Evaluation of Lipid composition and growth parameters of cold-adapted microalgae under different conditions

doi:10.21203/rs.3.rs-2694802/v1

In the current study, Cheshmeh-Sabz Lake located in northeastern Iran (36°20’N, 59°3’ E) with an average minimum annual temperature of -15˚C was screened to isolate native cold-adapted microalgae strains. Various isolation and purification methods (different freshwater culture media at 5 ºC, liquid serial dilution with streak plate methods, and then fluorescence-activated cell sorting) were used to find dominant cold-adapted microalgae strains. Three cold-adapted microalgae growth parameters (Scenedesmus sp., Ankistrodesmus sp., and Chlorella sp.) were investigated at 8 ºC and 25 ºC. Among isolates, Scenedesmus sp. (dominant in BG-11) had the same and relatively high biomass productivity (~ 0.54 ± 0.001 g L⁻¹ d⁻¹) at low and high temperatures. Fatty acid profile evaluation of three species at 8, 15, and 25 ºC indicated, the highest amount of α-linolenic acid was produced in Scenedesmus sp., and there was no significant difference between the amount of α-linolenic acid at 15 ºC and 25 ºC (10.96 ± 1.5% and 11.07 ± 0.31%, respectively). There were no significant differences between palmitic acid amount at 8, 15, and 25 ºC (41.05 ± 5.2, 38.48 ± 4.25, 39.82 ± 1.58% respectively) in Scenedesmus sp. Based on the results, Scenedesmus sp. is the proper choice for outdoor cultivation in different seasons due to its relatively high biomass productivity and the specific growth rate in low and high temperatures (8, and 25 ºC). In addition, the good ability of polyunsaturated fatty acids production (~27%) and rich in saturated fatty acids at low and high temperatures (~ 66%) makes this species susceptible to work in the biofuels field and feed supplements.

Cold-adapted

Fatty acid profile

Microalgae isolation

Lipid

Low temperature

Microalgae as photosynthetic (predominantly) unicellular eukaryotic organisms can produce active metabolites like lipids with applications in various fields [1, 2, 3]. Saturated fatty acids (SFA)(mainly neutral lipids) are used as biofuels due to their capacity for high-energy storage, while polyunsaturated fatty acids (PUFA) (polar or neutral) due to their beneficial effects on human health are used in food, feed, and pharmaceutical industries [4, 5]. Microalgae depending on the structure and cell organelle or various environmental conditions can produce different proportions of SFA and PUFA (between 20 and 70% of their fresh weight) [6, 7, 8].

Physical and chemical abiotic factors (such as water currents, temperature, access to light, carbon and nutrients content, pH value, dissolved oxygen, etc.) in aquatic ecosystems form scaffolds in which living organisms have achieved adaptations that prepare them to live in various environment [9]. Temperature is one of the most important factors affecting microalgae growth, lipid content, and fatty acid profiles [10]. The microorganisms from cold habitats can adapt well to low temperatures (between 0 and 15°C). Psychrotrophs/psychrotolerants as cold-adapted organisms expose the ability the growth at low temperatures but have optimal growth temperatures above 15°C. These cold strains are economical that have been adapted to culture in cold regions or cold seasons outdoors without expensive heating and artificial lighting equipment [11, 12].

In general, lowering the temperature increases the PUFA concentration to maintain their membrane fluidity at low temperatures, and high temperatures enhance the overall SFA content. Depending on the species, decreasing temperature and light intensity can increase PUFA[13]. The results of a study by Jo et al. in 2020 showed that the growth of the cold-resistant Chlorella vulgaris KNUA007, isolated from Antarctica, at 10 ºC increased unsaturated fatty acids in the form of hexadecatrienoic acid (C16:3)(17.31%), linoleic acid (C18:2)(8.52%), and α-linolenic acid (C18:3)(43.35%)[14].

