Effects of Selenite on Unicellular Green Microalga ... - ACS Publications

Article Cite This: J. Agric. Food Chem. 2017, 65, 10875−10883

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Effects of Selenite on Unicellular Green Microalga Chlorella pyrenoidosa: Bioaccumulation of Selenium, Enhancement of Photosynthetic Pigments, and Amino Acid Production Yu Zhong† and Jay J. Cheng*,†,‡ †

School of Environment and Energy, Peking University-Shenzhen Graduate School, Shenzhen 518055, China Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States

‡

ABSTRACT: Microalgae were studied as function bioaccumulators of selenium (Se) for food and feed supplement. To investigate the bioaccumulation of Se and its effects on the unicellular green alga Chlorella pyrenoidosa, the algal growth curve, fluorescence parameters, antioxidant enzyme activity, and fatty acid and amino acid profiles were examined. We found that Se at low concentrations (≤40 mg L−1) positively promoted algal growth and inhibited lipid peroxidation and intracellular reactive oxygen species. The antioxidative effect was associated with an increase in the levels of glutathione peroxidase, catalase, linolenic acid, and photosynthetic pigments. Meanwhile, a significant increase in amino acid and organic Se content was also detected in the microalgae. In contrast, we found opposite effects in C. pyrenoidosa exposed to >60 mg L−1 Se. The antioxidation and toxicity appeared to be correlated with the bioaccumulation of excess Se. These results provide a better understanding of the effect of Se on green microalgae, which may help in the development of new technological applications for the production of Se-enriched biomass from microalgae. KEYWORDS: amino acids, antioxidants, bioaccumulation, Chlorella pyrenoidosa, fatty acid profile, selenium

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INTRODUCTION

with high bioavailability. However, there have been only a few studies of the effect of Se on algae from the genus Chlorella. Chlorella showed superior performance in terms of biomass production or lipid production using wastewater for the cultivation of microalgae. Most of the microalgal products are sold on the market as animal feed (30%) or food supplements because of the high content of various metabolites, including polyunsaturated fatty acids, pigments, antioxidants, and other bioactive molecules.14 Therefore, the biomass of Chlorella sp. is considered to be a good alternative source of organically bound Se for food supplementation.15 Delineating the impact of Se on Chlorella could lead to future research that will expand the potential of microalgal biotechnology. In water, inorganic Se is present as selenate (SeO42−) and selenite (SeO32−). When Se was added to the culture medium, growth was stimulated in some algae,13,16 and such a growthstimulating effect is stronger for selenite ions than for selenate ions in Chlorella vulgaris.17 In addition, among both selected Chlorella species, Chlorella pyrenoidosa differs from C. vulgaris because of the presence of prominent pyrenoids within the chloroplast. A high protein content made C. pyrenoidosa an ideal protein resource. The objective of this study was to evaluate selenite biotransformation and the resulting effect on the growth curve, photosynthetic pigments, intracellular ROS generation, the activity of antioxidant enzymes, the profile of amino acids, and the fatty acid of green alga C. pyrenoidosa

Selenium (Se) is a natural trace element that acts either as an essential micronutrient or as a toxicant depending on the dose in biological and environmental systems. At low levels, Se shows beneficial effects for humans against multiple types of cancers, cardiovascular disease, and type 2 diabetes.1 However, Se at high concentrations can cause the generation of reactive oxygen species (ROS), which induce DNA oxidation, DNA double-strand breaks, and cell death.2 Dietary Se deficiency was found to result in reduced growth, vitality, and reproduction in animals and to cause severe disease in both livestock and humans (such as fatal Keshan disease).3 However, excess Se from the environment or industrial waste has a toxic impact on vertebrates and may cause the extinction of local fish populations.4 According to Lintschinger et al.,5 almost all European countries are classified as low-selenium regions. Selenium supplementation using microorganisms has received much attention in the past decade.6 In terms of microalgae, Se has been reported to be essential for at least 33 species, particularly for green algae, which can take up inorganic Se and incorporate it into amino acids through the sulfur assimilation metabolic pathway.7,8 In humans, 25 selenoproteins were identified.11 Compared with inorganic Se, organic Se has improved safety and bioavailability.9 Hence, green algae have become a target organism in Se bioaccumulation research.10 The metabolism and bioaccumulation of Se have been comprehensively studied in the green algae Chlamydomonas reinhardtii,10 Scenedesmus quadricauda,11 Dunaliella salina,12 and Ulva fasciata.13 These Se-enriched green algae are potential producers of organic Se © 2017 American Chemical Society

Received: Revised: Accepted: Published: 10875

September 12, 2017 November 23, 2017 November 27, 2017 November 27, 2017 DOI: 10.1021/acs.jafc.7b04246 J. Agric. Food Chem. 2017, 65, 10875−10883

