Cell density of Chlorella vulgaris in different light cycle .... Cell density of Chlorella vulgaris in different light intensity .... Growth rate of Chlorella vulgaris in different light
INTRODUCTION
Problem statement
Chlorella vulgaris microalgae are highly nutritious and serve as food supplements, while also being utilized in various industries such as pharmaceuticals, cosmetics, and aquaculture They are rich in proteins, carbohydrates, pigments, vitamins, and minerals Additionally, these microalgae play a crucial role in the environment by capturing sunlight and performing photosynthesis, contributing to approximately half of the Earth's atmospheric oxygen and absorbing significant amounts of carbon dioxide The growing emphasis on health and the increasing use of food additives are driving the rising demand for algal products among consumers.
Chlorella vulgaris has gained global popularity, with Japan leading in its consumption and medical applications due to its immune-modulating and anti-cancer properties In Vietnam, however, the production and consumption of Chlorella vulgaris remain limited due to minimal research and high costs To address this issue, the thesis titled “Study on the cultivation of Chlorella vulgaris and method for cell wall disruption” aims to identify the optimal growth conditions for Chlorella vulgaris.
Purpose and requirement
Study on cultivation of Chlorella vulgaris and method for cell wall disruption
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
LITERATURE REVIEW
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
Chlorella, derived from the Greek word "chloros" meaning green, and the Latin suffix "ella" indicating its microscopic size, is a member of the Chlorellaceae family.
Fig 2.1 Classification of Chlorella vulgaris
Chlorella vulgaris, a unicellular microalgae found in fresh water, boasts the highest chlorophyll content of any known photosynthetic plant at 28.9g/kg This remarkable organism has existed on Earth for approximately 2.5 billion years, maintaining its genetic integrity since the pre-Cambrian period.
Chlorella vulgaris is a unicellular eukaryote comparable in size to human red blood cells It thrives under optimal conditions, which include abundant light, clear water, and clean air.
Chlorella vulgaris can reproduces at a tremendous speed
Morphology and biology characteristics of Chlorella vulgaris
Chlorella vulgaris is a unicellular algae characterized by its spherical or oval cells, which typically range in size from 2 to 5 µm, depending on environmental conditions and developmental stages The cell membranes are composed of cellulose, providing resistance to light mechanical stress Environmental factors such as light, temperature, and chemical composition significantly influence the morphology and quality of these algae cells.
The cell wall plays a crucial role in maintaining cell integrity and providing protection against environmental threats and invaders Its structure varies throughout different growth phases Initially, during the formation of autosporangia, the newly created cell wall is delicate, consisting of a 2 nm thin electron-dense unilaminar layer As the daughter cell matures, the thickness of the cell wall gradually increases, ultimately reaching 17 nm.
At 21 nm post-maturation, a microfibrillar layer emerges, resembling a chitosan-like structure made of glucosamine, contributing to its rigidity In the mature stage, the thickness and composition of the cell wall vary, influenced by diverse growth and environmental conditions.
Chlorella vulgaris features a unique chloroplast with a double membrane structure made of phospholipids; the outer membrane allows the passage of metabolites and ions, while the inner membrane specializes in protein transport Under unfavorable growth conditions, starch granules composed of amylose and amylopectin can form within the chloroplast The pyrenoid, rich in ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), plays a crucial role in carbon dioxide fixation Additionally, the chloroplast contains a cluster of fused thylakoids where chlorophyll, the primary pigment, is synthesized, often overshadowing the colors of other pigments.
5 lutein During nitrogen stress, lipid globules mainly accumulate in the cytoplasm and the chloroplast [Van den Hoek C, Mann D, Jahns H 1995]
Chlorella vulgaris is a non-motile alga that reproduces asexually through a process called autosporulation, allowing one cell to multiply rapidly within 24 hours under optimal conditions This species does not engage in sexual reproduction, making autosporulation its primary method of reproduction.
Fig 2.2 The different phases of daughter cell-wall formation in Chlorella vulgaris
(a) Early cell-growth phase; (b) Late cell-growth phase; (c) Chloroplast dividing phase; (d) Early protoplast dividing phase; (e) Late protoplast dividing phase; (f) Daughter cells maturation phase; (g) Hatching phase [Yamamoto M, Kurihara I, Kawano S 2005]
The reproductive process is carried out by creating spores in the mother cell Depending on the environmental conditions, the number of spores can be 2,
Chlorella vulgaris can produce multiple spores, ranging from 4 to 64, within the cell wall of the mother cell Once division is complete, the spores break free by damaging the mother cell's wall, utilizing the remaining debris as nourishment These newly formed daughter cells then grow and enter the asexual reproduction stage, continuing the cycle The lifespan of Chlorella vulgaris is influenced by factors such as light intensity, temperature, and nutrient availability.
