Science of the Total Environment 654 (2019) 264–274

Contents lists available at ScienceDirect

Science of the Total Environment

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

Soil microbial community composition closely associates with specific

enzyme activities and soil carbon chemistry in a long-term nitrogen
fertilized grassland
Yue Li, Cheng Nie, Yinghui Liu ⁎, Wei Du, Pei He
State Key Laboratory of Earth Surface Processes and Resource Ecology, Faculty of Geographical Science, Beijing Normal University, Beijing 100875, 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

• N fertilization favors copiotrophic bacte-

ria and saprotrophic fungi.
• N fertilization increases specific N- and
P-acquiring enzyme activities.
• Bacterial phyla and fungal trophic guilds
closely link to enzyme activity.
• The shift of fungal phyla echoes the
change of soil aromatic C fractions.
• Microorganisms under N fertilization
affect soil C chemistry and nutrient
acquisition.

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

Article history: Due to the profound impact of nitrogen (N) input on soil systems, linking the activity and composition of soil mi-
Received 12 August 2018 crobial communities to soil organic carbon (SOC) is crucial to reveal the microbial-driven mechanisms underly-
Received in revised form 29 October 2018 ing SOC decomposition by nitrogen fertilization. A long-term nitrogen fertilization experiment with 6 urea
Accepted 2 November 2018
fertilizer gradients (0, 2, 4, 8, 16, and 32 g N m−2 yr−1) was conducted on a temperate grassland. The soil basic
Available online 07 November 2018
characteristics, microbial community DNA sequences, five soil enzymes including C, N, and phosphorus cycling,
Editor: Jay Gan and soil C fractions were measured after 14 years of N addition. N fertilization significantly modified both the bac-
terial and fungal community composition, with larger variations at higher N levels. N fertilization increased the
Keywords: proportion of copiotrophic bacteria and saprotrophic fungi. Specific enzyme activities standardized by microbial
Microbial community composition biomass carbon among N fertilizing gradients demonstrated that the potential of labile C acquisition was stable,
Enzyme activity but the potential of N and P acquisition and recalcitrant C degradation were increased. Recalcitrant soil C fractions
Soil carbon fraction including alkyl C and aromatic C significantly differed among N levels, despite the stable SOC concentration. The
Nitrogen fertilization variations of bacterial phyla and fungal trophic guilds were both associated with specific enzyme activities;
Grassland
meanwhile, fungal phyla were more related to soil C fractions, as the Basidiomycota abundance echoed the pro-
portion of aromatic C at 4–16 g N m−2 yr−1. In conclusion, this study indicates that the changes in microbial com-
munity composition by N fertilization can have far-reaching impacts on SOC turnover and nutrient acquisition.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. As a limiting factor for terrestrial ecosystem productivity, nitrogen

E-mail address: lyh@bnu.edu.cn (Y. Liu). (N) has been extensively studied by ecologists (Gruber and Galloway,

https://doi.org/10.1016/j.scitotenv.2018.11.031
0048-9697/© 2018 Elsevier B.V. All rights reserved.
 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
2008; Law, 2013; Sutton and Bleeker, 2013). N addition experiments
have been widely used to investigate the effects of N on plant-soil-
microorganism relationships in ecosystems (Treseder, 2004; Zhou
et al., 2014, 2017). N fertilization can significantly change the composi-
tion of soil microbial communities. For bacteria, N fertilization was
found to shift the bacterial communities from being dominated by oli-
gotrophic groups to being dominated by copiotrophic groups (Fierer
et al., 2012; Ling et al., 2017), which prefer to utilize labile carbon (C,
Eilers et al., 2010). For fungi, N fertilization was found to reduce the rel-
ative abundance of Basidiomycota (Zak et al., 2011), potentially de-
creased the production of oxidative enzymes to degrade lignin
(Sinsabaugh, 2010; Zak et al., 2017). In addition to the phylogenetic tax-
onomy, the life strategy for substrate acquirement in the fungal commu-
nity also predicts its metabolic activity (Eichlerová et al., 2015; Talbot
et al., 2015), and thus “trophic guilds” were classified and they were
proved to be sensitive to N fertilization (Cline et al., 2018). Studies
have reported that N fertilization increased the abundance of the
saprotrophs (Morrison et al., 2016), but decreased the abundance of
the symbiotrophs such as lichen (Johansson et al., 2012) and mycorrhi-
zal fungi (Treseder, 2004).
Soil microorganisms play crucial roles in ecosystem functioning and
fertility maintenance by regulating several key biogeochemical pro-
cesses in soil (Fierer, 2017). To bridge the soil microbial community dy-
namics and soil substrate availability (Nannipieri et al., 2018), enzyme
activities are indicative of microbial functioning in the soil system.
Among the soil hydrolytic enzymes frequently measured, β-
glucosidase (BG) is used to acquire C by hydrolysing cellulose, β-N-ace-
tyl-glucosaminidase (NAG) is indicative of a C and N-acquiring process
by decomposing N-acetyl-β-D-glucosamine, and acid phosphatase (AP)
is an indicator of a P-acquiring enzyme in acidic soil (Deng et al., 2011;
Acosta-Martínez et al., 2011). Two typical oxidative enzymes are phenol
oxidase which consume oxygen and oxidized phenol, and peroxidase
which use H2O2 as an acceptor to depolymerize lignin (Sinsabaugh,
2010). Both hydrolytic and oxidative enzymes are affected by environ-
mental conditions, such as N amendment (Sinsabaugh et al., 2002;
Sinsabaugh, 2010). The resource allocation theory suggests that micro-
organisms produce enzymes to acquire the most limiting nutrient
(Allison and Vitousek, 2005). Hydrolytic enzymes for obtaining C, N,
phosphate and their stoichiometric ratios have been used to reveal the
transitions in nutrient limitations for microorganisms in N fertilization
studies (Cusack, 2013; Jing et al., 2017; Wang et al., 2014). Microorgan-
isms exhausting labile C can result in enhanced BG activity (Cenini et al.,
2015; Zeglin et al., 2007), but a decreased overall microbial biomass
might explain the decrease of BG activity by N fertilization (Wang
et al., 2015). The activity of NAG can decrease after N fertilization, due
to the alleviation of soil N limitation (Sinsabaugh et al., 2002; Cusack
et al., 2011). The increased AP activity by N fertilization was considered
as the existence of P-limiting condition probably induced by plant P up-
take (Sinsabaugh et al., 2002). Due to the differences in plant-derived
lignin input and the structure of soil microbial decomposers, the activity
of oxidative enzymes was found to have large spatiotemporal variations
among ecosystems (Sinsabaugh, 2010). A latest review shows that
some hydrolytic and oxidative enzymes have weaker responses in
grasslands to N fertilization than in forests (Xiao et al., 2018). Contrary
to the relatively thorough discussion regarding oxidative enzymes in
lignin-dominant forests (Goodell et al., 2008), uncertainties remain in
cellulose-rich grasslands.
Grassland soil harbours 343 Pg C (in the top 1 m) at global scale
(FAO, 2007); thus, it has great potential to shift between acting as a C
pool and C sink, which can be affected by N fertilization. However, the
relationship between soil organic carbon (SOC) concentration and N
fertilization in grasslands has been found to be neutral in meta-
analyses (Lu et al., 2011; Yue et al., 2016). A growing body of evidence
has revealed that N fertilization significantly modified the chemical
fractions in SOC. Based on 13C nuclear magnetic resonance analysis
(13C NMR), labile C includes O-alkyl C (derived from cellulose, semi-
265
cellulose and carbohydrates) and carbonyl C (originated from fatty
acids, amino acids, and organic acids); meanwhile, recalcitrant C in-
cludes alkyl C (derived from chitin, cutin, and lipids) and aromatic C (as-
sociated with lignin degradation, Cheng et al., 2017; Pisani et al., 2015;
Zhang et al., 2015). There is frequently discovered a trade-off between
alkyl C and O-alkyl C by N fertilization: accumulated alkyl C can be the
result of microbial selective utilization of labile C (Wang et al., 2017)
and the by-product of microbial turnover (Liang et al., 2017) after N fer-
tilization; or increased O-alkyl C can attribute to root exudates and
microorganism-derived polysaccharides stimulated by N addition
(Cheng et al., 2017). Lignin derived aromatic C was found increased by
N fertilization (Cusack et al., 2011), likely due to the inhibition of N ad-
dition on the fungal community (Zak et al., 2011). C chemistry, rather
than C quantity, is considered to have a tighter association with soil mi-
crobial communities. Therefore, revealing the molecular and microbial
mechanisms underlying the response of the chemical fractions in grass-
land SOC to N fertilization can help to clarify the potential effect of N fer-
tilization on the grassland soil C balance.
Establishing the linkage among SOC, enzymes, and microbial com-
munity composition has been a long-term, yet elusive, objective in soil
ecology, let alone under an N fertilizing scenario. To comprehensively
understand the impact of N fertilization on soil C, compositions of bac-
terial and fungal communities, soil C fractions, and extracellular enzyme
activities were measured, and their associations were discussed in this
study. We specifically hypothesized: (1) N fertilization would shift mi-
crobial communities to the copiotrophic bacteria and the saprotrophic
fungi which incline to utilize labile C; (2) N fertilization would modified
the enzymatic stoichiometry by changing microbial acquisition for C
and P; (3) the proportion of aromatic C fractions would be significantly
modified by N fertilization, despite the stable concentration of SOC; and
(4) fungal communities had closer associations with soil C fractions
than bacterial communities, although both communities were closely
related to specific enzyme activities.
2. Materials and methods
2.1. Study site and experimental design
The study site is located in Duolun County, Inner Mongolia, China
(42.02°N, 116.17°E; 1341 m above sea level). The mean annual temper-
ature is 2.1 °C, and the average monthly temperatures range from a min-
imum of −17.5 °C in January to a maximum of 18.9 °C in July. Rainfall
mostly happens from May through September, and the mean annual
precipitation is 379.4 mm (Wang et al., 2015). The typical soil in this re-
gion is chestnut soil under the category of Aridosols (Chinese Soil
Taxonomy Research Group, 2001), and the vegetation is dominated by
Stipa sareptana Becker var. krylovii (Roshev.) P. C. Kuo et Y. H. Sun,
Leymus chinensis (Trin.) Tzvel., Artemisia frigida Willd., Agropyron
cristatum (L.) Gaertn. and Allium bidentatum Fisch. ex Prokh. et Ikonn.-
Gal.
The long-term N fertilization experimental site was established in
July 2003; dry urea N fertilizer (CO(NH2)2) was manually spread on
the surface of the plots (15 m × 10 m, 4 m-wide buffer zone between
each) in mid-July of each year since they were established. Bulk N depo-
sition in this area is approximately 5 g N m−2 yr−1 (Wang et al., 2015).
The N addition treatments included 6 N levels (0, 2, 4, 8, 16 and
32 g N m−2 yr−1) and 4 replicates for each level.
2.2. Soil sampling and analysis
On 1st July and 2nd July 2017, two soil cores (0–10 cm deep, 10 cm
diameter) were randomly collected from each plot and combined to
form one composite soil sample per plot. Soil was sieved through a
2 mm mesh and stored at −20 °C before lab analysis. Soil samples
were separated into two parts: one part was fresh soil and the other
was air-dried.
266 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274

