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Mechanism and kinetics of methylisothiazolinone removal by cultivation of Scenedesmus sp. LX1
Journal of Hazardous Materials 386 (2020) 121959
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Mechanism and kinetics of methylisothiazolinone removal by cultivation of Scenedesmus sp. LX1
T
Xiao-Xiong Wanga, Wen-Long Wangb, Guo-Hua Daob, Zi-Bin Xub, Tian-Yuan Zhangb, Yin-Hu Wub, Hong-Ying Hub,c,* a
Department of Chemical and Environmental Engineering, Yale University, New Haven, CT, 06520-8286, United States Research Institute for Environmental Innovation, Tsinghua University, Suzhou 215163, China c Shenzhen Environmental Science and New Energy Technology Engineering Laboratory, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, 518055, China b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: R Teresa.
Methylisothiazolinone (MIT) is a widely used non-oxidizing biocide for membrane biofouling control in reverse osmosis (RO) systems usually with high dosages. However, few investigations have focused on MIT removal through bio-processes, since it is highly bio-toxic. This study proposed a novel biotreatment approach for efficient MIT degradation by Scenedesmus sp. LX1, a microalga with strong resistance capability against extreme MIT toxicity. Results showed that MIT (3 mg/L) could be completely removed within 4 days’ cultivation with a halflife of only 0.79 d. Biodegradation was the primary removal mechanism and this metabolic process did not rely on bacterial consortia, soluble algal products secretion or algal growth. The main pathway was proposed as ring cleavage followed by methylation and carboxylation through the identification of MIT transformation products. MIT biodegradation followed the pseudo-first-order kinetics under growth control. A new kinetic model was presented to depict the MIT removal considering algal growth, and this model could be used for generally describing non-nutritive contaminants biodegradation. The algal biodegradation capability was independent of the initial biocide concentration, and MIT removal could be enhanced by increasing the initial algal density. Our results highlight the potential application of algal cultivation for MIT-containing wastewater biotreatment, such as RO concentrate.
Keywords: Methylisothiazolinone Microalgal biodegradation Non-Oxidizing biocide Reverse osmosis concentrate Removal kinetics
Abbreviations: a, constant in the logistic model; c, MIT concentration at time t; c0, initial MIT concentration; DOC, dissolved organic carbon; kobs, observed pseudofirst-order kinetics rate constant; kobsN, observed rate constant considering algal growth; K, maximum algal density; m/z, mass-to-charge ratio; MIT, methylisothiazolinone; N, algal density at time t; r, intrinsic growth rate; ROC, reverse osmosis concentrate; SAP, soluble algal products; t, cultivation time ⁎ Corresponding author at: Research Institute for Environmental Innovation, Tsinghua University, Suzhou 215163, China E-mail addresses: [email protected], [email protected] (H.-Y. Hu). https://doi.org/10.1016/j.jhazmat.2019.121959 Received 5 November 2019; Received in revised form 16 December 2019; Accepted 21 December 2019 Available online 23 December 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 386 (2020) 121959
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1. Introduction
disruption of cell metabolism till death (Williams, 2006). Notably, Scenedesmus sp. LX1 could survive and well recover its growth from extreme MIT toxicity by regulating chlorophyll content and balancing ROS and antioxidant enzymes, such as superoxide dismutase and catalase (Wang et al., 2018). The synthesis of glutathione (GSH), which can selectively react with MIT by its sulfhydryl (Morley et al., 1998), also played a key role in algal detoxification. As microalgae have the potential to remove many types of toxic organic compounds, some specific algal species might also be possible to biodegrade MIT. As mentioned above, Scenedesmus sp. LX1 could well adapt in ROC and has strong capability to resist against extreme MIT toxicity. The phenomena of MIT concentration decrease during Scenedesmus sp. LX1 cultivation (Wang et al., 2018) suggested that this species is a good candidate. However, whether algal biodegradation was the primary reason for this decrease was unclear, and the capability and mechanism of MIT removal were also unrevealed. If MIT could be completely biodegraded by the microalgae with high efficiency, cultivation of Scenedesmus sp. LX1 might offer a potential application for biological removal of MIT from ROC even though this biocide is highly bio-toxic. The aim of this study is to reveal the capability, mechanism, and kinetics of MIT removal by microalgal cultivation. Scenedesmus sp. LX1, which was used for ROC treatment and MIT toxicity assessment in our previous studies, was selected for this purpose. The mechanism of MIT removal was clarified through comparison of removal capacity under different conditions. Transformation products were identified, which indicated the proposed pathway for MIT metabolism by the microalgae. Moreover, MIT removal kinetics with and without algal growth control were formulated, and the effects of pH and temperature were investigated.
As water scarcity is among the most important global challenges, there is an increasing need to augment water supply through the purification of unconventional water sources, such as municipal wastewater (Shannon et al., 2008; Werber et al., 2016a). Reverse osmosis (RO) technology is a key step in advanced wastewater treatment schemes for water reclamation, and it has been widely used on over 50 % of the worldwide desalination installed capacity (Shannon et al., 2008; Werber et al., 2016b). However, though the RO technology has been proven to be useful and reliable for high-quality reclaimed water production (Pérez-González et al., 2012), it generates RO concentrate (ROC) of 25 − 50 %, which contains nearly all of the constituents from the RO feed at elevated concentrations (Dialynas et al., 2008). ROC treatment and disposal has become one of the major challenges for the wider application of RO-based water reclamation (Pérez-González et al., 2012; Fritzmann et al., 2007). As a broad-spectrum non-oxidizing biocide, methylisothiazolinone (MIT) is commonly used alone or with 5-chloro-2-methyl-4-isothiazolin-3-one in the RO system to avoid biological fouling of membranes (Majamaa et al., 2011). Industrial grade isothiazolinones can be added weekly at a high concentration of 160 mg/L to the RO system of wastewater reclamation plants (Tang et al., 2012). However, MIT is highly bio-toxic. MIT inhibited the growth of Vibrio fischeri and Daphnia magna at low half-maximal effective concentrations (EC50) of 1.6 and 2.1 mg/L, respectively (Arning et al., 2009; Li et al., 2016). In addition, 5 mg/L of MIT could significantly reduce the efficiency of the nitrification process from 90 % to 20 % (Amat et al., 2015). The concentrated MIT in the ROC can lead to an increased eco-risk if discharged directly into the environment and biotreatment difficulties if recycling in wastewater treatment plants. Some studies have been carried out on MIT degradation, such as ozonation and electrochemical methods though their facility construction and operation may represent high costs (Li et al., 2016; Han et al., 2011; Peng et al., 2019). MIT degradation by biological processes is rarely investigated because this highly toxic biocide can kill most microorganism with very low dosages. Hence, if applying bio-processes to remove MIT, the microorganism should have the capacities to (i) well adapt to ROC, (ii) resist MIT toxicity and survive, and (iii) completely biodegrade MIT with a high efficiency. Microalgae can adapt to wastewaters containing various kinds of pollutants (Wu et al., 2014; Li et al., 2019), and some algae-based processes have been investigated for treating ROCs from different sources. Studies of photobiological treatment of ROC from groundwater replenishment systems showed that nutrients, scaling constituents and some pharmaceuticals and personal care products (PPCPs) could be removed simultaneously (Ikehata et al., 2018, 2017). Our previous study proposed a novel biotreatment approach for effective removal of nitrogen, phosphorus and hardness precursors from municipal wastewater reclaimed ROC by cultivating Scenedesmus sp. LX1, a microalgae with strong wastewater adaptability (Wang et al., 2016). Microalgae-based technologies can degrade many kinds of PPCPs and antibiotics, despite them being highly toxic (Wang et al., 2017a; Ahmed et al., 2017). Xiong et al. (2017a) reported that 1 mg/L of levofloxacin was biodegraded with an efficiency of 91.5 % by Chlorella vulgaris within 11 days of cultivation. Biodegradation of diclofenac and ciprofloxacin by Scenedesmus obliquus and Chlamydomonas mexicana, respectively, were also reported (Escapa et al., 2016; Xiong et al., 2017b). The main mechanisms for algal xenobiotic resistance include hydrophilic compounds transformation and conjugation to facilitate excretion (Torres et al., 2008; Xiong et al., 2018). The primary inhibitory mechanism of MIT to microorganism is that the reductive sulfur in MIT molecules can deactivate several specific enzymes with protein thiols by forming into disulfide bonds (Collier et al., 1990). The inhibition of adenosine triphosphate synthesis and the excessive accumulation of reactive oxygen species (ROS) result in the
2. Materials and methods 2.1. Microalgal strains and chemicals Freshwater microalgae, Scenedesmus sp. LX1 (Collection No. CGMCC 3036 in the China General Microbiological Culture Collection Center), was used in this study. The strain was stored in smBG11 medium with nitrogen and phosphorus contents of 45 and 4.5 mg/L, respectively, as described in our previous studies (Wang et al., 2018; Zhang et al., 2013). To avoid bacterial impacts, the stored strain was firstly purified by streaking with a smBG11 agar plate, and subsequently cultivated in sterilized smBG11 medium till exponential phases (approximately 2 weeks). The inocula were examined by fluorescence staining (SYBR Green I, Sigma-Aldrich Co.) to ensure sterility before use. MIT was purchased from Sigma-Aldrich Company (CAS Number 26172-54-3, ≥99 %), and 1 g/L stock solutions were prepared using deionized water. The solutions were filter-sterilized through a 0.2 μm membrane filter (PALL Co., USA) before use. 2.2. Experimental design Two series of experiments were designed in this study. The first series was conducted to reveal the mechanism of MIT removal by microalgal cultivation. The second series was to determine the kinetics of algae-based MIT removal. For all the algal cultivation experiments in this study, 50 mL flasks with 25 mL smBG11 medium were autoclaved at 121 °C for 20 min before use. On a clean bench, the MIT stock solution was added first to the sterilized media, and then the strain was inoculated. Cultivation was performed in an artificial climate chamber (Yiheng Technical Co. Ltd., China) under the following conditions, as described in our previous studies (Wang et al., 2018): light intensity = 55–60 μmol protons/m2/s, light/dark ratio = 14:10, and temperature = 25 ± 1 °C. For the first series, the effects of MIT photolytic and hydrolytic degradation, soluble algal products (SAP) secretion, algal growth, and 2
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2.3.4. MIT transformation products identification To identify the main transformation products of MIT by algal cultivation, the samples were concentrated and desalinated by solid phase extraction (SPE) as follows: 100 mL of algal culture was filtered through a 0.45 μm membrane and then adjusted pH to 2 by dilute sulfuric acid to ensure most compounds existed in an undissociated state. The pHadjusted solution was passed through a SPE cartridge (HLB, 6cc, Waters, USA) at a flow rate of 2–4 mL/min. The compounds adsorbed on the SPE cartridge were eluted with 10 mL chromatographically pure methanol. Finally, the eluate was blown in pure nitrogen conditions to 1 mL by a pressure blowing concentrator (PHC-12R, I WILL T&D, China). The SPE concentrated samples were diluted and the transformation products were identified using ultra-performance liquid chromatography (UPLC; ACQUITY UPLC, Waters, USA) with a C18 column (1.8 μm, 2.1 mm × 100 mm; ACQUITY UPLC HSS T3, Waters, USA) and a quadrupole time-of-flight mass spectrometer (QTOF MS; Xevo G2 QTOF, Waters, USA). The mobile phase consisted of 0.2 % formic acid (A) and chromatographically pure acetonitrile (B), and the flow rate was 0.3 mL/min. The mobile phase elution gradient was: 0.0 ∼ 5.0 min, 0 % B; 5.0 ∼ 10.0 min, 10 % B; 10.0 ∼ 20.0 min, 40 % B; 20.0 ∼ 26.0 min, 100 % B; 26.0 ∼ 35.0 min, 0 % B. The QTOF MS was operated in the electrospray ionization (ESI) positive mode. Micromass MassLynx software (4.10.0, Waters, USA) was used for data analysis.
