PUBLICATIONS
Global Biogeochemical Cycles
RESEARCH ARTICLE
10.1002/2016GB005423
Key Points:
• Effects of N deposition on S cycling
were addressed using meta-analysis
and incubation experiment
• N inputs slow down S cycling by
suppressing soil arylsulfatase activity
• Soil buffering capacity mediates the
response extent of arylsulfatase to
N inputs
Supporting Information:
• Supporting Information S1
Correspondence to:
D. Li,
dejunli@isa.ac.cn
Citation:
Chen, H., L. Yang, L. Wen, P. Luo, L. Liu,
Y. Yang, K. Wang, and D. Li (2016),
Effects of nitrogen deposition on soil
sulfur cycling, Global Biogeochem.
Cycles, 30, 1568–1577, doi:10.1002/
2016GB005423.
Received 8 APR 2016
Accepted 8 OCT 2016
Accepted article online 11 OCT 2016
Published online 2 NOV 2016
©2016. American Geophysical Union.
All Rights Reserved.
CHEN ET AL.
Effects of nitrogen deposition on soil sulfur cycling
Hao Chen1,2, Liqiong Yang1,2,3, Li Wen1,2,3, Pan Luo1,2,3, Lu Liu1,2,4, Yi Yang1,2,3, Kelin Wang1,2,
and Dejun Li1,2
1
Huangjiang Observation and Research Station for Karst Ecosystem, Chinese Academy of Sciences, Huangjiang, China, 2Key
Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of
Sciences, Changsha, China, 3University of Chinese Academy of Sciences, Beijing, China, 4Department of Biological Sciences,
Guangxi Normal University, Guilin, China
Abstract Increased atmospheric nitrogen (N) deposition has been found to alter processes and functions
of terrestrial ecosystems including the biogeochemical cycling of N and other elements, e.g., phosphorus (P),
calcium (Ca), and potassium (K). Nevertheless, how N deposition changes sulfur (S) cycling is largely
unknown. Based on a meta-analysis and a lab N addition experiment, here we show that N addition
significantly suppresses the activity of soil arylsulfatase, which is a major enzyme involved in the
mineralization of organic S. The evidence suggests that N-induced decrease in soil pH is responsible for
the decrease of arylsulfatase activity. Soil buffering capacity plays a critical role in mediating the extent
of arylsulfatase activity response to N inputs via its regulation on soil pH. Our results suggest that
N deposition may slow down S cycling by suppressing soil organic S mineralization.
1. Introduction
Over the past decades, atmospheric nitrogen (N) deposition has substantially increased worldwide due to
increased fossil fuel combustion and widespread use of chemical fertilizer N [Galloway et al., 2008]. It is
well documented that increased N deposition has been altering the processes and functions of terrestrial
ecosystems including the biogeochemical cycling of N and other elements, e.g., phosphorus (P), calcium
(Ca), and potassium (K) [Bowman et al., 2008; Marklein and Houlton, 2012; Sardans and Peñuelas, 2015].
For instance, enhanced N deposition usually increases ecosystem demand for P and thus causes an accel-
eration in soil P cycling by increasing soil phosphatase activity, which controls the release of P from soil
organic matter (SOM) [Marklein and Houlton, 2012]. Similar to P, sulfur (S) is also a necessary element of
organisms, accounting for about 1% of the dry mass of living organisms, and is a required component
of proteins (cysteine and methionine), sulfolipids, and sulfate eater [Howarth, 1984]. Sulfur and N are clo-
sely associated in protein synthesis [Tabatabai, 1984]. In this sense, elevated N deposition will theoretically
increase the demand for S and accelerate the ecosystem S cycling in a similar way as P. Although it has
been well recognized that N inputs accelerate ecosystem P cycling, whether or not N deposition also
accelerates S cycling is largely unknown.
Soil enzymes control the rate-limiting steps in SOM decomposition and soil nutrient return [Sinsabaugh et al.,
2008]. For example, soil phosphatase activity has been used as a proxy for P mineralization and P demand by
microbes and plants [Marklein and Houlton, 2012]. Similarly, soil arylsulfatase is involved in mineralization of
ester sulfate, which accounts for 30–75% of total soil organic S and represents the most labile form of organic
S in soil [Scherer, 2001; Tabatabai, 2005]. Changes in soil enzyme activities have been widely used as a direct
evidence to show how N deposition affects soil nutrient cycling [Marklein and Houlton, 2012; Sinsabaugh
et al., 2008]. Many studies have reported the effects of N enrichment on soil phosphatase activities using
long-term N fertilization experiments, N deposition gradient experiments, and meta-analysis [Jones and
Power, 2012; Olander and Vitousek, 2000; Treseder and Vitousek, 2001]. Nevertheless, very limited studies have
been conducted in terms of the effects of N enrichment on soil arylsulfatase activities (Table S3 in the
supporting information). As a consequence, so far, there is no consensus regarding how N deposition affects
soil S cycling.
In this study, we used a meta-analysis technique to reveal the effect of N addition on soil arylsulfatase activity
by synthesizing data from published studies. Due to very limited data available (Table S3), especially in
tropical/subtropical regions (Figure S1), we conducted a lab N addition experiment including 89 soil samples
collected in both calcareous and noncalcareous subtropical areas in southwest China in order to test whether
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 Global Biogeochemical Cycles 10.1002/2016GB005423

