Effect of nitrogen on biomass and β-carotene accumulation by dunaliella sp

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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY EFFECT OF NITROGEN ON BIOMASS AND β-CAROTENE ACCUMULATION BY Dunaliella sp. SUPERVISOR STUDENT Assoc. Prof. NGUYEN HUU HIEP THAI TRAN PHUONG MINH Student code: 3082610 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPERVISOR Assoc. Prof. NGUYEN HUU HIEP STUDENT THAI TRAN PHUONG MINH Can Tho, May 01, 2013 PRESIDENT OF EXAMINATION COMMITTEE ABSTRACT Dunaliella sp. is halophilic unicellular microalgae with blue-orange color, and can be found in the sea, salt lakes and salt field. Dunaliella sp. has ability to synthesize large amount of carotenoids – substances known as one of the essential antioxidants. In this study, Dunaliella sp. was cultured in Walne’s medium and was tested with three different nitrogen sources (Potassium nitrate, Sodium nitrate and Urea) at four nitrogen concentrations 2 mM, 4 mM, 6 mM and 8 mM. Their cells proliferation rate, biomass and β-carotene production were studied. In order to determine the favorable source as well as concentration of nitrogen required for the highest biomass and β-carotene accumulation, direct microscopic counting, spectrophotometry methods were used. After 18 days of cultivation, the cell density of Dunaliella sp. ranged between 180.33x104 and 383x104 cells/mL, and biomass within the 0.2605 – 0.493 g/100mL range. The highest specific growth rate was obtained at 6 mM urea concentration (0.26 d-1). The concentration of β-carotene per 100 mL varied from 0.064 to 0.228 mg and β-carotene per cell ranged between 0.29 to 0.59 pg/cell. The results of the experiments showed that the maximum cell density and the highest β-carotene content were obtained at 6 mM urea concentration. Key words: β-carotene, biomass, Dunaliella sp., microalgae, nitrogen i CONTENTS Page APPROVAL ABSTRACT i CONTENTS ii 1. INTRODUCTION 1 2. MATERIALS AND METHODS 3 2.1 Microalgal strain and culture medium 3 2.2 Nitrogen sources and concentration experimental design 4 2.3 Growth analysis 5 2.3.1 Biomass 5 2.3.2 Growth rate 6 2.4 Determination of β-carotene content 6 2.5 Data analysis 7 3. RESULTS AND DISCUSSIONS 8 3.1 Effect of nitrogen source on the growth of microalgae Dunaliella sp. 8 3.1.1 NaNO3 8 3.1.2 KNO3 9 3.1.3 Urea 11 3.2 Effect of nitrogen source on biomass of microalgae Dunaliella sp. 14 3.3 The accumulation of β-carotene in Dunaliella sp. 16 4. CONCLUSIONS AND SUGGESTIONS 18 4.1 Conclusion 18 4.2 Suggestion 18 REFERENCES 19 ii 1. INTRODUCTION Vietnam is a developing country, food security and dietary habits of the people have been improved significantly in recent years. The diet with enough essential vitamins for the body always received special attention. However, vitamin deficiency especially vitamin A in the daily diet is hard to avoid. According to the World Health Organization a long time vitamin A deficiency can lead to disorders such as xeroma and ophthalmia that are the common cause of blindness in children. Anemia and reduction of the effectiveness of immune system in the body may also increase the severity of the infectious disease and lead to higher mortality risk. Recognizing the importance of vitamin A to the health of humans, scientists had studied and generated transgenic golden rice which is rich in β-carotene. However, the use of genetically modified foods has not been recognized and widely applied in a number of countries in the world yet. Natural β-carotene as a good alternative source is really necessary. β-carotene is one of more than 600 types of carotenoids exist in nature. Carotenoids are found in plants and do not appear in animals and animal origin foods. β-carotene is known to be a precursor of vitamin A (Paiva and Russell, 1999). In addition, βcarotene