Chemosphere 238 (2020) 124680

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Review

Use of microalgae based technology for the removal of antibiotics

from wastewater: A review
Lijian Leng a, Liang Wei a, Qin Xiong a, Siyu Xu a, Wenting Li a, Sen Lv a, Qian Lu a,
Liping Wan b, Zhiyou Wen a, c, **, Wenguang Zhou a, *
a
School of Resources, Environmental & Chemical Engineering and Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of
Education, Nanchang University, Nanchang, 330031, China
b
Zhenghe Environmental Group, Nanchang, 330001, China
c
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, 50011, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Antibiotic toxicity and removal per-

formance depend on antibiotic and
algal species.

Antibiotic removal mechanisms are
mainly adsorption, accumulation,
and degradation.

Combining microalgae with irradia-
tion and oxidation favors antibiotic
degradation.

a r t i c l e i n f o a b s t r a c t

Article history: The antibiotic resistance induced by the release of antibiotics to the environment has urged research
Received 17 March 2019 towards developing effective technologies for antibiotic removal from wastewater. Traditional technol-
Received in revised form ogies such as activated sludge processes are not effective for antibiotic removal. Recently, microalgae-
29 July 2019
based technology has been explored as a potential alternative for the treatment of wastewater con-
Accepted 25 August 2019
Available online 26 August 2019
taining antibiotics by adsorption, accumulation, biodegradation, photodegradation, and hydrolysis. In
this review, the toxicities of antibiotics on microalgae, the mechanisms of antibiotic removal by
Handling Editor: Chang-Ping Yu microalgae, and the integration of microalgae with other technologies such as ultraviolet irradiation
(photocatalysis), advanced oxidation, and complementary microorganism degradation for antibiotic
Keywords: removal were discussed. The limitations of current microalgae-based technology and future research
Microalgal needs were also discussed.
Alga © 2019 Elsevier Ltd. All rights reserved.
Pharmaceutical and personal care products
(PPCPs)
Antibiotic resistance
Inhibitor toxicity
Wastewater treatment

* Corresponding author.
** Corresponding author. Department of Food Science and Human Nutrition, Iowa
State University, Ames, IA, 50011, USA
E-mail addresses: lljchs@126.com (L. Leng), wenz@iastate.edu (Z. Wen),
wgzhou@ncu.edu.cn (W. Zhou).

https://doi.org/10.1016/j.chemosphere.2019.124680
0045-6535/© 2019 Elsevier Ltd. All rights reserved.
2 L. Leng et al. / Chemosphere 238 (2020) 124680

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Inhibition of antibiotics to microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Factors affecting antibiotic removal performance by microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Effect of algal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Effect of antibiotic classes and concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.3. Effects of algal growth conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Antibiotic removal mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2. Accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.3. Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4. Photodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.5. Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Integration of microalgae-based antibiotic removal with other technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. Ultraviolet (UV) irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Advanced oxidation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.3. Integration with other microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6. Further research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6.1. The simultaneous removal of multiple antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6.2. The toxicity of degradation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.3. Accumulation of antibiotic resistance gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction Microalgae-based technology has also been introduced to treat

water or wastewater containing pharmaceutical and personal care
Antibiotics are commonly used to treat and prevent bacterial products (PPCPs). Studies have shown that microalgae can effec-
infections. Antibiotics such as penicillins, sulphonamides, cepha- tively remove PPCPs such as antibiotics from wastewaters (Bai and
losporins, macrolides, and quinolones have been widely used Acharya, 2016; Hom-Diaz et al., 2017; Villar-Navarro et al., 2018;
mainly in developing countries with 36% increase between 2000 Xiong et al., 2017a). For example, a high-rate algal pond (HRAP)
and 2010 (Thung et al., 2016). However, the prevalence of bacterial system dominated by Coelastrum sp. removed 64 PPCPs including
resistance is emerging as a significant concern due to the resultant 33 antibiotics (with an average concentration of 223 mg/L) from
higher medical costs, prolonged hospital stays, and increased municipal wastewater with average antibiotic removal rates being
mortality (Brown and Wright, 2016; Liu et al., 2016). 5e50% higher than the conventional activated sludge process
Wastewater treatment plants (WWTPs) are major sources of during a six-month-long process (Villar-Navarro et al., 2018). Wang
releasing antibiotics to the environment (Lai et al., 2017; Rizzo et al., et al. (2017) and Xiong et al. (2018b) summarized the potential of
2013). Due to the antibacterial natures, many antibiotics cannot be using microalgae to remove PPCPs. In the past three years, there has
effectively removed in WWTPs by activated sewage sludge, which is been a rapid increase in the number of publications related to
comprised of a group of bacteria (Baena-nogueras et al., 2017; Liu microalgae-based processes for antibiotics removal (Fig. 1). The aim
et al., 2018; Villar-Navarro et al., 2018). Many processes such as of the present review is to provide a comprehensive summary of
adsorption, advanced oxidation, and photocatalysis have been the recent progress of microalgae-based antibiotics removal pro-
studied to remove antibiotic (Homem and Santos, 2011; Tang et al., cess, with particular focus on the removal efficiency and mecha-
2016; Ternes et al., 2003; Wang et al., 2016; Wen et al., 2018; Zhou nisms. The current limitation in this area and future research
et al., 2017c). The antibiotic removal efficiency of adsorption process perspectives are also discussed.
is highly adsorbent-dependent, and the adsorbent is commonly
expensive (Ahmed et al., 2015; Tan et al., 2015). Advanced oxidation 2. Inhibition of antibiotics to microalgae
and photocatalysis may be generally effective but need intensive
expensive chemical agents or catalyst (Tang et al., 2016; Wen et al., The inhibition of microalgae induced by antibiotic is an impor-
2018; Jiang et al., 2018), in addition to the probable production of tant factor to consider in the development of algae-based antibiotic
secondary pollutants (Kang et al., 2012; Wang and Lin, 2012). On the removal processes. Antibiotics may affect algal growth through
other hand, microalgae wastewater treatment process is a biological inhibition of the synthesis of chemicals such as chlorophyll-a and
process and it needs few chemical agents and can be engineered to pigments and the activities of enzymes such as catalase and su-
effectively remove the emerging contaminants such as antibiotics peroxide dismutase (Perales-Vela et al., 2016; Bashir and Cho,
(Wang et al., 2017; Xiong et al., 2018b). 2016). Bashir and Cho (2016) reported that kanamycin and tetra-
Recently, microalgae-based technology has been widely re- cycline inhibited the growth and photosynthetic activity of Dic-
ported as an effective method to treat municipal and industrial tyosphaerium pulchellum and Micractinium pusillum through
effluents, with advantages such as CO2 fixation, contaminant regulating their protein synthesis. The decreased photosynthetic
removal, saving nutrients input, and potential of developing algae- activity of Chlorella vulgaris was observed when the alga was
derived products (Chen et al., 2015a; Bai and Acharya, 2019; Lu exposed to streptomycin (Perales-Vela et al., 2016). Species such as
et al., 2015; Zhou et al., 2011, 2012b, 2014, 2017a, 2017b). P. subcapitata, C. vulgaris, S. vacuolatus, and Desmodesmus
 L. Leng et al. / Chemosphere 238 (2020) 124680 3

