Research on applications of flat plate photobioreactor model using microalgae for shrimp culture in ninh thuan province combined with biomass recovery masters thesis major sciences and management of the environment

THE JOINT ACADEMIC PROGRAM OF EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF THE ENVIRONMENT BETWEEN INDUSTRIAL UNIVERSITY OF HOCHIMINH CITY AND LIÈGE UNIVERSITY TRAN THI MO RESEARCH

THE JOINT ACADEMIC PROGRAM OF EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF THE ENVIRONMENT BETWEEN

INDUSTRIAL UNIVERSITY OF HOCHIMINH CITY

AND LIÈGE UNIVERSITY

TRAN THI MO

RESEARCH ON APPLICATION OF FLAT PLATE

PHOTOBIOREACTOR MODEL USING

MICROALGAE FOR SHRIMP CULTURE IN NINH THUAN PROVINCE COMBINED WITH

The project was completed at The Industrial University of Hochiminh City

(Write full name and signature)

COMMITTEE CHAIR DEAN OF INSTITUTE OF ENVIRONMENTAL

SCIENCE, ENGINEERING AND MANAGEMENT

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ACKNOWLEDGMENT

A completed study would not be done without any assistance In addition, the efforts of myselves, there is the enthusiasm of teachers, as well as the support of family and friends during the research master thesis

First of all, I would like to express my endless thanks and gratefulness to Prof Le Hung Anh, Director of the Institute of Insititute For Environment Science, Engineering and Management His kindly support and continuous advices went through the process

of completion of my thesis His encouragement and comments had significantly enriched and improved my work Without his motivation and instructions, the thesis would have been impossible to be done effectively

Thanks to Prof Jean-Luc Vasel, the consultant, was always ready to provide technical support to me during the course of the thesis

I would like to state my thanks to Renewable project where supporting and creating conditions for me to research easily and conveniently, laboratory equipment and a part

of the research cost of the thesis

Therefore, I gratefully gives acknowledgement to their support and motivation during the time of doing this research

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ABSTRACT

The objective of the research is to evaluate the ability of shrimp culture treatment of microalgae on flat plate photobioreactor models, from which large scale application in Ninh Thuan The research has designed and built the Flat Plate Photobioreactor model with four different types of fluxes and artificial lighting systems

The experiments have compared the efficiency of culturing these algae with the Flat Plate Photobioreactor model in the same laboratory conditions and made the following conclusions: The study examined a number of different lighting cycles The lighting period of 16 hourd and 8 hours produces better and better results in terms of culture and harvesting of algae, but in reality the outdoor lighting only reaches 8 hours the study was conducted on 4 different types of aeration tanks, with data suggesting that the Flat Plate Photobioreactor is the most efficient way of cultivating algae The removal efficiency of pollutants is as follows: Total phosphorus is about 80-85% and total nitrogen is about 65-70%

Research results show that Chlorella sp is capable of treating shrimp waste water, opening up the direction of large-scale study of the topic

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CONTENTS

ACKNOWLEDGMENT 1

ABSTRACT 2

LIST OF ABBREVIATIONS 6

CHAPTER 1 INTRODUCTION 7

1.1 Overview of shrimp culture 7

1.1.1 Situation of shrimp farming in the world 7

1.1.2 Situation of shrimp farming in Vietnam 8

1.1.3 Situation of shrimp farming in Ninh Thuan province 9

1.2 Origin, composition and characteristics of shrimp pond effluent 11

1.2.1 Origins arise 11

1.2.2 Composition and characteristics of wastewater 12

1.3 Biological methods to treat waste water 13

1.3.1 Use microorganisms 13

1.3.2 Use of animals and plants 14

1.4 Overview of Chlorella sp 15

1.4.1 Introduce 15

1.4.2 Some forms of farming Chlorella sp 17

1.4.3 Factors affecting the growth and development of microalgae 18

1.4.4 Application of microalgae 22

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1.5 Overview of microalgae application 25

1.6 Overview of research on Flat Plate Photobioreactor model 29

CHAPTER 2 MATERIALS AND METHODOLOGY 33

2.1 Time and place 33

2.2 Study materials 33

2.2.1 Shrimp waste water 33

2.2.2 Chlorella sp 33

2.2.3 Experimental materials 33

2.2.4 Chemicals 34

2.3 Research Methods 34

2.3.1 Specific research methods 34

2.3.2 Method of data collection 36

2.3.3 Data processing methods 36

2.3.4 Comparison method, data evaluation 36

2.3.5 Method of determining microbial density 37

2.4 Experimental design 38

2.4.1 Experiment 1: Construction of OD growth curve and linear correlation of microalgae 38

2.4.2 Experiment 2: Investigate the effect of the lighting cycle 38

2.4.3 Experiment 3: Survey of wastewater treatment capacity 39

2.4.4 Experiment 4: Study on microalgae biomass harvesting by centrifugation 39

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CHAPTER 3 RESULTS AND DISCUSSION 41

3.1 Experiment to build growth curve and linear correlation 41

3.1.1 Build growth curve 41

3.1.2 Linear correlation 43

3.2 Change lighting time cycle 44

3.2.1 Chlorella1 sp 44

3.2.2 Chlorella2 sp 47

3.3 Survey of wastewater treatment capacity 50

3.3.1 Chlorella1 sp 50

3.3.2 Chlorella2 sp 52

3.4 Biomass recovery experiment 53

CONCLUSIONS AND RECOMMENDATIONS 55

A CONCLUSIONS 55

B RECOMMENDATIONS 56

REFERENCES 58

APPENDIX 61

A Some images in the experiment 61

B Appendix table of results 63

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LIST OF ABBREVIATIONS

1 Chlorella sp in fresh water Chlorella1 sp

2 Chlorella sp in salt water Chlorella2 sp

3 Flat Panel Photobioreactor Tank 1

4 Air-Lift Photobioreactors Tank 2

5 Flat Panel Airlift Photobioreactors Tank 3

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1.1 Overview of shrimp culture

