Evaluation of contribution of salinity, irradiance, and nutrient deficiency into the yield of cells and P-carotene accumulation in the culture of Dunaliella salina (Chlorophyta)

Целью исследования было количественное определение вклада солености, освещенности, нитрата, фосфата и их взаимодействия в урожай Dunaliella salina по количеству клеток и накоплению р-каротина. Чтобы предотвратить смешение эффектов факторов-условий с исчерпанием факторов-ресурсов, водоросль выращивали в периодической культуре с подпиткой. В исследованном диапазоне факторов (освещенность 2-8 клк, соленость 1-4 M NaCl, KNO3 0-80 мг/л, K2HPO4 010 мг/л), нитрат и фосфат сильнее влияли на продуктивность культуры по количеству клеток и накоплению р-каротина, чем соленость и освещенность. Влияние солености о освещенности зависело от биогенов и предшествовашей обеспеченности инокулята ними. Суммарная сила действия ц2 биогенов на урожай клеток составляла 0,59 для неголодающего по биогенам инокулята и 0,43 для голодающего инокулята, тогда как суммарная сила действия факторов-условий - 0,10 и 0,12 соответственно. По содержанию р-каротина в клетках, суммарная сила действия биогенов на клетки, выращенные из неголодающего и голодающего инокулятов составила 0,71 и 0,58, а факторов условий - 0,8 и 0,5 соответственно. Остаточная дисперсия урожая клеток и содержания р-каротина в клетках была отнесена к взаимодействию солености и освещенности с биогенами. Сочетание высоких значений солености и освещенности имело свое собственное, не смешанное с исчерпанием питательных элементов, но меньшее индуцирующее влияние на накопление р-каротина. Наибольшее содержание р-каротина 53 пг на клетку наблюдалось в культуре, выращенной из голодающего инокулята при дефиците фосфора. Сочетание высоких значений солености и освещенности давало 17 пг р-каротина на клетку по сравнению с около 5 пг при оптимальных условиях культивирования. Дозирование биогенов должно быть самым мощным инструментом управления биосинтезом в культуре D. salina.

Ключевые слова: нитрат, фосфат, микроводоросли, методология культивирования

Microalga Dunaliella salina Teodor. lives in hyperhaline waters (continental salt lakes, coastal lagoons, solar saltern ponds). Its massive development and intracellular р- carotene accumulation often causes red “bloom” of these habitats/

The microalga is of great practical importance as a source of р-carotene. Food industry uses the biomass as a functional ingredient (provitamin A, antioxidant), and extracted mixed carotenoids (mainly р-carotene) - as the food colorant E 160 a (i) [EFSA..., 2012]. World market demand for these products is steadily growing [MArz, 2008].

Open pond culture remains the only cost-effective way to manufacture algal biomass [BEN-AMOTZ, 2009]. Currently D. salina is manufactured mostly in Australia - extensively, in natural lagoons separated into large ponds (100-250 ha), under wind mixing [Schlipalius, 1991; Curtain, 2000], and in Israel - intensively, in open artificial raceways (up to 3000 m2), under forced mixing and CO2 supply in relatively controlled environment [Ben-Amotz, 2004; Del Campo et al., 2007]. The larger the pond area, the lower the control [Ben-Amotz, Avron, 1990; Del Campo et al., 2007].

In Mediterranean and Black Sea basins many solar salterns have persisted, partially or fully abandoned, that could be restored for cultivating D. salina [Lopez et al., 2010; Rodrigues et al., 2011; Komaristaya et al., 2014]. The further to the North and the shorter warm and sunny season, the more culture feasibility dependent on optimization and control of culture conditions [Borowitzka, 1999].

Theory and practice proved the two step technology of D. salina cultivation, because of mismatch between the optima for biomass growth and 3-carotene accumulation. At the first step the biomass is grown, and at the second step 3-carotene accumulation is induced by elevated salinity, high irradiance and nutrient depletion [Massjuk, 1973; Ben-Amotz, 1995].

