Physiological and morphological responses and tolerance mechanisms of Isochrysis galbana to Cr(VI) stress

Bioresource Technology 302 (2020) 122860

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Case Study

Physiological and morphological responses and tolerance mechanisms of Isochrysis galbana to Cr(VI) stress

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Meng Jin, Xinfeng Xiao , Liguo Qin, Weiwei Geng, Yu Gao, Lin Li, Jianliang Xue College of Chemistry and Environment Engineering, Shandong University of Science and Technology, Qingdao 266510, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Isochrysis galbana Antioxidant enzyme Cr(VI) Cr(III) Tolerance

The effects of the initial concentrations of Cr(VI) on chlorophyll-a (Chl-a), soluble protein and ultrastructure were investigated. Results showed that < 0.5 and > 1.0 mg L−1 Cr(VI) stimulated and inhibited the growth of Isochrysis galbana, respectively. The tolerance mechanisms of I. galbana to Cr(VI) included the following: (1) increased activities of superoxide dismutase (SOD) and peroxidase (POX) for peroxidative damage resistance, (2) accumulation of Cr(VI) on the cell surface and inside the cell for detoxification and (3) conversion of intracellular Cr(VI) to less toxic Cr(III) as indicated by X-ray photoelectron spectroscopy (XPS) results. Cr(VI) enrichment by I. galbana may cause damage to marine ecology and human bodies through the food chain. The tolerance mechanisms of I. galbana to Cr(VI) may be potentially used to treat low-concentration Cr(VI) wastewater. Therefore, the responses and tolerance mechanisms of I. galbana to Cr(VI) must be further studied.

1. Introduction Algae are the primary producers in bodies of water and play an important role in the food chain of aquatic ecosystem. However, various pollutants in the ocean can harm microalgae, especially heavy metal pollution. Cr(VI) possesses a strong oxidizing capacity to impair the genetic materials in all living things, and has been classified as Group A of human carcinogens by United States Environmental Protection Agency (USEPA) (Shen et al., 2019). The enrichment of heavy metals by microalgae may cause damage to marine ecology and human bodies through the food chain. Therefore, the toxic effects of heavy metals on microalgae must be studied. The effect of heavy metals on microalgae has been extensively studied. The toxicity of the former influences the growth characteristics and biochemical composition of the latter. Many studies have shown that heavy metals inhibit the growth (Huang et al., 2009), hinder photosynthesis (Juneau et al., 2002), destroy protein structure and even change cell morphology (Urrutia et al., 2019). In addition, the toxicity of heavy metals also leads to the production of reactive oxygen species (ROS), which causing structural damage to proteins, lipids, DNA and other biomolecules, leading to loss of protein function and even cell death (Sun et al., 2018). Generally, the severity of oxidative damage can be expressed by the amount of oxidised proteins and lipids in microalga cells, especially in the most common oxidative damage marker, that is, malondialdehyde (MDA) (Li et al., 2018). Plants’ response to

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oxidative damage depends on the efficiency of their antioxidant defence systems, such as superoxide dismutase (SOD), superoxide dismutase (POX), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase and low molecular weight non-enzymatic antioxidants, such as ascorbic acid, polyphenols, proline, tocopherols, and glutathione (Terzi and Yıldız, 2015). Heavy metals at low concentration do not or slightly promote the growth of microalgae, whereas at high concentration inhibit the growth and cause the death of microalgae cells. For example, Cu, Fe, Mn and Zn promote the production of biodiesel in Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3 (Abinandan et al., 2019). Certain microalga species show tolerance to heavy metals. Zhang et al. (2014) reported that high Pb2+concentrations do not affect the growth of Chlorella vulgaris as indicated by the remarkably growth of cultures even after 72 h. Some microalga species are resistant to heavy metals. At present, many studies have reported the tolerance mechanisms of different microalga species to heavy metals. The adsorption of heavy metals by functional groups on the surface of cells, the effective absorption and transport of metals to intracellular organelles, and the defense system of various antioxidants against oxidative stress are the key mechanisms for reducing metal toxicity (Pereira et al., 2013; Zhang et al., 2014). Samadani et al. (2018) reported that Chlamydomonas CPCC 121 showed a tolerance for Cd due to the exclusion of Cd at the cell wall surface and the uptake of Cd. In addition, Fu et al. (2019) found that the concentration of MDA increased in Chlorella vulgaris cells under Cu stress, correspondingly, and the activity of SOD increased to

Corresponding author. E-mail address: [email protected] (X. Xiao).

https://doi.org/10.1016/j.biortech.2020.122860 Received 24 December 2019; Received in revised form 20 January 2020; Accepted 21 January 2020 Available online 22 January 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.

