Algal Research 61 (2022) 102599

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Critical assessment of the filamentous green microalga Oedocladium

carolinianum for astaxanthin and oil production
Yu Wang a, b, e, Jing Jia b, Qinglei Chi b, Yanhua Li a, Hongxia Wang a, Yingchun Gong a,
Guoxiang Liu c, Zhengyu Hu c, Danxiang Han a, *, Qiang Hu a, b, c, d, *
a
Center for Microalgal Biofuels and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China
b
SDIC Microalgae Biotechnology Center, SDIC Biotechnology Investment Co. Ltd, State Development and Investment Corporation, Beijing 100034, China
c
Center for Algal Biology and Applied Research, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China
d
Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China
e
University of Chinese Academy of Sciences, Beijing 100086, China

A R T I C L E I N F O A B S T R A C T

Keywords: The filamentous microalga Oedocladium carolinianum that was isolated from soil was found to be capable of
Astaxanthin producing large amounts of astaxanthin under stress culture conditions. When the culture conditions were
Filamentous microalgae optimized, the maximum specific growth rate of 0.37 d− 1 and the maximum biomass concentration of 10.12 g
Oedocladium carolinianum
L− 1 were obtained after 18 days of cultivation in 1-liter glass columns (inner diameter: 5 cm) under laboratory
Tubular photobioreactor
Inclined thin layer photobioreactor
conditions. When subjected to culture conditions of nitrogen starvation and 2 g L− 1 NaCl-induced salinity stress,
however, the cells produced up to 3.91% w/w astaxanthin and a high astaxanthin productivity of 24.2 mg L− 1
d− 1 was obtained. Analysis by HPLC-MS revealed that the majority of astaxanthin was in fatty acid esterified
forms with a typical molecular ratio of free, monoester and diester astaxanthin of 1:18:81. The biosynthesis of
astaxanthin coincided with that of fatty acids, and the total fatty acid content reached 40% w/w or more. The
technical feasibility of mass culture of O. carolinianum was tested in a 7000-L inclined thin-layer photobioreactor
and a 10,000-L tubular photobioreactor in a greenhouse. This demonstrated that O. carolinianum grew rapidly in
a nitrogen replete BG-11 culture medium and the maximum biomass concentrations obtained in the inclined
thin-layer photobioreactor and tubular photobioreactor were 3.74 g L− 1 and 3.07 g L− 1 respectively, resulting in
maximum biomass productivities of 276 mg L− 1 d− 1 and 198 mg L− 1 d− 1, respectively. Although small pop­
ulations of a few zooplankton species occurred in the two types of photobioreactors, none grazed on
O. carolinianum and they grazed on invading unicellular microalgae instead. It was therefore concluded that
O. carolinianum is a promising microalga for sustainable co-production of astaxanthin and fatty acids.

1. Introduction antioxidant, anti-inflammatory, and antiapoptotic effects, which are

Astaxanthin (3,3′ -dihydroxy-β,β-carotene-4,4′ -dione) is a reddish
carotenoid that is synthesized de novo in some microalgae, fungi, bac­
teria and higher plants [1,2]. It is responsible for the red color of many
marine animals, as they cannot synthesize astaxanthin de novo but ac­
quire it from foods in their diet that contain astaxanthin [3]. As such,
astaxanthin has been used as a coloring agent in the food, aquaculture
and poultry industries [4]. Astaxanthin contains both a hydroxy and a
keto group on each of its β-ionone rings, but the hydroxy group on one or
both rings can be esterified with different fatty acids. The unique
structure of the molecule and its derivatives confers powerful
desirable for nutraceutical, pharmaceutical and cosmetics applications
[5,6].
The green microalga Haematococcus pluvialis is the richest source of
natural astaxanthin found in nature, as it can synthesize and accumulate
up to 5.6% (w/w) astaxanthin under stress conditions [7,8]. In the
H. pluvialis industry, astaxanthin-rich algal biomass is produced mainly
with tubular photobioreactors and, to a lesser extent, open raceway
ponds outdoors or indoor culture vessels with artificial illumination
[9,10]. The global astaxanthin production yield in 2019 was about 20
metric tons (100% pure astaxanthin) with an estimated first sale value of
US$150 million. However, further expansion of H. pluvialis production

* Corresponding author at: Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China.
E-mail address: huqiang@szu.edu.cn (Q. Hu).

