Grassland Ecosystem

Soil Priming Effect Mediated by Nitrogen Fertilization Gradients in a Semi-arid Grassland, China

  • LI Yue 1 ,
  • NIE Cheng 1 ,
  • SHAO Rui 2 ,
  • DU Wei 1 ,
  • LIU Yinghui , 1, *
  • 1. State Key Laboratory of Earth Surface Processes and Resource Ecology, Faculty of Geographical Science, Beijing Normal University, Beijing 100875, China
  • 2. Department of Geography, Ghent University, Ghent 9000, Belgium
*Corresponding author: LIU Yinghui, E-mail:

Received date: 2018-10-17

  Accepted date: 2018-12-20

  Online published: 2019-03-30

Supported by

National Natural Science Foundation of China (31770519)

National Key Research and Development Program of China (2017YFC0503805).


All rights reserved


The priming effect is well acknowledged in soil systems but the effect of nitrogen (N) fertilization remains elusive. To explore how N modifies the priming effect in soil organic matter (SOM), one in situ experiment with 13C labeled glucose addition (0.4 mg C g-1 soil, 3.4 atom % 13C) was conducted on soil plots fertilized with three gradients of urea (0, 4 and 16 g N m-2 yr-1). After glucose addition, the soil CO2 concentration and phospholipid fatty acid (PLFA) were measured on day 3, 7, 21 and 35. The study found that N fertilization decreased soil CO2, PLFA and the fungi to bacteria ratio. Glucose triggered the strongest positive priming in soil at 0 g N m-2 yr-2, meanwhile N fertilization decreased SOM-derived CO2. Soil at 4 g N m-2 yr-2 released the largest amount of glucose-derived carbon (C), likely due to favorable nutrient stoichiometry between C and N. Stable microbial community biomass and composition during early sampling suggests “apparent priming” in this grassland. This study concludes that N fertilization inhibited soil priming in semi-arid grassland, and shifted microbial utilization of C substrate from SOM to added labile C. Diverse microbial functions might be playing a crucial role in soil priming and requires attention in future N fertilization studies.

Cite this article

LI Yue , NIE Cheng , SHAO Rui , DU Wei , LIU Yinghui . Soil Priming Effect Mediated by Nitrogen Fertilization Gradients in a Semi-arid Grassland, China[J]. Journal of Resources and Ecology, 2019 , 10(2) : 147 -154 . DOI: 10.5814/j.issn.1674-764X.2019.02.005

