Science of the Total Environment 615 (2018) 1535–1546

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Soil acid cations induced reduction in soil respiration under nitrogen

enrichment and soil acidification
Yong Li a,b, Jian Sun a, Dashuan Tian a, Jinsong Wang a, Denglong Ha c, Yuxi Qu c, Guangwei Jing c, Shuli Niu a,d,⁎
a
Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China
b
Beijing Key Laboratory of Wetland Services and Restoration, Institute of Wetland Research, Chinese Academy of Forestry, Beijing 100091, China
c
Jigongshan Natural Reserve, Xinyang 464000, China
d
Department of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Nitrogen enrichment and soil acidifica-

tion significantly decreased soil respira-
tion and its components.
• Increased concentration of acid cations
(H+ and Al3+) resulted in decreased Ra.
• Reduction of Rh was attributable to sup-
pressed cellulose degrading enzymes
activity.
• SEM model demonstrated that soil acid-
ification played more important role
than N enrichment in affecting Ra and
Rh.

a r t i c l e i n f o a b s t r a c t

Article history: Atmospheric nitrogen (N) deposition and soil acidification both can largely change soil microbial activity and root
Received 14 July 2017 growth with a consequent impact on soil respiration (Rs). However, it remains unclear which one, N enrichment
Received in revised form 13 September 2017 or soil acidification, plays more important role in impacting soil respiration. We conducted a manipulative exper-
Accepted 13 September 2017
iment to simulate N enrichment (10 g m−2 yr−1 NH4NO3) and soil acidity (0.552 mol H+ m−2 yr−1 sulfuric acid)
Available online 18 September 2017
and compared their effects on Rs and its components in a subtropical forest. The results showed that soil pH was
Editor: Jay Gan reduced by 0.4 similarly under N addition or acid addition after 3 years' treatment. Acid addition decreased au-
totrophic respiration (Ra) by 22–35% and heterotrophic respiration (Rh) by 22–23%, resulting in a reduction of
Keywords: Rs by 22–26% in the two years. N addition reduced Ra, Rh, Rs less than acid addition did. The reductions of Rs
Autotrophic respiration and its components were attributed to increase of soil acid cations and reduction of cellulose degrading enzymes
Heterotrophic respiration activity. N addition and soil acidification significantly enhanced fungal to bacterial ratio. All the cellulose
Nitrogen deposition degrading enzymes were reduced more by soil acidity (43–50%) than N addition (30–39%). The principal compo-
Soil acidification nent scores of degrading enzymes activity showed significantly positive relationships with Rh. Structural equa-
Microbial community composition
tion model showed that soil acidification played more important role than N enrichment in changing Rs and its
Cellulose degrading enzymes
components. We therefore suggest that soil acidification is an important mechanism underlying soil respiration
changes, and should be incorporated into biogeochemical models to improve the prediction of ecosystem C cy-
cling in the future scenarios of anthropogenic N deposition and acid enrichment.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of
Sciences, Beijing 100101, China.
E-mail address: sniu@igsnrr.ac.cn (S. Niu).

