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Sludge Conditioning Treatments Impact the Fate of Antibiotic Resistance

Genes in Agricultural Soils Amended with Sludge Composts

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DOI: 10.1021/acsestengg.2c00113

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 pubs.acs.org/estengg Article

Sludge Conditioning Treatments Impact the Fate of Antibiotic

Resistance Genes in Agricultural Soils Amended with Sludge
Composts
Yi Lu, Jiajun Wang, Xinxin Wang, Xiaoqing Meng, Su Yan, Yu Chen, Lixiang Zhou, and Guanyu Zheng*
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ABSTRACT: Sewage sludge is one of the major environmental

reservoirs of antibiotic resistance genes (ARGs), while its recycling
releases abundant ARGs into the agricultural soils. Sludge
conditioning treatment, as an indispensable step to improve sludge
dewatering, can enhance the dewaterability of sewage sludge and
meanwhile attenuate ARGs in sludge compost, but it remains
unclear how sludge conditioning treatments impact the ARG
profiles in different agricultural soils amended with sludge
composts. In the present study, the fates of 18 ARGs and 2
mobile genetic elements (MGEs) in two types of agricultural soils
(i.e., red soil and yellow−brown soil) were investigated for 170
days, after the land application of sludge composts derived from
different conditioning treatments, including bioleaching and chemical conditioning using Fe[III]/CaO or polyacrylamide (PAM).
The results showed that the absolute abundance of total ARGs and MGEs was the lowest in both the red soil and the yellow−brown
soil amended with bioleached sludge compost (Day 170), which were only 26.4−76.8% of that in the corresponding soils amended
with the compost products of raw, PAM-conditioned, or Fe[III]/CaO-conditioned sludge. Besides, in comparison with other
conditioning treatments, the bioleaching conditioning treatment more greatly limited the enrichment of typical sludge-borne ARGs
(sul2, aadA1, aadA2-01, aadA2-02, and aadA2-03) and their potential hosts. It was found that the much less enrichment of sludge-
borne ARGs achieved by bioleaching conditioning most probably resulted from both the low abundance of ARGs in bioleached
sludge compost and the limited growth of bacteria carrying ARGs in the amended agricultural soils. Therefore, bioleaching
conditioning is superior to the chemical conditioning using Fe[III]/CaO or PAM in mitigating antibiotic resistance in different
agricultural soils amended with sludge composts, which was contributed by the preremoval of ARGs in sludge compost and the
potentially limited growth of bacteria carrying ARGs after the land application of sludge compost.
KEYWORDS: sludge compost, conditioning treatment, agricultural soil, antibiotic resistance gene, land application

■ INTRODUCTION
The enrichment and dissemination of antibiotic resistance
genes (ARGs) are weakening the effectiveness of various
antibiotics, which has been one of the largest public health
issues in the 21st century.1−5 If the current situation of
antimicrobial resistance continues, a loss of approximately 11
million people is estimated to occur by 2050, and meanwhile,
the global economy size will probably be reduced by 0.1−
3.1%.6−8 Sewage sludge is one of the major environmental
reservoirs of ARGs, and its recycling is threatening public
health and environmental safety by releasing abundant ARGs
into the natural environment.2,9−14 The agricultural soils, as
the final destination of land-applied sewage sludge, contain
diverse environmental microbes and mobile genetic elements
(MGEs), thus providing a breeding ground for the further
enrichment and spread of sludge-borne ARGs.15−17 With the
increases in sewage sludge production and antibiotic usage, the
environmental burden from sludge-borne ARGs is becoming
more and more serious in the agricultural soils receiving
sewage sludge. Thus, it is vital to develop feasible approaches
for the management and attenuation of antibiotic resistance
during the land application of sewage sludge.
The explicit goal of mitigating the release of ARGs from
sewage sludge should focus on employing effective and
practical technologies within the treatment facility, rather
than depending on the natural attenuation of sludge-borne
ARGs subsequent to the land application of sewage sludge, due
to their extremely slow decay rates in agricultural soils.15,17
Received: April 4, 2022
Revised: June 14, 2022
Accepted: June 14, 2022