1.1.1 Situation of shrimp farming in the world

Total annual harvests in the world today, including fishing and aquaculture, increase over the years Shrimp output grew steadily in East Asia in 2011, an average annual growth of 6% from 2008 to 2011 According to the Food and Agriculture Organization (FAO) estimated total production - including both natural and farmed fisheries - by

2015 would be around 7.1 million tons, up from 6.9 million tons in 2014 Whereas shrimp stocks from the sea are near saturation, shrimp farming is playing an increasingly important role in providing this kind of seafood

Shrimp farming grows mainly in tropical countries, where favorable climatic conditions, especially high temperatures throughout the year About 50 countries around the world are less likely to breed shrimp However, large countries are concentrated in two regions: Eastern Europe, including: China, Thailand, Indonesia, Vietnam, India, the Philippines, Taiwan, Japan and the western hemisphere, including Colombia, Mexico, Peru , Bazil

In recent years, shrimp farming in the world has made great achievements, contributing significantly to boosting the economic development of developed countries, but accompanied with great problems are posed as The epidemic, especially early mortality syndrome (EMS), is explosive in large areas, causing massive losses, the destruction of mangrove forests, the pollution of water and agricultural land, and the environment degradation

According to GOAL production fell from 3.45 million tons to 3.25 million tons in 2012 (down 5.8%) and 3.21 million tons in 2013 (down 1.1%) due to the impact of EMS in

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Central China, Thailand, Vietnam and Malaysia Production rose significantly in 2014

to -3.49 million tons (up 8.5 percent) mainly due to larger harvests than reported in China, Vietnam and India Even so, output will fall again in 2015 due to low harvests

in China, Thailand, Vietnam and Indonesia [2]

1.1.2 Situation of shrimp farming in Vietnam

Large sea areas have created favorable conditions for the exploitation and development

of aquatic products in Vietnam Shrimp are suitable habitats in brackish water areas near the sea With this feature, the Central, South Central (Khanh Hoa, Phu Yen, Ninh Thuan, Ba Ria - Vung Tau ), the Mekong Delta (Long An, Tien Giang, Ben Tre, Tra Vinh, Soc Trang, Ca Mau, Kien Giang) is the most concentrated shrimp production in the country

The rate of surface water surface for aquaculture has increased sharply since the 2000s with the total area of water surface for aquaculture is nearly 650 thousand hectares to

1054 thousand hectares by 2014

According to the report, in 2016, the total output of aquaculture in the country is estimated at over 6.7 million tons, up 2.5% over the same period in 2015 Of which, the output of mining is nearly 3.1 million tons (up 1.7% over the same period in 2015), aquaculture output is over 3.6 million tons (up 3.3% over the same period in 2015) Seafood export turnover is estimated at $ 7 billion, up 6.5% over the same period last year Although in the early months of 2016, the fisheries sector has been affected by droughts and salt water intrusion in the Mekong Delta, which has affected the planned release of brackish water shrimp as well as the damage to farmers However, the area

of brackish water shrimp farming in the country is estimated at 700,000 ha increased 0.72% of the plan The production of brackish water shrimp is estimated at 650,000 tons

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Ninh Thuan is the large shrimp breeding center of the country with the production scale in 2016 reaching the output of 24.1 billion; Most of them are high value producers such as tiger shrimp (5.1 billion), white shrimp (19 billion), marine fish species However, lack of waste treatment facilities, water supply and waste water drainage systems; Situation of the disease over the years continuously increasing is an important issue of the province

However, due to the high economic efficiency from shrimp farming, the aquaculture area is currently developing spontaneously, the scale and diversified farming methods, lack of training, full guidance self-priming, leading to environmental pollution Spontaneous aquaculture changes the purpose of land use (converting farmland, desert land into fish and shrimp farming), using unstable water supplies (digging underground water in the area), releasing into the environment Surrounding a large amount of waste, polluting the soil, surface water and groundwater The effects of aquaculture wastewater have long been assessed but there is no effective solution for many reasons

In the first six months of 2015, shrimp farmers reduced their stocking density by 30%

to combat EMS and export prices fell VASEP industry associations accounted for a decrease of 1.6% in vannamei production during this period over the same period of

2014 [10]

1.1.3 Situation of shrimp farming in Ninh Thuan province

With advantages over the coast of more than 100 km, over the past ten years, the commercial production of high value seafood and marine products such as lobster, shrimp, white shrimp, snails in coastal communes The sea of Ninh Thuan province develops quite fast It is normal for farmers to earn billions every year [11]

Due to the geographic advantage surrounding the surrounding mountains, the sea in Ninh Thuan is less affected when the climate changes, including storms so farmers

is short, high productivity, farmers Interest is more, so many households are excited to invest in expanding cages to raise

In the first eight months of 2017, shrimp farming in Ninh Thuan has seen positive changes, shrimp farming area has increased, there is a change in the target species as well as forms of farming

Area of commercial shrimp farming in the first eight months was 789.4 hectares, reaching 92.9% of the plan and 151% of the same period last year, of which the area of shrimp farming was 107.8 hectares and 681 6 ha The specific area for raising fish is

as follows: Ninh Phuoc 77.0 ha, Thuan Nam 221.5 ha and Ninh Hai 482.6 ha The selling price of shrimp was stable so the stocking area increased

