BSysE 595 Class Projcet
Xin Gao 11212457
BSysE 595 Class Project
Project name
Maximize the yield of Astaxanthin in the Haematococcus pluvialis growth
Project description
Astaxanthin is a carotenoid. It belongs to a larger class of phytochemicals known as terpenes,
which are built from five carbon precursors; isopentenyl diphosphate and dimethylallyl
diphosphate [1]. It is currently used as a feed supplement for seafood and animals [2]. The
primary use for humans is as a food supplement [2], which is a high-value product. Researches
showed that, due to astaxanthin's potent antioxidant activity, it may be beneficial in
cardiovascular, immune, inflammatory and neurodegenerative diseases. Some research
supports the assumption that it may protect body tissues from oxidative and ultraviolet damage
through its suppression of NF-κB activation.
Astaxanthin can be found in microalgae, yeast, salmon, trout, krill, shrimp, crayfish, crustaceans,
and the feathers of some birds [1]. It is difficult to synthetize astaxanthin artificially, because
synthetic astaxanthin always contains a mixture of stereoisomers [3]. Another method is
extracting it from the shellfish [4]. However, this method is limited by the low content of
astaxanthin in the shellfish, also the product yield and purity are not satisfied enough, which
cause the high cost. Some researchers [5] use yeast to product the astaxanthin, such as Phaffia
rhodozyma, Rhodotorula rubra and Rhodotorula glutinis. But, now the highest yield is only 0.3%
of the dry cell weight and small condition changes will easily affect the accumulation of
astaxanthin during the fermentation, such as temperature, pH value, dissolved oxygen, carbon
and nitrogen sources and so on. All in all, these are not the perfect method for this project.
In this project, Haematococcus pluvialis, one kind of the microalgae, was chosen to product the
astaxanthin. Haematococcus pluvialis can produce the astaxanthin as high as 4% of the dry cell
weight [3]. The current limitations are: 1). this kind of microalgae grows slowly, requires longer
time; 2). it is also sensitive to environmental changes and susceptible to other algae, bacteria
and pollutions; 3). the yield of 4% of dry cell weight is not ideal enough. So, it is necessary to
optimize the culture conditions to grow the Haematococcus pluvialis faster and research the
maximum biomass and then improve the product yield by changing the light intensity,
temperature, nutrient, etc.
Objectives
1. Find the optimal environment for growing the Haematococcus pluvialis faster and reach the
maximum biomass.
2. Improve and maximize the product yield of the astaxanthin.
BSysE 595 Class Projcet
Xin Gao 11212457
Major findings

Light intensity
Haematococcus pluvialis is very sensitive to the light intensity, especially at the beginning of
the microalgae growth in the reactor and it can be cultured under the 30 μmol/m2/s of the
light intensity.

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pH value
From the pH value change during the cultivation, we recommend that at the first 3 days or
when the DCW is less than 0.4g/L, the percent of the CO2 in the air flow can be set at 1~1.5%;
then the percent of the CO2 need to be set around 3%; when the DCW reaches 0.8 g/L, 5%
or more of the CO2 is needed.
Optical density
Some researchers mentioned that they used the value of OD at 680nm to calculate the
microalgae growth. However, from the UV spectrum, at 680nm there is a big peak, which is
the absorption of one kind of the chlorophyll. If the OD value is collected at 680nm, here
exists the error. Based on our collected spectra data, the OD value is more accurate, when
collected at 750 nm than at 680 nm. So, at 750 nm is a good point to detect the OD value.
Air flow rate
Under the 1L/L/min, the differences of air flow rates didn’t affect the growth very much.
However, if the rate is larger than that value, it may damage or kill some microalgae
because of the fast and strong bubbles. Some further research about this will be studied.
Vitamins, concentration and the preparation of the BG 11 medium
The results showed that adding vitamins or double concentration will help the
Haematococcus pluvialis growth. And it is better to add the metal and trace elements after
autoclaved the rest contents of the BG 11 medium.
The content of the astaxanthin, which extracted from H. pluvialis, was 31.29 ± 1.88 mg/L
(dry weight), or 2.94%-3.32%.
Contamination issues
Haematococcus pluvialis is very sensitive, and it is easy to get contaminated by other algae,
bacteria and microbe.