20 antibiotics. The same algal species (P. subcapitata) is even more

resistant to ampicillin and amoxicillin as 2000 mg/L of these two
antibiotics only resulted in 10% algal growth inhibition (Magdaleno
et al., 2015). Table 1 summarizes the concentrations of antibiotics
15 commonly present in WWTPs effluent and the EC50 values of those
antibiotics to specific algal species. Since the antibiotic concentra-
Number of publications

tions in WWTPs effluents are several orders of magnitudes lower

10
5
0
2009 2010 2011 2012 2013 2014 2015
Publication year
Fig. 1. Number of publications on the topic of “(microalga*) and (antibiotic) and
(treatment or removal)” (data obtained from Web of Science on Jan. 10, 2019).
subspicatus are sensitive to antibiotics and thus are widely used as
biomarkers to monitor antibiotic contamination levels in the
environment (Va €litalo et al., 2017).
The inhibitory effect of antibiotics on the microalgae is
commonly quantified through the half maximal effective concen-
tration EC50, which is defined as the antibiotic concentration at
which 50% of algal growth is inhibited. Most antibiotics have EC50
values several orders of magnitudes higher than the concentrations
in wastewater or surface/groundwater, indicating microalgae
generally have good resistance to those antibiotics. For example,
the concentrations of ciprofloxacin, tetracycline, and sulfameth-
oxazole are approximately 1 mg/L in municipal wastewater, while
the EC50 values of these antibiotics are 65 mg/L (to C. mexicana),
3.31 mg/L (to P. subcapitata), and 0.146 mg/L (to P. subcapitata),
respectively (Va€litalo et al., 2017; Wang et al., 2017; Yang et al.,
2005). In general, the EC50 value of an antibiotic depends on the
specific antibiotic and microalgae species, for example, sulfamo-
nomethoxine has an EC50 value of 5.9 to C. vulgaris and 9.7 mg/L to
Isochrysis galbana (Huang et al., 2014). For the alga P. subcapitata,
the EC50 values of ciprofloxacin and gentamycin are 11.3 mg/L and
12.9 mg/L, respectively; while cephalotin and vancomycin show
much higher EC50 values (>600 mg/L) (Magdaleno et al., 2015),
indicating the stronger resistance of P. subcapitata to these two
than the EC50 value, the use of the microalgae for treating antibi-
otics may be feasible after process development.
In general, the inhibitory effect of combined antibiotics can be
more severe than that caused by a single antibiotic. For example,
the 96 h-EC50 values of erythromycin, enrofloxacin, and
erythromycin-enrofloxacin mixture to C. vulgaris were 85.7, 124.5
and 39.9 mg/L, respectively, indicating the synergistic joint effect of
the two antibiotics (Wang et al., 2019). The inhibition of sulfa-
methoxazole and trimethoprim mixture on N. oculate was also
2016 2017 2018
considerably higher than that of a single of the two antibiotics
(Teixeira and Granek, 2017). The adverse effects of tetracycline/
oxytetracycline/chlortetracycline mixtures on the algal community
were more significant than that of any single of these three anti-
biotics (Taşkan, 2016).
It should be noted that the use of growth-based EC50 value for
the assessment of antibiotic toxicity may not be universally appli-
cable to evaluate the real toxicity of the substance. Other microalgae
activities such as photosynthesis, antioxidant system activities, and
carotenoid biosynthesis can also be influenced by antibiotics and
may be more indicative than biomass growth. For example, the
photosynthesis activity of chlorophytes can be more sensitive than
growth, as a result, the EC50 value based on the photosynthetic rate
of Desmodesmus subspicatus was only half of the EC50 based on cell
number (growth-based) (Guo et al., 2016a). Therefore, additional
measures, along with cell growth, should be used in order to provide
a comprehensive insight into the impacts of antibiotics on algae and
reveal the removal potential of the antibiotics.
3. Factors affecting antibiotic removal performance by
microalgae
3.1. Effect of algal species
The antibiotic removal rate highly depends on algal species.
Chlorella is very effective for the removal of many antibiotics. For
example, C. pyrenoidosa is commonly used to remove 7-amino
cephalosporanic acid (7-ACA), amoxicillin, cefalexin, cefixime,
cefradine, and ceftazidime (Table 2). Xiong et al. (2017a) studied the

Table 1
Concentrations of selected antibiotics in the environment and ecotoxicological effects.

Antibiotic Concentrations of antibiotics in effluent from municipal EC50 value (mg/L), time, species (ref.)
wastewater treatment plant (mg/L) (ref.)

Amoxicillin 1.34 (V€

alitalo et al., 2017) >50, 96 h, P. subcapitata (Va €litalo et al., 2017)
Cefradine 0.105 (Kim et al., 2017) 1.38, 72 h, M. aeruginosa (Chen and Guo, 2012)
1.77, 72 h, S. obliquus (Chen and Guo, 2012)
Cefalexin 0.13 (V€alitalo et al., 2017) 2.5 (Verlicchi et al., 2012)
Ceftazidime 3.68 (Liu et al., 2017b) >1.3 (Bengtsson-Palme and Larsson, 2016)
Ciprofloxacin 1.05 (Va€litalo et al., 2017) 65, 96 h, C. mexicana (Xiong et al., 2017c)
113, 72 h, P. subcapitata (Va€litalo et al., 2017)
Enrofloxacin 0.27 (V€
alitalo et al., 2017) 18.7, 72 h, P. subcapitata (Va€litalo et al., 2017)
5.57, 72 h, D. subcapicatus (Va €litalo et al., 2017)
Sulfamethazine <0.16 (Verlicchi et al., 2012) 1.231, 96 h, S. obliquus (Xiong et al., 2018c)
Sulfamethoxazole 1.94 (V€
alitalo et al., 2017) 0.146, 96 h, P. subcapitata (Va €litalo et al., 2017)
1.54, 24 h, S. vacuolatus (V€alitalo et al., 2017)
0.108, 96 h, S. obliquus (Xiong et al., 2018c)
Tetracycline 1.40 (V€
alitalo et al., 2017) 3.31, 72 h, P. subcapitata (Va€litalo et al., 2017)
Tilmicosin 0.018 (Verlicchi et al., 2013) -a
Trimethoprim 1.15 (V€
alitalo et al., 2017) 80.3, 72 h, P. subcapitata (Va€litalo et al., 2017)
a
Data not available.
 4
Table 2
Removal of antibiotics by microalgae-based technology and removal mechanisms.

