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Environmental Pollution 287 (2021) 117644
Available online 23 June 2021
0269-7491/© 2021 Elsevier Ltd. All rights reserved.
Algicidal mechanism of Raoultella ornithinolytica against Microcystis
aeruginosa: Antioxidant response, photosynthetic system damage and
microcystin degradation
☆
Dongpeng Li
a
, Xin Kang
a
, Linglong Chu
a
, Yifei Wang
a
, Xinshan Song
a
, Xiaoxiang Zhao
a
,
Xin Cao
a
,
b
,
*
a
Textile Pollution Controlling Engineering Center of Ministry of Environmental Protection, College of Environmental Science and Engineering, Donghua University,
Shanghai, 201620, China
b
Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, China
ARTICLE INFO
Keywords:
Raoultella ornithinolytica
Algicidal mechanism
Photosynthetic system
MC-LR degradation
Chlorogenic acid
ABSTRACT
Water eutrophication caused by harmful algal blooms (HABs) occurs worldwide. It causes huge economic losses
and has serious and potentially life-threatening effects on human health. In this study, the bacterium Raoultella
sp. S1 with high algicidal efciency against the harmful algae Microcystis aeruginosa was isolated from eutrophic
water. The results showed that Raoultella sp. S1 initially occulated the algae, causing the cells to sediment
within 180 min and then secreted soluble algicidal substances that killed the algal cells completely within 72 h.
The algicidal activity was stable across the temperature range −85.0 to 85.0 ◦C and across the pH range
3.00–11.00. Scanning electron microscopy (SEM) revealed the crumpling and fragmentation of cells algal cells
during the occulation and lysis stages. The antioxidant system was activated under conditions of oxidative
stress, causing the increased antioxidant enzymes activities. Meanwhile, the oxidative stress response triggered
by the algicidal substances markedly increased the malondialdehyde (MDA) and glutathione (GSH) content. We
investigated the content of Chl-a and the relative expression levels of genes related to photosynthesis, verifying
that the algicidal compounds attack the photosynthetic system by degrading the photosynthetic pigment and
inhibiting the expression of key genes. Also, the results of photosynthetic efciency and relative electric transport
rate conrmed that the photosynthetic system in algal cells was severely damaged within 24 h. The algicidal
effect of Raoultella sp. S1 against Microcystis aeruginosa was evaluated by analyzing the physiological response
and photosynthetic system impairment of the algal cells. The concentration of microcystin-LR (MC-LR) slightly
increased during the process of algal cells ruptured, and then decreased below its initial level due to the
biodegradation of Raoultella sp. S1. To further investigate the algicidal mechanism of Raoultella sp. S1, the main
components in the cell-free supernatant was analyzed by UHPLC-TOF-MS. Several low-molecular-weight organic
acids might be responsible for the algicidal activity of Raoultella sp. S1. It is concluded that Raoultella sp. S1 has
the potential to control Microcystis aeruginosa blooms.
1. Introduction
Increasing economic development and the accompanying rise in
industrialization have led to the discharge of large amounts of waste-
water containing nitrogen and phosphorus into natural water bodies,
causing eutrophication worldwide in recent years (Huang et al., 2014;
O’Neil et al., 2012). Harmful algal blooms (HABs) have become a serious
threat to ecological environment, human healthy and economy during
this time (Saraf et al., 2018). As one of the most common harmful algae
species, Microcystis aeruginosa is the primary target for control in
eutrophic water (Mohamed et al., 2014; Pivokonsky et al., 2014). To
date, chemical, physical, and biological techniques have been performed
to eliminate the negative effects of HABs (Pal et al., 2020). The
physico-chemical control methods include UV irradiation (Dai et al.,
☆
This paper has been recommended for acceptance by Sarah Harmon.
* Corresponding author. College of Environmental Science and Engineering, Donghua University, Textile Pollution Controlling Engineering Center of Ministry of
Environmental Protection, Shanghai, 201620, China.
E-mail address: caoxin@dhu.edu.cn (X. Cao).
Contents lists available at ScienceDirect
Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
https://doi.org/10.1016/j.envpol.2021.117644
Received 9 February 2021; Received in revised form 21 May 2021; Accepted 21 June 2021
Environmental Pollution 287 (2021) 117644
2
2020; Xing et al., 2019), ultra-sonication (Jantasrirad et al., 2021;
Kurokawa et al., 2016) and algicides such as copper sulphate and
chlorination (Chen et al., 2021; Chen et al., 2020). However, these
measures are hardly applied in a large scale for their high cost and
possible secondary pollution (Kim et al., 2007; Zhou et al., 2010).
Therefore, the development of non-polluting and economical methods of
controlling HABs is of both theoretical and practical importance. Bio-
logical methods such as the use of protozoans (Rose et al., 2016), plants
(Qin et al., 2016; Zhang et al., 2019) or microorganisms (Choi et al.,
2005; Sun et al., 2018) are considered to be efcient and sustainable
approaches. In recent decades, studies on the biological control of HABs
have attracted widespread notice, particularly with regarding to mi-
croorganisms that exhibit algicidal activity (Guo et al., 2016; Pal et al.,
2020). A variety of algicidal bacterial species, such as Acinetobacter (Yi
et al., 2015), Alcaligenes (Manage et al., 2000), Bacillus (Jeong et al.,
2003; Yu et al., 2014), Pseudoalteromonas (MITSUTANI et al., 2001),
Pseudomonas (Ren et al., 2009; Wang et al., 2005) and Vibrio (Li et al.,
2014a), have been found in different natural environments and their
algicidal mechanism has been studied.
The mechanism of bacterial algicidal activity can involve either a
direct attack on algal cells by bacteria cells (Chen et al., 2017), or lysis of
the agal cells as a result of bacterial secretion of algicidal compounds
such as intracellular enzymes, peptides, biosurfactants, antibiotics and
other bioactive compounds (Cho, 2012; Park et al., 2011; Yu et al.,
2019). Compared with the direct attack, the lytic activity of bacteria
against algae has been the focus of increasing interest. For instance,
Salvia miltiorrhiza releases algicidal substance neo-przewaquinone A,
which is active against M. aeruginosa (Zhang et al., 2013), and an algi-
cidal strain of Streptomyces amritsarensis strain kills M. aeruginosa by
secreting stable bioactive compounds (Yu et al., 2019). In order to
achieve constant stable lysis of algae, a continuous fermentation was
performed using Vibrio species to generate algicidal activity. This
fermentation allowed the mass production of substances by microor-
ganisms that cause algal cell lysis (Wang et al., 2020). Previous studies
described that algicidal substances induce a physiological response and
morphological changes in algal cells, thus leading to cell death (Zhang
et al., 2020b). During lysis the algicidal metabolites affect oxidative
stress, protein levels and antioxidant enzyme activity (Zhou et al.,
2016). However, the changes that occur in the photosynthetic system
during algal cell death have not been studied extensively. Photosyn-
thesis is the main physiological process by which plants and certain
other organisms generate chemical energy, and is therefore essential for
cell growth and reproduction. Consequently, it is signicant to gain an
understanding of the algicidal mechanism underlying the effect of toxic
substances by analyzing the response of the photosynthetic system and
of photosynthetic efciency bioactive metabolites. Hahella sp. KA22 has
been reported to secret prodigiosin, which impairs photosynthesis by
decaying the content of Chlorophyll a (Chl-a) and disturbing the
photosynthetic system of M. aeruginosa (Yang et al., 2017). The algicide
released by the Sultobacter Porphyrae ZFX1 inhibits the photosynthetic
capacity of Prorocentrum donghaiense and simultaneously degrades the
Chl-a (Zhang et al., 2020a). These ndings suggest that photosynthetic
system damage caused by algicidal bacteria plays a signicant role in the
process of algal lysis. However, the specic relationship between the
inhibition of photosynthesis and cell damage has yet to be explored.