1 Introduction

The dramatic short-term changes in soil organic matter (SOM) caused by unstable factors (e.g. the input of the labile carbon) are called priming effects (Kuzyakov et al., 2000). The priming effect is a key factor affecting the soil carbon (C) cycle and relates to the regulation of SOM mineralization by root-derived C inputs (Cheng, 2009) and the process of nitrogen mining performed by soil microorganisms (Craine, 2007). Labile C flow is huge due to continuous inputs and outputs, despite the small net concentration (de Vries and Caruso, 2016). Labile C can be immediately utilized by the soil microbial community, thus its retention time is short and the products (e.g. CO2 and microbial biomass) are easy to detect (Creamer et al., 2014). Labile C such as glucose is often used in substrate addition experiments to study the decomposition process of different C sources and C utilization dynamics in microbial communities (Li et al., 2018b, Liu et al., 2017; Zhang et al., 2016).
Nitrogen (N) is one of the factors limiting ecosystem productivity, and increasing atmospheric N deposition has a profound impact on global ecosystems (Galloway et al., 2008). The effect of N fertilization on the priming effect has been found to be positive (Chen et al., 2014; Qiao et al., 2014) and negative (Zang et al., 2016; Zhang et al., 2016). As crucial factors in the priming effect (Li et al., 2018a; Liu et al., 2017), the ratio of glucose to microbial biomass carbon (MBC) determines the direction of priming. A higher ratio of glucose to MBC generally leads to a lower priming effect (Blagodatskaya and Kuzyakov, 2008). Therefore, N fertilization can regulate the priming effect via its impact on MBC. Different mechanisms of priming have been defined based on the activities of microbial communities. The priming effect can associate with microbial “N mining” (Craine et al., 2007) and microbial stoichiometry (Hessen et al., 2014) at the community level. Labile carbon is both the energy and C source for soil microorganisms. Favorable nutrient stoichiometry between C and N enhances the growth of soil microorganisms (Hessen et al., 2014) and increases the utilization of labile C. To sufficiently utilize different substrate-derived C in soil, microbial community composition may change during C decomposition. According to Blagodatskaya and Kuzyakov (2008), “apparent priming” is defined as an acceleration of internal microbial metabolism without effects on SOM decomposition; meanwhile “real priming” happens with the increase of K-strategists and extracellular enzymes. Previous studies have found that priming in N fertilized soil is a short-term effect lasting 3 to 7 days (Zang et al., 2016; Li et al., 2017). However, the type of soil priming under N fertilization scenarios is seldom mentioned.
Soil decomposition driven by microorganisms is an important regulator of C storage. Since fungi are often seen as K-strategists which decompose the relatively recalcitrant SOM, the ratio of fungi to bacteria (F:B ratio) is widely used to predict soil quality or the extent of SOM decomposition (Kaur et al. 2005; Rousk and Frey, 2015). Understanding the decomposition dynamics of the soil microbial community helps to improve the prediction of land ecosystem C cycles (Leff et al., 2015). Therefore, changes in microbial community composition during soil priming may reveal interactions between soil C and the N cycles.
The objectives of this study are to explore the priming effect under different N fertilization gradients and the association with microbial communities. A 13C labeled glucose addition experiment was conducted on a long-term N fertilization experimental site with three gradients of urea fertilization (0, 4 and 16 g N m-2 yr-1). After the day of glucose addition (day 0), substrate-derived CO2 and phospholipid fatty acid (PLFA) in soil samples were measured at day 3, 7, 21 and 35. We hypothesized: 1) that N fertilization reduces positive priming effects in semi-arid grassland soil; 2) that low gradient N fertilization favors microbial preference for labile C substrate; and 3) that different types of soil priming occur after N fertilization.

2 Methods

2.1 Study site and experimental design

The study site is located in Duolun County, Inner Mongolia, China (42.02°N, 116.17°E; 1341 m above sea level). The mean annual temperature is 2.1 °C and the average monthly temperatures peaks in July. Mean annual precipitation is 379.4 mm, with most rainfall from May to September (Wang et al., 2015). The typical soil in this region is chestnut soil, and the vegetation is dominated by Stipa krylovii, Leymus chinensis, Artemisia frigida, Agropyron cristatum and Allium bidentatum.
A long-term N fertilization experimental site was established in July 2003. Dry urea N fertilizer was manually spread on the surface of the plots (15 m × 10 m, 4 m-wide buffer zone between each) in mid-July each year. Bulk N deposition in this area is approximately 5 g N m-2 yr-1 (Wang et al., 2015). We selected N fertilization plots with 3 N levels (0, 4 and 16 g N m-2 yr-1) and 4 replicates for each level to add 13C labeled glucose. A relatively low N fertilizing gradient (4 g N m-2 yr-1) and high fertilizing gradient (16 g N m-2 yr-1) were chosen based on the critical N load for typical grassland in China (5 g N m-2 yr-1, Liu et al., 2011). Two 20 cm × 20 cm experimental subplots were randomly established in each plot, one for glucose treatment and the other as a control.
On 25 July, 2017 (one week after N fertilization), aboveground biomass in the subplots was removed. One day later, 10.48 g 13C labeled glucose (3.4 atom % 13C) dissolved in 25 mL of well water (<1% of the monthly rainfall) was sprayed evenly on the soil surface in each glucose treatment subplot; 25 mL of well water was added in each control subplot. One PVC collar (10 cm diameter, 10 cm height) was installed in each plot immediately, the collar was inserted 5 cm deep and left in place throughout the experiment. Three blanks (only to measure environmental CO2 concentration) were performed using an additional three collars isolated from the soil surface. The day of collar installation was treated as day 0 in this study.
Considering that nearly 80% of microbial biomass C is distributed at a soil depth of 0-20 cm in the study area (Peng and Wang, 2016), we supposed that microbial communities in this layer are fastest to respond to labile C addition and a reaction depth for glucose utilization was 20 cm from the surface in this study. Transformed by bulk density (1.31 g cm-3), 13C added in the subplot was 0.4 mg g-1 soil, which is roughly 30% higher than microbial biomass carbon (MBC) at this site (Peng and Wang, 2016). We expected such a concentration to induce a positive priming effect in soil (<200% MBC, Blagodatskaya and Kuzyakov, 2008).

