Environmental Technology & Innovation 32 (2023) 103415

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Environmental Technology & Innovation

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

Effects of enrofloxacin on soil nitrification and denitrification: A

microbiological study
Jianpeng Gao a, b, Chang Wei a, b, Tian-Jin Mo a, b, Yu Yan a, Yan Sun c, Huayi Chen d,
Yulong Zhang a, b, Jinjin Wang a, b, Yongtao Li a, b, Hui-Juan Xu a, b, *
a
Key Laboratory of Arable Land Conservation (South China), MOA, College of Natural Resources and Environment, South China Agricultural
University, Guangzhou 510642, China
b
Guangdong Province Key Laboratory for Land Use and Consolidation, College of Natural Resources and Environment, South China Agricultural
University, Guangzhou 510642, China
c
Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China
d
College of Tropical Crops, Hainan University, Haikou 570228, China

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

Keywords: Extensive use of enrofloxacin (ENR) leads to its widespread presence in soil, which seriously
Enrofloxacin threatens the soil ecological environment and biogeochemical cycles. Thus, it is very important to
Nitrification comprehend the fate of ENR and its effect on nitrogen cycling in farmland soil. In this investi­
Denitrification
gation, quantitative real-time PCR and 16 S rRNA genes amplicon sequencing were used to
Microbial community
Nitrogen-cycling functional genes
analyze the microbiological mechanism of the effects of ENR on the soil nitrification and deni­
trification processes of farmland. The results showed that the addition of ENR suppressed the
ammoniation process, leading to a decline in NH+ 4 -N content. Additionally, ENR led to a decrease
in the soil nitrification potential by decreasing the relative abundance of Nitrosomonas and
Nitrosospira. However, ENR inhibited the relative abundance of narG , as well as the activity of
nitrate reductase, which led to the accumulation of NO-3-N. Furthermore, ENR increased the
possibility of nitrous oxide emissions by increasing the relative abundances of Flavobacterium,
Bacillus and Aeromonas, as well as those of nirS and nosZ. This study provided data to support for
the ecological impact and risk assessment of ENR on nitrogen cycling in farmland soil.

1. Introduction

The widespread use of antibiotics, one of the most important medications in human and veterinary medicine, is enormous and
continues to rise (Sarmah et al., 2006; Van Boeckel et al., 2015).
China is currently the world’s largest consumer of antibiotics, with an estimated total consumption of around 160,000 t in 2013.
Veterinary antibiotics comprised 52% of the total while fluoroquinolones (FQs) accounted for 17% (Zhang et al., 2015b).
According to the statistics, it is predicted that global livestock antibiotic usage will increase by 67% between 2010 and 2030 (Van
Boeckel et al., 2015). Enrofloxacin (ENR), one of the most widely used FQs, is frequently used in animal husbandry for disease pre­
vention and animal growth promotion because of its widespread activity range and affordability. However, despite the favourable

* Correspondence to: College of Natural Resources and Environment, South China Agricultural University, 483 Wushan Road, Guangzhou 510642,
China.
E-mail address: hjxu@scau.edu.cn (H.-J. Xu).

https://doi.org/10.1016/j.eti.2023.103415
Received 3 August 2023; Received in revised form 4 September 2023; Accepted 16 October 2023
Available online 18 October 2023
2352-1864/© 2023 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Gao et al. Environmental Technology & Innovation 32 (2023) 103415

