Khóa luận tốt nghiệp Công nghệ sinh học: Evaluation the effects of NANO3 and NaCl on phycocyanin production by Arthrospira platensis

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATION THE EFFECTS OF NaNO; AND NaCl ON PHYCOCYANIN PRODUCTION BY Arthrospira plat

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATION THE EFFECTS OF NaNO; AND NaCl

ON PHYCOCYANIN PRODUCTION BY Arthrospira platensis

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

UNDERGRADUATE THESIS

EVALUATION THE EFFECTS OF NaNO; AND NaCl

ON PHYCOCYANIN PRODUCTION BY Arthrospira platensis

ADVISOR STUDENT

HUYNH VINH KHANG, PH.D TRAN THI HONG

NGUYEN THI VAN ANH, MSC

Thu Duc City, 03/2024

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

Faculty of Biological Sciences of Nong Lam University Ho Chi Minh for enthusiastically

supporting and creating favorable conditions for me to carry out my thesis ecently

completed my graduation thesis I would like to especially express my sincere thanks to

Ph.D Huynh Vinh Khang and Msc Nguyen Thi Van Anh dedicatedly guided andimparted knowledge and experience as well as supported and created conditions for me interms of facilities, equipment and chemicals throughout the project implementationprocess.

I would like to sincerely thank the teachers of the Department of BiologicalSciences, Biological Sciences of Nong Lam University Ho Chi Minh taught and impartedvaluable knowledge and experience to me throughout the process of studying and

implementing the project Thank you to the student body of BIO315 Environmental

Biology - Faculty of Biological Sciences as well as the DH19SHD class for alwaysencouraging, supporting and helping me during the time of completing my graduationthesis

I would like to thank my parents for not being afraid to work hard and alwayscreating the best conditions for me to study and practice

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, March, 2024

AFFIRMATION AND COMMITMENT

My name is Tran Thi Hong, student ID: 19126055, class: DHI9SHD (Phone:

0358818400, email: 19126055@st.hcmuaf.edu.vn), Faculty of Biological Sciences, Nong

Lam university Ho Chi Minh City I declared that all results presented in this graduate

thesis were conducted by myself All the data and information are entirely accurate andunbiased I fully accept responsibility for these commitments in front of the committee

Thu Duc City, March, 2024Student’s signature

Tran Thi Hong

This study was conducted to determine the concentrations of NaCl and NaNOs,which may increase the accumulation of phycocyanin in Arthrospira platensis In order tounderstand the accumulation of phycocyanin in Arthrospira platensis, researchers

conducted solvent investigations on phycocyanin extracts Solvents of 1.5% CaCh,distilled water, and phosphate buffer 0.1M, pH=7 were investigated at the ratio of 1 g : 10

ml solvent and 1 g : 100 ml solvent The algae sample was washed twice with distilledwater before the experiment After mixing with solvent, the algae sample was frozen for

24 hours at a temperature of 20 °C then defrosted, sonicated, and centrifuged to collect thesupernatant To determine the concentration of nutrients in the culture medium thataccumulate the most phycocyanin, the study used a response surface method (RSM) with

two factors: NaCl and NaNO; concentrations Under optimal conditions, phycocyanin

solution was determined for the antioxidant activity using DPPH reagent Research results

showed that the highest yield of phycocyanin obtained after 3 extractions at the ratio of 1

g : 100 mL was in the solvent 1.5% CaClz 19.71 mg/g And when the concentration of

mnaci was 7.56 g and mnano3 was 0.6 g in the culture medium, the optimal yield ofphycocyanin was 19.65 mg/g The free radical scavenging ability of phycocyanin solutionwas 49.77%, corresponding to 6.15 ppm vitamin C Research results showed that NaCland NaNO; concentrations affect phycocyanin accumulation in A platensis, and can beapplied experimentally in areas where nutrient concentrations are investigated in research

Keywords : Arthrospira platensis, phycocyanin, NaCl, NaNO3

TÓM TẮT

Nghiên cứu này được tiến hành dé xác định nồng độ NaCl va NaNO; có khả năng

làm tăng sự tích lũy phycocyanin trong vi tao Arthrospira platensis Dé tìm hiểu được sự tích liy phycocyanin trong vi tảo A platensis, nghiên cứu đã tiến hành khảo sát dung môi chiết xuất phyccoyanin Các loại dung môi CaCl 1,5%, nước cất, đệm phosphate được khảo sát với tỷ lệ 1 g : 10 ml dung môi và 1 g : 100 ml dung môi Mẫu tảo được rửa sạch

hai lần bằng nước cất trước khi thí nghiệm, sau khi trộn với dung môi, mẫu tảo được đông

lạnh trong 24 giờ ở nhiệt độ 20 °C sau đó được rã đông và đánh sóng siêu âm, ly tâm dé

thu chất nổi phía trên Dé xác định nồng độ các chất dinh dưỡng trong môi trường nuôi cấy tích lũy nhiều phycocyanin nhất, nghiên cứu đã sử dụng phương pháp bề mặt phản

