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SOILS, SEC 1 •SOIL ORGANIC MATTER DYNAMICS AND NUTRIENT CYCLING •RESEARCH
ARTICLE
Soil and microbial biomass stoichiometry regulate soil
organic carbon and nitrogen mineralization in rice-wheat rotation
subjected to long-term fertilization
Muhammad Nadeem Ashraf
1
&Cheng Hu
2
&Lei Wu
1
&Yinghua Duan
1
&Wenju Zhang
1
&Tariq Aziz
3
&Andong Cai
1,4
&
Muhammad Mohsin Abrar
1
&Minggang Xu
1
Received: 21 January 2020 /Accepted: 22 April 2020
#Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Purpose Soil microbial biomass (SMB), as the source and sink of soil nutrients, and its stoichiometry play a key role in soil organic
carbon (SOC) and nitrogen (N) mineralization. The objective of this study was to investigate the responses of SOC and N mineral-
ization to changes in microbial biomass and SOC, N, and phosphorus (P) stoichiometry resulted from long-term fertilization regimes.
Materials and methods Soil was sampled from a rice-wheat rotation system subjected to 37 years of nine fertilization treatments
with different nutrient input amounts: control (CK), N alone, N combined with mineral phosphorus (NP), NP plus potassium
(NPK), manure alone (M), and M combined with N (MN), NP (MNP), NPK (MNPK), and a higher rate of M with NPK
(hMNPK). The sampled soil was incubated for the determination of SOC and N mineralization, C, N, and P stoichiometry of
soil and SMB, and associated soil enzymes related to C and N cycling.
Results and discussion Relative to the CK and treatments with mineral fertilizers, treatments with manure (M, MN, MNP, MNPK, and
hMNPK) significantly increased SOC and N mineralization by 48–78% and 54–97%, respectively. Microbial metabolic quotient (qCO
2
)
decreased by 32–55% in treatments with manure compared to the N and NP treatments, but showed no effect on the qCO
2
when
compared to the NPK treatment. The leucine amino peptidase (LAP) enzyme showed significant positive correlation with SOC and N
mineralization, and negatively related to the qCO
2
. Significantly negative correlations were also observed between SOC and N miner-
alization and soil C:P and N:P ratio, as well as microbial biomass SMBC:SMBP and SMBN:SMBP stoichiometry, respectively. However,
the availability of N and P had limited effects on the qCO
2
after reaching a certain value (0.69–0.72 mg CO
2
-C g
−1
MBC h
−1
).
Conclusions Lower soil elemental (C:P and N:P) and microbial biomass stoichiometry (SMBC:SMBP and SMBN:SMBP) and
increase of LAP resulted from combined application of manure and mineral fertilizers, accelerated SOC, and N mineralization.
Mineral nutrient input with manure amendments could be an optimal strategy to meet the microbial stoichiometric demands and
enhance nutrient availability for crops in agricultural ecosystems.
Keywords Stoichiometry .SOC mineralization .Nitrogen mineralization .Microbial biomass .Nutrient availability .Long-term
fertilization
Responsible editor: Weixin Ding
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11368-020-02642-y) contains supplementary
material, which is available to authorized users.
*Wenju Zhang
zhangwenju01@caas.cn
1
National Engineering Laboratory for Improving Quality of Arable
Land, Institute of Agricultural Resources and Regional Planning,
Chinese Academy of Agricultural Sciences, Beijing 100081, People’s
Republic of China
2
Institute of Plant Protection and Soil Science, Hubei Academy of
Agricultural Sciences, Wuhan 430064, People’s Republic of China
3
Institute of Soil and Environmental Sciences, University of
Agriculture, Faisalabad 38040, Pakistan
4
Key Laboratory for Agro-Environment, Ministry of Agriculture and
Rural Affairs, Institute of Environment and Sustainable Development
in Agriculture, Chinese Academy of Agricultural Sciences,
Beijing 100081, People’sRepublicofChina
Journal of Soils and Sediments
https://doi.org/10.1007/s11368-020-02642-y
1 Introduction
Soil organic carbon (SOC) and nitrogen(N) mineralization are
fundamental processes influencing soil fertility (Zhang et al.
2012). Understanding the processes involved in SOC and N
mineralization is therefore indispensable to improve soil fer-
tility. Fertilization is one of the most popular recommended
practices to sustain and improve fertility of arable land, which
is reported to enhance the soil capacity of nutrient supply, as
well as maintaining SOC levels, thus sustaining high crop
production (Cui et al. 2020; Mohanty et al. 2013).
