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OecologiaDOI 10.1007/s00442-015-3290-x

GLOBAL CHANGE ECOLOGY - ORIGINAL RESEARCH

The priming effect of soluble carbon inputs in organic and mineral soils from a temperate forest

Hui Wang1,2 · Wenhua Xu1 · Guoqing Hu1,2 · Weiwei Dai1 · Ping Jiang1 · Edith Bai1

Received: 23 May 2014 / Accepted: 5 March 2015 © Springer-Verlag Berlin Heidelberg 2015

mineralization of forest soils and warrant further in situ studies of PE in order to better understand C cycling under global climate change.

Keywords Soil organic carbon mineralization · Substrate availability · Nutrient status · Microbial biomass · Carbon sequestration

Introduction

Soil organic C (SOC) is the largest C pool in terrestrial ecosystems, and stores three times as much C as in the atmosphere or in plant biomass (Schmidt et al. 2011). The CO2 efflux from the soil to the atmosphere is 98 ± 12 Pg C year−1, about ten times greater than that from fossil fuel combustion and deforestation combined (Bond-Lamberty and Thomson 2010), which means that small changes in soil C dynamics may induce a significant increase of the atmospheric CO2 concentration. With increasing atmos-pheric CO2 concentration and temperature, the input of exogenous substrate to soil via plant residue or root exuda-tion might increase (Cheng 1999; Raich et al. 2006). More-over, soil has many microbial hotspots which receive a high input rate of exogenous C, such as the rhizosphere, detri-tusphere, drillosphere and some other biopores (Kuzyakov 2010). Thus, understanding the relationship between sub-strate input rate and soil organic matter (SOM) decompo-sition is essential for better prediction of soil C dynamics under global climate change.

Most process-based models consider SOM decomposi-tion a function of first-order rate constants (Jenkinson and Coleman 2008; Manzoni and Porporato 2009). However, interactions between C input and output such as the prim-ing effect (PE), defined as changes in SOM decomposition

Abstract The priming effect (PE) is one of the most important interactions between C input and output in soils. Here we aim to quantify patterns of PE in response to six addition rates of 13C-labeled water-soluble C (WSC) and determine if these patterns are different between soil organic and mineral layers in a temperate forest. Isotope mass balance was used to distinguish WSC derived from SOC-derived CO2 respiration. The relative PE was 1.1–3.3 times stronger in the mineral layer than in the organic layer, indicating higher sensitivity of the mineral layer to WSC addition. However, the magnitude of cumulative PE was significantly higher in the organic layer than in the min-eral layer due to higher SOC in the organic layer. With an increasing WSC addition rate, cumulative PE increased for both layers, but tended to level off when the addition rate was higher than 400 mg C kg−1 soil. This saturation effect indicates that stimulation of soil C loss by exogenous sub-strate would not be as drastic as the increase of C input. In fact, we found that the mineral layer with an WSC addi-tion rate of 160–800 mg C kg−1 soil had net C storage although positive PE was observed. The addition of WSC basically caused net C loss in the organic layer due to the high magnitude of PE, pointing to the importance of the organic layer in C cycling of forest ecosystems. Our find-ings provide a fundamental understanding of PE on SOC

Communicated by Hormoz BassiriRad.

* Edith Bai baie@iae.ac.cn

1 State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China

2 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China

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after the inputs of exogenous substrate (Kuzyakov et al. 2000), have been suggested as crucial mechanisms which need to be incorporated into future ecosystem models (Cheng et al. 2014).

One of the most important reasons for not taking PE into consideration in most C cycling models is its variabil-ity and unpredictability (Paterson and Sim 2013). Quanti-tative relationships between the magnitude of PE and the substrate addition rate have been found in recent studies (Blagodatskaya and Kuzyakov 2008; Guenet et al. 2010; Paterson and Sim 2013), all of which showed a nonlinear response of PE to the substrate addition rate, indicating that the ratio of PE to substrate addition rate always decreases with an increasing amount of exogenous substrate. How-ever, using the substrate input rate as a predictor of PE is far from practical application in models because this rela-tionship might be variable for different soils (De Graaff et al. 2014; Salomé et al. 2010). A prevalent view is that PE is driven by exogenous substrates, but may be medi-ated by soil properties such as microbial characteristics, nutrient status, and SOC content (Fig. 1) (Chen et al. 2014; Chowdhury et al. 2014; Fontaine et al. 2011; Sullivan and Hart 2013). Addition of exogenous substrates could affect PE through changing soil microbial characteristics (Fig. 1). In addition, other soil properties such as soil texture and aggregate, soil pH, and soil moisture could affect PE indi-rectly (Fig. 1) (Blagodatskaya and Kuzyakov 2008; Sulli-van and Hart 2013).

Among those factors affecting PE, the impact of nutrient availability has recently been widely studied (Chen et al. 2014; Chowdhury et al. 2014; Fontaine et al. 2011; Sulli-van and Hart 2013). While soil microbial growth is stimu-lated by exogenous C addition, other nutrients such as N could become limiting, causing nutrient mining from SOM by microbes, and thereby a positive PE (the nutrient mining

theory) (Blagodatskaya and Kuzyakov 2008; Fontaine et al. 2003). Therefore, soil with higher nutrient availability is expected to have lower PE. However, contrasting results have been found (Chen et al. 2014; Chowdhury et al. 2014), which may be partly due to the interactions between soil C and N availability (Craine et al. 2007).

