Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina

Accepted Manuscript Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina Ines Dahmen-Ben Moussa, Haifa Chtourou, Fatma Karray, Sami Sayadi, Abdelhafidh Dhouib PII: DOI: Reference:

S0960-8524(17)30489-3 http://dx.doi.org/10.1016/j.biortech.2017.04.008 BITE 17903

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

9 January 2017 31 March 2017 1 April 2017

Please cite this article as: Dahmen-Ben Moussa, I., Chtourou, H., Karray, F., Sayadi, S., Dhouib, A., Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.04.008

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Nitrogen or phosphorus repletion strategies for enhancing lipid or

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carotenoid production from Tetraselmis marina

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Ines Dahmen-Ben Moussa*, Haifa Chtourou, Fatma Karray, Sami Sayadi and

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Abdelhafidh Dhouib

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Laboratory of Environmental Bioprocesses, Centre of Biotechnology of Sfax, University of

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Sfax. Sidi Mansour Road Km 6, PO Box «1177», 3018 Sfax, Tunisia

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*

Corresponding author.

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E-mail address: [email protected] (I. Dahmen-Ben Moussa)

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Tel: +216 55307047

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Fax: +216 74 874 452

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Abstract

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The objective of this study was to investigate the accumulation of lipid and photosynthetic

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pigments from Tetraselmis marina. When the cells were grown in F/2-medium for seven days

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in the first stage, the carotenoid and lipid contents, and productivity were 44 g/kg (DW), 27%

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and 31 mg/L/d, respectively. After second stage of cultivation of T. marina for further 3-days

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under N-replete condition (4.41 mM NaNO3) increased biomass concentration of 1900

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mg/L and lipid content of 50% were observed, with an enhanced lipid productivity of

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86.36 mg/L/d and SFA and MUFA fractions of 70.76 and 13.14%, respectively. However,

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under P-repletion (2.08 mM NaH2PO4), its carotenoid content increased to 89.23 g/kg and its

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PUFA for 65% of total lipids. Results showed that N and P-replete conditions decreased SOD

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activity and increased H2O2 and TBARS levels of T. marina. Thus, this native microalga

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strain could be a potent candidate for feed,  food or biofuel production.

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Keywords: Tetraselmis marina; carotenoid; lipid; PUFA; biodiesel; oxidative stress.

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1. Introduction

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Microalgal cultivation has been investigated for production of fine chemicals and health foods

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such as pigments, vitamins, long chain polyunsaturated fatty acids, antioxidants for cosmetic,

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human and animal food industry (Pal et al., 2011).

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Microalgae accumulate lipids and carbohydrates which make them good feedstocks for the

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production of biofuels, owing to the fast growth rate and high lipid productivity. Firstly,

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microalgal culture requires non-arable land and can use municipal wastewater as a nutrient

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source. Thus, production of microalgal biodiesel does not compete with crops for resources

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and it can recover water pollution (Chisti, 2007). Secondly, biodiesel contains high density

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energy in small volume and weight, which is required for aviation and long distance trucking.

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For high production of pigment and lipid, it is important to select an appropriate strategy for

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inducing cellular lipid accumulation during cultivation, but not decreasing the biomass

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concentration. One of the most typical methods for increasing lipid content of microalgae is

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nitrogen and phosphorus limitation (Scott et al., 2010). However, this strategy is accompanied

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by the reduction of cell growth rate or even a loss of biomass concentration (Singhet al., 2015;

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Kim et al., 2016). Also, they are not the universal stress factors that stimulate lipid and

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carotenoid accumulation. For some microalgae, lipid and carotenoid synthesis does not

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respond to nutrient limitation, and larger lipid and carotenoid production is observed at higher

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nutrient levels (Feng et al., 2011; Martins et al., 2011; Kimet al., 2016).

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The final composition of microalgae depends on the environmental conditions which can be

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used as effective stimulants for the accumulation of a particular compound (Bhosale, 2004).

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Pigments protect the microalgae from damage obtained during environment stress by

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preventing the formation of reactive oxygen species (ROS) and also, it acts as a filter (Telfer,

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2002).

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Tetraselmis species are green marine microalgae (Chlorophyta) commonly used in

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aquaculture, due to their high nutritional value. A number of species have been used as a

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model for physiological and biochemical studies, as well as for survival and adaptation

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mechanisms to diverse conditions (Singh et al., 2015; Kim et al., 2016). Tetraselmis sp. was

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used as a laboratory model strain to study the effect of salinity on growth, FA accumulation

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(Adarme-Vega et al., 2014) and was also reported to grow under a high nitrogen

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concentration, with a lipid productivity of 18.6-47.3 mg/L/d (Kim et al., 2016).

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Few studies have focused on the pigment, lipid production and the FA profile of Tetraselmis

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species under nitrogen and phosphorus conditions. Therefore, the aim of the present work is

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to investigate the effect of nitrogen and phosphorus concentration on the biomass, carotenoid

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and lipid production of the marine microalga, Tetraselmis marina, and its further impact on

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fatty acids profile and oxidative stress in a two-stage culture process.

