Khóa luận tốt nghiệp Công nghệ sinh học: Evaluation the growth and lipid accumulation of the microalgae Scenedesmus obliquus under different cultivation conditions

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITYFACULTY OF BIOLOGICAL SCIENCES EVALUATION THE GROWTH AND LIPID ACCUMULATION OF THE MICROALGAE Scenedesmus obliquus UNDER DIFFERENT CU

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATION THE GROWTH AND LIPID ACCUMULATION

OF THE MICROALGAE Scenedesmus obliquus UNDER

DIFFERENT CULTIVATION CONDITIONS

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITYFACULTY OF BIOLOGICAL SCIENCES

UNDERGRADUATE THESIS

EVALUATION THE GROWTH AND LIPID ACCUMULATION

OF THE MICROALGAE Scenedesmus obliquus UNDER

DIFFERENT CULTIVATION CONDITIONS

To complete this thesis, I would like to sincerely thank the Board of Directors, the

Department of Biological Sciences of Ho Chi Minh City University of Agriculture andForestry and the Institute of Biotechnology and Environmental Research for theirenthusiastic support Supported and created favorable conditions for me to complete my

graduation thesis recently

I would like to especially express my sincere thanks to Dr Huynh Vinh Khang and

Msc Nguyen Thi Van Anh has dedicatedly guided and advised me during the process ofimplementing, researching and completing the thesis

Thank you to the teachers of the Department of Biological Sciences for always

dedicatedly helping and supporting me in answering my questions throughout my

research and study process at school

Thank you to the student body of the Environmental Biology Department - Faculty

of Biological Sciences as well as the DH19SHD class for always encouraging, supportingand helping me during the time of completing my graduation thesis

I would like to thank my parents for not being afraid to work hard and always

creating the best conditions for me to study and practice

Because I am initially participating in scientific research and have many

limitations in knowledge, experience and skills that I cannot see, I look forward to

receiving more comments from teachers and you to make this thesis more complete

Finally, I wish all teachers good health and confidence to complete their mission of

leading and imparting knowledge to future generations Thank you sincerely

Thu Duc City, April 10, 2024

AFFIRMATION AND COMMITMENT

My name is Thach Thi Giang Huong, student ID: 19126062, class: DHI9SHD (Phone:

0982904116, email: 19126062@st.hcmuaf.edu.vn), Faculty of Biological Sciences, Nong

Lam university I declared that all results presented in this graduate thesis were conducted

by myself All the data and information are entirely accurate and unbiased I fully accept

responsibility for these commitments in front of the committee

Thu Duc City, April 10" ,2023

Student’s signature

Thach Thi Giang Huong

This study aimed to explore the influence of extraction methods and solvents onlipid extraction efficiency and to identify optimal culture conditions for enhancing the

growth and lipid accumulation of the microalga Scenedesmus obliquus Soxhlet extraction

methods, using hexane as a solvent, and the ultrasound-assisted extraction involved three

solvent systems: hexane, hexane:methanol (1:1), and chloroform:methanol (2:1) The

investigation included evaluating the effects of microalgae culture conditions through

response surface method to assess the growth and lipid accumulation potential of S.obliquus The highest lipid extraction efficiency (160.64 + 4.89 mg/g DW) was achievedusing the chloroform: methanol (2:1) solvent system with ultrasound assistance The

results indicated that the ultrasound-assisted organic solvent method was as effective as,

or superior to, the traditional Soxhlet method, offering time and energy savings in theextraction process The study revealed that the highest lipid content (163.22 + 0.83 mg/g

DW) was attained under culture conditions, including 75% nitrogen (NaNO3), 50%phosphorus (K2HPO,4 and KH;PO¿) compared to the original BBM nutrient medium, and

0.55 mg/L 3-indole-acetic acid (IAA) The findings contribute valuable insights foroptimizing lipid extraction processes and cultivating microalgae for lipid production

Key words: BBM, lipids, nitrogen, phosphorus, and Scenedesmus obliquus

dé đánh giá khả năng tăng trưởng và tích lũy lipid của S obliquus Hiệu suất chiết lipid cao nhất (160,64 + 4,89 mg/g DW) đạt được khi sử dụng hệ dung môi chloroform: methanol (2:1) có hỗ trợ siêu âm Kết quả chỉ ra rằng phương pháp dung môi hữu cơ có

sự hỗ trợ của siêu âm có hiệu quả tương đương hoặc vượt trội so với phương pháp

Soxhlet truyền thống, giúp tiết kiệm thời gian và năng lượng trong quá trình chiết xuất Nghiên cứu cho thấy hàm lượng lipid cao nhất (163,22 + 0,83 mg/g DW) đạt được trong điều kiện nuôi cấy, bao gồm 75% nitơ (NaNO3), 50% phốt pho (KzHPO¿ và KHzPO¿) so với môi trường dinh dưỡng BBM ban dau và 0,55 mg /L axit indole-3-axetic (IAA) Những phát hiện này đóng góp những hiểu biết có giá trị để tối ưu hóa quá trình chiết

xuât lipid và nuôi cay vi tao dé sản xuat lipid

Từ khóa: BBM, lipids, nito, photpho va Scenedesmus obliquus

: Bold's Basal Medium

: Central composite design

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3.3.3 Ultrasonic-assisted extraction with a mixture of hexane and methanol 21

3.3.4 Ultrasonic-assisted extraction with a mixture of chloroform and methanol 21

3.4 Investigation of the influences of culture conditions on the growth and lipid

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3.4.5 The response surface methodology - central composite design (RSM-CCD) 25