Factors affecting the growth and development of Chlorella vulgaris
Light significantly impacts microalgae growth, serving as the primary energy source for photosynthesis The effects of light are determined by its quality, intensity, and duration Both excessive and insufficient light intensity can hinder algal photosynthesis While laboratory settings allow for controlled light intensity using neon lights, outdoor cultivation often requires shading due to intense sunlight Some algae do not thrive under constant light, as continuous illumination can decrease yields and reduce protein, carbohydrate, and unsaturated fatty acid content Providing adequate light is crucial, as algae have a higher light energy coefficient than higher plants, enabling them to convert inorganic carbon into organic carbon However, high light intensity and prolonged exposure can lead to photo-inhibition, potentially harming algal cultures Blue light enhances protein content, while red light boosts carbohydrate levels, with the optimal light conditions varying by species and stocking density.
Temperature significantly influences the growth of all organisms, particularly microalgae It impacts various factors such as cell structure, metabolic reaction rates, photosynthesis, distribution density, respiration intensity, and cell size Microalgae thrive within a temperature range of 16-30ºC, but exposure to temperatures above 35ºC or below 16ºC can hinder their development and potentially lead to the death of certain species if sustained The optimal temperature for microalgae growth is between 20-25ºC, although this can vary based on environmental conditions and species.
Chlorella vulgaris thrives at temperatures between 25-30ºC, where optimal nutritional conditions, light intensity, and stirring promote rapid growth; however, it cannot survive below 15ºC or above 35ºC In aquaculture, temperature significantly impacts the yield and quality of microalgae Given Vietnam's tropical monsoon climate, which experiences winter temperatures as low as 5-7ºC and summer highs of 38ºC, developing purebred varieties with indigenous origins is a highly effective strategy for ensuring stable and long-term production.
Microalgae species exhibit remarkable adaptability to varying salinity levels, with most demonstrating high tolerance to salinity changes Notably, Chlorella vulgaris thrives in saline environments, capable of growing in salinities ranging from 5 to 30 ‰, although it achieves optimal growth under specific conditions.
Salinity levels between 25-30‰ significantly impact osmotic pressure, limiting photosynthesis, respiration, and growth rates while reducing glycogen accumulation Research indicates that varying salinity concentrations affect the lipid content in microalgae cultivation Specifically, Chlorella vulgaris exhibited increased lipid content of 4.60%, 1.84%, and 3.09% at NaCl concentrations of 0.023, 0.05, and 0.075 M, respectively Notably, the effluent from the frozen seafood industry, containing 0.023 M of NaCl, resulted in optimal growth conditions for Chlorella vulgaris, yielding a lipid content of 4.60%.
The effluent from the frozen seafood industry is conducive to the growth of microalgae, particularly Chlorella vulgaris, due to its adequate salinity levels Increased salinity has been shown to enhance the lipid content in the Chlorella vulgaris strain, although the enhancement varies only slightly across different salinity concentrations.
pH is a crucial environmental parameter that influences the solubility of \$CO_2\$ and mineral salts, impacting algal metabolism Most algae species thrive within specific pH ranges, and maintaining pH control is essential for optimal growth Research indicates that cell density increases with higher pH levels, peaking at a pH of 10 The culture broth's pH, when controlled at 10, fluctuates between 10 and 10.5, primarily due to daily adjustments that prevent excessive pH increases This suggests that light intensity does not significantly affect the optimal pH for algal growth, with the range of 10 to 10.5 being ideal for Chlorella vulgaris.
Nutritional ingredients of Chlorella vulgaris
Chlorella vulgaris is rich in amino acids, complex carbohydrates, vitamins, minerals, fat (85% unsaturated fatty acids), RNA (over 10%), DNA (over 3%), chlorophyll, carotenoids, enzymes and polysaccharides (Table 2.3) [Katharina J
Table 2.1 Nutritional components of Chlorella vulgaris
Chlorella vulgaris boasts the highest chlorophyll content among all green algae and plants, with approximately 7% of its total weight comprised of this vital pigment This concentration is 5 to 10 times greater than that found in other sources.
Spirulina, and 10 times more than alfalfa Due to its high level of Chlorophyll,
Chlorella vulgaris is considered a perfect food It provides a full range of essential amino acids and non-essential amino acids for humans Protein in
Chlorella vulgaris offers superior protein benefits compared to meat, as it supplies easily digestible amino acids essential for various bodily functions Additionally, it boasts a higher protein content than traditional food sources.