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2.2. Chromium treatments

resist oxidative damage. To mitigate Cu stress impact, Chlorella sorokiniana markedly increased proline, polyphenols, tocopherols, glutathione levels, as well as the activities of APX and SOD enzymes; Scenedesmus acuminatus exhibited significant increases in proline, polyphenol, and tocopherol contents, as well as the activities of POX, APX and SOD enzymes (Hamed et al., 2017). However, few studies have been reported to explore the tolerance mechanisms through the change of heavy metal valence and the molecular docking simulation simulation of heavy metals with macromolecular compounds in cells. The current studies on I. galbana mainly focuses on the effects of heavy metals on its growth, physiology and biochemical composition, but few studies have reported the mechanism of tolerance of I. galbana to heavy metals (Liu et al., 2011). Therefore, the XPS analysis and molecular docking simulation were performed in this study to gain insights into the tolerance mechanism of I. galbana to Cr(VI). On the other hand, the tolerance of microalgae to heavy metals may be potentially used to treat low-concentration heavy metals wastewater. The ability of different microalgae species to remove heavy metals has been extensively studied, and a general understanding of heavy metal removal has been established. Some functional groups on the cell surface generally adsorb heavy metals, and the single cell structure provides a large specific surface area. Cells can also actively consume heavy metals and then either bind them to intracellular proteins or be transported to organelles (Monteiro et al., 2012). Microalgae have a good effect on the bioremediation of wastewater with low heavy metal concentration and therefore have become an important biological material for water environment monitoring and heavy metal pollution control (Ammar et al., 2018; Miranda et al., 2012). However, different microalga species have varying capacities to absorb and consume heavy metals. I. galbana may have great potential for the treatment of wastewater with low heavy metal concentration (Kadimpati et al., 2013; Kumar et al., 2015). However, the enrichment of heavy metals by I. galbana may cause damage to marine ecology and human bodies through the food chain. Therefore, studying the response and tolerance of I. galbana to heavy metal stress for the bioremediation of marine ecological pollution is of great importance. In this study, the effects of initial concentrations of Cr(VI) on the chlorophyll-a (Chl-a), soluble protein and ultrastructure of I. galbana were evaluated, and the tolerance mechanism of this species to Cr(VI) stress was also explored. Firstly, the oxidative damage degree was determined by analysing the changes of oxidative damage marker, and the regulation of oxidative stress by SOD and POX was investigated. The adsorption and accumulation of Cr(VI) by I. galbana were then explored by characterising the presence of Cr(VI) on microalga cell surface and measuring the accumulated Cr(VI) content in the cells. It is worth noting that there are few studies have been reported to explore the tolerance mechanisms through the transformation of heavy metal valence and the molecular docking simulation simulation of heavy metals with macromolecular compounds in cells. In this study, the valence state of Cr(VI) in the cells was explored by XPS, and molecular docking simulation was performed to determine the optimal binding sites of Cr (VI) with intracellular macromolecular compounds to explore the tolerance mechanisms.

A series of concentrations of Cr(VI) was added to the alga culture at the exponential growth phase. The initial concentrations of Cr(VI) in the experimental group cultures were 0.5, 1.0, 5.0 and 10.0 mg L−1. The culture without Cr(VI) served as the control group. The chromium stock solution was configured by K2Cr2O7. All cultures were incubated in accordance with the above conditions and shaken twice a day at regular intervals. Each group consisted of three replicates. 2.3. Determination of Chl-a content Chl-a content was measured in accordance with the method of Zhou et al. (2018). The collected microalgae were centrifuged at 1000 rpm for 15 min at 4 °C and washed three times with deionised water. The microalga biomass was suspended in 99.8% (v/v) methanol, and the absorbance of the supernatant was measured after soaking for 24 h at 4 °C in the dark. The content of Chl-a is calculated by the following Eq. (1):

