Journal of Hazardous Materials 437 (2022) 129322

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Journal of Hazardous Materials

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

Research Paper

Coconut shell and its biochar as fertilizer amendment applied with organic
fertilizer: Efficacy and course of actions on eliminating antibiotic resistance
genes in agricultural soil
Houyu Li a, 1, Xiaolong Wang b, 1, Lu Tan a, Qian Li a, Chunxue Zhang a, Xiaocheng Wei a,
Qiang Wang a, Xiangqun Zheng a, *, Yan Xu a, *
a
Agro-Environmental Protection Institute, Ministry of Agriculture and Rural Affairs, Tianjin 300191, China
b
College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin
300071, 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

• Compared with coconut shell, adding its

biochar has a better inhibitory on soil
ARGs.
• Environmental factors drive ARGs
changes mainly by modulating bacterial
communities.
• Biochar can hinder species dispersal,
thereby inhibiting ARGs propagation.
• Biochar can enhance soil heterogeneity
that enable to suppress ARGs.

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

Editor: Lingxin Chen Biomass amendments have numerous benefits in reducing antibiotic resistance genes (ARGs) in the soil envi­
ronment. However, there are debatable outcomes regarding the effect of raw biomass and its pyrolytic biochar on
Keywords: ARGs, and the exploration of the influence mechanism is still in infancy. Herein, we investigated the changes in
Antibiotic-resistant genes soil ARGs under the organic fertilizer application with coconut shell and its biochar. The results showed that the
Coconut shell
coconut shell biochar could effectively diminish ARGs, with 61.54% reduction in target ARGs, which was higher
Biochar
than that adding raw coconut shells (p < 0.05). Structural equation modeling indicated that ARGs were
Inhibit
Mechanism significantly affected by changes in environmental factors, mainly by modulating bacterial communities. Neutral
community model and network analysis demonstrated that the coconut shell biochar can restrict the species
dispersal, thereby mitigating the spread of ARGs. Also, coconut shell biochar exhibited strong adsorption, with a
large specific surface area (476.66 m2/g) and pores (pore diameter approximately 1.207 nm, total pore volume:
0.2451 m3/g), which markedly enhanced soil heterogeneity that created a barrier to limit the resistant bacteria
proliferation and ARGs propagation. The outcome gives an approach to control the development of ARGs after
organic fertilizer application into soil.

* Corresponding authors.
E-mail addresses: Zhengxiangqun@126.com (X. Zheng), xuyan@aepi.org.cn (Y. Xu).
1
Houyu Li and Xiaolong Wang contributed equally to this work