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1.2 Origin, composition and characteristics of shrimp pond effluent

1.2.1 Origins arise

Brackish water and saltwater aquaculture has been quite developed, the technical level

of farmers and the level of intensive farming has increased, but the awareness of people about the use of chemicals and antibiotics in shrimp farming is not high; Extinguishing and treating wastes before discharging into the environment have not been paid due attention by shrimp farmers

The amount of waste generated is associated with feed production technology and shrimp culture systems Redundant food, shrimp faeces and nutrient metabolism are the main sources of contaminants There are also residues of antibiotics and medicines Wastewater carries a large amount of nitrogen, phosphorus and other nutrients, creating super-nutrient, blooming bacteria The presence of carbonic compounds and organic matter will reduce dissolved oxygen and increase BOD, COD, H2S, ammonia and CH4content in the natural basin

Most of the surplus products in shrimp culture accumulate in pond mud, which is harmful to shrimp and shrimp farming because the mud is very toxic, hypoxia and contains many harmful substances such as ammonia, nitrite, H2S, the direct effect of shrimp is always stress, poor eating, reduced growth rate and susceptible to bacterial disease and mass death

The discharge of pond water and pond solid waste in intensive and semi-intensive shrimp culture to natural canals without treatment will result in sedimentation of the canal system, polluted natural water environment serious If the discharge is continuous, there is no time for the environment to recover, the pathogen is cut, the organic humus will accumulate as the water environment becomes ephemeral, intensive shrimp farming and sale Intensive farming will be at greater risk On the

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other hand, the infrastructure serving the shrimp farming areas is not complete, the irrigation system is the system serving the needs of agricultural cultivation; Many shrimp farms have no canals, separate canals, even canal sections are sedimented, the bottom of the canal is higher than the bottom of shrimp ponds Consequently, the pathogen still exists in the shrimp pond when the infected ponds discharge into the environment, so the infection is very high

1.2.2 Composition and characteristics of wastewater

The composition of shrimp wastewater is not as big as industrial waste water, but due

to the large discharge volume plus the amount of sludge in the pond, the quality of the surrounding environment is greatly reduced The cause of environmental pollution in shrimp farming is due to the high concentration of aquaculture but no plans for water treatment and lack of attention of the state Shrimp culture wastewater is rich in organic matter, suspended solids, H2S, and NH3 produced by the decomposition of organic matter

Gases: In the process of raising the use of chemicals have released into the environment a number of gases under the action of bacteria appear as H2S, NH3, CH4 these substances are very toxic to ponds and lakes When shrimp is sick, it is necessary to use chemicals to remove toxins in ponds or to treat shrimp diseases Overdose or overdose will lead to overdose causing some bacteria to develop poorly for shrimp In addition, the process of running the explosion engine generates emission

of SO2, NOx, CO [16]

Mud: Contains many organic substances, antibiotics, chemicals, toxic gases (H2S,

NH3) and many pathogens The sludge is discharged directly to the untreated soil Most shrimp ponds have black or muddy soil in the bottom layer and discharge into nearby water sources such as rivers and streams after harvesting shrimp causing degradation of

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water quality without control of diseases When the black mud layer is taken to the shrimp farming area without good management of waste, they return to the pond when heavy rain

Water: In shrimp farming using antibiotics, chemicals (potassium, chlorine) they will

be present in waste water Water rich in nutrients should produce H2S, NH3 and waste water contains more SO42-, HCO3-, NO2- toxic if not treated before discharging into the surrounding environment Long-term exposure to water will be eaten, dry skin, cracked, hardened In water, H2S reaches 0.001ppm for a continuous time, reducing the reproductive capacity of the shrimp, while NH3 is converted to NO2- by nitrosomonat and forming methemoglobin, which reduces the amount of oxygen to the cell In shrimp wastewater contains organic matter such as N, P Total N, P produced per hectare for semi-industrial (yield 2 tons) about 13 and 43 kg, so the effluent discharge nourish and reduce the amount of oxygen in the water In addition, shrimp waste water has odor due to microorganisms decomposing organic substances, algae, phytoplankton, dead bacteria and antibiotics, chemicals present in wastewater Normally, mud is discharged directly to the land or river without treatment, causing the diseased shrimp to die soon after shrimp

1.3 Biological methods to treat waste water

1.3.1 Use microorganisms

Use of microorganisms, the collection of extracellular enzyme components of microbial growth; extracellular enzymes; Biological nutrients, minerals that activate the original growth and active catalysts have the effect of dissolving organic soluble and insoluble matter from shrimp faeces, the leftovers accumulate to create stability, Maintaining water quality and water color in ponds This solution has two directions:

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Aerobic method: Use aerobic microorganisms to treat Researchers Phan Thi Hong Ngan and Pham Khac Lieu (Hue University) applied aerobic treatment technology with submerge aerated fixed bed (SAFB), using modified aerobic activated sludge Good results of brackish water aquaculture were 73.7%, 97.4% NH4-N Aerobic methods have long retention times that facilitate the growth and activity of nitrifying bacteria Anaerobic method: use anaerobic microorganism for treatment This is a commonly used method for wastewater treatment, especially the Upflow Anaerobic Sludge Blanket (UASB) This technology distributes waste water from the bottom up through the anaerobic sludge layer to carry out the decomposition of organic matter by anaerobic microorganisms The phase separation system separates the solid-liquid-gas phase to remove the gases, transports the slurry to the bottom of the tank and leads the water back to the treatment Research by Mirzoyan N and Gross A Published at NCBI, the UASB reactor is well suited for brackish water aquaculture, reducing 81% suspended solids (TSS), 98% COD, 92% volatile