Chl − a (mg / L) = 16.72 × A6652 − 9.16 × A652

(1)

2.4. Soluble protein analysis After the collected microalgae were centrifuged, 1.0 ml of phosphate buffer solution (pH = 7.8) was added, followed by sonication for 10 min. After the extract was centrifuged at 1000 rpm for 15 min at 4 °C, the soluble protein content in the supernatant was determined by the Bradford method (Bradford, 1976). 2.5. Determination of MDA content and SOD and POX activity After the collected microalgae were centrifuged, 1.0 ml of phosphate buffer solution (pH = 7.8) was added, followed by sonication for 10 min. The solution was then centrifuged at 1000 rpm for 15 min at 4 °C, and the supernatant was stored at a low temperature. After extraction with 5% (w/v) trichloroacetic acid, the MDA content in the sample was determined by thiobarbituric acid method (Liu et al., 2017). SOD activity was measured in accordance with the method of Nounjan et al. (2012). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of the photochemical reduction of nitroblue tetrazolium per gram of dry weight. A reaction mixture containing phosphate buffer solution (pH = 7.8) and guaiacol was added to the extract, and 3 mmol L−1 H2O2 was then incorporated into the reaction to determine POX activity (Nounjan et al., 2012). 2.6. Transmission electron microscope (TEM) analysis The I. galbana cells exposed to 10.0 mg L−1 Cr(VI) for 8 days and the control sample were centrifuged and washed three times with deionised water. After fixation, dehydration and slicing, the biomass was observed using TEM (HITACHI H-7000FA). 2.7. Scanning electron microscope coupled with energy dispersive spectrometer (SEM-EDS) analysis of chromium on the cell surface

2. Materials and methods SEM-EDS was performed to characterise the presence of chromium on the cell surface. The I. galbana cells exposed to 1.0 mg L−1 Cr(VI) for 8 days were centrifuged at 1000 rpm for 15 min at 4 °C and washed three times with deionised water. After fixation, cleaning and dehydration, the biomass was analysed by SEM (JEOL JSM-6700F) – EDS (Oxford, Japan).

2.1. Microalgae strain I. galbana was provided by the Institute of Oceanology, Chinese Academy of Sciences and cultured in 500 ml volume Erlenmeyer flasks with f/2 medium (Guillard and Ryther, 1962). The seawater medium was collected from Tangdao Bay and sterilised at 121 °C for 20 min after filtering through a 0.45 μm mixed-filter membrane. Cultures were incubated at 23 °C ± 1 °C and illuminated with fluorescent lamps in a 12/12 dark/light cycle at 4000 Lux irradiance level.

