VIET NAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATE THESIS TOPIC: “STUDY ON THE CULTIVATION OF CHLORELLA VULGARIS AND METHOD FOR CELL WALL DISRUPTION” Student: CHU THI PHUONG HOA Class: K61CNSHE Code student: 610722 Major: Biotechnology Instuctor: Assoc Prof Dr Nguyen Duc Bach HA NOI – 3/2021 DECLARATION I declare that the information reported in the current thesis is the result of my own work, except where the reference is made I declare that all information and references in this thesis have been clearly stated the origin, name of author, name of work, time and place of study I full responsibility for this declaration Hanoi, March NAME i 2021 TABLE OF CONTENTS DECLARATION i TABLE OF CONTENTS ii LIST OF FIGURES iii LIST OF TABLES v ABSTRACT 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 Chlorella vulgaris 2.2 Morphology and biology characteristics of Chlorella vulgaris 2.2.1 Morphology of Chlorella vulgaris 2.2.2 Reproduction of Chlorella vulgaris 2.3 Factors affecting the growth and development of Chlorella vulgaris 2.3.1 Light 2.3.2 Temperature 2.3.3 Salinity 2.3.4 pH 2.4 Nutritional ingredients of Chlorella vulgaris 2.5 Functional of active ingredients in Chlorella vulgaris 11 PART 3: RESEARCH METHODOLOGY 17 3.1 Research subjects 17 3.2 Study time and place 17 3.3 Chemicals 17 3.4 Research equipments 20 ii 3.5 Research methods 21 3.5.1 Determination of the cell density by Neubauer chamber 21 3.5.2 Specific growth rate of Chlorella vulgaris 22 3.5.3 Preservation of Chlorella vulgaris 23 3.5.4 Factors affecting the growth of Chlorella vulgaris 24 3.5.4.1 Effects of culture media on the growth of Chlorella vulgaris 24 3.5.4.2 Effects of light cycle on the growth of Chlorella vulgaris 24 3.5.4.3 Effects of light intensity on the growth of Chlorella vulgaris 25 3.5.4.4 Effects of nitrogen concentration on the growth of Chlorella vulgaris 25 3.6 Method for cell wall disruption 26 3.7 Data analysis 27 PART 4: RESULT AND DISCUSSION 28 4.1 Investigation of optimal culture media on the growth of Chlorella vulgaris 28 4.2 Investigation of optimal light cycle on the growth of Chlorella vulgaris 30 4.3 Investigation of optimal light intensity on the growth of Chlorella vulgaris 33 4.4 Investigation of optimalnitrogen concentration on the growth of Chlorella vulgaris 35 4.5 Ultrasonication method for cell wall disruption 38 PART 5: CONCLUSION AND RECOMMENDATION 40 5.1 Conclusions 40 5.2 Recommendations 40 REFERENCES 41 iii LIST OF FIGURES Table 2.1 Nutritional components of Chlorella vulgaris Table 2.2 Compare the protein content of Chlorella vulgaris with other foods Table 2.3 Vitamins were found in Chlorella vulgaris 10 Table 2.4 Fatty acids were found in Chlorella vulgaris 11 Table 4.1 Cell density of Chlorella vulgaris in different media 28 Table 4.2 Cell density of Chlorella vulgaris in different light cycle 31 Table 4.3 Cell density of Chlorella vulgaris in different light intensity 33 Table 4.4 Cell density of Chlorella vulgaris in different nitrogen concentration 36 Table 4.5 Chlorella vulgaris cell disruption by ultrasonication in diferrent power 38 iv LIST OF TABLES Fig 2.1 Classification of Chlorella vulgaris Fig 2.2 The different phases of daughter cell-wall formation in Chlorella vulgaris Fig 3.1 Neubauer counting chamber 21 Fig 3.2 Preservation of Chlorella vulgaris 23 Fig 4.1 Growth rate of Chlorella vulgaris in different media 29 Fig 4.2 Specific growth rate of Chlorella vulgaris in diferent media 29 Fig 4.3 Growth rate of Chlorella vulgaris in different light cycle 32 Fig4.4 Specific growth rate of Chlorella vulgaris in different light cycle 32 Fig 4.5 Growth rate of Chlorella vulgaris in different light intensity 34 Fig 4.6 Specific growth rate of Chlorella vulgaris in different