Process Safety and Environmental Protection 186 (2024) 1273–1285

Available online 24 April 2024

0957-5820/© 2024 Institution of Chemical Engineers. Published by Elsevier Ltd. All rights reserved.

Enhancing wastewater remediation in microalga Euglena gracilis: The role of

trivalent cerium (Ce3

⁺) as a hormonal effect factor and its

metabolic implications

Feimiao Lu a

, Guichun Wu a

, Guimei Wu b

, Liangtao Zhang c

, Jiangxin Wang d

, Zhiyuan Liu a,*

,

Mingcan Wu b,e,f,g,**

a School of Marine Biology and Fisheries, Hainan University, Haikou 570228, China b College of Agriculture and Biological Science, Dali University, Dali, Yunnan 671003, China c XinKai Environment Investment Co.Ltd, Beijing 101100, China d College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China e Co-Innovation Center for Cangshan Mountain and Erhai Lake Integrated Protection and Green Development of Yunnan Province, Dali University, Dali, Yunnan

671003, China f Cangshan Forest Ecosystem Observation and Research Station of Yunnan Province, Dali University, Dali, Yunnan 671003, China g Research Center for Northwest Yunnan Biodiversity, Dali University, Dali, Yunnan 671003, China

ARTICLE INFO

Keywords:

Microalgae

Wastewater treatment

Euglena gracilis

Cerium

Hormonal effect

Metabolic regulation

ABSTRACT

This study explored the effects of trivalent cerium (Ce3

⁺) as a hormonal effect factor on microalga Euglena gracilis

in domestic wastewater treatment. It was discovered that 12.50 mg/L Ce3

⁺ markedly enhance E. gracilis dry

weight (by 63.44%, P < 0.05) and boost its efficiency in extracting key water quality indicators such as total

nitrogen (TN) by 66.08% (p < 0.05), total phosphorus (TP) by 37.17% (p < 0.05), ammonia nitrogen (NH4

+-N) by

39.90% (p < 0.05), and chemical oxygen demand (COD) by 97.17% (p < 0.05) from wastewater. Metabolomic

analysis revealed that Ce3

⁺ treatment significantly alters E. gracilis’s metabolic profile, notably in lipid metabolite

synthesis. This treatment increased the production of essential membrane lipid metabolites like phosphatidylglycerol and phosphatidic acid, vital for cellular membrane integrity. Additionally, Ce3

⁺ stimulated the synthesis

of certain antioxidant dipeptides, reducing wastewater-induced reactive oxygen species (ROS) levels. This

treatment improved not only nutrient removal efficiency from wastewater by E. gracilis but also its conversion to

high-value metabolic products, such as paramylon and lipids. These findings highlight Ce3

⁺’s pivotal roles in

augmenting E. gracilis’s wastewater treatment efficacy and environmental resilience, offering significant insights

for microalgae’s use in environmental management and bioenergy.

1. Introduction

Water pollution has become a pressing global environmental issue,

particularly due to increased wastewater discharges fueled by rapid

industrialization and urbanization (Fan and Fang, 2024; Zahoor and

Mushtaq, 2023). The nutrients abundant in wastewater, such as total

nitrogen (TN), total phosphorus (TP), ammonia nitrogen (NH4

+-N), and

chemical oxygen demand (COD), if discharged untreated, can lead to

eutrophication, triggering harmful algal blooms and threatening the

health of aquatic ecosystems and water quality (Oduor et al., 2023;

Mishra, 2023). Consequently, the development of efficient and

eco-friendly water purification technologies is of urgent necessity.

Microalgae, characterized as single-celled photosynthetic lower

plants, are emerging as cost-effective and sustainable tools for bioremediation. Their notable photosynthetic efficiency and robust nutrient

absorption capabilities have drawn considerable attention in the field of

wastewater treatment (Arbour et al., 2024; He et al., 2023; Wang et al.,

2024). For example, the Euglena gracilis strain CCAP 1224/5Z is distinguished for its rapid growth rate and exceptional pollutant removal

capabilities (Kim et al., 2022). This strain uniquely thrives in acidic

environments with pH levels ranging from 2 to 3.5, enabling it to efficiently absorb metal ions from aquatic systems, a feature rare among

* Corresponding author.

** Corresponding author at: College of Agriculture and Biological Science, Dali University, Dali, Yunnan 671003, China.

E-mail addresses: liuzhiyuan111@163.com (Z. Liu), 17744864080@163.com (M. Wu).

Contents lists available at ScienceDirect

Process Safety and Environmental Protection

journal homepage: www.journals.elsevier.com/process-safety-and-environmental-protection

https://doi.org/10.1016/j.psep.2024.04.098

Received 1 February 2024; Received in revised form 20 April 2024; Accepted 21 April 2024

Process Safety and Environmental Protection 186 (2024) 1273–1285

1274

microalgae (Wu et al., 2022). Additionally, E. gracilis is known for producing significant quantities of high-value metabolites such as paramylon and lipids. These compounds find widespread application in the

pharmaceutical, food, feed additive, and energy sectors, underscoring

the strain’s versatility and economic value (Chen et al., 2022; Huang

et al., 2023). However, the complex composition of wastewater, containing numerous toxic or oxidative substances, can induce excessive

reactive oxygen species (ROS) in microalgae (Liu et al., 2023), inhibiting

growth and nutrient absorption, leading to suboptimal wastewater

treatment outcomes. Therefore, enhancing microalgal growth,

improving antioxidant enzyme activities and oxidative stress resistance,

and efficiently converting absorbed nutrients into high-value metabolites have become hot research topics in microalgal wastewater

treatment.

