P addition weakens the positive effects of N addition on CH4 uptake and does not affect the effects of N addition on CO2 emission in alpine grasslands

doi:10.21203/rs.3.rs-4700494/v1

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Research Article

P addition weakens the positive effects of N addition on CH4 uptake and does not affect the effects of N addition on CO2 emission in alpine grasslands

https://doi.org/10.21203/rs.3.rs-4700494/v1

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Aims

Anthropogenic activities have substantially elevated nitrogen (N) deposition globally and affect ecosystem processes, including soil carbon (C) storage potential. Phosphorus (P) can become a limiting factor for plant production in instances of N deposition, yet the responses of ecosystem C cycles to P enrichment are poorly understood, particularly in sensitive alpine ecosystems.

Methods

We conducted a short-term field study to appraise the effects of N and P addition on ecosystem CO₂ emissions and CH₄ uptake in three typical alpine grasslands, alpine meadow, alpine steppe, and cultivated grassland on the Qinghai-Tibet Plateau (QTP). The closed chamber technique was employed to monitor the fluxes of CO₂ and CH₄. Environmental factors, including plant biomass and diversity and soil nutrients, and the abundance of C-cycling genes were analyzed to investigate the factors regulating CO₂ and CH₄ fluxes.

Results

The results showed that: (i) N and P addition tended to increase CO₂ emissions and CH₄ uptake. Furthermore, P addition weakened the positive effects of N on CH₄ uptake across the three grasslands, but the interaction of N and P addition on CO₂ emissions varied across the three grasslands. (ii) N and P addition affected the fluxes of CO₂ and CH₄ both directly and indirectly through their impacts on soil and plant factors rather than C-cycling functional genes.

Conclusions

These results indicate that in the context of increasing N deposition in the QTP, short-term P addition is not an effective method for mitigating global warming potential and improving soil C sequestration in alpine grassland ecosystems.

Carbon dioxide

Methane

functional genes

N addition

P addition

Qinghai-Tibet Plateau

As the largest carbon (C) repository (> 1500 Pg C) in terrestrial ecosystems, soil stores more C than terrestrial vegetation and atmosphere combined (Batjes 2014). Due to the huge size of soil C pool, any small change of this pool will result in a significant dynamic in atmospheric carbon dioxide (CO₂) concentration (Bond-Lamberty and Thomson 2010). Soil CO₂ and methane (CH₄) fluxes, two main source components of soil C fluxes, are important regulators of soil C sequestration (Raich and Schlesinger, 1992; Pirk et al. 2016). In the context of future climate change, environmental changes are expected to have a profound impact on soil C fluxes and belowground C storage, which could have a significant impact on the global C cycling.

N deposition can affect soil C fluxes by changing plant growth and soil microbial population and composition (Chen et al. 2013b). Numerous studies assessed N enrichment's influence on soil C fluxes, and obtained the results contradictory results including increase (Luo et al. 2019), decrease (Zeng and Wang 2015, Wang et al. 2019) and no change(Ambus and Robertson 2006). The contrasting responses might be associated with differences in ecosystem types, environmental conditions, soil conditions, N fertilizer duration and rates (Zhong et al. 2016, Deng et al. 2020). Such variable responses could also suggest that N effects on soil C fluxes may depend on the addition of other nutrients in soil, such as P (Fornara et al. 2013). However, compared to the widely studied effects of N addition, the responses of C fluxes to P input has received less focus. Moreover, the mechanistic understanding of P effects on soil C fluxes is still poor, which hampers our capacity to forecast the long-term sustainability of ecosystem. Thus, comprehending how ecosystem C fluxes respond to N and P input is pivotal for the prediction of terrestrial C cycle.

Soil is the largest natural source of key greenhouse gas (GHG) of CO₂ (Bahn et al. 2008), which is released during heterotrophic respiration processes of microbial communities and autotrophic respiration processes of live roots (Kuzyakov 2006). For soil CO₂ flux, the important regulators include soil temperature and moisture, soil properties and vegetation composition (Raich and Tufekciogul 2000, Smith et al. 2003, Robroek et al. 2015). N and P enrichment directly provide more substrates for plant growth thus stimulating root respiration (Ambus and Robertson 2006, Mbonimpa et al. 2015, Feng and Zhu 2019), and indirectly alter activity and community structure of soil microorganisms through alleviating N and P limitations thus changing heterotrophic respiration (Wang et al. 2019).

Soil also performs a pivotal function in the production and oxidation of CH₄, a GHG whose 100-year global warming impact outpaces CO₂ by 28-fold (IPCC 2013). In soil, CH₄ is produced by methanogenic bacteria during anaerobic decomposition, in aerobic conditions CH₄ can be oxidized by methanotrophic bacteria (Le Mer and Roger 2001). The controllers of soil CH₄ fluxes include soil moisture, temperature, microbial activity, organic matter content and N availability (Mbonimpa et al. 2015). Changes in soil N and P availability could influence both methanogens and methanotrophs directly and through plant growth indirectly, thus consequently stimulate or inhibit soil CH₄ flux (Fang et al. 2014, McEwing et al. 2015).

Ecosystem CO₂ and CH₄ fluxes under N and P input vary with different ecosystems (Liu and Greaver 2009, Feng and Zhu 2019). For example, N addition stimulated CO₂ emission and inhibited CH₄ uptake in an alpine steppe (Qu et al. 2021), reduced soil CO₂ emission in an alpine meadow (Wang et al. 2019), increased CH₄ uptake in alpine grassland (Li et al. 2012), but did not have a significant impact on the fluxes of CO₂ and CH₄ in a Southern California grassland (Aronson et al. 2019). There are also great discrepancies in the effects of P input. For example, P input increased soil CO₂ emission in tropical forest (Hui et al. 2020), decreased soil CO₂ emission in a subtropical secondary forest (Zhang et al. 2021), but did not affect soil respiration in a typical steppe (Liu et al. 2021). These findings indicate that the effects of N and P enrichments on CO₂ and CH₄ fluxes should be predicted separately for different biomes (Zhong et al. 2016).

