Effects of Selenite on Unicellular Green Microalga Chlorella

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Effects of selenite on unicellular green microalgae Chlorella pyrenoidosa: Bioaccumulation of selenium, enhancement of photosynthetic pigments and amino acids production Yu Zhong, and Jay J. Cheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04246 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Journal of Agricultural and Food Chemistry

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Effects of selenite on unicellular green microalgae Chlorella pyrenoidosa: Bioaccumulation

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of selenium, enhancement of photosynthetic pigments and amino acids production

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Yu Zhong1, Jay J. Cheng1, 2*

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School of Environment and Energy, Peking University-Shenzhen Graduate School, Shenzhen, 518055, China

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Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695,

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USA

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∗Author for correspondence.

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Tel: +86-755-26611617;

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Fax: +86-755-26035343;

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract Microalgae were studied as function bioaccumulators of selenium (Se) for food and feed

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supplement. To investigate the Se bioaccumulation and its effects on the unicellular green alga

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Chlorella pyrenoidosa, the algal growth curve, fluorescence parameters, antioxidant enzymes

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activity, fatty acid and amino acid profiles were examined. We found that Se at low

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concentrations (≤ 40 mg L-1) positively promoted algal growth and inhibited the lipid

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peroxidation (LPO) and intracellular reactive oxygen species (ROS). The antioxidative effect

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was associated with an increase in glutathione peroxidase (GSH-Px), catalase (CAT), linolenic

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acid, and photosynthetic pigments. Meanwhile, significant increase in amino acids and organic

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Se content was also detected in the microalgae. In contrast, we found opposite effects in C.

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pyrenoidosa exposed to Se higher than 60 mg L-1. The antioxidation and toxicity appeared to be

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correlated with the excess Se bioaccumulation. These results provide a better understanding of

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the effect of Se on green microalgae, which may help to develop new technological applications

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for the production of Se-enriched biomass from microalgae.

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Keywords: Amino acids, Antioxidants, Bioaccumulation, Chlorella pyrenoidosa, Fatty acid

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profile, Selenium

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Introduction

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Selenium (Se) is a natural trace element that acts either as an essential micro-nutrient or a

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toxicant depending on the dose in biological and environmental systems. At low levels, Se shows

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beneficial effects for humans against multiple types of cancers, cardiovascular disease, and

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Type-2 diabetes.1 However, Se at high concentrations can cause the generation of reactive

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oxygen species (ROS), which induce DNA oxidation, DNA double-strand breaks, and cell

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death.2 Dietary Se deficiency was found to result in reduced growth, vitality, and reproduction in

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animals, and to cause severe disease in both livestock and humans (such as fatal Keshan

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disease).3 However, excess Se from the environment or industrial waste has a toxic impact on

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vertebrates and may cause the extinction of local fish populations.4

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According to Lintschinger et al., almost all European countries are classified as

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low-selenium regions.5 Selenium supplementation using microorganisms has received much

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attention in the past decade.6 In microalgae, Se has been reported to be essential for at least 33

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species, particularly for green algae, which can uptake inorganic Se and incorporate it into amino

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acids through the sulfur assimilation metabolic pathway.7,8 In humans, 25 selenoproteins were

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identified.11 Compared with inorganic Se, organic Se has an increased safety and bioavailability.9

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Hence, green algae have become a target organism in Se bioaccumulation research.10 The

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metabolism and bioaccumulation of Se have been comprehensively studied in the green algae

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Chlamydomonas reinhardtii,10 Scenedesmus quadricauda,11 Dunaliella salina,12 and Ulva

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fasciata.13 These Se-enriched green algae are potential producers of organic Se with high

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bioavailability. However, there are only a few studies on the effect of Se on algae from genus

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Chlorella.