https://doi.org/10.1016/j.jhazmat.2022.129322
Received 20 March 2022; Received in revised form 30 May 2022; Accepted 6 June 2022
Available online 7 June 2022
0304-3894/© 2022 Elsevier B.V. All rights reserved.
H. Li et al.
1. Introduction
In China, the amount of rural organic waste produced annually is
continuously increasing (Xie et al., 2018). The Chinese government has
promoted the agricultural reuse of rural waste, such as livestock, poultry
manure, and straw to reduce environmental pollution and economic
consumption. Therefore, farmers are bound use organic fertilizers made
from manure compost for cultivation. However, a new concern, that is,
the bloom of bacterial resistance in agricultural environments, has
emerged with the application of organic fertilizers (Subirats et al.,
2021). Numerous studies have revealed that organic fertilizers can
enhance the levels of antibiotic resistance genes (ARGs) in soil (Wu
et al., 2020a). As reported by Chen et al. (2019a), the diversity and total
abundance of ARGs in the arable soil of a corn plantation increased after
organic fertilizer application. However, most organic fertilizers cannot
be effectively processed, which may directly introduce numerous path­
ogenic bacteria and ARG into the soil environment, causing their spread
to crops (Sandberg and Lapara, 2016; Xie et al., 2019). Xie et al. (2022)
emphasised the need to manage the risk of ARG dissemination from
compost-based organic fertilizers because of the high mobility of ARGs
in organic fertilizers. Exogenous antibiotic-resistant human conditional
pathogens have been detected in farmland soils, and their abundance
and diversity have been increasing annually (Zhang et al., 2020a). Some
potential pathogens and resistant bacteria have also been frequently
detected in crops (Li et al., 2022). Altogether, these data acknowledge
the risk of ARG proliferation in agro-ecosystems. Thus, developing
technical solutions for the effective elimination of soil ARGs is critical to
ensure food safety and protect human health.
Biochar is a carbon-rich material with a large specific surface area,
rich pore space, high stability, and high adsorption capacity (Xiao et al.,
2018). Currently, using biochars prepared from wood wastes (Zheng
et al., 2021), sludge (Ihsanullah et al., 2022), and crop straw (Cheng
et al., 2021) for treating pollutants have been widely documented.
Numerous studies have proposed the idea of improving the soil quality
using biochar, and some of these studies have attempted using biochar
to alleviate ARG pollution in soil (Duan et al., 2017; Li et al., 2020a; Lian
et al., 2020). As previously reported, biochar can reduce the abundance
of ARGs in the environment, which may be related to the adsorption of
free DNA in the soil through hydrogen bonding, hydrophobic in­
teractions, and mesoporous filling (Wu et al., 2022a). Similarly, Zhang
et al. (2022a) revealed that rice straw return significantly accelerated
the elimination of ARGs, particularly intI1 and sul1. However, rice straw
biochar addition yielded contrasting results for ARG removal in a study
by Cui et al. (2016). These contradictory results suggest that not all
biochars consistently exhibited a positive result. Therefore, the effect of
biochar on ARGs in the soil remains elusive. Yang et al. (2021) revealed
that biochar can inhibit the spread of ARGs, and the possible reason was
that biochar may reduce interspecific electronic connection compo­
nents. Another study found that biochar primarily affected ARG distri­
butions via altering the composition of bacterial communities (Bair
et al., 2020). Despite these speculations, we are still unable to determine
the mechanism through which biochar affects the changes in ARGs.
Herein, we selected coconut shells and its biochar as a fertilizer
amendment. Coconut is the major cash crop in southern China and
south-eastern Asia and has a large yield. Coconut shells are typical
biomass materials with highly developed porous structures, and their
biochar can be employed as an adsorbent (Liu et al., 2017a; Wu et al.,
2020b). Thus, coconut shell biochar was previously applied to remove
contaminants, such as phenol (Hao et al., 2018) and heavy metals
(Paranavithana et al., 2016), present in the soil environment (Zhang
et al., 2022b). Nonetheless, there is a knowledge gap regarding the
management of ARGs in soil.
The aims of this study were as follows: 1) analyse the alterations in
ARG profiles after the addition of coconut shells and its biochar to
organic fertilizer, 2) identify the key regulators of soil ARG changes, and
3) explore the preliminary influence mechanism of coconut shells and its
2
Journal of Hazardous Materials 437 (2022) 129322
biochar on soil ARG proliferation. This study provides new insights into
the mechanisms for inhibiting the spread of ARGs in soil with added
biochar and offers an opportunity for green reutilization of agricultural
wastes.
2. Materials and methods
2.1. Experimental design and sample collection
The experimental field was located in Ninghe County, on the out­
skirts of Tianjin, China. It belongs to the Tianjin corn seed farm
(N:39◦ 25′ 31.89′′ ; E:117◦ 29′ 40.94′′ ), which is located far from livestock
farms, wastewater treatment plants, and other domestic emission sour­
ces (at least 5 km). The climate in this region is cold temperate conti­
nental monsoon with annual precipitation of 642 mm and annual mean
temperature of approximately 11.1 ◦ C. Organic fertilizer has never been
applied on the field before, and the soil type is cinnamon soil. The
experiment included four treatments: 1) non-fertilized soil, which was
used as a control treatment (NF treatment); 2) soil treated with organic
fertilizer (OF treatment); 3) soil treated with shredded coconut shells
and organic fertilizer (SF treatment); and 4) soil treated with coconut
shells and biochar organic fertilizer (BF treatment). The treatments were
named according to the acronyms of the materials. The organic fertilizer
was prepared using swine manure, with a total carbon (TC) of 51.52%,
total nitrogen (TN) of 2.16%, and total phosphorus (TP) of 1.14%. It was
applied only once during planting and was uniformly distributed
throughout the field at a depth of 15 cm, according to a local peasant’s
experience and previous study (Chen et al., 2022a). Moreover, the
shredded coconut shells had particle sizes of 1–2 cm, and the coconut
shell biochar was made by shredding coconut shell at 700 ◦ C for
approximately 3.5 h (particle size: 1–2 mm, Source of green Co., Ltd.).
The total duration of the experiment was 50 days, with a growth cycle of
40 days for pakchoi and post-harvest period of 10 days. The soil samples
were collected on days 0, 20, 40, and 50. Samples were collected via a
five-point sampling method from 0 cm to 20 cm. The sampling points
were uniformly distributed throughout the experimental field. Subse­
quently, the five samples were mixed to form a composite sample to
ensure homogeneity. Soil (1 kg) was extracted from the composite
sample for subsequent experiments. The specific methods and processes
used are presented in Fig. S1. Finally, all the samples were taken in
triplicates, placed in an ice-filled thermal barrier, and stored at − 20 ◦ C.
2.2. DNA extraction and PCR amplification
After the soil samples were thawed, grinded and mixed, DNA was
extracted from the soil using the DNeasy® PowerSoil® Pro Kit (QIA­
GEN) according to the manufacturer’s instructions (additional details
are provided in Text S1). Subsequently, the DNA concentration was
determined using a NanoDrop 2000 UV–vis spectrophotometer (Thermo
Scientific, Wilmington, USA), and DNA quality was analysed via 1%
agarose gel electrophoresis. Finally, the extracted DNA samples were
stored at − 20 ◦ C until analysis. High-throughput sequencing for mi­
crobial community analysis and quality control of the data are provided
