Agriculture, Ecosystems and Environment 362 (2024) 108850

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Agriculture, Ecosystems and Environment

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

Biofertilizer supplements allow nitrogen fertilizer reduction, maintain

yields, and reduce nitrogen losses to air and water in China paddy fields
Mingcheng Hu a, Huaiwen Xue a, Andrew J. Wade b, Nan Gao c, Zijian Qiu a, Yaou Long a,
Weishou Shen a, *
a
Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Collaborative Innovation Center of Atmospheric Environment and Equipment
Technology, and School of Environmental Science and Engineering, Nanjing University of Information Science and Technology, Nanjing 210044, China
b
Department of Geography and Environmental Science, University of Reading, Reading RG6 6DW, UK
c
National Engineering Research Center for Biotechnology, School of Biological and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ammonia (NH3) volatilization, nitrous oxide (N2O) emission and mineral nitrogen leaching from paddy fields are
Reactive nitrogen closely related to nitrogen fertilizer application. Excessive nitrogen fertilizer application has exacerbated adverse
NH3 volatilization environmental effects, including global warming, atmospheric haze and groundwater pollution. Reducing the
N2O emission
nitrogen application rate could alleviate negative environmental effects, but simultaneously brings risks of yield
Nitrogen leaching
CH4 emission
reduction. Biofertilizers, also known as microbial fertilizers, utilize microorganisms to improve the effectiveness
of concurrent nitrogen fertilizers and have been shown to help mitigate the adverse effects of nitrogen fertilizer
while ensuring rice yield. This study, which is one of the first field-scale trials of N2O-mitigating biofertilizers,
focused on a typical paddy field in East China and investigated 9 treatments that combined mineral nitrogen
fertilizer and N2O-mitigating biofertilizer applications to investigate yield and nitrogen loss effects over three
years. The results showed reducing nitrogen combined with the N2O-mitigating biofertilizers increased rice yield
by up to 26%, and simultaneously reduced N2O emission, mitigated nitrogen leaching loss, and had no significant
impact on NH3 volatilization and methane (CH4) emissions. This is an important result and suggests that wider-
scale adoption of N2O-mitigating biofertilizers could help reduce the environmental footprint of rice production
whilst maintaining, or even improving, rice yield.

1. Introduction acidic compounds to produce other secondary pollutants including

Nitrogen fertilizer is a critical crop nutrient, promoting growth and
increasing yield. However, there is widespread excessive application of
mineral nitrogen fertilizer in the agricultural systems of China with
approximately 20 million tons (pure nitrogen) applied annually, ac­
counting for a quarter of the global nitrogen fertilizer usage (Lin et al.,
2007; Qiao et al., 2012), and the annual nitrogen fertilizer consumption
in the paddy fields of China is about 6 million tons, which is approxi­
mately 37% of the global nitrogen fertilizer consumption in paddy fields
(Lin et al., 2007; Qiao et al., 2012; Feng et al., 2017). Long-term
excessive nitrogen fertilizer use in paddy fields will reduce soil
fertility, worsen soil structure, and increase the loss of reactive nitrogen,
leading to an increase in ammonia (NH3) volatilization, greenhouse gas
emissions, and nitrogen leaching (Dillon et al., 2012; Zhao et al., 2014;
Chen et al., 2015). The NH3 released to the atmosphere can react with
NH4HSO4, which can subsequently enter wetland, forest, lake, grassland
and other ecosystems through atmospheric transport and sedimentation,
posing a risk of water eutrophication and soil acidification (Giltrap et al.,
2010; Chatterjee et al., 2018). NH3 can also react to generate particles,
including NH4NO3 and (NH4)2SO4, which act as condensation nuclei to
generate PM2.5, promoting the formation of atmospheric haze (Giltrap
et al., 2010; Liang et al., 2017; Lee et al., 2021). Paddy fields are an
important source of nitrous oxide (N2O) and methane (CH4) emission
(Bhattacharyya et al., 2013; Das and Adhya, 2014; Song et al., 2021) and
N2O is a greenhouse gas with a global warming potential (GWP) 273
times that of CO2 on a 100-year scale, contributing approximately 6% to
the global greenhouse effect (Shen et al., 2022; IPCC, 2023). N2O can
also disrupt O3 in the stratosphere, leading to an increased risk of human
skin cancer and other diseases (Ravishankara et al., 2009; Reay et al.,
2012). As with NH3, N2O in the atmosphere can produce secondary

* Corresponding author.
E-mail address: wsshen@nuist.edu.cn (W. Shen).