Concentration of nutrients in natural brines is very low [Oren, 2009]. In the open pond culture one could easier control nutrient supply than salinity and irradiance. Brine evaporation rate and light intensity depend on climate, and are subject to seasonal and diurnal variations and weather fluctuations. To some extent, irradiance could be adjusted by changing pond depth [Bhumibhamon et al., 2003], and salinity - by pumping in fresh water [Komaristaya et al., 2014] or bittern; but large pond area makes this technically difficult.

Salinity, irradiance, nitrate and phosphate supply might contribute unequally to biomass growth at the first step of cultivation, and to 3-carotene accumulation at the second one. Coming from a fundamental principle of living systems organization as the scale-free networks [Wolf et al., 2002], D. salina cells must react significantly to some small number of environmental factors, moderately - to some intermediate number of them, and minimally - to many the other that could be considered as noise. In biological systems factor effects very often interact, i.e. the response to one factor depends on the levels of the other factors [Jongman et al., 1995]. For economically feasible biosynthesis control in the open pond culture of D. salina, factors and their combinations must be chosen that are the most powerful and easily adjustable.

The literature disagrees on contribution of different factors into 3-carotene biosynthesis in D. salina. Some researchers attach much importance to the impact of salinity and irradiance [Loeblich, 1982; Borowitzka, 2013], some other - to nutrient depletion [Lerche, 1936/1937; Coesel et al., 2008]. Not all experiments confirm the effect of phosphorus deficiency [Ben-Amotz et al., 1982]. The literature data are fragmentary: some different combinations of some different factor level ranges were explored, sometimes on the natural, sometimes on the cultural material. As a rule, the alga was grown in the batch culture, that inevitably led to the confounding of the effects of factors-conditions (salinity and irradiance) with the effects of depleting factors-resources (nitrate and phosphate).

The purpose of this study was to quantify the contribution of salinity, irradiance, nitrate and phosphate and these factors interactions into the yield of cell number and 3- carotene accumulation in D. salina culture.

Effect size ^2 - factor contribution into overall variance of a variable - was proposed as a statistical index [Cohen, 1973]. To investigate all possible combinations of factors and evaluate their main effects and interactions, the full factorial experimental design was used. To cover maximal factor level ranges, the two contrast levels of each factor were chosen: favorable for culture growth and for 3-carotene accumulation. To avoid confounding of the effects of factors-conditions with the depletion of factors-resources, the alga was grown in fed-batch culture; nitrate and phosphate periodically supplied according to the known dynamics of their absorption [Komaristaya et al., 2010]. To account for potential influence of inoculum nutrient status, the experiments were carried out in the two variants: on non- starved inoculum, pre-grown in standard nutrient supplied laboratory medium, and on starved inoculum, supported in nutrient deficient medium close to natural brine in composition.

Materials and methods

Dunaliella salina, strain IBSS1 received from the collection of Institute of Biology of Southern Seas (Sevastopol, Ukraine) in 2005 and cultured in our laboratory, was the object of study. Non-starved inoculum was grown in Artari medium in the modification of N.P. Massjuk [1973] containing 116 g L-1 NaCl, 50 g L-1 MgSO4^O, 2,5 g L-1 KNO3, 0,2 g L-1 K2HPO4. Starved inoculum was grown in the solution of natural sea salt with the density 1,15 g cm-3. The inocula were cultivated under 5 klx irradiance, photoperiod of 16 hours, and the temperature 27 °C.

The culture media for the experiments were prepared using NaCl and MgSO4'7H2O. Nitrogen and phosphorus were supplied as KNO3 and K2HPO4, respectively.