Table 2.2 Compare the protein content of Chlorella vulgaris with other foods Other foods Quantity (mg)
Chlorella vulgaris is a nutrient-dense superfood, boasting high concentrations of essential vitamins It contains twice the amount of folic acid and more vitamin B12 than beef Additionally, this naturally derived food is rich in vitamin B3 and vitamin A, contributing to its ability to prevent oxidative free radical formation, thereby protecting the body against cancer and slowing the aging process Notably, Chlorella vulgaris ranks among the highest sources of chlorophyll and β-carotene globally.
Table 2.3 Vitamins were found in Chlorella vulgaris
Vitamin Quantity (mg) α-carotene 24 β-carotene 86
Organic minerals are easily absorbed by the human body and are rich in essential nutrients like potassium, calcium, magnesium, and iron, which support cardiovascular health, blood formation, and circulation Additionally, they provide valuable substances such as fatty acids, lutein, and xanthophyll.
Table 2.4 Fatty acids were found in Chlorella vulgaris
Functional of active ingredients in Chlorella vulgaris
Chlorophyll, a natural substance, is safe for long-term use without side effects or drug interactions It effectively eliminates body odors and neutralizes the scents of bodily secretions, providing an internal and systemic deodorizing effect.
Chlorophyll is effective in inhibiting the growth of anaerobic bacteria found in the digestive tract and oral cavity, thanks to the oxygen it generates It is commonly used in dentistry for anti-caries treatment and to prevent infections before and after dental procedures Additionally, chlorophyll is beneficial for gastrointestinal issues such as bloating, inflammation, vomiting, and infections, as well as conditions like purulent colitis, constipation, and prolonged diarrhea It also enhances liver detoxification and has therapeutic effects on cancer, acting as an antioxidant and forming stable mixtures with certain carcinogens to prevent their absorption into the bloodstream.
12 longer harmful to the body [Gouveia L, Raymundo A, Batista AP, Sousa I, Empis J, 2005] b Function of Omega 3
Omega 3 are unsaturated fatty acids, they include 3 main types: EPA, DHA, DPA DHA helps to slow down and prevent the aging process of the brain, the older people are, the lower DHA content in the brain For adolescents in adulthood, DHA helps maintain health, the normal growth of nerve cells, and memory enhances Especially those who are stressed, high work pressure, and intellectual workers, DHA is considered as a very good nutrition for the brain For children, DHA helps in nerve and vision development DPA is an essential substance for the complete development of the fetus and nerve cells of the fetus DPA plays a very important role throughout the baby's prenatal and postnatal development EPA help to create Prostaglandin in the blood This Prostaglandin works to inhibit platelet clumping, it reduce and prevent the formation of blood clots, it reduce cholesterol, it reduce blood triglycerides, it reduce blood viscosity, and it keep circulation, ventilation EPA also works to reduce atherosclerosis Therefore, EPA is effective for the prevention and treatment of cardiovascular diseases caused by atherosclerosis [Seyfabadi J, Ramezanpour
Z, Amini Khoeyi Z, 2011] c Function of β-carotene β-carotene is a precursor to vitamin A, and it is a rich natural source of vitamin A for the body Vitamin A plays a role in visual ability and children's development, so it also works to brighten the eyes and looks more refined, good for kids and the elder It also has the function of strengthening the immune system so it is good for new person recovered from illness Besides, β-carotene also possesses a preeminent antioxidant ability because it works to eliminate excess free radicals in the body This free radicals seriously damage cell membranes, it damages organelles, it is closely related to the aging process,
Beta carotene plays a crucial role in preserving beauty and youth while also serving as a protective agent against various diseases, including cancer, which often lack official treatments By safeguarding cell membranes and slowing the aging process, beta carotene can help mitigate the deterioration associated with aging.
Carbohydrates represent a group of reducing sugars and polysaccharides such as starch and cellulose Starch is the most abundant polysaccharide in
Chlorella vulgaris contains starch, primarily found in chloroplasts, composed of amylose, amylopectin, and sugars that serve as energy storage for the cells Additionally, cellulose, a structural polysaccharide located in the cell wall, acts as a protective fibrous barrier Notably, β1 to 3 glucan is another significant polysaccharide in Chlorella vulgaris, offering various health and nutritional benefits.