ứng với hai yêu tố: nồng độ NaCl và NaNO: Trong điều kiện tối ưu, dung dich phycocyanin được xác định hoạt tính chống oxy hóa bằng thuốc thử DPPH Kết quả nghiên cứu cho thấy hiệu suất phycocyanin cao nhất thu được sau 3 lần chiết với tỷ lệ 1 g: 100 mL là ở dung môi CaCh 1,5% 19,71 mg/g Và khi nồng độ mnaci là 7,56 g và

mwaNo là 0,6 g trong môi trường nuôi cấy thì năng suất tối ưu của phycocyanin là 19,65 mg/g Khả năng nhặt gốc tự do của dung dịch phycocyanin là 49,77%, tương ứng với 6,15 ppm vitamin C Kết quả nghiên cứu cho thấy nồng độ NaCl va NaNO; ảnh hưởng đến sự tích lũy phycocyanin trong A platensis, và có thé được ứng dụng ngoài thực nghiệm ở

những nơi có nồng độ chất dinh dưỡng được thực hiện khảo sát trong nghiên cứu.

Từ khóa: Arthrospira platensis, phycocyanin, NaCl, NaNO3

PaAFFIRMATION AND COMMITMENT - - - - S- 31s nh HH nh nh re ii

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LIST OF TABLE

PageTable 3.1 Chemical compositions of Zarrouke Gd o.3.3204ssccerxsessanovenanannarsnesvn 16

Tablé.3:2 MictoOnisttrerit 11 OfEdIGHE AS irccsanscasunaaaasarcaranndinanihed ẽ 17

Table 3.3 Levels of optimization experimental facfOFS -. - 19

Table 3.4 Optimized treatment matrIX -<+ 20

Table 4.1 Phycocyanin yields (mg/g) across 13 treatments for optimization of the culture

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Table 4.2 Results of the ANOVA test of the effects of two factors on the objectiveTROON: 1: ceseerereseenenccnenemnamneoaaw RTE Ee 30

viii

LIST OF FIGURES

Figure 2.1: Crystal Structure of C-Phycocyanin from Arthrospira pÏafensis 9

Figure 4.1 The phycocyanin yields in three extraction cycles of A platensis freshbiomass with CaCl, phosphate buffer, and wafer -‹-< 22

Figure 4.2 Phycocyanin yield of three investigation solvents (ratio 1 g algae: 100 mLsolvent) alter threeiextractiOns': « gà m go 86 8ã 686 GuUBgBSGBEBQE SE ĐÃ Suilà ea ER CR kh 23

Figure 4.3 Total phycocyanin yield of three investigation solvents (ratio 1 g algae: 100

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Figure 4.4 The phycocyanin extracts with three different solvents 25

Figure 4.5 Microscopic images of A platensis biomass extracted with three different50) 06) 0 ks ce 27

Figure 4.6 Optical density chart of 13 experiments in 10 days of culture 28

Figure 4.7 Optimal culture conditions as proposed by Minitab 21 software 31

Figure 4.8 Ascorbic acid's free radical scavenging ability in DPPH test 34

Chapter 1 INTRODUCTION

1.1 Problem statement

Among microalgae, Arthrospira platensis (also known as Spirulina platensis) standsout as one of the most promising This is due to its valuable biological content and itscontribution to green production chains, such as wastewater farming, which can helpreduce carbon dioxide emissions and create biofuels, etc (Lim, HR et al., 2021), andhigher sustainability, strong adaptability to different environments, high yield, synthesis

of high-value biological compounds (Subudhi, S et al., 2023) A platensis is a celled cyanobacterial microalgae that grows in a spiral-shaped filamentous structure andthrives at a pH range of 9 to 12 The Arthrospira family is rich in bioactive compounds,such as proteins, lipids, carbohydrates, microelements (zinc, magnesium, manganese,selenium), pigments, and amino acids Therefore, the microalgae Arthrospira is a goodcandidate for food, pharmaceuticals, biofertilizers, and pigment applications The maincomponents of lipids are omega-6 family y-linolenic acid (18:3, n-6) and palmitic acid(16:0), both of which are known for their medicinal potential to prevent cardiovasculardiseases, hypercholesterolemia blood and other disorders (Ragusa e/ ai, 2021) Amongvarious proteins found in the A platensis cells, the most abundant proteins, which accountfor approximately about 60% of dry weight, are phycobiliproteins, specificallyphycocyanin, allophycocyanin, and phycoerythrin A platensis also contains highamounts of natural pigments, including 14% phycocyanin (blue), 1% chlorophyll (green),and 0.5% carotenoids (yellow, orange, or red pigments) (Jung Friedrich ef al., 2019)

single-C-phycocyanin (Phycocyanin from Cyanobacteria, C-PC) is a blue and

water-soluble photosynthetic pigment used as a natural dye in foods, cosmetics, and medicine

In addition, there is an increasing interest in C-PC due to its potential for drugdevelopment Phycocyanin eliminates free radicals and has outstanding antioxidant and

anti-inflammatory effects Moreover, it can effectively inhibit diseases caused byinflammation.

