The eco-physiological characteristics of the environments, including temperature, light, and the availability of food resources, allow certain microorganisms to inhabit aquatic habitats. Native species of resilient and competitive strains are supposed to be adapted to local environmental changes. Therefore, it would be an appropriate choice for large-scale cultivation to improve bio-compound production.
The relatively high altitude of Cheshmeh-Sabz Lake, its proximity to the highest peak of the Binalud Mountains, and the effects of Polar and Siberian cold masses have led to unique climate characteristics. Hence, there is a decreasing temperature and significant atmospheric precipitation, (especially snowfall), which at high altitudes lasts up to early summer. Results of chemical analysis of lake water show this lake is Ultra-Oligotrophic, exposed to a large range of temperature fluctuations (-15 to 18 ° C). Due to the mentioned climatic characteristics, the microorganisms grown in this ecosystem are freshwater cold-adapted species. Hence, this area is suitable for the presence of cold-adapted organisms [26].
The microalgae that grew at 5 to 25 ºC in freshwater culture media were identified by molecular methods, using the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). Strains were identified as Ankistrodesmus sp. (dominant in WC medium), Chlorella sp. MN738559 (dominant in BBM medium), and Scenedesmus sp. MN738556 (dominant in BG-11 medium). In general, the three algae species isolated from Cheshmeh-Sabz Lake were able to grow at both high (25 ºC) and low (5 ºC) temperatures, which in this regard can be considered cold-adapted species. This observation was confirmed elsewhere [27, 28, 29, 30, 31]. For example, Lake Trnquil in Antarctica with annual temperatures between 0.1 and 3 ºC was dominant by freshwater species, Ankistrodesmus falcatus, and Ankistrodesmus sp. [30]. Scenedesmus sp. LX1 was reported to grow in a wide temperature range (10–30 ºC) [28]. Although the growth of most Chlorella species can be negatively affected by lower temperatures, there are also reports of cold-adapted Chlorella species in very cold regions [27, 29, 31].
Our findings about dominancy in the specific medium are supported elsewhere [32, 33, 34]. For instance, in comparison to WC and COMBO culture media, A. falcatus showed similar and highest specific growth of 1.34 day− 1 at 20 ° C [33]. C. vulgaris showed the highest biomass productivity (114.208 ± 0.850 mg L− 1 day− 1) and highest lipid content (17.640 ± 0.002%, day 12) in BBM by comparing with other selected growth media [35]. According to another research, native Scenedesmus sp. was isolated and characterized in BG-11 culture medium [32].
The result of growth phases at the low temperature (8 ºC) showed there was a lag phase at the beginning of cultivation. The QY in the delay phase was low and, then, after the cold adaptation, the amount of quantum yield increased when the strains entered the stationary growth phase, indicating the time required to adapt to the coldness. QY is defined as the efficiency of converting absorbed light into emitted light in the fluorescence form. The decline of the quantum yield is often indicative of cells entering stress conditions [36]. Based on the eco-physiological studies at low temperatures, the decreasing growth rate and biomass productivity of microalgae can be attributed to decreased enzyme activity and modifying or denaturing of photosynthesis-related proteins [37]. In the lag phase, the development of cold adaptive mechanisms in photosynthesis, biochemical composition, and even gene expression occurs [38]. As is shown in Fig. 3, after adaptation, the strains began to grow, and finally behaved similarly to growth at the higher temperature (25 ºC), which indicates that species can grow in cold areas.
Scenedesmus sp. MN738556 had the same specific growth rate (0.07 day− 1) at low and high temperatures (8 and 25 ºC). The specific growth rate of Scenedesmus sp. MN738556, at 8 ºC, was higher than that of other species. The biomass productivity of Scenedesmus sp. MN738556 growing at 25 ºC was more than that of other species in different conditions (0.54 ± 0.001 g L− 1 day− 1). Ankistrodesmus sp. and Chlorella sp. MN738559 a lower specific growth rate was observed at 8 ºC (0.006 ± 0.0001 and 0.032 ± 0.009 day− 1, respectively) in comparison to 25 ºC (0.05 ± 0.001 and 0.066 ± 0.002 day− 1, respectively). Scenedesmus sp. MN738556 and then Ankistrodesmus sp. at 25 ºC had the highest biomass productivity (0.2 ± 0.01 g L− 1 day− 1). The temperature has a critical role in the growth and photosynthetic activity of microalgae. Therefore, in the cultural process, enzyme activity, substance exchange, and cell division at different temperatures can influence microalgal growth and lipid accumulation [39]. Cao and et al. showed that with decreasing temperature at 9°C, the specific growth rate of psychrotolerant Arctic Chlorella sp. and Chlorella-Temp was decreased (0.31 and 0.05 day− 1 respectively) and, the specific growth rate of Chlorella-Temp, reached about 0.3 day− 1 at 27°C. it was found that the maximum quantum efficiency of PSII photochemistry had a downward trend by decreasing temperature, because of photoinhibition [38].