cell status (living and inactivated cells) were investigated to reveal the mechanism of MIT removal. The initial density of the strain was 106 cells/mL and the initial MIT concentration was 3 mg/L. Cultivation was performed for 4 d. Sterilized smBG11 medium with only MIT addition was considered as MIT blank. SAP of Scenedesmus sp. LX1 was acquired by filtering the algal culture with a 0.45 μm membrane (PALL Co., USA), as described by (Yu et al. (2015)). Algal growth was controlled using two different methods of without adding nitrogen and phosphorus to the culture medium and without illumination. Inactivated algal cells were obtained using two different methods of disruption and freeze-drying. Algal cultures were centrifuged (10,000 rpm ×10 min, 4 °C) and the deposited cells were washed twice with smBG11 medium to obtain the living cells. The disrupted algal cells were obtained by resuspending the living cells in smBG11 medium followed by disrupting the suspension using a high-pressure cell disrupter (JN-mini, JNBIO Co. Ltd., China). The living cells were pre-frozen to −80 °C and the freezedried algal cells were obtained using a freeze dryer (FDU-1100, EYELA) (6.4 pa, −55 °C, 24 h). For the second series, MIT removal kinetics were determined under both controlled and normal growth conditions and the effects of pH and temperature were investigated. Data of the MIT concentration changes in the first series were directly used to determine MIT removal kinetics when the algal growth was controlled. To investigate the MIT removal kinetics under algal growth, Scenedesmus sp. LX1 was cultivated in the culture medium with different initial MIT concentrations of 1, 1.5, 2, 2.5, 3 and 3.5 mg/L (initial algal density = 2 × 105 cells/mL) and different initial algal density of 9 × 104, 20 × 104, 45 × 104, 100 × 104 and 250 × 104 cells/mL (initial MIT concentration =3 mg/L) for 14 d. Different pH values of 4.5, 5.7, 7.0, 9.1 and 10.7 and different temperature values of 10, 25 and 37 °C were set to investigate the effects of culture conditions. Differences of the pH were adjusted by sulfuric acid and sodium hydroxide and controlled by using citratephosphate or carbonate-bicarbonate buffers. The artificial climate chamber could adjust the temperature. The initial algal density was 106 cells/mL and the initial MIT concentration was fixed at 3 mg/L. Algal growth was controlled in this part of the experiments through both nitrogen and illumination regulation to prevent the assimilation of the buffers, and cultivation was performed for 4 d.
2.3.5. Model fitting Origin 9 (OriginLab Corp., USA) was used to model the data with linear and nonlinear curves fittings. 2.3.6. Statistical analysis All experiments in this study were conducted in triplicate. Data are expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed for analysis of significant differences using Origin 9. Statistical significance was accepted at a probability of p < 0.05. 3. Results and discussion 3.1. Mechanism of MIT removal by microalgal cultivation
2.3. Analytical methods
3.1.1. Causes of MIT removal Effects of MIT hydrolytic and photolytic degradation, SAP secretion, algal growth, and cell status were investigated to reveal the primary mechanism of MIT concentration decrease when cultivating Scenedesmus sp. LX1. Strict sterilizing and aseptic procedures were carried out to avoid influence from bacteria, as described in Section 2.1 and 2.2. As shown in Fig. 1a, MIT could be efficiently removed by the algae. With an initial concentration of 3 mg/L, over 97 % of the MIT was removed within 4 d. Less than 4 % of the MIT concentration decrease was observed under light condition without adding the algae, indicating the hydrolytic and photolytic degradation were weak (Fig. 1b). The reduction of MIT was also low in the SAP-containing culture with a decreasing ratio of 13 %. As the MIT removal efficiency by algal cultivation was significantly higher (p < 0.05), it can be concluded that the existence of algal cell was essential for MIT removal. The growth of Scenedesmus sp. LX1 was regulated by nutrients (nitrogen and phosphorus) control and illumination control to identify whether growth or photosynthesis was the decisive factor for MIT removal. As shown in Fig. 1c, with a relatively high initial algal density of 106 cells/mL, MIT concentration changes in the media with and without nutrients addition were almost the same (p > 0.05), and the concentration decrease under dark condition was even greater than that under light (p < 0.05), probably due to the light-induced oxidative stress caused by MIT toxicity (Perreault et al., 2012). These results indicated that the effects of algal growth or photosynthesis were
2.3.1. MIT concentration The algal culture was filtered through a 0.45 μm membrane prior to MIT concentration measurement. MIT concentration was determined using a high-performance liquid chromatography (HPLC) system (Prominence LC-20CE, Shimadzu, Japan) with a UV absorbance detector (SPD-M20A, Shimadzu, Japan) and a C18 column (5 μm, 4.6 × 150 mm; J&K Chemical Ltd., China) at 40 °C. The mobile phase comprised a 40 % 20 mM phosphate buffer (pH = 7) and 60 % methanol using a reversed-phase column for separation at a flow rate of 1 mL/min. 2.3.2. Algal density Algal density was determined based on optical density calibration curves at 650 nm. Linear relationship for Scenedesmus sp. LX1 was determined using a spectrophotometer (UV-2700, Shimadzu Co., Japan) to calculate algal density from optical density as follows [Eq. (1)]:
DS. LX1 = 8.86 × 106⋅OD650
(1)
where DS. LX1 is the cell density of Scenedesmus sp. LX1, and OD650 is the optical density at 650 nm. 2.3.3. Dissolved organic carbon (DOC) The algal culture was filtered through a 0.45 μm membrane prior to the DOC measure by a TOC analyzer (TOC-VCPH, Shimadzu Co., Japan). 3
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Fig. 1. Concentrations of MIT after treatment under different conditions of (a) control, (b) cultural blank (sterilized medium with only MIT addition) and soluble algal products (SAP) under light, (c) algal growth regulated by nitrogen and phosphorus control and light control, and (d) algal inactivation through disruption and freeze-drying at 4 d. [MIT]/ [MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/mL. Strict sterilizing and aseptic procedures were carried out to avoid influence from bacteria (see Section 2.1 and 2.2). Significant differences between control and treatments are indicated with different letters (p < 0.05).