Table 1 guilds representing at least 0.1% of the overall OTUs were considered
Alpha diversity indices for bacterial and fungal operational taxonomic units among 6 ni- in further analysis. OTU abundance information were normalized
trogen fertilizing levels.
using 42,000 sequences, according to the rarefaction curves (Fig. S2).
OTUs N level Observed Chao1 Shannon Alpha diversity was calculated for the number of OTUs identified for
Bacteria 0 3828.50(116.29)a 6238.08(131.69)a 6.39(0.11)a each sample, and the diversity indices including Observed species,
2 3794.50(67.84)a 6133.33(162.10)a 6.44(0.02)a Chao1, and Shannon were applied. Beta diversity was visualized by
4 3613.25(115.74)a 5811.41(137.84)ab 6.44(0.06)a the non-metric multidimensional scaling (NMDS) ordination. Analysis
8 3251.25(54.16)ab 5309.70(82.49)b 6.30(0.09)ab
of similarities (ANOSIM) was performed, and the differences between
16 2819.75(118.64)bc 4496.28(172.68)c 6.02(0.14)bc
32 2511.67(338.91)c 4034.62(589.65)c 5.87(0.38)c the microbial community composition among samples was considered
Fungi 0 1518.50(58.30) 2134.28(66.49) 4.71(0.23)a significant if P b 0.05. All of the analyses mentioned above were per-
2 1545.25(66.01) 2079.60(71.58) 4.94(0.16)a formed with R (v3.2.4, https://www.r-project.org). Heatmaps were
4 1425.75(132.60) 2036.12(164.28) 5.30(0.14)a used to show the relative abundance of microbial phyla and classes
8 1361.00(109.51) 1843.81(134.60) 5.17(0.08)a
16 1297.50(134.73) 1805.08(171.79) 4.64(0.37)a
among N fertilizing gradients. Trophic classification of pathotrophs,
32 1280.25(67.03) 1845.76(81.98) 3.85(0.28)b saprotrophs and symbiotrophs for fungi were made in FunGuild
(http://www.stbates.org/guilds/app.php) using OTUs described by
Data in the columns are mean value and standard errors in parentheses (n = 4). Different
lower-case letters in a column for one microbial OTUs indicate significantly difference at P Nguyen et al. (2016).
b 0.05. Raw reads used in this study were uploaded to the NCBI Sequence
Read Archive under accession number: PRJNA498362.

The hydrolytic enzymes investigated in this study were β-

glucosidase (BG), N-acetyl glucosaminidase (NAG), and acid phospha-
tase (AP); meanwhile, the oxidative enzymes activity was the sum of
the activities of polyphenol oxidase (POX) and peroxidase (PER,
Table S1). Enzyme assays were conducted following the protocol de-
scribed in previous studies (German et al., 2011; Bach et al., 2013; Jing
et al., 2017). In brief, fresh soil (1.5 g) was added to 125 ml of 50 mM so-
dium acetate buffer at the optimal pH value (6.0). Soil slurries were
contained in 96-well micro-plates and incubated at 25 °C for 2.5 h (for
hydrolytic enzymes) or 24 h (for oxidative enzymes). The quantity of
fluorescence (for hydrolytic enzymes) or absorbance (for oxidative en-
zymes) was determined with a micro-plate reader (Biotek Synergy 2,
Winooski, USA).
Dissolved organic carbon (DOC), microbial biomass carbon (MBC)
and soil inorganic nitrogen (ION, the sum of ammonium nitrogen and
nitrate nitrogen) were also measured. Ten grams of soil was extracted
with 40 ml 0.5 M K2SO4 for each sample. The extract was used to mea-
sure DOC and MBC with a TOC analyser (Liqui TOCII, Elementar,
Germany). MBC was the difference of organic C between fumigated
soil (for 24 h) and unfumigated soil (DOC), with an extraction efficiency
of 0.45. ION was measured with a flow injection analyser (AA3, Bran
Luebbe, Germany).
Air-dried soil was used to measure 13C nuclear magnetic resonance
(13C NMR), soil total organic carbon (TOC), total nitrogen (TN), and
soil pH. Soil pH was determined with a pH analyser. TOC and TN were
measured with an elemental analyser (Vario MAX, Elementar,
Germany), and soil was treated with 10% HCl to remove inorganic car-
bon before the measurement.

