Morphological and Morphometrical Features in Dunaliella salina (Chlamydomonadales, Dunaliellaceae) During the Two-phase Cultivation Mode

The paper presents studies of morphological and morphometrical characteristics of green halophilic carotenogenic microalga Dunaliella salina (Dunal) Teodoresco, 1905, from the south-west region of Crimean Peninsula. D. salina was cultivated in two-phase mode under conditions of natural illumination at the premises of A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (IBSS), Sevastopol, Russia. The maximum D. salina cell density was 1.69·10 6 cell/ml in the “green” phase and 0.84·10 6 cell/ml in the “red” growth phase. The maximum productivity by cell number reached 0.15 ∙ 10 6 cell/(ml·day) in the “green” phase while it was lower by 73% in the “red” phase (0.04∙10 6 cell/(ml·day). Along with the maximum productivity, linear growth stage in the first phase was characterized by a maximum fraction of small (up to 500 μm 3 in volume) cells (about 15-29%) and a decrease in cell volume by 40-45% as compared with initial value. The mean of D. salina cell volume in the “red” phase was 30% higher than in the “green” phase. At the same time, the large cell fraction in the “red” phase was consistently high (15-35%). The patterns of change in morphological and morphometrical cell parameters were in accordance to stage and conditions of growth. Thus, cell elongation was noted in the stage of linear growth, while under unfavorable conditions at growth-declining stage cells became more round-shaped, with orange and tile-red coloration and granulation of cell content. It was shown that morphological and morphometric cell parameters can serve as additional criteria for assessment of physiological condition in D. salina culture. The experiment demonstrated the prospects for two-stage D. salina cultivation in Crimea.


Introduction
Crimean Peninsula (the south of Russia) is region of Dunaliella salina (Dunal) Teodoresco, 1905 natural habitat. The abundance of hypersaline reservoirs provides favorable conditions for mass reproduction of this species in the summer time, while the impact of stress factors (high salinity, temperature, solar radiation) promotes β-carotene accumulation in the cells of microalgae (Massyuk 1973;Oren 2005;Posudin et al. 2010). Studies of natural reservoirs, as well as salt evaporation ponds, show that the concentration of biomass and β-carotene in them is significantly less as compared with intensive Dunaliella cultivation (Massyuk 1973;Oren 2014;Borovkov & Gudvilovich 2015).
Dunaliella salina cells might belong to the "green" or the "red" type depending on environmental conditions. The cells of Dunaliella have green coloration and reproduce predominantly by vegetative propagation under favorable for growth and reproduction external factors (Massyuk, 1973). Under influence of the stress factors the β-carotene synthesis is induced and Dunaliella cells turn into a "red" phase (Ben-Amotz 1995;Borovkov & Gudvilovich 2015) that in turn is used for commercial production of β-carotene.
Dunaliella salina cells have monad structure with two identical flagella at the apical side. Cells sizes are varying: from 2.8 to 40 μm in length, from 1.5 to 20 μm in width and from 8 to 4500 μm 3 in volume (Massyuk 1973;Oren 2005;Borovkov 2005;Tafreshi & Shariati 2009;Posudin et al. 2010;Preetha et al. 2012). High species variability (plasticity level) is the main morphological characteristic of D. salina, so that its cells change form and size easily in response to environmental changes (Oren 2005;Preetha et al. 2012;Srirangan et al. 2015). Cells form can vary from ovoid, pear-shaped, ellipsoid to fusiform and almost spherical depending on growth and development stage and on environmental conditions (Massyuk 1973). There is one cup-like chloroplast surrounded by starch with pyrenoid in the center. The abundant oil drops containing dissolved carotenoids, predominantly β-carotene (Tafreshi & Shariati 2009, Achour et al. 2019 accumulate in the chloroplast stroma under extreme conditions. As a result the cells acquire a yellow-brown, orange or tile-red coloration (Fu et al. 2014;Lv et al. 2016, Wei et al. 2017. Such colored cells usually contain carotene lipid globules not only in chloroplast, but also in cytoplasm. The presence of the thin transparent plasmalemma lacking rigid cell wall that enables examining cell organelles during cytokinesis is one of sufficient features of genus Dunaliella (Massyuk 1973). This feature allows quick response control of changes in the morphometric and physiological parameters in culture of D. salina at different growth stages and under the action of various abiotic factors. This character allows the use of the microalga as a good model for studying the characteristics of carotenogenesis taking into account the growth of alga in a specific region. Two-phase cultivation in open ponds under natural illumination is convenient for study of morphometric and physiological characteristics in D. salina, as it provides the observation of different growth stages, as well as the influence of nutrient supply and illumination increase due to culture dilution. In this regard, the purpose of our study was to investigate the physiological, morphological and morphometric characteristics of carotenogenic microalgae D. salina during its two-phase cultivation in the south-west of Crimean Peninsula (Sevastopol region).