https://doi.org/10.1016/j.agee.2023.108850
Received 3 August 2023; Received in revised form 10 November 2023; Accepted 6 December 2023
Available online 19 December 2023
0167-8809/© 2023 Elsevier B.V. All rights reserved.
M. Hu et al.
pollutants such as nitrate particles through a series of oxidation re­
actions, which poses further soil and water acidification risk (Guo et al.,
2010; Qiao et al., 2012). CH4 is a greenhouse gas with a GWP 27 times
that of CO2 on a 100-year scale, contributing approximately 14% to the
global greenhouse effect (Shen et al., 2022). Nitrogen leaching from rice
fields mainly includes NO-3-N and NH+ 4 -N, which causes eutrophication
and can have long residence times in groundwater, creating legacy
pollution issues into the future (Zhao et al., 2009, Islam et al., 2021).
Given these adverse effects, reducing nitrogen fertilizer usage is essen­
tial. However, reducing nitrogen input alone will lead to yield reduction
which is undesirable given the competing need for food security and the
United Nations Sustainable Development Goal of zero hunger.
Biofertilizers, which contain microorganisms, have been demon­
strated to reduce the amount of nitrogen fertilizer used, improve soil
physicochemical properties, enhance crop yield, and alleviate the
environmental negative effects brought about by chemical fertilizers
(Habibi et al., 2014; Mia et al., 2010; Urashima et al., 2005). Common
biofertilizers include rhizobia biofertilizer, nitrogen-fixing bacteria
biofertilizer and antibiotic biofertilizer. Biofertilizers produced with the
inclusion of N2O-mitigating microbes (N2O-mitigating biofertilizer) are
novel and have received less attention than other biofertilizers. Initial
studies have shown that the application of N2O-mitigating biofertilizers
can not only increase crop yield, but simultaneously reduce soil N2O
emissions (Ishii et al., 2011; Tago et al., 2011; Itakura et al., 2013; Gao
et al., 2016). However, there are few studies on N2O-mitigating bio­
fertilizers in China with few, if any, field trials. N2O-mitigating bio­
fertilizers include microbes that directly or indirectly stimulate the final
denitrification step, promoting N2O to N2 conversion (Hayatsu et al.,
2008). The microbes used include bacteria with the nosZ gene that
synthesizes active nitrous oxide reductase (N2OR) and other microor­
ganisms without the nosZ gene, to promote complete denitrification to
N2. The microbes can be bacteria or fungi, though existing N2O-miti­
gating microbes are generally denitrifying bacteria (Ashida et al., 2010;
Ishii et al., 2011; Tago et al., 2011; Xu et al., 2018). For example, Ishii
et al. (2011) isolated an N2O-reducing bacterium Burkholderia sp.
TSO47–3 containing nirS and nosZ genes from paddy soil in Japan,
which exhibited extremely low denitrification activity for reducing NO-3
and NO-2, but strong N2O reducing activity. In recent years, microor­
ganisms with the ability to mitigate N2O have been discovered and
isolated sequentially and enhancement of their abundance and activity
in the soil seems key to decrease soil N2O emissions (Bakken et al.,
2020). There are two possible mechanisms underlying the N2O emission
mitigation. One mechanism is that, following biofertilizer application,
the bacteria in biofertilizer could directly reduce N2O to N2, thereby
decreasing the N2O emissions from agricultural soils. A second mecha­
nism is that the applied microbes alter the microbial communities of the
N2O-reducing bacteria, possibly recruiting N2O-reducing bacteria to
stimulate their reduction of N2O to N2. The applied microbes may also
change the N2O-generating microbial communities, leading to
decreased N2O emissions (Calvo et al., 2016; Gao et al., 2017; Nishizawa
et al., 2014).
Currently, research on biofertilizers with N2O mitigating effects is
typically done under microcosm and potted conditions; there is little
study under field conditions. Under microcosm and potted conditions,
biofertilizers with N2O mitigating effects have achieved excellent results
in reducing N2O emissions and promoting crop growth and yield (Ita­
kura et al., 2013; Nishizawa et al., 2014; Calvo et al., 2016; Gao et al.,
2016; Wu et al., 2018; Xu et al., 2018). Existing research also focuses on
yield and N2O emission and there is a need to quantify other potential
reactive nitrogen loss pathways, namely ammonia emission and
ammonium and nitrate leaching. Further, following a literature review,
there appears to be little research on the N2O-mitigating biofertilizers
applications in China, especially in paddy ecosystems. To help address
these knowledge gaps, this study aims to quantify the effects of applying
combinations of mineral nitrogen fertilizer and N2O-mitigating bio­
fertilizers on yield, nitrogen gaseous emissions (NH3 and N2O) and
2
Agriculture, Ecosystems and Environment 362 (2024) 108850
-
leaching (NH+4 -N and NO3-N) in a paddy field in East China for three
consecutive years.
2. Materials and methods
2.1. Experimental site
The experimental site was in Liuhe, Nanjing, China (118.69◦ E,
32.58◦ N), located in the Chuhe River Basin within the lower reaches of
the Yangtze River Plain in East China. The climate is temperate
monsoon, with high summer air temperatures and rainfall and cold, dry
winters. The mean annual precipitation is 942 mm and the mean annual
average temperature is 15.6 ℃ (Fig. 1). The rice variety used in this
experiment was Ninggeng 8 which is planted commonly by local farmers
with a growth period of 125–130 days. The soil is an Anthrosol with pH
(H2O) of 5.9, organic carbon of 25.4 g kg− 1 soil, total nitrogen of
1.68 g kg− 1 soil, available phosphorus of 32 mg kg− 1 soil, and available
potassium of 108 mg kg− 1 soil.
2.2. N2O-mitigating biofertilizers preparation
The bacteria selected for making N2O-mitigating biofertilizers were
inoculated into a culture medium, which was an improved beef extract
peptone medium (known as NBNS culture medium). The medium con­
sisted of 5.0 g l− 1 polypeptone, 3.0 g l− 1 beef extract, 0.3 mmol l− 1 so­
dium nitrate and 4.4 mmol l− 1 sodium succinate. The solvent was water.
The culture medium was placed in a shaker at 160 rpm and 28 ℃ for
more than 24 hours. To select strains with approximately the same
concentration, a sterile NBNS activated liquid culture medium was used
to adjust the bacterial solution under 600 nm ultraviolet visible light,
with an absorbance of 1.0. For each N2O-mitigating bacteria, 500 mL of
bacterial solution and 2 kg of organic fertilizer (with a nitrogen content
of 1%), which served as a carrier for the N2O-mitigating bacteria, were
thoroughly mixed to produce the final biofertilizer for each field plot
(20 m2).
2.3. Field plot experimental design and field management
This study used six types of N2O-mitigating biofertilizers, namely
TSA2S, N2, NRCB002, NRCB008, NRCB010, and NRCB026, whose N2O-
mitigating functional microorganisms were Azospirillum sp., Bacillus
subtilis, Bacillus subtilis subsp., Paenibacillus sp., Pseudomonas stutzeri and
Bacillus velezensis, respectively (Table 1). The experiment used 9 treat­
ments: no nitrogen fertilizer application (0%N), 100% of typical
farmers’ nitrogen fertilizer usage (100%N) (256 kg N ha− 1), 80% of the
typical farmers’ nitrogen fertilizer usage (80%N) (205 kg N ha− 1), 80%
N and N2O-mitigating biofertilizer with TSA2S (TSA2S), 80%N and N2O-
mitigating biofertilizer with N2 (N2), 80%N and N2O-mitigating bio­
fertilizer with NRCB002 (NRCB002), 80%N and N2O-mitigating bio­
fertilizer with NRCB008 (NRCB008), 80%N and N2O-mitigating
biofertilizer with NRCB010 (NRCB010), as well as 80%N and N2O-
mitigating biofertilizer with NRCB026 (NRCB026). The nitrogen fertil­
izer was urea, and the application amount of biofertilizer for each bio­
fertilizer treatment was a mixture of 250 L bacterial liquid and 1000 kg
commercial organic fertilizer per hectare (Table 1). All treatments were
distributed using a random grouping design. Each treatment had 4
replicates, resulting in a total of 36 field plots. Each plot size was 4 m ×
5 m, separated by a 0.6 m wide ridge to prevent one plot affecting those
adjacent.
The nitrogen fertilizer was applied in three separate stages: basal,
tillering and panicle, in the ratio 6:3:1 (Table 1). Phosphorus fertilizer
(calcium magnesium phosphate) and potassium fertilizer (potassium
chloride) were added also: 60 kg ha− 1 (as P2O5) and 105 kg ha− 1 (as
K2O), respectively. The phosphorus and potassium fertilizers and the
biofertilizers were applied during basal fertilization (Table 1). Since
each biofertilizer treatment plot included 2 kg organic fertilizer and
M. Hu et al.
Fig. 1. The dynamic variation of precipitation, daily maximum and minimum temperature during the rice-growing season from 2019 to 2021.
500 mL NBNS culture medium, the 80% N and 100% N treatments also
included 2 kg organic fertilizer and 500 mL sterile NBNS culture me­
dium to ensure equivalent nitrogen applications (Table 1). The nitrogen
content of the biofertilizers used in this study was very low and almost
negligible, and its fertilizer efficiency was achieved through the pro­
moting effect of functional microorganisms.Table 1
This experiment was done continuously for three years starting in
June 2019 (Fig. S1), and the planting and field management methods of
rice were done according to local farming practice, which were also the
optimal field management methods recommended by the local govern­
ment for farmers based on production practices. Firstly, this involves
plowing and irrigation, followed by basal fertilizer application and rice
planting. After planting, there was a mid-season drying period of
approximately 10 days to promote seedling rooting. Tillering fertilizer
was applied approximately 20 days after rice planting. The field was
then irrigated, and panicle fertilizer applied. The irrigation was a dry-
wet alternation. In the later stage of rice growth, irrigation was not
applied (Fig. S1).
2.4. The batch-type airflow enclosure method
A batch-type airflow enclosure method was used to measure NH3
volatilization (Zhou et al., 2016; Fig. S2). The method used boric acid to
absorb the NH3 volatilized and then the absorption solution was titrated
3
Agriculture, Ecosystems and Environment 362 (2024) 108850
with dilute sulfuric acid to obtain the volume of dilute sulfuric acid
consumed by titration. The NH3 volatilization flux was calculated ac­
cording to the sulfuric acid volume as (Zhou et al., 2016; Eq. 1):
F=V × 10− 3
× C × 0.014 × 104 × π− 1
× r− 2
×6 (1)
.
where F is the NH3 volatilization flux (kg N ha− 1 d− 1), V is volume of
sulfuric acid for titration (mL), 10− 3 is volume conversion factor, C is
calibration concentration of sulfuric acid for titration (mol l− 1), 0.014 is
relative atomic mass of nitrogen atom (kg mol− 1), 104 is area conversion
factor, r is radius of air chamber (m), and 6 is the ratio of 24 h to daily
NH3 volatilization collection time.
2.5. Static chamber-gas chromatographic techniques
The N2O and CH4 emissions were measured using static chamber-gas
chromatography (Li et al., 2020; Fig. S3). The sampling box was made of
opaque PVC with a length, width and height of 50, 50 and 60 cm,
respectively. A thermometer was installed through the box top to mea­
sure the air temperature inside and a sampling port was set on the side.
When sampling, the groove of the base was filled with water and the
static dark box was placed into the groove to ensure airtightness. Gas
samples were collected at 0, 15, and 30 minutes after the device was
sealed and the air temperature inside the box during the collection was
M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Table 1 Table 1 (continued )