pharmacokinetic properties characteristics of FQs, the drugs cannot be fully absorbed by animals following administration, and 17%−
90% of them are excreted as parent drugs or metabolites through feces and urine, and enter the environment (soil, water, etc.) through
surface runoff or manure (Zhu et al., 2013).
Photolysis is a significant transformation process of antibiotics in aquatic environments and surface soil (Knapp et al., 2005; Xu
et al., 2009). However, ENR is not easily degraded due to limited light penetration in soil, with a half-life ranging from 12.5 to 120 days
(Albero et al., 2018; Li et al., 2016; Zhang et al., 2015a). Continuous environmental exposure, long half-life and strong adsorption
result in extensive ENR detection in the environment (Chen et al., 2015; Golet et al., 2003; Hanna et al., 2018; Li et al., 2014). The
concentration of ENR in farmland reached 6.14 mg kg− 1 (Parente et al., 2019), and a maximum concentration of 1420.76 mg kg− 1 was
found in manure (Zhao et al., 2010). The residual ENR in soil can inhibit the replication of bacterial DNA by inhibiting the cleavage and
ligase function of DNA cyclase, showing an antibacterial effect, which will adversely affect the important biogeochemical cycle
mediated by bacteria (Zou et al., 2019).
Nitrogen is an essential element for all living organisms and is required for the biosynthesis of key cellular components such as
proteins and nucleic acids, and all organisms except nitrogen-fixing bacteria must rely on ammonium or nitrate to provide growth
(Kuypers et al., 2018). Nitrification and denitrification, two important processes of soil nitrogen cycling, are mainly driven by mi­
croorganisms (Francis et al., 2007; Kuypers et al., 2018). Nitrification includes two steps: ammonia oxidation and nitrite oxidation,
among which ammonia oxidation is the rate-limiting step (Xu et al., 2018). Ammonia oxidation is driven by composed of
ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) (Xu et al., 2018). The gene amoA encodes ammonia
monooxygenase (AMO), which is usually used as a gene marker for ammonia oxidation and nitrification. Denitrification mainly in­
cludes four processe: NO-3 → NO-2, NO-2 → NO, NO → N2O, and N2O → N2, catalyzed by nitrate reductase, nitrite reductase, nitric oxide
reductase and nitrous oxide reductase, respectively (Zhu et al., 2018). Generally, narG , nirS, nirK and nosZ are used as functional
marker genes of nitrate reductase, nitrite reductase and nitrous oxide reductase, respectively (Li et al., 2018). Previous research has
found that ENR can change the structure of microorganisms, inhibit the process of both nitrification and nitrification, though the
majority of studies have been conducted in aquatic environments (Dalkmann et al., 2014; Deng et al., 2022; Ilhan et al., 2011; Pashaei
et al., 2022). Currently, research on ENR’s effects on the nitrogen cycle in soil has primarily focused on nitrification processes through
the study of ammonia-oxidizing microorganisms and related enzyme activities (Wang et al., 2019). However, the molecular mecha­
nism of ENR on soil nitrogen cycling can be better reflected by the changes in microbial communities and functional gene abundance
during different nitrogen cycling processes, but there is a lack of organized research on this aspect.
In this study, the natural decay process of ENR in soil was monitored, and the effect of ENR on soil nitrification and denitrification
microbial community and related functional genes was studied by 16S rRNA genes amplicon sequencing and real-time quantitative
PCR. The objectives of this study are: (i) to clarify the natural decay law of ENR in soil at varying concentrations; (ii) to explain the
effect of ENR on the processes of nitrification and denitrification; (iii) to investigate the microbiological mechanism behind the impact
of ENR on soil nitrification and denitrification processes, and to provide data for enhancing the ecological environment assessment of
FQs.

2. Materials and methods

2.1. Soil and chemical reagents

The soil was collected from the South China Agricultural University Farming Training Center in Guangzhou, Guangdong, China
(23◦ 9′55″N, 113◦ 21′59″E), where no crops have been grown for several years. Surface soil (0–20 cm) was collected, natural air-dried in
cool and dark places and sieved through a 2-mm mesh for the subsequent use. The soil was classified as silty clay loam soil according to
the International soil texture classification system. The physical and chemical properties of the soil samples were listed in Table S1. The
ENR content in the soil was below the instrumental detection limit through HPLC analysis.
Enrofloxacin (CAS 93106–60–6; purity ≥ 99.9%) was purchased from Sigma-Aldrich Co. (MO, USA). Soil Nitrate Reductase (S-NR)
and Soil Nitrite Reductase (S-NiR) Activity Assay Kit were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing,
China).

2.2. Experimental design

Previous studies have shown that ENR is often frequently found in farmland soil at an average concentration of approximately 1 mg
kg− 1, with the highest detected concentration detected being around 6.14 mg kg− 1 (Li et al., 2014; Parente et al., 2019). Therefore, we
selected two ENR concentrations for our experiment: 1 mg kg− 1 to represent the average level in farmland soil and 10 mg kg− 1 to
represent the risk concentration. Polyethylene pots, which were 18 cm in diameter and 13 cm in height, were used for the experimental
setup. Each plastic pot was filled with 2 kg of soil (dry weight). The soil moisture content was adjusted to 60% of the maximum
water-holding capacity in the field. Each group was incubated in the dark at 25℃, and soil moisture was adjusted once every two days
to compensate for the weight loss. Three treatments were established for this experiment: natural soil (Control), 1 mg⋅kg− 1 ENR
treatment group (ENR1), and 10 mg⋅kg− 1 ENR treatment group (ENR10), with 4 replicates for each treatment, weighing and hydrating
every two days. Soil samples were collected using a 3 cm diameter and 20 cm length soil extraction auger with multi-point vertical
sampling on day 0, 4, 10, 20, and 40 during the incubation period. Part of the soil was air-dried naturally to determine its physico­
chemical properties, part was freeze-dried and stored in the cold and dark to determine ENR and the third part was stored at − 80℃ for
microbial analysis.