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Chlorella showed superior performance on biomass production or lipid production using

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wastewater for microalgae cultivation. Most of the microalgal products are sold on the market as

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animal feed (30%) or food supplements due to the high content of various metabolites, including

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polyunsaturated fatty acids, pigments, antioxidants and other bioactive molecules.14 Therefore,

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biomass of Chlorella sp. is considered to be a good alternative source of organically bound Se

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for food supplementation.15 Delineating the impact of Se on Chlorella could lead to future

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research that will expand the potential of microalgal biotechnology.

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In water, inorganic Se is present as selenate (SeO42-) and selenite (SeO32-). When Se was

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added to the culture medium, growth was stimulated in some algae 13,16 and such a

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growth-stimulating effect is greater for selenite than selenate ions in Chlorella vulgaris.17 What’s

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more, among both selected Chlorella species, Chlorella pyrenoidosa (C. pyrenoidosa) differs

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from Chlorella vulgaris (C. vulgaris) due to the presence of prominent pyrenoids within the

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chloroplast. High protein content made C. pyrenoidosa to be an ideal protein resources. The

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objective of this study was to evaluate selenite biotransformation and resulting effect on the

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growth curve, photosynthetic pigments, intracellular ROS generation, the activity of antioxidant

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enzymes, the profile of amino acid, and fatty acid of green algae C. pyrenoidosa cultivated in the

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laboratory. The production of Se-Chlorella biomass as a food supplement seems to have a great

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potential for the algal biotechnology.

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Materials and Methods

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Culture conditions and Se treatment

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Chlorella pyrenoidosa SZ 16 was obtained from the Research Center of Hydrobiology of

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Jinan University (Guangzhou, China). Unialgal stock cultures were propagated and maintained

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in Erlenmeyer flasks containing BG11 medium (pH 6.8) under incubation conditions of 25 °C, a

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photon flux density of 45 µmol m-2 s-1 provided by two white fluorescent tubes, and a light:dark

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photoperiod of 12 h:12 h. Flasks were continuously shaken at 100 rpm. The components of basal

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culture medium are as follows: KH2PO4 0.7 g L−1, K2HPO4 0.3 g L−1, MgSO4·7H2O 0.3 g L−1,

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FeSO4·7H2O 3 mg L−1, glycine 0.1 g L−1, vitamin B1 0.01 mg L−1, A5 trace mineral solution 1

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ml L−1 (A5: H3B03 2.86 g L−1, MnCl2·4H2O 1.86 g L−1, ZnSO4·7H2O 0.22 g L−1,

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Na2MoO4·2H2O 0.39 g L−1, CuSO4·5H2O 0.08 g L−1, Co(NO3)2·H2O 0.05 g L−1).

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C. pyrenoidosa cultures in the late exponential growth phase were decanted into 250 ml

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flasks containing 100 ml of medium at 25±0.5 °C and illuminated with fluorescent lights (60

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µmol m-2 s-1 photon flux intensity) under a 12 h:12 h light:dark photoperiod. The cultures were

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initiated at 6×107 cells mL-1, shaken periodically and used in triplicate. All solutions and

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experimental containers were autoclaved at 121 °C for 15 min. Sodium selenite (Na2SeO3) was

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added to the medium before inoculation at a concentration of 20, 40, 60, 80 mg L−1. Cultures

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grown without sodium selenite served as controls.

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Algal growth measurement Algal cells of 10 mL were collected by centrifugation (Eppendorf 5804R centrifuge,

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Eppendorf, Germany) at 8,000 rpm for 1 min at 22 °C. The supernatant was then discarded and

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the algal cells were resuspended in 1 mL sterilized de-ionized water. The resuspended cells were

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diluted for cell number count. Cell count was performed with a Hemocytometer and optical

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density (OD) values of the algae were measured under 750 nm absorbance with a FlexStation 3

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spectrophotometer (Molecular Devices, Sunnyvale, USA). Using the regression equation

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between OD750 and cell density, cell numbers were only calculated from the OD750 values of the

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algae. In each Se concentration, the density of the culture (OD750) was adjusted to 0.05 by

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control of the dilution rate until a steady state was reached.

