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Article

Taxonomic and Biochemical Characterization of Microalga Graesiella emersonii GEGS21 for Its Potential to Become Feedstock for Biofuels and Bioproducts

by
Nam Seon Kang
1,
Kichul Cho
1,
Sung Min An
1,
Eun Song Kim
1,
Hyunji Ki
1,
Chung Hyeon Lee
1,
Grace Choi
1 and
Ji Won Hong
2,3,*
1
Department of Microbial Resources, National Marine Biodiversity Institute of Korea, Seocheon 33662, Republic of Korea
2
Department of Hydrogen and Renewable Energy, Kyungpook National University, Daegu 41566, Republic of Korea
3
Advanced Bio-Resource Research Center, Kyungpook National University, Daegu 41566, Republic of Korea
*
Author to whom correspondence should be addressed.
Energies 2022, 15(22), 8725; https://doi.org/10.3390/en15228725
Submission received: 3 November 2022 / Revised: 14 November 2022 / Accepted: 17 November 2022 / Published: 20 November 2022
(This article belongs to the Special Issue Innovative Technologies for Biomass Valorization in Sustainable Energy System)
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Abstract

:
Graesiella emersonii is a commercially exploitable source of bioactive compounds and biofuels with potential applications in microalgae-based industries. Despite this, little taxonomical information is available. Therefore, proper identification and characterization are needed for the sustainable utilization of isolated microalgae. In this study, an axenically isolated unicellular green alga from the Geumgang Estuary, Korea was investigated for its morphological, molecular, and biochemical characteristics. The morphological characteristics were typical of G. emersonii. Molecular phylogenetic analysis of the 18S rDNA sequence verified that the isolate belonged to G. emersonii and was subsequently named G. emersonii GEGS21. It was isolated from brackish water, and its optimal growth temperature, salinity, and light intensity were at 28–32 °C, 0 M NaCl, and 130–160 µmol m−2 s−1, respectively. The strain thrived over a range of temperatures (5–40 °C) and withstood up to 0.5 M NaCl. The isolate was rich in omega-6 linoleic acid (C18:2 n-6, 26.3%) and palmitic acid (C16:0, 27.5%). The fuel quality properties were determined, and biodiesel from GEGS21 could be used as a biodiesel blend. Value-added carotenoids lutein (1.5 mg g−1 dry cell weight, DCW) and neoxanthin (1.2 mg g−1 DCW) were biosynthesized as accessory pigments by this microalga. The biomass of this microalga may serve as feedstock for biodiesel production as well as producing valuable ω-6 and carotenoids.

1. Introduction

Microalgae are ubiquitous protists found in rivers, lakes, oceans, soils, and even extreme environments such as fresh or saltwater bodies with a low pH [1,2,3,4,5,6]. They are the primary producers in aquatic ecosystems and play crucial roles in global carbon and nutrient cycles, such as nitrogen and phosphorus cycles [7]. Due to their ability to convert carbon dioxide into a variety of useful organic compounds via photosynthesis [8,9,10], microalgae have recently attracted increasing attention. A number of studies have been conducted on the potential use of the microalgal biomass for bioenergy, nutraceutical, pharmaceutical, food, and cosmetic products, as well as agriculture and bioremediation applications [11,12,13,14,15]. Recently, the microalgae biorefinery concept has been proposed [16,17,18,19]. In this approach, microalgal biomass grown in wastewater can be converted into a range of bulk chemicals and value-added biocompounds [20]. Other technologies have also been integrated into microalgal biorefinery to facilitate the full-scale utilization of algal biomass [21,22].
Among green algae, Chlorophyta is one of the largest phyla with many species and a wide geographical distribution. Some can cause blooming in lentic systems during the summer season under favorable conditions [23,24,25]. Additionally, several microalgal strains of Chlorophyta have been used as biotechnological resources [26,27,28,29]. However, Chlorella-like species have at times been misidentified because of their morphological simplicity [30]. Accurate species identification and delimitation have important implications for the safe consumption of commercial microalgal products [31,32,33]. Misidentified names of microalgae may potentially lead to economic losses and also pose a risk to public health [30]. Therefore, isolation and establishment of clonal cultures from a variety of environments are important steps to accurately identify species of interest. This will further our understanding of their potential commercial value.
The genus Graesiella was first established by Kalina and Puncochárová [34] and, to date, only one species (Graesiella emersonii) has been described in the genus [35]. G. emersonii is usually found in freshwater, but occasionally inhabits brackish water. As it is capable of water purification, G. emersonii plays an indispensable role in maintaining healthy aquatic ecosystems [36,37]. Moreover, G. emersonii is a promising resource for valuable pigments, lipids, and potential pharmaceuticals [38,39,40,41]. In addition, G. emersonii has been consumed in Europe and is an approved food supplement in France [42,43]. Furthermore, this species can be used to sustainably produce biodiesel during wastewater treatment [39]. Hence, G. emersonii is a commercially exploitable source of bioactive compounds and biofuels with potential applications in microalgae-based industries. However, despite their ecological and economic importance in marine ecosystems and biotechnology, not much information is available on their taxonomy because of the difficulties in culturing and observing their morphology using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Thus, their roles as essential components of aquatic ecosystems and their potential for biotechnology are often overlooked. Additionally, microalgal strains within the same species can exhibit different physiological activities and metabolite profiles [44,45]. It is expected that strains belonging to the same species but isolated from very different environments will demonstrate different physiological responses or exhibit different metabolite profiles. Hence, accurate identification followed by establishment of a clonal culture of this species is essential prior to further research and application.
In this study, we isolated a G. emersonii strain from brackish water in the Geumgang Estuary, Korea and characterized its morphological, molecular, and chemotaxonomic features.

2. Materials and Methods

2.1. Sample Collection and Isolation

Plankton samples from the brackish water were obtained from the Geumgang Estuary, Gunsan-si, Jeollabuk-do, Korea (36°0′8.2′′ N, 126°45′10.8′′ E) in May 2021, when the water temperature and practical salinity unit (PSU) were 18.4 °C and 21, respectively (Table 1 and Figure 1). The Geumgang Estuary has substantial variabilities in salinity as the freshwater is released irregularly and artificially from the coastal reservoir. To mimic eutrophication, the water sample was mixed with an equal volume of BG-11 medium (Sigma-Aldrich, St. Louis, MO, USA) and the mixture was allowed to stand for two days. Cultures were ascetically isolated using a streak plate technique on a 1.5% agar-containing BG-11 medium. The plates were incubated in a culture room at 28 °C under cool fluorescent light (~60 μmol m−2 s−1) and a light/dark cycle of 14 h/10 h until green microalgal colonies formed. Single colonies were aseptically transferred to fresh BG-11 plates and this step was repeated until a pure culture was obtained. The axenic algal culture was confirmed by spreading 50 μL liquid culture of the single colony onto 1.5% Luria–Bertani (LB) agar plates, and was verified by 16S rRNA-based colony PCR. The isolated algal strain was maintained at 28 °C in a plant growth chamber (JSR, Gongju, Korea) with 150 rpm of shaking under continuous 60 μmol photons m−2 s−1 of cool-white, fluorescent illumination.