Recent studies have focused on the degradation of MC-LR by microor-
ganisms as well as the removal of harmful algae. PG (a red pigment
prodigiosin) secreted by Serratia marescens LTH-2 down-regulated the
expression of mcyB which is in positive correlation with the synthesis of
MC-LR and inhibited the production of MC-LR (Wei et al., 2020).Yang
etc. isolated an MC-LR degrading bacterium Sphingopyxis sp. YF1 from
Lake Taihu and the biodegradation pathway was identied. Some spe-
cic proteins were found to be responsible for the complete degradation
of MC-LR (Yang et al., 2020). The isolation of microorganisms that
degrade MC-LR while removing harmful algae is of great importance as
it helps to reduce the ecological damage caused by the release of MC-LR.
During the last few decades, the alga M. aeruginosa has caused
massive HABs in aquatic environments, with catastrophic effects on the
local environments and human health, and leading to huge economic
losses. To prevent and control HABs caused by the M. aeruginosa, an
algicidal bacterium Raoultella sp. S1 has been isolated from surface
water of the eutrophic Jingyue Lake, Donghua University, where the
harmful algae thrived. The process of algal lysis by Raoultella sp. S1
consisted of two stages. First, bioactive compounds released by the
bacterium occulated the alga, causing the cells to sink to the bottom.
The algal cells remained structurally intact. During the second stage the
algal cells began to rupture and die. To determine the mechanism un-
derlying algicidal effects and assay the algicidal process, we investigated
its efciency, activity and stability and identied the response of the
antioxidant defense system M. aeruginosa. In addition, the algal cell
death process was identied by scanning electron microscopy (SEM),
and the photosystem damage was analyzed by measuring the Chl-a
content, photosynthetic efciency and the relative electric transport
rate. In order to further verify the relationship between algal cell death
and photosynthetic system damage, the expression level of key genes
related to photosynthesis were measured. A comprehensive analysis of
the Chl-a content, photosynthetic efciency and expression level of key
genes indicated the response of the photosynthetic system of the
M. aeruginosa cells under oxidative stress. The release and degradation of
MC-LR during the occulation and lysis stages was also investigated, as
this is of great signicance for the control and treatment of HABs
without causing adverse effects. At last, possible algicidal substances
were detected by UHPLC-TOF-MS. Due to the occulation properties,
high algicidal efciency and degradation of MC-LR, Raoultella sp. S1 has
incomparable superiority as algicidal microorganism. The results ob-
tained from this study of the algicidal mechanism of Raoultella sp. S1 can
contribute to the development of new bioassay methods for exploring
the algicidal mechanism of microorganisms in eutrophic water and
HABs.
2. Materials and methods
2.1. Algae culture
M. aeruginosa FACHB-905 was purchased from the Freshwater Algae
Culture Collection at the Institute of Hydrobiology (Wuhan, China). The
algal strain was cultured in sterile BG-11 medium (Rippka et al., 1979).
The algal strain was cultured at 25 ◦C, with a 12 h:12 h (light: dark) cycle
and 2000 lux of illumination intensity, and manually shaken twice a
day. The strain was cultured to reach the exponential growth phase
before it was used for experiments. To ensure that only exponential
growth phase cultures were used, the strain was transferred once a week.
2.2. Isolation and cultivation of the bacterium Raoultella sp.
Raoultella sp. S1 was isolated from surface water of Jingyue Lake at
Donghua University (Shanghai, PRC) during a M. aeruginosa bloom in
the summer of 2018, and was characterized by 16 S rDNA gene sequence
analysis. In brief, the eutrophic water was diluted 10 000 to 100 000
times and spread evenly on the LB solid medium. After the colonies grow
on the surface of the medium, we selected multiple colonies of different
forms to test their algicidal rate, respectively. Then the single bacteria S1
with the strongest algicidal efciency was chosen for the following
research. The gene sequences of the strain were submitted to the BLAST
database and EzBioCloud database for sequence alignment. Then the
phylogenetic trees were constructed with the neighbor-joining method
with bootstrap support of 1000 replicates using MEGA 7 software. The
sequencing work was commissioned by Shanghai Meiji Biomedical
Technology Co., Ltd, and the strain S1 was characterized as Raoultella sp.
Raoultella sp. S1 was cultured in modied mineral medium as
described by (Sun et al., 2016) containing glucose 6 g, K
2
HPO
4
⋅3H
2
O
2 g, MgSO
4
⋅7H
2
O 0.3 g, (NH
4
)
2
SO
4
0.2 g, CaCl
2
0.03 g, and NaCl 0.9 g, in
D. Li et al.
Environmental Pollution 287 (2021) 117644
3
1 L of distilled water at pH 6.8–7.2 and 35 ◦C with rotation at 180 rpm
for 36 h for fermentation. Bacterial growth was determined by
measuring the absorbance value of bacterial cultures at 600 nm every
2 h using a spectrophotometer (UV–1800PC, MAPADA, China). Strain
cultures in different growth phases were added to the algal cultures to
test their the algicidal efciency.
