Science of the Total Environment 914 (2024) 170002

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Science of the Total Environment

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

Motility behavior and physiological response mechanisms of aerobic

denitrifier, Enterobacter cloacae strain HNR under high salt stress: Insights
from individual cells to populations
Meng Cheng a, 1, Hui-Min Fu a, b, 1, Zheng Mao a, c, Peng Yan a, Xun Weng a, Teng-Fei Ma b,
Xiao-Wei Xu a, Jin-Song Guo a, Fang Fang a, You-Peng Chen a, *
a
Key Laboratory of the Three Gorges Reservoir Region's Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China
b
National Research Base of Intelligent Manufacturing Service, Chongqing Technology and Business University, Chongqing 400067, China
c
Chongqing Institute of Geology and Mineral Resources, Chongqing 400042, 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

• Rotation velocity and vibration ampli­

tude of HNR were decreased under high
salinity.
• As salinity increased, both ETSA and
ATP content gradually decreased in
synchrony.
• The ABC transporters were the limiting
factors of matter and energy metabolism
of HNR.
• The biofilm-related motility regulation
mechanism was activated under high
salinity.
• Genetic diversity in motility regulation
makes HNR population more adaptable.

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

Editor: Qilin Wang The motility behaviors at the individual-cell level and the collective physiological responsive behaviors of aer­
obic denitrifier, Enterobacter cloacae strain HNR under high salt stress were investigated. The results revealed that
Keywords: as salinity increased, electron transport activity and adenosine triphosphate content decreased from 15.75 μg O2/
Aerobic denitrifiers g/min and 593.51 mM/L to 3.27 μg O2/g/min and 5.34 mM/L, respectively, at 40 g/L, leading to a reduction in
Flagellar motility
the rotation velocity and vibration amplitude of strain HNR. High salinity stress (40 g/L) down-regulated genes
Single-cell motion
involved in ABC transporters (amino acids, sugars, metal ions, and inorganic ions) and activated the biofilm-
Chemotaxis
Salt stress related motility regulation mechanism in strain HNR, resulting in a further decrease in flagellar motility ca­
pacity and an increase in extracellular polymeric substances secretion (4.08 mg/g cell of PS and 40.03 mg/g cell
of PN at 40 g/L). These responses facilitated biofilm formation and proved effective in countering elevated salt
stress in strain HNR. Moreover, the genetic diversity associated with biofilm-related motility regulation in strain
HNR enhanced the adaptability and stability of the strain HNR populations to salinity stress. This study enables a
deeper understanding of the response mechanism of aerobic denitrifiers to high salt stress.

* Corresponding author.
E-mail address: ypchen@cqu.edu.cn (Y.-P. Chen).
1
Meng Cheng and Hui-min Fu contributed equally to all aspects of conceptualizing planning, sample and data collection, data analysis, and preparation of the
manuscript.