The degree of nutrient limitation of SOM mineralization has been found to increase with depth through the soil pro-file (Fierer et al. 2003). In boreal or temperate forests, the organic layer has a higher SOC content and could provide more C sources for priming, but the mineral layer might be more susceptible to exogenous substrate input according to the nutrient mining theory. Therefore, study of the differ-ences between these two layers provides an opportunity to explore how exogenous substrate input and soil properties interact to affect PE. Until now, comparative studies of PE in organic and mineral layers are scarce and the only exist-ing result, of stronger PE in topsoil than subsoil, still needs to be tested because forest soils have never been studied with regard to this (De Graaff et al. 2014; Paterson and Sim 2013).

The objectives of our study were to quantify the pat-tern of PE in response to water-soluble C (WSC) (extracted from birch leaves) addition, and to determine if this pat-tern is different between soil organic and mineral layers in a temperate old-growth broadleaf and mixed Korean pine forest. Isotope labeling was used to distinguish WSC derived from SOC-derived respired CO2, soil microbial biomass C (MBC) and dissolved organic C (DOC) during the 53-day incubation. We hypothesized that:

1. A higher WSC supply should cause a higher PE on SOM mineralization because more soil microorgan-isms are stimulated by labile C, inducing higher SOM mineralization. However, the relationship between WSC addition rate and PE may not be linear consider-ing substrate saturation for soil microbes.

2. The mineral layer with lower N availability should be more sensitive to exogenous substrate addition (i.e., higher relative PE) according to the nutrient mining theory.

Materials and methods

Site, soils and 13C‑enriched WSC

The study site is located int an old-growth broadleaf and mixed Korean pine forest in Mountain Changbai, Northeast China (42°23′N, 128°05′E and 800 m a.s.l.). Mean annual precipitation is 700 mm, mean annual temperature is 3.0 °C, and Pinus koraiensis, Tilia amurensis, Quercus mongolica, Betula platyphylla, Fraxinus mandshurica and Acer mono

Microbial biomass & microbial community

DOC and SOC contentNutrient status

Other soil properties such as aggregate,

texture, pH, moisture, etc.

Exogenous substrate properties

Substrate quantitySubstrate quality

Priming effect

Soil properties

Fig. 1 Conceptual diagram of factors influencing the priming effect (PE). Both exogenous substrate properties and soil properties could affect the PE. Solid lines represent direct effects and dashed lines rep-resent indirect effects. DOC Dissolved organic C, SOC soil organic C

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are the dominant tree species at the site. The soil is devel-oped from volcanic ashes and classified as Albic Luvisol, which has an obvious albic horizon below the organic layer. In this site, four plots (10 m × 10 m) were chosen for soil sampling on 19 August 2012. After removing the recent litter and decomposing litter, seven soil samples were ran-domly collected in each plot with a soil corer (4.7 cm in diameter) to 20-cm depth. The soil cores were hand-divided into the organic layer (distinguished by its morphology; about 7 cm) and the mineral layer. The sub-samples of each layer were thoroughly mixed, passed through a 2-mm sieve, plant residues removed, and air-dried prior to use. The details of these two soils are given in Table 1.

13C-enriched WSC was extracted with cold deionized (DI) water from birch (B. platyphylla) leaves collected from the final harvest of a 13C-labeling experiment in Changbai Mountain. Briefly, a glass chamber (2.5 m high, 1.8 m deep and 1.7 m wide) was designed to allow the fumigation labe-ling of a 2-year-old birch seedling. 13C-enriched Na2CO3 (99.9 %) was titrated with HCl to generate 10 L 13CO2 dur-ing the 4-h labeling (per time). The labeling was carried out twice: trees were grown normally for 30 days after the first labeling on 27 July 2012 and the second labeling was car-ried out on 26 August 2012 to label the fast-turnover labile C pool of birch leaves. After the second labeling, the birch

seedling was harvested and leaves were collected, air-dried, ground to pass a 0.5-mm sieve, and prepared for the extrac-tion. The extraction was conducted in a shaker at 100 r.p.m. for 1 h with a solid-to-water ratio of 1:6, followed by cen-trifugation at 3,650 r.p.m. for 30 min (Uselman et al. 2008; Yano et al. 2005). The supernatant was filtered through 0.45-μm glass fiber filter and stored frozen as stock solu-tion. Previous studies have shown that 13C-labeled WSC generally predominantly consists of carbohydrates and some water-soluble polyphenols (e.g., tannins) (Brunner et al. 1996; Girardin et al. 2009; Yano et al. 2005). It should be noted that these compounds may be labeled non-uni-formly, which could cause bias in the results. However, even with the best methodology, a certain compound-specific het-erogeneity in the 13C labeling is unavoidable (Girardin et al. 2009). A previous study showed that this compound-specific heterogeneity is within 13 % (Girardin et al. 2009), which should be acceptable for PE studies. WSC solutions of dif-ferent concentrations were prepared as a batch from this stock solution with a total organic C (TOC) concentration of 8,050 mg C L−1. Aliquots for each concentration were stored at −18 °C prior to use. The stable C isotope ratio (δ13C) value of the stock solution was +832 ‰, which cor-responds to 2.02 atom %. For easier presentation with more significant figures, we used ‰ for δ13C values in this paper.