Fertilization influences the activities and function of soil
microbial biomass (SMB), both of which play an important
role in SOC mineralization and nutrient cycling (Luo et al.
2018;Xuetal.2019). It is well documented that soil nutrient
availability and stoichiometry of SOC to nutrients have sig-
nificant effects on SOC and N mineralization (Kuzyakov and
Xu 2013; Wei et al. 2019). Soil microbial respiration per unit
microbial biomass (qCO
2
), which indicates the energy effi-
ciency that soil microbes required to sustain (Fließbach et al.
2007), is usually used to evaluate the effect of microbial bio-
mass stoichiometry of SOC to nutrients on SOC and N min-
eralization (Cui et al. 2020;Yuetal.2018). An increase or
decrease in the qCO
2
is thought to be the response of soil
microbes by lowing or increasing carbon use efficiency to soil
nutrient availability change caused by fertilization manage-
ment (Allison et al. 2010; Hartman and Richardson 2013).
Microbial biomass stoichiometry and microbial extracellu-
lar enzymes involved in C, N, and phosphorus (P) cycling are
recently used as indicators of SOC and N mineralization. The
stoichiometry of microbial biomass C, N, and P is primarily
regulated by soil nutrient availability, which is the food of soil
microbes (Li et al. 2014; Zhu et al. 2018). According to eco-
logical stoichiometric theory, nutrient limits microbial growth
when the particular nutrient relative to the C falls below the
threshold ratio vital for the optimum growth (Cleveland and
Liptzin 2007). It is reported that long-term fertilization pro-
vides abundant N and P nutrient to boost the microbial bio-
mass and may decrease C:N ratio (Ren et al. 2019). Enzyme
activities reflect microbial nutrient demands, which are deter-
mined by the elemental stoichiometry of microbial communi-
ties associated with nutrient availability (Wei et al. 2019; Zhao
et al. 2018). Soil microorganisms can obtain limiting nutrients
from soil organic matter decomposition by producing extra-
cellular enzymes (Bowles et al. 2014). For example, the activ-
ities of β-glucosidase, cellobiohydrolase, and phosphatases
indicate microbial efforts for energy and P exploitation.
Consequently, changes in the enzyme activities to a certain
extent can reveal nutrient limitation of soil microbes (Zhao
et al. 2018). Manure application can significantly increase
the enzyme activities because soil microorganisms are fre-
quently C-limited, but showing no more effect with higher
application rate of manure (Chakrabarti et al. 2000; Chang
et al. 2007). However, little is known about the response of
SOC and N mineralization to changes in microbial biomass
stoichiometry resulted from long-term manure and mineral N,
and P fertilizer application.
Globally, paddy fields cover approximately 165 million ha
(Ge et al. 2012), and rice-wheat cropping systems cover 9.5–
13.5 million ha in China (Hu et al. 2019). The rotation of
upland-paddy fields is a common land-use type surrounding
the South of the Yangtze River basin to feed the ever-
increasing Chinese population (Hu et al. 2015). Regular inter-
vals of aerobic and anoxic conditions in paddy soils reduce the
decomposition of freshly added organic matter (Qiu et al.
2018), thus leading to accumulation of C stock (Wang et al.
2019;Xuetal.2020). The SOC and N mineralization in rela-
tion to C:N:P stoichiometry remains elusive in this upland-
paddy rotation system subjected to long-term fertilization.
The objectives of the present study were to (i) evaluate the
response of SOC and N mineralization and associated qCO
2
to
long-term mineral fertilizers and manure regimes in wheat-
rice cropping, and (ii) evaluate the impacts of soil enzymes
and SMB stoichiometry on SOC and N mineralization. We
hypothesized that (i) long-term mineral fertilization with ma-
nure would decrease soil, SMB stoichiometry and qCO
2
by
enhancing soil N and P availability, and (ii) decreased micro-
bial biomass stoichiometry would accelerate the SOC and N
mineralization, whereas the enzymes involved in the hydroly-
sis of organic N compounds and protein N cycling would have
effect on qCO
2
due to potential N loss in environment and N
limitation in these fertilizers and manure-treated soil. The
study will contribute to an improved understanding of the
processes of soil C and N cycling driven by changes of mi-
crobial nutrient stoichiometry resulted from fertilization prac-
tices with intensively managed rice-wheat cropping system.