https://doi.org/10.1016/j.scitotenv.2024.170002
Received 23 October 2023; Received in revised form 20 December 2023; Accepted 6 January 2024
Available online 14 January 2024
0048-9697/© 2024 Elsevier B.V. All rights reserved.
M. Cheng et al.
1. Introduction
Seawater substitution and production by various industries
discharge a large number of high-salt wastewater containing high con­
centrations of inorganic salts (3.0–3.5 wt%), which is the typical
wastewater (Zhu et al., 2022). Compared with traditional physical and
chemical methods, biological treatment technology has become one of
the focuses of high-salinity organic wastewater treatment research due
to its unique advantages of environmentally friendly and inexpensive
(Chen et al., 2022). Prior research has indicated that elevated salinity in
wastewater typically suppresses the activity of degradation enzymes,
leading to reduced cellular activity. This condition can result in cell
dehydration, lysis, and direct interference with the processing efficiency
of functional microorganisms (Chen et al., 2023; Chen et al., 2016).
Meanwhile, most of the microorganisms in biological wastewater
treatment systems are usually non-halophilic bacteria. The high-salinity
wastewater may
Interfere with the relative abundance of functional microorganisms
and affect the removal of target pollutants (Qiu and Ting, 2013).
Therefore, high salinity is considered to be a limited factor for the
application of the biological wastewater treatment systems.
Wastewater, characterized by high concentrations of ammonia ni­
trogen and salt, is frequently generated in aquaculture, desulfurization,
salinization, and related industrial processes (Ji et al., 2021; Zhou et al.,
2020). The application of aerobic denitrification has garnered signifi­
cant interest as a prevalent method for treating such ammonia-rich
wastewater. The conventional nitrogen removal process typically com­
prises two continuous steps: nitrification and anaerobic denitrification.
Nevertheless, nitrification and anaerobic denitrification demand strin­
gent operating conditions and are conventionally conducted separately.
This separation imposes elevated investment costs and operational in­
tricacies in engineering applications of the process (Xi et al., 2022). The
aerobic denitrification process is widely recognized as a promising and
cost-effective alternative to the traditional nitrogen removal process (Ma
et al., 2022). However, aerobic denitrifiers are sensitive to environ­
mental conditions (Wang et al., 2023a), such as salinity, which signifi­
cantly impacts the nitrogen removal efficiency of saline wastewater.
Presently, research has primarily focused on screening salt-tolerant
aerobic denitrifiers and studying the effect of salinity on their nitrogen
removal performance (Chen et al., 2022; Xi et al., 2022). Limited
attention has been given to the physiological and biomolecular response
of aerobic denitrification under high salt stress. Furthermore, these
studies have primarily focused on aggregate-level analysis, neglecting
the role of individual bacterial motion behavior and bacterial chemo­
taxis in adverse environments (Colin et al., 2021). Analyzing the motion
behavior of single cells and exploring bacterial chemotaxis is crucial for
elucidating the authentic adaptation mechanisms employed by aerobic
denitrifiers in adverse environments. Such an investigation will provide
valuable insights into the physiological relevance of individual bacterial
cells and bacterial populations under adverse conditions.
Multiple recent studies have demonstrated that flagellar motility and
chemotaxis can play diverse roles in collective behaviors, even within
the same species. These roles are highly influenced by specific physio­
logical and environmental conditions (Colin et al., 2021). However, the
lack of in-situ, fast, and real-time analysis methods has impeded our
understanding of how the behavior of individual bacteria drives changes
in population characteristics. Fortunately, advancements in optical im­
aging technology have led to the widespread use of surface plasmon
resonance imaging (SPRI) in biomolecular research. SPRI offers high
spatial resolution detection capabilities and a precise image analysis
process (Zhou et al., 2020). Its key advantages include high-throughput,
lossless in-situ observation, and real-time monitoring of biomolecular
interactions (Ma et al., 2018). Importantly, SPRI enables real-time and
sensitive perception of single-cell movement. It accurately captures and
records the movement direction and velocity in the horizontal plane.
Additionally, its distance sensing capabilities in the vertical direction
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Science of the Total Environment 914 (2024) 170002
allow for precise detection of nano-level vibrations of bacteria in the Z
direction. Our previous study employed SPRI to characterize the
motility of aerobic denitrifying bacteria under high ammonia stress
(Weng et al., 2022). Consequently, SPRI is a powerful analytical tool for
analyzing individual cell behavior (Zhang et al., 2016).
In this study, SPRI was employed to observe and record the motion
behavior of aerobic denitrifier, Enterobacter cloacae strain HNR at the
single-cell level under high salt stress. Additionally, transcriptomic
methods, as well as physiological and biochemical characterization,
were utilized to investigate the responsive collective behaviors of HNR
in high salinity conditions. Exploring the diverse roles of motility and
chemotaxis in the collective behaviors of aerobic denitrifiers is instru­
mental in understanding their regulatory mechanisms in response to
salinity stress. These findings provide valuable theoretical insights and
suggestions for optimizing and regulating biological treatment processes
for high-salt wastewater.
2. Materials and methods
2.1. Bacterial culture
The aerobic denitrifier HNR (Ma et al., 2021) was chosen as the
experimental bacteria. HNR was isolated from activated sludge of a
wastewater treatment plant by our research group, exhibiting a high
nitrate removal performance of 109.92 mg N L− 1 d− 1. The components
of each liter of medium were detailed in the supplementary materials
and our previous study (Weng et al., 2022). The activated HNR was
cultured at 130 rpm and 28 ◦ C for subsequent experiments.
2.2. Experimental design
The experimental groups were subjected to varying salinity levels
(expressed in terms of sodium chloride) of 5 g/L, 10 g/L, 20 g/L, 30 g/L,
and 40 g/L, aiming to simulate high-salt wastewater typical of con­
ventional industrial settings (Shi et al., 2020; Yan et al., 2016). The
groups without adding chloride ions was set as the control group in the
batch experiment. For the salinity stress experiment, the glucose solu­
tion was sterilized in an autoclave at 110 ◦ C for 15 min, and the mixture
of other components was sterilized in an autoclave at 121 ◦ C for 25 min.
The mixture was thoroughly mixed in a super-clean workbench before
use. Several 250 mL conical bottles containing 100 mL of liquid medium
with different salt concentrations were added with appropriate amount
of bacterial solution and cultured at a constant temperature shaking
table at 130 rpm and 28 ◦ C. The culture in the logarithmic phase (OD
600 = 0.6) of different experimental groups was centrifuged at 6000
rpm for 10 min, washed twice with 1 × PBS (pH = 7.5), and 10 μL of the
resulting cell pellet was added to the microwell sample cell. Next, an
appropriate amount of salt solution was added to achieve the desired
salinity concentration in the mixed solution. Subsequently, the solution
was placed in the SPRI system to observe the single-cell motility
behavior. Statistical analysis was performed based on observations of six
individual bacterial cells per salinity condition.
In addition, HNR culture in the logarithmic phase (OD600 = 0.6)
were exposed to various salt solution environments for 20 min. After the
incubation period, a suitable volume of the bacterial solution was
collected for subsequent analysis of physiological and biochemical
characteristics.
2.3. SPRI system
The SPRI system was mainly composed of SPR light sources, a mi­