Article

Journal of Agricultural and Food Chemistry

The activities of antioxidant enzymes in the protein extract were examined using a spectrophotometric diagnostic kit (NanJing JianCheng Bio Inst, Nanjing, China). Basically, the catalase (CAT) activity was measured according to the ammonium molybdate spectrophotometric method, based on the fact that ammonium molybdate could rapidly terminate the H2O2 degradation reaction catalyzed by CAT and react with the residual H2O2 to generate a yellow complex that could be monitored by the absorbance at 405 nm. Glutathione peroxidase (GSH-Px) activity was measured by quantifying the rate of H2O2-induced oxidation of GSH to oxidized glutathione (GSSG), catalyzed by GSH-Px. The GSH-Px content in the supernatant was measured by reaction with dithionitrobenzoic acid (DTNB) and monitored by the absorbance at 412 nm. Determination of the Total and Organic Se Content. The total Se concentration was determined with an inductively coupled plasma mass spectrometer (ICP-MS).16 After cultivation for 6 days, a 100 mg dried algal sample was digested with concentrated nitric acid and H2O2 [3/1 (v/v)] in a digestive stove (Qian Jian Measuring Instrument Co., Ltd.) at 180 °C for 3 h. Samples were allowed to cool, dissolved in 0.6% HNO3, and filtered through Whatman filter paper. Prior to drying, the samples were observed under a microscope to remove any contamination of non-algal particulates. The volume of each sample was maintained at 10 mL with 0.6% HNO3 and analyzed for total Se content with the ICP-MS. For inorganic Se determination, a 100 mg dried algal sample was extracted with 15% HCl (three times, to make sure the extraction of inorganic Se was complete), and the extract was analyzed with the ICP-MS. The organic Se concentration was calculated from the difference between the total Se and inorganic Se concentrations. An inductively coupled plasma mass spectrometer (model 7500ce, Agilent Technologies) was used for the analysis using the following operational conditions: forward power of 1500 W, sampling depth of 7−8 mm, auxiliary gas flow rate of 0.10−0.15 mL min−1, extract I of 0−3 V, extract II of −1375 V, omega Bias-ce of −20 V, omega Lens-ce of −1.6 V, cell entrance of −40 V, QP focus of −15 V, cell exit of −44 V, octP RF of 190 V, octP bias of −18 V, H2 flow rate of 3.8 mL min−1, QP bias of −16 V, discriminator of 8 mV, and analog HV 1840V. For quantification of Se, a standard addition method was used to eliminate matrix effects of residual carbon and other matrix elements. Se isotopes 77 and 82 were used, as these isotopes did not suffer from Ar-based spectral interference.8 Lipid and Protein Extraction. The freeze-dried algal powder was suspended in 0.1 mol L−1 PBS buffer (pH 7), frozen in liquid nitrogen for 10 min, and then thawed in 40 °C water. The residues after centrifugation at 4000g for 10 min were treated twice more as described above. The protein-containing supernatant was precipitated by 30−70% (NH4)2SO4 and the obtained crude protein eluted on a Sephadex G-100 column (1650 cm) with 0.1 mol L−1 PBS (pH 7) at a rate of 0.5 mL min−1. The eluted protein solutions were collected and dialyzed against sterilized water, concentrated with polyglycol (Mr = 20000), and finally freeze-dried.20 The extraction of lipid from C. pyrenoidosa was performed using the Folch extraction method.21 Folch extraction was performed by homogenizing the freeze-dried algal biomass with a chloroform/ methanol mixture [2/1 (v/v)]. The mixture was agitated at room temperature for 15−20 min and centrifuged at 7000 rpm at 4 °C for 7 min to separate cell debris from supernatant. The supernatant was washed with 0.2 volume of a 0.9% NaCl solution. The mixture was vortexed for a few seconds and again centrifuged at 3000 rpm for 5 min. The upper phase was removed and the lower chloroform phase containing the lipid evaporated under vacuum by a rota evaporator using weighing bottles. Amino Acid Analysis. The hydrolyzed samples were analyzed according to the guidelines of the European Community (98/64/EG). Amino acid samples were separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm (440 nm for proline) using the automatic amino acid analyzer (model LC-3000, Eppendorf-Biotronik, Hamburg, Germany). An amino acid standard solution (A9781 from SigmaAldrich) (0.5 μmol mL−1) was injected to calibrate the analyzer and to calculate the amount of amino acid in the samples.

cultivated in the laboratory. The production of Se-Chlorella biomass as a food supplement seems to have great potential for algal biotechnology.