2 Materials and methods
2.1 Site description
The long-term fertilization experiment was initiated in 1981 at
the Nanhu experimental station of the Hubei Academy of
Agricultural Sciences, Wuchang City, Hubei Province,
China (114° 25′E, 30° 28′N). The area has a subtropical
climate with a mean annual temperature of 13 °C. The average
annual precipitation is 1300 mm, and the annual frost-free
period is 240 days. The yellow-brown paddy soil used in this
study belongs to Udalfs with a clay loam texture (USDA soil
classification). At the beginning of the experiment in 1981, the
soil had SOC of 16.9 g C kg
−1
, a pH (1:2.5 soil/water ratio) of
6.30, and total N, phosphorus (P), and potassium (K) contents
of 1.80 g N kg
−1
,1.00gPkg
−1
, and 30.23 g K kg
−1
,respec-
tively. The soil Olsen-P and available K contents were
5.0 mg P kg
−1
and 98.50 mg K kg
−1
,respectively.
J Soils Sediments
2.2 Experimental design
The long-term fertilization experiment consisted of nine treat-
ments. Each treatment was replicated three times in a random-
ized complete block design, and each plot was 40 m
2
(8 m ×
5 m) in size. The fertilization treatments were as follows: no
fertilizer control (CK), N alone, N and P (NP), NP and K
(NPK), organic manure (M), organic manure plus N (NM),
manure plus NP (NPM), manure plus NPK (MNPK), and
MNPK with higher rates of M (hMNPK). Treatments with
N, P, and K (alone or combined) were applied in the form of
urea (300 kg N ha
−1
year
−1
), superphosphate
(150 kg P
2
O
5
ha
−1
year
−1
), and potassium chloride
(150 kg K
2
Oha
−1
year
−1
), respectively. Pig manure, with wa-
ter content of 69%, was applied as organic manure
(22,500 kg ha
−1
year
−1
). The oven-dried manure contained
15.1 g total N kg
−1
,20.8gP
2
O
5
kg
−1
,and
13.6 g K
2
Okg
−1
.The cropping pattern at this site is a rice-
wheat rotation (wheat from early November to early June, and
rice from May to late September). Herein, 40% of inorganic
fertilizers were applied during the wheat growing season and
the remaining 60% were applied during the rice growing pe-
riod. Pig manure was applied equally (1:1) to each crop. The P,
K, and manure were applied once as a basal dose. Whereas in
the rice season, 40% of N fertilizer was applied as a basal
dose, 40% at the tilling stage, and 20% at the booting stage.
For wheat, 50% of the N fertilizer was applied as a basal dose,
25% for the vegetative period, and 25% at the jointing stage.
Prior to sowing, manure and mineral fertilizers were evenly
broadcasted by hand and immediately incorporated into the
soil plow layer (0–20 cm) by tillage.
2.3 Soil sampling and preparation
Soil samples (0–20 cm) were collected from each plot
after the rice harvest in October 2017. Samples were
transported to the laboratory in an icebox, and then
hand-picked to remove visible roots, debris, and gravel
from the soil after sieving (< 2 mm). Subsequently, the
soil was divided into two portions. One portion was air-
dried and passed through 0.25 mm sieve prior to chemical
analyses. The soil moisture content was determined gravi-
metrically by weighing the soil before and after drying at
105 °C. Soil pH was determined using a pH meter (Metro
pH 320; Mettler-Toledo Instruments Ltd., Shanghai,
China). The second portion of soil sample was stored at
4 °C to determine microbial biomass and enzyme activi-
ties, as well as for usage in the incubation experiments.
2.4 Soil chemical and microbiological analyses
Soil organic carbon (SOC) and total N (TN) were analyzed
using a CNHS EURO elemental analyzer (Milan, Italy). Soil
Olsen-P was determined using the 0.5 M NaHCO
3
extraction
method (Olsen et al. 1954). Available N was measured follow-
ing the methods of Magill and Aber (2000), and total P (TP)
was determined using the methods described by Murphy and
Riley (1962).
The SMB of C (SMBC) and N (SMBN) was determined
from pre-incubated, moist soil samples by the chloroform fu-
migation extraction method (Vance et al. 1987)followedby
0.5 M K
2
SO
4
extraction. The dissolve organic carbon (DOC)
was determined using 0.5 M K
2
SO
4
extracts (1:10 w/v) of the
samples and measured multi C/N analyzer (Multi N/C3100,
Analytik jena, AG, Germany). Soil microbial biomass P
(SMBP) was determined from the chloroform fumigation ex-
traction method according to Brookes et al. (1982). Briefly,
fumigated and un-fumigated samples were extracted at a ratio
of 1:20 (soil to 0.5 M NaHCO
3
) after shaking for 1 h. P-spiked
samples received a 1 mL of KH
2
PO
4
solution
(250 μgPmL
−1
) and extracted immediately. The SMBP
values were calculated as the difference between fumigated
and un-fumigated samples. The inorganic P recovery factor
(R) was calculated as 100 × (P
spiked
−P
UF
)/P
spiked
according
to Voroney et al. (2008).