Nutrients complete the life cycle of microalgae. In aquatic ecosystems, nitrogen and phosphorus are the most important elements that cause production changes of organic compounds in microalgae and phytoplankton according to the species’ requirements [40, 41]. Nitrogen is the most important nutrient affecting biomass production and lipid accumulation and it is noteworthy, that nitrogen consumption for optimal growth is strongly influenced by the culture temperature. [42, 43, 44]. In this study, nitrate was consumed equally by Scenedesmus sp. and Ankistrodesmus sp. at low and high temperatures, also Scenedesmus sp. had the same biomass productivity at 8 and 25°C. The membrane transport inhibition and decreased nutrient uptake at low temperatures lead to growth limitation. In addition to temperature, the availability of nutrients affects fatty acid profiles[45]. The psychrotolerant microalgae using adaptive mechanisms can increase nutrient uptake, grow well under low-temperature conditions, and increase lipid contents [11, 44]. The research showed that nitrogen availability in aqueous environments during the algal growth process and synthesis of new cell membrane compounds lead to unsaturated fatty acids production to participate in maintaining membrane fluidity with increased biomass productivity [46, 47]. Increases in PUFAs of the membrane lipids showed in psychrophiles and psychrotolerants at low temperatures [48].
In addition to affecting the growth of microalgae, cultivation temperature also affects their fatty acid profiles. In general, as the temperature decreases, the degree of unsaturation of fatty acids increases, and as the temperature raises the overall amount of SFAs increases. Low temperatures increase the viscosity of the cell membrane, slowing electron transfer, so the viscosity is further reduced by replacing PUFA with membrane-saturated lipids and allowing the cell to continue its vital functions. Hence, PUFAs play an important role in temperature acclimation and adaptation [13, 49, 50].
In the present study, fatty acid profiles of three species grown at temperatures of 8, 15, and 25 ºC with light conditions of 100 µmol photons m− 2 s− 1 (25 and 15 ºC) and 30 µmol photons m− 2 s− 1 (8 ºC) were investigated. The result showed that Ankistrodesmus sp. produced the highest amount of PUFA at the low temperature (8 ºC)(19%), while Chlorella sp. MN738559 and Scenedesmus sp. MN738556 PUFA reached 16.5% and 15.8%, respectively. Generally, in Ankistrodesmus sp. and Chlorella sp. MN738559 proportion of unsaturated fatty acids increases with decreasing temperature, whereas the percentage of unsaturated fatty acids in Scenedesmus sp. MN738556 was similar at three temperatures (about 16.5%). It was also found that α-linolenic acid (C18:3 n-3)(ALA) had the highest amount of unsaturated fatty acids at the low temperature (8 ºC) for the three strains.
it was reported that in different taxa of freshwater Chlorophyceae, fatty acids C16:0, C18:1, C18:2, and C18:3 were the common fatty acid types [51]. Also, in C. vulgaris, C16, and C18 are major fatty acids, and the sequential desaturation of these fatty acids usually involves the following steps [14]:
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Microalgae can produce linoleic (18:2n6) and α-linolenic acid (C18:3n-3)(ALA) de-novo from 18:0-ACP which are precursors for longer chain PUFAs. Δ12- and Δ15-desaturase enzymes are needed to synthesize LA and ALA from stearic acid (C18:0) [52]. It is also well known that at low temperatures, the concentration of dissolved oxygen in the environment increases, which allows oxygen-dependent enzymes such as desaturase to be activated [53].
The results of the previous research showed that with increasing light intensity, the amount of SFA production increases. However, low light has been shown to increase PUFA accumulation. At low light intensities, the thylakoid membrane increases the content of chlorophyll pigments to maintain photosynthesis efficiency and converts its membrane to a hexagonal phase. Increasing the amount of unsaturated fatty acids in the form of mono-galactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) causes a hexagonal phase in the thylakoid membrane [54], [55]. Depending on the species, decreasing the temperature and the light intensity (which happens naturally in the winter) can increase the amount of unsaturated fatty acids, and also prevent photoinhibition [56].
Microalgal strains with high PUFA proportion indicate their exceptional potential for application in the development of healthy food products [57]. Analysis of the fatty acid composition of the strains showed that they are rich in palmitic acid (C16:0) as saturated fatty acids. At the high temperature (25 ºC), Chlorella sp. MN738559 contained the highest percentage of palmitic acid (C16:0)(44.41 ± 7.56%), whereas, Ankistrodesmus sp. production was 35.37 ± 5.23%. The amount of palmitic acid (C16:0) in Scenedesmus sp. MN738556 was almost equal in all three temperatures (40%). It has been shown that Chlorella sp. MN738559 compared to the other two strains at different temperatures, had the highest amount of oleic acid (C18:1) at 25 ºC (0.89 ± 0.02%). As a result, Chlorella sp. MN738559 had the highest ratio of SFA among other strains (45.3%). The high proportion of three major fatty acids, palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) are suitable for biodiesel production [58]. Palmitoleic acid (C16:1) and oleic acid (C18:1 n-9) were two major MUFAs in three strains. Chlorella sp. MN738559 and Scenedesmus sp. MN738556 produced more MUFAs at 15 ºC (8.7%, and 6.4% respectively), while, Ankistrodesmus sp. comprised the highest amount at 25 ºC (9%). It has been reported that lipids with high MUFAs are globally suitable as a biofuel with good cold-flow properties and yet are particularly susceptible to oxidation [59].