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MATERIALS AND METHODS

Culture Conditions and Se Treatment. C. pyrenoidosa SZ 16 was obtained from the Research Center of Hydrobiology of Jinan University (Guangzhou, China). Unialgal stock cultures were propagated and maintained in Erlenmeyer flasks containing BG11 medium (pH 6.8) at 25 °C, a photon flux density of 45 μmol m−2 s−1 provided by two white fluorescent tubes, and a 12 h/12 h light/dark photoperiod. Flasks were continuously shaken at 100 rpm. The basal culture medium consisted of the following: 0.7 g L−1 KH2PO4, 0.3 g L−1 K2HPO4, 0.3 g L−1 MgSO4·7H2O, 3 mg L−1 FeSO4·7H2O, 0.1 g L−1 glycine, 0.01 mg L−1 vitamin B1, and a 1 mL L−1 A5 trace mineral solution [A5 consisted of 2.86 g L−1 H3BO3, 1.86 g L−1 MnCl2·4H2O, 0.22 g L−1 ZnSO4·7H2O, 0.39 g L−1 Na2MoO4·2H2O, 0.08 g L−1 CuSO4·5H2O, and 0.05 g L−1 Co(NO3)2·H2O]. C. pyrenoidosa cultures in the late exponential growth phase were decanted into 250 mL flasks containing 100 mL of medium at 25 ± 0.5 °C and illuminated with fluorescent lights (60 μmol m−2 s−1 photon flux intensity) under a 12 h/12 h light/dark photoperiod. The cultures were initiated at a density of 6 × 107 cells mL−1, shaken periodically, and used in triplicate. All solutions and experimental containers were autoclaved at 121 °C for 15 min. Sodium selenite (Na2SeO3) was added to the medium before inoculation at a concentration of 20, 40, 60, or 80 mg L−1. Cultures grown without sodium selenite served as controls. Measurement of Algal Growth. Algal cells (10 mL) were collected by centrifugation (Eppendorf 5804R centrifuge, Eppendorf, Hamburg, Germany) at 8000 rpm for 1 min at 22 °C. The supernatant was then discarded, and the algal cells were resuspended in 1 mL of sterilized deionized water. The resuspended cells were diluted for cell number count. Cell counting was performed with a Hemocytometer, and optical density (OD) values of the algae were measured under a 750 nm absorbance with a FlexStation 3 spectrophotometer (Molecular Devices, Sunnyvale, CA). Using the regression equation between OD750 and cell density, cell numbers were calculated from only the OD750 values of the algae. At each Se concentration, the density of the culture (OD750) was adjusted to 0.05 by control of the dilution rate until a steady state was reached. Measurement of Photosynthetic Pigments. Photosynthetic pigments were extracted from algal biomass after a 6 day cultivation. In brief, 40 mL of the culture solution was centrifuged at 10000g for 10 min. Pellets were then homogenized (Polytron homogenizer, ArtMiccra D-8) with 5 mL of cold acetone (80%), followed by centrifugation at 10000g for 10 min at 4 °C. Photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoid) in the supernatant were quantified spectrophotometrically at 663 and 646 nm18

y = 8 × 106x − 72800

R2 = 0.987

(1)

where y is the cell density and x = OD750. Chl a = 12.21A 663 − 2.81A 646

(2)

Chl b = 20.13A 646 − 5.03A 663

(3)

carotenoid concentration = (1000A470 − 3.27 × Chl a − 104 × Chl b)/229

(4)

where Chl a, Chl b, and carotenoid are given in units of milligrams per liter. Antioxidant Enzyme Assay. The C. pyrenoidosa cell suspensions were centrifuged, and then the pellets were resuspended in prechilled phosphate buffer (pH 7.0) and disrupted by ultrasonication. The homogenate was centrifuged at 10000g for 30 min, and the supernatant was collected. The total protein content in the supernatant of homogenate extracts was determined according to the Bradford method, using bovine serum albumin as a standard.19 10876

DOI: 10.1021/acs.jafc.7b04246 J. Agric. Food Chem. 2017, 65, 10875−10883

Article


Figure 1. (A) Effect of different concentrations of selenite on the growth curves of C. pyrenoidosa. (B) Total and organic Se contents of the dried C. pyrenoidosa biomass grown in medium at day 6. (C) Changes in organic Se contents of C. pyrenoidosa during cultivation time of days 3, 6, 9, and 12 at different Se concentrations. (D) Distribution of organic Se in biochemical components in C. pyrenoidosa. Values expressed are means ± the standard deviation (SD) of three replicates.

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Fatty Acid Analysis. In situ transesterification of algal lipids was performed with a modified protocol. The freeze-dried algal biomass was placed in a glass test tube and mixed with 1.7 mL of methanol, 0.3 mL of sulfuric acid, and 2 mL of hexane. The reaction mixture was heated at 90 °C for 1 h, and the samples were well-mixed while they being heated. After the reaction, the tubes were allowed to cool to room temperature, and 2 mL of distilled water was added, vortexed, and centrifuged for 30 min at 3220g. The hexane layer that contained FAME was collected and transferred to a preweighed glass vial. The solvent was evaporated using N2, and the masses of FAMEs were determined gravimetrically.22 FAME profiles were determined using gas chromatography (model 7890A, Agilent) with an autosampler, a flame ion detector (FID), and a DB5-MS column (60 m × 250 μm × 0.25 μm). The column oven was set to a 70 °C equilibration for 2.0 min, heated at a rate of 10 °C min−1 to 230 °C and at a rate of 3 °C min−1 to 290 °C, and then held for 5 min; the injection temperature was 280 °C (splitless injection). Helium was used as the carrier gas. Statistical Analysis. All treatments and controls were performed in triplicate. The significance of the difference between mean values obtained from four independent experiments was determined by oneway analysis of variance (ANOVA) at the 95% confidence interval by using SPSS software.

