVA database (Version 138) for 16S rRNA and the FunGene database (Version 1.0) for pmoA sequences. To ensure adequate representation of each community’s structure, we removed bacterial samples with fewer than 70,320 sequences per sample and methanotroph samples with fewer than 32,140 sequences per sample, leaving 60 sites for further analysis. Rarefaction curves confirmed that the sequencing depth was sufficient to assess the diversity and community composition of the microbial populations in the soil (Supplementary Fig. 7). Richness, which describes the microbial alpha diversity, was used as a key metric to quantify the diversity within each community⁶⁹.

Statistical analyzes

We divided five types of explanatory factors related to the temperature sensitivity of soil microbial CH₄ oxidation: climate factors (mean annual temperature, mean annual precipitation, annual maximum temperature and annual temperature variation), soil resources (total carbon content, total nitrogen content, soil C:N ratio, ammonium and nitrate content), soil properties (soil pH and clay content), bacterial community composition (relative abundances of Proteobacteria, Acidobacteriota, and Verrucomicrobia), and methanotroph traits (pmoA gene copy number, methanotroph richness, relative abundance of type I methanotrophs, relative abundance of type II methanotrophs, and the ratio of type II to type I methanotrophs). Spearman correlation analysis was conducted to identify the positive or negative correlations among the explanatory factors as well as between each factor and the temperature sensitivity of soil microbial CH₄ oxidation. Variance decomposition analysis was performed to quantify the relative importance of climate factors, soil resources, soil properties, bacterial community composition, and methanotroph traits in explaining the temperature sensitivity of soil microbial CH₄ oxidation, using the package of “glmm.hp”^70,71.

Additionally, independent random forest analysis was used to identify the key drivers of temperature sensitivity in soil microbial CH₄ oxidation⁷². The importance of each predictor was determined using the R package “spatialRF”⁷³, which reduces multicollinearity, identifies relevant variable interactions, and assesses model transferability via spatial cross-validation⁷⁴. To evaluate the relationships between the temperature sensitivity of soil microbial CH₄ oxidation and each variable, a mixed-effects model with the sampling site as a random factor was performed with “nlme” package⁷⁵. One-way ANOVA was conducted to examine differences in soil CH₄ oxidation rates and their temperature sensitivity across different climatic zones.

Structural equation model

To explore the complex and potentially simultaneous interactions between climate factors, soil resources, soil properties, bacterial community composition, and methanotroph traits influencing the temperature sensitivity of soil microbial CH₄ oxidation, we applied a structural equation model (SEM). Given the correlations among these factors (Fig. 2), we first performed principal components analysis (PCA) to create three new composite indices for climate factors, soil resources, soil properties. The first component (PC1), which explained 48.9–82.9% of the total variance across these groups, was introduced as a new variable in the SEM. We fit and evaluated the model using restricted maximum likelihood with the “nlme”⁷⁵ and “piecewiseSEM”⁷⁶ packages. After determining the best model, we conducted a goodness-of-fit evaluation for piecewise SEM based on Fisher’s C and chi-squared tests (P > 0.05). All statistical analyzes were performed using R statistical software (v.4.2.0).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.