Microalgae hold promise as producers of sustainable biomass for the production of biofuels and other biomaterials. However, the selection of strains with efficient and robust production of desirable resources remains challenging. In this study, we isolated a green microalga from Korea and analyzed its morphological, molecular, and biochemical characteristics. Microscopic and phylogenetic analyses demonstrated that the isolate could be classified into the genus
, and we designated the isolate
sp. KIOST -1. Compositions of protein, lipid, and carbohydrate in the microalgal cells were estimated to be 58.8 ± 0.2%, 22.7 ± 1.2%, and 18.5 ± 1.0%, respectively. Similar to other microalgae belonging to Chlorophyceae, the dominant amino acid and monosaccharide in
sp. KIOST-1 were glutamic acid and glucose. On the other hand, the proportions of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids clearly differed from other species in the genus
, and monounsaturated fatty acids accounted for a large portion (41.3%) of the total fatty acids in the isolate. Based on these results,
sp. KIOST-1 has advantageous characteristics for biomass production.
Since the late 1950s, studies have shown that microalgaebased biofuels are a potential replacement of fossil fuels
. The use of microalgae for the production of various renewable biofuels such as bioethanol
, and biohydrogen
has been reported. Moreover, microalgae are considered as one of the most promising producers of other high-value compounds and nutritional supplements for humans and animals
. The lipid and fatty acid (FA) composition and biomass productivity of several eukaryotic microalgae have been evaluated for biofuels and/or biomaterials production, and certain species such as
have been successfully cultivated at commercial scales
. Numerous previous studies on microalgae-based biofuel (especially biodiesel) production relied heavily on these species, but these efforts have not always yielded sufficient results. Thus, there is a critical need for the selection of strains from natural environments or through genetic/metabolic engineering with improved scientific performance
Members of the genus
(Chlamydomonadaceae, Chlorophyceae, Chlorophyta) are commonly found in diverse ecological habitats and even under extreme conditions
. To date, a total of 432 species have been taxonomically classified in AlgaeBase (
, of which the genome is completely sequenced, is considered as a type species and is commonly used as a eukaryotic microalgae model for physiological, biochemical, and genetic research
species have been relatively well investigated for the production of biodiesel under various environmental conditions with different pretreatment methods
. Likewise, a number of studies on the use of
for improved production of bioethanol
, hydrogen gas
, and bioremediation of heavy metal pollutants
were recently reported. It is also important to note that
has been successfully cultivated at large scales, and that commercial utilization of its biomass (
, whole-cell dietary supplements, pharmaceutical proteins, and biofuels) is currently well established
In this paper, we report a new strain of
sp. KIOST-1) isolated in Korea. We investigated the morphological and molecular characteristics of the isolated microalga, and focused on its growth characteristics and biochemical composition in order to evaluate its potential for biofuel and other biomaterials production.
Materials and Methods
- Isolation and Culture Conditions of Microalga
A unicellular green microalga was isolated from a stagnant rainwater sample collected at the KIOST building rooftop (latitude 37°29’N, longitude 126°83’E) in March 2012. The collected water sample was serially diluted and incubated in 96-well plates at 30℃ under 100 µmol photons/m
/sec light intensity (12 h:12 h light:dark cycle) in BG-11 medium. Unialgal cells were streaked and cultured on BG-11 agar medium (1.5% agar) under the same conditions described above, and single colonies were repeatedly subcultured until a pure isolate was obtained. All subsequent analyses in this study were performed using microalgal cells cultured in BG-11 medium under the conditions described above.