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4.2 Effects of culture conditions on the growth and lipid accumulation of the microalgae3 |

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4.2.6 The response surface method - central composite design (RSM-CCD) 44

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APPENDIX

LIST OF TABLE

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Table 3.2 Experimental setup to evaluate the effects of culturing time 22

Table 3.3 Experimental setup to evaluate the effects of nitrogen deficiency 23

Table 3.4 Experimental setup to evaluate the effects of phosphorus deficiency 24Table 3.5 Experimental setup to evaluate the effects of 3-indole acetic acid (IAA)

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Table 3.6 Levels of optimization for the three varIabÌes - ¿+55 +++<<£+ex+esseesss 26

Table 4.1 Results of the experimental setup for optimizing the culture conditions 45

Table 4.2 ANOVA analysis of the effects of the three factors on the objective function 47

Table 34.3 Optitnal culture CONGITIONG «sc sevcenccewsseesssvaessssseuseersoxvsoveenevanevessusenveresnseresveeweouss 48

LIST OE EIGURES

Trang

Figure 2.1 S obliquus microalgae cells under an optical microscope (Carlos et al., 2021).5

Figure 2.2 Chemical structure of chlorophyll (Mandal and Dutta, 2020) 12

Figure 4.1 Lipid content from S obliquus extracted using different methods C:chloroform, H: hexane, M: methanol Different letters indicated statistically significant

PTI [TH TD) scscssscrnasercsssanesicnoss eso a ces ac RO AS OR NEN 30Figure 4.2 Standard curve showing the correlation between cell density and the

absorbance at 680 nm wavelength of microalga S obliquus bliomass - 32

Figure 4.3 S obliquus cell density during 12 days of culture Jn the same sampling day,different letters indicated statistically significant differences (p < (.05) - 33Figure 4.4 Dry biomass of S obliquus after 12 days of culture Different letters indicated

StatistiGally SiONIfiCaTL-GIJEFENCES (TS O05)» cassnessaswxssxeanswancasusnennscexancnsastanawanesensousaeveemeess 34

Figure 4.5 Effect of nitrogen on lipid accumulation of S obliquus after 12 days of culture

Different letters have statistically significant differences (p < 0.05) . 35

Figure 4.6 S obliquus cell density during 12 days of culture Jn the same sampling day,different letters indicated statistically significant differences (p < 0.05) 37

Figure 4.8 Effect of phosphorus on lipid accumulation of S obliquus after 12-day ofculture Different letters have statistically significant differences (p < 0.05) 39

Figure 4.9 Effect of culturing time on cell density of S obliquus In the same treatment,

different letters indicated statistically significant differences (p < 0.05) 40Figure 4.10 Effect of culturing time on dry biomass of S obliquus In the same treatment,

different letters indicated statistically significant differences (p < 0.05) - 41

Figure 4.12 Effects of IAA concentrations on the dry biomass of S obliquus Differentletters indicated statistically significant differences (p < ().5) «-.«<s<<<x++ 43

Figure 4.13 Effect of IAA concentration on lipid content of S obliquus Different lettersindicated statistically significant differences (P~<0.05) c.ccsssnsssnssnsonssncancessvnrsvnesanenseannys 4-4

Figure 4.14 Optimal culture conditions as proposed by Minitab 21 software 48

Chapter 1 INTRODUCTION

1.1 Problem statement

Fossil fuels are finite resources Using fossil fuels for energy generation is the

leading cause of air pollution and global warming (Klass, 2004) One of the mostimportant dilemmas of the modern world is providing sufficient energy with minimalimpact on the environment The use of biofuels to replace fossil fuels will not only

contribute to reducing air pollution and preventing global warming but also help to be

proactive about energy sources (Thanh ef al., 2017) Since then, the issue of needing to

find bioenergy sources from this and other renewable biofuels is receiving more and moreattention, research, and use, in which the highlight is biodiesel obtained from organicmatter (Amaro et al., 2011; Chisti, 2007)

Microalgae are single-celled, photosynthetic organisms that use light energy andcarbon dioxide, with a higher photosynthetic efficiency than plants, for biomassproduction Recent research has shown that microalgae have the potential to be a source

of bioenergy due to their advantages such as easy cultivation, tolerance to harshconditions, rapid biomass accumulation, short growth cycle, and high oil content (Oncel,2013) Furthermore, exploiting microalgae to produce biodiesel does not affect the

production of food or animal feed, and does not compete with other crops for arable land

(Chisti, 2008)

The growth and lipid accumulation process of microalgae is influenced by manyfactors, including light, temperature, pH, and the composition of the nutritionalenvironment Among these factors, nitrogen (N) and phosphorus (P) have the greatestimpact on the lipid content and composition of microalgae cells (Brindhadevi et al., 2021;Yaakob et al., 2021) A deficiency in N/P can affect biochemical processes within cells,causing a shift in the carbon flow from photosynthesis and protein synthesis will shift to

the biosynthesis of energy-rich macromolecular compounds, mainly lipids Shen et ai.(2015) found that the microalga Chlorella vulgaris accumulated approximately three

times more lipids when cultured in a nitrogen-free environment On the other hand, under

conditions of high light intensity and N/P deficiency, microalgae cells enhance the

accumulation of large amounts of lipids in the form of triacylglycerol (TAG) (Chisti,