Currently, all of the cultured Haematococcus pluvialis in the reactors and flasks, and even
some of the Haematococcus pluvialis seeds were contaminated by one kind of unknown
microbe. Based on our experience, it may be one kind of cliates, which causes all the
microalgae died. And we believe that this microbe may swallow and digest the
Haematococcus pluvialis cell, if it is true, it could explain that why the dry cell weight and
the optical density decrease very fast just in one day. However, we haven’t observed the
process that this microbe swallowed the cell.
BSysE 595 Class Projcet
Xin Gao 11212457
Results of the first batch
Haematococcus pluvialis is very sensitive to the light intensity, especially at the beginning of the
microalgae growth in the reactor.
The above picture shows that after the seed loading, only less than about 15 hours later, under
the 60~70 μmol/m2/s of the light intensity, the Haematococcus pluvialis in the reactor died or
partly turned to the second stage. And based on our previous observation, the microalgae
would die after 3~5 days developed, if it was under the 50 μmol/m2/s of the light intensity.
So, based on our results, at the beginning the Haematococcus pluvialis can be cultured under
the 30 μmol/m2/s of the light intensity.
BSysE 595 Class Projcet
Xin Gao 11212457
Also, the original loading of the seeds into the reactors may be an important factor. Thus,
currently we are preparing more seeds for the experiment. Our loading of the first batch is
0.129g.
BSysE 595 Class Projcet
Xin Gao 11212457
Growth curve:
Original loading: 0.129g Haematococcus pluvialis into 1000mL BG11 medium.
Growth curve
Dry cell weight (g/L)
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
Time (day)
About the optical density (OD)
10
12
14
BSysE 595 Class Projcet
Xin Gao 11212457
Some researchers mentioned that they used the value of OD at 680nm to calculate the
microalgae growth. However, from the UV spectrum Tingting and I got, at 680nm there is a big
peak, which is the absorption of one kind of the chlorophyll. If the OD value is collected at
680nm, here exists the error.
The content of the astaxanthin from Haematococcus pluvialis
The content of the astaxanthin, which extracted from H. pluvialis, was 31.29 ± 1.88 mg/L (dry
weight), or 2.94%-3.32%.
Results of second batch
Growth curve:
Original loading: 0.370g Haematococcus pluvialis seed into each reactor with 1000mL BG11
medium.
Growth curve
Dry cell weight (g/L)
0.8
0.7
0.6
0.5
0.4
Double BG 11
0.3
BG 11
0.2
BG 11+vitamin
0.1
0
0
2
4
6
8
Time (day)
10
12
14
16
BSysE 595 Class Projcet
Xin Gao 11212457
pH value:
pH value
8
pH value
7.5
7
0
2
4
6
8
10
12
14
16
6.5
6
Time (day)
Here, the x axis is at pH 7.1, which is the optimal pH value and also the pH of the BG 11 medium.
During the cultivation, the percent of the CO2 in the air flow was 2%. So, from the pH value
change during the cultivation, we recommend that at the first 3 days or when the DCW is less
than 0.4g/L, the percent of the CO2 in the air flow can be set at 1~1.5%; then the percent of the
CO2 need to be set around 3%; when the DCW reaches 0.8 g/L, 5% of the CO2 is needed.
Optical density (OD) analysis
Based on our collected spectra data, the OD value is more accurate, when collected at 750 nm
than at 680 nm. So, we think at 750 nm is a good point to detect the OD value. As we can see
from the two figures about the linear equations of optical density vs. dry cell weight below, by
collecting the spectrum of the same microalgae samples, the R2 of the 750 nm OD value vs. DCW
is much better than the 680 nm obtained OD values vs. DCW (0.9243 vs. 0.8349).
BSysE 595 Class Projcet
Xin Gao 11212457
Optical density (680 nm) vs. dry cell weight
Optical density (750 nm) vs. dry cell weight
BSysE 595 Class Projcet
Xin Gao 11212457
Results of the third batch
Here, different air flow rates, medium with/without vitamins and different preparation
conditions were compared to see their influence on the growth rate.
Original loading: 0.051 g Haematococcus pluvialis seed into each 500mL glass flask
Cultivation under different conditions (3 days growth)
BG 11 BG 11 I BG 11 II BG 11 + Vitamins* BG 11 filter BG 11×2
Air flow rate
1.00
0.40
0.70
1.00
1.00
1.00
0.18
0.16
0.18
0.46
0.60
0.58
(L/L/min)
Dry cell weight
(g/L)
*Vitamins: Biotin 25 10 μg/L, B12 15 μg/L, Thiamine-HCl 17.5 μg/L
1) BG11, BG11 I, BG11 II, BG11+Vitamins and BG11×2, these mediums used the same
preparation conditions; for BG11 filter, metal and trace elements were added into the flasks
through the filter, after all other contents were autoclaved at 121℃ for 20mintues.