http://dx.doi.org/10.1016/j.scitotenv.2017.09.131
0048-9697/© 2017 Elsevier B.V. All rights reserved.
1536 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546
1. Introduction
Anthropogenic nitrogen (N) input, originating mainly from fossil-fuel
combustion and excessive N fertilizer application, has increased N10-fold
in the last 150 years and is predicted to increase another twofold or
threefold in the next decades (Galloway and Cowling, 2002; Gruber
and Galloway, 2008; Vitousek et al., 1997). The large amount of N depo-
sition has dramatically altered ecosystem functions, with particular
impacts on ecosystem carbon (C) cycling by providing more N nutrient
and stimulating plant C uptake (Liu and Greaver, 2010; Xia and Wan,
2008; Yue et al., 2016). Effects of nitrogen deposition on C and N cycling
processes have been extensively studied in terrestrial ecosystems by
Nitrogen Saturation Experiment (NITREX), Experimental Manipulation
of Forest Ecosystem in Europe (EXMAN), and The European Nitrogen As-
sessment in Europe (Butterbach-Bahl and Gundersen, 2011; Wright and
Rasmussen, 1998) and Harvard forest experiment in North America
(Bowden et al., 2004; Magill et al., 2004). Meanwhile, hotspot of sulfur
(S) deposition in Asia could cause soil acidification (Bowman et al.,
2008; Duan et al., 2016; Guo et al., 2010; Vet et al., 2014), leading to
the leaching of base cations (Tian et al., 2015), which may negatively
affect plant growth and thus ecosystem C cycle as well (Bobbink et al.,
2010). However, there remains a major gap in our integrated under-
standing about how N deposition and soil acidification play differential
roles in affecting ecosystem C cycling.
Soil respiration (Rs) is one of the largest C effluxes between terrestrial
ecosystems and atmosphere and plays an important role in regulating
the atmospheric CO2 concentration (Davidson et al., 2002; Kuzyakov,
2006). Soil respiration consists of autotrophic respiration (Ra) and
heterotrophic respiration (Rh), which mainly derives from plant roots
and decomposition of litter and soil organic matter, respectively
(Kuzyakov, 2006; Luo and Zhou, 2006). On average across all the studies
in forests, Rh and Ra declined by 15% and 21%, respectively, under N ad-
dition based on a global meta-analysis (Janssens et al., 2010; Zhou et al.,
2014), which suggest differential sensitivity and regulation mechanisms
of root and microbial processes. In specific, N enrichment could induce
ammonium toxicity (Van Den Berg et al., 2005), acid cation toxicity
(Tian et al., 2015) and loss of base mineral cations (Bowman et al.,
2008), and thus decrease belowground C allocation (Phillips and
Fahey, 2007), leading to reduction in Ra in some ecosystems with sensi-
tive root response. Meanwhile, several microbial mechanisms were pro-
posed in accounting for decreased Rh in some ecosystems that have
sensitive microbial response (Riggs and Hobbie, 2016). First, N addition
could cause microbial community composition shift towards dominance
by microorganisms that utilizing C more effectively for growth (Schimel
and Weintraub, 2003). Model studies have predicted that N addition
would induce more C to be allocated to microbial growth rather than mi-
crobial respiration, resulting in increases in carbon use efficiency (CUE)
and reduction in Rh under N addition (Schimel and Weintraub, 2003;
Thiet et al., 2006). This increase of CUE might result from microbial com-
munity shift from low CUE community to high CUE counterparts (e.g.
increase of fungi: bacteria ratio could increase microbial CUE) (Riggs
and Hobbie, 2016). Second, N addition could reduce Rh by decreasing mi-
crobial biomass (Liu and Greaver, 2010; Treseder, 2008), which is due to
microbial physiology change by lower soil pH (Riggs and Hobbie, 2016)
and microbial growth limitation by loss of base cations (e.g., Mg2 +,
Ca2+) (Bowman et al., 2008) and soil acid cations (e.g., Al3+) toxicity
(Tian and Niu, 2015). Finally, N addition could directly inhibit cellulose
degrading enzymes, leading to reduction in Rh (Edwards et al., 2011;
Keeler et al., 2009). Although those mechanisms are speculated
previously, few study comprehensively evaluates different regulation
pathways of N enrichment on Ra or Rh in forest ecosystems.
Soil acidification plays an important role in affecting Rs by changing
soil chemistry and biotic communities in forest ecosystem (Högberg et
al., 2006; Liang et al., 2016; Oulehle et al., 2011). Previous studies have
proposed that soil acidification may suppress plant and microbial com-
munities by increasing soil H+ and Al3 + concentration (Chen et al.,
2013; Rousk et al., 2009; Rousk et al., 2011). Other studies have reported
that soil acidification has negative effects on soil mineralization (Aciego
Pietri and Brookes, 2008), the concentration of base cations (Bowman et
al., 2008), plant and microbial communities (Chen et al., 2015b; Rousk
et al., 2010; Stevens et al., 2010) and thus reduces soil respiration
(Evans et al., 2012; Oulehle et al., 2011). The above-mentioned biotic
mechanisms have largely been studied separately in different case stud-
ies, the relative contributions of these mechanisms or pathways to N-
and/acid-induced changes in Rs, however, have not been explicitly stud-
ied yet with field experiments.
Subtropical forests in China take up large amount of carbon dioxide,
largely due to that N deposition stimulates tree growth (Yu et al., 2014).
Meanwhile, high S deposition was found in eastern China (Duan et al.,
2016). However, we are not clear on the responses of soil respiration to