light cycle 34 Fig 4.7 Growth rate of Chlorella vulgaris in different nitrogen concentration 36 Fig 4.8 Specific growth rate of Chlorella vulgaris in different nitrogen concetration 37 Fig 4.9 Cell disruption by ultrasonication of Chlorella vulgaris in different power 39 v ABSTRACT Chlorella vulgaris, a green alga that is widely used as a food supplement, has good antioxidant and therapeutic properties Studies on cultivation of the Chlorella vulgaris showed a high nutritional value in proteins of this species, that could represent a major source of food for human However, this goal has not yet been reached due to the very high cultivation costs, which lead to a noncompetitive price compared to other biomass protein sources, such as soy In this study, Chlorella vulgaris will be experimented to determine the optimal culture media, the optimal growth condition such as light cycle, light intensity and nitrogen concentration Then based on experimental results to cultivate the biomass of Chlorella vulgaris algae Some methods for disruption cell wall is microwave, enzymatic, grinding with liquid nitrogen, bead willing and more In this study, the ultrasonication method was chosen to disrupt the cell wall of Chlorella vulgaris PART 1: INTRODUCTION 1.1 Problem statement Chlorella vulgaris microalgae have high nutritional value Chlorella vulgaris are consumed as food supplement and its products are also used for different purposes like dyes, pharmaceuticals, animal feed, aquaculture and cosmetics It also proved to be a source of products such as proteins, carbohydrates, pigments, vitamins and minerais In addition, microalgae capture sunlight and perform photosynthesis by producing approximately half of atmospheric oxygen on earth and absorbing massive amounts of carbon dioxide as a major feed Overall, nutritional demand for algal products of human is increasing Stemming from a greater focus on health and wider use of food additives Nowadays, Chlorella vulgaris is popular all over the world, and Japan is the world leader in consuming Chlorella vulgaris and using it for medical treatment because it showed to have immune-modulating and anti-cancer properties In Vietnam, the consumption and production of Chlorella vulgaris is still limited because there is very little research and high cost Foreseeing this situation, the thesis “Study on the cultivation of Chlorella vulgaris and method for cell wall disruption” was chosen to determine the optimal conditions for the growth of Chlorella vulgaris 1.2 Purpose and requirement 1.2.1 Purpose Study on cultivation of Chlorella vulgaris and method for cell wall disruption 1.2.2 Requirement Determine the optimal culture media for the growth of Chlorella vulgaris Determine the optimal conditions for the growth of Chlorella vulgaris Method for cell wall disruption PART 2: LITERATURE REVIEW 2.1 Overview of the Chlorella vulgaris Hence, Martinus Willem Beijerinck, a Dutch researcher, first discovered Chlorella vulgaris in 1890 as the first microalga with a well-defined nucleus The name Chlorella comes from the Greek word chloros, which means green, and the Latin suffix ella referring to its icroscopie size It belongs to the family of Chlorellaceae [Beijerinck M 1890] Phylum • Chlorophyta Class • Trebouxiophyceae Order • Chlorellales Family • Chlorellaceae Genus • Chlorella Species • Chlorella vulgaris Fig 2.1 Classification of Chlorella vulgaris Chlorella vulgaris lives in fresh water and has the highest chlorophyll content (28.9g/kg) of any known photosynthetic plant lt is a unicellular microalgae that has been present on earth since the pre-Cambrian period 2.5 billion years ago and since then its genetic integrity has remained constant Chlorella vulgaris is part of a group of eukaryotes