Grassland ecosystems, covering about 40% of the Earth’s land surface (Morrison 2006), are important C sinks and exert strong controls on global C budget and climate change. Qinghai-Tibetan Plateau (QTP) is the world's largest and highest plateau, covering an area of 2.53×10⁸ km² (Li and Zhou, 1998). Alpine grasslands, the most dominant ecosystems on QTP, occupy 50.9% of the total area and store about 10% of soil organic C in China (Dong et al. 2007, Yang et al. 2008). Therefore, research on CO₂ and CH₄ fluxes from alpine grasslands on the QTP is crucial for assessing grassland ecosystem C budget. In recent years, the atmospheric N deposition in QTP have continuously increased, with an average N deposition rate of 8.7–13.8 kg N ha^− 1 year^− 1 (Lü and Tian 2007). Because of its unique climate and hydrology, the alpine ecosystems of the QTP are more prone to be disturbed by climate change (including N deposition) than other ecosystems (Chen et al. 2013a). Since plant and microbial growth are limited by low N and P availability in this region (Li et al. 2019), increase in soil N and P availability will not only affect the productivity and vegetation composition of plants (Li et al. 2019, Zheng et al. 2022), but also affect the composition and function of soil microbe (Zheng et al. 2022), and finally altering CO₂ and CH₄ fluxes (Zhao et al. 2017). However, the increasing trend of N deposition is ineluctable in current models of N climate change.

Here, we conducted N and/or P addition experiments in three typical alpine grasslands, alpine meadow, alpine steppe, and alpine cultivated grassland on the QTP, to investigate how N and P addition influence soil-atmosphere exchanges of CO₂ and CH₄. We hypothesized: (1) N and P addition would increase CO₂ emissions and CH₄ uptake due to the increased plant production resulting from the alleviation of N and P limitation; and (2) P addition would augment the effect of N addition on CO₂ emissions and CH₄ uptake due to the synergetic effects on plant growth of N and P. The aims of this study were to provide sound scientific basis for carbon management in alpine grassland ecosystems to cope with increasing N deposition

Study site

We conducted this study from 2019 to 2020 in Xihai Town (36°56′N, 100°57′E, 3100 m.a.s.l.) and Tiebujia Town (37°02′N, 99°35′E, 3270 m.a.s.l.) of Qinghai province, China (Fig. S1). In Xihai Town, mean annual precipitation (MAP) is 330–370 mm, mean annual temperature (MAT) is 0.9–2.2°C, and soil is Mat-Gryic Cambisols. The typical vegetation is alpine meadow (AM), dominated by Kobresia capillifolia and Carex melanantha. In Tiebujia Town, MAP is 377 mm, MAT is -0.4–1.2°C, and soil is predominately dark chestnut. The typical vegetation is alpine steppe (AS), with the dominant species of Stipa capillata and Poa crymophila. The experimented AM and AS were fenced in 2014, and the cultivated grassland (CG) was planted with Elymus nutans in 2012 next to AS in Tiebujia Town.

Experimental design

Nitrogen and phosphorus addition experiments were initiated in May 2019. In each type of grasslands, 4 treatments (in 3 replicates) were established: CK (without N or P added), N (72 kg N ha^− 1 yr^− 1), P (36 kg P ha^− 1 yr^− 1), and NP (72 kg N ha^− 1 yr^− 1 + 36 kg P ha^− 1 yr^− 1). Twelve 2 m ×5 m plots were established with each plot separated by 1 m wide buffer strips. All treatments were laid out randomly. N in the form of NH₄NO₃ and P in the form of Ca(H₂PO₄)₂ were applied in early May, July, and September from 2019 to 2020.

Measurement of CO₂ and CH₄ fluxes

The CO₂ and CH₄ fluxes were measured using static chambers daily (except during cloudy weather) from mid-July to early mid-August of 2019 and 2020. Polyvinylchloride collars (20 cm in internal diameter and 10 cm in height) were inserted 4–5 cm into soil in each plot. During sampling times, each base was covered with a removable top (20 cm in diameter and 25 cm in height). Gas samples were taken at time intervals of 0, 5, 15, and 30 min and were collected between 9:00 a.m. and 12:00 p.m. local time. Gas samples were collected from the chamber using 100 mL polypropylene syringes and injected into pre-evacuated polyethylene-coated aluminum bags (100 mL). The concentrations of CO₂ and CH₄ were analyzed using a gas chromatograph (Shimadzu, GC-2014C) fitted with a thermal conductivity detector for CO₂ and a flame ionization detector for CH₄. The plants within chambers were not disturbed and not exposed to light during measurement, thus the CO₂ flux reflected the ecosystem respiration. The fluxes of CO₂ and CH₄ were adjusted for chamber area, headspace volume and field sampling temperature (Robertson et al. 1999).

Soil sampling and analysis

Soil samples were collected in mid-August 2019 and 2020 to a depth of 20 cm randomly in each plot. Gravel and plant residue were removed. Soil samples were divided into two parts, one was preserved at 4°C for the determination of soil microbial biomass and properties, and the other was preserved at -80°C until DNA extraction.

Soil total nitrogen (TN) and total carbon (TC) were determined using an element analyzer (EA 3000, Italy). Soil available phosphorus (AP) was determined by an inductively coupled plasma spectrometer (ICP) (SPECTRO ARCOS EOP, Germany). Soil NH₄⁺ and NO₃⁻ were measured using an autoanalyzer (AACE, Germany). Soil pH value was determined using a combination glass electrode. Soil dissolved organic carbon (DOC) was determined using a TOC analyzer (TOC-L CPN, Japan). Soil microbial biomass carbon (MBC) and nitrogen (MBN) were measured using the chloroform fumigation method (Brookes et al. 1985, Vance et al. 1987).

Plant sampling and analysis

In mid-August each year from 2019 to 2020, plant community characteristics (plant coverage, species richness and diversity) were measured. In each plot, one 1 m × 1 m quadrat was selected. The plant coverage was measured using the grid method. The species richness was measured by counting the number of all species in the quadrat. Shannon-Wiener index (H') was calculated as:

$$\:{H}^{{\prime\:}}=-\sum\:_{i=1}^{S}{P}_{i}\text{ln}{P}_{i}$$

where S is the number of species, and P_i is the individual number of species i in proportion to the total number of individuals.

