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Enhancing Lipid Content in Chlorococcum Littorale Using in Situ Fluorescence Cell Sorting

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Enhancing Lipid Content in Chlorococcum Littorale Using in Situ Fluorescence Cell Sorting SIZE DOESN’T MATTER: THE INFLUENCE OF CELL DIAMETER ON LIPID PRODUCTIVITY OF Chlorococcum littorale USING FLUORESCENCE CELL SORTING Carolina Gomes Sanches Fernandes Thesis to obtain the Master of Science Degree in Biological Engineering Supervisors: Dr. ir. Dorinde Kleinegris Dr. Frederico Castelo Alves Ferreira EXAMINATION COMMITTEE Chairperson: Prof. Arsénio do Carmo Sales Mendes Fialho Supervisor: Dr. Frederico Castelo Alves Ferreira Member of the committee: Dra. Nídia Dana Mariano Lourenço de Almeida November, 2014 ACKNOWLEDGEMENTS In March I started my thesis project at AlgaePARC, as part of the Bioprocess Engineering group. The five months I spent in Wageningen were some of the best I had and definitely the ones when I learned more about microalgae of my life. A whole new theme, whole new people, whole new equipments (the FACS nightmare)... And the greatest supervisor to help me go through all this! Iago, thank you for the help provided during this time and for becoming more than just a supervisor: a friend. Thank you for all the conversions (usually in Portuguese, thank God!), the funny facts about Brasil and the guidance about the Netherlands, they made my work much easier. Obrigada por tudo! Also, to the other students, PhD’s, investigators, technicians, thank you for answering to all my questions and helping whenever I needed. Thank you, Dorinde, for your truly helpful advices. To the WUR, thank you for receiving me and helping with everything a student needs in a new country. I would also like to thank the financial support to AlgaePARC Research Program provided by: Ministry of Economic Affairs, Agriculture and Innovation (the Netherlands), Province of Gelderland (the Netherlands), and the companies: BASF, BioOils, Drie Wilgen Development, DSM, Exxon Mobil, GEA Westfalia Separator, Heliae, Neste Oil, Nijhuis, Paques, Cellulac, Proviron, Roquette, SABIC, Simris Alg, Staatsolie Suriname, Synthetic Genomics, TOTAL and Unilever. To Instituto Superior Técnico, my home university, thank you for teaching me everything I need to start a career, especially how to overcome any obstacle that comes in my way. In other words, thank you for teaching me how to be an engineer. To all my friends that have helped me go through these five years, a giant THANK YOU, you actually turned this into the best time of my life! Ana and Joana, thank you for caring and being always around. Finally, a special thanks to my family and boyfriend, who have supported me unconditionally, even when they were far away from me. I hope you enjoy this report, Carolina ii SUMMARY When performing cell sorting to isolate populations with high lipid content, and use them to start new cultures, the size of the cells has not been taken into account. This project had the objective of assessing the effect of the cells’ diameter in the final lipid productivity of Chlorococcum littorale, after the diameter was used as a sorting criterion in FACS (Fluorescence Activated Cell Sorting). Sorting gates were created to isolate cells in four groups with different diameters, named small (5-6 µm), medium (8-9 µm), large (11-14 µm) and control (5-14 µm). Two sets of experiments were done: before-sorting (BS) and after-sorting (AS). In both experiments the cells were grown under controlled conditions and then exposed to nitrogen starvation. The growth and photosynthetic activity were followed-up daily and the chlorophyll and lipid fluorescence and diameter were analysed daily with flow imaging. Simultaneously, a Single-Cell Experiment was carried out. It consisted of collecting 15 samples of each sorting gate, with just one cell. After recovery, the autofluorescence and diameter were analysed using flow imaging. The evolution of all parameters was similar in the four different cultures. The final lipid productivity of the four samples was not significantly different (P = 0.665 > 0.05). The results of the Single-Cell Experiment supported this fact, showing no significant difference (P = 0.628 > 0.05) in the average diameter of cells after the recovery period. So, the sorted cells’ diameter had no influence on the final lipid productivity of the new cultures. Key words: Microalgae, C. littorale, FACS, Lipid productivity, Diameter. iii RESUMO FACS (Fluorescence Activated Cell Sorting) é uma técnica utilizada com microalgas para isolar células com elevado conteúdo lipídico e criar novas populações com a mesma característica. No entanto, o efeito do diâmetro das células como critério de separação nunca foi estudado. Assim, o objectivo do presente trabalho foi avaliar o efeito do diâmetro das células na produtividade lipídica final das culturas. Foram desenvolvidas duas experiências: antes e depois da separação, com as mesmas condições experimentais, que incluem um período de stress por limitação de azoto. Para a separação foram criados quatro gates, de maneira a isolar diferentes diâmetros: pequeno (5-6 µm), médio (8-9 µm), grande (11-14 µm) e um de controlo (5-14 µm). O crescimento, a actividade fotossintética, a fluorescência da clorofila e dos lípidos e ainda o diâmetro foram seguidos diariamente, os três últimos através de fluid imaging. Simultaneamente foi desenvolvida a experiência Single-Cell, onde 15 amostras unicelulares de cada gate foram isoladas e após um período de recuperação analisados quanto à sua autofluorescência e diâmetro. As amostras dos quatro gates tiveram evoluções semelhantes no que diz respeito aos parâmetros seguidos. As diferenças na produtividade lipídica final entre os quatro tipos de amostras não foram estatisticamente significativas (P = 0.665 > 0.05), bem como no que toca ao diâmetro médio das células nas amostras da experiência Single-Cell (P = 0.628 > 0.05). Portanto, o diâmetro das células isoladas não tem influência na produtividade lipídica final das culturas. Palavras-chave: Microalgas, C. littorale, FACS, Produtividade lipídica, Diâmetro. iv CONTENTS List of tables ..................................................................................................................................................... 1 List of figures .................................................................................................................................................... 2 List of acronyms ................................................................................................................................................ 4 Greek letters................................................................................................................................................. 5 Introduction ..................................................................................................................................................... 6 Aim .............................................................................................................................................................. 7 Approach ...................................................................................................................................................... 7 Theoretical background .................................................................................................................................... 9 FACS (Fluorescence-Activated Cell Sorting) ................................................................................................... 9 The fundamentals..................................................................................................................................... 9 Scattering/fluorescence and setting gates................................................................................................. 9 Advantages and drawbacks .................................................................................................................... 10 Sorting microalgae since the 1980’s ........................................................................................................ 11 BODIPY 505/515 ......................................................................................................................................... 12 Lipid accumulation by nitrogen limitation ................................................................................................... 15 Chlorococcum littorale................................................................................................................................ 17 Appearance and reproduction ................................................................................................................ 17 Growth characteristics ............................................................................................................................ 20 Lipid production ..................................................................................................................................... 20 Materials and methods ................................................................................................................................... 22 Experimental set-up.................................................................................................................................... 22 Cultivation .................................................................................................................................................. 23 Stress experiments ..................................................................................................................................... 23 Optical density and quantum yield .............................................................................................................
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