Accepted Manuscript

Removing ethinylestradiol from wastewater by microalgae mutant Chlorella

PY-ZU1 with CO2 fixation

Jun Cheng, Qing Ye, Ke Li, Jianzhong Liu, Junhu Zhou

PII: S0960-8524(17)31855-2
DOI: https://doi.org/10.1016/j.biortech.2017.10.036
Reference: BITE 19077

To appear in: Bioresource Technology

Received Date: 25 July 2017

Revised Date: 30 September 2017
Accepted Date: 7 October 2017

Please cite this article as: Cheng, J., Ye, Q., Li, K., Liu, J., Zhou, J., Removing ethinylestradiol from wastewater by
microalgae mutant Chlorella PY-ZU1 with CO2 fixation, Bioresource Technology (2017), doi: https://doi.org/
10.1016/j.biortech.2017.10.036

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 1 Removing ethinylestradiol from wastewater by microalgae
2 mutant Chlorella PY-ZU1 with CO2 fixation
3 Jun Cheng, Qing Ye, Ke Li, Jianzhong Liu, Junhu Zhou

4 State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

5 Abstract:

6 Removal of endocrine-disrupting chemical ethinylestradiol (EE2) from

7 wastewater by microalgal mutant Chlorella PY-ZU1 under 15% CO2 were investigated.

8 Moreover, the effects of EE2 on microscopic structure and antioxidation ability of

9 microalgal cells were determined. EE2 concentrations in range of 0.01–5mg L−1

10 stimulated microalgal growth. Increasing the original EE2 concentration from 0 to

11 5mg L−1 increased the cell fractal dimension from 1.38 to 1.59 and reduced the cell

12 size from 5.18 to 3.41μm. Meanwhile, superoxide dismutase and catalase activities,

13 which represented cellular antioxidant capacity, first increased from 44.59 and 0.54U

14 mL−1 to peak values of 65.57 and 1.49U mL−1, respectively, and then correspondingly

15 decreased to 34.36 and 0.36U mL−1. Malondialdehyde content that indicated the cell

16 oxidation damage degree first decreased from 2.57 to 2.03nmol mL−1, then increased

17 to 2.59nmol mL−1. The highest EE2 removal efficiency of 94% by Chlorella PY-ZU1

18 was achieved at the original EE2 concentration of 5mg L−1.

19 Keyword: Endocrine-disrupting chemicals; Microalgae; Ethinylestradiol; Fractal

20 dimension; Antioxidation ability


Corresponding author: Prof. Dr. Jun Cheng, State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027,
China. Tel.: +86 571 87952889; fax: +86 571 87951616. E-mail: juncheng@zju.edu.cn

1
 1 1. Introduction

2 Endocrine-disrupting chemicals (EDCs) in aquatic environments have been

3 extensively investigated. They are exogenous chemicals that can disrupt hormonal

4 signaling systems. These chemicals exist at a concentration range of 0.1–100 μg L−1

5 in groundwater, wastewater, and sewage sludge. Although EDCs residual

6 concentrations in aquatic environment are usually not high, these chemicals are

7 deemed to be emerging micro-pollutants due to their direct and potential threat to

8 environment and organisms (Zhou et al., 2014). The occurrence of thousands of

9 organic micro-pollutants, such as pharmaceutical compounds (PCs), personal care

10 products (PCPs), and steroid hormones in secondary wastewater effluent, may cause

11 additional negative impact on human health and the environment (Bueno et al., 2012;

12 Guerra et al., 2014). Several MPs are EDCs that can interfere with the endocrine (or

13 hormone system) of invertebrates and vertebrates, including mammals, and may

14 trigger unwanted ecological effects (Cruz-Morato et al., 2014). EDCs can also

15 influence the endocrine systems of shellfish, fish, and even humans at

16 environmentally relevant concentrations. EDCs are toxic to aquatic organisms, such

17 as fish and marine diatom Navicula incerta (Liu et al., 2010).

18 EDCs in the aquatic environment mainly originate from the discharging water of

19 wastewater treatment plants (WWTPs). These compounds are often recalcitrant and

20 insufficiently removed in conventional WWTPs, because conventional biological

21 treatments have yet to be designed to remove EDCs. As such, techniques with

22 enhanced safety, effectiveness, and usefulness for the removal of EDCs from
2
 1 wastewater should be established. Several methods, such as ozonation and reverse

2 osmosis, have been proposed to treat EDCs in waste water. However, ozonation may

3 produce toxic byproducts in wastewater, thereby inducing secondary pollution.

4 Reverse osmosis also cause many problems, including membrane pollution and high

5 cost. Conversely, using microalgae to remove pollutants from waste water has many

6 advantages.