1.3.2 Use of animals and plants

It is possible to carry out the absorption of pollutants based on the process of metabolism in the ecosystem through the food chain Often people use phytoplankton, algae or algae to absorb nitrogen, phosphorus and carbon, etc in wastewater to increase biomass A study by the Nha Trang Institute of Materials Science on the ability of environmental treatment in shrimp farming ponds shows that seaweed can absorb large amount of ammonium salt at high speed After 24 hours, with a density of

400 g / m2, the ammonia content in the water was reduced by more than 20% As of Thursday, ammonia levels have dropped by more than 80% and by 10 am am 10% less than initially For phosphate, absorbed 30-60% after 24 hours The study by Ngo Thi Thu Thao and colleagues on the effect of combined culture of seaweed and white shrimp larvae concludes that algae helps improve the quality of the environment and

on the ability of organic matter processing of tilapia, mullet and snail in intensive waste water shrimp culture The results showed that, with effluents having dissolved oxygen parameters, NH3, BOD5, COD, TSS, coliform exceeded the limit of circular 44/2010/TT-BNNPTNT và QCVN 11:2008/BTNMT many times, using a suitable ratio

of subjects have helped the water after treatment meet the requirements, although the coliform indicator is still higher than the allowed threshold

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1.4.1.2 Characteristics

Chlorella is a genus of single-celled green algae belonging to the division Chlorophyta

It is spherical in shape, about 2 to 10 μm in diameter, and is without flagella Chlorella contains the green photosynthetic pigments chlorophyll-

a and -b in its chloroplast Through photosynthesis, it multiplies rapidly, requiring only carbon dioxide, water, sunlight, and a small amount of minerals to reproduce [21] Chlorella is a potential food source because it is high in protein and other essential nutrients; when dried, it is about 45% protein, 20% fat, 20% carbohydrate, 5% fiber, and 10% minerals and vitamins Mass-production methods are now being used to cultivate it in large artificial circular ponds

1.4.1.3 Role

Chlorella has the potential and can be used as a source of food and energy as it has the potential for photosynthesis, which in theory can reach 8%, which can compete with other crops such as plants Cane It is also an attractive food source because it has high levels of protein and other essential nutrients; When dried, it contains about 45% protein, 20% fat, 20% carbohydrate, 5% fiber, 10% minerals and vitamins

It is a rich and varied food supplement that helps the body regulate itself and balance its tolerance and excretion This helps the body to recover health quickly, creating good resistance to many diseases and environmental pollution

Characterized by single-celled microorganisms, photosynthetic growth through photosynthesis, or heterodox, or both Chlorella has the ability to accumulate heavy metals and thus remove toxic compounds from wastewater In addition to being a natural source of food for aquaculture, they also enhance oxygenation through photosynthesis under daylight In some cases algae also play a role in eliminating

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pathogens Therefore, Chlorella can be used primarily to remove nutrients (removal of nitrogen and phosphorus) in aquaculture waste water treatment

Today, algae are also used to feed cattle and poultry, produce biofuels to clean energy

1.4.2 Some forms of farming Chlorella sp

1.4.2.1 The open system

Natural ponds: using natural ponds and ponds for culturing ponds, not using agitators but low productivity

Figure 1.1 Open system [24]

Round tanks: Chlorella aquaculture in circular tanks with aeration system for high productivity but high construction costs, energy consumption

Pond system: Aquaculture in artificial pond system with water depth of more than 15cm Productivity is usually 20-25 g/ m3 /day High export value, however, depends

on the weather Dry season evaporates quickly, especially in the dry season, so it is difficult to control temperature

1.4.2.2 The closed system

This system has a high illumination rate of over 90%

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Figure 1.1 Closed system [25]

The system allows to limit direct contact with air and contaminated parts (dust, microorganisms ) between the algae and the external environment

Advantages:

Increased illumination efficiency due to increased surface contact area

Achieve high biomass

Increase the sterility of the culture system

Increases the efficiency of CO2 conversion

Reduce exposure

Disadvantages: high investment costs

1.4.3 Factors affecting the growth and development of microalgae

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Like other higher plants, light is one of the important contributors to photosynthesis Algae have the effect of increasing the intensity of light When the light intensity is low, the true rate of photosynthesis will be balanced with the respiratory rate This is called a point offset Depending on the type of algae, the optimal illumination range and the appropriate lighting time Typically, algae are fed in the light intensity of 1,000

- 10,000 lux, with lighting time of 16 - 24h / day

The light that acts on the microalgae through the pigment system in chloroplasts, the number and size of algae that play a major role in determining the chlorophyll content

of each microalgae, the intensity of light is The light intensity plays an important role, but the demand for it varies depending on the depth and density of the algae in the culture medium

1.4.3.2 Salinity

Sea algae have a high salt content, with a variety of algae that are capable of being domesticated and cultured in freshwater habitats, with large numbers of green algae (Fabregas, 1984) According to Coutteau (1996), algae can live and grow in new environments with a lower salinity of up to 15 ppt The optimum salinity for algae grows from 20 to 24 ppt According to Tran Suong Ngoc and colleagues (2007), Tetraselmis is thought to be algae with a salt capacity of 6 - 53 ppt Tetraselmis gracilis reproduces at salinity ranges from 9 to 30 ppt and Chlorella distributes broadly at salinity from 5 to 30 ppt However, in algae culture, to develop the best algae, the

salinity difference should be less different than where they are distributed

1.4.3.3 Temperature

In addition to environmental factors such as light, temperature also contributes significantly to the development of algae Algae grown in the appropriate temperature environment develop rapidly, the length of algae extends In contrast, the temperature

