TYPE Original Research

PUBLISHED 05 July 2023

DOI 10.3389/fpls.2023.1193668

Keystone microalgae species

OPEN ACCESS determine the removal efficiency
EDITED BY
ZhongQiang Li,
Hubei University, China
of sulfamethoxazole: a case
REVIEWED BY
Kalpana Bhatt,
study of Chlorella pyrenoidosa
Purdue University, United States
Bin Ji,
Wuhan University of Science and
and microalgae consortia
Technology, China

*CORRESPONDENCE Ruohan Huang 1†, Wan Liu 1†, Jinghua Su 2, Shihao Li 1,3,
Wei Zhang
[email protected] Liqing Wang 1, Erik Jeppesen 4,5,6,7 and Wei Zhang 1*
† 1
Key laboratory of Exploration and Utilization of Aquatic Genetic Resources of the Ministry of
These authors have contributed equally to
this work Education, Engineering Research Center of Environmental DNA and Ecological Water Health
Assessment, Shanghai Ocean University, Shanghai, China, 2 Research Institute of Natural Ecology
RECEIVED 25 March 2023 Conservation, Shanghai Academy of Environmental Sciences, Shanghai, China, 3 Shanghai Aquatic
ACCEPTED 19 June 2023 Technology Co., Ltd, Shanghai, China, 4 Department of Ecoscience, Aarhus University,
PUBLISHED 05 July 2023 Aarhus, Denmark, 5 Sino-Danish Centre for Education and Research (SDC), University of Chinese
Academy of Sciences, Beijing, China, 6 Limnology Laboratory and EKOSAM, Department of Biological
CITATION
Sciences, Middle East Technical University, Ankara, Türkiye, 7 Institute of Marine Sciences, Middle East
Huang R, Liu W, Su J, Li S, Wang L,
Technical University, Mersin, Türkiye
Jeppesen E and Zhang W (2023) Keystone
microalgae species determine the removal
efficiency of sulfamethoxazole: a case
study of Chlorella pyrenoidosa and
microalgae consortia.
In recent years, antibiotics pollution has caused serious harm to the aquatic
Front. Plant Sci. 14:1193668.
doi: 10.3389/fpls.2023.1193668 environment, and microalgae mediated degradation of antibiotics has attracted
COPYRIGHT
increasing attention. However, the potential toxicity of antibiotics to keystone
© 2023 Huang, Liu, Su, Li, Wang, Jeppesen microalgae species or their microalgae consortia, and the impact of microalgal
and Zhang. This is an open-access article
diversity on antibiotic removal need to be further studied. In this study, we
distributed under the terms of the Creative
Commons Attribution License (CC BY). The investigated the removal efficiency and tolerance of five freshwater microalgae
use, distribution or reproduction in other (Chlorella pyrenoidosa, Scenedesmus quadricauda, Dictyosphaerium sp.,
forums is permitted, provided the original
author(s) and the copyright owner(s) are
Haematoccocus pluvialis, and Botryococcus braunii) and their microalgae
credited and that the original publication in consortia to sulfamethoxazole (SMX). We found that the removal efficiency of
this journal is cited, in accordance with SMX by C. pyrenoidosa reached 49%, while the other four microalgae ranged
accepted academic practice. No use,
distribution or reproduction is permitted between 9% and 16%. In addition, C. pyrenoidosa, S. quadricauda, and
which does not comply with these terms. Dictyosphaerium sp. had better tolerance to SMX than H. pluvialis, and their
growth and photosynthesis were less affected. At 10 and 50 mg/L SMX, the
removal capacity of SMX by mixed microalgae consortia was lower than that of C.
pyrenoidos except for the consortium with C. pyrenoidos and S. quadricauda.
The consortia generally showed higher sensitivity towards SMX than the
individual species, and the biochemical characteristics (photosynthetic
pigment, chlorophyll fluorescence parameters, superoxide anion (O 2 - ),
superoxide dismutase activity (SOD), malondialdehyde (MDA) and extracellular
enzymes) were significantly influenced by SMX stress. Therefore, the removal of
antibiotics by microalgae consortia did not increase with the number
of microalgae species. Our study provides a new perspective for the selection
of microalgal consortia to degrade antibiotics.