7 Microalgae are utilized in wastewater treatment due to their fast growth rate,

8 high photosynthesis efficiency, and efficient adaptive ability and capacity to take

9 advantage of nutritions in wastewater. Microalgae is able to uptake and use inorganic

10 nutrients in waste water in their growth period, thereby reducing N, P, heavy metals

11 and other inorganic contaminants in waste water (Cheng et al., 2015; Sun et al., 2015).

12 Microalgae can degrade and transform antibiotics (Cheng et al., 2017), phenols

13 (Ghasemi et al., 2011), EDCs (Sole and Matamoros, 2016), and other organic

14 contaminants in waste water. Microalgae can treat waste water, and high-value-added

15 microalgal biomass can be further utilized as biodiesel feedstock (Levine et al., 2011)

16 or animal feeds (Cheng et al., 2016).

17 Among numerous EDCs, 17α-ethinylestradiol (EE2) possesses high endocrine

18 disrupting potency. EE2 is apparently recalcitrant and the main contributor to

19 estrogenic activity in environmental samples (Johnson and Williams, 2004; Thomas et

20 al., 2001). Hom-Diaz et al. (2015) examined EE2 removal (efficiency=60–95%) by

21 Selenastrum capricornutum and Chlamydomonas reinhardtii in anaerobic digester

22 centrate. Wang et al. (2017) investigated the removal of EE2 by Scenedesmus, and

3
 1 obtained a maximum EE2 removal efficiency of 75.3%. However, the removal

2 efficiencies of EE2 by microalgae remain low, and the influence of original EE2

3 concentrations on EE2 removal efficiencies by microalgae has yet to be evaluated.

4 Sole and Matamoros (2016) achieved a high EE2 removal efficiency (97%) by

5 using Chlorella sp.. However, antioxidative response of microalgae in EE2

6 wastewater has yet to be measured and discussed. Wang et al. (2013) investigated the

7 antioxidative response of S.capricornutum and Chlorella sp. in EE2 wastewater. Liu

8 et al. (2010) investigated EE2 removal by marine diatom Navicula incerta, and

9 quantified the superoxide dismutase (SOD) and catalase (CAT) activities of

10 microalgal cells. However, the effects of EE2 on the organic components and

11 microscopic structure of microalgal biomass have yet to be described, thereby could

12 not reveal the microscopic mechanism of EE2 removal and microalgae growth.

13 In this study, the removal efficiencies of EE2 from wastewater by the mutant

14 Chlorella PY-ZU1 under 15% CO2 and the cellular stress responses were investigated.

15 This study demonstrated the effects of EE2 concentration on microalgal growth and

16 EE2 removal efficiency, the influence of EE2 on microalgal microscopic structure and

17 antioxidation ability were also elucidated. The effects of various original EE2

18 concentrations on microscopic structure of microalgae were examined through

19 transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

20 Meanwhile, the influences of various original EE2 concentrations on the antioxidation

21 ability and degree of oxidative damages to the biomembrane system of microalgal

22 cells were examined. Furthermore, the organic components and lipid components of

4
 1 Chlorella PY-ZU1 biomass cultivated in EE2 wastewater were also measured.

2 2. Methods

3 2.1 Microalgal mutant and culture condition

4 Chlorella PY-ZU1 was obtained by the mutagenesis of Chlorella pyrenoidosa

5 with 500 Gy 60Co γ−ray radiation, then domesticated by continuous aeration with 15%

6 CO2 (Cheng et al., 2015). Chlorella PY-ZU1 cells were cultivated in Brostol’s solution

7 [also denoted as soil extract (SE)] with slight modifications as described in previous

8 study (Cheng et al., 2015).

9 Microalgal mutants were cultivated in a air-lift column bioreactor containing 300

10 mL of SE medium under continuous illumination (8000 lux) at 25 C. The inoculation

11 density was 0.4 g L−1. The growth medium was consecutively aerated with 15% CO2

12 at 30 mL min−1. The mixed gas of 15% CO2 was made by pure CO2 gas balanced with

13 N2. The cultivated condition described above was defined as the control.

14 2.2 EE2 addition

15 EE2 (purity ≥ 98%) was provided by Sigma-Aldrich (USA). The EE2 stock

16 solution was prepared with deionized water then diluted to different concentrations

17 before experiment. EE2 was added to sterilized SE medium before cultivation

18 experiment to obtain solutions with the concentrations as follows: 0 (control), 0.01,

19 0.1, 1, 2, and 5 mg L−1. Three replicates were established for each culture condition.

20 The experimental groups with activated or inactivated microalgae or without

5
 1 microalgae were used to compare the contributions of biodegradation, physical

2 absorption, and photodegradation to EE2 removal. All groups had an original EE2

3 concentration of 1 mg L−1. Inactivated microalgae were heat−killed in a drying oven

4 at 90 C for 12 h.