3.1.2. Proposed pathway for MIT transformation An UPLC-QTOF MS was used to determine the transformation products of MIT by Scenedesmus sp. LX1 biodegradation (total ions chromatograms see Fig. S2). The results of the blank algal culture solution and the algal-treated MIT solution were compared to eliminate the interferences of other organic compounds, such as SAP. One main transformation product (retention time =12.1 min) was identified and its MS spectra is shown in Fig. S3. Based on the accurate mass-to-charge ratios (m/z), empirical formula and structure of this transformation product were determined (Table S1). The main pathway for MIT degradation by Scenedesmus sp. LX1 was proposed, as shown in Fig. 3. The transformation product was formed through (i) loss of the nitrogenous group (−NH − CH3) by ring cleavage and methylation of sulfhydryl group, (ii) oxidization of aldehyde group to carboxyl group, and (iii) dissociation of carboxyl group under alkaline condition. This product seems to be stable in the algal culture because its peak area showed an increasing trend during cultivation, as mentioned in Fig. 2a. Similar pathway was also found for biodegradation of benzisothiazolinone by Scenedesmus sp. LX1 (details are shown in Fig. S4). Different types of enzymatic reactions such as ring cleavage, carboxylation, and oxidation have been reported for microalgal biodegradation and detoxification of endogenous organic compounds (Matamoros et al., 2016; Xiong et al., 2016, 2017c). The reaction between reductive sulfur of MIT and protein sulfhydryl results in ring cleavage of the MIT molecules (Morley et al., 1998; Carmellino et al., 1994). It was found that the synthesis of sulfhydryl-contained GSH was enhanced dramatically when increasing the MIT concentration (Wang
negligible. The primary mechanism for cellular activity inhibition by MIT is that the reductive sulfur in MIT molecules can deactivate cell protein thiols (Collier et al., 1990; Morley et al., 1998). The consumption of MIT through these inhibition reactions might also be a reason for the concentration decrease. Cell disruption and freeze-drying, two commonly used methods for inactivating cells without destructing the structure of cellular proteins, were used to identify the effects of different cell status on MIT removal. As a result, MIT concentrations decreased slightly with disrupted or freeze-dried algal cells addition (Fig. 1d). Therefore, it can be concluded that the biological removal effect of living cells was the main cause for the MIT concentration decrease when cultivating Scenedesmus sp. LX1. Biodegradation and sorption are two main mechanisms for the removal of toxic organic compounds by living algal cells (Wang et al., 2017a). For example, more than 88 % of the climbazole and nearly all of the 7-amino cephalosporanic acid have been efficiently removed by microalgae through biodegradation and adsorption, respectively (Pan et al., 2018; Guo et al., 2016). A new peak was detected through HPLC measurement when MIT was removed by the algae (HPLC chromatogram see Fig. S1). The peak area increased with the decreasing MIT concentration, as shown in Fig. 2a. This substance might be a MIT transformation product of microalgal biodegradation. Furthermore, no significant changes of the DOC difference between groups with and without MIT addition were observed after the first day of cultivation (Fig. 2b) (p > 0.05), indicating a weak algal sorption of the MIT. Therefore, it could be speculated that algal biodegradation was the primary reason for MIT removal by Scenedesmus sp. LX1.
Fig. 2. Changes of (a) MIT concentration and peak area of MIT transformation product with time and (b) dissolved organic carbon (DOC) difference between groups with and without 3 mg/L of MIT addition with time. The algae was under growth control with an initial density of 106 cells/mL. 4
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Fig. 3. Proposed pathway of MIT biodegradation by Scenedesmus sp. LX1. The formulae without box were detected by UPLC-QTOF MS (details are shown in Fig. S2, S3 and Table S1), and the formula in box was a proposed transformation product.
et al., 2018). This might be a possible reason for the ring cleavage during MIT biodegradation. Furthermore, methylation reaction is commonly observed in biological detoxification processes (Challenger, 1945; Golan-Rozen et al., 2015). Methyl could be transferred onto sulfhydryl to form a − S−CH3 bond by catalysis of enzymes, such as Sadenosyl methionine cosubstrate. Therefore, the biodegradation of MIT might be a result of algal detoxification since Scenedesmus sp. LX1 could completely recover from MIT toxicity if its growth was not completely inhibited (Wang et al., 2018). These results also indicated that the toxicity of this MIT transformation product on the regrowth of Scenedesmus sp. LX1 was weak. An increasing number of studies have focused on the biodegradation of toxic organic compounds by microalgae, such as climbazole, levofloxacin, and carbamazepine (Xiong et al., 2017a; Pan et al., 2018; Xiong et al., 2016). Independent from the consortia of bacteria or photodegradation, these contaminants could be efficiently removed via algal metabolism, which is considered as an important mechanism for algal resistance to toxicity (Wang et al., 2017a). Microalgae are considered as “green livers” for contaminant removal because their xenobiotic detoxification pathways are similar to that of the mammalian livers (Torres et al., 2008). The property of algal-based MIT biodegradation may point to the potential application of Scenedesmus sp. LX1 for the efficient biotreatment of MIT-containing ROC.
Fig. 4. Changes of MIT concentration and its logarithm with time under algal growth control. [MIT]/[MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/mL.
conditions (Fig. 5a and b). The removal trend of pH = 5.7 was similar to the neutral condition, and the kobs values of both conditions were roughly the same (p > 0.05). However, when the pH was further elevated to 9.1 and 10.7, slight decreases in both removal rates and kobs were observed. These results indicated that the neutral or weak acidic conditions were more favorable for MIT removal by Scenedesmus sp. LX1. As algal growth would cause certain pH elevation of the culture media (Wang et al., 2016), appropriate pH control would be beneficial to keep the MIT removal in an optimal rate. The MIT removal capability of Scenedesmus sp. LX1 showed a similar trend at normal and higher temperatures of 25 and 37 °C (no significant difference of the kobs values, p > 0.05), and the MIT were almost completely removed within 4 d under both temperatures (Fig. 5c and d). However, when the temperature dropped to 10 °C, about 30 % of the MIT still remained at the end of the cultivation because of the decrease of algal metabolic rate and enzymatic activities at lower temperatures. As Scenedesmus sp. LX1 could efficiently remove not only MIT, but also nitrogen and phosphorus from ROC (Wang et al., 2016), whereas excessive heat might cause significant reduction of nutrients removal or even complete collapse of the algae-based system (Wang et al., 2017b; Kosaric et al., 1974), a mild cultivation temperature, such as 25 °C, was suggested to ensure the rapid algal growth and efficient MIT and nutrients removal.
3.2. Kinetics of MIT biodegradation by microalgae 3.2.1. Determination of MIT biodegradation kinetics As mentioned above, the primary reason for the MIT removal by Scenedesmus sp. LX1 was algal biodegradation. Data on the MIT concentration changes were used to determine the biodegradation kinetics of MIT when the algal growth was controlled. If the biodegradation of MIT followed the observed pseudo-first-order kinetics, these data should be fitted with the following equation:
ln
c = −k obs⋅t c0
(2)
where c is the MIT concentration at time t (mg/L), c0 is the initial MIT concentration (mg/L), kobs is the observed rate constant (d―1), and t is the cultivation time (d). As shown in Fig. 4, the trend of MIT concentration decreases well fitted the observed pseudo-first-order kinetics with a high R2 value of 0.997. The kobs value was 0.88 d―1 and the half-life of MIT was 0.79 d. Much slower biodegradation rates were reported for climbazole by Scenedesmus obliquus and levofloxacin by Chlorella vulgaris with halflives of more than 4.4 and 5.8 d, respectively (Xiong et al., 2017a; Pan et al., 2018), suggesting that MIT could be biodegraded by Scenedesmus sp. LX1 with high efficiency.
3.2.3. Kinetics formulation considering algal growth As pointed in Section 3.2.1, MIT biodegradation by Scenedesmus sp. LX1 followed the pseudo-first-order kinetics when the algal growth was controlled. However, as the microalgae could recover from growth inhibition caused by MIT toxicity (Wang et al., 2018), changes on MIT concentration might not follow the same kinetic equation if the participated algal density for MIT biodegradation increased. Therefore, it is necessary to formulate the kinetics of MIT removal considering algal growth.
3.2.2. Effects of pH and temperature pH and temperature are two significant factors affecting the removal of contaminants by microalgae, since the algal metabolism and enzymatic activities are sensitive to these two factors (Wang et al., 2017b). Effects of pH and temperature on MIT removal were investigated under algal growth control, as shown in Fig. 5. MIT removal was not conducive when pH was too low (pH = 4.5), since the algal cells might be seriously damaged under strongly acidic 5
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Fig. 5. MIT concentration changes and observed pseudo-firstorder kinetics rate constants (kobs) under different conditions of (a, b) pH and (c, d) temperature under algal growth control. [MIT]/[MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/ mL. Significant differences between control and treatments are indicated with different letters (p < 0.05).
initial MIT concentrations and algal densities are shown in Fig. S5. The trend of MIT concentration changes under different initial MIT concentrations well fitted the removal kinetic model considering algal growth, and all R2 values were over 0.987 (Fig. 6a). As shown in Fig. 6b, the kobs,N values were basically the same in all lower concentration treatments (initial MIT ≤ 3 mg/L, p > 0.05), indicating the biodegradation of MIT was independent of the initial MIT concentrations if the algal growth was not severely inhibited. However, the kobs,N value became smaller when the initial MIT concentration was further increased to 3.5 mg/L, implying a serious affection of the biodegradation caused by MIT toxicity. Data of the MIT concentration changes in different initial algal densities also well fitted the kinetic model (R2 ≥0.989 for all groups, see Fig. 6c). kobs,N values decreased with the increasing initial algal density (Fig. 6d), indicating that the MIT biodegradation capability per algal cell dropped when the algal density increased. This might be because of the growth-mediated pH elevation when cultivating the algae with higher density, as alkaline condition was not beneficial for MIT removal by Scenedesmus sp. LX1 (see Section 3.2.2). Even so, the increase of initial density was favorable for MIT removal since the time used for 95 % of the concentration decrease was shortened from 12 to 3 d when the initial algal density increased from 9 × 104 to 250 × 104 cells/mL. Several studies claimed that the biodegradation of some toxic organic compounds, such as climbazole and ciprofloxacin, followed the pseudo-first-order kinetics during algal growth (Xiong et al., 2017b; Pan et al., 2018). However, this seems inaccurate because the algal biomass increased significantly during the removal processes. Algal density N should remain approximately constant to ensure an unchanged observed rate constant kobs during algal biodegradation. The increase of N did not match this condition even though the correlation
If the biodegradation capability of Scenedesmus sp. LX1 was not affected by its growth, considering the impact of algal density, the equation of the MIT biodegradation kinetics could be expressed as follows:
dc = −k obs,N ⋅N ⋅c dt
(3)
where N (cells/mL) is the algal density at time t, and kobs,N is the observed rate constant considering algal growth [mL/(cells‧d)]. Logistic model, a classic model to describe the population growth under limited environmental conditions, was used to represent the growth dynamics of Scenedesmus sp. LX1 (Li et al., 2010):
N=
K 1 + ea − rt
(4)
where K is the maximum algal density reached in the culture (cells/ mL), a is the constant that indicates the relative position from the origin, and r is the intrinsic growth rate (d−1). The values of K, r, and a was obtained through model fitting of algal growth curves. Substituting the logistic model [Eq. (4)] into Eq. (3),
dc K = −k obs,N c dt 1 + e−rt + a
(5)
The MIT removal kinetic equation was obtained by integrating Eq. (5) with respect to time t:
c = c0 (
1 + ea k obs,N K ) r ert + ea
(6)
Data of the MIT concentration changes under different initial MIT concentrations and algal densities were used to validate the model [Eq. (6)] and to further evaluate the impacts of model parameters on microalgal biodegradation. The algal density changes under different 6
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Fig. 6. Biodegradation kinetics of MIT and observed rate constants (kobs,N) for algal growth under different (a, b) initial MIT concentration (initial algal density = 2 × 105 cells/mL) and (c, d) initial algal density (initial MIT =3 mg/L). Lines are the results of model fitting with Eq. (6). Significant differences between control and treatments are indicated with different letters (p < 0.05).