croscope containing high NA object mirrors, beam mirrors, inductive
charge-coupled device cameras, sensor chips, sample microcardrons,
and trace injection pumps. The SPR light source was composed of a 680
NM ultra-radiated diode (QSDM-680-2, Qphotonics, the United States)
and light source drives (QSDIL-500, Qphotonics, the United States). The
M. Cheng et al.
details on the parameters were seen from our previous study (Weng
et al., 2022).
2.4. Observation and imaging of single-cell motion behavior
The microwell sample cell was fabricated using poly mailing silicone
(PDMS) and chip bonding techniques. Prior to use, the chip surface was
thoroughly cleaned with acetone, absolute ethanol, and pure water for 5
min respectively, followed by drying with nitrogen gas. To enhance
hydrophilicity for subsequent observations, the chip was then modified
with a 0.1 mM sodium alginate solution and incubated overnight at 4 ◦ C.
Subsequently, the laser generator, SPR microscope, and image sensor
were activated. The laser intensity was adjusted to 130 mA, and the
incident angle was optimized until the SPR intensity signal reached one-
third of the maximum value. After the SPRI system was set up, HNR and
high-salt solution were added through two channels at the same time,
and then mixed to a specific concentration to observe the movement
changes of HNR in 0 min. The bacterial motion was recorded using a
charge-coupled device camera (Pike-032B, Allied Vision Technology,
Germany) at a frame rate of 50 frames/s, with an image resolution of
320 pixels × 240 pixels.
Following image collection, the initial frame of the image sequence
was discarded to eliminate impurities and background noise signals.
Subsequently, denoising was performed using Gaussian filtering pro­
vided by Image J software. MATLAB (MathWorks Inc., USA) was utilized
for further processing and description of the bacterial motion informa­
tion under different salinity stress conditions.
It is worth noting that previous study found that by comparing SPR
imaging of live and dead bacteria, the error caused by Brownian motion
was within 1 nm (Syal et al., 2016). In this study, the motion fluctuation
range of HNR could reach up to 200 nm, so it was negligible.
2.5. Determination of physiological and biochemical characteristics of
HNR
Extracellular polymeric substances (EPS) were extracted and quan­
tified as reported by Yin et al. (2015). The biofilm-forming capacity of
bacteria was determined by crystal violet staining (CV). The adenosine
triphosphate (ATP) content was determined using the BacTiter-Glo™
reagent kit (Promega, USA). The electron transport system activity
(ETSA) was measured by iodonitrotetrazole (INT) (Tu et al., 2022). The
ratio of live to dead bacteria was measured by the apoptosis fluorescent
Hoechst 33342/PI double stain kit (Solarbio, China) (Wang et al., 2021).
All the experiments were done in triplicate.
2.6. Pretreatment and testing of transcriptome samples
After resuscitation, HNR was incubated for another 7 h and placed in
sterile pure water containing 40 g/L NaCl and NaCl-free for 20 min. The
bacteria were collected and washed twice with 1 × PBS (pH = 7.5). The
pellet was rapidly transferred to a − 80 ◦ C freezer to prevent bacterial
RNA degradation. Later samples were sent with dry ice for tran­
scriptomic testing.RNA extraction and transcriptome sequencing were
performed following previous work (Peng et al., 2019). Sequencing data
generated from the Illumina platform had been submitted to NCBI
(PRJNA1028167) and was used for bioinformatics analysis. All analyses
were performed using the Majorbio Cloud Platform.
3. Results and discussion
3.1. Single-cell movement behavior under high salt stress
3.1.1. Bacterial rotation
By analyzing the variation in SPR signal intensity associated with
bacteria at different locations along the horizontal axis, it is possible to
extract information about the horizontal movement of bacteria. When
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Science of the Total Environment 914 (2024) 170002
bacteria undergo reversible attachment, they loosely adhere via a single
pole, allowing for rapid rotation, vibration, or movement (Petrova and
Sauer, 2012). In this study, the rotational behavior of HNR over time
under different salinity conditions was investigated, with the rotation of
bacteria in the absence of salt stress serving as a control. The corre­
sponding results are presented in Fig. 1.
In the control group, the average rotation speed was measured to be
15.67 rad/Frame. Compared to the control group, under 5 g/L salt
stress, the average rotation speed of HNR initially increased by 18.8 %.
However, after 5 min, the average rotation speed of HNR decreased to
40.1 % of the control group, and by 10 min, rotation ceased entirely.
When exposed to a 10 g/L salt stress, the average rotation speed of HNR
dropped to 49.4 % of the control group within 5 min, reaching a minimal
0.25 rad/Frame, and subsequently halted. Similarly, under 20 g/L salt
stress, rotation slowed to 4.77 rad/Frame (17.6 % of control) and
stopped at 5 min. Exposure to 30 g/L and 40 g/L salt caused immediate
cessation of bacterial rotation.
The introduction of 5 g/L of salinity initially resulted in an
augmentation of rotational speed, attributable to the provision of Na+
ions from NaCl. These ions are known to enhance microbial flagellar
motility, promote enzyme synthesis, and facilitate ATP synthesis (Pis­
men, 2020; Xie and Cai, 2003). However, as the salinity increased to 10
g/L and beyond, the rotation speed of HNR gradually decreased until it
ceased. This result can be attributed to high salinity inhibiting metabolic
enzymes and suppressing HNR activity (Fu et al., 2019). This hypothesis
can be substantiated through an examination of the correlation between
bacterial motility and physiological responses under salinity stress,
coupled with an investigation into the associated alterations in meta­
bolic pathways.
3.1.2. Bacterial vertical movement
When a solitary bacterium adheres to the chip, distinct nano-scale
vertical motion ensues as an outcome of its metabolic processes (Weng
et al., 2022). Syal et al. (2016) have demonstrated that bacterial nano-
motion correlates with bacterial metabolism, and sub-micron move­
ment is indicative of favorable bacterial metabolic activity. To obtain
the movement information of bacteria in the vertical direction, Image J
software was utilized to analyze the exponential attenuation relation­
ship between the image light intensity and the vertical distance (Syal
et al., 2016; Yang et al., 2015). The results are shown in Fig. 2.
In the control group, the maximum amplitude of vertical movement
ranged from 75 to 104 nm. Under 5 g/L and 10 g/L salt stress, the
vertical motility of HNR exhibited slight inhibition compared to the
control group. As the salinity increased to 20 g/L and beyond, the bac­
terial vertical movement was significantly impeded. Under 40 g/L salt
stress, the vertical movement of bacteria nearly ceased. The metabolic
activity of living cells is associated with processes like cellular mem­
brane transfer, cytoplasmic fluidity, and changes in membrane lipid
components in response to environmental variations. These factors
contribute to the micro-movement of cells (Syal et al., 2016). The
decrease in vertical movement indicated that salinity affected the ac­
tivity of HNR.
3.2. Metabolic characteristics of HNR based on population
3.2.1. EPS analysis
EPS plays an important role in the formation and maintenance of the
biofilm structure (Fu et al., 2022a). Previous study has shown that EPS
can enhance the adaptability of bacteria to salt stress by regulating cell
penetration pressure (He et al., 2020). EPS is mainly composed of pro­
tein (PN) and polysaccharides (PS), and a small amount of DNA (He
et al., 2020). This study mainly analyzed the concentration changes of
PN and PS under different salinity stress. The results were shown in
Fig. 3(a).
The amino acid composition and secondary structure of PN basically
contribute to hydrophobic interactions. PN with high molecular weight
M. Cheng et al. Science of the Total Environment 914 (2024) 170002

