Accumulation of carotenoid, lipid, phenolic and antioxidant capacity of microalgae Dunaliella salina ccap 19/18 cultivated under temperature stress regimes

Introduction

The halophilic green microalga Dunaliella salina is receiving increasing attention through the provision of highly biologically active substances under appropriate inhibition conditions, for application in biofuel production, wastewater treatment, fertilizer, medicine and food. China, India and the United States are the leading countries for the research contribution of D. salina, where the main focus of research is on carotenoid (specifically βcarotene) and lipid production [1] . β-carotene is a component of photosynthesis and is produced in lipid droplets in the thylakoids of chloroplasts under inhibitory conditions such as temperature, nutrients, light, salinity, pH to protect the photosynthesis apparatus and membrane integrity [2,3] . Temperature is considered one of the factors that affects the growth process and composition of valuable substances in microalgae, the impact of temperature can cause changes in the synthesis of carotenoids or the composition of fatty acids. The studies have reported that increased temperature significantly reduces the amount of unsaturated fatty acids, affecting the maintenance and fluidity of algae cell membrances [4]. Research by Wu and colleagues (2016) investigated the effects of light, temperature and nutrition on the growth and pigment accumulation of three strains of Dunaliella salina KU XI, KU 10 and KU 31. The results showed that the optimal light and temperature for the growth of all three strains were 135,3 µmol.photons/m 2 /s and 22 o C while the condition of 245,6 µmol.photons/m 2 /s and 22 o C gave the highest amount of β-carotene. The optimal nutrient concentrations of KNO 3 , CO (NO 2 ) 2 and NaHCO 3 for the growth of the three strains are 0,5 g/L, 0,36 g/L and 1,5 g/L, respectively 0 g/L, 0,12 g/L and 1,5 g/L gave the highest β-carotene content [5] . Another study by Reshma and colleagues (2021) aimed to evaluate the effects of pH, salinity, temperature, photoperiod and light intensity on the synthesis and accumulation of chlorophyll and carotenoids of microalga D.salina [6] . The results showed that growth, biomass, chlorophyll a, b, total carotenoids and β-carotene increased under optimized conditions of pH=7, salinity 37 PSU, temperature 23 o C, photoperiod 12:12 and light intensity of 200 µm/m 2 /s when compared to normal conditions. Antioxidants (enzymatic or non-enzymatic) are classified depending on their mode of activity as primary antioxidants (hydrogen or electrons donors) or secondary antioxidants (oxygen scavengers or chelating agents) [7] . Phenols are important natural compounds with antioxidant and other biological properties. Phenolic compounds effectively scavenge hydroxyl and peroxyl radicals and prevent fats oxidation and have great advantages in food, chemistries, pharmaceutical and medical industries. Phenolic content of D. salina is increased at low temperatures. Low temperature along with light increase the oxidative stress by limiting photosynthetic enzymes thereby increasing the phenolics as photoprotective compounds [8] . Microalgae D. salina were cultivated at low temperature (13 °C) together with low light irradiation (20 µmol photons m -2 s -1 ) to enhance SOD, APX, MDHAR, DHAR activity compared to the unstressed controls, whilst cultivation at a very low temperature (5 °C) decreased CAT activity and less carotenoid and ascorbate contents at extremely low temperatures. SOD, APX, DHAR, activity were increased in D. salina (IR-1) when cells were grown for 2 days under high temperature (28 °C) under light irradiation ranging between 100-1200 µmol photons m -2 s -1 compared to a different strain (Gh-U) [7] . This study aimed to investigate the accumulation of carotenoid, lipid, phenolic compounds and antioxidant capacity of microalgae D. salina cultivated under the stress of temperature regimes.

From the results, we can build strageries for culturing D. salina in another wether conditions in Viet Nam. [9] .

