Bsyse 595 Class Projcet Xin Gao 11212457

BSysE 595 Class Projcet Xin Gao 11212457

BSysE 595 Class Project

Project name

Maximize the yield of Astaxanthin in the Haematococcuspluvialis 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; isopentenyldiphosphate and dimethylallyldiphosphate [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.

Astaxanthincan be found in microalgae, yeast, salmon, trout, krill, shrimp, crayfish, crustaceans, and the feathers of some birds [1]. It is difficult to synthetizeastaxanthin artificially, because synthetic astaxanthinalways 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 Phaffiarhodozyma, Rhodotorularubra and Rhodotorulaglutinis. But, now the highest yield is only 0.3% of the dry cell weight and small condition changes will easily affect the accumulation of astaxanthinduring 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, Haematococcuspluvialis,one kind of the microalgae, was chosen to product the astaxanthin.Haematococcuspluvialis 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 Haematococcuspluvialisfaster 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 Haematococcuspluvialisfaster and reach the maximum biomass.

2.Improve and maximize the product yieldof the astaxanthin.

Major findings

• Light intensity

Haematococcuspluvialis 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.

• 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 Haematococcuspluvialisgrowth. 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

Haematococcuspluvialis is very sensitive, and it is easy to get contaminated by other algae, bacteria and microbe.

Currently, all of the cultured Haematococcuspluvialis in the reactors and flasks, and even some of the Haematococcuspluvialisseeds 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 Haematococcuspluvialis 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.

Results of the first batch

Haematococcuspluvialis 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 Haematococcuspluvialis 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 Haematococcuspluvialis can be cultured under the 30 μmol/m2/s of the light intensity.

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.

Growth curve:

Original loading: 0.129g Haematococcuspluvialis into 1000mL BG11 medium.

About the optical density (OD)

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 Haematococcuspluvialis

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 Haematococcuspluvialis seed into each reactor with 1000mL BG11 medium.

pH value:

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).

Optical density (680 nm) vs. dry cell weight

Optical density (750 nm) vs. dry cell weight

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 Haematococcuspluvialis 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
(L/L/min) / 1.00 / 0.40 / 0.70 / 1.00 / 1.00 / 1.00
Dry cell weight
(g/L) / 0.18 / 0.16 / 0.18 / 0.46 / 0.60 / 0.58

*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 Haematococcuspluvialisgrowth. 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

Haematococcuspluvialis is very sensitive, and it is easy to get contaminated by other algae, bacteria and microbe.

Currently, all of the cultured Haematococcuspluvialis in the reactors and flasks, and even some of the Haematococcuspluvialisseeds 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 CliateJian Fang reported in the Jinjing 2012 annual report

Now, we believe that this microbe may swallow and digest the Haematococcuspluvialis 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 Haematococcuspluvialiscell. Below is the picture of the contaminated reactor.

The contaminated reactor

Conclusion

From this project, currently we understand some of the characteristics of Haematococcuspluvialis. 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 Haematococcuspluvialis 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 Haematococcuspluvialis 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, Haematococcuspluvialis 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 Phaffiarhodozyma. Microbiology. 1979, 115(1), pp 173-183.