2.8. Extracellular accumulation of chromium The intracellular accumulation of chromium in I. galbana was 2

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measured using the modified method of Abinandan et al. (2019). Culture suspensions were centrifuged and washed three times with 0.25 M EDTA to remove the adsorbed chromium on the cell surface. The samples were digested by HNO3 after vacuum freeze-drying, and the intracellular chromium content was measured using inductively coupled plasma mass spectrometer (optima 8000, America). 2.9. XPS analysis of chromium The microalga biomass obtained by centrifugation was dried using vacuum freeze dryer and then analysed by XPS (Thermo VG, America). The obtained spectra were analysed and processed using the XPS Peak 4.1 software. 2.10. Molecular docking simulation Molecular docking simulation was performed by using the molecular operating environment software (MOE) (Version 2009, Chemical Computing Group, Montreal, Canada) to determine the possible binding sites of dichromate with intracellular macromolecule compounds. The crystal structure of intracellular macromolecule compounds was obtained from RSCB Protein Data Bank (http://www.pdb.org/), and the 3D structure of dichromate was obtained from the zinc database. The potential low-energy binding sites were found by the site finder module. Finally, internal interactions within these complexes were visualised by MOE ligand module. 3. Results and discussion 3.1. Chl-a and soluble protein analysis The effect of the different initial concentrations of Cr(VI) on Chl-a content of I. galbana is shown in Fig. 1a. Although I. galbana could grow at all chromium concentrations, it exhibited distinct growth conditions. At 0.5–5.0 mg L−1 Cr(VI), the Chl-a content gradually increased with culture time; at 10.0 mg L−1 Cr(VI), the Chl-a content decreased significantly after 2 days of culture. Among them, Chl-a content was always higher than that of the control at 0.5 mg L−1 Cr(VI) and reached the highest value after 7 days of culture. At exposure to 1.0 mg L−1 Cr (VI), the Chl-a content was almost equivalent to the control. However, at 10.0 mg L−1 Cr(VI), the Chl-a content reached the lowest value after 7 days of culture, which was 78% lower than that of the control. Chl-a is the main photosynthetic pigment of I. galbana and plays the role of absorbing and transferring light energy to generate biomass in the photosynthesis of I. galbana. Therefore, the content of Chl-a represents the growth potential of microalgae (Wang et al., 2016). In this study, at exposure to 0.5 mg L−1 Cr(VI), a slight increase in Chl-a content was observed compared with the control. The increase of Chl-a production may be an effective way to improve photosynthetic efficiency, thereby promoting biomass production. Increased Chl-a production promoted CO2 fixation by microalgae and distributed carbon to sugars and carbohydrates (Alho et al., 2019). At exposure 5.0 mg L−1 Cr(VI), I. galbana maintained a certain growth rate even though the biomass was lower than the control. This finding indicated that microalgae were tolerant to Cr(VI) stress. However, at 10.0 mg L−1 Cr(VI) stress, the biomass of I. galbana decreased dramatically or even the cells died. Heavy metals might have induced the inactivation of certain functional proteins in photosynthesis, resulting in the reduction in photosynthetic electron transport (Samadani et al., 2018). In general, heavy metals induce the accumulation of ROS, which leads to an imbalance of intracellular redox state, thus causing damage to proteins (Nowicka et al., 2016). This result was just consistent with previous observation that cells were damaged by peroxidation under high Cr(VI) concentration. Therefore, the high concentration of Cr(VI) could significantly inhibit the growth of I. galbana.

Fig. 1. Effects of different initial concentrations of Cr(VI) on the Chl-a (a) and soluble protein (b) contents of I. galbana.

The effect of the different initial concentrations of Cr(VI) on the soluble protein content of I. galbana is shown in Fig. 1b. Changes in soluble protein levels of I. galbana exposed to different Cr(VI) concentrations were caused by heavy metal stress. By comparing Fig. 1a and b, it could be seen that the effects of different initial concentrations of Cr(VI) on Chl-a and soluble protein contents have similar trends. At 0.5–5.0 mg L−1 Cr(VI), the content of soluble protein gradually increased with culture time. However, at 10.0 mg L−1 Cr(VI), the content of soluble protein decreased significantly. Among them, the soluble protein content was always higher than that in the control at 0.5 mg L−1 Cr(VI) and reached the highest value after 8 days of culture. The addition of 1.0 mg L−1 Cr(VI) showed no effect on the soluble protein content. However, at 10.0 mg L−1 Cr(VI), the soluble protein content reached the lowest value after 8 days of culture, which was 57.15% lower than the control. A toxic effect of Cr(VI) to I. galbana was reflected in the interference of protein synthesis. Low concentrations of Cr(VI) promoted the growth of I. galbana and thus gradually increased the soluble protein content. 3