2.3. Determination of algicidal mechanism of strain S1
After cultivation of the bacterium to the stationary phase, the culture
was centrifuged (10 000 rpm, 4 ◦C, 10 min) to obtain the supernatant
and the precipitated cells. The supernatant was ltered through a
0.22
μ
m membrane lter to obtain cell-free supernatant. The precipi-
tated cells were collected and washed with sterilized BG-11 medium for
3 times, and were then resuspended in BG-11 medium and labelled as
bacterial cells. Either the bacterial cultures, cell-free supernatant or
bacterial cells were added (at a concentration 5%, v/v) to the algal
cultures. In addition, the same volume of sterilized mineral medium was
added to algal cultures as a control. In this study, the algicidal effects
were quantied by measuring the Chl-a concentration. Algal cultures
were shaken evenly after co-culture for 24, 48- and 72- h and were then
centrifuged (8000 rpm, 5min) to collect the cells. The cells were sus-
pended in 1 mL of deionized water and the suspension was boiled for
3 min. Then, after cooling to room temperature, 4 mL of acetone were
added to the suspension to extract Chl-a. After extraction for 30 min in
darkness, the mixture was centrifuged again (8000 rpm, 5 min). The Chl-
a content was investigated by measuring the absorbance value of the
supernatant at 665 nm and 645 nm using a spectrophotometer
(UP–1800PC, MAPADA, China), and then calculating the content based
on the method of (Marr et al., 1995). The algicidal ratio was determined
according to the following formulae (1):
ρ
(Chl −a) (mg /L) = 12.7×A665 −2.69 ×A645 (1)
Ar (%) = (
ρ
Chla1 −
ρ
Chla2)
ρ
Chla1 ×100% (2)
where Ar represents the algicidal effect of compounds secreted by the
bacteria Raoultella sp. S1 against M. aeruginosa,
ρ
Chla1 represent the
Chl-a content of the control group and
ρ
Chla2 represent the Chl-a con-
tent of the treatment group.
2.4. Algicidal activity and occulation
To quantify the algicidal activity of Raoultella sp. S1 supernatant, 1%,
3%, 5% and 7% (by volume) of the supernatant were added to the algal
cultures. The same volume of sterile mineral medium was added to algal
cultures as a control. After 24, 48 and 72 h the algal cultures were
shaken evenly prior to measurement of the Chl-a content. The following
formulae was used to calculate the algicidal activity.
Similarly, the occulation ratio was calculated by measuring the Chl-
a content of cultures at a depth of 1 cm below the surface of the liquid
after 30, 60, 90, 120, 150 and 180 min. The occulation ratio was
calculated as follows:
Fr (%) = (Cc −Ct)/CC×100% (3)
where Fr represents the occulation ratio of algal cells, and Cc and Ct
represent the Chl-a content of the supernatant from the control and
treatment groups, respectively.
2.5. SEM observations of algal cell morphology
M. aeruginosa cells were treated with 5% cell-free supernatant for 6,
36 and 72 h, and were then harvested by centrifugation (8000 rpm,
5 min). The collected cells were xed overnight at 4 ◦C in 0.1 M
phosphate-buffered saline (pH =7.4) containing 2.5% glutaraldehyde
(v/v), and were then washed with PBS for 3 times. Finally, the cells were
dehydrated using ethanol solution of different concentrations (30, 50,
70, 90 and 100%) and the xed cells were examined by SEM (Quanta
250, FEI, Czech Republic).
2.6. Measurement of zeta potential and pH
Five percent cell-free supernatant was added to the algal cultures,
and zeta potential and pH were then measured every 12 h. The surface
charge of the algal cells was determined using a nanoparticle size dis-
tribution analyzer (Brookhaven Instruments Corp., USA), and pH was
measured using a pH probe (ST3100, Ohaus, Shanghai). From 12 to
72 h, the zeta potential of the algal cell surface was determined using a
Zetasizer with 3 mL samples of co-cultivation cultures. The temperature
sampling probe was used to detect the ambient temperature of liquid
samples automatically and continuously. A computer was used to collect
and process the data automatically for calculation of the zeta potential.
Each sample was measured three times and the average zeta potential
and pH values were determined.
2.7. Characterization of algicidal substances
To determine the temperature stability of the algicidal substances,
the cell-free supernatant was prepared and incubated for 30 min at 0, 35,
45, 55, 75, 85 and 100 ◦C in a water bath, or at −85 ◦C in a freezer, and
was then returned to room temperature (25 ◦C). To measure the pH
stability, the pH of the cell-free supernatant was adjusted to 3, 5, 7, 9 or
11 using hydrochloric acid or sodium hydroxide, maintained at that
value for 2 h. Then the supernatant was reset to the initial pH value. All
of these treated cell-free supernatants were added to algal cultures at 5%
(v/v) concentration. In addition, the same volume of sterile mineral
medium was added to algal cultures as a control. The algicidal ratios
were calculated every 24 h by measuring the Chl-a content of the control
and treatment groups using the method described in Section 2.3.
2.8. Protein, GSH and MDA content, and antioxidant enzyme activity
The algal cultures were treated with Raoultella sp. S1 supernatant
(5%, v/v) for 12, 24, 36, 48, 60 or 72 h. The same volume of sterile
mineral medium was added to algal cultures as a control. The algal
cultures were then centrifuged (5000 rpm, 10min). After the superna-
tant had been discarded, the algal cells were washed with sterilized PBS
for 3 times. The algal cells were then resuspended in PBS and ultra-
sonicated in an ice water bath 100 times at 5 s intervals at 100 W using
an Ultrasonic Cell Disruption System (Ningbo Scientiz Biotechnology
Co., Ltd, Ningbo, China). The algal cell debris was discarded after
centrifugation (12 000 rpm, 10 min, 4 ◦C). The resulting supernatant
was the crude enzyme solution, which was used to determine the con-
tent of the protein, malondialdehyde (MDA), and glutathione (GSH). In
addition, superoxide dismutase (SOD), peroxidase (POD), catalase
(CAT) enzyme activities were assayed. The GSH content and activities of
SOD, POD and CAT were determined using diagnostic reagent kits
(Jiancheng Bioengineering Institute, Nanjing, China). The protein con-
tent was determined using Coomassie brilliant blue staining as described
by (Zhang et al., 2020b). A modied thiobarbituric
acid-malondialdehyde (TBA-MDA) assay was used to measure the MDA
content according to (Song et al., 2014), using a spectrophotometer
(UV–1800PC, MAPADA, China).
2.9. The expression level of key genes related to photosynthesis
To measure the expression level of key genes, M. aeruginosa cells
were treated with the bacterial supernatant (5% v/v) for 12, 36 and 48 h
and then collected by centrifugation (10 000 rpm, 10 min, 4 ◦C). Total
RNA was collected using a Plant RNA Extraction Kit (MJYHIVD, Meiji,
，Shanghai）), and cDNA was obtained using the HiScript Q RT
D. Li et al.
Environmental Pollution 287 (2021) 117644
4
SuperMix for qPCR (with gDNA wiper) (Vazyme, Nanjing). Real-time
quantitative PCR (RT-qPCR) was performed using a Real-time PCR
System (ABI7500, Applied Biosystems, USA) with ChamQ SYBR Color
qPCR Master Mix (2X) (Vazyme, Nanjing). The primers which were used
as an internal reference gene are provided in Table S1. The PCR con-
ditions consisted of 1 cycle of 95 ◦C for 5 min, 35 cycles of 50 ◦C for 30 s
and 72 ◦C for 60 s, and then raising the temperature from 60 ◦C to 95 ◦C.
