effect of nitrogen on biomass and βcarotene accumulation by 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..

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

APPROVAL

SUPERVISOR STUDENT

Assoc Prof NGUYEN HUU HIEP 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 180.33x10 4 and 383x10 4 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

CONTENTS

PageAPPROVAL

2.1 Microalgal strain and culture medium 3 2.2 Nitrogen sources and concentration experimental design 4

3.1 Effect of nitrogen source on the growth of microalgae

3.3 The accumulation of β-carotene in Dunaliella sp 16

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, β-carotene 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 β-carotene can reduce the free radicals that affect the physiological

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 β-carotene 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 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

Manganous chloride (MnCl2, 4H2O) 0.4 g

EDTA(b), di-sodium salt 45.0 g Sodium di-hydrogen orthophosphate (NaH2PO4,

2H2O)

20.0 g

Trace Mineral Supplement (TMS) 1.0 mL Make up to 1L with sea water

Trace Mineral Supplement (TMS)

Cobaltous chloride (CoCl2.6 H2O) 2.0 g Ammonium molybdate ((NH4)6Mo7O24.4H2O) 0.9 g Cupric sulphate (CuSO4.5H2O) 2.0 g

Concentrated HCl 10.0 mL Make up to 100mL with distilled water

Vitamin solution (0.1mL/1L medium)

Vitamin B12 (Cyanocobalamin) 10 mg

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 5x105cells/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

Table 4 Experimental design

Nitrogen

source Treatment

Nitrogen concentration (Mm)

Amount (g/l)

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

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 β-carotene 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 β-carotene (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

3 RESULTS AND DISCUSSIONS

3.1 Effect of nitrogen source on the growth of microalgae

Dunaliella sp

3.1.1 NaNO 3

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 particularly nitrogen to support their growth

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 (311.67x104cells/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

0 50 100

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)

Figure 5 Dunaliella sp in relation to NaNO3 nitrogen

source after 18 days cultivation 3.1.2 KNO 3

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

Figure 6 Dunaliella sp cell density in relation to KNO3

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

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 x104cells/mL, respectively After 18 days cultivation, Dunaliella cells began to decline (Fig 8)

Figure 8 Dunaliella sp cell density in relation to urea

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)

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)

Figure 10 Dunaliella sp after 18 days cultured

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)