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 discerned under a microscope. During the macrozooid stage (Figure 2A), cells are spherical, ellipsoidal, or pear-shaped and biflagillated with equal length flagella (Shah,
Laing, Cheng, & Daroch 2016). They have a distinctive extracellular matrix and are able to divide by mitosis into 2-32 daughter cells. When the macrozooid cells environment
2 becomes unfavorable, they begin their transition into palmella by losing their flagellum
(Figure 2B). Palmella stage cells have a thick cell wall with two layers of extracellular matrix and begin to form astaxanthin granules near the nucleus (Figure 2C). During this stage, thylakoids begin to degrade and chloroplast activity declines (Wayama et al.
2013). As palmella stage cells continue to stress, they enter into the hematocyst stage
(Figure 2D).
Figure 2: Green microalgae growth stages (A) Green vegetative motile cell in the macrozooid stage, (B) Green vegetative palmella cell, (C) Cell in transition to hematocyst stage accumulating astaxanthin, (D) Hematocyst cell. Figure reproduced from Shah, Laing, Cheng & Daroch (2016)
In this stage cells have a thick and rigid trilaminar sheath and a secondary cell wall made of acetylysis resistant material. At this stage, cells begin to fill with astaxanthin and other secondary carotenoids, making them appear red/orange. When conditions become too extreme, resulting in a metabolic imbalance, “ghost cells” (Figure 3) or empty sacs once containing cells, are observed. The amount of fully-bleached ghost cells increases in response to the level of adverse environmental conditions.
3
Figure 3 An empty “ghost cell” faintly visible within the box. Original photo.
During the hematocyst or aplanospore stage in which cells are exposed to growth- limiting conditions, Haematococcus pluvialis cells synthesize large amounts of astaxanthin. This is an evolutionary adaptation to protect the cells from extreme environmental conditions. The cells are able to remain in a vegetative state for long periods of time, provided they have adequate nutrients, but as the cells age and nutrients are depleted, they encyst and accumulate astaxanthin. Conditions promoting encystment include an increase in salinity and an increase in light intensity. During their transition from the palmella to hematocyst stage, cells undergo distinctive changes to their morphology, physiology and photosynthetic properties. Morphologic changes include losing their flagella, accumulating large, astaxanthin-containing lipid bodies, and thickening of the cell wall to prevent oxidative degradation.
During gametogenesis, cells can produce up to 64 small, high-motile gamete cells originally called microzooids. Gametogenesis in H. pluvialis is thought to be induced when a starved culture is supplemented with freshwater medium, although observation of conjugation of gametes has never been observed (Triki 1997). Nutrient dependency,
4 light, and temperature are largely responsible for the onset of reproduction, but varies by species of microalgae. Cells are able to survive inauspicious conditions, allowing them to grow with limited competition in ephemeral pools in the biflagellated state, allowing them to grow and divide rapidly. Under challenging conditions, the cells lose their flagella and undergo encystment. After encystment, gametes are observed in conjunction with the division of cysts (Triki 1997). There is still much debate on the formation and reproduction of H. pluvialis and further work is needed to elucidate the complete life cycle of the cell.
In H. pluvialis, the primary function of astaxanthin is thought to be a photo- protectant, filtering damaging light using pigment in the cells’ photosystem II as well as providing a physical and chemical barrier to prevent damage from reactive oxygen species (Régneir et al. 2015). The intensity of light to which algae was exposed has a significant impact on the level of astaxanthin synthesized by the cells. In cultures exposed to high light intensities, a higher rate of astaxanthin formation occurs, compared to cultures exposed to low intensity light (Harker, Tsavalos & Young 1996).
Accumulation of astaxanthin begins in the center of the cell, surrounding the nucleus before filling the rest of the protoplast (Lang NJ, 1968). It is thought accumulation begins in this way as a means to protect and stabilize the genetic material of the cell. It is important to note H. pluvialis is not the only species of microalgae that is a producer of hydroxyl groups containing secondary carotenoids. Various Oocystis and Chlorella species also accumulate astaxanthin as a mixture of mono- and bis-ester fatty acids
(Harker, Tsavalos, & Young 1996).