All cDNA samples were run in triplicate. The relative transcription levels
of key genes were calculated by the 2-ΔΔCt method (Pfaf, 2001). Ac-
cording to this formula, Ct (the cycle threshold) represents the cycle
number. ΔCt represents the difference in Ct values between the internal
reference gene and gene of interest, and ΔΔCt represents the difference
between the ΔCt values of the control group and treatment group.
2.10. Determination of chl-a content, Fv/Fm and rETR
To assay the damage to photosynthetic system, the M. aeruginosa
cells were treated with the cell-free supernatant (5%, v/v). The relative
electron transport rate (rETR) and the maximum quantum yield (Fv/Fm)
of PSII and were measured on a pulse-amplitude modulation uorom-
eter (PHYTO-PAM-II, WALZ, Germany). Chl-a content was measured
using the method described in Section 2.3. Algal cells had been incu-
bated in the dark for 15 min before the Fv/Fm was measured.
2.11. Detection of microcystin-LR (MC-LR)
To assay the release and degradation of MC-LR during the process of
occulation and destruction of algal cells, the Raoultella sp. S1 culture
(5%, v/v) was added to algal cultures. The same volume of mineral
medium was also added to algal cultures as a control. Then 5 mL samples
were collected and centrifugated (8000 rpm, 10 min) and supernatants
were obtained at 6, 12, 24, 48 and 72 h determination. The concentra-
tion of MC-LR was measured using an ELISA kit (Meimian Biotech-
nology, Jiangsu, China).
2.12. Algicidal substances analysis
The bacterial culture was ltered through a membrane lter (pore
size 0.22
μ
m). An Agilent UHPLC 1290II couple with a time-of-light
mass spectral system 6545 A was used for chemical analysis. The mo-
bile phase A was =0.1% formic acid in water, and B was =0.1% formic
in acetonitrile. The sample was washed through a column (Agilent 300
Extend-C-18, 4.6 ×150 mm, 3.5
μ
m) according to a time and ratio of
0–2 min 2% of B, which was increased to 100% at 40min, with a ow
speed of 0.35 mL/min.
The parameters of the ESI source consisted of a drying gas temper-
ature of 300 ◦C at a ow speed of 6 L/min, while the nebulizer pressure
was 25 Psi, the sheath gas temperature was 320 ◦C at a ow speed of
11 L/min, and the Vcap 3500 V nozzle voltage was 500 V. For mass TOF,
the fragmentor was 150 V, the skimmer was 65 V, and Oct RF Vpp was
750 V. Reference mass correction was set over the whole analyzed
course as 121.0508 and 922.0098 sending to auto recalibration.
2.13. Statistical analysis
We performed each experiment in triplicate and the data were
analyzed using OriginPro 2020 software, and the signicance of differ-
ences was tested using the SPSS 26.0 for windows.
Fig. 1. Algicidal process observed by scanning electron micrographs. M. aeruginosa cells were treated with Raoultella sp. S1 cell-free supernatant for (a) 0 h, (b) 6 h,
(c) 36 h and (d) 72 h. The damaged algal cells are marked by white arrows.
D. Li et al.
Environmental Pollution 287 (2021) 117644
5
3. Results and discussion
3.1. Bacterial growth phase and algicidal efciency of strain S1
The phylogenetic tree analysis categorized strain S1 as Raoultella
ornithinolytica (Fig. S1). We use Raoultella sp. S1 to refer to this strain in
the following. As shown in Fig. S2, the exponential growth phase of
Raoultella sp. S1 started after 3 h of fermentation, and lasted for 12 h.
During the lag and exponential growth phases, strain S1 cultures had
only weak algicidal activity, and death of 1.0% and 15.6%, respectively,
of the algal cells was observed. During the stationary growth phase, from
15 h to 36 h, the strain secreted sufcient fermentation products to exert
a strong algicidal effect (death of nearly 100% of algal cells). Therefore
the decision was taken to harvest after 36 h of fermentation for the
following experiment.
3.2. Algicidal action of Raoultella sp. S1 supernatant against M.
aeruginosa
To investigate the algicidal action of the cell-free supernatant against
M. aeruginosa, the morphological changes in the algal cells during lysis
were monitored by SEM observation. The treatment resulted in occu-
lation followed by lysis. As Fig. 2a shows, the control M. aeruginosa cells
were plump and spherical with an intact cell structure and smooth
surface. After treatment for 6 h, most of the algal cells settled at the
bottom of the vessel. In addition, the cells started to become shrunken
and deformed as shown in Fig. 1b. The sedimentation was followed by
algicidal action as the treatment time increased. After 36 h of exposure
to the supernatant, rupture and death of the algal cells began to occur.
During the lysis stage the cells swelled and adhered to each other, as
shown in Fig. 1c. Some algal cells showed loss of membrane integrity
and broke down into cell fragments. After 72 h of treatment (Fig. 1d)
death of almost all the algal cells had occurred and no intact cell
structures were visible. Only algal cell debris remained, which suggested
that the M. aeruginosa cells had been killed by the cell-free supernatant,
having remained structurally intact during the occulation stage and
then ruptured in the lysis stage.
3.3. Flocculation efciency of Raoultella sp. S1 and changes of zeta
potential and pH
Raoultella sp. S1 supernatant caused the algal cells to aggregate into
ocs at the bottom of the culture vessels (Fig.S2). With increasing vol-
ume of added supernatant and treatment time, the occulation ratio rose
signicantly. As shown in Figs. 2a, 1% supernatant treatments resulted
in a lower occulation efciency (43.7%) than in the groups that were
treated with 3% or 5% supernatant. The occulation efciency of 3%
and 5% supernatant (86.2% and 97.6%, respectively, within 3 h), was
found to be statistically signicant (P <0.05). Flocculation of algae
helps to reduce nutrient levels by removing algal debris, thereby sepa-
rating the algal biomass from eutrophic water.
The addition of the supernatant resulted in changes in the zeta po-
tential of the algal surface from an initial value of −30 mV to −10 mV
after 72 h of treatment. The zeta potential values for control algal cul-
tures (without addition of supernatant) were generally negative. During
treatment, the zeta potential of algal cultures continued to increase, and
the absolute value of zeta potential decreased continuously. In general,
the absolute value of zeta potential value reects the stability of algal
cultures. If this value is low, the algal cells tend to aggregate. Clearly the
addition of the supernatant leads to occulation by increasing the
attractive forces, causing the cells to clump together. The use of chem-
ical occulants for the removal of algae has been described in a previous
study (Sun et al., 2012), but there are only a few reports of biologically
active substances causing both occulation and algae lysis. The explo-
ration of biological occulants that also have algicidal activity offers a
potential new approach to the treatment of HABs.