Fig. 1. Rotation speed of HNR exposed to different salinities at 0, 5, 10, and 20 min. The acquisition t ime is 10 s. (a) the rotation speed of individual HNR under 5 g/
L salt stress; (b) the rotation speed of individual HNR under 10 g/L salt stress; (c) the rotation speed of individual HNR under 20 g/L salt stress; (d) the rotation speed
of individual HNR under 30 g/L salt stress; (e) the rotation speed of individual HNR under 40 g/L salt stress. (The control group was the rotation speed of individual
HNR in the same batch without adding salinity).

Fig. 2. Time domain measurement of vertical movement of HNR under salt stress of 0 g/L(a), 5 g/L(b), 10 g/L(c), 20 g/L(d), 30 g/L(e), and 40 g/L(f). The black color
represented the SPRI signal before the addition of the salt solution (control group), and the red color represented the SPRI signal after the addition of the salt solution
for 20 min (experimental group).

can provide more ion binding sites or polymer interaction sites to protect the increase of salinity, the PN content in EPS increased first and then
cells from external shocks (Dong et al., 2017). PN in EPS also partici­ decreased. At the salinity of 30 g/L, the PN content was the highest at
pates in the removal of some metal ions such as Na+, so that microor­ 42.74 mg/g cell. PS contained a large number of polar groups, which
ganisms maintain a normal physiological state (Hu et al., 2020). With had strong hydrophilicity. PS could thicken the hydration layer around

4
M. Cheng et al.
Fig. 3. Effect of salt stress on physiological and biochemical characteristics of HNR: The EPS content (a), biofilm formation capacity (b), ETSA content (c), and ATP
content (d) of HNR under salt stress of 5 g/L, 10 g/L, 20 g/L, 30 g/L and 40 g/L.
the cell and enhance the density of biofilm (Zhao et al., 2022b). With the
increase of salinity, the variation trend of PS concentration was similar
to that of PN. The maximum content of PS was 11.82 mg/g cell at 20 g
NaCl/L. Under low salinity conditions, HNR secreted more EPS to resist
the impact of salinity. However, the impact of high salinity might affect
the normal metabolic activity of HNR, thus reducing EPS secretion.
Previous study has shown that the changes in PS content are more
sensitive than PN content (He et al., 2020). Therefore, a certain amount
of salinity is more conducive to PS accumulation of HNR.
The ratio of PN/PS is an important representation of EPS properties,
and the change of the ratio can reflect the hydrophobic changes of
bacteria. It can be seen from Fig. 3(a) that the ratio was decreased first
and then increased, indicating that the hydrophobicity first decreased
and then increased, indicating that the adhesion ability of HNR might
increase after salinity stress of 20 g/L NaCl.
During the process of biofilm formation, reversible and irreversible
adhesion of bacteria to the surface is typically mediated by flagella, pili,
and EPS (Bowen et al., 2018). The initial step of adhesion involves
slowing down or stopping the rotation and vibration of flagella. High
salt stress may inhibit flagella-mediated motility by increasing intra­
cellular signaling molecules, while simultaneously promoting the pro­
duction of EPS in the biofilm, thereby facilitating the adhesion of HNR to
the substrate (Hickman and Harwood, 2008). Liu et al. (2023) demon­
strated that reduced motility in Pseudomonas aeruginosa, induced by low-
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Science of the Total Environment 914 (2024) 170002
temperature stress, promoted the transition from a planktonic to a bio­
film state, enhancing initial adhesion and subsequent biofilm develop­
ment. Additionally, the decreased motility under high salt stress
appeared to significantly alleviate the metabolic burden associated with
other physiological activities (such as, EPS secretion) during biofilm
formation.
3.2.2. Change of biofilm forming ability (BFA)
The biofilm is considered to be conducive to the ability of microor­
ganisms to overcome the adverse environment and improve the ability
of functional microorganisms to resist environmental fluctuations (Fu
et al., 2022a; Fu et al., 2022b). However, biofilm formation is affected
by many factors and salt stress is one of the important environmental
stress. High salinity may cause physiological changes in bacteria and
affect the formation of biofilms. As shown in Fig. 3(b), compared with
the control group, the biofilm forming ability showed an increasing
trend except that the biofilm forming ability was slightly decreased
under 5 g/L and 40 g/L salt stress. It can also be seen from the rotation
results of HNR (Fig. 1) that under short-term (<5 min) stress of 5 mg/L,
appropriate Na+ could enhance the movement ability of HNR, shorten
the residence time of HNR at the interface, which may further shorten
bacterial adhesion stage and inhibit biofilm formation of HNR (Fu et al.,
2021). When the salt concentration increased to 40 g/L, metabolic ac­
tivity and EPS secretion of HNR were significantly inhibited, thereby
M. Cheng et al. Science of the Total Environment 914 (2024) 170002

