See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/348464800

Isolation and optimization of a novel thraustochytrid strain for DHA rich and
astaxanthin comprising biomass as aquafeed supplement

Article in 3 Biotech · February 2021

DOI: 10.1007/s13205-020-02616-4

CITATIONS READS

15 201

5 authors, including:

Pratik Pawar Sujata Kumari

Institute of Chemical Technology, Mumbai Imperial College London
6 PUBLICATIONS 63 CITATIONS 6 PUBLICATIONS 38 CITATIONS

SEE PROFILE SEE PROFILE

Gunjan Prakash
Institute of Chemical Technology, Mumbai
37 PUBLICATIONS 671 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Regulation of carotenoid biosynthesis in Chlamydomonas reinhardtii View project

All content following this page was uploaded by Gunjan Prakash on 03 November 2022.

The user has requested enhancement of the downloaded file.

3 Biotech (2021) 11:71
https://doi.org/10.1007/s13205-020-02616-4

ORIGINAL ARTICLE

Isolation and optimization of a novel thraustochytrid strain for DHA

rich and astaxanthin comprising biomass as aquafeed supplement
Pratik R. Pawar1 · Sneha Velani1 · Sujata Kumari1 · Arvind M. Lali2 · Gunjan Prakash1

Received: 16 May 2020 / Accepted: 26 December 2020

© King Abdulaziz City for Science and Technology 2021

Abstract
A marine organism, belonging to the Thraustochytrids family was isolated from mangroves of Mumbai, India. The isolated
strain was identified as Aurantiochytrium limacinum by internal transcribed spacer sequence analysis. Optimization of pro-
cess parameters yielded 14.47 g/L dry cell weight containing 55–58% oil in 3.5 days’ cultivation on glucose, yeast extract,
and peptone in the bioreactor. Docosahexaenoic acid (DHA) was found to be the dominant long-chain polyunsaturated fatty
acid, accounting for 32–35% of total fatty acid content. The process parameter was tweaked to simultaneously synthesize
astaxanthin along with DHA. The concurrent synthesis of DHA and astaxanthin-containing biomass establishes the isolated
strain as a perfect choice for aquafeed.
Accession number: NCBI accession number MN046792.

Keywords Aurantiochytrium limacinum · Biomass · ITS · PUFAs · Astaxanthin

Introduction membranes. Docosahexaenoic acid (DHA; 22:6), one of the

LC-PUFA, comprises up to 60% of the total fatty acids of the
The growing global population has created a remarkable retina and 15–20% of the cerebral cortex (Jakobsen 2008;
challenge for the availability of the nutritious, safe, and high- Carlson and Colombo 2016). DHA has also been found use-
quality food supply to the masses. Seafood is a rich source of ful for the prevention of multiple ailments like arteriosclero-
protein, vitamins, minerals, and omega-3 (ω-3) long-chain sis, coronary heart disease, cardiovascular disease, cancer,
polyunsaturated fatty acids (LC-PUFA). LC-PUFA has a inflammation, schizophrenia, and Alzheimer’s (Das 2003;
vital effect on human and animal nutrition (Barclay et al. Mohebi-Nejad and Bikdeli 2014). The human body cannot
1994; Siriwardhana et al. 2012). They play an important synthesize LC-PUFA in the required amount. Plant-based
role in early neural as well as retinal development. They oils, a primary fats source in the human diet also does not
are one of the major structural components in animal cell contain these LC-PUFAs (Jakobsen 2008) and, therefore,
their inclusion from the external sources become imperative.
* Gunjan Prakash The major sources of LC-PUFA have been marine fish
g.prakash@ictmumbai.edu.in like salmon, sardines, tuna, and mackerel. Fish oil is cur-
Pratik R. Pawar rently the conventional and commercially available source
pratik.pawar35@gmail.com for pure DHA. However, extensive purification methods are
Sneha Velani needed for purifying DHA from fish oil. The usage of fish oil
sneha.sebiotech@gmail.com as a DHA source is also linked with problems like the typical
Sujata Kumari fishy smell, unpleasant taste, concern raised by vegetarians,
soni1507@gmail.com and possible contamination by heavy metals (Jakobsen 2008;
Arvind M. Lali Gupta et al. 2012). Fishmeal and fish oil are also two main
am.lali@ictmumbai.edu.in products contributing all the essential nutrients including
ω-3-PUFAs for aquaculture. Due to the decline in fishmeal
1
DBT‑ICT Centre for Energy Biosciences, Institute and fish oil production in recent years, aquaculture industries
of Chemical Technology, Mumbai, India
have started utilizing plant-derived oil and meals such as
2
Department of Chemical Engineering, Institute of Chemical soy-based products. However, plant-based oils are devoid
Technology, Mumbai, India