in Text S2.
2.3. Quantitative real-time PCR
The Wafergen SmartChip real-time PCR system was used to perform
qPCR, as described by Su et al. (2015). In this experiment, 15 genes were
amplified from soil samples using a pair of 16 S rRNA internal reference
primers. Based on previous studies (Luo et al., 2010; Zhu et al., 2013), 13
ARGs, including two β-lactam resistance genes (blaTEM and blaVIM), two
sulphonamide resistance genes (dfrA1 and sul2), two quinolone resis­
tance genes (acrA-01 and acrA-02), three tetracycline resistance genes
(tetQ, tetM-01, and tetX), and four macrolide resistance genes (ermB,
ermF, ermA, and ermX), and one integrator gene (intI1) were selected for
H. Li et al.
testing. The primer sequences for the target genes are presented in
Table S1. The screening conditions and calculation methods for the re­
sults are provided in Text S3.
2.4. Measurement of soil properties
2.4.1. Environmental factors in soil
Based on national standard methods and previous studies (Han et al.,
2018; Smal et al., 2019), soil pH was measured using a pH meter (pH 5 S
Spear pH Tester, Shanghai, China) at a fresh soil-to-water ratio of 1:2.5.
Electrical conductivity (EC) was determined by end-to-end shaking at a
soil-to-water ratio of 1:5 for 1 h. Total organic carbon (TOC) and TN
contents were determined by applying oxidative spectrophotometry to
potassium dichromate and the Kjeldahl methods, respectively. The TP
content was determined via the molybdenum blue colorimetric method.
The samples were first digested using concentrated nitric acid and 70%
perchloric acid, followed by leaching with hydrochloric acid (Liu et al.,
2017b; Cheng et al., 2019; Duan et al., 2019). Soil ammonium nitrogen
- strated by a bar plot generated using OriginPro 2021 (OriginLab, USA).
(NH+ 4 -N) and nitrate nitrogen (NO3-N) were extracted using 1 M po­
tassium chloride via indophenol blue colorimetry and dual-wavelength
(220 nm and 275 nm) methods, respectively (Nie et al., 2018). The
- component analysis (PCA) was performed using the R packages ‘Fac­
concentrations of TN, TP, NH+ 4 -N, and NO3-N were determined using a
mobile injection analyser (AA3). The limits of detection, precision, ac­
curacy, quality assurance, and quality control for these factors are pro­
vided in Text S4.
2.4.2. Soil enzyme activity
The activities of peroxidase (POD), catalase (CAT), and alkaline
phosphatases (AKP) were determined via the methods described by
Chen et al. (2019b) and Li et al. (2020b). These enzymes are most
commonly detected in soil, and previous studies have confirmed that the
activities of POD, CAT, and AKP can drastically change after the appli­
cation of organic fertilizers (Markowicz et al., 2021; Xu et al., 2022).
Additionally, POD and CAT play important roles in the oxidation and
metabolic activity of aerobic organisms, which determines the removal
efficiency of environmental pollutants (Sellami et al., 2022).
2.5. Characterization of organic fertilizer and coconut shells and its
biochar
The surface morphologies of the materials (organic fertilizer, coco­
nut shells, and biochar) were observed using a field-emission scanning
electron microscope (Zeiss Sigma 500, Germany) (Feng et al., 2021). The
specific surface area was calculated via the Brunauer–Emmett–Teller
(BET) method (3 Flex, Micromeritics, USA) (Kim et al., 2021), as shown
in Eq. (1):
P 1 C− 1 P
= + × (1)
V(P0 − P) Vm × C Vm × C P0
Where P is the nitrogen partial pressure, P0 is the saturation vapour
pressure of nitrogen at the adsorption temperature, P/P0 is the relative
pressure, V is the amount of nitrogen gas adsorbed on the surface of the
tested sample, Vm is the adsorption capacity of the saturated nitrogen
monolayer, and C is a constant related to the adsorption capacity.
The pore size distributions of the organic fertilizer and coconut shells
were determined using the Barret–Joyner–Halenda (BJH) algorithm
(Vincent et al., 2022), as shown in Eqs. (2–4):
Kelvin equation:
/ as sublethal concentrations of antibiotics and heavy metals, can create a
rk = −
2σ VL
In(P/P0 ) (2)
RT
Halsey equation:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
̅ selective stresses. The absolute abundances of acrA-01, blaTEM, ermB,
/
P ermF, sul2, and tetM-01 decreased by 63.13%, 67.14%, 62.95%, 71.86%,
(3)
3
t = 6.0533 × − 1 ln⁡( )
P0
3
Journal of Hazardous Materials 437 (2022) 129322
Pore size of mesopore:
rp = rk + t (4)
Where rk is the curvature radius of the adsorbed gas condensed in the
pore, σ is the surface tension of the liquid condensate, VL is the molar
volume of the liquid condensate, R is the gas constant, T is the analytical
temperature, P is the nitrogen partial pressure, P0 is the saturation
vapour pressure of nitrogen at the adsorption temperature, and P/P0 is
the relative pressure.
The pore size distribution of the coconut shell biochar was calculated
via the non-local density functional theory (NLDFT) method (Wei et al.,
2016) because it contains both micropores and mesopores, and the
formula was obtained from Landers et al. (2013).
2.6. Statistical analysis
The reduction rate of the absolute abundance of ARGs is demon­
The relative abundance of ARG and bacterial data were graphically
represented in a heatmap using TBtools (Chen et al., 2020). Principal
toextra’ and ‘FactoMineR’ (R 4.0.3). Structural equation modelling
(SEM) was performed using Smart PLS 3 based on partial least-squares
path modelling. The explanation rate of ARGs from environmental fac­
tors and enzymes was calculated via variation partitioning analysis
(VPA) in R (4.0.3) and using Vegan 2.0. Computations of the neutral
community model (NCM) were performed in R. Redundancy analysis
(RDA) was performed using CANOCO 4.5 (Microcomputer Power, USA)
to identify potential correlations between ARGs and environmental
factors. In addition, network analyses of the interactions among bacteria
and co-occurrence relationships between bacteria and ARGs were per­
formed using R language and Gephi 0.9.2.
3. Results and discussion
3.1. Alterations in ARG profile and intI1 in differential treatments
PCA revealed that the distribution of soil ARGs was clustered into
four groups based on different fertilizer treatments and application
times (Fig. 1A). This suggests that the absolute abundance of ARGs in the
soil was influenced by the amendment of the coconut shells and its
biochar. And there was observed that the absolute abundance of ARGs
after fertilizer application (fertilizer application: 2.00 ×103–2.64 ×105
copies/g) was higher than that of NF (ND–1.94 ×103 copies/g), sug­
gesting that the abundance of soil ARGs would increase following
organic fertilizer application. This is consistent with the findings of Xu
et al. (2021a). The reduction rate of the absolute abundance of ARGs is
shown in Fig. 1B, and the data are provided in Table S2. After 50 d, the
absolute abundances of partial ARGs, including acrA-01(50.94%), bla
TEM (59.80%), ermB (56.72%), and tetM-01 (48.11%), decreased under
the NF treatment owing to the lack of favourable survival conditions,
such as the influence of antibiotics or heavy metals, which assist in
resistant bacterial proliferation and horizontal gene transfer (HGT)
among bacteria. Notably, the conjugative transfer of genes requires
appropriate concentrations of carbon, nitrogen, and phosphorus to
provide the necessary energy (Guo et al., 2015; Li and Zhang, 2022).
Gullberg et al. (2014) also stated that some environmental stresses, such
continuous survival advantage to maintain the transferring of resistant
plasmids, which eventually promotes the development of ARGs.
Accordingly, the abundance would generally decline without external
66.97%, and 70.11%, respectively, in BF, which were higher than those
in the SF and OF treatments. The removal efficiency of these genes in SF
H. Li et al. Journal of Hazardous Materials 437 (2022) 129322