In recent years, rare earth elements (REEs), particularly trivalent

cerium (Ce3

⁺), have sparked widespread interest in the scientific community due to their potential in modulating metabolic and growth

characteristics of organisms. Ce3

⁺, as a potential biostimulant, is

believed to enhance adaptability to environmental stresses by regulating

metabolic pathways and boosting antioxidant capacity (Trejo-T´ellez

et al., 2023). While the growth-promoting effects of cerium on higher

plants have been studied (Haghighi et al., 2023; Tao et al., 2022), and its

hormone-like functions similar to plant hormones are recognized, the

role and mechanisms of cerium in microalgae, particularly in E. gracilis

wastewater treatment, remain largely unexplored.

The current study aims to investigate the role and metabolic mechanisms of Ce3

⁺ as a hormonal effect factor in the wastewater treatment

process of E. gracilis. The study analyzes the impact of Ce3

⁺ on the

growth, antioxidant enzyme activity, cell metabolism, and nutrient

removal efficiency of E. gracilis from wastewater. Using metabolomic

approaches, the focus is on understanding how Ce3

⁺ affects the metabolic network of E. gracilis, especially in promoting the synthesis of

medical and energy storage substances (such as paramylon and lipids)

and enhancing adaptability to environmental stresses. These findings

are expected to provide new insights into the application of E. gracilis in

environmental management and bioenergy sectors, as well as offer significant theoretical support for the application of cerium in microalgal

biotechnology.

2. Materials and methods

2.1. Algal strain and culture conditions

In this study, the Euglena gracilis strain CCAP 1224/5Z was kindly

provided by the Jiangxin Wang research group at Shenzhen University

(Wu et al., 2022). Initially, this strain was acquired from the Culture

Collection of Algae and Protozoa (CCAP). E. gracilis was cultured and

maintained in photoautotrophic Euglena medium (PEM) medium, as

detailed in previous literature (Wu et al., 2020a; Wu et al., 2022). The

trivalent cerium (Ce3

⁺) used in the experiments was sourced from

CeCl₃⋅7 H₂O, supplied by China’s Xilong Scientific Company. The domestic wastewater selected for this study was obtained from the submerged reclaimed water plant in Dali Ancient City, operated by China

Water Environment (CWE), Yunnan Province, China. After undergoing

filtration through a 0.45 μm membrane and sterilization at 121℃ for

30 minutes, the wastewater’s characteristics were measured, with the

findings presented in Table 1. At the start of the experiment, E. gracilis

cells in their exponential growth phase were inoculated into 100 mL

conical flasks at a 20% inoculation ratio, resulting in a final cell concentration of approximately 106 cells/mL, with 20 mL of medium in

each flask. Considering the wastewater’s high nutrient content, no extra

inorganic salts were supplemented, thereby establishing the control

group. To investigate the impact of Ce3

⁺, varying concentrations of

CeCl₃⋅7 H₂O were added to the wastewater to establish a series of

experimental groups with Ce3

⁺ concentrations set at 1, 2.5, 5, 10, 12.5,

25, 50, and 100. mg/L. All experiments were conducted under a 12-hour

light/dark cycle, light intensity of 150 μmol photons m− 2 s

− 1 at a constant temperature of 25◦C, with bi-daily shaking of the flasks to promote

adequate gas exchange and mixing. To monitor the accumulation of

biomass, both cell density and cell dry weight were measured at two-day

intervals and the 8th day culture, respectively. Under consistent culture

conditions, CeCl₃⋅7 H₂O was added to 500 mL conical flasks, expanding

the working volume to 200 mL to achieve a final concentration of

12.50 mg/L Ce3

⁺. Subsequently, 20% (v/v) of E. gracilis cells were

inoculated into the wastewater for cultivation, leading to a final cell

density of approximately 106 cells/mL. The chlorophyll fluorescence

(Fv/Fm) was measured at the commencement of the experiment (Day 0)

and on Day 8. In addition, comprehensive physiological and biochemical

analyses of the algae cells cultured for 8 days were conducted. These

included assessments of dry weight, cell morphology changes, pigment

concentration, activities of key antioxidant enzymes (SOD, POD, CAT),

levels of malondialdehyde (MDA), and observation of the intracellular

distribution of Ce3

⁺ using transmission electron microscopy. Moreover,

the content of intracellular paramylon, fatty acids, and proteins were

meticulously quantified. Further, the study evaluated the removal efficiencies of TN, TP, NH4

+-N, and COD from the wastewater, as well as the

residual concentration of Ce3

⁺. Finally, the collected cell samples were

sent to Shanghai Baiqu Biomedical Technology Co., Ltd. (Shanghai,

China) for metabolite measurement and metabolomic analysis, aiming

to comprehensively reveal the impact of Ce3

⁺ on the metabolic network

of E. gracilis.

2.2. Analysis methods

2.2.1. Measurement of cell density, dry weight, and morphological changes

To determine cell density, a 450 μL algae suspension was extracted

from the culture flasks and transferred into a 1.5 mL centrifuge tube. Cell

counting was then conducted under an Olympus CX21 optical microscope (Olympus Corporation, China) using a hemocytometer (Xiang Bo

25×16 type, China). The methodology for measuring cell dry weight

was based on the protocols described by Lee et al. (2013) and Wu et al.

(2020b). Simultaneously, the morphology of the algal cells was observed

using a microscope, and the quantities of cells exhibiting the three

predominant shapes - elongated, oval, and circular - were statistically

analyzed.