© XXXX American Chemical Society https://doi.org/10.1021/acsestengg.2c00113

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Aerobic composting and anaerobic digestion, the most
common technologies for sewage sludge treatment, are widely
utilized to stabilize sewage sludge before the land application
of sewage sludge, and their impacts on sludge ARG profiles
have already been elucidated.2,18−21 Although ARGs possess-
ing varied resistance mechanisms distinctly behaved during the
anaerobic digestion process of sewage sludge, anaerobic
digestion was still found to be useful to mitigate ARGs
conferring resistance to tetracycline, macrolides, and amino-
glycosides.22,23 In addition, both Su et al. (2015)24 and Zhang
et al. (2016)25 have found that the ARG subtypes and the
ARG abundance can even be increased by the sludge
composting process. In comparison with anaerobic digestion,
aerobic composting of sewage sludge is extensively employed
by wastewater treatment plants (WWTPs) in China. Prior to
aerobic composting, sewage sludge should be conditioned and
then mechanically dewatered to obtain dewatered sludge cakes
with low content of moisture.2,13,26,27 Thus, effective
conditioning, high-efficient dewatering, and aerobic compost-
ing form an important technical routine for sewage sludge
treatment in China.
The conventional conditioning could not effectively reduce
the ARG abundance in sewage sludge and the aerobic
composting even enriched the sludge-borne ARGs.2,13
Fortunately, our previous study found that bioleaching
conditioning treatment, which is a biological conditioning
approach driven byAcidithiobacillus ferrooxidansto improve the
dewaterability of sewage sludge,28,29 is much more effective
than chemical conditioning using polyacrylamide (PAM) or
Fe[III]/CaO in reducing the ARG abundance in compost
product of conditioned sludge. This mainly resulted from the
fact that ARGs in sewage sludge can be removed during
bioleaching conditioning and the horizontal transfer of residual
ARGs in conditioned sludge can be further limited during the
composting of bioleached sludge.2 Although some previous
study has reported the fates of ARG profiles in soils following
sludge compost application,7 few studies focused on the roles
of sludge conditioning treatments in limiting the transfer and
enrichment of sludge-borne ARGs during the land application
of sewage sludge composts. In fact, different sludge
conditioning treatments most probably exert distinct influences
on change trends of ARG profiles during the land application
of sludge composts, via prereducing ARGs in sludge composts
to different extents, changing soil physicochemical properties
to varying degrees, and impacting the soil bacterial community
in different ways. In addition, the soil type can also influence
the fates of ARGs in soils receiving sludge composts, mainly
due to the differences in the microbial community, environ-
mental factors, and soil texture among soil types. Munir and
Xagoraraki (2011)30 reported elevated levels of tetW, tetO, and
sul1 in loam soil amended with biosolid, but no significant
increase of ARGs in sandy loam soil amended with the same
biosolid. Similarly, Zhang et al. (2018)7 reported that changes
in ARG abundance were different among loess, red, and black
soils after they were amended with a sludge compost product.
Therefore, it is meaningful to investigate the impacts of
different conditioning treatments on ARG profiles in soils
following sludge compost application and thus to select
potential conditioning technologies that can be employed by
WWTPs to effectively attenuate the spread of sludge-borne
ARGs into agricultural soils.
Chemical conditioning using Fe[III]/CaO or PAM and
bioleaching conditioning are three conditioning approaches
B https://doi.org/10.1021/acsestengg.2c00113
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that are widely used in WWTPs of China.13,31 Red and
yellow−brown soils are representative agricultural soils in east
China, on which rice and vegetables are widely cultivated using
artificial agricultural irrigation systems.32 The present study
thus aims to investigate how the bioleaching conditioning and
chemical conditioning using Fe[III]/CaO or PAM impact the
profiles of ARGs and MGEs in red and yellow−brown soils
amended with different sludge composts and to reveal the
main factors influencing the dynamics of ARGs in a period of
170 days. The findings of the present study can guide the
WWTPs to select appropriate conditioning approaches to
attenuate the release of sludge ARGs into the soil environment.
■ MATERIAL AND METHODS
Conditioning, Mechanical Dewatering, and Aerobic
Composting Treatments of Municipal Sewage Sludge.
Municipal sewage sludge with a volume of around 600 L was
collected from the sludge thickening pond of Lucun WWTP
(Wuxi City, Jiangsu Province, China), and then their solid
content (2.72%), organic matter content (53.9%), and pH
(7.01) were immediately determined using the corresponding
standard methods.33 Subsequently, the sewage sludge was
conditioned, mechanically dewatered, and aerobically com-
posted according to our previous studies.2,19 Bioleaching
conditioning, chemical conditioning using PAM, and chemical
conditioning using Fe[III]/CaO were respectively applied to
treat sewage sludge. The detailed procedures of each
conditioning treatment kept consistent with that described in
our previous study and can also be found in Table S1 of the
Supporting Information.2 After the various sludge conditioning
treatments, the obtained conditioned sludge and uncondi-
tioned sludge (raw sewage sludge) were mechanically
dewatered using a small-scale mechanical filter press unit as
described in our previous study.19 There are three nylon filter
cloth-covered chambers in the filter press unit for sludge
pressing and the retrieval of dewatered sludge cake, and a
pressure of 20 MPa generated by a hydraulic pump was applied
to the chambers. The dewatered sludge cakes of unconditioned
and conditioned sludge were separately collected, mixed with
the mushroom residue, and then composted for 39 days in four
respective lab-scale reactors to obtain the corresponding
compost products. The detailed composting processes were
described in our previous publications.2,19 The organic
contents of the four sludge composts were 38.1−54.2%, and
their total organic carbon, total nitrogen, and total phosphorus
were 177−216, 21.8−37.8, and 27.7−34.8 g/kg dry solid,
respectively.
Land Application Experiments of Various Sludge
Composts. The red soil and yellow−brown soil were
respectively applied to carry out the land application
experiments of various sludge composts, including the compost
products of raw sludge, PAM-conditioned sludge, bioleached
sludge, and Fe[III]/CaO-conditioned sludge. The red soil with
a moisture content of 14% was collected from the Red Soil
Research Institute located in Jiangxi Province of China, and the
yellow−brown soil with a moisture content of 20% was
collected from the Baima Experimental Farmland of Nanjing
Agricultural University in Jiangsu Province of China. The soil
samples were air dried, crushed, and sieved through a 2 mm
mesh before use, and then, the basic properties of soil samples
were measured (Table S2). Sludge composts with a weight of
80 g (dry solid) were respectively added into these two types
of soils (2000 g, dry weight) once at the beginning. Soils with
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Figure 1. Changes in the absolute abundance of ARGs and MGEs in soils (a: red soil; b: yellow−brown soil) after the land application of composts
derived from raw and various-conditioned sludge. R1-R170: soil amended with raw sludge compost for 1−170 days; P1−P170: soil amended with
PAM-conditioned sludge compost for 1−170 days; B1−B170: soil amended with bioleached sludge compost for 1−170 days; F1−F170: soil
amended with Fe[III]/CaO-conditioned sludge compost for 1−170 days; CK1-CK170: soil without sludge compost for 1−170 days.