Fig. 1. (A) Principal component analysis (PCA) of antibiotic resistance genes (ARGs) under different treatments and times. (B) Bar plot: the reduction rate absolute
abundance of ARGs and intI1 among all treatments. (C) Heatmap: the relative abundance of ARGs and intI1 among all treatments, and the data of ARGs are
normalized by Log 10.

ranged between those of BF and OF and decreased by 49.33%, 47.18%,
39.59%, 58.86%, 56.42%, and 35.43%, for acrA-01, blaTEM, ermB, ermF,
sul2, and tetM-01, respectively. Thus, it was concluded that the addition
of coconut shell biochar was more effective in reducing soil ARGs.
Similarly, Qiu et al. (2021) revealed that biochar amendment could
effectively decrease the occurrence of extracellular and intracellular
ARGs in soil. In particular, the absolute abundance of partial ARGs (such
as blaTEM, ermB, and tetX) exhibited increasing trends after harvesting
(from 40 d to 50 d, Fig. S2), wherein a more conspicuous ascending
trend was observed under the SF treatment than under the BF treatment,
suggesting that biochar could more effectively suppress ARG persistence
than pure coconut shells.
Furthermore, to minimise the impact of the total biovolume, the
relative abundance of the ARGs was calculated, as shown in Fig. 1C.