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With increasing cadmium concentration, the growth inhibition of I. galbana became severe, and the production of soluble protein was significantly reduced. Heavy metal toxicity can inhibit protein activity or damage the structure of proteins, causing cell death. Chlorella vulgaris decreases its protein levels by 13% and 17% under Cd2+ and Cu2+ stress, respectively. This result again confirmed that Cd2+ stress inhibits the production of intracellular soluble protein and accelerates the degradation of protein (de Abreu et al., 2014). Metals cause changes in proteins, DNA and cells by inducing ROS production. Carfagna et al. (2013) studied the changes in the soluble protein content of Chlorella sorokiniana during exposure to Pb2+ or Cd2+, and the protein levels in the cells are reduced by 61% and 65%, respectively, and found that the reduction of protein content may also be attributed to the shortage of carbon skeleton from low photosynthetic rate. Although external environmental stress effects protein production, the specific mechanisms of extracellular stress signalling and intracellular protein production require further investigation. 3.2. Antioxidant enzyme analysis Heavy metal toxicity to microalgae is related to the production of ROS and the resulting imbalance of cellular redox states. Under normal circumstances, the production and elimination of ROS in microalgae is balanced. Under external environment stress, microalgae can locally produce ROS, such as superoxide anion (O2‾), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) (Shanker et al., 2009). ROS can inhibit photosynthesis and damage DNA, proteins, lipids and other biological molecules, leading to loss of protein function and even cell death (Sun et al., 2018). The severity of heavy metal stress can be expressed by the amount of oxidised proteins and lipids in microalga cells, and the most common oxidative damage marker is MDA (Li et al., 2018). In response to oxidative stress, microalgae provide cytoprotection through antioxidant mechanisms, including various enzymes, such as SOD, peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) and compounds, such as glutathione, carotenoids, and tocopherols (Ullah et al., 2019). Antioxidants can minimise oxidative damage by increasing the natural defense of cells and scavenging free radicals. Therefore, the degree of the oxidative damage of Cr(VI) to I. galbana cells was determined by measuring changes in MDA content, and the antioxidant mechanism was studied by the changes of POX activity and SOD activity in this study. With increasing Cr(VI) concentration, the content of MDA gradually increased (Fig. 2a). MDA represented the degree of membrane lipid peroxidation, which indicated that the higher the concentration of Cr (VI), the more the cells were damaged. The response of POX and SOD to oxidative stress was subsequently observed in this study (Fig. 2b and c). Although the POX activity of the I. galbana under Cr(VI) stress was higher than that of the control at almost all concentrations, the highest value reached was 1.0 mg L−1 Cr(VI) (Fig. 2b). The SOD activity of the I. galbana in 0.5, 1.0 and 5.0 mg L−1 Cr(VI) was higher than that of the control and reached the highest value at 1.0 mg L−1 Cr(VI) (Fig. 2c). This result occurred probably because SOD catalysed the disproportionation of O2.‾ to O2 and H2O2, and the resulting H2O2 could be further decomposed by POX and other enzymes (Mi et al., 2018). To mitigate Cu stress impact, when Cr(VI) was at 0.5, 1, and 5 mg L−1, the SOD activity and POX activity were significantly increased to eliminate ROS. Therefore, the enhancement of SOD activity and POX activity was an attempt to maintain the intracellular redox balance. However, when Cr(VI) was increased to 10.0 mg L−1, the SOD activity was significantly lower than that of the control, indicating the imbalance between the consumption of intracellular SOD and its production. The results showed that high concentrations of Cr(VI) may severely damage cells, which was almost consistent with the observation of the effect of Cr(VI) on the growth of I. galbana. Okamoto et al. (2001) found that Pb2+, Cu2+ and Cd2+ induce protein and lipid oxidation in the chloroplasts of Gonyaulax polyedra

Fig. 2. Effects of different initial Cr(VI) on MDA content (a), POX activity (b) and SOD activity (c) of I. galbana.

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and observed the response of SOD and APX. When Scenedesmus acuminatus was exposed to Cu2+, its POX, SOD and tocopherol levels are significantly increased to regulate intracellular oxidative stress (Hamed et al., 2017). The antioxidant enzymes are involved in the regulation of intracellular oxidative stress and are critical for the tolerance of microalgae to heavy metal stress. Therefore, the adaption of microalgae to the toxicity of heavy metals through an antioxidant system is a feasible strategy. 3.3. TEM analysis The culture with 10.0 mg L−1 chromium was analysed using TEM to reveal the effect of Cr(VI) on the ultrastructure of I. galbana. The TEM images of control and chromium-treated microalgae cells were shown. The control showed typical single-celled organisms, in which I. galbana possessed no cell wall, and the nucleus and cytoplasm showed a typical organisation. Compared with the control group, Cr(VI) caused the organelle structure indistinguishable, the cytoplasm vacuolated, some organelles gradually disappeared and the cell membrane destroyed. Esupplementary data of this work can be found in online version of the paper. Heavy metal stress on the ultrastructure of many microalga species has been studied. Zhang et al. (2013) observed that with the addition of 100.0 mg L−1 As3+, the cell morphology of Scenedesmus quadricauda is deformed, chloroplast and microalgae cell membranes are completely damaged, and other organelles gradually disappear. The amount of starch granules increases significantly in Chlorella pyrenoidosa cells exposed to copper, and the pyrenoid disappears in Scenedesmus obliquus cells (Zhou et al., 2012). Additionally, Samadani et al. (2018) observed that the organelle structure of microalgae cells under cadmium stress is difficult to distinguish, the cytoplasm is vacuolated and the relative cell size is increased. Electron-dense granules has appeared in the cells, presumably which may be cadmium absorbed by the microalgae. Some black granules were also found in the stressed cells and were presumed to be the accumulation of chromium. Therefore, the changes in cell ultrastructure are the species’ response to Cr(VI) stress.