RESULTS AND DISCUSSION Accumulation of Se and Its Effects on C. pyrenoidosa Growth. Studies have reported that some algae require Se for growth, such as Emiliania huxleyi.7 Therefore, we first studied the growth curves of C. pyrenoidosa under different Se concentration treatments. Figure 1 shows the result. In our study, an increase in biomass was obtained in cells exposed to low Se concentrations (≤40 mg L−1), with the highest biomass found at 40 mg L−1 Se. However, higher Se concentrations (>40 mg L−1) significantly decreased the rate of growth of C. pyrenoidosa (P < 0.05, compared to the control group), possibly because of the toxic effects of high Se stress, and few red dots can be found on the cell precipitate when C. pyrenoidosa was exposed to Se at concentrations of >80 mg L−1 at the end of the cultivating process. This result is in agreement with the previous study showing that Se is toxic at higher concentrations.8 The capacity of microalgae to assimilate dissolved inorganic Se is limited by prevailing ambient conditions. Above the tolerance dose, Se may result in a decreased growth rate, photosynthesis inhibition, and damage to the cell ultrastructure. For example, chloroplasts are responsible for photosynthesis, when structural damage caused by a high concentration of Se would negatively 10877


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Figure 2. Effect of different concentrations of selenite on the (A) chlorophyll fluorescence parameter Fv/Fm, (B) chlorophyll a content, (C) chlorophyll b content, and (D) carotenoids of C. pyrenoidosa. The measurements were performed every 3 days from day 0 to 12 of cultivation. Values expressed are means ± the standard deviation (SD) of three replicates.

impact biomass growth.23 Compared to Haematococcus pluvialis (13 mg L−1) and maize Zea mays L. (4.3 mg L−1),24,25 C. pyrenoidosa showed a better tolerance of Se (40 mg L−1), indicating that there is a large potential space for C. pyrenoidosa to be a good Se bioaccumulator. We next studied the amount of cellular Se. The control groups grown in the absence of Se possessed an extremely small amount of cellular Se (undetected by ICP). After selenite was added, the intracellular Se contents all significantly increased (P < 0.001) (Figure 1B). At selenite concentrations of 40 mg L−1, the level of bioaccumulated Se started to decline. Under 20−80 mg L−1 Se treatment, the amount of organic Se that accumulated in C. pyrenoidosa increased proportionately with cultivation time (0−3 days), and no lag time was observed. Approximately 74% of organic Se accumulated during the first 3 days of exposure under 40 mg L−1 Se treatment. A slower increase was found from day 3 to day 6. After day 6, a slow decrease in the rate of Se accumulation was found. The maximum accumulation of organic Se was recorded at 337 μg g−1 at 40 mg L−1 Se for 6 days (Figure 1B,C), indicating that most of the selenite was biotransformed into organic Se during the culture. In our study, the total Se accumulation is very close to that of S. quadricauda at a 10 mg L−1 selenite dosage and the percentage of organic Se in C. pyrenoidosa is double that in S. quadricauda.26 Organic Se (>73%) was the most abundant constituent of Se compounds in C. pyrenoidosa at 40 mg L−1). Effect of Se on Activities of Antioxidant Enzymes. The function of selenium in an organism is mediated mostly by selenoproteins, including glutathione peroxidase. Glutathione peroxidase is a potent antioxidative enzyme, scavenging a variety of peroxides. In a previous study, we revealed that the antioxidant effect of Se was associated with increased activities of GSH-Px in macroalga U. fasciata.13 Seppänen et al. confirmed that Se altered the accumulation of the transcript of GSH-Px in potato.35 In our study, CAT and GSH-Px activities were assayed (Figure 3). In comparison to the control, the activities of CAT and GSH-Px changed with the intracellular Se content. With the levels of intracellular total (or organic) Se increasing from 0 to 40 mg L−1 in the culture medium, the CAT and GSH-Px activities increased. However, the activities of CAT and GSH-Px in higher-Se concentration culture medium (>40 mg L−1) decreased with a decrease in intracellular total (or organic) Se content (Figures 1B and 3). The maximum activity of CAT was found under 40 mg L−1 Se treatment and was 1.57 ± 0.02 IU/μg of protein, consistent with the intracellular Se content. A sharp increase in GSH-Px activity was observed when cells were exposed to 40 mg L−1 Se, with the maximum activity (207.5 ± 11.8 IU/mg of protein) showing a positive correlation between Se concentration and GSH-Px activity. Higher Se concentrations (60−80 mg L−1) resulted in a significant decrease in CAT activity (P < 0.05) compared to that seen with the lower Se concentrations (20− 40 mg L−1), which reached their minimum (0.49 ± 0.02 IU/μg of protein) at 80 mg L−1 Se. Se acts as an antioxidant at low concentrations with its integration into selenoproteins, However, if the intracellular Se content is too high, it may result in oxidative stress and excess ROS production. In our study, inhibitory effects on CAT and GSH-Px activities caused by high intracellular Se contents were not observed, probably because the maximum intracellular Se level might be below the upper tolerance limit of C. pyrenoidosa. Effect of Se on Intracellular ROS Generation and Lipid Peroxidation (LPO). Environmental stress can increase the levels of ROS. For example, the toxicity of metals to marine algae may alter the cellular levels of ROS, resulting in cellular