4
H. Li et al. Journal of Hazardous Materials 437 (2022) 129322

There was no significant difference in the variance of relative abundance
and absolute abundance of ARGs within the groups (p > 0.05), and both
exhibited similar trends. The relative abundance of acrA-01, blaTEM,
ermB, ermF, ermX, and tetM-01 in the BF treatment decreased by 56.96%,
61.63%, 56.75%, 67.15%, 62.22%, and 65.10%, respectively, from
0 d to 50 d. Therefore, using coconut shell biochar as an additive with
organic fertilizer application can be considered a solution to control soil
ARG proliferation caused by the introduction of organic fertilizer.
3.2. Key regulators of ARG changes under different treatments
3.2.1. Relationships among ARGs and soil environmental factors, enzymes,
and bacterial community
SEM was conducted to examine the variations in ARGs under
different treatments in response to soil environmental factors, enzymes,
and bacterial communities (Fig. 2). As shown in Fig. 2A, bacterial
community, enzyme type, and environmental factors had significant
impacts on soil ARG variance, with path coefficients of − 0.464
(p < 0.05), − 0.853 (p < 0.001), and 0.591 (p < 0.001), respectively.
Enzyme type and environmental factors had a crucial role than the
bacterial community, which is consistent with a study by Cui et al.
(2022), who concluded that soil properties, rather than the bacterial
community, highly influenced ARG abundance. In addition, the impact
of the bacterial communities on ARGs was primarily dependent on in­
direct effects, as evidenced by the high path coefficients for the indirect
effect (− 0.650, p < 0.05) (Fig. 2B). This finding is supported by Yang
et al. (2022), who revealed that soil properties indirectly affect ARGs via
influencing the bacterial community. Notably, soil enzymes had the
major direct effect on ARGs, which has typically been overlooked in
previous studies. Enzymes are the metabolites of microorganisms

Fig. 2. Structural equation modelling (SEM) to analyse the relationship among environmental factors, bacterial community, enzymes, and ARGs. (A) Total effect; (B)
Direct effect and indirect effect. The numbers adjacent to blue arrows represent the standardised path coefficients, which are direct effects. The green arrows
represent indirect effects. The R2 values represent the proportion of the variance explanation. The p < 0.05: "* ", p < 0.01: "* *" p < 0.001: "* ** ". (C) Specific indirect
effect. Note: A direct effect+indirect effect=total effect.

