Downloaded from orbit.dtu.dk on: Oct 08, 2021 Novel microalgal based ingredients Ljubic, Anita Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Ljubic, A. (2020). Novel microalgal based ingredients. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Novel microalgal based ingredients Ph.D Thesis by Anita Ljubic National Food Institute Technical University of Denmark November 2020, Kgs. Lyngby Preface This PhD project entitled “Novel microalgal based ingredients” was carried out at the National Food Institute, Technical University of Denmark, under the supervision of Professor Charlotte Jacobsen as main supervisor and three co-supervisors, associate professor Susan L. Holdt, senior researcher Jette Jakobsen and senior researcher Anette Bysted. The project started in December 2017 and ended in November 2020. The project was funded by the National Food Institute as a strategic Ph.D project, which aimed at further development of the research field within microalgal utilization and application. The project included internal collaboration within the research group by applying the expertise in the algae, lipid and vitamin teams. The aim of this Ph.D project was to optimize existing technologies and develop new technologies for the production of high value bioactive ingredients from microalgae. Employment of different abiotic factors on the production of lipids, carotenoids and fat-soluble vitamins was studied in order to identify the tools for maximizing their production. Furthermore, optimization and evaluation of cell disruption and drying technologies for recovery of high value ingredients was carried out. Some of the novel findings of this Ph.D project were a base for building a start-up company Nordic Algae Nutrition in 2020, where I took a role as a co-founder. The company aims at commercializing microalgae as a new source of vegan vitamin D3. During the project, I have carried out a research stay at the Department of Biological Sciences, California State University of San Marcos, which was supported by a research grant from Otto Mønsted Foundation. During the last year of the Ph.D project, practical work was limited to a certain extent due to the COVID-19 pandemic. However, the limited number of performed experiments in the second part of the project was compensated by conducting a thorough literature review on the topic. Anita Ljubic 30th of November, 2020 Kgs. Lyngby, Denmark ii Acknowledgments I would like to express my appreciation to numerous people who contributed to the achievements of this project and who supported me on this journey. First, I wish to thank to my main supervisor Charlotte Jacobsen for her continuous support, encouragement and motivation during the project. I really appreciate your positive attitude, that you are easy to approach and willing to help in any situation. I would also like to thank to my co- supervisors, Susan L. Holdt, Jette Jakobsen and Anette Bysted for their support during the project. I appreciate you were able to find time whenever I needed and you were always ready to share your knowledge and experience. Moreover, I sincerely thank to all colleagues and friends who supported me during the project: Professor Betsy Read, for making my external stay at the California State University exciting and memorable. It was a pleasure learning from such an inspiring person. Claus Asperud Reesbøll, for sharing his technical expertise during the project. Without your help, it would be impossible to perform my experiments. Thanks to Jack Melbye from DTU Aqua for sharing some microalgae culture. Thanks to Eitan Solomon and Naama Segal for providing microalgae samples from the International Center for Mariculture in Israel. Heidi Jahn and Inge Holmberg, for sharing the office these years, for our fun office chats and always being ready to help in any situation. It feels amazing working in such a positive environment you create. Also, thank to all the trainees that were part of the Disco office, Marie, Claus and Emilie, it was a pleasure sharing the office with you. Hamed Safafar, thank you for introducing me to the world of microalgae and being ready to answer my questions any time during the project. To all my colleagues from the Bioactives group, I enjoyed being a part of such an amazing group of people. To all the students I had a chance to work with, Dona, Usman, Elisabeth, Rizwan and Kathrine, you were a great help during this project. Finally, I would like to thank to all my friends, especially to Dolores and Efthymios, for supporting me and making this period enjoyable. Big thank to my family in Croatia and in Denmark, especially to my iii sister and my parents for supporting me continuously throughout my life. Lastly, a special thanks to my amazing husband, without you this journey would be incomplete. Anita Ljubic iv Summary Microalgae are excellent sources of various bioactive compounds such as omega-3-fatty acids, carotenoids and vitamins. With greater consumer preferences toward naturally derived, non-animal ingredients over synthetically produced, it is expected that the market demand will continue to grow in the future. Microalgal based ingredients represent reliable and sustainable substitutes to high value bioactive ingredients from both animal and plant origin. However, for economically viable production of high value ingredients there are still challenges to be addressed in order to enhance the production of these ingredients. The nutritional quality of microalgae can be improved by optimizing abiotic factors such as irradiance, salinity, nutrient availability, pH or temperature, which may stimulate production of metabolites of interest. Besides maximizing the production of high value compounds, maintaining the functionality of compounds during the cell disruption, extraction and drying process, while obtaining high recoveries, is essential. The overall aim of this PhD study was to optimize existing technologies and develop new technologies for the production of high value ingredients from microalgae. Effect of the abiotic factors, including light intensity, spectral distribution, UVB radiation and salinity, on the production of bioactive ingredients in 8 selected microalgal species was evaluated. Furthermore, the aim was to identify tools that can be used for enhanced production of high-value lipophilic compounds - omega-3 fatty acids, various carotenoids, α-tocopherol, vitamin D and novel compounds, alkenones. Optimization of the downstream processing included optimizing the process parameters for an efficient cell disruption and metabolite extraction by high-pressure homogenization in combination with enzymatic treatment. Lastly, the effect of the novel swirl flash drying technology on the high value compounds present in the biomass was evaluated in order to assess the suitability of the novel drying technology for microalgae. Stimulating production of bioactive compounds by manipulating different abiotic factors was shown to be highly dependent on species and target compound. Different patterns were observed across the selected species and relatively weak correlation between the content of different metabolites was observed, which required selection of optimal stress tools for each individual species or individual compound. This research demonstrated that it was possible to manipulate different microalgae to produce various high value compounds by exposing them to different light treatments. Production of some of these compounds in microalgae had not been reported before. The most novel finding with the greatest potential was that exposing Nannochloropsis oceanica, N. limnetica and Dunaliella salina to artificial UVB radiation stimulated production of relatively high levels of the fat-soluble vitamin D3 v (up to 2.7 µg/g dry biomass), which is commonly found in foods of animal origin. UVB radiation was also shown to stimulate production of α-tocopherol in N. oceanica making this species an excellent alternative vegan source of vitamin D3, α-tocopherol and omega-3 fatty acids, ingredients commonly found in fish oil. Chlorella minutissima illuminated by green:red LED (50:50) was shown to contain up to 70% more lutein compared to control, making this species highly competitive to other lutein- rich sources. For Rhodomonas salina, a species rich in omega-3 fatty acids EPA and DHA, illumination by red LED in combination with salt stress (40 ppt) resulted in an increase in the relative proportion of omega-3 by 40% (nearly doubled proportion of EPA). Similarly, illumination by green LED in combination with salt stress increased production of β-carotene in R. salina by 75% compared to the control, which indicates that interaction of different abiotic