Algal cultures to which supernatant had not been added had a pH of
9.9. The strain supernatant is acidic (pH =3.8), and as anticipated the
pH of the algal cultures decreased (to pH 5.0) after addition of the su-
pernatant. The positive charge of the acidic supernatant neutralizes the
negative charge on the surface of the algal cells, causing them to oc-
culate (Nasser and James, 2006).The pH of the algal cultures then
increased to a nal value of 7.8, when cells death occurred at 72 h. Eq.
(4) shows that photosynthetic cyanobacteria generates net alkalinity by
consuming a weak base (bicarbonate) and producing a strong base
(hydroxyl ions) (Johnson and Hallberg, 2005). Hence, the presence of
living algal cells causes the pH of algal cultures to rise.
6HCO−
3(aq) + 6H20→C6H12O6+6O2+6OH−(4)
In brief, the addition of the acidic supernatant increased the pH value
of the cultures while neutralizing the negative charge on the surface of
the algae cells and increased the zeta potential at the same time. As
treatment time increased, the algae cells died, and the algal cultures
cannot recover to a stable state. During this period, the alkaline sub-
stances produced by the metabolism of algae kept the pH value
increasing.
Fig. 2. Flocculation rate and changes in zeta potential and pH of M. aeruginosa cultures treated with Raoultella sp. S1 supernatant. (a) Flocculation rate for different
concentrations of supernatant and (b) changes of zeta potential and pH during the algae-lysing process. The values represent the means of parallels ±SDs (n =3).
Different letters represent signicant differences between treatment groups at the same time point (P <0.05).
D. Li et al.
Environmental Pollution 287 (2021) 117644
6
3.4. Algicidal mode, activity and stability of Raoultella sp. S1
Untreated cultures, cell-free supernatant and bacterial cells and of
strain S1 were added at a concentration of 5% (v/v) to the algal cultures,
respectively, in order to determine the algicidal mode. The results are
shown in Fig. 3a. Both the cell-free supernatant and the bacterial cul-
tures exhibited strong algicidal activity which caused algicidal effects
(92.4% and 96.2%, respectively, after treatment for 72 h). In contrast,
the bacterial cells of Raoultella sp. S1 displayed negligible algicidal ac-
tivity (2.0% after 72 h). This nding demonstrates that the physiological
activity of the algal cells was not affected by the bacteria cells, and
therefore the algal cells continue to grow after treatment. These results
indicate that the algicidal bacteria destroy algal cells by releasing
bioactive compounds into the surrounding solution, rather than by
making direct contact with the M. aeruginosa cells. Consequently, sub-
sequent experiments were conducted using the cell-free supernatant to
further explore the stability and the mechanism of algicidal substances.
Fig. 3b shows data for the algicidal activity of cell-free supernatant of
Raoultella sp. S1 against M. aeruginosa. The algicidal efciency are
related to the concentration of supernatant and to the treatment time,
with increasing concentration and treatment time enhancing the algi-
cidal activity. The signicance test values indicate that there was no
signicant difference between the 5% and 7% supernatant addition
groups (p >0.05). From 24 to 72 h, the 5% and 7% supernatant
treatment groups had a strong algicidal ratio, with values of 95.9% and
97.6%, respectively. The dead algal cells sedimented as white ocs at the
bottom of the culture vessel (Fig. S4). However, the 1% treatment group
showed little algicidal activity (4.2%). The 3% treatment group
exhibited a lower algicidal activity than the 5% and 7% treatment
groups. Only 55.9% algal cells died after 72 h of exposure in the 3%
treatment groups. However, if the supernatant concentration exceeded
5%, the algicidal activity did not increased further. On the basis of these
data and the occulation efciency results, 5% cell-free supernatant was
chosen as the optimal dosage for subsequent experiments.
To investigate the temperature and pH tolerance of the algicidal
substances secreted by Raoultella sp. S1, the supernatant was exposed to
a range of temperatures conditions or pH values and then added to the
M. aeruginosa cultures. As Fig. 3c shows, the algicidal efciency of the
cell-free supernatant was stable within the pH range 3.00–11.00. The
algicidal effect ranged over 90%. This result demonstrates that the
algicidal substances are acid and alkaline resistant and that their algi-
cidal activity are not affected by changes of the pH. The data in Fig. 3d
show that the algicidal effect exceeded 90% if the supernatant was
subjected to temperatures in the range of −85.0 to 85.0 ◦C. However, it
decreased to 35.1% after incubation at 100.0 ◦C for 2 h. This result
suggests that the algicidal substances secreted by S1 strain can be stored
at low temperatures, but high temperatures will markedly impair their
activities. Previous studies have shown that the algicidal
Fig. 3. Algicidal activities and algicidal modes of Raoultella sp. S1 supernatant against M. aeruginosa. (a) Algicidal modes of Raoultella sp. S1 supernatant and (b)
algicidal activities of different concentrations of Raoultella sp. S1supernatant. (c) pH stability and (d) thermal stability. The values represent the means of parallels
±SD (n =3). Different letters across treatments represent signicant differences at P <0.05.
D. Li et al.
Environmental Pollution 287 (2021) 117644
7
microorganisms released more than one kind of algicidal substance
(Xuan et al., 2017; Yu et al., 2019).Therefore it is possible that some of
the algicidal substances synthesized by Raoultella sp. S1 were inacti-
vated by high temperatures, but some other bioactive compounds
maintained their algicidal activity and caused algal cell death. The wide
temperature and pH tolerance of the supernatant would favor its po-
tential application in the control of the HABs.
3.4.1. Effects of Raoultella sp. S1 supernatant on proteins, lipids and the
antioxidant defense response
Proteins and lipids, as biological macromolecules, have a vital role in
the physiological activity of cells. Changes in proteins and lipids content
reect the physiological status of algal cells (Zhang et al., 2020b). As
Fig. 4a shows, the initial protein content of the treatment group and the
control group were 0.28 mg/mL. Due to growth of the algae, the protein
level in the control group increased gradually to 0.6 mg/mL. In contrast,
the protein content of the treatment group decreased slowly from 12 h to
72 h, revealing that the growth of algal cells was inhibited. After a
treatment for 72 h almost all of algal cells had died, as was reected by
the signicant decline in protein content (to below 0.1
μ
mgmL).
Malondialdehyde (MDA) is produced when free oxygen radicals
attack unsaturated fatty acid in cell membranes. The accumulation of
MDA exacerbates the damage caused to the cell membrane. Thus MDA
content reaveals the degree of membrane lipid peroxidation, and
Fig. 4. Changes in (a) total protein content and (b) MDA content of M. aeruginosa cells after treatment with Raoultella sp. S1 supernatant. The algal cells were treated
with the Raoultella sp. S1 supernatant and then collected for activity analyses of (c) SOD, (d) POD, (e) CAT and (f) GSH content. The values represent the means of
parallels ±SD (n =3). Different letters represent signicant differences between control and treatment groups at the same time point. (P <0.05).