2.2.2. Measurement of pigment content

The methodology for quantifying the pigment content in algal cells

was adopted from Gu et al. (2023). During the procedure, 1.5 mL of algal

suspension was transferred to a 1.5 mL centrifuge tube, followed by

centrifugation at 12,000 rpm for 10 minutes. The supernatant was discarded under dark conditions, and 1.5 mL of methanol was then added

to the tube. The mixture was subsequently heated at 60◦C for 30 minutes. After heating, the sample was homogenized and centrifuged again

at 12,000 rpm for 10 minutes. Using methanol as a blank control, the

absorbance of the algal supernatant was measured at five wavelengths

(λ) - 665 nm, 750 nm, 644 nm, 662 nm, and 440 nm - using a spectrophotometer. The pigment content in the algal cells (mg/L) was calculated using the Eqs. (1–3), and based on this, the pigment content per

Table 1

Characteristics of the domestic wastewater sampled from the submerged

reclaimed water plant in Dali Ancient City, operated by China Water

Environment (CWE), Yunnan Province, China.

Water quality indicators Concentrations (mg/L)*

TN 22.65 ± 1.21

TP 5.21 ± 0.34

NH4

+-N 19.73 ± 0.62

COD 7.63 ± 0.81

Note: *, the concentrations of the wastewater were detected after filtrated

through a 0.45 μm membrane and sterilized by 121℃, 30 min.

F. Lu et al.

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1275

cell was determined relative to the cell density:

Chlorophyll a = 13.90 (λ665 - λ750) (1)

Chlorophyll b = 21.43 λ644 - 4.65 λ662 (2)

Carotenoid = 4.70 λ440 - (1.38 λ662 + 5.48 λ644) (3)

2.2.3. Measurement of chlorophyll fluorescence (Fv/Fm)

The measurement of maximum efficiency of chlorophyll fluorescence (Fv/Fm ratio) in this study was conducted following the method

outlined by Sha et al. (2019). The Fv/Fm ratio is calculated by dividing

the variable fluorescence (Fv) by the maximum fluorescence (Fm). Prior

to measurement, algal cell samples were placed in quartz cuvettes and

allowed to acclimate in a dark environment for 3 minutes. Subsequently,

the Fv/Fm ratio of the algal cells was determined at room temperature

using a PHYTO-ED fluorometer produced by Walz GmbH (Effeltrich,

Germany).

2.2.4. Measurement of SOD, POD, CAT activities, and MDA levels

The activity of superoxide dismutase (SOD) was measured based on

the consumption of Nitrotetrazolium Blue chloride (NBT). The specific

methodology was adapted from Ginnopolitis and Rice (1977), along

with detailed steps provided by Li et al. (2016). Peroxidase (POD) activity was assessed using the guaiacol method as proposed by Liu et al.

(2009). Catalase (CAT) activity was determined by measuring the consumption of potassium permanganate (KMnO4), following the protocol

described by Li et al. (2016). The content of malondialdehyde (MDA)

was quantified using the method outlined by Kong et al. (2010).

2.2.5. Transmission electron microscopy sample preparation and

observation

The samples were fixed overnight at 4◦C in 2.5% glutaraldehyde,

followed by three washes with 0.1 M phosphate buffer (pH 7.0) for

15 minutes each. Subsequently, they were fixed for 1–2 hours in 1%

osmium tetroxide solution and washed again. A graded series of ethanol

dehydration (ranging from 30% to 100%) was performed, with each step

lasting 15 minutes, and finally, the samples were treated with acetone

for 20 minutes. The samples were infiltrated with a mixture of Spurr’s

resin and acetone (ratios of 1:1 and 3:1), followed by overnight

embedding in pure resin at room temperature. The embedded samples

were then cured at 70◦C overnight. Ultra-thin sections of 70–90 nm were

prepared using a LEICA EM UC7 ultramicrotome (Leica Microsystems,

Wetzlar, Germany). Sections were stained with lead citrate and uranyl

acetate solutions for 5–10 minutes. Finally, the samples were observed

under a transmission electron microscope (TEM; FEI Tecnai Spirit G2,

Hillsboro, OR, USA).

2.2.6. Measurement of paramylon, lipid, and protein content

The determination of paramylon content was performed using the

method described by Takenaka et al. (1997) and Wu et al. (2021). The

total lipid content of E. gracilis was quantified through the n-hexane

soxhlet extraction method proposed by Kanda et al. (2020). The measurement of algal cell protein content followed the protocol outlined by

Slocombe et al. (2013). The brief procedure is as follows: 250 μL of 10%

(w/v) trichloroacetic acid was added to 5 mg of freeze-dried algal

powder. The suspension was sonicated for 15 minutes and then incubated at 65◦C for 30 minutes. After centrifugation at 15,000×g for

20 minutes, the pellet was resuspended in 0.5 mL of alkaline solution

containing 20 g/L Na2CO3 and 4 g/L NaOH to dissolve the proteins.

Protein quantification was carried out using the DC protein assay kit

from Bio-Rad (USA), based on the method by Lowry et al. (1951).

2.2.7. Measurement of TN, TP, NH4

+-N, COD, and residual Ce3

⁺

concentration

This study employed the methodology described by Qiu et al. (2023)

to determine the contents of TN, TP, NH4

+-N, and COD in the treated

water and to calculate their removal efficiencies. Cells and culture medium were separated by centrifugation, and the supernatant was filtered

through a 0.45-μm syringe filter. The filtered solution was then sent to

the Beijing Zhongke Guanghua Chemical Technology Research Institute

(Chemical Laboratory) for analysis of residual Ce3

⁺ concentrations using

Inductively Coupled Plasma Mass Spectrometry (ICP-MS; Thermo Scientific iCAP RQ, USA).