in the form of a single cell The size of Chlorella vulgaris is equal to the size of human red blood cells Under optimal living conditions: plenty of light, clear water and clean air, Chlorella vulgaris can reproduces at a tremendous speed 2.2 Morphology and biology characteristics of Chlorella vulgaris 2.2.1 Morphology of Chlorella vulgaris Chlorella vulgaris is a unicellular algae, with no capillary injection, and does not have active mobility Cells are spherical or oval in shape Cell size ranges from - 5µm, or even - 4µm depending on environmental conditions and development stage Cell membranes are encased in cellulose, and that can resistant to light mechanical stress Changes in environmental conditions such as light, temperature, chemical composition in the environment will affect the morphology and quality of algae cells [Tran Van Vy 1995] Cell wall preserves the integrity of the cell and is basically a protection against invaders and harsh environment It varies according to each growth phase During its early formation in its autosporangia, the newly formed cell wall remains fragile, forming a nm thin electron-dense unilaminar layer The cell wall of the daughter cell gradually increases in thickness until it reaches 1721 nm after maturation, where a microfibrillar layer is formed representing a chitosan-like layer composed of glucosamine, which accounts for its rigidity In the mature stage, cell wall thickness and composition are not constant because they can change according to different growth and environmental conditions [Yvonne N, Tomas K 2000] Chlorella vulgaris has a single chloroplast with a double enveloping membrane composed of phospholipids; the outer membrane is permeable to metabolites and ions, but the inner membrane has a more specific function on proteins transport Starch granules, composed of amylose and amylopectin, c an be formed inside the chloroplast, especially during unfavourable growth conditions The pyrenoid contains high levels of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and is the centre of carbon dioxide fixation The chloroplast also stores a cluster of fused thylakoids where the dominant pigment chlorophyll is synthesised masking the colour of other pigments such as Specific growth rate (generation/day) Fig 4.3 Growth rate of Chlorella vulgaris in different light cycle 12h 20 18 16 14 12 10 2 24h 16h Time (day) Fig4.4 Specific growth rate of Chlorella vulgaris in different light cycle In the first days, the density of algae increases slowly Day 5, the density of algae tends to increase rapidly At this time, the algae density changed to the growth phase, the increasing trend of algae density in experimental plots began to depend on the light cycle On day 5, the density of algae at cycle 12/24 was 4.56x106 (4.56±0.01) cells/ml, at cycle 16/24 reached 5.68x106(5.68±0.01) cells/ml, at cycle 24/24 reached 5.7x106(5.7±0.01) cells/ml Based on the chart, we find that the most appropriate light cycle for Chlorella vulgaris is 16 hours a day (CT3: 16/8) At cycle 12/12, algae reached a maximum density of 12.43x106 (12.43±0.01) cells/mL on day of the experiment, then the density decreased to 9.56x106 (9.56±0.01) cells/mL on day 14 At cycle 24/00, the algae density reached a maximum of 15.96x106 (15.96±0.017) cells/mL on day of the experiment, then the density decreased to 12.75x106 (12.75±0.017) cells/mL on day 14 At cycle 16/8, the algae density reached a maximum of 17.86x106 (17.86±0.01) cells/mL on day of the experiment, then the density decreased to 14.37x106 (14.37±0.02) cells/mL on day 14 Therefore, the most suitable lighting cycle for Chlorella vulgaris is 16 hours a day 32 4.3 Investigation of optimal light intensity on the growth of Chlorella vulgaris Light is a