13
Vol.:(0123456789)
 71 Page 2 of 10
of ω-3-PUFAs; therefore, their addition cannot fortify the
aquafeed with essential PUFA content. Therefore, there is an
imminent need for the inclusion of a source material which
is enriched in LC-PUFA for the aquaculture (Jaseera and
Kaladharan 2019).
A class of protist known as thraustochytrids is the source
of LC-PUFA in the marine ecosystem (Yang 2011). They
were formerly considered to be closely related to fungi and
protozoans. However, recent molecular phylogenetic stud-
ies have resulted in their classification to the class Laby-
rinthulomycota in phylum Heterokonta within the kingdom
Chromista (Mendes et al. 2009). A variety of these thraus-
tochytrids are known to produce LC-PUFAs and have gained
considerable importance in recent years. For example, the
heterotroph dinoflagellate Crypthecodinium cohnii is used
for the commercial production of DHA (Jakobsen 2008).
C. cohnii produces only DHA as PUFA in its intracellu-
lar lipid, which makes the purification process economical,
especially for pharmaceutical and nutraceutical applications
(Yang 2011). Different strains of thraustochytrids accumu-
late a high content of triacylglycerols (50–80% of dry bio-
mass) with a high amount of LC-PUFAs (Fan et al. 2001;
Jakobsen 2008). The oil derived from the thraustochytrids or
their biomass itself can be an effective alternative to fish oil
and aquafeed supplement, respectively. Thraustochytrids are
also capable of producing other high-value compounds like
squalene and carotenoids, most prominently the astaxanthin
(Raghukumar 2008). The astaxanthin is a natural colorant
and known to be a potent antioxidant molecule; therefore,
its presence in aquafeed can be helpful in enhancing the
visual appearance and nutritional value of fishes. However,
the pure astaxanthin is an expensive carotenoid (~ 200–250
$/kg (IndiaMart, India) and its inclusion at its current price
would disturb the overall fish feed economics. Therefore,
despite the known advantages, astaxanthin has not become
part of the aquafeed. Thraustochytrid biomass containing
both LC-PUFA and astaxanthin can be a valuable source as
aquafeed in the aquaculture industry.
For use in aquaculture, a strain of thraustochytrids should
have the characteristics of higher growth rate along with
lipid and preferably astaxanthin-accumulating capability. For
this, there is a need to screen different strains and optimize
their growth conditions to maximize biomass and oil con-
tent. There are multiple strains of thraustochytrids known
till date (Bajpai et al. 1991; Barclay et al. 1994; Singh and
Ward 1996; Burja et al. 2006; Jakobsen 2008); however,
most of these strains are patent protected, thus restricting
their use by the patent holder. Also, not all strains of thraus-
tochytrids produce astaxanthin and the culture conditions
needed to produce astaxanthin are different from that of
LC-PUFA synthesis. In the present study a screening exer-
cise for the identification and characterization of a novel
thraustochytrid strains from mangrove areas in India was
13
3 Biotech (2021) 11:71
undertaken. Further, process parameters were optimized for
simultaneous production of LC-PUFA and astaxanthin to
make it a potential aquafeed supplement.
Materials and methods
Thraustochytrid isolation and growth conditions
Thraustochytrid strains were isolated from different man-
grove areas in Mumbai, India. Leaves, soil, and water sam-
ples were collected from areas like Vikhroli, Marve, Bhay-
ander, Gorai, Bandra, Sewri, Nerul (Mumbai, India), and
Nargol (Gujarat, India) (Table 1). During collection, the
samples were kept on ice and were processed within 24 h.
After the water wash, the leaves were soaked in 0.1% mer-
curic chloride for 30 s. The samples were inoculated with
antibiotics; Rifampicin (50 mg/L), Nystatin (10 mg/L), Peni-
cillin G (300 mg/L), and streptomycin sulfate (500 mg/L) in
different combinations (Table 1). The flasks were incubated
in a shaker at 180 rpm and maintained at 28 °C. Simultane-
ously, isolates were also maintained on the solid medium
containing 1% glucose, 0.1% yeast extract and peptone
each, 0.01% ­KH2PO4 (GYPK), 0.8% agar and sub-cultured
monthly. After screening, the isolate was grown in 100 ml
flasks with GYPK liquid medium in 100% natural seawater.
Genetic identification
The genomic DNA of the selected strain was isolated
according to the cetyl trimethyl ammonium bromide (CTAB)
extraction protocol (Rathod et al. 2020). PCR isolation of
18S was done with FA1 and RA1 primers described by Mo
et al. (2002) with cycling conditions 95 °C for 5 min fol-
lowed by 35 cycles of 95 °C for 30 s, 45 °C for 30 s, and
72 °C for 30 s, followed by 72 °C for 5 min. ITS2 sequence
was isolated by PCR with primer pairs Aur F (GTA​GCA​TTC​
CTG​CTT​GAG​TGT​TTG​) and Aur R (CTT​CTT​TCC​AAC​
ATG​AGG​TTTGA), Sc F (GAG​TAT​TCC​TGC​ATG​AGT​
GTTTG) and Sc R (CTC​TTA​CCT​GAC​ATG​AGG​TTTGC)
or Th F (GAG​TAT​TCC​TGT​TTG​AGT​GTTTG) and Th R
(TCC​TCT​TTC​CTG​ATT​TGA​GGT​TTG​) with cycling con-
ditions 95 °C for 5 min followed by 35 cycles of 95 °C for
30 s, 45 °C for 30 s, 72 °C for 30 s, and subsequently 72 °C
for 5 min. Isolated fragments were cloned in pTZ57R/T and
sequenced by Sanger sequencing (SciGenom, India).
Substrate optimization
Various substrates such as glucose, xylose, lignin, glyc-
erol, and sodium acetate were screened for A. limacinum
growth and lipid accumulation. Initially, each substrate
was taken at 10 g/L concentration. Various organic (yeast
3 Biotech (2021) 11:71 Page 3 of 10 71