2.2.8. Metabolomic analysis

For metabolomic analysis, algal cell samples were collected with

each sample group comprising three biological replicates. Metabolites

were extracted from the algal cells following the method outlined by Wu

et al. (2020b). Metabolite detection and analysis were conducted using

an Ultra-High Performance Liquid Chromatography (UHPLC) system

equipped with a Phenomenex Kinetex C18 column (2.1 mm × 50 mm,

2.6 μm), coupled with an Orbitrap Exploris 120 Mass Spectrometer

(Orbitrap MS, Thermo Fisher Scientific). The mobile phase A consisted

of water with 0.01% acetic acid, and phase B was composed of isopropanol:acetonitrile (1:1, v/v). The autosampler temperature was set at

4◦C, and the injection volume was 2 μL. The Orbitrap Exploris 120 MS

operated in Information Dependent Acquisition (IDA) mode for MS/MS

spectrum acquisition, controlled by the Xcalibur software (Thermo

Fisher Scientific). In this mode, the software continuously evaluated

full-scan MS spectra. The conditions for the electrospray ionization (ESI)

source were as follows: sheath gas flow rate of 50 Arb, auxiliary gas flow

rate of 15 Arb, capillary temperature of 320◦C, full scan MS resolution of

60,000, MS/MS resolution of 15,000, collision energy: stepped

normalized collision energy (SNCE) 20/30/40, and spray voltage set at

3.8 kV (positive) or − 3.4 kV (negative). Preprocessing and annotation

analysis of the metabolomic data were performed following the methods

previously published by Wu et al. (2021), leading to the construction of

a comprehensive metabolic network.

2.3. Statistical analysis

All data are presented as mean ± standard deviation (SD) based on

three independent experimental replications. To ascertain the statistical

differences between the control group and experimental groups,

repeated measures one-way or two-way analysis of variance (ANOVA)

was employed. Post hoc comparisons were conducted using Sidak’s

multiple comparisons test and Student’s t-test. A p-value less than 0.05

(p < 0.05) was considered statistically significant, while a p-value

greater than 0.05 (p > 0.05) indicated no significant statistical difference. All analyses were performed using GraphPad Prism and Microsoft

Excel software.

Table 2

The effects of various concentrations of Ce3

⁺ on the dry weight of E. gracilis in the

8th day of wastewater cultivation.

Ce3+ concentrations Dry weight (g/L) IPCDW (%)

Control 0.93 ± 0.06 -

1 mg/L (7.14 μM) 1.62 ± 0.11a 74.19

2.5 mg/L (17.84 μM) 1.59 ± 0.16a 70.97

5 mg/L (35.69 μM) 1.56 ± 0.09a 67.74

10 mg/L (71.38 μM) 1.49 ± 0.03b 60.22

12.5 mg/L (89.22 μM) 1.52 ± 0.10a 63.44

25 mg/L (178.44 μM) 0.73 ± 0.06c -21.51

50 mg/L (356.89 μM) 0.67 ± 0.02d -27.96

100 mg/L (713.78 μM) 0.53 ± 0.07 h -43.01

Note: different letters within the same column mean significant differences between groups (p < 0.05, n = 3); IPCDW, the increase in the percentage of

E. gracilis cell dry weight.

F. Lu et al.

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Fig. 1. The effects of various concentrations of Ce3

⁺ on the growth, morphology, pigment content, and Fv/Fm of E. gracilis in wastewater. (A) Growth curves; (B) Cell

density of E. gracilis on the 8th day of cultivation; (C) Cell dry weight; (D) Morphological changes; (E) Pigment content in E. gracilis, including chlorophyll a (Chl a),

chlorophyll b (Chl b), and carotenoids (Caro); (F) Chlorophyll fluorescence efficiency (Fv/Fm); \"Ce3

⁺-treatment\" denotes the experimental group treated with Ce3

⁺,

while \"control\" represents the untreated control group. C, D, E, and F specifically represent conditions under a Ce3

⁺ concentration of 12.50 mg/L. The notation \"NS\"

signifies no significant difference, while an asterisk (\"*\") indicates statistical significance with P < 0.05. Data are presented as mean ± standard deviation (SD), based

on three independent experimental replicates (n=3).

F. Lu et al.

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3. Results and discussion

3.1. Impact of Ce3

⁺ on the growth and physiological characteristics of

E. gracilis in wastewater

Rare Earth Elements (REEs) predominantly occur as trivalent ions in

environments where the pH ranges from 4 to 8, and carbonate complexes in conditions where the pH exceeds 8 (Anastopoulos et al., 2016).

Given that the pH of the E. gracilis culture medium is around 3.5, the

addition of Ce3

⁺ in this study does not result in the formation of new

compounds. Thus, this study investigated the impact of Ce3

⁺ on the

growth and physiological characteristics of E. gracilis during wastewater

treatment. The results indicated a pronounced stimulatory effect on the

algal growth within a low Ce3

⁺ concentration range of 1.00–12.50 mg/L.

Notably, at a concentration of 1.00 mg/L, the growth rate and biomass

were significantly higher than those of the control group (Table 2;

Fig. 1A and B). Additionally, by the 8th day of cultivation, the dry

weight of the E. gracilis in the 12.50 mg/L Ce3

⁺ treatment group (named

Ce3

⁺-treated E. gracilis) notably exceeded that of the control (Table 2,

Fig. 1C, by 63.44%, p < 0.05), suggesting a stimulatory role of Ce3

⁺

within this concentration range. However, growth inhibition became

apparent as the concentration of Ce3

⁺ exceeded 12.50 mg/L, leading to a

notable decrease in cell density and dry weight at concentrations of

25.00, 50.00, and 100.00 mg/L (Table 2; Fig. 1A and B). This trend

underscores the inhibitory effects of higher Ce3

⁺ concentrations, aligning

with findings in the published literature (Cao et al., 2021), which also

reported toxic effects on algae at elevated concentrations. This phenomenon not only demonstrates the concentration-dependent nature of

Ce3

⁺ on algal growth but also highlights its potential as a growth promoter at optimal concentrations. Thus, to investigate the effects on algal

biochemical physiology and wastewater treatment efficiency under this

maximal tolerable concentration, and to explore related metabolic

mechanisms, we selected 12.50 mg/L of Ce3+ as a representative low

concentration for our study.