https://doi.org/10.1016/j.algal.2021.102599
Received 8 July 2021; Received in revised form 7 December 2021; Accepted 7 December 2021
Available online 27 December 2021
2211-9264/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Wang et al.
and the development of broader applications of H. pluvialis–derived
astaxanthin have been hampered by the high production cost and thus
high sale price. This stems mainly from the fact that H. pluvialis is
vulnerable to ozooplanktonic grazers because of its small cell size (5–20
μm diameter) and to a host-specific parasitic fungi. These microbial
contaminations often cause a reduction in biomass yield and sometimes
result in the loss of cultures altogether [2,11,12]. In addition, harvesting
and dewatering of the H. pluvialis cells requires centrifugation or a plate-
and-frame filter press that is capital- and energy-intensive.
Chlorella zofingiensis is another unicellular green microalga that has
the ability to produce fatty acid esterified astaxanthin. This organism
has attracted some attention due to its potential for rapid growth in a
heterotrophic or mixotrophic culture mode [13,14]. However, the
relatively low cellular astaxanthin content (0.07–0.7% dwt) (Table 2) of
C. zofingiensis prevents this organism from being commercially exploi­
ted. Therefore, additional astaxanthin-producing microalgae are sought
that have a high astaxanthin content, are highly resistant to microbial
contamination and are easy to harvest.
Recently, we introduced a novel, astaxanthin-producing filamentous
green microalga Oedocladium carolinianum [15]. We reported for the
first time that while little astaxanthin was detectable in O. carolinianum
under favorable growth conditions, it produced astaxanthin at a con­
centration of 1.62% w/w on a per dry weight basis under stress condi­
tions [16].
In this study, we aimed to determine the culture conditions that
would optimize the astaxanthin content and growth of the
O. carolinianum, first under laboratory-scale conditions and then in
larger photobioreactors, and the molecular species of astaxanthin and
lipids produced. By examining microbial contaminants and their
apparent effect on the growth of O. carolinianum, we also aimed to assess
the potential impact of contamination on mass culture of the alga. The
results of the study demonstrated that O. carolinianum holds promise as
an organism that can be mass cultured for production of astaxanthin and
lipids.
2. Materials and methods
2.1. Organism and preculture conditions
O. carolinianum and Haematococcus pluvialis were obtained from the
Freshwater Algae Culture Collection of the Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan, China. Stock cultures were
maintained in 250 mL flasks containing 100 mL BG-11 medium and
placed in a controlled environment: a plant growth chamber set at 25 ±
1 ◦ C and a photon flux density of 40 μmol photons m− 2 s− 1 in a 12 h:12 h
light-dark cycle.
2.2. Cultivation of O. carolinianum in glass columns
The growth characteristics and astaxanthin production of
O. carolinianum were studied in glass columns under laboratory condi­
tions. Stock cultures were inoculated into glass columns (height 80 cm,
inner diameter 5 cm) each containing 750 mL culture medium. The glass
columns were maintained at 25 ◦ C with continuous fluorescent light at
60 μmol photons m− 2 s− 1 and aerated with compressed air containing
1.5% CO2 (v/v) at a flow rate of 0.5 L compressed air per glass column
per minute. In order to determine an appropriate basal culture medium
for the organism, it was cultivated in four different culture media: BG-
11, BBM, a modified BG-11 with 3 mmol nitrogen and a modified
BBM with 18 mmol nitrogen (Table S1).
In order to determine the minimum amount of nitrogen that may
sustain the maximum growth and biomass production of the organism,
modifications of the BG-11 culture medium were also tested, with the
original amount of nitrogen (18 mmol N as NaNO3) being reduced by
half (9 mmol N), a quarter (4.5 mmol N) and an eighth (2.25 mmol N).
To determine the optimal light intensity, O. carolinianum was
2
Algal Research 61 (2022) 102599
cultivated at four light intensities: 60, 120, 250, and 500 μmol photons
m− 2 s− 1. Stock culture of the microalga was inoculated into the glass
columns at an initial biomass concentration of about 0.8 g L− 1. Each
column contained 750 mL of 9 mmol-N BG-11 medium. The cultures
were maintained at a temperature of 28 ◦ C and aerated with fine air
bubbles containing 1.5% CO2.
The effect of the temperature on the growth of O. carolinianum was
studied using culture conditions similar to those used for the light in­
tensity experiment, with five temperatures being tested: 19 ◦ C, 23 ◦ C
28 ◦ C, 33 ◦ C, and 38 ◦ C. The different temperatures were controlled via
different devices, with the 19 ◦ C and 23 ◦ C temperatures being
controlled by two circulating cooling devices and the 28 ◦ C, 33 ◦ C and
38 ◦ C were maintained by three temperature water baths. The photon
flux density was 500 μmol photons m− 2 s− 1.
2.3. An inclined thin-layer PBR and a tubular PBR
O. carolinianum was cultivated in two types of photobioreactors
(PBRs): a 7000-L inclined thin-layer PBR (I-PBR) and a 10,000-L tubular
PBR (T-PBR) located in the Microalgal Biotechnology Center at the State
Development and Investment Corporation in Beijing, China. The I-PBR
was a U-shape metal trough structure, tilted at an angle of 0.3%. It was
140 m long, 1 m wide, and 5 cm deep, with a culture volume capacity of
7000 L. A water tank was placed at the lowest point of the trough to
collect culture suspension. A water pump connected to the water tank
continuously pumped the culture back to the highest level of the trough,
from where the culture suspension flowed down the trough by gravity at
a flow velocity of ca. 60 cm min− 1. The culture temperature followed the
ambient temperature. The T-PBR consisted of glass tubes (5 cm in
diameter), a centrifugal pump and a degasser, and had a total culture
volume of 10,000 l. The flow rate of culture suspension was 0.7 m s− 1.
Carbon dioxide was introduced into the culture suspension through
bubbling of compressed air containing 1.5% CO2 in the water tank of the
I-PBR and in the degasser of the T-PBR. Temperature and light intensity
were not controlled in the cultures, but were dependent on the pre­
vailing weather conditions.
2.4. Measurement of biomass concentration
The algal biomass concentration was measured by a gravimetric
method. A volume (V) of 5–10 mL culture suspension was filtered
through a pre-dried (dried in an oven set at 105 ◦ C overnight) and pre-
weighed (W0) Whatman GF/C filter membrane (47 mm diameter, 1.2
μm pore size) and washed twice with 10 mL 0.5 M NH4HCO3. Cells on
the filter paper were dried at 105 ◦ C overnight in the oven and were
subsequently cooled to room temperature in a vacuum desiccator before
weighing. This weight was recorded as W1. The biomass concentration
(g L− 1) of algal culture was calculated as (W1 - W0)/V.
2.5. Pigment extraction and analysis
To extract the pigments from O. carolinianum, 10 mg of freeze-dried
algal powder was mixed with 0.4 g micro-glass beads (0.4–0.6 mm) and
2 mL solvent (3:1 mixture of methanol: dichloromethane, v/v). Algal
cells were disrupted by the Fast Prep System (FastPrep-24, MP Bio) that
vibrated at a speed of 6 m s− 1 for 3 cycles. The suspension was centri­
fuged at 14,000g for 10 min at 4 ◦ C and the supernatant was transferred
into a 5-mL brown volumetric vial. One mL solvent (3:1 mixture of
methanol: dichloromethane, v/v) was added into the vial to resuspend
the residues. The resuspension was centrifuged at 14,000g for 10 min,
and the supernatant was transferred into a 5-mL brown volumetric vial.
These steps were repeated until the color of the cell debris became
white. The solvent was then evaporated under N2 flux.
Cellular pigments were identified and quantified by a HPLC method
[17], using commercial pigment standards for chlorophyll a, chlorophyll
b, lutein, astaxanthin, β-carotene, zeaxanthin, canxathanthin,
Y. Wang et al.
violaxanthin and fucoxanthin (purchased from the Sigma company).