Table 1  Various media Location Isolation medium Antibiotics

concoctions used for
isolation and screening of
Thraustochytrid strains
Marve GYP a
Penicillin G (1000 U/ml)
Gorai Streptomycin sulphate (300 mg/L)
Amphotericin B (2.5 mg/L)
Sewri YP70b Penicillin G (1000 U/ml)
Nerul Streptomycin sulphate (300 mg/L)
Amphotericin B (2.5 mg/L)
Bandra YP70b Penicillin G (1000 U/ml)
Godrej Streptomycin sulphate (300 mg/L)
Amphotericin B (2.5 mg/L)
Kanamycin sulphate (50 mg/L)
Gentamicin sulphate (50 mg/L)
Godrej mangroves YP70c Penicillin G (300 mg/L)
Nargol Streptomycin sulphate (500 mg/L)
Vasai Rifampicin (50 mg/L)
Nystatin (10 mg/L)
Tetracycline hydrochloride (2 mg/L)

NSW Natural sea water

a
GYP: Glucose (0.3%), yeast extract and peptone (0.1%) each + Tween 80 (1%) + 50% NSW
b
YP70: Yeast extract and peptone (0.1%) each + Tween 80 (1%) + 70% NSW
c
YP100: Yeast extract and peptone (0.1%) each + Tween 80 (1%) + 100% NSW

extract, peptone) and inorganic (urea, sodium nitrate, ammo-
nium sulphate, ammonium acetate) nitrogen sources were
screened. Different C: N ratios were also studied, with glu-
cose as carbon source and yeast extract and peptone as a
nitrogen source.
Effect of salinity and different type of seawater
The selected strain was grown in liquid medium using sea-
water of different salinity percentage. 0% salinity indicates
freshwater and 100% salinity represents pure seawater. A.
limacinum was also cultivated using Natural Sea Water
(NSW), Sea Salt Water (SSW), and Artificial Sea Water
(ASW). NSW was obtained from the Arabian Sea, Mum-
bai coast, India. SSW was prepared by adding 3% raw salt
(obtained from CMSCRI, Bhavnagar, Gujrat, India) in
tap water. ASW contains (g/L): NaCl-24.54, ­MgCl2.H2O-
11.1, ­Na2SO4-4.09, ­CaCl2.6H2O-1.54, KCl-0.7, KBr-0.1,
­H3BO3-0.003, ­Na2SiO3.9H2O-0.005, ­SrCl2.6H2O-0.4, and
NaF-0.003.
Effect of pH
Experiments were executed with pH range from 5 to 8 to
determine the optimum pH for biomass growth and lipid
synthesis. Initially, the pH of the media was adjusted with
the help of 0.1 N HCl or 0.1 N NaOH. pH was measured at
the end of the experiment.
Analytical methods
Determination of cell dry weight
The samples were harvested at regular time intervals by cen-
trifugation at 8000 rpm for 5 min. Cell biomass was washed
at least thrice by distilled water to remove dissolved solids.
The pellet fraction was oven-dried at 60 °C till the constant
weight of biomass was achieved. Dried biomass was stored
at 15 °C for further analysis.
Lipid analysis
Lipid was extracted and quantified from oven-dried bio-
mass using a previously described protocol (Bligh and Dyer
1959). 40–60 mg dried biomass was added in 8 ml of a mix-
ture of solvents (chloroform, methanol, and water in 1:1:0.8
ratio) and kept for shaking in thermo-shaker at 58 °C for 1 h.
The lower phase was collected and placed in a petri plate
followed by drying in the oven at 60 °C. Lipid weight was
measured gravimetrically.
Fatty acid methyl ester (FAME) were prepared by pro-
cessing 40 mg of dried cells with the addition of 4 ml 5%
methanolic ­H2SO4 (v/v) and incubating the samples at 90 °C
for 1 h. After cooling, 4 ml hexane and 2 ml of water was
added and samples were vortexed and centrifuged. FAMEs
extracted in hexane were analyzed using Gas Chromatog-
raphy (7890A) equipped with Mass Spectroscopy (5975C)
system of Agilent Technology, USA. Samples were analyzed
by HP5MS capillary column (30 m × 0.20 mm, 0.33 μm
coating thickness). Helium was used as the carrier gas and