Fig. 3. The content of chromium accumulated inside the cells of I. galbana at different initial Cr(VI) concentration.

could be rapidly absorbed by the microalgae, which also explained that the addition of 0.5 mg L−1 Cr(VI) was beneficial for the growth of I. galbana. When the initial concentration of Cr(VI) increased to 10.0 mg L−1, the amount of chromium accumulated in the cells gradually decreased. This result may be due to the inhibition of protein synthesis by the enhancement of toxicity and even the damage of cell ultrastructure, which gradually invalidated cell defines system. In conclusion, I. galbana had a certain absorption capacity for chromium, which provided a potential strategy for the treatment of heavy metal wastewater. 3.6. XPS analysis of chromium in cells XPS was performed on I. galbana cells exposed to 1.0 mg L−1 Cr(VI) for 8 days to further explore the valence change of chromium in cells. The results showed that the peaks of the chromium-loaded microalgae at 587.2 and 578.4 eV binding energies were mostly attributed to Cr (VI), whereas the peaks of the binding energy of Cr(III) were mostly at 585.6 and 576.8 eV (Wang et al., 2019). The main forms of Cr(III) were Cr2O3 and Cr(OH)3. Therefore, the part of Cr(VI) that accumulated in cells may be converted into Cr(III). The toxicity of Cr(III) is 100 times lower than that of Cr(VI) (Pan et al., 2014), so the transformation of Cr (VI) into Cr(III) with low toxicity may be a possible strategy to improve the tolerance of I. galbana to Cr(VI). Perhaps it was the presence of various reducing enzymes in cells that made it possible for Cr (VI) to be reduced to Cr (III). E-supplementary data of this work can be found in online version of the paper. The capability of bacteria to reduce Cr(VI) to Cr(III) and its possible mechanism were also described (Ma et al., 2019; Jin et al., 2016). However, the migration and transformation of heavy metals in cells is so complicated that it is difficult to obtain a comprehensive understanding. Therefore, the specific migration mode of Cr(VI) into cells and the mechanism of the valency transition of Cr (VI) in cells need to be further studied.

3.4. SEM-EDS analysis of chromium on the cell surface SEM-EDS analysis of the cells of I. galbana after exposure to 1.0 mg L−1 Cr(VI) for 8 days was performed to evaluate the local elemental distribution on the cell surface. The chromium signal was detected in a localised area of the cell surface by SEM-EDS analysis, indicating that the chromium was adsorbed or accumulated on the cell surface. According to previous studies, the functional groups on the surface of microalgae cells can specifically bind to heavy metals. Thus, the cell surface is the first barrier for microalgae exposed to heavy metals (Kumar et al., 2015). Miranda et al. (2012) also performed energy dispersive X-ray analysis on the surface of Oscillatoria laete-virens cells under Pb2+ stress, indicating that Pb2+ is adsorbed to the cell surface. E-supplementary data of this work can be found in online version of the paper. Therefore, when exposed to heavy metals, the cell membrane of I. galbana acted as a barrier to protect cells from toxic damage. 3.5. Extracellular accumulation of chromium

3.7. Molecular docking simulation

The cell surface of microalgae adsorbs, actively consume and store heavy metals in organelles (Garnham et al., 1992). The content of chromium accumulated inside the cells of I. galbana is shown in Fig. 3. At initial 0.5 mg L−1 Cr(VI), the amount of chromium accumulated in the cells reached the maximum, indicating that chromium migrated into cells in a certain way. The direct binding of the small amounts of heavy metals to metallothionein was an important detoxification mechanism, and the compartmentalisation of heavy metals in cells was also an effective detoxification strategy (Zhou et al., 2018). Given the efficient metal storage system in microalgae, a small amount of Cr(VI)

