Science of the Total Environment 917 (2024) 170522

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Science of the Total Environment

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

Biochar application alters soil metabolites and nitrogen cycle-related

microorganisms in a soybean continuous cropping system
Xin Cui a, 1, Jun Yuan b, 1, Xu Yang a, *, Chaoqun Wei a, Yinghui Bi a, Qiang Sun a, Jun Meng a,
Xiaori Han a
a
Key Laboratory of Biochar and Soil Improvement of Ministry of Agriculture and Rural Affairs, Shenyang Agricultural University, Shenyang 110866, China
b
Liaodong University, Dandong 118001, China

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

• Biochar addition significantly improved

soil nitrogen supply.
• Biochar treatments markedly reduced
the Nitrospira abundance.
• Soil metabolites related to stress resis­
tance of soybeans were enhanced by
biochar.
• Biochar was beneficial for alleviating
soybean continuous cropping obstacles.

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

Editor: Ouyang Wei Biochar application is a promising practice to enhance soil fertility. However, it is unclear how field-aged biochar
affects the soil metabolites and microbial communities in soybean fields. Here, the rhizosphere soil performance
Keywords: after amending with biochar addition rates at 0 (CK), 20 (B20), 40 (B40), and 60 t ha− 1 (B60) was examined via a
Biochar aging five-year in-situ field experiment based on a soybean continuous cropping system. Untargeted metabolomics and
Continuous cropping obstacle
metagenomics analysis techniques were applied to study the regulatory mechanism of biochar on soybean
Soil metabolites
growth from metabolomics and N cycle microbiology perspectives. We found that the contents of soil total N
Microbial composition
Rhizosphere soil (TN), available N (Ava N), NH+ 4 -N, and NO3 -N were significantly increased with biochar addition amounts by
−

20.0–65.7 %, 3.6–10.7 %, 29.5–57.1 %, and 24.4–46.7 %, respectively. The B20, B40, and B60 triggered 259
(236 were up-regulated and 23 were down-regulated), 236 (220 were up-regulated and 16 were down-
regulated), and 299 (264 were up-regulated and 35 were down-regulated) differential metabolites, respec­
tively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and topology analysis
demonstrated that differential metabolites were highly enriched in seven metabolic pathways such as Oxidative
phosphorylation and Benzoxazinoid biosynthesis. Moreover, ten differential metabolites were up-regulated in all
three treatments with biochar. Biochar treatments decreased the Nitrospira abundance in soybean rhizosphere
soil while increasing Bradyrhizobium abundance significantly in B60. Mantel test revealed that as the biochar

* Corresponding author at: No.120 Dongling Road, Shenyang 110866, China.

