HỌC VIỆN NÔNG NGHIỆP VIỆT NAM KHOA CÔNG NGHỆ SINH HỌC ---------------- KHÓA LUẬN TỐT NGHIỆP ĐỀ TÀI: “STUDY ON THE CULTIVATION OF GREEN ALGAE DUNALIELLA SALINA IN RACEWAY POND SYSTEM” Người thực hiện: BÙI QUANG HIỆP Lớp: K61CNSHE Chuyên ngành: Công nghệ Sinh Học - Biotechnology Giáo viên hướng dẫn: PGS. Nguyễn Đức Bách HÀ NỘI – 3/2021 DECLARATION I hereby certify that this is my own research. The data and results in the thesis are true and they have never been published in any research work. I hereby certify that the information cited in the thesis has been clearly sourced.
Hanoi, March 2021 NAME i ACKNOWLEDGEMENT First of all, I would like to express my gratitude to my Assoc. Nguyen Duc Bach, who enthusiastically guided and helped me in the implement of the thesis. I would like to thank the Department of Molecular Biotechnology and Insitute for Research and Development of Microalgae of Vietnam National University of Agriculture for creating favorable conditions to allow me to conduct experiments in my graduation thesis. I also would like to send thanks to Ms.
Phi Thi Cam Mien, Mr. Hien and all interns for assist me for my thesis. My special thanks approve to my parents for their endless love, care and have most assistances and motivation me for the whole of my life. I also would like to explain my thanks to my siblings, brothers and sisters for their support and care me all the time.
ii TABLE OF CONTENS DECLARATION. ii LIST OF FIGURES. vi LIST OF TABLES. viii PART 1: INTRODUCTION.
Purpose and requirement. 2 PART 2: LITERATURE REVIEW. Overview of the Dunaliella salina. Taxonomic, morphological and biological characteristics of Dunaliella salina.
Taxonomy of Dunaliella salina. Morphology of Dunaliella salina. Biology characteristics of Dunaliella salina. Factors affecting the growth of Dunaliella salina.
Application of Dunaliella salina. Studies on Dunaliella salina. Studies on Dunaliella salina in Vietnam. Studies on Dunaliella salina in the world.
Algae biomass production by Raceway pond. Introduction of the Raceway pond. Typical configuration of the Raceway pond. 18 PART 3: RESEARCH METHODOLOGY.
Study time and place. Determination of cell density by Neubauer counting chamber. Determination of specific growth rate. Determination of the optimal culture medium.
Effect of temperature on the growth of Dunaliella salina. Effect of light intensity on the growth of Dunaliella salina. Effect of salinity on the growth of Dunaliella salina. The growth of Dunaliella salina in Raceway pond.
30 PART 4: RESULTS AND DISCUSSION. Preservation of Dunaliella salina. Determination of the optimal culture medium. Effect of temperature on the growth of Dunaliella salina.
Effect of light intensity on the growth of Dunaliella salina. Effect of salinity on the growth of Dunaliella salina. The growth of Dunaliella salina in raceway pond. 42 iv PART 5: CONCLUSIONS AND RECOMMENDATIONS.
45 v LIST OF FIGURES \ Fig 2. Taxonomic hierarchy of Dunaliella salina. Morphology of green microalgae Dunaliella salina. Morphology of some species in the genus Dunaliella salina.
The life cycle of Dunaliella salina. A top view of a raceway pond as typical used for algal biomass production. Neubauer counting chamber………………………………………. Investigation of the optimal medium for the growth of Dunaliella salina.
Investigation of the optimal temperature for the growth of Dunaliella salina. Investigation of the optimal light intensity for the growth of Dunaliella salina. Investigation of the optimal salinity for the growth of Dunaliella salina. Preservation of Dunaliella salina in liquid medium………………….
Growth rate of Dunaliella salina in different medium for 2 weeks. Specific growth rate of Dunaliella salina in different medium. Growth rate of Dunaliella salina in different temperature. Specific growth rate of Dunaliella salina in different temperature.