Bioprospecting new strains with high potential in biomass and lipid production is a major purpose of researchers and industries. These strains can grow on outdoor large-scale systems with tolerating a wide range of temperatures and light intensities. Different regions of Iran with unique ecosystems and different climatic conditions have a special potential for the study of native microalgal species. In this regard, due to the special eco-physiological conditions of Cheshmeh-Sabz lake located at the foot of the Binalud mountain range (Golmakan, Khorasan Razavi), with an average minimum temperature of -15˚C, fatty acid profiles of cold-adapted microalgae isolated from this lake has not been studied yet. The most important objectives of this project are to study the growth characteristics, biomass productivity, and fatty acid profiles of specie isolated from Cheshmeh-Sabz Lake and the effect of different environmental conditions on the quantity and quality of these indicators.

Sampling site

Cheshmeh-Sabz Lake (36°20’N, 59°3’ E) with an area of 384000 square meters (480×800 m²) is situated in northeastern Iran, Khorasan Razavi province, at an altitude of about 2500 m above sea level. This lake is located on the slope of the highest peak in the Binalud Mountains, i.e. Zardukh, with an altitude of 3245 meters. The satellite imagery of the location was retrieved using geographic information system (GIS) software (Fig. 1).

Sampling method

Water from Cheshmeh-Sabz Lake was sampled in January 2017. 700 mL of water were randomly collected at four points from different depths (surfaces to 12 m from top) using volume sampling from the lower and surface layers of the lake water (Fig. 1b), in sterile 1-liter glass bottles, and then transferred to the laboratory. The temperature was recorded directly during the field studies.

Chemicals

All analytical-grade organic reagents and solvents were purchased from Merk. The FAME standard was purchased from Supelco (Bellefonte, USA). All solutions were prepared based on the weight of the sample in 100 mL of solution (w/v %) unless otherwise stated.

Sample enrichment and isolation of microalgal strains

To grow freshwater microalgae, different culture media were prepared and used. Wright Chu culture medium (WC) [15], Bold's Basal Medium (BBM) [16], and Blue-Green culture medium (BG-11) [17]. To grow high biomass microalgae at a low temperature (5 ºC), 50 mL of water samples were centrifuged and transferred to the bottle containing 450 mL medium cultures (BBM, BG-11, and WC). The strains were cultured in the aerated bottles under white light (20 µmol photons m^− 2 s^− 1, 12 h light/12 h dark cycles) at 5 ± 0.5 ºC, for 25 days. In each medium, microalgae with the highest population were isolated using liquid serial dilution plates [18] and then cultured on agar media. To purify microalgae colonies, the streak plate method was used [19]. After growth, the colonies were transferred to the appropriate liquid medium. Eventually, fluorescence-activated cell sorting (FACS, SONY SH800, Japan) was used to sort cells. Two-dimensional profiles of the FL2 channel against FSC (Forward Scatter, indicating cell size) with the highest population in each medium were used by measuring chlorophyll contents (chlorophyll-dependent, auto-fluorescence, which was detected in the FL3 channel, 617/30 nm). Also, a PC channel as FL4, using a diode laser (665/30 nm), was used to monitor all chlorophyll contents present in the samples [20]. After the sorting, cells were immediately resuspended in their sterile growth culture medium from which they were isolated. The sorted cells in tubes were placed under constant light at 30 µmol photons m^− 2 s^− 1, 12 h light/12 h dark cycles under 8 ºC, allowing cells to grow. Then, the growing cells were transferred to conical flasks. Finally, the isolation of single strains was confirmed by visual and microscopic examination (Olympus/BX51).

Molecular identification

To identify the isolated species, DNA extraction was performed using the CTAB protocol [21]. Internal Transcribed Spacer (ITS) of ribosomal DNA was amplified by using ITS1/ITS4 or ITS5/ITS4 primers [22]. The temperature and duration of DNA denaturation, annealing, and extension stages in each cycle were at 95 ºC for 45 seconds, 55 ºC to 60 ºC for 30 seconds, and 72 ºC for 30 seconds, respectively. Seven minutes were considered for the final extension. Amplified PCR products were tested on 1% agarose gel. The PCR products were sequenced using Macrogen’s sequencing service (Macrogen Inc., Korea). Sequences were edited using sequencer 5.4 (Gene Codes Inc., Ann Arbor, Michigan, USA). Moreover, BLAST was performed to find related and similar sequences in GenBank to those in this work (https://www.ncbi.nlm.nih.gov/).