Materials and Methods

Methods

Determine cell density

Microalgae were counted directly using a Neubauer red blood cell counting chamber to determine cell density. Take 10 µL of algal solution fixed with Lugon solution (5% Iodine, 10% KI salt) into a red blood cell counting chamber with a depth of 0.1 mm, square area of 1 mm 2 and count cells under a 10X objective optical microscope, repeatability at least 3 times per sample [10] . Cell density per 1 mL was determined according to the formula:

N: total number of counted cells I: counting area D: cell density (cell/mL) Total carotenoid content Take 1 mL of algae solution and centrifuge it, discard the liquid and keep the algae. The algal residue was extracted with 3 mL of ethanol: hexane solvent mixture, then added to 15 mL of fancol and vortexed evenly. Continue adding 4 mL of hexane and 2 mL of distilled water to the fancol containing algae and then vortex until the color is gone.

After centrifugation, the mixture will separate into 2 layers, the solvent layer containing the pigment will be absorbed and measured photometrically at wavelengths of 450 nm, 645 nm and 662 nm [2] . Total carotenoid content was determined using the formula [11]: Total carotenoids (µg/ mL): A 450 × 25.2

The content of chlorophyll a and b was calculated according to the formula [12]: Chlorophyll a (μg/ml) = 11.75 (A 662 ) -2.35 (A 645 ) Chlorophyll b (μg/ml) = 18.61 (A 645 ) -3.96 (A 662 ) Total chlorophyll (μg/ml) = chlorophyll a + chlorophyll b A 645 is the absorbance at wavelength 645 nm A 662 is the absorbance at wavelength 662 nm

Determination of total lipid content by Sulfo-Phospho-Vanillin method

Prepare Phosphovanillin reagent: weigh 0.06 g of vanillin into a 50 mL volumetric flask covered with foil, add 2 mL of absolute ethanol and 8 mL of distilled water and shake well to dissolve. Then use phosphoric acid (H 3 PO 4 ) to the mark and shake well [13] . Building a lipid standard curve equation: Canola Oil (total lipid content 100 g/100 g) was mixed in chloroform to a standard lipid concentration range of 10-140 µg/mL and heated at 90 o C for 10 minutes on the stove water bath to evaporate all chloroform. Next, add 2 mL of concentrated sulfuric acid into the test tube, vortex well and boil at 95 o C for 10 minutes, then cool. Add 5 mL of phosphovanillin reagent and vortex until the mixture is homogeneous. The sample was measured photometrically at a wavelength of 530 nm [13] . From the results, proceed to build a linear regression equation y=ax+b showing the correlation between optical density (OD, y) and lipid concentration (x, µg/mL). Determination of total lipid content of microalga D. salina CCAP 19/18: take 1 mL of microalgae solution and centrifuge, discard the supernatant and keep the microalgal pellets. The pellet is extracted with 2 mL of concentrated sulfuric acid and followed by the above procedure. The sample was measured photometrically at a wavelength of 530 nm [14] . The total lipid content of the microalga Dunaliella salina was determined by a linear regression equation with (y) being the optical density (OD) value and (x) being the total lipid content (µg/mL). Total lipids (µg/ mL): (A 530 -b)/a a,b: constants in the lipid standard curve equation y: OD value measured at wavelength A 530 x: total lipid content (µg/mL) Determination of total phenolic content using Folin-Ciocalteu's phenol reagent Building a phenolic standard curve equation: gallic acid is mixed in methanol to create a standard gallic acid concentration range from 1-50 mg/L. Take 500 µL of each concentration into a 2 mL eppendorf, add 500 µL of Folin-Ciocalteu's phenol vortex reagent evenly. After 5 minutes, slowly add 500 µL of 10% Na 2 CO 3 , mix well and incubate for 90 minutes. Centrifuge and measure photometrically at 750 nm. From the results, proceed to build a linear regression equation y=ax+b showing the correlation between optical density (OD, y) and lipid concentration (x, µg/mL). Determination of total phenolic content of microalgae D. salina CCAP 19/18: take 1 mL of microalgae solution separate, discard the liquid and keep the algae. The bite part was extracted with 1 mL of absolute methanol and incubated for 30 minutes in the dark and followed by the above procedure. After, centrifugation and photometric measurement at 750 nm [15] . The total phenolic content of the microalga Dunaliella salina was determined by a linear regression equation with (y) being the optical density (OD) value and (x) being the total phenolic content (µg/mL). Total Phenolic (µg/ mL): (A 750 -b)/a a,b: constants in the phenolic standard curve equation y: OD value measured at wavelength A 750 x: total phenolic content (µg/mL)