different sludge composts were regarded as the treatment
group of raw, PAM, bioleaching, and Fe[III]/CaO, respec-
tively, while the soils without any sludge compost were
regarded as the control group. The deionized water was added
to the abovementioned soil samples to adjust their moisture
contents according to the field capacity of corresponding soils,
and the whole adding process was very slow to prevent soil
agglomeration. The treatment and control soil samples were
placed in a series of 1.95 L experimental boxes (20 × 15 × 6.5
cm), and all soil samples (2.71−2.76 kg) were cultured for a
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period of 170 days, in a climate incubator (Saifu prx-250c,
Ningbo) under the conditions of 20 °C and 70% relative
humidity. A total of 30 experimental boxes were used to ensure
that each treatment or control was conducted in triplicate.
During the experiment, 5 g of soil samples were collected
from each experimental box on Day 1, Day 15, Day 45, Day 70,
Day 120, and Day 170. Soils were collected from the three sites
along the diagonal line in each box with a depth of 2−4 cm,
and then the collected soils in each box were mixed thoroughly
as a representative sample. A total of 180 soil samples were
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obtained for all treatments. The pH, moisture content, water-
soluble organic nitrogen content (WSON), and water-soluble
organic carbon content (WSOC) of each soil sample were
characterized using their respective methods (Figures S1−
S3).2,19 In addition, 1 g of each soil sample was frozen at −20
°C and then freeze-dried by using a vacuum freeze dryer
(Scientz-N10, Ningbo Scientz Biotechnology Co., Ltd.,
China). The freeze-dried soil samples were utilized to extract
total genomic DNA in triplicate using a PowerSoil DNA
Isolation Kit (MOBIO Laboratories, CA, USA). After merging
the triplicate DNA extracts, their concentration and purity
were measured at A260 and A280 nm, using a NanoDrop 2000
(Thermo Scientific, USA) (Table S3).2,19 The total genomic
DNA was eluted in 100 μL of 10 mM Tris−HCl buffer (the
mixture of trimethylolmethylaminomethane and dilute hydro-
chloric acid, pH 8) and stored at −80 °C until further use.
Real-Time Quantitative PCR for the Measurement of
ARGs, MGEs, and 16S rRNA Genes. The 10 aminoglyco-
sides (aadA1, aadA-01, aadA-02, aadA2-01, aadA2-02, aadA2-
03, aadA5-01, strB, aac(6′)-Ib-03, and aac(6′)-II), 3 tetracy-
clines (tet(34), tetG-01, and tetM-01), 2 chloramphenicol
(catB3 and f loR), 1 multidrug (mexF), 1 sulfonamide (sul2),
and 1 vancomycin (vanC-03) resistance genes in the collected
soil samples were monitored to reflect changes in ARG profiles
during the land application of various sludge composts, and the
16S rRNA gene copies and 2 MGEs (plasmid IncQ oriV and
integron integrase intI1) were also measured to reflect changes
in total bacterial biomass and the transfer ability of ARGs,
respectively. The abovementioned ARGs and MGEs were
abundant in the tested sewage sludge according to the results
of our previous study,13 and they also kept relatively high
abundance in the compost products of sewage sludge.2,24,25
The LightCycler 480 II Real-Time PCR System (Roche, USA)
was applied to measure the target genes using the methods
mentioned in previous studies.2,26,34 The primers of bacterial
universal 16S rRNA gene, 2 MGEs, and 18 ARGs are shown in
Table S4, and the qPCR standard curves with their
amplification efficiencies (90.2−109.3%), R2 values (>0.997),
and specific detection limits are provided in Table S5. The
abundance of the target genes are expressed as copy numbers
per gram of dry soil (absolute abundance) and copy numbers
per 16S rRNA gene (relative abundance) in all soil samples,
respectively.
16S rRNA Gene Amplification, Sequencing, and Data
Processing. The 16S rRNA gene amplification, sequencing,
and data processing were performed according to previous
studies,2,13,35 and the details are described in the Supporting
Information.
Statistical Analysis. The Statistical Package for Social
Sciences (24, IBM, USA) was applied to conduct multiple
comparisons among various soil samples with one-way
ANOVA and Student−Newman−Keuls test at the probability
level of 0.05. R 4.1.2 environment with a vegan package was
applied to generate heatmaps of ARG subtypes and perform
Procrustes analysis between ARG profiles and bacterial
communities (based on OTU abundance) and Spearman’s
rank correlation analysis between the relative abundance of
ARG subtypes and bacteria at the genus level.2,34 The Gephi
platform was further applied to visualize the Spearman
correlation analysis between ARG subtypes and bacterial taxa
at the genus level, and the significant correlation (ρ > 0.80 and
p < 0.01) was selected to identify the ARG-associated
bacteria.13 In addition, redundancy analysis (RDA) was also
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performed using R 4.1.2 environment with a vegan package, in
order to reflect how the land application of compost products
of raw sludge and the various-conditioned sludge influenced
the antibiotic resistance in the two types of soils.34
■ RESULTS
Changes in the Profiles of ARGs and MGEs in Soils
Amended with the Compost Products Derived from
Raw Sludge or Various Conditioned Sludges. The
absolute abundance of total ARGs were 1.97 × 108 and 2.18
× 108 gene copies per gram of dry soil in the red and yellow−
brown soils (day 1), respectively, which were greatly increased
by 8.80−12.5 times by the addition of raw or PAM-
conditioned sludge composts (Figure 1a,b). However, the
absolute abundance of total ARGs was only increased by 4.29−
5.51 times in soils amended with bioleached or Fe[III]/CaO-
conditioned sludge composts (day 1). This phenomenon
resulted from the fact that the absolute abundance of total
ARGs in bioleached and Fe[III]/CaO-conditioned sludge
composts were only 15.8−39.8% of that in raw and PAM-
conditioned sludge composts (Table S6). Similarly, the
absolute abundance of IntI1 and InCQ were 1.91 × 108−
2.38 × 108 and 3.63 × 108−4.08 × 108 gene copies per gram of
dry soil in the red and yellow−brown soils (day 1), respectively
(Figure 1a,b). The addition of raw and PAM-conditioned
sludge composts respectively increased their absolute abun-
dance by 1.69−2.24 and 1.70−2.23 times, while the addition of
bioleached and Fe[III]/CaO-conditioned sludge just increased
their absolute abundance by 1.23−1.81 and 1.17−1.40 times,
respectively.
Changes in the absolute abundance of ARGs and MGEs
were continuously monitored for 170 days in red and yellow−
brown soils after the application of raw or various-conditioned
sludge composts (Figure 1). In the first 45 days, the absolute
abundance of total ARGs slightly decreased by 4.90−6.83% in
red soil amended with the compost products derived from raw,
PAM-conditioned, or bioleached sludges, while they were even
further increased by 1.96−3.38 times in red and yellow−brown
soils amended with Fe[III]/CaO-conditioned sludge compost
(Figures 1a,b). After that, the absolute abundance of total
ARGs evidently decreased by 27.5−49.1% from Day 45 to Day
70 and continuously decreased from Day 70 to Day 170 in the
abovementioned soils amended with different sludge composts.
As a result, the absolute abundance of total ARGs was
evidently higher in the first 45 days than that in Day 70−Day
170 in red and yellow−brown soils amended with raw or
various-conditioned sludge composts. Finally, the absolute
abundance of total ARGs were 4.52 × 108 and 6.05 × 108 gene
copies per gram of dry sludge at Day 170, respectively, in red
and yellow−brown soils amended with bioleached sludge
compost, which were significantly lower than that (7.75 ×
108−1.71 × 109 gene copies per gram dry soil) in the
corresponding soils amended with the compost products of
raw, PAM-conditioned, or Fe[III]/CaO-conditioned sludge
(Day 170, p < 0.05, Figures 1a,b). It is noteworthy that on Day
170, the absolute abundance of total ARGs was 1.16 × 109−
1.71 × 109 gene copies per gram of dry soil in soils amended
with Fe[III]/CaO-conditioned sludge compost, which was
even higher than that (8.72 × 108−1.20 × 109 gene copies per
gram dry soil) at Day 1. The absolute abundance of total ARGs
were 7.75 × 108, 9.65 × 108, and 4.52 × 108 gene copies per
gram of dry soil in red soils amended with the compost
products of raw, PAM-conditioned, or bioleached sludge (Day
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Figure 2. Heatmap of differences in the absolute abundance of 18 ARG subtypes between soil (a: red soil; b: yellow−brown soil) with and without
raw and various-conditioned sludge composts. Both of the color intensities show a log10 ratio of ARG absolute abundance between the soil with
and without sludge composts as the color key indicates at the top right. Raw: soil amended with raw sludge compost; PAM: soil amended with
PAM-conditioned sludge compost; Bioleaching: soil amended with bioleached sludge compost; Fe[III]/CaO: soil amended with Fe[III]/CaO-
conditioned sludge compost.