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2. Materials and Methods

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2.1. Microalgal strain, identification and culture condition

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Microalgae CTM 20015 was isolated from the sea water of the Cap-Bon region (Tunisia) as it

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was described in our previous work (Dahmen et al.,2014). The genomic DNA of the CTM

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20015 strain was extracted using the Favour Prep Plant genomic DNA extraction mini kit

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according to the manufacturer's instructions. Microalgal DNA preparation, sequencing and

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phylogenetic analyses were performed as described by Dahmen etal. (2014).

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CTM 20015 pure-culture at 60 mg/L (dcw) was used to inoculate 500 mL of optimized F/2

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Provasoli medium without sodium silicate (Provasoli et al., 1957) containing NaNO3, 0.88

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mM; NaH2PO4, 0.42 mM; Vitamin B1,0.66 pM; Vitamin B12, 0.0007 pM; Vitamin H, 0.004

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pM;FeCl3 6H2O, 0.019 mM; Na2EDTA,0.011 mM; CuSO4 5H2O,0.039 pM; Na2Mo4 2H2O,

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0.012 pM; ZnSO4 7H2O, 0.076 pM; CoSO4 6H2O, 0.038 pM and MnCl2 4H2O, 0.91 pM, into

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hermetic 1 L Erlenmeyer flasks.

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Flasks were placed on a Shaking Incubator at 120 rpm for 12 days at room temperature of

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26°C. Light was provided by cool white fluorescent tubes at an intensity of 60 µmoL photons

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m-2 s-1. The inoculums were transferred to15-Lglass air bubble photobioreactor (Isotherm,

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Germany) containing 8 L fresh optimized F/2 culture medium. T. marina culture was assessed

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for 11 days and was carried out under the same previously conditions and aerated

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continuously (0.03 vvm) with filtered (0.22 µm) air.

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2.2. Nitrogen and Phosphorus stressful conditions

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Effect of nitrogen and phosphorus on lipid content and productivity, polyunsaturated fatty

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acid profile, ROS and pigment accumulation by T. marina, were tested by transferring the

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microalga adopted at its phase of exponential growth (7 th day of the batch culture), after

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centrifugation at 4000×g, into a fresh optimized F/2 Provasoli medium without nitrogen or

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phosphorus (0 mM NaNO3 or NaH2PO4) for nitrogen and phosphorus depletion stress and

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fresh optimized F/2 Provasoli medium (Provasoli et al., 1957) at 2.65 (3-fold), 4.41 (5-

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fold)and 8.82 (10-fold) mM N-NO3 for nitrogen repletion stress or 1.25 (3-fold), 2.08 (5-fold)

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and 4.17 (10-fold) mM P-PO4 for phosphorus repletion stress. Cultures were re-incubated at

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the same conditions for 3 days.

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2.3. Determination of growth and biomass productivity

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Microalgal growth was monitored daily by measuring optical density at 750 nm and the dry

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cell weight (DCW) (mg/L). The biomass productivity (BP) are the presented values an

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average for the 3 days and was calculated according to equation BP (mg/L/day)= (X2-

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X1)/(t2-t1), where,X2 and X1 are the dry cell weight (DCW) (mg/L) at time t2 and t1,

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respectively.

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2.4. Determination of photosynthetic pigments

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Three samples of the same mass of the pellet (1 mg) of culture of T. marina obtained were

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freeze-dried and resuspended in 0.7 mL of DMSO to extract pigments (Hiscox and Isrealstam,

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1979). Mixture was incubated at 65°C for 1 h. After cooling, additional 0.3 mL of DMSO was

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re-homogenized with the mixture and the absorbance of the pigment extract was read in the

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supernatant using spectrophotometer (UV-1650PC Shimadzu, Japan). Corresponding

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maximum absorbance of chlorophyll a, chlorophyll b and total carotenoids are 662 nm, 646

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nm and 470 nm, respectively. The amount of these pigments was calculated according to the

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formulas of Wellburn (Wellburn, 1994). Data are means of triplicate determinations ±

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standard deviations.

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2.5. Determination of protein, carbohydrate and Lipid, and fatty acid analysis

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Cells were removed by means of centrifugation (5000×g at 4°C). The pellet was washed twice

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with distilled water and was analyzed for proteins and carbohydrates content as described by

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Bradford (1976) and Dubois et al. (1956) using the dinitrosalicylic acid (DNS), respectively.

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The total lipids were extracted according to the method of Bligh and dyer (Bligh and Dyer,

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1959). Lipid productivity (LP) is calculated as the product of the lipid content (LC) and the

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BP. Fatty acid methyl ester (FAME) was prepared by acid transesterification protocol and

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analyzed in GC-MS as described by Dahmen et al. (2016).

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2.6. Determination of stress biomarkers

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One g of humid biomass samples of T. marina were disrupted by alumina (1:1, w/w) in 2 mL

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of 0.05 M sodium phosphate buffer pH 7.8, at 4°C. Mixtures were crushed in mortar and

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pestle and the debris were removed from the algal extract by centrifugation at 5000×g for 10

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min at 4°C.