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lies within the tolerance of the algae, the cells of the algae are hypoechoic or hypotonic, leading to the algae being stunted or dead (Pham Thi Diem Phuong, 2012) Each algae has its own optimum temperature range According to Truong Sy Ky (2000), algae can be kept at around 20 - 30oC Lavens and Sorgeloos (1996) suggest that saltwater algae, including Tetraselmis, grows well in the range of 16 -270C for Chlorella vulgaris under natural light conditions of 25-300C However, algae grow slowly when they rise to 400C and below 250C Experiment on the effect of salinity and temperature on the growth rate of Tetraselmis tetratheles from Mohd Adib Fadhid B Azian in 2007, Tetraselmis tetratheles developed at a temperature between 20 and 300C

is quite stable and does not grow well At about 330C, at this temperature the algae grow slowly and decay rapidly According to Coutteau (1996), the appropriate temperature for algae development is 16 - 350C and the optimum temperature for algae growth is 20 - 240C Temperatures are lower than 160C, algae are slow to grow and algae will die when temperatures are above 350C In addition, raising algae in the room will easily control the temperature, while outdoor weather can change abnormally According to Diem (2011), the suitable temperature for Isochrysis galbana is 10 - 350C but the optimal temperature is about 25 - 300C When outdoor temperature algae were raised to optimum algae productivity of 270C, while temperatures higher than 320C or lower than 190C algae yield significantly decreased

1.4.3.5 Diet composition

The macronutrients (C, N, P, K, S, C, Mg, Na, Ca and Fe) và Trace elements (Zn, Cu, Ni)affect the growth of algae in which trace elements are required, Or stimulates the action of many enzymes, promotes biosynthesis of chlorophyll and reduces the chlorophyll breakdown by increasing the stability of the chlorophyll-linker complex with proteins.Just like higher plants, phytoplankton should have high concentrations of minerals such as N, P, S, Ca, Mg… for their simultaneous growth with other substances at lower concentrations such as Cu, Zn, Mn …Normally, these micronutrients are present in the natural environment in sufficient quantities for the

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normal growth of phytoplankton, But when other factors intervene, especially the pollution caused by human activity at levels higher than the normal level, it will cause

the inhibitory effect on growth

In the macronutrients, N and P are the most important N plays an important role in the composition and structure of proteins, a component of photosynthesis and enzyme systems N is a component of amino acids, nucleotides, hormones,….Carbon N deficiency is not used for the synthesis of N from nitrate and ammonium salts If ammonium is the only nitrogen source for algae, the pH environment will be significantly reduced, affecting their growth

1.4.4 Application of microalgae

1.4.4.1 Application of microalgae in food technology

Microalgae are a rich source of carbohydrates, protein, enzymes and fiber Besides, many vitamins and minerals like vitamin A, C, B1, B2, B6, niacin, iodine, potassium, iron, magnesium and calcium are abundantly found in microalgae Being such a rich source of essential nutrients, they are a major source of food, especially in Asian countries like China, Japan and Korea Green micro-algae have been used as nutritional supplement or food source in Asiatic countries for hundreds of years Nowadays, they are consumed throughout the world for their nutritional value [12]

Compared with traditional wastewater treatment methods, the use of algae for wastewater treatment has the following important benefits:

 Compared with sludge processing and other secondary processing processes, using algae is a low cost method of removing phosphate compounds as well as nitrogen compounds and pathogens

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 Traditional wastewater treatment processes include energy-consuming aeration, while the algae wastewater treatment produces the oxygen required by aerobic bacteria, using Algae are an effective way to digest nutrients in wastewater and provide oxygen from photosynthesis to aerobic bacteria

 In conventional wastewater treatment facilities, sludge often contains harmful solid waste, which will eventually be disposed of in the landfill Whereas the sewage treatment facilities by algae, which produce sludge are algae biomass with high energy content, can be further processed for the production of fertilizers or biofuels, Algae also do not use chemicals and the whole process is quite simple, producing only minimal amount of sludge

 Traditional wastewater treatment plants, which contribute significantly to the formation of greenhouse gases and algal waste water treatment facilities, also emit CO2but are much smaller in CO2 Algae consume, so that the whole process of algae processing does not arise but also consume CO2

1.4.4.2 Other environmentally friendly applications of microalgae

Algae can be used to seize fertilizers in agricultural wastewater, after collection, algae are again used as fertilizer

Because algae grow well under harsh conditions and do not require much nutrients, they can be cultivated in areas that are not suitable for agricultural production, so there is no competition for cultivated land Otherwise, when cultivating algae, one can use waste water without the use of agricultural water

Unlike other crops, algae do not depend on seasonal conditions, algae can grow well wherever the weather is warm and sunny

Algae can also be cultivated in seawater or in the desert Algae can also be cultured in wastewater and water containing phosphates, nitrates, or other contaminants

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Because algae farming is neutral in terms of CO2 emissions, it can help remove

CO2 from the air, and algae farms can be located near thermal plants or CO2 emitters

1.4.4.3 Other applications of microalgae

Algae can be cultivated for hydrogen production In 1939, German researchers discovered an alga that could shift from oxygen production to hydrogen production Algae is a protein that contains important metabolic-related amino acids, for example, producing enzymes, and producing energy, algae that contain complex and monohydric carbohydrates, which can supply the body with the source Nutritional supplements Specifically, sulphated cabon hydrate improves the immune system response It also contains omega 3 and omega 6

