4.1 Microbial response to the addition of root exudates
Artificial root exudates were designed by using three low-molecular organic compounds in four different C/N ratios, which are easily degradable and accessible by the microbial community (de Graaff et al., 2010; Bastida et al., 2013). When C and N are sufficient, microorganisms prefer to easily utilize compounds from root exudates than from native SOM (Shahbaz et al., 2017; Wei et al., 2019). We found that the addition of root exudates stimulated microbial activity, as indicated by large accumulative CO2 emissions. These emissions responded differently to various stoichiometric ratios of root exudates with the same amount of C input, indicating that microbial activity is modified by N demand. Higher activity of the N-acquiring extracellular enzyme (NAG) was observed in C-rich soil (N limit at high C/N ratio). Thus, a continuous supply of low C/N ratio artificial root exudates increased N availability and decreased N-acquiring enzymes (Fig. 3). This is consistent with previous studies that have applied stoichiometric root exudates exacerbate CO2 emission due to microbial N demand in paddy soils (Liu et al., 2020). However, in contrast to the observation of Liu et al. (2020), we found that a higher proportion of N in artificial root exudates stimulated more CO2 emission than at high C/N ratios (CN6 ≥ CN10 > CN80 ≥ C-only) (Fig. 1a, b), and the C-acquiring enzyme activity (BG and XYL) was increased by a higher C/N ratio of artificial root exudates. These differences due to the different forms of N nutrients [(NH4)2SO4 vs. alanine] added to paddy soils.
Furthermore, unlike labile inorganic forms of N (NH4+ and NO3-), N-containing low-molecular organic compounds (alanine in this study) can decrease microbial nutrient limitation from C/N ratios: carbohydrate hydrolase has been shown to increase with increasing C/N stoichiometric ratios of root exudates due to unbalanced nutrient addition (Allison and Vitousek, 2005; Sinsabaugh et al., 2008). On the other hand, higher N-rich artificial root exudates (CN6) showed higher qCO2 and lower CUE than other treatments at day 3 and day 12, whereas C-only caused low CUE (Fig. 2a, b). A higher proportion of organic N-containing root exudates may have promoted microbial catabolism relative to anabolism, as there is a higher energy demand for microorganisms in acquiring organic N compared to inorganic N (Näsholm and Persson, 2001; Czaban et al., 2016). Moreover, the positive relationship between BG/NAG ratio and qCO2 suggested that microbial catabolism of root exudates attributed to microorganisms enhancing C- and N-acquiring enzymes to obtain available C and N from both root exudates and SOM. Labile C from root exudates can be quickly utilized by microorganisms for nutrients acquiring to enhance microbial consumption of energy and released as CO2 (Zhu et al., 2018).
Along with the increase in CO2 emission, the continuous supply of artificial root exudates caused lower microbial CUE in all treated soils than in the control. This indicated that the addition of root exudates caused substrate and nutrient uptake by microorganisms (Blagodatskaya et al., 2014; Chen et al., 2020). Simultaneously, the higher CUE corresponded to a higher C/N stoichiometric ratio, except in C-only. This indicated that N-containing stimulates affected microbial catabolic activity, and that low N-containing formations of root exudates (i.e., with high C/N ratios) increased the CUE for SOC accumulation. A positive relationship between the MBC/MBN and CUE (Fig. 4d) suggested that the stoichiometry of microbial biomass played a key regulator in promoted microorganism utilize input labile C source and increased the soil C accumulation (Sinsabaugh et al., 2016; Soares and Rousk, 2019). These findings thus supported our hypothesis that the addition of a higher C/N ratio with decreasing N content increased microbial biomass with lower CO2 emissions and stimulated soil C sequestration.