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Enhanced production of carotenoids using a Thraustochytrid microalgal strain

containing high levels of docosahexaenoic acid-rich oil

Article in Bioprocess and Biosystems Engineering · September 2018

DOI: 10.1007/s00449-018-1963-7

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Bioprocess and Biosystems Engineering
https://doi.org/10.1007/s00449-018-1963-7

RESEARCH PAPER

Enhanced production of carotenoids using a Thraustochytrid

microalgal strain containing high levels of docosahexaenoic acid-rich
oil
Hansung Park1 · Minsoo Kwak2 · JeongWoo Seo3 · JeongHyun Ju3 · SunYeon Heo3 · SeungMoon Park1 ·
WonKyung Hong1

Received: 9 March 2018 / Accepted: 4 June 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Results to date suggest that microalgal Thraustochytrids family strains can be used to produce high-functional omega-3
rich oil (~ 30–70% of dry cell weight) and carotenoid-based antioxidant pigments simultaneously with value-added bioac-
tive potential. In the present study, we describe the isolation and characterization of a new Thraustochytrid Schizochytrium
sp. from the west coastal area of Korea. This newly isolated Thraustochytrid, identified as Schizochytrium sp. through 18S
rRNA analysis and named SH104, simultaneously produces high levels of DHA and carotenoid-based antioxidant pigments.
An improved Schizochytrium mutant, named SHG104, was obtained from the original host strain by γ-irradiation-induced
mutagenesis. Under combined temperature-shift cultivation conditions employing white-light LEDs (light-emitting diodes),
Schizochytrium sp. SHG104 yielded 10.8 g L ­ −1 of biomass comprising 45.8% total lipids (32.1% DHA) and 4.6 mg L ­ −1 of
astaxanthin. In addition to DHA, the main fatty acids produced by Schizochytrium sp. SHG104 were palmitic acid and a trace
of other long-chain fatty acids. The carotenoid profile of SH104 and SHG104 was β-carotene, astaxanthin, canthaxanthin,
pheonicoxanthin and echinenone, which analyzed by HPLC and LC/APCI–MS. Furthermore, genomic analysis of Schiz-
ochytrium and Aurantiochytrium microalgae confirmed that the presence of carotenogenesis pathway enzymes and genes
including geranylgeranyl diphosphate, phytoene synthase, lycopene cyclase, and cytochrome P450 hydroxylase that necessary
for the production of antioxidants via a complete biosynthetic KEGG synthesis pathway. This newly isolated Schizochytrium
microalga potentially have wide application as a source of antioxidants for astaxanthin-containing pigments, commercial
omega-3 lipids and feed additives, such as nutritional supplements for aquaculture.

Keywords Thraustochytrid Schizochytrium · Antioxidant · Astaxanthin · DHA · Carotenogenesis

Introduction

Microalgae are found in diverse habitats, including seas,

Hansung Park and Minsoo Kwak are co-first authors and ponds, rivers, coastal areas and polluted waters [1]. Thraus-
contributed equally. tochytrids are common marine heterotrophs, taxonomically
SeungMoon Park and WonKyung Hong are co-corresponding
classified with stramenopile (or heterokont) microalgae
authors and contributed equally. (photosynthetic protists) [2]. These microalgae are oleagi-
nous microorganisms well known for their production of
Electronic supplementary material The online version of this omega-3 fatty acids such as docosahexaenoic (DHA) and
article (https​://doi.org/10.1007/s0044​9-018-1963-7) contains
eicosapentaenoic (EPA) [3]. Thraustochytrids microalgae,
supplementary material, which is available to authorized users.
which are distributed widely, are now established candidates
* SeungMoon Park for commercial production of omega-3 polyunsaturated
[email protected] fatty acid (PUFA) and DHA, which are important in human
* WonKyung Hong health and aquaculture [4–8]. Thraustochytrid microalgae,
[email protected] the main commercial source of PUFA, have very simple
Extended author information available on the last page of the article PUFA profiles compared with other microalgal and fish oils

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investigated to date. Because of this simplicity, the use of sp. SH104 from the west coastal area of Korea and generated
Thraustochytrid-derived oils can reduce the cost of manu- γ-irradiation-induced mutant named SHG104 to increase
facturing high-purity microbial PUFA oils. With the wide- carotenoid production. We demonstrate the potential of this
spread recognition of the benefits of PUFA for human health heterotrophic eukaryotic microalga as a biofactory for the
and nutrition, the applications of microbial DHA and EPA large-scale production of omega-3 fatty acids and carote-
are rapidly expanding. noid-based antioxidants.
These microorganisms are also potential sources of other
beneficial products such as enzymes, polysaccharides, and
secondary metabolites [9, 10]. It has recently been reported Materials and methods
that Thraustochytrid microalgae also produce carotenoid
pigments, as bioactive substances in addition to omega-3 Isolation of Schizochytrium sp. strains
oils. Carotenoids are widespread yellow–red pigments, oxi-
dation or cyclization form of C40 tetraterpenes which syn- Thraustochytrid strains were obtained from soil, leaf, and
thesized from eight C5 isoprenoid units, are easily found in pneumatophore samples of a marine mangrove ecosystem.
nature including bacteria, yeasts, molds, green plants, and The Thraustochytrid family Schizochytrium sp. SH104
many animals. As natural pigments, carotenoids are used as strain was isolated from a coastal ecosystem in Southwest-
food additives, cosmetics, and pharmaceutical products for ern Korea. Screening was carried out by adding 10 mL of
antioxidants. Thus, in addition to producing PUFAs, Thraus- physiological saline to the sample and serially diluting the
tochytrids are a promising source of carotenoid-based anti- supernatant and plating on B1 solid media (1 g L−1 yeast
oxidant pigments [11, 12] such as astaxanthin, zeaxanthin, extract, 1 g L−1 peptone, 10 g L−1 agar, 300 mg L−1 penicil-
canthaxanthin, echinenone, pheonicoxanthin, and β-carotene lin G and 500 mg L−1 streptomycin sulfate in 1 L natural sea-
[13]. Canthaxanthin and astaxanthin, which have chemo- water) [13, 19]. Microalgae were further enriched by inocu-
protective properties, are representative antioxidants with lating samples into 50 mL of B1 culture media, incubating
potential for use as food additives; they also have been at 28 °C with shaking at 200 rpm for 3 days, followed by
reported to possess activity that suppresses the development serially diluting the culture broth and plating on B1 media.
of neurodegenerative disorders [14]. Because of its supe- After 2–4 days of incubation at 28 °C, numerous colonies
rior antioxidant properties, astaxanthin has been reported to appeared on the plates. Single, irregular, hyaline colonies
exhibit anticancer, anti-obesity, anti-diabetic, anti-inflam- were picked and cultivated on B1 plates to obtain axenic cul-
matory, and cardioprotective activities [15]. β-Carotene, a tures; each colony was further observed under a light micro-
precursor of vitamin A, helps prevent diseases caused by scope. Single colonies of screened isolates were inoculated
a vitamin A deficiency, including blindness, immune dys- into 15 mL of marine broth (Sigma-Aldrich, St. Louis, MO,
function, and skin disorders [15]. More than 80% of com- USA) or basal medium (50 g L−1 glucose as carbon source,
mercially used β-carotene is synthesized through chemical 10 g L−1 yeast extract as nitrogen source, 5 g L−1 artificial
processes. However, carotenoids produced via microorgan- sea salt, and 9 g L−1 ­KH2PO4 (Sigma-Aldrich, St. Louis,
isms are widely accepted and considered a safe drug com- MO, USA) and cultured at 28 °C with shaking (120 rpm)
ponent. The marine microalga Dunaliella sp. can produce for 3 days. Following incubation, 1 mL of pre-culture cells
β-carotene, but has a number of disadvantages including low were inoculated into 50 mL of the same media and incubated
production efficiency, high cost, the requirement of land for at the same condition. The cultured biomass was used for
culture, limits of mass culture, and poor production control characterization of fatty acid composition and estimation of
[16]. Thraustochytrids are a good alternative to Dunaliella dry cell weight (DCW) and lipid content.
sp. for the production of carotenoid-based pigmented anti-
oxidant that circumvents these shortcomings [17]. γ‑irradiation‑induced random mutagenesis
Recent studies have shown that some Thraustochytrid of Schizochytrium sp. strains
strains can be industrially cultured to produce high yields of
biomass containing substantial amounts of omega-3 oil and To isolate strains with improved antioxidant pigment pro-
antioxidants. The applications of microbial marine omega-3 duction, we induced mutations by irradiating the isolated
oils and carotenoid-based antioxidant pigments for human Schizochytrium sp. SH104 parent strain with γ-rays. For
health are expanding rapidly, and their efficacy has been γ-radiation-induced mutagenesis, strain SH104 was cultured
demonstrated in a large number of clinical trials [18]. The in 100 mL of basal medium in a 500 mL flask for 48 h with
development of an efficient large-scale cultivation system shaking at 120 rpm. The 50 mL of culture was transferred
for the commercial production of omega-3 fatty acids and to a 50 mL Falcon tube and centrifuged at 3500g at 4 °C.
carotenoid-based antioxidants would address a major global The cell pellet was recovered and suspended in 10 mL of
need. Here, we isolated the newly identified Schizochytrium phosphate-buffered saline (PBS), and then irradiated with