Algae can be used as sugars, algae that produce natural colorants, which can be used in place of synthetic dyes and dyes, under the influence of sunlight and heat The green algae that live in the water tanks will gradually turn red, which can be harvested and used as a natural coloring agent for food The food industry is seeking to replace coloring agents Are using with algae coloring agents

Current paper products are difficult to recycle because of the presence of ink, if using algae inks, the paper can easily be recycled because the ink is easy to decompose Algae can be cultivated in bioreactors for the purpose of producing biomass or reducing CO2 emissions Bio-reactors are also used to produce fuels such as bioethanol, or biodiesel, reducing emissions of gaseous pollutants such as CO2 and NOx in the exhaust gases of thermal power plants This device is based on the optical reaction of algae containing chlorophyll, a system of such biological reactors that can recycle waste into fuel, animal feed or biological fertilizers

 Application of microalgae in biotechnology

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Microalgae are applied to biotechnology through the extraction of lipids that produce biodiesel from microalgae, which is rated more sustainably and environmentally friendly than petroleum diesel The microalgae include the ability to accumulate large amounts of triacylgycerol, growth at high rates, atmospheric CO2 fixation, extensive adaptation including harsh environments and the ability to utilize nutrients from wastewater However, the effective lipid composition and lipid composition suitable for biodiesel production depends on many strains of microalgae The culture conditions include temperature, nutrient intensity, CO2 concentration

1.5 Overview of microalgae application

1.5.1 The world

In nature, microalgae are the first link in the food chain, so they are an indispensable source of food for many aquatic species such as in most stages of mollusc development, crustacean larval stage And fish In addition, they are also used to feed zooplankton (rotifers, copepods, artemia) as food for larval stages of crustaceans and fish [17]

In the United States, Thalasiossira pseudomonas, Skeletonema, Chaetoceros calcitrans, Chaetoceros mulleri, Nannochloropsis, Chlorella minutissima were fed to rotifers, bivalves, shrimp and fish larvae Or semi-continuous in composite tanks of 2,000-25,000 liters In Hawaii, Nannochloropsis yields about 2.2 million liters per year [3]

In Taiwan, the main target species are Tetraselmis, Nannochloropsis oculata, Chlorella

sp It is used for rearing larvae of Penaeus, Isochrysis galbana in clam For Skeletonema costatum, production can reach up to 9,000 tons per year.[3]

The history of microalgae farming is relatively short, starting with the early 1950s trials Today, the annual production of algae in the world produces more than 9,000 tons Mass production of Scenedesmus algae began in the 1960s in Czechoslovakia,

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Germany, Israel, Italy and some Eastern European countries Meanwhile, Spirulina is commercially grown in Mexico, USA, Taiwan, Israel, China, Thailand, India and Vietnam Dunaliella biomass production - the source of β - carotene began production

in Israel and expanded rapidly in Australia Porphyridium - a red algae rich in polysaccharides and arachidonic acid cultivated in France

In view of the observations discussed a highly defined mixing pattern that produces light–dark cycling at a given frequency is required for enhancing productivity through the flashing-light effect By contrast, chaotic mixing is not as effective in enhancing productivity as is organized mixing.-o¨rg Degen a,*, Andrea Uebele a, Axel Retze b, Ulrike Schmid-Staiger a,Walter Tro¨sch a (2001) “A novel airlift photobioreactor with baffles for improved light utilization through the flashing light effect” [8]

Study design of the flat model for microalgae farming, the disadvantage is that it causes hot surface when combined with aeration, low power consumption (53 W / m3) and high transfer capacity (0.007 1 / s).- E Sierra, F.G Acien, M Fern andez, Garc a,C Gonzalez, E Molina (200 ), “Characterization of a flat plate photobioreactor for the production of microalgae”, Department of Chemical Engineering, University of Almer a, E-040 1 Almer a, Spain [9]

The study designed a flat partition inside a flat model, which was tested on two microalgae species: Scenedesmus obliquus and Chlorella sorokiniana, which produced gas migration in the model The results show that the model does not affect the efficiency of light use can be applied on an industrial scale - Jian Li, Marisa Stamato, Eirini Velliou, Clayton effryes, and Spiros N Agathos (2015), “Design and characterizationl of a scalable airlift flat panel photobioreactor for microalgae cultivation”, Earth & Life Institute – Bioengineering Laboratory, Université Catholique

of novel internal mixers optimized with computational fluid dynamics”, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, PR China [14]

The study was conducted on two species of Chlorella sorokiniana and Scenedesmus obliquus because of their ability to absorb high organic matter, eliminate nutrients, eliminate pathogens and lipid accumulation with wastewater The results show that Chlorella sorokiniana is better suited to higher physiological and lipid stresses - Sanjay Kumar Gupta, Faiz Ahmad Ansari, Amritanshu Shriwastav, Narendra Kumar Sahoo, Ismail Rawat, Faizal Bux (2015), “Dual role of Chlorella sorokiniana and Scenedesmus obliquus for comprehensive wastewater treatment and biomass production for bio-fuels”, Institute for Water and Wastewater Technology, Durban University of Technology, P O Box1334, Durban 4000, South Africa [20]

The study investigated the effect of culture conditions at different temperatures and light regimes on growth, chlorophyll-a, chlorophyll-b, protein content and total free amino acid content of Chlorella vulgaris The results showed that culturing on the opposite side of daylight reception at 25-30°C, the best growth rate and biochemical factors will be higher - Rekha Sharma , Gajendra Pal Singh and Vijendra K Sharma (2012), “Effects of Culture Conditions on Growth and Biochemical Profile of Chlorella