The variation in photosynthetic pigment content corresponded to chlorophyll fluorescence (Figure 2B,C). A significant (P < 0.05) and continuous increase in carotenoid and chlorophyll contents was observed in C. pyrenoidosa when it was exposed to Se concentrations of ≤40 mg L−1. Maximum levels of carotenoid, chlorophyll a, and chlorophyll b reached 13.4, 52.8, and 21.5 mg L−1, respectively, after 12 days (Figure 2). Carotenoids, as some of the non-enzymatic antioxidants, play an important role in the cellular response to oxidative stress by reducing the level of ROS. The increase in carotenoid content in C. pyrenoidosa may be due to its antioxidant mechanisms.30 Changes in chlorophyll content may depend on the amounts of carotenoids. Because of their antioxidant properties, carotenoids are involved in the photosynthetic membranes protecting against photooxidation and neutralization of peroxide radicals, preventing lipid membranes of chloroplasts and chlorophyll degradation, and consequently increasing the amount of green pigments in cells.31 In addition, it has been established that Se is present in all lipid fractions, and the maximal amount of Se-containing lipids has been found in carotenoids.32 The increased content of photosynthetic pigments in C. pyrenoidosa is perhaps due to the need for chloroplasts, the photosynthetic efficiency of which partially decreased as a result of SeO32− ions bound by lipids and chlorophyll−protein complexes. However, at higher Se concentrations (>40 mg L−1), a significant decline (P < 0.05) in the overall content of photosynthetic pigments (including carotene and chlorophyll a and b) was observed (Figure 2). A similar inhibitory effect on chlorophyll production was reported in freshwater microalgae (Desmodesmus and Pseudokirchneriella) upon their exposure to high Se levels.33 Depending upon the Se concentration, the chloroplast was confirmed to be the organelle most affected by the ultrastructure modification, including fingerprint-like thylakoids, dense cytosol, less dense stroma, and autophagic vacuoles.8 This suggests that Se can substitute for sulfur in Rieske iron−sulfur protein, which is a constituent of the cytochrome of the complex located on the thylakoid membrane.34 Because the cytochrome of the complex acts as an electron carrier from PSII to PSI, a high level of Se inhibits photosynthetic electron transport on the thylakoid membrane and, therefore, photosynthesis and the subsequent decrease in energy supply and cell growth.29 This phenomenon can further explain the growth inhibition at high Se concentrations. In 10879


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Figure 4. Effect of selenite on (A) lipid peroxidation (LPO) and (B) intracellular reactive oxygen species (ROS) on day 6. Error bars represent standard deviations of triplicate cultures. A single * represents a significant difference compared to the control (P < 0.05), and a single ● represents a significant difference between 40 mg L−1 Se treatments and other Se treatments (P < 0.05).

Table 1. Amino Acid Composition of C. pyrenoidosa Cultured with Different Concentrations of Se after Cultivation for 6 Daysa amino acids (% of dry weight) −1

0 mg L

Se

−1

20 mg L

40 mg L−1 Se

Se

60 mg L−1 Se

80 mg L−1 Se

EAA isoleucine leucine lysine methionine phenylalanine threonine valine

0.39 2.65 1.13 0.34 1.09 0.96 1.07

± ± ± ± ± ± ±

0.01 0.00 0.01 0.01 0.00 0.01 0.02

0.38 3.11 1.53 0.34 1.16 0.99 2.03

± ± ± ± ± ± ±

0.00 0.10* 0.03* 0.00 0.02 0.01 0.00*

0.50 3.59 1.75 0.32 1.34 1.24 1.50

± ± ± ± ± ± ±

0.00* 0.00* 0.01* 0.00 0.02* 0.03* 0.03*

0.43 3.21 1.56 0.30 1.23 1.08 1.35

± ± ± ± ± ± ±

0.00 0.04* 0.00* 0.01 0.03* 0.02 0.01*

0.39 3.20 1.51 0.31 1.24 1.04 1.29

± ± ± ± ± ± ±

0.01 0.05* 0.01* 0.01 0.03* 0.01 0.03

alanine arginine aspartic acid glutamic acid proline cysteine glycine histidine serine tyrosine

1.42 ± 1.45 ± 1.79 ± 1.85 ± 1.21 ± 0.08 ± 1.53 ± 0.32 ± 0.81 ± 0.57 ± 9.73 8.93 18.66

0.05 0.04 0.01 0.06 0.00 0.00 0.01 0.00 0.01 0.01

1.65 ± 1.73 ± 1.74 ± 2.04 ± 1.08 ± 0.10 ± 1.47 ± 0.09 ± 0.84 ± 0.63 ± 12.07 8.84 20.91

0.02 0.05* 0.03 0.01 0.02 0.00 0.01 0.00* 0.01 0.01

2.03 ± 1.84 ± 2.19 ± 2.40 ± 1.32 ± 0.17 ± 1.85 ± 0.17 ± 1.02 ± 0.49 ± 12.67 11.04 23.71

0.06* 0.06* 0.06* 0.06* 0.02* 0.00 0.01* 0.00* 0.02 0.01

1.79 ± 1.74 ± 1.96 ± 2.24 ± 1.20 ± 0.13 ± 1.58 ± 0.14 ± 0.90 ± 0.44 ± 11.47 9.8 21.27

0.04* 0.01* 0.03* 0.01* 0.03 0.00 0.02 0.00* 0.03 0.01*

1.72 ± 1.88 ± 1.96 ± 2.21 ± 1.23 ± 0.11 ± 1.51 ± 0.13 ± 0.88 ± 0.49 ± 11.46 9.63 21.09