E-mail address: yangxu@syau.edu.cn (X. Yang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2024.170522
Received 13 December 2023; Received in revised form 15 January 2024; Accepted 26 January 2024
Available online 1 February 2024
0048-9697/© 2024 Elsevier B.V. All rights reserved.
X. Cui et al.
addition rate grows, the correlation between Nitrospira and soil properties other than NO−3 -N became stronger. In
conclusion, the co-application of biochar with fertilizers is a feasible and effective way to improve soil N supply,
even though biochar has undergone field aging. This work offers new insights into the variations in soil me­
tabolites and microbial communities associated with N metabolism processes under biochar addition in soybean
continuous cropping soils.
1. Introduction
Soybeans are globally recognized as a major agricultural crop, with
high nutritional value, occupying a particularly important position in
ensuring national grain and oil security (Wu et al., 2023). Due to limited
arable land, soybean continuous cropping is widespread in Northeast
China, posing challenges to both soybean yield and soil health and
hindering economic development (Liu et al., 2019b). Soil microbial
structure imbalance and pathogenic microorganism accumulation are
the leading causes of continuous cropping obstacles. As previously
described by Liu et al. (2019b), the continuous cropping resulted in a
significant reduction in the number of bacteria and actinomycetes, while
fungi display the opposite pattern. Researchers observed that contin­
uous cropping resulted in a soil pH decline, and acidic conditions
favored the survival of root rot pathogens, rendering soybeans more
susceptible to disease (Tian et al., 2019). Additionally, the allelopathy of
soybean root exudates also severely affects the growth of follow-planted
soybean and soil microorganisms, as well as the soybean-rhizobia
symbiotic interaction process (Haarith et al., 2020; Wang et al.,
2020a). Compared with gramineous crops, the N nutrition of soybeans is
more complex. In addition to assimilating ammonium N and nitrate N
from the soil, it also forms symbiotic relationships with rhizobia,
resulting in the development of nodules for the biological fixation of
atmospheric nitrogen. One notable feature of soybean growth is the
considerably extended parallel duration of vegetative growth and
reproductive development, and a large amount of N is needed to form
protein in soybean grain (Kelly et al., 2021). Thus, more research is
warranted to explore the relationship between soil metabolites, micro­
organisms, and N utilization of soybean.
It is a conventional practice in China’s soybean production to
enhance the yield by raising chemical fertilizer (CF) input. However, the
overuse of CFs poses environmental risks and degrades the soil quality,
which negatively impacts the sustainable development of China’s agri­
culture (Yan et al., 2017; Han and Zou, 2018). In recent years, the
Chinese government attaches great importance to the agricultural
pollution caused by the excessive application of CF, and co-application
of biochar with CF can achieve the target of reducing the CF input and
increasing the efficiency, which has been widely concerned in the
agricultural and environmental fields (Puga et al., 2020; Xia et al.,
2023). Biochar is the predominant product of high-temperature pyrol­
ysis of biological residues under anoxic conditions, which contains
abundant carbon (Zhao et al., 2022). Since it has a microporous struc­
ture, large specific surface area, and strong resistance to microbiological
decomposition, biochar has been extensively employed as a soil
amendment. Biochar has exhibited great potential to enhance soil pro­
ductivity, offset greenhouse gas emissions, and restore soil contami­
nated by heavy metals and pesticides (Jin et al., 2019; Khan et al., 2020;
Wang et al., 2021b, 2023a).
The effectiveness of biochar in improving soil has been demonstrated
in various crop systems. For instance, Yang et al. (2021) observed that
biochar could effectively alleviate soil acidification and boost the output
and quality of tea plants. Oladele et al. (2019) previously concluded that
CF combined with biochar promoted soil nutrient availability and grain
yield in rain-fed rice agroecosystems. Han et al. (2023) reported that
biochar altered the core microbial species, facilitating nutrient accu­
mulation and the final yields of maize and wheat. Likewise, numerous
investigations have also examined the impact and mechanism of biochar
on the growth of soybeans. According to Xiu et al. (2019), biochar
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Science of the Total Environment 917 (2024) 170522
application enhanced soybean yield by increasing soil pH, improving
soil structure, and promoting nutrient uptake. Some researchers believe
that biochar is beneficial to improve the root morphology and increase
the leaf gas exchange capacity of soybeans (Reyes-Cabrera et al., 2017;
Zhang et al., 2020a). Moreover, biochar application can also facilitate
nutrient absorption and metabolic efficiency by augmenting nodulation
and N2 fixation (Farhangi-Abriz and Torabian, 2018). It has been shown
that an enhancement in Mo and K supplies induced by the biochar
application boosted nodule weight per root system of soybean (Wang
et al., 2018). Soil microorganisms are essential for nutrients biogeo­
chemical cycling and plant growth (Beattie et al., 2018). Rhizosphere
soil is the most active zone for soil microorganisms to interact with
plants. Both root exudates and biochar are capable of functioning as
sources of energy for the growth of soil microbes (Yu et al., 2018).
Furthermore, the microbial metabolism of these organic compounds in
rhizosphere soil may also influence soil microbial species and root
physiological activities (Gong et al., 2023). Therefore, disclosing the
changes in microbial communities and metabolites in rhizosphere soil is
essential for comprehending the regulation mechanism of biochar to
soybean growth.
As a powerful technology, metagenomics sequencing targets the total
genome of all microorganisms contained in an environment sample.
However, it cannot provide us with information on actual microbial
functions, such as their metabolism. Metabolomics, which can analyze
all low molecular weight metabolites in organisms or cells qualitatively
and quantitatively, is a powerful method to explore the relationship
between metabolites and physiological and pathological changes
(Hayden et al., 2019). It can identify biochemical intermediates in
metabolic pathways, helping us advance our overall understanding of
biological processes in the soil. Soil microorganisms and metabolites are
a complex and changeable system, which is greatly affected by the
environment (Johns et al., 2017). It is necessary to combine meta­
genomics and metabolomics methods to examine the influence of bio­
char on soil metabolites and microbial communities. Thus, we
conducted a five-year study of soil metabolites and microorganisms in a
soybean continuous cropping system in Northeast China with different
application rates of biochar. This work aimed to: i) investigate the soil
metabolites and microbial communities variations in soybeans rhizo­
sphere soil under biochar addition; and ii) reveal the relationship be­
tween soil metabolites, microorganisms, and N forms. We hypothesize
that biochar amendment would significantly alter soil microbial struc­
ture and metabolites by increasing N levels in soybean rhizosphere soil.
Our work will provide theoretical support for biochar strategies to boost
soil fertility and soybean production.
2. Materials and methods
2.1. Trial site and setup
The field study with successive planting of soybeans was carried out