5 2.3 Analytical methods

6 When microalgae grew, 5 mL of each sample was centrifuged at 8000 rpm for 5

7 min and then washed thrice by deionized water. Microalgal biomass was obtained and

8 then dried at 90 C for 24 h to test the dry weight by gram per liter. Specific growth

9 rate (μ) of microalgae was calculated as follows:

10 (1)

11 where m2 is the dry weight at time t2 and m1 is the dry weight at time t1. CO2 fixation

12 efficiency was calculated as follows:

13 (2)

14 where is the carbon content (%) of microalgal dry weight m2 at time t2, is

15 the carbon content (%) of microalgal dry weight m1 at time t1, is the molecular

16 weight of CO2, and is the molecular weight of carbon.

17 On day 7 of growth, 35 mL of each sample was centrifuged at 6000 rpm for 20

18 min at 5 C to gather microalgal cells. Microalgal pellets were resuspended with 0.05

19 mol L-1 phosphate buffer (pH 7.8), placed in an ice-bath with ultrasonication (output

20 power=5 W) for 25 min, and then centrifuged at 11,000 rpm for 15 min at 5 C. The

21 supernate was collected to analyze CAT activity, SOD activity, and malondialdehyde

6
 1 (MDA) content. CAT and SOD activity were determined according to previously

2 described methods (Goth, 1991; Beauchamp and Fridovich, 1971). MDA, a kind of

3 lipid peroxidation product, was quantified according to the method of Hegedus et al.

4 (2001).

5 On day 7 of microalgal growth, 1.5 mL of samples from all conditions were

6 centrifuged at 8000 rpm for 5 min. The collected microalgae were immobilized with

7 2.5% glutaraldehyde solution and used for microstructural analysis by TEM (HT7700,

8 Hitachi, Japan) and SEM (SU-8010, Hitachi, The Netherlands). The microalgal

9 biomasses were centrifugally collected after 9 days' cultivation, and the supernates

10 were concentrated through solid-phase extraction. The concentrations of EE2 was

11 determined by HPLC-MS (UPLCIQPXE, Waters, America) with a column of Zorbax

12 SB C18. The mobile phases were water–acetonitrile (75:25 v/v) with the flow rate of

13 0.3 mL min−1. Mass spectroscopy was carried out on the model of MRM with an ESI+

14 source. Microalgal cells were freeze dried for Fourier transform infrared (FTIR)

15 spectrometry (Nocolet iS50, ThermoFisher, USA) and organic component analyses.

16 Lipid content was evaluated by the method of Bligh and Dyer (1959) with some

17 adjustments. Fatty acid methyl ester (FAME) content was measured using a gas

18 chromatograph (Agilent 7890, USA) with a column of HP-INNOWax and an FID

19 detector.

7
 1 3. Results and discussion

2 3.1 Growth and CO2 fixation by Chlorella PY-ZU1 in EE2 wastewater

3 Culture conditions like temperature, nutrients are essential for microalgal growth.

4 According to our previous studies (Cheng et al., 2015; Cheng et al., 2016; Cheng et al.,

5 2017), Chlorella PY-ZU1 specie highly tolerates harsh environment as long as carbon

6 and nitrogen sources are adequate; it can grow well in anaerobic digestion effluent

7 and other waste water environment. Fig. 1(a) illustrates the growth of Chlorella

8 PY-ZU1 in artificial wastewater with different concentrations of EE2. The final

9 biomass yield of Chlorella PY-ZU1 was 3.71 g L−1 in the control (0 mg L−1 EE2). The

10 final biomass yield of Chlorella PY-ZU1 initially increased and subsequently declined

11 when the original EE2 concentration was increased from 0 to 5 mg L−1. When the

12 initial EE2 concentration increased from 0 to 0.01 mg L−1, the final biomass yield

13 increased from 3.71 to 4.19 g L−1. However, further increase in the original EE2

14 concentration to 5 mg L−1 reduced the final biomass yield from 4.19 to 3.79 g L−1.

15 This biomass yield was still larger than that of control condition.

16 EE2 (0.01 mg L−1) greatly stimulated microalgal growth. However, 0.1−5 mg L−1

17 of EE2 reduced the biomass yield compared with 0.01 mg L−1 of EE2. This

18 phenomenon indicated that a high EE2 concentration inhibited microalgal growth.