When inoculating, trace elements were added to the following final concentrations: 3,1 mg L-1 H3BO4, 1,94 mg L-1 MnSO*5H2O, 0,287 mg L-1 ZnSO47№O, 0,088 mg L-1 (NH4)6Mot024'4H20, 0,146 mg L-1 Co(NO3)2'4H2O, 0,119 mg L-1 KBr, 0,083 mg L-1 KI, 0,048 mg L-1 NiSO47H2O, 0,08 mg L-1 CuSO4'5H2O.

The experiments were carried out according to the full factorial experimental design, 16 variants total. The design envisaged all possible combinations of 4 factors taken at 2 levels each: K2HPO4 - not added (0 mg L-1) and 10 mg L-1; KNO3 - not added (0 mg L-1) and 80 mg L-1; salinity - 1 M NaCl (0.1 M MgSO4^H2O correspondingly) and 4 M NaCl (0,4 M MgSO4.7H2O correspondingly); irradiance - 2 klx and 8 klx. Experimental design is shown in the legend to Fig. 1-4.

The cultures were periodically (every 3 days) fed by half initial doses of the nutrients according to previously studied dynamics of their absorption/

The light sources were "Maxus" lamps with the color temperature 2700 K: two 32 W lamps for 2 klx and four 50 W lamps for 8 klx.

The cultures were inoculated to the initial cell count of about 25 thousand per 1 mL and cultivated in 25 mL Erlenmeyer flasks, 15 mL of culture in each, at 16 hours photoperiod and the temperature 27 °C.

Cell yields were registered on the 42nd day of cultivation by counting the cells in Goryaev hemocytometer. Cell concentrations were calculated using the formula for the hemocytometer and expressed in millions per 1 mL.

The rapid method of total carotenoids quantification was used, because it is fast, low- cost and can be easily applied in the field and at outdoor cultivation. The results obtained were interpreted as P-carotene content, because D. salina is known to accumulate mainly P- carotene [Jimenez, Pick, 1994]. Extraction was performed on the 42nd day of cultivation by vigorous shaking culture aliquot (1 mL) with 2 mL of ethyl acetate. P-carotene content was quantified by absorbance of the extracts at 440 nm, calculated using the reference extinction value E1cm1% = 2500 [IARC, 1998] and expressed in pg per cell.

The experiments were carried out in triplicate. According to Shapiro-Wilk test the distribution of data differs insignificantly from the normal. The parametric statistic tests were used: multivariate analysis of variance and F value - to assess the significance of the effects of individual factors and their interactions, effect sizes ^2 (rounded to 2 decimals) - to assess their input into overall variance of the variables. The plots show mean values and Fisher's least significance differences (LSD), which were used to compare means. Discussed differences are significant at 95% level.

Dunaliella salina cells survived and remained in the vegetative phase during 42 days in all the experimental variants, including even starved inoculum in the nutrient deficient media. Starved and non-starved inocula had similarities and differences in the patterns of effect sizes of the factors and their interactions (tabl. 1).

yield carotene dyialidla saliia

Table 1. Effect sizes p2 of KNO3, K2HPO4, salinity, irradiance and their interactions onto concentration of cells and cellular p-carotene content in 42-day D. salina culture inoculated by non-starved and starved inocula

Note: * - F > Ftabl. (p < 0,05, df1 = 1, df2 = 32, Ftabl = 4,15)

In the culture grown from non-starved inoculum, all the factors and interactions influenced cell yield, though their majority contributed into the total variance no more than 3% (tab. 1). Nitrate, phosphate and their combination influenced cell yield most strongly (tabl. 1).

In the culture grown from starved inoculum, the nutrients led by their effect sizes on cell yield too. The contribution of salinity increased; the effects of nitrate and its interaction with salinity maintained the same level (tabl. 1). All the effects related to phosphate decreased compared with non-starved inoculum (tabl. 1). The main effects and interactions related to irradiance dropped out from the statistically significant (tabl. 1).

The data on cell yields confirmed and detailed the effect size pattern.

