Isolation, Identification, and Biochemical Characteristics of a Cold-Tolerant Chlorella vulgaris KNUA007 Isolated from King George Island, Antarctica

A cold-tolerant unicellular green alga was isolated from a meltwater stream on King George Island, Antarctica. Morphological, molecular, and biochemical analyses revealed that the isolate belonged to the species Chlorella vulgaris. We tentatively named this algal strain C. vulgaris KNUA007 and investigated its growth and lipid composition. We found that the strain was able to thrive in a wide range of temperatures, from 5 to 30 ◦C; however, it did not survive at 35 ◦C. Ultimate analysis confirmed high gross calorific values only at low temperatures (10 ◦C), with comparable values to land plants for biomass fuel. Gas chromatography/mass spectrometry analysis revealed that the isolate was rich in nutritionally important polyunsaturated fatty acids (PUFAs). The major fatty acid components were hexadecatrienoic acid (C16:3 ω3, 17.31%), linoleic acid (C18:2 ω6, 8.52%), and α-linolenic acid (C18:3 ω3, 43.35%) at 10 ◦C. The microalga was tolerant to low temperatures, making it an attractive candidate for the production of biochemicals under cold weather conditions. Therefore, this Antarctic microalga may have potential as an alternative to fish and/or plant oils as a source of omega-3 PUFA. The temperature tolerance and composition of C. vulgaris KNUA007 also make the isolate desirable for commercial applications in the pharmaceutical industry.


Introduction
Green microalgae (Chlorophyta) can be found in almost every conceivable environment from polar regions to deserts; they play a pivotal role in global carbon, nitrogen, and phosphorus cycles [1][2][3]. Due to their remarkable tolerance to harsh conditions combined with rapid growth rates, phototrophs are often dominant in the cryosphere, representing a large portion of the total ecosystem biomass [4][5][6]. m −2 s −1 ) under a 16:8 light/dark cycle until algal growth was apparent. Well-developed algal cultures (1.5 mL) were centrifuged at 3000× g for 15 min to harvest the algal biomass. The resulting pellets were streaked onto BG-11 agar supplemented with imipenem (20 µg mL −1 ) and incubated under the aforementioned conditions. A single colony was then aseptically re-streaked onto a fresh BG-11 plate to obtain an axenic culture.

Morphological and Molecular Identification
The isolate was grown in BG-11 medium for 20 days. Live cells were harvested by centrifugation at 3000× g for 5 min, washed twice with sterile distilled water, and examined at 1000 × magnification using a Zeiss Axioskop 2 light microscope (Carl Zeiss, Standort Göttingen, Vertrieb, Germany) equipped with differential interference contrast optics.
For scanning electron microscope (SEM) analysis, 10 mL of cells were fixed in osmium tetroxide (OsO 4 ; Electron Microscopy Sciences, EMS hereafter, Hatfield, PA, USA) for 10 min at a final concentration of 2% (v/v) in distilled water. Fixed cells were collected on a 3 µm pore polycarbonate membrane filter without additional pressure, then rinsed with distilled water to remove residual salts. Cells were dehydrated in an ethanol series (Merck, Darmstadt, Germany) and dried using a critical point dryer (CPD 300, Bal-Tec, Balzers, Liechtenstein). The dried filters were mounted on a stub and coated with gold-palladium using a sputter coater (SCD 005; Bal-Tec). Cell images were analyzed with a field emission (FE)-SEM (S-4800; Hitachi, Hitachinaka, Japan).
For molecular analysis, genomic DNA was extracted using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). The universal primers NS1 and NS8 described by White et al. [24] were used for 18S rRNA sequence analysis. The D1-D2 region of the large subunit (28S) rRNA gene was amplified using NL1 and NL4 primers [25], and the internal transcribed spacer (ITS) region was amplified using ITS1 and ITS4 primers [24]. The DNA sequences obtained in this study were deposited in the National Center for Biotechnology Information database under accession numbers KJ148623 to KJ148625 (Table 1). Phylogenetic analysis was performed with the 18S rDNA string sequences using the software package MEGA version X [26]. The combined datasets of C. vulgaris strains and Neochloris sp. (as an outgroup) were aligned with those of the 20 chlorellacean microalgae strains using ClustalW in MEGA X. The 18S rDNA sequence lengths ranged from 1697 bp to 1732 bp. The best-fit nucleotide substitution model K2 was selected using MEGA X based on the Bayesian information criterion. This model was used to build a maximum likelihood (ML) phylogenetic tree with 1000 bootstrap replicates [27]. The ITS-2 secondary structures were constructed using Mfold [28] according to Germond et al. [29].