Determination of antioxidant capacity by DPPH test

Mix reagent: weigh 0.004 g of 1,1-Diphenyl-2picrylhydrazyl (DPPH) reagent into a 100 mL volumetric flask covered with foil, use absolute methanol to dissolve and make up to the mark. Take 1 mL of microalgae solution and centrifuge, remove the supernatant and keep the pellet. The pellet was extracted with 1 mL of absolute ethanol, mixed well and incubated at 4 o C for 4 hours. After, centrifuge the algal solution, pipet 500 µL of algal solution into a 2 mL eppendorf covered with foil, add 1 mL of DPPH reagent and incubate in the dark for about 30 minutes, optical density wavelength at 517 nm [16] . Antioxidant capacity (I%/mL) is calculated based on the ability of DPPH to inhibit free radicals according to the formula [17,16]: 1).

Harvesting and analyzing microalgae: samples were collected once on day 12 before inhibition (Before stress) and days 3, 6, 9 and 12 during inhibition. According to Orset and Young, 2002, the main factor in stimulating the synthesis of carotene was determined to be exposure to lower than optimum temperatures for microalgal Dunaliella salina growth. A 7.5-fold increase in the levels of α-carotene was observed when the temperature was decreased from 34 to 17° C, whilst levels of β-carotene were unaltered [9,18] . Potijun and colleagues (2021) studied Chlamydomonas Reinhardtii and found that there was an increase in carotenoid content under the influence of cold temperature conditions [19] . Meanwhile, under inhibitory culture conditions of 45 o C for 3 and 6 hours a day, D. salina CCAP 19/18 does not grow. At this temperature, the cell membrane of CCAP 19/18 ruptures. In addition, cells tend to stick together in clusters and are unable to survive the inhibition process. Research by Ras and colleagues (2013) shows that when temperature conditions exceed the optimal threshold, the effects are more harmful than low temperatures, negatively impacting the decline in growth rate as well as causing increase the rate and death rate of microalgae [20] . and colleagues (1994) researched and discovered that cells produce PUFA to protect the photosynthetic apparatus from the effects of cold temperature stress [21] .

Total Phenolic Content

Linear regression equation y=0.0239+0.0283 with (y) being the optical density (OD) value and (x) is the total lipid content (µg/mL) with a linear range determined from 1-50 µg/ mL, correlation coefficient R 2 =0.9995. The equation meets the linearity requirements (Figure 1.5) The total phenolic content of D. salina CCAP 19/18 increased sharply after temperature inhibition. Specifically, phenolic content (µg/mL) increased sharply and reached its maximum on day 12 after inhibition, between the conditions Control+T25, T10+3h and T10+6h with no significant difference (p= 0.057) (Figure 1.6). The total phenolic content (pg/cell) of CCAP 19/18 increased and reached the highest value in the T10+6h inhibition condition from the 6th day after inhibition and was significantly different from the control and T10+3h conditions (p≤0.05) (Figure 1.6). Phenolic compounds are one of the substances that play a major role in antioxidant activity in microalgae. Under cold temperature culture conditions, it helps increase the accumulation of phenolic compounds in microalgae to resist stress of cold conditions, content under cold conditions in response to abiotic oxidative stress [22] . and phenolic content (355% increase), suggesting potential temperature dependent variations in the activity of different antioxidant systems. This may be due to the low temperature factor of 10 o C affecting the activity of the antioxidant enzyme system of D. salina. Free radical neutralization test of D. salina CCAP 19/18 shows the antioxidant capacity of substances in microalgae such as carotenoids, phenolic compounds and enzymatic antioxidants produced in chloroplasts and mitochondria under cold stress conditions to fight harmful free radicals of DPPH. Although cold conditions reduce enzyme activity, the antioxidant capacity of carotenoids or phenolic compounds plays their role very well [23] .

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