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2.3. Sample detection and analysis

2.3.1. Physico-chemical analysis of soils

Soil pH was determined using a combination glass electrode at a soil to water ratio of 1: 2.5 (w/v). Soil water soluble organic carbon
(WSOC) and total soluble nitrogen (TSN) were extracted using ultrapure water (1:4, soil/water) and detected by a total organic carbon
-
(TOC) analyzer (Elementar vario TOC, Germany) (Ghani et al., 2003). Soil mineral nitrogen (NH+ 4 -N and NO3-N) was extracted by 2 M
KCl solution and analysed within 7 days using the Skalar SAN Segmented Flow Analyzer (Skalar Analytic B. V., the Netherlands)
++

(Zhong et al., 2016).

2.3.2. Potential ammonia oxidation and denitrifying enzyme activity

Soil potential ammonia oxidation (PAO) was determined using the chlorate inhibition method (Xu et al., 2018). Briefly, 5.0 g of
fresh soil was weighed into a 50-mL centrifuge tube and 20 mL of induction solution containing 1 mM phosphate buffer (NaCl, 8 g⋅L− 1;
KCl, 0.2 g⋅L− 1; Na2HPO4, 0.2 g⋅L− 1; NaH2PO4, 0.2 g⋅L− 1; pH, 7. 4) and 1 mM (NH4)2SO4 was added. Then KClO3 (final concentration of
10 mM) was added to the tubes to inhibit nitrite oxidation. Soil slurries were incubated at 25℃ in the dark for 24 h with shaking at 180
rpm. NO-2-N was extracted with 5 mL of 2 M KCl with shaking at 180 rpm for 1 h, and spectrophotometrically determined at 540 nm
with N-(1-naphthyl) ethylenediamine dihydrochloride. The PAO (μg NO-2-N g− 1 dry soil d− 1) represented the quantity of NO-2-N
accumulated during 24 h. Soil denitrification enzyme activity was determined by the visible spectrophotometric method following
reference to the instructions of the S-NR and S-NiR Activity Assay Kit.

2.3.3. Determination of ENR residues in soil

The extraction and detection method of ENR referred to Wu et al. (2014) with appropriate adjustments. The brief summary was as
follows: 1,000 g of freeze-dried soil was weighed into a 10 mL Teflon tube, and 5 mL of a solution with 50% Mg(NO3)2-10% NH3⋅H2O
(96:4, v/v) was added, after which the tube was vortexed for 1 min, shaken at 200 r min− 1 for 5 min, ultrasonicated for 15 min, and
centrifuged at 4500 r min− 1 for 5 min. The above extraction steps were repeated twice more, and the extracted solutions were
combined to about 15 mL extraction in total. Subsequently, the concentrated solution was purified with an activated Oasis HLB
Cartridge (6 CC, 200 mg) purchased from Waters (MA, USA) that was pre-treated with 6 mL of methanol and 6 mL of deionized water.
The solution was passed through the cartridge at a flow rate of 1 mL/min, followed by washing with 6 mL of water. Finally, the
cartridge was vacuum-dried for 5 min. The cartridge was eluted with acidified acetonitrile (1% acetic acid, v/v). The resulting eluate
was dried near 40℃ using high-purity nitrogen and reconstituted in a 1 mL solution of acetonitrile. Subsequently, a high-performance
liquid chromatography (Agilent 1260, USA) analysis was conducted utilizing a CNW Athena C18-WP column (4.6 mm × 250 mm, 5
µm). The mobile phase A was acetonitrile (99.9% pure), while the B phase was 0.05 moL L− 1 phosphoric acid. An elution program of A:
B = 18:82 (v/v) was performed at a flow rate of 0.8 mL/min; keeping the column temperature at 30℃ and injecting 20 μL volume. The
detection wavelength of the UV detector was 280 nm. The calculation formula of the enrofloxacin residue rate was as follows:
Ct
RENR = × 100%
C0

Where Ct and C0 are the concentrations of enrofloxacin in soils at times 0 and t, respectively.

2.3.4. Soil DNA extraction

According to the degradation of ENR, soil samples were selected on day 4 and 20 for DNA extraction. Soil DNA was extracted from
0.5 g soil using the DNeasy® PowerSoil® Pro Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA
concentration and purity of the extracted soil samples were determined by Nanodrop 2000 (Thermo Scientific, USA). The purified DNA
was stored at − 80℃ until further analysis.