KEYWORDS

sulfamethoxazole, microalgae consortia, diversity, ecotoxicity, removal

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1 Introduction not enhance but potentially reduce the removal efficiency of SMX.
Our results contribute to a better understanding of the ecotoxicity
The relationship between biodiversity and ecosystem functioning of SMX and its removal by microalgae and microalgae consortia.
is a subject of widespread concern and has been studied in both
terrestrial and aquatic ecosystems (Diaz and Cabido, 2001). Increased
species diversity typically leads to higher functional differences in the 2 Article types
community, which may result in more effective access to and use of
resources in turn (Loreau and Hector, 2001; Fargione et al., 2007). Original Research.
Several studies have shown that high species diversity of plants,
animals, and microorganisms can resist the negative effects of a
changing external environment (Ghorbannezhad et al., 2018; 3 Materials and methods
Norberg, 2000; Vacek et al., 2021). In addition, species diversity
may have effect on the degradation of pollutants in the 3.1 Chemicals
environment. For example, inoculating microbial communities
isolated from in-situ contaminated soil can effectively improve the SMX was purchased from Sangon Biotech Co., Ltd (Shanghai,
degradation of polycyclic aromatic hydrocarbons (Li et al., 2008). And China) with >98% purity. We used 0.1% mol/L NaOH as a solvent
microalgae consortia have proven to significantly improve the to fully dissolve 0.1 g SMX. Then, we added 0.1 mol/L HCl to adjust
degradation of cefradine, p-chlorophenol, and other pollutants in the initial pH 7 of the test medium (Chen et al., 2020b). The
the water environment in comparison with monocultures (Lima et al., chemicals were used of analytical grade.
2004; Xiao et al., 2021). However, Choudhury et al. (2018) and Brisson
et al. (2020) pointed out that mixed cultivation of plants is not
necessarily better than single cultivation in resource utilization and 3.2 Algal cultures
pollutant removal.
Antibiotics, widely used in aquaculture andanimal husbandry, Five microalgae: Chlorella pyrenoidosa (FACHB-10),
have become an important class of contaminants of emerging Scenedesmus quadricauda (FACHB-44), Dictyosphaerium sp.
concern (Kümmerer, 2009; Liu et al., 2018; Li et al., 2020). (FACHB-2072), Haematoccocus pluvialis (FACHB-1928) and
Previous studies have shown that the removal of antibiotics Botryococcus braunii (FACHB-357) were purchased from the
depends on physical adsorption, advanced oxidation technology Freshwater Algae Culture Collection of the Institute of
and microbial degradation (Dutta and Mala, 2020; Huang et al., Hydrobiology (FACHB-Collection), Wuhan City, China. The
2021). Recently, microalgae-mediated biodegradation has attracted microalgae were cultivated in 3000 mL Erlenmeyer flasks
attention and proven to be an environmentally effective and safe containing BG-11 medium and incubated in a homoeothermic
antibiotic removal technology (Xiong et al., 2021; Yu et al., 2022). incubator at 25 ± 1°C under 3000 lux illumination with a light-
Microalgae such as Chlorella sp., Dictyostelium sp., and dark period of 12:12 h. To maintain the logarithmic growth of the
Scenedesmus obliquus have been shown to effectively degrade microalgae and reduce any effect caused by minor differences in
antibiotics (Chen et al., 2020a; Chen et al., 2020b; Yang et al., photo irradiance, the flasks were arranged randomly and gently
2020; Aydin et al., 2022). Nevertheless, microalgae have specific shaken three times a day (Wan et al., 2015). All experimental
preferences for antibiotic types, and different microalgae have devices used for the algal culture/for algal cultivation were
different cell hydrophobicity, which affect the degradation of autoclaved at 121°C for 30 min before use.
antibiotics (Bai and Acharya, 2016; Xiong et al., 2017c).
Therefore, recent studies have focused on the consortia of
microalgae and bacteria or other microalgae to improve the 3.3 Experimental design
removal efficiency of antibiotics (Rodrigues et al., 2020; Rodrigues
et al., 2021; Wang et al., 2022). However, whether the determinant To explore differences in toxicity to and removal efficiency of
of antibiotic removal efficiency of microalgae consortia is higher antibiotics by keystone microalgae species in monocultures or when
than for keystone species needs further studies. combined, we designed two experiments: microalgae screening and
We investigated the removal and tolerance capacity to microalgae consortia.
sulfamethoxazole (SMX) of five green microalgae (C. pyrenoidosa,
S. quadricaudas, Dictyostelium sp., H. pluvialis and Botryococcus 3.3.1 Microalgae screening
braunii) and then screened several combinations of these According to previous research and our pre-experiment results
microalgae for consortia effects. To explore the difference in the our preliminary experimental results on nine microalgae species
efficiency of removing antibiotics by different microalgae consortia, (Supplementary Figure 1), we selected five microalgae, C.
we measured a series of biochemical indicators. We hypothesized pyrenoidosa, S. quadricauda, Dictyosphaerium sp., H. pluvialis and
that the keystone microalgae species had a strong removal capacity B. braunii, which have shown tolerance and removal capacity to
for SMX. Besides, consortia of individual keystone microalgae with organic pollutants in the environment (Cheirsilp et al., 2016; Chen
a high removal efficiency of SMX and other microalgae species may et al., 2020a; Yang et al., 2020; Aydin et al., 2022), as experimental