D. Li et al.
Environmental Pollution 287 (2021) 117644
8
indirectly reects the severity of membrane damage caused to the
membrane system by the supernatant. The data in Fig. 4b show that the
initial MDA content in M. aeruginosa cells was about 0.66 nmol/mg
protein. The MDA content in the treatment group increased to 1.93
nmol/mg protein at 12 h, 2.16 nmol/mg protein at 36 h, and a peak of
3.62 nmol/mg protein at 48 h. It then decreased slightly from 60 h to
72 h. High MDA levels indicate strong lipid peroxidation. The accu-
mulation of MDA damages the membrane structure and impairs the
physiological activities of the cells, thereby accelerating the process of
cell death. Besides, the accumulation of lipid peroxidation products re-
sults in the inhibition of photosynthesis, which plays an important role
in blocking energy acquisition and transport.
The activities of antioxidant enzymes (SOD, POD and CAT) and the
content of the nonenzymatic antioxidant (GSH) were determined to
investigate the antioxidant defense response of M. aeruginosa to the
algicidal supernatant. Algae cells produce reactive oxygen species (ROS)
under adverse external conditions to resist oxidative damage. SOD cat-
alyzes the conversion of superoxide anion radicals to O
2
and H
2
O
2
,
which is less toxic than superoxide radicals. CAT can decompose H
2
O
2
to
form H
2
O and O
2
. POD uses other substances as hydrogen donors to
hydrogenate H
2
O
2
and reduce it to water without generating O
2
. Thus,
POD catalyzes the decomposition of H
2
O
2
while oxidizing other sub-
stances at the same time, such as phenols and amines. Thus the accu-
mulation of harmful H
2
O
2
in algal cells is avoided (Hegedüs et al., 2001;
Mates, 1999). Fig. 4c, d and e show that the enzymatic activities of SOD,
POD and CAT were markedly increased in the treatment groups
(P <0.05) after treatment with the supernatant for 12 h or 24 h, and
reached a maximum after 48 h. Compared with the control values, these
values were 5.5, 2.93 and 2.8-fold higher. A previous study has shown
that the SOD, CAT and POD activities of Prorocentrum donghaiense also
increased signicantly within a short time period after treatment with
the algicidal bacterium Heterosigma akashiwo (Zhang et al., 2020a). It is
worth noting that the SOD and CAT activities in the treatment groups
decreased slightly after 48 h, but still remained higher than those in the
control groups after 72 h. However, the POD activity in the treatment
group remained almost the same as that in the control group. These
results indicate that the antioxidant system was damaged by the effects
of supernatant and consequently failed to produce active enzymes. As
the nonenzymatic antioxidant, GSH can reduce and degrade toxic sub-
stances due to its redox properties. Reduced glutathione (GSH) is
consumed in the process of neutralizing excess ROS to generate oxidized
glutathione (GSSG). Fig. 4f shows that the GSH content increased during
treatment with the supernatant over the period from 12 h to 24 h,
reaching a peak at 24 h. These values were 3.6-fold higher than those for
the control group. After treatment with the supernatant for 36 h, the
GSH content decreased rapidly and remained lower than the control
value. A previous study reported that a large increase in GSH levels
enhances the degree of oxidative stress (Lusia et al., 1994). We therefore
propose that the algicidal supernatant induced the algal cells to produce
more GSH initially. These increased levels GSH led to an increase in the
activity of other antioxidant enzymes. Subsequently, however, con-
sumption of the GSH exceeded its production, due to the high levels of
Fig. 5. Relative expression level of (a) psbA1, (b) psbD1, (c) rbcL of M. aeruginosa after treatment with the Raoultella sp. S1 cell-free supernatant for 12, 36 and 48 h.
The values represent the means of parallels ±SD (n =3).
D. Li et al.
Environmental Pollution 287 (2021) 117644
9
oxidative stress. Liu et al. obtained similar results in a study of the
oxidative stress response of Bacillus licheniformis to M. aeruginosa (Liu
et al., 2019). These changes suggest that the antioxidant defense system
of M. aeruginosa cells was activated to protect the cells from oxidative
damage caused by the algicidal supernatant. Oxidative stress up- or
down-regulates antioxidant-related genes, thereby regulating enzyme
activity and directing the antioxidant system to function (Cui et al.,
2020). Nevertheless, the antioxidant system was not able to resist the
sustained damage and the accumulation of peroxide products such as
MDA. These conditions trigger the structural and functional disruption
of M. aeruginosa cells and ultimately result in membrane system
dysfunction.
3.4.2. Effects of the algicidal supernatant on gene expression of M.
aeruginosa
To conrm the damage to the photosynthetic system of M. aeruginosa
at the genetic level, the expression levels of three key genes, namely
psbA1, psbD1 and rbcL, were measured using RT-qPCR. The psbA1 and
psbD1 genes regulate the synthesis of D1 and D2 proteins, which consist
of the membrane of reaction center of photosystem II (PSII). The rbcL
gene is related to the xation of carbon dioxide. The expression levels of
psbA1 and psbD1 were both downshifted and decreased to 6.76% and
5.85% compared with the control group after treatment with the su-
pernatant for 12 h (Fig. 5a and b), respectively. After treatment for 48 h,
the transcriptional level of psbA1 and psbD1 increased somewhat, to
55.21% and 26.7% compared with the control group. These ndings
indicate that the M. aeruginosa cells were starting to repair the damaged
photosynthetic system. Commensurate with this, expression of the rbcL
gene was markedly suppressed by treatment with the algicidal super-
natant after 12 h, and remained at about 13.97% of the control group to
48 h (Fig. 5c). The transcriptional changes in psbA1, psbD1 and rbcL
indicate that the electron transport chain became affected, so in turn the
CO
2
xation process was inhibited. A similar result was reported by (Yu
et al., 2019), who described that expression of the genes psbA1, psbD1
and rbcL genes were strongly inhibited after 72 h of exposure to a su-
pernatant secreted by Streptomyces amritsarensis. Previous studies have
conrmed that components of bacterial secretions down-regulated the
expression levels of photosynthesis-related genes (Jin et al., 2017; Lin
et al., 2020), while the ROS triggered by the oxidative stress existed
across the plasma membrane and caused damage to the thylakoid
membrane (Knauert and Knauer, 2008). Antibiotics such as strepto-
mycin also restrict the transcription of genes related to photosynthesis in
M. aeruginosa cells (Qian et al., 2012). These negative effects eventually
led to damage of the photosynthetic system and hindered the process of
energy uptake in algal cells. Consequently, we hypothesize that the
algicidal substances in the supernatant blocked the pathway by which
algal cells obtain energy, and thus inhibited the accumulation of nutri-
ents and an associated increase in cyanobacteria biomass.
