Soils store approximately 1500 Pg of organic carbon (SOC) and 940 Pg of inorganic carbon (SIC) to a depth of 1 m (Batjes, 1996; Jobbágy and Jackson, 2000), which are the largest carbon (C) pool in the terrestrial ecosystem and play a critical part in the global C cycling (Darwish et al., 2018; Lal, 2004; Prietzel et al., 2016). To alleviate the elevated levels of atmospheric carbon dioxide (CO2), most previous studies concentrate on the SOC pool because it responds quickly to global climate change such as warming and nitrogen (N) deposition, and it is strongly linked with various ecosystem functions (Wang et al., 2002; Yang et al., 2012). Compared to the relatively short turnover time of SOC, SIC has a long residence time due to soil weathering (Monger et al., 2015; Zang et al., 2018), which is considered to be fairly stable and has less contribution to changes in terrestrial ecosystem C balance (Yang et al., 2012). Therefore, previous studies have paid little attention to SIC. However, recent studies suggest that SIC is also responsive to anthropogenic activities and global climate changes such as soil acidification, atmospheric N deposition, and global warming (Yang et al., 2010; Song et al., 2022), acting as a critical C source (Liu et al., 2020) or C sink (Gao et al., 2018; Liu et al., 2021). Thus, the preservation of SIC and its roles in climate mitigation should not be neglected, especially in arid and semiarid grasslands which store a large amount of SIC (Yang et al., 2012).

The SIC stock and stability can be fundamentally altered by an array of abiotic and biotic processes (Raza et al., 2020). High precipitation can promote the weathering of soil silicate minerals and removal of base cations (Ca2+, Mg2+, K+ and Na+) by leaching (Vicca et al., 2022). Soil acidification due to atmospheric nitrogen (N) and acid deposition and the nitrification of NH4+ may greatly accelerate soil carbonate dissolution and CO2 releases (Raza et al., 2020; Song et al., 2022). Plant growth can deplete soil carbonates by releasing proton and organic acids from root rhizosphere (Goulding, 2016; Kuzyakov and Razavi, 2019), and biological N2 fixation by some legumes are likely to cause SIC losses (Tang et al., 1999). Furthermore, plant autotrophic and microbial heterotrophic respiration often facilitate carbonate dissolution by enhancing CO2 partial pressures (An et al., 2019; Liu et al., 2021). Nevertheless, how these abiotic and biotic factors affect SIC stock and what the relative importance is of these confounding drivers remain largely uncertain.

Previous studies on SIC stock have mostly focused on the topsoil within 10 cm soil depth (Yost and Hartemink, 2020), which are relatively different from the subsoil (i.e., soils residing >20 cm below ground) in the aspect of biochemical processes, plant roots, soil properties, and microbial communities (Rumpel et al., 2012; Zhou et al., 2021), while the patterns of SIC stock in the subsoil on a large scale remain elusive. The predictors of SIC stock in the subsoil may differ from those in the topsoil due to distinct soil microenvironments, soil physicochemical properties, root exudates, and microbial abundance and functions (Jia et al., 2017). For instance, the topsoil has larger root biomass (RB) and higher microbial activity than the subsoil, but the subsoil tends to preserve soil parent material because of the weakened weathering by the isolation of heat and energy from the surface soil (Crowther et al., 2016). Thus, the abiotic and biotic variables may exhibit different effects on SIC stock in the subsoil compared to the topsoil due to the various importance of these variables.

The Tibetan Plateau has the largest alpine grassland on the Eurasian continent, which is a vital component of global terrestrial ecosystems, providing an ideal platform to explore SIC stock and its determinants (Wang et al., 2002; Yang et al., 2010). During the past several decades, the plateau has experienced significant warming (Wang et al., 2008) and pronounced atmospheric N deposition (Liu et al., 2013; Yu et al., 2019). This continuous warming and N deposition have resulted in a significant increase in plant growth and soil acidification (Ding et al., 2017; Yang et al., 2012), which could likely induce potential CO2 releases from soil carbonates by biogeochemical processes (Raza et al., 2020). However, a general understanding of SIC stock with soil depth across Tibetan alpine grasslands remains unexplored.

Here, we researched the relative importance of climatic, edaphic, plant and microbial variables to SIC stock at different soil layers along an approximately 3000 km transect of alpine grasslands on the Tibetan Plateau, spanning a broad range of climatic and geographical conditions. Specifically, two key questions are addressed in this study: (1) What are the differences of SIC stock between the topsoil and subsoil? (2) What is the relative importance of climatic, edaphic, plant and microbial variables to the variation of SIC stock along with soil depth?