Table 1 Main characteristics of the studied organic layer and mineral layer soils of the temperate forest ecosystem

Mean values (SD) are shown (n = 4). Significant differences (P < 0.05) between characteristics of the two soils are denoted with different lettersa An independent t-test between the two soils was not conducted

Organic layer Mineral layer

Soil organic C (SOC) (%) 14.98 (0.18)a 1.52 (0.07)b

Total N (TN) (%) 0.90 (0.09)a 0.12 (0.01)b

C/N ratio 16.62 (0.03)a 12.85 (0.59)b

Stable C isotope ratio (δ13C) (‰) −28.02 (0.01)a −27.46 (0.09)a

pH (H2O) 5.26 (0.23)a 5.13 (0.15)a

Water-holding capacity (WHC) (%) 102.14 (7.70)a 39.86 (2.12)b

Particle size fraction (%)

Clay (<0.002 mm) 32.70 (6.58)a 14.77 (2.56)b

Silt (0.02–0.002 mm) 23.17 (5.73)b 62.29 (3.50)a

Sand (2–0.02 mm) 44.13(2.82)a 22.94 (1.11)b

Water-stable aggregate size fraction (%)

Large macroaggregate (>2,000 μm)a 0 2.99 (1.23)

Small macroaggregate (250–2,000 μm) 82.80 (0.40)a 26.95 (2.82)b

Microaggregate (53–250 μm) 13.28 (0.29)b 30.17 (4.16)a

Silt plus clay particles (<53 μm) 3.92 (0.69)b 39.89 (0.11)a

Basal respiration (mg C kg−1 soil days−1) 137.74 (5.51)a 15.11 (0.77)b

SOC-specific respiration (mg C kg−1 soil C days−1) 919.50 (36.81)b 994.08 (51.61)a

Dissolved organic C (mg kg−1 soil) 220.65 (11.68)a 124.47 (2.56)b

Microbial biomass C (MBC) (mg kg−1 soil) 1,409.92 (47.73)a 90.39 (10.13)b

Fungi/bacteria ratioa 0.187 0.051

Inorganic N (NH4+ + NO3

−) (mg N kg−1 soil)a 29.25 3.72

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Experimental units and incubation

Sub-samples of the air-dried soil (50 g for the organic layer and 100 g for the mineral layer) were added to each incu-bation unit, which consisted of a 1.0-L Mason jar sealed with parafilm M (American Can). Several tiny holes were pricked on the parafilm to allow better gas exchange dur-ing incubation. Soil was rewetted to 50 % of water-holding capacity (WHC) and pre-incubated in an incubator (25 °C, dark) for 10 days prior to WSC application for soil equili-bration after the disturbance due to the preliminary prepa-ration. Seven treatments were carried out, including six WSC addition rates: 32 (WSC1), 64 (WSC2), 160 (WSC3), 320 (WSC4), 640 (WSC5), and 1,600 mg C kg−1 dry soil (WSC6) for the organic layer; and 16 (WSC1), 32 (WSC2), 80 (WSC3), 160 (WSC4), 320 (WSC5), and 800 mg C kg−1 dry soil (WSC6) for the mineral layer, and a control (where the same amount of DI water was added as in the above treatments). The highest WSC level (1,600 mg C kg−1 dry soil) applied to the organic layer is approximately equal to 16 % of annual fresh C input (litterfall plus fine roots) to the surface soil (approximately 450 g C m−2). The WSC addition rates for mineral soil were half those for the organic layer considering that the initial (after pre-incubation but before treatments) DOC concentration in the organic layer (221 mg kg−1 soil) is approximately double that in the mineral layer (124 mg kg−1 soil) (Table 1). This C input gradient for the mineral layer covered three impor-tant inflection points (20, 50 and 200 % of MBC) for induc-ing different PEs according to the meta-analysis by Bla-godatskaya and Kuzyakov (2008). A syringe was used to apply 10 ml WSC solutions or DI water evenly onto the soil surface. Each treatment had four replicates. The jars were then incubated for 53 days (after WSC addition). The soil water content was maintained at 70 % WHC throughout the incubation by adding DI water as necessary.

Measurements of respired CO2 and its δ13C

Soil CO2 effluxes and δ13CO2 were measured with proce-dures similar to those used by Garcia-Pausas and Pater-son (2011). In detail, jars were flushed with CO2-free air for 1 h, thereby reducing the headspace CO2 concentra-tion to <10 p.p.m. To accumulate enough respired CO2 for 13C analyses, jars were tightly sealed for 2 h for the organic layer and 4 h for the mineral layer with a two-hole silicone stopper attached to two three-way stopcocks. After the accumulation period, one gas sample was taken from each jar with a 150-ml syringe through the three-way stop-cock and injected into an evacuated aluminum foil airbag (200 ml). To avoid gas exchange during sampling, 150 ml CO2-free air was injected into each jar just before sampling. Gas sampling was carried out at 0.21, 1, 2, 3, 4, 6, 8, 10, 13,

17, 21, 26, 32, 39, 46 and 53 days after WSC addition. The 140-ml gas sample was analyzed for both CO2 concentra-tion and δ13C within 24 h after sampling using a CO2 iso-tope analyzer (CCIA-36d-EP; LGR, USA). Reference gas samples (CO2 383 p.p.m., Ar 0.941 %, O2 20.9 % and N2 as the balance gas) of known δ13C (−31.82 ‰) were used to calibrate the CO2 isotope analyzer before the measure-ments and at every 20 samples during the measurements.