For the both inocula one or two nutrients deficiency significantly decreased cell yield (Fig. 1, 2). In the variants supplied with the both nutrients, high salinity inhibited culture density increase but less than nutrient deficiency (Fig. 1, 2). High irradiance increased cell concentration at low salinity and the both nutrients supply, but this effect appeared statistically significant for non-starved inoculum only (Fig. 1).

As to 3-carotene accumulation, in the culture grown from non-starved inoculum, the number of significant main effects and interactions decreased compared with the cell yields. The nutrients kept the lead, especially phosphate, which contributed 56% into the variance of that variable (tabl. 1). Salinity influenced 3-carotene accumulation only combined with phosphate (tabl. 1).

Fig. 1. Concentrations of cells on the 42nd day of cultures inoculated with non-starved inoculum in the variants of full factorial experimental design

Fig. 2. Concentrations of cells on the 42nd day of cultures inoculated with starved inoculum in the variants of full factorial experimental design

Compared with the effect on cell yields, the main effect of irradiance more than doubled; interaction with phosphate remained the only significant interaction of irradiance (tabl. 1).

In the culture grown from starved inoculum, nutrients still kept the lead, though phosphate contributed 20% less, and nitrate affected insignificantly, but they interact more strongly (tabl. 1). On the contrary to non-starved inoculum, the main effect of salinity gained significance, and the main effect of irradiance lost it (tabl. 1). Salinity interacted more strongly with nitrate than with phosphate (tabl. 1). The effect of irradiance depended on nutrients, at that, irradiance interacted with phosphate more strongly, than with nitrate or with both the nutrients (tabl. 1).

For non-starved inoculum, P-carotene content values showed that the lack of any or both the nutrients induced significant P-carotene accumulation compared with corresponding variants supplied with the nutrients (Fig. 3). At phosphate or the both nutrients deficiency, elevated salinity negatively impacted P-carotene accumulation (Fig. 3).

Fig. 3. Cellular 3-carotene content on the 42nd day of cultures inoculated with non-starved inoculum in the variants of full factorial experimental design

Fig. 4. Cellular 3-carotene content on the 42nd day of cultures inoculated with starved inoculum in the variants of full factorial experimental design

In starved inoculum any or the both nutrients deficiency induced 3-carotene accumulation too (Fig. 4). Salinity impaired 3-carotene accumulation at the deficiency of nitrate or the both nutrients deficiency (Fig. 4). Stimulating effect of irradiance on 3-carotene accumulation disappeared in starved inoculum; at phosphate deficiency high irradiance even decreased 3-carotene content (Fig. 4).

At phosphate or the both nutrients supply, impact of elevated salinity changed to neutral or, at high irradiance, to positive (Fig. 3). At phosphorus deficiency, or the both nutrients deficiency, high irradiance stimulated 3-carotene accumulation (Fig. 3).

For the both inocula, the variants, supplied with the two nutrients, tended to accumulate 3-carotene at high salinity. This effect reached statistical significance (p < 0,05) at high irradiance (Fig. 3, 4).

Discussion

IARC Working Group on the Evaluation of Cancer Preventive Agents. (1998). IARC Handbook on Cancer Prevention, Vol.2: Carotenoids. Lyon, France: IARC, 326 p.

Jimenez C., Pick U. (1994). Differential stereoisomer compositions of P, P-carotene in thylakoids and in pigment globules in Dunaliella. Journal of plant physiology, 143 (3): 257-263. doi: 10.1016/S0176- 1617(11)81628-7

JONGMAN R.H.G., Ter BRAAK C.J.F., VAN Tongeren O.F.R. (1995). Data analysis in community and landscape ecology. Cambridge, UK: Cambridge university press, 324 p.

Komaristaya V.P., Antonenko S.P., Rudas A.N. (2010). Cultivation of Dunaliella salina Teod. at suboptimal concentrations and exclusion of nitrogen and phosphorus from the medium. Algologia, 20 (1): 4255. (in Russian).

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