2.2. Morphological Identification

The morphology of living cells that had been grown photosynthetically was examined using an inverted microscope (CKX53, Olympus, Tokyo, Japan). The length and width of the live cells were measured using a digital camera (Zeiss AxioCam MRc5; Carl Zeiss, Göttingen, Germany). Additionally, to corroborate the location and presence of cell nuclei, samples were stained for 15 min with a nuclear marker (4′,6-diamidino-2-phenylindole, DAPI 10 µg mL−1 stock; Sigma-Aldrich) and analyzed using a UV filter under a fluorescence microscope (Zeiss Axio Imager 2; Carl Zeiss).
For field emission (FE)-SEM, 10 mL aliquots of cultures—at a density of approximately 2 × 106 cells mL−1—were fixed for 10 min in osmium tetroxide (OsO4, Electron Microscopy Sciences, Hatfield, PA, USA) at a final concentration of 1% (v/v). The fixed cells were collected on polycarbonate membrane filters which had 3 µm pores (Whatman Nuclepore Track-Etched Membranes; Whatman, Kent, UK) and were washed thrice with 50% filtered seawater diluted with distilled water to remove the residual salts. The membranes with attached cells were dehydrated in a graded ethanol series (10, 30, 50, 70, 90, and 100% ethanol), followed by two changes in 100% ethanol (Merck, Darmstadt, Germany), and were immediately dried using an automated critical point dryer (EM CPD300, Leica, Wetzlar, Germany). The dried filters were mounted on an aluminum stub (Electron Microscopy Sciences) using copper conductive double-sided tape (Ted Pella, Redding, CA, USA) and coated with gold using an ion sputter (MC1000, Hitachi, Tokyo, Japan). The cells and surface morphologies were observed using high-resolution Zeiss Sigma 500 VP FE-SEM (Carl Zeiss). For TEM, cells were transferred to a 10 mL tube and fixed in 2.5% (v/v) glutaraldehyde (final concentration) for 1.5 h. The tube contents were placed in a 10 mL centrifuge tube and concentrated at 1610× g for 10 min in a centrifuge (VS-5500, Vision, Bucheon, Korea). The resulting pellet was subsequently transferred to a 1.5 mL tube and rinsed with 0.2 M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences). After several rinses with 0.2 M sodium cacodylate buffer, the cells were post-fixed for 90 min with 1% (w/v) OsO4 prepared in deionized water. The pellet was embedded in agar. Dehydration was performed in a graded ethanol series (50, 60, 70, 80, 90, and 100% ethanol), followed by two changes in 100% ethanol. The material was then embedded in Spurr’s resin (Electron Microscopy Sciences). Sections were prepared using an EM UC7 ultramicrotome (Leica, Wetzlar, Germany) and stained with 3% (w/v) aqueous uranyl acetate (Electron Microscopy Sciences), followed by 0.5% (w/v) lead citrate (Electron Microscopy Sciences). The sections were visualized using a Sigma 500/VP TEM (Carl Zeiss).

2.3. Molecular Identification

For molecular analysis, genomic DNA (gDNA) was extracted using an AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea), as per the manufacturer’s instructions. The rDNA was amplified using universal eukaryotic primers (Table 2). The reaction mixtures for PCR amplification comprised 5 µL of 10× F-Star Taq Reaction Buffer, 1 µL of 10 mM dNTP mix, 0.02 µM of primers, 0.25 µL of 5 U/µL BioFACT F-Star Taq DNA polymerase (BioFACT Co., Ltd., Daejeon, Korea), 38.75 µL of UltraPure DNAse/RNAse-Free Distilled Water (Invitrogen, Carlsbad, CA, USA), and 3 µL of the DNA template (ca. 10–30 ng DNA). PCR amplification was performed on an Eppendorf Mastercycler PCR machine (Eppendorf, Hamburg, Germany) under the following thermal cycling conditions: pre-denaturation at 94 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, the selected annealing temperature (AT) for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The AT of the primers was determined by gradient PCR. We optimized the ATs as follows: 56 °C (EukA-G18R), 56 °C (570F-EukB), 57 °C (ITSF2-ITSFR2), 57 °C (D1R-LSUB), and 57 °C (rbcL-192-rbcL-657). PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer) and subjected to Sanger sequencing (Macrogen, Daejeon, Korea). Nucleotide sequences were identified using NCBI BLAST. Alignments and phylogenetic and molecular evolutionary analyses of the small subunit (SSU) rDNA sequences were performed using Geneious Prime v.2022.2.2 (Biomatters Ltd., Auckland, New Zealand), with diverse assemblages using other species data available in NCBI GenBank. Bayesian analyses were run using MrBayes v.3.2.7 [46,47] with the default GTR + G + I model to determine the best available model for the data of each region. For all sequence regions, four independent Markov chain Monte Carlo runs were performed, as described by Kang et al. [48]. Maximum-likelihood (ML) analyses were conducted using RAxML v.8.2.10 [49]. Two hundred independent free inferences were allowed and the–# option was used to identify the best tree. Bootstrap values (MLBS) were calculated with 1000 replicates using the same substitution model.

2.4. Determination of Optimal Culture Conditions

Routine serial sub-culturing on the BG-11 agar slant was performed to maintain a pure culture of G. emersonii. A single colony of GEGS21 was streaked onto BG-11 agar plates containing glucose (1.0 g L−1) as the sole carbon source in triplicate and incubated for 21 days. Finally, a single colony was cultured in BG-11 medium, and a subsequent optimal culture test was conducted at a laboratory scale. Optimal salinity was determined using a daily growth test at 28 °C with BG-11 media at NaCl concentrations of 0, 0.5, 1.0, 1.5, and 2.0 M for 10 days in a shaking incubator (JSR). Daily growth was analyzed using a DHC-N01 hemocytometer (INCYTO, Cheonan, Korea). Furthermore, optimal temperature and illumination analyses were conducted simultaneously using a PhotoBiobox [55]. Briefly, a 200 µL algal culture aliquot was injected into 96-well black/clear bottle plates and covered using a well plate sealing film. After incubation for 72 h in PhotoBiobox controlled at 5–40 °C of temperature, and 0–300 µmol m−2 s−1 of illumination, 600 nm of absorbance was analyzed using a Synergy II microplate reader (Biotek, Winooski, VT, USA). Specific growth rate was determined, and a heat map was generated using R 4.2.1 (The R Project for Statistical Computing, https://www.r-project.org, accessed on 16 October 2022).

2.5. Analyses for Fatty Acid Composition of Lipids and Their Biodiesel Properties

Lipid extraction was performed using a modification of the Blight–Dyer method developed by Breuer et al. [56]. The fatty acid methyl ester (FAME) composition was analyzed using a 7890A gas chromatograph equipped with a 5975C mass selective detector (Agilent, Santa Clara, CA, USA), in accordance with the methodology used in our previous study [57]. Compound identification was performed by matching the mass spectra with those in the Wiley/NBS registry of the mass spectral data. A search with a match value greater than 90% was considered valid. Biodiesel properties, including saponification value (SV), iodine value (IV), degree of unsaturation (DU), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), long-chain saturation factor (LCSF), cold filter plugging point (CFPP), cetane number (CN), and oxidative stability (OS), based on the FAME profiles were calculated in accordance with the method described by Islam et al. [58].

2.6. Microalgal Pigment Extraction and Analysis

Algal photosynthetic pigment extraction was performed using the method described by Baek et al. [59] with slight modifications. Briefly, 1 mg freeze-dried biomass was extracted using 90% HPLC-grade acetone (Daejung, Siheung, Korea) and filtered through a Whatman polytetrafluoroethylene (PTFE) syringe filter with a pore size of 0.2 μm (Whatman, Florham Park, NJ, USA). Samples were then analyzed on an Agilent 1260 Infinity HPLC system (Agilent, Waldbronn, Germany) equipped with a Spherisorb 5.0 μm ODS1 4.6 × 250 mm cartridge column (Waters, St. Louis, MO, USA) at 33 °C. Pigment profiles were detected and quantified based on absorbance at 445 and 670 nm using a diode array detector. The mobile-phase gradient was programmed as described by Baek et al. [59] at a constant flow rate of 1.2 mL min−1 using a mixture of 2% methanol, 14% 0.1 M Tris-HCl (pH 8.0), and 84% acetonitrile (Solvent A, v/v) from 0 to 15 min, and a mixture of 32% acetonitrile 68% methanol (Solvent B, v/v) from 15 to 19 min, and the post-run was performed for 6 min using a Solvent A. The concentration of each pigment was analyzed using the HPLC profile determined using chlorophyll and carotenoid standard solutions (DHI, Hørsholm, Denmark).