helps the body prevent the vitamin A deficiency, blindness, and boosts the immune system. β-carotene not only keeps the role of vitamin A, but also has independent biological activity, β-carotene acts as an antioxidant. This means that βcarotene can reduce the free radicals that affect the physiological 1 processes taking place in cells, from which prevents the damage of organelles, the aging process, as well as against cancer-causing agents (Paiva and Russell, 1999). Thus, β-carotene is used as a component of cosmetics for skin, anti-aging and also widely used in the food industry. Currently, food safety is a matter of interest not only Vietnam but around the world. The extraction of βcarotene from the algae begins to receive attention from many countries. Natural color that is good for health was interest of food production. For more nutritional value as well as commercial, the high levels β-carotene production is promoting to research. To satisfy the high performance β-carotene, ease of culture, proliferative, utilizing of the available resources, scientists find an alternative production sources. In this case, Dunaliella sp. has ability to synthesize large amount of β-carotene. However, not only appropriate source of nutrients, but the costs for the production of Dunaliella sp. are also paid attention. Traditional sources of nutrients, especially nitrogen source rather expensive cost. Therefore, the "Effect of nitrogen on biomass and β-carotene accumulation by Dunaliella sp." project was conducted to find cheaper sources of nitrogen suited for the Dunaliella sp. production. The objective of this study is determining the source and concentration of nitrogen favorable for biomass and β -carotene accumulation in Dunaliella sp. 2 2. MATERIALS AND METHODS 2.1 Microalgal strain and culture medium Dunaliella sp. was provided by the College of Aquaculture and Fisheries, Can Tho University. Dunaliella sp. was cultured in 2.5% salinity Walne’s medium (Table 3) with sterilized sea water at pH 7 ± 0.2. Table 3. Modified Walne’s medium Chemicals Amounts Nutrient solution (1 mL/1L medium) NaCl 25 g Ferric chloride (FeCl3) 0.8 g Manganous chloride (MnCl2, 4H2O) 0.4 g Boric acid (H3BO3) 33.6 g EDTA(b), di-sodium salt 45.0 g Sodium di-hydrogen orthophosphate (NaH2PO4, 20.0 g 2H2O) Sodium nitrate (NaNO3) 100.0 g Trace Mineral Supplement (TMS) 1.0 mL Make up to 1L with sea water Trace Mineral Supplement (TMS) Zinc chloride (ZnCl2) 2.1 g Cobaltous chloride (CoCl2.6 H2O) 2.0 g Ammonium molybdate ((NH4)6Mo7O24.4H2O) 0.9 g Cupric sulphate (CuSO4.5H2O) 2.0 g 3 Concentrated HCl 10.0 mL Make up to 100mL with distilled water Vitamin solution (0.1mL/1L medium) Vitamin B1 10 mg Vitamin B12 (Cyanocobalamin) 10 mg Vitamin H (Biotin) 200 μg Make up to 100 mL with distilled water Walne’s medium with salinity level at 2.5% was prepared from sea water with 2.8% salinity by adding distilled water to get the desired salinity. Dunaliella sp. was inoculated at the ratio 1/10 (10 mL algae solution: 90 mL Walne’s medium) (Pisal and Lele, 2005), and continued to culture until 10 liters of algae at 5x105 cells/mL was reached (Pisal and Lele, 2005). 2.2 Nitrogen sources and concentration experimental design Dunaliella sp. was cultured in 1 liter plastic cans. There were 4 treatments in every nitrogen source and one control treatment (Table 4). In each treatment, 100 mL of algae were added to 900 mL Walne’s medium. Each treatment was repeated 3 times. Initial algae cell density was equal in every treatment 5x105 cells/mL. All treatments were kept under continuous light (24h photoperiod), at 29˚C and continuous aeration. The pH of the culture was adjusted to 7 ± 0.2 at the beginning of the experiment. 