at the Kalima Experimental Station (41◦ 54′N, 122◦ 68′E) of Liaozhong,
Liaoning Province, China, from 2019 to 2023. This region experiences a
temperate semi-humid continental climate characterized by a mean
temperature of 8.1 ◦ C and mean annual precipitation of 694 mm. The
growing period for soybeans is from May to October in this region. The
variety of soybean is named Tiefeng29. The soil is classified as meadow
soil with the dominant texture of sandy loam. Prior to this field test, the
0–20 cm soil had the following properties: a pH of 7.27, a total carbon
X. Cui et al.
(TC) of 5.13 g kg− 1, a total potassium (TK) of 32.75 g kg− 1, a total ni­
trogen (TN) of 0.45 g kg− 1, a total phosphorus (TP) of 0.25 g kg− 1, an
NH4OAc-extractable K of 102.50 mg kg− 1, an available N (Ava N) of
33.83 mg kg− 1, and an Olsen-P of 9.37 mg kg− 1.
The biochar for this field experiment was produced by pyrolyzing
corn stover under oxygen-limited conditions at 350–550 ◦ C (Jinhefu
Agricultural Development Company, Liaoning Province, China). After
cooling, biochar was sifted through a 2 mm sieve to evenly incorporate
with the soil. The biochar has a pH of 9.2, a surface area of 8.87 m g− 2,
and an average pore size of 16.23 nm. The TC, TN, ash, and volatile
matter contents of the biochar are 660 g kg− 1, 12.7 g kg− 1, 155.7 g kg− 1,
and 219.4 g kg− 1, respectively.
This field study assigned four treatments: no biochar applied (CK);
biochar application rates at 20 t ha− 1 (B20), 40 t ha− 1 (B40), and 60 t
ha− 1 (B60), which were considered as low, intermediate, and high
addition. Each treatment had three replicate plots (2 m × 4 m), which
were distributed in a randomized block design. During the experiment,
soybeans were planted with 30 cm row spacing and 20 cm plant spacing,
biochar was manually sprinkled into the test plots only once and mixed
with the soil well. All plots received identical basal fertilizers before
soybean sowing each year, including 30 kg N ha− 1, 39.3 kg P ha− 1, and
74.7 kg K ha− 1.
2.2. Soil sampling
In 2023, three rhizosphere soil samples were randomly collected in
each plot at the soybean fluorescence stage (R2 growth stage) to
generate a homogenized sample. Soils adhering to the root surface were
gently shaken off the root and then removed visible root pieces, which
were considered as the rhizosphere soil samples. Samples were kept in
an insulated container with ice and then shipped to the laboratory
immediately. Parts of the fresh samples were directly used for NH+ 4 -N
and NO−3 -N analysis. A portion of each soil sample was frozen at − 80 ◦ C
for metabolomics and microorganisms testing. The remains were air-
dried and sieved (<2 mm) for the pH, Ava N, and TN contents
measurements.
2.3. Determination of soil pH and N content
Soil TN was measured at combustion temperatures of 1150 ◦ C in an
elemental analyzer (Elementar Vario Max Analyzer, Germany). The
determination methods for soil NH+ 4 -N and NO3 -N concentrations draw
−
on the approach of Yang et al. (2022). Ava N contents were assayed
using the alkali-hydrolytic diffusion method (Bao, 2000), and soil pH
was determined in a 1:2.5 soil: water suspension (1:2.5 v/v) by glass
electrode pH meter.
2.4. Soil metabolite analysis
Accurately weigh 1000 ± 5 mg samples into a 2 mL centrifugal tube
with 1000 μL extracts (methanol: water = 4:1 v/v) and add a 6 mm
diameter grinding bead. The extracting solution contains four internal
standards (L-2-PCPA 0.02 mg mL− 1, etc.). After grinding for 6 min
(− 10 ◦ C, 50 Hz), ultrasonic extraction for 30 min (5 ◦ C, 40 Hz), standing
for 30 min at − 20 ◦ C, and centrifugation for 15 min (13,000 rpm), the
supernatant was taken and dried by N2, redissolved with 120 μL of
acetonitrile: water = 1:1 (v/v), ultrasonic extraction for 5 min, centri­
fugation for 15 min, and the supernatant was taken for the test.
Liquid chromatography with tandem mass spectrometry (LC-MS/
MS) analyses was performed with a UHPLC-Q Exactive HF-X system
(Vanquish, Thermo Fisher Scientific). The chromatographic column was
ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Mil­
ford, USA). In order to identify soil metabolites more efficiently, we
referred to Warren’s method and optimized it (Warren, 2018). The
gradient elution conditions are shown in Table S1. The temperature of
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Science of the Total Environment 917 (2024) 170522
the column was maintained at 40 ◦ C, with a flow rate of 0.4 mL min− 1
and a sample volume of 3 μL. The samples were separated by the liquid
chromatographic column, and the ion source of the high vacuum mass
spectrometer ionized the single component. The mass spectrogram was
obtained by separating the mass charge ratio (m/z). After analyzing the
mass spectrogram data of the samples, the qualitative and quantitative
analysis results of the samples were obtained.
2.5. Soil microorganism determination
Metagenomics method is used for soil microbial testing, DNA in soil
samples using FastDNA ® Spin Kit for Soil MP Biomedicals (USA)
extraction, PE library using NEXTFLEX ™ Rapid DNA-Seq Kit con­
struction (USA), sequencing process follows NovaSeq Reagent Kits/
HiSeq X Reagent Kits (USA). The detailed steps of microbial diversity
analysis are the same as those described by Zhu et al. (2022). According
to the OTUs in a sample, Chao, Simpson, and Shannon indices were used
in this study to assess microbial diversity. Chao index was computed as
detailed previously by Chao (1984):
n1 (n1 − 1)
SChao = Sobs + (1)
2(n2 + 1)
The calculation of the Shannon index was as follows (Shannon and
Weaver, 1949):
Sobs
∑ ni ni
Hshannon = − ln (2)
N N
i=1
Simpson index was calculated as follows (Simpson, 1949):
S∑
obs
ni (ni − 1)
Dsimpson = i=1
(3)
N(N − 1)
where Schao, Hshannon, and Dsimpson correspond to the Chao, Simpson, and
Shannon indices, respectively; Sobs indicates the number of observed
OTUs; n1 represents the number of OTUs containing only one sequence;
n2 represents the number of OTUs containing only two sequences; ni
corresponds the number of sequences contained in the ith OTU; N is the
total number of sequences.
2.6. Data processing and statistical analysis
The original data were imported into ProgenesisQI software
(WatersCorporation, Milford, USA) for the metabonomics processing.
Use this software to search and identify characteristic peaks, and iden­
tify metabolites based on matching scores from secondary mass spec­
trometry. Trimmomatic software was applied to perform quality control
steps such as sequence splicing and de-redundancy on sequencing raw
data, classify the obtained effective sequences, analyze the data through
the Illumina Miseq platform, and the microbial community composition
was calculated through the Non-Redundant Protein Sequence Database
(NR).
For a comprehensive analysis of differential metabolites, it is
necessary to combine various statistical methods to reduce the error rate
and improve the reliability of the analysis results. Therefore, a partial
least-squares discriminate analysis (PLS-DA) model was used to distin­
guish metabolites in the CK and biochar treatments, and R2X and Q2
were applied to assess the prediction accuracy of the model. The
significantly different metabolites met the two conditions of FC ≥ 1.2 or
≤0.8333 and p < 0.05. Topological analysis of significantly different
metabolite pathways and enrichment analysis of metabolite pathways
among the four treatments were performed by utilizing the Kyoto
Encyclopedia of Genes and Genomes (KEGG) database. The Mantel test
analysis was conducted utilizing the tools on OmicStudio at
https://www.omicstudio.cn/tool. The significant differences analysis of
soil pH, N contents, and the 10 soil metabolites enhanced by biochar
X. Cui et al. Science of the Total Environment 917 (2024) 170522