3. Results

3.1. Morphology or Morphological Characteristics

The single vegetative cells of G. emersonii GEGS21 in culture were nearly spherical to ellipsoidal in shape (Figure 2a,b). The cells exhibited variable sizes but were most commonly 8.8–14.4 μm (12 ± 0.3, n = 20) in diameter (Table 3). A cup-shaped chloroplast was observed along the cell periphery (Figure 2a), and a single pyrenoid was clearly visible in the vegetative cells as well as in the reproductive cells (Figure 2a,c). A single nucleus was located next to the pyrenoid (Figure 2a,b) and several nuclei (Figure 2b,c, Table 3) were observed in mature cells. Actively growing cultures showed asexual reproduction via autosporulation (Figure 2c–f). Parental and daughter cells were observed in all cultures examined by light microscopy, and most parental cells contained two, four, or six daughter cells (Figure 2c–f), but eight or more daughter cells were also observed (not shown). The daughter cells in the parental cell were globose or spheroidal to ellipsoidal (Figure 2g) and were 5.0–12.7 μm (6.9 ± 0.4, n = 20) in diameter (Table 3). The daughter cells with chloroplasts, nuclei, and prominent pyrenoids were released through parental cell wall rupture (Figure 2g).
Scanning electron micrographs of a group of vegetative and reproductive cells of G. emersonii GEGS21 are shown in Figure 3 and Figure 4. Figure 3a shows an example of variable forms, from globose to ellipsoidal cells, which were found in variable sizes. As shown in the micrographs, the parental and daughter cells exhibited characteristic cell wall sculptures in the form of a meridional or irregular network of fine ribs (Figure 4a–e). The number of fine ribs varied according to various stages of development of the parental and daughter cells (Figure 4a–e). Scanning electron micrographs showed that the parental cell wall was composed of multiple layers (Figure 4f,g).
Transmission electron micrographs of the vegetative and reproductive cells of G. emersonii GEGS21 are shown in Figure 5. Figure 5a shows an example of variable forms, from globose to ellipsoidal cells, which were found in variable sizes. Chloroplasts were observed along the cell periphery (Figure 5a–c). The pyrenoid matrix was surrounded by starch grains and was present in the chloroplasts (Figure 5b). The thylakoids that surround the pyrenoid never directly touched the surface of the pyrenoid and did not penetrate it (Figure 5b). A single nucleus was located adjacent to the pyrenoid (Figure 5b). Two nuclei were frequently observed in mature cells (Figure 5c); however, the occurrence of multiple nuclei per cell was also seen (not pictured). Some cells and electron-dense bodies (DB) were observed (Figure 5d). Thin TEM sections showed the structure of the G. emersonii GEGS21 cell wall, which was composed of a trilaminar sheath (TLS) and fibrillar wall (Figure 5f). The TLS appeared as a translucent line inserted between two electron-dense lines. The fibrillar wall was located between the TLS and plasma membrane (Figure 5f). Overall, the strain GEGS21 exhibited the typical morphology of G. emersonii.

3.2. Molecular Identification and Sequence Analysis

The SSU, ITS1, 5.8S, ITS2, and LSU rDNA sequences and rbcL genes (GenBank accession no. OP592224–OP592226, OP605746; Table 1) of the new isolate comprised 3139 nucleotides. When properly aligned, the sequence of SSU rDNA of G. emersonii GEGS21 was identical to that of G. emersonii strains CCAP 211/11N (Berlin, Germany), BEA0616B (Gran Canaria, Spain), BEA1005B (Cadiz, Spain), NT1e (Tennant Creek, Australia), CCAP 211/15 (England), CCAP 211/11M (unknown), CCAP 211/8H (unknown), and CCAP 211/8P (unknown), while G. emersonii strains NIES-2151 (Berlin-Dahlem, Germany), NIES-690 (Berlin, Germany), and unknown Indian strains have a 1–8 base substitution in the SSU compared to strain GEGS21 (Table 4). In the phylogenetic tree based on SSU rDNA sequences, G. emersonii GEGS21 formed a large clade (i.e., Graesiella emersonii) with strains NIES-2151, CCAP211/15, NIES-690, BEA1005B, BEA0616B, NT1E, CCAP211/8H, CCAP211/11N, CCAP211/11M, CCAP211/8P, and unknown strains (GenBank accession no. KJ725233 and MN877773) (Figure 6). Molecular characterization inferred from sequence analyses of SSU rDNA showed that the isolate was a member of the G. emersonii group (Table 4, Figure 6). Therefore, this microalga was identified as G. emersonii GEGS21 and deposited at the National Marine Biodiversity Institute of Korea (MABIK) and the Korean Collection for Type Cultures under the accession numbers MABIK LP00000155 and KCTC15115BP, respectively.

3.3. Verification of the Optimal Cultivation Conditions of the Isolated Strain

To verify the optimal cultivation conditions for the isolated algal strain, growth responses to different salinities, temperatures, and light intensities were determined under laboratory-scale conditions. As shown in Figure 7, a salinity-dependent decrease in the daily growth of G. emersonii was observed after 10 days of cultivation. Specifically, the exponential growth phase appeared after 3 days, and approximately 3.1 × 106 cells mL−1 were counted at a 0.5 M NaCl concentration. However, approximately 5.48-fold higher cell numbers (1.7 × 107 cells mL−1) were counted at 0 M NaCl after 10 days, and no growth was observed at an NaCl concentration of 1.0–2.0 M. As shown in Figure 8, G. emersonii GEGS21 grew at temperatures between 5 and 40 °C, and the highest growth rate was determined at 28–32 °C and 130–160 µmol m−2 s−1 of light intensity (white LED). In conclusion, the strain thrived over a wide range of temperatures (5–40 °C) and withstood up to 0.5 M NaCl.

3.4. Fatty Acid Composition of Lipids and Their Biodiesel Properties

Gas chromatography/mass spectroscopy (GC/MS) analysis revealed the FAME profiles of G. emersonii GEGS21 (Table 5). The major FAME profiles of G. emersonii GEGS21 were C16:0 (27.5%), C18:1 n-9 (22.2%), C18:2 n-6 (26.3%), and C18:3 n-3 (22.1%) (Table 5). In addition, trace amounts of saturated fatty acids (SFAs, C18:0, 0.6%), polyunsaturated fatty acids (PUFAs, C18:3 n-6, 1.0%), and monounsaturated fatty acids (MUFAs, C20:1 n-9, 0.3%) were also detected (Table 5). Eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3) were not detected in the strain GEGS21 (Table 6).
Biodiesel properties, including SV, IV, DU, MUFA, LCSF, CFPP, CN, and OS, based on FAME profiles were calculated using the biodiesel property equations [58]. As shown in Table 7, G. emersonii showed relatively higher IV (124.79) and PUFA (49.40), and lower CN (46.25), MUFA (22.50), LCSF (3.05), and OS values (4.98) compared to other plants and microalgae. However, G. emersonii GEGS21 showed relatively lower IV than those of C. minutissima UTEX 2341 and Microglena monadina NFW3, and higher CN values than those of M. monadina NFW3, respectively. The biodiesel standards EN14214 and ASTM D6751-02 establish the quality of biodiesel suitable for diesel engines. EN14214 and ASTM D6751-02 had slightly different biodiesel standards. Specifically, while a suitable standard IV was established lower than 120 and CN was higher than 51 in EN14214, the ASTM D6751-02 standard does not include IV as a standard specification. Moreover, ASTM D6751-02 suggests an upper limit for CN of 47, and a suitable standard OS was established above 6 and 3 in the EN14214 and ASTM D6751-02 standards, respectively.

3.5. Analysis of Microalgal Pigment Profile

The pigment profiles of G. emersonii GEGS21 are listed in Table 8. The pigment compositions of the isolate were chlorophyll a (19.1 mg g−1 DW), lutein (1.49 mg g−1 DW), neoxanthin (1.23 mg g−1 DW), chlorophyll b (Chl b, 0.85 mg g−1 DW), β-carotene (0.84 mg g−1 DW), zeaxanthin (0.46 mg g−1 DW), and α-carotene (0.16 mg g−1 DW). Other minor peaks were not identified owing to the lack of available commercial standard reagents.