The potential soil extracellular enzymes of β-glucosidase
(BG), cellobiohydrolase (CBH), N-acetyl-glucosaminidase
(NAG), leucine aminopeptidase (LAP), and acid phosphatase
(AP) were selected because of their closely linked to C and N
cycling and determined using method followed by DeForest
(2009). Briefly, a soil suspension was prepared by homoge-
nizing 1.0 g of fresh soil with 100 mL of freshly prepared
sodium acetate (C
2
H
3
NaO
2
) buffer (50 mM). The pH of the
buffer was adjusted to the mean of the tested soil (Table 1).
Subsequently, soil suspension, acetate buffer (200 μL), and
substrates (50 μL) were dispensed into 96-well black micro-
plates which were incubated in the dark at 25 °C for 4 h.
Finally, fluorescence intensity was quantified using the micro-
plate reader (SynergyH1, BioTek) with 365 nm excitation and
450 nm emission filters.
2.5 Incubation for carbon and nitrogen mineralization
Soil incubation for SOC mineralization was carried out for
28 days in the laboratory. For this purpose, moist sieved soil
subsamples (equivalent to 25 g of dry weight) were placed
into 250 mL Schott airtight jars. Small vials containing
10 mL of 0.5 M NaOH were placed at the soil surface in the
jars to trap evolved CO
2
. Samples were incubated at 25 °C,
and soil moisture was maintained at 55% of the soil water
holding capacity by weighing the jars and adding the quantity
of lost water to the soil. The evolved CO
2
trapped in the vials
was measured on days 1, 2, 3, 4, 7, 14, 21, and 28 after the
incubation. In order to measure CO
2
, vials were removed, and
the jars were opened for 30 min to refresh the air before new
vials with fresh NaOH solution were placed in the jars. The
J Soils Sediments
evolved CO
2
trapped in the NaOH solution was then analyzed
by a multi C/N analyzer (Multi N/C3100, Analytik Jena, AG,
Germany).
Soil N mineralization was carried out following the classic
method of Stanford and Smith (1972). Approximately 15 g of
soil and acid-washed sand quartz (1:1) was placed in polyvi-
nyl chloride leaching tubes. The leached samples were col-
lected after 0, 7, 14, 21, and 28 days with the addition of
100mLof0.01MCaCl
2
,followedby25mLofN-freesolu-
tion. Sample tubes were incubated at 25 °C for 28 days. After
leaching, samples were immediately analyzed for mineral N
(NH
4
+
-N and NO
3
−
-N) using a continuous flow analyzer
(Foss FIASTAR 5000 Analyzer).
2.6 Calculations and statistical analyses
Cumulative SOC mineralization was calculated as the sum of
evolved CO
2
over the 28 days. The SOC mineralization po-
tential (C
0
) was calculated by fitting the dynamics of C min-
eralization with the first-order kinetic model as follows:
Cmin ¼C01−e−kt
;ð1Þ
where C
min
is the cumulative mineralized C (mg kg
−1
), C
0
is
the mineralizable SOC potential (mg kg
−1
), and kis the rate
constant of SOC mineralization (day
−1
).
Soil cumulative N mineralization was calculated using the
sum of mineral N over the 28 days. Nitrogen mineralization
potential (N
0
) was also calculated using the first-order kinetic
model:
Nmin ¼N01−e−kt
;ð2Þ
where N
min
is the cumulative mineralized N (mg kg
−1
), N
0
is
the mineralizable N potential (mg kg
−1
), and kis the rate
constant of N mineralization (day
−1
). Model fitting for SOC
and N mineralization was performed using the global fit curve
wizard in SigmaPlot 13.0 software package (Systat Software,
Inc., Chicago, IL, USA).
Microbial metabolic quotient (qCO
2
) is an index of micro-
bial efficiency utilizing the available SOC and was calculated
as qCO
2
=CO
2
/SMBC and expressed as mg CO
2
-
Cg
−1
MBC h
−1
.
The stoichiometry of the microbial biomass of C, N, and P
was calculated as SMBC:SMBN, SMBC:SMBP, and
SMBN:SMBP, respectively. Similarly, the stoichiometry of soil
elemental C, N, and P was calculated as described previously.