2007; Hu et al., 2008) The TAG form is considered a highly suitable raw material source

for biofuel production However, achieving high lipid content and biomass yield are often

difficult to achieve simultaneously and can be challenging, as conditions that favor high

biomass yield often lead to low lipid content and vice versa (H Taher et al., 2015) For

example, when microalgae are deprived of nutrients during the cultivation phase, their

growth rates decrease significantly but lipid content increases (H Taher et al., 2014) A

new focus in diesel research on microalgae biology is to optimize ideal conditions forhigh growth rates of microalgae while producing high lipid content in stressful

environments This can be achieved through phytohormone supplementation, which has

also been considered a promising method for increasing lipid production from microalgae

in recent times There are various popular methods for extracting lipids from microalgae

biomass, including Soxhlet extraction, solvent extraction, carbon dioxide extraction

Supercritical, ultrasonic extraction (UAE), and microwave extraction (MAE) (Iqbal and

Theegala, 2013) Some common organic solvents used for lipid extraction include

chloroform, toluene, n-hexane, methanol, acetone, and ethanol (Mubaral et al., 2015).According to Abomohra et al (2016), when using solvent mixtures of different polaritiessimultaneously can extract lipids from the lipid-protein complex using a polar solvent andthen dissolving them into a nonpolar solvent The efficiency of lipid extraction frommicroalgae also depends on the nature of each type of solvent (Abomohra e¿ al., 2016)

Based on the above reasons, this project aims to evaluate the effects of nitrogen

concentration (NaNO3), phosphorus concentration (K2HPO4 and KH2POs), and Acetic Acid (IAA) concentration on the lipid accumulation in the microalga S obliquus

Indole-3-The results of this study could pave the way for further in-depth research and help inform

real-world manufacturing applications to increase lipid yields and reduce production costs

1.2 Objectives

To determine the culture conditions that increase growth and lipid accumulation

in the microalgae S obliquus

1.3 Contents

Content 1: Investigate different methods for lipid extraction from the microalgal

biomass

Content 2: Investigate the influences of culture conditions on the growth and lipid

accumulation of the microalgae

Chapter 2 LITERATURE REVIEW

2.1 Overview of Scenedesmus obliquus

Microalgae are single-celled organisms that can be classified as either prokaryotic

or eukaryotic They are found in a wide range of ecosystems on Earth and are typicallyobserved under a microscope These tiny algae are commonly found in freshwater andmarine environments and are capable of performing photosynthesis, which is responsible

for producing approximately half of the oxygen in our atmosphere (Robin Williams,2013) Additionally, microalgae play a crucial role in reducing greenhouse gasses byutilizing carbon dioxide to develop photoautotrophs Research has shown that algaefarming can significantly decrease the amount of CO; produced from agriculturalcultivation and help stabilize the global climate (Ha et al., 2019; Helen Onyeka et al.,2021) The biodiversity of microalgae is vast, with an estimated 200,000 - 800,000species belonging to various genera One of the most prominent genres 1s Scenedesmus,which falls under the order Sphaeropteris and the family Scenedesmaceae It is estimatedthat there are over 300,000 species of microalgae, and Scenedesmus is the third mostresearched genus in the world in terms of the number of published documents, surpassingeven popular species like Spirulina and Nannochloropsis (Garrido-Cardenas et al., 2018).Scenedesmus is known for its ability to treat pollutants in wastewater, produce biomass,and accumulate lipids for biofuel production Due to its high adaptability, minimal water

requirements compared to terrestrial crops, rapid growth rate, and environmentalfriendliness, microalgae have immense potential in various fields such as food, medicine,

fuel, and biological fertilizer

The scientific classification of Scenedesmus obliquus is as follows:

Kingdom: Plantae

Phylum: Chlorophyta

Class: Chlorophyceae

Order: Sphaeropleales

Family: Scenedesmaceae

Genus: Scenedesmus

Species: Scenedesmus obliquus

Scenedesmus is a widely recognized genus of freshwater algae, with a majority ofits species found worldwide However, there are also some species that are only found inspecific local populations, such as S intermedius and S serratus, which are exclusive toNew Zealand The genus Scenedesmus has a long history, dating back to 70 - 100 millionyears ago Currently, there are approximately 70 known species within the Scenedesmus

genus, making it a significant component of freshwater plankton One of the earliestknown species, Scenedesmus obliquus, was first isolated by (Turpin) Kiitzing in 1833

2.1.1 Morphological characteristics and cell structure

S obliquus is characterized by its spindle-shaped cells with pointed and inwardlybent ends These cells are typically arranged in groups of 2 — 4 - 8, attached to each other

in the middle and aligned in a row According to Duong Duc Tien and Vo Hanh (1997),

the length of S obliquus cells ranges from 4 - 30 um, while their width ranges from 2-9.5

um The cell walls are primarily composed of neutral sugars (glucose, mannose, fructose,and rhamnose) and amino acids Additionally, each cell contains a single chloroplast that

Figure 2.1 S obliquus microalgae cells under an optical microscope (Carlos et a/., 2021)

This type of microalgae has a fast growth rate and good resistance to adverseconditions S obliquus has the ability to grow in nitrogen-rich wastewater and convert

pollutants into biomass (Martinez et al., 2000) S obliquus is resistant to high temperature

(Yang et al., 2018) and high irradiation (Hurtado et al., 2019) This algae can also grow inheterotrophic and mixotrophic conditions These are outstanding advantages for large-

scale farming

2.1.2 Reproduction

S obliquus reproduces asexually by releasing spores through cell wall rupture(Oliveira et al., 2020) During the replication process, the mother cell grows andundergoes multiple divisions, resulting in a multinucleated cell The cell then separatesinto mononuclear daughter cells, which develop into non-motile spores These daughter

cells often join with other daughter cells to form a colony within the mother cell wall,

which is later released This process follows a mitotic cycle similar to other members of

the family Chlorophyceae, with the cytoplasm of the daughter cells becoming densely

packed Eventually, the mother cell wall ruptures and releases the daughter spores into the

surrounding environment It is worth noting that the cells at the ends of the colony have a

different morphology than those in the center (Pickett-Heaps and Staehelin, 1975)