reducing the biofilm-forming ability. mortality in HNR. This phenomenon also accounted for the observed
rapid decline in HNR rotational speed under conditions of elevated
3.2.3. Effects of salinity on ETSA and ATP salinity.
The carbon source metabolism and electronic transmission process
are closely related to bacterial movement and functional processes (Gu
3.3. Correlation between bacterial nano movement and metabolic
et al., 2022; Guo et al., 2022). By measuring the ETSA value and ATP
characteristics
content, the impact of NaCl on the metabolic process of HNR was
analyzed. The results are shown in Fig. 3 (c,d). The ETSA of the control
The relationship between bacterial movement and physiological re­
group was 15.75 μg O2/g/min. Under 5 g/L salt stress, the ETSA of HNR
sponses was analyzed by Pearson correlation analysis and shown in
increased to 27.32 μg O2/g/min, which was 173.5 % of the control
Fig. 5. First of all, relative vertical movement amplitude (RVMA) and
group. However, when the salinity was >10 g/L, the ETSA of HNR
relative average rotation rate (RARR) were significantly positively
decreased with the increase of salinity and finally decreased to 3.27 μg
correlated (R2 ≥ 0.61, p < 0.05), indicating that the vibration in the
O2/g/min at 40 g/L, which was only 20.8 % of the control group. These
vertical direction and the rotation in the horizontal direction were
results indicated that the electron transmission of respiratory chain was
relatively consistent. More importantly, RARR was significantly posi­
significantly inhibited, which was consistent with the rotational motion
tively correlated with ATP and ETSA (R2 ≥ 0.82, p < 0.05; R2 ≥ 0.94, p
of HNR.
< 0.05), significantly negatively correlated with PS and PN (R2 ≥ 0.69, p
ATP, as a source of energy for many basic reactions, is a key indicator
< 0.05; R2 ≥ 0.66, p < 0.05). RVMA was also significantly positively
of biological activity in all living cells (Zhao et al., 2022a). The changes
correlated with ATP (R2 ≥ 0.72, p < 0.05). ATP plays an important role
of the ATP content under different salt stress are shown in Fig. 3(d). The
in the bacterial flagellar rotation. The bacterial flagellar motility is
ATP content of HNR in the control group was 593.52 mM/L. Compared
mainly driven by protons, and the transmembrane potential is directly
with the control group, under salt stress of 5 g/L, 10 g/L, 20 g/L, 30 g/L,
proportional to the rotation rate of the flagellar motor. This mechanism
and 40 g/L for 20 min, the ATP contents of HNR were reduced by 49.7
requires the consumption of ATP to expel H+ outside the bacteria to
%, 49.2 %, 85.4 %, and 99.1 %, respectively, which meant the greater
form a transmembrane gradient that rotates the flagellar motor
the salt stress, the greater the effect on the ATP content in HNR. These
(Wadhwa and Berg, 2022). The electron transfer process of the bacterial
results were consistent with the changes in the vertical movement trend
respiratory system is coupled with the formation of ATP, and the whole
of HNR.
process of ATP formation is accompanied by the phosphorylation of ADP
(Lobritz et al., 2015). Therefore, the strength of ETSA will indirectly
3.2.4. Effects of salinity on the ratio of live to dead bacteria
affect the rotation behavior of bacteria by affecting the amount of ATP
In this study, under salt stress of 5 g/L, 10 g/L, 20 g/L, 30 g/L, and
production in microorganisms. In this study, increasing salinity was
40 g/L for 20 min, the ratio of live to dead bacteria of HNR was tested for
observed to cause a reduction in electron transport activity, resulting in
viability. The results are shown in Fig. 4. Under salt stress of 5 g/L, 10 g/
a concurrent decrease in ATP content. Consequently, these reductions
L, and 20 g/L, the ratio of live to dead bacteria of HNR was not affected
led to a reduction in both the rotational velocity and vibrational
by the salinity. When the salinity increased to 30 and 40 g/L, the pro­
displacement of HNR. In addition, salinity stress synchronously
portion of HNR in the Q3-UR and Q3-LR area increased more, indicating
decreased the flagellar motility capacity of HNR and increased EPS
that the number of early apoptotic bacteria and necrotic bacteria
secretion, inducing bacterial aggregation and adhesion (Tan et al.,
increased. High salinity was hypothesized to induce damage and
2019), which might be conducive to the biofilm formation of HNR.