Fertilization and N2O-mitigating bacteria in different experimental treatments. Treatment Name of Basal fertilization Tillering Panicle
Treatment Name of Basal fertilization Tillering Panicle bacterial fertilization fertilizer
bacterial fertilization fertilizer strain
strain
biofertilizer (A
0%N / Calcium magnesium / / mixture of 250 L
phosphate (P2O5 NRCB008 bacterial
content: 12 %) liquid and 1000 kg
500 kg ha− 1, commercial organic
potassium chloride fertilizer was applied
(K2O content: 60 %) per hectare)
175 kg ha− 1 NRCB010 Pseudomonas Urea 175 kg ha− 1, Urea 165 kg Urea
80%N / Urea (nitrogen Urea 165 kg Urea stutzeri calcium magnesium ha− 1 55 kg
content: 46 %) ha− 1 55 kg phosphate 500 kg ha− 1
175 kg ha− 1, calcium ha− 1 ha− 1, potassium
magnesium chloride 175 kg ha− 1,
phosphate 500 kg NRCB010
ha− 1, potassium biofertilizer (A
chloride 175 kg ha− 1, mixture of 250 L
sterile NBNS culture NRCB010 bacterial
medium 250 L ha− 1, liquid and 1000 kg
commercial organic commercial organic
fertilizer (nitrogen fertilizer was applied
content: 1 %) per hectare)
1000 kg ha− 1 NRCB026 Bacillus Urea 175 kg ha− 1, Urea 165 kg Urea
100%N / Urea 260 kg ha− 1, Urea 205 kg Urea velezensis calcium magnesium ha− 1 55 kg
calcium magnesium ha− 1 70 kg phosphate 500 kg ha− 1
phosphate 500 kg ha− 1 ha− 1, potassium
ha− 1, potassium chloride 175 kg ha− 1,
chloride 175 kg ha− 1, NRCB026
sterile NBNS culture biofertilizer (A
medium 250 L ha− 1, mixture of 250 L
commercial organic NRCB026 bacterial
fertilizer 1000 kg liquid and 1000 kg
ha− 1 commercial organic
TSA2S Azospirillum Urea 175 kg ha− 1, Urea 165 kg Urea fertilizer was applied
sp. calcium magnesium ha− 1 55 kg per hectare)
phosphate 500 kg ha− 1
ha− 1, potassium
chloride 175 kg ha− 1, recorded. The samples were sent to the laboratory for determination of
TSA2S biofertilizer N2O and CH4 concentrations using a gas chromatograph (Agilent
(A mixture of 250 L
GC-7890B, Agilent Technologies, USA). The column temperature of the
TSA2S bacterial
liquid and 1000 kg gas chromatograph was 60 ℃, and the carrier gas used was 99.999%
commercial organic high-purity N2. The N2O concentrations in the gas samples were
fertilizer was applied analyzed using an electron-capture detector of the gas chromatograph.
per hectare) The working temperature was 400 ℃, the make-up gas was 5% argon
N2 Bacillus subtilis Urea 175 kg ha− 1, Urea 165 kg Urea
calcium magnesium ha− 1 55 kg
methane standard gas (99.999%), and the flow rate was 2 mL min− 1.
phosphate 500 kg ha− 1 The CH4 concentrations were analyzed using a flame-ionization detector
ha− 1, potassium of the gas chromatograph. The working temperature was 250 ℃, the
chloride 175 kg ha− 1, combustion gas was 99.999% high-purity H2, and the flow rate was
N2 biofertilizer (A
60 mL min− 1. The N2O and CH4 emission fluxes were calculated using
mixture of 250 L N2
bacterial liquid and Eq. (2) (Li et al., 2020):
1000 kg commercial
organic fertilizer was F = ρ × H × (Δc/Δt) × 273/(273 + t) (2)
applied per hectare)
NRCB002 Bacillus subtilis Urea 175 kg ha− 1, Urea 165 kg Urea .
subsp. calcium magnesium ha− 1 55 kg where F is the emission flux of N2O (μg m− 2 h− 1) or CH4 (mg m− 2
phosphate 500 kg ha− 1 h− 1); ρ is the gas density of N2O (1340 μg cm− 3) or CH4
ha− 1, potassium (0.717 mg cm− 3) at standard conditions (0 ℃, 101 kPa); H is the height
chloride 175 kg ha− 1,
of the chamber above the water layer (m); Δc/Δt is the cumulative rate
NRCB002
biofertilizer (A of N2O (μg m− 3 h− 1) or CH4 (mg m− 3 h− 1) in the chamber, and t is the
mixture of 250 L mean temperature inside the chamber at each sampling (℃).
NRCB002 bacterial
liquid and 1000 kg
commercial organic
2.6. Leachate collection
fertilizer was applied
per hectare)
NRCB008 Paenibacillus Urea 175 kg ha− 1, Urea 165 kg Urea The leachate was collected using a porous PVC pipe with an outer
sp. calcium magnesium ha− 1 55 kg diameter of 10 cm and a small hole at the bottom (Fig. S4). The pipe was
phosphate 500 kg ha− 1 wrapped with a 100-mesh nylon to prevent soil entry. A hose was con­
ha− 1, potassium
chloride 175 kg ha− 1,
nected to the top of the PVC pipe to collect the leachate and, when the
NRCB008 hose was not connected, the hole was sealed to prevent rainwater
entering. During installation, the leachate collection devices were
inserted vertically into the soil, to collect leachate 30 cm, 60 cm, and