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objects and explored their tolerance to and removal capacity of sample was centrifuged at 6500 rpm for 15 min. After removing the
SMX. The SMX solution was prepared with BG-11 medium with supernatant, 5 mL of 95% methanol was added to the sample, which
the experimental concentration of 5 mg/L and added to the five was then placed in a 60 °C constant temperature water bath for
microalgae cultures. The experiment was then conducted in 500-mL 10 min. The mixed samples were centrifuged at 6500 rpm for
Erlenmeyer flasks containing 400 mL of BG-11 medium inoculated 15 min, and absorbance of the supernatant at 665, 652, and 470 nm
with 15 mg/L microalgal cell suspension. Besides, the control was measured with a spectrophotometer. Chlorophyll a, chlorophyll
comprised an algal medium without added SMX, and an abiotic b and carotenoids were calculated as follows:
control group contained only SMX ([CK]). Three replicates were set
up for each treatment and control group, and all flasks were Chlorophyll a (Ca, mg=L) = 16:82 A665 − 9:28 A652
incubated under the same conditions used for the inoculum
culture for 16 days. Samples were collected on days 2, 4, 6, 8, 10, Chlorophyll b (Cb, mg=L) = 36:92 A652 − 16:54 A665
12, 14, and 16 to determine each index.
Carotenoids (mg=L) = (1000 A470 − 1: 91Ca − 95: 15Cb)=225
3.3.2 Microalgae consortia Moreover, chlorophyll fluorescence parameters, including
Based on the results of our first experiment, we selected three of photosynthetic system II (PSII) maximum photosynthetic rate (Fv/
the five species: C. pyrenoidosa , S. quadricauda and Fm) and actual photosynthetic efficiency (Yield), were measured
Dictyosphaerium sp. C. pyrenoidosa, which had the strongest using a pulse amplitude-modulated fluorometer (Phyto-PAM,
tolerance and removal capacity to SMX, was selected as control Walz, Effeltrich, Germany) equipped with an emitter-detector-
(C), and groups where C. pyrenoidosa was combined with the other fiberoptic unit with an irradiance of 16 mmol photons/m2/sec PAR.
two microalgae respectively acted as treatments (Table 1). All
microalgae in the treatment group were inoculated at the same
fresh weight ratio, and the total content of microalgae in the final 3.6 Antioxidant responses
system was 15mg/L. Microalgae consortia experiments were carried
out with 10 and 50 mg/L SMX, respectively. According to the Samples of microalgae suspension (5 mL) were collected on day
microalgae screening experimental results, SMX can be mostly 3 and 7 to determine superoxide anion. The samples were
removed by microalgae in the first 7 days. Therefore, a 7-days centrifuged at 7000 rpm for 20 min, and then the supernatant
exposure experiment was conducted where photosynthetic was removed. Then 1 ml of extract was added, crushed with a cell
pigments, photosynthesis, and the activities of intracellular and crusher for 18 min, and centrifuged at 10000 rpm at 4 °C for 20 min.
extracellular enzymes of microalgae consortia were measured. The concentration of O2- was determined according to the assay kit
(Beijing Solabao Technology Co., Ltd., China).
In addition, the concentrations of SOD and MDA were
3.4 Measurement of cell growth determined using assay kits (Nanjing Jiancheng Bioengineering
Institute, China) following the manufacturer’s protocol. The
Algal cell growth in each treatment was measured according to extraction steps were as follows: 1) 10 mL samples were collected
Peng et al. (2014). The algal biomass of five microalgae was and centrifuged at 6500 rpm for 10 min at 4°C (Eppendorf,
calculated according to its linear relationship with an optical Centrifuge 5810R), 2) the biomass pellet was washed with 0.01 M
density of 680nm (OD680) (Supplementary Table 1). sodium phosphate buffer (pH 7.4), crushed and centrifuged, and 3)
the enzymes obtained from the disrupted microalgae were extracted
using 500mL of sodium phosphate buffer.
3.5 Measurement of photosynthetic
pigment and chlorophyll
fluorescence parameters 3.7 Determination of extracellular
enzyme content
The modified hot methanol method was used to determine the
cytochrome of microalgae (Xiong et al., 2016). Briefly, 5 mL of the The change characteristics of extracellular enzyme activity of
microalgae were determined by API ZYM (Merier, France), which
TABLE 1 Experimental setup of the combined experiment. is a laboratory kit for semi-quantitative analysis of the production of
hydrolytic enzymes. 3mL samples were collected and centrifuged at
Group name Microalgae species 7000 rpm for 15 min to obtain the supernatant containing
CK Abiotic control group extracellular enzymes. Supernatants (65 mL) were dispensed into
the 20 microcupules of API ZYM strips and incubated at 37°C for
C C. pyrenoidosa
4 h. After that, 30 mL of the commercial reagents ZYM A and ZYM
C+S C. pyrenoidosa + S. quadricauda B was added to all the microcupules to develop chromogenic
C+D C. pyrenoidosa + Dictyosphaerium sp. substrates. The color reactions were read after 5 min, and the
results were recorded at four grades from 0 to 4 according to the
C+S+D C. pyrenoidosa + S. quadricauda + Dictyosphaerium sp.
color rendering degree in the microcupules (Martinez et al., 2016).