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Bioprocess and Biosystems Engineering

γ-rays at a dose of 1–3 kGy for 1 h. After irradiation, the Accession Number KX379459. A phylogenetic tree was
suspension was diluted by 1­ 0−6, plated on a basal solid constructed and analyzed by the neighbor-joining method,
medium (adding 15 g L−1 agar) and cultivated at 28 °C for with a gamma distribution parameter of 1.0 and replicated a
2 days. The remaining strains was cultured in 50 mL of basal number of 100. To calculate the distance of matrix nucleo-
medium in a 250 mL flask for 2 days at 28 °C with shaking tide positions, containing gaps or missing data were deleted
at 120 rpm. The death rate was determined by counting the completely using CLC Main Workbench version 7.7.1 (Qia-
number of colonies on each plate after culturing the irradi- gen Bioinformatics, Hilden, Germany). The tree was rooted
ated (2 kGy) SH104 strain for 2 days; the death rate was using Schizochytrium sp. SH104 (GenBank Accession No.
confirmed to be greater than 99% compared with the non- KX379459). A total of 1781 bases of 18S rRNA were used
irradiated SH104 strain. After culturing with shaking for for the phylogenetic analysis.
2 days, the irradiated SH104 strain was diluted appropriately
and cultured on a basal solid medium. The stained plates Cell growth analysis
were cultured in an incubator under conditions of fluorescent
light illumination, after which orange-colored colonies were The 2 mL of cryopreserved cells were initially inoculated
selected and cultured in flasks. Mutant strains with improved into basal medium and pre-cultured for 2 days at 28 °C with
performance were selected from among the single colonies shaking at 130–165 rpm. As inexpensive alternatives to glu-
and named Schizochytrium sp. SHG104. cose, cane molasses purchased from a local company and
corn steep liquor (CSL) were tested as carbon and nitrogen
Cloning and sequencing of the 18S rRNA gene sources, respectively. Fermentation conditions were 28 °C,
shaking at 200 rpm with 1 v/v/min of air, and pH 6.8. Cul-
This novel oleaginous Thraustochytrid strain was identi- tures were inspected for contamination by microscopic
fied as Thraustochytrid family Schizochytrium sp. SH104 examination. The pre-culture was transferred to the main
strain through 18S rRNA analysis. The genomic DNA of culture with initial optical density of 0.1 at 600 nm ­(OD600).
the selected strain was extracted using a PureLink Genomic These cells were cultured in basal medium for 4–8 days at
DNA kit (Invitrogen, Waltham, MA, USA) according to the 28 °C with shaking at 165 rpm, with or without illumina-
manufacturer’s protocol. A DNA sequence containing the tion, with periodic measurement of OD, DCW, and carote-
18S rRNA gene was amplified using the forward primer P1 noid-based antioxidant pigments. OD was measured using a
(5′-TAC CTG GTT GAT CCT GCC AGT AGT CAT-3′) and UV–Vis spectrophotometer (Ultrospec 3100 pro; Amersham
reverse primer P2 (5′-CCT TCC GCA GGT TCA CCT ACG Biosciences, Buckinghamshire, UK). Light-emitting diode
GA-3′). Each 50 µL polymerase chain reaction (PCR) mix- (LED) panels (50 × 4.5 × 4.5 cm; LUXPIA Co., Ltd., Iksan,
ture contained 5 µL of 10 × PCR buffer, 20 mM each deoxy- Korea) were used as a broad-spectrum white-light source
ribonucleotide triphosphate, 0.5 µM each primer, 2 U of Taq to induce oxidative stress for the microalgae. Each of the
polymerase (Takara, Japan), and 1 µg of genomic DNA was four LED strips was spaced at a 20 cm interval in rows and
prepared for PCR. The PCR protocol consisted of 1 min of columns. The color of the LED square panels that consisted
denaturation at 94 °C, followed by 30 cycles of 0.5 min at of LED strips emitting at wavelengths of 420–680 nm was
94 °C, 0.5 min at 55 °C and 1.5 min at 72 °C. A final exten- evaluated for its effect on algal growth rate and astaxanthin
sion step was performed for 7 min at 72 °C. PCR products production. Schizochytrium sp. SH104 and SHG104 were
were resolved by agarose gel electrophoresis and visualized cultured on agar plates using the same nutrient medium con-
by ethidium bromide staining. The band corresponding to taining 15% agar.
the desired product was excised and purified using the Wiz-
ard SV Gel and PCR Clean-up System (Promega, Madison, Optimization by response surface methodology
WI, USA), and cloned into pGEM-T Easy vector (Promega,
Madison, WI, USA). The accuracy of the resulting construct The growth conditions for Schizochytrium sp. SHG104 were
was confirmed by sequencing from both strands using the optimized under flask culture conditions using response
primers P1 and P2 in conjunction with primers correspond- surface methodology (RSM), which is the most effective
ing to the internal region of the DNA template. The Big method for optimizing fermentation production processes.
Dye Terminator Cycle Sequencing Kit (Applied Biosystems, The optimum levels of significant variables were determined
USA) and an ABI PRISM 3100 Genetic Analyzer were used by RSM using a central composite design (CCD) adapted for
to determine the sequence. Nucleotide sequences of 18S DCW as a dependent variable (Y). Molasses (X1), CSL (X2),
rRNA genes from related microorganisms were obtained and sodium glutamate (X3) were tested as independent vari-
from the NCBI DNA databank (DDBJ; http://www.ddbj. ables, which was assessed at five coded levels (− 1.682, − 1,
nig.ac.jp). The obtained sequence of the Schizochytrium 0, + 1, and + 1.682), as shown in Table 3. Schizochytrium
sp. SH104 isolate was deposited in GenBank under the sp. SHG104 was cultivated in a 500 mL flask with shaking