2.3.5. High-throughput sequencing and bioinformatics analysis

The soil DNA samples were amplified using universal primers 341 F (5′-CCTACGGGCTGCAACAG-3′) and 806 R (5′-GGACTAC­
CAGGGTATCTAAT-3′) targeting the V3-V4 hypervariable regions of the bacteria 16S rRNA genes. Purified amplicons were pooled in
equimolar concentrations and paired-end sequenced on an Illumina NovaSeq PE300 platform (Illumina, USA) according to the
standard instructions of Novogene Co., Ltd. (Beijing, China). The downstream data was split from each sample based on the Barcode
and PCR amplified primer sequences. After truncating the Barcode and primer sequences, the samples were merged using FLASH and
filtered to obtain high-quality sequences. The final valid data was obtained by performing quality control through QIIME (v1.9.1). The
sequences were clustered into Operational Taxonomic Units (OTUs) through UPARSE (v7.0.1001) with 97% agreement. Species
annotation analysis was performed with the SSUrRNA database in SILVA138 through the MOTHUR (threshold: 0.8–1). Fast multiple
sequence comparison was performed using MUSCLE (V3.8.31) to obtain all OTUs representing the phylogenetic relationships of the
sequences. Finally, the data from each sample was homogenized using the least amount of data in the sample was used as the criterion.
Subsequent analyses were based on the homogenized data. Three nitrifying bacteria and 25 denitrifying bacteria were screened at the
genus level using literature review and 16S rRNA amplicon sequencing results (Abed et al., 2013; Angnes et al., 2013; Lu et al., 2019;
Peng et al., 2011).

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2.3.6. Real-time quantitative PCR

Bacterial 16 S rRNA genes, AOB-amoA (bacterial ammonia monooxygenase coding gene), AOA-amoA (archaeal ammonia mono­
oxygenase coding gene), narG (nitrate reductase encoding gene), nirK and nirS (nitrite reductase encoding gene), and nosZ (nitrous
oxide reductase encoding gene) were quantified by the QuantStudio™ 6 Flex Real-Time PCR Detection System (Applied Biosystems,
USA). Real-time PCR experiments were performed using SYBR Green fluorescent dye according to the SYBR® Premix Ex Taq™
(TaKaRa, Japan) kit. The PCR reaction volume is 10 μL, including 5 μL of SYBR Premix Ex Taq™, 0.2 μL of ROX Reference Dye, 0.2 μM
of forward and reverse primer and 0.5 μL of DNA template. The remaining volume is made up with ddH2O. The primers and ampli­
fication conditions for each gene are listed in Table S3. According to the slope of the standard curve, the amplification efficiencies of
16SrRNA gene, AOB-amoA, narG, nirK, nirS, and nosZ were 85.1%, 93.0%, 89.4%, 91.3%, 91.4%, and 90.7%, respectively. However,
AOA-amoA was not amplified successfully.

2.4. Statistical analysis

The mean and standard deviation of the original data were calculated using EXCEL2016 (Microsoft, USA). One-way ANOVA
(Duncan’s tests, p < 0.05) was conducted using SPSS Statistics 26 (IBM, USA). Line plots, column plots and principal component
analysis (PCA) were generated using Origin 2021 (OriginLab, USA). ImageGP (https://www.bic.ac.cn/ImageGP/), a data visualization
web server, was used to complete the heatmap. Redundancy analysis (RDA) was performed using Canoco 5 (Microcomputer Power,
USA).

3. Results and discussion

3.1. Natural degradation of ENR in soil

The degradation of ENR mainly was primarily observed during the early incubation period, from the beginning to the 20th day
(Fig. 1). The quickest degradation rate was recorded between the 4th and 10th day, and the degradation rate of the 1 mg kg− 1
treatment group was higher than that of the 10 mg kg− 1 treatment. Moreover, the ENR residue rate of ENR1 was significantly lower
than that of ENR10 on the 20th day. Subsequently, the degradation rate slowed down during the 20–40 day period. On the 40th day,
the residual rate of ENR in the 1 mg kg− 1 treatment group (49.8%) was lower compared to the 10 mg kg− 1 treatment group (54.0%).
This suggests that the natural degradation of ENR was more complete in the former.
ENR is a photodegradable sensitive organic pollutant. Its half-life is about 2 days when exposed to light, but it degrades slowly
under dark conditions (Wu et al., 2005). Li et al. (2016) studied the degradation of 0.1–10 mg kg− 1 ENR in soil, and found that ENR’s
half-life exceeded 56 d. Zhang et al. (2015a)found that the half-lives of 1, 5, and 20 mg kg− 1 ENR in soil were 12.5d, 16.2d, and 19.1d,
respectively; and the half-life was proportional to the concentration of antibiotics, which was consistent with the degradation trend of
ENR in this study. In this study, the half-lives of 1 and 10 mg kg− 1 ENR in soil were about 39.7 days and 44.8 days, respectively. These
values fell within the range of those reported by Li et al. (2016) and Zhang et al. (2015a). This difference may be caused by the
physicochemical properties of the cultured soil and the composition of the microbial community (Li et al., 2016; Zhang et al., 2015a).