5 Microalgae has the potential to become an environmentally-friendly alternative for compounds in many consumer products such as soy candles, biofuels, pharmaceuticals, and cosmetic products. Secondary carotenoids may be purple, yellow, or red. The pigments found within microalgae show promising treatments for health ailments caused by oxidative damage such as cancer and other age-related diseases
(Pignolet et al. 2013). As previously discussed, the secondary carotenoid astaxanthin produced from H. pluvialis is the most abundant natural source of the compound. It is synthesized through the carotenoid pathway glyceraldehyde-3-phosphate and pyruvate.
Astaxanthin is comparable to vitamin C (ascorbic acid) as it functions as an antioxidant, but it is about sixty-five times more powerful due to its longer carbon backbone (Shah,
Laing, Cheng & Daroch 2016). Currently over 95% of astaxanthin is produced synthetically. However, synthetic astaxanthin has a twenty-times-lower antioxidant capacity than the natural compound and is not currently approved for human consumption. The powerful antioxidant power of natural astaxanthin has driven producers of astaxanthin to explore different methods of growing and harvesting H. pluvialis on a large scale for this use.
Recently, microalgae have received considerable research interest for many of our environmental challenges such as carbon dioxide in the atmosphere and biofuels (Slade &
Bauen 2013). By capturing sunlight and carbon dioxide, it is thought that we may be able to make almost any bio-product such as fuel and plastics (Lundin 2014). Based on species and cultivation conditions, microalgae can produce polysaccharides and triglycerides which are converted into bioethanol and biodiesels (Slade & Bauen 2013).
Freshwater fish account for approximately 45% of world aquaculture and is ecologically
6 friendly and fully sustainable with microalgae being used as a sole component for basic nutrients in addition to being a color enhancement for the flesh of salmonids (Muller-
Feuga 2013). Additionally, many species of bivalve mollusks consume various species of microalgae throughout their lifespan (Becker 2013).
In the environment, open ocean cultivation of microalgae is thought to be a potential solution to address the buildup of carbon dioxide in the atmosphere in addition to providing an adequate protein for the world’s increasing population. Due to a heavy consumption of carbon-based energy sources combined with land-clearing practices needed for building and farming, there is a steadily increasing concentration of carbon dioxide in the atmosphere. A consequence of the rising carbon dioxide levels is the acidification of the ocean, which has a negative impact on coral reefs. By finding a way to filter large quantities of ocean water, saltwater microalgae can consume some of the excess carbon dioxide as well as lessen the concentrations of run-off nitrogen fertilizers which contribute to unwanted harmful algae blooms (Jones & Harrison 2013).
Present work with algae is also aimed at eliminating nitrates and phosphates from industrial wastewater. Studies have shown approximately 80% of nitrates and 60% of phosphates can be consumed by microalgae in 12 days, reducing the water from the industrial classification to irrigation classification (Gupta, Choi, & Lee 2016; Lee & Chen
2016; CWA. 2002). Recycling water is the only significant, currently applicable practice allowing us to help meet the domestic, industrial and environmental water demands that are increasing on a daily basis (Anderson 2003). All of the above-mentioned problems can be solved, in part, by taking advantage of microalgae’s unique properties. Current
7 and future studies using microalgae to reduce the impact of many anthropomorphic issues should receive greater attention and funding.
While commercially important, different stress parameters have not been fully investigated across various strains of Haematococcus spp. The objective of this project was to test three different strains of Haematococcus pluvialis in different known conditions that will induce a stress response by the cells and measure the average amount of astaxanthin produced by the different strains. The results of this experiment may be able to be applied to commercial natural astaxanthin producers so that they may use the most effective stressing conditions to produce a maximum amount of astaxanthin based on what strain of H. pluvialis they grow.
8
MATERIALS AND METHODS Cell culture
Strain 1 (CCAP 34/8) of Haematococcus pluvialis, was purchased from the
Culture Collection of Algae and Protozoa and was isolated by Droop in 1953 from
Tvärminne, Finland. Strain 2 of Haematococcus lacustris, was obtained from the
National Institute for Environmental Studies (NIES-144) and strain 3 of H. pluvialis was acquired from the Scandinavian Culture Collection of Algae and Protozoa (SCCAP-
0084) and was isolated by Pocock in 1950 from Trutbådan, Sweden. All three strains are freshwater species and were grown from a liquid culture and transferred to a 200mL aerated bottle for approximately 10 days. Cultures were kept on a 12:12 light/dark cycle while in the growth phase and 8mM of media was supplemented for volume added of an adapted version of Optimized Haematococcus Media (OHM) (Fabregas & Herrero 1990).