19 Perron and Juneau (2011) found that EDCs, such as 4−nonylphenol and β−estradiol,

20 can inhibit the photosynthesis of several kinds of green algae. Balina et al. (2015)

21 found that 8 µg L−1 EE2 can reduce algal growth by 10%, and approximately 80 µg

8
 1 L−1 can inhibit algal growth by 50%. Low EE2 concentration (0.01 mg L−1) promotes

2 microalgal growth, and this phenomenon is known as hormesis in toxicology

3 (Calabrese and Baldwin, 2003). High concentrations of toxic chemicals cause

4 negative impacts on plants and microorganisms, such as growth and development

5 inhibition. By contrast, low concentrations of toxic chemicals can stimulate biological

6 processes (such as promoting growth and development). Low EE2 concentrations

7 probably activate maintenance and repair functions of microalgal cells, such as the

8 upregulation of proteins involved in cell protection (e.g. antioxidant enzymes). EE2 in

9 high concentration inhibited the growth of Chlorella PY-ZU1 cells, because such

10 concentrations can damage the maintenance and repair functions of microalgal cells.

11 Moreover, EE2 may inhibit photosynthetic activity of microalgal cells because of its

12 negative effect on Photosystem II of these cells. The biomass yield of microalgae

13 increases when the inhibitory effect on microalgal growth is lower than the

14 stimulating effect. Increasing the original EE2 concentration gradually improved the

15 inhibitory effect compared with the stimulating effect. This phenomenon resulted in a

16 reduced biomass yield. The concentration of EE2 in SE medium was limited, thereby

17 preventing a further increase in the original EE2 concentration to values higher than 5

18 mg L−1. Thus, EE2 positively affected the growth of Chlorella PY-ZU1 cells.

19 Chlorella PY-ZU1 exhibits good tolerance to EE2 and can grow well in EE2

20 wastewater.

21 Fig.1(b) shows the specific growth rate and CO2 fixation rate of Chlorella

22 PY-ZU1 in wastewater with different concentrations of EE2. Chlorella PY-ZU1 grew

9
 1 rapidly within the first 3 days, resulting in a relatively high specific growth rate and

2 CO2 fixation rate. At 0 mg L−1 EE2, the mean specific growth rate in the first 3 days

3 was 0.591, and the mean CO2 fixation rate was 1.412 g L−1d−1. As the initial EE2

4 concentration increased from 0 to 1 mg L−1, the mean specific growth rate in the first

5 3 days increased from 0.591 to 0.617, and the increase of mean CO2 fixation rate in

6 the first 3 days from 1.412 to 1.526 g L−1d−1. Because various nutrients were

7 sufficient and growth conditions were excellent for microalgae in the first 3 days, the

8 effects of EE2 concentrations on microalgal growth were indistinct with only little

9 variations. When the original EE2 concentration further increased to 5 mg L−1, the

10 mean specific growth rate in first 3 days decreased from 0.617 to 0.600, mean CO2

11 fixation rate decreased from 1.526 to 1.457 g L−1d−1. With increase in the original EE2

12 concentration, the mean specific growth rate and CO2 fixation rate in 7 days also

13 initially increased first and subsequently decreased, but the maximum point was

14 achieved at 0.01 mg L−1 EE2. This phenomenon may be due to the high cellular

15 activity of Chlorella PY-ZU1 in the first 3 days and high hormesis caused by EE2,

16 resulting in the stimulation of very rapid growth of the microalgae. Moreover,

17 microalgal function of scavenging reactive oxide species (ROS) caused by EE2 was

18 very active, and the tolerance ability to EE2 concentration of microalgae was

19 enhanced. Thus, up to 2 mg L−1 of EE2 could cause inhibition in specific growth rate

20 of Chlorella PY-ZU1. After the first 3 days, microalgal growth gradually became

21 stable, and microalgae grew slowly. During this period, cellular activity decreased, the

22 capacity to scavenge ROS decreased，and the tolerance ability to EE2 decreased.

10
 1 Consequently, the maximum mean specific growth rate and CO2 fixation rate in 7

2 days of growth was achieved at a lower EE2 concentration than that in 3 days of

3 growth. However, for the 5 mg L−1 original EE2 concentration, the specific growth

4 rate and CO2 fixation rate were still higher than those of the control. This result

5 indicated that the stimulating effect on microalgal growth caused by EE2 was still

6 higher than the inhibitory effect.

7 3.2 EE2 removal by microalgae Chlorella PY-ZU1

8 The variation in the EE2 removal efficiencies along with growth time by

9 activated, inactivated, and without microalgae in 1 mg L−1 EE2 wastewater was

10 illustrated in Fig.2(a). The control showed the lowest final EE2 removal efficiency at

11 39%, which was removed by photodegradation. The maximum EE2 removal

12 efficiency by inactivated microalgae was 54%. In addition to photodegradation,

13 partial EE2 was adsorbed onto the cytoderm or passed into cells through millipores on

14 the cytoderm due to the certain porosity of the cytoderm of dead microalgae. The

15 maximum EE2 removal efficiency by activated microalgae was 88 % attributed to the

16 integrated effects of photodegradation, physical absorption and biodegradation. EE2 is

17 probably biodegraded into three weakly toxic products through glucosylation process:

18 ethinylestradiol glucoside, 3-b-D-glucopyranosyl-2-hydroxyethinylestradiol, and

19 3-b-D-glucopyranosyl-6b-hydroxyethinylestradiol (Della Greca et al., 2008). This was

20 a general detoxification pattern which conjugates biomolecules, e.g. sugars, amino

21 acids or glutathione to the activated sites of xenobiotics.

