Research on applications of tubular 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 THAN THI MAI RESEARCH

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

BETWEEN INDUSTRIAL UNIVERSITY OF HOCHIMINH CITY

AND LIÈGE UNIVERSITY

THAN THI MAI

RESEARCH ON APPLICATION OF TUBULAR

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

ACKNOWLEDGEMENTS

To complete a research project whether large or small, whether in the long time or short time, is hard time and need help During those hard times, I received a lot of help and would like to send my most sincere gratitude to those helpers

Special thanks to Dr Le Hung Anh, Director of the Insititute For Environment Science, Engineering and Management, who directly provide ideas and guidance I study completed thesis He always devoted his time listening and leading my ideas in the right direction as possible If not have great help in terms of expertise and motivation from my teacher, I could not finish this research well

Thanks to Prof Jean-Luc Vasel, the consultant, was always ready to provide technical support to me during the course of the thesis; Especially the person who is sympathetic

to the time delay and has many ideas in completing the thesis

Thanks to the Renewable Project, a collaborative project between the Industry University of Ho Chi Minh City and the University of Liège has provided a number of tools, laboratory equipment and a part of the research cost of the thesis

Finally, I would like to express my sincere thanks to my family, my friends who have loved and supported me materially and spiritually during the course of the thesis Sincerely thank you all!

ABSTRACT

In Vietnam, the aquaculture industry is still on the rise, bringing very high economic benefits, especially shrimp culture With high profitability, easy-to-grasp farming techniques, the shrimp farming area is rapidly expanding in both quantity and volume The most typical are Ninh Thuan, Can Gio, Ben Tre are localities with many favorable conditions of climate, location as well as hydrology However, besides the positive aspects of socio-economic development, too rapid development is not synchronous and lack of management leads to many negative impacts on the environment Disposal of wastes from untreated shrimp ponds involves a series of pollutants emanating into the surrounding environment, rapidly degrading the quality

of the environment, increasing disease outbreaks that occur in aquaculture ponds A number of research projects have been carried out to overcome and treat this source of pollutants, but the combination of treatment and recovery of renewable energy from

this source of pollution has not yet been addressed The thesis “Research on application of Tubular Photobioreactor model using microalgae for shrimp culture

in Ninh Thuan province combined with biomass recovery” has made for the above

purposes The thesis used two strains of algae net worth of biomass are Chlorellaa sp and Chlorellab sp, was obtained from the Academy of Science and Technology of Vietnam (18, Hoang Quoc Viet, Cau Giay, Hanoi) to conduct the survey of average annual lighting conditions in Ninh Thuan L8D16 and conditions optimal lighting L16D8; survey on shrimp wastewater treatment capacity in Ninh Thuan and combine biomass recovery Results showed that two strains of microalgae were able to grow in waste water in shrimp ponds and reduced pollutants by their metabolism After 10 days

of culture in the laboratory, microalgal Chlorellab sp was cultured at condition L16D8

in 100% wastewater on the Tubular photobioreactor with an internal loop air-lift (short

biomass obtained was 0,1g/100ml; The dry biomass after harvest is 9.2 times higher than that of the initial stocking; The effective treatment of pollutants: 69,58% T-N and 92% T-P With the initial results showing the feasibility of adopting the scale model, the premise for the use of microalgae for treating aquaculture wastewater and receiving microalgae for Targeted for the production of biofuels, fuel for the food industry and medicine