Prior to HPLC analysis, pigment samples were dissolved by 1 mL solvent
(3:1 mixture of methanol: dichloromethane, v/v) and filtered through a
nylon membrane with a pore size of 0.22 μm. A 10 μL aliquot of filtered
sample was then injected into a Waters e2695 HPLC system equipped
with a Symmetry C18 reverse phase column (5 mm; 150 × 4.6 mm) and
a Waters 2998 photodiode array detector. The mobile phase consisted of
an eluent A (dichloromethane: methanol: acetonitrile: water,
5.0:85.0:5.5:4.5, v/v) and eluent B (dichloromethane: methanol:
acetonitrile: water, 25.0:28.0:42.5:4.5, v/v). Separation of pigments was
achieved by the following gradient procedures: 0% of B for 8 min; a
linear gradient from 0 to 100% of B within 6 min; 100% of B for 40 min,
at a flow rate of 1.0 mL min− 1. The column temperature was 35 ◦ C and
the flow rate was 1 mL min− 1. Data were acquired by photodiode array
detector over a wavelength range of 270–750 nm. Concentrations of
individual pigments were determined by calibrating HPLC profiles at
664 nm and 445 nm with the chlorophyll and carotenoid standards.
For quantitative analysis of individual molecular species of astax­
anthin, 20 mg of freeze-dried algal biomass was ground in a mortar and
pestle with liquid nitrogen for 2 min, followed by mixing with 3 mL
methanol:dichloromethane (3:1, v/v) [18]. The extract was vigorously
agitated at room temperature for 30 min, and then centrifuged at 1000g
for 10 min. The pigment-containing supernatants were collected. The
extraction process was repeated twice to ensure complete extraction of
the pigments. The solvents were then evaporated from the combined
pigment extracts under nitrogen and the samples were kept in the dark
at − 20 ◦ C until analysis.
The pigment extracts for analysis of astaxanthin were dissolved in 2
mL methanol:dichloromethane (3:1, v/v) containing 0.1% formic acid
for mass spectrometry (MS) analysis. The MS analysis was performed by
using a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters,
US) equipped with an electrospray ion source in positive ion mode.
Nitrogen was used as a nebulizing and drying gas. The flow rate was set
at 450 L h− 1 and the desolvation temperature was 250 ◦ C. The capillary
voltage and cone voltage were 3.0 kV and 40 V, respectively. Argon was
used as the collision gas (25 V) for MS/MS fragmentation. The sample
was infused into the ESI source at a flow rate of 5 μL min− 1.
2.6. Measurement of total cellular fatty acids, carbohydrates and proteins
Algal biomass was harvested by filtration and lyophilized by a freeze-
dry method. The lyophilized algal powders were then stored in the dark
at − 80 ◦ C until their biochemical composition was analyzed. Total
carbohydrate was measured by an improved phenol‑sulfuric acid
method [19]. Total protein was determined by a modified Bradford
method using a Bio-Rad Protein Assay Kit (Cat no. 500-0002). Fatty
acids were determined by direct transmethylation of algal biomass with
methanol to form fatty acid methyl esters (FAMEs). The FAMEs were
identified using a gas chromatography mass spectrometer (GC–MS)
(Agilent 7890B-5977A) and quantified by gas chromatography with a
flame ionization detector (GC-FID) (Agilent 7890B) with a DB-23 col­
umn (60 m × 0.25 mm inner diameter) (Agilent Technologies, Cat no.
J&W 122-2361). The inlet temperature was set at 250 ◦ C with a nitrogen
flow rate of 24 mL min− 1 and a pressure of 18.293 psi. The split ratio was
20:1. The oven temperature was set initially at 50 ◦ C for 1 min and raised
to 175 ◦ C at a rate of 25 ◦ C min− 1. The temperature was set to increase
from 175 ◦ C to 190 ◦ C at a rate of 3.5 ◦ C min− 1 and then to hold at 190 ◦ C
for 5 min. Then the temperature increased from 190 ◦ C to 220 ◦ C at a
rate of 2 ◦ C min− 1 and was held at 220 ◦ C for 1 min, followed by a post
run at 200 ◦ C for 2 min. One run took approximately 35 min. The
temperature of the FID was 250 ◦ C with a hydrogen flow rate of 40 mL
min− 1, air flow rate of 400 mL min− 1 and nitrogen flow rate of 30 mL
min− 1. The FAMEs samples (1 μL) were loaded into the inlet of the
GC–MS using a 10 μL injector. Fatty acids were quantified using a FAMEs
standard (Sigma Aldrich, Cat no. 18919-1 AMP).
3
Algal Research 61 (2022) 102599
2.7. Identification and quantification of microzooplankton contaminants
The initial identification of protozoa and rotifers was conducted
based on morphological characteristics according to Shen et al. [20],
Patterson [21] and Wang [22]. Observations and photomicrography of
specimens were performed with differential interference contrast mi­
croscopy using an Olympus microscope BX53 with an attached Olympus
digital camera DP80 (Olympus, Japan). A 100 μL phytoplankton
counting chamber was used to count the abundance of micro­
zooplankton with an upright microscope (CX-31, Olympus, Japan). Each
sample was counted twice [23], and the following formula [24] was
used to determine final microzooplankton abundance: N = Vs x (N1 +
N2)/(2 x Vj x V), where N is the abundance of zooplankton in the
sample, Vs represents the concentrated volume, Vj represents the
counting volume, N1 and N2 are the two counts, and V is the sampling
volume.
2.8. Statistical analysis
All the experiments were conducted in triplicate cultures and all the
data shown in figures and tables are expressed as mean value ± SD.
3. Results
3.1. Effect of culture conditions on growth and biomass production of
O. carolinianum
Two popular microalgal culture media, BBM and BG-11, were eval­
uated for growth of O. carolinianum. In the case of BBM, in addition to
the original medium formula, which had 3 mmol N as NaNO3, a modi­
fied one with the nitrogen content six times greater was included. As
shown in Fig. 1A, O. carolinianum grown in BBM containing 3 mmol
NaNO3 reached its maximum biomass concentration of 3.47 g L− 1 on
day 16, and an increase in Na2NO3 to 18 mmol in BBM resulted in a
maximum biomass concentration of 4.48 g L− 1. A greater biomass yield
was obtained, however, when O. carolinianum was grown in BG-11
culture medium. The BG-11 medium containing 3 mmol or 18 mmol
NaNO3 resulted in the biomass yields of 4.33 g L− 1 and 5.48 g L− 1,
respectively, during the same time period. The BG-11 culture medium
was therefore chosen for further study.
Growth of O. carolinianum in BG-11 containing different amounts of
NaNO3 (2.3, 4.5, 9.0, and 18.0 mmol N as NaNO3) was investigated. As
shown in Fig. 1B, there were no significant differences in growth during
the early stage of cultivation (from Day 1 to Day 8) between the different
NaNO3 treatments. From the day 9 onwards, the growth in the 2.3 mmol
N cultures leveled off and reached a maximum biomass concentration of
4.6 g L− 1 on day 20. An increase in NaNO3 concentration to 4.5 mmol N
and to 9.0 mmol N resulted in the maximum biomass concentration
increasing to 5.2 and 6.1 g L− 1, respectively. However, a further increase
in NaNO3 concentration produced no more increase in maximum
biomass. As such, 9.0 mmol N as NaNO3 was adopted for further study.
Fig. 2A shows that light intensity significantly affected the growth of
O. carolinianum. Under a photon flux density of 60 μmol photons m− 2
s− 1, O. carolinianum grew fairly well and the final biomass concentration
was 7.3 g L− 1 after 20 days of cultivation. With an increase in photon
flux density from 60 to 120 μmol photons m− 2 s− 1 and then to 250 μmol
photons m− 2 s− 1, the maximum specific growth rate of the cells
increased from 0.60 d− 1 to 0.46 d− 1 and to 0.37d− 1. Further increase in
photon flux density to 500 μmol photons m− 2 s− 1 resulted in a reduced
maximum specific growth rate of 0.36 d− 1, but significantly increased
the final biomass concentration, which was 10.12 g L− 1 on day 20.
During the cultivation, O. carolinianum underwent gradual changes in
color and cell morphology. The color of the organism changed from
green to yellow-green to a reddish color. Yet the higher the light in­
tensity, the quicker the color changes that took place and the deeper the
color of red that occurred in the cells (Fig. 2B). When green, the
Y. Wang et al. Algal Research 61 (2022) 102599