4. Discussion

Microalgal biomass has attracted increasing attention because it produces useful value-added compounds for bioenergy, cosmeceuticals, nutraceuticals, and pharmaceuticals via environmentally friendly bioprocesses [85]. Microalgae have a variety of advantages over other biodiesel feedstocks because they do not compete with food crops and exhibit faster growth rates that can result in considerably higher carbon sequestration rates than terrestrial plants. To investigate the potential bioenergy and other biotechnological applications, we identified and analyzed the biochemical properties of the indigenous algal isolate.
The genus Graesiella is a unicellular microalga comprising ellipsoidal and globose cells. The cell wall exhibited a meridional or irregular network of fine ribs on its surface. Ultrastructurally, the vegetative cells were uninuclear, single cup-shaped, and had parietal chloroplasts, each with one pyrenoid surrounded by starch plates. The cell wall was double-layered, with an inner polysaccharide and outer trilaminar. In this genus, asexual reproduction occurs through 2–16 autospores released via the rupture of the parental cell wall [34,60]. Morphological and physiological characteristics determined by light, fluorescence, and electron microscopy suggested that the isolate shared typical morphological and asexual reproduction characteristics with those of the genus Graesiella (Table 3). In addition, G. emersonii GEGS21 shared morphological characteristics with Maryland Culture Collection No. 2, CCMP421, and NIES-226 (Table 3). In particular, the morphological features of this isolate resembled those of strain NIES-226 as it had a single cup-shaped chloroplast, numerous vacuoles, more than two nuclei in mature cells, more than two daughter cells in parental cells, a pyrenoid matrix surrounded by starch grains, and a trilaminar sheath cell wall (Table 3). However, the cell wall of the strain NIES-226 does not always have meridional ribs on its surface [60], whereas most cells of the strain GEGS21 had meridional ribs (Figure 3a). It would be worthwhile to examine whether other G. emersonii strains have meridional ribs on the surface of most cells. The size of vegetative and daughter cells of the G. emersonii GEGS21 (8.79–14.4 µm and 5.0–12.7 µm, respectively) were similar to those of other strains of G. emersonii reported at 3.1–17.0 µm and 5.0–7.1 µm, respectively (Table 3).
The SSU rDNA sequence was also identical to that of the G. emersonii strains CCAP211/11N, BEA0616B, BEA1005B, NT1e, CCAP 211/15, CCAP 211/11M, CCAP 211/8H, and CCAP 211/8P (Table 4). Moreover, phylogenetic analysis confirmed that the strain GEGS21 is a member of G. emersonii (Figure 6). Based on the morphological and genetic results, this green alga was identified as G. emersonii.
Graesiella species have trilaminar sheaths, fibrillar walls, and plasma membranes in their cell walls [60,86]. G. emersonii GEGS21 shares these morphological characteristics. Afi et al. [63] reported the presence of a classical polysaccharide wall and a thin trilaminar outer wall composed of highly aliphatic, non-hydrolyzable macromolecules of G. emersonii. These polysaccharide walls are an important source for the paper, food, and textile industry, and for biofuel production as a by-product of biorefinery [87,88]. Thus, the cell wall of the GEGS21 strain can be used for biotechnological applications as a biorefinery by-product.
Prior to this study, G. emersonii strains were reported to inhabit mainly freshwater in Australia, China, Germany, Korea, India, Ireland, and Portugal [66,89,90,91,92,93,94]. In this study, many strains that inhabit freshwater were investigated (Figure 6, Table 4). However, the BEA1005B strain has been reported to inhabit brackish waters in Spain (GenBank accession no. ON652615), and the strain MM0036 reported by Jo et al. [28] also inhabited brackish waters (27.2 PSU) in Korea. In this study, the strain GEGS21 was isolated from brackish water (21 PSU) in Korea. Strain CCAP211/11M was isolated from the soil (Figure 6). Therefore, G. emersonii is likely to be distributed in a variety of habitats, such as soil, freshwater, and brackish water, and this newly isolated GEGS21 strain could serve as a good example of a cosmopolite species found in different water environments. Indeed, G. emersonii showed the ability to grow in a range of salinities and was supported by physiological measurements taken on culture CCAP 211/11N, which showed that this strain grew optimally at ~2.5‰, but also grew at ~10‰ [62]. In addition, the maximal growth of G. emersonii GEGS21 was obtained at 0 M NaCl, but it also grew at ~0.5 M NaCl. The wide range of salinity tolerance may enable G. emersonii to survive in a wide variety of water environments around the world. Moreover, the salinity adaptability of G. emersonii GEGS21 facilitates large-scale cultivation, as it can be used as a growth medium in freshwater or seawater. Mandal and Chaurasia [92] reported that the lipid content of G. emersonii could be improved depending on the salt concentration added to the culture. In this report on salinity stress conditions, the maximum enhancement in lipid content (i.e., 58 ± 2% DCW) was obtained after 18 days incubation period under 0.3 M NaCl supplemented conditions [92]. Therefore, owing to the tolerance of G. emersonii GEGS21 to various salinity ranges, it can be used as a potential microalgal strain for commercial biodiesel production. In addition, G. emersonii GEGS21 could grow at temperatures of 5–40 °C; the highest growth rate was determined at 28–32 °C. The Indian G. emersonii strain can grow at temperatures ranging from 25–42 °C, with the highest growth observed at 38 °C [41,95]. It seems that the wide range of temperature tolerance of this species, related to G. emersonii’s survival and presence in a wide variety of water environments around the world, could facilitate large-scale cultivation under outdoor conditions. In conclusion, G. emersonii GEGS21 is easy to cultivate and can serve as a potential biological resource for various applications.
Analysis of the major cellular fatty acid composition of the strain GEGS21 revealed that it is rich in palmitic acid (C16:0, 27.5%), oleic acid (C18:1 n-9, 22.2%), linoleic acid (C18:2 n-6, 26.3%), and α-linolenic acid (C18:3 n-3, 22.1%) (Table 5). Previous studies have reported that palmitic acid could be used as an ideal competent source for biodiesel production owing to its high CN. In addition, it is used in pharmaceuticals, cosmetics, lube oils, water proofing, and food-grade additives [96,97]. Owing to these industrial uses, the global palmitic acid market was valued at USD 21.19 million in 2020 and predicted to have a 2.48% annual growth by 2027 [98].