Antibiotic Microalgae-based technology Initial antibiotic concentration and removal rate, Wastewater category Removal mechanisms Ref.
hydraulic retention time

7-amino Chlorella sp. 100 mg/L and 100%, 97 h BM medium Adsorption > hydrolysis, photolysis Guo et al.
cephalosporanic acid Chlamydomonas sp. 100 mg/L and 100%, 135 h BBM medium (2016b)
(7-ACA) Mychonastes sp. 100 mg/L and 100%, 135 h BG-11 medium
7-ACA C. pyrenoidosa 40 mg/L and 96.1%, 24 h (light) BG-11 medium Adsorption, biodegradation Yu et al. (2017)
Amoxicillin C. pyrenoidosa 100e300 mg/L and 100%, over 48 h BG-11 medium Algal action Li et al. (2015)
400e500 mg/L and 83e89%, 36e48 h
C. pyrenoidosa þ H2O2/Fe2þ 1 g/L and 93.3%, 48 h Algal action > Fenton oxidation
Amoxicillin S. obliquus þ UV irradiation 100 mg/L and 98.5%, 24 h (UV irradiation at 280 nm BG-11 media Photodegradation, biodegradation, and Yang et al. (2017)
for 5 min) hydrolysis
Selenastrum capricornutum þ UV irradiation 100 mg/L and 85.8%, 24 h (UV irradiation at 365 nm
for 5 min)
C. pyrenoidosa þ UV irradiation 100 mg/L and 85%, 24 h (UV irradiation at 365 nm
for 5 min)
Antibiotic mixtures a
Mixture of algae-bacteria consortia (dominanted 28e1054 mg/L, 38.6e97.8%, 144 h Real domestic wastewater -b Villar-Navarro
by Coelastrum sp.) in HRAP et al. (2018)
Cefradine C. pyrenoidosa 100e500 mg/L and 7e23%, 48 h BG-11 medium Algal action Li et al. (2015)
C. pyrenoidosa þ H2O2/Fe2þ 1 g/L and 92.2%, 48 h Algal action > Fenton oxidation
Cefalexin C. pyrenoidosa 10 mg/L and 94.9%,48 h BG-11 medium Algal action > self-degradation Guo and Chen

L. Leng et al. / Chemosphere 238 (2020) 124680

(2015)
Cefalexin Nonliving lipid-extracted Chlorella sp. 49.17 mg/L and 71.2%, 2 h Deionized water Adsorption Angulo et al.
Nonliving Chlorella sp. 49.17 mg/L and 82.8%, 7 h Deionized water Adsorption (2018)
Cefalexin C. vulgaris þ UV irradiation 40 mg/L and 97.15%, 24 h (UV irradiation at 280 nm EPA medium Photodegradation Liu et al. (2017b)
for 1 h)
C. pyrenoidosa þ UV irradiation 1.1 mg/L and 99.23% 24 h (UV irradiation at 185 nm Photodegradation Liu et al. (2017b)
for 1 h)
Cefixime C. pyrenoidosa 10 mg/L and 100%,48 h BG-11 medium Algal action > self-degradation Guo and Chen
(2015)
Cefradine C. pyrenoidosa 10 mg/L and 89.9%,48 h BG-11 medium Algal action > self-degradation Guo and Chen
(2015)
C. pyrenoidosa þ activated sludge 100 mg/L and 97.9%,48 h Algal action > activated sludge Guo and Chen
(2015)
Cefradine C. pyrenoidosa 30 mg/L and 75.9%, 48 h BG-11 medium Algal degradation Chen et al.
60 mg/L and 54.6%, 48 h (2015b)
Cefradine S. obliquus þ UV irradiation 100 mg/L and 62.5%, 24 h (UV irradiation at 365 nm BG-11 media Photodegradation, biodegradation, and Yang et al. (2017)
for 5 min) hydrolysis
Selenastrum capricornutum þ UV irradiation 100 mg/L and 61.8%, 24 h (UV irradiation at 365 nm
for 5 min)
C. pyrenoidosa þ UV irradiation 100 mg/L and 29.4%, 24 h (UV irradiation at 365 nm
for 5 min)
Ceftazidime C. pyrenoidosa 10 mg/L and 89.7%,48 h BG-11 medium Algal action > self-degradation Guo and Chen
(2015)
Ceftazidime C. pyrenoidosa 40 mg/L and 92.7%, 6 h (light) BG-11 medium Adsorption, biodegradation Yu et al. (2017)
Ceftazidime C. pyrenoidosa 40 mg/L and 80e99.2% in 6 h (depending on BG-11 media Biodegradation, photodegradation, Du et al. (2015b)
different light and nutrient supply) adsorption and hydrolysis
Ciprofloxacin Chlamydomonas mexicana 2 mg/L and 13.0%, 11 d Bold's Basal Medium Biodegradation, accumulation and Xiong et al.
adsorption (2017c)
C. pitschmannii 2 mg/L and 1.6%, 11 d
O. multisporus 2 mg/L and 2.0%, 11 d
C. vulgaris 2 mg/L and 0, 11 d
Chlamydomonas mexicana 2 mg/L and 56%, 11 d Bold's Basal Biodegradation > accumulation, adsorption
Medium þ sodium acetate
(4 g/L)
Ciprofloxacin Nannochloris sp. 57 ng/L and 100%, 7 d Water from Las Vegas Wash Direct photolysis Bai and Acharya
(2017)
Ciprofloxacin Mixture of algae-bacteria consortia in pilot high 1.31 mg/L and 20.1%, 24 h (8 h sunlight/16 h dark) Real domestic wastewater Photodegradation during daytime, and Hom-Diaz et al.
rate algae pond (HRAP) adsorption during night time (2017)
 Mixture of algae-bacteria consortia in 1-L HRAP 1.11 mg/L and 83.8%, 24 h Photodegradation
Enrofloxacin S. obliquus 1 mg/L and 23%, 11 d Bold's Basal Medium Biodegradation > accumulation, adsorption Xiong et al.
(2017a)
Chlamydomonas mexicana 1 mg/L and 25%, 11 d
C. vulgaris 1 mg/L and 26%, 11 d
Ourococcus multisporus 1 mg/L and 18%, 11 d
Micractinium resseri 1 mg/L and 20%, 11 d
Microalgae consortia of the above five species 1 mg/L and 26%, 11 d
Florfenicol Chlorella sp. 46 mg/L and 97% BG-11 medium Biodegradation > accumulation, adsorption Song et al. (2019)
159 mg/L and 74%
Levofloxacin C. vulgaris 1 mg/L and 91.5%, 11 d Bold's Basal Medium þ1% Accumulation and biodegradation Xiong et al.