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3.8 Determination of antibiotic pyrenoidosa exposed to SMX was significantly higher than that of
concentration the control group (p<0.05) (Figure 2A), Fv/Fm of Dictyostelium sp.
and S. quadricauda were not higher (Figures 2B, C), for H. pluvialis
The samples were filtered through a 0.22 mm polytetrafluoroethylene and B. braunaii it was significantly lower (p<0.05) (Figures 2D, E).
(PTFE) filter, and the residual SMX concentration was determined by SMX did not exert obvious stress on C. pyrenoidosa, S. quadricauda,
high performance liquid chromatography (HPLC, Alliance 2695 system, and Dictyostelium sp. but promoted the synthesis of chlorophyll a
USA). A total of 2 mL of the samples was separated using an ACQUITY (Supplementary Figure 2). In addition, during the whole
UPLC BEH C18 chromatography Column [250 × 4.6 mm, 5 μm, waters, experimental period, the Yield of the microalgae treatment group
USA) at a column temperature of 30°C. The mobile phase consisted of a was different from that of the control group, especially that of the B.
mixture of solution A (acetonitrile and LCMS-grade water (30: 70 v/v)] braunaii treatment group showed a very significant decrease
and solution B (0.1% formic acid in LCMS-grade water) at a flow rate of (p<0.01) (Supplementary Figure 3).
1.0 mL/min.
The removal rate (%) of SMX was calculated using the following 4.1.2 SMX removal of five microalgae
formula: C. pyrenoidosa had the highest removal rate of SMX (49%)
during the 16 days exposure. The removal rates of S. quadricauda
Removal rate ( % ) = (1 − Ct =C0 )  100 % and Dictyostelium sp. were 16% and 13%, respectively, and 10% and
where C0 (mg/L) is the initial concentration of antibiotics at 9% for H. pluvialis and B. braunii, respectively. (Figure 3).
time 0, and Ct (mg/L) is the concentration of antibiotics at time t. Compared with CK group, the removal rate of SMX by C.
pyrenoidosa was significantly increased (p<0.01), and higher than
that of the other four microalgae. Besides, the removal rate of SMX
3.9 Statistical analysis by S. quadricauda and Dictyostelium sp. was also significantly
higher than that of CK group (p<0.05).
To determine significant differences between the control and
treatment groups, a one-way analysis of variance (ANOVA) was
conducted using SPSS statistics software (version 22, Chicago, IL, 4.2 Microalgae consortia
USA). Values with p< 0.05 and p< 0.01 were considered significant
and very significant, respectively. To reveal the relationship between 4.2.1 Effects of SMX on pigment and
the variation of various indicators of microalgae and the photosynthetic parameters
experimental period, principal component analysis (PCA) was At different concentrations of SMX treatment, the content of
applied using R 4.1.1 statistical computation software. In addition, three photosynthetic pigments in each treatment group showed
hierarchy cluster analysis of the extracellular enzyme was upward trends (Figure 4). However, when the concentration of
performed using “pheatmap” package in R 4.1.1 statistical SMX increased to 50 mg/L, the total amount of photosynthetic
computation software. All figures were generated using Origin pigment synthesis decreased (Figure 4). When treated with 10 mg/L
2021 software (OriginLab, Northampton, MA, USA). SMX, there was no significant difference in chlorophyll a content
between the treatment and control groups at the end of the
experiment. However, when SMX reached 50 mg/L, the
4 Results chlorophyll a content of [C+S] group and [C+S+D] group was
significantly lower than in the control group (p<0.05) (Figures 4A,
4.1 Microalgal screening B). at the two SMX concentrations, the content of chlorophyll b
in [C+S] group fluctuated greatly, but the content of chlorophyll b
4.1.1 Effects of SMX on algal growth in [C+D] group and [C+S+D] group was significantly lower than in
and photosynthesis the control group (p< 0.05) (Figures 4C, D). In addition, the change
The growth ability of the five microalgae differed, and they also of carotenoids in the [C+S] group fluctuated more at the two SMX
had different responses to SMX (Figure 1). Compared with the concentrations. (Figures 4E, F). When exposed to SMX on day 2,
control groups, C. pyrenoidosa, S. quadricauda and Dictyostelium Fv/Fm of all groups was significantly inhibited (p<0.05) and then
sp. grew better in the medium containing SMX, especially the gradually recovered on day 3 (Supplementary Figures 4A, B). Yield
biomass of C. pyrenoidosa increased significantly compared with was significantly higher for all groups than in the control group
the control group (p<0.05) (Figures 1A, C, D). By contrast, the (p<0.05) (Supplementary Figures 4C, D).
growth of H. pluvialis and B. braunaii was significantly inhibited
(p<0.05) (Figures 1B, E). During the course of the experiment, the m 4.2.2 Effect of SMX on the intracellular
of five microalgae showed a decline to a varying degree, most antioxidant system of microalgae
pronounced for H. pluvialis. In the 10 mg/L SMX treatment, the concentrations of O2-, SOD,
After exposure to SMX, F v /F m of the microalgae was and MDA in the [C] group, [C+S] group, and [C+D] group showed
significantly reduced (p<0.05) except for S. quadricauda early in no significant change on day 3 and day 7 (Figures 5A, C, E). However,
the experiment (Figure 2). On day 14 and 16, Fv/Fm of C. the contents of O2-, SOD, and MDA in [C+S+D] group were