A 7 B 7 Fig. 1. Effects of culture medium type (A) and ni­

18 mmol N BG11
18 mmol N BBM
trogen concentration (2.25, 4.5, 9, and 18 mmol N as
3 mmol N BBM 9 mmol N BG11
NaNO3) in the BG-11 culture medium (B) on growth
6 18 mmol N BG11 6 4.5 mmol N BG11
of O. carolinianum grown in glass columns (5 cm
3 mmol N BG11 2.25 mmol N BG11
inner diameter) operated in a batch culture mode.
Biomass (g L-1)

Biomass (g L-1)
5 5
Data are mean values ± SD of three independent
experiments. Culture mixing was provided by com­
4 4
pressed air containing 1.5% CO2. Culture volume:
750 mL; photon flux density: 60 μmol photons m− 2
3 3
s− 1; temperature: 25 ± 1 ◦ C; pH: 7–7.5.

2 2

1 1

0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Cultivation time (day) Cultivation time (day)

A
12
60 µmol m ² s ¹
120 µmol m ² s ¹
10
250 µmol m ² s ¹
500 µmol m ² s ¹
Biomass (g L-1)

0
0 2 4 6 8 10 12 14 16 18 20 22
Cultivation time (day)

B Day 2 Day 4 Day 10 Day 12 Day 16 Day 18

-2 -1
60 mol m s

-2 -1
120 mol m s

-2 -1
250 mol m s

-2 -1
500 mol m s
Fig. 2. Effects of light intensity on growth (A), morphology and pigmentation (B) of O. carolinianum grown in glass columns (5 cm inner diameter) operated in a
batch culture mode. Data are mean values ± SD of three independent experiments. Culture mixing was provided by compressed air containing 1.5% CO2. Culture
volume: 750 mL; temperature: 25 ± 1 ◦ C; pH: 7–7.5. Scale bars = 20 μm.

4
Y. Wang et al. Algal Research 61 (2022) 102599

vegetative cells were cylindrical to subcylindrical, measuring 13–22 μm
in diameter and 80–140 μm in length. However, as the batch cultures
preceded, the cylindrical vegetative cells became shorter but larger in
diameter. As a result, the red vegetative cells in the filaments measured
25–38 μm in diameter and 40–90 μm in length at the end of the
cultivation.
The biomass concentration of O. carolinianum increased gradually
when the temperature was raised from 18 ◦ C to 28 ◦ C, and the final
biomass concentration increased from 6.4 g L− 1 at 18 ± 1 ◦ C to 9.9 g L− 1
at 28 ± 1 ◦ C on Day 18 (Fig. 3A). The maximum biomass concentration
dropped dramatically when the temperature was raised to 33 ± 1 ◦ C.
The growth ceased shortly after the cultures began when the tempera­
ture was raised up to 38 ◦ C. Temperature also affected the color and
cellular morphology of O. carolinianum. When the O. carolinianum fila­
ments maintained at 25 ◦ C were subjected to various culture tempera­
tures (18, 23, 28, and 33 ◦ C), the cell morphology underwent gradual
change from cylindrical to subcylindrical and then to round or oval in
shape and the color of the cells changed from green to yellowish green to
red. While apparent changes in these features didn't occur until 10 to 12
days of cultivation at 18 ◦ C, they were observed in the 23 and 28 ◦ C
cultures after 4 to 10 days and occurred in the 33 ◦ C cultures after only 2
to 4 days of cultivation (Fig. 3B).
Taken together, the optimal laboratory culture conditions for
obtaining the maximum biomass production of O. carolinianum culture
were as follows; a modified BG-11 medium with 9.0 mmol N as NaNO3, a
culture temperature of 28 ◦ C and continuous illumination at a light in­
tensity of 500 μmol photons m− 2 s− 1. Under such conditions, an average
biomass productivity of 0.5 g L− 1 day − 1 was obtained.
3.2. Biochemical composition of O. carolinianum during cultivation in
glass columns
The biochemical composition of O. carolinianum was studied during
its cultivation in the glass columns and the changes in culture colors are
shown in Fig. 4A. The biomass concentration increased gradually from
an initial concentration of 0.8 g L− 1 to 10.12 g L− 1 after 18 days of batch

A
12
18
23
10 28
33
Biomass (g L-1)

8 38

0
0 2 4 6 8 10 12 14 16 18 20 22
Cultivation time (day)

Day 2 Day 4 Day 10 Day 12 Day 16 Day 18

B 18

23

28

33
Fig. 3. Effects of temperature on growth (A), morphology and pigmentation (B) of O. carolinianum grown in glass columns (5 cm inner diameter) operated in a batch
culture mode. Data are mean values ± SD of three independent experiments. Culture mixing was provided by compressed air containing 1.5% CO2. Culture volume:
750 mL; photon flux density: 500 μmol photons m− 2 s− 1; pH: 7–7.5. Scale bars = 20 μm.