factor that has a great influence on the growth and development of microalgae, is the main source of energy for their photosynthesis The experiment was performed by formulations with different light intensity: 4Klux, 7Klux, 13Klux The results of the experiment to determine the effect of light intensity on algae growth are shown in the chart below: Table 4.3 Cell density of Chlorella vulgaris in different light intensity Day Cell density in different light intensity (Klux) (x𝟏𝟎𝟔 cells/mL) 13 1.90 2.10 1.95 2.23 2.35 2.22 2.64 3.74 2.95 3.64 2.55 3.24 6.91 9.43 7.25 10.26 5.78 7.23 11.95 15.65 13.85 16.94 9.86 12.74 17.88 19.68 14.94 10 11 17.65 16.44 19.23 18.86 14.34 13.82 12 13 15.86 14.34 17.55 15.86 13.21 12.65 14 12.98 15.22 11.89 33 4Klux 7Klux 13Klux 25 20 15 10 5 10 11 12 13 14 Fig 4.5 Growth rate of Chlorella vulgaris in different light intensity Specific growth rate (generation/day) 13 25 20 15 10 5 Time (day) Fig 4.6 Specific growth rate of Chlorella vulgaris in different light intensity Through the experimental results, the Chlorella vulgaris developed quite quickly, the algae density increased rapidly and reached the maximum density around days of culture In the first days, the density of algae increases slowly Day 5, the density of algae tends to increase rapidly At this time, the algae density changed to the growth phase, the increasing trend of algae density 34 in experimental plots began to depend on the light intensity On day 5, the density of algae at 4Klux was 6.91x106 (6.91±0.01) cells/ml, at 7Klux reached 7.25x106(7.25±0.01) cells/ml, at 13Klux reached 5.78x106(5.78±0.01) cells/ml Growth of Chlorella vulgaris at 7Klux light intensity was very strong, reaching a maximum density of 19.68x106 (19.68±0.01) cells/mL on day of the experiment, and decreasing to 15.22x106 (15.22±0.01) cells/mL on day 14 While algae cultured at 13 Klux light intensity showed the lowest results, the maximum density was only 14.94x106 (14.94±0.02) cells/mL on day and decreased to 11.89x106 (11.89±0.026) cells/mL on day 14 The maximum density of Klux light intensity was 17.88x106 (17.88±0.017) cells/mL on day 9, and decreased to 12.98x106 (12.98±0.01) cells/mL on day 14 At Klux light intensity, algae grow best, the amount of biomass obtained is also the most, so we use it in the next experiments 4.4 Investigation of optimalnitrogen concentration on the growth of Chlorella vulgaris The concentration of nitrogen added to the culture medium greatly affects the time on biomass as well as the growth and development of algae Nitrogen deficiency also increases lipid accumulation from algae and changes color The experiment was performed by formulations with different nitrogen concentration: 50mg/L NaNO3, 60mg/L NaNO3, 70mg/L NaNO3 and 80mg/L NaNO3 The results are shown in the chart below: 35 Table 4.4 Cell density of Chlorella vulgaris in different nitrogen concentration Day Cell density in diferent 𝑵𝒂𝑵𝑶𝟑concentration (mg/L) (x𝟏𝟎𝟔 cells/mL) 50 2.56 60 2.58 70 2.72 80 2.47 2.83 2.88 3.50 2.81 3.76 4.68 3.61 4.23 4.26 4.68 3.05 3.86 5.99 7.23 5.15 6.12 5.01 5.83 4.23 5.64 9.18 15.42 8.23 11.56 7.64 10.68 7.62 10.48 16.34 15.34 14.83 13.65 10 11 16.86 15.51 15.84 14.64 14.34 14.11 13.98 14.62 12 13 13.34 12.56 13.16 12.24 13.86 13.11 14.23 13.68 14 11.63 11.20 12.65 12.95 50mg/L 60mg/L 70mg/L 80mg/L 18 16 14 12 10 2 10 11 12 13 14 Fig 4.7 Growth rate of Chlorella vulgaris in different nitrogen concentration 36 Specific growth rate (generation/day) 50 18 60 70 80 16 14 12 10 2 Time(day) 10 11 Fig 4.8 Specific growth rate of Chlorella vulgaris in different nitrogen concetration In days of the experiment, the difference in algae density was not significant Day 8, the density of algae tends to increase rapidly At this time, the