2.3. DNA sequencing and analysis

Total genomic DNA was extracted using a PowerSoil DNA Isolation

Kit (MoBio Laboratories Inc., Carlsbad, USA). The DNA integrity, concen-
tration and purity were monitored on 1% agarose gels. The 16S rDNA
genes in the V4-V5 region from soil bacterial communities were ampli-
fied using the 515F and 907R primers (Ye et al., 2016), and the ITS genes
in the ITS2 region from soil fungal communities were amplified using
the ITS5-1737F and ITS2-2043R primers (Zhang et al., 2016). Sequenc-
ing libraries were generated using the NEBNext® Ultra™ DNA Library
Prep Kit for Illumina® (New England Biolabs, Ipswich, USA) following
the manufacturer's recommendations, and index codes were added.
The library quality was assessed on the Qubit@ 2.0 Fluorometer
(Thermo Scientific) and Agilent Bioanalyser 2100 system. The library Fig. 1. Non-metric multidimensional scaling ordination for the bacterial (a) and fungal
was sequenced on the IlluminaHiseq2500 platform, and 250-bp (b) OTUs responding to nitrogen fertilization (mean + se, n = 4). Clustering of soil
samples are based on the N fertilizing levels and are presented as circles
paired-end reads were generated. Sequences analyses were performed (0 g N m−2 yr−1), inverted triangles (2 g N m−2 yr−1), quadrates (4 g N m−2 yr−1), and
by usearch software (V8.0.1517), and sequences with ≥97% similarity crosses (8 g N m−2 yr−1). Significant differences in sample clustering are measured by
were assigned to the same operational taxonomic unit (OTU). The ANOSIM.
 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
2.4. Nuclear magnetic resonance analysis
Before the measurement, 5 g of air-dried soil sample was treated
with 50 ml of 10% (v/v) hydrofluoric acid (HF) to remove paramagnetic
compounds and concentrate organic carbon (Skjemstad et al., 1994).
The mixture was shaken for 2 h and centrifuged, and then, the residue
was mixed with HF. This procedure was repeated five times. The soil
sample was rinsed with deionized water three times before it was
oven-dried to a constant weight. Finally, the dry soil was ground
through a 0.25 mm sieve. The 13C cross polarization/total sideband sup-
pression (CP/TOSS) and CP/TOSS with dipolar dephasing experiments
were run using a Bruker AVANCE400 spectrometer at 100 MHz to mea-
sure 13C with 4 mm sample rotors.
The peak areas were integrated with MestRenova 11.0 (Mestrelabs
Research, Santiago de Compostela, Spain) under the following major
chemical shift regions: 0–45 (alkyl C), 45–110 (O-alkyl C), 110–160 (ar-
omatic C), and 160–190 (carbonyl C). The chemical shift region of
190–220 ppm (ketone C) was too small to trace in this study. The
index of hydrophobicity was calculated using the equation: HI =
[(0–45 ppm) + (110–160 ppm)]:[(45–110 ppm) + (160–190 ppm)].
Fig. 2. Heatmaps for the relative abundance of bacterial (a) and fungal (b) phyla and classes responding to nitrogen fertilization. N0–32 represents 0–32 g N m−2 yr−1. Dominant phyla in
bacteria and fungi were marked in bold font.
267
2.5. Statistical analysis
One-way ANOVA was used to analyse the variations in soil enzymes,
13
C NMR, alpha diversity indices for microbial OTUs among the different
N levels. LSD tests (equal variances assumed) or Dunnett's (equal vari-
ances not assumed) tests were used for pair-wise comparisons. A
distance-based redundancy analysis (dbRDA) was used to visualize
the associations between soil microbial communities and environmen-
tal variables (pH, ION, DOC, TOC, C:N ratio, MBC, enzyme activities and
soil C fractions). Microbial community data were Hellinger-
transformed, and environmental variables were normalized and de-
tected for multicollinearity prior to the analysis. Then the environmen-
tal variables were subjected to a forward-selection procedure to
develop a model to explain the variance in microbial community data.
The Akaike's information criterion (AIC) was used to select the most
parsimonious models. The significance of the final model and the axes
were tested, and the proportion of variance explained by the axes was
provided. To identify the dominant variables contributing to the vari-
ances of microbial data, the marginal effects of the selected environ-
mental variables were examined. Spearman correlations were further
268 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274

conducted to assess the correlations among soil characteristics. The c). Oxidative enzymes were significantly increased by N additions at
dbRDA was conducted with R (v3.2.4), other statistical analyses were 32 g N m−2 yr−1 (Fig. 4d). The enzymatic ratios were measured to in-
performed using IBM SPSS 20.0 (IBM, USA). Graphs were created vestigate the shifts in limiting nutrients for soil microorganisms. The
using Sigmaplot 12.5 (Systat Software, CA, USA). BG:NAG ratio and BG:AP ratio both significantly decreased at
16–32 g N m−2 yr−1; meanwhile, although NAG:AP ratio was signifi-
3. Results cantly lower at 2 g N m−2 yr−1, this ratio did not show a significant var-
iation among N levels (R = 0.06, P = 0.78, Fig. S5).
3.1. Soil bacterial and fungal communities
3.3. Soil carbon fractions
The alpha and beta diversities of OTUs were used to determine the
effect of N fertilization on microbial communities. The observed rich- N fertilization significantly modified the soil recalcitrant C fractions,
ness, chao1, and Shannon diversity indices of bacterial OTUs all signifi- but the effect was inconsistent on alkyl C and aromatic C. The relative
cantly decreased by N fertilization (Table 1). In comparison, the abundance of alkyl C significantly increased by an average of 39% by N
observed richness and chao1 indices of fungal OTUs showed no signifi- fertilization (Fig. 5a), but aromatic C at 4 and 8 g N−2 yr−1 was
cant response to N fertilization, but the Shannon diversity index was
significantly decreased by N addition (Table 1). The NMDS ordination
performed with ANOSIM showed significant microbial composition
groupings by N fertilization (Fig. 1). High similarities were demon-
strated in clusters between 0 and 2 g N m−2 yr−1 for both bacterial
and fungal OTUs (Fig. 1). By contrast, the clusters at 16 and
32 g N m−2 yr−1 were distributed farther from each other, and larger
error bars for the clusters at 4–32 g N m−2 yr−1 (except for fungi at
32 g N m−2 yr−1) suggested higher heterogeneity in microbial commu-
nity composition (Fig. 1).
Twenty three bacterial phyla were detected in this study site, and
the dominant bacterial phyla were Proteobacteria (23.6% of total OTUs,
averaged by N fertilizing gradients), Acidobacteria (16.7%),
Actinobacteria (15.2%), Bacteroidetes (13.9%), and Verrucomicrobia
(10.6%). Two groups of bacterial phyla responded divergently to N fertil-
ization in the heatmap (Fig. 2a). Phylogenetic taxa were summed to de-
scribe the responses of specific bacterial groups to N fertilization. The
relative abundances of copiotrophic bacteria such as Proteobacteia,
Baceroidetes and Firmicutes were increased by N fertilization, but oligo-
trophic bacteria such as Acidobacteria, Planctomycetes, and
Verrucomicrobia were decreased by N fertilization (Fig. 2a). Therefore,
the proportion of copiotrophic bacteria in this grassland were positively
correlated with increasing N level, yet oligotrophic bacteria showed the
opposite correlation (Fig. S2). However, Actinobacteria did not show a
clear trend by N fertilization, because two clusters of classes responding
to N fertilization existed in the phyla and obscured the overall trend
(Fig. 2a; Fig. S3).
With six fungal phyla being detected, Ascomycota (65.2%) domi-
nated the fungal communities in this study, followed by Basidiomycota
(23.5%). The heatmap showed that the relative abundance of Basidiomy-
cota was relatively high under N fertilization treatments from 4 to
16 g N m−2 yr−1 (Fig. 2b). Looking into the order level, the dominant
fungal communities generally exhibited inconsistent trends with in-
creasing N level, such as Auriculariales was substantially higher at
4–16 g N m−2 yr−1 than other N levels (Fig. 2b). Only Eurotiales was in-
creased by N fertilization, and Cantharellales showed a negative correla-
tion with increasing N level (Fig. 2b). Classified by trophic guilds, the
pathotrophs and the saprotrophs were positively correlated with in-
creasing N level, but the symbiotrophs had negative responses to N fer-
tilization (Fig. 3). Among the saprotrophic groups, undefined
saprotrophs were the dominant group and they were significantly in-
creased by N fertilization; meanwhile, the proportion of wood
saprotroph had no statistically significant feedback to N fertilization
(Fig. S4).