Total carbohydrates are typically measured using the sulphuric phenol method, which produces simple sugars through hydrolysis at 110 °C, followed by quantification via HPLC, particularly HPIC The enzymatic method is more effective for starch quantification than the acidic method Under nitrogen-limiting conditions, total carbohydrates can constitute 12-55% of dry weight.
Chlorella vulgaris features a strong cell wall primarily made up of a chitosan-like layer, cellulose, hemicellulose, proteins, lipids, and minerals Its sugar composition includes a blend of rhamnose, galactose, glucose, xylose, arabinose, and mannose, with rhamnose being the predominant sugar.
Vitamin E join the metabolism of cells, it protect vitamin A and fatty acids from oxidation, erythropoiesis, prevent cell damage, help the body use vitamin
K and prevent atherosclerosis by reducing oxidation of fat-soluble proteins, thereby preventing these proteins from participating in the process of clogging of the arteries Vitamin E prevents cardiovascular diseases, including
Vitamin E plays a crucial role in reducing the aggregation of low-density lipoprotein (LDL) cholesterol in blood vessels, which can help prevent myocardial infarction and cerebral vascular accidents It enhances immunity by protecting cells from damage, thereby increasing resistance to bacterial infections Additionally, its antioxidant properties lower the risk of cataracts Vitamin E is absorbed in the small intestine alongside vitamins A, D, and K, with about 35% entering the lymphatic system while the remainder is excreted It circulates in the bloodstream and is primarily stored in the liver and adipose tissue, with smaller amounts in the pituitary, adrenal glands, testicles, and uterus Notably, Vitamin E does not cause toxicity even in large doses.
2.6 Application of Chlorella vulgaris in functional foods
Chlorella vulgaris, a functional food widely embraced in Japan, is renowned for its comprehensive nutrient profile that supports human health This green algae not only provides essential nutrients but also offers various therapeutic benefits, enhancing the body's healing processes, promoting balance, and revitalizing overall well-being.
Chlorella vulgaris is a rich source of essential nutrients often missing from the human diet Its natural vitamins and minerals, along with amino acids, enhance the body's ability to absorb these nutrients and support metabolic functions.
Chlorella vulgaris, a functional food rich in chlorophyll, offers numerous health benefits, including intestinal and blood cleansing, as well as enhancing liver and kidney function This superfood boosts blood filtration, strengthens the immune system by stimulating lymphocytes and macrophages, and increases interferon levels, aiding in the body's defense against colds and flu Additionally, Chlorella vulgaris is packed with nucleosides and nucleotides that support brain development, making it a valuable addition to a healthy diet.
Chlorella vulgaris is effective in excreting toxins like cadmium, a heavy metal found in tobacco Scientific studies indicate that the vitamin A and β-carotene in Chlorella vulgaris enhance immune function and contribute to cancer prevention and treatment Additionally, β-carotene and vitamin E play a role in reducing early-stage cancer risk Diets high in sodium and low in potassium are linked to increased blood pressure, while those rich in potassium and low in sodium can help lower it To reduce sodium intake, it's advisable to avoid ready-to-eat foods and table salt, opting instead for potassium-rich foods like Chlorella vulgaris.
Chlorella vulgaris is a nutrient-dense food source, renowned for its high concentration of nucleic acids and rich antioxidant content, including carotenoids, vitamins C and E, chlorophyll, and selenium These antioxidants play a crucial role in preventing premature aging by neutralizing free radicals in the body Additionally, Chlorella vulgaris aids in weight management by supplying essential nutrients and reducing cravings, ultimately promoting a balanced endocrine system and enhancing weight loss efficiency.
Chlorella vulgaris is known to be a good source of Lutein, a group of
Carotenoids play a crucial role in preventing and treating macular degeneration Additionally, studies show that 80% of diabetics experience low magnesium levels, and increasing magnesium intake through Chlorella vulgaris can help regulate blood sugar levels.
RESEARCH METHODOLOGY
Research subjects
Determination of the optimal culture media for the growth of Chlorella vulgaris
Determination of the optimal conditions for the growth of Chlorella vulgaris
Method for cell wall disruption.
Study time and place
The thesis was done at the laboratory of Department of Biotechnology Vietnam National University of Agriculture Thesis was occur from August
Chemicals
BBM, or Bold's Basal Medium, is a specialized freshwater algae medium designed for cultivating various green algal species, including Trichosarcina, Chlorococcum, and Chlorella, without requiring soil extracts or vitamins.