2.2 Nutritional forms of S obliquus

2.2.1 Photoautotrophic

Photoautotrophs are the most common form of nutrition for microalgae, which

use photosynthesis to convert light into chemical energy This energy is then stored in theform of adenosine triphosphate (ATP) and nicotinamide and adenine dinucleotidephosphate (NADPH) These compounds are essential for the Calvin cycle, which isresponsible for synthesizing carbohydrates and other organic compounds (Xia andMurphy, 2016)

Photobioreactors are used to control the phototrophic biological processes andoptimize photosynthesis This includes monitoring factors such as pH, irradiance,

temperature, CO2 concentration, and culture medium These reactors are commonly used

for the production of biomass and high-value biological products, as they provide a closed

system that results in higher biomass yields (1.5 — 4.0 g/L) and a lower risk of

contamination compared to open systems like ditch ponds (Lee and Shen, 2003).However, the main drawback of photobioreactors is their high maintenance andproduction costs

2.2.2 Heterotrophic

In heterotrophs, organic carbon sources are utilized to fulfill both nutritional andenergy requirements (Venkata Mohan et al., 2015) Unlike photoautotrophic cultures, thegrowth of microalgae is not reliant on light and can thrive in high ratios of illuminatedarea to volume This leads to a substantial increase in biomass production Furthermore,

adopting heterotrophic farming methods can also reduce harvesting expenses (Mohan et

al., 2015)

2.2.3 Mixotrophic

Mixotrophy is the ability to utilize both organic carbon and light as sources ofenergy This presents a promising opportunity for cultivating microalgae at exceptionallyhigh cell densities (J Geider and Osborne, 1989) Studies have demonstrated that thegrowth rate of microalgae in this form is comparable to that of heterotrophic andphotoautotrophic forms combined However, the mixotrophic exchange process is morecomplex than other forms (Oliveira et al., 2021)

2.3 Factors affecting the growth and development of S obliquus

Converti et al., 2009; Taoka et a/, 2009), in many microalgae species, a decrease in

growth temperature increases the amount of unsaturated fatty acids while the amount of

saturated fatty acids increases as the growth temperature increases (Renaud et al., 2002)

Most microalgae species are capable of carrying out photosynthesis and cellular divisionrange of temperatures stated between 15 and 30°C, the temperature for maximum growth

is between 20 and 25°C for thermophilic species, low temperatures (15 — 20°C) can

reduce or limit growth On the other hand, relatively high temperatures (20 — 30°C)

accelerate metabolic rate, leading to cell multiplication, but can be increased up to 40°Cfor thermophilic strains or reduced to 17°C for psychrophilic strains (Ras et al., 2013)

According to Breuer et al (2013) the highest fatty acid yield in microalgae S obliquus

was achieved at a temperature of 27.5°C Martinez et al (1999) observed that the growth

of S obliquus at 30 or 35°C was almost the same and the lowest value was obtained at20°C According to Guedes et al (2011), the highest biomass yield of S obliquus wasachieved at a temperature of 30°C, nearly three times higher than at 20°C The above

studies have demonstrated that the microalgae S obliquus is heat-resistant and is

a potential candidate for growth in tropical, semi-arid and desert areas

2.3.2 pH

pH is a crucial factor that microalgae cells respond to almost instantaneously, byadjusting their physiological rate and nutritional needs This regulation of pH also affects

their biomass composition and antioxidant content In a study by Guedes ef z/ (2011), it

was found that the growth of S obliquus M2-1 was least impacted at pH 6 and the highest

at pH 8 The relationship between antioxidant production and pH was also significant,with an increase in antioxidant production as pH increased However, there was no

statistical difference between the results obtained at pH 7 and 8 (Guedes ef al., 2011).Optimal conditions for nitrogen - replete growth of S obliquus was found to be at pH 7(Hodaifa et al., 2010) Similarly, Breuer et al (2013) found that pH 7 was the optimalcondition for the accumulation of triacylglycerols (TAGs) in S obliquus

2.3.3 Light

Light intensity is also a significant factor that affects the growth of microalgae

Microalgae use light conditions to conduct metabolic activities, with a certain light

saturation limit (6,458 KLux) as an energy source for synthesizing cell protoplasm

(Difusa et al., 2015) The influence of light on microalgae growth is expressed through

light quality, lighting intensity, and lighting time (Bosence, 1976; Qin, 2005)

According to Al-Qasmi ef al (2012), light conditions directly affect the growth

and photosynthesis of microalgae (with both duration and intensity) playing a crucial role.Light is necessary for the photochemical phase, which produces essential molecules such

as Adenosine triphosphate (ATP) and Nicotinamide adenine dinucleotide

phosphate-oxidase (NADPH) On the other hand, darkness is needed for the biochemical phase,

which synthesizes molecules necessary for development

2.3.4 Nutrient

2.3.4.1 Nitrogen

Nitrogen is an essential nutrient for the growth of microalgae and plays a crucial

role in the synthesis of amino acids and lipids It makes up approximately 10% of the dryweight of cells (Wijffels et a/., 2010) Inorganic nitrogen is quickly absorbed by algae and

converted into biologically active compounds (Ankita Juneja ef al., 2013) The most

commonly used nitrogen sources for algal growth are ammonia, nitrate, and nitrite,each

having different effects on cells and biochemical composition (Lourenco ef al., 2004).According to Tang et al (2011), research has shown that using nitrate as a nitrogensource can result in 2-3 times higher biomass compared to using urea or ammonia.Therefore, nitrate is a preferred nitrogen source for the production of S obliquus algalbiomass However, under nitrogen-deficient conditions, S obliquus has the ability toincrease lipid accumulation but at the cost of reducing algal biomass (Breuer et al., 2013;