For aboveground (AGB) and belowground biomass (BGB), three 50 cm × 50 cm quadrats were randomly selected in each plot and all aboveground plants were sampled to assess AGB. At the same time, three soil cores were collected to a depth of 40 cm to assess BGB. The soil cores were sieved through 0.5 mm sieves soaked in gently flowing water to separate roots. Then, all plant samples were oven-dried (65°C for 48 h) to constant weight, weighted and grinded. The plant N concentration was measured using elemental analysis (EA 3000, Italy) and P concentration was measured using ICP (SPECTRO ARCOS EOP, Germany).

Soil DNA extraction and quantification of functional gene abundance

Soil DNA extraction and quantification of functional gene abundance were following Dong et al.'s (2020) approach (Table S1).

Data analysis

The statistical analyses were conducted using SPSS (v 25) and R (v 4.2.3). The significant differences were set at p < 0.05, unless otherwise stated. In all tests, normal distribution and homogeneity of variance were confirmed using Shapiro Wilk and Levene's test, respectively. Logarithmic or square root transformation was used to fulfil normality and homogeneity. Repeated measures analysis of variance (ANOVA) was performed to examine the effects of sampling mouth, grassland type, experimental treatment and their interactions on CO₂ and CH₄ fluxes. Repeated measures ANOVA was also performed to test the effects of N addition, P addition and their interactions on CO₂ and CH₄ fluxes in each grassland type. One-way ANOVA was used to test N and P effects on CO₂ and CH₄ fluxes and environmental factors. Multiple regression models were used to assess the joint effects of soil, plants, microbes (i.e. MBC and MBN), and climate on CO₂ and CH₄ fluxes. Firstly, a model selection procedure based on AIC was used to select the best predictors. This procedure was performed using the R package MuMIn. Second, reweights function in R was used to evaluate the relative importance of predictor variables. Structural equation models (SEM) were constructed with the Amos 21.0 (IBM, SPSS) to examine direct and indirect hypothetical relationships among N addition, P addition, environmental factors, and CO₂ and CH₄ fluxes. Using maximum likelihood estimation method, data were fitted to the model. An insignificant (p > 0.05) Chi-square test indicates adequate model fit.

Changes of CO₂ and CH₄ fluxes with N and P addition

The alpine grasslands acted as CO₂ source and CH₄ sink at the peak growing season (Fig. 1, 2). CO₂ emissions and CH₄ uptake were significantly affected by grassland type, experimental treatment and sampling year, and the interactions between them (Table 1). However, N and P showed inconsistent effects on CO₂ emissions in the three alpine grasslands (Fig. 2). The significant effects of N on CO₂ emissions were only found in AM, but significant effects of P on CO₂ emissions were found in AS and CG (Table 1). CO₂ fluxes increased with N addition alone in AM in 2019 and 2020 and in CG in 2019, but remained unchangeable with N addition in AS (Fig. 1). On the other hand, P addition alone promoted CO₂ fluxes in AS and CG, but had no effects on CO₂ fluxes in AM (Fig. 1). CH₄ uptake tended to increase with N and P addition and were significantly affected by the interactions between N addition and P addition in all grasslands (Fig. 2). Specifically, N addition alone increased CH₄ uptake in all grasslands in both 2019 and 2020, P addition alone increased CH₄ uptake in all grasslands in 2019 and only AM in 2020, and N and P co-addition increased CH₄ uptake except for AS in 2020 (Fig. 2). CO₂ emissions and CH₄ uptake were significantly affected by the interaction between N and P for all grasslands except for CO₂ emissions in AS in 2019 (Fig. 1, 2). Under combined N and P addition, CO₂ emissions decreased in AM, remained unchangeable in AS, and increased in CG (Fig. 1). CH₄ uptake decreased in AM and CG and remained unchangeable in AS (Fig. 2).

Table 1

Results of repeat-measurement ANOVA on the effects of sampling year (Year), grassland type (GT), experimental treatment (ET), and their interactions on CO₂ and CH₄ fluxes.
	Year	GT	ET	Year×GT	Year×ET	GT×ET	Year×GT×ET
CO₂	20.85^***	15.89^***	52.87^***	21.99^***	14.9^***	16.44^***	3.04^*
CH₄	12.6^**	35.59^***	58.62^***	9.01^***	1.05	4.74^**	0.88
Note: ^* p < 0.05; ^ p < 0.01; ^* p < 0.001.

Changes of C-cycling genes with N and P additions

In AM, the abundance of most C degradation genes (from labile to recalcitrant C, including starch, cellulose, chitin, pectin, and lignin) and all methane metabolism genes increased significantly under P addition alone (Fig. 3). N addition alone decreased most of the abundance of C degradation and methane metabolism genes, but significant difference was only observed in the abundance of pectin degradation genes (exoPG) (Fig. 3). P addition alone had significant positive effects on the abundances of most genes involved in starch, pectin, and lignin degradation and all methane metabolism genes (Fig. 3).

In AS, the abundance of C degradation and methane metabolism genes increased under N or P addition alone, but decreased under N and P co-addition (Fig. 3). However, significant differences were only observed in xylA (hemicellulose degradation) and exg (cellulose degradation) under N addition alone, and in the abundance of manA and xylA (hemicellulose degradation) under N and P co-addition (Fig. 3).

In CG, N addition alone decreased the abundance of C degradation and methane metabolism genes except for amyA, and reached the significant level except for CDH and exoPG (Fig. 3). P addition alone or N and P co-addition had positive or negative effects on the abundance of C degradation and methane metabolism genes, but did not reach the significant level (Fig. 3).

Factors impacting CO₂ and CH₄ fluxes under N and P addition

In AM, N addition directly contributed to CO₂ fluxes and indirectly contributed to CO₂ fluxes through plant richness, and P addition indirectly contributed to CO₂ fluxes through N:P_root (Fig. 4A). N addition adversely affected plant richness, and P addition positively affected N:P_root, which consequently affected CO₂ fluxes (Fig. 4A). Soil pH also directly contributed to CO₂ fluxes and indirectly contributed to CO₂ fluxes through plant richness (Fig. 4A). The SEM indicated that CH₄ fluxes was directly controlled by N addition, plant richness and MBC (Fig. 5A). P addition exerted an indirect effect on CH₄ fluxes by affecting N:P_root (Fig. 5A).

