Study on the cultivation of green algae dunaliella salina raceway pond system (khóa luận tốt nghiệp)

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.TS 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 NAME i 2021 ACKNOWLEDGEMENT First of all, I would like to express my gratitude to my Assoc Prof Dr 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 Tuan, Ms 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 i ACKNOWLEDGEMENT ii LIST OF FIGURES vi LIST OF TABLES vii ABSTRACT viii PART 1: INTRODUCTION 1.1 Problem statement 1.2 Purpose and requirement 1.2.1 Purpose 1.2.2 Requirement PART 2: LITERATURE REVIEW 2.1 Overview of the Dunaliella salina 2.1.1 Taxonomic, morphological and biological characteristics of Dunaliella salina 2.1.1.1 Taxonomy of Dunaliella salina 2.1.1.2 Morphology of Dunaliella salina 2.1.1.3 Biology characteristics of Dunaliella salina 2.1.2 Factors affecting the growth of Dunaliella salina 2.1.3 Application of Dunaliella salina 13 2.2 Studies on Dunaliella salina 16 2.2.1 Studies on Dunaliella salina in Vietnam 16 2.2.2 Studies on Dunaliella salina in the world 17 iii 2.3 Algae biomass production by Raceway pond 18 2.3.1 Introduction of the Raceway pond 18 2.3.2 Typical configuration of the Raceway pond 18 PART 3: RESEARCH METHODOLOGY 21 3.1 Research subjects 21 3.2 Study time and place 21 3.3 Chemicals 21 3.4 Research equipments 23 3.5 Research methods 24 3.5.1 Determination of cell density by Neubauer counting chamber 24 3.5.2 Determination of specific growth rate 25 3.5.3 Determination of the optimal culture medium 26 3.5.4 Effect of temperature on the growth of Dunaliella salina 27 3.5.5 Effect of light intensity on the growth of Dunaliella salina 28 3.5.6 Effect of salinity on the growth of Dunaliella salina 29 3.5.7 The growth of Dunaliella salina in Raceway pond 30 3.6 Data analysis 30 PART 4: RESULTS AND DISCUSSION 31 4.1 Preservation of Dunaliella salina 31 4.2 Determination of the optimal culture medium 32 4.3 Effect of temperature on the growth of Dunaliella salina 34 4.4 Effect of light intensity on the growth of Dunaliella salina 37 4.5 Effect of salinity on the growth of Dunaliella salina 39 4.6 The growth of Dunaliella salina in raceway pond 42 iv PART 5: CONCLUSIONS AND RECOMMENDATIONS 44 5.1 Conclusions 44 5.2 Recommendations 44 REFERENCES 45 v LIST OF FIGURES \ Fig 2.1 Taxonomic hierarchy of Dunaliella salina Fig 2 Morphology of green microalgae Dunaliella salina Fig Morphology of some species in the genus Dunaliella salina Fig The life cycle of Dunaliella salina Fig A top view of a raceway pond as typical used for algal biomass production 19 Fig Neubauer counting chamber……………………………………… 24 Fig Investigation of the optimal medium for the growth of Dunaliella salina 26 Fig 3 Investigation of the optimal temperature for the growth of Dunaliella salina 27 Fig Investigation of the optimal light intensity for the growth of Dunaliella salina 28 Fig Investigation of the optimal salinity for the growth of Dunaliella salina 29 Fig Preservation of Dunaliella salina in liquid medium………………….31 Fig Growth rate of Dunaliella salina in different medium for weeks 33 Fig Specific growth rate of Dunaliella salina in different medium 33 Fig 4 Growth rate of Dunaliella salina in different temperature 35 Fig Specific growth rate of Dunaliella salina in different temperature .36 Fig Growth rate of Dunaliella salina in different light intensity 38 Fig Specific growth rate of Dunaliella salina in different light intensity 38 Fig Growth rate of Dunaliella salina in different salinity 40 Fig Specific growth rate of Dunaliella salina in different salinity 41 vi LIST OF TABLES Table Cell density for weeks of Dunaliella salina in different medium 32 Table Growth density of Dunaliella salina in different temperature for weeks 35 Table Cell density of Dunaliella salina in different light intensity for weeks 37 Table 4 Cell density of Dunaliella salina in different salinity for weeks 40 Table 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 different temperature were 18-22℃, 24-28℃ and outdoor temperature.To investigation of the optimal light intensity, Dunaliella