EDTA solution see following recipe 1.0
Acidified iron solution see following recipe 1.0
Trace metals solution see following recipe 1.0
F/2 media is a common and widely used general enriched seawater medium designed for growing coastal marine algae, especially diatoms [Guillard and Ryther 1962]
Trace Metal Solution see following recipe 1
Vitamin Solution see following recipe 0.5
Walne media is the culture media conventionally used by various laboratories for culture of most algae, phytotlagellates, diatoms [Gopinathan
Trace metals solution see following recipe 1
Vitamin solution see following recipe 1
Research equipments
Laboratory equipment: PH meter, OD meter, electronic scale, sterile cabinet, autoclave, air conditioner, refrigerator, water distillation machine, clamp, implant knife, bottle, test tube, Petri dish,
Research methods
3.5.1 Determination of the cell density by Neubauer chamber
The Neubauer chamber is a thick crystal slide measuring 30x70 mm with a thickness of 4 mm, designed for cell counting It consists of a central part featuring a counting grid and two independent counting areas The counting grid measures 3mmx3mm and is divided into 9 square subdivisions, each 1mm wide Each of these squares is further divided into 25 smaller squares, each 0.2 mm (200 µm) in width, resulting in a total of 400 tiny squares within the central square.
Fig 3.1 Neubauer counting chamber Step 1: Sample preparation
Depending on the type of sample, a preparation of a dilution with a suitable concentration should be prepared for cell counting
Step 2: Introducing the sample into the Neubauer chamber
The micropipette was calibrated to draw 10 µL of the dilution prepared in step 1 A glass cover was placed over the central area of the Neubauer chamber, which was positioned on a flat surface like a table or workbench The micropipette tip was immersed in the dilution, and the plunger was pressed slowly until fully depressed Afterward, the pipette tip was removed from the dilution and brought to the Neubauer chamber for further analysis.
To ensure accurate loading of the pipette, always hold it vertically and position the pipette tip near the center of the glass cover edge of the Neubauer chamber Gradually release the plunger to allow the liquid to enter the chamber uniformly through capillarity If bubbles form or the glass cover shifts, repeat the loading process.
Step 3: Microscope set up and focus
To count cells using a Neubauer chamber, place it on the microscope stage and turn on the light Adjust the focus until the cells are clearly visible through the eyepiece Begin counting in the first grid square and record the number of cells observed Continue this process for all remaining squares, documenting the results for each.
To calculate the concentration of cells in a Neubauer chamber, use the formula: Concentration (cell/ml) = Number of cells / Volume (ml) The total number of cells is obtained by summing all counted cells across the designated squares Each large square has an area of 0.01 cm², and with a chamber depth of 0.1 mm, the volume is calculated as 0.0001 ml or 0.1 µl Therefore, the concentration formula for counting in the large squares is: Concentration = (Number of cells × 10,000) / Number of squares.
3.5.2 Specific growth rate of Chlorella vulgaris
The specific growth rate measures the increase in biomass of a cell population relative to its biomass concentration This growth rate is observed in the sigmoid curve during the transition between the lag and stationary phases, where cell growth can be represented by a linear equation.
𝐭 Where 𝛍 is the specific growth rate, A is the biomass at time t, and B is the initial biomass
Chlorella vulgaris is preserved at the Faculty of Biotechnology, Vietnam National University of Agriculture Prior to culturing, glass flasks and media are autoclaved at 121°C and 2 atm for 15 minutes to ensure sterility The transport and distribution of algae seeds are conducted in a highly sterile environment Algae are cultivated in flasks of varying sizes, including 500 mL, 1 L, 10 L, and 20 L, under continuous illumination of 24 hours with a light intensity ranging from 3000 to 4000 lux, while being aerated throughout the culture period.
Chlorella vulgaris can be preserved using two methods: storing algae seeds in agar plates, which maintains seed quality for 4-5 months, and keeping them in liquid medium, requiring transfers every two weeks While the liquid medium method allows for quicker seed availability, it leads to a gradual decline in seed quality after 8 to 9 transplants Algae harvesting occurs approximately two weeks into the culture when biomass growth stabilizes.
Fig 3.2 Preservation of Chlorella vulgaris
Liquid media (left) and Agar plate (right)
3.5.4 Factors affecting the growth of Chlorella vulgaris
3.5.4.1 Effects of culture media on the growth of Chlorella vulgaris
The experiment involved three formulations using different culture media: BBM, F/2, and Walne Chlorella vulgaris was cultured under continuous light and aeration at laboratory temperature, with daily cell counts monitored over a 14-day period.
The optimal culture media was determined based on the highest cell density observed in the experiment Algae biomass growth was quantified using a Neubauer chamber, and the findings were illustrated in a graph These results will inform the design of subsequent experiments.
