Faculty of Resource Science and Technology Improvement of NGP medium for mass culture of Chlorella vulgaris Cassy Cassandra anak Mitaha (18141) Bachelor of Science with Honours (Aquatic Resource Science and Management) 2010 Improvements of NGP medium for mass culture of Chlorella vulgaris Cassy Cassandra anak Mitaha A final report submitted in partial fulfillment of the Final Year Project STF 3014 Supervisor: Ass. Prof Dr. Norhadi Ismail Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak 3 May 2010 DECLARATION I hereby declare that no portion of the work referred to in this dissertation has been submitted in support of an application for another degree or qualification to this university or any other institution of higher learning. ________________________________________ Cassy Cassandra anak Mitaha Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak ACKNOWLEDGEMENTS First of all, I would like to thank God for His guidance and His strength, as well as time and wisdom that has given to me throughout all the way from the beginning until the final step in doing this Final Year Project Thesis. I am grateful to Him who has giving me the opportunity to learn and discover many challenges in different aspects, and taking this challenges in a positive manner. I would like to express my deepest gratitude and appreciation to my supervisor, Dr. Norhadi Ismail for his constant guidance, advice, patience and support throughout completing my thesis. The knowledge he imparted upon me, his generous assistance and concerns that contributed a lot in completing this study. Thanks also to Dr. Ruhana Hassan for her guidance and advices in writing this thesis since the beginning. Special thanks dedicated to Ms. Iqliema Afdalia, postgraduate student in Aquatic Botany Laboratory, who had given valuable technical advices and helped during laboratory work as well as their assistance. I also would like extend my sincere thanks to laboratory assistant, Mr. Zaidi Ibrahim and Mr. Mohd Norazlan Bujang for their cooperation and assistant. On the other hand, I also would like to thank my family especially my parents, Mr. Mitaha Mapang and Mdm. Selera Jiki as well as my beloved siblings, Goretty Mika, Gregory Rapin, Geraint Jenggi and Gwendoline Nilie for their moral support, patience and encouragement for me to complete my final year project. I also would like to thank my fellow classmates, housemates and friends for their continous encouragement and support. Without their support, assistance and advices, this project would never have been completed. I TABLE OF CONTENT Acknowledgements I Table of Content II List of Abbreviations IV List of Tables and Figures V Abstract………………………………………………………………… 1 1.0 Introduction and Objectives………………………………………….. 2 2.0 Literature Review 2.1 Potential of microalgae as biofuel production……………………... 4 2.2 Factors affecting growth rate of algae……………………………... 7 2.3 Mixotrophic and heterotrophic algae………………………………. 8 2.4 Batch culture……………………………………………………….. 10 3.0 Materials and Methods 3.1 Laboratory work 3.1.1 Establishment of stock culture of Chlorella vulgaris…….. 11 3.1.2 Improvement of NGP medium………………………….... 11 3.2 Outdoor mass culture………………………………………………. 12 3.2.1 Growth measurement…………………………………….. 13 3.3 Harvesting of algae ………………………………………………… 14 3.4 Lipid extraction………………………………………….................. 14 4.0 Result 4.1 Improvement of NGP medium for subcultures of Chlorella vulgaris…………………………………………………. 15 II 4.2 Chlorella vulgaris mass culture……………………………………. 19 4.3 Lipid content analysis……………………………………………… 20 5.0 Discussion 5.1 Factors affecting growth of Chlorella vulgaris……………………. 21 5.2 Chlorella vulgaris mass culture under outdoor conditions…………………………………………………………. 24 5.3 Lipid content analysis of Chlorella vulgaris………………………. 26 6.0 Conclusion……………………………………………………………... 27 7.0 References……………………………………………………………… 28 8.0 Appendix Appendix 1…………………………………………………………… 33 Appendix 2…………………………………………………………… 34 Appendix 3…………………………………………………………… 35 Appendix 4…………………………………………………………… 36 Appendix 5…………………………………………………………… 37 Appendix 6…………………………………………………………… 38 Appendix 7…………………………………………………………… 39 III LIST OF ABBREVIATIONS NGP Norhadi Growth Promoter N Nitrogen P Phosphate K Potassium L Liter g Gram ml Milliliter CO2 Carbon dioxide gas EOM Extracellular organic matter oC Degree Celcius µm Micrometer R & D Research and Development m Metres km Kilometers A, B, C, D, E Treatment for algae that have different concentration of salt and Organisol EU European Union USA United States of America ha Hectares SD Standard deviation wt Weight Vt Volt IV LIST OF TABLES Table 1 : The oil content of some microalgae those are potential in producing biofuel in future. (Extracted from Christi, 2007)……. 