The percentage of C16:0 in G. emersonii GEGS21 (27.5%) was the highest among the reported G. emersonii strains, except for the G. emersonii strain Maryland Culture Collection No. 2 (28.3%) and was higher than that of the second-generation oil sources such as jatropha, karanja, mahua, and rapeseed (3.49–21.5%), except for palm (47.9%) (Table 6). In general, microalgae show higher photosynthetic efficiency and greater oil production than terrestrial plant sources [99]. Therefore, G. emersonii GEGS21 could be considered as a potential alternative plant source for biodiesel production. To compare the biodiesel quality of the isolated microalgae with that of terrestrial plants, the biodiesel properties were calculated based on the FAME profiles. It has been reported that the quality of biodiesel depends on FAME profiles [100,101]. Thereafter, the biodiesel quality parameters can be calculated from the FAME compositions, which provide information on the chain lengths and unsaturation, as shown in Table 7 [58]. In particular, several biodiesel properties, including IV, CN, and OS, are considered important for the application of diesel engines. The European standard EN14214 and American standard ASTM D6751-02 provide specific standard values for qualitative biodiesel. Compared with other terrestrial plants such as jatropha, karanja, palm, mahua, and rapeseed and microalgal strains including Chlamydomonas hedleyi MM0020, Chlorella gloriosa MM0063, Coelastrum microporum IBL-C119, Dunaliella salina LIMS-PS-1511, Haematococcus pluvialisa, Jaagichlorella luteoviridis MM0014, and Tetradesmus obliquus MM0026, G. emersonii GEGS21 had relatively higher IV and lower CN and OS values. However, G. emersonii GEGS21 represented lower IV than those of Chlorella minutissima UTEX 2341 and Microglena monadina NFW3 and higher CN values than those of M. monadina NFW3, respectively. As shown in Table 7, whereas the established upper limits of IV and the lower limits of CN and OS are 120, 51, and 6 in EN14214, respectively, the lower limits of CN and OS in the ASTM D6751-02 biodiesel standard are 47 and 3, respectively. In this study, most terrestrial plants showed suitable IV, CN, and OS (Table 7). IV is defined as the amount of iodine (g) in a specific biodiesel and is related to the number of double bonds that affect the DU, OS, and cold flow of the biodiesel [58,100]. CN is an important parameter that reflects engine performance, the generation of nitrous oxide, and the combustion of biodiesel [58,100]. Although the biodiesel properties of the isolated alga G. emersonii showed a slightly higher value of IV (124.79) and a lower value of CN (46.25) than the standard values (IV: ≤ 120; CN: ≥ 47), it is expected that the biodiesel produced from G. emersonii can be used as a blending resource for the transportation of diesel fuels as an alternative energy source because biodiesel blends with fossil diesel have been widely offered as automotive fuels worldwide [102]. However, further studies are required to promote properties and quality of microalgal biodiesel by applying different cultivation techniques and using other biotechnological parameters because microalgal fatty acid profiles largely depend on nutritional status and multiple environmental factors such as temperature, light intensity, pH, substrate, and carbon and nitrogen ratio [103]. Additionally, numerous studies have shown that essential PUFAs have various beneficial health effects [104]. Moreover, signaling molecules that regulate inflammation, cardiac function, and tumor growth are biosynthesized by PUFAs [105,106,107]. These properties indicate the potential of PUFAs for nutraceutical and pharmaceutical purposes, and a variety of commercial products containing these PUFAs are available worldwide. Humans, like other mammals, are unable or poorly able to synthesize essential PUFAs, such as linoleic acid and α-linolenic acid. Therefore, direct uptake of these compounds from external sources is necessary. These sources include fish, mollusks, crustaceans, meat, milk, eggs, plants, and microalgae [108,109].
The percentage of omega-6 (C18:2 n-6, 26.3%) of G. emersonii GEGS21 was the highest among the reported G. emersonii strains (4.0–22.0%) and was higher than that of other microalgae species (0.6–25.2%), except for the Chlorophyta Chlamydomonas oblonga YSL07 (37.0%), Jaagichlorella luteoviridis MM0014 (35.6%), and Auxenochlorella protothecoides MM0012 (29.4%) (Table 6). In addition, the percentage of omega-6 in G. emersonii GEGS21 was the highest among the second-generation oil plant sources, including karanja, palm, mahua, and rapeseed (9.07-22.3%), except for jatropha (42.1%) (Table 6). Omega-6 PUFAs are primarily derived from plant sources, such as sunflower, corn, and soybean oils, and a large number of commercial products are available worldwide. Therefore, this isolate has the potential to be used as an alternative to plant-based sustainable PUFA sources.
Pigment analysis revealed that the major carotenoids found in G. emersonii GEGS21 include the antioxidants neoxanthin and lutein (Table 8). The neoxanthin (1.23 mg g−1) and lutein (1.49 mg g−1) contents of G. emersonii GEGS21 were the highest among the reported G. emersonii strains (0.09-0.4 mg g−1, neoxanthin; 0.56-1.34 mg g−1, lutein) (Table 9). Lutein has certain beneficial pharmaceutical effects on human health owing to its strong ocular-protective, antioxidative, and anti-inflammatory properties [110,111]. Thus, the lutein market is growing rapidly, and the market size was worth about USD 288 million in 2020, predicted to have a 6.1% annual growth by 2027 [112]. Currently, potent commercial sources are mainly extracted from marigold flower petals [113], but the lutein content in marigold flowers is very low (0.3 mg g−1), and the extraction process requires several steps which results in low yields. Compared with marigold, the concentration of lutein in microalgae is generally higher (>0.3 mg g−1, Table 8). In addition, though lutein exists in green vegetables, some seeds (such as corn), and fruits, the content is low (up to ca. 0.4 mg g−1) [111], and therefore they cannot be used as substitute materials for large-scale production. Improved productivity can be achieved by evaluating the effects of various culture conditions, including media components, on G. emersonii GEGS21 growth. Therefore, G. emersonii GEGS21 has the potential to be used as an alternative lutein source.
Several microalgae species have great potential to produce healthy food and feed supplements. G. emersonii is used as a raw food material in European countries, such as Austria, Germany, and Switzerland, and is authorized as a food supplement in France [42,43]. There are cases where it has been approved as a food additive, so it is more likely to be approved as a food or food additive in other countries in the future. Hence, the G. emersonii strain GEGS21 may have the potential to be used as a raw food material and aid future commercial applications in European countries.
In this study, we provided a record of G. emersonii in Korea based on morphological and molecular data. The accurate identification of the isolate was achieved using our findings. In addition, this microalga could serve as a promising candidate for further phylogenetic studies in research fields as well as a potential biological resource to produce biofuels and biochemicals of commercial interest in industrial fields.