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Measurement of photosynthetic pigments Photosynthetic pigments were extracted from algal biomass after 6-day cultivation. In brief,

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40 mL of the culture solution was centrifuged at 10,000 g for 10 min. Pellets were then

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homogenized (Polytron homogenizer, Art-Miccra D-8, Germany) with 5 ml of cold acetone

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(80%), followed by centrifugation at 10,000 g for 10 min at 4 oC. Photosynthetic pigments (Chl a,

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Chl b, and carotenoid) in the supernatant were quantified spectrophotometrically at 663 and 646

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nm, respectively, i.e.,18

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Chl a = 12.21A 663 – 2.81A646

(2)

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Chl b = 20.13A 646 – 5.03A663

(3)

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Carotenoid concentration = (1000A470 – 3.27Chla – 104Chlb)/229

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(4)

where the units of Chl a, Chl b, and carotenoid are mg L-1.

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Antioxidant enzymes assay

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The C. pyrenoidosa cell suspensions were centrifuged, and then the pellets were

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resuspended in pre-chilled phosphate buffer (pH 7.0) and disrupted by ultrasonication. The

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homogenate was centrifuged at 10,000 g for 30 min and the supernatant was collected. The total

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protein content in the supernatant of homogenate extracts was determined according to the

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Bradford method, using bovine serum albumin as a standard.19

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The activities of antioxidant enzymes in the protein extract were examined using

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spectrophotometric diagnostic kit (NanJing JianCheng Bio Inst, Nanjing, China). Basically,

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catalase (CAT) activity was measured according to the ammonium molybdate

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spectrophotometric method, based on the fact that ammonium molybdate could rapidly terminate

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the H2O2 degradation reaction catalysed by CAT and react with the residual H2O2 to generate a

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yellow complex which could be monitored by the absorbance at 405 nm.

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GSH-Px activity was measured by quantifying the rate of H2O2-induced oxidation of GSH

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to oxidised glutathione (GSSG), catalysed by GSH-Px. The GSH-Px content in the supernatant

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was measured by reaction with dithionitrobenzoic acid (DTNB) and monitored by absorbance at

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412 nm.

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Determination of total and organic Se content Total Se concentration was determined by inductively coupled plasma-mass spectrometer

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(ICP-MS).16 After cultivation for 6 days, 100 mg dried algal sample was digested with

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concentrated nitric acid and H2O2 (3:1, v/v) in a digestive stove (Qian Jian Measuring Instrument

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Co., Ltd., China) at 180 oC for 3 h. Samples were allowed to cool down, dissolved in 0.6% HNO3

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and filtered through Whattman filter paper. Prior to drying, the samples were observed under

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microscope for removing any contamination of non-algal particulates. The volume of each

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sample was maintained at 10 ml with 0.6% HNO3 and analyzed for the total Se content with the

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ICP-MS. For inorganic Se determination, 100 mg dried algal sample was extracted with 15%

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HCl (three times, to make sure extraction of inorganic Se completely) and the extract was

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analyzed with the ICP-MS. Organic Se concentration was calculated from the difference between

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the total Se and inorganic Se concentrations. An inductively coupled plasma–mass spectrometer

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Agilent 7500ce (Agilent Technologies, Japan) was used for the analysis using the following

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operational conditions: forward power 1500W, sampling depth 7-8 mm, auxiliary gas flow rate

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0.10-0.15mL⋅min−1, extract I: 0-3V, extract II: -1375V, omega Bias-ce -20V, omega Lens-ce

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-1.6V, cell entrance -40V, QP focus -15V, cell exit -44V, octP RF 190V, octP bias -18V, H2 flow

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3.8mL min-1, QP bias -16V, discriminator 8mV, and analog HV 1840V. For quantification of Se,

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a standard addition method was used to eliminate matrix effects of residual carbon and other

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matrix elements. Se isotopes 77 and 82 were used, as these isotopes did not suffer from Ar-based

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spectral interferences.8

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Lipid and protein extraction The freeze-dried algal powder was suspended in 0.1 mol L-1 PBS buﬀer (pH=7), frozen in