N enrichment and soil acidification in this region, even less on the
underlying mechanisms. In this study, we specifically addressed the
following questions: (1) how soil respiration and its components, Ra and
Rh, respond to N enrichment and soil acidification? And which one, N
addition or soil acidification, plays more important role in impacting soil
C cycle? (2) how soil biotic and abiotic factors control the responses of
Rs, Ra and Rh to N deposition and soil acidification? (3) what are the mech-
anism underlying Rs in response to N deposition and soil acidification?
2. Materials and methods
2.1. Site description and experimental design
This study was conducted at the Nature Reserve of Jigong Mountain
(31°51′58″N, 114°5′12″E), which is located in southern Henan Province,
central China. It is located within a climate-transitional region from
northern subtropical climate to warm temperate climate. The mean an-
nual surface air temperature at the reserve is 15.2 °C with the highest
and lowest monthly mean air temperatures being 27.5 °C in July and
1.9 °C in January, respectively. The mean annual rainfall was
1118.7 mm. The rock formations of Jigongshan are composed of
migmatitic granite and gneiss belonging to Early Precambrian Period.
The soil type belongs to the yellow-brown soil with layer thickness
0.3–0.6 m (Yan et al., 2014). The forest type in study area is deciduous
oak mixed forest, with Quercus acutissima and Q. variabilis as dominant
species in the canopy layer, and Liquidambar formosana, Lindera glauca,
Viburnum dilatatum as understory arborous layer. The stand is second-
ary forest with an age of about 50 years. The initial soil chemistry before
N and A addition treatments was listed in Table S1.
An experimental manipulation of N and acid additions was conducted
with a complete randomized block design. Four replicate blocks were
established in July 2013. Each block had four treatments which were ran-
domly assigned to 10 m × 10 m plots. Each plot was surrounded by a 10 m
buffer strip to the next plot. The treatments included control (CK, without
nitrogen and acid addition), N addition (N, 10 g m−2 yr−1 NH4NO3),
acid addition (A, 0.552 mol H+ m−2 yr−1 sulfuric acid) and N plus acid
addition (NA, 10 g m−2 yr−1 NH4NO3 + 0.552 mol H+ m−2 yr−1 sulfuric
acid). The additions were dissolved in 50 L water for each plot and were
sprayed onto the forest floor using a backpack sprayer monthly from
May to October each year. The control plot also received 50 L water
(equivalent to 0.5 mm precipitation) each time, and this could be
negligible due to the high mean annual precipitation of 1118.7 mm.
2.2. Soil respiration measurements
Three soil respiration collars (PVC tubes, 20 cm inner diameter and
8 cm height) were installed into the ground to 5 cm depth in each
plot for soil respiration measurements in July 2013. Trenching method
was used to separate soil respiration (Rs) into autotropic respiration
(Ra) and heterotropic respiration (Rh), which is widely used in previous
studies (Hanson et al., 2000; Kuzyakov, 2006; Sayer and Tanner, 2010).
Specifically, in July 2013, one subplot (1 m × 1 m) was trenched to 0.6 m
 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546
depth in each plot and pieces of thick polyethylene board were inserted
into the inner side of trenches to prevent roots from growing into the
subplots. Plants in the subplots were cut at the soil surface in order to
kill roots in the trenched subplots. Disturbance inside the subplots
was avoided as far as possible during trenching. In July 2014, soil cores
(3.5 diameter, 0.6 m depth) were collected from trenched subplots
and we found no visual living roots (0.6 m depth).
Three PVC tubes were also installed into each trenched subplot and
all the measurements of Rs, soil moisture and temperature at 5 cm
depth were conducted between 8:00 and 14:00 using a LiCor-8100
soil CO2 flux system (LI-COR, Lincoln, USA). The SR in the trenched sub-
plot excluded root respiration thus represents Rh (Kuzyakov, 2006).
2.3. Soil properties
Five soil cores (3.5 cm diameter) were collected randomly from each
plot at 0–15 cm depth and mixed to one composite sample in October
2014 and 2015. The samples were passed through a 2 mm sieve and di-
vided into two parts. One part of fresh soil was used for the analysis of
−
ammonium (NH+ 4 ), nitrate (NO3 ), microbial biomass carbon (MBC),
and dissolved organic carbon (DOC). The other part was air dried for
the determination of soil pH, available phosphorus and extractable cat-
−
ions (Al3+, Ca2+, Mg2+, and Na+). Soil NH+ 4 and NO3 concentrations
were determined by extraction with 2 M KCl solution followed by color-
imetric analysis on a FIAstar 5000 Analyzer (FIAstar 5000 Analyzer, Foss
Tecator, Hillerød, Denmark). Soil MBC was estimated by using a chloro-
form fumigation extraction method (Brookes et al., 1985). Soil DOC was
extracted by adding 50 mL of 0.5 M potassium sulfate to subsamples of
12.5 g homogenized soil, and by agitating on an orbital shaker at
120 rpm for 1 h. The filtrate was analyzed using a TOC analyzer (multi
N/C 3100, Analytik Jena, Germany). The soil pH was determined in a
1:2.5 soil:water solution (w/v). Available phosphorus was determined
using a spectrophotometer (UV2550, Shimadzu, Japan). The extractable
cations (Al3+, Ca2+, Mg2+, and Na+) were measured using a modified
extraction procedure (Rauret et al., 2000).
2.4. Microbial community structure
Phospholipid fatty acid (PLFA) analysis was used to evaluate soil mi-
crobial community composition. Phospholipid fatty acid was extracted