11
 1 Fig.2(a) illustrates that EE2 removal efficiencies increased with growth period.

2 High EE2 removal efficiencies were quickly achieved in the first 24 h, and they were

3 much bigger than those on the other days, perhaps for the reason that the

4 concentration difference of EE2 between culture medium and inside cell was the

5 biggest at the first 24 h. As result, the driving power of EE2 absorption onto cell wall

6 and diffusion across the cell wall into cells was the biggest. Afterward, EE2

7 concentration difference declined, so the rate of EE2 absorption onto cytoderm and

8 diffusion through the cytoderm into cells slowly reduced.

9 The results of EE2 removal were desirable after 9 days of Chlorella PY-ZU1

10 culture. The EE2 removal efficiencies for every original concentrations all reached

11 more than 80 % [Fig. 2(b)]. When the initial EE2 concentration increased from 0.01 to

12 5 mg L−1, the EE2 removal efficiency increased from 83% to 94%, and the residual

13 EE2 increased from 0.002 to 0.318 mg L−1. As the original EE2 concentration

14 increased, the concentration difference between the culture medium and the inner

15 microalgal cells was enlarged. Therefore, the driving power of EE2 absorption on the

16 cytoderm and diffusion across the cytoderm into cells improved. However, a bigger

17 original EE2 concentration in culture medium implied that EE2 balance

18 concentrations between culture medium and cytoplasm were bigger. Thus, EE2

19 residual concentration increased with the original EE2 concentration.

20 Actual wastewaters have complicated composition. Some types of actual

21 wastewater contain toxic chemicals, refractory high-molecular-weight organics, and

22 some exhibit dark color and low light transmittance. Thus, some pretreatment

12
 1 methods should be applied to make wastewater suitable for microalgal growth, such

2 as ozonation, Fendon method, which can degrade toxic chemicals to aviod microalgal

3 toxication, decompose high-molecular-weight organics to nutrients for microalgal

4 growth, and increase light transmittance to improve microalgal growth.

5 3.3 Microscopic structure of microalgae Chlorella PY-ZU1 in EE2 wastewater

6 The effects of original EE2 concentrations on the microscopic structure of

7 Chlorella PY-ZU1 cells were studied by SEM and TEM. The SEM images show the

8 smooth surface and integrity of Chlorella PY-ZU1 cells in the culture medium without

9 EE2. Mild wrinkles and deformations were monitored on Chlorella PY-ZU1 cells in 1

10 mg L−1 EE2 wastewater. While the original EE2 concentration increased to 5 mg L−1,

11 the wrinkles on Chlorella PY-ZU1 cells increased.

12 The deformation degree and surface roughness of Chlorella PY-ZU1 cells

13 increased with original EE2 concentration. The fractal dimension (FD) was employed

14 to quantify the changes in the microalgal cell surfaces. This parameter indicates the

15 validity of spaces occupation by complicated shapes, and serves as a measure of the

16 irregularity of complicated shapes (Li et al., 2009). The SEM pictures of cells were

17 binarized firstly with MATLAB and then calculated as follows [Equation (3)]:

18 lim 0 log log ( 1 (3)

19 where d denotes FD, corresponds to the side length of a grid and N represents the

20 grids' number that demanded to cover the fractal (Cheng et al., 2014). Fig. 3 shows

21 the changes in the FD of microalgae cultivated in EE2 wastewater with various

13
 1 original concentrations. The FD of microalgal cells increased from 1.38 to 1.59 when

2 increased original EE2 concentration from 0 to 5 mg L−1. This result indicated that

3 high concentration of EE2 enhanced cell corrugation and deformation. The TEM

4 pictures illustrated the structural integrity of the microalgal cells cultivated in the

5 culture without EE2. Abundant starch and lipid granules accumulated in the cells, and

6 microalgal cell size was about 5.18 μm. When the initial EE2 concentration increased

7 to 1 mg L−1, microalgal cells accumulated numerous lipid granules due to external

8 stress and stimulation, and the size of microalgal cell was mildly shranked to

9 approximately 4.45 μm. Further increasing the original EE2 concentration to 5 mg L−1,

10 the size of microalgal cell declined to 3.41 μm or by 34.2 % compared with the

11 control. This result indicated that the cytoplasm shrank severely.