6 Table 2 : The combinations of fertilizer and growth promoter per 1 L of improved NGP medium…………………………………………. 12 Table 3 : Growth rate, K’ of Chlorella vulgaris in subcultures under different concentration of fertilizer and Organisol (growth 17 promoter).………………………………………………………... Table 4 : The pH value of different concentration of fertilizer and Organisol before adding Chlorella vulgaris ……………………. 17 Table 5 : Summary of number of cells of Chlorella vulgaris under different concentration of fertilizer and growth promoter..……... 18 Table 6 : Summary on duration time (s) obtained by using flocculation harvesting technique at different concentration of Chitosan and 20 voltage (Volt)...………………………………………………….. V LIST OF FIGURES Figure 1 : The growth curves of Chlorella vulgaris in different concentration of fertilizer and Organisol ……………………….............................. 16 Figure 2 : The growth curves of Chlorella vulgaris in mass culture under outdoor conditions.…………………………………………………. 19 Figure 3 : Percentage weight of dry algae obtained from different concentration of Chitosan and voltage (Volt). (Total biomass = 36).. 20 VI Improvement of NGP medium for mass culture of Chlorella vulgaris Cassy Cassandra anak Mitaha Aquatic Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak ABSTRACT Chlorella vulgaris is a single-celled of green algae that are spherical in shape and do not have flagella, which can be found in freshwater. It is fast-growing green algae and has different lipid production capabilities (30–40% of dry weight) under natural conditions. In the present study, the microalgae were grown in NGP medium. It was tested to grow in different combinations amount of fertilizer and Organisol (growth promoter) as an improvement to the original NGP medium. Results showed that C. vulgaris had the highest growth rate when the NGP supplied with combination of 0.13ml L-1 growth promoter and 2.0g L-1 fertilizer. Using this nutrient combination, the algal was mass cultured outdoor in aquariums. The lipid yield produced after 26 days of cultivation was about 2.17%. Keyword: Chlorella vulgaris, out-door conditions, NGP medium, mass culture, lipid extraction ABSTRAK Chlorella vulgaris ialah alga hijau yang mempunyai sel tunggal berbentuk seperti sfera dan tidak mempunyai flagella, serta boleh ditemui di air tawar. Alga ini sangat cepat bercambah dan mempunyai berbeza keupayaan menghasilkan minyak (30-40% daripada berat alga kering) dalam keadaan semulajadi. Berdasarkan kajian ini, mikroalga telah bertumbuh di dalam NGP media. Ia telah dikaji untuk bertumbuh di dalam kombinasi baja dan Organisol (penggalak tumbesaran) yang berbeza sebagai penambahbaikkan daripada NGP media yang asal. Hasil kajian menunjukkan C. vulgaris mempunyai kadar tumbesaran yang tertinggi apabila NGP disediakan dengan kombinasi 0.13ml L-1 penggalak tumbesaran dan 2.0g L-1 baja. Dengan menggunakan kombinasi nutrisi ini, alga telah dikultur di persekitaran luar secara besar-besaran di dalam akuarium. Hasil minyak yang dihasilkan selepas 26 hari pengkulturan ialah 2.17%. Kata kunci: Chlorella vulgaris, persekitaran luar, NGP media, jisim kultur, rentapan minyak 1 1.0 INTRODUCTION AND OBJECTIVES Chlorella vulgaris is unicellular green algae that can be found in marine ecosystem, freshwater and terrestrial area. C. vulgaris (See Appendix 1) is a single-celled of green algae that are spherical in shape and do not have flagella. Their sizes are about 2-10μm in diameter and contain photosynthetic pigments including chlorophyll a and b. This algae is taxonomically placed under: Kingdom Protista, Division Chlorophyta, Order Chlorellales and Family Chlorellaceae. Many people believed that Chlorella vulgaris could serve as a potential source of food and energy because of its photosynthetic efficiency compare to other crops such as sugar cane. It is also an attractive food source because it is high in essential nutrients such as protein, lipid, carbohydrates and vitamin. Chlorella sp. has been the oldest commercial application of microalgae. Chlorella vulgaris is fast-growing green algae and has different lipid production capabilities (30–40% of dry weight) under natural conditions. Its heterotrophic growth mode in the presence of glucose or acetate has also been studied in the 1960s and 1970s (Pratt & Johnson, 1963; Nichols et al., 1967; Harris & James, 1969; Podojil et al., 1978; Liang et al., 2009). C. vulgaris can be cultivated on acetate in the dark and in the light with acetate being directly converted to fatty acids (Nichols et al., 1967; Harris & James, 1969; Liang et al., 2009). Based