3.5.4.2 Effects of light cycle on the growth of Chlorella vulgaris
The experiment involved three formulations of Chlorella vulgaris cultured under continuous lighting and aeration for 14 days, with varying light cycle times of 12 hours, 24 hours, and 16 hours Daily cell counts were monitored at laboratory temperature throughout the study.
The optimal light cycle was determined from the experiment that yielded the highest cell density Algae biomass growth was quantified using a Neubauer chamber, and the results were illustrated in a graph These findings will inform the design of subsequent experiments.
3.5.4.3 Effects of light intensity on the growth of Chlorella vulgaris
The experiment involved three formulations of Chlorella vulgaris cultured under continuous lighting and aeration at laboratory temperature The light intensities tested were 4 Klux, 7 Klux, and 13 Klux, with cell counts monitored daily over a 14-day period.
The optimal light intensity was determined based on the highest cell density observed in the experiment Algae biomass growth was quantified using a Neubauer chamber, and the findings were illustrated in a graph These results will inform the design of subsequent experiments.
3.5.4.4 Effects of nitrogen concentration on the growth of Chlorella vulgaris
The experiment involved four formulations with varying concentrations of NaNO₃: 50 mg/L, 60 mg/L, 70 mg/L, and 80 mg/L To enhance nitrogen concentration, the NaNO₃ concentration was increased accordingly Utilizing data from previous experiments, the cultures were monitored daily for 14 days to track cell counts.
The optimal nitrogen concentration based on the result with the largest cell density of experiment Growth of algae biomass was measured by using
Neubauer chamber and the results were plloted in graph The result of this experiment will be used for the next experiment.
Method for cell wall disruption
Breaking the cell wall of Chlorella vulgaris presents a significant challenge and incurs high costs Various techniques, including high-pressure homogenization, autoclaving, enzymatic lysis, bead milling, and grinding, have been employed for this purpose The choice of cell disruption method is crucial, as it can affect the quality of the target components, necessitating careful selection based on the intended application.
This research investigates the effectiveness of cell disruption in Chlorella vulgaris using ultrasonication methods Ultrasonication is a widely utilized laboratory technique known for its short processing time, high efficiency, and ease of operation, making it ideal for enhancing the extraction of oils, proteins, and other metabolites The study involved dispersing 2g of dry Chlorella vulgaris cells in 100 mL of distilled water and applying ultrasonic treatment for 60 minutes at three different power levels: 360W, 720W, and 1080W The proportion of cell disruption was measured at intervals of 5, 10, 30, and 60 minutes across the varying ultrasonic power settings.
Data analys is
Each experiment is independent, and repeated three times Data results are analysis mean value with standard deviation 𝒙̅± σ This is the formular of standard deviation:
The standard deviation, denoted as σ, measures the dispersion of a data set, where 𝑥1 represents the value of the ith observation, 𝜇 is the average of the data set, and n indicates the total number of observations The differences between the formulas are analyzed using a t-test, and statistical analyses along with data visualizations were conducted using Microsoft Excel.
RESULT AND DISCUSSION
Investigation of optimal culture media on the growth of Chlorella vulgaris
Algae exhibit varying growth rates depending on the culture media used In a study, Chlorella vulgaris was cultivated in three distinct media: Walne media, F/2 media, and BBM, with the results illustrated in the accompanying chart.
Table 4.1 Cell density of Chlorella vulgaris in different media
Day Cell density in different culture media
Fig 4.1 Growth rate of Chlorella vulgaris in different media
Fig 4.2 Specific growth rate of Chlorella vulgaris in diferent media
Research indicates that various culture media influence algal growth According to Figure 4.1 and Table 4.1, the acclimation phase in Walne, F/2, and BBM media lasted approximately 8 days After this period, no significant differences in algal growth and development were observed among the three experimental culture media On day 8, the algal density remained consistent across all media types.
S pe ci fic gr ow th ra te ( ge ne ra tio n/ da y)
30 in Walne was 11.2x10 6 (11.2±0.01) cells/ml, F/2 reached to 11.54x10 6 (11.54±0.01) cells/ml, BBM reached to 13.47x10 6 (13.47±0.01) cells/ml
Biomass of Chlorella vulgaris algae in different media peaked from days
The BBM medium exhibited the highest maximum density of 17.84x10^6 (17.84±0.01) cells/mL, surpassing the F/2 medium at 15.47x10^6 (15.47±0.01) cells/mL and the Walne medium at 14.55x10^6 (14.55±0.01) cells/mL However, from day 11 onwards, the concentration of algae began to decline, with the BBM medium decreasing to 15.35x10^6 (15.35±0.01) cells/mL On day 14, the Walne medium dropped to 12.97x10^6 (12.97±0.01) cells/mL, while the F/2 medium decreased to 12.56x10^6 (12.56±0.01) cells/mL Consequently, the BBM medium was selected for subsequent experiments.