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The level of lipids per-oxidation in cells was measured as the amount of thiobarbituric acid

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reactive substances (TBARS) according to Draper and Hadley (1990). Briefly, 1mL of

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thiobarbituric acid reagent (0.67%) was added to 0.5 mL of supernatant and heated at 90°C

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for 15 min. The mixture was then cooled and measured for absorbance at 532 nm. The

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malondialdehyde values were calculated using 1,1,3,3-tetraethoxypropane as standard and

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expressed as nmol of TBARS/mg protein.

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H2O2 content was determined according to the method of Velikova et al. (2000). 0.5 mL of

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algal extract was mixed with 0.5 mL of phosphate buffer (10 mM, pH 7.0) and 1 mL of KI (1

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M). Absorbance was recorded at 390 nm and expressed as µmol H2O2/mg protein.

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The superoxide dismutase (SOD) activity was assayed according to Asada and Takahashi

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(1987) method. Fifty µL of algal extract were mixed with 1000 µL of EDTA (0.1 mM)-Meth

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(13 mM), 1842.2 µL of sodium phosphate buffer (50 mM) and 85.2 µL of NBT (75 µM). In

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this assay, 22.6 µL of riboflavin (2 µM) was added to start the reaction. The absorbance was

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mesured at 560 nm after 30 min. SOD activity was expressed as units per milligram of

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proteins (U/mg protein).

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2.7. Statistical analysis

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One-way analysis of variance (ANOVA) was performed. The experiments were done in

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triplicate. Data are expressed as mean±SD. Values were considered statistically significant

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when p<0.05.

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3. Results and discussion

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3.1. Identification, growth and photosynthetic pigments and lipid production of T. marina

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Microalga strain was isolated from the sea water of the Cap-Bon region (Tunisia) and

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rendered axenic. Using PCR amplification and subsequent DNA sequencing, we determined

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almost full length (1636 pb) of 18S rDNA of these isolates. Phylogenetic analysis based on

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18S rDNA sequence data firmly placed the CTM 20015 isolate in the Chlorodendrophyceae

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Class, Chlorodendrales Order, Chlorodendraceae Family and Tetraselmis Genus (Fig.1).

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This isolate is most closely related to a green coastal microalga Tetraselmis marina

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(KT023599) (98.8% similarity) (Marin, 2012).

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The growth curve of T. marina was shown in Fig. 2A. Biomass content increased promptly in

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the media containing 0.88 mM N-NO3 and 0.42 mM P-PO4. In the microalgae cultures, after a

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lag phase of 2 days, typical exponential growth phase lasted up to 3 days. The maximum

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growth rate (µmax) was 0.6/day. Stationary phase occurred on day 5, with culture attaining a

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maximum biomass content of 1200mg/L. The recorded specific growth rate of T. marina was

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lower than Tetraselmis gracilis and Minutocellus polymorphus (0.87 and 1.49/day,

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respectively) reported by Sigaud-Kutner et al. (2002). However, it exhibited a higher specific

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growth rate than Lingulodinium polyedrum studied by the same author and Tetraselmis

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sp.studied by Kim et al. (2016).

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The variation of photosynthetic pigments content along the growth of microalgae was

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examined in Fig.2A. The content of carotenoid, chlorophyll a (Chla) and chlorophyll b (Chlb)

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in T. marina were recorded as mg/g (DW) (p<0.05). The carotenoid content of T. marina

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(Fig. 2A) was reduced from the beginning of its growth until day 5. However, an increase of

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1.6-fold (44 mg/g (DW)) in carotenoid concentration was detected on day 7 of growth,

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remaining at a concentration of 25 mg/g DW. Such improvement is related to its antioxidant

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role against nutrient deficiency. Likewise, Chla content increased to 80 mg/g DW, decreasing

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from day 5 during the Stationary phase (Fig. 2A). As shown in Fig.2A, Chlb was higher at the

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beginning of T. marina growth (28 mg/g (DW)). Moreover, it exhibited a second peak on

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stationary growth (day 9), with mean values about 1.4-fold higher than the initial

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concentration. T. marina showed considerable variability in its carotenoid and chlorophyll

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content related to the growth phase. Our results were in agreement with the findings of Chla

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content reported by Sigaud-Kutner et al. (2002) in Tetraselmis gracilis and Minutocellus

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polymorphus. Under standard condition, the maximal T. marina Chlb content was 30 fold

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higher than Picochlorum sp. (Dahmen et al., 2014). The decrease in Chlb, after the

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exponential phase of T. marina can be associated with the reduction of photosynthetic activity

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when nutrients were lacking, while the subsequent increase in the content of Chlb in the

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stationary phase of growth of T. marina could be related to photo-acclimation to shading

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conditions due to increases in cell culture density. Tsai et al. (2016) has demonstrated much

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lower amount of chla ranged from 1.92 to 3.21% DW of Tetraselmis sp. DS3 at different salt

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stress conditions.

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The standard method determines the triacylglycerol and cell membrane lipid (phospholipid) in

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algae cells (Bligh and Dyer, 1959). Fig.2B showed the variation of lipid content along the

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growth of microalgae curves. The initial lipid content of T. marina on day 0 for all the

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cultures was 5%. The total lipids concentration increases in terms of the culture duration; it

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rises from 18±0.07 at the exponential phase (day 5) to reach 27±0.09 at the stationary phase.