3.2. Soil enzyme activity

The specific enzyme activity (enzyme activity normalized to micro-

bial biomass carbon) is more representative of microbial physiology
since the microbial biomass also changed by N fertilization. The specific Fig. 3. Effects of N fertilization on the trophic guilds of the pathogens (a), saprotrophs
BG did not show significant variation among N levels, but the specific (b) and symbiotrophs (c, mean + se, n = 4). The different lower-case letters indicate
NAG and AP had positive correlations with increasing N level (Fig. 4a– significant differences between N levels at P b 0.05.
 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
significantly decreased by N fertilization by 94% and 98% of
0 g N m−2 yr−1, respectively (Fig. 5c). However, the labile C fraction,
as O-alkyl C and carbonyl C, had no consistent responses to N fertiliza-
tion (Fig. 5b and d). The index of hydrophobicity (HI) was calculated
to investigate the extent of decomposition of soil organic carbon by N
fertilization. Compared to 0 g N m−2 yr−1, HI was significantly higher
at 2 g N m−2 yr−1 (P = 0.043), while HI at other N levels did not signif-
icantly differ from 0 g N m−2 yr−1.
3.4. Correlations among soil C, enzymes and microbial communities
By N fertilization, the pH value and MBC significantly decreased, but
ION and DOC increased in the soil (Table 2). The TOC and C:N ratio did
not significantly differ among N levels (Table 2). dbRDA was conducted
to analyse the associations among soil microbial community and envi-
ronmental variables in 24 samples, and marginal effects of environmen-
tal variables for dbRDA were investigated (Fig. 6, Table 3). The vectors of
DOC, specific NAG, oligo- and copiotrophic bacteria directed along axis
1, while vectors of specific BG and aromatic C were closer to axis 2 for
bacteria (Fig. 6a). Both DOC and specific NAG had significant marginal
effects and relatively high scores in axis 1, indicating that the variation
in bacterial community was more closely associated with enzyme activ-
ity and DOC (Table 3). In comparison, the vectors of specific BG, aro-
matic C, carbonyl C and Ascomycota directed along axis 1, and vectors
of the saprotrophs and specific NAG were closely associated to each
other (Fig. 6b). The marginal effects of the environmental variables sug-
gested that the variation in fungal community closely linked to the
change of enzyme activity and soil C chemistry (Table 3).
Except for Actinobacteria, the abundance of bacterial phyla was gen-
erally correlated with N level and specific enzyme activities (Table 4).
Copiotrophic bacteria had a positive correlation with specific NAG, AP
Fig. 4. Effects of N fertilization on the activity of specific β-glucosidase (normalized to microbial biomass carbon, a), specific N-acetyl glucosaminidase (b), specific acid phosphatase (c) and
specific oxidative enzymes (d, mean + se, n = 4). The different lower-case letters indicate significant differences between N levels at P b 0.05.
269
and oxidative enzyme activities; meanwhile oligotrophic bacteria
were negatively correlated with specific NAG, AP and oxidative enzyme
activities (Table 4). The abundance of Ascomycota and Basidiomycota
were not impacted by either N level or specific enzyme activities. How-
ever, classified by trophic guilds, the richness of the saprotrophs had
positive correlations by N fertilization, specific NAG and oxidative en-
zyme activities, but the symbiotrophs demonstrated the opposite pat-
tern (Table 4). Ascomycota was positively related to aromatic C and
negatively to alkyl C, while Basidiomycota had a negative correlation
with aromatic C (Table 4). In trophic assignment, neither total nor unde-
fined saprotrophs have a significant response to soil C fractions. How-
ever, the abundance of wood saprotrophs had a negative correlation
with aromatic C (Table 4).
4. Discussion
4.1. Response of microbial communities to nitrogen fertilization
In accordance with previous studies (Riggs and Hobbie, 2016; Yue
et al., 2016), the grassland soil microbial biomass was inhibited by N
fertilization. The possible reasons for this inhibition include decreased
pH by soil acidification and increased toxicity by the accumulation of
Al3+, Fe3+ and Mn3+ (Tian and Niu, 2015). MBC was significantly de-
creased by N fertilization in this study, and alpha diversity for bacterial
and fungal OTUs also showed decreasing trends when the N gradient
was high. In addition, beta diversity (NMDS) showed that a larger
community-level variation occurred in both bacteria and fungi with in-
creasing N gradients. Due to the tradeoff between microbial growth and
nutrient acquisition (Cline et al., 2018; Ling et al., 2017), high nutrient
availability could lead to large diversity in the microbial composition.
270 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
A latest review found that long-term N fertilization favours
Proteobacteria and Actinobacteria, but inhibits Acidobacteria (Dai et al.,
2018). Ours result partially agreed with it. Considering the divergent liv-
ing strategies in bacterial communities, Proteobacteria, Bacteroidetes and
Firmicutes are considered to be copiotrophic bacteria (Fierer et al., 2012;
Ling et al., 2017), and Acidobacteria, Planctomycetes, and
Verrucomicrobia are considered to be oligotrophic bacteria (Eilers
et al., 2010). In line with Leff et al. (2015), the copiotrophic and oligotro-
phic groups in this study showed divergent responses to N fertilization
and shifted the bacterial community composition from oligotrophic-
dominant to copiotrophic-dominant. However, Actinobacteria did not
show a clear pattern responding to N fertilization in this study, since
two clusters of classes had opposite responses to N fertilization.
Actinobacteria can exist in low soil pH and have relatively high tolerance
to environmental stress (Dai et al., 2018), and have functional genes for
glycoside hydrolase enzymes and resemble the life style of fungi (Zhang
et al., 2017), thus it was more sensitive to the effect of substrate (de
Menezes et al., 2015) but less sensitive to the direct effect of N
fertilization.
Ascomycota predominates the fungal community in cellulose-rich
grasslands. Compared to the lignin-degrading Basidiomycota, Ascomy-
cota contains abundant soft rot and brown rot fungi, which are unable
to break the phenol rings in lignin (Goodell et al., 2008). Among domi-
nant orders in Ascomycota, only Eurotiales had a significant and positive
correlation with increasing N level in this study. Previous studies found
that N addition inhibited Basidiomycota (Leff et al., 2015; Zak et al.,
2017), but in this study, the relative abundance of Basidiomycota was
relatively high in the 4–16 g N m−2 yr−1 treatment, mainly caused by
a substantial increase of Auriculariales. From the trophic guilds aspect,
N fertilization was found to increase the abundance of the saprotrophs
but decrease the abundance of the symbiotrophs (Morrison et al.,
2016). As Nguyen et al. (2016) defined, the saprotrophs are the fungi
Fig. 5. Effects of N fertilization on the relative abundance of alkyl C (a), O-alkyl (b), aromatic C (c), and carbonyl C (d, mean + se, n = 4). The different lower-case letters indicate significant
differences between N levels at P b 0.05.
that receive nutrients by breaking down dead hosts. Wood saprotrophs
were dominated by Agaricomycetes and had a negative correlation with
aromatic C in this study. Meanwhile, some of undefined saprotrophs
prefer to utilize labile C, such as Penicillium and Aspergillus in Eurotiales
(Brabcova et al., 2016). Although wood saprotrophs had no significant
responses to N fertilization, the correlation between the abundance of
undefined saprotrophs and N level was positive. Lichen and mycorrhizal
fungi are major symbiotrophic fungi in the soil. N input can inhibit the
growth of lichen through physiological stress and a lack of colonization
(Johansson et al., 2012), and dampen the collaboration between hosts
and mycorrhizal fungi by reducing plant investment in roots
(Treseder, 2004), thus leading to a decrease in the symbiotrophs.
4.2. Response of soil enzyme activities to nitrogen fertilization
Compared to the total enzyme activity, specific enzyme activity is
more representative of the microbial physiology (Cusack et al., 2011;
Jing et al., 2017), and is valid to present a panorama of microbial phys-
iology responding to N fertilization in this grassland. Specific BG had
no significant response to N fertilization, regardless of the modification
in microbial community. Considering the larger variations in the micro-
bial composition at higher N gradients, microbial functional redundancy
might exist for acquiring labile C (Allison and Martiny, 2008). Above-
ground biomass was found significantly increased by N fertilization in
this study site (Tian et al., 2016), resulting in the immobilization of
ION. Specific NAG was enhanced by N fertilization, and the potential
mechanism is probably because enhanced plant growth by N fertiliza-
tion stimulates ION uptake and competes with microorganisms.
Since microbial N acquisition is coupled with different strategies of
soil C utilization and thus involved different N-acquiring enzymes
(Cenini et al., 2016; Li et al., 2018), increased specific NAG may also be
the consequence of microbial preferable utilization in glucosamine.
 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274 271

Table 2
Soil basic characteristics among 6 nitrogen fertilizing levels.

N level pH TOC TN C:N ION DOC MBC

(g kg−1) (g kg−1) (mg kg−1) (mg kg−1) (mg kg−1)

0 6.57(0.01)a 19.53(1.35) 1.88(0.15) 10.44(0.13) 4.34(0.35) 8.60(0.26)a 666.37(66.56)a

2 6.44(0.04)a 23.05(2.71) 2.18(0.03) 10.64(0.15) 7.15(0.37) 11.02(1.31)a 658.42(92.16)a
4 5.85(0.11)b 22.90(2.09) 2.18(0.19) 10.52(0.06) 12.45(2.12) 14.68(2.22)b 523.14(101.78)a
8 5.48(0.03)c 21.93(1.28) 2.05(0.01) 10.69(0.20) 15.06(4.07) 34.53(6.62)c 491.01(69.13)ab
16 5.04(0.07)d 22.45(0.71) 2.08(0.01) 10.83(0.18) 19.69(7.73) 26.06(5.06)c 307.32(21.14)b
32 4.76(0.06)e 20.98(3.32) 1.98(0.03) 10.55(0.24) 23.76(10.38) 29.92(8.72)c 268.11(36.65)b

TOC is total organic carbon, TN is total nitrogen, C: N is total organic carbon: total nitrogen ratio, ION is inorganic nitrogen, DOC is dissolved organic carbon and MBC is microbial biomass
carbon.
Data in the columns are mean value and standard errors in parentheses (n = 4). Different lower-case letters in a column indicate significantly difference at P b 0.05.