the pattern derived from the meta-analysis is applicable broadly. In order to facilitate detecting the mechan-
isms underlying the impacts of N input on soil arylsulfatase activity, the N addition experiment was divided
into two stages. In the first stage, only N was added (+N experiment), and in the second stage P and K were
added to soils previously treated with N (+NPK experiment). Therefore, major objectives of the current study
were (1) to explore the general pattern of responses of arylsulfatase activity to N addition and (2) to unravel
the mechanisms underlying the impacts of N addition on soil arylsulfatase activity.

2. Methods
2.1. Data Compilation
Peer reviewed publications (1990–2015) that reported the responses of soil arylsulfatase activity to N addition
were selected by searching Web of Science and Google scholar. The following criteria were applied to select
appropriate studies: (1) the N addition and control plots were deployed under the same climate, soil, and
vegetation conditions to avoid confounding factors; (2) the means, standard deviations or standard errors,
and sample sizes of the target variables were directly reported or could be calculated from data presented
in the paper; (3) measurements for different N application rates were considered as independent observa-
tions if more than one level of N addition were applied in the same study [Liu and Greaver, 2010]; and (4) data
from the latest measurements were used if more than one measurement at different time points was avail-
able for the same study [Treseder, 2008].
The raw data were either obtained from tables or extracted by digitizing graphs using the GetData Graph
Digitizer (version 2.24, Russian Federation). For each paper, the following information was collected: location,
ecosystem types, experiment methods (field or incubation study), sampling horizons (organic horizon or
mineral horizon soil), fertilization rates, fertilization duration, soil texture, concentrations of soil organic
carbon (SOC), total N (TN), and other soil properties (Table S3). The related references refer to the supporting
information [Ajwa et al., 1999; Hay et al., 2015; Hu et al., 2013; Jang et al., 2010; Kang and Lee, 2005; Klose et al.,
1999; Prietzel, 2001; Stursova et al., 2006; Wang et al., 2016; Zhang et al., 2013].
2.2. Lab Incubation Experiment
Soil samples were collected in Huangjiang county of Guangxi Zhuang Autonomous Region, southwest China
(24°44′–25°33′N, 107°51′–108°43′E; Figure 1). This region is located in the subtropical humid forest life zone
with a monsoon climate. Annual mean relative humidity is greater than 80%. Mean annual air temperature
is 15.0–18.7°C, with the lowest monthly mean in January (3.4–8.7°C) and the highest in July (23.0–26.7°C).
Mean annual precipitation ranges from 1530 to 1820 mm with a distinct seasonal pattern. The period from
April to August is wet season and that from September to March is dry season. This region is characterized
by a typical karst landscape with gentle valleys flanked by steep hills. The bedrock is mostly limestone nested
with dolomite and clasolite (Figure 1). The soil is calcareous lithosols (limestone soil) over limestone and dolo-
mite lithology and is Ferric Acrisols over the lithology of clasolite.
Soil sampling was conducted from the middle of May to early June 2015. The selected sampling sites (plots)
covered the three main lithology types, i.e., limestone, dolomite, and clasolite, and included the five major
land use types, i.e., secondary forest (SF), shrubland (SH), grassland (GR), cropland (CR), and plantation forest
(PL). The secondary forest denotes that regenerated naturally from abandoned cropland.
At each site, a plot of 20 m × 20 m was selected. Ten to 15 soil cores (0–15 cm in depth and 5 cm in diameter)
were collected from each plot after removing the organic layer and mixed to a composite sample. Roots and
stones were picked out using forceps, and soils were passed through a 2 mm mesh sieve on site. The sieved
soil samples were divided into portions for further processes. The samples for analyses of extracellular
enzyme activities were kept on ice in the field and were stored under
20°C in the laboratory. In total, 89
composite soil samples were collected in this study. The distribution of the 89 soil samples among the three
lithology types or the five land use types is presented in Table S1.
Physicochemical properties including SOC, TN, total P (TP), available P (AP), total sulfur (TS), available sulfur
(AS), soil pH, exchangeable cations (K+, Ca2+, Na+, and Mg2+), and soil texture (clay, silt, and sand) were ana-
lyzed for each composite soil sample [Carter and Gregorich, 2007; Butters and Chenery, 1959; Zhao and
McGrath, 1994]. SOC was determined by wet oxidation with KCr2O7 + H2SO4 and titrated with FeSO4. Soil
TN was analyzed using an elemental analyzer (EA 3000; EuroVector, Italy). Soil TP was determined by acid