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 Bioprocess and Biosystems Engineering

at 165 rpm in medium containing molasses, CSL, sodium where WL is the weight of the aluminum dish containing
glutamate and artificial sea salt (5 g L−1) and yeast extract dried lipid residue (g), WD is the weight of the empty alu-
(1 g L−1). The temperature was shifted from 28 to 20 °C at minum dish (g), WS is the weight of the sample (g), VC is the
day 2 and cultivation was ended at day 4. RSM experimental total volume of the chloroform layer in a graduated cylinder
results were fitted to a second-order polynomial equation (mL), and VP is the volume of chloroform transferred to the
by applying a response surface regression procedure using aluminum dish (mL). All chemicals used in this experiment
MINITAB 7 statistical software (Minitab Inc., Pennsylvania, were purchased from Sigma-Aldrich (Sigma-Aldrich, St.
USA), which was also used to plot response surface graphs Louis, MO, USA).
for determining optimal DCW conditions. Factors exhibiting
P values less than 0.05 were considered to have significant
effects on the response and were selected for further opti- Analysis of the fatty acid composition
mization studies. A total of 20 experiments were conducted,
and experimental runs were randomized. All variables were The dried cells were re-suspended in 3 mL of 5% (v/v)
taken at a central coded value, defined as zero, and minimum methanolic sulfuric acid and heated at 90 °C for 1 h in
and maximum ranges of variables were used; the full experi- sealed vials. Fatty acid methyl esters were extracted from
mental plan with regard to values in actual and coded form the cells with 0.6 mL of hexane and then applied to a gas
is provided in Table 3. The response values (Y) in each trial chromatograph (Hewlett Packard 6890N; HP Ltd, Califor-
are the average of duplicates. RSM data obtained for DCW nia, USA) equipped with a flame-ionized detector and an
were assessed statistically by analysis of variance (ANOVA). HP-5 capillary column (30 m × 0.32 mm, 0.25 mm; Agi-
lent Technologies, Santa Clara, CA, USA). The column
Dry cell weight analysis temperature was raised from 150 °C (2 min) to 270 °C
(2 min) at a rate of 7 °C per min. All chemicals used
DCW was analyzed by the following protocol. First, cell cul- in this experiment were purchased from Sigma-Aldrich
ture was harvested at 4500 rpm at 4 °C for 20 min, the super- (Sigma-Aldrich, St. Louis, MO, USA).
natant was discarded and the pellet was washed three times
with PBS (pH 7.2). The re-suspended cells were harvested
by centrifugation at 4500 rpm at 4 °C for 20 min. The pellet Preparation and analysis of carotenoid‑based
was re-suspended in 600 µL of distilled water and transferred antioxidant pigment extracts
to pre-weighed vials. After drying the cell pellet solution
at 60 °C for 12 h in a speed vacuum concentrator (Biotron The 10 mL of cultures were harvested by centrifugation
4080C; Ecopsin Ltd, Leicestershire, UK). The initial and at 3500 rpm for 10 min and washed twice with 0.5 M PBS
final weight of the vial was measured to calculate the DCW. to remove salt residue. Samples were stored at − 20 °C
before extraction of carotenoid-based antioxidant pig-
Lipid content analysis ments. For extraction, 1–10 mL of organic solvents [hex-
ane and chloroform/hexane 3:1 (v/v)] was added and the
Total lipid content was estimated using a modified sample was vortexed vigorously for 10 min. Samples were
Bligh–Dyer method, as described in the previous study then centrifuged at 13,000 rpm for 10 min, and the red-
[8]. Briefly, 125 mg of dried cells were placed in a tinted colored layer was obtained after washing two times with
screw-cap test tube, and 6.25 mL of chloroform, 12.5 mL of distilled water. Extracted carotenoid-based antioxidant
methanol, and 5 mL of 50 mM phosphate (­ K2HPO4) buffer pigments were stored at − 20 °C protected from light for
solution (pH 7.4) were added. Each sample was shaken at subsequent analysis. Compounds were analyzed by liquid
200 rpm for 1 h at 28 °C, and then additional 6.25 mL of chromatography-atmospheric pressure chemical ioniza-
chloroform and 6.25 mL of phosphate buffer were added. tion mass spectrometry (LC-APCI/MS) using an Agilent
After inverting tubes 30 times and allowing the contents system (Agilent Technologies, Santa Clara, CA, USA), as
to settle for 1 h, the bottom chloroform layer (~ 12.5 mL) shown in Supplementary Fig. 1a. For these analyses, wet
was transferred to a pre-weighed aluminum dish. Aluminum biomass was first frozen in liquid nitrogen and extracts
dish with solvent was placed in a drying oven at 80 °C for were separated by LC-APCI. The molecular weights (M)
30 min to evaporate the solvent. After cooling, the dish was of protonated compounds (M + 1) were assessed by MS,
re-weighed, and the total lipid extract was determined based and the molecular weights of carotenoid-based antioxidant
on the weight of the extracted lipid with following equation: pigments were determined by reference to the database.
[( ) ]/ [ ]
Total lipid (g of oil per 100 g sample) = WL − WD × VC × 100 VP × WS ,

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Carotenoid-based antioxidant pigment profiles in Results