Microalgae can accumulate various amounts of metabolites under the nutritionalstress conditions (i.e rich or lack of nutrients) (Manirafasha et al., 2017) Zeng andVonshak (1998) demonstrated that changing environmental factors such as nitrate,

phosphate, salt concentration, temperature, etc., strongly affected the biomass production

of A platensis High salt contents slowed down the photosynthetic systems I and II of

Arthrospira, leading to a decrease in starch accumulation (Shipton and Barber, 1994).Nitrates are essential for the biosynthesis of organic molecules, such as proteins andcarbohydrates, which play an important role in cell growth and biomass yields Previousstudies have reported that C-phycocyanin production in A platensis is affected by thenutrient compositions and concentrations in the media, especially nitrate, which can lead

to a reduction or inhibition of C-phycocyanin biosynthesis (Manirafasha e/ al., 2018;Mohy El-Din, 2020)

Therefore, this study aimed to investigate the influences of different nitrate and salt

concentrations in the culture media on biomass and phycocyanin production by themicroalgae A platensis

Content 2: Investigate the effects of different sodium nitrate (NaNO3) and sodiumchloride (NaCl) concentrations on the production of phycocyanin by A platensis.

Content 3: Investigate the antioxidant activity of phycocyanin extracts obtainedfrom the Content 2

Chapter 2 LITERATURE REVIEW

2.1 Overview of Arthrospira platensis

Arthrospira platensis, also known as Spirulina, is blue-green, filamentous,

spiral-shaped, multicellular, photosynthetic bacterium It is believed to be the oldest living plant

on Earth, dating back to 3.6 billion years ago (Koru, E (2012) This ancient creature was

the first photosynthetic life form that created our oxygenated atmosphere Although

commonly known as blue-green algae, A platensis has been used as a food source sinceancient times First reported in 1852 by Stizenberger, many other species of spiral-shaped

cyanobacteria in this genus have been described and isolated And Dangeard (1940),

Brandily (1959), and Léonard and Compére (1967) all recorded how African tribes livingalong Lake Chad collected and processed this algae into a daily food source Biomass is

harvested from the waters near the lake and then dried on the shore to create hard blackcakes called “dihé” (Sili, C., et al., 2012)

2.1.2 Classification

There are two types of filamentous cyanobacteria: Arthrospira maxima and

Arthrospira platensis Arthrospira maxima has a long and spiral shape and is commonly

found in freshwater environments On the other hand, Arthrospira platensis has a shorter

and rounder shape, making it better suited for survival in saltwater environments (Truong,P.N., et al., 2019) A platensis belongs to (Gomont, 1892):

Species: Arthrospira platensis

2.1.3 Structural morphology

A platensis is a filamentous photosynthetic cyanobacterium with a diameter ofapproximately 8 pm It is characterized by spiral filaments and multicellular structures,which are linked together to form trichomes that can reach lengths of up to 500 um

Arthrospira undergoes cell division for elongation but multiplies through fragmentation

(Chaiyasitdhi, A., et al., 2018)

2.2 Nutritional compositions of Arthrospira platensis

A platensis has been consumed by humans for thousands of years and isconsidered a functional food due to its diverse range of nutritional compounds Theseinclude antioxidants, minerals, fiber, proteins (up to about 70% by dry weight), and aminoacids, vitamins, and pigments such as chlorophyll, phycobiliprotein, and carotenoids

(Marzarati, S., et al, 2020) These properties make it a popular ingredient in health foods,cosmetics, pharmaceuticals, and even fuel production (Ashaolu, T.J, et a/, 2021)

2.2.1 Carbohydrates

Important components of cyanobacterial and microalgal biomass include

carbohydrates and lipids (De Philippis, R & Vincenzini, M., 1998; Materassi, R., et al.,

1980) These two compounds are crucial for energy storage and play a significant role in

an organism's ability to adapt to its environment (Chen, Y., et a/., 2013) Fats and oils are

the primary forms of energy storage in many organisms, while phospholipids,triglycerides, and sterols are essential structural components of biological membranes.Although present in smaller quantities, other lipids serve as enzyme cofactors, electroncarriers, light-absorbing pigments, and intracellular messengers, playing important roles

in various biological processes Furthermore, fatty acids are commonly used by organisms

to store energy in the form of fats and oils (Lehninger, AL, et al., 2005)