TABLE OF CONTENTS

ACKNOWLEDGEMENTS 1

ABSTRACT 2

LIST OF ABBREVIATIONS 7

LIST OF TABLES 8

LIST OF FIGURES 9

INTRODUCTION 11

CHAPTER 1 LITERATURE REVIEW 15

1.1 Overview of shrimp culture 15

1.1.1 Situation of shrimp culture in the world 15

1.1.2 Situation of shrimp culture in Viet Nam 16

1.1.3 Situation of shrimp culture in Ninh Thuan province 17

1.2 Origin, composition and properties of shrimp pond wastewater 18

1.2.1 Derived Origin 18

1.2.2 Composition, characteristics of shrimp pond wastewater 20

1.3 Biological methods for treating shrimp wastewater 22

1.3.1 Method of using microorganisms 22

1.3.2 Method of using plants and animals to absorb pollutants 23

1.3.3 Biology lake 23

1.3.4 Artivificial Wet land systems 24

1.4 Overview of microalgae Chlorella sp 25

1.4.1 General introduction of microalgae Chlorella sp 25

1.4.2 Development stages of algae populations 26

1.4.3 Factors influencing the development of the microalgae 27

1.4.4 Some forms of culturing microalgae 32

1.5 Model of the actual research 33

1.6 Overview of research on Tubular photobioreactor of microalgae culture model for wastewater treatment 38

1.6.2 In Viet Nam 40

CHAPTER 2 MATERIAL AND METHOD 42

2.1 Time and place 42

2.2 Material 42

2.2.1 Shrimp waste water 42

2.2.2 Microalgae 42

2.2.3 Materials and Chemicals 42

2.3 Method 43

2.3.1 Specific research methods 43

2.3.1.1 Sampling method 43

2.3.1.2 Methods of analysis 43

2.3.1.3 The method of data collection 44

2.3.1.4 Analytical methods for data processing 44

2.3.1.5 Comparative method 45

2.3.1.6 Microalgae culture method 45

2.3.1.7 Determination method of density, microalgae cell biomass 46

2.3.1.8 Harvesting method 46

2.3.2 Experimental design 46

2.3.2.1 Experiment 1: Determine the growth curve and linear correlation 47

2.3.2.2 Experiment 2: Investigate the effect of light on the growth of microalgae [33] 47

2.3.2.3 Experiment 3: Survey of wastewater treatment capacity 48

2.3.2.4 Experiment 4: Microalgae biomass harvesting by centrifugation 48

CHAPTER 3 RESULTS AND DISCUSSION 49

3.1 Determine the growth curve and linear correlation 49

3.2 Investigate the effect of light on the growth of microalgae 53

3.3 Survey of wastewater treatment capacity 61

3.4 Microalgae biomass harvesting by centrifugation 64

CHAPTER 4 CONCLUSIONS AND RECOMMENDATIONS 66

4.1 Conclusion 66

4.2 Recommendations 67

REFERENCES 68

APPENDIX 73

APPENDIX A SOME IMAGE DURING THE EXPERIMENT 73

APPENDIX B RESULT DURING THE EXPERIMENT 75

B.1 Optical measuring results and the density of microalgae 75

B.1.1 Construction of growth curve, linear correlation 75

B.1.2 Change the lighting cycle 76

B.2 Recovery biomass 80

B.3 Changes in pH and temperature of the medium during culture of Chlorella sp 81

LIST OF ABBREVIATIONS

Chlorella a sp Microalgae Chlorella sp in fresh water (sanility 0-8%o)

Chlorella a sp Microalgae Chlorella sp in salt water (sanility 20-35%o)

L8D16 Condition culture have 8 hours lingting and 16 hours dark

L16D8 Condition culture have 16 hours lingting and 8 hours dark

LIST OF TABLES

Table 1.1 The composition of shrimp wastewater after 3 months at the center of

aquatic seed production level 1, Ninh Thuan Province 21

Table 1.2 List of equipment in the model 36

Table 2.1 Parameter and Methods of analysis 43

Table 2.2 Nutrient composition F/2 45

Table 2.3 Measure the standard line OD 46

Table 3.1 Analysis results of wastewater through 2 times of culture of microalgal Chlorellaa sp 62

Table 3.2 Analysis results of wastewater through 2 times of culture of microalgal Chlorellab sp 63

LIST OF FIGURES

Figure 1.1 Close algae culture system 32

Figure 1.2 Open ditches system 33

Figure 1.3 Tubular photobioreactor Model 34

Figure 1.4 3D drawing design model (Tubular Photobioreactor) 34

Figure 1.5 Composition of each single tube (from left to right are type bubble; air-lift long – short; bulkhead) 35

Figure 1.6 The system of culture of algae Tubular Photobioreactor after installation 37 Figure 3.1 Chart absorbance microalgae Chlorellaa sp 49

Figure 3.2 Chart absorbance microalgae Chlorellab sp 50

Figure 3.3 Density chart of microalgae Chlorellab sp 51

Figure 3.4 Correlation linear Chlorellaa sp 52

Figure 3.5 Correlation linear Chlorellab sp 53

Figure 3.6 pH change, average temperatures during culture Chlorellaa sp 8h light cycle 54

Figure 3.7 pH change, average temperatures during culture Chlorellaa sp 16h light cycle 54

Figure 3.8 Growth chart of microalgae Chlorellaa sp in experiment 8 hours lighting through average 2 times cultural 55

Figure 3.9 Growth chart of microalgae Chlorellaa sp in experiment 16 hours lighting through average 2 times cultural 56

Figure 3.10 pH change, average temperatures during culture Chlorellab sp 8h light cycle 57

Figure 3.11 pH change, average temperatures during culture Chlorellab sp 16h light cycle 58

Figure 3.12 Growth chart of microalgae Chlorellab sp in experiment 8 hours lighting through average 2 times cultural 58

Figure 3.13 Biomass chart of microalgae Chlorellab sp in experiment 8 hours lighting through average 2 times cultural 59

Figure 3.14 Growth chart of microalgae Chlorellab sp in experiment 16 hours lighting through average 2 times cultural 60

Figure 3.15 Biomass chart of microalgae Chlorellab sp in experiment 16 hours lighting through average 2 times cultural 61

Figure 3.16 Growth chart of microalgae Chlorellaa sp in experiment wastewater

treatment through average 2 times cultural 62Figure 3.17 Growth chart of microalgae Chlorellab sp in experiment wastewater

treatment through average 2 times cultural 63Figure 3.18 Biomass chart of microalgae Chlorellab sp in experiment wastewater treatment through average 2 times cultural 64Figure 3.19 Dry biomass chart of microalgae Chlorellab sp 65