extracts were also analyzed by high-performance liquid
chromatography (HPLC). For HPLC analysis, 1–10 mL of Isolation and characterization of Schizochytrium sp.
a chloroform and methanol mixture (2:1 v/v) was added SH104 and its mutant SHG104
to lyophilized biomass for extraction of carotenoid-based
antioxidant pigments. Samples were applied to an HPLC For cloning and sequence analysis of 18S rRNA, a nearly
system (Thermo Scientific Dionex UltiMate 3000; Agi- full-length sequence of the 18S rRNA gene was isolated from
lent Technologies, Santa Clara, CA, USA) equipped with Schizochytrium sp. SH104 (deposited in GeneBank under
a YMC-Triart C18 column (150 × 4.6 mm ID, S-5 µm, accession number KX379459). The phylogenetic tree was
12 nm; YMC, Tokyo, Japan). Elution was performed at rooted using Schizochytrium sp. SH104; a total of 1781 bases
a flow rate of 1 mL ­min−1 and a column temperature of of 18S rRNA were used for the analysis (Fig. 1a, b). The reli-
40 °C. The following two-phase gradient system employ- ability of the phylogenetic tree was evaluated by neighbor-
ing the solvents acetone (A) and 90% methanol (B) was joining analysis, as described in “Materials and methods”. A
used for separation: B was run at 80–20% for 25 min, 20% comparison of the 18S rRNA gene sequence of Schizochytrium
for 10 min, and 20–80% for 5 min. Eluted compounds sp. SH104 with related microalgae in the GenBank database
were monitored with a UV–Vis detector at 460 nm, and positioned these organisms within the Thraustochytrids. Com-
commercial β-carotene, canthaxanthin, and astaxanthin bined with the resulting phylogenetic tree, BLAST analyses
(Sigma-Aldrich, St. Louis, MO, USA) were used as stand- of the 18S rDNA sequence of Schizochytrium sp. SH104 with
ards for quantification of each pigment. All chemicals used gene sequences deposited in NCBI identified Thraustochytrid
in this experiment were purchased from Sigma-Aldrich SH104 as a Schizochytrium sp. In the 18S rRNA phyloge-
(Sigma-Aldrich, St. Louis, MO, USA). netic tree (Fig. 1b), the Schizochytrium sp. SH104 strain was
located in a well-supported monophyletic group with the
Microscopic sample preparation Schizochytrium strain type; therefore, we named this strain as
Schizochytrium sp. SH104.
In this study, we compared the pictures obtained from scan- To isolate strains with improved antioxidant pigment
ning electron microscopy (SEM) with those obtained using production, we exposed the isolated Schizochytrium sp.
light microscopy. The successive steps for preparing the mate- SH104 parent strain to γ-irradiation to induce mutations. For
rial for observation using a scanning electron microscope are γ-irradiation-induced mutagenesis, the parent strain Schiz-
in next procedure. The cultured Schizochytrium sp. samples ochytrium sp. SH104 was recovered from culture, then sus-
were placed on a glass slide and fixed with 2% glutaraldehyde pended in 10 mL of PBS buffer, irradiated with γ-rays at a
at 4 °C for 2 h. Those washed 3 times with 0.05M cacodylate dose of 1–3 kGy for 1 h, then diluted by 1­ 0−6 and plated in
buffer, pH 7.2 for 10 min each. The specimen were post-fixed an appropriate medium (see “Materials and methods”). Colo-
with 1% OsO4 at RT for 2 h under the flow-hood then washed nies were selected from cultures irradiated with 2 kGy, which
2 times with distilled water for 10 min each. After fixation, exhibited a death rate greater than 99% compared with the
the material was dehydrated in alcohols of increasing concen- non-irradiated SH104 parent strain. The stained plates were
tration (30, 40, 50, 60, 70, 80, 90, 95, and 100%) and dried, cultured in an incubator under light irradiation conditions,
because samples have to be compatible with the vacuum in the and yield orange and yellow colonies. Under the high light
microscope. Finally, samples were dried in air more. The dried condition, short wavelength light in visible ray range induces
sample was placed on an aluminum stub with carbon tape and reactive oxygen species that occur in oxidative stress on Schiz-
coated with platinum using ION-COATER (COXEM Ltd., ochytrium sp., which induces carotenoids accumulation to
Daejeon, Korea). EMcraft (EMCRAFT Ltd., Gyeonggi-do, resist oxidative stress. Mutant strains with improved perfor-
Korea) scanning electron microscope was used to measure the mance were selected from among the single colonies grown
electron beam spot size of 10 pA, the working distance (WD) on the stained plate and named Schizochytrium sp. SHG104.
between the lens and the object was ~ 5 mm, the acceleration The morphology of Schizochytrium sp. SH104 and SHG104
voltage was 20 kV, prove was 3.00–4.00 and magnification were investigated by light and electron microscopic analysis
was about ×5000 (The scale bar was 2.0 μm). Light micros- (Fig. 2) and confirmed the formation of zoospore and limaci-
copy was performed normally. form amoeboid cells.

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 Bioprocess and Biosystems Engineering

Fig. 1  18S rDNA sequence of Schizochytrium sp. SH104 (a) and an analysis of its phylogenetic tree (b)

Production of antioxidants and omega‑3 oil as omega-3 fatty acids. To analyze the composition of carot-
by Schizochytrium sp. strains enoid-based antioxidant pigments, we cultured 100 mL of
Schizochytrium sp. SH104 and its mutant strain SHG104 at
Thraustochytrids can be used as alternative potential 28 °C in 500 mL baffled flasks with shaking at 130 rpm for
sources of carotenoid-based antioxidant pigments, as well 4–8 days under fluorescent light. The characterization of

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Bioprocess and Biosystems Engineering

Fig. 2  Light (left) and electron (right) microscopic analysis of the morphology of Schizochytrium sp. SH104 (a) and its mutant strain SHG104
(b). Scale bar of left (light) images shows 10 µm and Scale bar of right (electron) images shows 2 µm

pigments were performed by first analyzing pigment extracts production of antioxidant pigments. As a first step toward
using LC-ACPI/MS on day 8. Using hexane as the organic optimizing culture conditions for the production of antioxi-
solvent, β-carotene, echinenone, canthaxanthin, and phoeni- dants by the Schizochytrium sp. SHG104 mutant strain, we
coxanthin were detected with measured molecular weights assessed the effect of varying the agitation speed from 130
of 537.33, 551.31, 565.36, and 579.35, respectively. The val- to 200 rpm. A speed of 165 rpm was found to be optimal for
ues are about one Dalton greater than the original molecular the production of antioxidants (data not shown). Therefore,
weights of the pigments due to addition of H ­ + ion on the in subsequent experiments, carotenoid-based antioxidant
chemicals (Supplementary Fig. 1a). Astaxanthin was also pigments were produced by culturing Schizochytrium sp.
detected using a chloroform and hexane mixture (3:1 v/v) SH104 and its SHG104 mutant strains at 28 °C for 4 days
as an extraction solvent; its measured molecular weight was with agitation at 165 rpm. Table 1 shows the carotenoid
595.36 (Supplementary Fig. 1b). The profile of carotenoid- profile and detailed analysis of DCW, total lipid contents and
based antioxidant pigments had analyzed by HPLC of S. DHA composition. The mutant Schizochytrium sp. SHG104
SH104 on day 4. Cells had extracted with a chloroform and showed the dramatic increase of carotenoid production com-
methanol mixture (2:1 v/v) to confirm the production of pared with the parent Schizochytrium sp. SH104 (Table 1;
antioxidants pigments. The peaks detected at 7.93, 12.98, Fig. 3). The mutant strain produced 0.072 mg L−1 of astax-
20.29 and 26.54 min were determined to be astaxanthin, can- anthin (a 5.2-fold increase), 0.175 mg L−1 of β-carotene (a
thaxanthin, echinenone and β-carotene, respectively (Sup- 5.6-fold increase), and 0.339 mg L−1 of total carotenoids (a
plementary Fig. 2). Thus, this strain is capable of simultane- 5.3-fold increase). In addition, the mutant Schizochytrium sp.
ously producing a series of antioxidant pigments, including SHG104 strain showed no significant decrease in cell growth
canthaxanthin, echinenone, and β-carotene, in addition to compared with the parent strain and exhibited a similar total
omega-3 DHA oil and astaxanthin. lipid content (Table 1). The production of DHA omega-3 oil
It is well known that various factors, such as pH, oxygen by the Schizochytrium sp. SHG104 mutant accounted for
supply, temperature, and light, among others, influence the ~ 33% of DHA, a value slightly lower than that of the parent

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 Bioprocess and Biosystems Engineering