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A B

C D

FIGURE 1
Differences in the algal biomass of five microalgae (A: C. pyrenoidosa, B: S. quadricauda, C: Dictyosphaerium sp., D: H pluvialis and E: B braunii)
during 16 days of cultivation. Error bars represent standard deviation (n=3). Columns with different letters indicate significant differences (p<0.05)
between the control and treatment.

significantly higher than in the other groups, and the content on day 3 treatment (p<0.05), and the content on day 3 was 1.7-fold higher than
was significantly higher than on day 7 (p<0.05). When SMX reached on day 7. The content of MDA in [C+S+D] group was not
50 mg/L, the contents of O2- and SOD in the [C+S+D] group significantly different from that in the 10 mg/L SMX treatment,
increased significantly compared with those in the 10 mg/L SMX and the MDA content exhibited no significant change on day 3 and
treatment, and the contents on day 3 were 1-fold and 1.6-fold higher day 7 as the [C] group and the [C+D] group (Figure 5F).
than on day 7, respectively. In addition, the contents of O2- and SOD
in [C] group and [C+S] group were significantly higher on day 3 than 4.2.3 Effects of SMX on extracellular enzyme
on day 7 (p<0.05), but there was no significant change in [C+D] The heatmap revealed changes in the activity of extracellular
group (Figures 5B, D). However, the content of MDA in [C+S] group enzymes throughout the experiment. Overall, 7 of the 20 enzymes
was significantly higher than in the other groups below 50 mg/L SMX could be detected on day 3 and day 7 of the experiment, respectively,

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A B

C D

FIGURE 2
Effects of the SMX on the maximum photosynthetic efficiency (Fv/Fm) of five microalgae (A: C. pyrenoidosa, B: S. quadricauda, C: Dictyosphaerium
sp., D: H pluvialis and E: B braunii) during 16 days of cultivation. Error bars represent standard deviation (n=3). Asterisks indicate significant
differences between the control and treatment groups (p< 0.05*).