4
M. Hu et al.
90 cm below the soil surface. The top of the device was 30 cm higher
than the soil to prevent surface water from entering the pipe. During
sampling, the hose inserted into the bottom was used to extract leachate
at different depths using a handheld pump and the volume of the
leachate was recorded. The leachate filtered in the laboratory and NH+4-
N and NO-3-N concentrations measured using the indophenol blue
-
colorimetry for NH+4 -N and spectrophotometry for NO3-N.
2.7. Calculation of the cumulative N2O and CH4 emission, GWP and
GHGI of N2O and CH4
The cumulative emissions of N2O and CH4 were calculated using Eq.
(3) (Hu et al., 2023):
/
∑n
S= (Fi + Fi+1 ) 2 × (ti+1 − ti ) × 24 (3)
i=1
where S is the cumulative N2O emission (g N ha− 1) or CH4 emission (kg
ha− 1); i is the ith sampling; n is the total number of sampling times; Fi is
the N2O (μg m− 2 h− 1) or CH4 (mg m− 2 h− 1) emission flux for the ith
sampling; (ti+1 - ti) is the number of days between the ith sampling and
the (i + 1)th sampling.
The global warming potential (GWP) was estimated using the radi­
ative forcing potential of the two greenhouse gases compared to the
equivalent for carbon dioxide (eq-CO2) based on a 100-year time hori­
zon (Mosier et al., 2005). The GWP of 1 kg N2O is 273 kg eq-CO2, and
1 kg CH4 is 27 kg eq-CO2. The GWP of N2O and CH4 was calculated by
the following Eq. (4) (Hu et al., 2023; IPCC, 2023):
GWP = 273 × S (N2O) + 27 × S (CH4)
.
where S (N2O) is the cumulative N2O emission (kg ha− 1); and S (CH4)
is the cumulative CH4 emission (kg ha− 1).
The greenhouse gas intensity (GHGI, kg eq-CO2 kg− 1) which relates
the global warming potential to the rice yield was calculated as Eq. (5)
(Qi et al., 2020):
GHGI = GWP/Y
.
where Y is the rice yield (kg ha− 1).
2.8. Calculation of nitrogen loss ratio in leachate
The proportion of accumulated nitrogen loss in the leachate was
calculated as Eq. (6):
R=T/C
.
where R is the ratio of NO-3-N or NH+ 4 -N loss to nitrogen application
amount (%); T is the cumulative amount of NO-3-N or NH+ 4 -N (kg N
ha− 1); and C is the total nitrogen application amount (kg N ha− 1).
2.9. Statistical analyses
Least significant difference (LSD) of one-way ANOVA was used for
significance analysis, Pearson correlation was done, and two-way
ANOVA was used to determine the effects of biofertilizers and experi­
mental years on the experimental results. All statistical analyses were
done using IBM SPSS Statistics 21.0.
3. Results
3.1. Rice yields
In general, the application of N2O-mitigating biofertilizers enhanced
Agriculture, Ecosystems and Environment 362 (2024) 108850
rice yield during the rice-growing seasons from 2019 to 2021 (Fig. 2). In
2019, 2020 and 2021, the 80%N treatment resulted in yield reductions
of 11, 22 and 20% compared to the 100%N, respectively (all P > 0.05).
Compared to the 80%N treatments, those treatments with biofertilizers
had, in 2019, yield increases of 11–16%, with TSA2S producing the
highest total yield of 9291 kg ha− 1. In 2020, treatments with bio­
fertilizers increased yield by 5–54% relative to the 80%N treatment. Of
the biofertilizer treatments, NRCB002 resulted in the highest yield of
10368 kg ha− 1. In 2021, again compared with the 80%N treatment, all
biofertilizer treatments resulted in yield increases of 1–25%, with TSA2S
producing the highest yield of 11125 kg ha− 1 (Fig. 2).
3.2. NH3 volatilization
During the rice-growing seasons from 2019 to 2021, different
fertilization treatments all showed peak NH3 volatilization within 3 days
after fertilization, and NH3 volatilization was mainly concentrated
within the week following fertilization. NH3 volatilization from rice
fields was primarily concentrated in the basal and tillering fertilizer
stages because the nitrogen application rate during the panicle fertilizer
stage was lower (Fig. 3). Compared with the 0%N treatment, the NH3
volatilization for all treatments applying nitrogen was higher. For the
80%N treatment, NH3 volatilization was significantly lower than that for
the 100%N treatment, and the peak NH3 volatilization for the 80%N
treatment decreased from 2019 to 2021 by 66%, 48%, and 27% for the
three consecutive years. After applying biofertilizers, there was no sig­
nificant change in NH3 volatilization compared to the 80%N treatment.
From 2019–2021, the cumulative NH3 volatilization under various
(4)
(5)
(6)
Fig. 2. Rice yields under different fertilization treatments from 2019 to 2021
(0%N: no nitrogen fertilizer; 80%N: 80% of local traditional nitrogen applica­
tion amount; 100% N: 100% of local traditional nitrogen application amount;
TSA2S: 80%N with TSA2S biofertilizer; N2: 80% N with N2 biofertilizer;
NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008
biofertilizer; NRCB010: 80%N with NRCB010 biofertilizer; NRCB026: 80%N
with NRCB026 biofertilizer). Different small letters above bars meant signifi­
cant difference among treatments at 5% level.
5
M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Fig. 3. Ammonia volatilization flux and cumulative ammonia volatilization from different fertilization treatments during the rice-growing season from 2019 to 2021
(0%N: no nitrogen fertilizer; 80%N: 80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application amount; TSA2S:
80%N with TSA2S biofertilizer; N2: 80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008 biofertilizer;
NRCB010: 80%N with NRCB010 biofertilizer; NRCB026: 80%N with NRCB026 biofertilizer). The red arrows indicate fertilization events, and B, T and P represent
basal fertilizer, tillering fertilizer, and panicle fertilizer, respectively. Different small letters above bars meant significant difference among treatments at 5% level.

nitrogen application treatments all significantly increased compared to
the 0%N treatment (P < 0.05). Compared with 100%N, the cumulative
NH3 volatilization of 80% N decreased by 44% (P < 0.05), 17% (P >
0.05), and 40% (P < 0.05) through 2019, 2020 and 2021, respectively.
Comparing the cumulative NH3 volatilization among all the treatments
from 2019 to 2021 for all biofertilizers treatments, then relative to the
80%N treatment, there was no significant difference (P > 0.05). Overall,
based on the results of the three-year experiment, reducing nitrogen
appears to effectively reduce NH3 volatilization from rice fields, which
showed a positive correlation between NH3 volatilization and nitrogen
application rates, but the combination of nitrogen reduction and
applying biofertilizers has no significant effect on NH3 volatilization
from rice fields (Fig. 3).
3.3. N2O emissions
The N2O emissions were mainly concentrated during the dry-wet
alternation and the mid-season drying periods, with low N2O emis­
sions otherwise (Fig. 4 and S1). In 2019, the N2O emission fluxes of each
treatment ranged from − 42 to 674 μg N m− 2 h− 1. For the 80%N treat­
ment, the peak N2O emission flux decreased by 66% compared to the
flux for the 100%N treatment. After applying biofertilizers, then
compared with 80%N treatment, the peak N2O emission flux of the N2,
NRCB008 and NRCB010 treatments decreased by 37%, 15%, and 13%,
respectively. Furthermore, a smaller N2O emission peak was also
observed for the 100%N and 80%N treatments after applying panicle
fertilizer (Fig. 4a). In 2020, the N2O emission fluxes ranged from − 79 to
185 μg N m− 2 h− 1, and there was a significant decrease compared to
2019. N2O emission was mainly concentrated during the mid-season
drying period and a large N2O emission peak occurred 16 days after
the basal fertilizer application (July 8, 2020). After applying bio­
fertilizers, the N2O emission of different treatments decreased markedly
compared to 2019, and the N2O emission flux was negative for most of
the rice-growing period. On the eighth day after the basal fertilizer
application (June 30, 2020), a period of N2O absorption occurred.
Except for the TSA2S treatment, all biofertilizer treatments showed
peaks of N2O absorption, ranging from − 79 to − 12 μg N m− 2 h− 1
(Fig. 4b). In 2021, the N2O emission fluxes ranged from − 75 to
95.5 μg N m− 2 h− 1, further reduced compared to 2019 and 2020, and
there was only a notable peak of N2O emission during the dry-wet
alternation period (August 19, 2021). After applying biofertilizers,
N2O emission was also further reduced compared to 2019 and 2020, and
N2O flux was mostly negative. On the twentieth day after the application
of basal fertilizer (July 19, 2021), a discernible peak period of N2O
absorption occurred, with N2O absorption peaks ranging from − 75 to
− 8 μg N m− 2 h− 1 (Fig. 4c).
After reducing nitrogen and applying biofertilizers then, compared
to the 80%N treatment, in 2019, the cumulative N2O emissions from the
N2, NRCB008, NRCB026, and NRCB010 treatments decreased by 55, 27,
24, and 21% (all P > 0.05), respectively (Fig. 4d). In 2020, the cumu­
lative N2O emissions for N2, NRCB008, and NRCB010 treatments were
negative and therefore N2O sinks. Compared with the 80%N, the N2O
emissions from the N2, NRCB008, NRCB010, NRCB026, NRCB002 and
TSA2S treatments reduced by 216 (P < 0.05), 181 (P < 0.05), 135 (P <
0.05), 89 (P > 0.05), 78 (P>0.05) and 39% (P > 0.05), respectively
(Fig. 4e). In 2021, the N2, NRCB008, NRCB010, and NRCB026 treat­
ments all had a negative cumulative N2O emission. Compared with the
80%N, the N2O emission from the NRCB002, TSA2S, NRCB026,
NRCB008, N2 and NRCB010 treatments decreased by 16 (P > 0.05), 79
(P > 0.05), 120 (P > 0.05), 130 (P < 0.05), 142 (P < 0.05) and 168% (P
< 0.05), respectively (Fig. 4f). The N2O emission from paddy fields had
a decreasing trend year by year, and the effect of applying N2O-miti­
gating biofertilizers on mitigating N2O emission increased year on year
(Fig. 4). Among all the six biofertilizers, N2, NRCB008, and NRCB010