Specific AP had a positive response to N fertilization in this study. N ad- mask such relationship (Fierer, 2017), the other is the pre-treatment
dition was found to accelerate soil P cycling (Deng et al., 2017) and in- of 10% hydrofluoric acid might loss the labile C to some extent by mul-
duce P limitation (Sinsabaugh et al., 2002). In addition, Talbot et al. tiple times of rinsing (He et al., 2018). However, N fertilization signifi-
(2015) found that the saprotrophs had higher activities of acid phos- cantly shaped recalcitrant C; alkyl C was increased but aromatic C was
phatase than the symbiotrophs, while oxidoreductase activities were decreased by N fertilization in this grassland. Alkyl C was generally
similar between two fungal groups. The shift of symbiotrophic fungi to
saprotrophic fungi by N fertilization might be associated with the in-
crease of specific AP. Significantly decreased BG:NAG ratio and BG:AP
ratio at high N fertilizing gradients suggested that N and P were rela-
tively limiting for soil microorganisms.
In contrast to the ubiquity of hydrolytic enzyme production by soil
microorganisms, fungi are considered as the main source for oxidative
enzymes (Sinsabaugh, 2010). Some studies conducted on grasslands
found that N addition had no impact on oxidative enzymes
(Sinsabaugh, 2010; Zeglin et al., 2007), and others found that the effect
was positive (Riggs and Hobbie, 2016). To our surprise, despite no sig-
nificant response of wood saprotroph to N fertilization, specific oxida-
tive enzyme activities were drastically stimulated by high N gradients
at 32 g N m−2 yr−1. Two possible reasons to the abnormality of oxida-
tive enzyme activities were presumed. First, as enzymes can be
adsorbed to the surface of the clay particle and no longer controlled
by soil microorganisms (Nannipieri et al., 2018; Six et al., 2006), the ab-
normality of oxidative enzyme activities in this study might partially as-
sociated with the highest proportion of silt-clay fraction at
32 g N m−2 yr−1 (Fig. S6). Second, manganese (Mn) is the substrate
for Mn-peroxidase, and Mn2+ at 32 g N m−2 yr−1 is nearly four times
higher than it at 0 g N m−2 yr−1 in this site (Tian et al., 2016), thus po-
tentially increased the substrate availability for Mn-peroxidase
synthesis.

4.3. Response of soil carbon fractions to nitrogen fertilization

Different from the positive effect on SOC in forests (Frey et al., 2014;
Zak et al., 2017), N addition had a neutral effect on grassland SOC (Lu
et al., 2011; Yue et al., 2016). Neither TOC or the C:N ratio showed sig-
nificant differences among the N gradients in this study. Although N fer-
tilization did not change the grassland SOC quantity, it significantly
increased DOC and modified the SOC chemistry. Yue et al. (2016) in
their meta-analysis found that DOC in the O-horizon of terrestrial eco-
systems was enhanced by N addition. Suppressed microbial biomass, in-
creased belowground biomass and litter input could explain such
phenomenon (Rodriguez et al., 2014), which echoed the decreased
MBC by N addition in this study. Higher hydrophobicity represents a
higher degree of degradation and stability of SOM (Cheng et al., 2017; Fig. 6. Distance-based redundancy analysis for bacterial (a) and fungal (b) communities
with 6 nitrogen fertilizing levels. The environmental variables in the model for bacteria
Guo et al., 2017), and this index was significantly higher at
(23 phyla, oligotrophic bacteria and copiotrophic bacteria) capture 67.8% of the variation
2 g N m−2 yr−1 than 0 g N m−2 yr−1. in data. The environmental variables in the model for fungi (six phyla, pathogens,
N fertilization can alter the grassland SOC chemistry by decreasing saprotrophs and symbiotrophs) capture 58.4% of the variation in data. Sample
the labile C fractions and increasing the recalcitrant C fractions (Cheng distribution is labelled as white circle, factors of the environmental variable are labelled
et al., 2017; Pisani et al., 2015), but we did not observe these alterations as red vectors, and the microbial community as black vectors with the names of typical
groups demonstrated in blue. TOC is total organic C, BG is specific β-glucosidase, AP is spe-
in the study. Instead, no clear relationship was found between N fertili- cific acid phosphatase, Oxidative is specific oxidative enzymes, Aromatic is aromatic C and
zation and labile C fractions. One possible reason is that the large flux of Carboxyl is carboxyl C. (For interpretation of the references to color in this figure legend,
labile C in- and outflows in various soil biochemical processes might the reader is referred to the web version of this article.)
272 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274

Table 3 be impacted by N fertilization more directly (Leff et al., 2015). Spearman

Marginal effects in the distance-based redundancy analysis and the scores of the environ- correlations further demonstrated that aromatic C was more tightly re-
mental variables for the first axis.
lated to the fungi than bacteria. Through the change of plant composi-
Variable F Pr (NF) score tion (Leff et al., 2015) and litter chemistry (Cline et al., 2018), fungi
Bacteria was found to be regulated by the indirect effect of N fertilization.
DOC 18.235 0.001 0.700 By N fertilization, the plants in this study site shifted from Stipa
NAGspec 7.570 0.001 0.613 krylovii and Artmisia frigida co-dominant to Stipa krylovii dominant
Aromatic 6.730 0.010 −0.064
(Tian et al., 2016). Liu et al. (2010) found that either the decomposition
BGspec 3.535 0.043 −0.025
rate of Stipa krylovii litter or Artmisia frigida litter was slower than the
Fungi litter mixture of Stipa krylovii and Artmisia frigida at this study site.
Aromatic 10.183 0.001 −0.695
The succession of plant community would shape the litter chemistry,
NAGspec 3.705 0.036 0.371
Carbonyl 3.285 0.044 −0.250 and the recalcitrance of C substrate transferring into soil changed by N
BGspec 3.413 0.037 0.295 fertilization. Litter chemistry and N fertilization might interactively im-
Only environmental variables with significant marginal effects are presented. DOC is dis-
pact on the community composition and function of microorganisms,
solved organic carbon, BGspec is specific β-glucosidase, NAGspec is specific N-acetyl especially the fungi which can utilize recalcitrant substrates. Therefore,
glucosaminidase and Aromatic is aromatic C. litter chemistry should be considered in the further exploration on how
The score of environmental variables in axis 1 were presented. Models for bacteria (F = fungi participate in the soil C decomposition by N fertilization.
36.5871, Pr = 0.001) and fungi (F = 19.5378, Pr = 0.003) are only significant in axis 1.
Microbial community composition provided an underlying connec-
tion between microbial enzyme activities and SOC chemistry. Therefore,
higher in soil fertilized with urea in this study. Microbial necromass is a changes in microbial community composition by N fertilization can
major source of stable soil C sink (Liang et al., 2017), and the bacterial have far-reaching impacts on SOC turnover and nutrient cycling.
structure contains higher aliphatic C than fungi (Six et al., 2006). Since
N fertilization decreased the fungi: bacteria ratio in this grassland (Li
et al., 2015), it might lead to a higher alkyl C proportion by N fertiliza- 5. Conclusion
tion. Aromatic C is closely associated with lignin degrading enzymes
in the fungal community (Cusack et al., 2011). Consisting of white rot, N fertilization had a profound impact on this grassland soil system;
which completely decomposes phenolic lignin, Basidiomycota is the soil microbial communities, enzyme activity, and soil C fractions were
key player for decomposing aromatic C (Goodell et al., 2008). Therefore, all shaped by urea fertilization. The bacterial composition shifted from
a higher abundance of Basidiomycota occurring with 4–16 g N m−2 yr−1 oligotrophic-dominant to copiotrophic-dominant; the relative abun-
echoed the lower abundance of aromatic C in this study. dance of Basidiomycota in the fungal community was significantly stim-
ulated in the 4–16 g N m−2 yr−1 treatments, while the trophic guilds of
4.4. Relationship among soil organic carbon and microbial composition and the saprotrophs outcompeted the symbiotrophs. Specific NAG, AP and
function oxidative enzyme activities were increased by N fertilization, and the
enzymatic stoichiometry suggested the relative limitation of N and P
Both bacterial and fungal communities in this grassland were signif- for soil microorganisms. SOC chemistry rather than SOC quantity was
icantly associated with specific enzyme activities. The dbRDA ordination significantly modified by N fertilization. Both bacterial phyla and fungal
in this study showed that DOC and specific NAG had significant associ- trophic guilds closely associated with specific enzyme activities, but soil
ations with the variation in bacterial community. In fungal communi- C chemistry was more tightly related to fungal phyla. Our findings indi-
ties, the variation of phyla was closely related to soil C fractions, and cate that the microbial community composition closely links to soil C
the variation of trophic guilds to specific enzyme activities. Since DOC and nutrient cycling under N fertilizing scenarios. To further explore
and specific enzyme activities all have significant and positive correla- these linkages, litter chemistry needs to be investigated in the future
tions with increasing N level, the variation of bacterial phyla seems to N fertilization study.