The recA gene encodes the DNA repairing enzyme recA, which is
involved in the repair of damaged DNA. The expression level of recA was
decreased to 50.85% and 49.38% after 12 and 36 h treatment, respec-
tively, compared with the values for the control group (Fig. S5). This
suggests that the DNA repair system had been severely damaged, and the
DNA repair process was inhibited. A previous study also reported that
substances produced by the algicidal bacterium Bacillus licheniformis
decreased transcription of the recA gene in M. aeruginosa cells (Liu et al.,
2019).
3.4.3. Effects of the algicidal supernatant on the photosynthetic system of
M. aeruginosa
To determine the photosynthetic activity of M. aeruginosa, the rela-
tive electron transport rate (rETR), Chl-a content and maximum quan-
tum yield of photosystem II (Fv/Fm) were measured. The results are
shown in Fig. 6.Fv/Fm value indicates the maximum photosynthetic
efciency in PS II while the rETR reects the photosynthetic rate, which
is the rate of utilization of photons by algal cells. Changes in these values
Fig. 6. Changes in Chl-a content and photosynthetic system damage. Effect of
the Raoultella sp. S1 supernatant on (a) Chl-a content, (b) Fv/Fm and (c) rETR of
M. aeruginosa. The values represent the means of parallels ±SD (n =3).
Different letters represent signicant differences between control and treatment
groups at the same time point. (P <0.05).
D. Li et al.
Environmental Pollution 287 (2021) 117644
10
indicate damage to the photosynthetic system caused by the algicidal
supernatant (Schreiber et al., 1995). The Fv/Fm value was 0.56 for
control (untreated) algal cells. Then the Fv/Fm value decreased to 0.47,
0.36, 0.19 and 0.16, respectively, as the treatment time increased to 3 h,
6 h, 9 h and 12 h. After treatment for 24 h, the Fv/Fm value for
M. aeruginosa was only 0.08 (14.3% of the initial value). The decrease in
Fv/Fm indicates that the photosynthetic system had been seriously
damaged, resulting in inhibition of algal cell photosynthesis. Similarly,
the rETR value of the algal cells decreased signicantly to 64.2% of the
control value after 3 h of treatment, and to 3.9% after 24 h of treatment.
This nding indicates that the electron transport chain within the
thylakoid membrane was blocked. Photosynthetic pigments, such as
Chl-a, are essential for harvesting, capture and utilization of light and
therefore affect the primary photosynthetic reaction (Rowan, 1989). The
Chl-a content decreased gradually from 1.1 mg/L to 0.6 mg/L after 24 h
of treatment. The reduction of Chl-a is closely related to membrane
damage caused by lipid peroxidation (Huang et al., 2012), while it has
been noted that photosynthetic pigments are sensitive and easily
damaged by fungicides and organic xenobiotics (Du et al., 2019; Huang
et al., 2012).This decrease caused impairment of light energy absorption
and conversion. The results presented in Section 3.5.2 of this paper
suggest that the algicidal supernatant downshifted the expression of the
key genes related to photosynthesis. Therefore we suggest that the
photosystem dysfunction was caused by the inhibition of expression of
key genes and the simultaneous decrease in Chl-a content. In the rst
3 h, algal cells began to occulate and sink to the bottom of the vessel
with the Fv/Fm and rETR value dropped signicantly. From this we infer
that the inhibition of photosynthesis might also be related to the rapid
occulation of M. aeruginosa caused by the supernatant, as the aggre-
gation of algal cells reduces their utilization of light. Previous research
has demonstrated that photosynthetic systems are susceptible to adverse
environmental conditions (Takahashi and Murata, 2008; Wang et al.,
2011) such as oxidative stress. Moreover, many studies have conrmed
that the photosynthesis was signicantly inhibited under metal stress
(Wang and Ki, 2020) and excessive ROS-induced lipid peroxidation
damages thylakoid membranes and organelles associated with photo-
synthesis (Deng et al., 2019; Overmyer et al., 2003). Therefore, the
accumulation of lipid peroxidation products such as MDA might have
contributed to the impaired functioning of the photosynthetic system. In
addition, peroxidative damage destroys key proteins that constitute the
membrane structure of the PSII, such as protein D1 and D2, and inhibits
their synthesis (Nishiyama et al., 2006; Okada et al., 1996). Given that
the stress lipid peroxidation triggered by oxidative stress weakened the
photosynthetic system in several ways, we speculate that the compo-
nents in the supernatant attacked on the photosynthetic system of
M. aeruginosa via the same mechanism.
3.5. Release and degradation of microcystin-LR (MC-LR)
Algal toxins, including MC-LR, are synthesized by M. aeruginosa and
are present in its cells. MC-LR is released into eutrophic water when
apoptosis of algal cells occurs. Traditional methods of removing algae
cause the death of algae cells and the simultaneous release of large
amounts of MC-LR, which then enters the food chain and ultimately
poses serious risks to the environment and human health. Several MC-LR
degrading bacteria strains have been isolated from eutrophic water (Jin
et al., 2018; Maghsoudi et al., 2016; Yang et al., 2020; Yang et al., 2014).
The degradation mechanism of MC-LR by these bacteria is mainly the
metabolism of MC-LR as a nitrogen or carbon source to non-toxic forms
by intracellular enzymes or functional proteins (Maghsoudi et al., 2016;
Yang et al., 2020). However, the secretions released by microorganisms
have little degradation effect on MC-LR and only down-regulated the
expression levels of genes related to MC-LR thereby inhibiting their
production (Wei et al., 2020; Yu et al., 2019). Our pre-experimental
results also conrmed that the addition of the supernatant of Raoul-
tella sp. S1 did not decrease the MC-LR concentration while removing
M. aeruginosa. It was the action of the bacterial cultures that eliminated
the MC-LR by biodegradation. Therefore, we conducted the following
experiments using the bacterial cultures instead of the supernatant. As
Fig. 7 shows, the MC-LR concentration in control group had increased
gradually after 72 h, from 323
μ
g/L to 354
μ
g/L, as a result of the
accumulation of secondary metabolites produced during algal growth.