2.2.2 Protein

The level of total protein in a cell can indicate its rate of metabolic activity,particularly in cells that are actively growing Environmental stress can affect thedistribution of these components among cells (Chen, Y., et a/., 2013) Arthrospira algae,for example, can have a protein content as high as 60-70% when grown without nitrogen

(which is often stored as protein) This makes them a valuable source of food protein forboth humans and animals When cell membranes lack cellulose, they become easilydigestible and absorbable by the human body (Sassano, CEN, e¢ al., 2010)

2.2.3 Vitamins, minerals, and fatty acids

A platensis is a widely used source of both medicinal and nutritional benefits forboth humans and animals Numerous studies have shown that A platensis is renowned forits ability to scavenge free radicals and its strong antioxidant activity, thanks to its main

antioxidants such as vitamins, b-carotene, carotenoids, and vitamin E This species alsocontains a variety of vitamins, including vitamin B12 (8 ppm), B1, B2, and pro-vitamin A

(0.2%), as well as vitamin C and vitamin E Additionally, A p/a/ensis is rich in mineralssuch as iron (0.1%), calcium, copper, and magnesium It also contains high amounts ofamino acids and essential fatty acids, making it a valuable dietary source of y-linolenicacid, an essential unsaturated fat This omega-6 fatty acid (18:3, n-6) and palmitic acid(16:0) have been shown to have potential pharmaceutical benefits in preventing

cardiovascular diseases, hypercholesterolemia, and other disorders (Ragusa, L, et al.,2021)

2.2.4 Pigments

In addition to being rich in nutrients, A platensis 1S also a valuable source of plantpigments These pigments include chlorophyll a, water-soluble pigments such asphycoerythrin (PE), allophycocyanin (A-PC), and phycocyanin (C-PC), as well aslipophilic pigments like carotenoids (B-carotene and cryptoxanthin) (Vernès e/ al., 2015).The presence of these pigments in Arthrospira has been found to have numerous health

benefits, including anti-obesity, anti-inflammatory, anti-cancer, antioxidant, andneuroprotective effects (Pameswari & Lakshmi, 2022) These effects are attributed to the

presence of various pigment components in Arthrospira, such as phycobiliproteins,

phenolic compounds, B-carotene, and polyunsaturated fatty acids (PUFAs)

2.2.5 Chlorophyll

Chlorophyll is a non-polar green pigment containing a porphyrin or hydroporphyrin ring with a central magnesium atom bound to it and is found in all autotrophicalgae because of its ability to convert light into bioenergy (Osorio, C et al., 2020)

Arthrospira contains high amounts of chlorophyll, especially chlorophyll a (De

Morais, MG, & Costa, JAV, 2007) Considerations regarding chlorophyll in the foodindustry often focus on preventing the degradation of these compounds during storage andprocessing steps (Hynstova, V., et al., 2018)

Chlorophyll is a green pigment that is found in all autotrophic algae It contains aporphyrin or hydroporphyrin ring with a central magnesium atom bound to it, and it isresponsible for converting light into bioenergy (Osorio, C et al., 2020) Arthrospira, inparticular, contains high levels of chlorophyll, specifically chlorophyll a (De Morais, MG,

& Costa, JAV, 2007) In the food industry, there is a focus on preserving the integrity of

chlorophyll during storage and processing to prevent degradation (Hynstova, V., et al.,2018)

2.2.6 Carotenoids

Carotenoids are natural lipid-soluble pigments responsible for the red, yellow, andorange colors found in many plants and microorganisms (Park, WS, e/ z/., 2018) They

play a crucial role in regulating plant growth and development, acting as photosynthetic

pigments, hormone precursors, and attractants for other species during pollination and

seed distribution The most interesting feature of carotenoids is the alternating

7

arrangement of single and double bonds in the central part of the molecule (Hynstova, V.,

et al., 2018) While humans and other animals cannot synthesize carotenoids themselves,they can obtain them through food metabolism (Park, WS, et al., 2018) Out of the 50

compounds with pro-vitamin A activity, three are particularly important for the humandiet: a-carotene, B-cryptoxanthin, and B-carotene Some carotenoids found in foods can

provide pro-vitamin A, which is converted to vitamin A in the body (Hynstova, V., et al.,

2018) The role of carotenoids in humans and animals is becoming increasingly clear,

with evidence suggesting that these pigments may protect against serious disorders related

to oxidative stress, age-related skin, cardiovascular and eye diseases, and certain types ofcancer (Park, W.S., e ai, 2018)

pigment found in A platensis, making up more than 20% of its dry weight This pigment

is composed of two subunits, the œ chain and the B chain, which are both rich in a-helices

The o chain has one phycocyanobilin attached at cysteine 84, while the B chain has twophycocyanobilins attached at cysteines 84 and 155 (Figure 2.1)

Figure 2.1 Crystal Structure of C-Phycocyanin from Arthrospira platensis.

