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Soil microbial biomass is critical for biogeochemical cycling and serves as precursor for carbon (C) sequestration. The anthropogenic nitrogen (N) input has profoundly changed the pool of soil microbial biomass. However, traditional N deposition simulation experiments have been exclusively conducted through infrequent N addition, which may have caused biased effects on soil microbial biomass compared with those under the natural and continuous N deposition. Convincing data are still scarce about how the different N addition frequencies affect soil microbial biomass. By independently manipulating the frequencies (2 times vs. 12 times N addition yr–1) and the rates (0–50 g N m−2 yr−1) of N addition, our study aimed to examine the response of soil microbial biomass C (MBC) to different N addition frequencies with increasing N addition rates. Soil MBC gradually decreased with increasing N addition rates under both N addition frequencies, while the soil MBC decreased more at low frequency of N addition, suggesting that traditional studies have possibly overestimated the effects of N deposition on soil microbial biomass. The greater soil microbial biomass loss with low N frequency resulted from the intensified soil acidification, higher soil inorganic N, stronger soil C and N imbalance, less net primary production allocated to belowground and lower fungi to bacteria ratio. To reliably predict the effects of atmospheric N deposition on soil microbial functioning and C cycling of grassland ecosystems in future studies, it is necessary to employ both the dosage and the frequency of N addition.

Determining the abundance of N isotope (δ

We investigated whether plant resistance to soil impoverishment would depend on their flexibility in taking up nutrients and on maintaining elemental stoichiometry. We mixed sand with grassland soil in mass proportions of 0, 10, 30, 50 and 70% to simulate soil impoverishment as caused by a gradient of desertification intensity and examined how plant nitrogen (N) uptake (15NH4NO3 and NH415NO3 labelling) and nutrient stoichiometry were associated with plant growth responses of a sedge (Carex duriuscula) and a forb (Potentilla chinensis). With increasing intensity of soil impoverishment, plant biomass, cover and density decreased for C. duriuscula, but not for P. chinensis. Interestingly, uptake of both 15NO3− and 15NH4+ increased for the sedge but not for the forb, with the former species showing stronger preference of NO3−. However, the sedge showed decreasing nutrient contents and concentrations of phosphorus (P) and magnesium (Mg), and thus increasing stoichiometric ratios of N:P and calcium (Ca):Mg. In contrast, the forb maintained its biomass by showing strong stability in nutrient content, concentration and stoichiometry. Stability of N uptake and nutrient stoichiometry were associated with higher resistance to soil impoverishment of the forb than the sedge. Our study highlights the importance of stability in nutrient uptake and stoichiometry for determining plant-species resistance to nutrient-poor conditions.

Plant litter decomposition is critical for the carbon (C) balance and nutrient turnover in terrestrial ecosystems, and is sensitive to the ongoing anthropogenic biologically nitrogen (N) input. Previous studies evaluating the N effect on litter decomposition relied mostly on short-term experiments (

Trait-based approaches have been widely applied to uncover the mechanisms determining community assembly and biodiversity-ecosystem functioning relationships. However, they have rarely been used in forest-steppe ecotones. These ecosystems are extremely sensitive to disturbances due to their relatively complex ecosystem structures, functionings and processes. In this study, we selected seven sites along a transect from closed canopy forests (CF) to forest-steppe ecotones (FSE) and meadow steppes (MS) in northeast China. Six leaf functional traits (i.e. leaf nitrogen and phosphorus contents, leaf length and thickness, single leaf area and leaf mass per unit area, LMA) as well as the community composition and aboveground biomass at each site were measured. Both functional trait diversity indices (richness, evenness and divergence) and community-weighted mean trait values (CWMs) were calculated to quantify community trait distributions. We found that dominant species in the FSE communities showed acquisitive strategies with highest leaf nitrogen (Mean ± SE: 19.6 ± 0.5 mg g