In AS, N addition indirectly contributed to CO₂ fluxes through MBC. N addition adversely affected MBC, which consequently affected CO₂ fluxes indirectly (Fig. 4B). CO₂ fluxes was also directly influenced by P addition, plant Shannon-Wiener and BGB based on SEM analysis (Fig. 4B). N addition and P addition indirectly contributed to CH₄ fluxes through N:P_root and BGB based on SEM analysis (Fig. 5B). N addition positively affected N:P_root, and P addition adversely affected BGB, which consequently affected CH₄ fluxes indirectly (Fig. 5B). Soil AP also exerted a direct effect on CH₄ fluxes based on SEM analysis (Fig. 5B).

In CG, N addition exerted an indirect effect on CO₂ fluxes by affecting soil NO₃⁻ (Fig. 4C). P addition indirectly contributed to CO₂ fluxes through AGB and soil NO₃⁻. P addition positively affected AGB and soil NO₃⁻, which consequently affected CO₂ fluxes indirectly (Fig. 4C). CO₂ fluxes was directly affected by P addition and soil NO₃⁻ (Fig. 4C). CH₄ fluxes were directly affected by N addition, P addition, plant coverage, and soil NH₄⁺ based on SEM analysis (Fig. 5C). N addition mainly directly contributed to CH₄ uptake. In addition to the direct impact, P addition positively influenced plant coverage, which consequently affected CH₄ uptake indirectly (Fig. 5C).

Effects of N and P additions on CO₂ emissions

The response of CO₂ emissions to N and P addition varied depending on the grassland type. In alignment with our first hypothesis partially, N addition alone promoted CO₂ emissions in AM, and P addition alone promoted CO₂ emissions in AS and CG. Similar findings have been reported in other studies (Ambus and Robertson 2006, Fang et al. 2012, Mbonimpa et al. 2015, Feng and Zhu 2019). P addition increased the effects of N addition on CO₂ emission in CG in 2019, but decreased the effects of N addition on CO₂ emission in AM in 2020 (Fig. 1). This may be due to the different climate and soil conditions in the three grasslands (Fig. S2, S3).

In AM, the plant richness and root N:P ratio play an important role in the response of CO₂ emissions to N and P addition. Besides, the addition of N and P has no significant effects on soil properties, indicating that plants may have a greater impact on CO₂ emissions than soil in the peak growing season (Fig. S3). The significant promotion of CO₂ emissions by N addition confirms previous findings from N addition studies in N-limited natural ecosystems (Ambus and Robertson 2006, Fang et al. 2012, Mbonimpa et al. 2015). On the one hand, N addition directly increased CO₂ emissions. Because N addition can increase plant photosynthates, litter decomposition, and root exudation, which triggers soil microbial activity and subsequently stimulates CO₂ fluxes (Güsewell 2004, Cleveland et al. 2006, Bahn et al. 2008). For example, soil CO₂ emissions in a Moso Bamboo Plantation were significantly related to litter stoichiometry (Tian et al. 2021). However, we did not measure the stoichiometry of litter and root exudates, which requires further investigation. On the other hand, the increase in CO₂ emissions with N addition may be resulted from the decreased N:P ratio of shoot and root (Fig. S2). It has been reported that, root N content exert significant influence on the variance in CO₂ fluxes in a temperate grassland (Barneze et al. 2022), aboveground biomass was a main driver of CO₂ emissions from alpine meadow soil (Fang et al. 2012), and leaf N was the most important factor for the prediction of CO₂ emissions in Chinese fir plantations (Xie et al. 2023). The effect of P addition on CO₂ emissions is negligible, but P addition showed positive effect on plant aboveground biomass and shoot N:P ratio (Fig. S2, S4), indicating that the positive and negative effects of P addition on CO₂ emissions cancel each other out.

In AS, N addition showed little effects on CO₂ emissions. This is similar to other short-term N addition experiments (Ambus and Robertson 2006, Jiang et al. 2010). N addition is generally thought to increase CO₂ emissions when soil microbial activity was N-limited (Jiang et al. 2010). This, combined with no change in microbial biomass, indicates that there is no microbial N limitation in the alpine steppe we studied. The increase in CO₂ emissions with P addition was mainly a direct effect. P addition was demonstrated to increase CO₂ emissions by increasing soil organic matter decomposition in P-limited tropical forest (Kaspari et al. 2008). In this study, the abundance of C-cycling genes showed a significant correlation with plant N:P ratio, which is significantly influenced by P addition (Fig. S2, S5). Therefore, we speculated C degradation processes in soil may be stimulated by P addition via increasing plant N:P ratio (Fig. 3, Fig. S3), thus increasing CO₂ emissions. Changes in plant diversity caused by P addition could also affect CO₂ emissions by altering plant N:P ratio.

In CG, N addition only increased CO₂ emissions in 2019, while P addition increased CO₂ emissions in both 2019 and 2020. The SEM analysis also showed a minor effect of N addition on CO₂ emissions. Like in AS, the soil microbial activity in CG was not N-limited too, because their study areas are right next to each other. P addition promoted CO₂ emissions mainly via enhancing AGB and soil NO₃⁻. For the P-limited alpine ecosystems we studied (Xiao et al. 2022), P addition promotes plant N demand, and greater energy demands of root respiration for acquiring accessible N leads to augmented root respiration and subsequently higher CO₂ emissions (Bloom et al. 1992, Bowden et al. 2004). In this study, P addition increased plant aboveground biomass and N:P ratio, which were significantly correlated with CO₂ emissions (Fig. S6). P addition increased aboveground biomass by increasing soil P availability, thereby increasing CO₂ emissions. Just like the drivers of CO₂ emissions from AM, the plant N:P ratio also play an important role in driving CO₂ emissions from CG. Litter stoichiometry is a reliable indicator of soil respiration (Tian et al. 2021), thus as the source of litter, the stoichiometry of plant biomass also has significant implications for CO₂ emissions. Short-term nutrient addition may alleviate nutrient limitation to plant growth in alpine grassland ecosystems, while other environmental factors become more important for CO₂ emissions in the long term.