5
Y. Wang et al. Algal Research 61 (2022) 102599

A B 12

10

Biomass (g L-1)
8

0
0 30 days 0 2 4 6 8 10 12 14 16 18 20 22 30
Cuitivation time (day)
C 80 D 12
Carbohydrates (% dwt)

10
60

Proteins (% dwt)
8

40 6

4
20
2

0 0
0 8 14 18 22 30 0 8 14 18 22 30
Cultivation time (day) Cultivation time (day)
E 50 F 1.2
FAMEs (% dwt)

Astaxanthin (% dwt)

40
0.9
30
0.6
20
0.3
10

0 0
0 8 14 18 22 30 0 8 14 18 22 30
Cultivation time (day) Cultivation time (day)

Fig. 4. Growth and biochemical composition of O. carolinianum grown in glass columns. (A) Appearance of O. carolinianum cultures in glass columns during a 30-day
period; (B) growth curve; (C) total carbohydrates (% dw), (D) total proteins (% dw); (E) fatty acid methyl esters (FAMEs) content (% dw); (F) astaxanthin (% dw).
Data are mean values ± SD of three independent experiments. Culture volume: 750 mL; photon flux density: 500 μmol photons m− 2 s− 1, 25 ± 1 ◦ C.

culture and then leveled off thereafter (Fig. 4B). The total carbohydrate
increased gradually from 44.9% to 64.9% during the first 14 days of
cultivation and then reduced slowly to 48.6% on Day 30 (Fig. 4C). The
cellular protein content of the cells was 10.4% (w/w) at the start of the
culture and decreased rapidly to 1.5% during the first 14 days
decreasing further to 0.6% on Day 30 (Fig. 4D). In contrast, the total
lipid content, as represented by fatty acid methyl esters (FAMEs),
increased rapidly from 10.7% to 30.6% during the first 14 days and
continued increasing to 39.5% on Day 30 (Fig. 4E). The most stricking
feature, however, was the occurrence and accumulation of astaxanthin
during the batch culture. While little was detected in the green vege­
tative cells in the early stage of batch culture, astaxanthin occurred and
accumulated in the cells as the batch culture proceeded and reached an
astaxanthin content of 1.03% (w/w) after 30 days of cultivation
(Fig. 4F).
3.3. Determination of molecular species of astaxanthin
Free astaxanthin from O. carolinianum was detected as its protonated
ion [M+H]+ at m/z 597, accompanied by [MH-H2O]+ at m/z 579
(Fig. 5A). The ESI-MS/MS spectra of astaxanthin fatty acyl monoesters
(ME) displayed a precursor ion [ME+H]+ along with fragmentation
products [ME+H-FA]+. As an example, Fig. 5B shows a typical mass
spectrum of astaxanthin monoester ME-C16:0 in which m/z 835 and m/z
579 were assigned as the precursor [ME(C16:0)+H]+ and as a fragment
ion with the palmitic acid eliminated [MH- C16:0]+, respectively. The
fragment ion at m/z 817 resulted from the loss of a water molecule (i.e.
[MH-H2O]+). The fragmentation patterns of astaxanthin fatty acyl di­
esters (DEs) were similar to those of MEs. As shown in Fig. 5C, DE-
C16:0/C18:1 displayed [DE(C16:0/C18:1)+H]+ at m/z 1100, which
generated the fragment ions m/z 843 ([DH- C16:0]+) and m/z 817 ([DH-
C18:1]+), respectively by the neutral loss of one fatty acid. The ion at m/z
561 ([DH- C18:1- C16:0]+) and m/z 579 ([DH+H2O- C18:1- C16:0]+) were
yielded through the loss of two fatty acids. Moreover, the ions reflecting

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Y. Wang et al. Algal Research 61 (2022) 102599

Fig. 5. The product-ion spectrum of the [M + H]+ ion of free astaxanthin from O. carolinianum at m/z 597 (A), monoester-C16:0 at m/z 835 (B), and diester-C16:0/
C18:1 at 1100 (C).

the astaxanthin backbone were detected at m/z 147, 173, 201 and 219,
Table 1
which are therefore deemed as the characteristic ions for identification
Molecular species of astaxanthin in Oedocladium. carolinianum and Haemato­
of free astaxanthin, astaxanthin monoesters and diesters.
coccus pluvialis. ME, astaxanthin monoester; DE, astaxanthin diester.
By employing the ESI-MS/MS method developed in this study, the
O. carolinianum Haematococcus pluvialis
molecular species of astaxanthin in free and esterified forms were
identified from O. carolinianum and the unicellular green microalga m/z Compound m/z Compound
H. pluvialis (Table 1). The astaxanthin esters profile of O. carolinianum 597.4 Free astaxanthin 597.4 Free astaxanthin
was distinguished from that of H. pluvialis by its ME-containing odd- 801.5 ME 14:3 835.6 ME 16:0
numbered fatty acyl groups, such as C15:1, C15:2, C17:2 and C17:1. In 803.7 ME 14:2 855.6 ME 18:4
817.7 ME 15:2 857.6 ME 18:3
contrast, the major MEs of H. pluvialis were C16:0, C18:1, C18:2, C18:3
819.7 ME 15:1 859.6 ME 18:2
along with C18:4. The profile of DE was very similar between 841.7 ME 17:4 861.6 ME 18:1
O. carolinianum and H. pluvialis, both of which contained C16 and C18 843.8 ME 17:3 1074.1 DE 16:0/16:0
acyl groups. It is noteworthy that the relative abundances of MEs and 845.8 ME 17:2 1094.0 DE 16:0/18:4
DEs were dramatically different between O. carolinianum and 847.8 ME 17:1 1096.0 DE 16:0/18:3
1072.1 DE 16:0/16:1 1098.1 DE 16:0/18:2
H. pluvialis, with the former containing more DE than the latter (Fig. 6). 1074.0 DE 16:0/16:0 1100.1 DE 16:0/18:1
In a comparative study, the astaxanthin profiles of H. pluvialis red 1096.0 DE 16:0/18:3 1120.1 DE 18:2/18:3
cells, and green and red O. carolinianum cells were obtained. As shown in 1098.1 DE 16:0/18:2 1122.2 DE 18:2/18:2
Fig. 6, three forms of astaxanthin (i.e., free, mono- and di-ester forms of 1100.1 DE 16:0/18:1 1122.2 DE 18:1/18:3
1122.1 DE 18:1/18:3 1124.1 DE 18:1/18:2
astaxanthin) were detected in O. carolinianum cells, accounting for 1%,
1124.1 DE 18:1/18:2 1126.1 DE 18:1/18:1
18% and 81% of total astaxanthin, respectively. In H. pluvialis red cells, 1126.1 DE 18:1/18:2
however, the ratio of free, mono-esters and diesters of astaxanthin were 1128.0 DE 18:1/18:1
1%, 81% and 18%, respectively. In other words, O. carolinianum pro­
duces di-esters as the major astaxanthin species, whereas Haematococcus
synthesizes the predominant astaxanthin species as mono-esters. 3.4. Determination of fatty acids in O. carolinianum

