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

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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 SPIRULINA SP. SUPERVISOR: Ass. Prof. NGUYEN HUU HIEP STUDENT: NGUYEN THI HUYNH NHU Student Code: 3082618 Session: 34 (2008 - 2013) APPROVAL SUPERVISOR Ass. Prof. NGUYEN HUU HIEP STUDENT NGUYEN THI HUYNH NHU Can Tho, May 2013 PRESIDENT OF EXAMINATION COMMITTEE ii ABSTRACT 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. i 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 ii 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 iii 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). 1 1.2 Objectives Determination of pH concentration, dark – light cycle and light colours appropriate for biomass growth rate, chlorophyll and carotenoid production in Spirulina sp. 2 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): NaNO 3 (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. 3 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 4 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 x [chl b])/227 2.2.2 Study the effect of pH on Spirulina sp. chlorophyll and carotenoid production Objectives: Study the suitable pH 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. 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 5 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. 6 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 7 Figure 2. pH 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). Figure 3. Chlorophyll a production in different pH 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). 8 Figure 4. Chlorophyll b production in different pH Highest carotenoid was achieved in day 8 with 1.13 µg/mL for treatment pH = 8 and 1.43 µg/mL at pH = 9. While in day 12, pH = 10 and 11 these numbers were 1.11 µg/mL and 0.77 µg/mL, respectively (figure 5). For all treatments, carotenoid decreased in day 16 and 20 because of lack nutrition in culture and high density of algae. Figure 5. Carotenoid production in Spirulina 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 9 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). Figure 6. Biomass, chlorophyll and carotenoid in Spirulina under different light colours 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 10 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. Figure 7. Biomass production of Spirulina under different light colours 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 8. Carotenoid production in Spirulina under different light colours 11 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. Figure 9. Chlorophyll a in Spirulina under different light colours 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 10. Chlorophyll b in Spirulina under different light colours 12 pH value in this study had no significant difference for all treatments and remained at 9.8 – 9.9 in day 4 and 5 (figure 11). These results were similar to experiments before in this paper. It could be concluded that pH did not change much in algal growth. Figure 11. The effect of colour light on pH of algal culture 3.3 The effect of dark – light cycle on Spirulina sp. chlorophyll and carotenoid production In sufficient light regime (24/24 hour of illumination) Spirulina grew better than 12/24 hour of illumination. After 4 day, biomass was similar between two treaments but pigments of Spirulina were sighnificant difference. In day 5, both biomass and pigments of Spirulina at 24/24 hour of illumination were higher than 12/24 hour of illumination (1.08 times in biomass, 2.36 times in chlorophyll a, 1.2 times in chlorophyll b and 1.7 times in carotenoid) (figure 12). 13 Figure 12. The effect of dark – light cycle on chlorophyll a, chlorophyll b and carotenoid in spirulina at day 5 Biomass production of Spirulina was significant difference in day 5 (figure 13). For full light, biomass, chlorophyll a, chlorophyll b and carotenoid were 0.13 g/50 mL; 2.83 µg/mL; 3.63 µg/mL; 2.51 µg/mL, respectively, while these numbers were 0.11 g/50 mL; 1.35 µg/mL; 2.23 µg/mL; 1.50 µg/mL in the half day light. Figure 13. Spirulina biomass during 5 days under 12/24 and 24/24 hour illumination 14 Algal biomass dramatically increased during 5 days in 24/24 light illumination. However in 12/24 hour light illumination, the biomass increased in the first 3 day and increased slower in day 3, 4 and day 5 (biomass around 0.22 g/50 mL) (figure 13). At this time, algae density was very high so that they needed more light for photosynthesis. It could be explained that at high density of algae, light could not reached to all algal fibers, nutrition and carbohydrate decreased so that the photosynthesis was inhibited (Richmond and Grobbelaar, 1986). Figure 14. Chlorophyll a in Spirulina during 5 days under 12/24 and 24/24 hour illumination As from the first day, chlorophyll a was difference value between two treatments. At day 5 chlorophyll a production was 1.35 µg/ml for 12/24 light hour and 2.83 µg/ml for 24/24 light hour (figure 14). That mean chlorophyll a was more affected by light than chlorophyll b and carotenoid. 15
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