from the soil as described by Bossio et al. (1998). The extracted fatty
acid methyl esters were separated and quantified with a gas chromato-
graph (Agilent 6890, Agilent Technologies, Palo Alto, CA, USA) and iden-
tified by the MIDI Sherlock Microbial Identification System (MIDI Inc.,
Newark, DE, USA). Fatty acids were quantified by calibration against
internal standard (19:0 nonadecanoic methyl ester). Individual lipids
were used as biomarkers to indicate different microbial groups, with
i14:0, i15:0, a15:0, i16:0, 10Me16:0, i17:0, a17:0, 16:1ω7c, 17:1ω8,
18:1ω9, 18:1ω7c, cy17:0 and cy19:0 as bacteria, 18:2ω6,9 as fungi,
10Me 16:0, 10Me17:0 and 10Me 18:0 as actinobacteria (Frostegård
and Bååth, 1996; Frostegård et al., 2011).
2.5. Soil enzyme activity
Soil cellulose degrading enzymes, including β-D-Cellubiosidase (CB),
β-Glucosidase (BG), α-Glucosidase (AG) and β-Xylosidase (XYL), were
measured using fluoremetric methods described by Wallenstein et al.
(2009). Briefly, 1 g of fresh soil was homogenized with 250 mL of
50 mM acetate buffer (pH = 5) in blender. Then 800 μL of sample slurry
was added into 96-well microplate which contained 200 μL of 200 μM
substrate (4-methylumbelliferyl, MUB) for enzyme activity measure-
ment. Quench standard was created with 200 μL of MUB solution and
800 μL of sample slurry. Negative control received substrate solution
plus acetate buffer. Reference standard receive MUB solution and ace-
tate buffer. We used six analytical replicates for each soil sample. The
assay plates were incubated in the dark at 25 °C for 3 h and fluorescence
1537
was measured using a SynergyMX microplate reader (Biotek, VT, USA)
365 nm excitation and 450 nm emission filters. After correcting for
negative controls and quenching, enzyme activity was expressed as
nmol g soil dry weight−1 h−1.
2.6. Statistical analysis
Structural equation modeling (SEM) was performed using AMOS 7.0
(Amos Development, Spring House, Pennsylvania, USA) to analyze the
hypothetical pathways of N addition and soil acidification effects on
soil respiration components. Prior to SEM analysis, soil N availability
−
(NH+ +
4 and NO3 ), soil acid cations (H and Al
3+
), microbial community
composition (bacterial, fungal, and actinobacterial PLFAs) and cellulose
degrading enzymes activity (AG, BG, CB, and XYL) were subject to
principal component analysis (PCA) procedure to reduce the number
of variables (Chen et al., 2013). The first principal components (PC1)
were used in the subsequent SEM analysis to represent soil N availabil-
ity, soil acidity, soil microbes and cellulose-degrading enzymes (Table
S2). Data were fitted to the models applying the maximum likelihood
estimation method. Adequacy of the models was determined by using
χ2 tests, Akaike information criteria (AIC), and root square mean errors
of approximation (RMSEA) (Wei et al., 2013). Because there were no
significant interactions between year and treatments on the variables
(Rs, Ra, and Rh), we pooled the two years' data together in the SEM.
PCA was performed using CANOCO version 4.5 (Plant Research Interna-
tional, Wageningen, The Netherlands).
Analysis of variance (ANOVA) was performed using the SPSS 15.0
software package (SPSS, Chicago, IL, USA). Mixed linear models across
all response variables were conducted with N and acid addition as
fixed effects and block as random effects. Repeated-measure ANOVA
with Duncan's multiple-range tests were was performed to examine
the main and interaction effects of N, A and the measurement times
on the differences of Rs and its components in 2014 and 2015. Between-
subject effects were evaluated as N or A treatment; within-subject
effects were time of seasonal variation under four treatments. Two-
way ANOVA was performed with growing season mean soil respiration,
soil microbial composition and cellulose degrading enzymes activity to
determine the main and interactive effects of N enrichment and soil
acidification in 2014 and 2015, respectively. Three-way ANOVA was
conducted to determine effects of year, N enrichment, soil acidification
and interactions on soil properties, fine root biomass, microbial compo-
sition, cellulose degrading enzymes and soil respiration in 2014 and
2015 (Table S3).
3. Results
3.1. Soil properties and fine root biomass
Across the three years, acid (A) addition, nitrogen (N) addition and
nitrogen plus acid (NA) addition didn't statistically change soil moisture
(Fig. S1). Soil dissolved organic carbon (DOC) was significantly reduced
by 12.6% and 10.1% under A addition in 2014 and 2015, respectively;
while neither N addition nor NA treatment significantly changed DOC
−
(Table 1). Soil inorganic N concentration, NH+ 4 and NO3 , increased
under N addition (Table 1), but decreased (P b 0.05 for NO− 3 and P N
0.05 for NH+ +
4 ) under A addition, and increased (P b 0.05 for NH4 and
−
P N 0.05 for NO3 ) under NA addition (Table 1).
Soil pH significantly decreased by 0.2, 0.2 and 0.4, respectively,
under A, N and NA addition in 2014 and by 0.4, 0.4 and 0.6, respectively
in 2015 (Table 1). Soil Al3+ was increased by 38.7%, 32.1% and 44.8%, re-
spectively, under A, N, and NA addition in 2014 and by 61.4%, 57.4% and
75.2%, respectively in 2015 (Table 1). Acid addition, N addition and NA
addition decreased concentration of Ca2+, but did not change concen-
trations of Mg2+ and Na+ in 2014 and 2015 (Table 1). Fine root biomass
was reduced by 10.6% and 11.1% under A addition, and by 10.2% and
14.4% under NA addition, in 2014 and 2015, respectively, but didn't
1538 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546