Purpose Whether molybdenum (Mo) addition promotes Mo uptake by plants in nitrogen-fertilized ecosystems may depend on nutrient interactions. But this has rarely been paid attention to in continuously mown and nutrient-losing grassland ecosystems. Methods We investigated Mo uptake by three dominant species and how it varied with plant phosphorus and sulfur concentrations after 6-year combined nitrogen and Mo addition and mowing management in a cold meadow. Results Molybdenum addition increased Mo concentrations in the three plant species across all treatments due to increased soil available Mo. Without Mo addition, nitrogen addition significantly reduced Mo concentrations of Leymus chinensis irrespectively of mowing and of Stipa baicalensis with mowing. With Mo addition, the negative nitrogen-addition effect on plant Mo absorption was boosted for all plant species. This could be due to the fact that increases in plant sulfur absorption possibly induced antagonistic effects on plant Mo uptake. Additionally, reductions in plant phosphorus concentration weakened the synergistic uptake of Mo by plants with nitrogen addition, except for Carex duriuscula. Mowing only reduced Mo concentration of S. baicalensis without nitrogen addition under Mo addition. Conclusions Our results indicated that plant Mo deficiency could occur in nitrogen-fertilized ecosystems via nutrient interplay, facilitating it a necessity to supplement Mo.

Rhizodeposition plays an important role in below-ground carbon (C) cycling. However, quantification of rhizodeposition in intact plant-soil systems has remained elusive due to methodological issues. We used a 13 C-CO2 pulse-labelling method to quantify the contribution of rhizodeposition to below-ground respiration. Intact plant-soil cores were taken from a grassland field, and in half, shoots and roots were removed (unplanted cores). Both unplanted and planted cores were assigned to drought and nitrogen (N) treatments. Afterwards, shoots in planted cores were pulse labelled with 13 C-CO2 and then clipped to determine total below-ground respiration and its δ13 C. Simultaneously, δ13 C was measured for the respiration of live roots, soils with rhizodeposits, and unplanted treatments, and used as endmembers with which to determine root respiration and rhizodeposit C decomposition using two-source mixing models. Rhizodeposit decomposition accounted for 7-31% of total below-ground respiration. Drought reduced decomposition of both rhizodeposits and soil organic carbon (SOC), while N addition increased root respiration but not the contribution of rhizodeposit C decomposition to below-ground respiration. This study provides a new approach for the partitioning of below-ground respiration into different sources, and indicates that decomposition of rhizodeposit C is an important component of below-ground respiration that is sensitive to drought and N addition in grassland ecosystems.

Soil aggregates constitute spatially-separated microbial habitats and architectural units for biogeochemical reactions. However, little is known about how aggregates varying in size can affect soil carbon (C) storage and mediate the direction of plant-soil interactions. In a meadow steppe, we assessed soil aggregate C storage affected by nitrogen (N) addition and mowing and to what degree plant-soil interactions differed among soil aggregate-size classes. Nitrogen addition increased plant biomass, soil C concentration in macroaggregates and relative abundance of bacteria, but decreased fungal relative abundance and the activity of oxidative enzymes. However, mowing counteracted N effects on plant productivity and enzyme activity. In macroaggregates, close linkages with plant biomass and species richness were found for soil available nutrients and ratio of fungi to bacteria (F:B ratio). In contrast, strong physical protection and restricted fungal dominance and activity contributed to C stabilization in microaggregates, where plant community and soil properties were less linked with each other. Our study clearly demonstrated that stronger plant-soil linkages occurred in macro- vs. microaggregates. This linkage was associated with a rise in plant biomass and soil C accumulation but a drop in F:B ratio in macroaggregates under N addition.