Growth rate of Dunaliella salina in different light intensity. Specific growth rate of Dunaliella salina in different light intensity. 8 Growth rate of Dunaliella salina in different salinity. Specific growth rate of Dunaliella salina in different salinity .41 vi LIST OF TABLES Table 4.
Cell density for 2 weeks of Dunaliella salina in different medium. Growth density of Dunaliella salina in different temperature for 2 weeks. Cell density of Dunaliella salina in different light intensity for 2 weeks. Cell density of Dunaliella salina in different salinity for 2 weeks.
Total cell count of Dunaliella salina cultured in raceway pond. 42 vii ABSTRACT The microalgae Dunaliella salina is the best commercial source of natural -carotene. In this study, the growth of Dunaliella salina algae will be evaluated by culture medium and other conditions such as temperature, light intensity and salinity. To determine the optimal culture medium, three types of media commonly used to evaluate the growth of Dunaliella salina is F/2 media, Walne media and J/l media.
To investigation of the optimal temperature, Dunaliella salina was experimented at 3 different temperature were 18-22℃, 24-28℃ and outdoor temperature.To investigation of the optimal light intensity, Dunaliella salina was experimented at 3 different light intensity were 10Klux, 12Klux and 14Klux. To investigation of the optimal salinity, Dunaliella salina was experimented at 5 different salinity were 0. The result of experiment show that F/2 medium is best optimal media for cultivate Dunaliella salina algae and the optimal conditions of this algae is at temperature 24-28℃, light intensity 14Klux and 1M salinity. Then the experimental results was used to cultivate biomass in the raceway pond system.
viii PART 1: INTRODUCTION 1. Problem statement Nowadays, microalgae are used as nutrient supplements for human consumption because of high proteins, vitamins and polysaccharides content. Currently and in the future, the use of algae will develop in the energy sector, food production and many other applications in life. -carotene, protein, polysaccharide, unsaturated fatty acids are compounds in the composition of microalgae that have been exploited and used by humans.
Dunaliella salina is a rare single-celled algae found in high salt concentration environments. It adapted to survive in highly salty environments by accumulating large amounts of β-carotene to shield them from UV radiation. β-carotene has great potential in applying biotechnology for commercial purposes such as cosmetics or functional foods. Dunaliella salina contains the richest source of β-carotene.
Accumulating β-carotene requires high salinity, high temperature and high light intensity. Studies have shown that at salinity above 27% NaCl, Dunaliella salina can accumulate carotenoids up to 14% dry weight and active more than plant carotenoids. β-carotene plays an important role in preventing the development of cancer, enhancing human vision, ulcers, aging. Vietnam is a tropical country with great sea potential with a coastline of more than 3200km and famous salt fields such as Sa Huynh, Cam Ranh, Can Gio, Long Dien.
So Vietnam has economic potential are great for growing Dunaliella salina algae. However, in Vietnam, there is very little research on the optimal growth conditions of Dunaliella salina and β-carotene biosynthesis. So the thesis “Study on the cultivation of green microalgae Dunaliella salina in raceway pond system” was chosen to determine the optimal conditions for the growth of Dunaliella salina and cultivation of them in raceway pond system. Purpose and requirement 1.
Purpose Study on cultivation of the green algae Dunaliella salina in raceway pond system. Requirement Determination of the optimal culture medium for the growth of Dunaliella salina algae. Determination of the optimal conditions for the growth of Dunaliella salina algae. Cultivation of algae biomass in the raceway pond system.
2 PART 2: LITERATURE REVIEW 2. Overview of the Dunaliella salina 2. Taxonomic, morphological and biological characteristics of Dunaliella salina 2. Taxonomy of Dunaliella salina Dunaliella salina is a green algae that now belong to the genus Dunaliella and phylum Chlorophyta.