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Vulgaris”, Department of Botany, University of Rajasthan, aipur-302 055, Rajasthan, India [18]

The research has optimized algal culture conditions for producing high levels of lipid

in FPBRs The results showed that after culture medium nitrogen levels of 25-75 mg NaNO3 mg /l, better fat and biomass - Yongjin He, Langjun Chen, Youcai Zhou, Han Chen, Xiuli Zhou, Fan Cai, Jian Huang, Mingzi Wang, Bilian Chen, Zheng Guo (2015), “Analysis and model delineation of marine microalgae growth and lipid accumulation in flat-plate photobioreactor”, Biochemical Engineering Journal

[22]

1.5.2 In Viet Nam

Research topic The model of wastewater treatment of industrial shrimp ponds by Tetraselmis sp And Duong et al., (2012) have been conducted on a pilot scale of the shrimp pond effluent industry using Tetraselmis sp And bivalve molluscs (blood cockle, riverbed), resulting in more than 70% of water after treatment can be cultured

again

Tetraselmis sp With a culture medium in the laboratory culture tetraselmis sp Dang Thi Thanh Hoa and Pham Thi Dieu aromatics of aquatic biology and fisheries management, aquaculture, agro-forestry and aquaculture universities Aquaculture activities have been and are Will grow more and more in the world, so the demand for children is increasing In hatchery production, feed for larvae is very important because the small size of the mouth, the body's sense of taste and the digestive system are not developing enough to limit the choice and use of the right food During this period Feed for larvae needs to: (1) be easily digested whole or in part (food must contain free amino acids and less peptides), (2) contain enzymatic systems that allow digestion ( Self-destroying food particles) and (3) providing all the necessary nutrients In general,

29

processed foods are hard to meet all these requirements, while some organisms are capable of satisfying them In addition, continuous movement can attract larvae and swimming activities will help to distribute food in the water column There are currently three widely used live food groups: microalgae, rotifer Brachionus and nauplii of artemia species (Lavens and Sorgeloos, 1996) About the algal blooms, there are over 40 different species that are isolated and cultivated for food including algae, green whorls and green algae ranging in size from a few micrometers to over 100 micrometers Some algae species are most commonly used in saline water such as Skeletonema costatum, Thalassiosira speudonana, Chaetoceros gracilis, Isochrysis galbana, Tetraselmis suecica, etc Microorganism of microalgae using clean inorganic media The experiment was expensive, so some studies were conducted to find organic substitutes that were easier to find and less expensive such as garden fertilizers, soil extracts…(Fabregas, 1987)

Tran Chan Bac (2013), "Research on the efficiency of Chlorella sp using waste water from catfish pond ", Faculty of Environment and Natural Resources, Can Tho University [5]

Truong Thi Thuyen, Nguyen Thi Thanh Than (2015), "Study on nitrogen and phosphorus absorption of chlorella vugaris on aquatic wastewater", Faculty of Chemical Engineering, College of Technology [6]

Nguyen Van Thoi (2013), “Research culture and recory biomass of Chlorella sp”, Faculty of Chemistry and Food Science, University of Technical Education [4]

1.6 Overview of research on Flat Plate Photobioreactor model

The algae culture model that is designed and installed in the laboratory is a photomicrographic photovoltaic device The light is supplied daily with fluorescent lamps arranged around the device

• Flat Panel Photobioreactor: Designed as a flat rectangular box without baffles

and is a regular aerobic style as in aquariums, but if aeration is strong enough to produce water downwards, it forms an oval shape Cycle through the length of the tank

• Air-Lift Photobioreactors: This is a aerobic form that helps the microalga

follow an oval in the width of the tank, a rectangular box with a baffle running along the bottom of the tank and a water level of 3cm Aeration is placed on one side of the partition to facilitate mixing of water

• Flat Panel Airlift (FPA) Photobioreactor: a spherical aerosolized balloon

bubble that is blown from the bottom to the top and to the right of the planks arranged

in a zigzag shape to cause the air bubble to go up in a circular shape Water tank on the one side is the water down on the side of the zigzag plate is arranged in order to disturb the inside of the tank

• Frame And Plastic Bag: This is a rectangular rectangle shaped rectangular

enclosure, but it is enclosed by a steel frame and has a plastic bag inside to hold water and allow light to pass through it Aeration is almost like the Flat Panel Photobioreactor

The model used in laboratory experiments using artificial lighting requires an appropriate lighting system to ensure algae growth

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The design should ensure that the algae culture temperature is in the range of 350C, which is the appropriate growth temperature range for algae

20-Figure 1.1 Flat Plate Photobioreactor model [23]

The experimental design consists of 4 tanks arranged in a rectangular box with a support frame 500 cm above the ground The lighting system is placed in four corners and in the middle of the model

There are two types of tanks:

Type 1: made of transparent glass (3 tanks)

Type 2: transparent plastic bag (1 tank) with outside grid

Table 1.2 Parameter size of each tank

Flat Panel Photobioreactor Length: 40cm

Width: 8cm Height: 70cm

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Air-Lift Photobioreactors Length: 40cm

Width: 8cm Height: 70cm High partition: 56cm, in the middle of the tank 3 cm from the bottom

Flat Panel Airlift (FPA)

Photobioreactor

Length: 40cm Width: 8cm Height: 70cm The partition is 5x10cm in size Frame And Plastic Bag Length: 40cm

