INFLUENCE OF THE CONDITIONS OF CULTIVATION OF A NEW SPECIES OF THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS FOR THE SAMARA REGION ON THE TRANSITION FROM THE HEMIMONAD STAGE TO THE MONAD STAGE

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During the analysis of periphytic organisms on a suburban area in the village of Novosemeikino in October 2020, we discovered a new for the Samara Region species of green algae Haematococcus pluvialis. At the time of the study it looked like large, stationary, spherical, red cells with hematochrome in the hemimonad stage. It is known that in the stationary (hemimonad) stage it waits out unfavorable conditions, and in the mobile (monad) stage it actively reproduces and accumulates biomass.

Systematic position. Haematococcus pluvialis Flotow [1] is located in the kingdom of Chloroplastida and belongs to the department of Chlorophyta [2].

This microalgae is the best natural source of astaxanthin. Its cells contain up to 80% of hematochrome, containing astaxanthin, which is used as:

– biologically active additives;

– medicines;

– cosmetic products;

– animal feed/feed additive (for example, salmon, flamingos and shrimp).

Due to its ability to accumulate a large amount of astaxanthin, Haematococcus pluvialis is being actively studied and cultivated around the world. In the PubMed database Central.org we have found 1717 articles mentioning this substance [3].

 

Research methods.

For the transition of H. pluvialis to the active monad stage we have prepared two media (BBM-1, BBM-2), which we consider the most suitable for the "awakening" of hematococcal cells.

After placing small samples in the flasks with media (three flasks per a medium), they were closed with cotton plugs and cultured at 400–500 LUX illumination at room temperature (approximately 18–20 °C), in a cycle of light and darkness of 12:12 hours respectively (later 16:8).

 

Results and their discussions.

After 4 days in the BBM-1 medium we found small cells up to 10 microns in diameter. It is 40% of the total number of cells, whose contents are light green granular (fig.1). In large cells with a diameter of more than 40 microns, the process of astaxanthin disappearance began from the periphery of the cell to the center, in about 25% of cells of this type (fig.2). The remaining 35% of the cells were green with average size 25 microns in diameter.

Fig. 1 H. pluvialis on day 4th of cultivation                               

Fig. 2 H. pluvialis on day 4th of cultivation                                                          

We have observed the disappearance of hematochrome and the manifestation of a bright green chromatophore with a granular structure in 50% of the cells in the BBM-2 medium (fig.3). In other cells the hematochrome occupies a clearly limited position at the edge of the cell (fig.4). Larger single cells still remain uniformly red.

Fig. 3 H. pluvialis on the 4th day of cultivation         

Fig. 4 H. pluvialis on the 4th day of cultivation                                      

It should be noted, that in two quite similar in composition media being studied, morphological changes are different at this stage of cultivation.

After nine days of cultivation, the differences became minimal, and in both media 95% of individuals turned green, but monadic forms had not been detected yet (fig.5).

Fig. 5 General view of the sample on the 9th day of cultivation

Fig. 6 H. pluvialis cells in BBM-1 medium       

Fig. 7 H. pluvialis cells in BBM-2 medium

On the 26^th day a monad stage with a length of 10 microns and width of 8 microns was detected in the BBM-1 flask. Inside there was a clear pyrenoid and a cup-shaped chromatophore.

In addition to the monad stage, there were all green autospores, but among them some dead colorless autospores were also found (fig.8). Bright green divided cells, 6 pieces per division (fig.9) were observed as well.

Fig. 8 H. pluvialis cells in BBM-1 medium on the 26th day of cultivation             

Fig.9 H. pluvialis cells in the BBM-1 on the 26th day of cultivation       

In another flask with BBM-1 medium, there were single bright red cells. A hematochrome appeared in each cell of the sample, because the cultivation conditions might have become somewhat worse.

Fig. 10 H. pluvialis cells with hematochrome manifestation in BBM-2 medium on day 26th of cultivation

In one of the 6 flasks with BBM-1 medium, an active transition to the monadic stage of Haematococcus pluvialis was observed after 33 days, with up to 1-2 cells in one field of view in the water column, which gave a uniform yellow-green color to the medium. Dead cells were also found (fig.11).

Fig.11 H. pluvialis cells in BBM-2 medium on day 33 of cultivation

After 10 months of cultivation, the changes were as follows.

The sonata form might become hemimonad again because of the hot summer season, conditions worsened. As a result, Haematococcus pluvialis became an aplanospore again, but without the formation of a hematochrome. Staying in hemimonad form, H. pluvialis continued to divide mitotically. Unfortunately, because of the fact that the culture is not pure, the number of its cells has not increased much, as it is known that microalgae Scenedesmus sp. divide more actively.

In the 10th month of cultivation, Haematococcus pluvialis began to divide mitosis more actively (fig.12) and increased in size. It reached 40 microns in diameter again (fig.13). Most likely, this is due to the fact that in the winter season the brightness is sufficiently lowered so as not to get into the hematocyst stage. As for the other types of microalgae, it is too low for active reproduction.

Fig. 12 H. pluvialis cells in BBM-2 medium after 10 months of cultivation

Fig. 13 H. pluvialis cells in BBM-2 medium after 10 months of cultivation

Conclusion.

Having observed the cultivation of Haematococcus pluvialis, we can argue that this microalgae is quite sensitive to seasonal transitions, namely to natural light. It has also been experimentally proven that H. pluvialis is not a dominant species in a mixed medium with other species. While cultivating, we poured carbonated water for bubbling of the culture, when the medium changed from 150 ml to 100 ml. After the infusion, the cells divided more actively, which indicates that the presence of CO₂ in the medium has a beneficial effect on the growth and development of all algae, including Haematococcus pluvialis.

Summarizing the data on cultivation of a liquid nutrient medium, we have not been able to obtain a pure culture yet, since other concomitant species we have studied reproduce more actively than Haematococcus pluvialis, which leads to clogging of the culture.

To obtain a pure culture, it is necessary to take individual cells of Haematococcus pluvialis under a microscope and place them in a sterile nutrient medium.

Now we can only draw preliminary conclusions, since the work is still at the beginning.

1. BBM medium is suitable for the cultivation of Haematococcus pluvialis;

1. For the transition from the hemimonade to the monad stage, it is necessary to use the nutrient medium BBM-1, and BBM-2 for the accumulation of biomass. 

In the future, we are going to isolate a pure culture of algae, as well as tocontinue to study necessary conditions not only for its active growth, but also for the increased accumulation of astaxanthin in it. Then we need to develop ways to the best extraction and use of the active ingredient.

About the authors

Анастасия Павловна Касьянова

Самарский национальный исследовательский университет имени академика С.П. Королёва

Author for correspondence.
Email: anastasiakasyanova22@mail.ru
Russian Federation

Evgeny Sergeevich Korchikov

Samara University

Email: evkor@inbox.ru
443086, Russia, Samara, Moskovskoye Shosse, 34

References

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