5
H. Li et al.
regulated by environmental factors and communities; however, such
processes predate the effect of environmental factors on ARGs (Brzostek
and Finzi, 2012; Min et al., 2014; Zuo et al., 2021). We believe that the
chain of action appears to be from environmental factors to bacterial
communities to enzymes to ARGs. Therefore, it is deduced that envi­
ronmental factors might be major drivers of ARG alterations in soil.
3.2.2. Effect of abiotic factors on ARGs: environmental factors and
enzymes
VPA was performed to quantitatively analyse the importance of
environmental factors and enzymes on ARG changes. As shown in
Fig. 3A, the total effects of environmental factors and enzymes on ARG
changes accounted for 63.53% of all ARG changes. The rates for envi­
ronmental factors and enzymes were 42.48% and 13.81%, respectively,
and the collective rate was 28.67%. This result corresponded to our
previous inference that environmental factors primarily drive soil ARG
changes, among which TOC and EC were identified as the major
explanatory variables, whereas POD was the most critical enzyme
affecting ARGs. This was corroborated by RDA (Fig. 3B), in which the
cumulative interpretation rate of the first and second axis was 60.1%. A
significant positive correlation existed between TOC and ermA
(p < 0.05) and between POD and arcA-02 (p < 0.05). Most ARGs
exhibited a significant positive correlation with EC. The t-test analysis
only exhibited a significant difference (p < 0.05) in the TOC across the
OF, SF, and BF treatments, suggesting that biochar amendment pri­
marily affected the TOC in the soil, thereby modulating the microbial
community when combined with the above SEM analyses. A similar
finding reported by Li et al. (2021) was that organic matter could affect
ARG patterns by shifting bacterial communities. Studies have revealed
that the migration of ARGs in soil is related to EC (Liu et al., 2022). POD
can participate in the oxidative/precipitation biodegradable process
required for ARG elimination (Chen and Wan, 2017). However, in most
cases, TOC and POD were negatively correlated with ARGs, consistent
with a recent finding that the abundance of ARGs had a negative effect
on the TOC content in soil (Zhang et al., 2019). Therefore, controlling
TOC and POD at appropriate concentrations in the soil environment may
mitigate the risk of ARGs proliferation. To date, most studies have been
conducted on the control methods of POD and TOC in the soil. Peroxi­
dase can be a biological catalyst for bioremediation in soil (Sellami et al.,
2022). TOC can be enhanced by biochar application (Zhang et al., 2021),
straw returning (Huang et al., 2021), and land conversion (Chen et al.,
2022b).
3.2.3. Effect of biotic factors on ARGs: microbial community
Numerous studies have revealed a close relationship between ARGs
and microbial communities (Lu et al., 2018; Cerqueira et al., 2019b).
The variations in microorganisms can affect the behaviour of ARGs in
the environment. However, in this study, the role of soil microbes in
ARG changes remained unclear. The evolution of microorganisms de­
pends on their habitat preferences and fitness, which are commonly
affected by abiotic and biotic factors, including environmental factors
(such as pH and temperature) and species interactions (such as
competition and predation). This is referred to as the deterministic
process. In addition, neutral theory suggests that the structure of mi­
crobial communities is shaped by stochastic processes, including birth,
death, migration, species formation, and limiting dispersal (Chen et al.,
2019c). Numerous studies have revealed the succession of microor­
ganisms based on these two processes using an NCM (Mo et al., 2021).
Herein, we describe these processes using NCM based on operational
taxonomic units (OTUs) to determine the role of microbes in ARG var­
iations. As illustrated in Fig. 4, the relative contributions of stochastic
processes under the SF (R2 =0.582) and BF (R2 = 0.594) treatments were
lower than that under the OF treatment (R2 =0.664). This indicates that
either coconut shell biochar or pure coconut shell supplementation can
hinder the bacterial community reshaping. The impact of the deter­
ministic process enhanced the bacterial community that developed
6
Journal of Hazardous Materials 437 (2022) 129322
under the SF and BF treatments. The effects of environmental factors
were validated, and interspecific interactions were expressed using a
network analysis of co-occurrence relationships among the bacteria
(Fig. 5). The 95, 134, and 160 edges, which represented negative
co-occurrence relations (R<− 0.6, p < 0.05), were obtained under the
OF, SF, and BF treatments, respectively. This indicates that biochar
addition caused a significant increase in competitive relationships in the
soil. For example, significant negative relationships were observed
among Nocardioides, Agromyces, and Nitrospira (p < 0.05). Among these,
Nitrospira is the dominant genus in the soil microbiota, all of which are
typically considered indigenous soil bacteria (Bartosch et al., 2002).
Nocardioides and Agromyces were previously considered as the bio­
markers of the thermophilic phase during composting (Chavez-Romero
et al., 2016; Zhao et al., 2019), and they were introduced into the soil by
fertilizer to become exogenous bacteria. The relative abundances of
Nocardioides and Agromyces decreased by 59.70% and 50.05%, respec­
tively; however, the abundance of Nitrospira increased by 30.42%
(Fig. S3). These findings suggest that there is a competitive relationship
between indigenous and exogenous bacteria, and the structure of the soil
bacterial community was changed by exogenous bacteria. This is
consistent with a study by Yang et al. (2022), who reported that exog­
enous microorganisms may not colonise successfully because of the
competitive nature of indigenous microbes. As described by Xu et al.
(2021b), exogenous bacteria could alter the composition of the soil
bacterial community, and Mallon et al. (2018) revealed the response of
the indigenous bacterial community to invasion. Additionally, the m
values of the different treatments in NCM were in the order of
OF>SF>BF (0.045, 0.041, and 0.039, respectively), indicating that the
species dispersal of the bacterial community in the BF treatment was
limited. This could be explained by the positive co-occurrence rela­
tionship in the network analysis (Fig. 5), which was lower in the BF
treatment (56.16%) than in the other treatments (OF: 69.94%; SF:
70.22%). Thus, this largely restricted the HGT of ARGs among bacteria,
which partly explained the lowest ARG abundance in the BF treatment,
as concluded based on a similar finding obtained by Zheng et al. (2021)
that the combined application of biochar and pyroligneous acid can
reduce the level of ARGs via weakened HGT.
Overall, these results indicate that fertilizer application with coconut
shell biochar can reduce ARGs by influencing HGT and bacterial com­
munity interaction. However, the effect of biochar addition on ARG
reduction is also determined by the properties of biochar, and different
properties of biochar have different effects (Sun et al., 2014a), such as
pore size, surface area, etc. As described by previous studies, those
properties generally depend on the carbonization temperature, feed­
stock type and pyrolysis operating conditions (Kavitha et al., 2018; Das
et al., 2021). For instance, Ding et al. (2019) selected six types of biochar
to impact the ARGs compositions and abundance in the soil, included
sludge, soybean, bark, rice, bamboo or manure biochar. And results
signified that the differences among the raw caused the properties of
biachar variation, which performed various effects on ARGs. In Xiao
et al. study, it was found that changes in biochar surface area can affect
the removal of ARGs. These findings all illustrated that biochar prop­
erties played a pivotal role in affecting the behavior of ARGs in different
systems. Therefore, coconut shell biochar is compared with either straw
biochar or other types of biochars, there will occur certain differences in
the impact on ARGs. At present, the commonly used pyrolysis temper­
ature is mainly in the range of 300 ℃− 600 ℃, with a few reaching
700 ℃. And the study on the effect of ARGs reduction after coconut shell
biochar addition was lacking. Based on these, we elaborate the prop­
erties of organic fertilizer, coconut shell and its biochar below.
3.3. Influence of material properties on ARGs
To determine whether the proliferation of ARGs was inhibited in the
soil after the application of fertilizer with biochar amendment, scanning
electron microscopy was performed to characterise the morphologies of
H. Li et al. Journal of Hazardous Materials 437 (2022) 129322