Chemical analyses

After pre-incubation but before any treatments, four soil samples of each type were collected. For the control, WSC3 and WSC6 treatments in the organic layer, four additional samples were collected, respectively, after an incubation of 2 days. Soil was not collected for other treatments due to inadequate amounts. At the end of the incubation, all soils in the experimental units were recovered to measure SOC, DOC, MBC and δ13C.

The δ13C value of SOC was determined using a sta-ble isotope ratio mass spectrometer (Thermo Finnigan, DELTA Plus XP) linked to an elemental analyzer (Flash EA 1112). MBC was determined by a fumigation-extrac-tion method (Vance et al. 1987). Two subsamples [fumi-gated and non-fumigated (NF)] of each soil sample were extracted with 0.5 M K2SO4 at a soil-to-water ratio of 1:4. The TOC and δ13C of K2SO4 extracts were analyzed by a Picarro iTOC-CRDS isotopic C analyzer (WCO-CRDS; Picarro-OI, USA). Briefly, 5 % phosphoric acid was used to remove inorganic C at 70 °C, and 10 % sodium per-sulfate was used for wet chemical oxidation at 98 °C to convert organic C into CO2. The concentration and δ13C of CO2 were determined by a solid-state non-dispersive infrared detector and a wavelength-scanned cavity ring-down spectroscopy detector, respectively. The TOC and δ13C results from NF soils were used as TOC and δ13C of DOC.

Calculations

CO2 concentration in gas samples was corrected by multi-plying an index of 1.135 (calculated according to the vol-ume of total accumulated air before and after substitution) to offset the decrease of CO2 concentration during gas sub-stitution. CO2 efflux was calculated following Eq. 1

where Rr is CO2 efflux (or respiration rate) in μg C g−1 soil day−1; C is the recorded CO2 concentration in p.p.m. (mmol CO2 mol−1); V is the effective volume (L) of a 1-L Mason jar; M is the molar mass of C, which is 12 g mol−1; W is gram dry weight of the soil; and t is the amount of air accumulation time (days).

(1)Rr = (1.135 × C × V × M)/(22.4 × W × t)

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The contribution of added 13C-enriched WSC to CO2 respiration (PWSC) was estimated according to Eq. 2:

where δ13[CO2]W+S and δ13[CO2]S are the δ13C of CO2 respired in the WSC + soil treatments and bare soil treatment (control), respectively; δ13CW is the initial δ13C value of WSC and δ13CS is the initial δ13C value of SOC. 13C isotopic fractionation during WSC decom-position (δ13CW – δ13[CO2]W) and SOM decomposition (δ13CS – δ13[CO2]S) was assumed to be equal (Waldrop and Firestone 2004; Werth and Kuzyakov 2010).

The contribution of WSC-derived C to TOC in K2SO4 extracts from fumigated and NF soils could also be calcu-lated following Eq. 2. WSC-derived MBC was calculated in the same way as in Paterson and Sim (2013):

PE induced by WSC was calculated by comparing the amount of SOC-derived CO2 in samples from WSC treat-ments to the amount of CO2 in the control (Kuzyakov et al. 2000), according to the following equations:

where, [CO2] represents the CO2 efflux or cumulative CO2 respiration and (1 – PWSC) represents the contribution of SOC to CO2 respiration.

Cumulative CO2 respiration, cumulative SOM miner-alization and cumulative PE for a specific time span could be estimated by integrating CO2 efflux, SOM-derived CO2 efflux and absolute PE over time, respectively. The ratio of cumulative PE to the WSC addition rate is used to indi-cate the efficiency of an exogenous substrate to trigger microbial PE on SOM mineralization (Blagodatskaya and Kuzyakov 2008; Paterson and Sim 2013).

Statistical analysis

Statistical analysis was carried out with the SPSS 16.0 package (SPSS, Chicago, IL). Homogeneity of variances of data was tested prior to analyses and data were log trans-formed when necessary (P < 0.05). An independent t-test (n = 4) was applied to examine the differences in soil char-acteristics between the organic layer and mineral layer. Repeated-measures ANOVA was used to examine the dif-ferences in CO2 respiration and PE with time among WSC treatments. A two-way ANOVA was conducted to compare the combined effects of soil layer and WSC treatment on

(2)PWSC =

(

δ13[CO2]W + S − δ13

[CO2]S

)

/

(

δ13CW − δ13

CS

)

(3)

MBCWSC =[

(PWSC × TOC)F − (PWSC × TOC)NF

]

/

KEC(KEC = 0.45)

(4)PE = [CO2]W+S × (1 − PWSC) − [CO2]S

(5)Relative PE (%) = PE/[CO2]S

cumulative CO2 respiration, cumulative PE, and MBC at the same WSC addition rate (WSC1, WSC3, WSC4 for the organic layer corresponding to WSC2, WSC4, WSC5 for the mineral layer, respectively). An α-value of 0.05 was chosen to indicate statistical significance.

