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.