Chu et al., 2014; Shen et al., 2018)

2.3.4.2 Phosphorus

Phosphorus is an essential component necessary for the normal growth anddevelopment of microalgae, typically making up 1% of the cell’s dry weight (Chist,

2007) A deficiency in phosphorus can hinder the synthesis and recombination of

substrates in the Calvin cycle, ultimately impacting photosynthesis Additionally, a lack

of phosphorus can lead to an increase in lipid, astaxanthin, and carbohydrate content incells, while decreasing the levels of chlorophyll a, 13 protein and biomass (Ankita ef al.,

2013) Research has shown that a phosphorus concentration of 0.1-2.0 mg/L can increase

lipid content in Scenedesmus sp from 23% to 53% (Juneja et al., 2013)

2.3.4.3 Carbon

Carbon is another crucial nutrient for photosynthesis and the growth anddevelopment of biomass, accounting for approximately 50% of the cell's dry weight(Carlos eft al., 2021) Microalgae require an inorganic of carbon for photosynthesis to

occur This can come in the form of CO2, carbonate, or bicarbonate for autotrophic

growth, and acetate or glucose for heterotrophic growth (Ankita e ai, 2013) Previousstudies have reported that using sodium carbonate as a carbon source can increase S

obliquus biomass by 9%, or increase biomass by 2.3 g/L at a 15% CO; concentration

(Singh et al., 2014; Mansouri et al., 2018)

2.4 Componds accumulate in the alga S obliquus

2.4.1 Lipid

The primary lipid categories found in algae encompass membrane lipids

(glycosylglycerides, phosphoglycerides, betaine ether lipids) and storage lipids

(triacylglycerol) Additionally, algae contain minor quantities of various other lipid types

including terpenoids, sphingolipids, hydrocarbons, sterols, and pigments, with varying

proportions depending on the algal class (Li-Beisson, Y., Nakamura, Y., & Harwood, J

2016) Lipids are extracted from microalgae in the form of triacylglycerol (TAG) andserve as substitutes for palm oil and long-chain polyunsaturated fatty acids (n3 LC-PUFA)

such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Maria J Barbosa

et al., 2023)

Lipids found in algae can be categorized as follows (Pohl and Zurheide, 1982): (1)

Neutral lipids (NLs), including triacylglycerols (TAGs), wax esters, hydrocarbons, fatty

acids (FAs), and sterols (1) Phospholipids (PLs), such as phosphatidylcholine (PC),

phosphatidylethanolamine (PEA), phosphatidylserine (PS), phosphatidylglycerol (PG),and phosphatidylinositol (PI) (iii) Glycolipids (GLs), which encompass

sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and

digalactosyldiacylglycerol (DGDG) TAGs are typically considered as energy storage

compounds, while PLs and GLs serve as structural components primarily found in cellmembranes Additionally, the predominant fatty acids are saturated and cis isomers ofunsaturated fatty acids, comprising carbon chains ranging from 12 to 22 atoms and

containing 0 to 6 double bonds

2.4.2 Pigment

Photosynthesis is the process by which plants, algae, and some bacteria capture

and convert sunlight energy into organic compounds for their own use and as a foodsource for other living organisms on Earth In photosynthetic cells, pigments make upapproximately 5% of the mass of dry matter These Pigments can be divided into three

main groups: chlorophyll, carotenoids, and phycobilins Among these, chlorophyll a,

chlorophyll b, and carotenoids are the most abundant in green microalgae species(Nguyen Thi Kim Dung, 2020)

Chlorophylls absorb light most strongly in the high-frequency and high-energy

wavelengths of 450 to 495 nm, which is in the blue region of the electromagnetic

spectrum In addition, synthetic photosynthetic pigments also absorb frequency, energy wavelengths from 620 to 750 nm, which is in the red region of the electromagneticspectrum Chlorophyll comes in many different forms, with each type having a structure

low-attached to a chlorine ring with a magnesium ion in the center (Donald et al., 2020)

Figure 2.2 Chemical structure of chlorophyll (Mandal and Dutta, 2020).

The most common form of chlorophyll is chlorophyll a, with a molecular formula

of CssH720sNaMg Its structure consists of a porphyrin ring attached to a protein

backbone By replacing the functional groups at positions C2, C3, C7, C8, and the C17 —C18 bond, the structure of a specific chlorophyll can be determined Chlorophyll b, foundonly in green algae and plants, has a molecular formula of CszH;oOsNaMg and is an

accessory photosynthetic pigment It Structure is similar to that chlorophyll a, with the

only difference being at position C-7 of the second pyrrole ring (ring B) Chlorophyll ahas a methyl group (-CH3), while chlorophyll b has a formyl group (-CHO) This change

allows for better absorption of shorter wavelengths Carotenoids are another of accessory

pigments that capture light energy during photosynthesis and also promote biochemical

processes that protect organisms from the harmful effects of sunlight (Donald et al., 2020)

Carotenoids can absorb light in a wide range of wavelengths, from 400 nm to 550 nm.The quantity of pigments extracted from various species may differ based on themicroalgae's traits, cultivation conditions, extraction techniques, and methods of analysis(Parag et al., 2020)