Author Contributions

Conceptualization, N.S.K., K.C. and J.W.H.; methodology, N.S.K., S.M.A., H.K., C.H.L. and E.S.K.; software, N.S.K. and S.M.A.; validation, K.C., S.M.A. and G.C.; investigation, H.K. and K.C.; resources, E.S.K., H.K. and C.H.L.; data curation, N.S.K. and K.C.; writing—original draft preparation, N.S.K.; writing—review and editing, J.W.H.; visualization, N.S.K. and K.C.; supervision, J.W.H.; project administration, K.C.; funding acquisition, K.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Development of Useful Materials Derived from Marine Microorganisms and Microalgae (2022M00600), funded by the National Marine Biodiversity Institute of Korea (MABIK). This study was also supported by the Project for Establishment and Operation of Marine Biobank funded by the MABIK.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Location and photographs of the sampling site in the Geumgang Estuary, Korea. (a) Map of the sampling site; location of site in the Geumgang Estuary. The Geumgang Estuary is located at the mouth of the Geum River on the west coast of Korea. (b) The surrounding environment of the Geumgang Estuary in Korea; image acquired using Google Earth. (c) The image shows the sampling site of Graesiella emersonii GEGS21.
Figure 1. Location and photographs of the sampling site in the Geumgang Estuary, Korea. (a) Map of the sampling site; location of site in the Geumgang Estuary. The Geumgang Estuary is located at the mouth of the Geum River on the west coast of Korea. (b) The surrounding environment of the Geumgang Estuary in Korea; image acquired using Google Earth. (c) The image shows the sampling site of Graesiella emersonii GEGS21.
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Figure 2. Light and epifluorescence micrographs of the vegetative and reproductive cells from Graesiella emersonii GEGS21. (a) Vegetative cells are green, unicellular, and grow in variable forms from spheroidal to ellipsoidal. A single pyrenoid (P), nucleus (N), and cup-shaped chloroplast (C) is visible. (b) Uninuclear cells were stained with DAPI and observed under fluorescence. The arrow indicates the nucleus (N). (c,d) Vegetative cells in different stages of development, autospores. Micrographs showing a nucleus (N) and pyrenoid (P). (e,f) Light (e) and epifluorescence (f) micrographs of several daughter protoplasts are formed in the parental cell wall. (g) Light micrograph of daughter cells released from the parental cell. The arrow indicates the ruptured parental cell wall. Micrographs showing a nucleus (N) and pyrenoid (P). Scale bars: (a–g) = 10 μm.
Figure 2. Light and epifluorescence micrographs of the vegetative and reproductive cells from Graesiella emersonii GEGS21. (a) Vegetative cells are green, unicellular, and grow in variable forms from spheroidal to ellipsoidal. A single pyrenoid (P), nucleus (N), and cup-shaped chloroplast (C) is visible. (b) Uninuclear cells were stained with DAPI and observed under fluorescence. The arrow indicates the nucleus (N). (c,d) Vegetative cells in different stages of development, autospores. Micrographs showing a nucleus (N) and pyrenoid (P). (e,f) Light (e) and epifluorescence (f) micrographs of several daughter protoplasts are formed in the parental cell wall. (g) Light micrograph of daughter cells released from the parental cell. The arrow indicates the ruptured parental cell wall. Micrographs showing a nucleus (N) and pyrenoid (P). Scale bars: (a–g) = 10 μm.
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Figure 3. Scanning electron micrographs of the strain Graesiella emersonii GEGS21 cells. (a) Various shapes and sizes of G. emersonii GEGS21. (b) Vegetative cell. (c) Parental cell (PC) and daughter cell (DC). Scale bars: (a) = 10 μm, (c) = 2 μm, (b) = 1 μm.
Figure 3. Scanning electron micrographs of the strain Graesiella emersonii GEGS21 cells. (a) Various shapes and sizes of G. emersonii GEGS21. (b) Vegetative cell. (c) Parental cell (PC) and daughter cell (DC). Scale bars: (a) = 10 μm, (c) = 2 μm, (b) = 1 μm.
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Figure 4. Scanning electron micrographs of the strain Graesiella emersonii GEGS21 cells. (a–c) Vegetative cell with fine ribs on the surface. The arrow indicates the fine ribs. (d,e) Parental cell with fine ribs on the surface. The arrow indicates the fine ribs. (f) SEM of the ruptured parental cell wall. (g) Enlarged SEM of (f), showing the outer layer of the parental cell wall. Scale bars: (c) = 3 μm, (f) = 2 μm, (a,b,d,e,g) = 1 μm.
Figure 4. Scanning electron micrographs of the strain Graesiella emersonii GEGS21 cells. (a–c) Vegetative cell with fine ribs on the surface. The arrow indicates the fine ribs. (d,e) Parental cell with fine ribs on the surface. The arrow indicates the fine ribs. (f) SEM of the ruptured parental cell wall. (g) Enlarged SEM of (f), showing the outer layer of the parental cell wall. Scale bars: (c) = 3 μm, (f) = 2 μm, (a,b,d,e,g) = 1 μm.
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Figure 5. Transmission electron micrographs of Graesiella emersonii GEGS21 cells. (a) Various shapes and sizes of G. emersonii GEGS21. (b) Micrograph showing a chloroplast (C), nucleus (N), pyrenoid (P), starch (S), and vacuoles (V). (c) TEM of vegetative cell showing two nuclei (N). (d,e) TEM showing the formation of autospores, two (panel d) and four (panel e) cells are visible. DB; electron-dense body. (f) TEM micrograph of cell wall structure shows two main layers: trilaminar sheath (TLS) and fibrillar wall. The plasma membrane is also visible. Scale bars: (a,c) = 2 μm, (b,d,e) = 1 μm, (f) = 0.5 μm.
Figure 5. Transmission electron micrographs of Graesiella emersonii GEGS21 cells. (a) Various shapes and sizes of G. emersonii GEGS21. (b) Micrograph showing a chloroplast (C), nucleus (N), pyrenoid (P), starch (S), and vacuoles (V). (c) TEM of vegetative cell showing two nuclei (N). (d,e) TEM showing the formation of autospores, two (panel d) and four (panel e) cells are visible. DB; electron-dense body. (f) TEM micrograph of cell wall structure shows two main layers: trilaminar sheath (TLS) and fibrillar wall. The plasma membrane is also visible. Scale bars: (a,c) = 2 μm, (b,d,e) = 1 μm, (f) = 0.5 μm.
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Figure 6. Maximum-likelihood and Bayesian inference phylogenetic tree based on the 18S rDNA sequence. The values on each node indicate maximum-likelihood bootstrap and Bayesian posterior probabilities (%), respectively. Bootstrap values lower than 50 and Bayesian posterior probabilities lower than 75 are not shown. * = 100. Our strain is shown in bold.
Figure 6. Maximum-likelihood and Bayesian inference phylogenetic tree based on the 18S rDNA sequence. The values on each node indicate maximum-likelihood bootstrap and Bayesian posterior probabilities (%), respectively. Bootstrap values lower than 50 and Bayesian posterior probabilities lower than 75 are not shown. * = 100. Our strain is shown in bold.
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Figure 7. Effect of salt (NaCl, M) supplementation on the density of Graesiella emersonii GEGS21.
Figure 7. Effect of salt (NaCl, M) supplementation on the density of Graesiella emersonii GEGS21.