Table 1  Analysis of antioxidants with carotenoid-based pigments of of some microalgae [20, 21]. To improve the production
Schizochytrium sp. SH104 and its mutant SHG104 of antioxidants with carotenoid-based pigments, we used
Antioxidant pigments S. SH104 S. SHG104 Relative production white-light LED to cultivate Schizochytrium sp. SHG104
[mutant/host] (folds) mutant. Although one advantage of an LED light source is
its ability to emit at a single wavelength, we used a broad-
Astaxanthin (mg L−1) 0.014 0.072 5.2
spectrum LED, which includes blue wavelengths as well
Canthaxanthin 0.01 0.045 4.4
(mg L−1) as the weaker green and red wavelengths. Some reports
Echinenone (mg L−1) 0.009 0.047 5.5 have suggested that the pea leaves illuminated with red-
β-Carotene (mg L−1) 0.031 0.175 5.6 light LED contained higher levels of β-carotene than those
Total carotenoids 0.064 0.339 5.3 grown under blue or white LED light [22]. The broad-
(mg L−1) spectrum white-light LED used in this study emitted light
DCW (g L−1) 10.9 10.4 having wavelengths of 420–680 nm, which is broader than
Total lipid (% of 54.5 51.4 blue or white LED lights and containing red LED lights.
biomass) Schizochytrium sp. SHG104 were cultured in basal
DHA (% of TFA) 40.8 33.2 medium for 4 days with shaking at 165 rpm under white-
light LED, and DCW and carotenoid content was meas-
ured. As shown in Table 2, the production of β-carotene
strain. Other saturated and unsaturated fatty acids were simi- and carotenoid-based antioxidant pigments by Schiz-
lar between mutant Schizochytrium sp. SHG104 and SH104 ochytrium sp. SHG104 under LED-illumination conditions
except for the docosapentaenoic acid (DPA, C22:5, n-6), were superior to those obtained under LED-off conditions
which was about 3% higher in the mutant strain (data not (previous data in Table 1). At a constant temperature of
shown). The advantage of this strain is that it can protect 28 °C, astaxanthin and β-carotene content were increased
each other by producing omega-3 unsaturated fatty acids dramatically to 1.119 and 3.685 mg L−1, respectively; can-
and antioxidants. thaxanthin (0.141 mg L−1) and echinenone (0.135 mg L−1)
production were also increased. Cell growth was slightly
Enhancement of antioxidant production higher under white-light LED illumination than under
in Schizochytrium sp. strains LED-off conditions. Total lipid content of the Schiz-
ochytrium sp. SHG104 mutant constituted about 43% of
Effect of LED lighting biomass, with DHA omega-3 oil accounting for 29.8% of
total fatty acid (Table 2), a percentage slightly less than
Improved growing systems using artificial lighting such that in the parent strain. The production of antioxidants
as LED technology have recently been adopted to increase was also slightly increased under white-light LED for
the production capacity of target compounds. LEDs have Schizochytrium sp. SH104 strain (data not shown); how-
been reported to enhance lipid production and cell growth ever, the change was smaller than that of SHG104 mutant
strain.

Fig. 3  Production of carotenoid-based antioxidant pigments (a), DCW (b) and total lipid and DHA (c) in the Schizochytrium sp. SH104 parent
and SHG104 mutant strain at day 4 with shaking (165 rpm). Error bars had calculated from three independent experimental data

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Bioprocess and Biosystems Engineering

Table 2  Production of Antioxidant pigments 28 °C 28 °C → 20 °C Relative production

antioxidants of carotenoid-based [temp shift/normal]
pigments such as astaxanthin, (folds)
canthaxanthin, echinenone, and
β-carotene in Schizochytrium Astaxanthin (mg L−1) 1.119 4.598 4.1
sp. SHG104 under LED lighting
Canthaxanthin (mg L−1) 0.141 0.598 4.3
and cultivation temperature shift
Echinenone (mg L− 1) 0.135 0.552 4.1
β-Carotene (mg L−1) 3.685 6.707 1.8
Total carotenoids (mg L−1) 5.079 12.456 2.5
DCW (g L−1) 11 10.8
Total lipid (% of biomass) 43 45.8
DHA (% of TFA) 29.8 32.1

Effect of cultivation temperature shift SHG104 was decreased slightly under temperature-shifting
condition from 11 to 10.8 g L−1. Total lipid content con-
Temperature is one of the most important environmental stituted about 45.8% of biomass, with DHA omega-3 oil
factors affecting the biosynthesis of lipids containing func- production representing 32.1% of total fatty acids under the
tional oils, such as DHA. In general, low temperature slows combined LED illumination and temperature-shifting con-
strain growth but it promotes the accumulation of lipid and dition. We noted that Schizochytrium sp. SHG104 mutant
carotenoid. To further optimization of culture conditions, showed no significant decrease in cell growth and exhib-
we investigated the effects of a temperature shift (2 days ited similar total lipid content. Although the cultivation
at 28 °C followed by 2 days at 20 °C) on the production of temperature shift did not affect DCW or lipid content, it
carotenoid by Schizochytrium sp. SHG104. The color of the greatly affect the overall carotenoid production. In the Schiz-
colonies was changed from white to orange by temperature ochytrium sp. SH104 strain, the combined effect of LED
shift, as shown in Fig. 4 and the data of the comparative lighting and temperature shift increased the production of
color scale are shown in Supplementary Fig. 3. The temper- antioxidants about 2.5-fold compared with the only LED
ature-shifting strategy was performed on flask cultures of lighting conditions.
Schizochytrium sp. SHG104, and was performed under con-
ditions of white-light LED illumination. Combining LED Effect of renewable medium on the production
lighting and a temperature shift exerted a significant posi- of antioxidants, determined by the responsive surface
tive effect on carotenoid production. As shown in Table 2, methodology
astaxanthin and β-carotene content increased to 4.598 and
6.707 mg L−1, respectively, under these conditions; canthax- Thraustochytrids microalgae are capable of using organic
anthin and echinenone showed corresponding increases to resources as raw materials to accumulate high lipid contents
0.598 and 0.552 mg L−1. The growth of Schizochytrium sp. including valuable omega-3 lipids more than 40 wt% and

Fig. 4  Color change of Schiz-

ochytrium sp. SHG 104 colonies
from white to red–orange at low
temperature. a 28 °C for 4 days.
b 28 °C for 2 days followed by
20 °C for 2 days

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 Bioprocess and Biosystems Engineering

Table 3  Outline of experimental Design point Independent variables in the Indent. Vars. in their natural form DCW (g L−1)
design by central composite coded form (g L−1)
design (CCD) matrix for
the experimental design and Run order X1 X2 X3 X1 (mL) X2 (mL) X3 Observed Predicted
predicted responses for DCW
1 −1 −1 −1 30 (9.56) 10 (2) 10 8.4 11.7
2 1 −1 −1 70 (22.3) 10 (2) 10 21 20.4
3 −1 1 −1 30 (9.56) 30 (6) 10 12.4 12
4 1 1 −1 70 (22.3) 30 (6) 10 24.9 24.2
5 −1 −1 1 30 (9.56) 10 (2) 30 8.9 8.4
6 1 −1 1 70 (22.3) 10 (2) 30 19.1 18.3
7 −1 1 1 30 (9.56) 30 (6) 30 11.7 11.2
8 1 1 1 70 (22.3) 30 (6) 30 25 24.6
9 − 1.682 0 0 16.36 (5.23) 20 (4) 20 6.7 7.3
10 1.682 0 0 83.64 (26.6) 20 (4) 20 24.9 25.9
11 0 − 1.682 0 50 (15.9) 3.18 (0.636) 20 13.2 14.2
12 0 1.682 0 50 (15.9) 36.82 (7.364) 20 19.1 19.7
13 0 0 − 1.682 50 (15.9) 20 (4) 3.18 15.8 16.6
14 0 0 1.682 50 (15.9) 20 (4) 36.82 13.5 14.2
15 0 0 0 50 (15.9) 20 (4) 20 13.8 15.5
16 0 0 0 50 (15.9) 20 (4) 20 16.6 15.5
17 0 0 0 50 (15.9) 20 (4) 20 17.8 15.5
18 0 0 0 50 (15.9) 20 (4) 20 14.4 15.5
19 0 0 0 50 (15.9) 20 (4) 20 15.7 15.5
20 0 0 0 50 (15.9) 20 (4) 20 14.9 15.5

