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A Comparison of Stressing Techniques of Haematococcus A COMPARISON OF STRESSING TECHNIQUES OF HAEMATOCOCCUS PLUVIALIS by Lauren Smiarowski A Thesis Submitted to the Faculty of The Wilkes Honors College in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in Liberal Arts and Sciences with a Concentration in Biology Wilkes Honors College of Florida Atlantic University Jupiter, FL December 2017 A COMPARISON OF STRESSING TECHNIQUES IN HAEMATOCOCCUS PLUVIALIS By Lauren Smiarowski This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Jon Moore and has been approved by the members of her/his supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Science in Liberal Arts and Sciences. SUPERVISORY COMMITTEE: ___________________________ Emily Hopkins, Thesis Supervisor ___________________________ Dr. Jon Moore, First Reader ___________________________ Dr. Catherine Trivigno, Second Reader ___________________________ Dean Ellen S. Goldey, Wilkes Honors College ___________ Date ii ACKNOWLEDGEMENTS To everyone at Avespa: Thank you for everything you’ve done to help me the past two years and to grow as a scientist and a person. Emily and Megan, it was a pleasure to work along side you and I’ll always appreciate your advice and insights when it comes to school and life. To Dr. Trivigno: Thank you for recommending me to work at Avespa back when I was an enthusiastic freshman looking for laboratory experience. I was so glad you were able to be my second reader on my thesis, as it wouldn’t have been possible without your connection. To Dr. Moore: Thank you for taking a chance on me when I asked you to be my advisor. Despite not have ever taking a class with you, I still learned a lot from having you as a mentor. To everyone who dealt with me while I worked on my thesis: Thank you for your patience and putting up with listening to something you might not have cared about, information you didn’t understand, or my complaining. iii ABSTRACT Author: Lauren Smiarowski Title: A Comparison of Stressing Techniques of Haematococcus pluvialis Institution: Wilkes Honors College of Florida Atlantic University Thesis Advisor: Dr. Jon Moore Degree: Bachelor of Science in Liberal Arts and Sciences Concentration: Biology Year: 2017 Haematococcus pluvialis, a freshwater species of microalgae, is one of the most important sources of natural astaxanthin, a keto-carotenoid of high value to the pharmaceutical industry. Astaxanthin possesses antioxidant and anticancer properties as well as serving as food coloration. Production of astaxanthin from natural sources is limited, and microalgae is a promising source to meet the increasing demand. Three strains of H. pluvialis from various culture collections, Culture Collection of Algae and Protozoa (CCAP 34/8), Nation Institute of Environmental Studies (NIES-144), and Scandinavian Culture Collection of Algae and Protozoa (SCCAP K-0048), were tested in different conditions to compare the synthesis of astaxanthin. The strains were compared in three different conditions, high light (control), low pH, and addition of salt water, and the amount of astaxanthin produced was compared using a 2-way T-test. This research is of interest to explore different methods for producing astaxanthin for the growing market. iv TABLE OF CONTENTS INTRODUCTION .............................................................................................................. 1 MATERIALS AND METHODS ........................................................................................ 9 Cell culture ...................................................................................................................... 9 Control experiment ......................................................................................................... 9 Stress condition 1: pH ................................................................................................... 10 Stress condition 2: Salt ................................................................................................. 10 Astaxanthin extraction & saponification ...................................................................... 11 RESULTS ......................................................................................................................... 13 Control .......................................................................................................................... 13 Experiment 1: low pH ................................................................................................... 14 Experiment 2: salt ......................................................................................................... 15 DISCUSSION ................................................................................................................... 18 CONCLUSION ................................................................................................................. 21 REFERENCES ................................................................................................................. 22 v LIST OF TABLES Table Page Table 1: Quantitative astaxanthin composition (in pg cell-1) of cells stressed under control conditions, obtained by HPLC analysis of H. pluvialis cells of different strains ........................................................................................................................ 14 Table 2: Quantitative astaxanthin composition (in pg cell-1) of cells stressed under low pH conditions, obtained by HPLC analysis of H. pluvialis cells of different strains 15 Table 3: Quantitative astaxanthin composition (in pg cell-1) of cells stressed under 1% salt conditions, obtained by HPLC analysis of H. pluvialis of SCCAP-0084 .......... 16 LIST OF FIGURES Figure Page Figure 1: Astaxanthin structure ........................................................................................... 2 Figure 2: Green microalgae growth stages ......................................................................... 3 Figure 3 An empty “ghost cell” faintly visible within the box ........................................... 4 Figure 4 Sample readout from HPLC analysis of CCAP-34/8 in the control experiment 12 Figure 5: Physiological changes of Haematococcus cells from stress day 0 to fully stressed ...................................................................................................................... 13 Figure 6: Bar graph showing the difference in astaxanthin formation in the averages of the control experiment and the low pH experiment .................................................. 15 Figure 7: SCCAP-0084 lysed cell wall due to 1% salt concentration .............................. 16 Figure 8: Bar graph showing the difference in astaxanthin formation average of the control and salt experiments ..................................................................................... 17 vii INTRODUCTION Microalgae are often single-celled protists which live in aquatic environments: freshwater, salt water, and brackish water (Raven & Giordano 2014). They are mostly found within the top 300 meters of water, where the photon flux density is between 400- 700nm, and contain chlorophylls A and C (Raven & Giordano 2014). Even though algae undergo photosynthesis, this does not allow them to be classified as a “higher plant.” Algae do not have specialized tissues, which is a distinguishing characteristic from plants. The size of a single cell can range from one micrometer to approximately eight meters long. While the number of species is uncertain, it is estimated there are between 30,000 to over a million different species of algae. Most algae are autotrophic, using external organic materials for their growth in addition to light and inorganic materials such as nitrates, phosphates, iron, trace metals and vitamins B1, B7, and B12 (Raven & Giordano 2014). This characteristic allows them to exist independently, but some species are found living in symbiosis with other organisms. Most algae are found in areas of moderate temperature around 20-22˚C, with a pH between 5-7 (Andersen 2005). Each species and strain of algae has a unique biochemical makeup consisting of carbohydrates, lipids, and proteins. Many lipids found in algae are commercially useful when taken as dietary supplements, such as astaxanthin and beta-carotene. In the chlorophyte species Haematococcus pluvialis, when the cells are exposed to an environment causing them to become stressed, their biochemical makeup changes from 1 approximately 16% lipid and 28% fatty acid to approximately 33% lipid and 30% fatty acid (Becker, 2013). Under stress they also produce a ketocarotenoid known as astaxanthin (Figure 1), which is used as a defense mechanism by thickening their cell walls. Astaxanthin is commonly manufactured as a food supplement capsule and is valued for its high antioxidant properties as well as its use to supplement the omega-3 fatty acid eicosapentaenoic acid (EPA) in food for aquaculture. It is a fat-soluble compound and in humans, absorption can be increased by consuming it along with dietary oils (Ambati, Phang, Ravi, & Aswathanarayana 2014). Figure 1: Astaxanthin structure (National Center for Biotechnology Information) Many species of microalgae, including Haematococcus species, follow a similar life cycle consisting of the macrozooid, palmella, and hematocyst (also called aplanospore) stages. The characteristics of these stages are unique and are easily
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