Fig. 4. Results of HNR staining of Hoechst 33342/PI under salt stress of 0 g/L(a), 5 g/L(b), 10 g/L(c), 20 g/L(d), 30 g/L(e), and 40 g/L(f), respectively. (Q3-LL, Q3-
LR, Q3-UR, and Q3-UL respectively, represent the areas of living bacteria, early-apoptotic bacteria, non-viable apoptotic bacteria, and necrotic bacteria).

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M. Cheng et al.
Fig. 5. The nitrogen removal activity of HNR under salinity stress (a); Correlation between bacterial motility and physiological indicators of HNR (b). (Abbreviations:
relative vertical movement amplitude (RVMA); relative average rotation rate (RARR); biofilm formation ability (BFA); adenosine triphosphate (ATP); electron
transport system activity (ETSA); protein (PN) and polysaccharides (PS); Specific nitrogen removal activity (SNRA)).
There were also some correlations between physiological and
biochemical characteristics. ATP is significantly positively correlated
with ETSA (R2 ≥ 0.64, p < 0.05) and negatively correlated with PN (R2
≥ 0.83, p < 0.05). PS is significantly positively correlated with BFA (R2
≥ 0.77, p < 0.05) and negatively correlated with ETSA (R2 ≥ 0.58, p <
0.05). Previous studies showed that biofilm formation capacity was
positively correlated with PN and PS content (Fu et al., 2022a; Tang
et al., 2016). PN and PS contributed to the formation of biofilms,
especially PN (Fu et al., 2022b). However, under high salt stress, the
correlation between biofilm formation and PN was not significant (p >
0.05), and the correlation with PS was higher than that of PN, which
might be related to the adaptive adjustment of bacterial metabolic
pathways under salt stress.
SNRA exhibited positive correlations with RVMA, RARR, ATP, and
ETSA, while displaying negative correlations with PN and PS. These
findings suggest a significant association between nitrogen removal
activity and the movement capacity and physiological characteristics of
HNR. Bacterial chemotaxis serves a crucial role in exploring nutrient
sources and identifying optimal growth niches, influencing chemo-
effector preferences and flagellar gene regulation. Raina et al. (2023)
investigated the impact of bacterial motility behavior on metabolic ex­
changes at the single-cell level between the picophytoplankton Syn­
echococcus and the heterotrophic bacterium Marinobacter adhaerens.
Utilizing motility and chemotaxis-deficient bacterial mutants, stable-
isotope tracking revealed that chemotaxis increased nitrogen and car­
bon uptake by both partners up to 4.4-fold. In the context of this study,
high salt stress inhibited the movement performance and metabolic
activity of HNR, leading to reduced substrate uptake capacity and ni­
trogen removal performance.
3.4. Transcriptome analysis
In this part, transcriptomics techniques were used to analyze and
compare differentially expressed genes of two representative culture
conditions (Control, 40 g/LNaCl) to reveal the response mechanism of
HNR to high salt stress at the gene level.
According to the overall analysis of KEGG enrichment, the main
pathways involved in up-regulated genes were in ribosomes, bacterial
secretion systems, biofilm formation-Pseudomonas aeruginosa, lipopoly­
saccharide biosynthesis, mismatch repair, purine metabolism,
aminoacyl-tRNA biosynthesis (Fig. S1). While the main pathways
involved in down-regulated genes were ABC transporters, quorum
sensing, nitrogen metabolism, lysine degradation, etc. (Fig. S1), which
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Science of the Total Environment 914 (2024) 170002
were closely related to the physiological and biochemical indicators
discussed earlier.
3.4.1. Tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and ABC
transporters
The TCA cycle and oxidative phosphorylation are crucial for
providing intermediates, ATP synthesis, and metabolic energy during
microbial growth (Guan et al., 2014). A previous study found that genes
related to the TCA cycle and oxidative phosphorylation were down-
regulated in Nitrosomonas europaea under CeO2 nanoparticle exposure
(Wu et al., 2019). In this study, high salinity stress also decreased the
ATP content and might have an inhibitory effect on microbial energy
metabolism. While, TCA cycle-related genes (fumA, sucACD, frdAB, aceE,
acnB, mqo, and pckA) and oxidative phosphorylation-related genes
(atpABEGH, cydAB, cyoB, frdAB, nuoCDM, ppa, and ppk) were signifi­
cantly up-regulated under 40 g/L NaCl stress (Figs. S2 and S3). The
decline in ATP content can be attributed to the pronounced down-
regulation of multiple genes associated with ABC transporters. ABC
transporters play a crucial role in regulating cellular defenses, including
ion homeostasis, nutrient absorption, and signaling molecule output in
bacteria (Feng et al., 2022). Under 40 g/L NaCl stress, it was observed
that genes involved in the transport of amino acids, sugars, metal ions,
and inorganic ions were markedly down-regulated (Fig. 6(a)), leading to
the inhibition of corresponding signal transduction, substrate, and
metabolite transport. Besides, the ABC transporter might be involved in
ATP synthesis by influencing ATP synthase activity (Fan et al., 2023).
Consequently, this down-regulation diminished the ETSA and ATP
synthesis of the entire bacterial population.
3.4.2. Chemotaxis, flagellar assembly, and biofilm formation
Chemotaxis is a fundamental mechanism utilized by bacteria to
locate and navigate toward nutrient sources. Bacteria achieve this by
modulating the direction of flagellar rotation, enabling them to move
away from unfavorable environments and toward favorable ones
(Keegstra et al., 2022). Bacterial flagella serve as organelles crucial for
bacterial propulsion and play a pivotal role in various functions asso­
ciated with bacterial pathogenicity. These functions encompass biofilm
formation, protein export, and adhesion (Weng et al., 2022). Under 40
g/L NaCl stress, most of the genes related to chemotaxis (aer, cheAB,
cheR, cheZ, gene1541, gene2372, mcp, tap, and tar) (Fig. S4), flagellar
synthesis (fliCD, fliI, fliOZ, fliP, fliR, fliT, flgABD, flgK, and flgL), and
regulation (csgD, flhCD, fliG, motAB, rcsA, and ycgR) were significantly
up-regulated (Table S1). In addition, a large number of genes related to
M. Cheng et al. Science of the Total Environment 914 (2024) 170002