170), respectively, which were significantly lower than that with Fe[III]/CaO-conditioned sludge compost (Day 170),
(1.21 × 109, 1.68 × 109, 6.05 × 108 gene copies per gram dry which was higher than that (1.16 × 109) in the corresponding
soil) in the corresponding yellow−brown soils (Day 170, p < yellow−brown soils (Day 170, p < 0.05). In addition, the
0.05). Contrarily, the absolute abundance of total ARGs was relative abundance of total ARGs at Day 170 was 0.199−0.235
1.71 × 109 gene copies per gram of dry soil in red soil amended gene copy per 16S rRNA gene in the soils amended with
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Figure 3. Network analysis showing the co-occurrence of ARGs and their associated bacteria (based on Spearman correlation analysis) in soils in
the first 45 days and the following 125 days, after the land application of (a) raw sludge composts, (b) PAM-conditioned sludge compost, (c)
bioleached sludge compost, and (d) Fe[III]/CaO-conditioned sludge compost. Nodes stand for ARG subtypes and bacterial species at genus, and a
connection (i.e., edge) stands for an extremely significant (p < 0.01) and strong (Spearman’s r > 0.80) pairwise correlation. Node size is
proportional to the number of connections.

F https://doi.org/10.1021/acsestengg.2c00113
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bioleached sludge compost, which was also significantly lower
than that (0.261−0.457 gene copy per 16S rRNA gene, p <
0.05) in the corresponding soils amended with other sludge
composts (Day 170, Figure S4a,b). The absolute abundance of
IntI1 and InCQ were 2.80 × 108−8.37 × 108 gene copies per
gram of dry soil in red and yellow−brown soils amended with
bioleached sludge compost at Day 170, which were 59.9−
76.8% of that in the corresponding soils amended with
composting products of raw, PAM-conditioned and Fe[III]/
CaO-conditioned sludge (Day 170, Figures 1a,b).
The differences in the absolute abundance of various ARG
subtypes were further compared between the soils with and
without sludge composts (Figure 2). In the red soil, the
absolute abundance of 9 ARG subtypes in subgroup A did not
evidently increase in 170 days after the land application of
most sludge composts, while the absolute abundance of 9 ARG
subtypes in subgroup B drastically increased after 170 days,
especially sul2, aadA2-02, aadA2-03, aadA-01, aadA1, and
aadA2-01 (Figure 2a). After 170 days, the absolute abundance
of these abovementioned six ARG subtypes in red soil
increased by 19.7−88.3 times by the land application of raw
and PAM-conditioned sludge composts, while their absolute
abundance in red soil even increased by 38.2−210 times by the
land application of Fe[III]/CaO-conditioned sludge compost.
In comparison, after 170 days, the absolute abundance of these
six ARG subtypes just increased by 5.86−21.2 times by the
land application of bioleached sludge compost. In the yellow−
brown soil, the absolute abundance of 13 ARGs in subgroup A
slightly increased 170 days after the land application of
different sludge composts, while the absolute abundance of
sul2, aadA1, aadA2-01, aadA2-02, and aadA2-03 in subgroup B
largely increased (Figure 2b). Finally, after 170 days, the
absolute abundance of these abovementioned five ARG
subtypes increased by 17.7−730 times by the land application
of raw, PAM-conditioned, and Fe[III]/CaO-conditioned
sludge composts, while their absolute abundance only
increased by 6.06−146 times by the bioleached sludge
compost application. It was noteworthy that the sul2, aadA1,
aadA2-01, aadA2-02, and aadA2-03 were the typical sludge-
borne ARGs, all of which were largely enriched in the red and
yellow−brown soils due to the land application of various
sludge composts.
Impact of Sludge Compost Application on Soil
Bacterial Communities and Their Correlation with the
Relative Abundance Profiles of ARGs. In total, 11,973,229
high-quality sequences were obtained from all samples, with
44,777−80,728 sequences per sample, which were uniformed
to the minimum numbers of quality sequences per community
across all of the samples for subsequent analysis. These
sequences were clustered into 13122 OTUs at a 97% identity
level. As shown in Figure S5, the bacterial diversity dropped to
a varying degree in different treatments with the duration of
sludge compost land application. In particular, the bacterial
diversity in soils amended with bioleached sludge compost
showed the largest drop, with only 2327 OTUs (red soil) and
2727 OTUs (yellow−brown soil) at Day 170, which were
significantly (P < 0.05) lower than those of other treatment
soils. Principal coordinate analysis (Figure S6a,b) showed that
the bacterial community compositions were largely different in
the control red and yellow−brown soils and the corresponding
soils amended with various sludge composts (Day 1−Day
170). It is noteworthy that the bacterial community
compositions were similar in soils amended with raw, PAM-
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conditioned, and bioleached sludge composts, especially at
Day 170, which were largely different from that in soils
amended with Fe[III]/CaO-conditioned sludge compost. In
detail, the total abundance of bacteria belonging to the phylum
of Proteobacteria, Actinobacteria, and Chloroflexi accounted
for 64.7−82.5% of total bacterial abundance in red and
yellow−brown soils in the 170 days following the application
of the composting products of raw, PAM-conditioned, and
bioleached sludge (Figure S7a−c), while their total abundance
accounted for 52.9−72.2% of total bacterial abundance in soils
amended with Fe[III]/CaO-conditioned sludge compost
during the 170 days (Figure S7d). In comparison, the total
abundance of bacteria belonging to the phylum of
Planctomycetes, Gemmatimonadetes, and Bacteroidetes ac-
counted for 19.0−37.5% of total bacterial abundance during
the 170 days in soils amended with Fe[III]/CaO-conditioned
sludge compost, which were evidently higher than that (6.11−
22.3%) in the corresponding soil amended with other sludge
composts within the same period. Furthermore, similar
bacterial composition profiles at the genus level can be
found in soils amended with most sludge composts, except for
Fe[III]/CaO-conditioned sludge compost, during the 170
days. In red and yellow−brown soils, the 43 genera in
subgroup A kept relatively lower abundance in 170 days after
the land application of sludge composts, while the 71 genera in
subgroup B kept relatively higher abundance, especially
including the bacteria belonging to the genus of Actinomodura,
Mycobacterium, Rhodanobacter, Kaistobacter, Planctomyces,
Gemmata, and Rhodoplanes, which were largely detected in
all sludge composts (Figures S8 and S9).
A Mantel test confirmed that ARG profiles significantly
correlated with bacterial community compositions based on
the Bray−Curtis distance (R = 0.5037−0.6712, p = 0.0001, and
permutation N = 9999) in red and yellow−brown soils
amended with the various sludge composts. A network analysis
was further utilized to reveal the co-occurrence between ARG
subtypes and bacterial taxa in soils in the first 45 days and the
following 125 days after the land application of various sludge
composts (Figure 3). It can be found that 15−28 bacterial taxa
at the genus level (Table S7) significantly correlated with 10−
12 ARG subtypes (Spearman; r > 0.80, p < 0.01) in soils
amended with various sludge composts in the first 45 days.
From Day 1 to Day 45, the abundance of these identified
ARGs and their associated bacteria increased in soils receiving
raw and PAM-conditioned sludge composts (Figures 3a,b),
while their abundance mostly decreased in soils amended with
bioleached sludge compost, except mexF, vanc-03, tet(34), and
their associated bacteria (Figure 3c). The abundance of 60% of
identified ARGs and their associated bacteria decreased and
that of other identified ARGs and their associated bacteria
increased in the first 45 days, in soils amended with Fe[III]/
CaO-conditioned sludge compost (Figure 3d). It is note-
worthy that sul2 was the main ARG that increased, the
abundance of which accounted for 47.2−50.9% of the
abundance of total ARGs. From Day 45 to Day 170, 6−14
bacterial taxa at the genus level (Table S7) significantly
correlated with 5−9 ARG subtypes (Spearman; r > 0.80, p <
0.01) in soils amended with various sludge composts. The
abundance of most identified ARGs and their associated
bacteria decreased in soils amended with raw, PAM-
conditioned, and Fe[III]/CaO-conditioned sludge composts,
except tetM-01, aadA5-01, flor, and their associated bacteria. In
soils amended with bioleached sludge compost, the abundance
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ACS ES&T Engineering pubs.acs.org/estengg Article

Figure 4. RDA analysis results showing the relationships between environmental factors including the potential hosts carrying ARGs, MGEs (intI1
and inCQ), soil properties (pH, MC, and WSOC/WSON), and ARGs profiles in red and yellow−brown soils amended with (a) raw sludge
compost, (b) PAM-conditioned sludge compost, (c) bioleached sludge compost, and (d) Fe[III]/CaO-conditioned sludge compost. MC: moisture
content; WSOC: water soluble organic carbon content; WSON: water soluble organic nitrogen content.