All culture bottles were shaded so they received 1.2 photons of light. The pH of the cultures was kept controlled using added CO2 and was monitored using a standard pH meter to ensure the pH was between 7.0 and 7.7.
Control experiment
Once cultures grew in density, 60mL were subaliquoted into sterile glass culture tubes fixed with an aerated stopper system. The tubes were connected to HEPA filtered
air and enriched with CO2 every 5 minutes to control pH. The starting densities of each tube was calculated using a Muse® Cell Analyzer (EDM-Milipore, Darmstadt,
Germany). Although no nitrates were added after the growth phase ended, nitrate
9 concentration was monitored using MQuant™ colorimetric nitrate test strips (EDM-
Milipore, Darmstadt, Germany) until each culture measured 0 ppm. pH, culture density, and nitrate levels were recorded at the beginning of each day. In addition, daily photos were taken from the first through last day to monitor the visual changes in astaxanthin formation.
Stress condition 1: pH
A similar procedure to the control was followed except for CO2. The air/CO2 line was opened more to allow additional CO2 to flow into the culture than the control experiment. Culture tubes received CO2 every 5 minutes for 3 seconds to maintain a slightly acidic pH of approximately 6.25-6.75. All other procedures remained the same.
Stress condition 2: Salt
In stress experiment 2, 40 mL of NIES and SCCAP strains were sub-aliquoted into the glass culture tubes. CCAP was not able to reach a high enough cell density in the original culture to be split into three replicates and diluted. They were then brought up to 60 mL total using 3% salt water resulting in each tube being 1% saltwater. Culture tubes were corrected daily using sterile freshwater and the same procedure was used as the control experiment. It should be noted an original dilution of the cells was tried at
4% salt, however the cells did not survive and the study was restarted using a 1% salt environment.
10 Astaxanthin extraction & saponification
Astaxanthin was extracted from the cells by mechanically cracked the cells using a mini bead beater, in which each sample was processed for three minutes in acetone.
After extraction, the extracted pigment/acetone solution was recovered from the beads and the astaxanthin standard was added to the solution. Samples were then dried under a nitrogen flux in a dry bath of approximately 40 degrees Celsius, until all acetone had evaporated. The dried pigment was then resuspended in dicholoromethane and put through a saponification reaction using sodium hydroxide and ammonium chloride. The mixture was then centrifuged for five minutes at 3100 rpm. Once finished in centrifuge, the pigments were recovered from the separated reaction and dried under nitrogen flux, until all dichloromethane was evaporated. The pigment was then resuspended in acetone, filtered, and injected into HPLC for analysis. Resulting graph from HPLC analysis is shown below in figure 4. Picograms of astaxanthin was calculated using cell density and
HPLC analysis. The weight was then compared between strains and stressing conditions using a two-way T-test to determine if there was an interaction between the variables on the formation of astaxanthin.
11
Figure 4 Sample readout from HPLC analysis of CCAP-34/8 in the control experiment; Peak labelled 7.430 is free-astaxanthin peak
12 RESULTS
Control
The Haematococcus cultures were monitored through daily visual observation under a light microscope in order to determine the progression of stressing. In order to declare a sample fully stressed, the following criteria had to be met: a red appearance occupying most of the cell, thick cell walls, no flagella, and non-motile, with more than
85% of cells sharing these characteristics. The change from the beginning of the experiment (day 0) to the end of stressing can be seen in figure 5. CCAP 34/8 and NIES-
144 showed all these characteristics and were considered fully stressed on stress day 3 and were then sampled for analysis, while SCCAP-0084 still had 50 ppm nitrates left in the sample. Having 0 ppm nitrates was not a requirement of being considered fully stressed because if there is low cell density, cells will stress and die before they would fully consume all nutrients in media. SCCAP-0084 reached 0 ppm nutrients on day 4 and was sampled at that time.