2.2 CO2 sampling and analysis

CO2 sampling was conducted on day 3, 7, 21 and 35. A water sealed chamber (15 cm height) was used to measure CO2 concentration for 24 h in the collar. The daily mean soil CO2 flux is approximately 3.5 μmol m-2 s-1 (Li et al., 2015), thus 20 mL 1 g NaOH resolution was prepared to guarantee CO2 trap validity for 24 h. One 5 cm-diameter plastic petri dish containing the NaOH resolution was put at 5 cm above the soil surface, quickly followed by the close of the chamber.
The alkaline solution in the collar was collected and stored in a sealed plastic vial at -20 °C before lab analysis. The respired CO2 concentration from soil was measured as inorganic C in the soil using a TOC analyser (Liqui TOCII, Elementar, Germany). The δ13C of the CO2 was determined by adding 2 M SrCl2 to the solution for precipitation, and analyzing the δ13C of SrCO3 precipitate (0.25 mg) using a continuous-flow gas isotope ratio mass spectrometer (MAT253, Finnigan MAT, Bremen, Germany).

2.3 Soil sampling and analysis

In each CO2 sampling day, one soil core (0-20 cm deep, 3 cm diameter) was randomly collected outside of the PVC collar but inside the subplot. Soil was sieved through a 2 mm mesh and measured for phospholipid fatty acids (PLFA). PLFAs were extracted based on the protocol of Bligh and Dyer (1959) and analyzed via gas chromatography (Agilent, CA, USA). Saturated FAMEs were 15 : 0, 16 : 0 and 18 : 0; PLFA biomarkers indicating gram-positive bacteria were i15:0, a15:0, i16:0, i17:0 and a17:0; gram-negative bacteria biomarkers were 16:1w7c, cy17:0, 17:1w8c and 18:1w7c; saprophytic fungal biomarkers were 18:2w6c and 18:1w9c (Frostegard and Baath, 1996; Bossio and Scow, 1998). The proportion of bacteria and saprophytic in total PLFA were measured, and the ratio of saprophytic fungi to bacteria (F:B ratio) calculated.

2.4 13C-CO2 calculation

Due to the existence of environmental CO2, correction is needed for the δ13C of respired CO2 (Harris et al., 1997):
(Ma+Mb) × δ13C = Ma × δ13Ca + Mb × δ13Cb (1)
δ13C is measured by the isotope ratio mass spectrometer, (Ma+Mb) is the measured CO2 concentration in the glucose treatment or control (mg C m-2 d-1), Mb is the CO2 in the blank, δ13Cb is the δ13C of environmental CO2 (﹣8 ‰), and δ13Ca is the corrected δ13C of CO2 in the glucose treatment or control.
The total CO2 from each collar is partitioned into its glucose- and SOM-derived components using a mass balance model (Garcia-Pausas and Paterson, 2011):
P-glucose = (δ13C-sample - δ13C-control)/(δ13C-glucose - δ13C-control) (2)
P-glucose is the fraction of glucose-derived CO2 to the total P-glucose is the fraction of glucose-derived CO2 to the total CO2, δ13C-sample is the δ13C of CO2 in the glucose treatment sample, δ13C-control is the δ13C of CO2 in the control sample, and δ13C-glucose is the δ13C of the added glucose.
CO2-glucose= P-glucose × [CO2] (3)
CO2-SOM = (1 - P-glucose) × [CO2] (4)
Primed C = CO2-SOM - (CO2 flux in the control sample)
CO2-glucose is the glucose-derived CO2, CO2-SOM is the SOM-derived CO2, and [CO2] is the CO2 concentration in the glucose treatment sample.