( Padyana, AK, et al., 2001)Phycobiliproteins are water-soluble, highly fluorescent protein complexes that can

be easily isolated These proteins are brightly colored and can be classified into threegroups: phycocyanin (dark green), phycoerythrin (dark red), and allophycocyanin (blue)

(Jian-Feng, N.I.U, et al., 2007)

One of the advantages of these proteins is their stable color and rheologicalproperties, making them a potential replacement for synthetic dyes In recent years, therehas been a growing interest in the use of phycobiliproteins as nutraceuticals, particularly

in health foods This is due to their antioxidant and free radical scavenging activities,which have potential health benefits Research has shown that phycobiliproteins may have

therapeutic potential in the treatment of diseases related to oxidative stress In addition totheir use in nutraceuticals, phycobiliproteins also have applications in diagnostics Theapoprotein chains of these proteins contain amino and carboxyl groups that can formbonds with other molecules, such as immunoglobulin, protein A, and avidin This makes

them useful as fluorescent probes for cellular and molecular analysis, and they arecommonly used in techniques such as histochemistry, fluorescence microscopy, flowcytometry, and immunofluorescence assays (Vernés, L., et al., 2015).

In the extracted form, the concentration of these water-soluble molecules is noteasily determined by chromatographic methods suitable for solvent-soluble pigments The

concentration of phytochemical compounds was determined by UV-Visspectrophotometry (Sobiechowska-Sasim, M., et al., 2014) The concentration and purity

of phycocyanin were determined spectrophotometrically as described by Bennet and

Bogard (1973) The concentration was calculated according to the formula below usingthe absorbance at 620 nm and 652 nm using a spectrophotometer

highest growth rate of 0.091 day ! was observed at 35°C, but as the temperatureincreased, the growth rate decreased For instance, at 40°C, the doubling rate dropped to

0.041 day !, which is almost half of the growth rate at 35°C Moreover, pigment

accumulation is also affected by high temperatures When the temperature exceeds 35°C,

there is no growth and the microalgae start to lose their pigmentation At 35°C, the culturemedium contains the highest levels of chlorophyll a, carotenoids, and phycobiliprotein(Kumar et al., 2011)

10

2.3.2 Light

Studies have shown a correlation between the growth of microalgae (including

productivity, maximum volume concentration, and growth rate) and light sources thatemit higher levels of protons in the red spectral range (specifically at 660 nm) This isbecause the presence of chlorophyll b in the optical system is effective in absorbingprotons at this wavelength (Wang, C.Y., et a/., 2007) However, for A platensis and other

cyanobacteria that lack chlorophyll b in their photosynthetic system, their light absorptionspectrum is expected to be influenced by other accessory pigments such as betacarotenoids, phycoerythrin, and phycocyanin (with a wavelength of approximately 620nm) These pigments are able to capture light components closer to the red region of the

spectrum, rather than the blue region (Schulze, P.S., et al., 2014)

2.3.3 pH

The growth and synthesis of substances in A platensis are significantly influenced

by the pH of the growth medium While this species is able to tolerate a wide pH range(from 8.5 to 9.5), its growth is hindered when the pH exceeds 10, causing the cells tobecome pale This is due to the fact that high pH levels inhibit the synthesis of essentialnutrients, such as chlorophyll and carotenoids, which are crucial for algae growth (Del

Campo, J.A., et a/., 2000) In extreme pH conditions, such as 10.5 and 11.0, all cellularsystems are unable to function properly, leading to a complete halt in algae growth andthe breakdown of protective systems As a result, biomass production is significantlyreduced, pigment content is low, and phenolic content decreases (Ismailel., et al., 2016)

2.4 Recent studies on nitrate and salt stress on biomass and phycocyanin production

by A platensis

In 2017, Nhung et al studied and applied a method for measuring the UV-Vis

optical absorption index and SDS-PAGE denaturing electrophoresis to evaluate thequality of phycocyanin Their results showed that purifying phycocyanin from fresh algae

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using a two-step process (precipitation with ammonium sulfate and chromatographythrough an LH20 column) resulted in a purity of A 620/280 of 1.2, making it suitable foruse in the food and chemical cosmetics industry.