Culture conditions

The purified microalgae were cultured at three temperatures of 8, 15, and 25 ºC. Each strain was aerated in a 250-mL Erlenmeyer flask containing 150 mL of the corresponding culture medium (BBM, BG-11, or WC) under white light conditions of 30 µmol photons m^− 2 s^− 1 (8 ºC) and 100 µmol photons m^− 2 s^− 1 (15 and 25 ºC)(12 h light / 12 h dark cycles) aerated.

Growth parameters

Microalgae growth was monitored (every two days) by measuring the optical density (OD) at 680 nm (with three replications) by a spectrophotometer (Awareness Stat Fax-2100), and then the growth curves of the studied strains were plotted versus time.

To monitor nitrate concentration during growth, semi-quantitative test strips (Quantofix Nitrate / Nitrite, Macherey-Nagel) were used for rapid estimation of nitrate/nitrite availability in exponential and stationary phases of microalgae growth. Chlorophyll fluorescence was used to study the effect of environmental stresses. Accordingly, the quantum yield (QY) was measured at 455 nm by a portable fluorometer (AquaPen AP 110/C). The samples were harvested at the stationary growth phase [23], and biomass was lyophilized and stored at -20 ºC for lipid analysis.

The specific growth rate (SGR)(indicating the amount of growth and biomass production in a given time) was determined for the samples taken from the exponential growth phase according to Eq. (1). In this regard, X₁ and X₂ are the dry weight of strains at time t₁ and t₂ (at the exponential growth phase), respectively [24].

\(\text{S}\text{G}\text{R}=\frac{ \mathbf{l}\mathbf{n}{{X}}_{2}-\mathbf{l}\mathbf{n}{{X}}_{1}}{{{t}}_{2}-{{t}}_{1}}\) Eq. (1)

Biomass productivity is based on which the amount of biomass produced per unit of time is calculated and Eq. (2) was used to calculate this index. In this regard, X₂ and X₁ microalgae's dry weight of biomass concentrations at times t₂ and t₁ (at the exponential growth phase),(g L^− 1 day^− 1), respectively [24].

BP=\(\frac{{{X}}_{2}-{{X}}_{1}}{{{t}}_{2}-{{t}}_{1}}\) Eq. (2)

Lipid profile

Gas chromatography (GC)(6890, Agilent, USA) was used to determine fatty acid profiles. To chemically convert triacylglycerol to fatty acid methyl esters, the direct acidic transesterification method was used based on the procedure reported by Lari [25]. Dried biomass was harvested in the stationary phase (4 mg) and 450 µL of methanol containing 2% (v/v) solution of concentrated H₂SO₄ was used as extraction solvent. The mixture was then incubated at 70 ºC in a shaker incubator for 4 h at 250 rpm. The reaction mixture was added to 150 µL hexane and vortexed. Then 600 µL of 1% (w/v) NaCl solution was added to the mixture. The hexane phase was collected after centrifuging at 7000 g for 5 min. The fatty acid profile was determined using a GC equipped with a flame ionization detector (FID). The sample (1 µL) with a split ratio of 20:1 was injected on an H88 capillary column (100 m, 0.25 mm I.D., film thickness 0.2 µm) for GC analysis. The temperatures of the injector and detector were maintained at 260 ºC and finally, for 5 minutes the oven temperature was kept at 140 ºC, and then increased to 240 ºC at a rate of 4 ºC min^− 1. Helium (1.5 mL min^− 1) was utilized as the carrier gas. Fatty acid profiling was determined by a comparison of retention times with those of a mixture of 37 fatty acids methyl ester (FAME) standards (Supelco, USA, Catalog No: 18919-1AMP)(g L^− 1). The steps taken to find native cold-adapted species and determine their fatty acid profiles from Cheshmeh-Sabz Lake are summarized in Fig. 2.