- Morphological Identification
Morphological characteristics of the isolated microalga were investigated by light microscopy (LM) (Eclipse 80i; Nikon Co.). Images were taken using a camera (DXM 1200C; Nikon Co.), and the sizes of cells were calculated with an image analyzer (NIS-Elements BR 3.0; Nikon Co.). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for ultrastructural analysis of the microalga were performed as follows: cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, and post-fixed with 2% osmium tetroxide. The fixed material was dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%), dried with 100% hexamethyldisilazane, mounted on an aluminum stub, and lightly gold-coated for 90 sec in a Sputter Coater (SCD050; BAL-TEC, USA). Coated samples were examined directly under the Field Emission SEM (AURIGA; Carl Zeiss, Germany). For TEM analysis, cells were fixed, washed, post-fixed, and dehydrated as described above for the SEM procedure, and embedded in Spurr’s resin
. The samples were sectioned at 70 nm using an ultramicrotome (EM UC7; Leica Microsystems, Germany), stained with 2% uranyl acetate and lead citrate, and examined by TEM (JEM1010; JEOL Ltd., Japan).
- Molecular Identification and Phylogenetic Analysis
Genomic DNA of the microalga was extracted from a pure cultured single colony using the plant DNA isolation reagent (Takara, Japan), according to the manufacturer’s instructions. The isolated DNA was used as a PCR template to amplify the 18S rRNA gene or 18S-28S internal transcribed spacer (ITS) regions, including the D1/D2 region (ITS1-5.8S rRNA-ITS2-28S rRNA, hereinafter referred as ITS region). The 18S rRNA gene was amplified using NS1 (5’- GTA GTC ATA TGC TTG TCT C-3’), NS3 (5’-GCA AGT CTG GTG CCA GCA GCC-3’) and NS8 (5’-TCC GCA GGT TCA CCT ACG GA-3’), and the ITS regions were amplified using ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’), ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’), and LR3R (5’-GGT CCG TGT TTC AAG AC-3’). The amplified PCR products were cloned into pGEM-T Easy Vector (Promega), and both strands of the products were sequenced by Macrogen Inc. (Korea) using an ABI 377 Sequencer (Applied Biosystems). The obtained sequences were assembled as a single contig using the AlignX tool in the Vector NTI program (Invitrogen). The 18S rRNA and ITS regions were compared against the GenBank database at the National Center for Biotechnology Information using the BLASTN algorithm (
) and aligned with other relatives of the phylum Chlorophyta. Alignments were performed using Clustal X (ver. 1.83)
and edited using the BioEdit Sequence Alignment Editor (ver. 188.8.131.52)
. Phylogenetic analysis of the 18S rRNA gene was conducted using the neighbor-joining (NJ) tree with Jukes-Cantor distance matrixes and the maximumlikelihood (ML) method using the Hasegawa-Kishino-Yano model of nucleotide substitution. The ITS region was subjected to phylogenetic analyses involving NJ and maximum parsimony (MP) methods. The phylogenetic trees were reconstructed using Molecular Evolutionary Genetic Analysis (ver. 5.0) software
, and the reliability of the trees was accessed by performing 1,000 bootstrap replicates.
- Growth Characteristics
sp. KIOST-1 was cultivated in BG-11 medium for 9 days with gentle agitation at 30℃ under a 100 µmol photons m
light intensity (12:12 light and dark cycle). During cultivation, culture densities were measured based on direct cell counting using the Sedgwick-Rafter chamber, and the maximum specific growth rate (µ
) and doubling time (h) were calculated according to the method of Levasseur
. For chlorophyll-
analysis, 20 ml of culture samples were filtered through filter paper (GF/F; Whatman, Germany). The paper was then soaked with 5 ml of 90% acetone, sonicated for 1 min, and stored in a dark room at 4℃ for 24 h. The supernatant was centrifuged and analyzed using a UV/Vis spectrophotometer (Optizen POP; Mecasys, Korea) at specific wavelengths (664 and 630 nm) based on the method of Jeffrey and Humphrey
- Cellular Composition and Fatty Acid Profile
The proportions of the major components (carbohydrate, lipid, and protein) in lyophilized microalgal cells were determined according to the methods of the AOAC