Investigation of optimal light cycle on the growth of Chlorella vulgaris
The light and dark cycle significantly influences photosynthesis, nutrient synthesis, and cellular metabolism Variations in lighting conditions lead to differing levels of algal biomass growth The experiment utilized three formulations to assess these effects.
3 different lighting cycle times in a day: 12h, 24h and 16h The results are shown in the chart below:
Table 4.2 Cell density of Chlorella vulgaris in different light cycle
Day Cell density in different light cycle (hours/24h)
Fig 4.3 Growth rate of Chlorella vulgaris in different light cycle
Fig4.4 Specific growth rate of Chlorella vulgaris in different light cycle
In the initial four days, algae density increases gradually; however, by day five, it experiences a rapid surge as it transitions into the growth phase The rate of increase in algae density becomes influenced by the light cycle On day five, the algae density recorded at a 12/24 light cycle was 4.56x10^6 (4.56±0.01) cells/ml, while at a 16/24 cycle, it reached 5.68x10^6 (5.68±0.01) cells/ml, and at a 24/24 cycle, it peaked at 5.7x10^6 (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 12.43x10 6 (12.43±0.01) cells/mL on day 9 of the experiment, then the density decreased to 9.56x10 6 (9.56±0.01) cells/mL on day
14 At cycle 24/00, the algae density reached a maximum of 15.96x10 6 (15.96±0.017) cells/mL on day 9 of the experiment, then the density decreased to 12.75x10 6 (12.75±0.017) cells/mL on day 14 At cycle 16/8, the algae density reached a maximum of 17.86x10 6 (17.86±0.01) cells/mL on day 9 of the experiment, then the density decreased to 14.37x10 6 (14.37±0.02) cells/mL on day 14 Therefore, the most suitable lighting cycle for Chlorella vulgaris is 16 hours a day
S pe ci fic gr ow th ra te ( ge ne ra tio n/ da y)
Investigation of optimal light intensity on the growth of Chlorella vulgaris
Light significantly impacts the growth and development of microalgae, serving as the primary energy source for their photosynthesis An experiment was conducted using three formulations under varying light intensities of 4 Klux, 7 Klux, and 13 Klux The results, illustrating the effect of light intensity on algae growth, are presented in the chart below.
Table 4.3 Cell density of Chlorella vulgaris in different light intensity
Day Cell density in different light intensity (Klux)
Fig 4.5 Growth rate of Chlorella vulgaris in different light intensity
Fig 4.6 Specific growth rate of Chlorella vulgaris in different light intensity
The experimental results indicate that Chlorella vulgaris exhibits rapid growth, with algae density peaking around day 9 of cultivation Initially, the density increases slowly during the first 4 days, but by day 5, a significant acceleration in growth is observed, marking the transition to the growth phase.
S pe ci fic gr ow th ra te ( ge ne ra tio n/ da y)
The density of algae in experimental plots was found to be influenced by light intensity On day 5, at a light intensity of 4 Klux, the algae density measured 6.91 x 10^6 (6.91±0.01) cells/ml At 7 Klux, the density increased to 7.25 x 10^6 (7.25±0.01) cells/ml, while at 13 Klux, it decreased to 5.78 x 10^6 (5.78±0.01) cells/ml.
Chlorella vulgaris exhibited robust growth at a light intensity of 7 Klux, achieving a peak density of 19.68 million cells/mL on day 9, which subsequently declined to 15.22 million cells/mL by day 14 In contrast, algae grown under 13 Klux light intensity demonstrated the lowest growth, with a maximum density of only 14.94 million cells/mL on day 9, decreasing to 11.89 million cells/mL on day 14 Additionally, the maximum density recorded at 4 Klux light intensity was 17.88 million cells/mL on day 9.
On day 14, the cell concentration decreased to 12.98 x 10\(^6\) (12.98±0.01) cells/mL The optimal light intensity for algae growth was found to be 7 Klux, resulting in the highest biomass yield, which was utilized in subsequent experiments.