Effects of N and P additions on CH₄ uptake

Our results showed that the alpine grassland ecosystems are net sinks of CH₄. CH₄ uptake by soil are the balance between the production of CH₄ by methanogenesis and oxidation of CH₄ by methanotrophs (Le Mer and Roger 2001). Our study found that N and/or P addition increased CH₄ uptake irrespective of the type of alpine grasslands, consistent with our first hypothesis. The increased CH₄ uptake could result from stimulated CH₄ consumption or inhibited CH₄ production from soil after P addition (Zheng et al. 2016). Studies have more commonly observed inhibiting effects of N addition on CH₄ uptake (Gao et al. 2017, Chen et al. 2022, Yang et al. 2022), which is primarily caused by the following mechanisms: toxic inhibition by hydroxylamine (NH₂OH) and nitrite (NH₂⁻) on methanotrophs, NH₄⁺ acting as the competitor for CH₄ mono-oxygenase enzyme, and osmotic stress caused by high concentrations of soil inorganic N (Reay and Nedwell 2004). However, there are also contradictory reports regarding N input effects on CH₄ uptake (Yue et al. 2016, Stiles et al. 2018, Xia et al. 2020, Chen et al. 2023). For example, N input stimulated CH₄ uptake in a saline-alkaline grassland soil (Chen et al. 2023), and showed no effects on CH₄ uptake in alpine grassland (Yue et al. 2016). N availability was a limiting factor for CH₄ oxidation in the N-limited ecosystem, because mineral N was necessary for the stimulation of methanotrophs (Bodelier and Laanbroek 2004). Therefore, N fertilization would be favorable for CH₄ oxidation and uptake, which may explain the positive response of CH₄ uptake to N addition in our study. The positive effects of P addition on CH₄ uptake may be due to the amelioration of P availability which stimulates soil methanotrophy activity and root growth (Zhang et al. 2011). In previous studies, stimulated, inhibited and unchanged CH₄ uptake after P addition have been reported (Lund et al. 2009, Song et al. 2012, Zheng et al. 2016). The inconsistent effects of N or P addition on CH₄ uptake was dependent on the duration and dose of nutrients addition, and also biome specific (Stiles et al. 2018, Xia et al. 2020).

In AM, plant richness, root N:P ratio and soil MBC are major factors in increasing soil CH₄ uptake. CH₄ is produced by methanogenic bacteria and eliminated by methanotrophic bacteria (Le Mer and Roger 2001), thus CH₄ flux is closely related to soil microbes. P addition affected soil MBC through root N:P ratio (Fig. S2, S4). As aforementioned, plant N:P ratio correlates with plant growth, litter decomposition rates, and root exudates (Güsewell 2004, Cleveland et al. 2006), which have significantly exerted impacts on soil microbial community and activity and ultimately changed the CH₄ uptake.

In AS, N addition promoted CH₄ uptake mainly through increasing root N:P ratio and P addition promoted CH₄ uptake mainly through decreasing plant belowground biomass. The mechanism of root N:P ratio CH₄ uptake has been mentioned previously. For belowground biomass, and higher biomass means higher availability of C sources such as root litter for CH₄ production, thus decreasing CH₄ uptake (Wang and Adachi 2000).

In CG, plant coverage and soil NH₄⁺ play key roles in regulating soil CH₄ uptake. P addition increased CH₄ uptake by increasing plant coverage. CH₄ exchange between the atmosphere and soil involves three complex processes: CH₄ production, oxidation, and transport in the soil (Le Mer and Roger 2001). Higher coverage of plants usually means more substrates for methanogenesis and transport pathways for CH₄, thus increasing CH₄ emissions (Eriksson et al. 2010). For example, Eriksson et al. (2010) reported that N deposition induced changes in vegetation cover play an important role in the increase in CH₄ emissions from a boreal peatland. However, research has also found that N addition inhibited CH₄ emissions in spite of the promoted plant growth (Granberg et al. 2001). They believe that the inhibiting effect of added N were dependent on the sedge density. Although we did not measure the density of sedges, we still believe that this inference can partially explain the promoting effect of P addition on CH₄ uptake. Because P addition suppresses sedges growth (Luo et al. 2019). As for soil NH₄⁺, it is closely related to the abundance of C-cycling genes, which may directly regulate CH₄ uptake (Fig. S6). Soil NH₄⁺ and NO₃⁻ are the alternative electron acceptors for methanogens responsible for CH₄ production, so increased CH₄ uptake are usually correlated with higher soil NH₄⁺ and NO₃⁻ concentration after N addition (Le Mer and Roger 2001, Reay and Nedwell 2004).

We found that the CO₂ and CH₄ fluxes in alpine grassland ecosystems are sensitive to N and P addition. Specifically, N addition alone elevated CO₂ fluxes in AM, P addition alone elevated CO₂ fluxes in AS and CG, but the interactions between N and P on CO₂ emissions vary significantly among the three grasslands. The addition of N and P alone or together increased CH₄ uptake, and P addition weakened the positive effects of N addition on CH₄ uptake across the three grasslands. N and P addition affected CO₂ and CH₄ fluxes both directly and indirectly through their impacts on soil and plant factors rather than C-cycling functional genes. These results indicate that in the context of increasing N deposition in the QTP, short-term P addition is not an effective method for mitigating global warming potential and improving soil C sequestration in alpine grassland ecosystems.

Acknowledgments

This study was funded by National Key R&D Program of China (2021YFE0112400；2023YFE304303), and National Natural Science Foundation of China (32361143870).