1 Necessity of the subject

Ninh Thuan with has 105 kilometers long coastline that can be exploited all year round, the fisheries sector is identified as one of the four key fishing grounds of the country with total 120,000 tonnes of fish and shrimp stocks Mining capacity is 60,000 tons per year with many kinds of high economic value seafood, it can be exploited for processing industry and export

On culture, Ninh Thuan sea is the habitat of many marine species, with abundant parent seed sources and clean sea environment It’s the ideal place to produce high quality varieties, especially shrimps and sweet snail Currently the province has produced more than 6 million pieces of post/year The Ministry of Fisheries has established a center for producing and testing high quality shrimp seed in Ninh Thuan There are many investors who have been to Ninh Thuan to produce shrimp for region and whole country; such as Minh Phu Seafood Import Export Company has invested 5 billion post/year Grobest & Imei has invested 2.4 billon post/year … [1]

However, along with the strong and rapid development of the aquaculture industry, especially in the field of shrimp breeding caused problems to environmental concerns Specifically, shrimp breeding waste water mainly contains organic substances, nitrogen, phosphorus, suspended solids, some toxic gases such as NH3, H2S are produced by the decomposition of organic matter in pond bottom along with pathogenic microorganisms, algae and some chemicals are used to eradicate plague in shrimp… The source of waste has highly centralized and no thorough treatment are the main causes of soil and water pollution In addition, this source has many sources of disease that affect the quality of shrimp in the next breeding

The conduct of project: “Research on application of Tubular Photobioreactor model using microalgae for shrimp culture in Ninh Thuan province combined with biomass recovery” helping to address the pressing environmental problem mentioned

above, while promoting economic development by utilizing microalgae biomass after harvest to produce biodiesel, pharmaceuticals, In addition, the successful research project is the basis to promote the advantages of nature as well as creating jobs for people in here

2 Research purposes

- Evaluation of microalgae culture capacity on 20 liters Tubular Photobioreactor model

- Use of microalgae to treat shrimp waste water in Ninh Thuan

- Investigation of some parameters affecting microalgae culture in Tubular Photobioreactor model such as aeration, light cycle

- Research on the ability and technology to recover microalgae biomass

4 Object and scope of project

Research Objects

- Tubular Photobioreactors model

- Shrimp wastewater in Ninh Thuan

- Microalgae Chlorella Sp

Research Scope

- Laboratory scale on the 20 liters Tubular Photobioreactor model

- Shrimp wastewater at Aquaculture Center Level 1- Ninh Thuan Province

- Venue: Institute of Science, Technology and Environmental Management – Industrial of University in Ho Chi Minh City

5 Signification

Scientific Signification

The subject inherits the achievements of previous microalgae wastewater treatment, used for research in shrimp wastewater treatment in Ninh Thuan The results of the research are the basis, the foundation for further research on the microalgae’s ability to treat shrimp wastewater, as well as practical application of large-scale cultivation of microalgae in Tubular Photobioreactor model

Practical Signification

The project of using Tubular Photobioreactor model for cultivating microalgae for treating shrimp wastewater in Ninh Thuan has not only achieved high effluent treatment efficiency, environmental friendliness, consistent with the trend of the world

but it also takes advantage of the microbial biomass used for research, transforming it into bioenergy

With the advantages of cultivating microalgae, Tubular photobioreactor model helps to meet the requirements of higher quality microalgae in the field of food, cosmetics, pharmaceutical …

CHAPTER 1 LITERATURE REVIEW

1.1 Overview of shrimp culture

1.1.1 Situation of shrimp culture in the world

Prior to 2000, many Southeast Asian countries have sought to limit the development of white leg shrimp due to fear of spreading disease to black tiger shrimp But after that, people in many countries spontaneous started shrimp culture because high profitability and clear advantages of it Shrimp productivity increased rapidly and stably in Asia at that time due to white leg shrimp, which contributed to double the world shrimp

production in 2000

Prior to 2003, the countries with the largest shrimp production (Thailand, China, Indonesia) mainly cultured black tiger shrimp or indigenous shrimp After that, they concentrated in white leg shrimp China’s white leg shrimp production in 2003 reached 600,000 tonnes (76% of total shrimp production) By 2018, the output will reach 1.2 million tons (in total 1.6 million tonnes of shrimps) Indonesia imported white leg shrimp for breeding in 2002 and in 2005 reached 40 thousand tons In 2007 reached

120 thousand tons (in total 320 thousand tonnes of shrimps)

In 2004, white leg shrimp leaded the shrimp production, contributed over 50% of total shrimp production in the world In 2007, white leg shrimp accounted for 75% of global shrimp production, and it’s the main target species in three Asian countries (Thailand,

China, Indonesia) They are leading countries in shrimp production in the world

About value, in 1997, shrimp production reached 700 thousand tons, equal with 3,5 billion USD, average price is 5USD/kilogram In the last 10 years, the growth rate of world shrimp production is about 20% per year, that has brought the world 3.2 million tons of shirmp with the current value of shrimp is $11 billion, average price is 3,4-3,5