According to Trung et al (2019), the concentration of NaNO; in the culture mediahad a significant impact on the growth and protein accumulation of Arthrospira Theyfound that increasing the concentration of NaNOa in the Zarrouk medium from 1.25 g/L

to 5 g/L led to an increase in biomass, specific growth rate, and total protein content of

Arthrospira The highest biomass (0.60 g/L) and protein content (34.41%) were achieved

with a NaNO; concentration of 5.0 g/L, compared to lower concentrations of 1.25 g/L and2.5 g/L Algae grow well and accumulate a large amount of protein and amino acids under

cultivation conditions with a NaNO3 concentration of 5.0 g/L

Pan-utai et al (2019) conducted a study on the preparation and concentration ofArthrospira platensis biomass using various methods such as physical drying, and freeze-

drying with the aim of extracting and purifying C-phycocyanin (C-PC) The mosteffective processing conditions for C-PC extraction and purification were found to bedrying the biomass in an oven at 70°C for 4 hours, followed by extraction at 25°C for 24hours using a phosphate buffer of 0.01M and assisted homogenization at a biomass

concentration of 0.02 g/mL This resulted in a C-PC purity of 0.67 and an antioxidantactivity of 54% The purified C-PC was further treated with activated carbon (70-80 g/L

for 24 hours) to achieve a purity index of 1.2, which yielded a satisfactory yield of

approximately 80% This increase in purity index also helped to reduce the unpleasant

odor of proteins in the solution

The study conducted by Chaiklahan, R., et al., (2022) focused on the cultivation of

A platensis using a semi-continuous mode to maintain the cell concentration at an opticaldensity (OD) of 0.4, 0.6, and 0.8 The results showed that the highest biomass yield (0.62

gL! d') was achieved when the cells were grown under a light intensity of 2300 umol

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approximately 2.26 - 2.31 g (KW/h)! đ 2 s1 at OD 0.8 This highlights the significant

s at OD 0.6 At this concentration, the energy consumption for algae biomass was

impact of light intensity, cell concentration, and light-dark conditions on enhancing

biomass and phycocyanin production in a well-processed BP photobiological reactor, with

the most optimal results observed at light intensities of 635, 980, 1300, and 2300 umol/m

and a phycocyanin production of 123 mg/L Additionally, the maximum photosyntheticefficiency achieved was 8.02% under a light intensity of 635 umol/m

Freire Balseca (2024) investigated how two nitrogen sources for the Zarrouk

environment (urea and potassium nitrate) affect the accumulation of phycocyanin in the

microalga A platensis, to reduce production costs on a large scale A platensis cannot fix

Na so when nitrate enters the cell, it is reduced to nitrite by the enzyme nitrate reductase(Nar) and then converted to ammonia via the enzyme nitrite reductase (Nir) And urea ishydrolyzed by the enzyme urease found in A platensis The products obtained from thisenzymatic process are ammonia and carbon dioxide Ammonia end products will also be

incorporated into the protein structure However, high ammonia concentrations will

inhibit the assimilation of nitrogen sources leading to cell toxicity and death due toinhibition of the photosynthetic system, and adverse effects on biomass production,protein, and phycocyanin production Freire Balseca (2024) showed that in treatmentswith high initial urea concentrations, ammonia concentrations were proportional andphycocyanin production was negatively affected This indicates that low ureaconcentration (0.098 g/L) and high KNO3 concentration (3.1 g/L) have a positive effect on

production Based on the described findings, the study concluded that higher ureaconcentrations are harmful to A platensis growth and phycocyanin production

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Chapter 3 MATERLALS AND METHODS

3.1 Research time and location

This study was conducted from August to December 2023 in the EnvironmentalBiology laboratory (BIO 315), Faculty of Biological Sciences, Nong Lam University Ho

Chi Minh city

3.2 Research materials

A platensis stock culture was obtained from the Aquaculture Research Institute 2(Ho Chi Minh city, Vietnam) and stored at 4 °C Before each experiment, the microalgaewas inoculated for 10 - 14 days in Zarrouk medium (Table 3.1 and Table 3.2) in 500 mL

glass containers to achieve the optical density (OD) of approximately 1.0 at 680 nm Allcontainers were continuously aerated (2.7 L/min) and maintained at 30 + 2 °C under

continuous white fluorescent illumination at an intensity of 3500 lux The light bulbs were

placed at a distance of 30 cm from the glass containers

Table 3.1 Chemical compositions of Zarrouk medium

K2HPO4.3H20 (CAS#16788-57-1, China), NaCl (CAS#7647-14-5,

MgSOu.7H2O (CAS#10034-99-8, China),

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China),

CaCl2.2H20 (CAS#10035-04-8, China),

FeSOx.7HzO (CAS#7782-63-0, China), NaNO; (CAS#7631-99-4, China), K2SO,(CAS#7778-80-5, China), EDTA-Na (CAS#6381-92-6, China), NaHCO; (CAS#144-55-