a b
Amino acid Sugar Metal ion Inorganic ion
CsrA PGA PgaABD
livK gene1420
7.0
gene1029
livM cysP
6.0
5.0
urtA gene1421 gene1030 csgC
5.0
urtB cysU
OmpR FlhDC RcsAB 4.0
gene1422 csgD
gene1031 4.0
urtC
iatP
3.0
urtD 3.0 pgaA
gene1769 cysW
urtE ibpA 2.0
pgaB 2.0

aapJ gene1033 Colanic acid

malE
nrtA 1.0 CsgD pgaD 1.0
aapM
sitB 0.0
aapP malF flhC
Flagella 0.0

C
metI nrtB −1.0
sitC
metN malG −2.0
flhD −1.0

sitD
Curli
metQ nrtC flhE
malK −3.0 −2.0
opuA
afuA −4.0 rcsA
opuBD mglA −3.0
phnC YcgR
−5.0
opuC mglB afuB ycgR
−4.0
proX −6.0
afuC phnD yhjH
tauC mglC
−7.0 c-di-GMP YhjH −5.0

log2|FC log2|FC

Fig. 6. The down-regulated genes associated with ABC transporters of HNR under 40 g/L NaCl stress (a) (ABC transporters mainly transport amino acids, sugars,
metal ions and inorganic ions); Potential network of HNR biofilm-related motility regulation (b) (Abbreviations: ß-1,6-poly-N-acetylglucosamine (PGA); cyclic-di-
guanosine monophosphate (c-di-GMP)).