Duplicates runs were carried out all design point and the average recorded. The experimental runs were
randomized
X1 molasses, X2 Corn steep liquor, X3 Sodium glutamate

antioxidant pigments. Therefore, to produce high-value- The RSM analysis with ANOVA test was performed
added antioxidants while minimizing media costs, we tested using MINITAB 7 statistical software (Minitab Inc., Penn-
the effects of cultivation using low-cost alternative media sylvania, USA). The regression equation used to determine
with Schizochytrium sp. SHG104. the optimal conditions for DCW production was calcu-
Growth conditions for Schizochytrium sp. SHG104 were lated by performing a multivariate regression analysis
optimized using response surface methodology (RSM) with using a model reaction formula based on a full factorial
central composite design (CCD), which is the most effective design. Factors exhibiting P values less than 0.05 were
method for optimizing fermentation production processes. considered to have significant effects on the response and
As cheaper nutrient sources, cane molasses and corn steep were selected for further optimization studies. Molasses,
liquor (CSL) were tested as alternatives to glucose (carbon with a P value of 0.0004, was determined to be the most
source) and yeast extract (nitrogen source), respectively. significant factor, followed by CSL (0.001) and sodium
Independent variables (Xn), media components that might glutamate (0.070). The lower probability values indicate
most affect the growth of Schizochytrium sp. SHG104, were that the corresponding factor is a more significant determi-
cane molasses (X1), CSL (X2), and sodium glutamate (X3). nant of DCW. Sodium glutamate exerted a negative effect
For RSM analyses in flasks, the values of the independent on DCW, whereas molasses and CSL exerted positive
variables considered were molasses (X1), ~ 30–70 g L−1 effects. The highest DCW (25.005 g L−1) was obtained at
(as total reduced sugar); CSL (X2), ~ 4–12 mL L−1; and a molasses concentration of 70 g L−1 (22.3 mL), a CSL
sodium glutamate (X3), ~ 10–30 g L−1. The central com- concentration of 30 g L−1 (6 mL) and a sodium glutamate
posite design (CCD) used these three variables coded in concentration of 30 g L−1 (Run 8 in Table 3); the theo-
five steps (− 1.682, − 1, 0, 1, and 1.682) relative to the base retically expected DCW value was 24.6 g L−1. Overall,
condition. Using the coded levels, we calculated the actual the results were generally consistent with model predic-
values in each of 20 experiments (eight-factor scores, six tions. The lowest DCW was found to be 6.68 g L −1 at a
axis points, and six center points), which shown in Table 3 molasses concentration of 16.36 g L−1 (5.23 mL), a CSL
with observed DCW (Y) value.

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Bioprocess and Biosystems Engineering

concentration of 20 g L−1 (4 mL) and a sodium glutamate Table 5  Antioxidants with carotenoid-based pigments of Run 8
concentration of 20 g L−1 (Run 9 in Table 3). Antioxidant pigments S.
Using molasses and CSL as low-cost sources of carbon SHG104
and nitrogen sources, overall medium cost was reduced (Run 8)
to about 15–20% of those for glucose and yeast extract.
Astaxanthin (mg L−1) 0.0041
Detailed production profile of fatty acid and carotenoids of
Canthaxanthin (mg L−1) 0.0127
Run 8 is analyzed in Tables 4 and 5, respectively. The results
β-Carotene (mg L−1) 0.0237
showed that DHA was 43.7% of total fatty acid, which is
Total carotenoids (mg L−1) 0.0405
good, whereas total lipid content was slightly decreased
Total lipid (% of biomass) 25.5
(25.5%). Other unsaturated fatty acids, including arachidonic
DHA (% of TFA) 43.7
acid (C20:4, n-6), EPA (C20:5, n-3), DPA (C22:5, n-6) and
DPA (C22:6, n-3), were also produced, achieving maximum
values of 1.21, 4.64, 10.96 and 1.12%, respectively. The sat-
urated palmitic acid (C16:0) maximally constituted 30.3% synthesis: the classical mevalonate pathway and the alter-
of total fatty acids (Table 4). The maximum total polyun- native, non-mevalonate (MEP/DOXP) pathway [25–27]. In
saturated fatty acid (PUFA) and saturated fatty acid (SFA) the mevalonate pathway, whose enzymes and genes have
obtained were ~ 61.15 and ~ 34.54%, respectively. The total been well studied, acetyl-CoA is converted to IPP through
carotenoid yield was 0.0405 mg L−1, and the dominant carot- mevalonate [27]. This pathway typically found in most
enoid pigment was β-carotene (0.0237 mg L−1). The lowest eukaryotes cells and some bacteria [26, 27]. Otherwise, the
amounts of astaxanthin and canthaxanthin produced were non-mevalonate pathway found in plant cells that synthesize
0.0041 and 0.0127 mg L−1, respectively (Table 5). This low isoprenoid in their chloroplast, a photosynthetic organelle.
lipid and carotenoid content is attributed to high biomass In the mevalonate pathway, two molecules of acetyl-CoA
productivity of the microalgae. In general, carotenoids accu- produced through glycolysis are combined to form acetoa-
mulated at harsh stress condition such as oxidative stress cetyl-CoA, which is converted to HMG-CoA, mevalonate,
or nitrogen stress with low biomass concentration. Run 8 mevalonate-5-phosphate, mevalonate-5-diphosphate, iso-
condition is optimized to maximize biomass yield under rich pentenyl-diphosphate in series reaction. Finally, isopentenyl-
nutrient condition compared to experimental results, as dis- diphosphate converted to IPP (C5) and its isomer, dimethy-
played in Table 1, which caused to reduction of lipids and lallyl diphosphate (DMAPP), which are used as the building
carotenoid content. These results indicating that control of blocks in terpenoid synthesis. These IPP and DMAPP are
nutrient condition is critical for production of both biomass combined to form geranyl-pyrophosphate (geranyl-PP, C10)
and value-added products. by farnesyl diphosphate synthase and continuously convert
into farnesyl-PP (C15). Another IPP is added to farnesyl-PP
Carotenoid synthesis (carotenogenesis) pathways, by geranylgeranyl pyrophosphate synthase (CrtE, GGPS) to
enzymes, and genes in Thraustochytrid strains yield geranylgeranyl-PP (C20). In a head-to-head condensa-
tion of the two C20 compounds by phytoene synthase (CrtB,
Terpenoid biosynthetic metabolism produces the basic Psy) formed the first carotene, phytoene (C40) [28, 29].
building blocks called isopentenyl pyrophosphate (IPP), a A genomic analysis was performed to assess terpenoid
C5-compounds, which needed for the synthesis of C30, C40, biosynthetic pathways in Thraustochytrid strains with Schiz-
and higher, carbon complexes such as isoprenoids, terpenes, ochytrium sp. and Aurantiochytrium sp. (Tables 6, 7). The
quinones, phytol of chlorophylls, carotenoids and sterols [23, genomic analysis revealed that the Thraustochytrid strains
24]. There are two known independent pathways for IPP use the mevalonate pathway, which is reflecting their lack of

Table 4  Fatty acid composition Saturated fatty acids Unsaturated fatty acids
of microalgal oil of Run 8
Lipid name Content (%) Lipid name Content (%)

Myristic acid (MA), C14:0 2.36 Arachidonic acid (ARA), C20:4 n-6 1.21
Pentadecylic acid, C15:0 1.91 Eicosapentaenoic acid (EPA), C20:5 n-3 4.64
Palmitic acid (PA), C16:0 30.27 Docosapentaenoic acid (DPA), C22:5 n-6 10.96
Margaric acid, C17:0 Trace Docosahexaenoic acid (DHA), C22:6 n-3 43.22
Stearic acid (SA), C18:0 Trace Docosapentaenoic acid (DPA), C22:5 n-3 1.12
Others Trace
Sum 34.54 Sum 61.15