13
 71 Page 4 of 10
the flow rate was maintained at 1 ml/min. The oven was
programmed from 100 °C (1 min hold) to 200 °C (1 min
hold) at a rate of 25 °C/min and from 200 °C to 250 °C
at 5 °C/min (7 min hold). Fatty acid peaks were identified
with chem-station chromatography software (Agilent Tech-
nologies, Santa Clara, CA, USA) using the retention time
of standards (Sigma–Aldrich, St. Louis, MO, USA) as the
reference.
Substrate utilization
The residual glucose concentration was analyzed using the
GOD-POD enzymatic method (Eco-Pak glucose 500 kits)
procured from Accurex Biomedical Pvt. Ltd. Mumbai, India
and by high-performance liquid chromatography (HPLC)
(Agilent Technologies, USA). The supernatant of the sample
was filtered with 0.2 μm syringe filters before being ana-
lyzed by HPLC. Substrate utilization was estimated using
HPLC with an Aminex 87H column (Bio Rad, Richmond,
CA) at 65 °C with RI detector (G1362A) at 35 °C with
5 mM ­H2SO4 as mobile phase at the flow rate of 0.6 ml/
min. EZchrome software (Agilent Technologies, USA) was
used for data processing.
Carotenoid analysis
Experiments were performed with optimized media compo-
nents at 20 °C and 100 rpm in 250 ml baffled flask. Carot-
enoid extraction was done with dimethyl sulfoxide. Samples
were analyzed using the method described by Sarnaik et al.
(2018). Commercial β-carotene, canthaxanthin, and astax-
anthin (Sigma-Aldrich, St. Louis, MO, USA) were used as
standards for quantification of each pigment.
Results and discussion
Isolation and screening of thraustochytrids strain
Mumbai coastal region is an excellent bio-resource hot-
spot for marine organisms. The soil, decayed leaves, and
water were collected from different locations as mentioned
in Table 1. The contamination is the biggest bottleneck for
isolation of the thraustochytrids strain. The bacterial and
fungal load was considerably decreased when leaf samples
were soaked in 0.1% mercuric chloride solution after sterile
water wash. Mercuric chloride treatment was most effec-
tive in the removal of fungal contamination. In general, the
soil samples led to much higher contamination load as com-
pared to that of leaf samples. The soil sample of the Nargol
site did not yield thraustochytrid strains due to the over-
growth of contaminants. The leaf samples from Nargol site
did yield thraustochytrid-like culture in the liquid medium;
13
3 Biotech (2021) 11:71
nonetheless no isolates could be recovered on plates due to
contamination. Colonies started appearing after 5–6 days
of incubation for samples collected from other sites. The
isolates obtained were identified based on their cell mor-
phology by light microscope. Colonies obtained from Vasai
(leaves and soil) and Godrej sample (soil) were small and
cream-colored while those from Bhayander (soil and leaves)
produced large and light pink-colored colonies. The different
isolates obtained from the various sites were observed under
the microscope as shown in Fig. 1a. The lipid and DHA
percentage of different isolates are depicted in Fig. 1b. The
culture obtained from Bhayander site (BY S3) was most pro-
liferating on the agar medium and showed the highest lipid
percentage (Fig. 1b) translating into the highest DHA con-
centration. BY S3 was thus selected as a promising isolate
based on higher growth as well as lipid content and named
as ICTSG-17. Figure 1c depicts the ICTSG-17 undergoing
binary fission.
Application of pine pollen is one of the well-known
techniques for the isolation of thraustochytrids (Bongiorni
et al. 2005; Berryman 2012). However, in the present study,
thraustochytrid strains could also be isolated without the
application of pine pollen technique. This was contradic-
tory to the reports where pine pollen technique was found to
be the only method for thraustochytrid isolation (Bongiorni
et al. 2005; Berryman 2012; Gupta et al. 2013). Therefore,
it can be concluded that inclusion of mercuric chloride and
use of antibiotic concoction can also lead to thraustochytrid
isolation without the application of pine pollen technique.
Genetic identification of isolated strain
BLAST analysis of isolated 18S sequence from ICTSG-17
(Fig. 2b) showed similarity to species of Aurantiochytrium,
Schizochytrium, and Thraustochytrium suggesting the local
isolate could belong to any of the three genera. The sequence
similarity was due to the conserved nature of 18S sequences
across different genera. The non-coding nature of internal
transcribed spacers (ITS) allows them to diverge during
evolution, thus leading to variation in the ITS sequence in
the strains of even the same species and making it a better
molecular marker for identification of novel isolates. BLAST
analysis of the ITS2 sequence suggested the ICTSG-17 iso-
late to be A. limacinum. The ITS2 sequence (Fig. 2d) has
been deposited in NCBI with accession number MN046792.
Identification and characterization of intracellular
lipid
ICTSG-17 A. limacinum was initially grown on GYPK
medium. Figure 3 shows a wet mount of ICTSG-17 A.
limacinum, in which the oil globules can be seen. This
was further confirmed by Nile red staining (Fig. 3) where
3 Biotech (2021) 11:71
Fig. 1  a Isolates obtained from different screening sites; from left
to right; first row: Bhayander/Leaves (BY L1), Bhayander/Leaves
(BY L5), Bhayander/Soil (BY S2), Bhayander/Soil (BY S3), Godrej/
Soil (GDS1); second row: Vasai Leaves (VL2), Vasai Soil (VS2),
yellowish golden stain indicates the presence of intracellular
lipids inside the cells. The Nile red dye is specifically used
for intracellular lipid staining confirming the intracellular
oil synthesis (Velmurugan et al. 2014; Cooksey et al. 1987).
Figure 4 illustrates the fatty acid profile of oil obtained
from ICTSG-17 A. limacinum. DHA was principal unsatu-
rated fatty acid (34.2%) along with docosapentanoic acid
(7.94%). Palmitic acid was present as major saturated fatty
acid (44.97%) followed by myristic acid (5.74%). Eicosap-
entaenoic acid was also present in minute quantity but could
not be quantified. The fatty acid profile of ICTSG-17 A.
limacinum resembles that of other thraustochytrid strains as
described in the literature (Havermale et al. 2006; Lippmeir
et al. 2009).
Process parameter optimization for ICTSG‑17 A.
limacinum
The nutrient requirements of every strain vary according to
its genetic make-up and metabolism and are further influ-
enced by its physical environment. Therefore, it becomes
Page 5 of 10 71
Vasai Soil (VS3), Nargol Leaves (NG L1); b total lipid content (%)
and DHA (%) of isolated thraustochytrids strain; c ICTSG-17 iso-
late showing the binary fission. Arrow indicates the cell undergoing
binary fission
imperative to standardize the nutrient and physical param-
eters for every newly isolated strain for bioprocess optimiza-
tion. Some strains of thraustochytrids are reported to grow
on varied carbon substrates like glucose, fructose, glyc-
erol, acetate, ethanol, molasses (Yokochi et al. 1998; De
swaaf et al. 2003a, b; Gong et al. 2015). The ICTSG-17 A.
limacinum was thus screened for its growth and lipid produc-
tion on different carbon sources. ICTSG-17 A. limacinum
showed marginal growth when xylose, lignin, or sodium
acetate was used as the carbon substrate (biomass < 2 g/L,
data not shown). Glucose, glycerol, fructose, and hydro-
lyzed molasses could support growth and lipid production
of ICTSG-17 A. limacinum. Maximum biomass and lipids
were obtained for glucose and glycerol (Table 2). Both glu-
cose and glycerol can be obtained from agriculture residue
and as a byproduct of the biodiesel industry, respectively
(Lali et al. 2012; Lee Chang et al. 2015). Biomass and lipids
were comparatively lower on molasses (Table 2). However,
molasses is the byproduct of the sugar industry and is avail-
able in abundance at very nominal cost (0.07–0.14 $ per
kg). Therefore, it is promising to use molasses to produce
13
 71 Page 6 of 10
Fig. 2  Isolation and cloning of 18S and ITS2 from the Thraus-
tochytrid strain. a Colony PCR depicting positive TA clones of FA1.
M = 1 Kb ladder, lanes 1–5 = 5 putative clones of FA1 screened by
PCR, NTC no template control, PC PCR product used as positive
control. b 18S sequence of thraustochytrid strain. BLAST analysis
of the 18S sequence showed 80% similarity with Aurantiochytrium
limacinum. c Isolated fragments of ITS2 with different pairs of prim-
Fig. 3  ICTSG-17 A. limaci-
num images; from left to right:
Microscopic view, Nile red
staining
DHA-rich biomass which can be used as an aquafeed supple-
ment. Production of the DHA-rich biomass, will bypass the
expensive lipids/DHA extraction, isolation, and purification
steps, thus compensating for lower biomass and lipid yields
on molasses. The usage of waste-derived substrates will lead
13
3 Biotech (2021) 11:71
ers. AuITS = ITS2 isolated with Aur F and Aur R primer pair; NTC
AuITS = NTC for Aur F and Aur R primer pair; ScITS = ITS2 iso-
lated with Sc F and Sc R primer pair; NTC ScITS = NTC Sc F and
Sc R primer pair; ThITS = ITS2 isolated with Th F and Th R primer
pair; NTC ThITS = NTC Th F and Th R primer pair; M = 1 Kb DNA
ladder. d ITS2 sequence of thraustochytrid strain submitted to NCBI
with accession number MN046792
to a major reduction in the nutrient cost, thus allowing the
thraustochytrid biomass production process to be sustain-
able and cost-competitive. There are also few reports for
assimilation of acetic acid and ethanol as a carbon source by
thraustochytrids (De swaaf et al. 2003a, b), but ICTSG-17 A.
3 Biotech (2021) 11:71 Page 7 of 10 71