Statistical analysis

All experiments were performed with three independent biological replications for every treatment. Biochemical measurements were performed on each replication (three technical replications) and the data are presented as mean ± standard deviation (s.d.). Statistical analysis was performed by using SPSS Statistics version 22 software (IBM, USA). One-way analysis of variance (ANOVA) and therefore Duncan´s test at a 99% confidence level was used to compare the differences among treatments.

Sampling

The physico-chemical properties of the lake and measuring the water depth (with maximum a depth of 12 meters) show that Cheshmeh-Sabz Lake is characterized as one of the freshwater lalakeTable 1).

Isolation and purification of microalgae

As the results show in Fig. 2a and Table 2, at the low temperature (5 ºC), Ankistrodesmus sp. grows in WC medium as the dominant spice. Chlorella sp. MN738559 prevailed in BBM medium (Fig. 2b) (Table 2), while in the BG-11 medium, Scenedesmus sp. MN738556 was dominated (Fig. 2c) (Table 2).

Table 1

The physico-chemical parameters of sampling the Cheshmeh-Sabz Lake water.
Sampling	pH	Temperature(ºC)	EC(µs)	TDS(mg L^− 1)	Max depth(m)
January 2017	7.46	5.5	112.6	67	12

Table 2

Identification of three dominant microalgae strains (at 5 ºC), isolated from Cheshmeh-Sabz Lake in various culture media, based on the closest recorded sequence in the National Center for Biotechnology Information (NCBI).
Medium cultures	Closest species (GenBank)	Class
WC	Ankistrodesmus sp.	Chlorophyceae
BBM	Chlorella sp. MN738559	Trebouxiophyceae
BG-11	Scenedesmus sp. MN738556	Chlorophyceae

Growth measurement

The dominant species grown in the WC medium at the low temperature (5 ºC) was Ankistrodesmus sp. (Fig. 3a). The results of the growth parameter measurements in Fig. 4, and Fig. 5a show that, in the WC culture medium, at 25 ºC, Ankistrodesmus sp. was able to grow faster (0.05 ± 0.001 day^− 1) than that at 8 ºC (0.006 ± 0.0001 day^− 1), with biomass productivity (about the sevenfold increase, 0.2 ± 0.01 day^− 1)(Fig. 4). Control of photoluminescence quantum yield (Fig. 5a) and semi-quantitative measurement of nitrogen contents (Fig. 4) at different growth stages showed that with changes in nutrient composition and temperatures in the culture medium, a change in quantum yield and consequently a change in photosynthetic efficiency was observed. Initially, at 8 ºC, the QY was 0.45, which reached 0.69 on the eighth day after inoculation. In addition, the decrease in QY at 8 ºC from 0.69 to 0.45 on the 18th day in the stationary phase was associated with the removal of nitrate. At 25 ºC, from the early growth phase to the 18th day, the QY increased from 0.45 to 0.65, due to a decrease in nitrate concentration, and finally, the microalgae stopped growing on the 18th day (Fig. 4 and Fig. 5a).

Chlorella sp. MN738559 was dominant in BBM culture medium at 5 ºC (Fig. 3b). As shown in Fig. 5c at 8 ºC, the growth of Chlorella sp. MN738559 after inoculation had a ten-day lag phase. As can be seen, in this phase, the change in QY delay was less than that of the exponential growth phase (0.35)(Fig. 5b). This value increased to 0.65 on the tenth day. At the high temperature (25 º C), the specific growth rate of Chlorella sp. MN738559 was about twice that at 8 ºC and reached a stationary phase at high temperature on the fourth day. The biomass productivity at 8 ºC was about six times higher than that at 25 ºC. The change in nitrate availability in exponential and stationary phases was insignificant at 8 ºC whereas it was 80% at 25 ºC (Fig. 4).