2.5 Statistical analysis

Repeated-measures ANOVAs were used to analyze the effect of N level, substrate addition or sampling day on CO2 concentration, total PLFA and the F:B ratio. Independent T tests were conducted to test for differences between control and glucose treatments. One-way ANOVAs were used to measure variations in CO2, total PLFA and the F:B ratio among treatments for each sampling days. LSD tests (homogeneity of variance reached) or Dunnet tests (homogeneity of variance did not reach) were used for pair-wise comparisons. Spearman correlations were performed to assess the relationship between total PLFA, bacteria, fungi, F:B ratio and N levels. Statistical analyses mentioned above were conducted using IBM SPSS 20.0 (IBM, NY, USA).
To analyze groupings of microbial communities in different treatments on day 3 and 7, data were analyzed using non-metric multidimensional scaling ordination (NMDS). The significance of sample clustering in N level, substrate addition or sampling day were investigated by the analysis of similarities (ANOSIM). NMDS and ANOSIM were performed using R (version 3.5.0). Graphs were created using Sigmaplot 12.5 (Systat Software, CA, USA).

3 Results

3.1 Dynamics of sampled CO2

Repeated-measures ANOVA showed that the soil CO2 concentration was different across N level, glucose treatment and sampling day (Table 1). N fertilization substantially reduced soil CO2 compared to the control. Averaged across sampling days, soil CO2 was 167.68, 55.95 and 50.48 mg C m-2 d-1 in the control subplot at 0, 4 and 16 g N m-2 yr-1, respectively. For glucose treatment soil, the average CO2 across sampling days was 431.42, 505.70 and 398.90 mg C m-2 d-1 at 0, 4 and 16 g N m-2 yr-1, respectively. Soil CO2 added with glucose peaked at 4 g N m-2 yr-1 and was generally higher than control soil. Soil CO2 declined significantly on day 7 and then steadied (Fig. 1). Independent T tests showed that the addition of glucose significantly enhanced soil CO2 compared to the control for day 3 (P < 0.001) and day 7 (P = 0.027) (Fig. 1).
Table 1 Repeated-measures ANOVA for soil CO2, total PLFA, bacteria, fungi and the F:B ratio among treatments in four sampling days.
Source of variation Soil CO2 Total PLFA Bacteria Fungi F:B ratio
Day 1.13 44.53*** 40.16*** 28.55*** 0.41
Nitrogen 8.21** 23.43*** 0.73 22.24*** 19.77***
Glucose 134.83*** 142.39*** 0.01 8.57** 6.58*
Day × Nitrogen 1.66 1.17 7.87*** 2.94** 0.85
Day × Glucose 20.01*** 19.16*** 63.38*** 15.93*** 14.19***
Nitrogen × Glucose 1.90 2.01 1.58 0.01 0.02
Day × Nitrogen ×Glucose 1.94 1.86 7.00*** 2.37 0.38

Note: * indicates significant difference at P < 0.05, ** indicates significant difference at P < 0.01, *** indicates significant difference at P < 0.001.

Fig. 1 Dynamics of soil CO2 among treatments in four sampling days
Note: N0, N4 and N16 represent nitrogen levels of 0, 4 and 16 g N m-2 yr-1, respectively.
Soil CO2 concentration derived from various C sources had different dynamics during the sampling. The priming effect was defined as the difference between SOM-derived CO2 and control soil CO2 (Fig. 2). After 13C glucose addition, the priming effect was generally positive in the early stage of sampling (day 3 and 7, Fig. 2a and b). However, the SOM-derived CO2 was lower than the control soil CO2 on day 21 and 35 (Fig. 2c and d). Due to heterogeneity in field conditions, the difference between SOM-derived CO2 and control soil CO2 at each N level was not statistically significant for day 3 (Fig. 2a). Nonetheless, SOM-derived CO2 was of a higher concentration than the control, and SOM-derived CO2 was highest at 0 g N m-2 yr-1 (Fig. 2a). Soil CO2 decreased by day 7 at the three N levels. Although the SOM-derived CO2 was still higher than the control at 0 and 4 g N m-2 yr-1, positive priming diminished at 16 g N m-2 yr-1 on day 7 (Fig. 2b). From day 21, SOM-derived CO2 was significantly lower than the control (Fig. 2c; Fig. 2d).
Fig. 2 Soil CO2 in the control treatment and soil CO2 and soil organic matter derived CO2 in the 13C labeled glucose treatment on day 3(a), 7(b), 21(c), and 35(d).
Note: A one-way ANOVA is conducted for all CO2 concentrations in the control treatment and SOM-derived CO2 among nitrogen levels in each sampling day. Different lowercase letters indicate significant difference in CO2 at P < 0.05.