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 Global Biogeochemical Cycles 10.1002/2016GB005423

Figure 1. Map showing the sampling locations.

digestion with a H2SO4 + HClO4 solution. Soil AP was extracted with 0.5 M NaHCO3 and measured by molyb-
denum blue colorimetric method. Soil TS was determined by oxidation with Mg(NO3)2. Soil AS was
extracted with 0.01 M CaCl2 and analyzed by inductively coupled plasma atomic emission spectroscopy
(ICP-AES). Soil pH value was measured with a pH meter (Mettler Toledo, China) using a 1:2.5 soil/water ratio.
Soil texture was determined using a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, UK).
According to the U.S. Department of Agriculture classification system, soil particles were divided into three
fractions, i.e., clay (<0.002 mm), silt (0.05–0.002 mm), and sand (2.0–0.05 mm). Exchangeable K+, Ca2+, Na+,
and Mg2+ were displaced via compulsive exchange in 1 mol L
1 ammonium acetate at pH 7.0 and analyzed
by ICP-AES. The background soil properties for different lithology types and land use types are listed in
Table S2.
The 89 composite soil samples were used for the lab incubation experiment. For each composite soil, two
portions (30 g dry weight equivalent each) were weighed into 200 mL plastic bottles, respectively. One batch
of soils was treated with 10 mg N as NH4NO3 solution (equivalent to 50 kg N ha
1yr
1), while another batch
was used as controls, to which no N was added but the same amount of water was spayed (+N experiment).
All samples were adjusted to 35% water-holding capacity and incubated at 25°C for 30 days in an incubation
chamber [Ramirez et al., 2012].
After 30 days of incubation, soil alkaline phosphatase and arylsulfatase activities were assayed using fluoro-
genic substrates (i.e. 4-methylumbelliferyl phosphate and 4- methylumbelliferyl sulfate potassium salt for
the alkaline phosphatase and arylsulfatase, respectively) based on the microplate protocols [Saiya-Cork
et al., 2002; Sinsabaugh et al., 2008]. Soil suspensions were prepared by homogenizing 1 g of fresh soil in
125 mL of buffer, which was 50 mM sodium bicarbonate (pH 8.0) for alkaline phosphatase assay and 50 mM
sodium acetate (pH 5.0) for arylsulfatase assay. Aliquots of suspension were dispensed into 96 well micro-
plates with 8 replicate wells per sample per assay. After 4 h of incubation under 20°C black environment,
fluorescence was measured using 365 nm excitation and 450 nm emission filters. Soil pH and soil AP were
measured using the methods as described above.

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 Global Biogeochemical Cycles 10.1002/2016GB005423

To determine the effects of N, P,

and K addition (+NPK experi-
ment) on soil arylsulfatase and
phosphatase activities, 44 mg
KH2PO4 (10 mg P and 12 mg K;
equivalent to 50 kg P ha
1 yr
1
and 60 kg K ha
1 yr
1, respectively)
was added to the soils which have
been incubated for 30 days with N
addition alone. The soils were con-
tinuously incubated for another
20 days. At the end of incubation,
soil arylsulfatase and phosphatase
activities were assayed again using
methods as described above.