Fig. 4  GC–MS chromatogram of fatty acids profile of oil obtained from ICTSG-17 A. limacinum

Table 2  Cell biomass, lipid content, and DHA concentration of ICTSG-17 A. limacinum on different substrates
DCW (g/L) Lipid content (%) Lipid DHA Biomass Lipid yield (g/g) Biomass produc- Lipid produc-
conc. conc. yield (g/g) tivity (g/L h) tivity (g/L h)
(g/L) (g/L)

Glucosea 14.47 55.11 7.97 2.72 0.45 0.25 4.13 2.28

Glycerola 14.37 52.32 7.52 2.41 0.45 0.24 3.60 1.88
Fructosea 13.21 54.87 7.25 2.35 0.42 0.23 3.77 2.07
Molassesb 8.58 31.89 2.73 0.49 0.30 0.09 2.45 0.026
a
At 3%
b
At 3% reducing sugars

limacinum did not utilize acetate or xylose as carbon source
effectively (data not given).
Nitrogen is an essential nutrient for cell functioning.
High nitrogen is favorable for cell growth while its star-
vation with excess carbon triggers oil synthesis in oleagi-
nous microorganisms (Ratledge 2004). Thraustochytrids
are well known to grow on organic and inorganic nitrogen
sources (Goldstein 1963; Chen et al. 2010); hence ICTSG-
17 A. limacinum was cultivated on different organic and
inorganic nitrogen sources. ICTSG-17 A. limacinum
could not grow when cultivated on inorganic nitrogen
sources like ammonium sulfate, sodium nitrate, and urea,
whereas it could utilize organic nitrogen sources such as
yeast extract, peptone, etc. This was contrary to Yokochi
et al. (1998) report, which has reported that some Schiz-
ochytrium strains can utilize inorganic nitrogen sources
such as ammonium sulfate and ammonium hydroxide. Fig-
ure 5 presents ICTSG-17 A. limacinum growth data on
GYPK media with different nitrogen concentrations.
Low C/N ratio favored high biomass (12.39 ± 1.32 g/L),
but low lipid content (34.91%) while at higher C/N ratio
cell growth reduced (6.73 ± 0.57 g/L) but high lipid con-
tent (69.42%) was obtained. Similar results have been
obtained by Bowles et al. (1999) and Jakobsen (2008).
The C/N ratio did not have any significant effect on the
total fatty acid profile. This information is useful in devel-
oping a bioprocess for higher biomass and lipid content
simultaneously.