3.2 Cumulative C from different sources

On day 7, the cumulative primed C at 0 g N m-2 yr-1 was significantly higher than at 16 g N m-2 yr-1 (P = 0.027); there was no difference in cumulative primed C among N levels on day 35 (P = 0.358), although the trend was 0 > 4 > 16 g N m-2 yr-1 (Fig. 3a). The SOM-derived C on day 35 was 2468.17, 1384.87 and 1033.41 mg C m-2 at 0, 14 and 16 g N m-2 yr-1, respectively, and was higher at 0 g N m-2 yr-1 than the other two levels (P = 0.008, Fig. 3b). Glucose- derived C on day 35 was 1636.25, 3118.79 and 2500.70 mg C m-2 at 0, 14 and 16 g N m-2 yr-1, respectively (Fig. 3c). Higher glucose-derived C was found at 4 and 16 g N m-2 yr-1 than 0 g N m-2 yr-1 (P = 0.031), indicating that the application of N fertilizer promoted the consumption of soluble C by soil microorganisms.
Fig. 3 Cumulative primed carbon (a), soil organic matter derived carbon (b) and glucose-derived carbon (c) in four sampling days

3.3 Microbial community composition

According to repeated-measures ANOVA, total PLFA and fungal PLFA were significantly affected by sampling day but the bacterial PLFA and the F:B ratio were relatively stable (Table 1). Averaged by sampling days, total PLFA, fungi and the F:B ratio were negatively associated with increasing N, but bacteria were not correlated with N levels (Table 2). Day 3 and 7 were treated as early sampling stages. No significant differences in mean PLFA biomass of day 3 and 7 were found among treatments (Fig.4a), and ANOSIM in NMDS showed that microbial community composition was relatively stable between day 3 and 7 (Fig. 4b).
Table 2 Spearman correlations between N levels and total PLFA, bacteria, fungi and the F:B ratio averaged by sampling days.
Treatment Variable Coefficient
Control Total PLFA -0.621*
Bacteria -0.443
Fungi -0.857**
F:B ratio -0.828**
Glucose Total PLFA -0.680*
Bacteria 0.266
Fungi -0.710**
F:B ratio -0.917**

Note: * indicates significant difference at P < 0.05, ** indicates significant difference at P < 0.01.

Fig. 4 Mean PLFA biomass on day 3 and 7 (a) and non-metric multidimensional scaling ordination of PLFA biomarkers (b) among treatments on day 3 and 7
Note: A one-way ANOVA is conducted for PLFA among treatments. Different lowercase letters indicate significant difference in PLFA at P < 0.05