12 3.4 The antioxidation ability of microalgae Chlorella PY-ZU1 in EE2 wastewater

13 Fig. 4 shows the CAT activity, SOD activity and MDA content of Chlorella

14 PY-ZU1 cells cultivated in EE2 wastewater with various original concentrations.

15 External stress, e.g. organic acids, salinity, and heavy metals, can trigger massive ROS

16 production in plants (Mallick and Mohn, 2000). Excess cell ROS induces damage to

17 the membrane system of microorganism and finally hinders growth. Organisms

18 possess sophisticated systems, such as various antioxidant enzymes (e.g. CAT and

19 SOD), as defense against and to eliminate ROS (Tripathi et al., 2006). CAT can

20 catalyze the decomposition of H2O2 into H2O and O2 to alleviate the oxidative

21 damage caused by H2O2. SOD is an important antioxidative enzyme that can transfer

14
 1 O2− to H2O2 and O2, thus avoiding the production of O2− radicals. At 0 mg L−1 EE2,

2 the CAT and SOD activities were 0.54 and 44.59 U mL−1, respectively. Increasing the

3 original EE2 concentration in the culture medium resulted in the original increase and

4 subsequent decrease in the CAT and SOD activities of Chlorella PY-ZU1 cells. The

5 CAT activity of Chlorella PY-ZU1 cells reached the peak value of 1.49 U mL−1 when

6 the original EE2 concentration was 0.01 mg L−1. Increasing the original EE2

7 concentration to 5 mg L−1 reduced the CAT activity of microalgal cells to 0.36 U mL−1.

8 The SOD activity of Chlorella PY-ZU1 cells reached the peak value of 65.57 U mL−1

9 when original EE2 concentration was 0.1 mg L−1. Further increase in the original EE2

10 concentration to 5 mg L−1 reduced the SOD activity of microalgal cells to 34.36 U

11 mL−1. While Chlorella PY-ZU1 were exposed to EE2 with low concentrations, they

12 can enhance antioxidant enzyme activities to eliminate excess free radicals, thereby

13 avoiding or reducing oxidative damage. Therefore, the activities of CAT and SOD

14 increased. Once EE2 concentration increased over a critical level, the antioxidant

15 system failed to maintain the dynamic equilibrium of ROS generation and elimination.

16 This case would result in oxidative damage to cells, including antioxidant system,

17 thereby reducing the CAT and SOD activities. The CAT activity began to decrease at a

18 lower original EE2 concentration than SOD activity, did probably because CAT and

19 its synthetic pathway were less tolerant to EE2 than SOD.

20 Under external stress, membrane system of microalgae will be attacked by extra

21 cell ROS. MDA is the peroxidation end-product of membrane lipids, and MDA

22 content is a relevant parameter to represent cellular oxidation damage (Wan et al.,

15
 1 2014). In our study, the MDA content was 2.57 nmol mL−1 at 0 mg L−1 EE2. However,

2 MDA content of microalgal cells decreased from 2.57 nmol mL−1 to 2.03 nmol mL−1

3 when original EE2 concentration was increased from 0 mg L−1 to 0.1 mg L−1. Further

4 increase in the original EE2 concentration to 5 mg L−1 increased the MDA content of

5 microalgal cells from 2.03 to 2.59 nmol mL−1. This phenomenon may be due to the

6 fact that at original EE2 concentration of 0−0.1 mg L−1，antioxidant enzymes, such as

7 CAT and SOD, were promoted by the stimulation of EE2. This process eliminated

8 ROS and reduced the attacks on the membrane lipid, thereby reducing MDA content.

9 However, when the original EE2 concentration was ≥1 mg L−1, the equilibrium

10 between ROS producing and clearing inside cells was broke, causing excessive ROS

11 accumulation. Thus the membrane lipids peroxidation degree exacerbated, so the

12 MDA content of microalgal cells increased.

13 3.5 FTIR spectroscopy and lipid analysis of Chlorella PY-ZU1 biomass cultivated

14 in EE2 wastewater

15 Functional groups that could be associated with EE2 removal on microalgal cells

16 were analyzed by FTIR. Maximum absorption peak in the FTIR spectrum of

17 Chlorella PY-ZU1 cells appeared between 3400 and 3500 cm−1. It corresponds to the

18 –OH group and implies the existence of water or carbohydrates in microalgal cells

19 (Murdock and Wetzel, 2009). The peaks at 1745 cm−1 and between 2750 and 3000

20 cm−1 correspond to the C=O and –CH2/–CH3 groups, respectively, and stands for

21 lipids inside cells. The peaks at 1654 and 1544 cm−1 correspond to amides I and II,

16
 1 respectively. The peaks at 1400 cm−1 correspond to the symmetric vibration of COO–.