. Crude lipid and protein contents were determined using the Soxhlet method (official method 920.39) and the Kjeldhal method (official method 976.05), respectively. The amino acid profile of the isolate was determined using an amino acid analyzer (Sykam S433; Sykam, Germany) with the ninhydrin method, and the monosaccharide composition was determined using high-performance anion-exchange chromatography coupled with a pulsed amperometric detection system (HPAEC-PAD; Dionex Co., USA). Lipid in the isolate was extracted three times with a mixture of dichloromethane and methanol (1:1 (v/v)) by the Bligh and Dyer procedure
. Internal standard (
-nonadecanoic acid; Sigma-Aldrich Co., USA) was added to the samples, and the remaining solvent was dried under the presence of N
gas. The extracted lipids were subjected to alkaline hydrolysis using 0.5 N KOH with heating at 70℃ for 30 min. After cooling at room temperature, the lipid fraction containing the FAs was separated following acidification to pH 2, dried under nitrogen gas, and then converted to the FA methyl ester (FAME) using boron trifluoride methanol (BF
-MeOH), with heating at 70℃ for 30 min. FAMEs were isolated by partitioning into hexane: diethyl ether (9:1). A subsample of FAMEs was treated with dimethyl disulfide to determine the position of double bonds in unsaturated FAs
. FAMEs were quantified using gas chromatography (GC; Agilent 7890A; Agilent Technologies, USA) equipped with a flame ionization detector with a ZB-5MS column (60 m × 0.32 mm × 0.25 µm; Phenomenex, USA) and helium as the carrier gas. The samples were injected in split-less mode at an initial oven temperature of 50℃, an injector temperature 250℃, and a detector temperature of 320℃. The oven temperature was ramped at 10℃/min to 120℃ and 4℃/min to a final temperature of 300℃. Individual FAs were identified using a gas chromatography-mass spectrometer detector (GC-MSD; Agilent 7890A GC-Agilent 5975C MSD; Agilent Technologies, USA) operating at 70 eV with a mass range acquisition of 50–700 amu. The column and its setting conditions for GC-MSD were the same as those of GC-FID.
- Nucleotide Sequence Accession Number and Strain Deposition
The 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and 28S rRNA gene sequences of
sp. KIOST-1 were deposited in GenBank under the accession number JX911252. A living axenic culture of
sp. KIOST-1 was deposited in the Korean Collection for Type Cultures (KCTC) at the Korean Research Institute of Bioscience and Biotechnology under the accession number KCTC 12379BP.
Results and Discussion
- Cell Morphology and Ultrastructure
In LM and SEM analysis, biflagellate vegetative cells with ellipsoidal or spherical shapes (4–6 µm in diameter) were predominantly observed, with cells in the zygote (11–14 µm in diameter) or palmella (4–7 µm in diameter) stages occasionally appearing in the culture. Vegetative cells had an oval-shaped single pyrenoid embedded within the chloroplasts and possessed a pair of flagella (8–10 µm in length and 0.2 µm in width) on each side of the basal body (
). Cellular structures of the isolate were further investigated using TEM, and several cellular organs such as the nucleus, golgi apparatus, chloroplast, pyrenoid, mitochondria, starch granule, and lipid droplets were observed. The nucleus, which was surrounded by golgi apparatus, was typically observed at the center of the cell. The cup-shaped chloroplasts, which consisted of several thylakoids, were located on the edge of the cell and occupied the majority of the inner cellular matrix, and the oval-shaped pyrenoid was surrounded by large starch granules. During asexual reproduction, biflagellate vegetative cells divided into two zoospores, and the sporangia in the palmella stage usually possessed four zoospores enclosed within the parent cell wall. Several relatively large starch granules were observed within cells in the dividing stages, whereas rounded lipid droplets were mostly found in the cytosol of vegetative cells. Furthermore, detailed structures of the anterior flagellum of the microalga were also investigated. In the cross-section of the flagellum, nine doublet microtubules and a central pair of microtubules representing the well-characterized “9+2” structure
were found in the axoneme. A pair of flagella was respectively connected to the two basal bodies, which were anchored to the cytoplasm by flagella roots and their microtubular and fibrous components (
Light micrograph of Chlamydomonas sp. KIOST-1 in BG11 medium. Pyrenoid (black arrow) and eye-spot (white arrow) were visible in the microalgae cells. (A, B) Scanning electron micrographs of biflagellated Chlamydomonas sp. KIOST-1 (vegetative cell) and (C) highmagnification view of the flagella (D).