0.02* 0.00* 0.06* 0.01* 0.00 0.00 0.00 0.00* 0.01 0.01

NEAA

∑EAA ∑NEAA total AA

Abbreviations: EAA, essential amino acid; NEAA, non-essential amino acid; AA, amino acid. Values represent the means ± the standard error (SE) of three replicates (N = 3). Values followed by an asterisk in the same column are significantly different from the control at the P < 0.05 level.

a

ambient Se concentration (>40 mg L−1). The maximum LPO level (91.4 ± 1.4 nmol of MDA/mg of protein) was obtained at 80 mg L−1 Se. During oxidative stress, excess ROS production causes membrane damage, eventually leading to cell death. For protection against ROS, plants contain antioxidant enzymes such as CAT and GSH-Px as well as a wide array of nonenzymatic antioxidants (such as carotenoid).37 As shown in Figures 2 and 3, the higher carotenoid content and GSH-Px activity could reduce the level of ROS caused by a high Se concentration during cultivation. Effect of Se on the Intracellular Amino Acid and Fatty Acid Composition. Selenium can significantly influence the amino acid content of plant tissues.38 The total content of selected amino acids was higher in cells of all Se-treated C.

injury. ROS and LPO are well-known as indices for determining the degree of oxidative stress and considered main contributors to growth retardation.36 In previous studies, high concentrations of selenium from the environment caused oxidative stress in plants, such as S. quadricauda, U. fasciata, and C. vulgaris, which led to the induction of ROS.11,13,16 In our study, the LPO level decreased significantly in cells treated with increasing levels of ambient Se from 0 to 40 mg L−1 and reached the minimum (70.9 ± 1.7 nmol of MDA/mg of protein) at 40 mg L−1 Se (Figure 4A). However, high concentrations of Se may cause oxidative stress and membrane damage through the production of ROS. Compared to that of the low-Se concentration group, the LPO and ROS levels increased significantly (P < 0.05) at a high 10880


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Table 2. Fatty Acid Profile of C. pyrenoidosa Cultured with Different Concentrations of Se after Cultivation for 6 Daysa fatty acid profile (% of total fatty acid) C16:0 C16:1 C17:1 C18:1 C18:2 C18:3 other n6/n3

control

20 mg L−1 Se

40 mg L−1 Se

60 mg L−1 Se

80 mg L−1 Se

11.4 ± 0.6 2.1 ± 0.1 8.3 ± 0.0 4.3 ± 0.1 17.9 ± 0.0 26.7 ± 0.8 29.3 ± 0.0 0.67 ± 0.0

11.1 ± 0.0 2.1 ± 0.1 8.9 ± 0.7 2.0 ± 0.0* 17.8 ± 0.8 30.4 ± 1.7* 27.7 ± 3.3 0.58 ± 0.0

11.3 ± 0.3 2.1 ± 0.3 9.0 ± 1.0 2.1 ± 0.0* 18.2 ± 0.3 30.0 ± 0.7* 27.2 ± 2.4 0.60 ± 0.0

11.9 ± 0.1 2.3 ± 0.2 8.6 ± 0.3 2.6 ± 0.0* 17.9 ± 0.5 30.5 ± 0.5* 26.1 ± 2.3 0.58 ± 0.0

11.4 ± 0.1 2.5 ± 0.1 8.5 ± 0.3 2.1 ± 0.1* 19.2 ± 0.3 26.9 ± 1.6* 29.4 ± 2.3 0.71 ± 0.1

Values represent the means ± the standard error (SE) of three replicates (N = 3). Values followed by an asterisk in the same column are significantly different from the control at the P < 0.05 level.

a

pyrenoidosa than in the control (Table 1). This confirms the findings of Ježek.39 In the study presented here, application of a small dose of Se had a positive effect on the cell total amino acid content, increasing by 12.1% in 20 mg L−1 Se and by 27.1% in 40 mg L−1 Se compared with the controls. However, higher levels of selenite supplementation did not result in further increases in the total amino acid content. After application of the larger dose of Se (>40 mg L−1), a small decrease in the total content of amino acids was detected compared to that seen with the 40 mg L−1 medium, but still larger than the control. The significant influence of amino acid content in plant tissues can be caused by nutrition and fertilization, such as magnesium and selenite.38 We next investigated the amino acid composition in C. pyrenoidosa with Se treatment. The amino acid composition (percent of dry weight) is illustrated in Table 1. In terms of essential amino acids, levels isoleucine, leucine, lysine, phenylalanine (Phe), threonine (Thr), and valine increased 28, 35, 54, 23, 29, and 40%, respectively, because of the application of 40 mg L−1 Se. The one with a large increase after Se treatment at all concentrations was Phe. Munshi et al. also detected an increase in Phe content in potato tubers from 20 to 53% with an increasing rate of Na2SeO3 application.38 Adámková et al. reported that one of the defense mechanisms of plants against stress is the production of phenylpropanoids, which are derived from Phe.40 The positive correlation observed here between Se application and Phe synthesis is probably another mechanism that is triggered under conditions of stress. With regard to the essential amino acids, the content of Thr also underwent some noteworthy changes. The Thr level increased significantly after application of 40 mg L−1 Se, by 29.1% compared with the controls. Methionine (Met) was the limiting essential amino acid, and application of Se in our study did not affect the Met content of C. pyrenoidosa, confirming previous results reported by Ježek.39 The results for non-essential amino acids are also listed in Table 1. The application of Se markedly influenced the content of aspartic acid (Asp); the level of Asp increased on average by 22.3% (40 mg L−1 Se) compared with the controls. Asparagine (Asn) results from amidation of aspartic acid (Asp). Munshi et al. discovered that the total content of Asn increased on average by 13% in Se-treated potato tubers.38 Aspartic acid and Asn are important in the transport and storage of nitrogen in plant tissues. They perform this function even under stress when the plant cannot ensure the standard level of proteosynthesis (nutrient deficiency, drought, salting, heavy metal contamination, and/or pathogen attack).40