Ambus, P., and G. P. Robertson. 2006. The Effect of Increased N Deposition on Nitrous oxide, Methane and Carbon dioxide Fluxes from Unmanaged Forest and Grassland Communities in Michigan. Biogeochemistry 79:315-337.
Aronson, E. L., M. L. Goulden, and S. D. Allison. 2019. Greenhouse gas fluxes under drought and nitrogen addition in a Southern California grassland. Soil Biology and Biochemistry 131:19-27.
Bahn, M., M. Rodeghiero, M. Anderson-Dunn, S. Dore, C. Gimeno, M. Drösler, M. Williams, C. Ammann, F. Berninger, C. Flechard, S. Jones, M. Balzarolo, S. Kumar, C. Newesely, T. Priwitzer, A. Raschi, R. Siegwolf, S. Susiluoto, J. Tenhunen, G. Wohlfahrt, and A. Cernusca. 2008. Soil Respiration in European Grasslands in Relation to Climate and Assimilate Supply. Ecosystems 11:1352-1367.
Barneze, A. S., J. Whitaker, N. P. McNamara, and N. J. Ostle. 2022. Interactions between climate warming and land management regulate greenhouse gas fluxes in a temperate grassland ecosystem. Science of the Total Environment 833:155212.
Batjes, N. H. 2014. Total carbon and nitrogen in the soils of the world. European Journal of Soil Science 65:10-21.
Bloom, A. J., S. S. Sukrapanna, and R. L. Warner. 1992. Root Respiration Associated with Ammonium and Nitrate Absorption and Assimilation by Barley 1. Plant Physiology 99:1294-1301.
Bodelier, P. L. E., and H. J. Laanbroek. 2004. Nitrogen as a regulatory factor of methane oxidation in soils and sediments. Fems Microbiology Ecology 47:265-277.
Bond-Lamberty, B., and A. Thomson. 2010. Temperature-associated increases in the global soil respiration record. Nature 464:579-582.
Bowden, R. D., E. Davidson, K. Savage, C. Arabia, and P. Steudler. 2004. Chronic nitrogen additions reduce total soil respiration and microbial respiration in temperate forest soils at the Harvard Forest. Forest Ecology and Management 196:43-56.
Brookes, P. C., A. Landman, G. Pruden, and D. S. Jenkinson. 1985. Chloroform fumigation and the release of soil nitrogen: A rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology and Biochemistry 17:837-842.
Chen, H., H. Chen, Q. Zhu, C. Peng, N. Wu, Y. Wang, X. Fang, Y. Gao, D. Zhu, G. Yang, J. Tian, X. Kang, S. Piao, H. Ouyang, W. Xiang, Z. Luo, H. Jiang, X. Song, Y. Zhang, G. Yu, X. Zhao, P. Gong, T. Yao, and J. Wu. 2013a. The impacts of climate change and human activities on biogeochemical cycles on the Qinghai-Tibetan Plateau. Global Change Biology 19:2940-2955.
Chen, W., X. Zheng, Q. Chen, B. Wolf, K. Butterbach-Bahl, N. Brüggemann, and S. Lin. 2013b. Effects of increasing precipitation and nitrogen deposition on CH₄ and N₂O fluxes and ecosystem respiration in a degraded steppe in Inner Mongolia, China. Geoderma 192:335-340.
Chen, X., H. Diao, S. Wang, H. Li, Z. Wang, Y. Shen, A. A. Degen, K. Dong, and C. Wang. 2023. Plant community mediated methane uptake in response to increasing nitrogen addition level in a saline-alkaline grassland by rhizospheric effects. Geoderma 429:116235.
Chen, Y., S. Yin, Y. Shao, Q. Zhang, and K. Zhang. 2022. Greenhouse gas fluxes from riparian forest soil depend on the responses of microbes to nitrogen and phosphorus additions. Applied Soil Ecology 173:104365.
Cleveland, C. C., S. C. Reed, and A. R. Townsend. 2006. Nutrient regulation of organic matter decomposition in a tropical rain forest. 87:492-503.
Deng, L., C. Huang, D. G. Kim, Z. Shangguan, K. Wang, X. Song, and C. Peng. 2020. Soil GHG fluxes are altered by N deposition: New data indicate lower N stimulation of the N₂O flux and greater stimulation of the calculated C pools. Global Change Biology 26:2613-2629.
Dong, S. K., H. Gao, G. Xu, X. Hou, R. Long, M. Kang, and J. Lassoie. 2007. Farmer and professional attitudes to the large-scale ban on livestock grazing of grasslands in China. Environmental Conservation 34:246-254.
Eriksson, T., M. G. Öquist, and M. B. Nilsson. 2010. Effects of decadal deposition of nitrogen and sulfur, and increased temperature, on methane emissions from a boreal peatland. Journal of Geophysical Research: Biogeosciences 115.
Fang, H., S. Cheng, G. Yu, J. Cooch, Y. Wang, M. Xu, L. Li, X. Dang, and Y. Li. 2014. Low-level nitrogen deposition significantly inhibits methane uptake from an alpine meadow soil on the Qinghai–Tibetan Plateau. Geoderma 213:444-452.
Fang, H., S. Cheng, G. Yu, J. Zheng, P. Zhang, M. Xu, Y. Li, and X. Yang. 2012. Responses of CO₂ efflux from an alpine meadow soil on the Qinghai Tibetan Plateau to multi-form and low-level N addition. Plant and Soil 351:177-190.
Feng, J., and B. Zhu. 2019. A global meta-analysis of soil respiration and its components in response to phosphorus addition. Soil Biology and Biochemistry 135:38-47.
Fornara, D. A., L. Banin, and M. J. Crawley. 2013. Multi-nutrient vs. nitrogen-only effects on carbon sequestration in grassland soils. Global Change Biology 19:3848-3857.
Gao, W., H. Yang, L. Shenggong, and L. Kou. 2017. Responses of Soil CO₂, CH₄ and N₂O Fluxes to N, P, and Acid Additions in Mixed Forest in Subtropical China. Journal of Resources and Ecology 8:154-164.
Granberg, G., I. Sundh, B. H. Svensson, and M. Nilsson. 2001. Effects of Temperature, and Nitrogen and Sulfur Deposition, on Methane Emission from a Boreal Mire. Ecology 82:1982-1998.
Güsewell, S. 2004. N:P ratios in terrestrial plants: variation and functional significance. 164:243-266.
Hui, D., W. Porter, J. R. Phillips, M. P. M. Aidar, S. J. Lebreux, C. W. Schadt, and M. A. Mayes. 2020. Phosphorus rather than nitrogen enhances CO₂ emissions in tropical forest soils: Evidence from a laboratory incubation study. European Journal of Soil Science 71:495-510.
IPCC. 2013. Climate Change 2013 – The Physical Science Basis: Working Group I Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge.
Jiang, C., G. Yu, H. Fang, G. Cao, and Y. Li. 2010. Short-term effect of increasing nitrogen deposition on CO₂, CH₄ and N₂O fluxes in an alpine meadow on the Qinghai-Tibetan Plateau, China. Atmospheric Environment 44:2920-2926.
Kaspari, M., M. N. Garcia, K. E. Harms, M. Santana, S. J. Wright, and J. B. Yavitt. 2008. Multiple nutrients limit litterfall and decomposition in a tropical forest. Ecology Letters 11:35-43.
Kuzyakov, Y. 2006. Sources of CO₂ efflux from soil and review of partitioning methods. Soil Biology and Biochemistry 38:425-448.
Le Mer, J., and P. Roger. 2001. Production, oxidation, emission and consumption of methane by soils: A review. European Journal of Soil Biology 37:25-50.
Li, K., Y. Gong, W. Song, G. He, Y. Hu, C. Tian, and X. Liu. 2012. Responses of CH₄, CO₂ and N₂O fluxes to increasing nitrogen deposition in alpine grassland of the Tianshan Mountains. Chemosphere 88:140-143.
Li, S., S. Dong, H. Shen, Y. Han, J. Zhang, Y. Xu, X. Gao, M. Yang, Y. Li, Z. Zhao, S. Liu, H. Zhou, Q. Dong, and J. C. Yeomans. 2019. Different responses of multifaceted plant diversities of alpine meadow and alpine steppe to nitrogen addition gradients on Qinghai-Tibetan Plateau. Science of the Total Environment 688:1405-1412.
Liu, L., and T. L. Greaver. 2009. A review of nitrogen enrichment effects on three biogenic GHGs: the CO₂ sink may be largely offset by stimulated N₂O and CH₄ emission. Ecology Letters 12:1103-1117.
Liu, Y., C. Zhao, J. Guo, L. Zhang, J. Xuan, A. Chen, and C. You. 2021. Short-term phosphorus addition augments the effects of nitrogen addition on soil respiration in a typical steppe. Science of the Total Environment 761:143211.