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Fig. 4. Dynamic variation of nitrous oxide emission flux and cumulative nitrous oxide emission from different fertilization treatments during the rice-growing season
from 2019 to 2021 (0%N: no nitrogen fertilizer; 80%N: 80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application
amount; TSA2S: 80%N with TSA2S biofertilizer; N2: 80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008
biofertilizer; NRCB010: 80%N with NRCB010 biofertilizer; NRCB026: 80%N with NRCB026 biofertilizer). The red arrows indicate fertilization events, and B, T and P
represent basal fertilizer, tillering fertilizer, and panicle fertilizer, respectively. Different small letters above bars meant significant difference among treatments at
5% level.

have the largest N2O alleviation effect.
3.4. CH4 emissions
The CH4 emissions were concentrated during the flooding period. For
the 80%N treatment, the peak CH4 emission flux did not change mark­
edly compared to the 100%N treatment. After applying biofertilizers,
the CH4 emission flux of each treatment did not show obvious changes
(Fig. S5). Compared with the 0%N treatment, the cumulative CH4
emission for the different fertilization treatments increased significantly
increased compared to that for the 80%N treatment in 2019 (Fig. 6). In
2020 and 2021, the response was different, with decreases in NO-3-N
leaching at all three depths for the biofertilizer treatments relative to the
leaching for the 80%N treatment. Some of the reductions were large,
especially for 90 cm during 2020 (a 41–60% reduction) (Fig. 6). Overall,
the 80%N treatment with the application of N2O-mitigating bio­
fertilizers reduced NO-3-N leaching and the effect was enhanced over the
three years.
3.6. NH+
4 -N leaching

(P < 0.05). Compared with the 100%N treatment, the cumulative CH4
emission from the 80%N treatment was not significantly different (P > NH+4 -N leaching occurred mainly during the basal fertilizer period

0.05). For the biofertilizer treatments, the cumulative CH4 emission and was significantly smaller than the NO-3-N leaching (Figs. 7 and 8).
similarly did not change markedly (P > 0.05) from that resultant from The NH+ 4 -N concentrations were lower as depth increased and the NH4 -
+

the 80%N treatment (Fig. S5). The CH4 emission had an increasing trend N leaching in 2019 was significantly higher than that in 2020 and 2021.
year by year, which was opposite to that for N2O emission. In summary, the NH+ 4 -N leaching among different treatments had no
regular change, and the observations from the three-year field experi­
ment did not show regular patterns with either treatment or year. For
3.5. NO-3-N leaching
the 80%N treatment, compared to the 100%N treatment, the NH+ 4 -N
concentration increased slightly in most cases which suggests that
From 2019–2021, NO-3-N leaching occurred after fertilization
reducing the nitrogen fertilizer input did not significantly reduce NH+ 4 -N
mainly, and the leachate NO-3-N concentration gradually decreased with
leaching. After applying N2O-mitigating biofertilizers, NH+ 4 -N leaching
depth (Fig. 5). Compared to the 0%N treatment, the NO-3-N leachate
among the different treatments still did not display any regular patterns,
concentration increased significantly for the other fertilization treat­
and the effect of biofertilizers on NH+ 4 -N leaching was insignificant
ments. After applying biofertilizers, the NO-3-N concentration in most
(Fig. 8). The experimental results reflected the complexity of NH+ 4 -N
treatments increased compared to the 80%N treatment in 2019 at all
leaching and the low concentrations. The proportion of NO-3-N leaching
depths yet decreased in 2020 and 2021. The cumulative NO-3-N leaching
to total nitrogen application was much greater than that of NH+ 4 -N, and
for all treatments reduced with increasing depth. For the 80%N treat­
NO-3-N was the main form of nitrogen leaching (Tables 2 and 3).
ment, the NO-3-N leaching at 30, 60 and 90 cm depths decreased by 35,
17 and 61% compared of that for the 100%N treatment, respectively.
After applying biofertilizers, the NO-3-N leaching in most treatments

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Fig. 5. Dynamic variation of nitrate nitrogen concentrations of leachate at different depths during the rice-growing season from 2019 to 2021 (0%N: no nitrogen
fertilizer; 80%N: 80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application amount; TSA2S: 80%N with TSA2S
biofertilizer; N2: 80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008 biofertilizer; NRCB010: 80%N with
NRCB010 biofertilizer; NRCB026: 80%N with NRCB026 biofertilizer).

Fig. 6. Cumulative leaching amount of nitrate nitrogen at different depths during the rice-growing season from 2019 to 2021 (0%N: no nitrogen fertilizer; 80%N:
80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application amount; TSA2S: 80%N with TSA2S biofertilizer; N2:
80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008 biofertilizer; NRCB010: 80%N with NRCB010 bio­
fertilizer; NRCB026: 80%N with NRCB026 biofertilizer).Different small letters above bars meant significant difference among treatments at 5% level.

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Fig. 7. Dynamic variation of ammonium nitrogen concentrations of leachate at different depths during the rice-growing season from 2019 to 2021 (0%N: no nitrogen
fertilizer; 80%N: 80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application amount; TSA2S: 80%N with TSA2S
biofertilizer; N2: 80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008 biofertilizer; NRCB010: 80%N with
NRCB010 biofertilizer; NRCB026: 80%N with NRCB026 biofertilizer).