addition were conducted with SPSS 21.0 using variance analysis 1.2, P < 0.05. Venn diagram was used to compare the differences be­
(ANOVA), and multiple comparisons used the least significant difference tween metabolic sets (Fig. 1e, f). Under the positive ion mode, 114, 134,
(LSD) at p < 0.05. and 186 kinds of significantly different metabolites were detected in the
three metabolic sets, and 27, 9, and 46 kinds of unique metabolites were
3. Results detected in B20, B40, and B60, respectively. Under the negative ion
mode, 115, 102, and 113 metabolites were detected in the three meta­
3.1. Soil N content and pH bolic sets, and 36, 10, and 21 unique metabolites were detected in B20,
B40, and B60, respectively. Table S2 showed that 4 unique metabolites
The soil N contents and pH are shown in Table 1. Relative to CK, the in biochar treatments were Terpenoids (neg), and 4 unique metabolites
soil TN was significantly increased by 24.3 %, 20.0 %, and 65.7 % in of B60 (pos) were classified into Alkaloids, which are nitrogen-
B20, B40, and B60, respectively. For the B40 and B60, the Ava N con­ containing organic compounds. There were 6, 1, and 3 unique metab­
tents were strongly increased by 7.97 % and 9.37 %, respectively, while olites in B20, B40, and B60 under the negative ion mode, respectively,
no significant difference was observed between B20 and CK. The NH+ 4 -N
and classified into Flavonoids (Table S2). Compared with CK, B60 had
contents in B20, B40, and B60 were 29.48 %, 36.30 %, and 57.11 % the most significantly different metabolites (299 kinds), B40 had the
higher than that in CK, respectively. Biochar treatments dramatically least (236 kinds), and B20 had 259 kinds. In the positive and negative
improved NO−3 -N contents by 24.35 % to 46.68 %, compared with CK. ion modes, B40 vs CK and B60 vs CK have 200 identical metabolites, and
Soil pH gradually changed from neutral to weak alkaline, and increased the metabolites are similar between the groups.
with the addition of greater biochar amounts (Table 1). The difference
between the biochar treatments and CK was significant, but no signifi­ 3.2.2. Metabolites analyzed by volcano plots
cant difference was found between B40 and B60. Fig. 2 showed the volcano plots of metabolites with significant dif­
ferences between the biochar treatments and CK. Under the positive ion
mode, there were 127, 125, and 170 kinds of significantly differentiated
3.2. Soil metabolites
metabolites with an up-regulation effect and 17, 9, and 16 kinds of
significantly differentiated metabolites with a down-regulation effect.
In the positive and negative ion mode, 5050 and 4480 effective peaks
Under the negative ion mode, 109, 95, and 95 significantly different
were identified respectively, and 4359 and 3870 effective peaks were
metabolites were up-regulated, and 6, 7, and 18 significantly different
retained after the data preprocessing process. 742 metabolites were
metabolites were down-regulated.
annotated based on KEGG classification; 1159 metabolites were anno­
tated by the Human Metabolome Database (HMDB). 345 metabolites
3.2.3. Analysis of soil metabolites by KEGG
have been annotated in the KEGG pathway.
Through KEGG (Figs. 3 and S1), compounds classification, metabolic
pathway topology, and enrichment were analyzed for three metabolic
3.2.1. PLS-DA and Venn diagram analysis
sets (B20 vs CK, B40 vs CK, B60 vs CK). From Fig. 3a, c and f, six me­
The PLS-DA score chart of samples can intuitively show the classi­
tabolites were classified in KEGG secondary metabolism level both in the
fication effect of groups (Fig. 1). The greater the classification degree of
three sets. They are Terpenoids, Polyketides, Phenylpropanoids, Flavo­
each sample in the figure, the more significant the classification effect
noids, Amino acid related compounds, and Alkaloids. Among them, the
between treatments. As can be seen from Fig. 1a and b, the separation
B20 vs CK metabolic set classified 1 Fatty acid compound and 13
effect between biochar treatments and CK was obvious under the posi­
Flavonoid compounds, with a significantly higher number of Flavonoids
tive and negative ion mode. The model with biochar addition as a
compounds compared to the other two metabolic sets. As the biochar
grouping factor is reliable. In other words, from the perspective of
application rate increases, the types of compounds in the soil gradually
metabonomics, biochar changed the metabolites composition of soy­
increase, except for fatty acids, the number of compounds classified by
bean rhizosphere soil. In the positive ion mode, all groups were within
the B60 vs CK set is greater than the other two metabolic sets.
their confidence intervals, but the confidence intervals of the treatment
KEGG topology analysis was presented in Fig. 3b, d, and f, and it
groups were repeated. B40 was relatively discrete, and one sample was
mainly focused on the influence of each pathway and used a rectangular
located in the overlap area with B20. B20 and B60 had a good separation
tree diagram to display each metabolic pathway. As shown in Fig. 3b, d,
trend. In the negative ion mode, the separation degree of B40 and B60
and f, five identical metabolic pathways can be obtained in the three
was not readily apparent. Fig. 1c and d were permutation tests for PLS-
metabolic sets by analyzing the important pathways of each metabolic
DA model validation, with the decrease of replacement retention, R2 and
set according to the influence, which are: Isoflavonoid biosynthesis,
Q2 decrease, and the tropic showed an upward trend, which indicated
Phenylpropanoid biosynthesis, Phenylalanine metabolism, Benzox­
the model was reliable without an overfitting problem.
azinoid biosynthesis and One carbon pool by folate.
Metabolites in each biochar treatment were compared with CK, and
Significant differences in metabolite enrichment for each pathway
three metabolic sets (B20 vs CK, B40 vs CK, B60 vs CK) were obtained
were identified by KEGG and the top 20 metabolic pathways between
according to the significantly different screening conditions of Log2FC ≥
biochar treatments and CK were presented in Fig. S1. The results indi­
cated that the metabolic pathways shared by the three groups of meta­
Table 1 bolic sets are: Phenylpropanoid biosynthesis, Oxidative
Effects of biochar addition on soil pH and N contents. phosphorylation, and Biosynthesis of phenylpropanoids. It is noted that
Treatment pH TN Ava N 4 -N
NH+ NO−3 -N isoflavonoid biosynthesis as an important pathway for the synthesis of
(g kg− 1) (mg kg− 1) (mg kg− 1) (mg kg− 1) effective components in soybean is apparent enrichment in B20 vs CK
CK 7.00 ± 0.70 ± 49.32 ± 8.65 ± 12.36 ± and B60 vs CK.
0.10c 0.05c 0.33b 0.16c 0.31c In summary, based on KEGG topological analysis (Fig. 3b, d, and f)
B20 7.37 ± 0.87 ± 51.10 ± 11.20 ± 15.37 ± and pathway enrichment analysis (Fig. S1), we have identified seven
0.05b 0.05b 0.57b 0.50b 0.62b
B40 7.44 ± 0.84 ± 53.90 ± 11.79 ± 17.52 ±
pathways shared by all biochar treatments. These pathways were Phe­
0.03a 0.04b 0.71a 0.85b 0.05a nylpropanoid biosynthesis, Oxidative phosphorylation, Isoflavonoid
B60 7.49 ± 1.16 ± 54.60 ± 13.59 ± 18.13 ± biosynthesis, Biosynthesis of phenylpropanoids, Phenylalanine meta­
0.02a 0.15a 0.14a 0.44a 1.06a bolism, Benzoxazinoid biosynthesis, and One carbon pool by folate.
Values represent means with standard error (n = 3), and different lowercase
letters indicate significant differences among treatments at p < 0.05.