4 Table 4. Experimental design Nitrogen source Nitrogen Treatment concentration (Mm) No nitrogen supplement KNO3 NaNO3 CO(NH2)2 Amount (g/l) DC 0 0 K1 2 0.202 K2 4 0.404 K3 6 0.606 K4 8 0.808 N1 2 0.17 N2 4 0.34 N3 6 0.51 N4 8 0.68 U1 2 0.06 U2 4 0.12 U3 6 0.18 U4 8 0.24 2.3 Growth analysis 2.3.1 Biomass Algae at the density of 5x105 cells/mL was divided into small volume, and then diluted at five different rates to form a series of five different OD values. Each OD value repeated 3 times. Optical density values 5 were measured by spectrophotometer at wavelength of 680 nm. The linearity of the relationship between the optical density at wavelength 680 nm and dry biomass had been established for Dunaliella sp. Dunaliella sp. samples were obtained on days 3, 6, 9, 12, 15, 18 and 21. The samples for measuring OD value have to be collected at the same time (Payer, 1971) 2.3.2 Growth rate To compare cell growth in different nitrogen treatment, cell counting was done using a light microscope and Neubauer haemocytometer. Lugol’s iodine solution was added for fixing (Andersen, 2005). Dunaliella sp. samples were collected on days 3, 6, 9, 12, 15, 18 and 21, sampling for counting should be at the same time every day. Cell densities were followed by three-day counts for three replicates. Number of cells was calculated following algae cells (per mL) = 104.a.k. In this formula, a was the number of Dunaliella sp. cells counted in the large square (total volume of 0.1 mm3) and k was dilution factor. Specific growth rate µ was calculated according to equation (Garcia et al., 2007) Where y1, y2 is the cell number at day x1 and x2 respectively 2.4 Determination of β-carotene content Dunaliella sp. samples were collected on days 3, 6, 9, 12, 15, 18 and 21. An aliquot (2 mL) of Dunaliella sp. cell suspension was centrifuged at 4000 rpm for 10 minutes. The 6 pellet obtained was washed with distilled water and after removal of water by centrifugation again suspended in acetone-hexane (4:6) and vortex the mixture for 1 – 2 minutes. The cell membrane gets ruptured because of organic solvent, and βcarotene was extracted. Acetone-Hexane extract separated from cell debris by centrifuging at 4000 rpm for 10 minutes. OD values of extracted solution were determinated by spectrophotometer at wavelengths of 453, 505, 645 and 663 nm. β-carotene content was calculated based on the Nagata and Yamashita (1992) formula βcarotene (mg/100mL) = 0.216A663 – 1.22A645 – 0.304A505 + 0.452A453. The assay were carried out in triplicate. 2.5 Data analysis Data were analyzed statistically using SPSS program version 13.0 7 3. RESULTS AND DISCUSSIONS 3.1 Effect of nitrogen source on the growth of microalgae Dunaliella sp. 3.1.1 NaNO3 The development of Dunaliella sp. in medium containing NaNO3 as the nitrogen source could be divided into 3 stages (Fig. 4). The first stage, six days after inoculation, algae density in all treatments increased slowly and did not differ significantly. The reason was that algae had to be adapted to the new environment. From the day of 9th to 18th, microalgae used available nutrients Cell density (x104 cells/mL) particularly nitrogen to support their growth. 350 300 250 200 150 100 50 0 N1 N2 N3 N4 0 3 6 9 12 15 18 21 Day Figure 4. Dunaliella sp. cell density in relation to NaNO3 nitrogen source Algal density increased dramatically, significantly and reached their peaks at the 18th day on N3 treatment (311.67x104 cells/mL), followed by the treatments to N4, N2 and lowest N1. The third stage, after 18 days cultivation, all treatments had the decreasing in algal density. This could be explained by the 8 running out of nutrient, and substances produced by algae that affected the growth and caused inhibition of the development. During development, growth rate of all treatments were statistically significant difference. The treatments N1 (µ=0.217), N2 (µ=0.234) and N3 (µ=0.247) have growth rate proportional to the nitrogen concentration. However, N4 (µ=0.237) recorded lower compared to N3, this could be due to the inhibition by high nitrogen concentration (Fig. 5). N1 N2 N3 N4 Figure 5. Dunaliella sp. in relation to NaNO3 nitrogen source after 18 days cultivation 3.1.2 KNO3 The treatments with nitrogen source were KNO3, approximately 6 days undergoing lag phase (Fig. 6). In this period, algae had to adapt to new environment, there were a slow growth and no significant differences among treatments. 