Fig. 1. The residual rate of enrofloxacin in soil. Natural soil + 1 mg⋅kg− 1 enrofloxacin (ENR1), natural soil + 10 mg⋅kg− 1 enrofloxacin (ENR10).
The data are represented with average ± standard deviation (n = 4). The “*” on the error bar represent significant difference between different
group in the same period (ANOVA, Duncan’s test, p < 0.05).

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3.2. Soil physicochemical properties and nitrogen transformation

During the incubation period of 40 days, the concentration of soil NH+ 4 -N increased in the first 10 days, and then decreased to a level
even lower than the initial concentration (Fig. 2a). The length of the incubation period had a negative correlation with the ammonium
nitrogen content (p < 0.05, Fig. S4). Soil NH+ 4 -N contents in the ENR10 treatment were significantly lower than those in the Control
treatment. The early accumulation of soil ammonium nitrogen could be attributed to the mineralization of soil organic nitrogen. As the
consumption of soil organic nitrogen progressed, the production of ammonium nitrogen from mineralization decreased (Zhou et al.,
2008). Meanwhile, the ammonia oxidation process continued, which lead to a decrease in soil ammonium nitrogen from the 10th day
to the end of the incubation period. The addition of ENR led to lower soil ammonium nitrogen content compared to the control without
ENR at the same sampling time. There may be two potential reasons for this result: firstly, ENR might have promoted the process of
ammonia oxidation; secondly, ENR might have inhibited soil ammoniation to form ammonium. It has been reported that ENR can
inhibit ammoniation by suppressing the abundance of nifH gene (Ma and Chen, 2010). At the later stage (40 days), there was no
significant difference in the concentration of NH+ 4 -N among the treatment groups, indicating that ENR chiefly impacted NH4 -N during
+

the initial and intermediate stages.

As the incubation period increased, there was a noticeable decrease in the concentration of nitrate nitrogen in the soil, with a
statistically significant difference (p < 0.05, Fig. S4). This may be attributed to the reduction in the level of ammonium nitrogen, which
resulted in a slower rate of nitrification (Liu et al., 2020). During the same time period, it was observed that the nitrate nitrogen content
showed a significant increase in the ENR1 and ENR10 treatments in comparison to the Control treatment (Fig. 2b). However, it should
be noted that there was no significant variation in the nitrate nitrogen levels between ENR1 and ENR10 (except for 20d). This increase
in nitrate nitrogen levels during the same culture period could be attributed to ENR’s promotion of nitrification or inhibition of
denitrification (Kuypers et al., 2018). Combined with the results of soil ammonium nitrogen, it is likely that ENR probably promoted
-
the process of soil ammonia oxidation and subsequently nitrification, resulting in the lower levels of NH+ 4 -N and higher levels of NO3-N
in ENR10 and ENR1.
There were no significants difference in the concentration of total soluble nitrogen (TSN) among the treatments at 4d, 10d and 40d.
However, on the 20th day, TSN was significantly different between the three treatments, with the TSN content order of ENR10 >
ENR1 > Control (Fig. 2c). It is possible that ENR stimulated the mineralization of soil organic nitrogen or inhibited the assimilation of
soluble nitrogen by certain microorganisms.

3.3. Potential nitrification rate and denitrification enzyme activity

Potential ammonia oxidation (PAO) is a commonly used method for evaluating potential nitrification rate (PNR) (Xu et al., 2018).
The soil PAO ranged from 0.89 to 1.29 μg NO-2-N g− 1 dry soil d− 1 (Fig. 3a). During the early stages of the incubation (4d and 10d), the
soil PAO of ENR10 was lower than that of ENR1, which was also significantly lower than that of Control. On the 10th day, the PAO in
ENR10 was significantly lower than that in Control, but no statistical difference was found either between ENR10 and ENR1or between
ENR1 and Control. Afterward, no significant difference in PAO between the three treatments was observed. The results of PAO showed
that the inhibitory effect of ENR on nitrification mainly occurred in the early stage (4d and 10d), and returned to the level of the
Control in the middle and late stages of the incubation (20d and 40d). The short-term effect may be due to two possible reasons: first,
FQs are easily adsorbed by soil, resulting in a decrease in their effective concentration (Conkle et al., 2010; Yu et al., 2012); and second,
the soil’s functional microorganisms are potentially restored.
Soil nitrate reductase activity is shown in Fig. 3b. It is evident that ENR inhibited the abundance of nitrate reductase on day 10, and
the enrofloxacin-added treatments displayed a significant difference from the Control treatment. Nevertheless, the effect of ENR on
nitrate reductase on the other days did not result in significant differences from the Control. Enrofloxacin barely affected nitrite
reductase in the initial phase of the trial (0–4d). However, ENR10 significantly increased nitrite reductase activity in comparison to the