of sul2, aadA-01, and their associated bacteria continuously
decreased; the abundance of aac(6′)-II, aadA5-01, tetM-01,
and their associated bacteria increased, which just accounted
for 0.065−0.190% of the abundance of total ARGs. It was
noteworthy that the bacteria belonging to the genus of dyella,
rhodanobacter, and/or dermacoccus were identified as ARG-
associated bacteria in both the first 45 days and the following
125 days, in red and yellow−brown soils amended with raw
and PAM-conditioned sludge composts (Figure 3a,b); the
bacteria belonging to the genus of cellulosimicrobium,
saccharopolyspora, and pedomicrobium were identified as
ARG-associated bacteria in both the first 45 days and the
following 125 days in soils amended with bioleached sludge
compost (Figure 3c); the bacteria belonging to the genus of
streptomyces and catellatospora were identified as ARG-
associated bacteria in both the first 45 days and the following
125 days in soils amended with Fe[III]/CaO-conditioned
sludge compost (Figure 3d).
H https://doi.org/10.1021/acsestengg.2c00113
Contributing Factors to the ARG Profiles in Agricul-
tural Soils Amended with the Composting Products of
Raw Sludge or Various Conditioned Sludges. The RDA
was applied to investigate the relationships among soil
properties (pH, MC, and WSOC/WSON), potential hosts
carrying ARGs, MGEs (intI1 and inCQ), and ARG profiles in
soils amended with various sludge composts. Bacteria that were
identified as ARG-associated ones in both the first 45 days and
the following 125 days were selected as the potential hosts
carrying ARGs in red and yellow−brown soils following the
application of raw, PAM-conditioned, bioleached, or Fe[III]/
CaO-conditioned sludge compost. As shown in Figure 4, the
first ordination axis of RDA analysis could explain the 32.4−
50.4% variance of ARGs, which account for 70.8−80.0% of the
total explained variance. The points of control soils in all RDA
analyses distinctly clustered apart from those of soils amended
with different sludge composts (Figure 4a−d), and the
absolute distances were 0.575−1.05 between the control soils
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ACS ES&T Engineering pubs.acs.org/estengg Article

Figure 5. RDA analysis results showing the individual and common contributions of the growth of potential bacteria carrying ARGs (Host), the
transfer of ARGs among bacteria (MGEs), and the change in soil environmental properties (Environment) based on the shift of ARG profiles in red
and yellow−brown soils amended with (a) raw sludge compost, (b) PAM-conditioned sludge compost, (c) bioleached sludge compost, and (d)
Fe[III]/CaO-conditioned sludge compost.