salina was experimented at different light intensity were 10Klux, 12Klux and 14Klux To investigation of the optimal salinity, Dunaliella salina was experimented at different salinity were 0.5M, 1M, 1.5M, 2M, 2.5M 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 1.1 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 density of algae is 2.61x106 cells/mL (2.61±0.31) At 18-22℃, the maximum density of algae is 3.18x106 cells/mL (3.18±0.17) The results show that, at different temperatures, the maximum algal density is different Based on the results obtained, Dunaliella salina is a species that is able to adapt to a wide temperature spectrum But in the experimental temperature range we see that the higher the temperature is, the more limited the growth of this algae is The suitable temperature for growth of Dunaliella algae is 22-28℃ This is the optimal temperature for this algae to reach high density for biomass 4.4 Effect of light intensity on the growth of Dunaliella salina Light is an important factor, determining the existence and development of phototrophs Therefore, in order to find optimal conditions for the growth of Dunaliella salina algae, this experiment conducted a culture experiment in the different light intensity of 10Klux, 12Klux and 14Klux The growth of Dunaliella salina in three different light intensity were presented in below: Table Cell density of Dunaliella salina in different light intensity for weeks Day Cell density in different light intensity (x𝟏𝟎𝟔 cells/mL) 10Klux 12Klux 14Klux 0.20 0.20 0.20 0.65 0.66 0.69 2.13 1.92 1.72 2.69 2.36 2.32 5.20 2.63 4.80 10 6.52 3.95 7.03 12 6.13 3.28 4.20 14 6.05 3.14 4.08 37 10Klux 12Klux 14Klux 0 10 12 14 Time (day) Fig Growth rate of Dunaliella salina in different light intensity 10Klux 12Klux 14Klux Specific growth rate (generation/day) 2 Time (day) 10 Fig Specific growth rate of Dunaliella salina in different light intensity Through the experimental results, the Dunaliella salina developed quite quickly, the algae density increased rapidly and reached the maximum density around 10 days of culture The corresponding 10Klux decreased from 6.52x106 38 cells/ml (6.52±0.22) to 6.13x106cells/ml (6.13±0.18), 12Klux decreased from 3.95x106cells/ml (3.95±0.26) to 3.28x106cells/ml (3.28±0.3), and 14Klux decreased from 7.03x106cells/ml (7.03±0.17) to 4.2x106cells/ml (4.2±0.39) in just day In the first days, the growth rate is uniform Until day 10 onwards, the growth rate starts to increase differently with different light intensity At the light intensity of 14Klux and 10Klux, the increase in the number of algae was most evident Algae adapt very well at these lighting intensities Although the maximum algae density at 14Klux was higher than that of 10Klux, the algae density in the growth phase and the equilibrium phase was always lower than that at 10Klux The maximum density fell on the 10th day of culture, with quantities up to 6.52x106 cells/mL (6.52±0.22) to 7.03x106 cells/mL (7.03±0.17) At 12Klux light conditions, Dunaliella algae grows relatively slowly, algae density increases slowly, there is no high jump in density as at other light intensities and the highest density on day 10 is 3.95x106 cells/ml (3.95±0.26) This cycle of growth at this light level is relatively slow This is explained because under this light condition the energy metabolism of microalgae cells is relatively low, so the growth and development of algae is also weaker From the results of the study, we found that Dunaliella salina is able to adapt and grow at light intensity of 10Klux – 14Klux when raised at a volume of 140mL, the best growth at light intensity is 10Klux and 14Klux and reach maximum density under 14Klux condition 4.5 Effect of salinity on the growth of Dunaliella salina Variations in salinity also influence several biochemical and physiological mechanisms such which are essential in Dunaliella salina algae Therefore, in order to find optimal conditions for the growth of Dunaliella salina algae, this experiment conducted a culture experiment in the different salinity of 0.5M, 1M, 1.5M, 2M, 2.5M The growth of Dunaliella salina in five