4
X. Cui et al. Science of the Total Environment 917 (2024) 170522

Fig. 1. PLS-DA and Venn diagrams analysis of soil metabolites. (a) is the positive mode, and (b) is the negative mode; the abscissa component 1 and ordinate
component 2 in the figure represent the first and second principal component scores, respectively. Different color scatter points represent different samples, and the
ellipse is 95 % confidence interval. (c) and (d) are permutation tests for the PLS-DA validation in positive mode and negative mode, respectively, and the abscissa
represents the degree of permutation retention for permutation testing, and the ordinate represents the values of R2 (blue dot) and Q2 (red triangle). (e) and (f) are
Venn diagrams of soil metabolites in different group sets under positive mode and negative mode, respectively, and different colors represent different group sets.

5
X. Cui et al. Science of the Total Environment 917 (2024) 170522

Fig. 2. Volcano plots of different metabolites between biochar treatments and CK. (a) and (b) represent the metabolite changes between B20 and CK under positive
mode and negative mode, respectively. (c) and (d) represent the metabolite changes between B40 and CK under positive mode and negative mode, respectively. (e)
and (f) represent the metabolite changes between B60 and CK under positive mode and negative mode, respectively. Each dot denotes a specific metabolite, and the
size of the dot represents the Vip value. The red dot indicates this metabolite has significant up-regulation, blue dot indicates it has significant down-regulation.

6
X. Cui et al. Science of the Total Environment 917 (2024) 170522

Fig. 3. KEGG compound classifications in B20 (a), B40 (c), and B60 (e) treatments. (b), (d), and (f) are KEGG topology analyses of B20, B40, and B60, respectively.
The size of the box denotes the influence degree, while the color of the box denotes the enrichment degree for a particular metabolic pathway.