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 Bioprocess and Biosystems Engineering

Table 6  Terpenoid synthetic Enzyme Aurantiochytrium sp. (KRS101) Schizochytrium sp. (SH103)
genes of Thraustochytrid
microalgae Acetyl-CoA acetyl transferase KRS_SC14CG01170 SH103_SC7CG01430
HMG-CoA synthase KRS_SC10CG01270 SH103_SC15CG02470.1
HMG-CoA reductase KRS_SC60CG00070 SH103_SC17CG01770
Mevalonate kinase KRS_SC83CG00220 –
Phosphomevalonate kinase KRS_SC6600100 SH103_SC18CG02980
Diphosphomevalonate decarboxylase KRS_SC71CG00250 SH103_SC07CG00340.1
Isopentenyl-diphosphate isomerase KRS_SC24CG01690 SH103_SC09CG01720.1
Farnesyl diphosphate synthase KRS_SC115CG00240 SH103_SC10CG00530
Geranylgeranyl diphosphate synthase KRS_6CG02110 SH103_SC2CG04310

Table 7  Carotenoid synthetic Enzyme Aurantiochytrium sp. KRS101 Schizochytrium sp. SH103
genes of Thraustochytrid
microalgae Phytoene synthase KRS_SC50CG00180 SH103_SC04CG04410.1
Phytoene desaturase KRS_SC125CG00180 SH103_SC21CG00660
Lycopene cyclase_CrtY KRS_SC125CG00190 SH103_SC21CG00660
Cytochrome P450 hydroxylase (Asta- KRS_SC39CG00890 SH103_SC25CG02010
xanthin synthase)
β-Carotene hydroxylase_CrtZ KRS_SC90CG00300 SH103_SC9CG00830
β-Carotene ketolase_CrtW KRS_SC42CG00290 SH103_SC42CG00290

a photosynthetic organelle. This analysis revealed the pres- and canthaxanthin [12]. No genomic analysis of astaxanthin
ence of phytoene synthase activity in these Thraustochytrid biosynthetic metabolism in Thraustochytrids has been previ-
strains. It also further confirmed that both Aurantiochytrium ously reported.
sp. and Schizochytrium sp. possess the series of terpenoid Our genomic analysis identified phytoene synthase, phy-
biosynthetic metabolic genes encoding acetyl-CoA, acetyl- toene desaturase and lycopene cyclase, which are involved in
transferase, HMG-CoA synthase, HMG-CoA reductase, β-carotene metabolism in both Thraustochytrid microalgae
mevalonate kinase, diphosphomevalonate decarboxy- strains (Table 7). These results demonstrated that Thraus-
lase, phosphomevalonate kinase, isopentenyl-diphosphate tochytrid microalgae are capable of carotenoid synthesis.
isomerase, farnesyl diphosphate synthase and geranylgera- Carotenoid ketolase and carotenoid hydroxylase enzymes
nyl diphosphate synthase (Table 6). Terpenoid biosynthetic are commonly used in astaxanthin-producing strains, such
pathways and the enzymes involved for each Thraustochytrid as Haematococcus pluvialis, which synthesizes canthaxan-
microalgae strain are shown in Fig. 5, together with path- thin, zeaxanthin, and astaxanthin [33]. Our genome analy-
ways identified through genomic analysis. sis identified ketolase or hydroxylase in Thraustochytrid
Among oxygenic phototrophs, carotenoid synthesis path- strains, also. On the other hand, the astaxanthin-synthesiz-
ways and their enzymes have mainly been investigated in ing yeast, Xanthophyllomyces dendrorhous (Phaffia rho-
cyanobacteria and land plants [30, 31]. Algae also have dozyma), uses a cytochrome P450 3A type hydroxylase as
pathways in common with land plants as well as additional a β-carotene 3-hydroxylase/4-ketolase [34]. Our BLAST
algae-specific pathways. Some common carotenogenesis analysis confirmed a high degree of similarity between the
genes in algae are suggested based on homology of known cytochrome P450 gene in X. dendrorhous and those in both
genes [32], but most genes and enzymes for algae-specific Thraustochytrid microalgae strains. Thus, these observa-
pathways are still unknown due to its high complexity. tions suggested that Thraustochytrid microalgae strains
Related enzymes are known mostly from cyanobacteria and use a cytochrome P450 hydroxylase (astaxanthin synthase)
green algae [31]. The phytoene synthesized from GGPP con- for astaxanthin synthesis, same as X. dendrorhous. In sum-
verted into lycopene by phytoene desaturase through series mary, genomic analysis of Thraustochytrid microalgae
of reaction. Then, pathway divided into lutein synthesis Aurantiochytrium sp. and Schizochytrium sp. suggested the
pathway and astaxanthin synthesis pathway starting from carotenoid synthesis pathway that described in Fig. 6. Phy-
lycopene via δ and β-carotene, respectively. Only astaxan- toene, the first carotenoids are synthesized from GGPP via
thin synthesis has been reported in Thraustochytrid strains, a typical mevalonate pathway. Phytoene is first converted
where astaxanthin is produced via β-carotene, echinenone into lycopene by phytoene desaturase, and then converted

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Bioprocess and Biosystems Engineering

Fig. 5  Terpenoid synthetic path-

ways and associated enzymes in
Thraustochytrid microalgae

into β-carotene by lycopene cyclase. A serial conversion high blood pressure [35–37]. They have also been reported
is occurred by cytochrome P450 hydroxylase, to produce to offer beneficial effects against depression, rheumatoid
astaxanthin from β-carotene; β-carotene is converted into arthritis, and asthma [38, 39]. Microalgae can produce long-
echinenone, canthaxanthin, and astaxanthin at the end. chain fatty acids, mainly DHA, EPA, palmitic acid and other
long-chain fatty acids. Heterotrophic microalgae are well
established as an alternative source of DHA and are included
Discussion as a supplement in infant milk formulas. Other microalgal
products are used as food additives, ingredients in animal
Long-chain omega-3 fatty acids such as DHA and EPA pro- feed (including aquaculture), vitamins, pigments, pharma-
vide significant benefits for the human health, in particular ceutical compounds, cosmetics and potentially as a biofuel
by reducing cardiac diseases such as arrhythmia, stroke and source [40]. The fatty acids produced by our newly isolated

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 Bioprocess and Biosystems Engineering