Fig. 8. Cumulative leaching amount of ammonium nitrogen at different depths during the rice-growing season from 2019 to 2021 (0%N: no nitrogen fertilizer; 80%
N: 80% of local traditional nitrogen application amount; 100%N: 100% of local traditional nitrogen application amount; TSA2S: 80%N with TSA2S biofertilizer; N2:
80%N with N2 biofertilizer; NRCB002: 80%N with NRCB002 biofertilizer; NRCB008: 80%N with NRCB008 biofertilizer; NRCB010: 80%N with NRCB010 bio­
fertilizer; NRCB026: 80%N with NRCB026 biofertilizer).Different small letters above bars meant significant difference among treatments at 5% level.

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Table 2
The ratio of nitrate nitrogen loss to nitrogen application amount in different fertilization treatments. Data are presented as the mean ± standard error. The different
small letters following each mean and standard error refer to the significant difference among treatments at 5 % level.
Treatment Ratio of NO-3-N loss to nitrogen application in 2019 Ratio of NO-3-N loss to nitrogen application in 2020 Ratio of NO-3-N loss to nitrogen application in
(%) (%) 2021 (%)

30 cm 60 cm 90 cm 30 cm 60 cm 90 cm 30 cm 60 cm 90 cm

80%N 5.82±2.09b 5.66±2.92b 3.40±0.88a 7.91±1.88ab 11.00±3.33ab 9.55±4.77a 9.87±1ab 6.95±4.25a 5.87±2.45a
100%N 9.04±1.71ab 5.49±1.31b 7.02±2.57a 11.86±6.09a 17.30±11.25a 12.46±12.98a 10.17±2.5a 7.55±3.42a 6.92±2.16ab
TSA2S 16.70±7.05a 15.65±9.49a 3.94±2.22a 7.32±3.54ab 8.84±6.40ab 4.97±2.36a 8.94±2.86a 6.15±3.5a 3.61±2.23ab
N2 11.0±8.13ab 5.97±1.73b 7.16±5.42a 6.15±1.65b 4.98±2.09b 3.89±2.21a 9.28±4.09a 4.83±4.09a 2.93±2.18b
NRCB002 13.63±7.96ab 4.85±3.54b 3.62±1.92a 5.69±1.33b 4.30±2.14b 3.83±2.44a 7.95±3.1ab 5.99±3.1a 2.82±1.51b
NRCB008 14.96±6.85ab 13.67±4.52a 6.23±3.76a 5.68±3.45b 10.84±8.25ab 5.66±4.81a 8.63±3.56ab 4.94±1.97a 3.36±1.97ab
NRCB010 14.37±6.29ab 10.63±7.34ab 5.73±5.28a 6.73±1.91b 7.04±4.99b 4.40±2.44a 9.73±2.93ab 5.5±2.93a 4.23±2.93ab
NRCB026 5.29±1.70b 4.03±1.78b 2.30±0.45a 5.71±1.96b 8.40±2.76ab 8.99±6.79a 7.46±2.27b 4.02±1.63a 3.07±1.63ab

Table 3
The ratio of ammonium nitrogen loss to nitrogen application amount in different fertilization treatments. Data are presented as the mean ± standard error. The
different small letters following each mean and standard error refer to the significant difference among treatments at 5 % level.
Treatment Ratio of NH+
4 -N loss to nitrogen application in 2019 Ratio of NH+
4 -N loss to nitrogen application in 2020 Ratio of NH+
4 -N loss to nitrogen application in 2021
(%) (%) (%)

30cm 60cm 90cm 30cm 60cm 90cm 30cm 60cm 90cm

80%N 0.21±0.79a 1.60±1.40ab 1.38±1.11a 0.66±0.41a 0.43±0.40a 0.20±0.24a 0.18±0.04bc 0.53±0.07a 0.27±0.25a
100%N 2.51±3.43a 0.09±0.20b 2.52±1.22a 0.34±0.19ab 0.22±0.29a 0.23±0.39a 0.53±0.2a 0.26±0.16b 0.12±0.02ab
TSA2S 0.95±2.17a 0.01±0.16b 2.87±2.80a 0.26±0.23ab 0.19±0.23a 0.03±0.02a 0.07±0.04c 0.18±0.06b 0.08±0.03b
N2 3.76±3.91a 0.67±0.73ab 0.44±0.52a 0.57±0.42ab 0.18±0.18a 0.03±0.04a 0.08±0.01c 0.25±0.14b 0.1±0.05ab
NRCB002 6.54±9.21a 0.82±0.31ab 1.36±1.56a 0.35±0.37ab 0.04±0.01a 0.01±0.01a 0.1±0.04bc 0.2±0.14b 0.12±0.08ab
NRCB008 2.33±2.66a 3.85±5.03a 1.70±1.78a 0.13±0.09b 0.19±0.10a 0.12±0.16a 0.4±0.03ab 0.2±0.11b 0.05±0.01b
NRCB010 1.11±0.98a 2.51±2.78ab 6.16±9.17a 0.26±0.24ab 0.64±1.17a 0.21±0.23a 0.25±0.17ab 0.24±0.14b 0.06±0.03b
NRCB026 0.29±0.41a 0.08±0.07b 0.89±1.08a 0.10±0.03b 0.16±0.17a 0.15±0.15a 0.3±0.21ab 0.27±0.11b 0.07±0.04b

4. Discussion thereby potentially achieving yield increase. Furthermore, Bacillus sub­

4.1. Effects of N2O-mitigating biofertilizers on rice yields
This study found that reducing nitrogen input by 20% (the 80%N
treatment) resulted in rice yield reduction. This may be because the
nitrogen application rate selected by local farmers (100%N) is the
optimal nitrogen application rate recommended by the local govern­
ment. Reducing nitrogen limited the growth of rice plants, leading to a
decrease in rice yield. However, applying N2O-mitigating biofertilizers
had an apparent growth promoting effect with increased rice yields
evident for TSA2S, NRCB002, NRCB008 and NRCB026 in 2019,
NRCB002 in 2020 and 2021, and the yield exceeded that of the 100%N
treatment (Fig. 2). The results suggest applying N2O-mitigating bio­
fertilizers could replace chemical nitrogen fertilizer dosage by 20%, with
no significant difference in rice yield (Fig. 2). The yield increases are
likely due to the rhizosphere promoting effect of the biofertilizers used
(Calvo et al., 2016; Gao et al., 2016) though this remains to be
demonstrated through process-based studies. The results of the
three-year field experiment showed that TSA2S and NRCB002 had the
best growth promoting effect among the six biofertilizers (Fig. 2). TSA2S
and NRCB002 are Azospirillum sp. and Bacillus subtilis subsp., respectively
(Table 1). Azospirillum sp. has been shown to produce crop growth reg­
ulators and has nitrogen-fixing capacity (Tailor et al., 2014). Bacillus
subtilis also has plant growth enhancement capability and produces a
variety of compounds that are resistant to bacteria, fungi, and viruses,
strengthening the ability of crops to resist pathogenic microorganisms,
tilis also has certain effects of dissolving phosphorus, calcium, and po­
tassium, which can thereby improve soil fertility and increase crop yield
(Verma et al., 2018). Table 4
4.2. Effects of N2O-mitigating biofertilizers on NH3 volatilization from
rice paddy fields
In this study, NH3 volatilization typically occurred within one week
of fertilization and the NH3 volatilization peak occurred within three
days of fertilization, which was likely a response to surface water NH+4 -N
concentration increases (Zhou et al., 2016; Uddin et al., 2021). After
urea was applied to the paddy, it was rapidly hydrolyzed into NH+ 4 -N
under the action of urease, causing a rapid NH+ 4 -N concentration in­
crease in the field surface water, and subsequent NH3 volatilization in
the first three days after fertilization. NH3 volatilization rapidly
decreased after reaching a peak because, as volatilization progressed,
the concentration of NH+ 4 -N in surface water continuously reduced, and
therefore NH3 volatilization also rapidly decreased (Fig. 3 and S6). After
applying N2O-mitigating biofertilizers, there was no obvious change in
NH3 volatilization compared to 80%N in the three-year experiment (P >
0.05) most likely because NH3 volatilization, being a physical-chemical
process, was affected mainly by the field surface water NH+ 4 -N concen­
tration (Table 5) (Sun et al., 2019; Lee et al., 2021). In this study, the
effect of applying N2O-mitigating biofertilizers on the concentration of
NH+ 4 -N in surface water was insignificant (Fig. S6), thereby resulting in
no remarkable impact on NH3 volatilization compared to the 80%N