8, China), H3BO3 (CAS#10043-35-3, China), MnSOz.7H20 (CAS#10034-96-5, China),

ZnSO4.7H20 (CAS#7446-20-0, China), CuSO4.SH2O (CAS#7758-99-8, China),NaMoO¿.2HzO (CAS#7631-95-0, China), KOH (CAS#1310-58-3, China), NaHzPOa.HzO

(CAS#13472-35-0, China), NaxHPOx.12H»O (CAS#10039-32-4, China)

3.3.2 Laboratory tools and equipment

Refrigerators, ultrasonic generators (WUC-32, China), molecular absorption

spectroscopy equipment (Model 752N, England), centrifuge (DLAB DM0412, China) andlaboratory instruments

3.4 Investigation of the suitable solvents for the extraction of phycocyanin from A.platensis

The experiment was conducted under room temperature of 30 + 2 °C, at a light

intensity of 3500 lux, continuous aeration (2.7 L/min), and continuous illumination Thelight intensity was measured with a Tenmars TM - 209 light meter, and the solution pH

was adjusted to 9 - 10, which are optimal for the growth of A platensis (Ogbonda et al,

2007), using 2M KOH or 2.55% HCI solution when necessary Each container contained

470 mL of the Zarrouk medium and 30 mL of the microalgae The microalgal suspensionwas cultivated for 10 days, during which 30 - 50 mL of the culture medium were added toeach container every 2 days to compensate for water loss due to evaporation and providenutrients for the microalgae growth

The OD values of the microalgal suspension were checked on days 3, 5, 7, and 10.The microalgal biomass was then harvested and subjected to phycocyanin extraction

using different solvents

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3.4.1 Investigation of phycocyanin extraction solvent

a Principle: Phycocyanin, a pigment found in algae, can be extracted using

solvents and ultrasound waves The concentration of phycocyanin in the solution can then

be measured using UV-Vis spectroscopy

The microalgae suspension was centrifuged (4000 rpm) at room temperature and

washed twice with distilled water Then, | g of fresh sample and solvent were sequentially1.5% CaCl2 solution, phosphate buffer pH=7; 0,1M and distilled water at a ratio of 1 galgae: 10 mL solvent and a ratio of 1 g algae: 100 mL solvent into separate falcons Thetubes were then frozen at -20 °C for 24 hours After thawing, the sample was sonicated at

120 W for 10 minutes, 20 °C and then centrifuged at 4000 rpm for 10 minutes, threetimes The supernatant was collected

b Determination of Phycocyanin content

The concentration of phycobiliprotein (specifically C-phycocyanin) was

determined using UV-Vis spectrophotometry (Bennett and Bogorad, 1973; Silveira et al.,

2007) The extracts in the solvents were measured for absorbance at wavelengths of 620and 652 nm The concentration and content of phycobiliprotein were then calculated using

the following formula:

Bennett and Bogorad equation, 1973:

PC (mg/mL) = (OD s¿o— 0.474 x OD 652) / 5.34

Silveira equation et al., 2007:

PCC (mg/g) = (PC (mg/mL) x SV (mL)) / m biomass (g)

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Where: ODs¿o, OD 652: optical density at wavelengths 620, and 652 nm,

respectively; PC: Phycocyanin concentration in the extract (mg/mL); PCC: Phycocyanin

content in the extract (mg/g)

3.4.2 Effects of different sodium nitrate (NaNOs) and sodium chloride (NaCl)

concentrations on the production of phycocyanin by A platensis

3.4.2.1 Design of the response surface treatment matrix

The experimental model was implemented to investigate the impact of two factors:NaCl (Xi) and NaNO; (X2), on phycocyanin accumulation in microalgae This wasachieved through CCD statistical analysis using MiniTab 21 software The total number

of experiments designed was N = 2‘ +2k + n, with a = 1.0; k is the number of factors; n

is the number of repetitions of the mental experience The design model consists of 13

treatments, including four combination treatments, four axial treatments, and five

repetitions of the mental experience The factors selected for optimization were theconcentration of NaCl and NaNO: concentration The ranges of variation for these twofactors and the treatment matrix are presented in Table 3.2 and Table 3.3, respectively

Table 3.2 Levels of optimization experimental factors

Table 3.3 Optimized treatment matrix

No NaCl concentration NaNOs3 concentration

According to the optimized formula matrix design in Table 3.3, the extraction and

quantification of phycocyanin are performed as Content 1, and the investigated solvents

and ratios are apply to Content 1 This result is statistically process on Minitab 21 to

determine the optimal conditions for the growth process of microalgae, namely the

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highest accumulation of phycocyanin, and expressed in a response surface model in theform of a quadratic equation.