7
X. Cui et al.
3.2.4. Metabolites information in KEGG
Metabolites in these seven metabolic pathways were summarized in
Table S3. Twenty-two metabolites exist in these seven pathways, ten of
these were common in the three biochar treatments: Yangonin, 1,3-
Dihydro-(2H)-indol-2-one, (6R)-5,10-Methylenetetrahydrofolate,
Methyl salicylate, Demethylsuberosin, Ubiquinone-1, Estragole, 4-Hy­
droxy-3-methoxycinnamaldehyde, Xenognosin B and Phenyl Alanine.
Fig. 4 showed the relative abundance of the screened signature dif­
ferential metabolites among the treatments. Biochar treatments
increased the relative abundance of these 10 different metabolites, and
the highest values appeared in B60, which were all significantly higher
than CK. In B20, the abundance of the other 8 metabolites showed sig­
nificant differences compared to CK with the exception of Yangonin and
(6R)-5,10-Methylenetetrahydrofolate. Moreover, the abundances of 8
metabolites in B40 were significantly higher than CK except for Yan­
gonin and Methyl salicylate. There is no significant difference between
B20 and B40, B40 and B60 for these 10 metabolites. However, the
abundance of 1,3-Dihydro-(2H)-indol-2-one, Ubiquinone-1, Estragole,
4-Hydroxy-3-methoxycinnamaldehyde, and Phenyl Alanine in B60 were
significantly higher than those in B20.
3.3. Analysis of soil microbial community
Fig. 5 showed the relative abundance of the different microbial
communities. According to the results of microorganism classification at
the gate level, the main type after biochar addition was still bacterial. At
the phylum level, the top 8 dominant microbial communities in terms of
abundance were Actinobacteria, Proteobacteria, Chloroflexi, Acid­
obacteria, Thaumarchaeota, Candidatus_Rokubacteria, Gemmatimonadetes,
and Nitrospirae. The abundances of Nitrospirae in B40 and B60 were
significantly lower than CK and B20. At the genus level, the dominant
species in the soil still belong to the dominant phylum species. The
dominant genera of known names in the figure were Bradyrhizobium,
Sphingomonas, Nocardioides, Arthrobacter, and Nitrospira. The abundance
of Bradyrhizobium in B60 was notably greater than that in CK. The
abundance of Nitrospira at the genus level in the three biochar treat­
ments was significantly lower than in CK.
The α diversities of bacteria and fungi at phylum level in soybean
rhizosphere soil after biochar addition were studied separately by
calculating Chao, Shannon, and Simpson indices (Table 2). The com­
munity coverage index in each treatment was 1, indicating that the
sequencing results accurately reflected the functional microbial struc­
tures. As shown by Chao, Shannon, and Simpson indices, biochar has
little effect on fungal richness, uniformity, and diversity in soil regard­
less of the biochar addition rate. The B20 significantly reduced Chao and
Shannon indices of bacterial communities compared with CK.
3.4. Mantel test analysis
To investigate the relationship between soil properties, metabolites,
and microbial species, we performed a co-analysis of 5 soil properties, 2
microbial communities, and 10 differential metabolites, resulting in a
correlation heatmap matrix (Fig. 6). In B20, Estragole, 4-Hydroxy-3-
methoxycinnamaldehyde, Phenyl Alanine, 1,3-Dihydro-(2H)-indol-2-
one, Methyl salicylate, and Demethylsuberosin were significantly
negatively correlated with Nitrospira. Bradyrhizobium did not associate
significantly with the metabolites. Nitrospira had a significant correla­
tion with TN and pH, but Bradyrhizobium had no correlation with soil
properties. The NH+ 4 -N was the most predominant factor affecting me­
tabolites (Fig. 6a). In B40, the 10 metabolites were significantly nega­
tively correlated with Nitrospira, and (6R)-5,10-
Methylenetetrahydrofolate, 4-Hydroxy-3-methoxycinnamaldehyde, and
Xenognosin B were significantly positively correlated with Bradyrhi­
zobium. In B60, Nitrospira and Ubiquinone-1 were significantly corre­
lated with NH+ 4 -N, Xenognosin B had a significant correlation with
8
Science of the Total Environment 917 (2024) 170522
NO−3 -N, and Yangonin was significantly correlated with Ava N. Ac­
cording to Mantel tests, it could be found that as the biochar addition
rate increases, the correlation between soil properties, metabolites, and
microorganisms also gradually increases. The correlation coefficient and
significance level among metabolites, microorganisms, and environ­
mental factors in B60 were the highest.
4. Discussion
4.1. Biochar application changed the N composition of soybean
rhizosphere soil
In our study, biochar amendment has a significant impact on the
contents of different forms of N (Table 1). Borchard et al. (2019) pointed
out that the influence of biochar on N dynamics of soil may vary with
time. Biochar undergoes a series of physical and chemical changes over
time as it ages, affecting its properties such as surface area, pore volume,
surface functional groups, and elemental composition (Chang et al.,
2018; Mia et al., 2017; Yang et al., 2023). This may lead to a degradation
of biochar’s positive effects in a few years following its soil application
(Hagemann et al., 2017). It should be mentioned that the biochar was
added to the soil in spring 2019 and its positive effects on soil nitrogen
retention persisted until 2023 (Table 1). Previous studies also confirmed
that soil TN and fertilizer N retention were facilitated significantly by
biochar addition in a long term (Liu et al., 2019c; Wang et al., 2020b).
Nevertheless, biochar may also increase ammonia volatilization by
raising soil pH levels, which will reduce the soil NH+ 4 -N. Both the soil pH
and NH+ 4 -N content exhibited an enhancement under biochar addition in
our study (Table 1). Biochar surface can structurally carry enriched
oxygen-containing functional groups like carbonyl, carboxylic, hydrox­
yl, and phenolic during field aging, which could possibly strengthen the
adsorption of NH+ 4 -N and reduce its loss (He et al., 2019; Mia et al.,
2017; Tan et al., 2020). This is likely due to (i) more available
exchangeable sites enhanced by oxygen-containing functional groups,
which resulted in electrostatic interaction, and (ii) chemical bonding
between ammonium and O of oxygen-containing functional groups
(Esfandbod et al., 2017; Wang et al., 2016).
In addition, field aging of biochar particles is beneficial to the for­
mation of organo-mineral complexes (OMC), thereby promoting the
retention of NO−3 -N and reducing the loss of N leaching (Hagemann
et al., 2017; Liu et al., 2018). OMC makes an enormous contribution to
the process of soil aggregate formation (Ibrahim et al., 2023). Though
bonding directly of biochar functional groups to soil minerals or
adsorbing soil organic matter by biochar, OMC can form on biochar
surfaces with soil minerals bind (Han et al., 2020). Biochar may enrich
hyphae, microbial polysaccharides, and root exudates, which provide a
binder and stabilizer for the formation of soil aggregates (Islam et al.,
2021; Sale et al., 2021). Soil aggregates can act as temporary N pools by
retaining N within their structure (Srivastava et al., 2016). Improved soil
structure is also conducive to the N retention of microorganisms.
Generally, soils with low organic C often display a weak nutrient
retention power due to insufficient OMC (Thakur et al., 2023). The TC of
the experimental soil was only 5.13 g kg− 1, therefore biochar played a
more immediate and effective role in minimizing soil N leaching in the
present study.
4.2. Influence of biochar addition on soil metabolites
Soil metabolites originate mainly from microorganisms and plant
roots, and their composition and abundance can sensitively reflect the
responses of plants and microorganisms to changes in soil management
measures (Hartman et al., 2018). In this experiment, biochar treatments
significantly altered soil metabolites of the soybean rhizosphere soil
(Fig. 2). The same metabolite can be involved in different metabolic
pathways, such as (6R)-5,10-Methylenetetrahydrofolate, Genistein, and
X. Cui et al. Science of the Total Environment 917 (2024) 170522

Fig. 4. Changes in the relative abundance of the ten screened signature differential metabolites. *0.01 < p ≤ 0.05, **0.001 < p ≤ 0.01, ***p ≤ 0.001.

9
X. Cui et al. Science of the Total Environment 917 (2024) 170522

Fig. 5. Response of microbial communities to different treatments. (a) and (b) are the changes in the relative abundance of different microbial communities at the
phylum level and genus level, respectively. The bubble size represents the relative abundance of the microbe, and the bubble color denotes the classification
information.

