the effect of ph, dark – light cycle and light colour on the chlorophyll and carotenoid production of spirulina sp.

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY THE EFFECT O

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY

THE EFFECT OF pH, DARK – LIGHT CYCLE AND LIGHT COLOUR ON THE CHLOROPHYLL AND CAROTENOID PRODUCTION OF

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APPROVAL

SUPERVISOR STUDENT

Ass Prof NGUYEN HUU HIEP NGUYEN THI HUYNH NHU

Can Tho, May 2013

PRESIDENT OF EXAMINATION COMMITTEE

Spirulina sp., multicellular filament algae, is helically coiled This is a rich nutrition microalgae with protein, carbohydrate, vitamin, chlorophyll and carotenoid Many researches and applications of Spirulina sp have been studied by interested scientist, especially, pigment production The purpose

of this research was determination the effect of pH, dark – light cycle and light colour on the growth and pigment production of Spirulina sp The results showed that pH = 9, white light and continuous illumination 24/24 hours were appropriate conditions for biomass increasing, chlorophyll a, chlorophyll b and carotenoid production in Spirulina sp

At pH = 9, biomass and carotenoid were highest in day 8 (0.16 g/ 50 mL and 1.43 µg/mL, respectively), the highest production of chlorophyll a and chlorophyll b were collected in day 12 (2.72 µg/mL and 3.35 µg/mL) The growth of Spirulina sp was slow at green colour and limited at red colour

Compared to 12/24 hour illumination, growing algae under continuous illumination 24/24 hour was higher 1.08 times in biomass, 2.36 times in chlorophyll a, 1.2 times in chlorophyll b and 1.7 times in carotenoid When white light was applied, continuous illumination 24/24 hour and aeration, pH remained from 10 to 10.8, decreased in day 16 and 20 and no significant differences between treatments during the experiment Besides, Spirulina algae recovered quickly in appropriate conditions

Key words: biomass, carotenoid, chlorophyll a, chlorophyll

b, Spirulina sp

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CONTENTS APPROVAL

ABSTRACT i

CONTENTS ii

1 INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives 2

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.1.1 Time and location 3

2.1.2 Tools and equipments 3

2.2 Methods 3

2.2.1 Microalgae biomass increasing, chlorophyll and carotenoid extraction method 3

2.2.2 Study the effect of pH on Spirulina sp chlorophyll and carotenoid production 5

2.2.3 Study the effect of light colour on Spirulina sp chlorophyll and carotenoid production 5

2.2.4 Study the effect of dark – light cycle on Spirulina sp chlorophyll and carotenoid production 6

2.2.5 Study the growth recovering of Spirulina sp 6

2.2.6 Data analysis method 6

3 RESULTS AND DISCUSSIONS 7

3.1 The effect of pH on Spirulina sp chlorophyll

and carotenoid production 7

3.2 The effect of light colour on Spirulina sp chlorophyll and carotenoid production 9

3.3 The effect of dark – light cycle on Spirulina sp chlorophyll and carotenoid production 13

3.4 The growth recovery of Spirulina sp 18

CONCLUSIONS AND SUGGESTIONS 20

4.1 Conclusions 20

4.2 Suggestions 20

REFERENCES 21

1

1 INTRODUCTION

1.1 Introduction

Microalgae has been chosen as food for many years (Jensen,

2011) Spirulina sp contains bio - elements such as beta -

caroten, vitamine E, carotenoid, chlorophyll and phycocyanin pigment which can prevent oxidation, old age and cancer About the structure, the width is 6 – 12 µm, length is 0.5 – 1 µm, cylinder cell The algae can change from curly to helically coiled base on hydration and dehydration of oligopeptide in

peptidoglycan (Genene Tefera, 2009) Spirulina sp dry biomass

contains 60 – 70 % protein, more than 40% essential amino acid but small nonessential amino acid, sulphur such as methionine

and cysteine (Borowitzka, 1988) Beside that Spirulina also

contain vitamin A, B1, B2, B3, B12 and minerals such as iron, phosphor, magie and calci…(Pandey et al., 2010) Moreover, chlorophyll is a photosynthesis pigment which only find in autotrophic organisms or algae, chlorophyll content depends on biomass production (Norbert Wasmund, 2006) Carrotenoid is provitamine A which prevent natural oxidation (Goodwin, 1980) The accumulation and isomer of β - carotene were controlled by light intensity and quality (Senger et al 1993) Temperature plays

an important role in the growth of algae, biomass production, protein and chlorophyll concentration (Pandey, 2010) According

to Dylan (2011), Spirulina sp growth well at pH = 9 - 11 High

pH leads to prevent the infection of other green algae (Richmond

et al., 1982)