Table 1
Soil available carbon and nitrogen, soil cations and plant fine root biomass under different treatments. Values are means of four replicate block with SE for each treatment. Different letters
within the same year indicate significant differences among the four treatments (P b 0.05). CK = Control, A = acid addition, N = nitrogen addition, NA = nitrogen and acid addition.

Year CK A N NA

Available C & N
DOC (mg kg−1) 2014 299.16 ± 8.28 a 261.41 ± 11.47 b 281.58 ± 6.10 ab 297.59 ± 8.12 a
2015 294.81 ± 2.79 a 265.07 ± 0.96 b 288.86 ± 5.46 a 288.29 ± 2.32 a
−1
MBC (mg kg ) 2014 571.73 ± 3.78 a 526.89 ± 4.63 b 557.11 ± 3.48 b 549.82 ± 4.37 b
2015 563.41 ± 15.04 a 504.90 ± 1.83 b 545.02 ± 10.30 a 559.78 ± 4.51 a
−1
NH+
4 (mg kg ) 2014 8.08 ± 0.2 b 7.83 ± 0.35 b 10.91 ± 0.59 a 10.59 ± 0.49 a
2015 7.67 ± 0.30 b 7.57 ± 0.57 b 9.44 ± 0.28 a 9.89 ± 0.69 a
NO−
3 (mg kg
−1
) 2014 38.36 ± 0.92 b 28.96 ± 1.02 c 42.44 ± 1.12 a 39.32 ± 0.92 ab
2015 40.13 ± 2.63 a 30.21 ± 0.92 b 43.06 ± 3.99 a 44.95 ± 2.28 a
Soil cations
Soil pH 2014 4.72 ± 0.04 a 4.52 ± 0.03 b 4.52 ± 0.07 b 4.44 ± 0.06 b
2015 4.63 ± 0.08 a 4.21 ± 0.03 b 4.21 ± 0.04 b 4.02 ± 0.05 c
3+ −1
Al (mmol kg ) 2014 37.99 ± 1.03 b 52.69 ± 2.16 a 50.17 ± 1.00 a 55.02 ± 1.99 a
2015 38.36 ± 3.22 c 61.90 ± 1.45 ab 60.39 ± 0.40 b 67.22 ± 0.82 a
Ca2+ (mmol kg−1) 2014 33.62 ± 3.09 a 23.13 ± 1.14 b 25.14 ± 1.77 b 24.41 ± 2.87 b
2015 30.27 ± 2.90 a 18.85 ± 0.64 b 20.10 ± 0.53 b 19.39 ± 0.46 b
Mg2+ (mmol kg−1) 2014 4.71 ± 0.38 a 3.73 ± 0.28 a 4.31 ± 0.16 a 4.23 ± 0.28 a
2015 4.16 ± 1.01 a 3.29 ± 0.37 a 3.48 ± 0.58 a 3.01 ± 0.10 a
Na+ (mmol kg−1) 2014 1.53 ± 0.18 a 1.32 ± 0.13 a 1.44 ± 0.04 a 1.38 ± 0.05 a
2015 1.22 ± 0.24 a 1.06 ± 0.15 a 1.32 ± 0.35 a 1.38 ± 0.21 a
Plant
Fine root biomass (g m−2) 2014 174.95 ± 2.40 a 156.36 ± 2.67 b 177.62 ± 2.52 a 157.06 ± 1.71 b
2015 181.35 ± 4.17 a 161.23 ± 0.64 b 175.07 ± 4.02 a 155.22 ± 2.25 b

significantly change under N addition in either 2014 or 2015 (Table S3).
Microbial biomass carbon (MBC) was decreased by 7.8%, 2.6% and 3.8%,
respectively, under A, N, and NA addition in 2014 and by 10.4% (P b
0.05), 3.3% (P N 0.05) and 0.6% (P N 0.05) in 2015, respectively (Table 1).
3.2. Soil respiration and its components
The temporal dynamics of soil respiration was consistent with the
seasonal pattern of soil temperature in both years, in which the maxi-
mum rates of soil respiration occurred in July and August (Fig. 1). In
2014 and 2015, A addition decreased the growing season mean Rh by
23% and 22%, and also reduced Ra by 22% and 35%, resulting in a signif-
icant decrease in Rs by 22% and 26%, respectively (Fig. 2, Table S3).
N addition significantly decreased Rs, Rh and Ra by 15%, 11% and 22%,
respectively on average across the two growing seasons (Fig. 2, Table
S3). In 2014, a 16% reduction of Rh and a 22% reduction of Ra led to a
significant decrease of 19% in Rs (all P b 0.05). However, Rs under N
addition slightly reduced by 12% (P N 0.05) in 2015, due to a significant re-
duction in Ra (22%) but little change in Rh (P N 0.05, Fig. 2). There were sig-
nificant interactive effects between A addition and N addition treatments
on Rs, Rh and Ra in 2014 (P b 0.001) but not in 2015 (P N 0.05, Fig. 1).
Over the growing season, mean Rh/Rs ratio in the control plot was
58% in 2014 and 61% in 2015 (Fig. 2). NA addition increased the ratio
of Rh/Rs to 63% but either A addition or N addition did not change the
ratio of Rh/Rs (Fig. 2) in 2014. However, A addition, N addition and NA
addition increased the Rh/Rs ratio to 64%, 65% and 67%, respectively, in
2015 (Fig. 2).
3.3. Soil microbial community composition and cellulose-degrading
enzymes
N addition, A addition and NA addition did not significantly change
bacterial, fungal and actinobacterial PLFA in 2014 and 2015 (Fig. 3a–c,
e–g, Table S3). N addition increased fungi: bacterial ratio (Fig. 3d), and
A addition did not significantly change it (P = 0.38, Fig. 3d), but the in-
teraction of N and A addition was significant (Fig. 3d) in 2014. N addi-
tion and A addition increased fungi: bacterial ratio (Fig. 3h), but the
interaction of N and A addition was not significant (Fig. 3h) in 2015.
All the three treatments did not change α-Glucosidase (AG) activity
(Fig. 4a, e). In 2014, A addition significantly reduced β-Glucosidase (BG)
activity but N addition did not (Fig. 4b). Neither N nor A addition
changed BG activity in 2015 (Fig. 4f). β-D-Cellubiosidase (CB) activity
was significantly decreased by 43%, 30% and 39% under A, N, and NA ad-
dition, respectively in 2014 (Fig. 4c), and by 32%, 29%, and 34%, respec-
tively in 2015 (Fig. 4g, Table S3). Acid addition significantly suppressed
β-Xylosidase (XYL) activity by 50% but N addition did not significantly
change it in 2014 (Fig. 4d). Both N and A addition significantly
decreased XYL activity in 2015 (Fig. 4h).
3.4. Pathways determining the responses of soil respiration and its
components
Structural equation model explained 77% of the variation in soil N
availability, 56% of the variation in soil acid cations, 85% of the variation
in fine root biomass, 27% of the variation in soil microbial community
composition, 61% of the variation in cellulose degrading enzymes, 65%
of the variation in soil autotrophic respiration (Ra) and 44% of the
variation in soil heterotrophic respiration (Rh), respectively (Fig. 5). N
addition elevated soil N availability and soil acid cations, shifted soil
microbial community composition and consequently decreased cellu-
lose-degrading enzymes activity, but did not affect fine root biomass.
The pathway of soil acidification directly increased soil acid cations
(H+ and Al3+) and decreased soil N availability, plant fine root biomass
and cellulose-degrading enzyme activities.
Sixty five percent of the total variance in Ra was directly explained by
soil N availability, soil acid cations and fine root biomass, but only soil
acid cation pathway was significant (pathway coefficient = −0.78, P
b 0.05, Fig. 5). Linear regression revealed that principal component
(PC1) scores of acid cations was significantly correlated with Ra (R2 =
0.64, P b 0.001, Fig. S2a), while that of cellulose degrading enzymes
was significantly correlated with Rh (R2 = 0.39, P b 0.001, Fig. S2b).
Forty four percent of the total variance in Rh was directly explained by
soil N availability (pathway coefficient = 0.34, P b 0.05) and cellulose
degrading enzymes (pathway coefficient = 0.50, P b 0.05, Fig. 5). More-
over, soil acid cations significantly altered soil microbial community
composition (pathway coefficient = 0.75, P b 0.05). There was a signif-
icant correlation between soil microbial community composition and
cellulose degrading enzymes (R2 = 0.46, P b 0.05).
Standardized total effects from SEM showed that soil acidification
had stronger effects on Ra and Rh compared to N enrichment (Fig. 6).
Specifically, acid cations showed the most powerful negative effect on
Ra. Cellulose degrading enzymes had stronger positive effect on Rh
 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546 1539