Transmission electron micrographs of ultrasectioned Chlamydomonas sp. KIOST-1 cells. (A, B) General ultrastructure. (C, D) Ultrastructural images of microalgal cells during asexual reproduction stage. Several relatively large starch granules were found from the dividing cells. (E) Ultrastructural image of the vegetative cell. Several lipid droplets were scattered throughout the cell. (F) General ultrastructure of a pair of flagella possessed by the cell. (G) High-magnification view of the microalgal flagellum with verticalsection and (H) cross-section. A total of nine doublet microtubules (black arrow) and a central pair of microtubules (white arrow) were observed. c, Chloroplast; p, Pyrenoid; m, Mitochondria; g, Golgi apparatus; s, Starch granule; l, Lipid droplet; f, Flagella; mt, Microtubules; n, Nucleus; cm, Central pair microtubules; dm, Doublet microtubules; fr, Flagellar roots; bb, basal body.
- Molecular Identification and Phylogenetic Analysis
The obtained 18S rRNA (1,717 bp) and ITS region (626 bp) of the isolate were analyzed against the GenBank database. According to the BLASTN search using the 18S rRNA as query, the closest related species was
CCAP 11/77 (GenBank Accession No. FR865613) with 97.7% sequence identity, followed by
CCAP 11/31 (GenBank Accession No. FR865573) with 97.4%,
CCAP 11/55A (GenBank Accession No. FR865592) with 97.2%, and
SAG 48.72 (GenBank Accession No. U70790) with 97.0% identity. Our isolate also showed similarity (95.9%) to
CCAP 11/32C (GenBank Accession No. FR865575), which is known as the type species in the genus
. In addition, the ITS region was compared with other ITS region sequences of
species in GenBank, and was most similar to
CCAP 11/77 (GenBank Accession No. FR865613) with 84.2% sequence identity, followed by
C. rapa f. vasta
CCAP 11/73 (GenBank Accession No. FR865611) with 83.9% and
CCAP 11/55A (GenBank Accession No. FR865592) with 83.5%. Similar to the 18S rRNA sequence comparison results, the ITS region showed similarity to the type species
CCAP 11/32C (GenBank Accession No. FR865575) with 80.2% identity. Based on these results, the newly isolated unicellular green microalga was classified into the genus
, and was designated as
According to recent molecular phylogenetic analyses, the genus
is highly polyphyletic and thus there is a need for reclassification
. To determine the precise phylogenetic position of our isolate, detailed phylogenetic analyses were conducted using a data set of twenty-six 18S rRNA and sixteen ITS region sequences derived from the genus
. Our resultant tree using the ML method also suggested that the genus
was polyphyletic as reported previously
, and could be divided into three major groups (groups I–III) (
A). Group I was further divided into two subgroups; subgroup I contained type species
CCAP 11/32C and other species such as
SAG 7 73,
SAG 26 72, and
UTEX1341, and its sister subgroup II contained
CCAP 11/55A, and
CCAP 11/7. In subgroup II,
sp. KIOST-1 was most closely related to
CCAP 11/31 and
CCAP 11/55A; however, it clearly formed different lineages from the related species. Similar results were obtained based on the phylogenetic trees inferred using the NJ method (data not shown). Phylogenetic analysis of ITS regions using the MP method clustered
sp. KIOST-1 together with the
CCAP 11/77 strain, but the isolate formed a separate lineage, showing divergence (
B). In addition, a similar tree topology was obtained when inferred using the NJ method (data not shown). Based on these results,
sp. KIOST-1 was clearly differentiated from other related species in the genus
Phylogenetic analyses of 18S rRNA and ITS region sequences from Chlamydomonas species. (A) Maximum-likelihood tree reconstructed using a data set of twenty-three 18S rRNA sequences derived from the genus Chlamydomonas. Members of the genus Chlamydomonas were distributed in all three major groups, and group I was divided into two subgroups. The type strain, C. reinhardtii (FR865575), was included in subgroup I, and Chlamydomonas sp. KIOST-1 (JX911252) is indicated in bold in subgroup II. Scenedesmus acutus UTEX 72 (AJ249508) was used as an outgroup. The scale bar indicates the number of nucleotide substitutions. (B) Maximum-parsimony tree reconstructed using a data set of sixteen ITS region sequences derived from the genus Chlamydomonas. The determined ITS region sequence of Chlamydomonas sp. KIOST-1 (JX911252) is indicated in bold in group II. Scenedesmus acutus UTEX 72 (AJ249508) was used as an outgroup. The scale bar indicates the number of nucleotide substitutions.