USD/kilogram

At present, the fishery sector is growing in diversified forms such as pangasius, basa, shrimp, clam, crab, tortoise… In particular, shrimp is considered as a potential product and widely cultivated

The world’s largest shrimp culture areas are the Western Hemisphere (including Latin American countries) and East Hemisphere (including South Asia and South East Asia countries) In the East Hemisphere, shrimp production accounts for nearly 70% of the world shrimp production, with Thailand the leading country, the next is Indonesia, China, India, Bangladesh and VietNam The most cultured shrimp are white leg shrimp, black tiger shrimp, China shrimp

Shrimp culture in Asian countries, although developed very strong, achieved initial results but had to face early disease and environmental degradation problems Normally, shrimp culture areas are only highly profitable in the first two to four years Then, due to outbreaks and environmental degradation, shrimps are prone to diseases causing productivity decline The main cause of this severe degradation was determined by the rapid development of aquaculture, where the focus was on cultivating the area and increasing yields in the ponds without neglecting the waste treatment incurred during the culture process After a period of effective culture, the environment gradually degraded shrimp culture leading to disease

1.1.2 Situation of shrimp culture in Viet Nam

In 2015, brackish water shrimp farming area reached 680.000 hectares throughout the country, reaching a total output of 600.000 tons, exports $ 3 billion Particularly in the Mekong Delta, shrimp farming area is 621.000 ha, accounting for 91,2% of the total area of the country (of which, shrimp farming area is 554.392 ha, white shrimp is 66.428 ha) Shrimp yield reached 484.000 tons, accounting for 81% of total shrimp production of the country At present, the area of intensive/semi-intensive shrimp

farming is mainly white leg shrimp (90.704 ha), while the area of extensive farming, extensive farming is mainly shrimp (542.764 ha)

In terms of market demand, in 2015 shrimp prices are lower than in 2014 due to declining production In 2016, shrimp production is expected to recover after EMS(Early Mortality Syndrome) but with slow growth On the other hand, shrimp production in large countries (eg Ecuador, India .) may be reduced due to the diminished stocking density due to the EMS epidemic In addition, demand for frozen shrimp remains stable and may increase as the economy recovers In the long run, shrimp is still a favorite product of the market; FTAs (Free Trade Agreement) and TPPs(Trans-Pacific Partnership) are removing tariff barriers

1.1.3 Situation of shrimp culture in Ninh Thuan province

There are over 472 establishments producing shrimp seed, therein, over 272 ones white leg shrimp seed and over 200 ones black tiger shrimp seed Learning from the results

of production activities, business in 2015, all facilities are linked to the market demand, the shrimp production and trading activities in 2016 have improved

In 2015, shrimp seed production reached over 21,800 million postlarvae, of which, shrimp breed on 17,000 million postlarvae, prawn on 4.800 million postlarvae, reaching 101.4% compared to the year plan

During the year, shrimp farming areas in the province is over 860 ha, mainly white leg shrimp In particular, Ninh Hai is 422 hectares, Thuan Nam 280 hectares, Ninh Phuoc

150 hectares and the lowest is Phan Rang - Thap Cham 8 hectares It is worth mentioning that many households in the lagoon are changing form, from intensive shrimp farming, semi-intensive to improved extensive farming, multi-polyculture, low density shrimp farming, both suitable for business conditions At the same time, they have a quick turnaround (2-4 crops/year) As in the shrimp culture area, most of the shrimp farming area is concentrated in large farms and mainly stocked in the form of

rolling, releasing and monitoring the situation, not farming series on the main stocking However, due to the influence of the weather, climate, shrimp growth retardation, fragmentation phenomenon is common, high Feed Consumption Ration (FCR), no economic efficiency calculation, even lose money if get disease Therefore, fishermen stocked low stocking density and small areas Another point is to overcome difficulties

in commercial shrimp culture, some businesses, fishermen have shifted from commercial shrimp culture to developing some types of sea fish such as grouper, cobia interspersed in lobster rafts; Pacific oyster culture in shrimp ponds is ineffective, culture snail in sand and etc [2]

1.2 Origin, composition and properties of shrimp pond wastewater

1.2.1 Derived Origin

Leftovers, shrimp feces and the process of nutritional metabolism are main origin of the pollutants in poorly-managed shrimp culture Observations have shown that in shrimp intensive systems, only 15 - 20% of feed is used to develop animal tissue, up to 15% of total food due to not eating and loss, only 40 – 45% is used in the nutritional process, maintaining live and peeled [3], [4]

Nitrogen contamination accounts for a large proportion of the feed waste It is estimated that about 63 – 78% nitrogen and 76 – 80% phosphorus for feeding shrimps

is lost to the environment [3], [4], [5], [6], [7], [8] The nitrogen in the form of protein

in absorbed by the shrimp and excreted in the form of ammonia Total amount of nitrogen and phosphorus produced per hectare of semi-intensive shrimp culture with a yield of 2 tons, equal to 113 kg and 43 kg In the intensive culture systems, this volume increased from 7 – 31 times