algae density changed to the growth phase, the increasing trend of algae density in experimental plots began to depend on the light intensity On day 8, the density of algae at concentration of 50mg/L NaNO3 was 15.42x106 (15.42±0.01) cells/ml, at concentration of 60 mg/L NaNO3 reached 11.56x106 (11.56±0.01) cells/ml, at concentration of 70 mg/L NaNO3 reached 10.68x106 (10.68±0.01) cells/ml, at concentration of 80 mg/L NaNO3 reached 10.48x106 (10.48±0.01) cells/ml The graph show that from day 8-10, most of the algae density is high At concentration of 50mg/L NaNO3, the highest maximum density is 16.86x106 (16.86±0.01) cells/mL on day 10, and decreased to 11.63x106 (11.63±0.036) cells/mL on day 14 The lowest maximum density is obtained at 80mg/L NaNO3 with a maximum density is 14.62x106 (14.62±0.2) cells/mL on day 11 and declining gradually to 12.95x106 (12.95±0.29) cells/mL on day 14 At concentration of 60mg/L NaNO3, the maximum density is 15.84x106 37 (15.84±0.017) cells/mL on day 10, and decreased to 11.20x106 (11.20±0.026) cells/mL on day 14 At concentration ofmg/L NaNO3, the maximum density is 14.83x106 (14.83±0.165) cells/mL on day 9, and decreased to 12.65x106 (12.65±0.16) cells/mL on day 14 Therefore, the best nitrogen concentration for growth of Chlorella vulgaris is 50mg/L NaNO3 4.5 Ultrasonication method for cell wall disruption Based on the result of experriment, cultivation biomass of Chlorella vulgaris in BBM media, 7Klux light intensity, 50 mg/L NaNO3 nitrogen concentration, light cycle in 16h a day to Chlorella vulgaris entrifugured reach 20 liters of the meidum at 3500rpm for minutes to recover the biomass.Ultrasonication method was used for disruption 2g of dry Chlorella vulgaris was dispersed in 100 ml of distilled water Time processing of ultrasonication method is 60 minutes with different power is 360W, 720W and 1080W The results are shown in the chart below: Table 4.5 Chlorella vulgaris cell disruption by ultrasonication in different power Proportion of cell disruption in different Processing ultrasonic power (% ) time (min) 360W 720W 1080W 6.80 17.5 23.6 10 18.3 22.4 41.5 30 17.7 36.8 59.4 60 19.5 43.6 83.8 38 360W Cell disruptin (%) 90 720W 1080W 80 70 60 50 40 30 20 10 10 30 Processing time (min) 60 Fig 4.9 Cell disruption by ultrasonication of Chlorella vulgaris in different power As depicted in Figure 4.9, the disruption rate increased as the processing time and ultrasonic power increasing with a distinct tendency By the time, the ultrasonic power of 360W obtained 6.8% cell disruption in min, 18.3% cell disruption in 10 min, 17,7% cell disruption in 30 and 19.5% highest cell disruption in 60 The ultrasonic power of 720W obtained 17.5% cell disruption in min, 22.4 cell disruption in 10 min, 36.8 cell disruption in 30 and 43.6 highest cell disruption in 60 The highest Chlorella vulgaris cell disruption proportion was obtained at the ultrasonic power of 1080W: 23.6% in min, 41.5% in 10 min, 59.4% in 30 and 83.8% in 60 Based on the result, ultrasonication method for disruption cell wall worked best at the processing time of 60 and 39 the ultrasonic power of 1080W PART 5: CONCLUSION AND RECOMMENDATION 5.1 Conclusion • Chlorella vulgaris is suitable for culture under BBM medium • The optimla light intensity of Chlorella vulgaris is 7Klux • The optimal light cycle of Chlorella vulgaris is 16 hours a day • The optimal nitrogen concentration Chlorella vulgaris is 50 mg/L 𝐍𝐚𝐍𝐎𝟑 • The ultrasonication method for disruption Chlorella vulgaris cell wall worked best at the processing time of 60 and the ultrasonic power of 1080W 5.2 Recommendation • Research to optimize growing conditions to maximize lipid-collecting yield • Research on building 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