MNT_ELSEVIER_JOURNAL_HAZMAT_121959_110
application of microalgae for efficient biotreatment of MIT-containing wastewater, such as ROC.
coefficients were high in these researches. The removal kinetic model [Eq. (6)] proposed in the present study was demonstrated to be effective to describe the kinetic process of MIT biodegradation by Scenedesmus sp. LX1. Moreover, it could also be considered as a reference to depict the removal of other non-nutritive organics during algal cultivation, as pseudo-second-order kinetics [Eq. (3)] and logistic model [Eq. (4)], the only two equations used for deriving this kinetic model, are widely applicable for describing growth-independent contaminant biodegradation and phototrophic algal growth. In general, up to 160 mg/L of industrial isothiazolinones are added to municipal wastewater reclamation RO processes, with effective constituent of about 1.6 % (Tang et al., 2012). As one-fourth of the RO influent is converted to ROC (Dialynas et al., 2008; Xu et al., 2019), a maximum concentration of 10 mg/L would be obtained if the effective constituent of the industrial isothiazolinone was only MIT. Though Scenedesmus sp. LX1 could hardly tolerate 10 mg/L of MIT, such high concentration is seldom contained in ROC since isothiazolinones are normally added for only several hours per week (Tang et al., 2012). As reported in our previous study, MIT lower than 3 mg/L did not have significant effect on its biodegradation by Scenedesmus sp. LX1 even at a lower initial density of 2 × 105 cells/mL (Wang et al., 2018). The commonly used density of over 106 cells/mL for Scenedesmus sp. (Gutierrez et al., 2016; Prandini et al., 2016) would surely be capable to further enhance the algal resistance and removal capability of MIT. Hence, cultivation of algae such as Scenedesmus sp. LX1 would be a possible approach to treat the ROC containing high concentration of MIT after a few times of dilution.
1) MIT could be completely removed with high efficiency by Scenedesmus sp. LX1. Algal biodegradation was the primary reason for the removal. The effects of MIT hydrolytic and photolytic degradation were weak. The removal process also did not rely on bacterial consortia, sorption, SAP secretion, algal growth or photosynthesis. MIT transformation product was identified and its effect on subsequent algal growth was negligible weak. The main biodegradation pathway was proposed as ring cleavage of the MIT molecule followed by methylation and carboxylation. 2) The biodegradation of MIT followed the pseudo-first-order kinetics under algal growth control. A novel non-nutritive biodegradation kinetic model was proposed for the removal of MIT considering algal growth. The algal biodegradation capability for MIT was independent from the initial MIT concentration if the algae was not severely inhibited. The increase of initial algal density could accelerate the MIT removal process even though the biodegradation capability of a single algal cell decreased. Alkaline and low temperature conditions were disadvantageous for MIT removal.
CRediT authorship contribution statement Xiao-Xiong Wang: Conceptualization, Methodology, Investigation, Data curation, Writing - original draft, Visualization. Wen-Long Wang: Methodology, Data curation. Guo-Hua Dao: Validation. Zi-Bin Xu: Investigation. Tian-Yuan Zhang: Visualization. Yin-Hu Wu: Writing review & editing. Hong-Ying Hu: Supervision, Project administration.
4. Conclusions This study revealed the mechanism and kinetics of MIT removal by cultivation of Scenedesmus sp. LX1, which emphasizes the potential 7
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Declaration of Competing Interest
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We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the submitted work titled “Mechanism and kinetics of methylisothiazolinone removal by cultivation of Scenedesmus sp. LX1″.Signed by all authors as follows: Xiao-Xiong Wang, Wen-Long Wang, Guo-Hua Dao, Zi-Bin Xu, Tian-Yuan Zhang, Yin-Hu Wu, Hong-Ying Hu Acknowledgments This study was supported by the National Key Research and Development Program of China for International Science & Innovation Cooperation Major Project between Governments (No. 2016YFE0118800), Key Program of the National Natural Science Foundation of China (No. 51738005). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121959. References Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in the biological and chemical treatment technologies for emerging contaminant removal from wastewater: a critical review. J. Hazard. Mater. 323, 274–298. Amat, A.M., Arques, A., López-Pérez, M.F., Nacher, M., Palacios, S., 2015. Effect of methylisothiazolinone on biological treatment: efficiency of SBRs and bioindicative studies. Environ. Eng. Sci. 32, 479–485. Arning, J., Matzke, M., Stolte, S., Nehen, F., Bottin-Weber, U., Boschen, A., Abdulkarim, S., Jastorff, B., Ranke, J., 2009. Analyzing cytotoxic effects of selected isothiazol-3one biocides using the toxic ratio concept and structure-activity relationship considerations. Chem. Res. Toxicol. 22, 1954–1961. Carmellino, M.L., Pagani, G., Pregnolato, M., Terreni, M., Pastoni, F., 1994. Antimicrobial activity of fluorinated 1,2-benzisothiazol-3(2H)-ones and 2,2’-dithiobis (benzamides). Eur. J. Med. Chem. 29, 743–751. Challenger, F., 1945. Biological methylation, in: advances in Enzymology and related areas of molecular biology. Chem. Rev. Collier, P.J., Ramsey, A., Waigh, R.D., Douglas, K.T., Austin, P., Gilbert, P., 1990. Chemical-reactivity of some isothiazolone biocides. J. Appl. Bacteriol. 69, 578–584. Dialynas, E., Mantzavinos, D., Diamadopoulos, E., 2008. Advanced treatment of the reverse osmosis concentrate produced during reclamation of municipal wastewater. Water Res. 42, 4603–4608. Escapa, C., Coimbra, R.N., Paniagua, S., García, A.I., Otero, M., 2016. Comparative assessment of diclofenac removal from water by different microalgae strains. Algal Res. 18, 127–134. Fritzmann, C., Löwenberg, J., Wintgens, T., Melin, T., 2007. State-of-the-art of reverse osmosis desalination. Desalination 216, 1–76. Golan-Rozen, N., Seiwert, B., Riemenschneider, C., Reemtsma, T., Chefetz, B., Hadar, Y., 2015. Transformation pathways of the recalcitrant pharmaceutical compound carbamazepine by the white-rot fungus pleurotus ostreatus: effects of growth conditions. Environ. Sci. Technol. 49, 12351–12362. Guo, W.Q., Zheng, H.S., Li, S., Du, J.S., Feng, X.C., Yin, R.L., Wu, Q.L., Ren, N.Q., Chang, J.S., 2016. Removal of cephalosporin antibiotics 7-ACA from wastewater during the cultivation of lipid-accumulating microalgae. Bioresour. Technol. 221, 284–290. Gutierrez, R., Ferrer, I., Gonzalez-Molina, A., Salvado, H., Garcia, J., Uggetti, E., 2016. Microalgae recycling improves biomass recovery from wastewater treatment high rate algal ponds. Water Res. 106, 539–549. Han, W., Chen, Y., Wang, L., Sun, X., Li, J., 2011. Mechanism and kinetics of electrochemical degradation of isothiazolin-ones using Ti/SnO2–Sb/PbO2 anode. Desalination 276, 82–88. Ikehata, K., Zhao, Y., Kulkarni, H.V., Li, Y., Snyder, S.A., Ishida, K.P., Anderson, M.A., 2018. Water recovery from advanced water purification facility reverse osmosis concentrate by photobiological treatment followed by secondary reverse osmosis. Environ. Sci. Technol. 52, 8588–8595. Ikehata, K., Zhao, Y., Maleky, N., Komor, A.T., Anderson, M.A., 2017. Aqueous silica removal from agricultural drainage water and reverse osmosis concentrate by brackish water diatoms in semi-batch photobioreactors. J. Appl. Phycol. 29, 223–233. Kosaric, N., Nguyen, H.T., Bergougnou, M.A., 1974. Growth of spirulina-maxima algae in effluents from secondary wastewater treatment plants. Biotechnol. Bioeng. 16, 881–896. Li, A., Wu, Q.Y., Tian, G.P., Hu, H.Y., 2016. Effective degradation of methylisothiazolone biocide using ozone: kinetics, mechanisms, and decreases in toxicity. J. Environ. Manage. 183, 1064–1071. Li, K., Liu, Q., Fang, F., Luo, R., Lu, Q., Zhou, W., Huo, S., Cheng, P., Liu, J., Addy, M., Chen, P., Chen, D., Ruan, R., 2019. Microalgae-based wastewater treatment for
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Mechanism and kinetics of methylisothiazolinone removal by cultivation of Scenedesmus sp. LX1
T
Xiao-Xiong Wanga, Wen-Long Wangb, Guo-Hua Daob, Zi-Bin Xub, Tian-Yuan Zhangb, Yin-Hu Wub, Hong-Ying Hub,c,* a
Department of Chemical and Environmental Engineering, Yale University, New Haven, CT, 06520-8286, United States Research Institute for Environmental Innovation, Tsinghua University, Suzhou 215163, China c Shenzhen Environmental Science and New Energy Technology Engineering Laboratory, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, 518055, China b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: R Teresa.