2.3. Data Analysis

Figure 2. N-induced changes of soil arylsulfatase activity, phosphatase activ-
Meta-analysis was conducted using
ity, soil pH, and soil available phosphorus (AP). (a) Results from meta-analysis,
(b) results from +N experiment, and (c) results from +NPK experiment. METAWIN version 2.0 (Sinauer
Values are presented as means ±95% CIs (n = 41 and 9 for changes of Associates, Inc., Sunderland, MA,
arylsulfatase activity and soil pH, respectively, in meta-analysis; n = 89 for +N
USA) with the mixed model
and +NPK experiments).
selected. The response ratio (RR),
weighted response ratio (RR++),
and 95% bootstrap confidence intervals (CIs) for the overall data set and subgroups divided according to
land use type, experimental method, and soil horizon were calculated. N-induced change of soil arylsulfa-
tase activity or other variables was calculated by equation (1):


Change ð%Þ ¼ eRRþþ
1  100 (1)

The change was considered significant if the 95% CI of the percentage change did not overlap zero [Hedges
et al., 1999]. Linear regression analysis was used to build the relationships between N-induced change of soil
arylsulfatase activity and N addition rate, N addition duration, soil pH, SOC, and TN.
For the incubation experiment, N-induced change of soil arylsulfatase or other variables was calculated by
equation (2) (Xt and Xc denote values for N addition and control treatments, respectively):

Xt
Xc
Change ð%Þ ¼  100 (2)
Xc
Additionally, paired-samples T test was used to compare the difference in soil pH, AP, and enzyme activities
between control and N addition treatments. Since our experiment was not a full factor design as the samples
were not evenly distributed among the land use or lithology types (Table S1), direct comparisons among land
use or lithology types were not applicable. Nevertheless, in order to discern the possible effects of land use or
lithology, we divided the data into different subgroups by land use or lithology type when it was possible for
comparison. In this case, we compared the difference among (i) the four land use types (cropland, grassland,
shrubland, and secondary forest) over dolomite and limestone and (ii) the two land use types (cropland and
plantation) over limestone and clasolite (Table S1). In both cases, two-way analysis of variance (ANOVA) was
used to examine the effects of land use, lithology, and their possible interactions on the change of arylsulfa-
tase activity or other variables. Linear regression analysis was used to build the relationships between soil
properties and the change of arylsulfatase activity or the relationship between the change of soil pH and
change of arylsulfatase activity. Path analysis was used to explore relationships between soil properties
and change of soil arylsulfatase activity [Sefcik et al., 2007]. Only soil properties that were significantly corre-
lated with change of soil arylsulfatase activity were included in the path analysis. Standardized regression
coefficients and significant level of each path were calculated. The path analysis was performed using
AMOS 21.0 (IBM, Armonk, NY, USA). Other statistical analyses were performed in SPSS 16.0 (SPSS Inc.,
Chicago, IL, USA). The difference was regarded as statistically significant if P value was less than 0.05.

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 Global Biogeochemical Cycles 10.1002/2016GB005423

Figure 3. Effects of land use type, lithology, experimental method, and soil horizon on the N-induced change of soil
arylsulfatase activity. (a–c) Results from meta-analysis, numbers for the forest, grassland, cropland, filed, incubation,
organic horizon, and mineral horizon are 22, 17, 2, 31, 10, 8, and 33, respectively. (d and e) Results from the incubation
experiment (the number of observations are shown in Table S1). CR: cropland; GR: grassland; SH: shrubland; SF: secondary
forest; and PL: plantation. Values are presented as mean ± standard error. Two-way ANOVA indicates no significant
effects of lithology, land use, or their interaction on change of soil arylsulfatase activity for Figure 3d but shows significant
effect of lithology on change of soil arylsulfatase activity for Figure 3e.

3. Results
3.1. Meta-Analysis
The final data set contained 10 studies with 41 observations. These studies were only conducted in boreal or
temperate climate zone (Table S3 or Figure S1), with three ecosystem types involved, i.e., forest, grassland,
and cropland (Figure S1). Soil pH, SOC, and TN ranged from 2.4 to 5.7, 238.0 to 552.0 g C kg
1, and 8.7 to
15.0 g N kg
1, respectively, for the organic horizon and from 3.2 to 8.5, 5.7 to 69.6 g C kg
1, and 0.4 to
4.6 g N kg
1, respectively, for the mineral horizon (Table S3).
Among the 41 observations, 37 showed N-induced decrease of soil arylsulfatase activity, while four observa-
tions showed N-induced increase (Table S3 and Figure S2). Across all the observations, soil arylsulfatase
activity was significantly decreased following N addition by an average of 32.6 ± 9.1% (mean ± 95% CI here-
after unless otherwise pointed out, Figure 2a). Soil pH was significantly decreased by 25.2 ± 0.2% (Figure 2a).