- Growth Characteristics
Different from the most common reference strains of
(CC-620 and CC-621), which grow very poorly in BG-11 medium
sp. KIOST-1 could be cultivated well on BG-11 medium in microwell plate, and therefore, the growth rate was determined using 9-day continuous cultures in BG-11 medium. When the fresh cultures of microalgal cells (approximately 1.7 × 10
cells/ml) were inoculated into BG-11 medium, the numbers of cells were exponentially increased within 5 days after inoculation (up to approximately 5.5 × 10
cells/ml) and retained its growth during the rest of the culture period, whereas the chlorophyll-
contents increased continuously during 9-day culture (
). The maximum specific growth rate of
sp. KIOST-1 was observed during the 0 to 2-day culture period, and its value was estimated to be 1.95/day. Moreover, the doubling time of
sp. KIOST-1 was calculated to be 8.5 h Therefore, the newly isolated
sp. KIOST-1 has advantageous characteristics for biomass production.
Variation of cell density and chlorophyll-a content of Chlamydomonas sp. KIOST-1 cultured in BG-11 medium.
- Cellular Composition and Fatty Acid Profile
To evaluate the potential of
sp. KIOST-1 as a microalgal bioresource, the proportions of cellular components were determined. The overall compositions of crude cellular components in
sp. KIOST-1 were similar to those reported in other green microalgae belonging to the class Chlorophyceae, including
). In the lyophilized microalgal cells, the carbohydrate, lipid, and protein contents were measured to be 18.5 ± 1.0%, 22.7 ± 1.2%, and 58.8 ± 0.2%, respectively. Several microalgal species such as
have been used as dietary supplements, and their protein contents are considered as a major factor determining their nutritional value
. In this study,
sp. KIOST-1 also contains a relatively high proportion of protein, making it a potential candidate for use as dietary supplements for human and/or animal consumption, similar to other commercially utilized
species to date
Comparison of cellular components in lyophilizedChlamydomonassp. KIOST-1 andChlamydomonasreinhardtii.
Comparison of cellular components in lyophilized Chlamydomonas sp. KIOST-1 and Chlamydomonas reinhardtii.
We also examined the compositions of amino acids, monosaccharides, and FAs in
sp. KIOST-1 (
). The total amino acid content in
sp. KIOST-1 was composed of 47.5% essential amino acids. The dominant amino acids were glutamic acid (11.6%), aspartic acid (9.9%), and leucine (9.8%), whereas methionine and cysteine were not detected. D-Glucose (49.7%) and D-galactose (21.4%) were the dominant types among the seven monosaccharides ( L-fucose, L-rhamnose, D-arabinose, D-galactose, D-glucose, D-mannose, and D-xylose). These results were in agreement with a previous report
, which showed that the dominant amino acid and monosaccharide in Chlorophyceae were glutamic acid and glucose, respectively.
Compositions (%) of amino acids in lyophilizedChlamydomonassp. KIOST-1.