13
 71 Page 8 of 10 3 Biotech (2021) 11:71

Total Lipid content (%) DCW (g/L) Thraustochytrid strains can be grown on a wide range of
80 14 pH from 4 to 8. The highest biomass and lipid content were
12 obtained at pH range 6–7 for the growth of ICTSG-17 A.
Total Lipid content %

60
10 limacinum (Fig. 7). Equivalent dry cell weight (13.69 g/L)
and lipid content (52.34%) were obtained when cultivation

DCW (g/L)
8
40
6 was done at uncontrolled pH (final pH of 7.10 from an initial
4 pH of 6.6). This led to the omission of pH control easing the
20
2
bioprocess development.
0 0
5 7.5 10 20 50 Astaxanthin optimization in ICTSG‑17 A. limacinum
C/N ratio

Metabolic pathway of thraustochytrids shows microorgan-

Fig. 5  Effect of different C/N ratio on growth and lipid accumulation isms to be capable of producing natural antioxidants like
of ICTSG-17 A. limacinum
carotenoids. Depending upon the published literature, these
organisms mainly produce astaxanthin and β-carotene
Figure 6 depicts dry cell biomass and lipid content of A. (Armenta et al. 2006; Aki et al. 2003). The reason for the
limacinum at different salinity percentage. ICTSG-17 A. simultaneous carotenoid synthesis could be to circumvent
limacinum could produce the highest cell biomass and lipid the oxidation of ω-3 fatty acids. Figure 8 shows the color
content at 100% saline water but was also able to grow with- variation among the two cultures cultivated in the baffled
out inhibition at different salinity tested. For the present flask at different process conditions. The color of the culture
strain, maximum DHA concentration was achieved at 100% changed from white to orange after 5 days in the baffled
salinity. This was in contrast with Perveen et al. (2006) who flask incubated at 20 °C. HPLC analysis confirmed astax-
have reported lower unsaturated fatty acid at low salinity as anthin to be the principal carotenoid along with β-carotene
compared with that of high salinity. by ICTSG-17 A. limacinum. The astaxanthin concentra-
Studies were also performed with optimized GYPK tion was determined to be 3.8 µg/L while β-carotene was
media (3% glucose, 0.5% yeast extract and peptone each, below detection level. The probable reason for undetect-
0.01% ­KH2PO4) by using NSW, SSW, and ASW. Dry cell able β-carotene could be its conversion to astaxanthin in the
biomass and lipid content using SSW was 14.34 g/L and carotenoid pathway (Rathod et al. 2020). The β-carotene is
49.51%, respectively, which was comparable to that of NSW known to be converted to astaxanthin due to genetic and
(DCW 14.79 g/L; lipid content 53.1%). The ASW resulted culture condition variations (Kumari et al. 2020). Absence
in 13.03 g/L DCW and 45.49% lipid content. Therefore, it of astaxanthin in the non-baffled flask at both the tempera-
can be inferred that the addition of sea salts to freshwater tures and in a baffled flask at 30 °C confirms that interaction
can effectively replace NSW for ICTSG-17 A. limacinum of temperature and aeration play critical role in astaxanthin
cultivation. From a commercial point of view, it is bene- synthesis in ICTSG-17 A. limacinum. The positive influ-
ficial where seawater is not easily available or difficult to ence of lower temperature shift and LED light exposure on
transport. carotenoid production has also been confirmed by Park et al.
(2018) in Schizochytrium sp. The chemical mutagenesis of

DCW (g/L) Total Lipid content (%) 25 80

DCW (g/L) Total Lipid content (%)
17 60
20
Total Lipid content (%)

15 50
60
Total Lipid content %

DCW (g/L)

13 40
15
DCW (g/L)

40
11 30 10

9 20 20
5
7 10

5 0 0 0
0 25 50 100 5 6 7 8 Uncontrolled
% Salinity pH

Fig. 6  Effect of seawater concentration on growth and lipid accumu- Fig. 7  Effect of different pH on growth and lipid accumulation of
lation of ICTSG-17 A. limacinum ICTSG-17 A. limacinum

13
3 Biotech (2021) 11:71 Page 9 of 10 71

Fig. 8  Production of astaxanthin by ICTSG-17 A. limacinum; from
left to right: 30 °C, 20 °C
acids. In crude microbial oil, DHA and DPA were the chief
unsaturated fatty acids and palmitic acid was dominant satu-
rated fatty acid (Table 3). DHA content in microbial oil from
ICTSG-17 A. limacinum strain was substantially higher than
fish oil. Table 3 also shows the total saturated and unsatu-
rated fatty acid percentage both in fish and microbial oil. The
saturated fatty acid content was lesser in the commercial fish
oil due to refining and purification (winterization process),
but the unsaturated fatty acid content was nearly equivalent
to that of ICTSG-17 A. limacinum derived oil. Significantly,
the DHA content of fish oil was much lesser as compared
to that of ICTSG-17 A. limacinum produced microbial oil
(Table 3). Therefore, it can be postulated that after refining,
ICTSG-17 A. limacinum derived oil can be an equivalent
alternative to fish oil specifically for DHA and can lower the
reliance on fish oil in aquaculture. Alternatively, high DHA
containing biomass can be a viable alternative as fish feed
and offer value addition because of the presence of astax-
anthin as antioxidant and natural colorant for aqua culture.

Table 3  Comparison of total saturated and unsaturated fatty acid con- Author contributions Experiments were planned by GP and AML. Iso-
tent in fish oil and microbial oil obtained from ICTSG-17 A. limaci- lation work was executed by SV. SK planned and executed the strain
num by GC–MS analysis identification studies. PRP did the growth optimization experiments.
Data were analysed by PRP, SK, AML and GP. The manuscript is
Fatty acids Fish oil (%) Microbial oil (%) written by PRP and GP. Funding for the work was facilitated by AML.