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 Global Biogeochemical Cycles 10.1002/2016GB005423

Figure 4. Relationships between N addition rate, experiment duration or background soil properties, and N-induced
change of soil arylsulfatase activity. (a–e) For meta-analysis (only data for mineral horizon are presented, while data for
2
organic horizon are not presented due to limited data), with significant correlation only for soil pH (R = 0.50; P < 0.001,
2 2
n = 25). For the incubation experiment: (f) pH (R = 0.32; P < 0.001, n = 89); (g) SOC (R = 0.18; P < 0.001, n = 89); (h) TN
2 2 2
(R = 0.25; P < 0.001, n = 89); (i) Ca (R = 0.19; P < 0.001, n = 89); (j) Mg (R = 0.21; P < 0.001, n = 89); and (k) N-induced
2
change of soil pH (R = 0.22; P < 0.001; n = 89).

The N-induced change of soil arylsulfatase activity was not significantly affected by land use type, experimen-
tal method, and soil horizon (Figures 3a–3c). Change of soil arylsulfatase activity was positively related to
background soil pH but showed no clear relationship with N addition rate, N addition duration, SOC, or TN
level (Figures 4a–4e).
3.2. Lab Incubation Experiments
After 30 days of N addition, soil arylsulfatase activity was significantly decreased by 45.3 ± 5.3% on average
compared to the control (Figure 2b). In contrast, soil phosphatase activity was significantly increased by

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 Global Biogeochemical Cycles 10.1002/2016GB005423

Figure 5. Path diagram showing the effects of soil properties on N-induced change of arylsulfatase activity (ASA). Solid
and dashed lines denote significant and insignificant effects, respectively. Black and grey lines represent positive and
negative effects, respectively. Width of lines is proportional to the strength of the relationship. Values in the inserted
table show standardized direct, indirect, and total effects of the selected properties on N-induced change of arylsulfatase
activity. The values of N-induced change of soil pH or arylsulfatase activity are all negative, so the larger the values of
change, the less soil pH or ASA decreases.

69.3 ± 19.6% on average (Figure 2b). For the four land use types over dolomite and limestone (Table S1 and
Figure 3d), no significant effects was found of lithology, land use, or their interaction on the change of soil
arylsulfatase activity. For the two land use types over limestone and clasolite (Table S1 and Figure 3e), lithol-
ogy was found to have a significant effect on the change of soil arylsulfatase activity, but no significant effect
of land use or the interaction of lithology and land use was found.
Soil pH was significantly decreased by 16.9 ± 0.5%, but soil AP was significantly increased by 32.9 ± 6.2%
relative to the control (Figure 2b). Regression analysis showed that change of soil arylsulfatase activity was
positively related to the background values (i.e., values in control) of soil pH, SOC, total N, exchangeable
Ca, and Mg, and change of soil pH (Figures 4f–4k). Path analysis showed that significant direct effect on
change of arylsulfatase activity was only found for change of soil pH (R2 = 0.37, P < 0.001, Figure 5).
Exchangeable Ca exerted significant total effect on change of arylsulfatase activity (R2 = 0.51, P < 0.001,
Figure 5). Nevertheless, the effect of Ca on arylsulfatase is largely indirect, i.e., via its direct effect on change
of soil pH (R2 = 0.65, P < 0.001, Figure S3). For the other soil physical variables, no significant direct or indirect
effects were found on change of arylsulfatase activity (Figure 5).
After KH2PO4 addition to the soils previously treated with N, the activities of both soil phosphatase (by
63.8 ± 17.3%) and arylsulfatase (by 64.5 ± 6.1%) were significantly suppressed relative to the control
(Figure 2c). The change of arylsulfatase activity after NPK addition was not significantly different from that
after N addition alone (Figures 2b and 2c).