L. Leng et al. / Chemosphere 238 (2020) 124680

NaCl w/v (2017b)
Sulfamethazine Scenedesmus obliquus 0.025e0.25 mg/L and 31.4e62.3%, 12 d Bold's Basal Medium e Xiong et al.
(2019)
Sulfamethoxazole Scenedesmus obliquus 0.025e0.25 mg/L and 27.7e46.8%, 12 d Bold's Basal Medium e Xiong et al.
(2019)
Sulfamethoxazole Nannochloris sp. 10 mg/L and 32%, 14 d (12 h light/12 h dark) F/2 medium Algae-mediated photolysis Bai and Acharya
(2016)
Sulfamethoxazole Nannochloris sp. 360 ng/L and 11%, 14 d Water from Las Vegas Wash Algae-mediated photolysis, accumulation Bai and Acharya
(2017)
Sulfamethoxazole C. pyrenoidosa 2e8 mg/L and 48.5e69.9%, 13 d F/2 medium Accumulation and biodegradation Sun et al. (2017)
Tetracycline Algae with activated sludge 2 mg/L and 97%, 24 h (12 h light and 12 h dark) Real domestic wastewater Photodegradation, biodegradation, Norvill et al.
adsorption and hydrolysis (2017)
Tilmicosin Chlorella PY-ZU1 0.01e50 mg/L and 90.2e99.8%, 9 d SE medium Adsorption, photodegradation, and Cheng et al.
biodegradation (2017)
Trimethoprim Nannochloris sp. 1.6 ng/L and 11%, 350 h Water from Las Vegas Wash Biodegradation, photolysis Bai and Acharya
(2017)
Trimethoprim Nannochloris sp. 10 mg/L and 0, 14 d F/2 medium e Bai and Acharya
(2016)
a
The concentrations (mg/L) and removal rates (%) for some major antibiotics are: ofloxacin, 704 and 83.9; ciprofloxacin, 918 and 73.4; norfloxacin, 513 and 88.7; azithromycin, 290 and 55.9; erythromycin, 173 and 91.3;
clarithromycin, 1054 and 97.8; spiramycin, 321 and 96.9; tetracycline, 296 and 43.6; chloramphenicol, 46 and 91.3; sulfadiazine, 44 and 38.6; trimethoprim, 252 and 40.5; metronidazole, 28 and 39.3.
b
No removal or not reported.