Table 4
Spearman correlation between specific enzyme activity, soil carbon fraction and microbial relative abundance.

Community N level DOC Specific enzymes Carbon fraction

BG NAG AP Oxidative Alkyl O-alkyl Aromatic Carbonyl

Bacteria
Proteobacteria 0.91⁎⁎ 0.64⁎⁎ 0.20 0.76⁎⁎ 0.64⁎⁎ 0.71⁎⁎ 0.20 0.31 −0.24 −0.21
Acidobacteria −0.94⁎⁎ −0.70⁎⁎ −0.20 −0.74⁎⁎ −0.68⁎⁎ −0.73⁎⁎ −0.16 −0.34 0.26 0.12
Actinobacteria −0.19 −0.21 −0.07 0.02 0.06 −0.08 −0.51⁎ 0.19 0.28 0.24
Bacteroidetes 0.72⁎⁎ 0.48⁎ −0.02 0.54⁎⁎ 0.33 0.47⁎ 0.20 0.42⁎ −0.35 −0.09
Verrucomicrobia −0.62⁎⁎ −0.43⁎ −0.16 −0.53⁎⁎ −0.43⁎ −0.53⁎⁎ −0.01 −0.21 0.02 0.20
Planctomycetes −0.67⁎⁎ −0.57⁎⁎ −0.02 −0.64⁎⁎ −0.32 −0.57⁎⁎ −0.19 −0.22 0.24 0.09
Oligotrophic −0.91⁎⁎ −0.71⁎⁎ −0.13 −0.73⁎⁎ −0.59⁎⁎ −0.69⁎⁎ −0.18 −0.37 0.28 0.16
Copiotrophic 0.90⁎⁎ 0.68⁎⁎ 0.22 0.80⁎⁎ 0.67⁎⁎ 0.71⁎⁎ 0.14 0.39 −0.28 −0.12

Fungi
Ascomycota −0.14 −0.28 0.03 −0.06 −0.06 0.07 −0.52⁎⁎ −0.20 0.71⁎⁎ 0.09
Basidiomycota 0.12 0.25 0.12 0.14 0.09 0.00 0.45⁎ 0.24 −0.66⁎⁎ −0.08
Pathotrophs 0.57⁎⁎ 0.51⁎ 0.13 0.44⁎ 0.38 0.39 0.33 0.30 −0.46⁎ −0.16
Symbiotrophs −0.88⁎⁎ −0.71⁎⁎ −0.14 −0.61⁎ −0.51⁎ −0.66⁎⁎ −0.25 0.27 0.34 0.09
Saprotrophs 0.69⁎⁎ 0.59⁎⁎ 0.08 0.42⁎ 0.37 0.46⁎ 0.34 0.16 −0.36 −0.22
Undefined 0.70⁎⁎ 0.60⁎⁎ 0.08 0.44⁎⁎ 0.44⁎⁎ 0.47⁎⁎ 0.33 0.17 −0.36 −0.21
Wood 0.23 0.32 −0.07 0.17 0.16 0.09 0.29 0.16 −0.42⁎ −0.12