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(day 7) Owing to lipid content, lipid productivity was also significantly increased from 1.64 to

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31 mg/L/d on the 7th day and then decreased due to the significant decrement of lipid content

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and cell concentration. Therefore, it is important to select an appropriate harvest time to

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achieve higher biomass concentration, carotenoid and lipid content, ultimately enabling the

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achievement of maximum lipid productivity.

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3.2. Effect of nitrate and phosphorus availability in 2nd stage cultivation

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3.2.1. Effect of nitrate and phosphorus on the biomass concentration and productivity

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In order to enhance the lipid and carotenoid content on the final day of cultivation, the two-

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stage culture process was utilized. In the first stage, T. marina cells were cultured in the

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standard F/2 medium with 0.88 mM nitrate and 0.42 mM phosphorus (control level) for 7

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days, and the cell concentration was more than 1100 mg/L. The second stage for

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accumulating lipids and carotenoids was started with or without different high nitrate or

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phosphorus levels or concentrations for 3 days.

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As shown in Fig. 3, at the higher concentration of nitrate, the cell biomass was more

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produced. The biomass concentration (DCW) and the biomass productivity (BP) of the T.

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marina strain were higher in the N1, N2, N3, P1, P2and P3 treatments than the control. The

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results show that the T. marina cells can survive for 3 days even under nitrogen or phosphorus

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deplete conditions, although the DCW and BP decrease due to nitrogen and phosphorus

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deficiency. In fact, many microalgae species were reported to decrease their DCW and BP

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significantly under complete nitrogen or phosphorus depletion. However, Li et al. (2008) have

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reported that some microalgae species are able to sustain their growth by utilizing intracellular

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nitrogen and phosphorus storage, such as chlorophyll, by conversion to proteins, nucleic acid,

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and cell wall materials.

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3.2.2. Effect of nitrate and phosphorus on pigments composition of T. marina

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Photosynthetic pigments like chlorophyll a and b play an important role in photosynthesis.

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The present investigation indicates that nutrient concentration affected the pigment content of

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T. marina cultures (Table 1). A significant decrease was observed in carotenoid content (0.8-

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to 2.1-fold) with a concomitant increase in Chla (1.4- to 2.1-fold) and Chlb (1.0- to 1.4-fold)

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with the supplementation of nitrate. Conversely, Table 1 showed the accumulation of

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carotenoid content (1.2 and 2.3-fold) under phosphorus-replete, nitrate and phosphorus

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deplete conditions, with a concomitant decrease in Chla (1.2- to 2.8-fold) and Chlb (1.1- to

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2.2-fold). The decrease in chlorophyll a and b content was detected in the N and P-deplete

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cultures. This decrease in chlorophyll a content due to nutrient stress is a well-known

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phenomenon, also known as ‘bleaching’. Goiris et al. (2015) showed a lack of photosynthetic

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pigments under N deficiency in comparison with Pi deficiency in Phaeodactylum

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tricornutum, Chlorella vulgaris and Tetraselmis suecica. Thus, we can conclude the strong

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relationship between pigment synthesis and N metabolism.

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Carotenoids play a key role against oxidative stress during cellular senescence in T. marina.

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In general, nitrogen deficiency has a greater impact than excess nitrogen on carotenoids

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production (Minhas et al., 2016), because a culture growing in a nitrogen-rich medium

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requires carbon to assimilate the nitrogen. Low concentration of nitrogen, on the other hand,

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leads to greater competition for carbon, which is required for the synthesis of both carotenoids

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and proteins (Borowitzka et al., 1991). Furthermore, when microalgae are nutrient-limited, the

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flow of electrons from the photosystems to the electron transport chain is impaired and

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reactive oxygen species are formed (Apel and Hirt, 2004). Therefore, an increase in

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carotenoids content is expected under nutrient limited conditions. According to Belghith et al.

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(2016) and Lamers et al. (2012), an accumulation of carotenoids marks a deficient nutrition.

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Mendoza et al. (1999) found that the maximum of carotenoids was observed with nutriment

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depletion. Moreover, absence or low levels of nutrients stimulate rapid physiological

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responses, which further trigger the secondary biosynthetic pathways (Touchette and

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Burkholder, 2000).

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Phosphorus has a key role in the conveyance of metabolic energy. For the strains of T.

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marina, there was a positive correlation between the increase of phosphate in the medium and

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the content of carotenoids. Similarly, Martins et al. (2011) have shown that phosphorus and

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nitrogen supplementation increased photosynthetic pigment contents in Hypnea musciformis.

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3.2.3. Effect of nitrate and phosphorus on metabolic composition of microalgae T. marina

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Nitrogen and phosphorus repleted or depleted conditions affect all basic metabolic pathways

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of microalgae because it is the major constitute of proteins and nucleic acids (Singh et al.,

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2015). This has been studied extensively for optimization of lipid accumulation in various

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microalgal strains (Singh et al., 2015).