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liquid nitrogen for 10 min and then thawed in 40 °C water. The residues after centrifugation at

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4000 g for 10 min were treated as above for two more times. The protein containing supernatant

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was precipitated by 30-70% (NH4)2SO4 and the obtained crude protein eluted on a Sephadex

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G-100 column (1650 cm) with 0.1 mol L-1, pH=7 PBS at a rate of 0.5 mL min-1. The eluted

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protein solutions were collected and dialyzed against sterilized water, concentrated with

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polyglycol (Mr = 20000) and ﬁnally freeze-dried.20

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The extraction of lipid from C. pyrenoidosa was carried out using Folch extraction

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method.21 Folch extraction was done by homogenizing freeze-dried algal biomass with

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chloroform/methanol (2:1 v/v). The mixture was agitated at room temperature for 15-20 min and

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centrifuged at 7000 rpm at 4 °C for 7 min to separate cell debris from supernatant. The

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supernatant was washed with 0.2 volumes of 0.9% NaCl solution. The mixture was vortexed for

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a few seconds and again centrifuged at 3000 rpm for 5 min. The upper phase was removed and

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the lower chloroform phase containing lipid evaporated under vacuum by rota evaporator using

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weighing bottles.

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Amino acid analysis The hydrolysed samples were analysed according to the guidelines of the European

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Community (98/64/EG). Amino acid samples were separated by ion exchange chromatography

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and determined by reaction with ninhydrin with photometric detection at 570 nm (440 nm for

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proline) using the automatic amino acid analyser LC-3000 (Eppendorf-Bio-tronik, Germany).

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Amino acid standard solution (A9781 from Sigma-Aldrich, Germany) (0.5 µmol mL-1) was

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injected to calibrate the analyser and to calculate the amount of amino acid in the samples.

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Fatty acid analysis In situ transesterification of algal lipids was performed with modified protocol. Freeze-dried

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algal biomass was placed in a glass test tube and mixed with 1.7 mL of methanol, 0.3 mL of

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sulphuric acid and 2 mL of hexane. The reaction mixture was heated at 90 °C for 1 h and the

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samples were well-mixed during heating. After the reaction, the tubes were allowed to cool to

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room temperature, and 2 mL distilled water was added, vortexed and centrifuged for 30 min at

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3220 g. The hexane layer that contained FAME was collected and transferred to a pre-weighed

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glass vial. The solvent was evaporated using N2, and the mass of FAMEs were determined

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gravimetrically.22

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FAME profiles were determined using gas chromatography Agilent 7890A (Agilent, US)

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with auto-sampler, flame ion detector (FID) and DB5-MS column (60 m × 250 µm × 0.25 µm).

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The column oven was set to 70 °C equilibration for 2.0 min and then 10 °C min−1 increase to

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230 °C, 3 °C min−1 to 290 °C then hold 5 min; splitless injection; injection temperature 280 °C.

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Helium was used as the carrier gas.

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Statistical analysis All treatments and controls were performed in triplicates. The significance of the difference

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between mean values obtained from four independent experiments was determined by one-way

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analysis of variance (ANOVA) at 95% confidence interval by using SPSS software.

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Results and Discussion

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Se accumulation and its effects on C. pyrenoidosa growth

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Studies have reported that some algae require Se for growth, such as Emiliania huxleyi. 7

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Therefore, we first studied the growth curves of C. pyrenoidosa under different Se concentration

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treatments. Figure 1 shows the result. In our study, an increase in biomass was obtained in cells

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exposed to low Se concentrations (≤40 mg L-1), with the highest biomass found at a Se

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concentration of 40 mg L-1.

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However, higher Se concentrations (>40 mg L-1) decreased the growth of C. pyrenoidosa

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significantly (P 73%) was the most abundant constituent of Se compounds in C.

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pyrenoidosa below 40 mg L-1. It indicated that C. pyrenoidosa was an efficient Se accumulator.