The content of Pro, the free form of which is connected with the resistance of plants to drought and salinization, increased by ≤9% after the 40 mg L−1 Se treatment. This confirms the findings of Munshi.38 However, many researchers believe that the accumulation of proline is simply an indicator of several stresses and that it is not involved in protecting plants from metal toxicity. With regard to cysteine (Cys), because of the chemical analogue of sulfur, green microalgae incorporate selenite preferentially into selenoproteins and various Se-amino acids, particularly Se-Cys and Se-Met.6 These amino acids can participate in protein synthesis as well as S-amino acids. In our study, the application of either 20 or 40 mg L−1 selenite to C. pyrenoidosa increased the level of Cys by 1.25- or 2.12-fold, respectively (Table 1). This confirms the findings of Munshi, but Munshi also reported the free amino acid cysteine content was decreased significantly by selenium fertilization.38 The Cys content of leaves was also influenced by the form of Se applied. ́ et al. with lettuce plants (Lactuca sativa In experiments by Rios L.), the Cys content decreased with an increasing rate of selenite (SeO32−) application but increased with an increasing rate of selenate (SeO42−) application.41 Se-Cys and Se-Met are also the main Se compounds that accumulated in Se-enriched C. pyrenoidosa, accounting for >50% of total accumulated organic Se in our study. Polyunsaturated fatty acids (PUFAs) are important nutrients, because they are essential constituents of cell membranes or precursors for eicosanoids. Microalgae are potential sources of many highly valuable products such as PUFAs. The fatty acid composition of algae can vary quite markedly throughout the growth cycle, as the biosynthesis of lipids is significantly influenced by the physiological state of the organism and environmental conditions.42 High levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been detected in many species of both microscopic and macroscopic marine algae. In contrast, very few species of freshwater algae contain significant amounts of EPA and DHA.43 Chlorophyceae (green algae) can grow very fast, and a high level of lipids can accumulate; however, polyunsaturated fatty acids are generally limited to various isomers of linolenic acid. In C. pyrenoidosa, most of the fatty acids were not significantly affected by Se treatment except oleinic acid (C18:1) and linolenic acid (C18:3) (Table 2). In these profiles, long chain polyunsaturated fatty acids, palmitic acid (C16:0, 11.1−11.9%), linoleic acid (C18:2, 17.8−19.2%), and linolenic acid (26.7−30.5%), were the dominant components, accounting for >50% of the TFA under either control or Se treatment. Thus, it opens dietary opportunities for C. 10881