Saturated fatty acids (SFA)

Myristic acid (C14:0) 5.71 5.76
Compliance with ethical standards
Palmitic acid (C16:0) 14.94 44.97
Conflict of interest The authors declare that they have no conflict of
Stearic acid (C18:0) 2.61 1.1 interest.
Monounsaturated fatty acids (MUFA)
Oleic acid (C18:1) 28.32 –
Polyunsaturated fatty acids (PUFA)
Linoleic acid (C18:2) 2.34 –
References
Eicosapentaenoic acid (C20:5) 5.21 –
Aki T, Hachida K, Yoshinaga M, Katai Y, Yamasaki T, Kawamoto
Docosapentanoic acid (C22:5) – 7.94 S, Ono K (2003) Thraustochytrid as a potential source of carot-
Docosahexaenoic acid (C22:6) 8.28 34.2 enoids. J Am Oil ChemSoc 80(8):789
TFA (%) 23.26 51.83 Armenta RE, Burja A, Radianingtyas H, Barrow CJ (2006) Critical
assessment of various techniques for the extraction of carotenoids
TUFA (%) 44.15 42.14
and co-enzyme Q10 from the Thraustochytrid strain ONC-T18. J
Agric Food Chem 54(26):9752–9758
Bajpai PK, Bajpai P, Ward OP (1991) Optimization of production of
docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC
Aurantiochytrium sp. has been done to isolate high carot- 34304. J Am Oil ChemSoc 68(7):509–514
enoid producing mutant strain (Watanabe et al. 2018). Fur- Barclay WR, Meager KM, Abril JR (1994) Heterotrophic production
of long chain omega-3 fatty acids utilizing algae and algae-like
ther, the metabolic analysis of the mutant strain revealed
microorganisms. J ApplPhycol 6(2):123–129
negative impact of oxidative stress on astaxanthin accumu- Berryman KT (2012) Isolation of marine protists for production of
lation, which was overcome by supplementing ferrous ions, polyunsaturated fatty acids. https:​ //docpla​ yer.net/864818​ 64-Kevin​
thus resulting in enhanced astaxanthin productivity. -thoma​s-berry​man-submi​tted-in-parti​al-fulfi​lment​-of-the-requi​
remen​ts-for-the-degre​e-of-maste​r-of-scien​ce.html#show_full_
text. Accessed 11 Jan 2021
Comparison of commercial fish oil and Microbial oil Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and
purification. Can J Biochem 37(8):911–917
Table 3 represents a comparison of the fatty acid profile of Bongiorni L, Jain R, Raghukumar S, Aggarwal RK (2005) Thraus-
tochytrium gaertnerium sp. nov.: a new thraustochytridstrameno-
fish oil and crude microbial oil isolated from ICTSG-17 A.
pilanprotist from mangroves of Goa, India. Protist 156(3):303–315
limacinum. Commercial fish oil mainly contains oleic acid, Bowles RD, Hunt AE, Bremer GB, Duchars MG, Eaton RA (1999)
EPA, and DHA which are the principal unsaturated fatty Long-chain n-3 polyunsaturated fatty acid production by members