different salinity were presented in below: 39 Table 4 Cell density of Dunaliella salina in different salinity for weeks Cell density in different salinity (x𝟏𝟎𝟔 cells/mL) Day 0.5M 1M 1.5M 2M 2.5M 0.20 0.20 0.20 0.20 0.20 0.69 0.96 0.66 0.70 0.17 1.66 2.05 1.23 0.78 0.16 3.74 4.59 3.24 2.81 0.11 4.81 5.73 4.42 3.86 0.15 10 5.31 6.22 4.59 4.32 0.16 12 4.42 5.00 3.22 2.64 0.08 14 4.38 4.83 3.14 2.52 0.10 Growth density ( 10^6 cells/mL) 0.5M 1M 1.5M 0 Time (Day) 10 12 14 Fig Growth rate of Dunaliella salina in different salinity 40 Specific growth rate (generation/day) 0.5M 1M 1.5M 2M 2 Time (day) 10 Fig Specific growth rate of Dunaliella salina in different salinity Dunaliella salina algae are very salty species, but when changing from 0.5M to 2.5M, algae can grow at 0.5M to 2M salinity At 2.5M salinity in general, the number of algae decreases gradually and after 14 days there are no algae cells The death of algae cells at 2.5M salinity can be caused by salt shock, high salinity is too stressful for algae, maybe the cells cannot accumulate glycerol in time to change the osmotic pressure of the cell membrane adapting to the environment So water goes out from the cell, causing cells to shrink and algae die In days of the experiment, the difference in algae density at salinity was not significant Density of Dunaliella salina reached the maximum after 10 days of culture, of which the highest was at 1M salinity with 6.22x106 cells/mL (6.22±0.25), and the lowest at 2M salinity was 4.32x106 cells/mL (4.32±0.21) After day 10, the erosion at salinity 0.5M, 1M was slower than salinity 1.5M, 2M The growth curve of the algae population at 1M salinity is relatively uniform and stable, after reaching the magnitude, the algae density did not decrease as sharply as the other experimental groups The maximum density of algae at 1M salinity is 6.22 x 106 cells/ml (6.22±0.25), which is higher than 41 that of 1.5M salinity is 5.31 x 106 cells/mL Dunaliella salina has a wide salinity range from 1M-2M, but the most suitable salinity is 1M 4.6 The growth of Dunaliella salina in raceway pond Based on result of experiment, Dunaliella salina was cultivated biomass in F/2 medium, at 24-28℃ temperature, 14Klux light intensity and 1M salinity.After reaching maximum density, continue diluting and cultivating the biomass until the volume of 20 liters is reached The cultivation of algae biomass in the raceway pond system at the rate of 20 liters of the algae and 100 liters of the medium When kept in raceway pond, Dunaliella salina use natural light and ambient temperature to cultivate biomass Table Total cell count of Dunaliella salina cultured in raceway pond Days Cell count (x106 cells/ml) 0.80 1.50 2.30 3.50 8.50 11.0 44.0 95.0 109 10 125 11 150 12 175 13 195 14 210 15 180 42 The culture was sampled daily, the cell numbers were measured using a Nebauer chamber The growth pattern showed gradual increase in the cell number up to 2nd day (1.5x106 cells/ml) and thereafter the increase was steep from 3rd day to 7th day (2.3x106 cells/ml to 44x106 cells/ml) Then the cell number reach to maximum density in14th day (210x106 cells/ml).In the 15th day cell numbers decrease (180x106 cells/ml) After 15 days, Dunaliella salina will be centrifuged at 3500rpm for minutes to recover biomass After separating algae from the medium, drying must be conducted immediately to keep biomass from damage by microorganisms There are many drying methods: exposure drying, sun-drying, spray-drying, and freeze-drying The most suitable method for drying Dunaliella salina is the spray-drying method because it is fast, effective, and energyconsuming 43 PART 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions • Dunaliella salina can be preserved in liquid medium for 4-6 months • The F/2 medium is best optimal media for cultivate Dunaliella salina algae • The most suitable temperature for Dunaliella salina is 24-28℃ • Under artificial light (with intensity from 10 to 14 Kux), artificial light intensity is suitable for algae growth 14 Klux • Dunaliella salina can grow at 0.5M – 2M salinity 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