Temperature Testing
Late exponential-phase cultures of C. vulgaris KNUA007 (1 mL each) were inoculated into BG-11 medium in triplicate and incubated for 20 days. The survival and growth of KNUA007 cells maintained at temperatures ranging from 5 to 35 • C (at intervals of 5 • C) were examined to determine the optimum culture temperature. Algal cell density was determined by measuring the optical density (OD) of the cultures at 680 nm with an Optimizer 2120UV spectrophotometer (Mecasys, Daejeon, Korea). We also incubated samples in BG-11 medium in a PhotoBiobox (Shinhwa Science, Daejeon, Korea) at temperatures ranging from 6 to 28 • C and light intensity ranging from 50-400 µmol photon m −2 s −1 with cycles of 24 h light for 3 days to monitor the optimal conditions. OD at 680 nm was measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA, USA) and OD values were visualized on a heat map by SigmaPlot 13 software (Systat Software, San Jose, CA, USA) [30].

Biomass Characterization
The isolates were autotrophically grown in BG-11 medium for 20 days and cells were harvested by centrifugation at 2063 g (1580R, Labogene, Daejeon, Korea). The freeze-dried biomass samples were pulverized with a mortar and pestle and sieved through ASTM No. 230 mesh (63 µm holes). Ultimate analysis was conducted to determine the carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) contents using a Flash 2000 elemental analyzer (Thermo Fisher Scientific, Milan, Italy). Gross calorific value (GCV) was estimated using the following equation developed by Friedl et al. [31], and protein content was calculated from the N content using a conversion factor of 6.25 ×.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
Extracted fatty acid methyl ester (FAME) composition was analyzed using a gas chromatograph-mass spectrometer (GC/MS 7890A; Agilent, Santa Clara, CA, USA) equipped with a mass selective detector (5975C; Agilent) and DB-FFAP column (30 m, 250 µm ID, 0.25 µm film thickness; Agilent). The initial temperature of 50 • C was maintained for 1 min. The temperature was then increased to 200 • C at a rate of 10 • C min −1 for 30 min, then increased to 240 • C at a rate of 10 • C min −1 and held for 20 min. The injection volume was 1 µL, with a 20:1 split injection ratio. Helium was supplied as the carrier gas at a constant flow rate of 1 mL min −1 . For the mass spectrometer parameters, the injector and source temperatures were 250 and 230 • C, respectively, and the electron impact mode was used for sample ionization at an acceleration voltage of 70 eV, with an acquisition range of 50-550 mass to charge ratio [32]. All compounds were identified by analyzing their mass spectra with the Wiley/NBS libraries.

Identification of Strain KNUA007
The cells were solitary, non-motile, and spherical, with a diameter of approximately 4-6 µm ( Figure 1) and a prominent cup-shaped chloroplast. The cell walls were composed of a single smooth layer. The strain KNUA007 exhibited morphology typical of the genus Chlorella. Daejeon, Korea). We also incubated samples in BG-11 medium in a PhotoBiobox (Shinhwa Science, Daejeon, Korea) at temperatures ranging from 6 to 28 °C and light intensity ranging from 50-400 µmol photon m −2 s −1 with cycles of 24 h light for 3 days to monitor the optimal conditions. OD at 680 nm was measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA, USA) and OD values were visualized on a heat map by SigmaPlot 13 software (Systat Software, San Jose, CA, USA) [30].