2 The peak at 1080 cm−1 denotes the elastic vibration of C–O (Benning et al., 2004).

3 After cultivation in artificial EE2 wastewater, the absorption peak at 1745 cm−1 and

4 between 2750 and 3000 cm−1 increased. This result indicated that the addition of EE2

5 stimulated the accumulation of more lipids in the microalgal cells. The decrease in the

6 peaks at 1400 cm−1 and 1080 cm−1 indicated a relationship between EE2 removal and

7 COO−/C–O functional groups on microalgal cytoderms. The peaks at 1654 and 1544

8 cm−1 also decreased, indicating that functional groups containing nitrogen may be

9 involved in EE2 removal by microalgal cells.

10 Table 1 shows the organic components of Chlorella PY-ZU1 biomass cultivated

11 in EE2 wastewater. The addition of EE2 has little effect on the protein content of

12 microalgal biomass. However, increasing the EE2 concentration from 0 to 5 mg L−1,

13 increased the lipid content from 29.4 % to 36.2 % but decreased soluble sugar content

14 from 12.6 % to 5.1 %. More carbon elements were metabolized to synthesize lipids

15 rather than carbohydrates because of the EE2-induced stimulation and stress to the

16 microalgae. This finding was consistent with the TEM results. Therefore, Chlorella

17 PY-ZU1 biomass cultivated in EE2 wastewater could be used as feedstock to produce

18 biodiesel.

19 Table 2 showed that the fatty acids in Chlorella PY-ZU1 cells mainly consisted of

20 C16:0, C17:0, C18:0, C18:2, and C18:3. Approximately 95% of FAMEs were covered

21 with C16−C18. Thus, fatty acids of Chlorella PY-ZU1 are appropriate to produce

22 biodiesel (He et al., 2013). Different EE2 concentrations all caused the increase in

17
 1 saturated fatty acid contents and decreased unsaturated fatty acid contents, especially

2 polyunsaturated fatty acid contents. This observation may be attributed to the increase

3 in ROS in microalgal cells caused by EE2 and easy oxidation of unsaturated fatty

4 acids by ROS, thereby reducing the unsaturated fatty acid contents (Pinto et al.,

5 2011).

6 4. Conclusions

7 EE2 concentrations ranging from 0.01−5mg L−1 stimulated microalgal growth.

8 Increasing original EE2 concentration from 0 to 5mg L−1 increased cell fractal

9 dimension from 1.38 to 1.59, reduced cell size from 5.18 to 3.41μm. Meanwhile, SOD

10 and CAT activities first increased to 65.57 and 1.49U mL−1 then decreased to 34.36

11 and 0.36U mL−1, respectively. MDA content first decreased to 2.03 then increased to

12 2.59nmol mL−1. The maximum EE2 removal efficiency of 94% by microalgae was

13 obtained at 5mg L−1 EE2. It is a continuable and efficient way to remove EE2 and the

14 microalgal biomass was a good material to produce biodiesel.

15 Acknowledgements

16 This study was supported by the National key research and development

17 program−China (2016YFB0601003)，National Natural Science Foundation−China

18 (51476141).

19

18
 1 Appendix A. Supplementary data

2 E-supplementary data for this work can be found in e-version of this paper

3 online.

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 1 List of Figures:

2 Fig.1 Growth curves of microalgae Chlorella PY-ZU1 in ethinylestradiol (EE2)

3 wastewater under 15% CO2: (a) Biomass dry weight; (b) Average specific growth rate

4 and carbon fixation rate in 3 days and 7 days.

5 Fig.2. Ethinylestradiol (EE2) removal from wastewater by microalgae Chlorella

6 PY-ZU1 under 15% CO2. (a) Removal efficiencies of EE2 by activated and inactivated

7 Chlorella PY-ZU1 (original EE2 concentration was 1 mg L-1); (b) Effects of original

8 EE2 concentration on EE2 removal by Chlorella PY-ZU1.

9 Fig. 3. Effects of original EE2 concentration in wastewater on cell sizes and fractal

10 dimensions of microalgae Chlorella PY-ZU1 after cultivation for 7 days. (Note: fractal

11 dimension is a parameter to describe irregular degree of microalgal cells)

12 Fig. 4. Effects of original EE2 concentration in wastewater on superoxide dismutase

13 (SOD) activity, catalase activity (CAT) and malondialdehyde (MDA) content of

14 microalgae Chlorella PY-ZU1 cells.

15 Table 1 Organic compositions of Chlorella PY-ZU1 biomass cultivated in EE2

16 wastewater under 15% CO2.

17 Table 2 Fatty acid methyl esters (FAME) compositions of lipids in Chlorella PY-ZU1

18 biomass cultivated in EE2 wastewater under 15% CO2.