Fig. 2. Physical and chemical indexes of soil in different treatment groups; Natural soil (Control), Natural soil + 1 mg⋅kg− 1 enrofloxacin (ENR1),
Natural soil + 10 mg⋅kg− 1 enrofloxacin (ENR10); (a)Ammonia nitrogen content; (b)Nitrate nitrogen content; (c)Total water-soluble nitrogen; The
data are represented with average ± standard deviation (n = 4). The different lowercase letters on each column represent significant difference
between different group in the same period (ANOVA, Duncan’s test, p < 0.05).

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Fig. 3. Effects of different concentrations of enrofloxacin on potential ammonia oxidation (a) soil nitrate reductase (b) and soil nitrite reductase (c)
in the soil during an incubation of 40 days. The data are represented with average ± standard deviation (n = 4). The different lowercase letters on
each column represent significant difference between different group in the same period (ANOVA, Duncan’s test, p < 0.05).

Control starting from day 10 (Fig. 3c). Combining the effects of ENR on PNR and denitrifying enzyme activities, it can be seen that the
increasing effect of ENR on nitrate levels is mainly caused by its inhibition of the conversion of nitrate to nitrite (Kuypers et al., 2018).
The inhibition of ammonium levels by ENR, on the other hand, may be attributed to its inhibition of ammonia oxidation (Ma and Chen,
2010).

3.4. Changes in the structure of nitrifying and denitrifying bacteria in soil

The changes in the abundance of three genera of nitrifying bacteria, Nitrolancea, Nitrosomonas, and Nitrosospira, are shown in
Fig. 4a. Nitrosospira and Nitrosomonas were observed to have decreased in relative abundance in the initial stage of the experiment (4d)
with the addition of ENR, and the suppressive effect of Nitrosomonas persisted in high-concentration ENR treatment (ENR10) up to the
20th day. Compared to the Control and ENR1 treatment, the ENR10 treatment significantly suppressed the total abundance of ni­
trifying bacteria on the 4th day, but there was no significant difference in the total abundance of nitrifying bacteria among the
treatment groups on the 20th day. Nitrosomonas and Nitrosopira belong to two autotrophic ammonia-oxidizing bacteria (Li et al., 2018).
The results showed that ENR can inhibit soil nitrification by reducing the relative abundance of soil ammonia-oxidizing bacteria.
However, its effects were mainly short-term, such as nitrification potential (Wang et al., 2018; Wei et al., 2018).
The twenty-five denitrifying bacterial genera identified were classified into four phyla, with Actinobacteriota consisting of five,
Bacteroidota having two, Firmicutes having four, and Proteobacteria having fourteen. The five denitrifying bacteria genera with the
highest degree were Streptomyces, Flavobacterium, Bacillus, Aeromonas, and Sphiningomonas. On the 20th day, the addition of ENR
significantly increased the total relative abundance of denitrifying bacteria (Fig. 4b). ENR significantly increased the relative abun­
dance of Aeromonas and Flavobacterium (p < 0.05) during the experimental culture phase. However, the inhibitory effect of ENR on
Sphingomonas only occurred on the 4th day (p < 0.05), and had returned to the level of the Control treatment’s levels by on the 20th

Fig. 4. Relative abundance of nitrifying bacteria (a) and nitrifying bacteria (b) in each treatment group (genus level). The data are represented with
average ± standard deviation (n = 3). Significant differences in the total relative abundance of nitrifying and nitrifying bacteria (ANOVA, Duncan’s
test) between different group are shown by “* ” on the column which represent p < 0.05. Similarly, “* ” in the column represents significant dif­
ference (ANOVA, Duncan’s test, p < 0.05) between each nitrifying or denitrifying bacteria at the same sampling date.

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Fig. 5. Relative abundance of nitrogen cycle functional genes；(a)AOB-amoA, (b)narG, (c)nirK, (d)nirS, (e)nosZ, (f) (nirK+nirS)/nosZ. The data are
represented with average ± standard deviation (n = 4). The different lowercase letters on each column represent significant differences (ANOVA,
Duncan’s test, p < 0.05) among the treatments.

day. Flavobacterium, Bacillus and Aeromonas are three common antibiotic-resistant bacteria and antibiotic-degrading bacteria often
found in antibiotic contaminated soil (Alexandrino et al., 2017; Tan et al., 2021). The increase abundance of these bacteria indicates
the microbial degradation process of ENR (Table S5, p < 0.05). At the same time, Flavobacterium, Bacillus and Aeromonas are the three
important genera of denitrifying bacteria genera, and the increase of these genera indicates that ENR promotes certain processes in soil
denitrification (Han et al., 2020). Flavobacterium has been shown to be closely associated with nitrous oxide emissions (Horn et al.,

Fig. 6. PCA analysis of denitrifying bacteria (genus level). The colors represent sampling times and the shapes represent treatment groups.