9 Cell density (x104 cells/mL) 350 300 250 200 150 100 50 0 K1 K2 K3 K4 0 3 6 9 12 Day 15 18 21 Figure 6. Dunaliella sp. cell density in relation to KNO3 nitrogen source th From the 9 day to 18th day, algal densities increased in all treatments. However, there was statistically significant difference of K3 treatments compared to three other treatments at the 12th day. Dunaliella cell density of K3 treatment was the highest after 18 days cultivation (305.33x104 cells/mL). The number of cells in K1, K2 and K4 were at 180.33x104 cells/mL, 201.33x104 cells/mL and 212x104 cells/mL, respectively. After 18 days, all treatments were on the death phase, cell numbers began to decline. The growth rate of algae increased when nitrogen concentration increased, and the highest rate of growth was obtained in K3 (µ=0.247). However, in higher nitrogen concentration of K4 (8 mM), the growth of Dunaliella sp. was inhibited (Fig. 7). 10 K1 K2 K3 K4 Figure 7. Dunaliella sp. in relation to KNO3 nitrogen source after 18 days cultured 3.1.3 Urea Similar to the use of nitrate nitrogen source, in 6 days adaption period, the density increased slowly. After 9 days, the growth of algae increased faster and there was a significant difference among treatments, especially, U3 had a high growth rate (μ = 0.26), and reached highest cells number at the 18th day (383x104 cells/mL), The high cell counts were also recorded in the treatments U4, U2 and U1 302x104, 218x104 and 183.33 x104 cells/mL, respectively. After 18 days cultivation, Dunaliella cells began to decline (Fig. 8). Cell density (x104 cells/mL) 500 400 300 U1 200 U2 100 U3 U4 0 0 3 6 9 12 Day 15 18 21 Figure 8. Dunaliella sp. cell density in relation to urea nitrogen source 11 Generally, after 21 days of development, U3 had the highest growth rate and number of cells, followed by treatments U1 and U2. Higher nitrogen concentrations (U4) caused negative effect, and the growth was low (Fig. 9). U1 U2 U3 U4 Figure 9. Dunaliella sp. in relation to urea nitrogen source after 18 days cultured 3.1.4 Comparison the density increasing in different nitrogen sources In all treatments, algae demanded to adapt to new culture conditions in the period of 6 days, the result was similar with Mishra's study (2008) on a Dunaliella sp. in Mexico. Algae population increased slowly in lag phase. By adapting stage, density of algae in all treatments had rapidly growth and significant differences from the day of 9th. The highest cell number was reached at the 18th day with 6 mM nitrogen concentration (N3, K3 and U3) (Fig. 10) N3 K3 U3 Figure 10. Dunaliella sp. after 18 days cultured 12 Nitrogen is an important source of nutrients for algae. Dunaliella sp. density results indicated that suitable nitrogen source for algae growth was urea. In terms of algal growth rates as well as density, urea treatments were preponderant compared to other treatments. When urea was used as a source of nitrogen, algae had to have an enzyme that catalyzes the hydrolysis of urea. Urea amidolyase which was found in D. primolecta is an ATP dependent enzyme could break down urea into NH3 and CO2 (Leftley and Syrett, 1973). This might lead to the high growth rate of Dunaliella sp. in urea-containing medium. Besides NH3, algae received additional CO2 together with aeration CO2 that increased photosynthetic capacity and promoted the growth of algae. The