Methylisothiazolinone (MIT) is a widely used non-oxidizing biocide for membrane biofouling control in reverse osmosis (RO) systems usually with high dosages. However, few investigations have focused on MIT removal through bio-processes, since it is highly bio-toxic. This study proposed a novel biotreatment approach for efficient MIT degradation by Scenedesmus sp. LX1, a microalga with strong resistance capability against extreme MIT toxicity. Results showed that MIT (3 mg/L) could be completely removed within 4 days’ cultivation with a halflife of only 0.79 d. Biodegradation was the primary removal mechanism and this metabolic process did not rely on bacterial consortia, soluble algal products secretion or algal growth. The main pathway was proposed as ring cleavage followed by methylation and carboxylation through the identification of MIT transformation products. MIT biodegradation followed the pseudo-first-order kinetics under growth control. A new kinetic model was presented to depict the MIT removal considering algal growth, and this model could be used for generally describing non-nutritive contaminants biodegradation. The algal biodegradation capability was independent of the initial biocide concentration, and MIT removal could be enhanced by increasing the initial algal density. Our results highlight the potential application of algal cultivation for MIT-containing wastewater biotreatment, such as RO concentrate.
Keywords: Methylisothiazolinone Microalgal biodegradation Non-Oxidizing biocide Reverse osmosis concentrate Removal kinetics
Abbreviations: a, constant in the logistic model; c, MIT concentration at time t; c0, initial MIT concentration; DOC, dissolved organic carbon; kobs, observed pseudofirst-order kinetics rate constant; kobsN, observed rate constant considering algal growth; K, maximum algal density; m/z, mass-to-charge ratio; MIT, methylisothiazolinone; N, algal density at time t; r, intrinsic growth rate; ROC, reverse osmosis concentrate; SAP, soluble algal products; t, cultivation time ⁎ Corresponding author at: Research Institute for Environmental Innovation, Tsinghua University, Suzhou 215163, China E-mail addresses: [email protected], [email protected] (H.-Y. Hu). https://doi.org/10.1016/j.jhazmat.2019.121959 Received 5 November 2019; Received in revised form 16 December 2019; Accepted 21 December 2019 Available online 23 December 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
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1. Introduction
disruption of cell metabolism till death (Williams, 2006). Notably, Scenedesmus sp. LX1 could survive and well recover its growth from extreme MIT toxicity by regulating chlorophyll content and balancing ROS and antioxidant enzymes, such as superoxide dismutase and catalase (Wang et al., 2018). The synthesis of glutathione (GSH), which can selectively react with MIT by its sulfhydryl (Morley et al., 1998), also played a key role in algal detoxification. As microalgae have the potential to remove many types of toxic organic compounds, some specific algal species might also be possible to biodegrade MIT. As mentioned above, Scenedesmus sp. LX1 could well adapt in ROC and has strong capability to resist against extreme MIT toxicity. The phenomena of MIT concentration decrease during Scenedesmus sp. LX1 cultivation (Wang et al., 2018) suggested that this species is a good candidate. However, whether algal biodegradation was the primary reason for this decrease was unclear, and the capability and mechanism of MIT removal were also unrevealed. If MIT could be completely biodegraded by the microalgae with high efficiency, cultivation of Scenedesmus sp. LX1 might offer a potential application for biological removal of MIT from ROC even though this biocide is highly bio-toxic. The aim of this study is to reveal the capability, mechanism, and kinetics of MIT removal by microalgal cultivation. Scenedesmus sp. LX1, which was used for ROC treatment and MIT toxicity assessment in our previous studies, was selected for this purpose. The mechanism of MIT removal was clarified through comparison of removal capacity under different conditions. Transformation products were identified, which indicated the proposed pathway for MIT metabolism by the microalgae. Moreover, MIT removal kinetics with and without algal growth control were formulated, and the effects of pH and temperature were investigated.
As water scarcity is among the most important global challenges, there is an increasing need to augment water supply through the purification of unconventional water sources, such as municipal wastewater (Shannon et al., 2008; Werber et al., 2016a). Reverse osmosis (RO) technology is a key step in advanced wastewater treatment schemes for water reclamation, and it has been widely used on over 50 % of the worldwide desalination installed capacity (Shannon et al., 2008; Werber et al., 2016b). However, though the RO technology has been proven to be useful and reliable for high-quality reclaimed water production (Pérez-González et al., 2012), it generates RO concentrate (ROC) of 25 − 50 %, which contains nearly all of the constituents from the RO feed at elevated concentrations (Dialynas et al., 2008). ROC treatment and disposal has become one of the major challenges for the wider application of RO-based water reclamation (Pérez-González et al., 2012; Fritzmann et al., 2007). As a broad-spectrum non-oxidizing biocide, methylisothiazolinone (MIT) is commonly used alone or with 5-chloro-2-methyl-4-isothiazolin-3-one in the RO system to avoid biological fouling of membranes (Majamaa et al., 2011). Industrial grade isothiazolinones can be added weekly at a high concentration of 160 mg/L to the RO system of wastewater reclamation plants (Tang et al., 2012). However, MIT is highly bio-toxic. MIT inhibited the growth of Vibrio fischeri and Daphnia magna at low half-maximal effective concentrations (EC50) of 1.6 and 2.1 mg/L, respectively (Arning et al., 2009; Li et al., 2016). In addition, 5 mg/L of MIT could significantly reduce the efficiency of the nitrification process from 90 % to 20 % (Amat et al., 2015). The concentrated MIT in the ROC can lead to an increased eco-risk if discharged directly into the environment and biotreatment difficulties if recycling in wastewater treatment plants. Some studies have been carried out on MIT degradation, such as ozonation and electrochemical methods though their facility construction and operation may represent high costs (Li et al., 2016; Han et al., 2011; Peng et al., 2019). MIT degradation by biological processes is rarely investigated because this highly toxic biocide can kill most microorganism with very low dosages. Hence, if applying bio-processes to remove MIT, the microorganism should have the capacities to (i) well adapt to ROC, (ii) resist MIT toxicity and survive, and (iii) completely biodegrade MIT with a high efficiency. Microalgae can adapt to wastewaters containing various kinds of pollutants (Wu et al., 2014; Li et al., 2019), and some algae-based processes have been investigated for treating ROCs from different sources. Studies of photobiological treatment of ROC from groundwater replenishment systems showed that nutrients, scaling constituents and some pharmaceuticals and personal care products (PPCPs) could be removed simultaneously (Ikehata et al., 2018, 2017). Our previous study proposed a novel biotreatment approach for effective removal of nitrogen, phosphorus and hardness precursors from municipal wastewater reclaimed ROC by cultivating Scenedesmus sp. LX1, a microalgae with strong wastewater adaptability (Wang et al., 2016). Microalgae-based technologies can degrade many kinds of PPCPs and antibiotics, despite them being highly toxic (Wang et al., 2017a; Ahmed et al., 2017). Xiong et al. (2017a) reported that 1 mg/L of levofloxacin was biodegraded with an efficiency of 91.5 % by Chlorella vulgaris within 11 days of cultivation. Biodegradation of diclofenac and ciprofloxacin by Scenedesmus obliquus and Chlamydomonas mexicana, respectively, were also reported (Escapa et al., 2016; Xiong et al., 2017b). The main mechanisms for algal xenobiotic resistance include hydrophilic compounds transformation and conjugation to facilitate excretion (Torres et al., 2008; Xiong et al., 2018). The primary inhibitory mechanism of MIT to microorganism is that the reductive sulfur in MIT molecules can deactivate several specific enzymes with protein thiols by forming into disulfide bonds (Collier et al., 1990). The inhibition of adenosine triphosphate synthesis and the excessive accumulation of reactive oxygen species (ROS) result in the
2. Materials and methods 2.1. Microalgal strains and chemicals Freshwater microalgae, Scenedesmus sp. LX1 (Collection No. CGMCC 3036 in the China General Microbiological Culture Collection Center), was used in this study. The strain was stored in smBG11 medium with nitrogen and phosphorus contents of 45 and 4.5 mg/L, respectively, as described in our previous studies (Wang et al., 2018; Zhang et al., 2013). To avoid bacterial impacts, the stored strain was firstly purified by streaking with a smBG11 agar plate, and subsequently cultivated in sterilized smBG11 medium till exponential phases (approximately 2 weeks). The inocula were examined by fluorescence staining (SYBR Green I, Sigma-Aldrich Co.) to ensure sterility before use. MIT was purchased from Sigma-Aldrich Company (CAS Number 26172-54-3, ≥99 %), and 1 g/L stock solutions were prepared using deionized water. The solutions were filter-sterilized through a 0.2 μm membrane filter (PALL Co., USA) before use. 2.2. Experimental design Two series of experiments were designed in this study. The first series was conducted to reveal the mechanism of MIT removal by microalgal cultivation. The second series was to determine the kinetics of algae-based MIT removal. For all the algal cultivation experiments in this study, 50 mL flasks with 25 mL smBG11 medium were autoclaved at 121 °C for 20 min before use. On a clean bench, the MIT stock solution was added first to the sterilized media, and then the strain was inoculated. Cultivation was performed in an artificial climate chamber (Yiheng Technical Co. Ltd., China) under the following conditions, as described in our previous studies (Wang et al., 2018): light intensity = 55–60 μmol protons/m2/s, light/dark ratio = 14:10, and temperature = 25 ± 1 °C. For the first series, the effects of MIT photolytic and hydrolytic degradation, soluble algal products (SAP) secretion, algal growth, and 2
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2.3.4. MIT transformation products identification To identify the main transformation products of MIT by algal cultivation, the samples were concentrated and desalinated by solid phase extraction (SPE) as follows: 100 mL of algal culture was filtered through a 0.45 μm membrane and then adjusted pH to 2 by dilute sulfuric acid to ensure most compounds existed in an undissociated state. The pHadjusted solution was passed through a SPE cartridge (HLB, 6cc, Waters, USA) at a flow rate of 2–4 mL/min. The compounds adsorbed on the SPE cartridge were eluted with 10 mL chromatographically pure methanol. Finally, the eluate was blown in pure nitrogen conditions to 1 mL by a pressure blowing concentrator (PHC-12R, I WILL T&D, China). The SPE concentrated samples were diluted and the transformation products were identified using ultra-performance liquid chromatography (UPLC; ACQUITY UPLC, Waters, USA) with a C18 column (1.8 μm, 2.1 mm × 100 mm; ACQUITY UPLC HSS T3, Waters, USA) and a quadrupole time-of-flight mass spectrometer (QTOF MS; Xevo G2 QTOF, Waters, USA). The mobile phase consisted of 0.2 % formic acid (A) and chromatographically pure acetonitrile (B), and the flow rate was 0.3 mL/min. The mobile phase elution gradient was: 0.0 ∼ 5.0 min, 0 % B; 5.0 ∼ 10.0 min, 10 % B; 10.0 ∼ 20.0 min, 40 % B; 20.0 ∼ 26.0 min, 100 % B; 26.0 ∼ 35.0 min, 0 % B. The QTOF MS was operated in the electrospray ionization (ESI) positive mode. Micromass MassLynx software (4.10.0, Waters, USA) was used for data analysis.