Further analysis revealed that at Ce3

⁺-treated E. gracilis predominantly exhibited elongated and oval morphologies, similar to observations in our previously published article (Wu et al., 2021), indicating

higher vitality compared to the control group (Fig. 1D). Such morphological changes could reflect the positive regulation of Ce3

⁺ on the

physiological state of the cells. Moreover, the observed decrease in

Fig. 2. The impact of Ce3

⁺ treatment on E. gracilis’s antioxidant enzyme activities and oxidative stress response in wastewater. (A)Superoxide dismutase (SOD)

activity; (B) Peroxidase (POD) activity; (C) Catalase (CAT) activity; (D) Malondialdehyde (MDA). The experimental group, labeled as Ce3

⁺-treatment, is contrasted

with the control group. The experiments were conducted with E. gracilis cultured in a medium containing 12.50 mg/L of Ce3

⁺. The asterisk (*) indicates a significance

level of P < 0.05. Data are presented as mean ± standard eviation (SD), based on three independent experimental replicates (n=3).

F. Lu et al.

Process Safety and Environmental Protection 186 (2024) 1273–1285

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chlorophyll a (Chl a) and b (Chl b) content, coupled with an increase in

carotenoid levels in this treatment group (Fig. 1E), suggests a role for

Ce3

⁺ in modulating algal pigment synthesis. Notably, despite the

reduced chlorophyll content, the Fv/Fm was significantly higher in the

Ce3

⁺ treated group compared to the control (Fig. 1F), implying that Ce3

⁺,

while reducing Chl a and Chl b levels, did not impair photosynthetic

efficiency. Instead, it may have enhanced the activity of Photosystem II,

thereby promoting photosynthesis. This finding contrasts with previous

studies on higher plants’ response to rare earth elements (REEs) (Wang

et al., 2006), suggesting distinct responses between higher plants and

microalgae to Ce3

⁺-mediated regulation, possibly linked to different

photosynthetic pathways modulated by REEs. Such differences warrant

further investigation. Previous research has predominantly focused on

the low-concentration stimulation and high-concentration inhibition

effects, known as the hormesis effect, of REEs in higher plants (Agathokleous et al., 2019; Hu et al., 2004; Sun et al., 2018; Tao et al., 2022).

This study is the first to observe a similar phenomenon in microalgae,

which we term the \"Microalgal Hormesis Effect,\" or \"Ce3

⁺ Hormesis

Effect Factor (HEF)\". This finding suggests that microalgae’s response to

REEs may involve complex physiological and biochemical regulation

mechanisms, particularly in photosynthesis and energy metabolism.

In summary, the impact of Ce3

⁺ on E. gracilis exhibits a clear concentration dependency, with growth promotion at low concentrations

and inhibition at high concentrations. This discovery provides vital

theoretical support for the application of microalgae in environmental

governance, particularly in determining the optimal concentration

range for Ce3

⁺.

3.2. Modulatory role of Ce3

⁺ treatment on antioxidant enzyme activity

and oxidative stress in E. gracilis

Wastewater contains a plethora of harmful substances that challenge

the capability of microalgae to treat wastewater, particularly concerning

antioxidant enzyme activity and oxidative stress (Li et al., 2016; Xiong

et al., 2022). This study delves into the role of Ce3

⁺, as a HEF, in

modulating the growth adaptability of E. gracilis under wastewater

Fig. 3. Distribution of Ce3+ accumulation within E. gracilis cells by scanning electron microscopy. (A-B) The structures observed in normal cells, control group. (C-D)

The experimental group, showing the intracellular accumulation and distribution of Ce3+ in Ce3+-treated E. gracilis; Key cellular components are labeled as follows:

CP represents chloroplasts; MT indicates mitochondria; P denotes paramylon; and L symbolizes lipid droplets.

F. Lu et al.

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treatment conditions. Our focus centered on the impact of Ce3

⁺ on the

antioxidant defense system of E. gracilis. The findings demonstrate that

Ce3

⁺ treatment significantly enhanced the activities of antioxidant enzymes such as SOD, POD, and CAT, while notably reducing MDA levels

(Fig. 2). Specifically, Ce3

⁺-treated E. gracilis’s SOD activity showed a

substantial increase (by 99.42%, p < 0.05) compared to the control

group (Fig. 2A), reflecting an enhanced capability of E. gracilis to scavenge superoxide radicals. Similarly, Ce3

⁺-treated E. gracilis’s POD activity was significantly augmented (by 63.46%, p < 0.05) (Fig. 2B),

indicating that Ce3

⁺ boosted the metabolism of hydrogen peroxide in

E. gracilis. Additionally, Ce3

⁺-treated E. gracilis’s CAT activity also

showed a significant rise (by 34.24%, p < 0.05) (Fig. 2C), suggesting that

Ce3

⁺ effectively enhanced the enzyme-mediated decomposition of

hydrogen peroxide. Concurrently, the marked reduction in MDA levels

(by 49.21%, p < 0.05) as shown in Fig. 2D implies that Ce3

⁺ treatment

notably mitigated lipid peroxidation, possibly due to the elevated antioxidant enzyme activities reducing oxidative stress.

These results indicate a potential protective role of Ce3

⁺ in the antioxidant defense system of E. gracilis. This phenomenon aligns with

similar observations in higher plants (Sun et al., 2018), suggesting its

universality. The findings elucidate that Ce3

⁺ can effectively improve the

oxidative stress state of E. gracilis and enhance its environmental

adaptability in wastewater treatment, crucial for enhancing treatment

efficiency and stability. However, while Ce3

⁺ offers protective benefits,

its potential toxicity and environmental impact require further in-depth

examination.