Fig. 3. (A) Variation partitioning analysis (VPA) to calculate the contributions of environmental factors and enzymes for ARGs. The bar graphs show the explanation
of each environmental factor and enzyme. (B) Redundancy analysis (RDA) of environmental factors, enzymes, and ARGs under all treatments, where the red arrows
indicate the environmental variables (explanatory variables), and the blue arrows represent the ARGs (explanatory variables).

7
H. Li et al.
Fig. 4. Fit of the neutral community model (NCM) of the bacterial community. The solid blue lines indicate the best fit to the NCM, and the dashed blue lines
represent 95% confidence intervals around the model prediction. Operational taxonomic units (OTUs) that occur more or less frequently than predicted by the NCM
are shown in different colours. Nm indicates the meta community size time immigration; R2 indicates the fit to this model.
the swine manure fertilizer, coconut shell, and biochar. As shown in
Fig. 6A, considerably different morphologies existed among the three
materials, among which the surface of the organic fertilizer was smooth,
with the formation of regular sheets. Evidently, the pores of coconut
shells were larger than those of organic fertilizers, and the surface of the
debris remained smooth. In contrast, coconut shell biochar primarily
comprised numerous stacked small fragments, with loose surface
structure, and a mass of minuscule pores could be observed. This implies
that coconut shell biochar had better adsorption properties than organic
fertilizers and coconut shells. Consistent with the present results, Sme­
bye et al. (2016) demonstrated that the high specific surface area of
biochar could promote adsorption. Similarly, Wu et al. (2022a) also
concluded that ARG could be reduced by the adsorption of cell-free
DNA. Furthermore, a strengthened spatial heterogeneity of the soil
was observed after the application of fertilizer with biochar (Fig. S5). A
previous study also described that straw biochar applied to soil could
induce the reorganization of the pore size distribution and aggregation
processes, resulting in an increase in soil porosity (Sun and Lu, 2014b).
This may weaken the communication between bacteria, thereby inhib­
iting the horizontal transfer of genes that could reduce ARG proliferation
in different environments. As stated in previous studies, biochar can
make microorganisms get rid of interference from the external
environment.
More than that, N2 adsorption–desorption curves and pore size dis­
tributions were mapped to further define the adsorption properties of
these materials. In the N2 adsorption–desorption curves (Fig. 6B), the
adsorption capacity was stronger when P/P0 was larger during the self-
acceleration of the adsorption process (Lin et al., 2013). There was a
higher quantity adsorbed in the curves of the coconut shell biochar
under the same P/P0, suggesting that the coconut shell biochar had a
better adsorption capacity than the others. This may be attributed to the
large specific surface area of the biochar. The specific surface areas of
the organic fertilizer, coconut shell, and biochar were 1.69 (C: 9.176;
correlation: 0.9995), 0.98 (C: 31.193; correlation: 0.999, and 476.66 (C:
676.6; correlation: 0.9999) m2/g, respectively. Wu et al. (2022b) re­
ported that the large specific surface area of biochar provides shelter for
microorganisms, and it can adsorb more soil microorganisms located
away from the original soil environment. This was also a cause of ARG
reduction via blocking communication among microorganisms and
inhibiting their spread.
Moreover, the pore size distribution was calculated using BJH
(organic fertilizer and coconut shell) and NLDFT (coconut shell biochar)
(Fig. 6C). The pore diameters of the highest frequency in the organic
fertilizer and coconut shells ranged from 2 to 20 nm. However, the main
pore diameter of the biochar was approximately 1.207 nm. The total
pore volume of the biochar (0.2451 m3/g) was larger than those of the
organic fertilizer (0.0044 m3/g) and coconut shells (0.0029 m3/g),
8
Journal of Hazardous Materials 437 (2022) 129322
indicating that coconut shell biochar is more microporous. Devens et al.
(2018) reported that the pores of biochar can partly reflect the removal
capacity of the contaminants. The porous structure of biochar could
provide room for the growth and reproduction of microorganisms and
provide a small amount of nutrition to resist the impact of external
environmental factors on the growth of indigenous microorganisms
(Lam et al., 2019; Zhang et al., 2020b). These pores also enhance steric
hindrance among the microorganisms. These results support the
conclusion that biochar can inhibit ARG proliferation via building more
barriers between bacteria and the external environment.
Consequently, coconut shell biochar could be more conducive to
hinder the transmission of ARGs among bacterial communities in the
soil. However, there is an evident disadvantage, which is that the bio­
char could not completely remove ARGs during the adsorption process,
thereby potentially inducing emission risks (Yao et al., 2021). In
particular, the partially adsorbed ARGs on biochar or the colonised
microbial community harbouring ARGs may persist in soil. The present
results were based on a short-term field trial; therefore, the long-term
effects of the biochars derived from coconut shells should be further
studied. This also could determine the length of the biochar usage period
and whether the disinfection process should be executed during the
growth of plant cycle.
4. Conclusions
This study demonstrated that the application of organic fertilizer in
combination with coconut shell biochar could effectively inhibit ARG
proliferation in agricultural soil. The shifts of ARG were significantly
affected by environmental factors via regulating bacterial communities.
And coconut shell biochar could hinder soil bacterial community
reshaping, limit species dispersal, and strengthen competitive actions
among bacteria, thereby mitigating the proliferation of ARGs. Moreover,
coconut shell biochar exhibited a strong adsorption capacity and
increased soil heterogeneity which may restrict the reproduction of
antibiotic-resistant bacteria and partly suppress the spread of ARGs. This
study provides a strategy for the efficient and safe utilization of rural
waste, which could contributes to the mitigation of agricultural soil ARG
to sustain the health of farming environments, thereby reducing public
health risks.
Environmental Implication
The effect of biochar on the elimination of soil antibiotic resistance
genes (ARGs) remains controversial. Herein, we selected coconut shells
and their biochar as research objects to investigate the effect of differ­
ences between biomass and biochar applied with organic fertilizers on
the elimination of soil ARGs. We also dissected the key influencing
H. Li et al. Journal of Hazardous Materials 437 (2022) 129322