Lü, C., and H. Tian. 2007. Spatial and temporal patterns of nitrogen deposition in China: Synthesis of observational data. Journal of Geophysical Research 112.
Lund, M., T. R. Christensen, M. Mastepanov, A. Lindroth, and L. Ström. 2009. Effect of N and P fertilization on the greenhouse gas exchange in two northern peatlands with contrasting N deposition rates. Biogeosciences 6.
Luo, R., J. Fan, W. Wang, J. Luo, Y. Kuzyakov, J. S. He, H. Chu, and W. Ding. 2019. Nitrogen and phosphorus enrichment accelerates soil organic carbon loss in alpine grassland on the Qinghai-Tibetan Plateau. Science of the Total Environment 650:303-312.
Mbonimpa, E. G., C. O. Hong, V. N. Owens, R. M. Lehman, S. L. Osborne, T. E. Schumacher, D. E. Clay, and S. Kumar. 2015. Nitrogen fertilizer and landscape position impacts on CO₂ and CH₄ fluxes from a landscape seeded to switchgrass. Global Change Biology Bioenergy 7:836-849.
McEwing, K. R., J. P. Fisher, and D. Zona. 2015. Environmental and vegetation controls on the spatial variability of CH₄ emission from wet-sedge and tussock tundra ecosystems in the Arctic. Plant and Soil 388:37-52.
Morrison, J. 2006. Grasslands of the World. Edited by J. M. Suttie, S. G. Reynolds and C. Batello. Rome: Food and Agriculture Organization of the United Nations, 2005, pp. 514. ISBN 92-5-105337-5. Experimental Agriculture 42:254-255.
Pirk, N., M. P. Tamstorf, M. Lund, M. Mastepanov, S. H. Pedersen, M. R. Mylius., and T. R. Christensen. 2016. Snowpack fluxes of methane and carbon dioxide from high Arctic tundra. Journal of Geophysical Research: Biogeosciences 121:2886–900.
Qu, S., R. Xu, J. Yu, F. Li, D. Wei, and A. Borjigidai. 2021. Nitrogen deposition accelerates greenhouse gas emissions at an alpine steppe site on the Tibetan Plateau. Science of the Total Environment 765:144277.
Raich, J. W., and W. H. Schlesinger. 1992. The global carbon dioxide flux in soil respiration and its relationship to vegetation and climate. Tellus 44B, 81-99.
Raich, J. W., and A. Tufekciogul. 2000. Vegetation and soil respiration: Correlations and controls. Biogeochemistry 48:71-90.
Reay, D. S., and D. B. Nedwell. 2004. Methane oxidation in temperate soils: effects of inorganic N. Soil Biology and Biochemistry 36:2059-2065.
B, J. M. Robroek., V. E. J. Jassey, M. A. R. Kox, R. L. Berendsen, R. T. E. Mills, L. Cécillon, J. Puissant, M. Meima-Franke, P. A. H. M. Bakker, and P. L. E. Bodelier. 2015. Peatland vascular plant functional types affect methane dynamics by altering microbial community structure. Journal of Ecology 103:925-934.
Robertson, G. P., D. C. Coleman, C. S. Bledsoe, and P. Sollins. 1999. Standard soil methods for long-term ecological research. 185–201, Oxford University Press, Oxford, United Kingdom.
Smith, K. A., T. Ball, F. Conen, K. E. Dobbie, J. Massheder, and A. Rey. 2003. Exchange of greenhouse gases between soil and atmosphere: interactions of soil physical factors and biological processes. European Journal of Soil Science 54:779-791.
Song, C., G. Yang, D. Liu, and R. Mao. 2012. Phosphorus availability as a primary constraint on methane emission from a freshwater wetland. Atmospheric Environment 59:202-206.
Stiles, W. A. V., E. C. Rowe, and P. Dennis. 2018. Nitrogen and phosphorus enrichment effects on CO₂ and methane fluxes from an upland ecosystem. Science of the Total Environment 618:1199-1209.
Tian, X., X. Ge, B. Zhou, and M. Li. 2021. The Linkage of Soil CO₂ Emissions in a Moso Bamboo (Phyllostachysedulis (Carriere) J. Houzeau) Plantation with Aboveground and Belowground Stoichiometry. 12:1052.
Vance, E. D., P. C. Brookes, and D. S. Jenkinson. 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19:703-707.
Wang, B., and K. Adachi. 2000. Differences among rice cultivars in root exudation, methane oxidation, and populations of methanogenic and methanotrophic bacteria in relation to methane emission. Nutrient Cycling in Agroecosystems 58:349-356.
Wang, J., B. Song, F. Ma, D. Tian, Y. Li, T. Yan, Q. Quan, F. Zhang, Z. Li, B. Wang, Q. Gao, W. Chen, and S. Niu. 2019. Nitrogen addition reduces soil respiration but increases the relative contribution of heterotrophic component in an alpine meadow. Functional Ecology 33:2239-2253.
Wei, D., Xu-Ri, Tenzin-Tarchen, Y. Wang, and Y. Wang. 2015. Considerable methane uptake by alpine grasslands despite the cold climate: in situ measurements on the central Tibetan Plateau, 2008–2013. Global Change Biology 21:777-788.
Xia, N., E. Du, X. Wu, Y. Tang, Y. Wang, and W. de Vries. 2020. Effects of nitrogen addition on soil methane uptake in global forest biomes. Environmental Pollution 264:114751.
Xiao, J., S. Dong, H. Shen, S. Li, Y. Zhi, Z. Mu, and C. Ding. 2022. Phosphorus addition promotes Nitrogen retention in alpine grassland plants while increasing N deposition. Catena 210:105887.
Xie, J., M. Chen, X. Zhang, S. Wang, X. Fang, M. Xie, and L. Zhang. 2023. Understory vegetation altered soil CO₂ and N₂O emissions and the correlation with plant and soil stoichiometry following N and P addition in Chinese fir plantations. Plant and Soil.
Yan, G., Y. Xing, G. Liu, B. Huang, and Q. Wang. 2021. Precipitation Pattern Regulates Soil Carbon Flux Responses to Nitrogen Addition in a Temperate Forest. Ecosystems 24:1608-1623.
Yang, Y., H. Fang, S. Cheng, L. Xu, M. Lu, Y. Guo, Y. Li, and Y. Zhou. 2022. Soil Enzyme Activity Regulates the Response of Soil C Fluxes to N Fertilization in a Temperate Cultivated Grassland. Atmosphere.
Yang, Y., J. Fang, Y. Tang, C. Ji, C. Zheng, J. He, and B. Zhu. 2008. Storage, patterns and controls of soil organic carbon in the Tibetan grasslands. Global Change Biology 14:1592-1599.
Yue, P., K. Li, Y. Gong, Y. Hu, A. Mohammat, P. Christie, and X. Liu. 2016. A five-year study of the impact of nitrogen addition on methane uptake in alpine grassland. Scientific Reports 6:32064.
Zeng, W., and W. Wang. 2015. Combination of nitrogen and phosphorus fertilization enhance ecosystem carbon sequestration in a nitrogen-limited temperate plantation of Northern China. Forest Ecology and Management 341:59-66.
Zhang, J., Y. Li, J. Wang, W. Chen, D. Tian, and S. Niu. 2021. Different responses of soil respiration and its components to nitrogen and phosphorus addition in a subtropical secondary forest. Forest Ecosystems 8:37.
Zhang, T., W. Zhu, J. Mo, L. Liu, and S. Dong. 2011. Increased phosphorus availability mitigates the inhibition of nitrogen deposition on CH₄ uptake in an old-growth tropical forest, southern China. Biogeosciences 8:2805-2813.
Zhao, Z., S. Dong, X. Jiang, S. Liu, H. Ji, Y. Li, and Y. Han. 2017. Effects of warming and nitrogen deposition on CH₄, CO₂ and N₂O emissions in alpine grassland ecosystems of the Qinghai-Tibetan Plateau. 592:565-572.
Zheng, M., T. Zhang, L. Liu, W. Zhang, X. Lu, and J. Mo. 2016. Effects of nitrogen and phosphorus additions on soil methane uptake in disturbed forests. Journal of Geophysical Research: Biogeosciences 121:3089-3100.
Zheng, M., T. Zhang, Y. Luo, J. Liu, X. Lu, Q. Ye, S. Wang, J. Huang, Q. Mao, J. Mo, and W. Zhang. 2022. Temporal patterns of soil carbon emission in tropical forests under long-term nitrogen deposition. Nature Geoscience 15:1002-1010.
Zhong, Y., W. Yan, and Z. Shangguan. 2016. The effects of nitrogen enrichment on soil CO2 fluxes depending on temperature and soil properties. Global Ecology and Biogeography 25:475-488.