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2006).
According to the heat map of the relative abundance of denitrifying bacteria, it can be seen that the 4d and 20d treatments were
clustered into separate groups. Meanwhile, the ENR1 and Control treatments were clustered together in a group and differentiated
from ENR10 during the same period (Fig. S5). It showed that both time and ENR concentration had a joint impact on the relative
abundance of denitrifying bacteria. Additionally, it was observed that the relative abundance of denitrifying bacteria exhibited a dose-
effect relationship with the concentration of ENR. PCA analysis was used to analyze the distribution changes of denitrifying bacteria at
the genus level (Fig. 6). PC1 and PC2 explained 28.0% and 12.7% of the total variance, respectively. The 0d and 4d treatments were
separated at the PC2 level, while the 4d treatment group and the 20d treatment group as a whole were separated at the PC1 level.
However, on the 4th day, the treatment groups clustered into one group, and the change rule with the antibiotic concentration was not
obvious. But on the 20th day, the changes on PC1 were observed among the treatment groups with regard to antibiotic concentration.
It indicated that prolonged exposure to ENR increased the succession of denitrifying bacterial genera.

3.5. Abundance of nitrogen cycle functional genes

Real-time PCR analysis was used to quantify the abundance of 16 S rRNA genes, AOB-amoA, narG, nirK, nirS and nosZ of bacteria
before and after cultivation (Fig. 5). The 16 S RNA genes were used as reference genes, and the abundance of functional genes/16 s
RNA genes were used to represent the relative abundance of different functional genes. The relative abundance of AOB-amoA
decreased as sampling time increased in the same treatment, but there were no significant differences in abundance between different
treatments at the same sampling time (Fig. 5a).
The addition of ENR reduced the relative abundance of narG after 4 days, but the ENR1 and ENR10 treatments returned to control
levels after 20 days (Fig. 5b). The results showed that the inhibition of ENR on the transformation of nitrate to nitrite mainly occurred
in the early stage. We demonstrated that the inhibitory effect of ENR on Sphingomonas was an important reason for the decrease in the
relative abundance of narG, because Sphingomonas and narG showed a significant positive correlation (p < 0.01, Table 1). The addition
of ENR to soil increases nitrate levels by inhibiting the conversion of nitrate to nitrite through reducing the abundance of Sphingomonas
and narG. To our knowledge, studies have reported that other FQs (e.g., ciprofloxacin, ofloxacin, norfloxacin) also can reduce narG
abundance and consequently inhibit nitrate reduction (Chen et al., 2021; Ma et al., 2014; Zhang et al., 2022).
At the same sampling time, there was no significant difference in the relative abundance of nirK among the treatment groups,
indicating that ENR had a minor impact on the nirK (Fig. 5c). On the 4th and 20th day, the addition of ENR increased the relative
abundance of nirS gene and displayed a dose-dependent connection though the difference between the ENR1 and the Control treatment
was not noticeable (Fig. 5d). Changes in the nirS gene indicated that ENR promoted the conversion of nitrous nitrogen to nitric oxide.
The selectivity of heme-containing CD1 Nitrate reductase (cd1-NIR, encoded by nirS) and copper-containing Nitrate reductase (cu-NIR,
encoded by nirK) for ENR may be responsible for the different changes in the nirK and nirS genes (Kuypers et al., 2018).
The relative abundance of nosZ showed a similar trend to that of nirK. Compared to the Control treatment, the ENR10-treated group
exhibited a significant increase in the relative abundance of the nosZ gene and increased the reduction tendency of N2O (Fig. 5e). In this
study, (nirK+nirS)/nosZ was used to characterize the potential of ENR for N2O emission (Fig. 5 f). It can be found that the ENR1
treatment group raised the value of (nirK+nirS)/nosZ in comparison to the Control treatment on the 20th day, indicating that ENR may
pose a risk of increasing N2O emissions. Lin et al. (2022) have also confirmed our view. We concluded that ENR1, which is related to
environmental concentration, may increase the risk of N2O emission more than the high-concentration ENR-contaminated soil
(ENR10).