Table 4
A two-way ANOVA for rice yield, ammonia volatilization, nitrous oxide emission, methane emission, nitrate nitrogen and ammonium nitrogen leaching losses. *, **
and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. NS: not significant.
Yield NH3 N2O CH4 NO-3-N (30 cm) NO-3-N (60 cm) NO-3-N (90 cm) NH+
4 -N (30 cm) NH+
4 -N (60 cm) NH+
4 -N (90 cm)

Biofertilizer *** NS *** NS NS * NS NS NS NS

Year *** NS *** *** ** NS NS *** ** ***
Biofertilizer × Year NS NS *** NS NS * NS NS NS NS

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Table 5
Correlation analysis between ammonia volatilization, greenhouse gas emissions, nitrogen leaching from rice fields and field surface water, soil physicochemical
properties as well as environmental factors during the rice-growing season from 2019 to 2021. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively.
Precipitation Air Soil Soil NO-3-N in NH+ 4 -N in NO-3-N in field NH+4 -N in field pH in field
temperature temperature moisture soil soil surface water surface water surface water

NH3 -0.482* 0.552* 0.236 -0.004 0.174 0.204 0.371 0.874*** 0.678*
volatilization
N2O emission -0.792** 0.203 0.207 -0.773*** 0.437* 0.096 0.137 0.112 0.236
CH4 emission 0.933*** 0.452 0.562* 0.852*** 0.312 -0.107 0.012 0.211 0.145
NO-3-N leaching 0.124 0.017 -0.118 0.204 -0.009 0.127 0.109 0.083 0.052
NH+4 -N 0.560* 0.133 0.067 0.115 0.043 0.072 0.092 0.243 -0.064
leaching

treatment.
4.3. Effects of N2O-mitigating biofertilizers on N2O emission from rice
field
The field experiment outcomes showed that the application of N2O-
mitigating biofertilizers could significantly reduce N2O emission from
rice fields (Fig. 4). There are four major production pathways of soil
N2O: nitrification, denitrification, denitrification by nitrifiers and
dissimilatory nitrate reduction to ammonium (DNRA) (Howarth, 2008;
Stein, 2020; Timilsina et al., 2020). The only known biological sink of
N2O is the biochemical reduction of N2O to N2 through N2O reductase
produced by denitrifying bacteria with the nosZ gene, which is the last
step in the complete denitrification process of
NO-3→NO-2→NO→N2O→N2 (Gao et al., 2017). Among the six N2O-mi­
tigating biofertilizers, the N2O-mitigating bacteria in TSA2S and
NRCB010 possess the nosZ gene that encodes N2OR, which could
directly promote the process of N2O reduction to N2 and decrease N2O
emission. By contrast, the N2O-mitigating bacteria in N2, NRCB002,
NRCB008, and NRCB026 do not possess the nosZ gene, but they may
promote the growth and reproduction of soil N2O-reducing bacteria with
the nosZ gene through microbial collaboration, and improve overall
microbe community abundance and activity, indirectly enhance the N2O
reduction process, thereby decreasing N2O emission (Gao et al., 2016;
Timilsina et al., 2020). It is worth noting that the N2O mitigation effect
of TSA2S with nosZ gene is weaker than that of N2, NRCB002, NRCB008,
and NRCB026 without nosZ gene (Fig. 4). This indicates that the pres­
ence or absence of the nosZ gene is not an absolute control factor for
alleviating N2O emission. Factors such as the colonization of functional
microorganisms, soil properties, crops, and meteorological conditions
may affect the normal functioning of N2O-mitigating bacteria, and
further work is required to quantify the relative importance of each. The
microbial function in the selected biofertilizers must be suitable for the
soil-crop-environment, to promote crop growth and reduce adverse
environmental effects.
In this study, the cumulative N2O emission from rice fields treated
with N2, NRCB008, and NRCB010 in 2020 and NRCB010, N2,
NRCB008, and NRCB026 in 2021 were all negative (Fig. 4). This indi­
cated that rice paddy fields can absorb N2O from the atmosphere as a
sink of N2O, which is consistent with previous studies (Chapuis-Lardy
et al., 2007; Hu et al., 2023). The results suggested that, for the
N2O-mitigating biofertilizer treatments, the N2O emission reductions
increase year on year (Fig. 4). A possible reason is that biofertilizers
directly or indirectly stimulated an increased abundance of soil
N2O-reducing microbial communities, enhancing N2O reduction to N2,
and not only consume N2O produced by the soil, but also consume N2O
absorbed from the atmosphere. A second reason might be that excessive
precipitation in 2020 and 2021 resulted in long-term high levels of soil
moisture, enhancing soil N2OR activity and promoting N2O reduction to
N2 and limiting N2O transport and diffusion, and ultimately led to low
N2O emission (Abdalla et al., 2009; Banerjee et al., 2016; Dowhower
et al., 2020).
The three-year field experiment showed that applying biofertilizers
had no significant effect on CH4 emissions (Table 4 and Fig. S5). This is
possibly because CH4 emissions from rice fields were mostly affected by
soil moisture conditions (Bharali et al., 2018). This study showed a
highly significant positive correlation (P < 0.001) between CH4 emis­
sion and soil moisture condition (Table 5 and Fig. S7). Moreover, the
results of this study indicated that CH4 emission from rice fields was
primarily concentrated during the flooding period, while N2O emission
was mainly concentrated during the mid-drying period; CH4 emissions
increased year on year, while N2O emission was decreased and therefore
there was a significant negative correlation between N2O and CH4 (P <
0.01; Table 6) consistent Hu et al., (2023). Paddy soil in the flooded
period is an anaerobic environment with low redox potential and this is
conducive to methanogenic archaea growth and CH4 production. The
soil moisture conditions during the mid-drying period were suitable for
the coupling of soil microbial nitrification and denitrification processes
which, in turn, was conducive to N2O emission (Cheng et al., 2021;
Maier et al., 2021; Serrano-Silva et al., 2014). However, with the
application of N2O-mitigating biofertilizers, the global warming poten­
tial (GWP) and greenhouse gas emission intensity (GHGI) of N2O in rice
fields was reduced (Table 7).
4.4. Effects of N2O-mitigating biofertilizers on nitrogen leaching from rice
fields
The results show that NO-3-N was the major form of nitrogen leaching
and NH+ 4 -N leaching was relatively small. This is likely because posi­
tively charged NH+ 4 is easily adsorbed by negatively charged soil colloids
and is less likely to move vertically down the soil profile or leach from
the soil, thus remaining in the upper and middle soil layers. NO-3-N is
repelled by the negative charges of soil mineral colloids and humus, and
NO-3-N accumulated in soil is easily leached with downward drainage
(Zhao et al., 2009, Islam et al., 2021). In this study, there was no reg­
ularity of NH+ 4 -N leaching under different treatments though patterns
are hard to discern given the low concentrations. Under the same ni­
trogen application rate, compared to the 80%N treatment, the bio­
fertilizer applications appeared to reduce NO-3-N leaching (Figs. 5, 6).
This is likely because the functional microorganisms in the biofertilizers
are N2O-mitigating bacteria with N2O emission mitigation effects, which
could enhance the soil denitrification process, improve the utilization
rate of soil NO-3-N and thereby reduce NO-3-N leaching. Additionally, the
effect of applying N2O-mitigating biofertilizers on reducing NO-3-N
leaching increased year on year. The primary reason was thought to be
that, over time, the adaptation of functional microorganisms to the soil
environment increased, promoting an abundance of soil N2O-reducing
bacteria and improving the utilization rate of soil NO-3-N. Furthermore,
biofertilizers promoted rice growth, ameliorated the rhizosphere envi­
ronment of rice, and potentially stimulated the absorption of more
NO-3-N by the rice roots (Calvo et al., 2016; Gao et al., 2016). After
applying biofertilizers, the soil NO-3-N concentration in 2019 was higher
than that in 2020 and 2021 (Fig. S8), indicating that more NO-3-N in the
soil was utilized by N2O-mitigating bacteria in 2020 and 2021, namely
the effect of N2O-mitigating biofertilizers on reducing NO-3-N leaching
increased year on year.