Nitrogen (N) addition and precipitation increment can greatly influence soil phosphorus (P) dynamics, with much emphasis on total and available P, yet little is known about their interactive effects on soil mineral‐bound inorganic P (Pᵢ) fractions under different historical land uses of grasslands and old fields. Thus, we compared (i) plant readily available P, (ii) less available Pᵢ (sum of NH₄F‐extractable Pᵢ, NaOH‐Na₂CO₃‐extractable Pᵢ and NaHCO₃‐extracted Pᵢ), (iii) refractory forms of Pᵢ (NH₄Ac‐extractable Pᵢ, H₂SO₄‐extractable Pᵢ and Na₃(citrate)‐dithionite‐extractable Pᵢ) and (iv) organic P under the same urea and water treatments in an old‐field grassland with a semi‐arid steppe. Soil total P remained unchanged with 10‐year urea addition in both sites, with lower organic P but higher Pᵢ concentrations in the old field. Urea addition promoted transformation of refractory Pᵢ into less available Pᵢ in both sites, which potentially was related to higher plant productivity and thus enhanced plant P accumulation. Specifically, urea addition increased less available Pᵢ fractions by as much as 42%, but decreased refractory Pᵢ fractions by as much as 24%, for two sites under ambient precipitation. Water addition decreased less available Pᵢ in the steppe under higher urea addition rates, whereas it increased less available and refractory Pᵢ but only in the control plot of the old field. Irrespective of urea and water treatments, the old field had a higher Pᵢ pool, which could replenish plant readily available P in the longer term and be less prone to P‐limitation than the steppe. We conclude that conversion of semi‐arid steppe to farmland causes long‐term increases in the soil Pᵢ pool, possibly by enhancing organic P mineralization and anthropogenic fertilization during cultivation. Urea addition accelerated soil P cycling via promoting refractory Pᵢ, transforming into less available Pᵢ, with the process being strongly mediated by water availability, whereas the projected precipitation increment could decrease less available Pᵢ via promoting plant P uptake and P leaching out of the plant–soil system. HIGHLIGHTS: Urea addition promoted transformation of refractory Pᵢ into less available Pᵢ fractions. Water addition counteracted the positive urea effect on NH₄F‐Pᵢ in both steppe and old field. Less responsive organic P suggested that Pᵢ fractions were more essential in soil P cycling. Water addition decreased NH₄F‐Pᵢ in both sites, potentially due to higher plant uptake and leaching. Conversion of steppe to farmland increased the soil Pᵢ pool by enhancing organic P mineralization.

The continuing nitrogen (N) deposition observed worldwide alters ecosystem nutrient cycling and ecosystem functioning. Litter decomposition is a key process contributing to these changes, but the numerous mechanisms for altered decomposition remain poorly identified. We assessed these different mechanisms with a decomposition experiment using litter from four abundant species (Achnatherum sibiricum, Agropyron cristatum, Leymus chinensis and Stipa grandis) and litter mixtures representing treatment-specific community composition in a semi-arid grassland under long-term simulation of six different rates of N deposition. Decomposition increased consistently with increasing rates of N addition in all litter types. Higher soil manganese (Mn) availability, which apparently was a consequence of N addition-induced lower soil pH, was the most important factor for faster decomposition. Soil C : N ratios were lower with N addition that subsequently led to markedly higher bacterial to fungal ratios, which also stimulated litter decomposition. Several factors contributed jointly to higher rates of litter decomposition in response to N deposition. Shifts in plant species composition and litter quality played a minor role compared to N-driven reductions in soil pH and C : N, which increased soil Mn availability and altered microbial community structure. The soil-driven effect on decomposition reported here may have long-lasting impacts on nutrient cycling, soil organic matter dynamics and ecosystem functioning.

Phosphorus (P) limitation is expected to increase due to nitrogen (N)-induced terrestrial eutrophication, although most soils contain large P pools immobilized in minerals (P

Purpose: In grassland ecosystems, plant functional group (PFG) is an important bridge connecting individual plant to community system. Grassland ecosystem is the main ecosystem type on the Qinghai-Tibet Plateau, so the change of community structure of grassland vegetation.Methods: The Altun Mountains in the northern part of the Qinghai-Tibet Plateau were used as the study area to investigate the PFGs of a high-altitude (> 3700m) grassland in desert areas and their response to temperature and moisture.Results: The main functional groups were forbs and grasses, and the importance values (IV) accounted for more than 50%. Plant species diversity of the community was influenced by the functional groups of legumes IV, and the increase of legumes would promote the increase of plant community diversity. The C, N, P of plant communities were mainly influenced by forbs and grasses, and the relationship between forbs and C, N, P was opposite to that of grasses. There was a positive correlation between forbs and soil TP; a negative correlation between grasses and soil TP; a positive correlation between legumes with soil SOC and TN; and a positive correlation between sedge and soil SOC. However, under the influence of different hydrothermal conditions, forbs and grasses as dominant functional groups had stronger correlation with community and soil nutrients. Conclusions: This indicated that the PFGs with the largest proportion in the community had the greatest influence on the community. This provides a basis for the study of alpine grassland community development and ecosystem function under alpine grassland.