and the soils amended with raw, PAM-conditioned, or
bioleached sludge composts, which were evidently lower than
that (0.747−1.18) between the control soils and the soils
receiving Fe[III]/CaO-conditioned sludge compost (Table
S8). The absolute biplot scores of WSOC/WSON, 16SrRNA
gene, and the bacteria belonging to the genus of
Rhodanobacter, Catellatospora, Streptomyces,and/orDyellawere
0.5187−0.6952 in the first ordination axis, which were
evidently higher than that of other constraining variables
(0.0098−0.4397), in the soils amended with raw, PAM-
conditioned, and Fe[III]/CaO-conditioned sludge composts
(Table S9). In addition, the absolute biplot scores of Intl1 were
as high as 0.5050 in the first ordination axis in the soils
amended with bioleached sludge compost, which were close to
that (0.5241−0.5707) of WSOC/WSON, 16SrRNA gene, and
I https://doi.org/10.1021/acsestengg.2c00113
the bacteria belonging to the genus of Saccharopolyspora
(Table S9).
The individual and shared contributions of the growth of
potential bacteria carrying ARGs (Host), the transfer of ARGs
among bacteria (MGEs), and the change of soil environmental
properties (Environment) on the shift of ARG profiles were
further compared to reveal the key contributors in driving
ARG profiles in soil amended with the various sludge composts
(Figure 5). The 16S rRNA gene and ARG-associated bacteria
were used to evaluate the growth of potential bacteria carrying
ARGs, and the transfer of ARGs among bacteria was reflected
by the intI1 and inCQ. The total contribution of the Host,
MGEs, and Environment accounted for 79.5−89.9% of the
total variance in soils amended with the various sludge
composts, and the Host individually contributed 16.9−27.0%
to the ARG profiles, which were evidently higher than that (0−
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ACS ES&T Engineering
4.39 and 0.16−16.5%) of MGEs and Environment. The
interaction between the Host and Environment contributed to
a variation of 39.3−42.3% in soil amended with raw and PAM-
conditioned sludge compost (Figures 5a,b), while it even
contributed to a variation of 56.2% in the soil amended with
Fe[III]/CaO-conditioned sludge compost (Figure 5d), which
were greatly higher than the interactions between any two of
the abovementioned three factors (0−3.46%). Although the
interaction between the Host and Environment only
contributed to a variation of 3.76% in soils amended with
bioleached sludge compost, the interaction among the Host,
MGEs and Environment contributed to a variation of 38.4%
and the Environment individually contributed 16.5% to the
ARG profiles (Figure 5c), both of which were largely higher
than that (0−20.0 and 0.16−2.50%) in soils amended with
other sludge composts.
Disscussions. The absolute abundance of IntI1, InCQ, and
total ARGs increased in red and yellow−brown soils after the
addition of different conditioned sludge composts, which
means that significant amounts of ARGs and MGEs in sewage
sludge were introduced into the investigated two types of soils
during the utilization of these sludge compost products. In fact,
sewage sludge compost has been recognized as a significant
reservoir of antibiotic resistance determinants,2,11,24,36 and
substantial quantities of ARGs and MGEs were discharged
from sewage sludge compost into agricultural soil during the
land application of sludge compost products.7,16,37 Interest-
ingly, a much lower abundance of ARGs and MGEs were
introduced into soils by the addition of bioleached and
Fe[III]/CaO-conditioned sludge composts compared with that
induced by the addition of raw and PAM-conditioned sludge
composts, because the much higher ARGs and MGEs
attenuation during the former two sludge conditioning
processes over the latter ones.2,13 In addition to the direct
introduction of ARGs from sludge composts into the soil, the
decay extents of sludge-borne ARGs after the land application
of sludge compost is also vital because they are directly
correlated with the potential environmental risks of ARGs in
the soil environments.16 After the land application of various
sludge composts, more abundant ARGs in soils were kept in
the first 45 days compared with that in the following 125 days
because ARGs decay relatively slowly, following sludge
compost application into agricultural soils.15,37 Zhang et al.
(2018)7 also reported that disturbances in ARGs induced by
the land application of sludge composts primarily occurred in
an early stage, and greater measures are needed to control the
environmental risk of ARGs. Although both the bioleaching
and Fe[III]/CaO conditioning treatments reduced the
abundance of ARGs introduced by sludge composts
application, much greater enrichment of ARGs occurred in
the first 45 days and highly abundant ARGs lasted for 170 days
in red and yellow−brown soils after the application of Fe[III]/
CaO-conditioned sludge compost. After the land application of
various sludge composts for 170 days, a lower abundance of
ARGs was found in red and yellow−brown soils amended with
bioleached sludge compost rather than that in the correspond-
ing soils amended with other sludge composts, which can
ascribe to the less introduction of ARGs and the limited
enrichment of ARGs in the early stage during bioleached
sludge compost application. In addition, ARGs were more
easily enriched in yellow−brown soils after the land application
of the compost products of raw, PAM-conditioned, or
bioleached sludge for 170 days, than that in the corresponding
pubs.acs.org/estengg Article
red soils. Contrarily, ARGs were more easily enriched in red
soil after the land application of Fe[III]/CaO-conditioned
sludge compost for 170 days, than that in the corresponding
yellow−brown soil.
The land application of sludge compost products not only
increased ARG abundance in red soil and yellow−brown soil
but also impacted the diversity of ARG subtypes in the soils.
The sul2, aadA1, aadA2-01, aadA2-02, and aadA2-03, as
sludge-borne ARGs, kept low abundance in red soil and
yellow−brown soils, but they were introduced and persistent