Culturing conditions
The investigation was carried out at the premises of Biotechnology and Phytoresources Department of IBSS, Sevastopol, Russia (located at 44°36'54.6"N 33°30'11.7"E) from May 24, 2018 to July 13, 2018. The unialgal culture of D. salina (strain IBSS-2 from IBSS common use center "World Ocean hydrobionts collection") was grown in modified culture medium of Shaish et al. (1990). The modification involved adding of sea salt ("Galit", Russia) up to 120 g/L concentration. Algae were cultivated in algabiotechnological greenhouse module made of polycarbonate that provided temperature maintaining and protection from possible atmospheric precipitation. Square ponds (1 × 1 m) were covered with polyethylene film and layed on the leveled ground. Culture of D. salina maintained in greenhouse module during previous winter and spring period was used as the inoculate, algal cells were at the "red" form. Temperature and illumination level were natural: the maximum illumination at the surface of the cultivators reached 80-90 klx on sunny days and 40-50 klx on the cloudy days; temperature in the ponds varied from 24 to 35 °С. Cell suspension was continuously stirred by aquarium pump Atman AT-201 (Chuangxing Electrical Appliances Co., Ltd, China).
The first phase duration was 29 days, the second phase duration was 21 days. In the first cultivation phase the inoculate and culture medium were mixed (with the ratio 1:1.5). In the second cultivation phase, culture of Dunaliella was diluted twofold with fresh culture medium lacking nitrogen and phosphorous salts. Culture layer depth was 9 cm in the first stage and 4 cm in the second stage, culture volume was 90 L and 40 L respectively.
Maximum productivity (P m ) was calculated through the approximation of growth curve in the linear growth stage area on the basis of cell number: where N lcell density at the beginning of linear growth phase; t l is the time at the beginning of linear growth phase (Lelekov &Trenkenshu 2007).
In the linear growth phase cell division number and rate were determined during the night. Cell division number (n) was calculated by equation: where N and N 0 are cell density in the end and in the beginning of the period.

Microscopy
Morphometrical characteristics of D. salina in culture were analyzed using a microphotographs obtained with the help of light microscope Carl Zeiss Axiostar Plus (Carl Zeiss company, Oberkochen, Germany) equipped with digital camera Canon PowerShot a620 (Canon Inc., Tokyo, Japan), and software Micam (Science4all, 2009). The morphological characters such as size, shape and coloration of the cell, flagella occurrence, characteristics of stigma, pyrenoids, chloroplasts and other cytoplasmic inclusions, such as starch granules were estimated based on 100 randomly chosen cells. Cell length (L) and width (D) were measured and cell volume (V) was calculated by the formula of prolate spheroid (Sun & Liu 2003): where Н is the third axis of ellipse and ellipsoid. Dividing cells were determined by the occurrence of appropriate physiological changes, including pyrenoid, chloroplast and cytoplasm fission (cytokinesis).

Statistical analyses
Statistical data analysis was performed using Microsoft Excel software. Arithmetic mean ( ), standard deviation (S), error of the mean, confidence intervals for the mean (Δ ) were estimated. The significance of differences was determined by Student's t-test (if the variances of the samples were assumed to be equal) or by unequal variances t-test (if samples have unequal variances). All calculations were made for the significance level α 0.05. The mean and confidence intervals (x Δ x ) are given in the text and at the diagrams.