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Figure 8. Matrix of growth rate (as shown in the legend) under different temperature and light regimes for Graesiella emersonii GEGS21.
Figure 8. Matrix of growth rate (as shown in the legend) under different temperature and light regimes for Graesiella emersonii GEGS21.
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Table
Table 1. Strain, location of collection (LC), water temperature (T, °C), salinity (S, PSU), and GenBank accession number (GBAN) for rDNA sequences of Graesiella emersonii GEGS21 isolated from Geumgang Estuary, Gunsan-si, Jeollabuk-do, Korea.
Table 1. Strain, location of collection (LC), water temperature (T, °C), salinity (S, PSU), and GenBank accession number (GBAN) for rDNA sequences of Graesiella emersonii GEGS21 isolated from Geumgang Estuary, Gunsan-si, Jeollabuk-do, Korea.
SpeciesStrainLCDateT (℃)S (PSU)Marker GeneGBAN
G. emersoniiGEGS21Geumgang
Estuary		May 202118.421SSUOP592224
ITSOP592225
LSUOP592226
rbcLOP605746
Table
Table 2. Primers used in this study to amplify the SSU, ITS, LSU region of rDNA, and the rbcL genes of Graesiella emersonii GEGS21.
Table 2. Primers used in this study to amplify the SSU, ITS, LSU region of rDNA, and the rbcL genes of Graesiella emersonii GEGS21.
Primer NamePrimer RegionSequence (5′-3′)References
EukAForward, SSUAACCTGGTTGATCCTGCCAG[50]
G18RReverse, SSUGCATCACAGACCTGTTATTG[51]
570FForward, SSUGTAATTCCAGCTCCAATAGC[52]
EukBReverse, SSUTGATCCTTCTGCAGGTTCACCTAC[50]
ITSF2Forward, ITSTACGTCCCTGCCCTTTGTAC[51]
ITSFR2Reverse, ITSTCCCTGTTCATTCGCCATTAC[51]
D1RForward, LSUACCCGCTGAATTTAAGCATA[53]
LSUBReverse, LSUACGAACGATTTGCACGTCAG[51]
rbcL-192Forward, rbcLGGTACTTGGACAACWGTWTGGAC[54]
rbcL-657Reverse, rbcLGAAACGGTCTCKCCARCGCAT[54]
Table
Table 3. Comparison of morphological and ultrastructural characteristics among Graesiella emersonii strains.
Table 3. Comparison of morphological and ultrastructural characteristics among Graesiella emersonii strains.
Character TraitsGEGS21NIES-226Maryland Culture Collection No. 2CCAP211/11N
Strain localityKoreaJapanUSAGermany
Cell shapeSpherical to ellipsoidalSphericalSpherical or ellipsoidalSpherical
Cell size (μm; vegetative cells)8.79–14.4 (11.6)~174–163–17
Cell size (μm; daughter cells)5.0–12.7 (6.9)5.4–7.1 (5.9) *NDND
PyrenoidPresent, surrounded by the starch grainsPresent, surrounded by the starch grains PresentPresent, surrounded by the starch grains
Numbers of nuclei (mature cell)More than two nuclei, sometimes remained singleMore than two nuclei, sometimes remained singleNDMore than two nuclei, sometimes remained single
Numbers of daughter cells (in parental cell)2, 4, 8, or 164, 8, or 16ND2, 4, 8, or 16
VacuolesNumerousNumerousNDND
Cell wallComposed of a trilaminar sheath and fibrillar wallComposed of a trilaminar sheathNDComposed of a trilaminar sheath
Cell wall ribsMost cells have meridional
or irregular network ribs		Lacking meridional ribs, but sometimes developing minute ribsNDND
References This study[60][61][60,62]
ND, information not available; * Not mentioned, but measured from figures. Mean values are shown in parentheses.
Table
Table 4. Comparison of small subunit rDNA sequence of Graesiella emersonii GEGS21 isolated from the Geumgang Estuary of Korea and other strains.
Table 4. Comparison of small subunit rDNA sequence of Graesiella emersonii GEGS21 isolated from the Geumgang Estuary of Korea and other strains.
Collection LocationStrain Habitat
(Isolation Source)		Strain NameGenBank Accession No.Graesiella emersonii
GEGS21 *
Berlin, GermanyFreshwaterCCAP 211/11NMK5417940 (0)
Gran Canaria, SpainFreshwaterBEA0616BON6526160 (0)
Cadiz, SpainBrackishBEA1005BON6526150 (0)
Tennant Creek, AustraliaFreshwaterNT1eKF2862730 (0)
EnglandFreshwaterCCAP 211/15FR8656610 (0)
NDSoilCCAP 211/11MFR8656570 (0)
NDFreshwaterCCAP 211/8HMG0227180 (0)
NDFreshwaterCCAP 211/8PFR8656870 (0)
Berlin-Dahlem, Germany FreshwaterNIES-2151AB4885622 (0.1)
Berlin, GermanyFreshwaterNIES-690AJ2427611 (0.2)
IndiaFreshwaterNDMN8777738 (0.9)
Mumbai, IndiaFreshwaterNDKJ7252337 (1.3)
* The numbers indicate the number of base pairs that differ from G. emersonii GEGS21. The numbers in parentheses indicate dissimilarity (%), including gaps. ND, information not available.
Table
Table 5. Lipid profile of Graesiella emersonii strain GEGS21.
Table 5. Lipid profile of Graesiella emersonii strain GEGS21.
ComponentContent (%)Note
Palmitic acid (C16:0)27.5SFA (major)
Stearic acid (C18:0)0.6-
Oleic acid (C18:1 n-9)22.2Omega-9 MUFA (major)
Linoleic acid (C18:2 n-6)26.3Omega-6 PUFA (major)
g-linolenic acid (C18:3 n-6)1.0-
α-linolenic acid (C18:3 n-3)22.1Omega-3 PUFA (major)
Eicosenoic acid (C20:1 n-9)0.3-
Table
Table 6. Fatty acid percentage comparisons of Graesiella emersonii strains, other microalgae, and selected second-generation oil sources. BACI, bacillariophyte (diatom); BIG, bigyra; CHL, chlorophyte; DHA, docosahexaenoic acid; DINO, dinoflagellate; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; HAP, haptophyta; OCH, ochrophyta; RHO, rhodophyta.
Table 6. Fatty acid percentage comparisons of Graesiella emersonii strains, other microalgae, and selected second-generation oil sources. BACI, bacillariophyte (diatom); BIG, bigyra; CHL, chlorophyte; DHA, docosahexaenoic acid; DINO, dinoflagellate; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; HAP, haptophyta; OCH, ochrophyta; RHO, rhodophyta.
SpeciesGroupStrainIndividual Fatty Acids as Percentages of Total Fatty AcidsReferences
Palmitic PalmitoleicStearicOleicLinoleicGamma-
Linolenic 		Alpha-LinolenicStearidonicEicosenoic EPADPADHA
C16:0C16:1 n-7C18:0C18:1 n-9C18:2 n-6C18:3 n-6C18:3 n-3C18:4 n-3C20:1 n-9C20:5 n-3C22:5 n-3C22:6 n-3
Graesiella emersoniiCHLGEGS2127.5-0.622.226.31.022.1-0.3---This study
G. emersoniiCHLCCAP
211/8P		191.62.353.84.86.7 *------[63]
G. emersoniiCHLMaryland Culture Collection No. 228.32-315.917 *-2.1----[64]
G. emersoniiCHLMAUA00118.7-4.1547.7413.72-------[40]
G. emersoniiCHLMM003616.80.91.717.610.7-27.2-----[28]
G. emersoniiCHLNFW220.933.410.198.44.01-45.04--16.39--[65]
G. emersoniiCHLNT1e18.792.392.0423.7911.0418.36--0.29---[66]
G. emersoniiCHLSXND-2514.86.53.244.510.112.9 *------[67]
Amphidinium carteraeDINOUTEX LB 100230.97.110.50.35.63.1 *---15.11.317[68]
Auxenochlorella protothecoidesCHLMM00127.111.83.829.4-21.9-----[28]
Chaetoceros affinisBACIB022633.93.22.81.3-0.8--0.6-0.1[69]
C. calcitransBACICS-I7810.7300.82.80.80.4-0.5-11.1-0.8[70]
Chlamydomonas hedleyiCHLMM002018.32.61.2-9.8-16.4-----[28]
C. oblongaCHLYSL0740-2337216-----[71]
Chlorella gloriosaCHLMM006322.50.412.210.6-------[57]
C. minutissimaCHLUTEX 234112.519.40.44.52.13.6 *---31.8--[72]
C. pyrenoidosaCHLSJTU-227.90.70.82.25.935.8 *------[73]
C. sorokinianaCHLMM003427.71.21.45.99.4-23.1-----[28]
C. vulgarisCHLCCAP 211/8K20.34.21.44.215.521.7 *------[63]
Coelastrum microporumCHLIBL-C11925.6612.9144.248.5811.12------[74]
Crypthecodinium cohniiDINOUTEX L164920.622.690.32.31.1 *----219.9[68]
Dunaliella salinaCHLLIMS-PS-151119.3-1.63.75.6-31.7-----[28]
D. tertiolectaCHLCS-I7514.70.10.424.82.743.51----[70]
Effrenium voratumDINOMABIKLP8822.19.30.73.30.60.90.315.2-10.9-25.4[75]
Haematococcus pluvialisaCHL-22.490.643.1519.3620.230.8616.18-0.130.57--[76]
Heterosigma akashiwoOCHQ0144.816.10.51.61.6-4.17.1-8.4-0.7[69]