5
6 L. Leng et al. / Chemosphere 238 (2020) 124680
removal of levofloxacin by various species including C. vulgaris,
Chlamydomonas mexicana, Chlamydomonas pitschmannii, Our-
ococcus multisporus, Micractinium resseri, and Tribonema aequale
and reported that C. vulgaris achieved a higher removal rate than
other species. In another study, the same group also reported that
C. vulgaris was more effective to remove enrofloxacin than
S. obliquus, Chlamydomonas mexicana, Ourococcus multisporus, and
Micractinium resseri (Xiong et al., 2017b). The antibiotic removal by
other algal species may be less effective. For example, the removal
rates of spiramycin and amoxicillin by Microcystis aeruginosa were
12.5e32.9% and 30.5e33.6%, respectively (Liu et al., 2012). The
removal of ciprofloxacin by Chlamydomonas mexicana was also
limited to 13% (Xiong et al., 2017c). Nannochloris sp. had limited
removal rate for trimethoprim (0e11%) and sulfamethoxazole
(11e32%) (Bai and Acharya, 2017, 2016). However, studies have also
found some antibiotics more vulnerable to other algal species than
to Chlorella. For example, cefradine can be removed by more than
60% using S. obliquus, while the removal by C. pyrenoidosa was less
than 30% (Yang et al., 2017).
Development of robust algal strains for antibiotic removal can
be accomplished through various approaches. A large pool of algae
species in the nature provides the possibility to isolate robust
strains that can tolerate antibiotics (Leng et al., 2018; Zhou et al.,
2017a). The polyculture of multiple algal strains with comple-
mentary antibiotic removal capabilities may also increase the effi-
ciency and stability of the microalgae-based antibiotic removal
system (Godwin et al., 2017). In addition, genetic modification and
metabolic evolution of the algal strain can also enhance antibiotic
removal efficiency (Shen et al., 2015). It was reported that genetic
adaptation and physiological acclimatization contributed to the
increased tolerance of N. oceanica to Zeocin (Lin et al., 2017) and the
enhanced levofloxacin removal by C. vulgaris (Xiong et al., 2017a).
3.2. Effect of antibiotic classes and concentration
Microalgae-based antibiotic removal also highly depends on the
antibiotic concentrations and classes. Most antibiotics pose in-
hibitions on algal growth when the concentration is close to or
higher than the algal tolerable level. As the antibiotic concentra-
tions commonly studied are in the magnitude of several mg/L to
hundreds of mg/L (Table 2), which are comparable to or even higher
than the corresponding EC50 values (Table 1), low antibiotic
removal rates would be expected (Chen et al., 2015b; Li et al., 2015;
Sun et al., 2017). However, it should be noted that the increasing
antibiotic concentration within a certain range may have little in-
fluence on its removal when the concentration is within algal
tolerable level. For example, the critical toxic concentration for
amoxicillin to C. pyrenoidosa was 300e400 mg/L (Li et al., 2015).
Different antibiotic has diverse critical concentration, and that of
tilmicosin to Chlorella PY-ZU1 was ~30 mg/L (Cheng et al., 2017).
Interestingly, greater removal rates of sulfamethazine and sulfa-
methoxazole in higher concentration mixtures were observed than
those in lower ones possibly because of the efficient adaptive
mechanism of Scenedesmus obliquus to these antibiotics (Xiong
et al., 2019).
The removal efficiency of antibiotics by microalgae also highly
depends on antibiotic classes. Different antibiotic kinds, even at a
similar concentration level, can result in significantly different
removal efficiencies. For example, microalgae C. pyrenoidosa can
remove 83e100% amoxicillin, but can only remove 7e23% cefradine
(Li et al., 2015) even the two antibiotics were at the same concen-
tration. When 7-ACA (40e100 mg/L) was treated by Chlorella sp.,
Chlamydomonas sp., Mychonastes sp., or C. pyrenoidosa, 96e100%
removal rate can be achieved (Guo et al., 2016b; Yu et al., 2017).
However, only 0e13% of ciprofloxacin was removed by strains such
as Chlamydomonas mexicana, C. pitschmannii, O. multisporus, or
C. vulgaris (Xiong et al., 2017c) even the concentration was at a
relatively low level (2 mg/L). Similarly, only 18e25% of enrofloxacin
(1 mg/L) was removed by S. obliquus, Chlamydomonas mexicana,
C. vulgaris, Ourococcus multisporus, or Micractinium resseri (Xiong
et al., 2017b). Clearly, ciprofloxacin and enrofloxacin are more
difficult to be removed than 7-ACA. It is interesting that the pres-
ence of multiple antibiotic classes may enhance their removal ef-
ficiency by using microalgae. For example, when S. obliquus was
used to remove the antibiotic sulfamethazine, the removal rate
increased from 18.5-23.6% to 38.2e51.8% when another antibiotic
sulfamethoxazole was added to the system (Xiong et al., 2018a).
Sulfamethoxazole may have increased the activities of cytochromes
P450 enzyme, such as aminopyrine N-demethylase and aniline
hydroxylase (Xiong et al., 2018a), which may have higher affinity
toward sulfamethazine than sulfamethoxazole.
3.3. Effects of algal growth conditions
The growth conditions of microalgae including nutrients, pH,
illumination, temperature, CO2 concentration, salinity, design of
the photobioreactor, dissolved oxygen, and algal growth inhibitors
are critical to the antibiotic removal efficiency (Norvill et al., 2016).
In general, favorable algal growth conditions are beneficial to the
removal of antibiotics.
Addition of appropriate nutrients can promote the algal growth
metabolisms, leading to enhanced antibiotic removal performance.
For example, addition of 4 g/L sodium acetate to Chlamydomonas
Mexicana cultures enhanced ciprofloxacin removal by more than 3
times (Xiong et al., 2017c) and addition of NaCl (1%, wt) to
C. vulgaris culture resulted in 3-fold higher levofloxacin removal
than the control (Xiong et al., 2017a). The same group also reported
that the removal of levofloxacin by S. obliquus can be increased
from 4.5% to 93.4% with the increase of salinity from 0 to 171 mM
NaCl (Xiong et al., 2017d). NaCl (salinity stress) promoted the bio-
accumulation and subsequent intracellular biodegradation of lev-
ofloxacin, due possibly to the increased quantity of ferroxidase-like
protein, which can direct electrons toward cytochrome P450-
catalyzed hydroxylation of levofloxacin (Xiong et al., 2017d).
The pH of the algal growth system can mediate the hydrolysis of
some ionic antibiotics, and thus pH is crucial to the removal effi-
ciency for those antibiotics. An alkaline pH can change the ionic
state of tetracycline due to the hydrolysis of this antibiotic,
enhancing its removal efficiency (Norvill et al., 2017). Trimethoprim
and sulfamethoxazole were in a stable form when pH was slightly
basic and thus underwent slow direct photolysis (Bai and Acharya,
2017). However, Guo et al. (2016b) reported that the pH variation
did not affect 7-ACA removal rate, due to the reason that 7-ACA is
relatively stable at the pH range (6.3e8.0) studied.
Illumination causes photodegradation of antibiotics, and thus it
can be used to improve antibiotic removal efficiency (Du et al.,
2015a). Strong light irradiation can also increase dissolved oxygen
and pH due to the elevated photosynthesis of microalgae, which in
turn induced the generation of reactive oxygen species and
enhanced tetracycline removal (Norvill et al., 2017).
Other algal growth conditions also play important roles in
antibiotic removal. It is reported that during a six-month operation
in HRAP, increasing temperature by 10  C and irradiance by 20 MJ/
m2/d considerably promoted the removal of antibiotics such as
ofloxacin, ciprofloxacin, and norfloxacin (Villar-Navarro et al.,
2018). The inoculum size and the hydraulic retention time of the