4 Discussion

High N application reduces microbial activity and the loss of soil CO2, and is beneficial to SOM accumulation (Reay et al., 2008). N fertilization suppressed soil CO2 in the study area regardless of N gradient, based on the results of control CO2. With the addition of glucose, SOM-derived CO2 at all N levels was higher than the control on day 3, leading to a positive priming effect (Kuzyakov et al., 2000). Glucose-derived CO2 in the soil at 4 and 16 g N m-2 yr-1 on day 3 was 3.05 times and 2.32 times that of soil at 0 g N m-2 yr-1, indicating rapid decomposition of labile C with N fertilization. The positive priming effect disappeared after day 7 at 0 and 4 g N m-2 yr-1, and after day 3 at 16 g N m-2 yr-1. Consistent with Li et al. (2017), cumulative primed C dynamics changed from positive to negative. At the late sampling stage a higher proportion of 13CO2 in total CO2 in the glucose-adding group may be the result of cross-predation of soil microorganisms (Creamer et al., 2014; Garcia-Pausas and Paterson, 2011).
The soil priming effect is an important factor in the regulation of the soil C balance (Zhang et al., 2016), and this process is regulated by nitrogen addition (Chen et al., 2014). According to the “N mining” theory (Craine, 2007), due to the limited availability of nitrogen, microorganisms use labile C to synthesize extracellular enzymes to accelerate SOM decomposition, leading to an increase in soil CO2. The addition of 13C labeled glucose stimulated soil CO2 at all N levels in this study, and the cumulative primed C was highest at 0 g N m-2 yr-1, suggesting that the strongest priming effect exists when N is most limited (Garcia-Pausas and Paterson, 2011).
Soil microorganisms switched to labile carbon instead of SOM in the N fertilization treatments, revealed by cumulative SOM-derived C and glucose-derived C at the three N levels. Soil at 0 g N m-2 yr-1 released the largest amount of SOM-derived C on day 35; the smallest amount was at 16 g N m-2 yr-1. The cumulative glucose-derived C on day 35 was highest at 4 g N m-2 yr-1, and showed a trend of 4 > 16 > 0 g N m-2 yr-1. Lower levels of N fertilization stimulated larger consumption of labile C, likely due to the favorable stoichiometry for microbial growth (Hessen et al., 2014; Li et al., 2018b).
N fertilization exerted an inhibitive impact on microbial PLFA biomass and the F:B ratio, in accordance with other studies (Wei et al., 2013; Liu et al., 2018). Fungi are considered a major player in recalcitrant C decomposition because of massive oxidative enzyme production (Sinsabaugh, 2010). A reduced F:B ratio following N fertilization might lead to a decrease in oxidative enzymes and consequently the inhibition of SOM-derived CO2. However, no significant variation in F:B ratios among sampling days suggests poor linkage between the F:B ratio and SOM mineralization (Rousk and Frey, 2015). Since glucose is a universal C and energy source for microbial communities, activation of dormant microorganisms (Blagodatskaya and Kuzyakov, 2008), quick response of r-strategists in saprotrophic fungi (Lemanski and Scheu, 2014; Li et al., 2018b) and functional diversity within bacterial taxa (Lonardo et al., 2017) might obscure the transition between fungal and bacterial communities. To be better indicative of SOM mineralization, 13C labeled PLFA rather than total PLFA biomass should be considered in each fungal and bacterial community.
Microbial community composition at the early sampling stage was relatively consistent despite the drastic change in the priming effect in soil, suggesting that microbial successional dynamics did not significantly relate to the priming effect (Li et al., 2017; Rousk et al., 2016). “Apparent priming” is defined as a quick increase in the respiratory activity by the acceleration of microbial metabolism without further composition alteration (Blagodatskaya and Kuzyakov, 2008). Such priming could be associated with the activation of dormant microorganisms (Blagodatskaya and Kuzyakov, 2008) and the catabolic response of active microorganisms (Rousk et al., 2016). However, enhanced SOM decomposition at the early sampling stage echoed the definition of “real priming” (Blagodatskaya and Kuzyakov, 2008). Since N addition was found to regulate microbial carbon and nitrogen use efficiency along with the priming effect (Rousk et al., 2016; Liu et al., 2018), multiple mechanisms of priming effect under N fertilization scenarios need to be classified thoroughly, and microbial functions should be emphasized in future studies on the priming effect.

5 Conclusions

N fertilization substantially reduced soil CO2 concentration, and N fertilization shifted microbial utilization of C substrate from bulk SOM to labile C. Glucose addition induced a positive priming in soil with N fertilization in semi-arid grassland soil, and the priming effect diminished after 7 days. Cumulative primed C was highest at 0 g N m-2 yr-1, and cumulative glucose-derived C was highest at 4 g N m-2 yr-1, in accordance with the theory of “N mining” and “microbial stoichiometry”. The F:B ratio was decreased by N fertilization in this study, echoing the inhibition of SOM-derived CO2 by N fertilization. However, the relatively stable F:B ratio during sampling suggests that the F:B ratio is a poor indicator of the extent of SOM decomposition. The “apparent priming” was likely to occur at the early stage of sampling, since there was no significant alteration in microbial community biomass or composition. Considering the diverse functions in microbial communities, future studies should look into the different mechanisms of priming when mediated by N fertilization.


We thank the Duolun Restoration Ecology Research Station (part of the Institute of Botany, Chinese Academy of Sciences) for providing access to the study site.

The authors have declared that no competing interests exist.

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