soil. These unique compounds are secondary metabolites produced by

Table 2
plants, such as terpenoids, flavonoids, and alkaloids. They play a vital
Alpha diversity analysis of microbial communities in different treatments.
role in the mitigation of harmful effects of diseases on crops. Terpenoids
Treatment Fungal communities Bacterial communities are the largest and most diverse group of specialized metabolites found
Chao Shannon Simpson Chao Shannon Simpson in plants (Christianson, 2017). Song et al. (2011) reported that terpe­
CK 6.33 1.17 ± 0.40 ± 135.33 1.65 ± 0.28 ± noids derived from the roots exhibited a broad antifungal action against
± 0.01a 0.03a ± 4.04a 0.11a 0.03ab 7 pathogenic fungi. Li et al. (2021) have proved that the enhancement of
0.58a terpenoids was conducive to the mitigation in continuous cropping ob­
B20 6.33 1.22 ± 0.39 ± 128.67 1.60 ± 0.29 ± stacles. Thus, the promoted terpenoids induced by biochar addition may
0.13a 0.07a ± 2.52b 0.04b 0.01a
be favorable for alleviating the disadvantages of continuous soybean
±
0.58a
B40 6.33 1.12 ± 0.44 ± 136.00 1.76 ± 0.25 ± planting. Flavonoids have a crucial role in facilitating interactions be­
± 0.08a 0.04a ± 1.00a 0.04a 0.01b tween plants and symbiotic microorganisms, particularly in the nodu­
0.58a lation of rhizobium in leguminous crops (Wang et al., 2022). In recent
B60 6.67 1.20 ± 0.38 ± 136.67 1.61 ± 0.30 ±
decades, it has been confirmed that flavonoids are vital in resistance to
± 0.05a 0.03a ± 4.04a 0.10b 0.02a
0.58a soybean cyst nematode, which causes devastating damage to the soy­
bean yield globally (Klink et al., 2010). The application of biochar
The small letters in a single column indicate significant differences among
possibly led to increased defensiveness and nodulation during soybean
treatments at p < 0.05.
growth, as evidenced by unique flavonoid metabolites in this study.
The 10 metabolites enhanced in all biochar treatments were involved
Coumesterol (Table S3). Even metabolites in the same metabolic in 7 metabolic pathways, most of which were related to plant roots
pathway may not respond identically to biochar. Hence, the effects of (Table S3). Zhang et al. (2020b) also reported that metabolites released
different biochar treatments on a certain metabolite depend not only on by plant roots may dominate the metabolite profiles in rhizosphere soil.
the metabolic pathway in which it participates but also on its specific In addition, we collected the soil samples at the soybean fluorescence
function in that metabolic pathway. Our results suggested that the re­ stage, and the root metabolism was exuberant in this period. As shown in
sponses of metabolite enrichment to different biochar treatments were Fig. 6, soil TN, NH+
4 -N, and NO3 -N contents were tightly linked to the 10
−
not completely uniform (Fig. S1 and Table S3). Moreover, there was an
enhanced soil metabolites. This was likely due to the increased nitrogen
overlap between B40 and B60 in the negative mode according to PLS-DA
supply from the biochar, which promoted the metabolism of soybeans
analysis (Fig. 1b), which means diminishing differences between B40
and microorganisms, thus boosting the abundance of these metabolites.
and B60. This trend can be explained by the following two aspects.
Furthermore, the KEGG pathway topology analysis and enrichment
Firstly, no significant difference was found in the 10 differential me­
analysis indicated that pathways associated with growth regulation and
tabolites between B40 and B60 (Fig. 4), although biochar treatments
stress resistance of soybeans were enriched significantly in biochar
increased the abundance of the 10 metabolites. Secondly, the KEGG
treatments (Figs. 3 and S1). For example, Phenylpropanoid biosynthesis
compound classification analysis displayed that the chemical composi­
is an important upstream pathway for plant synthesis of phenols, fla­
tion of metabolites in B40 and B60 had a great deal of convergence
vonoids, lignin, and other secondary metabolites (Khatkar and Sharma,
(Fig. 3c and e). Topology analysis revealed that the metabolic pathways
2020). The increase of the Phenylpropanoid biosynthesis pathway can
involving the metabolites of B40 and B60 were nearly identical (Fig. 3d
protect cells from damage under abiotic adversities such as drought, salt
and f). Therefore, we inferred that the metabolite composition and
stress, and pathogen infestation (Wang et al., 2023b; Zhang et al., 2021).
metabolic pathways of soybean rhizosphere soil had reached a stable
Isoflavones are secondary metabolites of flavonoids widely existing in
state under B40.
soybeans, and glyceollins are the terminal products of Isoflavonoid
Venn diagram (Fig. 1e and f) and Table S2 revealed that biochar
biosynthesis (Akashi et al., 2009). Glyceollins exert important defense
addition triggered some unique metabolites in the soybean rhizosphere
responses against pathogens such as Macrophomina phaseolina,