Purpose Sulfur (S) deposition as a global change issue causes worldwide soil acidification, nutrient mobilization and marked changes in plant nutrition. Here, we investigated how S deposition would affect leaf nutrient resorption and how this effect varies with yearly fluctuations in precipitation. Methods In a semiarid meadow exposed to S addition, we measured nitrogen (N), phosphorus (P) and S concentrations in green and senescent leaves of a grass and a sedge and calculated nutrient resorption efficiencies (NuRE) across two years with contrasting precipitation (13% higher and 27% lower than long-term mean annual precipitation). Results Concentrations of N, P, and S in green and senescent leaves generally increased with S addition across the two years, with the exception of N and P concentrations in green leaves of the grass that showed no response or even decreased with S addition. The coupling relationships between N and P concentrations showed interannual variations and tightened by nutrient resorption, as evidenced by stronger N and P correlations in senescent leaves than in green leaves in the wet year. Leaf NuRE convergently decreased with S addition across the two years congruent with soil acidification and increased soil N, P and S availability, while NuRE was higher in the wet year due to lower soil nutrient availability herein. Conclusions This study provides new evidence on the role of nutrient resorption in tightening stoichiometric N:P relationships, and a three-dimensional feedback framework that plant nutrient resorption was favored by higher precipitation to sharpen its tradeoff with soil nutrient availability.

Determining base cation and trace element dynamics in plant-soil systems is important for sustainable utilization of grasslands affected by global ecosystem nitrogen (N) enrichment. A 4-year field experiment was conducted with urea addition rates of 0, 2.5, 5, 7.5, 10, 15, 20, and 30 g N m−2 yr.−1 to investigate the dynamics of base cations (Ca, Mg, K) and trace elements (Fe, Mn, Cu and Zn) in soil and a dominant species Leymus chinensis in a semi-arid meadow grassland of northeast China. Soil showed a significant decrease in exchangeable Ca and increases in extractable Fe, Mn and Zn concentrations, while plant Ca and Zn remained unchanged and plant Fe decreased with N addition. For both plant and soil compartments, stoichiometric ratios of trace elements were more variable than base cations, mainly driven by plant physiological requirements, plant nutrient uptake intensity, and elemental interactions. Excessive soil Mn mobilization and plant uptake affected concentrations of Fe, Cu and Zn in the plant and even inhibited Fe absorption (antagonistic interactions), resulting in a plant Fe:Mn ratio lower than 1.5 with high N addition rates, indicating Mn phytotoxicity or Fe deficiency. As a result, plant elemental stoichiometry did not remain homeostatic and was even more variable than in soil. Our results provided direct evidence of decoupled plant-soil elemental responses, stricter homeostatic control of macronutrients than trace elements in plants, and altered stoichiometric variability in the plant-soil system, and suggest that N addition enhances metal nutrient imbalances in the meadow steppe grassland.