Fig. 1. Seasonal variability of soil respiration (Rs), heterotrophic soil respiration (Rh) and autotrophic soil respiration (Ra) under four different treatments from 2014 to 2015. Vertical bars
represent the standard error of the mean (n = 4). CK, control plots; A, acidification plots; N, N addition plots; NA, both nitrogen and acid addition plots. Repeated-measure ANOVA was
conducted to determine the effects of N enrichment and soil acidification on soil respiration and its components.

than soil N availability and acid cations exerted negative effect on Rh
(Fig. 6). The standardized total effect of soil acidification on cellulose
degrading enzymes was higher than N enrichment and only acid cations
exerted negative effect on cellulose degrading enzymes (Fig. S3). Linear
regression revealed that PC1 scores of cellulose degrading enzymes was
positively correlated with PC1 scores of acid cations (R2 = 0.42, P b
0.001, Fig. S4).
4. Discussion
4.1. Different roles of N enrichment and soil acidification in affecting soil
properties, fine root biomass and microbial composition
In line with previous studies (Wang et al., 2017; Wei et al., 2013),
−
soil NH+ 4 and NO3 were increased under N enrichment (Tables 1, S3).
Meanwhile, N enrichment increased soil acid cations (Al3+) by 44.7%
and decreased Ca2 + concentration by 29.4% on average across two
growing seasons (Tables 1, S3). Although N enrichment increased soil
N availability, it had no significant effect on fine root biomass (Tables
1, S3), probably due to the occurrence of N saturation in this forest
(Tian et al., 2016). We found that N enrichment significantly shifted mi-
crobial community composition (i.e. fungi: bacterial ratio), which is
consistent with previous studies (Chen et al., 2015a; D. Chen et al.,
2016). However, N enrichment significantly decreased cellulose
degrading enzymes activity (Fig. 4). The results are in contrast with
the previous studies, which showed that added N stimulated soil
cellubiosidase (CB) and β-Glucosidase (BG) activity (Carreiro et al.,
2000; J. Chen et al., 2016; Saiya-Cork et al., 2002). This stimulation
may be attributable to the increase of C acquiring enzymes (cellulose
degrading enzymes) resulted from the increase of microbial demand
1540 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546

Fig. 2. Growing season mean total soil respiration (Rs), heterotrophic soil respiration (Rh), autotrophic soil respiration (Ra), and heterotrophic soil respiration contribution to soil
respiration (Rh/Rs, %) under four treatments in 2014 and 2015. Vertical bars represent the standard error of the mean (n = 4). CK, control plots; A, acidification plots; N, N addition
plots; NA, both nitrogen and acid addition plots. Two-way ANOVA was conducted to determine the effects of N enrichment and soil acidification on soil respiration and its components.
 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546 1541