10
X. Cui et al.
Fig. 6. Relationships between soil properties, microorganisms, and metabolites
in B20 (a), B40 (b), and B60 (c). Line color corresponds to the significance level.
Line width represents Mantel’s r value. Pearson’s r is the correlation coefficient
between metabolites and microorganisms, and it is denoted by the size and
color shade of the single grid. *0.01 < p ≤ 0.05, **0.001 < p ≤ 0.01, ***p
≤ 0.001.
11
Science of the Total Environment 917 (2024) 170522
Phytophthora sojae, etc. (Lygin et al., 2010). Benzoxazinoids are also a
type of secondary metabolites known for their effectiveness in defense
and allelopathy (Frey et al., 2009). Oxidative phosphorylation is the
final metabolic pathway of cellular respiration and a crucial process in
the production of “energy currency” ATP. Biochar treatments signifi­
cantly enriched the Oxidative phosphorylation pathway by elevating the
metabolite of Ubiquinone-1 substantially (Fig. 4 and Table S3). These
metabolic pathways produce resistance signaling molecules or growth
regulators, which are needed for plant and microorganism growth.
Biochar improved soil microecology and promoted the production of
these metabolites, potentially demonstrating the promotion effect of
biochar on soybean growth from the perspective of metabonomics.
4.3. Impact of biochar on microbial species in the soybean rhizosphere soil
As demonstrated in numerous studies, biochar was able to alter the
microbial community structure through direct and indirect pathways
(Huang et al., 2022; Zhang et al., 2023). Microbial communities in soil
are influenced by the soil environment as well as their interactions (Li
et al., 2022). On the one hand, biochar can directly affect microorgan­
isms via its unique physical and chemical properties (Mukherjee et al.,
2022). On the other hand, biochar can reshape microbial communities
by interacting with soil and roots (Bolan et al., 2023), which is of great
significance for the study of rhizosphere soils. Soil health is closely tied
to the microbial community (Wang et al., 2021a; Zhang et al., 2020b).
The microbial community structure in soybean continuous cropping soil
changed from bacteria type to fungi type, which was one of the reasons
for the decline in soybean yield and quality (Song et al., 2016). This
tendency was not observed in this experiment, and it was found that
biochar treatments maintain the dominance of Actinobacteria and Pro­
teobacteria in soybean rhizosphere soil at the phylum level (Fig. 5).
During nitrification, nitrite oxidation is the second and crucial step in
the conversion of ammonium to nitrate in the soil and is catalyzed by
nitrite oxidoreductase (Liu et al., 2023). Nitrospira is the main genus of
nitrite-oxidizing bacteria, which mediates nitrite oxidoreductase and
regulates the nitrification process (Daims et al., 2015). Our results
indicated that Nitrospira was the most abundant nitrifier in the genus
level and biochar incorporation dramatically lowered the abundance of
Nitrospira at the genus level in the rhizosphere (Fig. 5). Similar results
were also found by Li et al. (2020) and Jiang et al. (2021), who noted
that biochar inhibited nitrification by reducing Nitrospira abundance.
However, Luo et al. (2020) and Nielsen et al. (2014) have shown that
biochar addition can increase the abundance of Nitrospira by improving
soil pH levels in acid soils. It was found that Nitrospira was sensitive to
soil pH in acidified terrestrial environments, and its abundance could be
reshaped by soil pH variation (Han et al., 2017). The pH value of the
tested soil (pH 7.27) was elevated by biochar application but remained
within the suitable range for Nitrospira growth in all treatments. Thus,
the decrease in Nitrospira abundance in this experiment cannot be solely
attributed to the increase in pH due to biochar addition. Nitrospira,
however, as K-strategists, substrate concentrations were more note­
worthy than pH (Daims et al., 2001). It has a high affinity to the sub­
strate and plays a stronger functional role in oligotrophic environments.
Compared to CK, biochar treatments had higher NH+ 4 -N contents
(Table 1), which may weaken Nitrospira’s comparative superiority.
Nevertheless, the effectiveness of biochar in retaining NH+ 4 -N or
balancing soil pH largely depends on its properties, addition rate, as well
as soil environments. In addition, Shi et al. (2019) pointed out that
phenolic compounds in biochar, which depend on the feedstock and
pyrolysis process, may inhibit soil nitrification by acting as nitrifier
toxins. Based on our experimental data, we speculate that biochar
application did not facilitate nitrification in the soybean rhizosphere
soil, despite increasing NO−3 -N content. The nitrogen fixation of rhizo­
bium and the presence of biochar substantially enhanced the complexity
of nitrogen turnover in soybean rhizosphere soil. Future studies are
X. Cui et al.
necessary to verify this hypothesis and further explore the response of
Nitrospira to biochar.
Bradyrhizobium is a representative type of N2-fixing bacteria that
forms symbiotic relationships with legumes through nodules, increasing
soil N content through atmospheric N fixation (Anderson et al., 2011;
Telles et al., 2023). Bradyrhizobium was the dominant genus of the
rhizobia populations in the present study (Fig. 5), consistent with pre­
vious agricultural soil investigations (Yao et al., 2017; Zhalnina et al.,
2013). Several studies have previously reported the influence of biochar
addition on the relative abundance of Bradyrhizobium. Most of these
observations found that biochar depressed the abundance of Bradyrhi­
zobium in soil (Khan et al., 2014; Yao et al., 2017; Liu et al., 2019a). They
inferred that biochar addition impeded the utilizability of NO−3 -N or
NH+ 4 -N, which can function as the N source for the growth of Bradyrhi­
zobium. Nevertheless, similar phenomena were not observed in this
study. Conversely, our results suggested that both NO−3 -N and NH+ 4 -N
contents were boosted in biochar treatments (Table 1), and even the
abundance of Bradyrhizobium in B60 was significantly enhanced (Fig. 5).
As a result, the abundance of Bradyrhizobium was heavily influenced by
the mineral N content present in the soil. Bradyrhizobium growth was
promoted by abundant N with biochar addition, which further increased
soil N content. Biochar seemed to turn on the engine of soil N accu­
mulation in this experiment. On the other hand, our previous study has
shown that biochar can retain soil moisture, enhance aeration, and
provide a habitat for bacterial growth due to its porous structure (Yang
et al., 2017). Additionally, we have also previously demonstrated that
biochar contains labile organic C that can serve as an additional C source
for microbial growth (Yang et al., 2017; Yang et al., 2018). All of these
findings provide compelling evidence that Bradyrhizobium can colonize
the pores of biochar and thrive. More investigations should be con­
ducted in the future regarding the synergy of biochar and N2-fixing
bacteria in soil N cycling.
5. Conclusions
Determining the metabolites and microbial communities in soybean
rhizosphere soil under biochar field aging is helpful in revealing the
micro-mechanism of biochar to alleviate soybean continuous cropping
obstacles. Biochar application increased the TN, Ava N, NH+ 4 -N, and
NO−3 -N contents in soybean rhizosphere soil, and the higher the biochar
addition rate, the greater the increment. This study identified 7 signif­
icantly enriched metabolic pathways (e.g. phenylpropane and oxidative
phosphorylation) via topological analysis of KEGG metabolic pathways
in the presence of five-year field-aged biochar. There were 10 different
upregulated metabolites involved in the above pathways possessed by
all three biochar treatments, and they were closely related to the growth
regulation and stress resistance of soybeans. All three biochar treatments
markedly reduced the Nitrospirae abundance, and B60 lifted the Bra­
dyrhizobium abundance, thus confirming that biochar changed the N
cycling-related microbial communities in soybean rhizosphere soil. Our
research integrated metagenomics and metabolomics to verify that
biochar altered soil metabolites and microbial communities involved in
the N cycle in soybean rhizosphere soil, which contributes to a better
understanding of biochar’s role in soybean continuous cropping
agroecosystem.
CRediT authorship contribution statement
Xin Cui: Writing – original draft, Investigation, Data curation,
Conceptualization. Jun Yuan: Writing – original draft, Investigation,
Data curation, Conceptualization. Xu Yang: Writing – review & editing,
Supervision, Funding acquisition. Chaoqun Wei: Investigation. Ying­
hui Bi: Investigation. Qiang Sun: Software, Formal analysis. Jun Meng:
Conceptualization. Xiaori Han: Writing – review & editing.
12
Science of the Total Environment 917 (2024) 170522
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 availability
Data will be made available on request.
Acknowledgements
Gratitude is expressed to all our colleagues engaged in the field
management of this experiment. We are also grateful for Xinmei Jiang’s
assistance in grammatical editing and similarity detection of the
manuscript. This work was supported by the National Natural Science
Foundation of China (42207382) and the Science and Technology
Planning Project of Shenyang, China (22-317-2-08).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2024.170522.
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