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2 MATERIALS AND METHODS

2.1 Materials

2.1.1 Time and location

Time: from January 2013 to April 2013

Location: Laboratory of Microbiology, Biotechnology Research and Development Institute, Can Tho University

2.1.2 Tools and equipments

Microalgae: Spirulina sp was received from Microbiology

Laboratory from Biotechnology Research and Development Institute

Laboratory equipments: Sterile cabinet, cabinet for incubating algae, Autoclave, Microscope, Centrifuge, Electronic scales, Micropipette

Chemicals: acetone, alcohol 90, alcohol 70, Zarrouk media (Zarrouk, C 1966 and Bharat Gami et all, 2011): NaNO3 (2,5 g/L), NaCl (1g/L), MgSO4.7H20 (0,2 g), CaCl2.H2O (0,04 g/L), FeSO4.7H2O (0,01 g/L), Na2EDTA (0,08 g/L), NaHCO3 (16,8 g/L), K2HPO4 (0,5 g/L), Na2CO3 (7,6 g/L), H3BO3 (2,86 g/L), MnCl2.4H2O (1,82 g/L), ZnSO4.7H2O (0,22 g/L), CuSO4.5H2O (0,08 g/L), Na2MoO4.2H2O (0,018 g/L),NiSO4.7H2O (0,048 g/L)

2.2 Methods

2.2.1 Microalgae biomass increasing, chlorophyll and carotenoid extraction method

Microalgae biomass increasing

Increasing the biomass of Spirulina sp in order to have enough

microalgae for next experiments

Two 300 mL flasks were prepared, each flask contained 100 mL sterilized medium and were covered by sterilized cotton button Then, each flask was inoculated with 3 stored microalgae tubes equivalent to 20% of the new medium volume

These flasks were grown in the cabinet for incubating microalgae

at room temperature, 24 hours of light and aerated continuously for 5 days

After 5 days, 140 mL of microalgae from the 300 mL flask were transferred to 1 liter - flask that contained 700 mL sterilized Zarrouk medium The last 60 mL were tranferred to two 100 mL flasks to store for next experiment These flasks were kept aerating in one week

After one week, 500 mL of microalgae were transferred from 1 liter - flask to 5 liter - bottle The last 200 mL were transferred to other 1 liter - flask to store The 5 liter - plastic bottle and 1 liter - flask were aerated continously in one week at room temperature and 24 hours of light

Biomass collection: Algae biomass was collected by Whatman

filter – paper 50 mL algae was took out, dry at 75 C in 24 hour, weight and detemine biomass

Clorophyll and carotenoid extraction (M.Henriques et al., 2007)

2 mL algae was tranfered from treatments to eppendorf, centrifuged at 6000 rpm in 10 munites, washing the algae two times by distilled water and extraction by 2 mL acetone 80 % Then, the extraction of chlorophyll and carotenoid production was measured by spectrophotometer (at wave length 663 nm, 646 nm

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and 470 nm) The calculation of chlorophyll and carotenoid were caculated based on Lichtenthaler Welburn (1983)

Chlorophyll a (µg/ml) = 12.21 x (A663) – 2.81 x (A646) Chlorophyll b (µg/ml) = 20.13 x (A646) – 5.03 x (A663) Carotenoid (µg/ml) = (1000A470 – 3,27 x [chl a] – 104

Spirulina sp was grown in 3 liter bottle with inoculated ratio 20

% and repeated three times for each treatment Using NaOH 1M and HCl 1M to adjust pH = 8, 9, 10, 11 The change of pH, increasing of biomass, chlorophyll and carotenoid production were determined in day 0, 4, 8, 16, 20 after algae inoculation

2.2.3 Study the effect of light colour on Spirulina sp

chlorophyll and carotenoid production

Objectives: Study the suitable light colour for chorophyll and

carotenoid production

Procedure

Spirulina sp was grown in 3 liter bottle with inoculated ratio 20

% and repeated three times for each treatment Algae were grown under red, green and white light in shelf The change of pH, increasing of biomass, chlorophyll and carotenoid production were determined in day 0, 4, 8 after algae inoculation

Next experiment was carried out in the same way but used green, blue, and white light to consider the change of pH, increasing of

biomass, chlorophyll and carotenoid production in 6 continuous days from day 0 to day 5

2.2.4 Study the effect of dark – light cycle on Spirulina sp

chlorophyll and carotenoid production

Objectives: Testing and comparing the effect of illumination

time 12/24 and 24/24 hour on the growth of biomass, chlorophyll and carotenoid production

Procedure

Spirulina sp was grown in 3 liter bottle with inoculated ratio 20%

and repeated three times for each treatment Controlled the light

to make 12/24 and 24/24 hour time illumination and the most important condition was restriction the outside light The change

of pH, increasing of biomass, chlorophyll and carotenoid production were determined in day 0, 1, 2, 3, 4 and day 5 after algae inoculation