Results

Soil characteristics

SOC, total N (TN), and C/N in the organic layer were 9.9, 7.5, and 1.3 times as high as those in the mineral layer, respectively (Table 1). There was no difference in soil δ13C and pH between the two layers (Table 1). WHC and parti-cle size fraction were significantly different (all P < 0.05) between the two layers. The organic layer had more clay (<0.002 mm) and sand (2–0.02 mm) particles, and less silt (0.02–0.002 mm) compared with the mineral layer (Table 1). The water-stable macroaggregate (>250 μm) plus microaggregate (53–250 μm) fraction was higher in the organic layer than in the mineral layer (P < 0.05; Table 1). Inorganic-N (NH4

+ plus NO3−) and the fungi/bac-

teria ratio in the organic layer were 6.9 and 2.7 times higher than those in the mineral layer, respectively (Table 1). After 10 days of pre-incubation, the basal respiration rate was 8.1 times higher (P < 0.05) in the organic layer than in the mineral layer on a per gram dry soil basis, but SOC-spe-cific respiration (the respiration rate expressed as per gram SOC) in the mineral layer was slightly higher than that in the organic layer (Table 1). From this point forwards, CO2 respiration is expressed as SOC-specific respiration if not noted otherwise.

CO2 production

Compared with basal respiration, the respiration rate decreased by 43.6–49.0 % in the organic layer and by 40.6–53.1 % in the mineral layer after the 53-day incu-bation. For the organic layer, the 53-day cumulative CO2 production was only increased (P < 0.05) by WSC4 (8.5 %), WSC5 (15.5 %) and WSC6 (29.7 %) treatments compared with the control (Fig. 2a). For the mineral layer, the 53-day cumulative CO2 production of WSC1–WSC6 treatments was 4.3, 13.9, 15.7, 27.8, 46.1 and 114.7 % higher (all P < 0.05 except for WSC1) than the control, respectively (Fig. 2b). At the same WSC addition rate, SOC-specific CO2 production in the mineral layer was 5.9–27.3 % higher than that in the organic layer (Fig. 2), although the absolute CO2 production in the organic layer was 6.7–8.3 times higher than that in the mineral layer. For the WSC-treated organic layer soil, SOM-derived CO2 accounted for 81.6–99.7 % of cumulative CO2

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production over the 53-day incubation, while the corre-sponding values for the mineral layer were 54.3–98.1 % (Fig. 3). The proportion of SOM-derived CO2 to total CO2 in the organic layer was 2.7–21.8 % higher than that in the mineral layer when the same WSC addition rate was applied (Fig. 3).

Priming effect

The relative PE generally increased with an increasing WSC addition rate at most time points. For both layers, WSC6 had a peak relative PE at 1 day after treatment while the relative PE under other treatments decreased with incu-bation time (Fig. 4). For the organic layer, the relative PE of WSC1, WSC2, WSC3 was positive before 13 days of incu-bation and then fluctuated around zero after that, whereas WSC4, WSC5 and WSC6 induced a positive relative PE

during the whole 53-day incubation (Fig. 4a, b). For the mineral layer, the positive PE generally lasted during the whole 53-day incubation for all treatments (Fig. 4c, d). At the same WSC addition rate, the relative PE was relatively stronger (1.1–3.3 times stronger at the highest relative PE, all P < 0.05) in the mineral layer than in the organic layer (Fig. 4).

Generally, both 4 and 53 days’ cumulative PE increased with increasing WSC addition rate and both were sig-nificantly different (P < 0.05) between the two layers (Fig. 5a, b; Table 2). For both time intervals and both lay-ers, there was a first-order exponential decay relationship (y = y0 + A e−x/t, where y0 is the y offset, A is the ampli-tude and t is the decay constant) between WSC addition rates and the ratio of cumulative PE relative to the WSC addition rate (Fig. 5c, d), indicating that the efficiency of WSC to trigger PE decreased with an increasing WSC addition rate. The fitted relationship for the first 4 days was better (as indicated by a higher R2 and lower P-value)

Fig. 2 Cumulative CO2 production (mg C g−1 SOC) over a 53-day (d) incubation for a the organic layer and b the mineral layer, which were treated with different water-soluble C (WSC) addition rates [32 (WSC1), 64 (WSC2), 160 (WSC3), 320 (WSC4), 640 (WSC5), and 1,600 mg C kg−1 dry soil (WSC6) for the organic layer; and 16 (WSC1), 32 (WSC2), 80 (WSC3), 160 (WSC4), 320 (WSC5), and 800 mg C kg−1 dry soil (WSC6) for the mineral layer]. Mean val-ues ± SD are shown (n = 4)

Fig. 3 Contribution of soil organic matter (SOM) mineralization to cumulative CO2 production over a 53-day incubation for a the organic layer and b the mineral layer. Controls are not shown because all CO2 respired in these treatments is SOM derived. Results are mean values ± SD (n = 4). For other abbreviations, see Fig. 2

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than that for the whole incubation period (Fig. 5c, d). The curve of the organic layer generally had a higher y0 and A but a lower decay constant, t, than the curve of the mineral layer (Fig. 5c, d).