The amount of waste generated is related to the technology of feed production anh the shrimp culture system Nitrogen and phosphorus are the main constituents of food waste Too much food, unstable water quality, soluble food, food which is hard to

absorb, and nitrogen retention are factors related to wastewater containing nitrogen and phosphorus

Other sources of organic waste are phytoplankton or fibrous algae (lab-lab) and sediment or dissolved organic matter, suspended solids, etc … which are carried by the water Waste from aquaculture contain a little residue of antibiotica, pharmaceuticals, medications and hormones

The wastewater carries a large amount of nitrogen compounds, phosphorus and other nutrients, resulting in supper nutritive and nutrient richness accompanied by an increase in the initial production and the proliferation of bacteria The presence of carbonic compounds and organic matter will reduce dissolved oxygen and increase BOD, COD, sulfide hydrogen, ammonia and the methane content in the natural water Another problem caused by shrimp culture is the sedimentation (of the slump) in neighbourhoods, such as many mangroves and where water is stagnant

Much of the surplus product in shrimp culture accumulate at the bottom of the pond This is a very damaging source for shrimp and shrimp culturing The mud floor is very toxic, lacks oxygen and contains many harmful substances such as Ammonia, nitrite, hydrogen sulfide Shrimp always tend to avoid the area and will move to cleaner areas Because focusing on one area will reduce the area of feeding as well as the shrimp are trapped in polluted environments The mud layer also affects the water in the pond, which reduce the water quality

Water quality and the quality of contaminated pond bottom will directly affect the shrimps Shrimps are always stressed, shown by poor eating, reduced growth rate and susceptible to bacterial diseases, such as Vibriosis, which leads to the death of the shrimp The majority of diseases, occur in the environment where they live

The environment outside of shrimp culture, where dirty waste is not well managed, can affect coastal ecosystems This affects not only the land but also coastal resource values, including shrimp culture The reuse of polluted or dispose into surrounding environment will facility polluting water sources and impact on coastal activities The accumulation of heavy organic matter to the end of the crop also cause major spontaneous contamination in ponds, causing adverse effects on shrimps due to lack of oxygen and congestion with shrimp Increased disease pressure on the host Leakage of waste water as well as shrimp pond water to salinized agricultural land around the land and round area (living/eating)

1.2.2 Composition, characteristics of shrimp pond wastewater

The pond waste water carries a large amount of nitrogen, phosphorus and other nutrients, causing super-nutrient and nutrient-rich conditions, accompanied by a rapid increase of bacterial and eutrophication by the algae thieves in this environment

Many studies have identified 63 – 78% nitrogen and 76 – 80% phosphorus in shrimps that are lost to the environment [3]-[8] This wastewater is highly concentrated and does not have a through treatment model, which is the main cause of environmental pollution of land and water Moreover, direct discharges into the environment lead to epidemic, spreading and reducing the quality of the latter crop

Most of the excess product in shrimp ponds is accumulated at the bottom of the pond This is a source of harm to shrimp and if directly discharged into the environment with waste stream will have a great impact on the quality of natural water The pond mud is very toxic, lacks oxygen and contains many harmful substances such as ammonia, nitrite, hydrogen sulfide The presence of H2S, NH3, the combination of carboniferous and organic matter will reduce the amount dissolved oxygen, increase BOD, COD, and methane content in the natural water The use of variety of chemicals to eradicate

antibiotics will beneficial species in nature, causes ecological imbalance In addition, the accumulation of heavy organic substances to the end of the crop also cause self-pollution in ponds, increased disease causing shrinkage of shrimp production and degradation of the water environment

Table 1.1 The composition of shrimp wastewater after 3 months at the center of

aquatic seed production level 1, Ninh Thuan Province [9]

Ordinal

Analytical Results

1 st Phase

2 nd Phase

3 rd Phase

01 TSS (a)(b) mg/L Vietnam Standard

- Phase 3: Harvest, when the shrimp are 3 months old

1.3 Biological methods for treating shrimp wastewater

Many biological methods have been applied in the treatment of water pollution, especially shrimp wastewater containing many organic substances Biological treatment consisting of two main directions are the use of microorganisms to decompose organic matter in wastewater and use aquatic fauna and flora to absorb organic matter

1.3.1 Method of using microorganisms

Some microorganisms have the ability to use organic substances and some minerals as

a source of nutrition and energy creation, thus increasing their biomass These microorganisms are used to decompose the organic and inorganic pollutants present in the waste from aquatic wastewater This process of decomposition is called biochemical oxidation decomposition Some microorganisms are used to improve the water environment of shrimp and fish ponds This biological constituents of this composition include many types of microorganisms, the extracellular matrix of microbial growth, extracellular enzymes, bioactive nutrients and activator minerals and catalytic activity They are able to consume the organic matter generated during the growth and development of animals in ponds In other words, they have the effect of dissolving dissolved organic matter and insoluble matter from shrimp, feces, the leftovers accumulate the pond bottom, creating stability, maintaining water quality and water color in ponds On the other hand, this preparation also helps to reduce the pathogenic microorganisms such as Vibrio, Aeromonas, E.coli…, , increase the amount

of oxygen dissolved in the pond water environment and reduce the amount of ammonia