In contrast, the MC-LR content in the treatment group showed only a
marginal increase during the rst 12 h, and then decreased signicantly
to 72.83% of the maximum value after 72 h. Compared with the control
group, the MC-LR content in the treatment group decreasedled by
25.09% when the rupture and death of almost all the algae cells
occurred. Previous studies have mentioned that damage to algal cells by
foreign substances causes the release of intracellular MC-LR, resulting in
the increasing extracellular MC-LR concentrations (Wei et al., 2020;
Yang et al., 2017). The rapid increase in MC-LR levels is due to its release
from algal cells during the process of occulation and cell death caused
by Raoultella sp. S1. However, Raoultella sp. S1 caused the breakdown of
a proportion of the MC-LR, thereby eliminating the potential risk of
harm associated with its release. In short, the algicidal effects of
Raoultella sp. S1 initially accelerate the release of MC-LR. Subsequently,
however, the MC-LR could be degraded by the bacteria, which would
account for the nding that the nal concentration of MC-LR in the
culture was lower than the concentration prior to algal cell death. The
removal of MC-LR relies on the inhibition of expression level of genes
related to MC-LR synthesis or biodegradation controlled by functional
genes (e.g., mlrA mlrB, mlrC and mlrD) that encode specic proteins
(Wang et al., 2018; Zhu et al., 2016). The presence of biodegradation in
this research was conrmed by the signicant decrease in MC-LR con-
centration. In addition, the release of photosynthetic pigments (e.g.,
chlorophyll a) due to cell lysis also contribute to the degradation of
MC-LR to some extent (Tsuji et al., 1994; Zhang et al., 2001). This in-
dicates that the algal lysis caused by Raoultella sp. S1 does not produce
more MC-LR, but in fact reduces MC-LR level compared with their
original value, so avoiding the risk of further ecological harm.
Previous studies have reported algicidal microorganisms which
degrade MC-LR. Streptomyces amritsarensis strain HG-16 only hindered
the synthesis of MC-LR, and could not remove the existing MC-LR (Yu
et al., 2019). Over a period of 30 days, Pseudomonas aeruginosa
UCBPP-PA14 removed 91% of MCs produced by M. aeruginosa (Kang
et al., 2012). Bacillus sp. AF-1 downshifted the mcyB gene expression
level, but its degradative ability was not documented (Wu et al., 2014).
Fig. 7. Changes in MC-LR concentration during the algicidal process. The
values represent the means of parallels ±SD (n =3). Different letters represent
signicant differences between control and treatment groups at the same time
point. (P <0.05).
D. Li et al.
Environmental Pollution 287 (2021) 117644
11
Thus Raoultella sp. S1 signicantly has excellent characteristics over
these reported bacteria strains. Moreover, unlike physical or chemical
methods, the microbial method provides a solution to the problem of
how to control the mass release of MC-LR. However, the mechanism of
biodegradation of MC-LR by Raoultella sp. S1 and the specic pathway of
MC-LR degradation are still unclear, which is the direction of our future
research. Although the specic degradation mechanism of MC-LR has
yet to be explored, it is feasible to use Raoultella sp. S1 for the control of
HABs in eutrophic water and for the simultaneous inhibition of MC-LR
contamination.
3.6. Algicidal substances in the supernatant
To investigate the algicidal mechanism of Raoultella sp. S1, the
algicidal substances in the S1 culture supernatant were identied by
UHPLC-TOF-MS. The results are shown in Table S2. The main compo-
nents of the supernatant include D-Gluconic acid, Chlorogenic acid, L-
Malic acid, 5-Hydroxy-2,4-dioxopentanoate and 2-Methyl-3-oxopropa-
noic acid, etc. Five of the six most abundant compounds are low-
molecular-weight organic acids in the supernatant which might be
responsible for the algicidal activity. Among these, chlorogenic acid
(Fig. 8) has been reported to increase the permeability of cytomem-
branes and plasma membranes of bacteria, resulting in damage to their
barrier function, and allowing it to exert its antibacterial effect (Li et al.,
2014b; Lou et al., 2011). In addition, some studies have noted that
chlorogenic acid can slow down the migration of bacteria by impairing
the structure of bacterial cell walls, affecting the stability of bacterial
cell membranes and inducing the production of reactive oxygen species
(Lee and Lee, 2018; Ren et al., 2015). Other substances detected in this
research have not been reported to have algicidal efciency.
As M. aeruginosa is a type of cyanobacterium, its physiological
function may be affected by chlorogenic acid, although, the toxicolog-
ical effect of the latter on this species has yet to be fully investigated.
4. Conclusions
Raoultella sp. S1 has been isolated from eutrophic water, and exhibits
strong algicidal activity against M. aeruginosa. Raoultella sp. S1 occu-
lates the algal cells within 180 min initially and then secret extracellular
bioactive compounds that cause cell death in 72 h. During the lysis stage
the antioxidant system of the algal cells was activated to resist external
oxidative stress. Persistent environmental stress and an accumulation of
lipid peroxidation products result in the crumpling and fragmentation of
cells algal cells. Subsequently, photosynthetic efciency, relative elec-
tric transport rate and the expression level of photosynthesis-related
genes (psbA1, psbD1 and rbcL) decreases signicantly, indicating the
severe damage of photosynthetic system in M. aeruginosa cells caused by
the algicidal supernatant. Strain S1 also partially degrades the MC-LR
that is released by the algicidal activity, and so prevents contamina-
tion of the algal metabolites. According to the results of UHPLC-TOF-MS,
Several low-molecular-weight organic acids may be responsible for the
algicidal activity of the supernatant. Since the algicidal substances
released by Raoultella sp. S1 that were present in the supernatant are
resistant to acid and alkaline environments and are stable below
100.00 ◦C, Raoultella sp. strain S1 might have a potential role in the
control of HABs caused by M. aeruginosa.
Author statement
Dongpeng Li had developed the methodology, conducted a research
and investigation process, and prepared the rst draft of the article. Xin
Kang, Linglong Chu, and Yifei Wang had helped the experiment testing
and data collection. Xinshan Song had improved experiments and had a
contribution to conceptualization. Xiaoxiang Zhao had oversight and
leadership responsibility for the research activity planning and execu-
tion. Xin Cao had designed experiments, reviewed and edited the orig-
inal draft.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgement
We thank Dr. Yining Liu in the Core Facility Center of the Institute of
Plant Physiology and Ecology for mass spectrometry assistance. The
authors acknowledge the nancial support from the Fundamental
Research Funds for the Central Universities (Grant No. 2232019D3-21);
the National Key Research and Development Project (Grant No.
2019YFC0408603 and 2019YFC0408604); the National Natural Science
Foundation of China (Grant No. 51909034); the Shanghai Sailing Pro-
gram (Grant No. 19YF1401900); the Research project of ecological
environment protection and restoration of Yangtze River in Zhoushan
(SZGXZS2020068); and the special fund from the Hubei Provincial En-
gineering Research Center of Systematic Water Pollution Control (China
University of Geosciences, Wuhan, PR China) (Project No. 20190813).
Fig. 8. MS spectra and fragmentation pattern of chlorogenic acid, a possible algicidal compound, in Raoultella sp. S1 supernatant by UHPLC-TOF-MS with a retention
time of 4.463–4.874 min.
D. Li et al.
Environmental Pollution 287 (2021) 117644
12
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envpol.2021.117644.
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