3.3. Selective intracellular accumulation of Ce3

⁺ in E. gracilis and its

impact on metabolic product synthesis

This study, utilizing transmission electron microscopy (TEM) analysis, has revealed the selective accumulation pattern of Ce3

⁺ within

E. gracilis cells and its consequential effects on the synthesis of key

metabolic products. In the control group, not treated with Ce3

⁺, the

chloroplast interthylakoid spaces appeared relatively sparse, with lower

levels of paramylon granules and lipid droplets (Fig. 3A and B).

Conversely, in the Ce3

⁺ treated group, these spaces within the

chloroplasts exhibited a notable increase in color depth, and there was a

significant augmentation in the quantity of paramylon granules and

lipid droplets (Fig. 3C and D). This indicates a concentration of Ce3

⁺

within the interthylakoid spaces of the chloroplasts, corroborating with

findings from Rezanka et al. (2016).

Further quantitative analysis of metabolic products confirmed that

Ce3

⁺ treatment significantly increased the content of paramylon (by

111.70%, P < 0.05) and lipids (by 22.74%, P < 0.05), while the protein

content noticeably decreased (by 30.51%, P < 0.05, Fig. 4). It suggests

that Ce3

⁺ might have stimulated the synthesis of energy storage forms,

thereby enhancing the energy reserves and environmental adaptability

of E. gracilis. Simultaneously, the decrease in protein content might

reflect an inhibitory effect of Ce3

⁺ on protein synthesis pathways or a

cellular energy-saving strategy under Ce3

⁺ stress.

In our previous studies, we observed that the starch-deficient mutant

strains of Chlorella sorokiniana, when subjected to high light stress,

exhibited a reduction in protein content. The carbon skeletons resulting

from protein degradation were channeled through the TCA cycle,

leading to the synthesis of lipids (Wu et al., 2019). Similarly, in

high-density heterotrophic fermentation cultures, E. gracilis demonstrated a decrease in protein and lipid content, while the levels of paramylon and lipids increased (Wu et al., 2021). Our current research

mirrors these findings, suggesting that under Ce3

⁺ treatment, E. gracilis

undergoes a metabolic pathway remodeling. This involves rerouting the

carbon flow from protein carbon skeletons towards the synthesis pathways of paramylon and lipids, thus enhancing the production of these

high-value metabolites. These findings shed light on the intricate role of

Ce3

⁺ in metabolic regulation within E. gracilis, potentially influencing

photosynthesis, energy allocation, and stress response mechanisms.

Notably, the increase in paramylon and lipid content under Ce3

⁺ treatment suggests that Ce3

⁺ treatment not only boosts the capacity of

E. gracilis for wastewater treatment but also yields high biomass content.

This positions Ce3

⁺ treatment as a pivotal factor in enhancing the sustainability of bioenergy, pharmaceuticals, and food sources, thereby

underscoring its significance in resource utilization.

Fig. 4. Impact of Ce3+ on the synthesis of paramylon, lipids, and proteins in E. gracilis under the wastewater culture. (A) Paramylon content; (B) lipid content; (C)

Protein content. Ce3+-treatment represents the experimental group, while control indicates the control group. E. gracilis was cultured in an environment with a Ce3+

concentration of 12.50 mg/L. An asterisk (*) denotes P < 0.05. Data are presented as mean ± standard deviation (SD), with experimental group data based on three

independent experiments (n=3).

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Process Safety and Environmental Protection 186 (2024) 1273–1285

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3.4. Ce3

⁺ treatment enhances nutrient removal and resource utilization by

E. gracilis in wastewater

In typical wastewater, high concentrations of TN, TP, NH4

+-N, and

COD are present. If discharged untreated into lakes and reservoirs, these

pollutants can lead to eutrophication and harmful algal blooms, severely

impacting ecological environments (Boonbangkeng et al., 2022; Oduor

et al., 2023). Thus, to further explore the capability of E. gracilis mediated by cerium (Ce3

⁺) in removing key nutrients from wastewater, this

study primarily assessed changes in these nutrient levels. As shown in

Fig. 5A, under Ce3

⁺ treatment, the removal rates of TN, TP, NH4

+-N, and

COD by E. gracilis were significantly increased by 66.08% (p < 0.05),

37.17% (p < 0.05), 39.90% (p < 0.05), and 97.17% (p < 0.05),

respectively, compared to the control group. This clearly indicates that

Ce3

⁺ significantly enhanced the efficient absorption of these nutrients by

E. gracilis.

We have previously examined the removal efficiency of TN, TP, NH4

+-

N, and COD by E. gracilis during its exponential growth phase (on the

fourth day of cultivation) under the Ce3+ treatment, but compared to the

control group, only the removal rate of NH4

+-N showed a significant

difference. There were no significant differences in the removal rates of

the other indicators (Supplementary Figure S1a), and cell growth did not

significantly differ either (Supplementary Figure S1b). Meanwhile, the

removal rates of wastewater quality indicators were only reached ~30%

by E. gracilis. This suggests that at this growth phase, Ce3+ does not

effectively enhance the growth of E. gracilis or improve removal rate of

wastewater quality indicators significantly. In other words, Ce3+ did not

exhibit its potential as a hormesis effect factor in wastewater treatment

until the early stationary phases of algal cell growth, where its effects

became notably apparent. Furthermore, on the eighth day of cultivation

(early stationary phases), the illumination of cultivation was increased

from 150 μmol photons m− 2 s

− 1 to 250 μmol photons m− 2 s

− 1

. We

observed a significant increase in cell growth in both the experimental

and control groups (Supplementary Figure S1b), especially under Ce3+

mediation, where the cell growth rate was markedly more pronounced.