cell status (living and inactivated cells) were investigated to reveal the mechanism of MIT removal. The initial density of the strain was 106 cells/mL and the initial MIT concentration was 3 mg/L. Cultivation was performed for 4 d. Sterilized smBG11 medium with only MIT addition was considered as MIT blank. SAP of Scenedesmus sp. LX1 was acquired by filtering the algal culture with a 0.45 μm membrane (PALL Co., USA), as described by (Yu et al. (2015)). Algal growth was controlled using two different methods of without adding nitrogen and phosphorus to the culture medium and without illumination. Inactivated algal cells were obtained using two different methods of disruption and freeze-drying. Algal cultures were centrifuged (10,000 rpm ×10 min, 4 °C) and the deposited cells were washed twice with smBG11 medium to obtain the living cells. The disrupted algal cells were obtained by resuspending the living cells in smBG11 medium followed by disrupting the suspension using a high-pressure cell disrupter (JN-mini, JNBIO Co. Ltd., China). The living cells were pre-frozen to −80 °C and the freezedried algal cells were obtained using a freeze dryer (FDU-1100, EYELA) (6.4 pa, −55 °C, 24 h). For the second series, MIT removal kinetics were determined under both controlled and normal growth conditions and the effects of pH and temperature were investigated. Data of the MIT concentration changes in the first series were directly used to determine MIT removal kinetics when the algal growth was controlled. To investigate the MIT removal kinetics under algal growth, Scenedesmus sp. LX1 was cultivated in the culture medium with different initial MIT concentrations of 1, 1.5, 2, 2.5, 3 and 3.5 mg/L (initial algal density = 2 × 105 cells/mL) and different initial algal density of 9 × 104, 20 × 104, 45 × 104, 100 × 104 and 250 × 104 cells/mL (initial MIT concentration =3 mg/L) for 14 d. Different pH values of 4.5, 5.7, 7.0, 9.1 and 10.7 and different temperature values of 10, 25 and 37 °C were set to investigate the effects of culture conditions. Differences of the pH were adjusted by sulfuric acid and sodium hydroxide and controlled by using citratephosphate or carbonate-bicarbonate buffers. The artificial climate chamber could adjust the temperature. The initial algal density was 106 cells/mL and the initial MIT concentration was fixed at 3 mg/L. Algal growth was controlled in this part of the experiments through both nitrogen and illumination regulation to prevent the assimilation of the buffers, and cultivation was performed for 4 d.
2.3.5. Model fitting Origin 9 (OriginLab Corp., USA) was used to model the data with linear and nonlinear curves fittings. 2.3.6. Statistical analysis All experiments in this study were conducted in triplicate. Data are expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed for analysis of significant differences using Origin 9. Statistical significance was accepted at a probability of p < 0.05. 3. Results and discussion 3.1. Mechanism of MIT removal by microalgal cultivation
2.3. Analytical methods
3.1.1. Causes of MIT removal Effects of MIT hydrolytic and photolytic degradation, SAP secretion, algal growth, and cell status were investigated to reveal the primary mechanism of MIT concentration decrease when cultivating Scenedesmus sp. LX1. Strict sterilizing and aseptic procedures were carried out to avoid influence from bacteria, as described in Section 2.1 and 2.2. As shown in Fig. 1a, MIT could be efficiently removed by the algae. With an initial concentration of 3 mg/L, over 97 % of the MIT was removed within 4 d. Less than 4 % of the MIT concentration decrease was observed under light condition without adding the algae, indicating the hydrolytic and photolytic degradation were weak (Fig. 1b). The reduction of MIT was also low in the SAP-containing culture with a decreasing ratio of 13 %. As the MIT removal efficiency by algal cultivation was significantly higher (p < 0.05), it can be concluded that the existence of algal cell was essential for MIT removal. The growth of Scenedesmus sp. LX1 was regulated by nutrients (nitrogen and phosphorus) control and illumination control to identify whether growth or photosynthesis was the decisive factor for MIT removal. As shown in Fig. 1c, with a relatively high initial algal density of 106 cells/mL, MIT concentration changes in the media with and without nutrients addition were almost the same (p > 0.05), and the concentration decrease under dark condition was even greater than that under light (p < 0.05), probably due to the light-induced oxidative stress caused by MIT toxicity (Perreault et al., 2012). These results indicated that the effects of algal growth or photosynthesis were
2.3.1. MIT concentration The algal culture was filtered through a 0.45 μm membrane prior to MIT concentration measurement. MIT concentration was determined using a high-performance liquid chromatography (HPLC) system (Prominence LC-20CE, Shimadzu, Japan) with a UV absorbance detector (SPD-M20A, Shimadzu, Japan) and a C18 column (5 μm, 4.6 × 150 mm; J&K Chemical Ltd., China) at 40 °C. The mobile phase comprised a 40 % 20 mM phosphate buffer (pH = 7) and 60 % methanol using a reversed-phase column for separation at a flow rate of 1 mL/min. 2.3.2. Algal density Algal density was determined based on optical density calibration curves at 650 nm. Linear relationship for Scenedesmus sp. LX1 was determined using a spectrophotometer (UV-2700, Shimadzu Co., Japan) to calculate algal density from optical density as follows [Eq. (1)]:
DS. LX1 = 8.86 × 106⋅OD650
(1)
where DS. LX1 is the cell density of Scenedesmus sp. LX1, and OD650 is the optical density at 650 nm. 2.3.3. Dissolved organic carbon (DOC) The algal culture was filtered through a 0.45 μm membrane prior to the DOC measure by a TOC analyzer (TOC-VCPH, Shimadzu Co., Japan). 3
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Fig. 1. Concentrations of MIT after treatment under different conditions of (a) control, (b) cultural blank (sterilized medium with only MIT addition) and soluble algal products (SAP) under light, (c) algal growth regulated by nitrogen and phosphorus control and light control, and (d) algal inactivation through disruption and freeze-drying at 4 d. [MIT]/ [MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/mL. Strict sterilizing and aseptic procedures were carried out to avoid influence from bacteria (see Section 2.1 and 2.2). Significant differences between control and treatments are indicated with different letters (p < 0.05).