2.7 Statistical analyses
Statistical analyses were performed using SPSS v21.0 (IBM
SPSS Statistics; Chicago, NY, USA) and graphs were plotted
using SigmaPlot 13.0. A one-way ANOVA was used to ana-
lyze the data, and Duncan’s multiple comparisons were per-
formed to identify significant differences between the treat-
ment means at P< 0.05. Regression analysis was used to an-
alyze relationships among determined soil parameters.
3 Results
3.1 Response of soil nutrients, microbial biomass,
and enzymes to long-term fertilization
Soil chemical properties after long-term fertilization are sum-
marized in Table 1. The SOC, TN, and TP were higher under
the combined treatments (MN, MNP, MNPK, hMNPK) rela-
tive to the CK and applications of mineral fertilizer treatments
(N, NP, NPK). Available nitrogen (AN) and Olsen-P signifi-
cantly increased in these combined treatments compared to the
CK and mineral fertilizer treatments (P< 0.05). Moreover,
Table 1 Soil chemical properties after 37 years of various fertilization regimes
Treatments SOC (g kg
−1
)TN(gkg
−1
)TP(gkg
−1
)AP(mgkg
-1)
AN (mg kg
−1
)pH(1:2.5)
CK 17.92 ± 0.66d 1.83 ± 0.04d 0.61 ± 0.05d 5.28 ± 0.18g 27.34 ± 0.50g 6.92 ± 0.02cd
N 19.41 ± 0.69cd 1.97 ± 0.02cd 0.55 ± 0.01d 4.45 ± 0.42g 29.22 ± 0.65f 6.99 ± 0.06bcd
NP 17.30 ± 1.97d 1.93 ± 0.11cd 0.72 ± 0.03d 17.37 ± 0.77f 32.6 ± 0.76e 6.93 ± 0.02cd
NPK 19.50 ± 1.34cd 2.13 ± 0.09c 0.86 ± 0.07cd 31.90 ± 0.61e 42.37 ± 0.58d 6.91 ± 0.04d
M 23.79 ± 1.36bc 2.59 ± 0.12b 1.42 ± 0.52bc 83.63 ± 2.86d 66.46 ± 0.53a 7.19 ± 0.04a
MN 25.09 ± 1.74ab 2.76 ± 0.08b 2.02 ± 0.29b 159.25 ± 1.32b 60.87 ± 0.70b 7.19 ± 0.04a
MNP 28.10 ± 0.38ab 3.12 ± 0.04a 2.76 ± 0.06a 179.58 ± 1.88a 65.57 ± 0.50a 7.06 ± 0.06abc
MNPK 29.44 ± 1.98a 3.17 ± 0.04a 2.78 ± 0.09a 183.08 ± 1.88a 46.72 ± 0.68a 7.13 ± 0.08ab
hMNPK 29.52 ± 0.53a 3.28 ± 0.07a 3.26 ± 0.11a 123.75 ± 1.32c 61.66 ± 0.49b 7.14 ± 0.02a
Data are the mean values ± SE (n= 3); different lower case letters show the significant difference among the treatments at P<0.05
SOC, soil organic carbon; TN, total nitrogen; TP, total phosphorus; AP, available phosphorus; AN, available nitrogen; pH (1:2.5), measured with soil to
water ratio as 1:2.5; CK, control; N, nitrogen; NP, nitrogen and phosphorus; NPK, nitrogen, phosphorus, and potassium; M, manure; MN, nitrogen plus
manure; MNP, manure plus NP; MNPK, manure plus NPK; hMNPK, high rate of manure and NPK
J Soils Sediments
soil pH ranged from 6.92 to 6.99 under the CK and mineral
fertilizer treatments, while the M and combined treatments
improved pH to 7.06–7.19 (Table 1).
Long-term fertilizer applications significantly increased the
soil microbial biomass (Fig. 1). The M and combined treat-
ments with manure had significant higher SMBC and SMBP
than the mineral fertilizer treatments (e.g., N, NP, and NPK)
(P<0.05). The combined treatments (MN, MNP, MNPK, and
hMNPK) led to 977–1323 mg kg
−1
for SMBC and 115–
279 mg kg
−1
for SMBN, which were 1.3–2.2 and 1.3–4.0
times higher than those for the mineral fertilizer treatments,
respectively (Fig. 1a, b). The SMBP was increased by 3.8
times under mineral fertilizer treatments, and by 5–12 times
under the combined treatments, compared to the CK treatment
(Fig. 1c). The microbial metabolic quotient (qCO
2
) varied
from 0.70 to 1.18 mg CO
2
-C g
−1
MBC h
−1
among treatments
(Fig. 1d). Relative to CK, qCO
2
significantly decreased
(P< 0.05) under the NPK and manure treatments.