DOC is dissolved organic carbon, BG is β-glucosidase, NAG is N-acetyl glucosaminidase, AP is acid phosphatase, Oxidative is oxidative enzyme activities, Undefined is undefined saprotroph
and wood is wood saprotroph.
⁎ Significant at the 0.05 probability level.
⁎⁎ Significant at the 0.01 probability level.
 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
Competing financial interests
The authors declare no competing financial interests.
Acknowledgments
This work was funded by National Natural Science Foundation of
China (31770519) and the National Key R&D Program of China
(2017YFC0503805). We thank the Duolun Restoration Ecology Re-
search Station (part of the Institute of Botany, Chinese Academy of Sci-
ences) for providing access to the study site.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.11.031.
References
Acosta-Martínez, V., Tabatabai, M.A., Dick, R.P., 2011. Phosphorus cycle enzymes. In: Dick,
R.P. (Ed.), Methods of Soil Enzymology. SSSA Book Series, pp. 161–183 https://doi.
org/10.2136/sssabookser9.c8.
Allison, S.D., Martiny, J.B.H., 2008. Resistance, resilience, and redundancy in microbial
communities. Proc. Natl. Acad. Sci. U. S. A. 105 (Suppl), 11512–11519. https://doi.
org/10.1073/pnas.0801925105.
Allison, S.D., Vitousek, P.M., 2005. Responses of extracellular enzymes to simple and com-
plex nutrient inputs. Soil Biol. Biochem. 37, 937–944. https://doi.org/10.1016/j.
soilbio.2004.09.014.
Bach, C.E., Warnock, D.D., Van Horn, D.J., Weintraub, M.N., Sinsabaugh, R.L., Allison, S.D., et
al., 2013. Measuring phenol oxidase and peroxidase activities with pyrogallol, l-
DOPA, and ABTS: effect of assay conditions and soil type. Soil Biol. Biochem. 67,
183–191.
Brabcova, V., Novakova, M., Davidov, A., Baldrian, P., 2016. Dead fungal mycelium in forest
soil represents a decomposition hotspot and a habitat for a specific microbial com-
munity. New Phytol. 210, 1369–1381.
Cenini, V.L., Fornara, D.A., McMullan, G., Ternan, N., Lajtha, K., Crawley, M.J., 2015. Chronic
nitrogen fertilization and carbon sequestration in grassland soils: evidence of a mi-
crobial enzyme link. Biogeochemistry 126, 301–313. https://doi.org/10.1007/
s10533-015-0157-5.
Cenini, V.L., Fornara, D.A., McMullan, G., Ternan, N., Carolan, R., Crawley, M.J., Clement, J.,
Lavorel, S., 2016. Linkages between extracellular enzyme activities and the carbon
and nitrogen content of grassland soils. Soil Biol. Biochem. 96, 198–206. https://doi.
org/10.1016/j.soilbio.2016.02.015.
Cheng, S., He, S., Fang, H., Xia, J., Tian, J., Yu, G., Geng, J., Yu, G., 2017. Contrasting effects of
NH4+ and NO3− amendments on amount and chemical characteristics of different
density organic matter fractions in a boreal forest soil. Geoderma 293, 1–9. https://
doi.org/10.1016/j.geoderma.2017.01.023.
Chinese Soil Taxonomy Research Group, Institute of Soil Science, Academia Sinica (ISSAS),
2001. Keys to Chinese Soil Taxonomy. (in Chinese). 3rd edn. University of Science and
Technology of China Press, Hefe.
Cline, L.C., Hobbie, S.E., Madritch, M.D., Buyarski, C.R., Tilman, D., Cavender-Bares, J.M.,
2018. Resource availability underlies the plant-fungal diversity relationship in a
grassland ecosystem. Ecology 99, 204–216. https://doi.org/10.1002/ecy.2075.
Cusack, D.F., 2013. Soil nitrogen levels are linked to decomposition enzyme activities
along an urban-remote tropical forest gradient. Soil Biol. Biochem. 57, 192–203.
https://doi.org/10.1016/j.soilbio.2012.07.012.
Cusack, D.F., Silver, W.L., Torn, M.S., Burton, S.D., Firestone, M.K., 2011. Changes in micro-
bial community characteristics and soil organic matter with nitrogen additions in two
tropical forests. Ecology 92, 621–632. https://doi.org/10.1890/10-0459.1.
Dai, Z., Su, W., Chen, H., Barberan, A., Zhao, H., Yu, M., Yu, L., Brookes, P.C., Schadt, C.W.,
Chang, S.X., Xu, J., 2018. Long-term nitrogen fertilization decreases bacterial diversity
and favors the growth of Actinobacteria and Proteobacteria in agro-ecosystems
across the globe. Glob. Chang. Biol. 24, 3452–3461. https://doi.org/10.1111/
gcb.14163.
Deng, S., Popova, I., Dick, R.P., 2011. Carbohydrate HYDROLASES. In: Dick, R.P. (Ed.),
Methods of Soil Enzymology. SSSA Book Series, pp. 185–209 https://doi.org/
10.2136/sssabookser9.c9.
Deng, Q., Hui, D., Dennis, S., Reddy, K.C., 2017. Responses of terrestrial ecosystem phos-
phorus cycling to nitrogen addition: a meta-analysis. Glob. Ecol. Biogeogr. 26,
713–728. https://doi.org/10.1111/geb.12576.
Eichlerová, I., Homolka, L., Žifčáková, L., Lisá, L., Dobiášová, P., Baldrian, P., 2015. Enzymatic
systems involved in decomposition reflects the ecology and taxonomy of
saprotrophic fungi. Fungal Ecol. 13, 10–22. https://doi.org/10.1016/j.
funeco.2014.08.002.
Eilers, K.G., Lauber, C.L., Knight, R., Fierer, N., 2010. Shifts in bacterial community structure
associated with inputs of low molecular weight carbon compounds to soil. Soil Biol.
Biochem. 42, 896–903. https://doi.org/10.1016/j.soilbio.2010.02.003.
FAO, 2007. State of the World's Forests 2007. Food and Agricultural Organization of the
United Nations, Rome, Italy.
273
Fierer, N., 2017. Embracing the unknown: disentangling the complexities of the soil
microbiome. Nat. Rev. Microbiol. 15, 579–590. https://doi.org/10.1038/
nrmicro.2017.87.
Fierer, N., Lauber, C.L., Ramirez, K.S., Zaneveld, J., Bradford, M.A., Knight, R., 2012. Compar-
ative metagenomic, phylogenetic and physiological analyses of soil microbial com-
munities across nitrogen gradients. ISME J. 6, 1007–1017. https://doi.org/10.1038/
ismej.2011.159.
Frey, S.D., Ollinger, S., Nadelhoffer, K., Bowden, R., Brzostek, E., Burton, A., Caldwell, B.A.,
Crow, S., Goodale, C.L., Grandy, A.S., Finzi, A., Kramer, M.G., Lajtha, K., LeMoine, J.,
Martin, M., McDowell, W.H., Minocha, R., Sadowsky, J.J., Templer, P.H., Wickings, K.,
2014. Chronic nitrogen additions suppress decomposition and sequester soil carbon
in temperate forests. Biogeochemistry 121, 305–316. https://doi.org/10.1007/
s10533-014-0004-0.
German, D.P., Weintraub, M.N., Grandy, A.S., Lauber, C.L., Rinkes, Z.L., Allison, S.D., 2011.
Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies.
Soil Biol. Biochem. 43, 1387–1397.
Goodell, Barry, Qian, Y., Jellison, J., 2008. Fungal decay of wood: soft rot—brown rot—
white rot. ACS Symp. Ser. 9–31.
Gruber, N., Galloway, J.N., 2008. An Earth-system perspective of the global nitrogen cycle.
Nature 451, 293–296. https://doi.org/10.1038/nature06592.
Guo, H., Ye, C., Zhang, H., Pan, S., Ji, Y., Li, Z., Liu, M., Zhou, X., Du, G., Hu, F., Hu, S., 2017.
Long-term nitrogen & phosphorus additions reduce soil microbial respiration but in-
crease its temperature sensitivity in a Tibetan alpine meadow. Soil Biol. Biochem. 113,
26–34. https://doi.org/10.1016/j.soilbio.2017.05.024.
He, Y., He, X., Xu, M., Zhang, W., Yang, X., Huang, S., 2018. Long-term fertilization increases
soil organic carbon and alters its chemical composition in three wheat-maize
cropping sites across central and south China. Soil Tillage Res. 177, 79–87. https://
doi.org/10.1016/j.still.2017.11.018.
Jing, X., Chen, X., Tang, M., Ding, Z., Jiang, L., Li, P., Ma, S., Tian, D., Xu, L., Zhu, J., Ji, C., Shen,
H., Zheng, C., Fang, J., Zhu, B., 2017. Nitrogen deposition has minor effect on soil extra-
cellular enzyme activities in six Chinese forests. Sci. Total Environ. 607–608, 806–815.
https://doi.org/10.1016/j.scitotenv.2017.07.060.
Johansson, O., Palmqvist, K., Olofsson, J., 2012. Nitrogen deposition drives lichen commu-
nity changes through differential species responses. Glob. Chang. Biol. 18, 2626–2635.
https://doi.org/10.1111/j.1365-2486.2012.02723.x.
Law, B., 2013. Nitrogen deposition and forest carbon. Nature 496, 308–309. https://doi.
org/10.1038/nature12092.
Leff, J.W., Jones, S.E., Prober, S.M., Barberán, A., Borer, E.T., Firn, J.L., Harpole, W.S., Hobbie,
S.E., Hofmockel, K.S., Knops, J.M.H., McCulley, R.L., La Pierre, K., Risch, A.C., Seabloom,
E.W., Schütz, M., Steenbock, C., Stevens, C.J., Fierer, N., 2015. Consistent responses of
soil microbial communities to elevated nutrient inputs in grasslands across the
globe. Proc. Natl. Acad. Sci. 112, 10967–10972. https://doi.org/10.1073/
pnas.1508382112.
Li, Y., Liu, Y.H., Wu, S.M., Niu, L., Tian, Y.Q., 2015. Microbial properties explain temporal
variation in soil respiration in a grassland subjected to nitrogen addition. Sci. Rep.
5, 18496. https://doi.org/10.1038/srep18496.
Li, Y., Liu, Y., Wu, S., Nie, C., Lorenz, N., Lee, N.R., Dick, R.P., 2018. Composition and carbon
utilization of soil microbial communities subjected to long-term nitrogen fertilization