3.1.2. Proposed pathway for MIT transformation An UPLC-QTOF MS was used to determine the transformation products of MIT by Scenedesmus sp. LX1 biodegradation (total ions chromatograms see Fig. S2). The results of the blank algal culture solution and the algal-treated MIT solution were compared to eliminate the interferences of other organic compounds, such as SAP. One main transformation product (retention time =12.1 min) was identified and its MS spectra is shown in Fig. S3. Based on the accurate mass-to-charge ratios (m/z), empirical formula and structure of this transformation product were determined (Table S1). The main pathway for MIT degradation by Scenedesmus sp. LX1 was proposed, as shown in Fig. 3. The transformation product was formed through (i) loss of the nitrogenous group (−NH − CH3) by ring cleavage and methylation of sulfhydryl group, (ii) oxidization of aldehyde group to carboxyl group, and (iii) dissociation of carboxyl group under alkaline condition. This product seems to be stable in the algal culture because its peak area showed an increasing trend during cultivation, as mentioned in Fig. 2a. Similar pathway was also found for biodegradation of benzisothiazolinone by Scenedesmus sp. LX1 (details are shown in Fig. S4). Different types of enzymatic reactions such as ring cleavage, carboxylation, and oxidation have been reported for microalgal biodegradation and detoxification of endogenous organic compounds (Matamoros et al., 2016; Xiong et al., 2016, 2017c). The reaction between reductive sulfur of MIT and protein sulfhydryl results in ring cleavage of the MIT molecules (Morley et al., 1998; Carmellino et al., 1994). It was found that the synthesis of sulfhydryl-contained GSH was enhanced dramatically when increasing the MIT concentration (Wang
negligible. The primary mechanism for cellular activity inhibition by MIT is that the reductive sulfur in MIT molecules can deactivate cell protein thiols (Collier et al., 1990; Morley et al., 1998). The consumption of MIT through these inhibition reactions might also be a reason for the concentration decrease. Cell disruption and freeze-drying, two commonly used methods for inactivating cells without destructing the structure of cellular proteins, were used to identify the effects of different cell status on MIT removal. As a result, MIT concentrations decreased slightly with disrupted or freeze-dried algal cells addition (Fig. 1d). Therefore, it can be concluded that the biological removal effect of living cells was the main cause for the MIT concentration decrease when cultivating Scenedesmus sp. LX1. Biodegradation and sorption are two main mechanisms for the removal of toxic organic compounds by living algal cells (Wang et al., 2017a). For example, more than 88 % of the climbazole and nearly all of the 7-amino cephalosporanic acid have been efficiently removed by microalgae through biodegradation and adsorption, respectively (Pan et al., 2018; Guo et al., 2016). A new peak was detected through HPLC measurement when MIT was removed by the algae (HPLC chromatogram see Fig. S1). The peak area increased with the decreasing MIT concentration, as shown in Fig. 2a. This substance might be a MIT transformation product of microalgal biodegradation. Furthermore, no significant changes of the DOC difference between groups with and without MIT addition were observed after the first day of cultivation (Fig. 2b) (p > 0.05), indicating a weak algal sorption of the MIT. Therefore, it could be speculated that algal biodegradation was the primary reason for MIT removal by Scenedesmus sp. LX1.
Fig. 2. Changes of (a) MIT concentration and peak area of MIT transformation product with time and (b) dissolved organic carbon (DOC) difference between groups with and without 3 mg/L of MIT addition with time. The algae was under growth control with an initial density of 106 cells/mL. 4
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Fig. 3. Proposed pathway of MIT biodegradation by Scenedesmus sp. LX1. The formulae without box were detected by UPLC-QTOF MS (details are shown in Fig. S2, S3 and Table S1), and the formula in box was a proposed transformation product.
et al., 2018). This might be a possible reason for the ring cleavage during MIT biodegradation. Furthermore, methylation reaction is commonly observed in biological detoxification processes (Challenger, 1945; Golan-Rozen et al., 2015). Methyl could be transferred onto sulfhydryl to form a − S−CH3 bond by catalysis of enzymes, such as Sadenosyl methionine cosubstrate. Therefore, the biodegradation of MIT might be a result of algal detoxification since Scenedesmus sp. LX1 could completely recover from MIT toxicity if its growth was not completely inhibited (Wang et al., 2018). These results also indicated that the toxicity of this MIT transformation product on the regrowth of Scenedesmus sp. LX1 was weak. An increasing number of studies have focused on the biodegradation of toxic organic compounds by microalgae, such as climbazole, levofloxacin, and carbamazepine (Xiong et al., 2017a; Pan et al., 2018; Xiong et al., 2016). Independent from the consortia of bacteria or photodegradation, these contaminants could be efficiently removed via algal metabolism, which is considered as an important mechanism for algal resistance to toxicity (Wang et al., 2017a). Microalgae are considered as “green livers” for contaminant removal because their xenobiotic detoxification pathways are similar to that of the mammalian livers (Torres et al., 2008). The property of algal-based MIT biodegradation may point to the potential application of Scenedesmus sp. LX1 for the efficient biotreatment of MIT-containing ROC.
Fig. 4. Changes of MIT concentration and its logarithm with time under algal growth control. [MIT]/[MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/mL.
conditions (Fig. 5a and b). The removal trend of pH = 5.7 was similar to the neutral condition, and the kobs values of both conditions were roughly the same (p > 0.05). However, when the pH was further elevated to 9.1 and 10.7, slight decreases in both removal rates and kobs were observed. These results indicated that the neutral or weak acidic conditions were more favorable for MIT removal by Scenedesmus sp. LX1. As algal growth would cause certain pH elevation of the culture media (Wang et al., 2016), appropriate pH control would be beneficial to keep the MIT removal in an optimal rate. The MIT removal capability of Scenedesmus sp. LX1 showed a similar trend at normal and higher temperatures of 25 and 37 °C (no significant difference of the kobs values, p > 0.05), and the MIT were almost completely removed within 4 d under both temperatures (Fig. 5c and d). However, when the temperature dropped to 10 °C, about 30 % of the MIT still remained at the end of the cultivation because of the decrease of algal metabolic rate and enzymatic activities at lower temperatures. As Scenedesmus sp. LX1 could efficiently remove not only MIT, but also nitrogen and phosphorus from ROC (Wang et al., 2016), whereas excessive heat might cause significant reduction of nutrients removal or even complete collapse of the algae-based system (Wang et al., 2017b; Kosaric et al., 1974), a mild cultivation temperature, such as 25 °C, was suggested to ensure the rapid algal growth and efficient MIT and nutrients removal.
3.2. Kinetics of MIT biodegradation by microalgae 3.2.1. Determination of MIT biodegradation kinetics As mentioned above, the primary reason for the MIT removal by Scenedesmus sp. LX1 was algal biodegradation. Data on the MIT concentration changes were used to determine the biodegradation kinetics of MIT when the algal growth was controlled. If the biodegradation of MIT followed the observed pseudo-first-order kinetics, these data should be fitted with the following equation:
ln
c = −k obs⋅t c0
(2)
where c is the MIT concentration at time t (mg/L), c0 is the initial MIT concentration (mg/L), kobs is the observed rate constant (d―1), and t is the cultivation time (d). As shown in Fig. 4, the trend of MIT concentration decreases well fitted the observed pseudo-first-order kinetics with a high R2 value of 0.997. The kobs value was 0.88 d―1 and the half-life of MIT was 0.79 d. Much slower biodegradation rates were reported for climbazole by Scenedesmus obliquus and levofloxacin by Chlorella vulgaris with halflives of more than 4.4 and 5.8 d, respectively (Xiong et al., 2017a; Pan et al., 2018), suggesting that MIT could be biodegraded by Scenedesmus sp. LX1 with high efficiency.
3.2.3. Kinetics formulation considering algal growth As pointed in Section 3.2.1, MIT biodegradation by Scenedesmus sp. LX1 followed the pseudo-first-order kinetics when the algal growth was controlled. However, as the microalgae could recover from growth inhibition caused by MIT toxicity (Wang et al., 2018), changes on MIT concentration might not follow the same kinetic equation if the participated algal density for MIT biodegradation increased. Therefore, it is necessary to formulate the kinetics of MIT removal considering algal growth.
3.2.2. Effects of pH and temperature pH and temperature are two significant factors affecting the removal of contaminants by microalgae, since the algal metabolism and enzymatic activities are sensitive to these two factors (Wang et al., 2017b). Effects of pH and temperature on MIT removal were investigated under algal growth control, as shown in Fig. 5. MIT removal was not conducive when pH was too low (pH = 4.5), since the algal cells might be seriously damaged under strongly acidic 5
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Fig. 5. MIT concentration changes and observed pseudo-firstorder kinetics rate constants (kobs) under different conditions of (a, b) pH and (c, d) temperature under algal growth control. [MIT]/[MIT]0 refers to the normalized MIT concentration (initial MIT =3 mg/L). The initial algal density was 106 cells/ mL. Significant differences between control and treatments are indicated with different letters (p < 0.05).
initial MIT concentrations and algal densities are shown in Fig. S5. The trend of MIT concentration changes under different initial MIT concentrations well fitted the removal kinetic model considering algal growth, and all R2 values were over 0.987 (Fig. 6a). As shown in Fig. 6b, the kobs,N values were basically the same in all lower concentration treatments (initial MIT ≤ 3 mg/L, p > 0.05), indicating the biodegradation of MIT was independent of the initial MIT concentrations if the algal growth was not severely inhibited. However, the kobs,N value became smaller when the initial MIT concentration was further increased to 3.5 mg/L, implying a serious affection of the biodegradation caused by MIT toxicity. Data of the MIT concentration changes in different initial algal densities also well fitted the kinetic model (R2 ≥0.989 for all groups, see Fig. 6c). kobs,N values decreased with the increasing initial algal density (Fig. 6d), indicating that the MIT biodegradation capability per algal cell dropped when the algal density increased. This might be because of the growth-mediated pH elevation when cultivating the algae with higher density, as alkaline condition was not beneficial for MIT removal by Scenedesmus sp. LX1 (see Section 3.2.2). Even so, the increase of initial density was favorable for MIT removal since the time used for 95 % of the concentration decrease was shortened from 12 to 3 d when the initial algal density increased from 9 × 104 to 250 × 104 cells/mL. Several studies claimed that the biodegradation of some toxic organic compounds, such as climbazole and ciprofloxacin, followed the pseudo-first-order kinetics during algal growth (Xiong et al., 2017b; Pan et al., 2018). However, this seems inaccurate because the algal biomass increased significantly during the removal processes. Algal density N should remain approximately constant to ensure an unchanged observed rate constant kobs during algal biodegradation. The increase of N did not match this condition even though the correlation
If the biodegradation capability of Scenedesmus sp. LX1 was not affected by its growth, considering the impact of algal density, the equation of the MIT biodegradation kinetics could be expressed as follows:
dc = −k obs,N ⋅N ⋅c dt
(3)
where N (cells/mL) is the algal density at time t, and kobs,N is the observed rate constant considering algal growth [mL/(cells‧d)]. Logistic model, a classic model to describe the population growth under limited environmental conditions, was used to represent the growth dynamics of Scenedesmus sp. LX1 (Li et al., 2010):
N=
K 1 + ea − rt
(4)
where K is the maximum algal density reached in the culture (cells/ mL), a is the constant that indicates the relative position from the origin, and r is the intrinsic growth rate (d−1). The values of K, r, and a was obtained through model fitting of algal growth curves. Substituting the logistic model [Eq. (4)] into Eq. (3),
dc K = −k obs,N c dt 1 + e−rt + a
(5)
The MIT removal kinetic equation was obtained by integrating Eq. (5) with respect to time t:
c = c0 (
1 + ea k obs,N K ) r ert + ea
(6)
Data of the MIT concentration changes under different initial MIT concentrations and algal densities were used to validate the model [Eq. (6)] and to further evaluate the impacts of model parameters on microalgal biodegradation. The algal density changes under different 6
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Fig. 6. Biodegradation kinetics of MIT and observed rate constants (kobs,N) for algal growth under different (a, b) initial MIT concentration (initial algal density = 2 × 105 cells/mL) and (c, d) initial algal density (initial MIT =3 mg/L). Lines are the results of model fitting with Eq. (6). Significant differences between control and treatments are indicated with different letters (p < 0.05).