Fig. 3. Responses of soil microbial community to nitrogen and/or acid additions in 2014 and 2015. Vertical bars represent the standard error of the mean (n = 4). CK, control plots; A, acidification
plots; N, N addition plots; NA, both nitrogen and acid addition plots. Two-way ANOVA was conducted to determine the effects of N enrichment and soil acidification on soil microbial community.
1542 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546

Fig. 4. Responses of cellulose degrading enzyme activity to nitrogen addition and acid additions in 2014 and 2015. Vertical bars represent the standard error of the mean (n = 4). CK,
control plots; A, acidification plots; N, N addition plots; NA, both nitrogen and acid addition plots. Two-way ANOVA was conducted to determine the effects of N enrichment and soil
acidification on cellulose degrading enzymes activity.
 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546 1543

Fig. 5. Structural equation model considered plausible pathways through which N enrichment and soil acidification influence soil autotrophic respiration (Ra) and heterotrophic
−
respiration (Rh) using data in 2014 and 2015. Prior to SEM analysis, soil N availability (NH+ +
4 and NO3 ), soil acid cations (H and Al
3+
), microbial community composition (bacterial,
fungal, and actinobacterial PLFAs) and cellulose degrading enzymes (AG, BG, CB, and XYL) were subject to principal component analysis (PCA) procedure to reduce the number of
variables. Black and red arrows represent significant positive and negative pathways, respectively, and grey dashed arrows indicate nonsignificant pathways. Numbers at arrows are
standardized path coefficients and arrow width is proportional to the strength of the relationship. R2 values on top of response variables indicate the proportion of variation explained
by relationships with other variables. The final results of model fitting: χ2 = 16.24, df = 14, P = 0.30, root mean square error of approximation (RMSEA) = 0.44, Akaike information
criteria (AIC) = 266.28.

for C under N addition, especially in N-limited ecosystems (Keeler et al.,
2009). However, the effect of N addition on enzyme activity is not al-
ways consistent due to site-specific variations in nutrient availability
(Keeler et al., 2009). Some studies demonstrated that experimental N
deposition leads to lower soil CB activity due in part to reduced micro-
bial biomass, low pH and soil moisture (Edwards et al., 2011; Keeler et
al., 2009). Finally, N enrichment increased soil N availability and acid
cations, shifted soil microbial composition and decreased cellulose
degrading enzymes in this forest.
Our results demonstrated that soil acidification increased soil acid
cations (H+ and Al3+), and decreased soil N availability, fine root bio-
mass, and cellulose degrading enzymes (Table 1, Fig. 5). First, high con-
centration of H+ could directly alter soil N availability for plant and
microbial growth (Aciego Pietri and Brookes, 2008; Van Den Berg et
−
al., 2005). In this study, the significant decline of NH+ 4 and NO3 concen-
tration was observed under A addition treatment, leading to significant
reduction in fine root biomass and microbial biomass (Table 1, Fig. 5).
Second, soil acidification could lead to loss of soil base cations (Rousk
et al., 2010; Van Den Berg et al., 2005). In this study, soil acidification in-
duced significant decrease in Ca2+ concentration and fine root biomass
but slightly declined Mg2+ and Na+ concentrations (Table 1). Finally, in
contrast to the previous studies (Chen et al., 2013), soil acidification had
no significant effects on fungal, bacterial and actinobacterial PLFA but
increased the fungi: bacterial ratio in the two growing seasons (Fig. 3,
Table S3). This increase of fungi: bacterial ratio indicates that soil acidi-
fication induced shifts in the composition of the microbial community
decrease the soil enzyme activities and hence the decomposition of lit-
ter and SOM (Franklin et al., 2003; Waldrop and Zak, 2006). The signif-
icant decreases in cellulose degrading enzyme activities (i.e. CB, BG and
XYL enzymes) are in concert with increase of H+ and Al3+ concentra-
tion, suggesting that soil acid cations directly decreased cellulose
degrading enzyme activities (Fig. S4). Overall, our results suggest that
soil acidification decreased fine root biomass and cellulose degrading
enzymes activities mainly by increasing soil acid cations (H+ and
Al3+) in this forest ecosystem.
4.2. Mechanisms underlying reductions in soil respiration and its
components
Nitrogen addition and soil acidification decreased Rs in this study
(Fig. 2), which is in line with some previous researches in forest ecosys-
tems (Bowden et al., 2004; Burton et al., 2004; Olsson et al., 2005). Sev-
eral mechanisms behind the observed decline in Ra under N addition or
soil acidification have been proposed, including decreased belowground
C allocation theory (Litton et al., 2007; Phillips and Fahey, 2007), ammo-
nium toxicity (Van Den Berg et al., 2005) and loss of base mineral cat-
ions by lower pH (Bowman et al., 2008). Contrary to the C allocation
theory, we did not find decrease in fine root biomass under N addition
(Table 1). Meanwhile, our results showed that N availability had no sig-
nificant relationship with Ra (Fig. 5), suggesting ammonium toxicity
theory could not explain the decline of Ra. Remaining possible reasons
for the decline of Ra include increased concentration of H+ and Al3 +
(Table 1). Furthermore, SEM results showed that soil acid cations (H+
and Al3+) caused significant reduction in Ra (Figs. 5, 6). So, we conclude
that increased H+ and Al3+ concentration in combination contributed
to the decline of Ra.
The reduction of Rh under nitrogen addition and soil acidification is
in line with previous studies (Bowden et al., 2004; Olsson et al., 2005).
Meanwhile, several hypotheses about the mechanisms underlying re-
duction in Rh are partially supported in this study. First, N addition
and soil acidification significantly changed microbial community com-
position (i.e. increase in fungi: bacterial ratio, Table S3) (Chen et al.,
2015a; D. Chen et al., 2016). Although we did not directly measure mi-
crobial carbon use efficiency (CUE), increase in fungi: bacterial ratio
would induce increase in microbial CUE at community level and thereby
reduction in Rh (Schimel and Weintraub, 2003). Second, N enrichment
and soil acidification reduced Rh by decreasing microbial biomass
(Table 1). It is well documented that N addition and soil acidification
could result in decreased microbial physiology (Riggs and Hobbie,
2016) and microbial growth limitation due to loss of base cations (e.g.,
Mg2+, Ca2+) (Bowman et al., 2008), soil acid cations (e.g., Al3+) toxicity
1544 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546