4. Discussion
The meta-analysis clearly indicates that N addition induced significant decrease in soil arylsulfatase activity.
Nevertheless, since all observations available for the meta-analysis were obtained from boreal and tempe-
rate regions, whether findings from the meta-analysis were also applicable to tropical/subtropical regions
was uncertain. To fill in this gap, soil samples from a variety of ecosystems in a subtropical region were

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 Global Biogeochemical Cycles 10.1002/2016GB005423

used in a N addition incubation experiment. As the findings based on the incubation experiment are con-
sistent with those from the meta-analysis, decrease of soil arylsulfatase activity induced by N addition may
happen widely.
There are two plausible mechanisms underlying the decrease in arylsulfatase activity following N input
(Figure S5). The first is related to nutrient acquisition strategy (the so-called biological mechanism in
Figure S5) adopted by microbes. Phosphorus is the second major limiting nutrient next to N in most terrestrial
ecosystems [Schachtman and Shin, 2007]. Under the circumstances of elevated N inputs, P becomes in turn
the first limiting nutrient. For example, it has been reported that in many temperate and tropical ecosystems,
long-term N addition has altered the ecosystems from N limitation to P limitation [Bobbink et al., 2010;
Vitousek et al., 2010]. In order to relieve P limitation, microbes may invest a large portion of their resources
to acquire P, which may subsequently reduce the resource allocated for S acquisition. This strategy is applic-
able to P acquisition because we found that soil phosphatase activity was increased with N addition alone but
decreased when both N and P were added (Figure 2). This is consistent with previous studies [Marklein and
Houlton, 2012; Rejmánková and Snyder, 2008], which showed that elevated N inputs stimulate phosphatase
activity while P inputs inhibit soil phosphatase activities with the effect of P being stronger than the N effect.
The mechanisms underlying substantial increase of soil phosphatase activity following N addition include
large N requirement involved in P acquisition [Houlton et al., 2008] and high N content in phosphatase
enzymes [Marklein and Houlton, 2012].
However, this strategy seems not applicable to the acquisition of S based on our experiment. Sulfur is among
the six macronutrients (N, P, K, Ca, S, and magnesium (Mg)) of organisms [Barker and Pilbeam, 2007]. If the
limitations by the other five nutrients are eliminated, S should theoretically be the major limiting nutrient
to organisms. In the present study, most of the soil samples were collected from the karst region where
the soil is abundant with Ca and Mg [Du et al., 2013]. Therefore, it is highly unlikely that the organisms are
limited by these two elements. Under this condition, if we remove the limitation of N, P, and K by adding
KH2PO4 to the soils previously treated with N, soil arylsulfatase activity should be increased because S
becomes the major limiting element. Nevertheless, in conflict with the nutrient acquisition strategy, we found
a continuing decrease of soil arylsulfatase activity after KH2PO4 addition (Figure 2), which implies that some-
other mechanisms may play the role or the effect of other mechanisms exceed that of nutrient acquisition
strategy.
We propose that N-induced decrease of soil arylsulfatase activity is caused by changes of soil pH after N
addition (the so-called nonbiological mechanism in Figure S5). It has been widely reported that N addi-
tion lowers soil pH via the nitrification and leaching of base cations [Bowman et al., 2008; Tian and Niu,
2015]. For laboratory incubation, nitrification may be more important in soil pH decrease, since leaching
of base cations does not likely occur in a closed system. We found that soil arylsulfatase activity was
positively and significantly correlated with soil pH according to analysis of the data from the control
(Table S4), which suggests that decrease of pH would negatively affect soil arylsulfatase activity. This
was supported by both the meta-analysis and incubation experiment in the current study; i.e., the
decrease of pH caused by N addition was accompanied by decrease of soil arylsulfatase activity
(Figures 2 and 4k). In other words, whether arylsulfatase activity changes following N addition depends
on N-induced change of soil pH, which is largely determined by soil buffering capacity, the ability of soils
to resist change in pH [Bowman et al., 2008]. Soil buffering capacity is mainly controlled by carbonate
when soil pH is greater than 7.5, while by base cations such as exchangeable Ca, Mg, and K in the pH
range from 4.5 to 7.5 [Tian and Niu, 2015]. Soil pH values ranged from 4.1 to 8.1 with an average of
6.2 (data not shown), so exchangeable cations were likely the major determinant on soil buffering capa-
city and pH variation in the current study. Consistently, we found that higher exchangeable Ca level and
thus higher soil buffering capacity resulted in less N-induced decrease of soil pH under conditions of ele-
vated N inputs (Figures S3 and 5). Due to the strong regulation of change of soil pH on the response of
arylsulfatase activity to N addition, exchangeable Ca in turn indirectly controls N-induced change of aryl-
sulfatase activity via its control on soil buffering capacity (Figure 5). In contrast, the regulation of soil pH,
SOC, and TN on responses of arylsulfatase activity to N addition was largely apparent, since exchangeable
Ca was also found to be the strongest controller of these variables in our study (Figure S4). This was
supported by the path analysis, which showed no significant effects of these variables on change of
arylsulfatase activity (Figure 5).