Temperate mixed forests, along with other high latitudinal ecosystems, are more vulnerable to global warming in comparison with warm sites, because of the slower carbon (C) turnover and higher soil organic carbon (SOC) accumulation. Lignin and cellulose are two major components of plant litter, and usually make contributions to the recalcitrant and labile SOC pool, respectively. Because the chemical composition of SOC plays key role in regulating the bioavailability of soil C pool, understanding the relationship between soil lignin or cellulose content and temperature are of great significance in evaluating the feedbacks between SOC pool and the future scenarios of global warming. The biological degradation of soil lignin or cellulose is mainly dependent on soil enzymatic activities, and thus, the response of ligninolytic and cellulolytic enzymes to increased temperature would determine C release under future warming scenarios. However, the responses of the soil lignin/cellulose content and the activity of cellulolytic and ligninolytic enzymes to increased mean annual temperature (MAT) have rarely been studied, and the factors driving these changes are not fully understood. Latitudinal gradients are often used for monitoring global-warming-related problems, because of its natural gradients of temperature. In this study, we demonstrate the latitudinal pattern of lignin/cellulose content and the activities of cellulose- and lignin- degrading enzymes in a temperate Broad-leaved Korean pine mixed forests distributed along a latitudinal gradient (with MAT ranging from −1.9 to 5.1 °C) in northeastern China. The linear mixed model revealed that soil lignin content was negatively correlated with MAT (Slope = −7.604, t = −2.608, P = 0.011), whereas soil cellulose content showed no response to increased MAT. The activity of soil polyphenol oxidase (PPO), one of the enzymes catalyze lignin decomposition, was higher in high-latitude sites, in contrast, the activity of the cellulase (CEL) complex was higher in low-latitude plots. Structural equation model (SEM) analysis indicates that MAT can directly influence soil lignin or cellulose content, and indirectly through changing NRCB, plant litter C/N, microbial biomass, and degrading enzymatic activities. The value of soil lignin/(lignin + cellulose) ratio and soil lignocellulose index (LCI, lignin/(lignin + holocellulose) ratio), varied between 0.8–0.9, and 0.6–0.8, respectively, indicating that the SOC pool in this temperate ecosystem is dominated by recalcitrant components. The negative correlations between MAT and LCI, soil lignin/(lignin + cellulose), and log (PPO + PER)/log(CEL) (Slope = −0.008, t = −2.363, P = 0.021; Slope = −0.004, t = −3.134, P = 0.003, and Slope = −0.057, t = −4.477, P

We examined the effect of nitrogen and water addition on soil carbon pool dynamics in a semi–arid grassland field experiment. Soils were sampled over a four–year period at different soil depths and analyzed for total soil organic carbon (SOC), oxidizable C (OxC), lignin and total nitrogen (TN) in bulk soil and the silt and clay fraction (

Background Soil microbial communities cope with an imbalanced supply of resources by adjusting their element acquisition and utilization strategies. Although soil pH has long been considered an essential driver of microbial growth and community composition, little is known about how soil acidification affects microbial acquisition and utilization of carbon (C) and nitrogen (N). To close the knowledge gap, we simulated soil acidification and created a pH gradient by adding eight levels of elemental sulfur (S) to the soil in a meadow steppe. Results We found that S-induced soil acidification strongly enhanced the ratio of fungi to bacteria (F:B) and microbial biomass C to N (MBC:MBN) and subsequently decreased the C:N imbalance between microbial biomass and their resources. The linear decrease in the C:N imbalance with decreasing soil pH implied a conversion from N limitation to C limitation. To cope with enhanced C versus N limitation, soil microbial communities regulated the relative production of enzymes by increasing the ratio of β-glucosidase (BG, C-acquiring enzyme) to leucine aminopeptidase (LAP, N-acquiring enzyme), even though both enzymatic activities decreased with S addition. Structural equation modeling (SEM) suggested that higher C limitation and C:N-acquiring enzyme stimulated microbial carbon-use efficiency (CUE), which counteracted the negative effect of metal stress (i.e., aluminum and manganese) under soil acidification. Conclusions Overall, these results highlight the importance of stoichiometric controls in microbial adaption to soil acidification, which may help predict soil microbial responses to future acid deposition.