microalgae growth system should also be adjusted based on the
antibiotic concentration and algal species (Bengtsson-Palme and
Larsson, 2016; Heidari et al., 2016). In addition, algae can use an
extended lag phase to adapt the algae to an extremely hostile
 L. Leng et al. / Chemosphere 238 (2020) 124680
environment such as the sharp increase of the tetracycline con-
centration from 1 mg/L to 20 mg/L (Xiong et al., 2018d) and the
presence of inhibitory compounds such as N-heterocyclic com-
pounds (Godwin et al., 2017; Tommaso et al., 2015).
4. Antibiotic removal mechanisms
When exposed to antibiotics, microalgae display a series of re-
sponses to survive and remove the toxic antibiotics. During the
removal process, adsorption, accumulation, biodegradation, pho-
todegradation, and hydrolysis of antibiotics may take place (Table 2
and Fig. 2).
4.1. Adsorption
Adsorption is the process of removing pollutants by passive
binding of the pollutants to a solid material (Bai and Acharya, 2016;
Davis et al., 2003). The removal of antibiotics by adsorbent such as
biochar, activated carbon, and nano-materials has been extensively
reported (Ahmed et al., 2015; Huang et al., 2017; Xiong et al., 2018c;
Zhang et al., 2016; Zhou et al., 2017d). Microalgae can be an effec-
tive adsorbent for antibiotics removal. The adsorption can be
accomplished by the functional groups and polymer assemblages
(similar to cellulose, hemicelluloses, and proteins, etc.) on the algal
cell walls and it is an extracellular process (Davis et al., 2003; Xiong
et al., 2018b). The effectiveness of the adsorption process may be
assessed by using dead algal biomass to adsorb antibiotics, and
adsorption performance varies significantly depending on specific
antibiotics and microalgae due to their different hydrophilicity,
functionality, and structures. In general, it is desirable when the
antibiotic is more hydrophobic rather than hydrophilic and when it
carries an opposite charge to microalgae (Leng et al., 2015; Xiong
et al., 2018b).
Some antibiotics can be removed effectively by adsorption. For
example, 82.7% and 71.2% of the cefalexin (at 50 mg/L) in modeling
wastewater can be removed by Chlorella sp. biomass and the de-
fatted biomass of Chlorella sp., and the calculated theoretical
adsorption capacities were 129 and 63 mg/g, respectively (Angulo
et al., 2018). Very high adsorption capacity (295 mg/g) was ob-
tained when using lipid-extracted S. quadricauda biomass to
remove tetracycline (Daneshvar et al., 2018). Adsorption was also
found to be one of the main mechanisms for the removal of
Fig. 2. Antibiotic degradation and detoxification mechanisms by microalgae.
7
tetracycline in HRAP (Godos et al., 2012; Norvill et al., 2017). The
adsorption process is generally fast, e.g., 7-ACA can be removed by
microalgae adsorption within 10 min (Guo et al., 2016b). The
adsorption of antibiotic by microalgae biomass is mainly achieved
through hydrogen bonds, electrostatic attraction, partition, and
hydrophobic effect (Ahmed et al., 2015; Tan et al., 2015) (Fig. 3). In
some other studies, adsorption was not a dominant antibiotic
removal mechanism, but it can act as the first process for the af-
terward microalgae-based antibiotic removal mechanisms such as
accumulation and biodegradation (Yu et al., 2017).
4.2. Accumulation
Unlike adsorption which is extracellular, accumulation is an
intracellular process to remove pollutant from water (Bai and
Acharya, 2016; Davis et al., 2003). Some antibiotics can cross algal
cell membranes and then possibly be assimilated by the cells. The
intracellular accumulated antibiotics may be extracted by sonicat-
ion coupled with dichloromethane/methanol mixture (v:v ¼ 1:2)
extraction (Song et al., 2019). Algae accumulation has been re-
ported played an important role in the removal of antibiotics such
as trimethoprim, sulfamethoxazole, and doxycycline (Bai and
Acharya, 2017; Prata et al., 2018).
Some accumulated antibiotics can induce the production of
reactive oxygen species which is essential to control cellular
metabolism at normal concentrations, but resulting in severe
damage to cells or eventually death if in excess (Xiong et al., 2018a).
On the other hand, the algal cell can counteract to deplete the
antibiotics by metabolism. In this case, accumulation becomes a
pre-step for biodegradation (described in Section 4.3), and the
combination of accumulation and biodegradation in algal cells
contribute considerably to the completion of the assimilation of
some antibiotics. The accumulation of sulfamethazine in
C. pyrenoidosa was observed and then followed by biodegradation
(Sun et al., 2017). Levofloxacin was also removed by C. vulgaris
through accumulation and subsequent intracellular biodegradation
(Xiong et al., 2017a). Otherwise, antibiotics will be accumulated in
the organisms, and the antibiotics can further accumulate and
magnify through the food chain and eventually cause antibiotic
resistance in the human body.
8 L. Leng et al. / Chemosphere 238 (2020) 124680
Fig. 3. Possible mechanisms during the adsorption of antibiotics, adapted from (Tan et al., 2015) with permission.
4.3. Biodegradation
Biodegradation describes the process of breaking down antibi-
otics by algae within or outside the cells, with some broken-down
derivatives being further consumed by the algal cells (Gao and Chi,
2015; Ke et al., 2010; Naghdi et al., 2018; Zhou et al., 2013a). An
example of this mechanism is the intracellular degradation of cef-
tazidime and its basic parent structure 7-ACA by C. pyrenoidosa. In
this process, the removal of antibiotics was proceeded in three
steps: ceftazidime was firstly adsorbed on algae, then it was slowly
transmitted algal cell wall, and finally, it was broken down by algal
enzymes (Yu et al., 2017). Other intracellular degradation-based
antibiotics removal cases include levofloxacin removal by
S. obliquus (Xiong et al., 2017d) and its removal by C. vulgaris (Xiong
et al., 2017a). For the extracellular degradation process, the anti-
biotic is broken down by algal metabolites such as extracellular
enzymes, and the biodegraded intermediates/end-products may be
further metabolized by algal cells (Naghdi et al., 2018). The con-
tributions of the intracellular and extracellular mechanisms for
antibiotic removal may be differentiated by analyzing the antibiotic
intermediates/end-products in the medium and in biomass or by
using extra- and intra-cellular enzymes extracted from algae to
degrade antibiotics.
Algal metabolism for antibiotic degradation can be classified as,
co-metabolic degradation and metabolic degradation patterns. Co-
metabolic degradation uses non-specific enzymes to degrade the
antibiotics with additional carbon and energy input is needed,
while in the metabolic degradation process, algae utilize antibiotics
as the sole carbon and energy sources with specific enzymes. Both
of these two patterns contributed to levofloxacin biodegradation by
C. vulgaris (Wang et al., 2017; Xiong et al., 2017a). Augmentation by
the addition of organic substrates such as methanol or sodium
acetate can promote the biodegradation of ciprofloxacin because of
co-metabolism (Xiong et al., 2017c).
Based on enzyme functionalities, the antibiotic biodegradation
can be treated as a two-phase enzymatic catalysis process. In the
first phase, antibiotics can be enzymatically hydrolyzed, oxidized,
or reduced to more hydrophilic compounds using enzymes such as
monooxygenase and dioxygenase (Xiong et al., 2016), while in the
second, the hydrophilic compounds can be catalyzed by another
group of enzymes such as glutathione-S-transferases to produce
smaller molecules with lower toxicity (Xiong et al., 2018b), e.g.,
resulting in the opening of the epoxide ring of antibiotics to protect