Biomass Characterization
The isolates were autotrophically grown in BG-11 medium for 20 days and cells were harvested by centrifugation at 2063 g (1580R, Labogene, Daejeon, Korea). The freeze-dried biomass samples were pulverized with a mortar and pestle and sieved through ASTM No. 230 mesh (63 µm holes). Ultimate analysis was conducted to determine the carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) contents using a Flash 2000 elemental analyzer (Thermo Fisher Scientific, Milan, Italy). Gross calorific value (GCV) was estimated using the following equation developed by Friedl et al. [31], and protein content was calculated from the N content using a conversion factor of 6.25 ×.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
Extracted fatty acid methyl ester (FAME) composition was analyzed using a gas chromatograph-mass spectrometer (GC/MS 7890A; Agilent, Santa Clara, CA, USA) equipped with a mass selective detector (5975C; Agilent) and DB-FFAP column (30 m, 250 µm ID, 0.25 µm film thickness; Agilent). The initial temperature of 50 °C was maintained for 1 min. The temperature was then increased to 200 °C at a rate of 10 °C min −1 for 30 min, then increased to 240 °C at a rate of 10 °C min −1 and held for 20 min. The injection volume was 1 µl, with a 20:1 split injection ratio. Helium was supplied as the carrier gas at a constant flow rate of 1 ml min −1 . For the mass spectrometer parameters, the injector and source temperatures were 250 and 230 °C, respectively, and the electron impact mode was used for sample ionization at an acceleration voltage of 70 eV, with an acquisition range of 50-550 mass to charge ratio [32]. All compounds were identified by analyzing their mass spectra with the Wiley/NBS libraries.

Identification of Strain KNUA007
The cells were solitary, non-motile, and spherical, with a diameter of approximately 4-6 µm ( Figure 1) and a prominent cup-shaped chloroplast. The cell walls were composed of a single smooth layer. The strain KNUA007 exhibited morphology typical of the genus Chlorella. Molecular characterization inferred from sequence analyses of the genes for 18S rRNA, 28S rRNA, and the ITS region confirmed that the isolate belonged to the C. vulgaris group ( Table 1). As illustrated in Figure 2, strain KNUA007 was clustered with C. vulgaris in a different clade from Molecular characterization inferred from sequence analyses of the genes for 18S rRNA, 28S rRNA, and the ITS region confirmed that the isolate belonged to the C. vulgaris group ( Table 1). As illustrated in Figure 2, strain KNUA007 was clustered with C. vulgaris in a different clade from Micractinium and Parachlorella. Therefore, the isolate was tentatively identified as C. vulgaris KNUA007.

KNUA007.
To accurately differentiate between the morphologically ambiguous genera Micractinium and Chlorella, the secondary structure of ITS-2 was examined. Helix 3 of ITS-2 has a distinct molecular signature that separates Chlorella and Micractinium. Specifically, in Figure 3, the paired nucleotides indicated in the orange box are a synapomorphy of Micractinium [22]. Therefore, strain KNUA007 was tentatively identified as C. vulgaris KNUA007.

Cold Tolerance of Strain KNUA007
C. vulgaris KNUA007 could grow at temperatures ranging from 5 to 30 °C; maximum growth was observed at 20 °C. The cells grew and survived at 5, 10, 15, and 25 °C, but relatively slow growth was observed (Figure 4). However, the isolate did not survive at 35 °C. This strain also grew at a wide range of light intensity, from 50 to 400 µmol photon m −2 s −1 ; maximum growth was observed at 150 µmol photon m −2 s −1 , and the strain preferred relatively low light intensity. To accurately differentiate between the morphologically ambiguous genera Micractinium and Chlorella, the secondary structure of ITS-2 was examined. Helix 3 of ITS-2 has a distinct molecular signature that separates Chlorella and Micractinium. Specifically, in Figure 3, the paired nucleotides indicated in the orange box are a synapomorphy of Micractinium [22]. Therefore, strain KNUA007 was tentatively identified as C. vulgaris KNUA007. Micractinium and Parachlorella. Therefore, the isolate was tentatively identified as C. vulgaris KNUA007.
To accurately differentiate between the morphologically ambiguous genera Micractinium and Chlorella, the secondary structure of ITS-2 was examined. Helix 3 of ITS-2 has a distinct molecular signature that separates Chlorella and Micractinium. Specifically, in Figure 3, the paired nucleotides indicated in the orange box are a synapomorphy of Micractinium [22]. Therefore, strain KNUA007 was tentatively identified as C. vulgaris KNUA007.

Cold Tolerance of Strain KNUA007
C. vulgaris KNUA007 could grow at temperatures ranging from 5 to 30 °C; maximum growth was observed at 20 °C. The cells grew and survived at 5, 10, 15, and 25 °C, but relatively slow growth was observed (Figure 4). However, the isolate did not survive at 35 °C. This strain also grew at a wide range of light intensity, from 50 to 400 µmol photon m −2 s −1 ; maximum growth was observed at 150 µmol photon m −2 s −1 , and the strain preferred relatively low light intensity.