Results
"Green" phase At the first, "green" phase, the favorable conditions for high cell division rate in D. salina culture are established. Hence, the conditions, provided in our experiment, enabled increasing Dunaliella cell density from 0.23·10 6 cell/ml to 1.69·10 6 cell/ml (7.3-fold) during the first 13 days (Fig. 1 A). It should be noted that exponential growth phase was absent whereas linear growth phase lasted 6 days. In the latter phase the maximum productivity (P m ) was constant and reached 0.15 • 10 6 cell / (ml · day) by cell number. After 13 days, the gradual decrease in cell density of D. salina was observed. By the end of the "green" phase cell density fell by a factor of 1,4 compared to the maximum value ( Fig. 1 A).
Mean dividing cell percentage at the linear stage (0 -6 days) decreased from 8 to 4 % and after that varied within 2 -6 % until the end of the first growth phase (Fig. 1 A).
At the linear growth stage in the "green" culture, 0.51 cell divisions occurred during the night (average value for five days of linear phase) that correspond to growth rate of 0.039 divisions/h. Study of D. salina morphometric parameters has demonstrated the decrease in mean cell size in 1.2 times during linear growth phase: from 17.8 to 15.34 μm in length and from 11.01 to 9.18 μm in width (Fig. 2 A). In the course of subsequent culturing (phase of growth declining and stationary phase) gradual increase of mean cell length and width was observed. The mean cell size in the "green" phase was 16.88 μm in length and 10.11 μm in width.
Mean cell volume in D. salina both dividing and non-dividing cells rapidly declined from the first to the 6 th day of cultivation (Fig 2 C). Thus, non-dividing cells volume significantly decreased from 1159.7 ± 6 μm 3 to 691.5 ± 5.4 μm 3 (t=11,24 > t 05 =1,66), and dividing cells from 1477.7 ± 4 μm 3 to 830.9 ± 3.9 μm 3 (t=3,91 > t 05 =1,81) (1.7 and 1.8 times in comparison with initial cell volume, respectively). Mean cell volume exhibited increase gradually from the 7th day when the phase of of growth declining in culture was attained. Thus, non-dividing D. salina cells significantly expanded up to 1185.5 ± 5 μm 3 (1.7 times) by the end of "green" phase and dividing cells increased their volume up to 1485.7 ± 3.8 μm 3 (1.8 times) (t=2,37 > t 05 =1,66) in comparison with the minimal values in the linear growth phase. The mean cell volume in the "green" phase was 1.3 times higher in dividing then in non-dividing cells (Fig 2 C).
It has been found that small cells (less than 500 μm 3 ) fraction in D. salina increased significantly (from 1 to 29%) while large cells (more than 1500 μm 3 ) fraction decreased (from 16 to 0%) during linear growth phase (Fig. 2 E). After linear growth phase small cells fraction progressively diminished and was not visible after the 28 th day. At the same time, large cells fraction increased from 0 to 20% in the course of stationary growth phase.
Also, morphological changes were noted in D. salina culture in the "green" phase ( Fig. 3 A). Early in the phase, the cells were green, with tight cell wall, the inclusions were absent in chloroplast, pyrenoid was clearly visible. After the 10 th day of exposition (in the phase of growth declining and stationary phase) as the culture grew and got senescent, the small granules of lipid globules became visible in the cells. At the end of the phase, the granular chloroplast stood out in the cell structure while pyrenoid became invisible and cells gained orange color.
"Red" phase At the second, "red" phase, the cell density of D. salina increased by 25% compared to initial number and reached the maximum value of 0.84·10 6 cell/ml during the first 5 days (Fig. 1 B). The maximum productivity of D. salina (P m ) calculated based on cell number, was 0.04•10 6 cell/ (ml·day). At the end of the second phase, the density of D. salina cells reduced by 78% in comparison with the maximum value ( Fig. 1 B).