This indicates that on the eighth day, the main issue was the high cell

density, which hindered light penetration into the medium and, consequently, photosynthesis, leading to reduced cell growth rates. This also

means that on the eighth day of cultivation (early stationary phases),

despite the removal rate reaching 97.34% (Fig. 5A), nitrogen was still

present in the medium, suggesting that the algal cells had not yet

reached a nitrogen-limiting stress condition. It’s possible that the algal

cells absorbed nitrogen ions extensively for storage within the cells,

preparing for subsequent growth.

This result not only reduces the risk of eutrophication in water bodies

but also enhances the recycling potential of these elements, facilitating

the transformation of pollutants into biomass (paramylon and lipids).

Additionally, the study found that E. gracilis could effectively adsorb and

remove Ce3

⁺ added to the water (Fig. 5B), thus reducing the residual

REEs in the treated water. This outcome suggests that algae treatment

technology mediated by Ce3

⁺ offers a new strategy for water restoration

and resource utilization.

Given the potential accumulation of Ce3

⁺ in E. gracilis, the study also

underscores the importance of examining the safety of these algae as

feed or food. Specifically, it is necessary to assess whether the accumulation of Ce3

⁺ in algae can be transferred through the food chain to

animal or human, and its potential impact on environmental and food

safety. These investigations are crucial for ensuring the sustainability

and safety of algal wastewater treatment technology.

3.5. Metabolic mechanisms of E. gracilis in efficient nutrient absorption

and conversion to high-value metabolites under Ce3

⁺ mediation

This study employed metabolomics to explore how Ce3

⁺, acting as a

HEF, enhances the growth of E. gracilis in wastewater treatment and

efficiently converts wastewater nutrients into high-value metabolites

(paramylon and lipids). Metabolomic analysis revealed that Ce3

⁺-treated

E. gracilis displayed a significantly different metabolite profile compared

to the untreated control group (Fig. 6A). This divergence indicates that

Ce3

⁺ profoundly influences the metabolism of E. gracilis, leading to the

upregulation of numerous metabolites and downregulation of a few

(Fig. 6B; Supplementary Table 1S, 2S), suggesting an active role of Ce3

⁺

in promoting specific metabolic pathways. Notably, under Ce3

⁺ treatment, there was a significant increase in lipid metabolites in E. gracilis

(Fig. 6C), consistent with an increase in lipid content. Additionally, Ce3

⁺

treatment affected organic acids, proteins, and amino acid metabolites,

albeit to a lesser extent. Compared to the control group (Fig. 6D), Ce3

⁺

treatment significantly altered the metabolite spectrum of E. gracilis,

particularly enhancing the synthesis of lipid metabolites.

Further metabolomics research, through heatmap and differential

metabolite analysis, revealed a significant enhancement in the synthesis

of various membrane lipid metabolites in E. gracilis under Ce3

⁺ treatment. This includes phosphatidylglycerol (LPG), phosphatidic acid

(LPA), Phosphatidylethanolamine (LPE), Phosphatidylcholine (LPC),

Digalactosyldiacylglycerol (DGDG), Phosphatidylethanolamine (PE),

and Phosphatidylcholine (PC) (Fig. 7A; Supplementary Table 1S, 2S).

Fig. 5. Removal rate of TN, TP, NH4

+-N, and COD by Ce3+-treated E. gracilis under the wastewater culture and the residual concentration of Ce3+ in treated water. (A)

The removal rates of total nitrogen (TN), total phosphorus (TP), ammonia nitrogen (NH4

+-N), and chemical oxygen demand (COD) from wastewater; (B) The residual

concentration of Ce3+ in the treated water. The Ce3+-treatment group is the experimental group, while the group of initial concentration is the comparison

benchmark. E. gracilis was cultured at a Ce3+ concentration of 12.50 mg/L. An asterisk (*) indicates P < 0.05. Data are presented as mean ± standard deviation (SD),

based on three independent experimental repeats (n=3).

F. Lu et al.

Process Safety and Environmental Protection 186 (2024) 1273–1285

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Fig. 6. Differential metabolite changes and classifications in Ce3+-treated E. gracilis under wastewater culture. (A) The principal component analysis (PCA) score

plot. (B) The number of upregulated and downregulated metabolites. (C) Categorizes and enumerates the differential metabolites in Ce3+-treated E. gracilis

(experimental group). (D) Categorizes and enumerates the differential metabolites in no-Ce3+-treated E. gracilis (control group).

F. Lu et al.

Process Safety and Environmental Protection 186 (2024) 1273–1285

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Fig. 7. Cluster analysis and bubble chart of select differential metabolites in Ce3+-treated E. gracilis under wastewater culture. (A) The cluster analysis chart of

selected differential metabolites. (B) The bubble chart. FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.

F. Lu et al.

Process Safety and Environmental Protection 186 (2024) 1273–1285

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Particularly, DGDG and PE, being signature lipids of chloroplast membranes (Holzl ¨ and Dormann, ¨ 2019), their increased levels indicate that

Ce3

⁺ could enhance the synthesis of these crucial lipids, potentially

improving photosynthetic efficiency and increasing CO2 fixation.