To the best of our knowledge, this study was the first to study large-scale evidence of the relative contribution of abiotic and biotic drivers to the variation of SIC stock at different soil depths, which has considerable implications for grasping the importance of SIC in the ecosystem C cycling. Due to considerably stable characteristics and the long turnover time (Mi et al., 2008; Yang et al., 2010; Zamanian et al., 2018), SIC stock is traditionally considered to be dominated by abiotic factors including soil moisture, soil pH, CO2 partial pressure, and Ca2+ concentrations according to the equilibrium of carbonate precipitation–dissolution reactions (CaCO3 + H2O + CO2 → Ca2+ + 2HCO3- and Ca2+ + 2HCO3- → CaCO3 + H2O + CO2) and mineral carbonation (MgSiO4 + 2CO2 → 2MgCO3 + SiO2 and CaMgSi2O6 + CO2 + H2O → Ca2Mg5Si8O22(OH)2 + CaCO3 + SiO2) (Mi et al., 2008; Rey, 2015; Yang et al., 2012; Yang and Yang, 2020). These abiotic factors were proved to have large impacts on the dissolution and deposition processes of inorganic C and ultimately determined the reservation and distribution of SIC (Rey, 2015; Rowley et al., 2018).

However, many biological processes and factors were not quantitatively considered in previous studies. In this study, based on the approach of large-scale field samplings across Tibetan alpine grasslands, we estimated the predominant drivers of SIC stock in the topsoil and subsoil. Our results found the predominant roles of microbial and plant factors in determining SIC stock in both topsoil and subsoil. More importantly, the effects of biotic factors on SIC stock weakened with soil depth (Fig. 4). These results were different from those demonstrating the critical influence of abiotic processes on SIC stock (Mi et al., 2008; Yang et al., 2010).

We found that SIC density showed a declining trend with the increasing plant aboveground biomass, plant coverage, and root biomass (Figs. 2 and 3). Plant factors could contribute to the decline of SIC stock by three pathways including uptakes of exchangeable cations, plant organic matter inputs, and rhizosphere processes. First, a large decline in soil base cations is likely to be induced by plant uptake with increasing plant biomass; and the losses of soil exchangeable base cations can cause the transformation of SIC to CO2, which is ultimately released into the atmosphere (Huang et al., 2015). Second, increasing plant residue inputs can enhance carbonic and organic acid production into soil water solutions via microbial decomposition, which reduces the availability of soil base cations through cation exchange in the soil (Sartori et al., 2007) and increases the dissolution and leaching of carbonates, resulting in a decrease in the SIC. Third, the plant rhizosphere effect on releasing CO2 from carbonates should not be ignored, especially in alkaline soils. By releasing organic acids and protons as well as CO2, plant roots can reduce soil pH and increase CO2 in the rhizosphere (Lenzewski et al., 2018), both of which dissolve carbonates by neutralization (Harley and Gilkes, 2000). In addition, organic compounds from plant root exudates, such as malate or citrate, can stimulate mineral weathering by dissolving silicate minerals (Dontsova et al., 2020).

Furthermore, the topsoil has a larger quantity and higher quality of plant residues than the subsoil, which indicates a greater potential for carbonate dissolution by biological processes for the surface soil (Liu et al., 2020). The large root biomass in the topsoil can increase the uptake of base cations and result in increasing proton and organic acids in root exudates (Li et al., 2007), thus reducing the soil carbonate content for maintaining the charge balance. In addition, the larger plant roots exuded more organic compounds in the topsoil that can stimulate parent mineral weathering and dissolve silicate minerals by chelating reaction products (Doetterl et al., 2018; Dontsova et al., 2020).

Also, the SIC density in both soil depths appears to have an increasing or decreasing trend from the alpine meadow to the alpine steppe and alpine desert (Figs. 2 and 3) sites. In the present study, for example, the alpine meadow has larger plant productivity than the alpine steppe, which implies that more plant above- and belowground residues are deposited in alpine meadow soils compared to alpine steppe soils. Therefore, from the perspective of the whole ecosystem, the grassland type would be a better predictor for the quantity and distribution of SIC density.

Previous studies reported that microbial properties may not be important in mediating SIC accumulation (Liu et al., 2021; Wang et al., 2015). However, our results found that microbial factors including microbial biomass and bacterial and fungal gene abundance showed significant associations with SIC stock (Figs. 2 and 3), which could be due to microbes driving the carbonate dissolution processes, including microbial respiration, organic matter mineralization, and releases of proton and organic acids by microbial metabolic activity. First, the increase in microbial respiration can improve CO2 production and enhance the partial pressure of CO2, leading to a decline in pH and further dissolution of carbonates (Chang et al., 2012). In addition, soil organic matter mineralization and litter decomposition by microbes can induce the dissolution of CO2 and the release of organic acids (Goulding, 2016; Kuzyakov and Razavi, 2019), both of which decrease the SIC stock. Meanwhile, chelates and enzymes excreted by microbes may contribute to enhancing mineral dissolution rates and organic matter decomposition (Xiao et al., 2015; Zaharescu et al., 2020).