The main pigments found in S obliguus are chlorophyll (a and b) and lutein(Wiltshire et al., 2000) S obliquus also contains significant amounts of carotenoids such

as B-carotene and astaxanthin, which have various industrial applications in aquacultureand pharmaceuticals (Guedes et al., 2011)

2.4.3 Antioxidant compounds

Polyphenols are a group of secondary compounds known for their antioxidantproperties They consist of thousands of compounds with diverse structures, including

phenolic acids, flavonoids, stilbene, and lignans (Rusandica ef al., 2013) These

compounds are characterized by the presence of at least one phenyl ring with one or more

hydroxyl groups (-OH) attached (Vincenzo, 2014) Polyphenols have various biologicalactivities, such as protecting against ultraviolet radiation and pathogen attack, and havebeen linked to potential health benefits, including protection against chronic inflammation,atherosclerosis, cancer, and cardiovascular disorders As a result, it has gained attention inthe fields of medicine, pharmaceutical production, and cosmetics (Rusandica et al., 2013)

2.5 Lipid extraction methods and solvents

Lipids are a diverse group of biological compounds primarily consisting of polar substances such as triglycerides, diglycerides, monoglycerides, and sterols, as well

non-as more polar compounds like free fatty acids, phospholipids, and sphingolipids They

have the ability to covalently bind to carbohydrates and proteins, forming glycolipids andlipoproteins, respectively The notion of "total lipid extract" and "extractable lipid" stemsfrom their capacity to bind with other molecules and the effectiveness of solvent mixtures

in solubilizing different lipid classes Solvents chosen for lipid extraction must possess

high solubility for all lipid compounds and adequate polarity to detach them from their

binding sites with cell membranes, lipoproteins, and glycolipids Several methods have

been developed for total lipid extraction, including the Bligh and Dyer Method (1959),

the Christie Method (1976), Gardner et al.'s approach (1985), the de Boer Method (1988),

Booij and Van den Berg's method (1994), and the Smedes Method (1999) It has been

demonstrated (de Boer, 1988; Randall et al., 1991) that the use of different methods

results in different lipid recovery

Extraction of lipids from biological tissues is a crucial step in lipid analysis The selection

of appropriate solvent is the most critical factor in the efficient extraction of lipids Amixture of polar (to disrupt the protein-lipid complexes) and nonpolar (to dissolve the

neutral lipids) solvents are precisely selected to extract lipids efficiently In addition, the

disintegration of complex and rigid cell-wall of plants, fungi, and microalgal cells by

various mechanical, chemical, and enzymatic treatments facilitate the solvent penetration

and extraction of lipids

Proficiency in extracting lipids from microalgae relies on the utilization of both polar andnonpolar solvents (Abomohra et al., 2016) The Folch method and the Bligh and Dyermethod stand as the two most conventional approaches to lipid extraction These methods

employ solvents such as chloroform and methanol in ratios of 2:1 and 1:2 v/v,

respectively (Guo et al., 2019) Typically, lipid extraction involves the use of solvents

such as ethanol, methanol, chloroform, and hexane The combination of polar and

nonpolar solvents ensures the comprehensive extraction of all lipids, including

free-standing globules Selection of solvents depends on the lipid class targeted for extraction

from algae However, some solvents like chloroform and hexane pose environmental risks

and toxicity, thereby being excluded from large-scale lipid extraction processes (Mubarak

et al., 2015) To expedite and enhance lipid extraction from algae within a shorterduration, solvent extraction processes can be augmented with heat or pressure.Consequently, alternative non-mechanical lipid extraction methods have been devised

2.6 Applications of S obliquus

2.6.1 Biology energy

In recent years, there has been a growing interest in using microalgae as a source

of third-generation biofuels, as an alternative to fossil fuels and fuels derived from food

crops rich in lignocellulose In addition, S obliquus has been found to have the ability to

accumulate large amounts of lipids, which can be converted into biodiesel, and

carbohydrates, which can be fermented to produce bio-ethanol (Oliveira et al., 2020).Previous studies have shown that S obliquus can achieve a conversion rate of 37.92%into ethanol (El-Sheekh et al., 2014) and 90.81% into biodiesel (Guldhe et al., 2015).Additionally, the biomass remaining after lipid or carbohydrate extraction is rich inprotein (Oliveira et al., 2020)

2.6.2 Biological activity

Microalgae are a rich source of bioactive compounds that possess anti-cancer,anti-diabetic, anti-inflammatory, and antioxidant properties (Lauritano et al., 2016;

Novoveska et al., 2019) According to Montone et al (2018), 25 peptides extracted from

proteins found in S obliquus algae have been identified to have antioxidant properties and

the ability to inhibit angiotensin-converting enzymes (ACE) Four of these peptides haveshown significant free radical scavenging activity, ranging from 56% to 70% with 2,2-diphenyl-1-picrylhydrazyl (DPPH)

2.6.3 Wastewater treatment

The traditional method of treating wastewater by removing pollutants is costly,complex, and often results in the production of unwanted by-products (Christenson andSims, 2011; Ruiz et al., 2012) In contrast, the use of microalgae for wastewater treatmenthas gained widespread popularity due to its high efficiency, low cost, and environmental

safety (Christenson and Sims, 2011; Hoffmann, 1998)

2.7 Recent studies on the impacts of nutritional stress on the growth and lipidaccumulation by the microalgae S obliquus

Pham Duy Thanh et al (2017) conducted a study to investigate the effects ofnitrogen and phosphorus deficiency on the oil accumulation process of the microalga

Scenedesmus deserticola This type of microalgae was cultivated in Bold's Basal Medium