Soil MBC

Total MBC in the control decreased by 24.8 and 10.8 % after the 53-day incubation in the organic and mineral layers, respectively. After 2 days of incubation, both total and SOC-derived MBC were significantly higher (all P < 0.05) under the WSC3 and WSC6 treatments than those under the control in the organic layer. At the end of the incubation, total and SOC-derived MBC did not dif-fer (both P > 0.05) among the seven treatments. WSC-derived MBC increased with increasing WSC addition rates in both layers. In addition, SOC-derived MBC was higher than WSC-derived MBC for all treatments, and the ratio of WSC-/SOC-derived MBC increased with increas-ing WSC rate (Fig. 6). Generally, at the same WSC addi-tion rate, WSC-/SOC-derived MBC were significantly higher (P < 0.05) in the mineral layer than in the organic layer (Fig. 6).

Fate of labeled WSC

The allocation pattern of recovered labeled WSC was simi-lar in different treatments, so all six treatments for each soil layer were lumped for this analysis. For organic layer, about 75 % WSC was recovered in the three labile C pools: CO2-C (65.29 %), MBC (9.31 %) and DOC (0.74 %), leav-ing 25 % remained in the soil matrix (Fig. 7a). However, the percentage of C remaining in the soil matrix for the mineral layer was much higher (32 %), and the percent-age of WSC recovered in the three labile C pools was much lower (63 %) (Fig. 7b). Total 13C recoveries in CO2-C, MBC, DOC and the soil matrix were 101.1 for the organic layer and 95.7 % for the mineral layer.

To assess the C budget affected by WSC addition, we subtracted SOC primed by WSC addition from the amount of WSC remaining in the soil (DOC, MBC, and soil matrix) (Table 3). For the organic layer, after the 53-day incubation, this value was positive under the WSC6 treatment while it was negative under the other treatments. For the mineral layer, this value was positive for the highest three WSC levels and was negative for the rest. At the same addition rate of 32 mg C kg−1 soil, both layers showed a loss of C

Fig. 4 Dynamics of the relative PE in the organic layer (a, b) and the mineral layer (c, d) over the incubation period. Results are mean val-ues ± SD (n = 4) at every single timepoint. For abbreviations, see Figs. 1 and 2

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affected by WSC addition, while at the same addition rate of 160 mg C kg−1 soil or 320 mg C kg−1 soil, the organic layer showed a loss of C, whereas the mineral layer showed a gain of C, which was affected by WSC addition (Table 3).

Discussion

WSC addition rate and PE

Our results showed that cumulative PE for the first 4 days and the whole incubation period generally increased with increasing WSC addition rate (Fig. 5a, b). There was a first-order exponential decay relationship between WSC addi-tion rate and the ratio of cumulative PE relative to WSC addition rate (Fig. 5c, d), which confirmed the possibility of using substrate input rate as a predictor of PE in soil C models. These patterns are consistent with the results of Blagodatskaya and Kuzyakov’s (2008) meta-analysis

Fig. 5 a, b Relationships between WSC addition rates (mg C kg−1 soil) and cumulative CO2-C primed (mg C kg−1 soil) during 0–4 and 0–53 days of the incubation. Results are presented as mean val-ues ± SD. c, d Fitted results of first-order exponential decay curves

between the WSC addition rate and the ratio of cumulative PE rela-tive to the WSC addition rate (mg C kg−1 soil). Equation 1 represents the fitted result of the organic layer and Eq. 2 represents the fitted result of the mineral layer. For abbreviations, see Figs. 1 and 2

Table 2 Results of two-way ANOVA [three water-soluble C (WSC) treatments × 2 soil layers] on four indicators related to C cycling

Two-way ANOVA was conducted between the two soils at the same WSC addition rate: 32 (WSC1), 160 (WSC3), 320 mg C kg−1 dry soil (WSC4) for the organic layer corresponding to WSC2, WSC4, WSC5 for the mineral layer, respectively

PE Priming effect; for other abbreviations, see Table 1

Soil Treatment Soil × treatment

CO2 production (mg C g−1 SOC)

0–4 days 0.000 0.000 0.000

0–53 days 0.000 0.000 0.000

Soil organic matter (SOM) mineralization (mg C g−1 SOC)

0–4 days 0.000 0.000 0.219

0–53 days 0.381 0.886 0.321

Priming effect (PE) (mg C kg−1 soil)

0–4 days 0.000 0.008 0.067

0–53 days 0.020 0.179 0.124

MBC (mg C kg−1 soil) 0.000 0.005 0.002

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of studies on plant residues. Because SOM can be stabi-lized by various mechanisms [e.g., chemical recalcitrance, physical protection (Kemmitt et al. 2008)] and microbial

communities are generally C limited in soils (Demoling et al. 2007), even a small amount of added WSC could trig-ger a strong response of soil microbes and induce PE. This stimulation effect of WSC was more efficient at lower addi-tion rates, and increasing WSC supply did not increase the stimulation of SOM mineralization in equal proportions (Fig. 5). We present possible reasons for this phenomenon:

1. Factors other than exogenous C (e.g., water, O2) may become limiting to soil microbes with an increase in the supply of WSC.

2. SOM, the substrate of priming, is not always accessi-ble to microbial communities due to spatial separation (Feeney et al. 2006; Salomé et al. 2010) or its insolu-bility (Kemmitt et al. 2008). The indirect evidence in our study was that only 3.6 % of SOC in the organic layer was mineralized after the 53-day incubation.