namely acid phosphatase, naphthol-AS-BI-phosphate, esterase (C4), phosphatase gradually decreased in all treatment groups but the
lipase esterase (C8), leucine aminopeptidase, valine aminopeptidase activity on day 7 of the experiment was higher than that on day 3.
and cystine aminopeptidase (Figure 6). At both SMX concentrations,
C4 and C8 did not appear on day 3 of the experiment but were 4.2.4 Removal of SMX by different
recorded on day 7, and the number and activity decreased with the microalgae consortia
increasing SMX concentration. The activities of the three The antibiotic content of treatment groups decreased
aminopeptidases were greater on day 3 than on day 7, being higher significantly during the first two days of the experiment (p<0.05).
in the [C+D] and [C+S+D] groups and lower in the [C+S] group. In With enhanced SMX concentration, the removal capacity of all
addition, with the increased SMX concentration, the activity of treatment groups decreased. [C] group and [C+S] group had a

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FIGURE 3
The removal rate (%) of SMX for five microalgae during 16 days of exposure. CK was an abiotic control containing 5 mg/L SMX. Error bars represent
standard deviation (n=3). Asterisks indicate significant differences between the control and treatment groups (p< 0.05*; p< 0.01**).

stronger removal capacity than the other groups whose removal even decompose and absorb antibiotics as organic substances (Kiki
rates reached 11.1% and 10.9%, respectively, at 10 mg/L SMX. et al., 2020). Some studies have shown that Dictyosphaerium sp. can
However, although [C+D] and [C+S+D] had the capacity to remove combine with the photocatalyst bismuth vanadate (BiVO4) to
the different concentrations of SMX, SMX was significantly lower improve the removal of sulfamethazine (SM2), while H. pluvialis
than for the [C] and [C+S] groups (p<0.05) (Figure 7). can efficiently degrade nearly 10 kinds of antibiotics through
membrane photobioreactors (Chen et al., 2020c; Claude et al.,
4.2.5 PCA 2022). Due to the different modes of action and drug affinity of
On day 3 and 7 of the SMX treatment, PC1 constituted 50.4% of microalgae, these microalgae did not show high degradation rates
the total variance in all groups, and PC2 accounted for an additional for SMX in this study. Therefore, C. Pyrenoidosa is a keystone
30.3% (Figure 8). The distribution of each microalgae consortium in microalgae species for degrading SMX. However, when we
PCA was significantly different on day 3 and day 7 after exposure to combined C. pyrenoidosa with two other microalgae, the
SMX. On day 3, the contents of O2-, SOD, and MDA of the microalgae consortia showed a higher photosynthetic pigment
microalgae consortia had changed most significantly, while on content and stronger photosynthesis than in the C. pyrenoidosa
day 7 mainly photosynthetic pigment and chlorophyll monoculture after the SMX treatment, and the activity of O2- and
fluorescence parameters of microalgae consortia had changed. antioxidant enzymes in the antioxidant system of the microalgae
consortia was higher than for the individual microalgae. The PCA
results also revealed that the effects of SMX on antioxidant enzymes
5 Discussion and photosynthesis of microalgae consortia were stronger than that
of individual microalgae species C. pyrenoidosa. Chlorophyll is an
We found that the tolerance and removal efficiency of SMX by important component of light capture and energy transduction in
the five different microalgae varied. C. pyrenoidosa showed the photosynthesis and photosynthetic reaction, including light
highest removal capacity, followed by S. quadricauda and reaction, Calvin cycle, and starch synthesis. Some emerging
Dictyosphaerium sp. Previous studies have shown that Chlorella pollutants can affect the accumulation of chlorophyll content in
sp. has excellent dissipation to many antibiotics, so it is often the microalgae (Telfer, 2002; Del Campo et al., 2007). And the increase
preferred microalgae to remove antibiotics (Song et al., 2019; Chen in chlorophyll content in cells can serve as a protective mechanism
et al., 2020b; Xiong et al., 2020). Besides, C. pyrenoidosa had better to eliminate the accumulation of reactive oxygen species in
growth and higher photosynthesis in the culture system containing chloroplasts (Kasahara et al., 2002; Tsiaka et al., 2013; Xiong
SMX. Other studies have shown increases in the biomass of C. et al., 2016). In addition, the antioxidant properties of carotenoids
pyrenoidosa treated with both chloramphenicol and florfenicol (Lai can prevent lipid peroxidation by inhibiting the production of
et al., 2009). Stimulated growth might reflect that C. pyrenoidosa singlet oxygen and free radicals in cells, thereby promoting the
can resist the persecution of antibiotics at low concentrations and stability of photosynthesis and protecting cells (Jahns and

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A B

C D

E F

FIGURE 4
Effects of two concentrations of SMX on the cytochromes of different microalgae consortia during 7 days. (A, C, E) 10 mg/L; (B, D, F) 50 mg/L. Error
bars represent standard deviation (n=3). Asterisks indicate significant differences between the control and treatment groups (p< 0.05*).