development of Dunaliella sp. depended on the nitrogen concentration. Growth rate (μ = 0.26) and the highest density of Dunaliella sp. (383x104 cells/mL) were recorded at 6 mM urea. This approximation of the Celekli and Donmez’s research results (2006) on a species of Dunaliella sp. with cell numbers peaked 4.2 x106 cells/mL at a concentration of 5 mM and another study also suggested that the appropriate of nitrogen was 5 mM (Hosseini Tafreshi and Shariati, 2009). However, high nitrogen concentration caused inhibition. This was seen in the treatments with 8 mM nitrogen (N4, K4 and U4) compared to recent studies also noted the same results when Dunaliella sp. cultured in 7 mM nitrogen concentrations (Kim, Park et al., 2012). 13 3.2 Effect of nitrogen source on biomass of microalgae Dunaliella sp. According to Massart and Hantson (2010), there was a linear regression correlation between OD680nm value and dry biomass (Fig. 11). Therefore, dry biomass was inferred from OD680nm value by equation dry biomass (g/100mL) = Dry biomass (g/100mL) 0.2765.OD680nm – 0.004. 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 y = 0,2765x - 0,004 R² = 0,9908 0 0,1 0,2 0,3 0,4 OD680nm 0,5 0,6 Figure 11. Correlation between optical density at 680 nm and dry biomass of Dunaliella sp. Similar to the density results, algal dry biomass increased slowly and no significant differences among treatments in the same day after 6 days cultivation. After 9 days, there were significant differences in the dry biomass of microalgae among treatments in same day. The highest dry biomass was recorded at 18th day in all treatments. After 21 days, dry algal biomass reduced. That could be due to the algae had gone to dead phase and the decomposition of the cells. 14 In NaNO3 and KNO3 supplement treatments, biomass increased directly proportion with nitrogen concentration, the highest values were recorded in the 18th day, 0.407 g/100mL, 0.402 g/100mL and 0.493 g/100mL in K3, N3 and U3 treatments, respectively. Dry biomass was reduced when culturing with 8 mM nitrogen. Dry biomass of Dunaliella sp. would increase when the concentrations of nitrogen in the medium increased and decreased with statistical significance when nitrogen increasing to 8 mM (Fig. 12). Dry-weight biomass (mg/100mL) 0,6 a 0,5 b 0,4 0,3 b f e de d f c de b f 0,2 0,1 0 N1 N2 N3 N4 K1 K2 K3 K4 U1 U2 U3 U4 Treatments Figure 12. Dry-weight biomass of Dunaliella sp. at 18th day Dry biomass of Dunaliella sp. was directly influenced by density. The results of previous experiments showed that U3 had the highest density and the dry biomass should be higher than those of other treatments (0.493 g/100mL). 15 3.3 The accumulation of β-carotene in Dunaliella sp. Nitrogen affected the growth and accumulation of βcarotene. Besides, obtained β-carotene content depended on algal biomass and β-carotene accumulated in individual cells. Similar to density, β-carotene accumulation of algae represented the significant difference among treatments on the 9th day. The high amount of β-carotene was recorded in all treatments on the 18th day ranged from 0.064 mg/100mL (K1) to 0.228 mg/100mL (U3), and urea-supplement treatments gave the β-carotene (mg/100mL) highest β-carotene compared to the nitrate treatments. 0,3 a 0,25 0,2 b 0,15 0,1 de d c d e d c c d c 0,05 0 N1 N2 N3 N4 K1 K2 K3 K4 U1 U2 U3 U4 Treatments Figure 13. β-carotene content in Dunaliella sp. at 18th day High levels of β-carotene of Dunaliella sp. was obtained at 6 mM nitrogen concentration compared to the results of Fazeli (2006) on microalgae D. teriolecta DCCBC26 (highest amount of β-carotene at 5 mM nitrogen with 4% medium salinity). Another study also found that the highest level of β-carotene was accumulated by microalgae Dunaliella sp. at 5 mM nitrogen 16
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