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You are reading this latest preprint version

P addition weakens the positive effects of N addition on CH4 uptake and does not affect the effects of N addition on CO2 emission in alpine grasslands

Status:

Version 1

Abstract

Aims

Methods

Results

Conclusions

Figures

Introduction

Material and methods

Study site

Experimental design

Measurement of CO₂ and CH₄ fluxes

Soil sampling and analysis

Plant sampling and analysis

Soil DNA extraction and quantification of functional gene abundance

Data analysis

Results

Changes of CO₂ and CH₄ fluxes with N and P addition

Changes of C-cycling genes with N and P additions

Factors impacting CO₂ and CH₄ fluxes under N and P addition

Discussion

Effects of N and P additions on CO₂ emissions

Effects of N and P additions on CH₄ uptake

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

P addition weakens the positive effects of N addition on CH4 uptake and does not affect the effects of N addition on CO2 emission in alpine grasslands

Status:

Version 1

Abstract

Aims

Methods

Results

Conclusions

Figures

Introduction

Material and methods

Study site

Experimental design

Measurement of CO2 and CH4 fluxes

Soil sampling and analysis

Plant sampling and analysis

Soil DNA extraction and quantification of functional gene abundance

Data analysis

Results

Changes of CO2 and CH4 fluxes with N and P addition

Changes of C-cycling genes with N and P additions

Factors impacting CO2 and CH4 fluxes under N and P addition

Discussion

Effects of N and P additions on CO2 emissions

Effects of N and P additions on CH4 uptake

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

Measurement of CO₂ and CH₄ fluxes

Changes of CO₂ and CH₄ fluxes with N and P addition

Factors impacting CO₂ and CH₄ fluxes under N and P addition

Effects of N and P additions on CO₂ emissions

Effects of N and P additions on CH₄ uptake