Extracellular enzyme activities varied among fertilization
treatments (Fig. 2). Under the mineral fertilizer treatments, the
BG and CBH activities were significantly increased by 8–67%
and 96–276%, respectively, compared to the CK (Fig. 2a). In
contrast, compared to the CK, the M alone significantly in-
creased NAG and LAP enzyme activities, whereas the mineral
fertilizers and the combined treatments showed little effect on
the LAP and NAG except the M and hMNPK (Fig. 2b). In
addition, mineral P fertilizer and manure addition significantly
increased AP activity compared to the CK, while manure ad-
dition (e.g., the MNP and MNPK) showed no effect on AP
activity compared to the NP (Fig. 2c).
3.2 Soil organic carbon and nitrogen mineralization
The dynamics of SOC and N mineralization over the 28 days
of incubation was shown in Fig. 3. At the end of the incuba-
tion period, cumulative CO
2
evolved was 6% and 12% lower
under the N and NP treatments, respectively, relative to CK.
The M and combined treatments (MN, MNP, MNPK, and
hMNPK) significantly increased cumulative CO
2
evolved by
35% and 48–78% as compared to CK, respectively (Fig. 3b).
The pattern of N mineralization over the incubation time
showed a similar trend as SOC mineralization (Fig. 3c).
Results of N mineralization revealed that the mineral fertilizer
treatments showed an increase of 9–27% in N mineralization,
whereas the M and combined treatments exhibited an increase
of 54–97% relative to the CK (Fig. 3d).
In contrast, SOC and N mineralization data fitted the first-
order kinetic equations significantly (R
2
=0.98–0.99 and R
2
=
0.93–0.99, respectively) (Table S1). Soil organic carbon min-
eralization potential (C
0
) increased significantly (P< 0.05) un-
der the combined treatments compared to the mineral fertil-
izers (e.g., N, NP, NPK), M, and CK. The N mineralization
(N
0
) potentials ranged from 43.9 to 45.7 mg N kg
−1
under the
combined treatments, 30.2–34.9 mg N kg
−1
under the mineral
fertilizer treatments, and 32.5 mg N kg
−1
under the CK.
The rate constant for SOC mineralization (K
C
)varied
among these fertilization treatments, while the lowest K
C
val-
ue was recorded in the MNPK. In contrast, the rate constant
for N mineralization (K
N
) showed an increasing trend in the
mineral fertilized treatments, while manure treatments showed
uneven trends. However, the highest K
N
was recorded in the
MNPK treatment (Table S1).
Fig. 1 Effect of different treatments on asoil microbial biomass carbon
(SMBC), bsoil microbial biomass nitrogen (SMBN), csoil microbial
biomass phosphorus (SMBP), and dmicrobial metabolic quotient
(qCO
2
) subjected to 37 years of various fertilizer regimes. Different
lowercase letters show significant differences among the treatments at
P<0.05
J Soils Sediments
3.3 Effects of soil and microbial biomass
stoichiometry on SOC and N mineralization
Soil and microbial biomass stoichiometry of C, N, and P had
significant effects on SOC and N mineralization (Fig. 4). SOC
and N mineralization significantly decreased with an increase
in soil C:P and N:P ratios (Fig. 4a–d). The increased
SMBC:SMBP ratio also resulted in an exponential decreasing
tendency with SOC mineralization (R
2
=0.61,P<0.001)and
N mineralization (R
2
=0.57, P< 0.001) (Fig. 4e, f).
Correspondingly, SOC and N mineralization suggested a sig-
nificant decline with increase in SMBN:SMBP (Fig. 4g, h).
Further analysis suggested that SOC and N mineralization
were significantly positively correlated with soil C:P and
N:P ratios, as well as the availability of SOC, N, and P
(Table S2,P< 0.001). Moreover, qCO
2
showed significantly
negative response to increase in SOC and N mineralization
(Fig. 5a, b) and DOC, N, and P availability (Fig. 5b, c, d,
P< 0.001), suggesting increased soil nutrient availability de-
creased the qCO
2
.
Regarding soil enzyme activities, results showed that
SOC and N mineralization, and qCO
2
were significantly
correlated to LAP enzyme, which was involved in the
hydrolysis of organic N compounds and proteins
(Fig. 2d–f). A significantly increasing trend was observed
between SOC (R
2
=0.69, P< 0.01) and N mineralization
(R
2
=0.58, P< 0.01) with the increase of LAP enzyme
activity (Fig. 2d, e). As expected, increase in LAP obtain-
ed a decreasing tendency in the qCO
2
(R
2
= 0.42,
P< 0.01; Fig. 2f).