(BBM) lacking nitrogen and BBM lacking phosphorus The results revealed that the oil

content in the microalgae under phosphorus-deficient and nitrogen-deficient conditions

was higher compared to that in the BBM environment Specifically, after 7 days of culture,the oil content reached 18.85% and 38.39% of biomass, respectively Analysis of the fattyacid composition of Scenedesmus deserticola algae oil indicated the presence of fattyacids with carbon chains ranging from 12 to 24, along with elevated levels of unsaturated

fatty acids such as oleic acid (10.24%), linoleic acid (7.98%), and linolenic acid (7.82%)

These findings suggest the potential of Scenedesmus deserticola microalgae as a viablesource of raw materials for biodiesel production

Phan Thi Diem My ef al (2020) conducted a study to explore the impact ofvarious culture environments on the growth and carotenoid accumulation of the microalgaTetradesmus obliquus (also known as Scenedesmus obliquus) during its growth phase.Their findings revealed that 7 obliquus exhibited the most robust growth in BGIImedium with nitrogen concentrations of 120 mg/L and phosphorus concentrations of 5.43mg/L, as well as in BGII medium with nitrogen concentrations of 260 mg/L and

phosphorus concentrations of 12.21 mg/L Correspondingly, the growth rates were

measured at 0.298 + 0.01 cells/mL/day and 0.252 + 0.20 cells/mL/day, respectively.Additionally, under these conditions, the total carotenoid accumulation was maximized,with average yields reaching 0.80 + 0.13% and 0.49 + 0.18% of dry biomass, respectively.Furthermore, the study noted that the presence of small amounts of NaCl (0.01 - 0.2M)

had an adverse effect on 7 obliquus during the growth phase, and a concentration of 0.6

M completely inhibited its growth

In 2016, Esakkimuthu ef al conducted a study examining the impact of calcium

(Ca), magnesium (Mg), and phosphorus (P) deficiency and supplementation on thegrowth and lipid accumulation of the microalga Scenedesmus obliquus in a BBM (Bold'sBasal Medium) nutritional medium The addition of magnesium resulted in a notableenhancement in lipid accumulation, rising from 37.5 mg (12.5% of dry weight) to 163.8

mg (54.6% of dry weight) Similarly, calcium deficiency within the culture medium led to

a substantial increase in lipid accumulation, reaching a maximum of 142.6 mg (47.5% of

dry weight) in 300 mg of algal biomass, which was 280% higher than the control (25mg/L CaCl2.2H2O) Furthermore, phosphorus deficiency also contributed to a significant

rise in lipid accumulation, peaking at 159 mg in 300 mg of biomass This represented a

cumulative increase of 323% compared to the control (75 mg/L K2HPOs, and 175 mg/LKH:PO¿)

Cuéllar-Garcia et al (2019) investigated the influence of the carbon/nitrogen ratio

on the total lipid content in algae by adjusting the proportions of NaHCO3 and NaNOs.The research utilized S obliquus cultured in a BBM (Bold's Basal Medium) environmentwithout pH adjustment, under a light intensity of 200 wmol.m-2.s-1, a 12-hour

photoperiod, and a constant temperature of 30°C Results revealed that a ratio of 1,500

g/L NaHCO; and 0.125 g/L NaNOs led to a significant increase in lipid accumulation,

reaching up to 66.0% Furthermore, the study identified the presence of stearic acid

(C18:0, 22.63%) and oleic acid (C18:1, 77.38%) in the algae, with no fatty acidscontaining two or more double bonds detected

Chapter 3 MATERLALS AND METHODS

3.1 Time and location of the study

This study was carried out from August to December 2023 in the EnvironmentalBiology laboratory (BIO 315), Faculty of Biological Sciences, Nong Lam University HoChi Minh city

3.2 Materials and methods

3.2.1 Source of Scenedesmus obliquus

The microalgae S obliquus was obtained from the Institute of AquacultureResearch and Culture 2 (Ho Chi Minh city, Vietnam) Prior to each trial, the microalgae

were inoculated in the Bold's Basal Medium (BBM, Table 3.1) for 12 days to reach anOD680 value of approximately 1.0

Nitrogen and phosphorus were supplemented to the culture media in the forms ofNaNOs, and K2HPOs and KH2POs, respectively

Table 3.1 Bold's Basal Medium (BBM) (CCAP)

3.3 Investigation of different extraction methods.

3.3.1 Soxhlet extraction

Aliquots of 1 + 0.0001 grams of oven-dried S obliquus biomass were weighed,wrapped in the filter papers with pore size 15 - 20 um and placed in a glass cup Then, 50

mL of hexane was added and the samples were sonicated 120W, 40KHz for 30 minutes at

room temperature After sonication, the samples were allowed to soak overnight Next,the samples were extracted using a Soxhlet apparatus for 6 to 8 hours Following

extraction, the solvent extractants were evaporated by a rotary evaporator (Heidolph,Germany) to remove the solvent The residues were oven-dried at 50-60°C for 8 hours

After drying, the samples were cooled down to room temperature in a desiccator, and the

mass of lipid residues were recorded The lipid content was calculated using the following

3.3.2 Ultrasonic-assisted extraction with hexane

Weigh | gram of dry S obliquus algae biomass and wrap it in filter paper, placing

it inside a glass cup To this, add 50 ml of hexane solvent, and subject the mixture tosonication 120W, 40KHz for 30 minutes at room temperature Subsequently, allow the

mixture to soak overnight Filter the resultant extract and repeat this process two

additional times using fresh solvent and ultrasound Combine the extracts obtained from

the three repetitions and transfer the mixture to a flask with a known mass Next, employ

a rotary vacuum evaporator to remove the solvent Finally, recover the product, completethe procedure, and calculate the results in a manner consistent with Method 1