Isochrysis galbanaHAPSW223.1832.13-.4.010.39-5.82--9.07-0.99[65]
Jaagichlorella luteoviridisCHLMM001420.7-1.47.135.6-16.2-----[77]
Microglena monadinaCHLNFW325.093.280.4-14.47-53.01-----[65]
Nannochloropsis oculataOCHCCAP 849/126.6538.122.429.14-----12.13--[78]
Phaeodactylum tricornutumBACIB24 A22.332.71.71.54.10.22.80.2-12.5-0.8[69]
Porphyridium purpureumRHOR0142.5-1.41.42.50.26.60.9-8.3-0.1[69]
Prorocentrum cordatumDINOS133.452.045.052.512.234.092.2812.337.32.91-20.87[79]
P. micansDINOD0661.90.63.23.20.7-4.53.3-0.1-2.8[69]
Schizochytrium aggregatumBIGATCC 2820915.318.6814.715.10.6 *---15.7--[68]
Scrippsiella acuminataDINOD0847.60.55.37.40.8-23.2-0.1-4.2[69]
Skeletonema costatumBACICS-18116.528.60.81.42.20.30.32.2-6-2[70]
Tetradesmus dimorphusCHLNT8c22.211.91.5924.456.2917.71--0.33---[66]
T. obliquusCHLMM00261821.316.45.2-28.3-----[28]
Tetraedron caudatumCHLNT57.161.430.466.133.4511.77------[66]
Tetraselmis chuiiCHLSW424.981.90.135.4312.85-29.96--18.01--[65]
T. suecicaCHLP03A36.80.9311.45-11.56.71.94.2-0.8[69]
T. marinaCHLP02D28.83.82.16.21.60.2168-5.8--[69]
Thalassiosira pseudonanaBACICS-17311.2180.70.50.40.20.15.30.219.3-3.9[70]
Second-generation oil sources
Jatropha--13.40.86.436.542.1-0.2-0.1---[80]
Karanja--7.4-3.865.615.44.4------[81]
Mahua--21.5-1939.119.60.16------[82]
Palm--47.90.044.23379.070.26------[83]
Rapeseed--3.49-0.8564.422.38.23------[84]
* Not clear whether it is alpha-linolenic acid (C18:3 n-3) or gamma-linolenic acid (C18:3 n-6).
Table
Table 7. Biodiesel properties calculated from the FAME compositions of the isolated algal strain and other crops, and biodiesel standard EN 14214 and ASTMD6751-02. SV, saponification value; IV, iodine value; DU, degree of unsaturation; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; LCSF, long-chain saturation factor; CFPP, cold filter plugging point; CN, cetane number; OS, oxidative stability.
Table 7. Biodiesel properties calculated from the FAME compositions of the isolated algal strain and other crops, and biodiesel standard EN 14214 and ASTMD6751-02. SV, saponification value; IV, iodine value; DU, degree of unsaturation; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; LCSF, long-chain saturation factor; CFPP, cold filter plugging point; CN, cetane number; OS, oxidative stability.
SourceSVIVDUMUFAPUFALCSFCFPPCNOS
Jatropha190.98105.42122.1037.3042.404.54−2.2151.165.37
Karanja184.0594.22105.2065.6019.802.64−8.1854.768.55
Mahua191.5867.7278.6239.1019.7611.6520.1259.558.56
Palm194.8248.0555.7037.049.336.915.2263.5015.23
Rapeseed188.61115.07125.4664.4030.530.77−14.0549.356.45
Graesiella emersonii GEGS21194.74124.79121.3022.5049.403.05−6.8946.254.98
Chlamydomonas hedleyi MM002095.6062.0955.002.6026.202.43−8.8489.427.09
Chlorella gloriosa MM0063132.19100.2685.002.6041.202.75−7.8465.035.45
Chlorella minutissima UTEX 2341142.74162.7998.9023.9037.501.45−11.9247.9123.28
Coelastrum microporum IBL-C119181.8382.6184.6445.2419.704.02−3.8457.738.58
Dunaliella salina LIMS-PS-1511121.2995.4178.303.7037.302.73−7.9069.835.75
Haematococcus pluvialisa162.7098.8695.8120.1337.843.82−4.4657.605.75
Microglena monadina NFW3188.54166.15138.243.2867.482.71−7.9737.864.34
Jaagichlorella luteoviridis MM0014157.60109.69110.707.1051.802.77−7.7756.254.87
Tetradesmus obliquus MM0026138.9298.6385.4018.4033.502.45−8.7863.406.11
EN14214-≤120----(≤−20~5)≥51≥6
ASTM D6751-02-------≥47≥3
Table
Table 8. Photosynthetic pigment composition of isolated algal strain Graesiella emersonii GEGS21.
Table 8. Photosynthetic pigment composition of isolated algal strain Graesiella emersonii GEGS21.
PigmentsRetention TimePeak AreaAmount (mg g−1)
Neoxanthin6.52483.041.23
Unidentified6.792154.9-
Lutein10.654152.391.49
Zeaxanthin10.86330.6930.46
Chlorophyll b14.21523.7840.85
Chlorophyll a15.643388.6519.1
α-carotene18.6618.7730.16
β-carotene18.87283.9270.84
Table
Table 9. Photosynthetic pigment composition comparisons of Graesiella emersonii strains and other microalgae. DCW: dry cell weight; NE: neoxanthin; VI: violaxanthin; AS: astaxanthin; MY: myxoxanthophyll; AN: antheraxanthin; LU: lutein; ZE: zeaxanthin; CA: canthaxanthin; EC: echinenone; AC: α-carotene; BC: β-carotene; CHA: chlorophyll a; CHB: chlorophyll b; FU: fucoxanthin; CHL: chlorophyte; BACI: bacillariophyte (diatom).
Table 9. Photosynthetic pigment composition comparisons of Graesiella emersonii strains and other microalgae. DCW: dry cell weight; NE: neoxanthin; VI: violaxanthin; AS: astaxanthin; MY: myxoxanthophyll; AN: antheraxanthin; LU: lutein; ZE: zeaxanthin; CA: canthaxanthin; EC: echinenone; AC: α-carotene; BC: β-carotene; CHA: chlorophyll a; CHB: chlorophyll b; FU: fucoxanthin; CHL: chlorophyte; BACI: bacillariophyte (diatom).
SpeciesGroupStrainPigment Composition in mg g−1 DCWReferences
NEVIASMYANLUZECAECACBCCHACHBFU
Graesiella emersoniiCHLGEGS211.23----1.490.46--0.160.8419.10.85-This study
G. emersoniiCHLCCNM10010.130.180.03000.890.340.06000.11.882.06-[114]
G. emersoniiCHLCCNM 10110.090.080.03000.560.170.1300.010.080.971.18-[114]
G. emersoniiCHLCCNM 10150.40.140.08001.340.590.1100.150.594.464.6-[114]
Botryococcus brauniiCHLUTEX572-----0.70.1--0.10.2---[115]
Bracteacoccus pseudominorCHLCCNM 10180.230.090001.160.38000.040.172.852.86-[114]
Chlorella minutissimaCHLMCC-27-----7.05--------[116]
C. sorokinianaCHLFZU60-----11.22--------[117]
C. variabilisCHLCCNM 10170.30.08000.071.130.360.0300.040.172.42.43-[114]
C. vulgarisCHLUTEX 265-----9.82--------[118]
Desmodesmus subspicatusCHLCCNM 10082.7300.3700.071.980.230.0400.040.266.67.21-[114]
Ettlia oleoabundansCHLUTEX 1185-----3.40.10.5-0.60.6---[115]
Monoraphidium minutumCHLCCNM 10420.590.430.2700.162.21.110.1500.120.530.846.66-[114]
Phaeodactylum tricornutumBACICCMP 1327-----2.10.10.6-0.11.6--24.3[115]
Tetradesmus dimorphusCHLCCNM 10454.130.020.0700.0220.540.0600.10.322.3710.79-[114]
T. obliquusCHLUTEX 2016-----1.61.60.3-0.20.4---[115]
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Kang, N.S.; Cho, K.; An, S.M.; Kim, E.S.; Ki, H.; Lee, C.H.; Choi, G.; Hong, J.W. Taxonomic and Biochemical Characterization of Microalga Graesiella emersonii GEGS21 for Its Potential to Become Feedstock for Biofuels and Bioproducts. Energies 2022, 15, 8725. https://doi.org/10.3390/en15228725

AMA Style

Kang NS, Cho K, An SM, Kim ES, Ki H, Lee CH, Choi G, Hong JW. Taxonomic and Biochemical Characterization of Microalga Graesiella emersonii GEGS21 for Its Potential to Become Feedstock for Biofuels and Bioproducts. Energies. 2022; 15(22):8725. https://doi.org/10.3390/en15228725

Chicago/Turabian Style

Kang, Nam Seon, Kichul Cho, Sung Min An, Eun Song Kim, Hyunji Ki, Chung Hyeon Lee, Grace Choi, and Ji Won Hong. 2022. "Taxonomic and Biochemical Characterization of Microalga Graesiella emersonii GEGS21 for Its Potential to Become Feedstock for Biofuels and Bioproducts" Energies 15, no. 22: 8725. https://doi.org/10.3390/en15228725

APA Style

Kang, N. S., Cho, K., An, S. M., Kim, E. S., Ki, H., Lee, C. H., Choi, G., & Hong, J. W. (2022). Taxonomic and Biochemical Characterization of Microalga Graesiella emersonii GEGS21 for Its Potential to Become Feedstock for Biofuels and Bioproducts. Energies, 15(22), 8725. https://doi.org/10.3390/en15228725

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