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In addition to this, 26.6% (of total Se) selenium was bound with protein and 12.1% (of total Se)

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with lipids (Figure 1D, P < 0.05). The superfluous 38.8% (of total Se) perhaps bound with the

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other ligands such as free amino acids, polypeptides, or other similar compounds. Although

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protein-bound Se was the major form of organic Se, lipids had the greatest ability to integrate

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with Se.20

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Effect of Se on photosynthesis of C. pyrenoidosa In microalgae, the effect of Se on photosynthesis can be assessed by monitoring the

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photochemical performance of the photosystem II (PS II), which is determined by means of the

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chlorophyll fluorescence measurement techniques, evolution of photosynthetic oxygen and

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content of pigments. We therefore studied the Effect of Se on photosynthesis of C. pyrenoidosa.

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The maximum fluorescence yield of PS II is calculated as Yop = Fv / Fm where Yop is the

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maximum fluorescence yield.27 Exposure of microalgae to various common stress factors, such

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as, excessive light, herbicides, osmotic stress, or Se salts among others, generally results in

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decrease of Yop.28 By monitoring the photochemical performance of the photosystem II (PSII),

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the effects of Se on C. pyrenoidosa photosynthesis were assessed using the chlorophyll

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fluorescence parameters and the cellular pigment determination. For the actual photochemical

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efficiency of PSII and the maximal photochemical efficiency of PSII (Fv/Fm), no significant

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differences were observed between the 20 and 40 mg L-1 selenite treatment groups. In healthy

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microalgae cultures, the Fv/Fm values are in the range of 0.7–0.8, supporting the finding that

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20-40 mg L-1 Se treatment has no or little effect on algal cells.23 In comparison, Fv/Fm values

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revealed significant differences in the photochemical efficiency of PSII, which was observed in

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the 60 and 80 mg L-1 treatment groups (P < 0.001) (Figure 2A). Based on the decrease observed

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in the chlorophyll fluorescence parameters at 60-80 mg L-1 Se concentrations, we suggested that

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Se exposure the structure of the PS II complex of C. pyrenoidosa result in strong inhibition of the

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photosynthetic electron transport.29

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The variation in photosynthetic pigments content corresponded with chlorophyll

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fluorescence (Figure 2B & 2C). Significant (P 40 mg L-1), a significant decline (P 40 mg L-1) decreased with the reduction of

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intracellular total (or organic) Se content (Figure 1B and Figure 3). The maximum activity of

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CAT was found under the Se treatment of 40 mg L-1, and was 1.57±0.02 IU/µg protein,

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consistent with the intracellular Se content. A sharp elevation in GSH-Px activity was observed

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when cells were exposed to Se at 40 mg L-1, with maximum activity, 207.5±11.8 IU/mg protein,

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showing a positive correlation between Se concentration and GSH-Px activity. Higher Se

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concentrations (60-80 mg L-1) resulted in significant decrease in CAT activities (P 40 mg L-1), a small decrease in the total content of amino

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acids was detected compared to the 40 mg L-1 medium, but still higher than control. The

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significantly influence of amino acid content in plant tissues can be caused by nutrition and

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fertilization, such as magnesium and selenite.38

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We next investigated the amino acid composition in C. pyrenoidosa with Se treatment. The

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amino acid composition (% of dried weight) is illustrated in Table 1. In terms of essential amino

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acids, isoleucine, leucine, lysine, phenylalanine (Phe), threonine (Thr) and valine increased 28%,

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35%, 54%, 23% 29% and 00%, respectively, due to the application of 40 mg L-1 Se. The one

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with intensive increase after Se treatment in all concentrations was Phe. Munshi et al. also

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detected an increase of Phe content in potato tubers from 20% to 53% with an increasing rate of

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Na2SeO3 application.38 Adámková et al. reported that one of the defense mechanisms of plants

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against stress is the production of phenylpropanoids, which are derived from Phe.40 The positive

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correlation observed here between Se application and Phe synthesis is probably another

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mechanism which is triggered under conditions of stress. With regards to the essential amino

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acids, the content of Thr also underwent some noteworthy changes. The Thr level increased

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significantly after application of 40 mg L-1 Se, by 29.1% compared with the controls. Methionine

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(Met) was the limiting essential amino acid, application of Se in our study did not affect the Met

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content in C. pyrenoidosa, confirming previous results reported by Ježek.39.