Fig. 6  Carotenoid synthetic

(carotenogenesis) pathways
and their enzymes in Thraus-
tochytrid microalgae

Thraustochytrid Schizochytrium sp. mainly produced pal- of carotenogenesis pathway, is synthesized by cytochrome
mitic acid, DHA and trace amounts of other long-chain fatty P450 hydroxylase. Our investigation of the microalgae
acids. The production profile of microalgae also includes Thraustochytrid Schizochytrium thus revealed the presence
carotenoid-based antioxidant pigments, such as β-carotene, of carotenogenesis pathway and related enzymes and genes
astaxanthin, canthaxanthin, and echinenone. Each of these that necessary for the production of the antioxidants via a
carotenoid pigments was also produced by our newly iso- complete biosynthetic synthesis pathway.
lated Schizochytrium sp. strain. Some partial genome data are available from the Joint
Carotenoid pigments are widely distributed in nature, Genome Institute (JGI; http://genome​ .jgi.doe.gov/) [42] and a
occurring in bacteria, yeasts, molds, all green plants, and few transcriptome studies of Thraustochytrids have also been
many animals. Carotenoids contribute to the yellow, orange, published [43]. The methods described above have mainly
and red colors of the skin, shell, or exoskeleton of aquatic been used to elucidate the role of enzymes related to fatty
animals. Astaxanthin, in particular, is found in microalgae, acid synthesis, but the information obtained to date is insuf-
yeast, and diverse marine organisms, notably including ficient for purposes of strain improvement. Despite more than
salmon, where it is responsible for the distinctive red color. 20 years of research on DHA production by Thraustochytrids,
For this reason, astaxanthin production has been exploited to our knowledge of the biochemistry, genetics, and regulation
improve the appearance of salmon as well as trout and other of synthesis pathways is still incomplete. The few reported
commercial fish products first. However, its superior antioxi- studies on squalene and carotenoids indicate high produc-
dant capacity, astaxanthin is used as a material of high-value tion potential for these compounds. DHA-rich oils produced
products as medicines and cosmetics. Notably, astaxanthin by Thraustochytrids have already been commercialized for
has been shown to possess anticancer, anti-obesity, anti- the human market. The production of DHA for other, lower
diabetic, anti-inflammatory, and cardioprotective activities. priced markets, such as feed ingredients, as well as the pro-
With the exception of animals, these organisms are capable duction of squalene and carotenoids by Thraustochytrids, will
of synthesizing many types of carotenoids through diverse depend on the availability and price of current feedstocks
carotenogenesis pathways. Carotenoids are usually produced for these products. Squalene and carotenoids could also be
in higher plants, vegetables, and fruits, where tetraterpenoids potential value-added co-products from the future large-scale
are synthesized from eight C5 isoprenoid units. Investigations production of DHA for use as a fish feed ingredient, thereby
of genes encoding carotenogenesis enzymes have reported improving the economy of the process.
that many such genes are highly similar from bacteria to The development of astaxanthin production processes
land plants, but others exhibit low similarity. Some homolo- using Thraustochytrid microalgae is currently ongoing. The
gous genes have been proposed [41, 42], but in some cases, market value of natural astaxanthin extracted from green
carotenogenesis enzymes and genes, especially algae-specific microalgae is 3900 (92%) to 23,000 (98%) USD per kilo-
genes, have not been found. Our genomic analysis confirmed gram. However, commercial cultivation of green microalgae
the carotenoid synthesis pathway in the Thraustochytrid is performed in outdoors so it is affected by weather and cli-
Schizochytrium sp., showing that GGPP is synthesized first mate; accordingly, the production process is less reliable. An
through a typical mevalonate pathway, after which β-carotene alternative approach suitable for generating a stable astaxan-
is synthesized through phytoene synthase, phytoene desatu- thin supply would be to culture Thraustochytrid microalgae
rase, and lycopene cyclase. Astaxanthin, the final product in an indoor fermenter. The production of astaxanthin by

13
Bioprocess and Biosystems Engineering

Thraustochytrid microalgae is affected by nutrient composi- long-chain polyunsaturated fatty acids in marine microalgae.
tion, oxygen concentration, light supply, and various stress Mar Drugs 11:2259–2281. https​://doi.org/10.3390/md110​72259​
4. Campoy C, Escolano-Margarit MV, Anjos T, Szajewska H,
conditions. Culture conditions that maximize astaxanthin Uauy R, Br (2012) Omega 3 fatty acids on child growth, visual
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fied, and additional increases in production capacity can be https​://doi.org/10.1017/S0007​11451​20014​93
attained through fermentation in fermentation tanks. Thraus- 5. Jacobson TA (2006) Secondary prevention of coronary artery
disease with omega-3 fatty acids. Am J Cardiol 98(4A):61i–70i.
tochytrid microalgae are relatively low in astaxanthin con- https​://doi.org/10.1016/j.amjca​rd.2005.12.028
tent compared with astaxanthin-producing strains currently 6. Kris-Etherton PM, Harris WS, Appel LJ (2002) Fish con-
used in industrial processes. Therefore, an important direc- sumption, fish oil, omega-3 fatty acids and cardiovascular dis-
tion for future research is to develop a strain that enhances ease. Circulation 106:2747–2757. https​://doi.org/10.1161/01.
CIR.00000​38493​.65177​.94
carotenoid synthesis to increase the astaxanthin productiv- 7. Pottala JV, Garg S, Cohen BE, Whooley MA, Harris WS (2010)
ity of Thraustochytrid microalgae. By combining traditional Blood eicosapentaenoic and docosahexaenoic acids predict all-
γ-ray breeding methods and molecular genetic methods, it cause mortality in patients with stable coronary heart disease:
should be possible to obtain high-astaxanthin–producing the heart and soul study. Circ Cardiovasc Qual Outcomes 3:406–
12. https​://doi.org/10.1161/CIRCO​UTCOM​ES.109.89615​9
strains with improved production yields. 8. Riediger ND, Othman RA, Suh M, Moghadasian MH (2009)
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disease. J Am Diet Assoc 109:668–79. https​://doi.org/10.1016/j.
jada.2008.12.022
Conclusion 9. de Morais MG, da Silva Vaz B, de Morais EG, Costa JAV (2015)
Biologically active metabolites synthesized by microalgae.
This study reports the newly isolated microalgae Thraus- Biomed Res Int 2015:1–15. https​://doi.org/10.1155/2015/83576​1
10. Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez
tochytrid Schizochytrium sp. SH104, which produce high DL, Ornelas-Soto N, Romero-Ogawa MA, Parra-Saldivar
DHA content and antioxidants including β-carotene, astax- R (2015) Extraction and purification of high-value metabo-
anthin, canthaxanthin, and echinenone. SHG104 strain was lites from microalgae: essential lipids, astaxanthin and phy-
obtained by traditional γ-ray breeding methods resulted in cobiliproteins. Microb Biotechnol 8:190–209. https ​ : //doi.
org/10.1111/1751-7915.12167​
dramatic increase of carotenoid productivity. Stress tests 11. Yokoyama R, Honda D (2007) Taxonomic rearrangement of the
reveal that temperature and LED lighting has a significant genus Schizochytrium sensu lato based on morphology, chemot-
effect on the accumulation of carotenoids. Medium com- axonomic characteristics, and 18S rRNA gene phylogeny (Thraus-
position was optimized with cane molasses, CSL, and tochytriaceae, Labyrinthulomycetes): emendation for Schiz-
ochytrium and erection of Aurantiochytrium and Oblongichytrium
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Acknowledgements This research was supported by a grant from the moting effects of astaxanthin: a high-value carotenoid mostly
Next Generation BioGreen 21 project through the Animal Genome from microalgae. Mol Nutr Food Res 55:150–65. https​://doi.
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Affiliations

Hansung Park1 · Minsoo Kwak2 · JeongWoo Seo3 · JeongHyun Ju3 · SunYeon Heo3 · SeungMoon Park1 ·
WonKyung Hong1

1 3
Division of Biotechnology, College of Environmental Applied Microbial Research Center, Jeonbuk Branch
and Bioresource Science, Chonbuk National University, Institute, Korea Research Institute of Bioscience
Iksan, Jeonbuk 570‑752, South Korea and Biotechnology (KRIBB), Jeongeup, Jeonbuk 580‑185,
2 South Korea
Department of Chemical and Biomolecular Engineering,
Korea Advanced Institute of Science and Technology
(KAIST), Daejeon 305‑701, South Korea

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