Figure 5: Physiological changes of Haematococcus cells from stress day 0 to fully stressed. (A) CCAP 34/8 day 0, (B) NIES-144 day 0, (C) SCCAP-0084 day 0, (D) CCAP 34/8 day 3, (E) NIES-144 day 3, (F) SCCAP-0084 day 4
13 HPLC analysis was performed on the fully-stressed samples. Differences were observed in the amount of astaxanthin measured in the control samples. CCAP 34/8 had an average amount of 62.97 picograms of astaxanthin per cell, NIES-144 had an average of 2.1 picograms of astaxanthin per cell, and SCCAP-0084 had an average of 23.83 picograms of astaxanthin per cell.
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 ______Replicate 1 Replicate 2 Replicate 3 Average Strain CCAP 34/8 57.85 93.27 37.81 62.98
NIES-144 2.79 3.51 0 2.10
SCCAP-0084 23.83 22.09 26.12 24.01
______
Experiment 1: low pH
HPLC analysis of the samples in low pH conditions showed significant results concerning NIES-144. The average amount of astaxanthin for NIES-144 was 33.09 picograms per cell, a statistically significant increase from the control average of 2.1 picograms per cell (p=0.006). To determine whether samples had a significant change in the amount of astaxanthin produced, a two-way T-test was used. The amount for the individual samples can be seen in table 2.
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 ______Replicate 1 Replicate 2 Replicate 3 Average Strain CCAP 34/8 63.63 60.13 97.25 73.67
NIES-144 30.47 31.01 37.80 33.09
SCCAP-144 79.73 3.01 8.40 30.38
______
Comparison of astaxanthin formation in control vs. low pH
80 70 60 ) 1
- 50 40 30 (in pg cellpg (in 20 Amount of Astaxanthinof Amount 10 0 CCAP 34/8 NIES-144 SCCAP-0084 Strain
Control Low pH
Figure 6: Bar graph showing the difference in astaxanthin formation in the averages of the control experiment and the low pH experiment
Experiment 2: salt
Originally, this experiment was attempted using a dilution of the cultures in three percent salt water. This experiment failed as none of the cultures could handle such a shock to their osmotic systems. The experiment was then repeated using a dilution of one
15 percent salt water, in which cultures were able to survive, with the exception of CCAP
34/8. Although cultures were able to survive, many of the cells were unhealthy and caused degradation due to cell wall instability as seen in figure 6.
Figure 7: SCCAP-0084 lysed cell wall due to 1% salt concentration
The two surviving strains, NIES-144 and SCCAP-0084, lost significant cell density when diluted with salt water. NIES lost approximately 78% of cell density from the time it was inoculated to the time the sample was taken. Due to this, the sample was not dense enough for HPLC analysis to take place. A similar situation arose concerning SCCAP-
0084, losing approximately 43% of cell density. As it had a much higher starting density than NIES-144, HPLC analysis was still able to be performed on the sample and results are shown in table 3.
Table 3: Quantitative astaxanthin composition (in pg cell-1) of cells stressed under 1% salt conditions, obtained by HPLC analysis of H. pluvialis cells of SCCAP-0084 ______
Replicate 1 Replicate 2 Replicate 3 Average Strain SCCAP-0084 34.25 206.07 237.39 159.23
16
Comparison of astaxanthin formation in control and salt conditions
180 160 140 )
1 120 - 100 80 60 (in pg cellpg (in 40
Amount of Astaxanthinof Amount 20 0 SCCAP-0084 Strain
Control Salt
Figure 8: Bar graph showing the difference in astaxanthin formation average of the control and salt experiments
17 DISCUSSION
HPLC analysis of saponified algae samples allowed the quantification of astaxanthin to be found in three different strains of Haematococcus pluvialis microalgae under three different stressing conditions. While most results were similar to other studies using the customary way of stressing microalgae- using high light and the cessation of nutrients (Shah, Laing, Cheng, & Daroch 2016; Ambati, Phang, Ravi, &
Aswathanarayana 2014)- the result of NIES-144 showed a statistically significant increase in astaxanthin amount when stressed in a low pH environment. The results of
SCCAP-0084 were expected to be statistically significant due to the high calculated amounts of astaxanthin, but due to the large variance between the replicates, it was not a significant result.