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(5) Lintschinger, J.; Fuchs, N.; Moser, J.; Kuehnelt, D.; Goessler, W. Selenium-enriched sprouts. A raw material for fortified cerealbased diets. J. Agric. Food Chem. 2000, 48, 5362−5368. (6) White, P. J. Selenium accumulation by plants. Ann. Bot. 2015, 117, 217−235. (7) Araie, H.; Shiraiwa, Y. Selenium utilization strategy by microalgae. Molecules 2009, 14, 4880−4891. (8) Gojkovic, Z.; Garbayo, I.; Ariza, J. L. G.; Márová, I.; Vílchez, C. Selenium bioaccumulation and toxicity in cultures of green microalgae. Algal Res. 2015, 7, 106−116. (9) Rayman, M. P. Food-chain selenium and human health: emphasis on intake. Br. J. Nutr. 2008, 100, 254−268. (10) Vriens, B.; Behra, R.; Voegelin, A.; Zupanic, A.; Winkel, L. H. Selenium uptakeand methylation by the microalga Chlamydomonas reinhardtii. Environ. Sci. Technol. 2016, 50, 711−720. ̌ (11) Vítová, M.; Bisová , K.; Hlavová, M.; Zachleder, V.; Rucki, M.; Č ížková, M. Glutathione peroxidase activity in the selenium-treated alga Scenedesmus quadricauda. Aquat. Toxicol. 2011, 102, 87−94. (12) Reunova, Y. A.; Aizdaicher, N. A.; Khristoforova, N. K.; Reunov, A. A. Effects of selenium on growth and ultrastructure of the marine unicellular alga Dunaliella salina (Chlorophyta). Russ. J. Mar. Biol. 2007, 33, 125−132. (13) Zhong, Y.; Chen, T. F.; Zheng, W. J.; Yang, Y. F. Selenium enhances antioxidant activity and photosynthesis in Ulva fasciata. J. Appl. Phycol. 2015, 27, 555−562. (14) Stengel, D. B.; Connan, S.; Popper, Z. A. Algal chemodiversity and bioactivity: sources of natural variability and implications for commercial application. Biotechnol. Adv. 2011, 29 (5), 483−501. (15) Doucha, J.; Lívanský, K.; Kotrbácě k, V.; Zachleder, V. Production of Chlorella biomass enriched by selenium and its use in animal nutrition: a review. Appl. Microbiol. Biotechnol. 2009, 83 (6), 1001−1008. (16) Sun, X.; Zhong, Y.; Huang, Z.; Yang, Y. Selenium accumulation in unicellular green alga Chlorella vulgaris and its effects on antioxidant enzymes and content of photosynthetic pigments. PLoS One 2014, 9 (11), e112270. (17) Hu, M.; Yang, Y.; Martin, J. M.; Yin, K.; Harrison, P. J. Preferential uptake of Se(IV) over Se(VI) and the production of dissolved organic Se by marine phytoplankton. Mar. Environ. Res. 1997, 44, 225−231. (18) Lichtenthaler, H. K. Chlorophylls and carotenoids: pigments of photosynthetic biomembrane. Methods Enzymol. 1987, 148, 350−382. (19) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (20) Li, Z. Y.; Guo, S. Y.; Li, L. Bioeffects of selenite on the growth of spirulina platensis and its biotransformation. Bioresour. Technol. 2003, 89 (2), 171−176. (21) Folch, J.; Lees, M.; Sloane-Stanley, G. H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957, 226, 497−509. (22) Johnson, M. B.; Wen, Z. Y. Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass. Energy Fuels 2009, 23, 5179−5183. (23) Rodríguez, M. C.; Barsanti, L.; Passarelli, V.; Evangelista, V.; Conforti, V.; Gualtieri, P. Effects of chromium on photosynthetic and photoreceptive apparatus of the alga Chlamydomonas reinhardtii. Environ. Res. 2007, 105 (2), 234−239. (24) Zheng, Y.; Li, Z.; Tao, M.; Li, J.; Hu, Z. Effects of selenite on green microalga Haematococcus pluvialis: Bioaccumulation of selenium and enhancement of astaxanthin production. Aquat. Toxicol. 2017, 183, 21−27. (25) Jiang, C.; Zu, C.; Lu, D.; Zheng, Q.; Shen, J.; Wang, H.; Li, D. Effect of exogenous selenium supply on photosynthesis, Na+ accumulation and antioxidative capacity of maize (Zea mays L.) under salinity stress. Sci. Rep. 2017, 7, 42039. ̌ (26) Umysová, D.; Vítová, M.; Doǔsková, I.; Bisová , K.; Hlavová, M.; Č ížková, M.; Machát, J.; Doucha, J.; Zachleder, V. Bioaccumulation

pyrenoidosa. Nevertheless, the influence of Se on the fatty acid profile was not clear. Se caused a significant decrease in oleinic acid content and an increase in linolenic acid content (P < 0.05) (Table 2). There was a negative association between linolenic and oleinic acids in saline stress showing that the selection scheme for improving either of these fatty acids will lead to a reduction in the other. Linolenic acids are common constituents of plant membranes and are major substrates for lipoxygenase (LOX). The hydroperoxides produced by the actions of LOX on linolenic acid result in changes in membrane fluidity and permeability, eventually causing malfunction of the lipid bilayer. These hydroperoxides are also precursors of the signaling molecule and may play a regulatory role in the expression of Se stress inducible proteins associated with defense responses. Therefore, it is possible to infer from these findings that the composition of fatty acids produced by C. pyrenoidosa is substantially influenced by metabolic pathways that are governed by a tolerance mechanism activated under a specific range of Se concentrations. In addition, the ratio of ω-6 to ω-3 (n6/n3) ranged between 0.58 and 0.71 based on the very high content of the n3 fatty acid α-linolenic acid. According to the recommendations of the Food and Agriculture Organization (FAO),44 the n6/n3 ratio should not be higher than 5. However, a high n6/n3 ratio (15/ 1 to 16.7/1) is found in today’s Western diets and promotes pathogenesis of many diseases, including cardiovascular disease, cancer, osteoporosis, and inflammatory and autoimmune diseases.45 C. pyrenoidosa total fat is very rich in the n3 αlinoienic acid, which results in low n6/n3 ratios. Addition of C. pyrenoidosa to human food would therefore correct the unfavorable n6/n3 ratio of fatty acids, which is especially present in the Western diet.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-755-26611617. Fax: +86-755-26035343. Email: [email protected]. ORCID

Jay J. Cheng: 0000-0003-2254-4933 Funding

This research was mainly funded by a Key Project of Shenzhen Emerging Industries (JC201104210118A) and partially supported by Public Science and Technology Research Funds Projects of Ocean (201305022). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Jiasi Wang and Xian Sun from the University of Washington and Sun YatSen University, respectively.

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REFERENCES

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Effects of Selenite on Unicellular Green Microalga ... - ACS Publications

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