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Nitrate and phosphate as stress factors influence not only carotenoids production but also

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affects lipid accumulation in green algae. Fig. 4 shows the effect of two stage nutrient stress

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on biochemical composition of microalgae T. marina.

293

The lipid content and productivity of microalgae T. marina were significantly affected by

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both depletion and repletion phosphorus and nitrogen stresses. As shown in Fig. 4A, the

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higher concentration of nitrate or phosphate supplied, the greater the increment of cellular

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lipid content. At higher phosphorus concentration of 4.17 mM, the lipid content increased

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from 21% to 35% (DW) within 3 days of stress, and reached the highest content of 50% at

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high nitrate concentration of 4.41 mM. As expected, the lipid content increased under nitrate

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and phosphorus deplete conditions (Fig. 4A). However, the results show lower lipid content

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than nitrate replete condition. No significant difference (p<0.05) in the lipid content was

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found in cells grown at 8.82 mM nitrogen (41.25%) and 0 mM phosphorus (41.59%) and in

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cells grown at 0 mM nitrogen, 2.08 and 4.17 mM phosphorus. The maximal lipid productivity

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of this species, 78.75 mg/L/d, occurred at 4.41 mM nitrate but its maximal lipid productivity

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decreased to 13 and 29 mg/L/d when cultivated 0 mM nitrogen and phosphorus, respectively.

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Carbohydrates and proteins are intracellular metabolites, and cellular levels in algal cells vary

306

with physiological state stands and depend on abiotic conditions. Fig.4B clearly shows that

307

only the carbohydrate content decreased under both nitrate and phosphorus repletion, while

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the protein contents continuously increased (Fig. 4C); it rises from 25% (DW) under control

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condition to 41 and 39% at N3 and P3 conditions, respectively. These results indicate that the

310

carbon flux was converted from carbohydrate to lipid and protein synthesis under nitrogen

311

and phosphorus replete conditions and the N2 replete condition is favorable for lipid

312

production of T. marina.

313

Conversely, under nitrogen and phosphorus deplete conditions (N0 and P0), protein contents

314

decreased, while the carbohydrate content significantly enhanced. The decrease in total

315

proteins may be explicated by the slowdown of the phenomenon of DNA replication (Wong

316

et al., 2016) In fact, in this nitrogen and phosphorus-starvation state, T. marina diverted the

317

inorganic carbon fixation form DNA and protein synthesis toward lipid synthesis.

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The enhancement of lipid content in this study is in accordance with previous reports nitrogen

319

and phosphorus limited conditions. However, some microalgae species have been found to

320

increase their lipid content at higher N concentrations, compared to N deplete conditions. Xu

321

et al. (2001) and Kim et al. (2016) have found that lipid content of the marine microalga

322

Ellipsoidion sp. and Tetraselmis sp., increased with increasing nitrate concentrations. The

323

accumulation of lipids under N repletion was attributed to the enhanced activity of acetyl-

324

CoA carboxylase or other key enzymes related to the conversion of carbohydrates into lipids

325

(Feng et al., 2011; Bellou et al., 2014).

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3.2.4. Effect of nitrate and phosphoruson fatty acids profiles of T. marina

329 330

The results in Fig. 5, showed the fatty acid methyl ester (FAME) in algal cultures grown

331

under NaNO3 and NaH2PO4 stresses. There were distinct differences in the composition of

332

saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty

333

acids (PUFA) when the cells were cultivated under the different treatments.

334

The percentage of saturated fatty acids increased by 1.4 folds (SFA = 70.76%) in culture

335

grown under N2 stress medium (4.41mM N-NO3) compared to the control (SFA =51.7%). In

336

contrasts to that present in the control culture, polyunsaturated fatty acid (PUFA) showed a

337

remarkable decrease. However, PUFA exhibited the reverse patterns, where it increased to be

338

59.41% for culture grown under P2 stress medium (2.08 mM NaH2PO4) and was significantly

339

higher compared to the control (PUFA =36.8%).

340

The results in Table 2 showed the individual fatty acids presented in algal culture grown

341

under 4.41 mM N-NO3 and 2.08 mM P-PO4 in comparison to the fatty acid composition of T.

342

marina grown on F/2 medium. In the algal culture grown under(4.41mM Nitrate), Palmitic

343

acid (C16:0), Stearic acid (C18:0), Eicosanoic acid (C20:0) and Oleic acid (C18:1) increased

344

to be 41.28, 3.5 and 24.16 and 13.14%,respectively.These changes in fatty acid composition

345

resulted in a reduced degree of unsaturation of the total fatty acid pool.

346

However, the most pronounced increase was noticed in Linoleic acid (C18:2), β-Linolenic

347

acid (C18:3), Arachidonic acid (C20:4) and Eicosapentaenoic acid (C20:5) content which

348

increased from 0.14.5, 12, 1.8 and 4.5%, respectively, in the control condition to 17.2, 13.31,

349

14.5 and 19.9% in the algal culture grown under 2.08 mM P.

350

Under the conditions tested, growing on 2.08 mM P-PO4 is the best choice for the production

351

of PUFA while growing on 4.41 mM N-NO3 condition is recommended as optimal for the use

352

of T. marina as a biofuel resource.