Asp: aspartic acid; Thr: threonine; Ser: serine; Glu: glutamic acid; Gly: glycine; Ala: alanine; Cys2: cystine-cystine dimer; Val: valine; Met: methionine; Ile: isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: histidine; Lys: lysine; NH4: Ammonia; Arg: arginine; Pro: prolin. aNot detected.
Compositions (%) of monosaccharides in lyophilizedChlamydomonassp. KIOST-1.
Compositions (%) of monosaccharides in lyophilized Chlamydomonas sp. KIOST-1.
The total FAs in
sp. KIOST-1 were composed of saturated fatty acids (SFAs) (27.9%), MUFAs (41.4%), and polyunsaturated fatty acids (PUFAs) (30.7%). The detailed FAs compositions in the microalga were further examined and compared with other species in the genus
. Similar to a previous report on
, lipids in
sp. KIOST-1 contained mainly hexadecanoic and octadecanoic FAs such as C16:0, C16:4ω3, C18:1ω9, and C18:2ω6, and only traces of other FAs such as C14:0, C16:1ω7, C16:1ω9, C16:2ω6, C16:3ω3, C18:0, C18:1ω7, C18:3ω3, C20:1ω9, C20:4ω6, and C20:5ω3 (
). Different from Prasinophyceae, microalgae belonging to Chlorophyceae are known to lack C20 and C22 PUFAs
, and microalgal FA compositions are considered to be related to its systematics
. However, similar to the report of An
, the isolate classified into the genus
distinctly contained C20 PUFAs (C20:4ω6 and C20:5ω3) as its fatty acid components, thus indicating that FA compositions are not related to microalgal systematics, at least in Chlorophyceae.
Interestingly, the composition of FAs in
sp. KIOST-1 clearly differed from the
strain, which mainly possessed SFAs and PUFAs as its dominant FAs (
KNUA021, SFA (C16:0, 16.3%) and PUFAs (C18:2ω6, 16.1%; and C18:3ω3, 19.9%) were dominant
. On the other hand, MUFA (C18:1ω9, 33.1%) was detected as the major FA in
sp. KIOST-1, and the sum of SFAs and PUFAs was much lower than in
KNUA021. Owing to the distinct FAs composition in
sp. KIOST-1 cultured in BG-11 medium, the expected biofuel produced from this new microalga will have strong potential as a microalgae-based biodiesel due to the following three characteristics. First, the pour and cloud points of manufactured biodiesel by the isolate will be increased owing to its relatively low level of SFAs compared with other
species. Second, fuels rich in these FAMEs will have an adequate cetane number, cold-flow parameters, and viscosities. The ideal biodiesel feedstock would be composed entirely of monounsaturated methyl octadecenoate (C18:1) and methyl hexadecenoate (C16:1)
; the composition of MUFAs in
sp. KIOST-1 was significantly higher than other
species studied for biodiesel production, and the majority of its MUFAs were composed of C16:1 and C18:1 FAs. Lastly, the oxidative stability of its manufactured biodiesel will be increased owing to the relatively low levels of PUFAs in
sp. KIOST-1. Moreover, MUFAs have been recently considered an attractive and alternative food source that can reduce the level of total and low-density lipoprotein cholesterol
, and can also decrease the incidence of the metabolic syndrome and cardiovascular disease
. Specifically, oleic acid (C18:1ω9), which can regulate the signaling pathway of the adrenoreceptor, is known to be responsible for blood pressure reducing effects
, and 80.1% of the MUFAs in
sp. KIOST-1 is composed of oleic acid.
Based on these results, it can be suggested that the newly isolated
sp. KIOST-1 has considerable potential as a microalgal bioresource due to its relatively high accumulation of total protein and MUFAs compared with other reported microalgal species, including the genus
Comparison of fatty acid compositions (%) in lyophilizedChlamydomonassp. KIOST-1 and another microalga belonging to the genusChlamydomonas.
This study was supported by a research grant from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries of Korean Government (PM58220), and also partially supported by a research grant funded by the Korea Institute of Ocean Science and Technology (PE99213).
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