Fig. 6. Structural equation model (SEM) derived standardized total effects of N enrichment and soil acidification, and variables including root biomass, cellulose degrading enzymes, N
availability and acid cations on (a) soil autotrophic respiration (Ra) and (b) heterotrophic respiration (Rh).

(Tian and Niu, 2015) and physic-chemical protection of C (Mueller et al.,
2012). Finally, decreased cellulose degrading enzymes activities leads to
reduction in Rh (Fig. 5). In this study, the increases in soil acid cations
(H+ and Al3+) contributed largely to the alteration of microbial com-
munity composition and consequently decreased cellulose degrading
enzymes activities under N addition and soil acidification (Figs. 5, 6).
4.3. Implications of soil acidification and the uncertainties of soil respiration
partitioning
Although most previous studies have addressed the impacts of N de-
position and soil acidification on soil respiration separately, this study
goes a step further, compares the different roles of N enrichment and
soil acidification in impacting Rs and its components. Our results suggest
that Rs reduced more in response to soil acidification than N enrichment
(Figs. 2, 6), which is mainly due to that soil acidification induced more
decrease of cellulose degrading enzymes activity (CB, BG and XYL)
than N enrichment (Fig. 4). Moreover, the structural equation model
(SEM) showed that soil acidification contributed more than N enrich-
ment in impacting Ra and Rh (Fig. 6). Since N addition and A addition
treatments decreased soil pH with similar magnitude (Table 1), we
suggest that soil acidification plays more important role than N enrich-
ment in subtropical forest.
The findings have important implication for terrestrial ecosystem C
cycle. Excessive mineral N fertilization leads to soil acidification in ter-
restrial ecosystem, especially in China (Guo et al., 2010; Liu et al.,
2013). Currently, 11% of world's natural vegetation receives N deposi-
tion in excess of 10 kg N ha− 1 yr− 1 (Dentener et al., 2006), and soil
pH has decreased by 0.63 unit in North China's grassland under increas-
ing N deposition over last two decades (Yang et al., 2012). Because soil
acidification play more important role than N enrichment in changing
Rs and its components, we need to take into account of the effect of
soil acidification and incorporate it into biogeochemical model in
order to more accurately predict terrestrial C cycling in the future sce-
narios of increasing N deposition.
The estimation of Ra and Rh in this study may have some uncer-
tainties. First, the trenched subplots may still allow root ingrowth un-
derneath the polyethylene board (N 0.6 m depth) into the subplots,
which might cause an overestimation of Rh and an underestimation of
Ra (Sayer and Tanner, 2010). Second, trenched subplots might shift mi-
crobial community composition, resulting in changes in Rh (Chen et al.,
2016). Finally, the conclusions are drawn from growing season soil
 Y. Li et al. / Science of the Total Environment 615 (2018) 1535–1546
respiration. It is insightful for further study to compare the mechanisms
accounting for reduction of Rs and its two components in response to N
enrichment and soil acidification between growing season and non-
growing season.
5. Conclusion
Our results showed that N deposition and soil acidification both sig-
nificantly decreased Ra and Rh, resulting in reduction of Rs in the two
growing seasons. The reductions of Rs and its components were attrib-
utable to the increased soil acid cations (H+ and Al3 +) and the de-
creased cellulose degrading enzymes activity. The reduction of
cellulose-degradation related enzymes activity is the main mechanism
underlying the impacts of N and acid enrichment on soil respiration.
Moreover, soil acidification played more important role than N enrich-
ment in decreasing Rs and its components. We therefore suggest that
soil acidification should be incorporated into biogeochemical model to
improve the prediction of ecosystem C cycling in the future scenarios
of anthropogenic N deposition and acid enrichment.
Acknowledgment
The authors would like to thank Wei Zhang, Mengjun Hu and
Yanchun Liu for their help in the field station. This study was financially
supported by National Natural Science Foundation of China (41403073,
31625006) and the Thousand Young Talents program to S. Niu.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.09.131.
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