3.3.3 Ultrasonic-assisted extraction with a mixture of hexane and methanol

The procedure begins by weighing | gram of dry S obliquus microalgae biomass.Next, the dry biomass is wrapped in filter paper and placed in a glass cup To this, 50 ml

of hexane:methanol in a 1:1 ratio are added The mixture is then sonicated 120W and40KHz for 30 minutes at room temperature Subsequently, the mixture is allowed to soakovernight before filtering the extract This process is repeated two more times, with freshsolvent added each time and using ultrasound The resulting extracts from the threerepetitions are combined and transferred to a flask with a known mass The solvent is thenevaporated from the mixture using a rotary vacuum evaporator Finally, the product can

be calculated and analyzed using the same method as described in Method 1

3.3.4 Ultrasonic-assisted extraction with a mixture of chloroform and methanol

To carry out the procedure, start by weighing | gram of S obliquus algal biomassand wrap it in filter paper Place the dried biomass in a glass cup and add 50 ml of achloroform:methanol solvent mixture in a ratio of 2:1 Proceed to sonicate 120W and40KHz the mixture for 30 minutes at room temperature After sonication, allow themixture to soak overnight, then filter the extract to separate the liquid from the solid

biomass Repeat this process two more times, adding fresh solvent and utilizing

ultrasound each time to ensure thorough extraction Once the extractions are complete,

transfer the combined extract mixture to a flask with a known mass Use a rotary vacuum

evaporator to evaporate the solvent from the mixture, leaving behind the desired product.Finally, the resulting product can be quantified and analyzed using the same method asdescribed in Method 1

3.4 Investigation of the influences of culture conditions on the growth and lipidaccumulation of the microalgae

3.4.1 Culturing time

In this experiment, four treatments were completely randomly arranged withnitrogen concentrations (NaNOa) of 25%, 50%, 75% and 100% according to the BBM

environmental formula Each treatment was repeated 3 times, a total of 12 bottles Each

flask contained 100 mL of microalgae (density 18.96 x 10° cells/mL) and 2900 mL of

nutrient medium Biomass was harvested at 3 times: 7, 9 and 12 days after culture The

obtained biomass was used for lipid content analysis

Table 3.2 Experimental setup to evaluate the effects of culturing time

No Coding NaNO; concentrations (mg/L)

standard BBM solution and the N-deprived media, respectively The experiment included

five treatments in triplicates, resulting in a total of 15 of 5-L polyethylene terephthalate(PET) containers Each container consisted of 50 mL of microalgal stock biomass

(density 4.9 x 10° cells/mL) and 3900 mL of the corresponding nutrient media.

Table 3.3 Experimental setup to evaluate the effects of nitrogen deficiency

No Coding NaNO; concentrations (mg/L)

in the standard BBM solution and the P-deprived media, respectively The experimentincluded five treatments in triplicates, resulting in a total of 15 of 5-L polyethylene

terephthalate (PET) containers Each container consisted of 50 mL of microalgal stock

biomass (density 4.9 x 10° cells/mL) and 3900 mL of the corresponding nutrient media.

Table 3.4 Experimental setup to evaluate the effects of phosphorus deficiency

K2HPOs and KH2PO4

3.4.4 3-indole acetic acid (LAA) concentrations

In this experiment, the microalgae were cultured in various nutrient media

supplemented with different 3-indole acetic acid (IAA) concentrations (i.e., 0.05, 0.10,0.50, 1 and 2 mg/L) The experiment included seven treatments in triplicates, resulting in

a total of 21 of 5-L polyethylene terephthalate (PET) containers Each container consisted

of 30 mL of microalgal stock biomass (density 18.8 x 10° cells/mL) and 970 mL of the

corresponding nutrient media

Table 3.5 Experimental setup to evaluate the effects of 3-indole acetic acid (IAA)

amendment

IAA

No Coding soncentations NaNOs concentrations

(mg/L) (mg/L)

3.4.5 The response surface methodology - central composite design (RSM-CCD)

Process of optimizing culture conditions for growth and lipid accumulation of

microalga S obliquus using response surface method was performed using response

surface methodology (RSM) combined with central composite design (CCD) (George EP

Box and KB Wilson 1951)

The experimental model was built to investigate the influences of three factors,including nitrogen concentrations (NaNO2) (X11), phosphorus concentrations (KzHPO¿a and

KH2POs) (X›), and 3-indole acetic acid (IAA) concentrations (X3) on the lipid content of

S obliquus The total number of experiments designed is N = 2‘ + 2k + n, with value ơ =

1.0; k is the number of factors; n is the number of repetitions of the mental experience

The design model included 20 treatments with eight combined experiences, six axial

experiences, and six repetitions of the center experience

The variation ranges of the three factors and the treatment matrix are presented in

Table 3.6 and Table 3.7

Table 3.6 Levels of optimization for the three variables

The results were statistically processed using Minitab 21 software to determine

the optimal conditions for the highest accumulation of lipid by the microalga S obliquusand were expressed by a response surface model in the form of a quadratic equation

Table 3.7 Optimization treatment matrix

NaNO; KzHPO¿ and KH>PO, IAA concentration

No concentration (%) concentration (%) (mg/L)

3.4.6 Check optimal conditions

Based on the optimal culture conditions obtained from the surface matrix, anempirical study was conducted using the theoretical estimations from the obtained model,with three replicates The average total lipid content was recorded and compared to the