2) For BG11+Vitamins medium, the vitamins were added into the flask through the filter, after
all other contents were autoclaved at 121℃ for 20mintues.
3) BG11×2 is the double concentration of the normal BG 11 medium.
4) The difference between BG11，BG11 I and BG11 II is the air flow rate. BG11 is 0.5L/min,
BG11 I 0.2L/min and BG11 II 0.35L/min.
From the table we can see that:
 Under the 1L/L/min, the differences of air flow rates didn’t affect the growth very much.
However, if the rate is larger than that value, it may damage or kill some microalgae
because of the fast and strong bubbles. Some further research about this will be studied.
 The growth under the conditions, like BG11+Vitamins, BG11 filter and BG11×2, is much
better than the normal BG 11 medium. It means that adding vitamins or double
concentration will help the Haematococcus pluvialis growth. And in the future, it is better to
add the metal and trace elements after autoclaved the rest contents of the BG 11 medium.
Current major problems and issues
Haematococcus pluvialis is very sensitive, and it is easy to get contaminated by other algae,
bacteria and microbe.
BSysE 595 Class Projcet
Xin Gao 11212457
Currently, all of the cultured Haematococcus pluvialis in the reactors and flasks, and even some
of the Haematococcus pluvialis seeds were contaminated by one kind of unknown microbe,
based on our experience; it may be one kind of cliates, which causes all the microalgae died.
Under the observation of the microscope, we found that this microbe is different from the one
which Jian Fang reported in the Jinjing 2012 annual report.
The Cliate Jian Fang reported in the Jinjing 2012 annual report
Now, we believe that this microbe may swallow and digest the Haematococcus pluvialis cell.
Based on this hypothesis, it could explain that why the dry cell weight and the optical density
decrease very fast. However, we haven’t observed the process that this microbe swallowed the
Haematococcus pluvialis cell. Below is the picture of the contaminated reactor.
The contaminated reactor
BSysE 595 Class Projcet
Xin Gao 11212457
Conclusion
From this project, currently we understand some of the characteristics of Haematococcus
pluvialis. By optimizing the light intensity, pH value, air flow rate and adding vitamins in the
medium, we can promote the growing of the cell in the first stage. Also, double concentration
and the new way to prepare the BG 11 medium can help increase the growth. Besides, obtain
the optical density at 750 nm is better than at 680 nm based on our results, and the content of
the astaxanthin, which extracted from H. pluvialis, was 31.29 ± 1.88 mg/L (dry weight), or 2.94%3.32% measured by the calculation. However, the most serious issue is the contamination, and
different treatment methods (i.e. acetic acid solution) are tried to solve the problem.
Due to the time limitation, only the first objective, find the optimal environment for growing the
Haematococcus pluvialis faster and reach the maximum biomass, is completed. Next, we will
focus on improving and maximizing the product yield of the astaxanthin from H. pluvialis. In the
future, the contamination treatment will be one of the major emphases in this work, also, by
understanding Haematococcus pluvialis metabolic pathway, maybe we can increase the yield of
astaxanthin by modifying some pathways in the molecular or genetic level.
Reference
1. Yousry M. A. Naguib. Antioxidant Activities of Astaxanthin and Related Carotenoids. J. Agric.
Food Chem., 2000, 48 (4), pp 1150–1154.
2. P. Stepnowski, G. Ólafsson, H. Helgason, B. Jastorff. Recovery of astaxanthin from seafood
wastewater utilizing fish scales waste. Chemosphere. 2004, 54(3), pp 413–417.
3. Makio Kobayashi, Toshihide Kakizono, Shiro Nagai. Astaxanthin production by a green alga,
Haematococcus pluvialis accompanied with morphological changes in acetate media.
Journal of Fermentation and Bioengineering. 1991, 71(5), pp 335–339.
4. W. Miki, K. Yamaguchi, S. Konosu. Comparison of carotenoids in the ovaries of marine fish
and shellfish. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry.
1982, 71(1), pp 7–11.
5. Eric A. Johnson and Michael J. Lewis. Astaxanthin Formation by the Yeast Phaffia rhodozyma.
Microbiology. 1979, 115(1), pp 173-183.