Fig. 5. Network analysis among the bacteria based on operational taxonomic units (OTUs). All include negative and positive relationships (R> |0.6|, p < 0.05). Here,
the blue and grey dots represent the classified and unclassified bacteria, respectively, and the magnitude correlations were represented by the degree of thickness in
the black lines. In the region denoting a negative correlation, different colours represented different genera, and a larger dot means that it was associated with
more genera.

factors driving ARG changes and explored their influence mechanisms
on soil ARG proliferation. This study provides a comprehensive under­
standing about the effect of biochar on ARG removal in soil and offers an
approach to control soil ARGs caused by organic fertilizer application.
CRediT authorship contribution statement
Houyu Lia: Conceptualization, Methodology, Formal analysis,
Writing – original draft. Xiaolong Wang: Conceptualization, Method­
ology, Formal analysis, Writing – original draft. Lu Tan: Methodology.
Qian Li: Methodology. Chunxue Zhang: Formal analysis, Visualization.

9
H. Li et al. Journal of Hazardous Materials 437 (2022) 129322

Fig. 6. (A) Scanning electron microscope images of organic fertilizer, coconut shell, and coconut shell biochar and their surface plot. (B) Nitrogen adsorption and
desorption curves of swine manure fertilizer, coconut shell, and coconut shell biochar. (C) Pore size distribution of organic fertilizer, coconut shell, and coconut
shell biochar.

10
H. Li et al.
Xiaocheng Wei: Supervision, Project administration, Conceptualiza­
tion. Qiang Wang: Supervision, Project administration, Conceptuali­
zation. Xiangqun Zheng: Writing – review & editing, Funding
acquisition. Yan Xu: Writing – review & editing, Funding acquisition.
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.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (41807369 and 42107456), Fundamental Research Funds for
the Central Universities (2021-jbkyywf-xy) and China Postdoctoral
Science Foundation (2021M691662).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2022.129322.
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