Fig. 2 Soil enzymes and its effect on soil organic carbon and nitrogen
mineralization and qCO
2
subjected to 37 years of various fertilizer
regimes. Different lowercase letters show significant differences among
the treatments at P<0.05.β-glucosidase (BG); cellobiohydrolase (CBH);
N-Acetyl-glucosaminidase (NAG); leucine aminopeptidase (LAP); acid
phosphatase (AP); microbial metabolic quotient (qCO
2
)
J Soils Sediments
4 Discussion
Long-term manure and mineral fertilizer application consider-
ably increased SOC and N accumulation, as well as soil mi-
crobial biomass, obtaining averaged SMBC consisted of 3.7%
of SOC, SMBN constituted 4.5% of total N, and SMBP com-
prised 2.1% of total P. These accumulations of SOC and N
were due to slow decomposition of native soil C (Qiu et al.
2018) and abundant supply of exogenously added manure
(Boye et al. 2017; Luo et al. 2018;Wangetal.2019).
During soil organic matter mineralization, the most crucial
fractions of SOC, N, and P become part of the microbe, such
as phospholipids and proteins which were released at the later
stages upon microbial turnover (Aira and Domínguez 2014;
Singh and Gupta 2018). Correspondingly, the significant in-
crease in the microbial biomass under long-term fertilization
in this study indicated higher microbial metabolic activities
(Geisseler and Scow 2014). In addition, a higher soil N avail-
ability resulted from manure addition boost microbial metab-
olism and decomposition of added manure, thereby enhancing
SOC and N mineralization (Liu et al. 2019). The rates of SOC
and N mineralization were relatively high than those observed
Fig. 3 Cumulative mineralization
of organic carbon (a,b)and
nitrogen (c,d) from soil subjected
to 37 years of various fertilizer
regimes. Different lowercase
letters show significant
differences among the treatments
at P<0.05
J Soils Sediments
in previous research with rice-rice cropping (Mohanty et al.
2013). The reason was mainly due to the unique upland-paddy
rotation in our study which has shown the effect to accelerate
soil organic matter turnover (Cai et al. 2016). Thus, boosted
microbial biomass size benefited soil enzyme activities related
to SOC and N mineralization.
Our results suggested that long-term manure amendments
in paddy soil increased the SOC and N mineralization poten-
tial as well as rate constant for SOC and N mineralization. The
variations in the mineralization constant for SOC and N min-
eralization resulted from long-term fertilizer regimes were
mainly due to the stability of SOC under various protection
mechanisms of SOC fractions (Wang et al. 2019;Xuetal.
2019). Our findings convinced that SOC and N mineralization
were also enhanced by the dissolved organic C (DOC), Olsen-
P, and N availability, which was strongly improved by manure
and mineral fertilizer application (Mooshammer et al. 2014).
Our result indicated that increase in soil pH after manure ad-
dition had a positive effect on SOC and N mineralization. The
fact that soil pH tended to increase after organic manure
applied due to cation enrichment contributes to high SOC
and N mineralization (Murugan and Kumar 2013). And also,
the optimal soil pH in the present study (6.92–7.19) was ben-
eficial for soil nutrient (especially P) availability in paddy soil
(Aziz et al. 2011; Shi et al. 2015). The current study showed
that the manure application decreased the proportion of C-
acquiring enzymes but increased N-acquiring enzymes, indi-
cating that the addition of specific nutrients decreased the
activity of particular enzymes (Zhou et al. 2017). In addition,
increasing soil N availability could boost the production of C-
degrading enzymes and decreased the N-acquiring enzyme in
response to N fertilization (Dunn et al. 2014; Lin et al. 2017).
The leucine amino peptidase (LAP) enzyme showed signifi-
cant positive correlations with SOC and N mineralization,
suggesting its significant role in the depolymerization of pro-
teins (Mooshammer et al. 2014). Considering that protein was
comprised of 60% of the total N in plant and microbial cells
(Geisseler et al. 2010), LAP could be the limiting step for N
mineralization. One of the reasons for increased aminopepti-
dase activity was the labile constituents after manure amended
Fig. 4 Relationships among mineralization of soil organic carbon (SOC) and nitrogen (N) and stoichiometry of soil C:P, N:P and microbial biomass
SMBC:SMBP, SMBN:SMBP
Fig. 5 Correlations among microbial metabolic quotient (qCO
2
) and soil organic carbon (SOC) and nitrogen (N) mineralization, dissolved organic
carbon (DOC), and NH
4
+
-N + NO
3
−
-N and P availability (Olsen-P)
J Soils Sediments
(Caruso 2010). Thus, the long-term combined application of
manure and mineral fertilization increased enzyme activities,
promoted SOC, and N accumulation and resulted in increased
soil fertility.