A total of 12 fatty acid species were detected in O. carolinianum. The

major fatty acid species were C16:0, C18:1trans (n9), C18:2cis and

7
Y. Wang et al.
Fig. 6. Mass spectra of astaxanthin esters of O. carolinianum (A) and Haematococcus pluvialis (B) identified with parent scanning for m/z 173.
C18:3n3, and the minor ones were C14:0, C16:1, C16:2, C16:3, C18:0,
C18:1trans (n11) and C18:3n6. The monounsaturated fatty acids
(MUFA) and polyunsaturated fatty acids (PUFA) made up over 80% of
the total fatty acids (TFA) and yet the MUFA increased from ca. 13% to
ca. 53% of TFA during the cultivation period (Table S2).
3.5. Effect of nitrogen availability and salinity on total lipid content of
O. carolinianum
When O. carolinianum was grown in a BG11 culture medium con­
taining 9.0 mmol N as NaNO3, the cellular lipid content in vegetative
cells increased from below 15% (w/w) to 41.4% (w/w) during a 16 day
batch culture (Table S2). However, when the alga was grown in a BG-11
medium in the absence of nitrogen, the total lipid content after 16 days
of cultivation was lower: 32.16% (w/w) under otherwise the identical
culture conditions (Table S3). Likewise, a BG11 culture medium con­
taining 2 g L− 1 NaCl also resulted in a lower lipid content to 30.81% (w/
w) after 16 days of cultivation (Table S4). On the other hand, the general
trends in the lipid content and fatty acid profiles of the vegetative cells
over the 16 days of cultivation under these culture conditions were
essentially the same.
3.6. Preliminary trials of the mass culture of O. carolinianum in an
inclined thin layer PBR (I-PBR) and a tubular PBR (T-PBR)
We hypothesized that the astaxanthin productivity of O. carolinianum
may be maximized by adopting a two-stage cultivation. The first stage
should be to sustain rapid growth of the organism under the favorable
culture conditions to maximize biomass productivity. As the vegetative
cells were green, we also called this stage the green stage. The second
stage would be to induce astaxanthin biosynthesis under stress condi­
tions to obtain a maximum cellular content of astaxanthin as quickly as
possible. Since the biomass produced at the end of this second stage was
red in color, we also called the second stage the red stage.
To test this hypothesis, the first, green-stage cultivation was carried
out both in a 7000-liter I-PBR (Fig. 7A) and in a 10,000-liter T-PBR
(Fig. 7B) in a greenhouse. The daylight solar intensities in the green­
house and daily culture temperatures are shown in Fig. 7C & D. During
the cultivation period, the mean solar irradiance in the greenhouse was
293 μmol photons m− 2 s− 1, which was just one-third of that measured
outdoors during the same time period (mean value of 925 μmol photons
m− 2 s− 1; data not shown). The culture temperatures varied from 12 ◦ C to
35 ◦ C, with a mean value of 15 ◦ C during the same period of time.
8
Algal Research 61 (2022) 102599
Compared to the T-PBR, the I-PBR experienced lower temperatures
during the day and lower night temperatures due to faster heat loss from
the culture suspension in this type of open culture system. Under such
circumstances, the alga grew well in both types of photobioreactors, but
the growth was more rapid in the I-PBR than in the T-PBR, probably due
to the shorter light path (ca. 3 cm) and greater flow rate (~80 cm s− 1) of
the I-PBR compared to the T-PBR (light path 5 cm and flow rate 50 cm
s− 1 for the T-PBR). As a result, the maximum biomass concentrations in
the I-PBR and the T-PBR were 3.74 g L− 1 and 3.07 g L− 1, respectively
(Fig. 7E).
3.7. Induction of astaxanthin production at the red stage of cultivation
In order to maximize astaxanthin production at the red-stage culti­
vation of O. carolinianum we determined nitrogen deficiency could
induce or accelerate astaxanthin biosynthesis in this organism.
Compared to a nitrogen replete (i.e., 9.0 mmol N as NaNO3) culture, the
final algal biomass concentration in a nitrogen depleted culture grown in
glass columns was lower by ca. 30% at the end of cultivation. However,
the cellular astaxanthin content in the nitrogen depleted culture
increased by ca. 70%, from 1.2% to 2.1% astaxanthin (w/w) (Fig. 8).
A common strategy for induction of carotenoid biosynthesis in
microalgae is to introduce salinity stress in the culture. In this study, we
conducted a preliminary study on the effect of various NaCl concen­
trations (0.5, 1, 2, 4, and 6 g L− 1) on growth and astaxanthin accumu­
lation of O. carolinianum and revealed that 2.0 gL− 1 NaCl induced
massive accumulation of astaxanthin at the minimum expense of growth
reduction, whereas the other NaCl concentrations either stimulated less
cellular astaxanthin production or caused more severer growth inhibi­
tion. Therefore, we chose 2 g L− 1 NaCl as the astaxanthin inducer for the
experiment. When 2 g L− 1 NaCl was added to a nitrogen depleted cul­
ture, growth of O. carolinianum was reduced further by ca. 70%,
compared to the nitrogen replete culture, indicating that the culture was
under salinity-induced stress (Fig. 8A). On the other hand, the maximum
cellular astaxanthin content was 3.91% (w/w), which was an increase of
more than 3-fold in astaxanthin content in the cells relative to the ni­
trogen replete culture (Fig. 8B). We concluded that both nitrogen
depletion and salinity stress can significantly stimulate astaxanthin
biosynthesis at the expense of growth in the O. carolinianum cultures.
From a quantitative standpoint, a reverse correlation between algal
growth and astaxanthin biosynthesis was evident: the more severe the
stress-induced growth reduction, the greater the formation of astax­
anthin in O. carolinianum cells. From a practical application perspective,
Y. Wang et al. Algal Research 61 (2022) 102599

A B

C D
1200 42
Light intensity (µmol m-2 s-1)

1000 35

Temperature ( )
800 28

600 21

400 14

200 7

0 0

Cultivation time (date) Cultivation time (date)

E 4
7000 L thin-layer PBR
10000 L tubular PBR
Biomass (g L-1)

Cultivation time (date)

Fig. 7. A 7000-liter inclined thin layer photobioreactor (A) and a 10,000-liter glass tubular photobioreactor (B) in a greenhouse, as used for mass culture of
O. carolinianum. The design details of the two culture systems are provided in the ‘Materials and methods’ section. (C) Solar irradiance during the daylight periods;
(D) culture temperature throughout the experimental period;(E) biomass concentration of O. carolinianum grown in a 7000-liter inclined thin layer photobioreactor
and a 10,000-liter glass tubular photobioreactor in a greenhouse. Error bars represent standard deviation of two independent pilot-scale cultivation trials (n = 2).