3.4.2.2 Testing optimal culture conditions

Two optimal extraction conditions were established and conducted experimentally.The average phycocyanin yield of three replicates will be recorded and evaluated for

significant differences with the predicted value obtained from the regression modelthrough a /-/es¿ at the 5% significance level

3.4.3 Investigation of the antioxidant activity of the phycocyanin extracts

a Principle: Antioxidant activity is investigated through the ability to neutralize1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals (Blois, 1958; Bondet et al., 1997).This method is based on the reaction of neutralizing free radicals and reducing the colorintensity of the DPPH reagent of substances with antioxidant effects The change in colorfrom purple to yellow of DPPH corresponds to the free electron of DPPH pairing with ahydrogen atom from the antioxidant to form reduced DPPH-H Ascorbic acid was used asreference material

b Study on the antioxidant activity of phycocyanin extract

The formula for calculating the percentage of antioxidant activity is as follows:

%AA = (OD - ODs)/ODe x 100

In the formula, OD, is the absorbance of DPPH and ethanol solutions; OD, is the

optical the absorbance of the C-PC and DPPH solutions at 517 nm; AA is a free radicalscavenging activity (%)

A linear regression correlation equation was constructed based on the percentage ofDPPH free radical removal activity: y = ax + b, where the y-axis represents the percentage

of DPPH free radical removal activity and the x-axis represents the sample concentration

Then, this equation is used to determine the antioxidant activity of phycocyanin extract

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The antioxidant reaction was performed according to the method of Chanda andDave (2009) with some modifications Briefly, aliquots of 0.5 mL of the reaction reagent

ascorbic acid were added into the test tubes containing 3 mL of 96% ethanol, followed by

the addition of 1 mL of 0.5 mM DPPH and the mixture was thoroughly mixed Themixture was left to stand in the dark for 30 minutes and the absorbance was measured at

517 nm A negative control sample was prepared by replacing the test solution withdistilled water The positive control is ascorbic acid dissolved in distilled water at a

concentration of 10 - 50 mg/L

3.5 Data analyses

One-way ANOVA with Tukey’s HSD post-hoc tests (p < 0.05) using Minitab 21software were performed to determine significant differences among treatments (p <0.05) The data are presented as mean + SD of three replicates Graphs were created usingExcel 2010 software

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Chapter 4 RESULTS AND DISCUSSION

4.1 Solvents for extraction of phycocyanin in A platensis

The fresh biomass of A platensis was extracted by different solvents, including 1.5

% CaCl solution, 0.1 M phosphate buffer (pH = 7) (Dianursanti, D., et a/, 2021), anddistilled water at a ratio of 1g biomass per 10 mL of each solvent with ultrasound-assistedextraction after three extraction cycles The phycocyanin yields in A platensis extracts arepresented in Figure 4.1

C-PC Yield (mg/mL)

Solvents

Figure 4.1 The phycocyanin yields in three extraction cycles of A platensis

fresh biomass with 1.5% CaCh, phosphate buffer, and water In the same

solvent, different letters have statistically significant differences (p< 0.05).

The yields of phycocyanin when extracted with 1.5 % CaCl solution after threeextractions were 3.41 + 0.04; 2.62 + 1.03; 2.54 + 0.41 mg/g, respectively For distilledwater, the yields were 1.12 + 0.59; 0.56 + 0.25; 0.76 + 0.56 mg/g, respectively Forphosphate buffer solution, the yields were 2.41 + 0.49; 3.35 + 0.21; 3.32 + 0.30 mg/g,respectively There was no represent statistically significant difference among the threesolvents after three extractions (p<0.05) After three extractions at the ratio of 1g algae: 10

22

mL solvent, the phycocyanin in all three solvents was not exhausted and needed to beinvestigated at a higher solvent ratio.

Figure 4.2 Phycocyanin yield of three investigation solvents (ratio 1 g algae:

100 mL solvent) after three extractions 7n the same solvent, different letters

have statistically significant differences (p < 0.05)

Figure 4.2 show the results of phycocyanin contents in A platensis biomass extractedwith ultrasound-assisted extraction after three extraction cycles at a ratio of 1 g algae per

100 mL of each solvent After three extractions with 1.5% CaCl2 solution, phycocyanincontents were 14.38 + 3.31; 4.28 + 1.52; 2.15 + 0.55 mg/g, respectively When using

distilled water, the yields were 2.97 + 0.77; 10.68 + 1.68; 1.01 + 0.57 mg/g, and withphosphate buffer solution, the corresponding yields were 0.92 + 0.18; 9.78 + 0.11; 3.08 +1.05 mg/g, respectively Phycocyanin yields in the first extraction with 1.5% CaClz solutionwere significantly higher than those obtained in the second and third cycles And

phycocyanin yields in the second extraction with distilled water and phosphate buffersolution were significantly higher than those obtained in the first and third cycles

23