Moreover, the increase in other lipid substances suggests that Ce3

⁺ may

activate lipid metabolic pathways, promoting biosynthetic processes

related to cell membrane structure and function, improving membrane

fluidity and signal transduction, reducing the levels of ROS produced

under wastewater stress, and enhancing the efficient absorption of TN,

TP, NH4

+-N, and COD from wastewater by E. gracilis. The study also

found certain dipeptides, such as valvl-lsoleucine, with significantly

increased relative content under Ce3

⁺ treatment (Fig. 7A, Supplementary

Table 1S). This finding aligns with a previous study/report (Ozawa et al.,

2022), suggesting that these dipeptides may possess antioxidative

properties to reduce intracellular ROS levels, meriting further in-depth

investigation. Additionally, the upregulation of riboflavin metabolism

and the citric acid cycle (TCA cycle) related metabolites (Fig. 7B) indicates that Ce3

⁺ enhances the energy metabolism and antioxidative

capacity of E. gracilis. Conversely, the downregulation of alanine,

aspartate, and glutamate metabolism may indicate the impact of Ce3

⁺ on

the synthesis or utilization of amino acids, thereby redirecting carbon

flow towards the production of high-value metabolites such as

paramylon and lipids. A similar phenomenon has also been observed in

high-density heterotrophic cultures of E. gracilis (Wu et al., 2021), suggesting that under Ce3

⁺ treatment, the microalgae can remodel its

metabolic pathways during phototrophic autotrophy, efficiently converting photosynthetically fixed carbon into high-value metabolic

products. The findings of this study significantly highlight the crucial

role of Ce3

⁺ in regulating the metabolic activities of E. gracilis, particularly in enhancing its resistance to environmental stress and elevating its

environmental remediation capabilities. Furthermore, Ce3

⁺ promotes

the efficient conversion of nutrients in wastewater into high-value metabolites like paramylon and lipids by E. gracilis. The regulatory role of

Ce3

⁺ not only contributes to a more stable structure of chloroplast

membranes but also protects cells from oxidative damage by lowering

the levels of ROS (Fig. 8). This mechanism supports the growth of

E. gracilis and significantly improves its efficiency in removing key nutrients from wastewater, providing a vital biochemical foundation for

the application of E. gracilis in water treatment and ecological

restoration.

Future research should continue to delve into how Ce3

⁺ affects the

overall metabolic network of E. gracilis and assess the long-term impact

of these metabolic changes on the physiological state and environmental

adaptability of E. gracilis. Moreover, the application and safety of Ce3

⁺ in

Fig. 8. Schematic representation of the potential metabolic mechanism in E. gracilis for the removal of TN, TP, NH4

+-N, and COD in wastewater, and promotion of

growth mediated by the HEF (Ce3+). Red font indicates upregulation, blue font represents downregulation; solid lines signify direct chemical reactions; dashed lines

denote indirect chemical reactions. TN, total nitrogen; TP, total phosphorus, NH4

+-N, ammonia nitrogen; COD, chemical oxygen demand; HEF, hormone effect factor

(Ce3+); ROS, reactive oxygen species; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DGDG,

digalactosyldiacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LysoPC,

lysophosphatidylcholine; PEtOH, phosphatidylethanol; Acar, acyl carrier protein; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; GMP, guanosine

monophosphate; TN, total nitrogen; TP total phosphorus; NH4

+-N, ammonia nitrogen; COD, chemical oxygen demand.

F. Lu et al.

Process Safety and Environmental Protection 186 (2024) 1273–1285

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environmental management must also be considered to ensure the sustainability and eco-friendliness of this technology. Through these

studies, we can better understand and optimize the potential application

of REEs-mediated E. gracilis in efficient wastewater treatment.

4. Conclusion

The study confirms that a concentration of 12.50 mg/L Ce3

⁺ significantly boosts the growth of E. gracilis, concurrently improving the

removal efficiencies of key wastewater indicators such as TN, TP, NH4

+-

N, and COD. This underscores the potential of Ce3

⁺ as a highly effective

enhancer in wastewater treatment processes. Ce3

⁺ treatment markedly

alters the metabolite profile of E. gracilis, notably enhancing the synthesis of lipid metabolites essential for cell membrane integrity and

function. This adjustment in metabolic pathways is pivotal for the algae’s adaptation and stress response in wastewater environments. These

discoveries highlight how Ce3

⁺ regulates the metabolic pathways of

E. gracilis through impacts on photosynthesis, energy allocation, and

stress response. Overall, this study confirms the vital role of Ce3

⁺ in

enhancing the efficiency of wastewater treatment by E. gracilis,

bolstering its environmental adaptability, and facilitating its conversion

to biomass, such as paramylon and lipids. However, it only investigated

the impact of a Ce3

⁺ concentration of 12.50 mg/L on wastewater treatment. Therefore, this research still has limitations. Future studies are

needed to assess the effects of other low concentrations of Ce3

⁺-mediated

E. gracilis on wastewater treatment to determine whether 12.50 mg/L

Ce3

⁺ is indeed the optimal concentration for wastewater treatment.

Additionally, it will be necessary to examine whether the components of

wastewater influence the efficacy of Ce3

⁺-mediated algae cell treatment

in practical applications.

CRediT authorship contribution statement

Mingcan Wu: Writing – review & editing, Supervision, Funding

acquisition, Data curation. Feimiao Lu: Writing – original draft, Methodology, Formal analysis, Data curation. Guichun Wu: Methodology,

Data curation. Jingxin Wang: Supervision. Zhiyuan Liu: Supervision,

Funding acquisition. Guimei Wu: Supervision, Resources. Liangtao

Zhang: Supervision, 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.

Acknowledgements

This work was supported by the Hainan Provincial Natural Science

Foundation of China (Grant No. 420RC520), High-level Talent Project of

Hainan Natural Science Foundation (Grant No. 323RC426), Foundation

of Yunnan Province Science and Technology Department (Grant No.

202305AM070003), and Foundation of Yunnan Provincial Education

Science Research Department (Grant No. 2024J0827). We thank

Shanghai Biotree Biomedical Biotechnology Co., Ltd., for metabolomic

data acquisition and data analysis.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the

online version at doi:10.1016/j.psep.2024.04.098.

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