Cold Tolerance of Strain KNUA007
C. vulgaris KNUA007 could grow at temperatures ranging from 5 to 30 • C; maximum growth was observed at 20 • C. The cells grew and survived at 5, 10, 15, and 25 • C, but relatively slow growth was observed (Figure 4). However, the isolate did not survive at 35 • C. This strain also grew at a wide range of light intensity, from 50 to 400 µmol photon m −2 s −1 ; maximum growth was observed at 150 µmol photon m −2 s −1 , and the strain preferred relatively low light intensity.

Biomass Properties
The C, H, N, and S contents were determined by elemental analysis ( Table 2). C and N decreased in inverse proportion to the temperature increase, S increased with temperature, and H content was independent of the temperature gradient. This result affected the GCV values determined based on elemental analysis, and consequently, the GCV decreased with increasing temperature.

GC/MS Analysis of Strain KNUA007
The FAME profile of C. vulgaris KNUA007 is summarized in Table 3; values are presented as the average ± standard deviation of three determinations. The major cellular fatty acids of the microalgae were C16:3 ω3, C16:0, C18:2 ω6, and C18:3 ω3. The C18:3 composition was approximately 43% at 10 °C, decreasing to 36 and 20% with increasing temperatures. C16:3 also decreased more than five-fold with the temperature increase, similar to C18:3. Meanwhile, C16:2 and C18:2, which have two unsaturated chains, increased two and three times, respectively, and the other fatty acid contents did not change. The protein contents calculated using ultimate analysis were 38.56, 36.5, and 32.81% at 10, 20, and 30 °C, respectively.

Biomass Properties
The C, H, N, and S contents were determined by elemental analysis ( Table 2). C and N decreased in inverse proportion to the temperature increase, S increased with temperature, and H content was independent of the temperature gradient. This result affected the GCV values determined based on elemental analysis, and consequently, the GCV decreased with increasing temperature.

GC/MS Analysis of Strain KNUA007
The FAME profile of C. vulgaris KNUA007 is summarized in Table 3; values are presented as the average ± standard deviation of three determinations. The major cellular fatty acids of the microalgae were C16:3 ω3, C16:0, C18:2 ω6, and C18:3 ω3. The C18:3 composition was approximately 43% at 10 • C, decreasing to 36 and 20% with increasing temperatures. C16:3 also decreased more than five-fold with the temperature increase, similar to C18:3. Meanwhile, C16:2 and C18:2, which have two unsaturated chains, increased two and three times, respectively, and the other fatty acid contents did not change. The protein contents calculated using ultimate analysis were 38.56, 36.5, and 32.81% at 10, 20, and 30 • C, respectively.