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Acknowledgments
This work was funded by the National
Natural Science Foundation of
China (31500405), the National Key
Basic Research Program of China
(2015CB452703), the Chinese
Academy of Sciences through its One-
Hundred Talent Program to Dejun Li
(Y523101030 and Y351025090), and the
Chinese Academy of Sciences through
its “Light of West China” Program to
Hao Chen. Additional information is
presented in the supporting informa-
tion as four tables and five figures. The
data are available from the authors
upon request (dejunli@isa.ac.cn).
CHEN ET AL.
10.1002/2016GB005423
Changes of soil phosphate level after N addition may also contribute to the decrease of soil arylsulfatase
activity. Previous evidence showed that soil phosphate inhibited arylsulfatase activity [Al-Khafaji and
Tabatabai, 1979; Tabatabai and Bremner, 1970], probably because phosphate can form a covalent bond with
the active site of 3-oxoalanine [Chruszcz et al., 2003]. Negative relationship between soil AP and arylsulfatase
activities was found for the control (Table S4), which supports that an increase of soil AP level inhibits arylsul-
fatase activity. Nonetheless, a significant correlation was not found between changes of AP levels and
changes of arylsulfatase activities in the present study, although soil AP levels were significantly enhanced
after N addition (Figure 2b). The possible reason for the lack of significant correlation may be due to the
weaker regulation of soil AP than soil pH on arylsulfatase activity.
Our findings indicate that elevated N deposition may slow down soil S cycling by inhibiting soil organic
S mineralization. This may result in S deficiency of ecosystems experiencing elevated N deposition.
Considering the important role of S in plant growth, S deficiency may have huge implications in the
responses of ecosystems to N deposition. It has been well documented that N deposition would impact eco-
system structure and function in many aspects, including decrease of biodiversity, alteration of community
composition, damage of plant health, and reduction of ecosystem productivity [Phoenix et al., 2012]. The
underlying mechanisms for these impacts have been attributed to depletion of Ca, Mg, and other base
cations, enhanced mobility of toxic metals (e.g., Al3+ and Mn2+), which are induced by soil acidification
[Bowman et al., 2008]. No studies have been conducted to evaluate whether S deficiency would occur and
how S deficiency would impact ecosystem structure and function with N enrichment. Since sign of sup-
pressed S cycling revealed in our study and since N deposition-induced decrease of pH is occurring globally
[Tian and Niu, 2015], it is imperative to investigate the possible impacts of elevated N deposition on ecosys-
tem S cycling, especially for the ecosystems with low soil buffering capacity, where the impact of N inputs on
S cycling may be stronger. This was supported by our incubation experiment, showing that N addition led to
greater decrease of arylsulfatase activity in the noncalcareous soils over clasolite than in the calcareous soils
over limestone (Figure 3), due to lower exchangeable Ca level and thus soil buffering capacity in soils over
clasolite (Table S2).
Although the results from field and incubation experiments are comparable and consistently support
N-induced decrease of S cycling (Figure 3b), interpretation of the findings of the current study should be care-
ful largely due to inherent limitations for incubation study, including alteration of soil aggregates, elimination
of labile C inputs to soil by exclusion of plants, potential limitation of microbes by carbon, and nitrogen avail-
ability during incubation. However, change of arylsulfatase activity was estimated by comparing the control
and treatment. If the inherent limitations of incubation occurred, their effects should be similar for the control
and treatment. In this sense, impacts of the inherent limitations of incubation on the findings were likely mild
in the current study. Nevertheless, considering the limited data available, more studies are undoubtedly
needed in order to derive the general patterns of soil S cycling responses to N inputs. Furthermore, based
on our analysis, suppression of S cycling was mainly due to decrease in soil pH. Therefore, both N and S
deposition have the potential to inhibit S cycling by decreasing soil pH. This is partly supported by the only
study which includes both N with S addition experiment [Hu et al., 2013]. Nevertheless, it should be noted
that S deposition may not only decrease soil pH but also increases soil S level. Whether the combination of
decrease in soil pH and increase in soil S level induced by S addition may exacerbate the negative effect of
soil pH decrease alone on S cycling need further investigation.
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