The mean percentage of dividing cells in the "red" phase generally averaged about 5% (Fig. 1 B). However, after the 15 th day the portion of dividing cells was 4 times less than it was in linear growth phase that corresponds to the pattern of D. salina cell density change over this period. In the conditions of "red" culture and in the phase of linear growth, 0.47 cell divisions occurred during the night that corresponds to growth rate of 0.036 divisions/h. The estimation of D. salina morphometrical parameters in culture has demonstrated that cell length did not vary significantly at the beginning and in the end of "red" phase, averaged about 17,3 μm. At the 5 th day of cultivation (at the end of linear growth phase), this parameter increased to 20.2 μm, by 20% in comparison with the initial value (Fig 2 B). The cell width remained almost constant over 21 cultivation days and averaged 11 μm. The D. salina cells became of oblate spheroid at the 5 th day of cultivation and obtained the spheroidal shape at the end of the second growth phase.
The average volume both of dividing and non-dividing cells of D. salina increased from the beginning of the phase to the 5 th day of cultivation. At the same time the volume of non-dividing cells significantly increased from 1188.5 ± 5 μm 3 to 1366.6 ± 6.1 μm 3 (t=1,37 > t 05 =1,65), i.e. by 20%, whereas the volume of dividing cells changed from 1349.7 ± 1.8 μm 3 to 1659.0 ± 3.6 μm 3 , but mean difference was not significant (Fig. 2 D). When algal culture had reached stationary phase the decreasing of cell volume was observed (981.6 ± 4.7 μm 3 for the cells in growth phase and 1106.4 ± 0.1 μm 3 for the cells in division phase, i.e. 1.4 and 1.5 times, respectively). It has been noted that the volume of D. salina dividing cells in the "red" phase was 20% higher than that of non-dividing cells.
As was shown the small cells fraction of D. salina (less than 500 μm 3 ) was practically absent at the second cultivation phase, comprising about 1%. The large cells fraction (more than 1500 μm 3 ), on the contrary, comprised about 30-35 % at the end of linear phase (3 rd -7 th days) (Fig. 2 F).
Significant morphological changes in cells of D. salina were observed in the "red" phase, when stress conditions for the algae growth and reproduction take place (Fig. 3 B). The granulation of cytoplasm in the form of colored lipid globules had been seen from beginning of the second phase, had been pronounced at the 9 th day and got distinct at the end of the experiment. At the same time the D. salina cells became more round-shaped and assumed the orange and tile-red coloration. In addition, some Dunaliella cells formed the pseudopodia-like processes (Fig. 3 B).

Discussion
The peculiarities of morphology, morphometry and physiology in D. salina during its two-stage cultivation depend on the conditions at the first and second stages of cultivation.
It is known that illumination is a leading factor responsible for carotenogenesis induction in D. salina (Ben-Amotz 1995), therefore at the second growth phase we used earlier approved procedure of raising the irradiance level of cells. The method consists in dilution of culture media and reducing of culture layer depth twofold (Borovkov & Gudvilovich 2017) that enabled increasing the irradiance level by a factor of four. It should be noted that the linear growth of the D. salina culture is not always observed at the second stage of growth because the stress factors might significantly reduce the rate of cell division. However, in our experiment the linear growth was exhibited during the first five days of the "red" phase, which is possibly due to the cloudy weather during this stage of cultivation. At the same time, the cells of D. salina apparently used residual amounts of mineral nutrients for its growth (Fig. 1 B).
Biogenic elements deficiency, especially in old cultures of algae, may disrupt the process of some cell organelles division, resulting in reduction of the cell division rate (Massyuk 1973;Davis et al. 2015;Lv et al. 2016). This circumstance allows explaining the fact, that dividing cells ratio did not decrease during 15 days of cultivation in the "red" phase, at the expense of prolonged cytokinesis of D. salina cells (Fig. 1 В).
It was observed that cell division in the linear growth phase took place both under the light and in the dark conditions (at "green" and "red" phases). This corresponds to the literature data on the division of D. salina cells under the conditions of a daily light-dark regime (Xu et al. 2016). According to our data, the cell division rate of D. salina by night did not differ significantly between the first and the second culturing stage (t=0,099 < t 05 =2,306), amounting to 0.036-0.039 div/h despite the difference in the average apparent daily productivity. This may be due to the circumstance that the stress effect leading to cell death occurred only during the daytime under high illumination.