however, a compromise may need to be applied in order to obtain
maximum sustainable astaxanthin production from mass culture of this
organism. In this study, the maximum astaxanthin productivity of 24.2
mg L− 1 d− 1 was obtained from O. carolinianum cultures operated in the
two-stage culture mode.
3.8. Biodiversity and impact of zooplankton contaminants in
O. carolinianum cultures in a greenhouse
During the course of the study, a few zooplankton were observed in
the mass culture of O. carolinianum in both types of photobioreactors in
greenhouse and outdoors. In totally, 8 species of zooplanktonic

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Y. Wang et al. Algal Research 61 (2022) 102599

A B
10 0 mmol N + 2 g L ¹ NaCl 5 0 mmol N + 2 g L ¹ NaCl
0 mmol N 0 mmol N
9 mmol N 9 mmol N
8 4

Astaxanthin content (%dw)

Biomass (g L-1)
6 3

4 2

2 1

0 0
0 2 4 6 8 10 12 14 16 0 4 8 12 14 16
Cultivation time (day) Cultivation time (day)

Fig. 8. Effects of nitrogen availability (0 or 9 mmol/L nitrogen as NaNO3) and salinity (2 g L− 1 NaCl) on growth (A) and astaxanthin content (B) of O. carolinianum
grown in glass columns (5 cm inner diameter) operated in batch culture mode. Data are mean values ± SD of three independent experiments. Culture volume: 750
mL; photon flux density: 500 μmol photons m− 2 s− 1; temperature: 28 ± 1 ◦ C; pH: 7–7.5; and 1.5% CO2.

contaminants were observed, including 2 amoebae, 3 ciliates, and 3
rotifers (Fig. 9). However, the populations of these zooplankton
remained small, with the numbers of rotifers and amoebae being less
than 50 and 150 counts mL− 1, respectively, while those of Vorticella and
ciliates were less than 150 and 450 counts mL− 1. Interestingly, none of
these grazers grazed on O. carolinianum filaments.
4. Discussion
In this study, we provide the first report of a filamentous microalga,
O. carolinianum (Oedocladium, Oedogoniales, Chlorophyta), producing
astaxanthin in quantities as high as 3.91% (w/w) under nitrogen
depleted or 2 g L− 1 NaCl-induced stress conditions. Among a limited
number of microalgae found in nature that possess the ability to produce
astaxanthin, O. carolinianum is the second most productive species after
the unicellular microalga H. pluvialis, and the astaxanthin content of
O. carolinianum can be 3 to 20 times greater than that reported in the
unicellular microalgae Chlorella zonfigiensis and Chlorococcum sp.
(Table 2).
Multiple forms of astaxanthin-free, mono-esters and di-esters-have
been found in astaxanthin-producing microorganisms. For a given
microorganism, however, the form and composition of astaxanthin are
relatively stable. While astaxanthin in the heterobasidiomycetous yeast
Xanthophyllomyces dendrorhous (formerly named Phaffia rhodozyma) is
in a free form with the (3R,3′ R)- isomer [25], the majority of astaxanthin
in microalgae is in fatty acid esterified forms [26,27]. It has been re­
ported that astaxanthin esters have a higher thermal stability and higher
bioavailability than free-form astaxanthin [28]. In the unicellular
microalga H. pluvialis, the ratio of monoesters, diesters and free form of
astaxanthin was found to be 81:18:1, whereas in our study

A B C D

E F G H I

Fig. 9. Zooplankton that occurred in O. carolinianum cultures grown in a 7000-liter inclined thin layer photobioreactor and 10,000-liter glass tubular photo­
bioreactors in a greenhouse, as observed under light microscopy with differential interference contrast. A-B. Centropyxis sp.; C. Mayorella sp.; D. Epistylis sp.; E.
Vorticella convallaria; F. Sterkiella sp.; G. Lecane sp.; H. Philodina sp.; I. Monostyla sp.. Scale bars: 10 μm for images A, B, C, E, I; 50 μm for images D, F, H.

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Y. Wang et al. Algal Research 61 (2022) 102599

Table 2
Comparison of trophic mode, culture volume, biomass yield, and content and productivity of astaxanthin in O. carolinianum and other astaxanthin-producing
microalgae (H. pluvialis, Chlorella zofingiensis and Chlorococcum sp.).
Organism Culture Culture volume Cultivation time Dry weight (g Astaxanthin content (% Astaxanthin productivity (mg References
conditions (L) (days) L− 1) dwt) L− 1 d− 1)

H. pluvialis
NIES-144 M, perfusion 3 38 12.3 4.9 15.8 [13]
ZY-18 SHDP 0.005 32/40 26 4.6 6.4/4.5 [14]
UTEX2505 P, M, fed - batch 0.15 28 7.57 4.25 10.2 [15]
JUN35 P, two-stage 0.3 30 20.1 2.7 18.1 [8]
batch

C. zofingiensis
ATCC30412 H, fed - batch 3 8 45.6 0.12 5.6 [16]
ATCC30412 M, two-stage 4 18 98.4 0.08 5.2 [12]
batch
ATCC30412 P, batch 0.2 6 7.2 0.6 7 [17]
Chlorococcum P, batch 1 16 9 0.2 1.2 [18]
sp.
Chlorococcum M, batch 0.5 7 10.1 0.7 10.2 [19]
sp.
O. carolinianum P, batch 1.0 16 4.10 3.91 24.2 This study

P, photoautotrophic culture; H, heterophic culture; M, mixotrophic culture; batch, a culture process where all the nutrients are added into the culture medium at once
and no extra nutrients are fed to the culture vessel from the beginning to the end of the cultivation; fed-batch, a batch culture mode where one or more nutrients were
fed to the culture vessel during cultivation and in which the algal biomass remains in the culture vessel until the end of the culture; two-stage batch, the first stage under
normal culture conditions, followed by the second stage under stress of some kind.