Scenedesmus sp. MN738556 at low temperatures (5 ºC) grew mainly in the BG-11 culture medium (Fig. 3c). The growth in the first ten days of this species at low and high temperatures (8 and 25 ºC) was not very high. On the tenth day, the growth of microalgae accelerated and at 25 ºC, reached a stationary growth phase, and finally, its growth stopped on the eighteenth day. While at low temperatures (8 ºC), the growth of microalgae increased with a gentle slope (about 35% lower than that in comparison to 25 ºC) until the eighteenth day. During growth at 25 ºC, the QY value did not change significantly, but at 8 ºC, it was low on the first to fourth days (0.59) but increased on the sixth day (0.69). As shown in Fig. 4 and Fig. 5c, the nitrate availability in the BG-11 culture medium in the stationary phase did not change in comparison to the exponential phase. The results showed that biomass productivity and specific growth rate at 8 ºC and 25 ºC are relatively similar.

Lipid profile

Lipids in microalgae grown in three media, WC (Ankistrodesmus sp.), BG-11 (Scenedesmus sp. MN738556), and BBM (Chlorella sp. MN738559), at three temperatures of 8, 15, and 25 ºC, were extracted at the stationary growth phase, and their fatty acid profile was determined by GC-FID.

The fatty acids profile of Ankistrodesmus sp., at low and, high temperatures (8 and 25 ºC) showed the highest proportion of PUFA at 8 ºC, which was related to α-linolenic acid (C18:3n-3)(ALA) as omega-3 (9.98 ± 0.10%). In addition, at this temperature, the highest amount of SFA was related to palmitic acid (C16:0)(35.37 ± 6.1%). The highest amount of MUFA (16.84%) was obtained at 25 ºC in the form of palmitoleic acid (C16:1)(8.56 ± 0.24%)(Fig. 6, Table 3).

Table 3. Fatty acid composition of Ankistrodesmus sp., Scenedesmus sp. MN738556, and Chlorella sp. MN738559 at 8, 15, and 25 ºC. Values are mean ± s.d. of three replicates. Different letters (P ≤ 0.01) denote the significance of differences. n.d: not detected.

		*Ankistrodesmus* sp.			*Chlorella* sp. MN738559			*Scenedesmus* sp. MN738556
		8° C	15° C	25° C	8° C	15° C	25° C	8° C	15° C	25° C
PUFA	C18:2–trans-Linolelaidic acid	3.88±0.06^b	0.69±0.004ⁱ	0.48±0.001^g	1.37±0.06^h	1.97±0.009^d	4.83±0.1^a	2.48±0.008^c	1.49±0.007^f	1.76±0.01^e
	C18:2 (n-6)–Linoleic acid	5.12±0.12^a	3.98±0.04^c	2.51±0.09^g	4.36±0.02^b	3.02±0.007^f	3.63±0.06^e	3.64±0.004^e	3.86±0.001^d	4.03±0.08^c
	C18:3 (n-3)–a-Linolenic acid (ALA)/ω3FA	9.98±0.10^d	7.80±0.15^e	6.06±.12^f	10.10±0.15^c	10.22±0.52^c	3.13±0.1^g	10.43±0.23^b	10.96±1.5^a	11.07±0.31^a
MUFA	C16:1–Palmitoleic acid	6.29±0.15^c	7.98±0.52^b	8.56±0.24^a	4.97±0.05^f	7.80±0.03^b	5.60±0.12^d	3.50±0.07^e	5.69±0.78^d	3.36±0.08^e
MUFA	C18:1 (n-9)–Oleic acid/ω9FA	0.44±0.003^e	0.69±0.02^d	0.48±0.01^e	0.96±0.01^b	0.95±0.001^b	1.27±0.052^a	0.39±0.005^g	0.78±0.06^c	0.57±0.001^f
SFA	C16:0–Palmitic acid	35.37±6.1^c+d	31.58±2.35^d	35.37±5.23^c+d	40.19±4.85^b	33.46±2.54^c+d	44.41±7.56^a	41.05±5.21^b	38.48±4.25^b+c	39.82±1.58^b
SFA	C18:0–Stearic acid	0.47±0.004^b	0.21±001^d	0.25±0.001^d	0.29±0.001^d	0.40±0.002^c	0.89±0.02^a	n. d.	0.11±0.006^e	0.15±0.0002^e