2.2.5 Study the growth recovery of Spirulina sp

Objectives: Spirulina was broken into many small fragments in

the disavantage condition Therefore, the next eperiment was

done to observe the recovery of Spirulina

Procedure

Small fragments of algae were suported in the appropirate culture

in 0.5 liter media which adjust pH = 9, continuous aeration, inoculation ratito in 20 % and 24/24 illumination light Followed

in 5 continuous days, the growth of Spirulina was determined by

miroscopy and the biomass increasing

2.2.6 Data analysis method

Microsoft Excel and SPSS software were used for data analyzed

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3 RESULTS AND DISCUSSIONS

3.1 The effect of pH on Spirulina sp chlorophyll and

carotenoid production

The results showed that biomass, chlorophyll and carotenoid production of all treatments were highest in day 8 and pH = 9 was

the better condition for the growth of Spirulina In day one, algae

was shock when transfer from pH = 9 to pH = 11 and they settled down to the bottle bottom However, they grew again in day 4 in case of green colour In all treatments, biomass increased from day 0 to day 8 and decreased to day 20 The highest biomass in day 4 was 0.14 g/ 50 mL at pH = 10, comparing to 0.12 g/ 50 mL and 0.11 g/ 50 mL at pH = 8 and 9, respectively In day 8, treatment at pH = 9 was highest biomass (0.16 g/ 50 mL) and significant difference at 5 % according to Duncan test among treatments (figure 1) These results were similar to the research about the effect of pH on biomass of Kemka et al (2004)

Figure 1 Biomass production changed in Spirulina culture in

different pH

During experiment, pH fluctuated from 10 to 10,18, decreasing

in day 16 and 20 (10.04 to 10.08) compared to day 12 (10.11 to

10.18) and no significant difference between treatments (figure

2) Another study also showed that pH was small change and

remained from 9.98 to 10.01 during algal living (Ngakou Albert

et al., 2012)

Like biomass, chlorophyll at pH = 9 was highest at day 12 and 8

(chlorophyll a: 2.72 µg/mL, chlorophyll b: 3.15 µg/mL) and

significant difference at 5 % according to Duncan test among

treatments (figure 3 and figure 4) According to Pandey et al

(2010), pH = 9 was better condition for chlorophyll accumulation

(among pH from 7 to 12)

Figure 3 Chlorophyll a production in different pH

Figure 2 pH changed in Spirulina culture in different pH

3.2 The effect of light colour on Spirulina sp chlorophyll and

carotenoid production

Light affected directly on the photosynthesis So this was a strong factor which controlled the growth of algae In this experiment, 3

Figure 5 Carotenoid production in Spirulina in different pH

Figure 4 Chlorophyll b production in different pH

different colours green, red and white had significant different

effect in all treatments In this case, Spirulina grew slow under

green light and limited under red light (figure 6)

The results of this experiment showed that light played the most

important roles on Spirulina growth Algae were broken into very small fragments in red and green light condition Spirulina also

grew to day 4 so that next experiment should be carried out to check the effect of light colour on 5 continuous days In this study, white, green and blue light were chosen As the results

obtained before, Spirulina grew well at white light From day 0 to

day 4, biomass in three treatments showed no difference while pigments had significant difference From day 1 to day 3, algae biomass increased under blue and white light It slow rose under green colour from day 2 because biomass was similar in first 4 days (figure 7) In day 5, highest biomass was collected at white

Figure 6 Biomass, chlorophyll and carotenoid in Spirulina under

different light colours

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light (0.13 g/ 50 mL) while these numbers were 0.09 g/ 50 mL

and 0.08 g/ 50 mL for blue and green light, respectively

Spirulina did not synthesize carotenoid under green and blue light

from day one (figure 8) In treatment applying white light, carotenoid grew up fast from day 2 (0.77 µg/mL) to day 4 (1.24 µg/mL)

Figure 7 Biomass production of Spirulina under different

light colours

Figure 8 Carotenoid production in Spirulina under different

light colours

In treatments controlled with white light, chlorophyll a accumulated rapidly on from day 2 (1.02 µg/mL) to day 3 (2.15 µg/mL) while day 4 (2.42 µg/mL) and day 5 (2.71 µg/mL) the accumulation of the pigment were slower (figure 9) Madhyastha and Vatsala (2007) demonstrated that white light was better

condition for chlorophyll accumulation in Spirulina compared to

blue and green light

Clorophyll b production of Spirulina under white light was 3.35

µg/mL in day 4 while this number was 0.27 µg/mL for green and 0.26 µg/mL for blue light (figure 10)

Figure 9 Chlorophyll a in Spirulina under different light colours

Figure 10 Chlorophyll b in Spirulina under different

light colours