against oxidative damage to algal cell (Ding et al., 2017).
Irrespective of the different antibiotic removal mechanisms
discussed above, hydrolysis, side chain breakdown, hydroxylation,
ring cleavage, demethylation, decarboxylation, and dehydrox-
ylation of antibiotics may take place when biodegrading antibiotics
(Chen and Xie, 2018; Xiong et al., 2017d, 2018a), e.g., biodegrada-
tion mechanisms of levofloxacin as shown in Fig. 4 include several
of these reactions.
4.4. Photodegradation
Photodegradation of antibiotics includes direct photolysis of
antibiotics contributed by light and their indirect photodegradation
which is induced by the reactive components produced by algae in
the presence of light.
Many antibiotics can be removed by direct photolysis in the
presence of light in algae-free conditions (Chen et al., 2016; Jiang
et al., 2018). By comparing the removal rate between daytime and
night, it was found that 40% of tetracycline in water can be removed
by direct sunlight (Norvill et al., 2017). The removal of tetracycline
(Godos et al., 2012), ciprofloxacin (Bai and Acharya, 2017), cefazolin
and cephapirin (Wang and Lin, 2012) in different microalgae-based
wastewater treatment systems were also reported to be a result
from direct photolysis.
The removal of certain antibiotics can be enhanced through
indirect photodegradation when algae present in the system. Dur-
ing indirect photodegradation, reactive oxygen species such as
 L. Leng et al. / Chemosphere 238 (2020) 124680
Fig. 4. Possible metabolic pathways of levofloxacin, reprinted from (Xiong et al.,
2017d) with permission.
hydroxyl radical can be generated from algal components, leading
to the break-down of the antibiotics. For example, when light
presented in the C. vulgaris culture, hydroxyl radical was generated,
resulting in rapid oxidation of norfloxacin (Zhang et al., 2012a). In
algae-bacteria based HRAP, tetracycline was removed almost
completely due to the indirect photodegradation mediated by dead
or active algal cells (Norvill et al., 2017). Indirect photodegradation
also contributed to the removal of ciprofloxacin (Hom-Diaz et al.,
2017), cefalexin and cephradine (Wang and Lin, 2012). Both direct
and indirect photodegradation contributed to cephradine removal
and the photodegradation products and degradation pathways of
cephradine are shown in Fig. 5.
However, some antibiotics are recalcitrant to light and the light-
induced algal reactive species. In these circumstances, photo-
degradation is not effective for antibiotic removal. For example,
sulfamethoxazole was only removed slightly (11%) in the presence
of Nannochloris sp. and light after 14-day cultivation (Bai and
Acharya, 2017). No photodegradation was observed when levo-
floxacin was treated with S. obliquus (Xiong et al., 2017d).
Fig. 5. Possible direct and indirect photodegradation products and degradation path-
ways of cephradine, reprinted from (Wang and Lin, 2012) with permission.
9
4.5. Hydrolysis
The break-down of some antibiotics can also be accomplished
by hydrolysis. It should be noted that hydrolysis reactions induced
by algal metabolites such as enzymes (enzymatic hydrolysis) can
also be regarded as biodegradation processes. Hydrolysis of 7-ACA
contributed to the removal of the b-lactam antibiotic due to the
ring-opening reactions of the b-lactam structure (Guo et al., 2016b).
Hydrolysis pathways for b-lactam antibiotic ampicillin varied
significantly depending on aqueous pH and temperature (Fig. 6);
increasing pH and temperature considerably increased the hydro-
lysis rates (Mitchell et al., 2014). Note that growth of algae may
change the pH of the culture, e. g, pH increase because of the
photosynthetic activity of microalgae, hydrolysis of pH-sensitive
antibiotics can be greatly influenced by these algae.
Hydrolysis reactions increased the polarity and hydrophilicity of
antibiotics and therefore facilitated antibiotics solubilization and
subsequent biodegradation by microalgae (Wang et al., 2018). Some
antibiotics such as fluoroquinolones and sulphonamides were
resistant to hydrolysis, e.g., sulphadiazine and sulphaguanidine
 ska et al.,
were hydrolytically stable at pH as low as 4.0 (Białk-Bielin
2012). Hydrolysis of tetracycline barely contributed to its removal
by microalgae (Norvill et al., 2017).
5. Integration of microalgae-based antibiotic removal with
other technologies
5.1. Ultraviolet (UV) irradiation
UV irradiation is a commonly used disinfection step in the
wastewater treatment and can enhance antibiotic removal by direct
and indirect photodegradation. Light-sensitive antibiotics can be
removed by UV irradiation through direct photolysis. During UV
irradiation, antibiotics absorb photons which can break some
chemical bonds of antibiotics (Du et al., 2015b; Liu et al., 2017b).
The resulted products were vulnerable to microalgae biodegrada-
tion (Liu et al., 2017b). Yuan et al. (2011) reported a rapid degra-
dation of doxycycline in the presence of UV light. More than 73% of
the cefradine (initial concentration of 150 mg/L) was removed with
UV irradiation (Du et al., 2015b).
UV irradiation can be combined with microalgae treatment to
enhance antibiotic degradation through indirect photodegradation.
Since UV light attenuates rapidly in wastewater within the range of
several centimeters to decimeters (Curtis et al., 1994), indirect
photodegradation can be the dominant mechanism for antibiotic
removal. Although individual algae C. pyrenoidosa was inefficient to
remove cefradine (<20%), the integrated algae-UV irradiation ach-
ieved a high cefradine removal rate (>78%) and reduced the toxicity
of the effluent by more than 50% (Du et al., 2015b). UV irradiation
was also a trigger for S. obliquus treatment of cefradine and amox-
icillin, a complete removal of these antibiotics were obtained when
coupling S. obliquus with UV irradiation (Yang et al., 2017). However,
it should be noted that UV irradiation (280e320 nm) may pose a
negative effect on algal growth and biochemical compositions
(Kumar et al., 2018). The use of UV-mutated algal species may be
favorable to avoid the negative effect of UV on algae (Liu et al., 2015).
5.2. Advanced oxidation processes
Advanced oxidation processes (AOPs) have been developed as a
pretreatment step to enhance the efficiency of the treatment of
wastewater containing recalcitrant organic compounds such as
antibiotics (Cheng et al., 2018; Guan et al., 2018; Liu et al., 2017a).
During the AOPs-algae treatment, the antibiotics can be removed
10 L. Leng et al. / Chemosphere 238 (2020) 124680
Fig. 6. Possible hydrolysis pathways for ampicillin in water at pH 4, 7, and 9 and 60  C: (a) amide hydrolysis and decarboxylation; (b) lactam hydration with acetic acid; (c)
ampicillin isomer; (d) lactam hydration; (e) lactam hydration isomer; (f) lactam hydration and decarboxylation, reprinted from (Mitchell et al., 2014) with permission.
directly by oxidation reactions. For example, the removal of
amoxicillin and cefradine by microalgae increased from 7.4 to 22.5%
to over 90% when coupling H2O2/Fe(II)-based Fenton reactions (Li
et al., 2015). Fenton reactions enhanced the biodegradability of
intermediates produced by the advanced oxidation of the antibi-
otics (Li et al., 2015).
On the other hand, antibiotics can also be removed by AOPs-
induced indirect photodegradation. For example, the presence of
photochemically reactive Fe(III) can induce reactive oxygen species
formation which in turn promoted the antibiotic removal by
microalgae (Batista et al., 2014). The addition of Fe(III) can also
enhance the photodegradation of norfloxacin by microalgae (Zhang
et al., 2012a). Ge and Deng (2015) reported the use of Fe(III)-
microalgae system to effectively remove the two fluoroquinolone