Chlorella sp. MN738559, as shown in Table 3, at 15 ºC in comparison to the other two temperatures (8 and 25 ºC), had the highest amount of polyunsaturated fatty acids (26.31%)(Fig. 6). In general, omega 3 as α-linolenic acid (C18:3n-3)(ALA) were detected in Chlorella sp. MN738559 at low temperature (8 ºC) at its highest percentage (10.10 ± 0.15%)(Table 3). Meanwhile, MUFA reached its maximum value at 15 ºC, which was the highest concentration of palmitoleic acid (C16:1) with a concentration of 7.80 ± 0.03% (Table 3). Palmitic acid (C16:0), at 8, 15, and 25 ºC, was the dominant fatty acid of SFAs (Table 3).

Scenedesmus sp. MN738556 grown in BG-11 culture medium showed different amounts of fatty acid after extraction. The levels of SFA and PUFA among the three temperatures were approximately equal (about 40% and 16.5%, respectively)(Fig.6). Palmitic acid (C16:0) had the highest amount of saturated fatty acid at three temperatures (~40%). Palmitoleic acid (C16:1) as MUFA had the highest amount (5.69±0.78%) at 15 ºC. The amount of PUFAs at three temperatures was relatively equal (8, 15, and 25 ºC), which was related to α-linolenic acid (C18:3n-3)(ALA)(~11%)(Table 3). In general, the highest percentage of PUFA was detected in Ankistrodesmus sp. at 8 ºC (30.84%), while Chlorella sp. MN738559 comprised the largest amount of SFAs at 25 ºC (71.03%).

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]:

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].

In the present study, three cold-adapted species, Ankistrodesmus sp., Chlorella sp. MN738559, and, Scenedesmus sp. MN738556 were isolated from Cheshmeh-Sabz Lake using fluorescence-activated cell sorting at different culture media (WC, BBM, and BG-11 respectively). The result showed that Scenedesmus sp. MN738556 had the same specific growth rate at low and high temperatures (8 and 25 ºC)(about 0.07 day^− 1). The specific growth rate of Scenedesmus sp. MN738556, at 8 ºC, was higher than that of other species. The biomass productivity for 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).

The study of their fatty acid profile showed that at low temperatures, the unsaturated fatty acids percentage increased relative to saturated fatty acids in all three species. Ankistrodesmus sp. produced the highest amount of PUFA at the low temperature (8 ºC)(30.84%). It was found that α-linolenic acid (ALA)(C18:3 n-3) had the highest amount of unsaturated fatty acids in the low temperature (8 ºC) at three strains and palmitic acid (C16:0) was the major saturated fatty acid in all temperatures. Among three cold-adapted microalgal strains which were isolated from Cheshmeh-Sabz Lake, Scenedesmus sp. MN738556 introduce as a native cold-adapted strain with relatively high biomass productivity and specific a growth rate with the highest amount of α-linolenic acid (C18:3 n-3)(ALA).

Acknowledgment We would like to thank prof.dr.ir. RH (Rene) Wijffels and prof.dr. MJ (Maria) Barbosa from Wageningen University, Netherlands, for using their fluorescence-activated cell sorting to isolate and purify strains. The authors would also like to thank Dr. Zahra Lari for her valuable comments.

Funding Financial support was provided by Ferdowsi University of Mashhad (grant no. 3/45727).

Data availability The raw data is proprietary but can be disclosed by sending a motivated request to the corresponding author.

Declarations The authors declare there is no conflict of interest regarding the publication of this article.

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Evaluation of Lipid composition and growth parameters of cold-adapted microalgae under different conditions

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material And Methods

Sampling site

Sampling method

Chemicals

Sample enrichment and isolation of microalgal strains

Molecular identification

Culture conditions

Growth parameters

Lipid profile

Statistical analysis

Result

Sampling

Isolation and purification of microalgae

Growth measurement

Lipid profile

Discussion

Conclusion

Declarations

References

Status:

Journal Publication

Version 1