Natural abundance of carbon (C) and nitrogen (N) stable isotope ratios (I´13 C and I´15 N) has been used to indicate ecosystem C and N status and cycling; however, use of this approach to infer plant and microbial N preference under projected ecosystem N enrichment is limited. Here, we investigated natural abundance I´13 C and I´15 N of five dominant plant species, and soil I´15 N of microbial biomass and available N forms under N addition in a meadow steppe. Additional N, applied as urea, led to decreases in I´15 N of soil NO3 - (I´15 Nnitrate , from 3.0 to 0.4‰) and increases in I´15 N of soil NH4 + (I´15 Nammonium , from -1.3 to 11‰) and dissolved organic N (I´15 NDON , from 8.5 to 15‰) that reflected increased net nitrification rates, a possible increase in NH3 volatilization, and greater availability of the three N forms. An overall increase in I´15 N of soil total N (I´15 NTN ) from 7.1 to 7.9‰ indicated accelerated and greater openness of soil N cycling that was also partially revealed by enhanced net N mineralization rates. Plant I´15 N, which ranged from -1.8 to 2.1‰, generally decreased with N addition, indicating a greater reliance on soil NO3 - under N-enrichment conditions. Nitrogen addition decreased I´15 N of microbial biomass N (from 14 to 2.8‰), possibly due to a shift in preferential N form (DON to NO3 - ), that indicated a convergence of plant and microbial preferential N forms and an increase in plant-microbial N competition. Microbes were thus more flexible than plants in the use of different forms of N. Addition of N decreased plant litter I´13 C, while plant species I´13 C remained unaffected, likely due to a shift in the abundance of dominant species with a greater proportion of biomass coming from I´13 C-depleted species. Enrichment factor (the difference in plant I´15 N relative to I´15 NTN ) of four non-legume species was negatively related to soil inorganic N availability, net nitrification rate, and net N mineralization rate, and was proven to be a good indicator of ecosystem N status. Our study highlights the importance of natural abundance of 15 N as an indicator of plant-microbial N competition and ecosystem N cycling in meadow steppe grasslands under projected ecosystem N enrichment.

Increased inputs of ecosystem nitrogen (N) and phosphorus (P) may affect the activity of soil enzymes that play essential roles in the metabolization of carbon (C), N and P for microbial growth. However, the associations between soil enzymatic activities and N and P availability remain poorly understood. We conducted a study in a meadow steppe to elucidate the effects of the addition of N, as ammonium nitrate (NH₄NO₃3), and two forms of P with contrasting solubility, comprising monopotassium phosphate (KH₂PO₄) and the less soluble triple superphosphate (Ca(H₂PO₄)₂, on activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG) and acid and alkaline phosphomonoesterases (PMEs). In general, there was a positive effect of N on BG, NAG and alkaline PME activity as a result of enhanced soil N availability, plant-microbe nutrient competition and plant P uptake. Addition of KH₂2PO₄ increased activity of BG, NAG and alkaline PME, but had no impact on acid PME activity. Addition of Ca(H₂PO₄)2 increased NAG activity, but only increased activity of BG and alkaline PME with the addition of N. Concentration of soil available P and microbial biomass P increased with added P, particularly KH₂PO₄. These results provide the first evidence for the N- and P-mediated stimulation of microbial activity depending on the chemical form of added P in this ecosystem. Relationships between activity of BG and NAG, and between that of NAG and PME, were allometric, indicating disproportionate changes in activity of these soil enzymes. This further suggests shifts in microbial acquisition of C, N and P along with increases in availability of N and P that may potentially affect plant productivity. We conclude that scenarios of global environmental change, in which ecosystem availability of N and P is affected, may result in variable activity responses among soil enzymes, and the chemical form of P input should be considered as an important factor influencing meadow steppe grassland ecosystem function.

Additional file 1 Appendix 1- Additional methods (Detailed priori hypothetical pathways and a priori structural equation model); Appendix 2- Additional results (Soil biochemical characteristics, SMA regression for enzymes and correlation between variables and element-use efficiency); Appendix 3- Reliability test for estimated CUE (Soil respiration and its relationship with estimated CUE).