19

24
 Original ethinylestradiol concentration (mg L-1)
0
4
0.01
Biomass dry weight (g L-1) 0.1
1
3
2
5

0
0 2 4 6 8 10
Microalgal growth time (d)
1
2 (a)

1.8
Specific growth rate in 3 days
0.64
Specific growth rate in 7 days
1.7
CO2 fixation rate in 3 days
0.62 CO2 fixation rate in 7 days 1.6

CO2 fixation rate (g L-1d-1)

Specific growth rate (μ)

0.60
1.5
0.58 1.4

0.56

0.32
0.31 0.9

0.30
0.29 0.8

0 0.01 0.1 1 2 5
Original EE2 concentration (mg L-1)
3
4 (b)
5 Fig.1 Growth curves of microalgae Chlorella PY-ZU1 in ethinylestradiol (EE2)
6 wastewater with 15% CO2: (a) Biomass dry weight; (b) Average specific growth
7 rate and carbon fixation rate in 3 days and 7 days.
8
9

25
 Activated microalgae
100 Inactivated microalgae
Without microalgae
Removal efficiency of EE2 (%)
80

60

40

20

0
0 50 100 150 200 250
Microalgal growth time (h)
1
2 （a）

0.4
94 Removal efficiency
Residual concentrations

Residual EE2 concentration (mg L-1)

92
Removal efficiency of EE2 (%)

0.3

90

88 0.2

86
0.1
84

82
0.0
80
0.01 0.1 1 2 5
Original EE2 concentration (mg L-1)

3
4 （b）
5 Fig.2 Ethinylestradiol (EE2) removal from wastewater by microalgae Chlorella
6 PY-ZU1 with 15% CO2. (a) Removal efficiencies of EE2 by activated and
7 inactivated Chlorella PY-ZU1 (original EE2 concentration was 1 mg L-1); (b)
8 Effects of original EE2 concentration on EE2 removal by Chlorella PY-ZU1.
9

26
 5.5
Cell fractal dimension
1.60

Fractal dimensions of microalgal cells

Cell Size

Sizes of microalgal cells (μm)

5.0
1.55

1.50 4.5

1.45
4.0

1.40
3.5

1.35
0 0.01 0.1 1 2 5
Original concentration of ethinylestradiol (mg L-1)
1
2 Fig. 3 Effects of original EE2 concentration in wastewater on cell sizes and
3 fractal dimensions of microalgae Chlorella PY-ZU1 after cultivation for 7 days.
4 (Note: fractal dimension is a parameter to describe irregular degree of
5 microalgal cells)
6

27
 75
SOD activity 1.8
2.7
70 CAT activity
MDA content 1.6

MDA content (nmol mL-1)

65 2.6
SOD activity (U mL-1)

1.4

CAT activity (U mL-1)

60 2.5
1.2
55 2.4
1.0
50 2.3
0.8
45
2.2
40 0.6
2.1
35 0.4
2.0
30 0.2
0 0.01 0.1 1 2 5
Original EE2 concentrations (mg L-1)
1
2 Fig.4 Effects of original EE2 concentration in wastewater on superoxide
3 dismutase (SOD) activity, catalase activity (CAT) and malondialdehyde (MDA)
4 content of microalgae Chlorella PY-ZU1 cells.
5

28
1 Table 1
2 Organic compositions of Chlorella PY-ZU1 biomass cultivated in EE2 wastewater
3 under 15% CO2.
Original EE2 concentration (mg Protein Lipid Soluble
L-1) (%) (%) carbohydrates (%)
0 17.3 29.4 12.6
0.01 16.9 30.5 12.4
0.1 17.1 33.8 10.3
1 17.6 34.4 8.4
2 16.6 35.5 6.7
5 16.9 36.2 5.1
4
5

29
1 Table 2
2 Fatty acid methyl esters (FAME) compositions of lipids in Chlorella PY-ZU1 biomass
3 cultivated in EE2 wastewater under 15% CO2.
FAME Original EE2 concentration (mg L-1, % of Total FAME)
compositions 0 0.01 0.1 1 2 5
C16:0 27.78 28.96 28.73 29.62 31.53 31.8
C16:1 2.63 2.83 2.86 2.39 1.98 1.53
C16:3 2.83 2.44 2.38 2.24 1.89 1.75
C17:0 8.36 10.5 10.22 10.89 11.23 11.37
C18:0 15.21 15.42 15.88 15.72 15.93 16.11
C18:1 2.24 2.25 2.36 2.19 2.15 2.09
C18:2 19.31 19.16 19.04 18.75 18.23 18.07
C18:3 16.86 16.5 16.7 16.11 15.8 15.35
Rest 4.78 1.94 1.83 2.09 1.26 1.93
4
5

30
1 >EE2 concentrations of 0-5 mg L-1 all stimulated the growth of microalgae mutant

2 >Cell fractal dimension increased and cell size decreased when EE2 increased

3 >Cellular MDA content initially decreased and subsequently increased

4 >Cellular CAT and SOD activity initially increased and subsequently decreased

5 >Maximum removal efficiency of EE2 by microalgae mutant reached 94%

31