Our result showed that SOC and N mineralization had de-
creasing trends with increase in soil and microbial biomass
stoichiometry of C:P and N:P. This result was proven that
microbial biomass stoichiometry depended on soil stoichiom-
etry in arable land (Mooshammer et al. 2014). Therefore, soil
stoichiometry could be considered as an indicator for SOC
and N mineralization, implying that long-term manure appli-
cation decreased soil elemental and microbial stoichiometry
by enhancing the microbial biomass pool via abundant nutri-
ent input. Moreover, a lower C:N ratio which was to be used
by soil microbes under the combined treatments also contrib-
uted to the increased SOC and N mineralization (Soares and
Rousk 2019; Zhu et al. 2018). Moreover, the observed de-
creasing trend of SOC and N mineralization with increase in
soil and microbial C:P and N:P ratio highlighted the impor-
tance of soil P for augmenting microbial activities.
Additionally, stoichiometric constraints boosted the microbial
C utilization efficiency which allowed more SOC to be con-
verted into microbial biomass (Zhu et al. 2018). Increased
mineralization with higher nutrient availability was mainly
due to the shift in microbial composition and enzyme activi-
ties (Schloter et al. 2018). It was observed that with lower
microbial biomass stoichiometry, SOC and N mineralization
increased. Concomitantly, an increase of sufficient C supply
(DOC) through manure and mineral fertilization in the present
study met the microbial stoichiometric requirements which
was confirmed by high available N, DOC, and Olsen-P, which
mostly likely decreased the microbial mining of existing soil
organic matter (Wang et al. 2019). Therefore, long-term ma-
nure and mineral fertilizers enhanced nutrient availability and
decreased soil and microbial nutrient stoichiometry to accel-
erate SOC and N mineralization.
Our result indicated that the microbial metabolic quotient
(qCO
2
) decreased by the combined mineral fertilization with
manure relative to CK. The lower qCO
2
value resulted from
manure application suggested higher microbial C utilization
efficiency which was related to high quantities of available
nutrients. These findings indicated that soil microorganisms
could uptake available nutrients to enhance microbial growth
such as protein synthesis rather than overflow respiration
(Chen et al. 2019). The decreased qCO
2
after manure and
mineral fertilization indicated that microorganisms could alle-
viate soil nutrient mining the native SOC (Luo et al. 2019).
Therefore, long-term nutrient (N and P) application played an
important role in improving the microbial C use efficiency
(low qCO
2
). This finding was consistent with the finding from
Zang et al. (2017), where nutrient-rich conditions increased
microbial C use efficiency in wheat rhizospheres. Our result
reported that the qCO
2
showed negative correlations with
increase in available N (NH
4
+
-N + NO
3
−
-N), DOC, and
Olsen-P, indicated that increase in the microbial nutrient sup-
plying capacity enhanced the microbial C use efficiency in
terms of the decreased qCO
2
(Wang et al. 2019). However,
the less sensitive response from non-linear regressions be-
tween the qCO
2
and Olsen-P and mineral N suggested that
microbial metabolic quotient was less limited by N and P
availability after reaching at a certain level.
5 Conclusion
Manure application enhanced SOC and N mineralization via
enhancing the size of microbial biomass and enzyme activi-
ties. Mineral P fertilizer and manure addition increased AP
activity, while manure addition showed no more effect on
AP activity. Soil organic carbon mineralization potential in-
creased with long-term combined mineral fertilizer and ma-
nure application. We found that the SOC and N mineralization
were negatively regulated by the soil elemental C:P and N:P
ratios, and microbial biomass stoichiometry (SMBC:SMBP
and SMBN:SMBP). The leucine amino peptidase activity
had positive effect on SOC and N mineralization, whereas it
showed a negative effect on qCO
2
. Moreover, increase in
DOC, N, and P availability in response to fertilization en-
hanced the microbial C utilization in terms of qCO
2
.
Therefore, long-term manure amendments could be an opti-
mal strategy to meet microbial stoichiometric demands and
enhance nutrient availability for crops in agricultural
ecosystems.
Acknowledgments We gratefully acknowledge the financial support pro-
vided by the National Natural Science Foundation of China (Grant num-
bers 41877105, 41907093) and The Fundamental Research Funds for
Central Non-profit Scientific Institution (1610132019044,
1610132019013). We also acknowledged the anonymous reviewers for
their constructive suggestions on this research.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Informed consent No informed or ethical consent was required for this
research.
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