It should be noted that the length and width of D. salina cells at the first and the last days of batch cultivation did not differ significantly at the "green" and the "red" stages ( Fig. 2 А, B). This was because the culture at "red" stage with large cells was used as inoculum. Cell length and width decreased by a factor of 1.2 at the first cultivation stage and increased by a factor of 1.2 at the second cultivation stage, which corresponded to the linear phase of culture growth in both cases. The differences of patterns of Dunalialla cell size changes are probably the result of significant differences in growth conditions at two experimental stages. As many authors (Massyuk 1973;Preetha et al. 2012;Davis et al. 2015) noted, the rounding of cells, i.e. a decrease in the length-to-width ratio, it is a typical response to stressful environmental conditions and the initial marker of algae depressed condition, whereas the elongation of cells (increase in the length-towidth ratio) is characteristic response to increasing of nutrient concentrations. The cell length of D. salina increased during the first five days of "red" phase along with the increasing of its cell number (at linear growth phase). It is our point that this testifies that some amount of biogenic elements was present in the medium at the beginning of above-mentioned stage (Fig. 2 В). The cells mainly had oblate spheroid form at this growth phase that transformed to spheroid at the close of the cultivation stage. The observed rise in the cell size in the linear phase is consistent with the data of other authors about increase of D. salina cells mean diameter along with increasing in levels of irradiance and temperature (Lv et al. 2016;Srirangan et al. 2015).
The mean D. salina cell volume was 30% higher in the "red" phase than in the "green" phase. The fraction of large cells in the "red" phase was consistently high (15-35%) (Fig. 2 D, F). The ratio between groups of cells of different volume changed over the entire time of cultivation, suggesting that changing of the age structure of the Dunaliella cell populations took place both at the "green" and the "red" growth phases. Hence, at the linear stage of the "green" phase, when productivity is constant, the small "young", actively dividing cells dominate, which are at the growth stage. While large "old" cells are prevalent at limiting factor change and under negative growth rate.
The morphological changes in Dunaliella cells evident as manifestation of granulation and gaining of orange and tile-red coloration point to the increasing of carotenoids content value in culture (Fig. 3 В).
The optimal duration of cultivation phases was determined by way of experimental registration of morphological and morphometrical parameters in D. salina culture grown at two-phase mode of cultivation in greenhouse complex and amounted about 10-12 days both for the first and the second phase. The concentration of Dunaliella cells reached the maximal levels in particular at 10-12 th days of each culturing stage ( Fig. 1 A, B); at that the "large" cells fraction started increasing at the "green" stage and was enough high (18%) at the "red" stage, while "small" cells fraction was close to minimal values at both stages (Fig. 2  E, F). After 10 days of cultivation at the second stage the mean cell volume and "large" cells fraction decreased, the reduction of motor activity was noted, and some cells had formed the pseudopodia-like processes. The mentioned modification is typical to old D. salina cultures as a response to extreme environmental conditions, such as the changes of salinity, mineral composition of the medium, temperature and illumination, as well as combination of specified parameters (Massyuk 1973;Oren, 2014;Lv et al. 2016).
The prolongation of the duration of first and second cultivation stages at more than 15 days was unfavourable, because the cell density did not increase at this period in the "green" phase and was significantly reduced in the "red" stage (by 75%). Moreover, just as the fraction of dividing cells significantly decreased (by 80%), so did their volume (by 15% from 10th to 22nd day) (Fig. 2 B, D, F).

Conclusion
Morphological and morphometrical characteristics of carotenogenic microalga D. salina have been studied during its two-phase cultivation in the south-west of Crimean Peninsula in summer. The obtained experimental data on the dynamics in cell density and size structure have indicated the significant influence of growth conditions in "green" or "red" phase as well as culture growth stage on the studied parameters. The relationship among Dunaliella cell size groups varied over all cultivation period, which indicates changes in the age structure of cell population both in the "green" and in the "red" growth phase. Changes in morphological and morphometrical parameters of D. salina cells can serve as an additional criterion for determining of durations in cultivation stages during the commercial cultivation of this alga. In conclusion, Crimea is not only a part of natural D. salina areal but also a prospective region for development of its biotechnology.