antibiotics, enrofloxacin and ciprofloxacin hydrochloride. However,
when using Fenton reagents in the algae-based antibiotic removal,
the dosage of Fenton reagents should be adjusted delicately as high
concentrations of H2O2 or Fe(II) can be lethal to microalgae (Li et al.,
2015).
5.3. Integration with other microorganisms
The performance on antibiotic removal by microalgae can be
enhanced by coupling with other microorganisms such as bacteria
and fungi. Many bacterial strains are capable of removing antibi-
otics (Wang et al., 2017). For example, sulfapyridine and sulfathia-
zole can be biodegraded effectively by bacteria Escherichia sp. HS21
or Acinetobacter sp. HS51 (Zhang et al., 2012b). Sulfamethoxazole
was removed effectively by Pseudomonas psychrophila HA-4 due to
the efficient complementary algal metabolic biodegradation (Jiang
et al., 2014). The fast biodegradation of ciprofloxacin by Thermus sp.
isolated from pharmaceutical sludge was also reported (Pan et al.,
2018).
The use of algal-bacterial consortia for removal of hazardous
contaminants has attracted increasing interests (Mun ~ oz and
Guieysse, 2006). Within the algal-bacterial symbiotic relationship,
Oxygen needed for bacteria can be photosynthetically produced by
microalgae in the presence of light and CO2, which is produced by
bacteria when oxidizing organic matter (Quijano et al., 2017). Other
interactions between these microorganisms include cooperative
interactions such as the use of microalgae as a protecting habitat for
bacteria to enhance bacterial growth, in addition to possible
competitive interactions such as the adverse effects on each other,
e.g., the excretion of algicidal or bactericidal substances (Gonçalves
et al., 2017). A C. pyrenoidosa-activated sludge integration system
was applied to effectively remove cefradine, and a removal effi-
ciency of 97.9% was obtained (Guo and Chen, 2015). Pretreatment of
wastewater containing amoxicillin as high as 150 mg/L by Chlorella
sp.-bacteria consortium in intertidal wetland sediment or activated
sludge resulted in >99% removal of the antibiotic (Shi et al., 2018).
The antibiotic biodegradation products from algae may be more
biodegradable to bacteria and vice versa. The complementary
degradation characteristics of multiple microorganisms and the
possible exchange of antibiotic degradation products between
algae and bacteria may have promoted the effectiveness of antibi-
otics removal (Guo and Chen, 2015; Mun ~ oz and Guieysse, 2006).
However, the photo-bioreactors for the microalgae-bacteria mixed
culture were not effective for tetracycline removal at high con-
centrations (Xiong et al., 2018d). In addition to bacteria, fungus is
another candidate to assist antibiotic removal with algae (Naghdi
et al., 2018). For example, the removal of antibiotic ranitidine by
C. vulgaris-Aspergillus niger biopellets was reported effective (Bodin
et al., 2016).
Co-culture of multiple microalgae species and the co-culture of
microalgae with bacteria or fungi may be more cost-effective for
antibiotic wastewater treatment. Note that the microalgae may be
easily harvested after co-culturing with bacteria or fungi (Chen
et al., 2018; Zhou et al., 2012a, 2013b), the integrated use of
microalgae with other microorganisms worth further in-
vestigations. On the other hand, whether if the co-culture can
contribute to the antibiotics resistance genes reducing is also a
research front of this technology.
6. Further research needs
6.1. The simultaneous removal of multiple antibiotics
Many studies have focused on the removal of a single antibiotic
class. The removal of the multiple antibiotics by microalgae is only
occasionally studied. In real wastewater, however, multiple anti-
biotics and other contaminants will most likely to co-exist. In
general, antibiotic removal efficiency is algal strain dependent and
antibiotic class-specific (Li et al., 2015; Ribeiro et al., 2018). In-
teractions between different antibiotics or between antibiotics and
 L. Leng et al. / Chemosphere 238 (2020) 124680
other substances make the microalgae removal process more
complicated. Future investigations are needed to assess the
possible adverse effects of multi-antibiotic.
6.2. The toxicity of degradation products
The removal of antibiotic compounds may not necessarily lead
to the elimination of toxicity. Depending on the structures of the
parents and the degradation pathways, some secondary com-
pounds may be even more toxic than the original compounds. For
example, the hydrolysis products of the oxytetracycline and tetra-
cycline were found more toxic than their parent antibiotics, as
indicated by the results from the acute toxicity test of these anti-
biotics and their hydrolysis metabolites to Vibrio fischer (Kang et al.,
2012). Photodegradation of cephalosporin resulted in compounds
with higher toxicity to Vibrio fischeri (Wang and Lin, 2012). Since
Vibrio fischeri commonly seen colonizing the light-producing organ
(photophore) of certain types of squids and fish as a mutualistic
symbiont (Abbas et al., 2018), the toxicity results obtained from
Vibrio fischeri test can be used to assess the toxicity to the related
organisms within the food chain. Therefore, the antibiotic degra-
dation products can be of significant concern. The toxicity results
obtained using microalgae also indicated the similar phenomenon.
The toxicity of cefapirin to Daphnia magna increased after UV/algae
treatment (Ribeiro et al., 2018), while the toxicity of cefradine to
Brachionus calycifloru remained unchanged even UV treatment was
further added to the treatment (Du et al., 2015b). The diverse
antibiotic degradation pathways and degradation products,
together with the complexity of wastewater usually impede the
complete antibiotic removal efficiency by microalgae. Systematic
studies on degradation pathways (removal mechanisms) and
resultant degradation products are needed in future investigations.
6.3. Accumulation of antibiotic resistance gene
Treatment or utilization of the microalgae biomass after anti-
biotic treatment is another issue. Antibiotic resistance, the spread
and accumulation of antibiotic resistance gene in the environment
and food chains, is emerging as a worldwide crisis as it can be
transferred to the human body and deteriorate human health. The
harvested biomass is not recommended to enter the food chain and
is advised to be processed by thermochemical methods for the
complete destruction of the antibiotics (Leng et al., 2019; Leng and
Huang, 2018; Zhuang et al., 2018).
7. Conclusions
The overall antibiotic removal performance suggests the great
potential of using microalgae-based technology to treat antibiotics-
containing wastewater. Antibiotics can be effectively removed by
adsorption, accumulation, hydrolysis, biodegradation, and photo-
degradation. The process optimization and its integration with
other treatment such as UV irradiation and advanced oxidation and
co-cultivation with bacteria/fungi can lead to an efficient antibiotic-
removal process.
Currently, the use of microalgae for antibiotic removal is still in
its infant stage. The potential of using microalgae-based technology
for antibiotic removal needs to be further evaluated using real
wastewater. The simultaneous removal of multi-antibiotic and
other pollutants, the toxicities of the intermediate/end-products
from antibiotics degradation, and the utilization of harvested
algae biomass remain to be addressed. Long-term operation per-
formance is also needed to evaluate the cumulative ecosystem
effects.
11
Acknowledgments
The study was supported by the major research and develop-
ment program of Jiangxi, China (No. 20182ABC28006) and the
National Natural Science Foundation of China (Nos. 51808278 and
51668044).
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