in a temperate grassland in northern China. Appl. Soil Ecol. 124, 252–261. https://doi.
org/10.1016/j.apsoil.2017.11.009.
Liang, C., Schimel, J.P., Jastrow, J.D., 2017. The importance of anabolism in microbial con-
trol over soil carbon storage. Nat. Microbiol. https://doi.org/10.1038/
nmicrobiol.2017.105.
Ling, N., Chen, D., Guo, H., Wei, J., Bai, Y., Shen, Q., Hu, S., 2017. Differential responses of
soil bacterial communities to long-term N and P inputs in a semi-arid steppe.
Geoderma 292, 25–33. https://doi.org/10.1016/j.geoderma.2017.01.013.
Liu, P., Huang, J., Sun, O.J., Han, X., 2010. Litter decomposition and nutrient release as af-
fected by soil nitrogen availability and litter quality in a semiarid grassland ecosys-
tem. Oecologia 162, 771–780. https://doi.org/10.1007/s00442-009-1506-7.
Lu, M., Zhou, X., Luo, Y., Yang, Y., Fang, C., Chen, J., Li, B., 2011. Minor stimulation of soil
carbon storage by nitrogen addition: a meta-analysis. Agric. Ecosyst. Environ. 140,
234–244. https://doi.org/10.1016/j.agee.2010.12.010.
de Menezes, A.B., Prendergast-Miller, M.T., Poonpatana, P., Farrell, M., Bissett, A.,
MacDonald, L.M., Toscas, P., Richardson, A.E., Thrall, P.H., 2015. C/N ratio drives soil
actinobacterial cellobiohydrolase gene diversity. Appl. Environ. Microbiol. 81,
3016–3028. https://doi.org/10.1128/AEM.00067-15.
Morrison, E.W., Frey, S.D., Sadowsky, J.J., van Diepen, L.T.A., Thomas, W.K., Pringle, A.,
2016. Chronic nitrogen additions fundamentally restructure the soil fungal commu-
nity in a temperate forest. Fungal Ecol. 23, 48–57. https://doi.org/10.1016/j.
funeco.2016.05.011.
Nannipieri, P., Trasar-Cepeda, C., Dick, R.P., 2018. Soil enzyme activity: a brief history and
biochemistry as a basis for appropriate interpretations and meta-analysis. Biol. Fertil.
Soils 54, 11–19. https://doi.org/10.1007/s00374-017-1245-6.
Nguyen, N.H., Song, Z., Bates, S.T., Branco, S., Tedersoo, L., Menke, J., Schilling, J.S., Kennedy,
P.G., 2016. FUNGuild: an open annotation tool for parsing fungal community datasets
by ecological guild. Fungal Ecol. 20, 241–248. https://doi.org/10.1016/j.
funeco.2015.06.006.
Pisani, O., Frey, S.D., Simpson, A.J., Simpson, M.J., 2015. Soil warming and nitrogen depo-
sition alter soil organic matter composition at the molecular-level. Biogeochemistry
123, 391–409. https://doi.org/10.1007/s10533-015-0073-8.
Riggs, C.E., Hobbie, S.E., 2016. Mechanisms driving the soil organic matter decomposition
response to nitrogen enrichment in grassland soils. Soil Biol. Biochem. 99, 54–65.
https://doi.org/10.1016/j.soilbio.2016.04.023.
Rodriguez, A., Lovett, G.M., Weathers, K.C., et al., 2014. Lability of C in temperate forest
soils: assessing the role of nitrogen addition and tree species composition. Soil Biol.
Biochem. 77, 129–140. https://doi.org/10.1016/j.soilbio.2014.06.025.
274 Y. Li et al. / Science of the Total Environment 654 (2019) 264–274
Sinsabaugh, R.L., 2010. Phenol oxidase, peroxidase and organic matter dynamics of soil.
Soil Biol. Biochem. 42, 391–404. https://doi.org/10.1016/j.soilbio.2009.10.014.
Sinsabaugh, R.L., Carreiro, M.M., Repert, D.A., 2002. Allocation of extracellular enzymatic
activity in relation to litter composition, N deposition, and mass loss. Biogeochemistry
60, 1–24. https://doi.org/10.1023/A:1016541114786.
Six, J., Frey, S.D., Thiet, R.K., Batten, K.M., 2006. Bacterial and fungal contributions to car-
bon sequestration in agroecosystems. Soil Sci. Soc. Am. J. 70, 555. https://doi.org/
10.2136/sssaj2004.0347.
Skjemstad, J.O., Clarke, P., Taylor, J.A., Oades, J.M., Neman, R.H., 1994. The removal of mag-
netic materials from surface soils - a solid-state C-13 CP/MAS NMR study. Aust. J. Soil
Res. 32, 1215–1229. https://doi.org/10.1071/SR9941215.
Sutton, M.A., Bleeker, A., 2013. Environmental science: the shape of nitrogen to come. Na-
ture 494, 435–437. https://doi.org/10.1038/nature11954.
Talbot, J.M., Martin, F., Kohler, A., Henrissat, B., Peay, K.G., 2015. Functional guild classifi-
cation predicts the enzymatic role of fungi in litter and soil biogeochemistry. Soil Biol.
Biochem. 88, 441–456. https://doi.org/10.1016/j.soilbio.2015.05.006.
Tian, D., Niu, S., 2015. A global analysis of soil acidification caused by nitrogen addition.
Environ. Res. Lett. 10, 24019. https://doi.org/10.1088/1748-9326/10/2/024019.
Tian, Q., Liu, N., Bai, W., Li, L., Chen, J., Reich, P.B., Yu, Q., Guo, D., Smith, M.D., Knapp, A.K.,
Cheng, W., Lu, P., Gao, Y., Yang, A., Wang, T., Li, X., Wang, Z., Ma, Y., Han, X., Zhang,
W.H., 2016. A novel soil manganese mechanism drives plant species loss with in-
creased nitrogen deposition in a temperate steppe. Ecology 97, 65–74. https://doi.
org/10.1128/JVI.01755-15.
Treseder, K.K., 2004. A meta-analysis of mycorrhizal responses to nitrogen, phosphorus,
and atmospheric CO2 in field studies. New Phytol. 164, 347–355.
Wang, R., Filley, T.R., Xu, Z., Wang, X., Li, M.H., Zhang, Y., Luo, W., Jiang, Y., 2014. Coupled
response of soil carbon and nitrogen pools and enzyme activities to nitrogen and
water addition in a semi-arid grassland of Inner Mongolia. Plant Soil 381, 323–336.
https://doi.org/10.1007/s11104-014-2129-2.
Wang, R., Dorodnikov, M., Yang, S., Zhang, Y., Filley, T.R., Turco, R.F., Zhang, Y., Xu, Z., Li, H.,
Jiang, Y., 2015. Soil Biology & Biochemistry Responses of enzymatic activities within
soil aggregates to 9-year nitrogen and water addition in a semi-arid grassland. Soil
Biol. Biochem. 81, 159–167. https://doi.org/10.1016/j.soilbio.2014.11.015.
Wang, H., Nie, Y., Butterly, C.R., Wang, L., Chen, Q., Tian, W., Song, B., Xi, Y., Wang, Y., 2017.
Fertilization alters microbial community composition and functional patterns by
changing the chemical nature of soil organic carbon: a field study in a Halosol.
Geoderma 292, 17–24. https://doi.org/10.1016/j.geoderma.2017.01.006.
Xiao, W., Chen, X., Jing, X., Zhu, B., 2018. A meta-analysis of soil extracellular enzyme ac-
tivities in response to global change. Soil Biol. Biochem. 123, 21–32. https://doi.org/
10.1016/j.soilbio.2018.05.001.
Ye, J., Song, Z., Wang, L., Zhu, J., 2016. Metagenomic analysis of microbiota structure evo-
lution in phytoremediation of a swine lagoon wastewater. Bioresour. Technol. 219,
39–444. https://doi.org/10.1016/j.biortech.2016.08.013.
Yue, K., Peng, Y., Peng, C., Yang, W., Peng, X., Wu, F., 2016. Stimulation of terrestrial eco-
system carbon storage by nitrogen addition: a meta-analysis. Sci. Rep. 6, 19895.
https://doi.org/10.1038/srep19895.
Zak, D.R., Pregitzer, K.S., Burton, A.J., Edwards, I.P., Kellner, H., 2011. Microbial responses to
a changing environment: implications for the future functioning of terrestrial ecosys-
tems. Fungal Ecol. 4, 386–395. https://doi.org/10.1016/j.funeco.2011.04.001.
Zak, D.R., Freedman, Z.B., Upchurch, R.A., Steffens, M., Kögel-Knabner, I., 2017. Anthropo-
genic N deposition increases soil organic matter accumulation without altering its
biochemical composition. Glob. Chang. Biol. 23, 933–944. https://doi.org/10.1111/
gcb.13480.
Zeglin, L.H., Stursova, M., Sinsabaugh, R.L., Collins, S.L., 2007. Microbial responses to nitro-
gen addition in three contrasting grassland ecosystems. Oecologia 154, 349–359.
https://doi.org/10.1007/s00442-007-0836-6.
Zhang, Y., Yao, S., Mao, J., Olk, D.C., Cao, X., Zhang, B., 2015. Chemical composition of or-
ganic matter in a deep soil changed with a positive priming effect due to glucose ad-
dition as investigated by 13C NMR spectroscopy. Soil Biol. Biochem. 85, 137–144.
https://doi.org/10.1016/j.soilbio.2015.03.013.
Zhang, L., Zhang, H., Wang, Z., Chen, G., Wang, L., 2016. Dynamic changes of the dominant
functioning microbial community in the compost of a 90-m3 aerobic solid state fer-
mentor revealed by integrated meta-omics. Bioresour. Technol. 203, 1–10. https://
doi.org/10.1016/j.biortech.2015.12.040.
Zhang, Q., Liang, G., Guo, T., He, P., Wang, X., Zhou, W., 2017. Evident variations of fungal
and actinobacterial cellulolytic communities associated with different humified
particle-size fractions in a long-term fertilizer experiment. Soil Biol. Biochem. 113,
1–13. https://doi.org/10.1016/j.soilbio.2017.05.022.
Zhou, L., Zhou, X., Zhang, B., Lu, M., Luo, Y., Liu, L., Li, B., 2014. Different responses of soil
respiration and its components to nitrogen addition among biomes: a meta-
analysis. Glob. Chang. Biol. 20, 2332–2343. https://doi.org/10.1111/gcb.12490.
Zhou, Z., Wang, C., Zheng, M., Jiang, L., Luo, Y., 2017. Patterns and mechanisms of re-
sponses by soil microbial communities to nitrogen addition. Soil Biol. Biochem. 115,
433–441. https://doi.org/10.1016/j.soilbio.2017.09.015.