1.3.2 Method of using plants and animals to absorb pollutants

The nature of the use of plants and animals to remove pollutants is based on the process of metabolism in the ecosystem through the food chain It is common for people to use plants as an organism that absorbs nutrients such as nitrogen, phosphorus and carbon to synthesise organic matters, that boost biomass (self-sustaining), that is algae or phytoplankton, seaweed and other mangrove species

Next to the food chain are first order animals For example, the first animals in coastal waters are clams, mussels, oysters, which can consume phytoplankton and improve bottom sediment conditions Fish species that eat plankton and organic humus such as luce and mullet are also tested for use in drainage channels [10]

In fact, in order to achieve high performance of pollutants with minimal operating costs, people often use a combination of different methods, systems and agents Depending on the amount of pollutants in the waste water and the specific conditions

of each area

There are many biological methods that can be used to treat environmental pollution caused by coastal aquaculture, each with its own advantageous in terms of both economic and environmental aspects, especially the scale of culturing is not high, the systems is still small retail culturing characteristics, the cycle of waste is from 3 to 15 days

1.3.3 Biology lake

Consisting of a chain of 3 to 5 ponds, the wastewater is purified by natural processes through algae and bacteria The relationship between microorganisms and vegetation in lakes is one through oxygen and basic nutrients

In the lake there is always the process of photosynthesis, oxygen diffusion into water But photosynthesis occurs only in light conditions, the light in the water depends on

two basic factors: depth of water and the presence of more or less suspended organic matter

This model can be applied to large areas where waste water in treatment in shrimp culture is environmentally and economically feasible

1.3.4 Artivificial Wet land systems

Coastal aquaculture is practised in saltwater and brackish water so it is possible to use wetland systems to treat environmental pollution, especially in mangrove areas

Mangroves are a very common wetland ecosystem in Vietnam It is possible to use mangroves as a biofilter of organic pollutants from urban, industrial and aquaculture waste According to the theoretical calculations in Vietnam, one hectare of mangroves grow 56 tons of biomass each year and can absorb 21 kg of nitrogen and 20 kg of phosphorus (Jesper Clausen, 2002) According to Robertson and Phillips, 1995, to treat one hectare of shrimp it is necessary to have a mangrove area of at least 22 hectares Mangroves can absorb large amount of organic matter from coastal aquaculture The plant system in this system has the role as follows:

- Reduces the light to the water surface, reduces photosynthetic, restricts the growth of algae

- Creates conditions for microclimate regulation, especially heat insulation in winter, light temperature at the bottom will increase the speed of organic decomposition

- Submaged underwater elements work to provide surface for bacterial adhesion, oxygen, supply for photosynthesis, nutrient uptake Roots help to stabilize and reduce erosion, sedimentation, making condition for sedimentation of mud

- Besides, flora and fauna in mangrove ecosystems such as mollusks, mussels, crabs and fish are agents eradicating organic/pollutants

In addition, the mangroves have a particularly well-structured root system that traps sediments containing heavy/metals and plant protection chemicals The mangrove plant along with the entire ecosystem in the flooded forest is a biofilter for wastes from coastal aquaculture In sustainable shrimp culture, this form is encouraged to develop

in order to protect the aquatic environment and flooded forest systems

1.4 Overview of microalgae Chlorella sp

1.4.1 General introduction of microalgae Chlorella sp

 Morphological characteristics and structure

The first pure scientific works of Chlorella culture in 1890 is about species Chlorella vulgaris, by M N Beijerinck [11] Chlorella is widely distributed in both fresh water and brackish environments Its scientific classification is as follows:

Chlorella is in the shape of a sphere, with a diameter of 2-10 μm and without pillory Chlorella is green by photosynthetic pigment chlorophyll a và b in the conjugate Cell membrane has cellulose enclosure, can stand light mechanical impacts Through photosynthesis it develops rapidly only by CO2, water, sunlight and a small amount of minerals for reproduction

 Nutritional ingredients

Chlorella has the potential and can be used as a source of food and energy; as a source

of food because of its high protein, content and other essential nutrients When dried is contains 50-60% protein, many essential amino acids, 20-30% glucid and 10-20% lipid with unsaturated saturated fatty acids Chlorella contains most vitamins: A, B1, B2, B6, B12, C, D, K [12]

 Growth

Algae Chlorella reproduces rapidly, for 3 hours freshwater algae can double the density The algae Chlorella is not sexually reproduced Reproduction occurs through the formation of spores in the parental body Depending on the algae and environmental conditions, the number of spores may be 2, 4, 8, 16, 32 (even only creating 64), after the separation ends, the spore separates itself from the mother by damaging the mother cell membrane [12]