[Avron and Ben-Amotz 1992; Garcia et al. The current taxonomy of the genus is based on morphological and physiological attributes including the ability of some species to grow over wide salinity ranges and at extreme salinities, as well as the accumulation of high levels of β- carotene. Taxonomic hierarchy of Dunaliella salina In the early 18th century, scientists thought that Dunaliella resembled Haematococcus, but in the second half of the 19th century, they found that the genus was different from Haematococcus and named it Dunaliella [Avron and Ben-Amotz 1992]. Up to now, 28 Dunaliella species have been recognized.
3 These include five species that live in freshwater and are rarely present, while 23 of these species are present in salty environments. Morphology of Dunaliella salina Dunaliella salina is vegetative motile cells. The cell shape of Dunaliella salina varies from ellipsoid, ovoid, cylindrical and pyriform to almost spherical. Adult green cells range from 5.4μm in length and 3.6μm in width; Cells of this algae may change shape with changing conditions, often becoming spherical under unfavorable conditions.
A rigid wall is lacking, but there is a distinctive mucilaginous cell coat. The two flagella are apically inserted, equal in length, and usually exhibit a homodynamic pattern of beating. The two basal bodies are displaced against each other and carry microtubular flagellar roots. The single chloroplast occupies most of the cell body.
This body maybe shaped like a cup, dish, and bell and have a thicker section containing a pyrite (kernels, starch granules, inclusions) [Borowitzka and Siva, 2007]. Anteriorly, the chloroplast is sometimes incised into several lobes. The thylakoids of the chloroplast are sometimes arranged in dense stacks of up to 10 units. Stacking of thylakoids was found to be particularly pronounced in cells grown at high light intensity and high salt concentration.
Starch grains usually surround the pyrenoid, but may also be found at other places of the chloroplast. The β- carotene globules of Dunaliella salina were found to be composed of practically only neutral lipids, more than half of which were β-carotene. Most of the reddish forms may lose their red color when grown at low light intensities [Sarmad et al. Morphology of green microalgae Dunaliella salina (A) Light micrograph of vegetative cell with a high content of β-carotene (cell length = 23μm); (B) Two cells in the early stage of mating showing appressed flagella and mating tube between the two cells (cell length 22μm); (C) Transmission electron micrograph of a section of a cell of Dunaliella salina.
Morphology of some species in the genus Dunaliella salina Asteromonas gracilis redrawn [Ruinen, 1938]; Brachiomonas submarina, Brachiomonas simplex, Chlamydomonas Pulsatilla, Dunaliella maritima, and Dunaliella salina [Butcher, 1959]; Pyramichlamys vectensis [Carter, 1937]. Biology characteristics of Dunaliella salina Dunaliella salina is a green algae that now belongs to the phylum Chlorophyta [Avron and Ben-Amotz 1992; Garcia et al.It lacks a rigid 6 cell wall nevertheless it can grow in aquatic environments varied salinities from 0. Under stress condition, Dunaliella salina can accumulate significant amounts of valuable chemical matter such as carotenoids, glycerol, vitamins and proteins [Hadi et al. Dunaliella salina can adapt to a wide range of salt concentrations was shown to be based on the ability of the alga to change its intracellular concentration of glycerol [Raja et al.
In fact, the accumulation of glycerol in this algae is regulated by external water activity rather than the specific solute effect [Shariati & Lilley, 1994]. The eyespot is usually distributed in the anterior part of the chloroplasts. In Dunaliella salina, the eyespot is almost difficult to see under an optical microscope [Avron & Ben-Amotz, 1992]. The nucleus is often obscured by a large number of particles.
It occupies most of the anterior part of the cell and is surrounded by the anterior lobes of the plastids. Superstructure research has shown it has a porous envelope and a single, convex nucleus surrounded by chromatin structures. Mitochondrial structures can be observed in different parts of the cell. The number and size of mitochondria varies greatly between cells at different growth stages.
Cells have 2 to 4 golgi forms. The endoplasmic reticulum typically underlies the plasmalemma over most parts of the cell.