Discussion
In this study, a cold-tolerant C. vulgaris strain, KNUA007, was axenically isolated using a combination of imipenem treatment and physical separation techniques. We analyzed the morphological, molecular, physiological, and biochemical characteristics of the isolates to identify the strain. The small and rounded cells lacked flagella and possessed the morphological features typical of C. vulgaris, including smooth cell wall layers and the presence of chloroplast pyrenoids (Figure 1). In addition, the phylogenetic position of the isolate was confirmed through sequence analysis of the SSU, ITS, and LSU regions ( Table 1). As shown in Figure 2, the isolated strain was clearly separated from the genus Micractinium and was closely related to C. vulgaris. However, Micractinium has similar morphological features to the isolated strain [22], so the Helix 3 structure of the ITS-2 region was compared for further classification (Figure 3). Our analysis of the Helix 3 structure revealed that the strain used in this experiment did not possess the characteristic paired-nucleotide synapomorphy of Micractinium [22,29,33]. The isolate had a 100% sequence homology with C. vulgaris CCAP 211/63 (accession no. FR865681) ( Figure 2) and the same ITS-2 overall sequence (data not shown); therefore, the strain used in this experiment was named C. vulgaris KNUA007.
The GCV was calculated to estimate the potential of this microalga as a biofuel feedstock. The results indicated that the GCV was similar to terrestrial energy crops and cellulosic biomass (17.0−20.0 MJ kg −1 ) (  [34,35]. However, given the high photosynthetic efficiency and growth rate of this C. vulgaris strain, it could be a superior bioenergy source for biofuel production. We also measured protein using ultimate analysis; the protein content was over 30% at all temperature ranges and was highest at 10 • C. These results demonstrate the potential of C. vulgaris KNUA007 microalgal biomass for producing animal feedstock or bio-fertilizer. Antarctica is one of the most extreme environments on the planet. Microalgae in Antarctic ecosystems are continuously exposed to direct sunlight during the summer months and to freezing temperatures during the winter months. Thus, Antarctic microalgae have developed a number of protection strategies to survive under these harsh conditions. In general, microalgae protect themselves from excessive light and oxidative damage by dissipating excess energy as heat, which has been termed non-photochemical quenching of chlorophyll fluorescence [36,37]. Moreover, some Chlorella species have been reported to express anti-freezing genes in response to low-temperature stress, enhancing their tolerance to freezing temperatures [38][39][40]. One of the most common self-defense mechanisms observed in microalgae to acquire cold tolerance is to increase the fluidity of lipid membrane, carried out by fatty acid desaturation, which increases the production of unsaturated fatty acids with lower chain lengths that remain in a liquid state at low temperatures [41,42]. The majority of fatty acids in C. vulgaris are C16 and C18, and the sequential desaturation of these fatty acids usually involves the following steps: C16:0 → C18:0 → C18:1 → C18:2 → C18:3 [43,44]. Analysis of the cellular fatty acid composition of the strain KNUA007 revealed that it was rich in C16:3, C18:2, and C18:3 unsaturated fatty acids (Table 3). In particular, C16:3 and C18:3, which have three unsaturated chains, increased in inverse proportion to the temperature; the C16:3 and C18:3 contents were more than twice as high at 10 • C than at 30 • C. As mentioned above, the content of polyunsaturated fatty acids is thought to increase as a defense mechanism to cope with low-temperature stress. Meanwhile, decreases in C18:1 and C18:2 were observed in the 10 • C culture of KNUA007 compared to those of the 20 • C culture of KNUA007. We presume that these slight decreases in C18:1 and C18:2 accounted for the degree of unsaturation, since C18:1 and C18:2 are intermediate forms of desaturation in the biosynthetic pathway of the major fatty acids by desaturases. On the other hand, the most unsaturated form of the major C18 fatty acids, C18:3, showed a significant increase in response to the low temperature. We assume C18:3 plays an important role in the fluidity modification capacity of C. vulgaris KNUA007. This kind of trend was also observed in Scenedesmus acutus [45]. Its fatty acid profile changed in response to temperature changes and the same pattern (increase in C18:3 composition in lower temperatures and increase in C18:1 and C18:2 compositions in higher temperatures, respectively) as our study with C. vulgaris was also observed in their study. Numerous studies demonstrated that these essential PUFAs also have many beneficial health effects [46] and various commercial products are available worldwide. Omega-3 PUFAs are typically derived from fish oils and omega-6 PUFAs are primarily obtained from plant sources such as sunflower, corn, and soybean oils. Therefore, this isolate may have the potential to be used as an alternative to fish-and/or plant-based oil sources. In addition, the biomass itself may serve as an excellent animal feed because of its adequate protein content.
Lastly, it should be stated that most polar microalgae are known to be psychrotolerant; however, truly psychrophilic microalgae are relatively rare [47,48], and temperature flexibility is more common than psychrophily [49,50]. Likewise, strain KNUA007 was shown to be a psychrotolerant and mesophilic microalga that could grow at temperatures as low as 5 • C; optimum growth was observed at 20 • C ( Figure 4). Furthermore, the strain grew over a wide temperature range (5-30 • C). This wide temperature tolerance is similar to those previously reported for Antarctic Chlorella strains [38,51]. The eurythermal properties of Chlorella may be advantageous for outdoor mass cultivation. The C. vulgaris KNUA007 isolate may be used for the production of value-added products under unfavorable weather conditions, including the cold temperatures that occur in autumn and winter seasons since ambient temperatures (approximately 20-35 • C) were the optimal growth conditions for most of C. vulgaris cultures for PUFA production [52]. In general, cell growth is suppressed at lower temperatures and the metabolic rate is accelerated at higher temperatures, but it is reported that temperatures above 35 • C usually inhibit growth [13].
In conclusion, this Antarctic microalga could serve as a potential biological resource to produce compounds of biochemical interest. The real potential of the isolate described in this paper should be evaluated through further cultivation studies at molecular, laboratory, and field scales.