1.4.2 Development stages of algae populations

The development stages of the algae population are divided into 4 phases:

 Phase lag or slow-growth phase

In this phase the algae population is increasing in size and weight, the increase in cell density can occur but is very slow The slow-growing algae is the physiological adaption of the cellular metabolism to develop, such as increased levels of enzymes and metabolites involved in cell division and carbon fixation

 Exponential growth phase

In this phase the cells divide and grow very fast, continuously, depending on cell size, light intensive and temperature

 Stable phase or balanced phase

In this phase the growth of microalgae in balanced by the limiting factors of growth

As a result, algae density is kept stable

 Decayed phase

Depletion of nutrients makes the algae population decay rapidly

1.4.3 Factors influencing the development of the microalgae

 Light intensity

The appropriate light intensity varies according to the culture conditions If algae is grown in small glass vats, the intensity needed is about 1,000 lux, in large tanks with the light about 5,000 to 10,000 lux, artificial lighting time is at least 18 hours/day If cultured in green water process improved by tilapia, light intensity needs about 4.000-30.000 lux

 Temperature

For microalgae, temperature affects cell structure, metabolic rate, photosynthesis, distribution density, respiratory intensity, cell size, and species adaption Most of the microalgae live in the range of 16-30ºC, if the temperature is over 35ºC or lower than 16ºC the algae will be less developed, some species maybe be killed if this threshold lasts

The temperature suitable for growth and development of microalgae is in the range of 20-25ºC This temperature can vary by species and environmental composition

At temperatures below 15ºC and over 35ºC algae grow poorly At temperatures of 28ºC with good nutritional conditions, appropriate light intensity and stirring, microorganisms will grow fast In large-scale production of microalgae in aquaculture farms, the temperature is very decisive to productivity and quality of microalgae

25-In addition, Vietnam is located in the tropical zone, the temperature fluctuation in the year is very high, winter temperatures can reach 5-7ºC, summer temperature can be up

to 38ºC Therefore to ensure the stability and long-term in production, the solution to create pure native varieties is considered to be very effective

 Light

Light is the source of energy used by photosynthesic organisms to convert carbon dioxite into carbon source in organic compounds Light intensity and duration affect the growth of algae through optical process

The light intensity is suitable for the development of algae in the range of 1000 –

10000 lux, depending on the depth of the environment and the density of the algae The light can be supplied naturally (sunlight) or artificialy (fluorescent)

The appropriate light intensity varies according to the culture conditions If algae is grown in small glass vats, the intensity needed is about 1,000 lux, in large tanks with the light about 5,000 to 10,000 lux, artificial lighting time is at least 18 hours/day If cultured in green water process improved by tilapia, light intensity needs about 4.000-30.000 lux

Especially pH has a great influence on the growth and development of microalgae by indirectly affecting some factors such as alkalinity, toxic gas (mainly H2S, NH3) The most appropriate pH for the development of two species of microalgae in the research

Salinity changes to alter osmotic pressure, restrict photosynthesis, respiration, growth rate and decrease in Glucogen accumulation

Silicium), micro-nutrients (to help in the exchange, absorption of nutrients), and some vitamins to improve the quality of microalgae

Different microalgaes have different nutritional needs For Tetraselmis, Chlorella, the increase of KNO3, MgSO4, KH2PO4 salt content 20 times (from 0.001 to 0.02M) did not affect their growth (Myers, 1935 quoted by Vu Thuy Minh, 2005) It is even assumed that high yields of Tetraselmis are obtained when the salt concentration in the solution

in increased to 0,063M (according to Speckis, Milner, 1949) (quoted by Vu Thi Thuy Minh, 2005) Actually there is a difference in nutrient content in culture media for the same species in different places The difference comes from the nutrient availability in the natural waters that they live For microalgae, the two most important nutritional factors are N, P, but other factors cannot be overlooked other

 Nitrogen

Nitrogen plays an important role in plants in general and microalgae in particular, since nitrogen is a major component of structural proteins In addition, it is involved in the composition of many vitamins B1, B6, B12, PP, is part of the enzyme system catalyzing many important reactions of the body

Nitrogen content in algae can range from 1 to 10% and there are differences in algae groups Nitrogen demand is highest in green algae, next up is the blue algae and the lowest is the silicium algae According to Mudresop (1995) (quoted by Hoang Thi Bich Mai , 1995) in green algae with light intensity 2000 lux will develop well in protein content 57mg/L Nitrogen demand varies within the species depending on the supply and nitrogen content available in cultured water, typical responses to nitrogen depletion are cell depletion (reduction and shrinkage Chlorophyll and Carotenoid ) and the accumulation of organic compounds such as lipids (most algae have the ability to use nitrogen in three forms Ammonium (NH4+), nitrate (NO3-), nitrite (NO2-), but

Ammonia protein is more easily absorbed (Muzapharow, Taybaer, 1975) Nitrogen into the algae cells need to convert Ammonia protein in the following scheme:

6 CO2 + 6 H2O + light  C6H12O6 + O2

 Other nutritional elements

There is about 30 elements and many organic compounds used as nutrients for algae Beside important elements such as C, N, P there are other elements such as S, K, Na,

Fe, Mg, Ca and trace elements Mn, Zn, Mo, Co, V, Se These elements are in the basic composition of algae, which Fe is the most abundant It is a supportive agent that builds up the enzyme system, engages in electron transport, water dissociation and synthetic phosphorylation Addition of vitamins increases algae’s ability to grow The main vitamins are B1, B6, B12, C vitamin

In addition, the growth of algae depends on the volume and density of the culture If the stocking density is too low, the algae will grow slowly and can die On the contrary, if the stocking density is high, the maximum time will be fast and the time of decay will be very fast

1.4.4 Some forms of culturing microalgae

According to John R Benemann (2009), there are many methods for algae culture such

as open, close systems, pond culture [14]… the culturing area is very diverse depending on the investment, culture purpose and other factors

 Close system [6] [7] [8]

The tube is designed with a 5cm diameter, design in hatch bag with the lens instead of around 10cm There are many different designs type like arch, hemisphere, pockets, flat screen

Figure 1.1 Close algae culture system [10]

 Open ditches system, flowing water, pond in combination with other systems

In water system, water depth from 6 – 16 inches (15 – 40 cm), to be built by cement or

disturbance The system is used to culture algae like: spirulina, dunaliella salina, Tetraselmis, Chlorella and Haematococcus pluialis

Figure 1.2 Open ditches system [6]

1.5 Model of the actual research

 Introduce the Tubular photobioreactor model

Tubular photobioreactor is a tubal algae culture model, with a total working volume of

80 liters (consisting of 4 single tubes, each with a working volume of 20 liters)

Designed with periodic lighting control work; aeration 24/24; Working temperature is between 25oC - 28oC [15], [16], [17], [18]

Figure 1.3 Tubular photobioreactor Model [19]

 Structural Modeling

The model consists of 4 acrylic transparent tubes of the 93% illumination, each tube has 180mm diameter and 1m height On each tube has bottom discharge valve layout and one sample valve at the bottom of 1/3 working height

The working height of the pipe is 0.8m, corresponding to the volume of 20L of water The tube's frame made of iron, tube support is made of good insulated wood, foot frame height 0.6m Height from support to prop is 0.9m Total pipe length 1.5 m

The bulb is arranged around 4 tubes, 20 cm from each tube, and a light bulb is located

at the center of the four tubes

Structural detail of 4 single tube[20]:

Figure 1.5 Composition of each single tube (from left to right are type bubble; air-lift

long – short; bulkhead) Each single tube corresponds to a model type

- Tubular model with bubble type

- Tubular model with air-lift type: Survey two types of air-lift:

+ Tube height of 60cm and a diameter of 80mm, placed 10cm below the bottom of the water 10cm

+ Tube height of 35cm and a diameter of 80mm, placed at a bottom 22,5cm, water level 22,5cm

- Tubular model with bulkhead type (partition wall 180mm wide, 600mm long)

 Model size [15]-[18]

Table 1.2 List of equipment in the model [19]

Ordinal

1 Acrylic tube Thailand 4 Φ180mm Thickness: 3mm

Figure 1.6 The system of culture of algae Tubular Photobioreactor after installation

[19]

 Advantages[20]:

- Closed model, easy to control temperature

- High transparency model enhances penetration of light

- Easy to install

- Produce high quality products in terms of biomass and quantity

- Closed model to limit bacterial contamination

 Disadvantages[20]:

- High initial investment costs

- The materials used in the model production are limited because the available acrylic tubes in Vietnam are limited in size and can not meet the demand for larger tubes

1.6 Overview of research on Tubular photobioreactor of microalgae culture model for wastewater treatment

1.6.1 In the world

Some of the research on Tubular photobioreactors (PBR) model in algae culture in the world

- Paula Peixoto Assemany, Maria Lúcia Calijuri, Mariana Daniel Tango, Eduardo

Aguiar Couto, (2016), Energy potential of algal biomass cultivated in a

photobioreactor using effluent from a meat processing plant Algal Research 17

53–60 [21]

Research on potential evaluation of energy, fat and biogas, lipit in algae biomass

- Sanjay Pawar, Effectiveness mapping of open raceway pond and tubular

photobioreactors for sustainable production of microalgae biofuel 640–653

[20]

Study of wastewater and sunlight cultivating microalgae in two open ditches (ORP, PBRs) and tube model (horizontal and vertical), conducted at the National Institute of Environmental Engineering – India Quality results of algae culture is the form of pipes (PBR) much higher than that of open algae (ORP), but the cost of producing biodiesel

is higher than the production of open ponds (ORP) However, the problem of temperature control and pipe hygiene of horizontal tube models are relatively difficult

The term ORP maybe confusing : in many papers ORP is oxydo reduction potential