3. The added WSC might be quickly used first by soil microorganisms (Werth and Kuzyakov 2010), thus the rapid exhaustion of newly added substrate might prevent the PE from increasing in an equal propor-tion.

Fig. 6 Ratio of WSC-/SOC-derived C in microbial biomass C (MBC) under different WSC treatments in the organic layer and min-eral layer. For other abbreviations, see Fig. 2

Fig. 7 Percentages of added WSC recovered in different C pools at the end of the incuba-tion (n = 6). For abbreviations, see Figs. 1, 2 and 6

Table 3 The budget of added WSC remaining in the soil (mg C kg−1 soil) compared with primed SOM mineralization (mg C kg−1 soil)

For abbreviations, see Table 2

Treatment Organic layer Mineral layer

WSC remaining in the soil

Cumulative PE (53 days)

C budget WSC remaining in the soil

Cumulative PE (53 days)

C budget

WSC1 12.78 (5.76) 71.76 (31.65) −58.98 6.80 (2.17) 11.87 (10.32) −5.07

WSC2 26.34 (8.74) 46.74 (171.45) −20.40 11.62 (3.70) 52.99 (23.28) −41.37

WSC3 50.97 (6.78) 101.10 (153.71) −50.13 30.49 (1.72) 35.41 (15.04) −4.92

WSC4 122.91 (22.24) 228.93 (117.05) −106.02 64.49 (8.79) 50.03 (23.22) 14.45

WSC5 208.28 (8.37) 332.41 (41.19) −124.13 115.38 (14.20) 44.16 (13.46) 71.22

WSC6 493.78 (58.00) 309.64 (89.24) 184.14 242.91 (5.53) 84.18 (5.68) 158.73

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With increasing incubation time, the first-order exponen-tial relationship between WSC addition rate and the ratio of cumulative PE to WSC addition rate became weaker (Fig. 5c, d). This indicated that the effect of the amount of added WSC on PE decreased with time after C input. This temporal effect on PE measurement might be one important reason for the variability of PE in many previous studies (see Blagodatskaya and Kuzyakov 2008). Therefore, clari-fication of the time span of the cumulative PE and determi-nation of the duration of PE are important issues needing more attention.

According to our results, the non-linear saturation rela-tionship between cumulative PE and WSC addition rate suggested that the increase of SOM mineralization would not be as drastic as the increase of the C input. In the con-text of global change, the total input of plant-derived labile C to soil via residue decomposition and rhizosphere exuda-tion might increase (Heath et al. 2005; Wang et al. 2013). Therefore, it is likely that SOC mineralization induced by PE would increase; but this increased soil C loss would not be proportional to the increased labile C input, indicating that the soil C pool might be a buffer against future climate change. More in situ studies are needed to assess these predictions.

Comparison between the organic layer and the mineral layer

We found weaker relative PE in the organic layer than in the mineral layer after the addition of WSC (Fig. 4), which indicates that the mineral soil was more sensitive to WSC addition. Since soil pH and moisture were the same between the two layers, this pattern could be attributed to several other factors (see Fig. 1): (1) nutrients status, (2) microbial biomass and community structure, (3) aggregate and particle size fractions, and (4) DOC content (Table 1).

First, the higher mineral N availability in the organic layer (Table 1) may be one of the major reasons for a higher relative PE in the mineral layer. This finding confirmed our second hypothesis and was in line with previous investi-gations which found that higher relative PE occurred with lower soil N availability (Paterson and Sim 2013; Sullivan and Hart 2013). This result also supported the conceptual model proposed by Chen et al. (2014), which suggested that the nutrient mining theory is the dominant mechanism governing relative PE under low nutrient availability due to the contribution of SOM feeding K-strategists. Therefore, with increasing N deposition, the PE on SOM mineraliza-tion might become weaker, which means C loss from the soil might become smaller and thus mitigate global climate change.

Second, the significant differences in microbial biomass and microbial community composition may help explain

the distinct relative PE between the organic layer and min-eral layer. Whether there is microbial growth or not after exogenous substrate addition depends on the pool size of MBC (Blagodatskaya and Kuzyakov 2008; Paterson and Sim 2013; Sullivan and Hart 2013). In our study, MBC in the organic layer was 14.6 times larger than that in the min-eral layer (Table 1). The WSC addition rate of 320 mg C kg−1 soil equaled 22.7 % of MBC in the organic layer and 354.0 % of MBC in the mineral layer, which means that more food was supplied to microbes in the mineral layer at the same WSC addition rate, and thereby a stronger relative PE. In addition, as deeper soil layers are usually resource poor (Fierer et al. 2003; Fontaine et al. 2007), there might be a greater proportion of slow-growing K-strategists in a mineral layer characterized by a higher diversity of meta-bolic pathways and more utilization of recalcitrant SOM. Thus, deeper soils are expected to be more sensitive to inputs of fresh C. We found that the mineral layer had a lower fungi/bacteria ratio than the organic layer (Table 1). However, we do not know which microbial group contrib-uted more to the observed PE and which group had more slow-growing K-strategists. More research is needed to bet-ter understand the effects of the microbial community on PE.