for 170 days in these two types of soils after the land
application of various sludge composts. Similarly, the
occurrence and persistence of aminoglycoside and sulfonamide
resistance genes have been found in soils amended with sewage
sludge or its compost products. Yang et al. (2018)38 found that
the aminoglycoside resistance genes were largely enriched in
soil with a 3% sewage sludge application rate (dry matter
basis), and the sul1 can be detected for 6 months in soil
amended with differently treated sludge, including air drying,
aerobic digestion, anaerobic digestion, pasteurization, and
alkaline stabilization.15,16,39 It is vital to develop feasible
technologies within the treatment facility for mitigating the
release of typical sludge-borne ARGs during the utilization of
sewage sludge, rather than just depending on their natural
attenuation subsequent to land application. In fact, the present
study suggested that the bioleaching conditioning attenuated
the enrichment of aminoglycoside and sulfonamide resistance
genes to a great extent, in red and yellow−brown soils
amended with sludge composts, which were not attained by
the chemical conditioning with PAM or Fe[III]/CaO. Thus,
bioleaching conditioning was superior to chemical condition-
ing using PAM or Fe[III]/CaO in attenuating the enrichment
of sludge-borne ARGs in soils amended with sludge compost
products.
The land application of various sludge composts evidently
impacted the bacterial community compositions in red and
yellow−brown soils because of the introduction of sludge-
borne bacteria into the soil environments. Similar bacterial
community compositions were found in red and yellow−
brown soils amended with raw, PAM-conditioned, or
bioleached sludge composts, but the sludge product derived
from Fe[III]/CaO conditioning treatment largely changed the
bacterial community compositions in soils, and such changes
lasted for 170 days. The mantel test and network analysis
indicate that changes in ARGs profile were significantly related
to the bacterial community compositions in red and yellow−
brown soils after the land application of various sludge
composts, due to the introduction and persistence of sludge-
borne bacteria carrying ARGs. In the abovementioned soils
amended with various sludge composts (Day 1−Day 170), the
relationships between ARG profiles and bacterial community
composition were further analyzed in the first 45 days (Day 1−
Day 45) and the following 125 days (Day 45−Day 170), due
to the obvious differences of ARG changes between these two
periods. After the addition of raw and PAM-conditioned sludge
composts, 11−12 ARG subtypes and their associated bacteria
continuously enriched for 45 days in soils, including aadA2-01,
aadA2-02, aadA2-03, aadA-01, aadA-01, aadA1, aadA5-01,
tetM-01, tetG-01, strB, catB3, sul2, and/or f lor, which led to the
persistence of ARGs for 170 days in these two types of soils.
Typical sludge-borne ARGs (sul2) and its associated bacteria
largely enriched in the first 45 days in soils amended with
Fe[III]/CaO-conditioned sludge compost, which contributed
J https://doi.org/10.1021/acsestengg.2c00113
ACS EST Engg. XXXX, XXX, XXX−XXX
ACS ES&T Engineering
to the further increase of soil ARG levels in the following 125
days. Interestingly, the typical sludge-borne ARGs and their
associated bacteria continuously decayed within the whole 170
days, especially sul2, in soils amended with bioleached sludge
composts, and thus bioleaching conditioning treatment not
only reduced the introduction of typical sludge-borne ARGs
but also limited the enrichment of their potential hosts in these
two types of soils after the application of the corresponding
sludge compost. In addition, the bacteria belonging to the
genus of dyella, rhodanobacter, dermacoccus, cellulosimicrobium,
saccharopolyspora, pedomicrobium, streptomyces, and catellato-
spora were the potential hosts carrying ARGs in red and
yellow−brown soils following the application of raw, PAM-
conditioned, bioleached, and/or Fe[III]/CaO-conditioned
sludge composts. In fact, our previous study found that the
bacteria belonging to the genus ofdyella, rhodanobacter,
cellulosimicrobium, saccharopolyspora, and streptomyces, as the
potential host carrying ARGs, were detected in the compost
products of raw, PAM-conditioned, bioleached, and/or
Fe[III]/CaO-conditioned sludges,2 and Zhang et al. (2018)7
reported that the bacteria belonging to the genus of
cellulosimicrobium was the potential host carrying ARGs,
which were detected in black soil amended with sludge
composts.
RDA analysis indicated that the application of these sludge
composts exerted a profound impact on the ARG profiles in
red and yellow−brown soils, and the land application of
Fe[III]/CaO-conditioned sludge compost product exerted a
much larger impact than that of the raw, PAM-conditioned, or
bioleached sludge compost products. The changes in environ-
mental properties and the growth of potential host carrying
ARGs, including the bacteria that belong to the genus of
Catellatospora, Streptomyces, Rhodanobacter, and/or Dyella,
mainly drove the changes in soil ARG profiles during the
170 days following the application of raw, PAM-conditioned,
and Fe[III]/CaO-conditioned sludge composts. In compar-
ison, the changes in environmental properties, the transfer of
ARGs, and the growth of potential bacteria carrying ARGs,
including the bacteria belonging to the genus of Saccharopo-
lyspora, mainly drove the changes in soil ARG profiles during
the 170 days after the application of the bioleached sludge
composts. In fact, our previous studies have reported that the
growth of bacteria carrying ARGs was related to the transfer of
ARGs and soil properties, which directly impact the ARG
profiles of soils amended with biogas slurry.34 The individual
and shared contributions of the abovementioned factors on the
shift of ARG profiles were further analyzed in red and yellow−
brown soils amended with different types of sludge compost
products. After the land application of raw, PAM-conditioned,
and Fe[III]/CaO-conditioned sludge composts, both the