O. carolinianum synthesized mostly astaxanthin diesters with a mono­
ester, diester and free astaxanthin ratio of 18:81:1. As the thermal sta­
bility of astaxanthin diesters is known to be greater than monoesters and
free astaxanthin [29–32], it is anticipated that astaxanthin derived from
O. carolinianum could be more stable and thus offer a longer product
shelf life than that from H. pluvialis.
Like other astaxanthin-producing unicellular microalgae,
O. carolinianum produces astaxanthin only in the late stage of a batch
culture when the nutrient availability is no longer adequate to sustain
rapid algal growth, and yet greater amounts of astaxanthin can be
produced under conditions of stress, such as under high light intensity,
high salinity, or nitrogen starvation. Under nitrogen depleted condi­
tions, the maximum volumetric astaxanthin productivity of 24.2 mg L− 1
d− 1 obtained from O. carolinianum is the highest among the astaxanthin-
producing microalgae reported thus far under phototrophic, heterotro­
phic or mixotrophic culture conditions (Table 2).
With the exception of H. pluvialis, which is commercially produced in
large-scale open raceway pounds and closed photobioreactors of various
designs with solar irradiance or artificial light as the energy source
[33,34], the exploitation of all other astaxanthin-producing microalgae,
such as Chlorella zofingiensis and Chlorococcum sp., has never gone
beyond small-scale cultures of several to tens of liters in volume under
laboratory conditions. In this study, we have moved one step further by
growing O. carolinianum in the 7000-liter inclined thin layer PBR and the
10,000-liter tubular PBR in a greenhouse. It appeared that this fila­
mentous alga could withstand the shear force generated by the me­
chanical pump circulating the algal suspensions in the two different
culture systems. We observed that there was no apparent difference
between the cultivation of O. carolinianum and H. pluvialis in terms of
effectiveness and energy consumption associated with culture mixing.
This demonstrated the technical feasibility of mass culture of
O. carolinianum in both open and closed photobioreactors. Moreover,
O. carolinianum forms branched filaments and can be cost-effectively
harvested and dewatered by a simple filtration or sedimentation
method. This is in contrast to the unicellular astaxanthin-producers (e.
g., H. pluvialis, Chlorella zofingiensis and Chlorococcum sp.), for which
expensive centrifugation or use of a plate-and-frame filter press is
required for algal cell harvesting and dewatering [2,11,35].
Zooplankton contamination (e.g., by amoebae, ciliates, rotifers,
parasitic fungi) is a major threat to the mass culture of unicellular
microalgae [36–38]. For example, H. pluvialis is extremely susceptible to
infection by the parasitic fungus Paraphysoderma sedebokerense (Blasto­
cladiomycota). Once this fungus occurs in a culture of H. pluvialis, the
culture may deteriorate rapidly, resulting in a considerable reduction in
biomass and astaxanthin production or, in some cases, culture crash
within a few days if not sooner [12,39]. In this study, O. carolinianum
grown both in the 7000-liter I-PBR and in the 10,000-liter T-PBR was
highly resistant to zooplankton contamination, which we assume is due
to the multicellular filamentous nature of the organism. The fact that
some of the zooplankton (Fig. 9A–C, E–G) did indeed grazed on uni­
cellular microalgal cells but not O. carolinianum suggests that these
zooplankton may actually help to ensure a healthy culture of the fila­
mentous organism by aggressively grazing invading unicellular micro­
algae. This is another advantage of the mass culture of O. carolinianum
for sustainable production of astaxanthin over its unicellular microalgal
counterparts. A greater resistance of filamentous microalgae to micro­
bial contamination has also been observed in cultivation of the fila­
mentous yellow-green microalgae Tribonema spp. [40] and the
filamentous green microalga Klebsormidium sp. LGX80 [41].
As well as being a producer of astaxanthin, O. carolinianum is also an
oleaginous organism and the lipid content in the cells can be as high as
41.4% (w/w). Unlike many unicellular oleaginous microalgae that
synthesize and accumulate large amounts of lipids only under nitrogen
depleted or salinity stress conditions [19,42], nitrogen repletion facili­
tated lipid production in O. carolinianum. Nitrogen depletion or salinity
stress induced by 2 g L− 1 NaCl resulted in 22% and 25% reduction in the
lipid content, respectively, compared to that obtained from the nitrogen
replete culture of O. carolinianum. This unusual phenomenon has also
been observed in some Tribonema species (Xanthophyceae) [43], in
which the cellular lipid content was found to be considerably higher in
the nitrogen replete cultures than that in the nitrogen depleted ones
[44].
The potential for a high lipid content in O. carolinianum may offer a
great opportunity for this organism to be used as an oil-rich feedstock for
animal feed application. Recently, Chen et al. (2019) investigated the
effects of O. carolinianum as a feed ingredient on growth performance,
tissue fatty acid profiles, and immunity of gibel carp. Compared to the
control, the treatment diet containing 40 g Kg− 1 O. carolinianum had
significant effects on tissue fatty acid profiles, antioxidant capacity and
immunity without compromising growth and body composition. The
addition of O. carolinianum significantly increased the contents of n-3
polyunsaturated fatty acids EPA and DHA and the ratio of n-3/n-6

11
Y. Wang et al.
polyunsaturated fatty acids, and simultaneously decreased the n-6
polyunsaturated fatty acids profile in fish muscles. Furthermore, the
addition of O. carolinianum significantly increased the total superoxide
dismutase activity level and the complement 3 and immunoglobulin M
contents in the plasma of the fish [45]. More research along these lines
will be carried out in order to confirm the value of O. carolinianum in
improving the quality of fish flesh quality and increasing the immunity
of fish and perhaps also other aquaculture animals.
5. Conclusion
O. carolinianum is the first filamentous microalga found to produce
astaxanthin in quantities as high as 3.91% (w/w) under nitrogen
depleted and salinity stress conditions. It is also the second richest
natural source of astaxanthin after H. pluvialis in the alga kingdom. The
volumetric astaxanthin productivity of 24.2 mg L− 1 d− 1 obtained from
O. carolinianum culture is the highest among the astaxanthin-producing
microalgae reported thus far. Owning to its potential for rapid growth,
high astaxanthin and lipid contents, resistance to microbial contami­
nation and apparent ease for algal harvesting, O. carolinianum could be a
superior strain for production of astaxanthin and lipids.
CRediT authorship contribution statement
Yu Wang: Investigation and writing- Original draft preparation.
Jing Jia: investigation – biochemical analyses of Oedocladium
carolinianum.
Qinglei Chi: Investigation: photobioreactor designs and fabrication.
Yanhua Li: investigation – LC/MS analysis of molecular astaxanthin
species.
Hongxia Wang: investigation – algal culture.
Yingchun Gong: investigation – microzooplankton contamination
assessment.
Guoxiang Liu: isolation and classification of Oedocladium
carolinianum.
Zhengyu Hu: Conceptualization and investigation – microalgal
taxonomy.
Danxiang Han: methodology – development of quantitative GC/MS
and LC/MS methods.
Qiang Hu: Conceptualization, supervision, manuscript reviewing
and Editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the State Development & Investment
Corporation (SDIC) of China. Thanks are due to Baolong Fu and Fenghai
Wang at the SDIC Microalgae Biotechnology Center, SDIC Biotechnology
Investment Co. Ltd., for helping in the mass culture of O. carolinianum in
the greenhouse and outdoors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.algal.2021.102599.
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