Investigation of optimalnitrogen concentration on the growth of Chlorella
The concentration of nitrogen in the culture medium significantly influences biomass production and the growth of algae A deficiency in nitrogen not only enhances lipid accumulation in algae but also alters their color The experiment utilized four formulations with varying nitrogen concentrations: 50 mg/L, 60 mg/L, 70 mg/L, and 80 mg/L of NaNO₃ The results are illustrated in the chart below.
Table 4.4 Cell density of Chlorella vulgaris in different nitrogen concentration
Day Cell density in diferent 𝑵𝒂𝑵𝑶 𝟑 concentration (mg/L)
Fig 4.7 Growth rate of Chlorella vulgaris in different nitrogen concentration
Fig 4.8 Specific growth rate of Chlorella vulgaris in different nitrogen concetration
In 7 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 NaNO 3 was 15.42x10 6 (15.42±0.01) cells/ml, at concentration of 60 mg/L NaNO 3 reached 11.56x10 6 (11.56±0.01) cells/ml, at concentration of 70 mg/L NaNO 3 reached 10.68x10 6 (10.68±0.01) cells/ml, at concentration of 80 mg/L NaNO 3 reached 10.48x10 6 (10.48±0.01) cells/ml
The data indicates that between days 8 and 10, algae density peaks significantly At a concentration of 50 mg/L NaNO₃, the maximum density reaches 16.86 x 10⁶ (16.86±0.01) cells/mL on day 10, subsequently declining to 11.63 x 10⁶ (11.63±0.036) cells/mL by day 14 In contrast, the lowest maximum density occurs at 80 mg/L NaNO₃, where it peaks at 14.62 x 10⁶ (14.62±0.2) cells/mL on day 11, followed by a gradual decrease to 12.95 x 10⁶ (12.95±0.29) cells/mL on day 14 Additionally, at a concentration of 60 mg/L NaNO₃, the maximum density recorded is 15.84 x 10⁶ cells/mL.
S pe ci fic gr ow th ra te ( ge ne ra tio n/ da y)
On day 10, the cell density of Chlorella vulgaris was measured at (15.84±0.017) cells/mL, which decreased to 11.20x10^6 (11.20±0.026) cells/mL by day 14 At a concentration of 50 mg/L NaNO₃, the maximum cell density reached 14.83x10^6 (14.83±0.165) cells/mL on day 9, followed by a decline to 12.65x10^6 (12.65±0.16) cells/mL on day 14 Thus, the optimal nitrogen concentration for the growth of Chlorella vulgaris is determined to be 50 mg/L NaNO₃.
Ultrasonication method for cell wall disruption
The experiment demonstrated that cultivating Chlorella vulgaris in BBM media under a light intensity of 7 Klux, with a nitrogen concentration of 50 mg/L NaNO3 and a light cycle of 16 hours per day, successfully produced 20 liters of biomass.
Chlorella vulgaris was centrifuged at 3500 rpm for 5 minutes to recover the biomass For disruption, 2g of dry Chlorella vulgaris was dispersed in 100 ml of distilled water and subjected to ultrasonication for 60 minutes at three different power levels: 360W, 720W, and 1080W The results are illustrated in the accompanying chart.
Table 4.5 Chlorella vulgaris cell disruption by ultrasonication in different power
Proportion of cell disruption in different ultrasonic power (% )
Fig 4.9 Cell disruption by ultrasonication of Chlorella vulgaris in different power
The disruption rate of Chlorella vulgaris cells increased with both processing time and ultrasonic power, as illustrated in Figure 4.9 At 360W, cell disruption reached 6.8% after 5 minutes, 18.3% after 10 minutes, 17.7% after 30 minutes, and peaked at 19.5% after 60 minutes In contrast, at 720W, the disruption rates were 17.5% at 5 minutes, 22.4% at 10 minutes, 36.8% at 30 minutes, and 43.6% at 60 minutes The highest cell disruption was achieved at 1080W, with a maximum of 23.6%.
The ultrasonication method for cell wall disruption proved most effective at a processing time of 60 minutes and an ultrasonic power of 1080W, achieving a disruption rate of 83.8% In comparison, the disruption rates were 41.5% at 5 minutes, 59.4% at 10 minutes, and 83.8% at 30 minutes.
C el l di sr upt in (% )
CONCLUSION AND RECOMMENDATION
Conclusions
• 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
• The ultrasonication method for disruption Chlorella vulgaris cell wall worked best at the processing time of 60 min and the ultrasonic power of 1080W.
Recommendations
• Research to optimize growing conditions to maximize lipid-collecting yield
• Research on building models of algae culture with a serial tube system
• Experiments on algae culture by wastewater environment
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