353

PUFAs play important roles in processes including thermal adaptation, regulation of

354

membrane fluidity and permeability, and oxygen and electron transport in cellular and tissue

355

metabolism (Cardozo et al., 2007). Particularly, ALA, AA and EPA are essential fatty acids

356

for humans and are involved in the skin function and the prevention of cardiovascular disease

357

(Belghith et al., 2016). According to Yu et al. (2014), high levels of SFAs and MUFAs

358

combined with low levels of PUFAs are essential for biodiesel. In the lipids produced by T.

359

marina, fatty acids containing more than four double bonds were non-detectable, and its

360

C18:3, ω3 was 7.5% produced under N2 condition (4.41 mM N-NO3). Based on these results,

361

it can be concluded that altered conditions can be selected according to various applications.

362 363

3.2.5. Effect of nitrate and phosphorus on stress biomarkers of T. marina

364 365

Under unfavourable environment conditions microalgae produce various reactive oxygen

366

species (ROS) like H2O2, O2·-, OH·- etc. A primary measure of oxidative damage is lipid

367

peroxidation, where TBA is measured as the index of lipid peroxidation (Yousef et al., 2006).

368

Antioxidant enzymes are considered to be the first line of cellular defense against oxidative

369

damage. Thus, SOD is an essential antioxidant enzyme, which counteracts free radical

370

generation. Therefore, carotenogenesis and lipid content are enhanced by reactive oxygen

371

species (ROS), under stress conditions such as high light intensity, salt stress (Kobayashi et

372

al., 1993). Therefore, carotenoid is believed to protect the body from free- radical-linked

373

diseases as oral, colon, and liver cancers (Guerin et al., 2003).

374

In the present study, to understand the role of ROS in lipid and carotenoid accumulation, we

375

studied the H2O2 content, lipid peroxidation along with activity of antioxidative enzyme SOD

376

under nutriment stress conditions. Table 3 shows the effect of various nitrate and phosphate

377

concentrations on H2O2, TBARS and SOD activity of microalgae T. marina. The highest

378

amount of H2O2 was observed in cells grown at P0 (138.19 µmol/mg protein) followed by P3

379

(122.17 µmol/mg protein) and N3 (105.31 µmol/mg protein), which were significantly higher

380

than the control. The sudden increase in H2O2 in 3 days stressed culture is mainly due to

381

sudden change in the nutrients concentration of the culture medium.

382

During two stages of cultivation, cells under both nitrate and phosphorus repletion had

383

marginal increase in TBARS level compared to control. However, phosphorus depletion

384

stressed culture had significantly higher TBARS (39.01 nmol/mg protein) compared to

385

control (3.20 nmol/mg protein).

386

These changes were accompanied by a significant depletion in SOD activity. It is clear that

387

although cells were stressed in all tested treatments, decreased activity of SOD was observed

388

with the increase of the nitrate and phosphate concentration in the growth medium. However,

389

lower SOD activity of about 28% was found in cells cultivated in 8.82 mM N compared to

390

control culture, (Table 3) followed by 0 mM P (24.21 U/mg protein), 0 mM N (28.50 U/mg

391

protein) and 4.17 mM P (31.78 U/mg protein), stressed conditions. Such decrease in enzymes

392

activity can result from either inhibition of protein synthesis or impairment of enzymatic

393

activity. The increase in TBARS level and the decrease in the activity of SOD, during all

394

treatments may indicate an increase in the peroxides levels. Thus, the inhibition of SOD

395

involved in free radical removal, has led to the accumulation of H2O2, which promoted lipid

396

peroxidation and modulation of DNA, altered gene expression and cell death (Yousef et al.,

397

2006). Actually, oxidative stress tolerant microalgae are more efficient for biofuel production

398

than non oxidative tolerant microalgae (Osundeko et al., 2013). Under unfavorable

399

environment conditions microalgae produce various reactive oxygen species (ROS) which are

400

very toxic of cells composition. To protect cells from these toxic effects, microalgae have

401

several defense systems such as antioxidative enzymes like SOD (Asada and Takahashi,

402

1987). In this study, induction of oxidative stress by phosphorus depletion or repletion was

403

studied by measuring H2O2 content. Superoxide dismutase (SOD) is an important antioxidant

404

enzyme, and is the first line of defense against oxidative stress in plants. Superoxide radicals

405

and other ROS are formed in the chloroplasts during photosynthetic light reactions at the

406

acceptor side of PS I, reducing site of PS II and at the oxygen evolving complex. It seems that

407

T. marina has a pool of stored phosphate and nitrogen that may support cell growth when

408

extracellular nutrients are limited and maintain metabolic activity (e.g., lipid and carotenoid

409

synthesis) when nitrogen or phosphorus are in excess (Yao et al., 2013). Thus, the decrease in

410

SOD activity can be explained by the increase in synthesis of lipid and carotenoid induced by

411

stress-nitrogen and phosphorus supplementation, respectively.