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The results for non-essential amino acids are also shown in Table 1. The application of Se

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markedly influenced the content of Aspartic acid (Asp), Asp increased on average by 22.3% (40

383

mg L-1 Se) compared with the controls. Asparagine (Asn) results from amidation of aspartic acid

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(Asp). Munshi et al. discovered that the total content of Asn increased on average by 13% in

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Se-treated potato tubers.38 Aspartic acid and Asn are important in the transport and storage of

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nitrogen in plant tissues. They perform this function even under stress when the plant is not able

387

to ensure the standard level of proteosynthesis (nutrient deficiency, drought, salting, heavy metal

388

contamination, and/or pathogen attack).40

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The content of Pro, the free form of which is connected with the resistance of plants to

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drought and salinisation increased by up to 9% after the 40 mg L-1 Se treatment. This confirms

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the findings of Munshi.38 However, many researchers believe that proline accumulation is simply

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an indicator of several stresses and that it is not involved in protecting plants against metal

393

toxicity.

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Refer to Cysteine (Cys), due to the chemical analog of sulfur, green microalgae incorporate

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selenite preferentially into selenoproteins and various Se-amino acids, particularly Se-Cys and

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Se-Met.6 These amino acids can participate in protein synthesis as well as S-amino acids. In our

397

study, the application of either 20 or 40 mg L-1 selenite to C. pyrenoidosa increased Cys by 1.25

398

and 2.12 folds (Table 1). This confirms the findings of Munshi, but Munshi also reported the free

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amino acid cysteine content was decreased significantly by selenium fertilization.38 The Cys

400

content in leaves was influenced by the form of Se applied too. In experiments by Ríos et al. with

401

lettuce plants (Lactuca sativa L.), the Cys content decreased with the increasing rate of selenite

402

(SeO32–) application, but increased with the increasing rate of selenate (SeO42–) application.41

403

Se-Cys and Se-Met are also the main Se-compound accumulated by Se-enriched C. pyrenoidosa,

404

accounting for more the 50% of total accumulated Se in our study.

405

Polyunsaturated fatty acids (PUFA) are important nutrients, because they are essential

406

constituents of cell membranes or precursors for eicosanoids. Microalgae are potential sources of

407

many highly valuable products such as PUFAs. The fatty acid composition of algae can vary

408

quite markedly throughout the growth cycle, as the biosynthesis of lipids is significantly

409

influenced by the physiological state of the organism and environmental conditions.42 High

410

levels of Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been detected in

411

many species of both microscopic and macroscopic marine algae. In contrast, very few species of

412

freshwater algae contain significant amounts of EPA and DHA.43 Chlorophyceae (green algae)

413

can grow very fast and accumulate a high level of lipids, but polyunsaturated fatty acids are

414

generally limited to various isomers of linolenic acid.

415

In C. pyrenoidosa, most of the fatty acids were not significantly affected by Se treatment

416

except oleinic acid (C18:1) and linolenic acid (C18:3) (Table 2). In these profiles, long chain

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polyunsaturated fatty acids, palmitic acid (C16:0, 11.1-11.9%), linoleic acid (C18:2, 17.8-19.2%)

418

and linolenic acid (26.7-30.5%), were the dominant components, accounting for >50% of the

419

TFA under either control or Se treatment. Thus, it opens dietary opportunities for C. pyrenoidosa.

420

Nevertheless, the influence of Se on the fatty acids profile was not clear. Se caused a significant

421

decrease of oleinic acid and increase of linolenic acid (P

Effects of Selenite on Unicellular Green Microalga Chlorella

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