potential host carrying ARGs and soil properties drove the
shift of ARG profiles in red and yellow−brown soils, which
means that the changes in soil environments mainly impacted
the growth of host carrying ARGs to further drive the shift of
soil ARG profiles. Obviously, the environments of soils
amended with Fe[III]/CaO-conditioned sludge compost
product were suitable for the growth and enrichment of
some potential hosts carrying ARGs, especially sul2. However,
the ARG profiles were determined by the potential host
carrying ARGs, the transfer of ARGs, and the soil properties in
red and yellow−brown soils amended with bioleached sludge
compost product. Thus, the attenuation of soil ARGs induced
by the bioleaching conditioning most probably resulted from
K https://doi.org/10.1021/acsestengg.2c00113
pubs.acs.org/estengg Article
the limited growth of bacteria-carrying ARGs in soils amended
with bioleached sludge compost, which possibly resulted from
the role of soil environments (i.e., relatively lower soil pH) in
limiting the transfer of ARGs among bacteria. In fact, some
previous studies reported that the low pH environment could
inactivate some MGEs, which was helpful for controlling the
potential environmental risks from the ARG transfer.40,41
■
CONCLUSIONS
The present study evaluated the effect of sludge conditioning
on the attenuation of ARGs in two types of agricultural soils
(i.e., red soil and yellow−brown soil) amended with sludge
composts. In detail, the absolute abundance of total ARGs and
MGEs was the lowest in both the red soil and the yellow−
brown soil amended with bioleached sludge compost (Day
170), and the enrichment of typical sludge-borne ARGs and
their potential hosts were more effectively limited by
bioleaching conditioning in comparison with the other
conditioning treatments. As a result, bioleaching conditioning
is superior to the chemical conditioning using Fe[III]/CaO or
PAM in mitigating antibiotic resistance in different agricultural
soils amended with sludge composts, which was contributed by
the preremoval of ARGs in sludge compost and the potentially
limited growth of bacteria carrying ARGs after the land
application of sludge compost. Thus, it is inferred that sludge
conditioning, as an indispensable step for sludge disposal, can
be an important sludge treatment process in attenuating the
release of sludge-borne ARGs into the environment. Further
work is still needed to focus on the fates of ARGs in
agricultural soil and its crops amended with the compost
product of conditioned sludge, which is treated using
bioleaching combined with hyperthermophilic composting.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsestengg.2c00113.
16S rRNA gene amplication, sequencing, and data
processing; detailed procedures for different sludge
conditioning treatments; physicochemical properties of
the various sludge composts and the tested soils; DNA
yield and purity; primer sets and target classification;
quality control of the real-time qPCR methods; Absolute
abundance of 16S rRNA gene, MGEs, and genes
conferring resistance to seven major classes of antibiotics
in the composting product of raw sewage sludge
(unconditioned sludge) and the three types of
conditioned sludge; microbial taxa of ARG-associated
bacteria at genus level based on the network analysis;
distances of site scores between soils amended with and
without different sludge composts in the RDA analysis;
biplot scores for constraining variables in different
ordination axis; changes in soil properties and the
relative abundance of ARGs during the land application
of sludge composts; microbial species richness and alpha
diversity analysis; change in total bacterial biomass
during the land application of sludge composts;
evolution of bacterial community at the phylum level
in red and yellow−brown soils after the land application
of sludge composts; heatmap of the main genera in soils
amended with different sludge composts (PDF)
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ACS ES&T Engineering
■
pubs.acs.org/estengg
AUTHOR INFORMATION
Corresponding Author
Guanyu Zheng − Department of Environmental Engineering,
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China; Jiangsu
Collaborative Innovation Center for Solid Organic Waste
Resource Utilization, Nanjing 210095, China; orcid.org/
0000-0002-0940-0559; Phone: +86-25-84395160;
Email: gyzheng@njau.edu.cn
Authors
Yi Lu − Key Laboatory of Recycling and Eco-treatment of
Waste Biomass of Zhejiang Province, School of
Environmental and Natural Resources, Zhejiang University of
Science and Technology, Hangzhou 310023, China;
Department of Environmental Engineering, College of
Resources and Environmental Sciences, Nanjing Agricultural
University, Nanjing 210095, China
Jiajun Wang − Department of Environmental Engineering,
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China
Xinxin Wang − Department of Environmental Engineering,
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China
Xiaoqing Meng − Department of Environmental Engineering,
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China
Su Yan − Department of Environmental Engineering, College of
Resources and Environmental Sciences, Nanjing Agricultural
University, Nanjing 210095, China
Yu Chen − Department of Environmental Engineering, College
of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China
Lixiang Zhou − Department of Environmental Engineering,
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Nanjing 210095, China; Jiangsu
Collaborative Innovation Center for Solid Organic Waste
Resource Utilization, Nanjing 210095, China; orcid.org/
0000-0002-9978-1163
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsestengg.2c00113
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors would like to thank the National Natural Science
Foundation of China for their financial support (21976091,
42107421).
■
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M https://doi.org/10.1021/acsestengg.2c00113
ACS EST Engg. XXXX, XXX, XXX−XXX

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