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M. Hu et al. Agriculture, Ecosystems and Environment 362 (2024) 108850

Table 6
Correlation analysis for ammonia volatilization, greenhouse gas emission, and nitrogen leaching from paddy fields. * and ** indicate P < 0.05 and P < 0.01,
respectively.
N2O CH4 NH3 NO-3-N (30 cm) NO-3-N (60 cm) NO-3-N (90 cm) NH+
4 -N (30 cm) NH+
4 -N (60 cm) NH+
4 -N (90 cm)

N2O 1
CH4 -0.353** 1
NH3 0.398** 0.125 1
NO-3-N (30 cm) 0.407** 0.037 0.232* 1
NO-3-N (60 cm) 0.272** -0.012 0.233* 0.364** 1
NO-3-N (90 cm) 0.187 0.141 0.145 0.386** 0.361** 1
NH+4 -N (30 cm) 0.370** -0.209* 0.216* 0.293** -0.046 -0.023 1
NH+4 -N (60 cm) 0.132 -0.214* 0.108 0.271** 0.145 -0.025 0.064 1
NH+4 -N (90 cm) 0.408** -0.196* 0.051 0.315** 0.178 0.191* 0.039 0.143 1

Table 7
Global warming potential (GWP) and greenhouse gas emission intensity (GHGI) of different fertilization treatments. Data are presented as the mean ± standard error.
The different small letters following each mean and standard error refer to the significant difference among treatments at 5 % level.
Treatment N2O-GWP (kg eq-CO2 ha− 1) CH4-GWP (kg eq-CO2 ha− 1) Total GWP (kg eq-CO2 ha− 1)

2019 2020 2021 2019 2020 2021 2019 2020 2021

0% N -120±19d -105±24c -70±32c 118±58b 3817±523b 2245±1362b -2±123d 3712±578b 2176±1236c

80% N 305±123bc 69±22b 79±29ab 698±338ab 4735±820ab 9085±2170a 1003±225bc 4804±783ab 9164±2092a
100% N 752±155a 355±121a 134±14a 760±388ab 5199±1194ab 7480±3084ab 1512±433ab 5555±1092ab 7614±2983ab
TSA2S 670±299ab 42±33b 16±5b 254±179b 4816±599ab 8383±1679ab 924±257c 4858±764ab 8399±1579ab
N2 138±84c -80±31c -33±10c 2211±762a 4148±1027ab 8061±1837ab 2349±673a 4068±879ab 8028±1943ab
NRCB002 459±223bc 15±68b 66±13ab 667±373ab 5572±1007ab 9128±1817a 1126±327bc 5587±984ab 9194±1793ab
NRCB008 224±45bc -56±76c -24±33bc 817±360ab 5955±734a 5683±2354ab 1041±278bc 5899±689a 5659±2138b
NRCB010 242±125bc -24±35c -54±20c 916±471ab 4461±892ab 8467±2512ab 1158±386bc 4437±1021ab 8413±2349ab
NRCB026 231±50bc 8±41bc -16±12c 1525±722ab 5086±487ab 7927±1297ab 1756±784ab 5093±764ab 7911±1194ab

N2O-GHGI (g eq-CO2 kg-1) CH4-GHGI (g eq-CO2 kg-1) Total GHGI (g eq-CO2 kg-1)

2019 2020 2021 2019 2020 2021 2019 2020 2021

0% N -28±11c -33±13c -13±7c 27±3c 1209±237a 428±135c 0±1c 1175±324a 414±109c

80% N 38±18ab 10±3b 9±4ab 87±23bc 703±258ab 1024±327a 125±32ab 713±216ab 1033±312a
100% N 83±34a 41±8a 12±3a 84±17bc 600±135ab 692±238abc 167±45ab 641±135ab 704±187ab
TSA2S 72±23a 5±2b 2±1b 27±7c 566±157ab 838±328ab 99±33b 571±134b 840±258ab
N2 16±7b -11±12c -4±1c 249±27a 589±129ab 889±298ab 265±57a 578±112b 886±239ab
NRCB002 50±19ab 1±1b 6±3ab 73±14bc 537±157b 820±257ab 123±45ab 539±165b 826±302ab
NRCB008 24±8b -7±5c -2±1bc 89±19bc 731±269ab 549±138bc 113±21ab 724±147ab 547±122c
NRCB010 27±7ab -3±4c -6±4c 102±25ab 574±121ab 947±237ab 129±19ab 571±126b 941±198ab
NRCB026 25±11ab 1±1bc -2±1c 167±28ab 692±231ab 810±268ab 192±27ab 693±103ab 809±174ab

5. Conclusions Data availability

After reducing nitrogen by 20% combined with the application of
N2O-mitigating biofertilizers then, compared to an addition of the
equivalent amount of nitrogen fertilizer, rice yield was enhanced for
each year for three years. Over the same period, the cumulative N2O
emissions and nitrogen leaching decreased and there was no significant
impact on NH3 volatilization and CH4 emissions. There was a negative
correlation between N2O and CH4 emission from rice fields. The nitro­
gen leaching in paddy fields was mostly as NO-3-N, while NH+ 4 -N leaching
was relatively small. Overall, the results suggest that the application of
N2O-mitigating biofertilizers at the field scale can maintain or even
enhance rice yields whilst reducing the environmental impact. The use
of biofertilizers appears to provide a means to reduce mineral nitrogen
application, thereby helping the move to a cleaner economy of using
N2O-mitigating biofertilizers for crop production whilst simultaneously
reducing the environmental footprint of agriculture. Further work to
determine the processes operating and the potential for large-scale
adoption in different environmental settings is required.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data will be made available on request.
Acknowledgments
This study was funded by the National Natural Science Foundation of
China (42377311 and 41771291) and Jiangsu Agricultural Science and
Technology Innovation Fund (CX(21)3183). Andrew Wade was sup­
ported by an internal joint award from the University of Reading and the
Nanjing University of Information Science and Technology.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.agee.2023.108850.
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