MNT_ELSEVIER_JOURNAL_HAZMAT_121959_110
application of microalgae for efficient biotreatment of MIT-containing wastewater, such as ROC.
coefficients were high in these researches. The removal kinetic model [Eq. (6)] proposed in the present study was demonstrated to be effective to describe the kinetic process of MIT biodegradation by Scenedesmus sp. LX1. Moreover, it could also be considered as a reference to depict the removal of other non-nutritive organics during algal cultivation, as pseudo-second-order kinetics [Eq. (3)] and logistic model [Eq. (4)], the only two equations used for deriving this kinetic model, are widely applicable for describing growth-independent contaminant biodegradation and phototrophic algal growth. In general, up to 160 mg/L of industrial isothiazolinones are added to municipal wastewater reclamation RO processes, with effective constituent of about 1.6 % (Tang et al., 2012). As one-fourth of the RO influent is converted to ROC (Dialynas et al., 2008; Xu et al., 2019), a maximum concentration of 10 mg/L would be obtained if the effective constituent of the industrial isothiazolinone was only MIT. Though Scenedesmus sp. LX1 could hardly tolerate 10 mg/L of MIT, such high concentration is seldom contained in ROC since isothiazolinones are normally added for only several hours per week (Tang et al., 2012). As reported in our previous study, MIT lower than 3 mg/L did not have significant effect on its biodegradation by Scenedesmus sp. LX1 even at a lower initial density of 2 × 105 cells/mL (Wang et al., 2018). The commonly used density of over 106 cells/mL for Scenedesmus sp. (Gutierrez et al., 2016; Prandini et al., 2016) would surely be capable to further enhance the algal resistance and removal capability of MIT. Hence, cultivation of algae such as Scenedesmus sp. LX1 would be a possible approach to treat the ROC containing high concentration of MIT after a few times of dilution.
1) MIT could be completely removed with high efficiency by Scenedesmus sp. LX1. Algal biodegradation was the primary reason for the removal. The effects of MIT hydrolytic and photolytic degradation were weak. The removal process also did not rely on bacterial consortia, sorption, SAP secretion, algal growth or photosynthesis. MIT transformation product was identified and its effect on subsequent algal growth was negligible weak. The main biodegradation pathway was proposed as ring cleavage of the MIT molecule followed by methylation and carboxylation. 2) The biodegradation of MIT followed the pseudo-first-order kinetics under algal growth control. A novel non-nutritive biodegradation kinetic model was proposed for the removal of MIT considering algal growth. The algal biodegradation capability for MIT was independent from the initial MIT concentration if the algae was not severely inhibited. The increase of initial algal density could accelerate the MIT removal process even though the biodegradation capability of a single algal cell decreased. Alkaline and low temperature conditions were disadvantageous for MIT removal.
CRediT authorship contribution statement Xiao-Xiong Wang: Conceptualization, Methodology, Investigation, Data curation, Writing - original draft, Visualization. Wen-Long Wang: Methodology, Data curation. Guo-Hua Dao: Validation. Zi-Bin Xu: Investigation. Tian-Yuan Zhang: Visualization. Yin-Hu Wu: Writing review & editing. Hong-Ying Hu: Supervision, Project administration.
4. Conclusions This study revealed the mechanism and kinetics of MIT removal by cultivation of Scenedesmus sp. LX1, which emphasizes the potential 7
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Declaration of Competing Interest
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We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the submitted work titled “Mechanism and kinetics of methylisothiazolinone removal by cultivation of Scenedesmus sp. LX1″.Signed by all authors as follows: Xiao-Xiong Wang, Wen-Long Wang, Guo-Hua Dao, Zi-Bin Xu, Tian-Yuan Zhang, Yin-Hu Wu, Hong-Ying Hu Acknowledgments This study was supported by the National Key Research and Development Program of China for International Science & Innovation Cooperation Major Project between Governments (No. 2016YFE0118800), Key Program of the National Natural Science Foundation of China (No. 51738005). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121959. References Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in the biological and chemical treatment technologies for emerging contaminant removal from wastewater: a critical review. J. Hazard. Mater. 323, 274–298. Amat, A.M., Arques, A., López-Pérez, M.F., Nacher, M., Palacios, S., 2015. Effect of methylisothiazolinone on biological treatment: efficiency of SBRs and bioindicative studies. Environ. Eng. Sci. 32, 479–485. Arning, J., Matzke, M., Stolte, S., Nehen, F., Bottin-Weber, U., Boschen, A., Abdulkarim, S., Jastorff, B., Ranke, J., 2009. Analyzing cytotoxic effects of selected isothiazol-3one biocides using the toxic ratio concept and structure-activity relationship considerations. Chem. Res. Toxicol. 22, 1954–1961. Carmellino, M.L., Pagani, G., Pregnolato, M., Terreni, M., Pastoni, F., 1994. Antimicrobial activity of fluorinated 1,2-benzisothiazol-3(2H)-ones and 2,2’-dithiobis (benzamides). Eur. J. Med. Chem. 29, 743–751. Challenger, F., 1945. Biological methylation, in: advances in Enzymology and related areas of molecular biology. Chem. Rev. Collier, P.J., Ramsey, A., Waigh, R.D., Douglas, K.T., Austin, P., Gilbert, P., 1990. Chemical-reactivity of some isothiazolone biocides. J. Appl. Bacteriol. 69, 578–584. Dialynas, E., Mantzavinos, D., Diamadopoulos, E., 2008. Advanced treatment of the reverse osmosis concentrate produced during reclamation of municipal wastewater. Water Res. 42, 4603–4608. Escapa, C., Coimbra, R.N., Paniagua, S., García, A.I., Otero, M., 2016. Comparative assessment of diclofenac removal from water by different microalgae strains. Algal Res. 18, 127–134. Fritzmann, C., Löwenberg, J., Wintgens, T., Melin, T., 2007. State-of-the-art of reverse osmosis desalination. Desalination 216, 1–76. Golan-Rozen, N., Seiwert, B., Riemenschneider, C., Reemtsma, T., Chefetz, B., Hadar, Y., 2015. Transformation pathways of the recalcitrant pharmaceutical compound carbamazepine by the white-rot fungus pleurotus ostreatus: effects of growth conditions. Environ. Sci. Technol. 49, 12351–12362. Guo, W.Q., Zheng, H.S., Li, S., Du, J.S., Feng, X.C., Yin, R.L., Wu, Q.L., Ren, N.Q., Chang, J.S., 2016. Removal of cephalosporin antibiotics 7-ACA from wastewater during the cultivation of lipid-accumulating microalgae. Bioresour. Technol. 221, 284–290. Gutierrez, R., Ferrer, I., Gonzalez-Molina, A., Salvado, H., Garcia, J., Uggetti, E., 2016. Microalgae recycling improves biomass recovery from wastewater treatment high rate algal ponds. Water Res. 106, 539–549. Han, W., Chen, Y., Wang, L., Sun, X., Li, J., 2011. Mechanism and kinetics of electrochemical degradation of isothiazolin-ones using Ti/SnO2–Sb/PbO2 anode. Desalination 276, 82–88. Ikehata, K., Zhao, Y., Kulkarni, H.V., Li, Y., Snyder, S.A., Ishida, K.P., Anderson, M.A., 2018. Water recovery from advanced water purification facility reverse osmosis concentrate by photobiological treatment followed by secondary reverse osmosis. Environ. Sci. Technol. 52, 8588–8595. Ikehata, K., Zhao, Y., Maleky, N., Komor, A.T., Anderson, M.A., 2017. Aqueous silica removal from agricultural drainage water and reverse osmosis concentrate by brackish water diatoms in semi-batch photobioreactors. J. Appl. Phycol. 29, 223–233. Kosaric, N., Nguyen, H.T., Bergougnou, M.A., 1974. Growth of spirulina-maxima algae in effluents from secondary wastewater treatment plants. Biotechnol. Bioeng. 16, 881–896. Li, A., Wu, Q.Y., Tian, G.P., Hu, H.Y., 2016. Effective degradation of methylisothiazolone biocide using ozone: kinetics, mechanisms, and decreases in toxicity. J. Environ. Manage. 183, 1064–1071. Li, K., Liu, Q., Fang, F., Luo, R., Lu, Q., Zhou, W., Huo, S., Cheng, P., Liu, J., Addy, M., Chen, P., Chen, D., Ruan, R., 2019. Microalgae-based wastewater treatment for
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