Third, soil aggregates and texture were considered to have an indirect influence on PE due to their impacts on SOM stability and microbial activity (Sullivan and Hart 2013). In this study, the organic layer contained more clay (<0.002 mm) and more small macroaggregates (250–2,000 μm) (Table 1). Our results agreed with previous findings which showed intermediate size aggregates (250–1000 μm) and finer soil experienced weaker, absent, or even negative net priming on SOM (Degens and Sparling 1996; Sullivan and Hart 2013). This was probably because these physical properties are associated with higher SOM content and/or higher microbial activity, which could pro-mote nutrient supply and weaken relative PE.

Finally, the SOC content is important to PE because it serves as the C source for PE. In particular, soils with higher DOC content usually have a higher respiration rate because DOC is the major form of C used by microbes (Kemmitt et al. 2008). If the soil respiration rate is high, even if the relative change is small, the absolute magnitude could be high. This is why we observed a greater (P < 0.05) absolute magnitude of cumulative PE in the organic layer than in the mineral layer (Table 2), although the latter had a stronger relative PE (Fig. 4). For organic layer soil, the first-order exponential decay-fitting curve between the WSC addition rate and the ratio of cumulative PE relative to the WSC addition rate was always above that for the mineral layer (Fig. 5c, d). DOC was, on average, 1.5 times greater in the organic layer than in the mineral layer at 53 days from the start of the incubation (data not shown). This

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finding echoes the observation of Chowdhury et al. (2014) who also reported a positive correlation between the DOC content and the absolute magnitude of PE.

In conclusion, when comparing the PE in different soils, it is important to be clear about which definition of PE is used. The mineral layer had higher relative PE than the organic layer, indicating a higher sensitivity of the former to exogenous inputs. This sensitivity was mainly affected by the soil nutrient status and the soil microbial community. The absolute magnitude of PE was higher in the organic layer than in the mineral layer because the former layer had more DOC or SOC, which is the source for the PE. Due to the large size of the SOC pool in the organic layer, even a small change in the PE could cause a significant increase in CO2 emission, pointing to the importance of the organic layer soil in forest ecosystems on global C cycling.

Effects of WSC addition on the soil C budget

After the 53-day incubation, the soil C budget affected by WSC addition (calculated as WSC remaining in the soil minus primed SOM mineralization) was negative at low levels and positive at high levels of WSC addition rate for both layers (Table 3). This finding suggested that higher positive PE does not necessarily lead to higher net C loss because the soil might also sequestrate more exogenous C simultaneously. When comparing the two layers at the same WSC addition rate, this budget was negative for the organic layer whereas it was positive for the mineral layer at WSC levels of both 160 mg C kg−1 soil and 320 mg C g−1 soil, which indicated that the mineral layer might have a greater potential for C sequestration than the organic layer. Our result of higher WSC-/SOC-derived MBC in the mineral layer than the organic layer (Fig. 6) confirmed this view-point, because microbial biomass is thought to be closely related to the formation of a stable SOM pool (Kirkby et al. 2013; Miltner et al. 2012), particularly in soils with higher clay plus silt (<0.02 mm) content (Creamer et al. 2014). Thus, while an increase of the WSC addition rate is likely to solely stimulate SOM decomposition in an organic layer, it could increase both SOM decomposition and SOM sta-bilization in a mineral layer and the net effect might be C sequestration. Therefore, deeper soil may be more efficient at C sequestration, mitigating climate change. These com-peting processes call for the separation of soil horizons in ecosystem C models for better prediction of soil C seques-tration and CO2 emission.

Conclusion

Our study demonstrated that mineral layer soil of the tem-perate forest was more sensitive to WSC inputs, reflected by the higher relative PE compared with organic layer soil.

However, the organic layer had a higher absolute magnitude of cumulative PE than the mineral layer due to its higher SOC content. There was a saturation effect of cumulative PE with an increasing WSC addition rate, indicating that stimulation of soil C loss by exogenous substrate would not be as drastic as the increased C input. There was a first-order exponential relationship between the WSC addition rate and the ratio of cumulative PE relative to the WSC addition rate, whose parameters varied with the soil layer and got weaker with increasing incubation time. This finding indi-cated that PE was also affected by other factors, e.g., soil microbial community and soil nutrients, besides character-istics of exogenous inputs. We found that a higher positive PE did not necessarily lead to a higher net C loss because the soil might have simultaneously sequestrated more exog-enous C. Mineral layer soil with an WSC addition rate of 160–800 mg C kg−1 soil showed net C storage after the 53-day incubation. The addition of WSC basically caused net C loss in the organic layer due to the high magnitude of PE, pointing to the importance of the organic layer in the C cycling of forest ecosystems and in global climate change.

Author contribution statement E. B., H. W., and W. X. conceived and designed the experiments. H. W., G. H., W. D., and P. J. performed the experiments. H. W., W. X., G. H., and E. B. analyzed the data. H. W., W. X., W. D., and E. B. wrote the manuscript; the other authors provided editorial advice.

Acknowledgments This work was supported by the National Basic Research Program of China (973 program; 2014CB954400), the National Natural Science Foundation of China (41175138), the State Key Laboratory of Forest and Soil Ecology, and the Doctoral Scien-tific Research Foundation of Liaoning Province (20121083). We are grateful to Sun, Jianfei and Li, Shanlong for their assistance in field soil sampling. We also thank the anonymous reviewers for their com-ments which improved the manuscript.

Conflict of interest The authors declare no conflict of interest. The experiments comply with the current laws of China, in which the experiments were performed.

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