4. The practical applications and future research prospects

The effect of antibiotic pollution on the nitrogen cycle is an important area in research. This research investigates the degradation
pattern of ENR in soil, which can help to develop agricultural practices and policies to reduce antibiotic residues in agricultural
products. The examination of nitrogen conversion processes in soil under ENR pressure can provide farmers with relevant data to
precisely manage nitrogen fertilizer application, improve fertilizer application practices, and reduce nitrogen loss to prevent water
body pollution. By understanding the effects of ENR on the microbial communities associated with the nitrogen cycle, the stability of
the ecosystem can be better managed and maintained. It is well known that the use of antibiotics has an impact on the prevalence and
distribution of antibiotic resistance genes (ARGs). However, the link between ARGs and the nitrogen cycle has yet to be fully examined
and will be the main focus of the forthcoming study.

5. Conclusion

ENR degrades slowly in soil with a half-life of approximately 40 days. For soil nitrification, ENR reduced the relative abundance of
Nitrosospira, Nitrosomonas and PAO, and inhibited nitrification at the beginning of the experiment. The impact of ENR on soil deni­
trification varied at different stages. ENR treatment inhibited the conversion of nitrate to nitrite by reducing the relative abundance of
Sphingomonas and narG, which resulted in the accumulation of nitrate. ENR treatment stimulated the process of nitrite to nitric oxide
and N2O reduction to nitrogen by increasing the relative abundance of denitrifying bacteria (Aeromonas, Flavobacterium) and deni­
trifying function genes (nirS, nosZ). Treatment with low-concentration ENR may increase the N2O emission potential during deni­
trification. Overall, ENR contamination affected microbial nitrogen cycling and had adverse effects on farmland soils. We hope this
study will raise awareness of the impact of antibiotic contamination on soil nutrient cycling.

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Table 1
Correlation analysis between top ten denitrifying bacteria and the functional genes of nitrogen cycle.
` AOB-amoA narG nirS nirK nosZ

Pseudomonas R -.601** -0.226 -0.248 0.158 -0.39

P 0.004 0.324 0.278 0.495 0.081
Comamonas R -.580** -0.353 -.442* -0.06 -0.385
P 0.006 0.116 0.045 0.796 0.085
Acinetobacter R -.541* -0.261 -.523* 0 -.527*
P 0.011 0.254 0.015 0.998 0.014
Hyphomicrobium R -0.001 -0.295 -.516* -.476* -0.039
P 0.996 0.194 0.017 0.029 0.868
`Paenibacillus R 0.073 -.504* 0.356 -.628** .554**
P 0.752 0.02 0.113 0.002 0.009
Streptomyces R -0.115 -0.212 -0.232 -0.272 0.318
P 0.62 0.357 0.311 0.233 0.16
Flavobacterium R -.531* -0.312 .462* 0.18 0.218
P 0.013 0.169 0.035 0.434 0.343
Bacillus R -0.034 -0.357 0.224 -0.186 0.307
P 0.884 0.112 0.328 0.419 0.176
Aeromonas R -0.408 -0.117 0.161 0.104 -0.241
P 0.066 0.614 0.486 0.655 0.292
Sphingomonas R .619** .845** -0.063 0.432 -0.344
P 0.003 0 0.786 0.05 0.127
Nitrolancea R 0.216 0.299 -0.131 0.25 -0.019
P 0.347 0.188 0.57 0.275 0.934
Nitrosomonas R -0.381 -0.003 -.457* 0.415 -0.352
P 0.089 0.991 0.037 0.061 0.118
Nitrosospira R 0.001 0.134 -.454* 0.082 -.838**
P 0.998 0.563 0.038 0.725 0
*
Significant correlation in p < 0.05 level;
**
significant correlation in p < 0.01 level; N = 21.

CRediT authorship contribution statement

Jianpeng Gao: Investigation, Sample testing, Data curation, Writing – original draft. Chang Wei: Investigation, Sample testing.
Tian-Jin Mo: Investigation, Sample testing. Yu Yan: Investigation, Sample testing. Yan Sun: Investigation, Sample testing. Huayi
Chen: Investigation, Sample testing. Yulong Zhang: Investigation, Sample testing. Jinjin Wang: Conceptualization, Methodology.
Yongtao Li: Supervision, Project administration, Funding acquisition. Hui-Juan Xu: Conceptualization, Methodology, Supervision,
Project administration, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Data Availability

Data will be made available on request.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China - Guangdong Joint Fund (No.
U1901601), the Natural Science Foundation of Guangdong Province (No. 2023A1515010593), the National Natural Science Foun­
dation of China (No. 41877063), the open competition program of top ten critical priorities of Agricultural Science and Technology
Innovation for the 14th Five-Year Plan of Guangdong Province (No. 2022SDZG08), the Double First-class Discipline Promotion Project
(No. 2021B10564001).

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.eti.2023.103415.

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