Width: 8cm Height: 70cm

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2.1 Time and place

The research was conducted at the laboratory of Renewable Project; Institute of Science, Technology and Environmental Management of Ho Chi Minh City University

of Industry from January 2017 to July 2017

2.2 Study materials

2.2.1 Shrimp waste water

The shrimp waste water sample was taken from Ninh Thuan and transported by bus to the laboratory

2.2.2 Chlorella sp

Two types of algae: Chlorella sp in fresh water and Chlorella sp in salt water

The algae culture is from the Vietnam Academy of Science and Technology from the Vietnam Academy of Science and Technology, No 18, Hoang Quoc Viet Street, Cau Giay District, Hanoi, Vietnam

2.2.3 Experimental materials

Micro-organisms: algal aquariums with a capacity of 1 liter (plastic bottles) and 5 liters (plastic bottles), fluorescent lamps, thermometers, aerators and pH meters

Algae densitometry: optical, microscope, Neubauer counters

Algae biomass recovery equipment: centrifuge, centrifuge tube, membrane, oven, electronic scale

Some equipment used: autoclave, oven, light intensity measurement equipment, smoke

cabinets

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2.2.4 Chemicals

Guillard's (F/2) medium was used to propagate Chlorella spp This medium added NaCl to create salt concentrations such as seawater (0.35% NaCl) Alcohol and chemicals analyzes the indicators of wastewater to be monitored

2.3 Research Methods

2.3.1 Specific research methods

2.3.1.1 Sampling process

 Sampling procedure in Ninh Thuan

Sample Bottle: Clean, 30 liter plastic lid with tight lid

Sampling location: Shrimp pond waste water at Ninh Thuan province's 1st grade seafood breeding center will be located in the middle of the line, taking the sample at a depth of 0.1m

 Optical sampling procedure, density determination

Bottles of glass are washed several times with distilled water

Samples in the OD probe for optical measurement will be obtained with sterilized

equipment, avoiding the effects of external environment

2.3.1.2 Methods of analysis

Sample analysis was performed according to the methods of analysis in the APHA, Eaton DA, and AWWA (Joint eds.)

Output standard

phosphorus

spectral absorption method

-

Vietnam Standard 6202:2008

Nitrogen

Kjeldahl method

Automatic Kjeldahl condenser and burette

Vietnam Standard 6624-2:2000

spectral absorption method

Spectrophotometer -

K2Cr2O7 Drying cabinet, burette APHA 5220C

2.3.1.3 Method of biomass harvesting

After the end of the algae cycle, algae biomass is recovered by centrifugal method Centrifuge rotates at high speed to extract water from algae Because the algae weighs more than water, the algae is pressed into the wall of the tube, while the water is outside and separated from the algae Membrane filter works according to the principle

of pressure

Take 100ml algae with maximum growth rate (experiment 2) and proceed to centrifugal water at 6000 rpm for 10 minutes with centrifuge tube Then, bring the centrifuge tube containing algae biomass at 1050C for 2 hours and weigh the mass

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2.3.2 Method of data collection

Conducting the search and gathering materials on related subjects has been carried out

at home and abroad as a basis for research The content of the research process will be based on inheritance and further development on existing results

Materials are collected from prestigious international e-journals such as:

- Journal of Applied Phycology

- Journal of Hospital Infection

- Plant Pathology & Microbiology

- International Journal of Biotechnology for Wellness Industries

- www.energyeducation.ca

- www.gso.gov.vn

- www.fao.org

2.3.3 Data processing methods

Using Excel software:

- Enter the collected data

- Statistics data

- Drawing growth curve

- Evaluate data results based on the requirements of the research content

2.3.4 Comparison method, data evaluation

- The obtained data assess the efficiency of microalgae wastewater treatment

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- From the results of laboratory experiments, compare the advantages and disadvantages of aeration types and environmental conditions to set microbiological treatment conditions

- Choosing suitable cultivation conditions for Chlorella sp To improve the treatment efficiency and achieve high biomass

2.3.5 Method of determining microbial density

Use a counting chamber or an absorption spectrophotometer to determine the number

of microalgae Chlorella Sp at wavelength 420 nm

Use a direct method of counting red blood cells and counting the number of microalgae that develop over time

Dilute the sample so that in each cell of the chamber there are about 5 to 10 microorganisms To achieve such dilution it is necessary to estimate the number of microorganisms in the sample and to try several times during the dilution Place a diluted sample drop into the counter on the counting chamber at the counting chamber Adjust the glass so that one market contains a large square (4 x 4 = 16 small squares) Counts the number of cells present in a large cell Then, adjust the market to find another big box Counts the number of cells in at least five large cells, taking the mean Microbial density based on quantity obtained The calculation of cell density is as follows:

Thus, the cell density of the sample suspension is:

N/ml = x 106 cells / ml (where a is the average number of cells in a large cell)

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2.4 Experimental design

Propagation of Chlorella sp Micropropagation of Chlorella sp in liquid F / 2 medium with volume of 1.5 liters and 5 liters; with light intensity and continuous aeration throughout the culture cycle, maintaining temperature: 25-28oC

2.4.1 Experiment 1: Construction of OD growth curve and linear correlation of microalgae

Use of Chlorella sp has been bred, using direct counting and photometric methods to determine initial stocking density and developmental stage From there, we determine the growth curve for different time

We then diluted the algae solution with maximum growth at different concentrations according to the values: 0; 20%; 40%; 60%; 80% and norm with distilled water in 50ml volumetric flask Simultaneously, photometric measurements of each dilution level at

2.4.2 Experiment 2: Investigate the effect of the lighting cycle

The experiment was performed with the Flat Plate Photobioreactor model, continuous aeration The lighting period is L16 (includes 16 hours of lighting and 08 hours of