412 413

4. Conclusion

414 415

The cellular lipid and carotenoid content of T. marina were found to increase on the 7th day of

416

growth. When the cells were cultivated through a two-stage culture process, T. marina cope

417

with nitrate and phosphate nutritional stress by altering the metabolic pathways of pigments

418

and altering lipids, including a shift in FA. Phosphate replete condition (2.08 mM) led to an

419

increase in carotenoid and lipid content with high level of PUFA. However, nitrate replete

420

condition (4.41 mM) led to a high lipid content and productivity, and affected the

421

physiological mechanism and lipid composition, that induce the suitable feedstock for

422

biodiesel production.

423 424

Abbreviations

425

Chl, chlorophyll; DCW, dry cell weight; FA, fatty acids; MUFA, monounsaturated fatty

426

acids; N, nitrogen; P, phosphorus; PUFA, polyunsaturated fatty acids; ROS, Reactive oxygen

427

species; SFA, saturated fatty acids; SOD, superoxide dismutase; TBARS, Thiobarbituric acid-

428

reactive substances.

429 430

Acknowledgments

431

This study was supported by the Ministry of Higher Education and Scientific Research of

432

Tunisia under Contract Program of the Environmental Bioprocesses Laboratory.

433 434

Disclosure

435

The authors declare no conflict of interest.

436 437

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438

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555

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558

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559

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560

altitudes. Fuel 115, 220−226.

561

562 563 564 565 566 567 568 569 570 571 572

Fig. 1. Dendrogram based on the sequence of 18S rDNA gene. Bootstrap values are given at

573

the nodes. Scale bar represents the substitution percentage. Desmochloris halophila was used

574

as outgroup. GenBank accession numbers follow species name in parenthesis.

575 576 577 578 579 580 581 582 583 584 585 586

588 589 590 591 592

Pigments content (mg/g DW)

Ca rotenoids

Chlorophyll (a)

Chlorophyll (b)

Biomass 1400

90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0

1200 1000 800 600 400 200 0 0

593

1

2

3

4

5

6

7

8

9

10

11

(A) Biomass concentration (mg/L)

587

12

Cultivation time (days)

594 30

596 597 598 599

Lipid contetnt (% DW)

595

(B)

LP 35

a

30

25

b c

20

b a

d

15

20 15

10

10

a 5

5

0

600

25

0 0

3

5

7

9

Lipid productivity (mg/L/d)

LC

11

Days

601 602 Fig. 2 The change of (A) cell concentration (Biomass), carotenoids, chlorophyll a and chlorophyll b 603 accumulation, and (B) cellular lipid content and productivity in the first stage of cultivation of T. marina in 604 F/2 medium. Values are mean±SD. [Different lower case letters above the bars indicate statistically 605 significant difference between treatment means by a Fisher LSD test (p<0.05).] 606 607 608 609 610 611

612 613

BP

2500

250

a

614

616

DCW (mg/L)

615

200

c

d 1500 1000

617

500

618

0

150

d e

f g

100 50 0

Control

619

b

b

2000

N1

N2

N3

N0

P1

P2

P3

Biomass productivity (mg/L/d)

DCW

P0

Treatments

620 621

Fig. 3 Effect of different concentrations of NaNO3 and NaH2PO4 on the cell concentration and

622

productivity in the second stage of cultivation. N1, N2 and N3 contained 2.65 (3-fold), 4.41

623

(5-fold) and 8.82 (10-fold) mM N-NO3, P1, P2 and P3 contained 1.25 (3-fold), 2.08 (5-fold)

624

and 4.17 (10-fold) mM P-PO4 and N0 and P0 contained neither nitrate nor phosphate. Values

625

are mean±SD. [Different lower case letters above the bars indicate statistically significant

626

difference between treatment means by a Fisher LSD test (p<0.05).]

627 628 629 630 631 632 633 634 635 636 637 638

Lipid content (% DW)

60

(A)

LP

100 90 80 70 60 50 40 30 20 10 0

a b

50

b c

40

d

30

c

c

d

e 20 10 0 Control

N1

N2

N3

N0

P1

P2

P3

P0

Treatments

(B)

50

a

Carbohydrate (% DW)

45

a

40 35 30

b

25

c

20

c d

d e

f

15 10 5 0 Control

N1

N2

N3

N0

P1

P2

P3

P0

Treatments 45

(C)

a

a

a

40 35 30

b b b

c

25 20

d

15

d

10 5 0 Control

N1

N2

N3

N0 Treatments

P1

P2

P3

P0

Lipid productivity (mg/L/d)

LC

Protein (% DW)

639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688

689

Fig. 4 Effect of different concentrations of NaNO3 and NaH2PO4 on (A) the cellular lipid

690

content and productivity, (B) carbohydrate and (C) proteins content in the second stage of

691

cultivation. N1, N2 and N3 contained 2.65 (3-fold), 4.41 (5-fold) and 8.82 (10-fold) mM N-

692

NO3, P1, P2 and P3 contained 1.25 (3-fold), 2.08 (5-fold) and 4.17 (10-fold) mM P-PO4 and

693

N0 and P0 contained neither nitrate nor phosphate. Values are mean±SD. [Different lower

694

case letters above the bars indicate statistically significant difference between treatment

695

means by a Fisher LSD test (p<0.05).]