Holzwarth, 2012). When the antioxidant defense system is When C. pyrenoidosa was combined with other microalgae, the
stimulated under stress conditions, its antioxidant enzyme activity removal efficiency of SMX was never higher than that of the
will increase (Qian et al., 2008; Singh et al., 2018). Throughout the individual keystone microalgae C. pyrenoidosa, and it even declined
entire experimental cycle, the changes in photosynthetic pigments for some of the consortia: the removal efficiency of [C+S] to SMX was
and antioxidant enzyme activity of microalgae consortia were close to that of keystone microalgae C. pyrenoidosa, but the removal
significantly stronger than those of individual microalgae. Our capacity of [C+S+D] group was weaker than that of the keystone
results, therefore, indicate that the microalgae consortia were microalgae and the consortia containing two microalgae species ([C
more sensitive to SMX than the individual microalgae species, +S] and [C+D]). Thus, the increase in the richness of the different
which is consistent with the findings of Xiong et al. (2017b) in microalgae taxa did not improve the removal rate of SMX. Lima et al.
their study of the effects of enrofloxacin on microalgae consortia. (2003) found that the higher the proportion of the core microalgae

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A B

C D

E F

FIGURE 5
Effects of different concentrations of SMX on the intracellular antioxidant system of microalgae consortia during 7 days of exposure. Superoxide
anion (O2-) content (A, B) superoxide dismutase (SOD) activity (C, D) malondialdehyde (MDA) content (E, F). Error bars represent standard deviation
(n=3). Columns with different letters indicate significant differences (p<0.05) between the control and treatment groups.

Coenochloris pyrenoidosa in the microalgae consortia, the higher the cultivation of multiple microalgae may lead to competition for
degradation rate of the microalgae consortia to p-nitrophenol. organic matter in the substrate, thereby affecting the metabolism of
Therefore, the lower removal rate of SMX in the [C+S+D] group SMX by the microalgae itself. In co-culture systems, there are
may likely be due to the decreased proportion of the keystone interactions between microalgae species, such as competition or
microalgae C. pyrenoidosa in the consortia because of resource symbiosis, so their capacity to degrade pollutants may also be
competition. Besides, In the study on the co- metabolism of SMX affected by such interactions (White et al., 2014; Goncalves et al.,
by C. pyrenoidosa, it was found that the addition of organic substrate 2017). The reported mechanisms of antibiotic removal by microalgae
or other nutrients can accelerate the potential of microbial include photolysis, biosorption, bioaccumulation, and intracellular
biodegradation of antibiotics, which is related to the characteristics and extracellular biodegradation (Liu et al., 2021; Yu et al., 2022). In
of additional substrates that can maintain biomass production and our study, photodegradation to remove SMX was negligible, and
act as an electron donor for co-metabolism of non-growth substrates other studies (Xiong et al., 2016; Xiong et al., 2017a) have revealed
(Xiong et al., 2020; Gao et al., 2022). Therefore, we speculate that co- that the biosorption and bioaccumulation of emerging pollutants

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Huang et al. 10.3389/fpls.2023.1193668

A B

FIGURE 6
Heat map analysis of the extracellular enzyme of different microalgae consortia on day 3 and day 7 of exposure to SMX at three concentrations: 10
mg/L (A) and 50 mg/L (B). The names of the different types of extracellular enzymes are distinguished by letters (C4, esterase; C8, lipase esterase; C,
Cystine aminopeptidase; A, Acid phosphatase; N, Naphthyl-AS-BI-phosphate; L, Leucine aminopeptidase; V, Valine aminopeptidase). The color of
each section is proportional to the significance of change in the biochemical parameters (red, relatively high; blue, relatively low).