A small amount of heavy metals enters the cell through a certain migration mode, and the combination with macromolecular compounds in the cell might be an effective detoxification method. Molecular docking simulation using software was conducted to explore the mechanism of interaction with macromolecular compounds in I. galbana cells. MOE is an effective means for studying the optimal binding sites for Cr(VI) and intracellular macromolecular compounds. The selected macromolecular compounds were POX, Alr0975 protein, 5

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Fig. 4. Molecular docking simulation of dichromate and macromolecular compounds in I. galbana cells. (a: peroxidase; b: Alr0975 protein; c: glucosidase BGLN1; d: membrane protein LCI1 channel).

Supervision.

glucosidase BGLN1 and membrane protein LCI1 channel in microalgae. The results of the molecular docking of dichromate and macromolecular compounds in microalga cells are shown in Fig. 4. Dichromate could react with TyrB95, GluA179, ArgA180 and LysA206 of Alr0975 protein to form hydrogen bonds. Dichromate could form hydrogen bonds with the LysA471, LysE471, LeuB488 and LeuF488 of glucosidase BGLN1 and with LysA105 and ArgC108 of the membrane protein LCI1 channel. However, dichromate could react with Ser188 and Arg52 of POX to form hydrogen bonds, which might hinder the formation of hydrogen bonds between H2O and amino acid residues, resulting in a steric hindrance (Li et al., 2017). Steric hindrance might affect the biological activity of the protein, which provided a reasonable explanation for the slight decrease in the activity of POX with the addition of 5.0 mg L−1 Cr (VI). In conclusion, stable hydrogen bond formation appeared between Cr(VI) and each protein. Therefore, the stable hydrogen bond between Cr(VI) and proteins could be an effective detoxification mechanism.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Thanks to the experimental team for their help. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.122860.

4. Conclusion References High Cr(VI) conditions and 0.5 mg L−1 Cr(VI) levels severely inhibited and stimulated the growth of I. galbana, respectively. The tolerance mechanism of I. galbana to Cr(VI) included the (1) increased activities of SOD and POX for peroxidative damage resistance; (2) accumulation of Cr(VI) on the cell surface and inside the cell for detoxification; (3) conversion of the intracellular Cr(VI) to the less toxic Cr (III), as indicated by the XPS results. Molecular docking simulation also showed that Cr(VI) was bounded with proteins in microalga cells in the form of hydrogen bonds to serve as detoxification mechanism.

Abinandan, S., Subashchandrabose, S.R., Panneerselvan, L., Venkateswarlu, K., Megharaj, M., 2019. Potential of acid-tolerant microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, in heavy metal removal and biodiesel production at acidic pH. Bioresour. Technol. 278, 9–16. Alho, L.D.O.G., Gebara, R.G., Paina, K.D.A., Sarmento, H., Melão, M.D.G.G., 2019. Responses of Raphidocelis subcapitata exposed to Cd and Pb: mechanisms of toxicity assessed by multiple endpoints. Ecotoxicol. Environ. Saf. 169, 950–959. Ammar, S.H., Khadim, H.J., Mohamed, A.I., 2018. Cultivation of Nannochloropsis oculata and Isochrysis galbana microalgae in produced water for bioremediation and biomass production. Environ. Technol. Inno. 10, 132–142. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Carfagna, S., Lanza, N., Salbitani, G., Basile, A., Sorbo, S., Vona, V., 2013. Physiological and morphological responses of lead or cadmium exposed Chlorella sorokiniana 211–8K (Chlorophyceae). SpringerPlus 2, 147–153. De Abreu, F.C.P., da Costa, P.N.M., Brondi, A.M., Pilau, E.J., Gozzo, F.C., Eberlin, M.N., Trevisan, M.G., Garcia, J.S., 2014. Effects of cadmium and copper biosorption on Chlorella vulgaris. Bull. Environ. Contam. Toxicol. 93, 405–409.

CRediT authorship contribution statement Meng Jin: Methodology, Investigation, Writing - original draft. Xinfeng Xiao: Conceptualization, Writing - review & editing, Formal analysis. : . Liguo Qin: Data curation. Weiwei Geng: Validation. Yu Gao: Resources. Lin Li: Project administration. Jianliang Xue: 6

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