696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735

745

SFA

MUFA

PUFA

P3 P2 P1 Treatments

736 737 738 739 740 741 742 743 744

P0 N3

N2 N1 N0

746 747

Control 0,00

20,00

40,00

60,00

80,00

100,00

% weight

748 749 750

Fig. 5 Saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acid

751

content of T. marina as affected by different concentrations of NaNO3 and Na2HPO4 in the

752

second stage of cultivation. N1, N2 and N3 contained 2.65 (3-fold), 4.41 (5-fold) and 8.82

753

(10-fold) mM N-NO3, P1, P2 and P3 contained 1.25 (3-fold), 2.08 (5-fold) and 4.17 (10-fold)

754

mM P-PO4 and N0 and P0 contained neither nitrate nor phosphate.

755 756 757

758 759 760 Table 1 Effect of different concentrations of NaNO3 and NaH2PO4 on the pigments 761 composition content of T. marina in the second stage of cultivation. (Different superscript 762 letters within column indicates significant differences at p<0.05.) 763 764 Treatments Element concentration Car (mg/g Wt) Chla (mg/g Wt) Chlb (mg/g Wt) 765 Control N (0.88 mM), P (0.42 mM) 40.34±0.07c 32.21±0.05d 39.12±1.34c 766 NaNO3 N1 (2.65 mM: 3-fold) 35.67±0.55c 47.87±0.06c 41.23±0.58c d b 767 N2 (4.41 mM: 5-fold) 28.80±0.08 65.82±0.41 54.12±0.09b e b 768 N3 (8.82mM: 10-fold) 19.79±0.13 64.07±0.05 52.87±0.25b b f 769 N0 (0 mM) 45.89±0.54 11.87±0.14 17.97±0.52d 770 Na2 HPO 4 P1 (1.25 mM: 3-fold) 51.54±0.11c 17.14±0.11c 21.94±0.17c a d 771 P2 (2.08 mM: 5-fold) 89.23±0.21 29.34±0.08 25.32±0.28a a e 772 P3 (4.17 mM: 10-fold) 87.25±1.28 38.67±0.21 35.97±0.11a 773 P0 (0 mM) 64.92±0.53a 25.95±0.07e 22.86±0.65d 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 Table 2 Fatty acid methyl ester profile of T. marina grown under nitrogen (4.41 mM N-NO3) 791 and phosphorus (2.08 mM P-PO4) repletion in the second stage of cultivation. 792 793 FA1% of total lipid C14:0 C16:0 C16:3 C18:0 C18:1 C18:2 C18:3 C20:0 C20:4 C20:5 C24:0

794 795

1)

Fatty acid.

Control 1.24 36.00 4.00 0.69 11.50 14.50 12.00 10.77 1.80 4.50 3.00

N2 (4.41 mM: 5-fold) 0.50 41.28 0.00 3.50 13.14 5.20 7.50 24.16 1.20 2.20 1.32

P2 (2.08 mM: 5-fold) 2.00 18.69 0.50 1.00 12.00 17.20 13.31 0.00 14.50 19.90 0.90

796 797 798 799

Table 3 Effect of different concentrations of NaNO3 and NaH2PO4 on H2O2, TBARS level and SOD activity of T. marina. (Different superscript letters within column indicates significant differences at p<0.05.)

Control

N (0.88 mM), P (0.42 mM)

H2O2 (µmol/mg protein) 80±0.55d

NaNO3

N1 (2.65 mM: 3-fold)

81.23±0.32d

N2 (4.41 mM: 5-fold)

85.57±0.13 d

Treatments

Na2HPO4

Elements concentration

808

SOD (U/mg protein) 78.13±1.68a

10.41±0.45cd

64.20±0.26b

12.15±0.94c

55.51±0.31c

c

N3 (8.82mM: 10-fold) N0 (0 mM)

105.31±1.41 94.84±0.96cd

14.50±0.52 21.98±1.12b

21.73±0.46d 28.50±0.35d

P1 (1.25 mM: 3-fold)

84.21±0.51d

9.52±0.12cd

54.14±0.49c

cd

93.35±0.74

P3 (4.17 mM: 10-fold)

122.17±1.87b

18.36±0.89b

a

a

138.19±0.63

10.84±0.51

cd

P2 (2.08 mM: 5-fold) P0 (0 mM)

800 801 802 803 804 805 806 807

c

TBARS nmol/mg protein) 3.20±0.06d

39.01±0.13

38.23±1.23e 31.78±2.9e 24.21±0.57f

809

Highlights

810 811

•

A two-stage process was applied to enhance the lipid productivity of T. marina.

812

•

Nutrients stress affect physiological and biochemical composition of T. marina.

813

•

Phosphorus and nitrogen depletion resulted in the lowest lipid productivity from T.

814

marina.

815

•

N repletion enhanced both the cell growth and lipid content for biodiesel production.

816

•

P repletion increased both carotenoid and the fraction of PUFA.

817

•

Two-stage N and P-stress change the H2O2, TBARS level and SOD activity of T.

818

marina

819

45.