contribute little to microalgae-mediated bioremediation. Therefore, microorganisms. Therefore, there may be a symbiotic relationship
the main mechanism of SMX removal by microalgae is the between C. pyrenoidosa and S. quadricauda (Johnson and Admassu,
biodegradation induced by the enzyme system of microalgae. The 2013; Koreiviene et al., 2014), which can maintain a stable growth
results of the API-ZYM reagent strip demonstrated that the state under SMX stress. The symbiotic relationship between them
extracellular enzyme activity of [C+S] group was always low even may have been destroyed when adding Dictyosphaerium sp., because
with the increase of SMX concentration, while the extracellular they need to mobilize more extracellular enzymes to decompose and
aminopeptidase and phosphatase of [C+D] group and [C+S+D] cycle nutrients. SMX can promote gene expression by affecting the
group were active. Nitrogen compounds are the products of the metabolic process of non-coding RNA, leading to the destruction of
nitrogen cycle, which begins with the hydrolysis of protein by the ultrastructure of microalgae cells, affecting the permeability of cell
aminopeptidase (Mayer, 1989), and phosphatase participates in the membranes and the activity of antioxidant enzymes (Xu et al., 2022).
phosphorus cycle (Martinez et al., 2016; Hu et al., 2022). The In this study, the intracellular antioxidant enzymes of the [C+S+D]
substances produced by these cycles can maintain the growth of group were always in a more active state, and the content of O2- was

A B

FIGURE 7
The removal rate (%) at different concentrations of SMX in three different microalgae consortia treatment groups (A) 10 mg/L; (B) 50 mg/L. Error bars
represent standard deviation (n=3). Asterisks indicate significant differences between the control and treatment groups (p< 0.05*; p< 0.01**).

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Huang et al. 10.3389/fpls.2023.1193668

FIGURE 8
Biplot from PCA integrating all the measured variables (Chl a, Chl b, carotenoids, Fv/Fm, Yield, MDA, O2-, SOD) for the two sampling times (days 3
and 7) and eight different treatments (shapes and colors interact in pairs: ◼-10 mg/L, ▲-50 mg/L; green-[C] group, red-[C+S] group, blue-[C+D]
group, orange-[C+S+D] group).

always at a higher level. This shows that under the stress of SMX, the review and editing. JS: Supervision, Writing-review and editing. SL:
balance between the generation and elimination of O2- was destroyed, Experiment, Analysis and Writing-review. LW: Supervision, Project
and the antioxidant enzyme could not effectively remove excessive administration. EJ: Writing-review and editing. WZ: Supervision,
O2-, so the physiological function and growth of the [C+S+D] group Project administration, Writing-review and editing. All authors
were under more severe stress. Additionally, when the SMX reached contributed to the article and approved the submitted version.
50 mg/L, the MDA content of [C+S] group significantly increased
and exceeded that of the other microalgae consortia. MDA is an
aldehyde product produced by lipid peroxidation caused by ROS,
which reflects the degree of lipid peroxidation of cell membranes and Funding
the tolerance of plants to stress (Celekli et al., 2013). MDA can also be
used as an indicator of NADPH-dependent oxidase and peroxidase, This work was supported by the Science and Technology
Commission of Shanghai Municipality (Grant No.19DZ1203404
which can effectively transform the accumulated pollutants in cells
through various enzyme systems (Apel and Hirt, 2013). Therefore, and No. 19DZ1204504). EJ was supported by the TÜBITAK
differently from the control group [C], the increase of MDA may be a program BIDEB2232 (project 118C250).
way to improve the antibiotic removal efficiency in the [C+S] group.

Acknowledgments
Data availability statement
We thank Anne Mette Poulsen for English edition and the
The original contributions presented in the study are included Shanghai Municipal Bureau of Ecology and Environment for its
in the article/Supplementary Material. Further inquiries can be support and assistance in this project.
directed to the corresponding author.

Author contributions Conflict of interest

RH: Experiment, Analysis, Visualisation, Writing draft, Writing- Author SL was employed by the company Shanghai Aquatic
review and editing. WL: Experiment, Analysis, Visualisation, Writing- Technology Co., Ltd.

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Huang et al. 10.3389/fpls.2023.1193668

The remaining authors declare that the research was conducted reviewers. Any product that may be evaluated in this article, or
in the absence of any commercial or financial relationships that claim that may be made by its manufacturer, is not guaranteed or
could be construed as a potential conflict of interest. endorsed by the publisher.

Publisher’s note Supplementary material

All claims expressed in this article are solely those of the authors The Supplementary Material for this article can be found online
and do not necessarily represent those of their affiliated at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1193668/
organizations, or those of the publisher, the editors and the full#supplementary-material

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