Three percent salt water was chosen in this study to match the salinity of ocean water. When this stress test was repeated this experiment using one percent saltwater,
NIES-144 and SCCAP-0084 were able to survive the stress of being mixed with salt water. Although NIES-144 was able to survive the change in salinity, the density of the culture was so low on the final day that it was too dilute to be sampled and quantified on
HPLC. Previous literature suggested the use of 0.2% NaCl as being the most successful in stressing H. pluvialis (Cordero et al. 1996) as cultures with higher salinities than 0.8% can be lethal to cultures. The results obtained for using salt as a stressing condition were consistent with what was shown in previous literature as all of the CCAP 34/8 culture died, and both NIES-144 and SCCAP-0084 had significant losses in culture density.
Boussiba and Vonshak (1991) reported exposing H. pluvialis cells to a condition of 0.8%
NaCl which led to the complete cessation of growth along with a large accumulation of
18 astaxanthin. While this experiment did not include using 0.8% salinity, it would be expected that an experiment involving 0.8% salinity would have a higher survival of cells when salt conditions are induced and an equal, if not higher amount of astaxanthin be expected per cell.
Another factor to take into account for the difference in results between the three strains tested is their innate ability to produce astaxanthin. The control experiment showed CCAP 34/8 had an increase in production of astaxanthin, but was not consistent between replicates. SCCAP-0084 had a moderately high but also consistent production of astaxanthin. All three strains used in this research originate from the same branch of the Haematococcus phylogenic tree based on rDNA results (Allewaert et al. 2015).
While all three have an ancestrally similar origin, they are genetically distinct strains and their genetic differences could possibly cause them to react to stress conditions differently as shown through differing amounts of astaxanthin produced (Allewaert et al.,
2015, Buchheim et al. 2013). This difference would be of interest to explore in future research.
Some samples yielded an inaccurate amount of astaxanthin because the range fell near the top of the standard curve for the test performed. Unfortunately, excess sample was not available for the test to be repeated using a different dilution. Another possible explanation for the discrepancy between HPLC results is that each lab has its own protocol for HPLC analysis and unless the protocol has been verified by other labs, it is unlikely that other labs will be able to reproduce the results exactly. Although this explanation addresses why these results may not align with previous research, all sample
19 analysis was performed on the same instrument using the same protocol, which gives a measure of internal validity.
The stressing experiment of NIES-144 under 1% salt condition yielded a significantly higher amount of astaxanthin than under standard conditions. This result is applicable to the aquaculture and nutraceutical industry for producers of astaxanthin who use the strain NIES-144. The standard protocol in the industrial growth and production of natural astaxanthin is to stress the cells using high light and cessation of nutrients, but producers may benefit from an addition of salt to the cultures. Further testing would need to be done to better establish what percent salinity will produce the highest amount of astaxanthin. A better stressed biomass would benefit natural astaxanthin producers for the aquaculture industry; as the food given to their fish would be more enriched, inferring that the general health of the fish could be increased due to the antioxidant properties of the astaxanthin, and would result in healthier looking salmon for consumers.
Although my data suggests that 1% salt caused the highest production of astaxanthin in NIES-144, this model of stressing is not ideal for the other two strains of
H. pluvialis used in my research and may not yield similar results for strains. If I were to repeat this experiment, I would compare the three strains used under the traditional stressing conditions against different salinities from the literature such as 0.2%, 0.4%, and 0.8% NaCl. Additionally, I would also ensure that each strain and replicate would have the same starting density to account for difference in survival when transferred to salt conditions. Further research should be done in order to assess if the other strains would have survived the shock of being transferred into a salt environment and investigate the astaxanthin production had they survived.
20 CONCLUSION
The powerful antioxidant capacity of natural astaxanthin, found in
Haematococcus pluvialis, is of interest as a nutraceutical, an additive for fish food, and for food coloring. The potency of natural astaxanthin is twenty times higher than synthetic astaxanthin and five hundred times stronger than Vitamin E (Ambati, Phang,
Ravi, & Aswathanarayana 2014). This compound shows great potential to impact several industries, if able to be produced naturally and cost effectively. Based on my findings, low pH conditions may promote astaxanthin formation in the NIES-144 strain.
Additionally, work to further elucidate optimal conditions for astaxanthin production in
H. pluvialis may lead to more efficient production of this important compound.
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