biofilm formation (Table S2), such as type II (gspCDEFGHJKL) and type
VI secretion protein (hcp, impABC, and impHJL), cyclic-di-guanosine
monophosphate (c-di-GMP) (mucR, yedQ, yegE, yhjH, and CdgR), and
EPS (bcsA, csgBC, wcaE, wcaLM, wecBC, and wza) were significantly up-
regulated. Wang et al. (2023b) also reported a significant increase in the
expression of flagella genes and chemotaxis genes in planktonic cells
subjected to nutritional stress.
Based on the analysis and summary of transcriptome data and related
literature, it was found that HNR had a biofilm-related motility regu­
lation mechanism similar to E. coli (Fig. 6(b)) (Guttenplan and Kearns,
2013; Weng et al., 2022). Motility plays a vital role in the initial stages of
biofilm formation, enabling bacteria to identify suitable environments
or surfaces. High salt stress may inhibit flagellate-mediated movement
by increasing intracellular signaling molecules, while promoting EPS
production in the biofilm, thus promoting adhesion process in the early
stage of HNR biofilm formation. Once biofilm formation is initiated, an
increase in c-di-GMP levels is triggered through the action of phospho­
diesterases such as YhjH. Elevated c-di-GMP levels activate YcgR, which
interacts with the flagellar rotor, resulting in decreased rotation speed
and increased direction reversals. Concurrently, c-di-GMP activation of
CsgD stimulates curli biosynthesis while inhibiting the expression of
flagellar genes. Moreover, as a global regulator exerting post-
transcriptional control, CsrA directly binds to mRNA, regulating the
transcription of flhDC. CsrA also influences the expression of ß-1,6-poly-
N-acetylglucosamine (PGA) polysaccharide synthesis operon by binding
to mRNA and obstructing the ribosome-binding site. Another potential
factor involved in motility inhibition is the Rcs phosphorelay system.
The RcsB protein, in conjunction with the accessory protein RcsA, ac­
tivates the expression of colanic acid synthesis genes while directly
repressing the flhDC promoter, consequently impeding motility. As the
biofilm matures, the accumulation of post-transcriptional regulators,
such as CsrA, may lead to the reactivation of motility, suppression of
matrix expression, and ultimately facilitate cellular dispersal, initiating
a new round of biofilm formation.
Upon exposure to high salinity, the activation of the biofilm-related
motility regulation mechanism in HNR proves advantageous in coun­
tering high salt stress. This activation facilitates bacterial aggregation,
and adhesion, and expedites biofilm formation. Notably, these findings
align with the observed decrease in motility and the increase in EPS
content documented in this experiment.
3.4.3. Other regulation
After 40 g/L NaCl stress, the nitrogen metabolism-related genes of
HNR (map00910), narGHI, nasAB, and nirBD were significantly down-
regulated compared with the control (Table S3), and the proteins
encoded by these genes were responsible for the reduction of nitrate
nitrogen to nitrous hydrogen and ammonia nitrogen. These results may
indicate that high salinity stress may inhibit the transcription of genes
related to nitrogen removal, thereby affecting the efficiency of nitrogen
removal of HNR. Previous study found that high salinity negatively af­
fects nitrogen removal and sludge settling performance by hindering
microbial metabolism and reducing the transport of compounds be­
tween the medium and cells (Chen et al., 2022).
KEGG enrichment results also showed that HNR necrotizing
apoptotic genes (CypA, Hsp90, GLUL, and PYGL) were upregulated
(Fig. S5). These adjustments also explained the increase in the number of
bacterial premature apoptosis and deaths as a result of the ratio of live to
dead bacteria exposed to high salinities.
3.5. Implications
Under short-term salinity stress, bacteria perform a series of physi­
ological responses and movement behaviors. Limited by the small size
and lower technical resolution of bacteria, previous studies of bacterial
movement and physiological characteristics are usually performed at
the population level (Arora et al., 2007; Colin et al., 2021). In this study,
horizontal and vertical movements of individual bacteria were observed
and analyzed through SPRI, and related physiological characteristics
were also investigated thoroughly, revealing information that begins to
bridge the gap between the behavior of individual cells and populations.
In this study, we place particular emphasis on the lesser understood
aspect of bacterial chemotaxis driven by flagellar motility, focusing on
its physiological relevance for both individual bacterial cells and bac­
terial populations. It is noteworthy that flagellar motility and chemo­
taxis within the same bacterial species can serve multiple roles
contingent on varying physiological and environmental conditions
(Colin et al., 2021). One prominent function of bacterial chemotaxis is
the exploration of nutrient sources and the identification of optimal
niches for growth, which may account for the preferences in chemo
effector selection and flagellar gene regulation. Moreover, chemotaxis
can generally enhance the efficiency of environmental colonization by
motile bacteria, which involves intricate interactions between individ­
ual and collective behaviors and necessitates trade-offs between growth

8
M. Cheng et al.
and motility (Colin et al., 2021). In this study, high salinity stress acti­
vated the biofilm-related motility regulation mechanism in HNR,
decreased the flagellar motility capacity of HNR, and increased EPS
secretion, inducing bacterial aggregation and adhesion, which was
conducive to biofilm formation of HNR and proved advantageous in
countering high salt stress.
The adaptability of bacteria to environmental stimulation is based on
a variety of regulatory systems operating through the selectivity
expression of related genes (Adhikary et al., 2020). Such genetic di­
versity likely facilitates the evolutionary adaptation of the chemotaxis
pathway to new environmental niches with different chemo effector
requirements (Colin et al., 2021). In this study, the genetic diversity of
HNR in biofilm-related motility regulation makes HNR populations
more adaptable and stable to salinity stress or other adverse
environment.
4. Conclusion
As salinity increased, electron transport activity and ATP content
decreased, leading to a reduction in the rotation velocity and vibration
amplitude of HNR at the single-cell level. High salinity stress down-
regulated genes involved in ABC transporters and activated the
biofilm-related motility regulation mechanism in HNR, resulting in a
further decrease in flagellar motility capacity and an increase in EPS
secretion. These responses were advantageous for biofilm formation.
Moreover, the genetic diversity associated with biofilm-related motility
regulation in HNR enhanced the adaptability and stability of the HNR
populations to salinity stress.
CRediT authorship contribution statement
Meng Cheng: Writing – original draft. Hui-Min Fu: Writing – orig­
inal draft, Writing – review & editing. Zheng Mao: Methodology,
Investigation. Peng Yan: Conceptualization. Xun Weng: Investigation.
Teng-Fei Ma: Methodology. Xiao-Wei Xu: Conceptualization. Jin-Song
Guo: Validation. Fang Fang: Validation. You-Peng Chen: Conceptu­
alization, Funding acquisition, Project administration, Resources.
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.
Acknowledgments
This research was financially supported by the National Natural
Science Foundation of China (21876016 and 42207022), the Funda­
mental Research Funds for the Central Universities (2023CDJXY-034),
the Science and Technology Research Program of the Chongqing
Municipal Education Commission (KJQN202000846 and
KJQN202300813), Chongqing Natural Science Foundation project
(CSTB2022NSCQ-MSX1121), and the Chongqing Technology and Busi­
ness University Research Program (2056021 and 2356010).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2024.170002.
9
Science of the Total Environment 914 (2024) 170002
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