Natural abundance of carbon (C) and nitrogen (N) stable isotope ratios (δ

Rhizodeposition plays an important role in below-ground carbon (C) cycling. However, quantification of rhizodeposition in intact plant-soil systems has remained elusive due to methodological issues. We used a

Carbon (C), nitrogen (N) and phosphorus (P) are the most important elemental components of an ecosystem. Nutrient stoichiometry reflects their cycling and distribution with ecosystems. The changes in nutrient stoichiometry in different ecosystems are not consistent. The altitudinal gradient within the Altunshan Nature Reserve can be used as a natural experiment to verify the effect of different altitudes gradients on nutrients changes. Therefore, we studied the changes in plant and soil nutrients from alpine desert ecosystem to alpine grassland ecosystem in this region. The results showed that there was a linear correlation for plant C, N and P, as well as soil C and N with altitude gradient. The C: N and C: P of plants and C: P and N: P of soils were linearly correlated with the elevation gradient. Plant C and N were mainly affected by soil C and N content, but there was no significant correlation between plant P and soil nutrient concentration. Soil nutrients were affected by soil moisture content and pH. Soil moisture content increased gradually with altitude, promoting nutrient mineralization and availability to the ecosystem. The increase of soil nutrient availability and soil water content significantly increased the species diversity, promoting the plant community assembly and the succession from alpine desert ecosystem to alpine grassland ecosystem. This study analyzed the relationship between nutrient cycling and distribution in different ecosystem types on the Qinghai-Tibet Plateau and found that nutrient changes were greatly affected by soil moisture in the arid and cold regions.

Atmospheric nitrogen (N) deposition can result in soil acidification and reduce soil acid buffering capacity. However, it remains poorly understood how changes in precipitation regimes with elevated atmospheric N deposition affect soil acidification processes in a water-limited grassland. Here, we conducted a 9-year split-plot experiment with water addition as the main factor and N addition as the second factor. Results showed that soil acid buffering capacity significantly decreased with increased N inputs, mainly due to the decline of soil effective cation exchange capacity (ECEC) and exchangeable basic cations (especially Ca 2+ ), indicating an acceleration of soil acidification status in this steppes. Significant interactive N and water effects were detected on the soil acid buffering capacity. Water addition enhanced the soil ECEC and exchangeable base cations and thus alleviated the decrease of soil acid buffering capacity under N addition. Our findings suggested that precipitation can mitigate the impact of increased N deposition on soil acidification in semi-arid grasslands. This knowledge should be used to improve models predicting soil acidification processes in terrestrial ecosystems under changing environmental conditions.

Global nitrogen (N) deposition has been influencing the structure of soil microbial communities, which play fundamental roles in modifying most biogeochemical processes. However, to date, our understanding of how long-term N deposition affects soil microbial communities, particularly those in subsurface soil, is largely incomplete. In this study, we examined soil microbial phospholipid fatty acids at different depths in the soil profile, including in topsoil (0–10 cm), midsoil (30–40 cm), and subsoil (70–100 cm), in a 10-year N addition experimental site in a semiarid steppe. We collected soil samples at four N addition treatment levels, including 0, 2, 10, and 50 g m−2 year−1, which represented control, low-N, medium-N, and high-N inputs, respectively. Our results showed that N addition remarkably shifted soil microbial communities by increasing the relative abundances of bacteria and Gram-positive (GP) bacteria, and decreasing Gram-negative (GN) bacterial relative abundance across the three soil layers by affecting soil pH and NO3−-N, particularly under the medium- and high-N addition rates. Moreover, the effects of N addition tended to increase with the N addition rate but diminished with soil depth. Soil pH, NO3−-N, and NH4+-N were the most important driving factors for changes in microbial community composition in topsoil, whereas soil total N (TN) and total carbon were important in midsoil, and TN, dissolved organic N, and soil moisture were important in subsoil. The relative abundances of bacteria and GN bacteria also decreased, whereas those of fungi and GP bacteria increased with soil depth regardless of N addition. Soil TN and pH were the most important factors in shaping the vertical distribution of soil microbial communities. Our results suggest that both N addition and soil depth may cause similar microbial community shifts, through which fungi and GP bacteria become dominant, but through different mechanisms. These results suggest that it is important to distinguish among the N-deposition effects on soil microbial communities at different soil depths when building soil system models.