We also revealed that bacterial and fungal gene abundance were significantly correlated with SIC stock (Figs. 2 and 3), which was likely to account for decreasing soil pH in the involvement of microbial biological reactions. For instance, nitrifying bacteria can oxidize ammonium to nitrate (NH4+ + OH- + 2O2 → NO3- + 2H2O + H+), and the increase in acidity is finally neutralized through accelerating carbonate dissolution (Zamanian et al., 2016). Also, some nitrogen-fixing bacteria that lived in symbiosis with leguminous plants can acidify the soil by excreting protons during N2 fixation (Vicca et al., 2022). Furthermore, fungi are likely to accelerate carbonate neutralization by exuding protons and organic acids (van Hees et al., 2006; Wild et al., 2021).

Microbial factors could also be better predictors for SIC stock in the topsoil than in the subsoil. The large plant residues incorporated into the topsoil provided substantial amounts of organic matter for microbial living and decomposition (Oelkers et al., 2015; Ven et al., 2020), which can stimulate microbial abundance and activities and promote microbial extracellular enzymes. These extracellular excretions play a fundamental role in microbial respiration and CO2 production, both of which stimulate silicate weathering and carbonate dissolution (Vicca et al., 2022). Meanwhile, the higher CO2 flux and CO2 partial pressure resulting from the biological activities of roots and soil microorganisms in the topsoil could enhance carbonate dissolution and formations of pedogenic inorganic C (Chang et al., 2012; Zamanian et al., 2016).

Different from plant and microbial factors, the prediction of edaphic factors on SIC stock strengthened with soil depth, with soil pH being the most important predictor among edaphic variables (Fig. 4). The buffering capacity in soil solutions determines the equilibrium of ion inputs and outputs by soil pH (Huang et al., 2015). In this study, soil pH in the subsoil (7.85) was much higher than that in the topsoil (7.66) (Table 1). The higher pH could buffer the replacement of the exchangeable cations with protons (Frank and Stuanes, 2003) and increase the preservation of base cations (Gandois et al., 2011). Given that base cations and carbonates provide the major buffering capacity in the alkaline soil (Yang et al., 2012), the topsoil could be subject to a larger loss of base cations and SIC due to the lower soil pH compared to the subsoil.

Taken together, our results revealed that SIC stock was closely linked with biotic factors, which highlights the roles of biological processes in predicting SIC dynamics (Hong et al., 2019). These results imply that the widespread enhancement of vegetation productivity under global environmental changes (e.g., warming and rewetting) (Ding et al., 2017; Wang et al., 2008) may aggravate the depletion of SIC stock (Raza et al., 2020). Meanwhile, previous studies have urged the need for incorporating microbial processes and indicators into Earth system models (ESMs) to reduce the uncertainty in predicting soil C dynamics, especially SOC decomposition (Allison et al., 2010; Moorhead and Sinsabaugh, 2006; Todd-Brown et al., 2013). However, our findings highlighted the vital role of microbial factors in regulating soil C balance from inorganic C preservation. Thus, incorporating microbial processes into the models can aid the understanding of overall soil C responses, because SOC and SIC are formed, protected, and lost in different ways.

More importantly, the predictions of biotic factors on SIC stock weakened with soil depth, which implies that SIC may be susceptive to environmental changes in the topsoil which is the hotspot of root and microbial activities. Even though biotic factors in the subsoil played a less important role in predicting SIC stock compared with the topsoil, an increase in rooting depth is expected in response to climate warming and land-use change (Liu et al., 2018), which is likely to cause SIC losses in the deep soil by root growth. Therefore, it is a necessity to further explore the effects of biotic factors on SIC stock in the deep soil in the context of global changes. Although most of the variations in SIC density were explained by our measured explanatory variables, some other potential variables may also predict SIC density (Fig. 5). Then, understanding the effects of other potential abiotic and biotic factors on SIC density with soil depth is urgently needed when predicting the response and feedback of SIC to climate change in the future. Overall, the contribution of SIC to CO2 is not ignored and SIC maintenance has a considerable effect on soil C losses and is important to maintain the health and ecosystem functions (Raza et al., 2020; Zamanian et al., 2018). Our study provides robust evidence that biotic factors are correlated with SIC stock in the Tibetan plateau and that topsoils and subsoils should be considered separately when modeling SIC dynamics and its feedbacks on climate change (Yang et al., 2012; Zamanian and Kuzyakov, 2019).

Our findings showed that SIC stock had no significant differences between the topsoil and subsoil in the Tibetan grasslands; the climatic, edaphic, plant and microbial variables jointly predicted SIC stock in the Tibetan grasslands; and the biotic factors had a larger contribution than abiotic factors to the variation of SIC stock. Furthermore, the relative importance of explanatory variables to the variation of SIC stock varied with soil depth, the predictions of microbial and plant variables on SIC stock weakened with soil depth, while the predictions of edaphic variables strengthened with soil depth. Our results revealed that biotic factors should be considered seriously for predicting SIC stock due to their regulating roles in biological processes. The contrasting responses and drivers of SIC stock between the topsoil and subsoil highlight differential mechanisms underlying SIC preservation with soil depth, which is crucial to understanding and predicting SIC dynamics and its feedbacks to environmental changes.