13
 71 Page 10 of 10
of the marine protistan group the thraustochytrids: screening of
isolates and optimization of docosahexaenoic acid production. J
Biotechnol 70:193–202
Burja AM, Radianingtyas H, Windust A, Barrow CJ (2006) Isola-
tion and characterization of polyunsaturated fatty acid producing
Thraustochytrium species: screening of strains and optimization of
omega-3 production. ApplMicrobiolBiotechnol 72(6):1161–1169
Carlson SE, Colombo J (2016) Docosahexaenoic acid and arachidonic
acid nutrition in early development. AdvPediatr 63(1):453
Chen G, Fan KW, Lu FP, Li Q, Aki T, Chen F, Jiang Y (2010)
Optimization of nitrogen source for enhanced production of
squalene from thraustochytridAurantiochytrium sp. N Biotech-
nol 27:382–389
Cooksey KE, Guckert JB, Williams SA, Callis PR (1987) Fluorometric
determination of the neutral lipid content of microalgal cells using
Nile Red. J Microbiol Methods 6(6):333–345
Das UN (2003) Long-chain polyunsaturated fatty acids in the growth
and development of the brain and memory. Nutrition 19(1):62–65
De Swaaf M, Pronk J, Sijtsma L (2003a) Fed-batch cultivation of the
docosahexaenoic-acid-producing marine alga Crypthecodinium
cohnii on ethanol. ApplMicrobiolBiotechnol 61(1):40–43
De Swaaf ME, Sijtsma L, Pronk JT (2003b) High cell density fed-batch
cultivation of the docosahexaenoic acid producing marine alga
Crypthecodinium cohnii. BiotechnolBioeng 81(6):666–672
Fan KW, Chen F, Jones EB, Vrijmoed LL (2001) Eicosapentaenoic and
docosahexaenoic acids production by and okara-utilizing potential
of thraustochytrids. J IndMicrobiolBiot 27(4):199–202
Goldstein S (1963) Development and nutrition of new species of
Thraustochytrium. Am J Bot 50(3):271–279
Gong Y, Liu J, Jiang M, Liang Z, Jin H, Hu X, Hu C (2015) Improve-
ment of omega-3 docosahexaenoic acid production by marine
dinoflagellateCrypthecodinium cohnii using rapeseed meal
hydrolysate and waste molasses as feedstock. PLoS ONE
10(5):e0125368
Gupta A, Barrow CJ, Puri M (2012) Omega-3 biotechnology: thraus-
tochytrids as a novel source of omega-3 oils. BiotechnolAdv
30(6):1733–1745
Gupta A, Wilkens S, Adcock JL, Puri M, Barrow CJ (2013) Pollen
baiting facilitates the isolation of marine thraustochytrids with
potential in omega-3 and biodiesel production. J IndMicrobiolBiot
40(11):1231–1240
Hauvermale A, Kuner J, Rosenzweig B, Guerra D, Diltz S, Metz JG
(2006) Fatty acid production in Schizochytrium sp.: involvement
2 of a polyunsaturated fatty acid synthase and a type I fatty acid
synthase. Lipids 41:739–747
Jakobsen AN (2008) Compatible solutes and docosahexaenoic acid
accumulation of thraustochytrids of the Aurantiochytrium group
Jaseera K, Kaladharan P (2019) Thrauastochytrids in aquaculture—can
it replace fish meal in aquaculture? AquacSpectr 2:25–27
Kumari S, Vira C, Lali AM, Prakash G (2020) Heterologous expres-
sion of a mutant Orange gene from Brassica oleracea increases
carotenoids and induces phenotypic changes in the microalga
Chlamydomonas reinhardtii. Algal Res 47:101871
Lali AM, Nagwekar PD, Varavadekar JS, Wadekar PC, Gujarathi SS,
Valte RD, Birhade SH, Odaneth AA, Inventors (2012) Chemi-
cal. Engr. Department. Inst. of Chemical Tech. (Deemed Univ.),
assignee. Method for production of fermentable sugars from bio-
mass. United States patent US 8,338,139
13
View publication stats
3 Biotech (2021) 11:71
Lee Chang KJ, Paul H, Nichols PD, Koutoulis A, Blackburn SI (2015)
Australian thraustochytrids: potential production of dietary
long-chain omega-3 oils using crude glycerol. J Funct Foods
19:810–820
Lippmeier JC, Crawford KS, Owen CB, Rivas AA, Metz JG, Apt KE
(2009) Characterization of both polyunsaturated fatty acid biosyn-
thetic pathways in Schizochytrium sp. Lipids 44:621–630
Mendes A, Reis A, Vasconcelos R, Guerra P, da Silva TL (2009)
Crypthecodinium cohnii with emphasis on DHA production: a
review. J ApplPhycol 21(2):199–214
Mo C, Douek J, Rinkevich B (2002) Development of a PCR strategy
for thraustochytrid identification based on 18S rDNA sequence.
Mar Biol 140(5):883–889
Mohebi-Nejad A, Bikdeli B (2014) Omega-3 supplements and cardio-
vascular diseases. Tanaffos 13(1):6
Park H, Kwak M, Seo J, Ju J, Heo S, Park S, Hong W (2018) Enhanced
production of carotenoids using a thraustochytridmicroalgal strain
containing high levels of docosahexaenoic acid-rich oil. Bio-
procBiosystEng 41(9):1355–1370
Perveen Z, Ando H, Ueno A, Ito Y, Yamamoto Y, Yamada Y, Tak-
agi T, Kaneko T, Kogame K, Okuyama H (2006) Isolation and
characterization of a novel thraustochytrid-like microorganism
that efficiently produces docosahexaenoic acid. BiotechnolLett
28(3):197–202
Raghukumar S (2008) Thraustochytrid marine protists: production
of PUFAs and other emerging technologies. Mar Biotechnol
10(6):631–640
Rathod JP, Vira C, Lali AM, Prakash G (2020) Metabolic engineering
of Chlamydomonas reinhardtii for enhanced β-carotene and lutein
production. Appl. Biochem Biotech 190(4):1457–1469
Ratledge C (2004) Fatty acid biosynthesis in microorganisms being
used for single cell oil production. Biochimie 86(11):807–815
Sarnaik A, Nambissan V, Pandit R, Lali A (2018) Recombinant Syn-
echococcuselongatus PCC 7942 for improved zeaxanthin produc-
tion under natural light conditions. Algal Res 36:139–151
Singh A, Ward OP (1996) Production of high yields of docosahexae-
noic acid by Thraustochytrium roseum ATCC 28210. J IndMicro-
biol 16(6):370–373
Siriwardhana N, Kalupahana NS, Moustaid-Moussa N (2012) Health
benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid
and docosahexaenoic acid. Adv Food Nutr Res 65:211–222
Velmurugan N, Sathishkumar Y, Yim SS, Lee YS, Park MS, Yang JW,
Jeong KJ (2014) Study of cellular development and intracellular
lipid bodies’ accumulation in the thraustochytridAurantiochytrium
sp. KRS101. BioresourTechnol 161:149–154
Watanabe K, Arafiles KHV, Higashi R, Okamura Y, Tajima T, Mat-
sumura Y, Nakashimada Y, Matsuyama K, Aki T (2018) Isolation
of high carotenoid-producing Aurantiochytrium sp. mutants and
improvement of astaxanthin productivity using metabolic infor-
mation. J Oleo Sci 67(5):571–578
Yang ST (ed) (2011) Bioprocessing for value-added products from
renewable resources: new technologies and applications. Elsevier,
Amsterdam
Yokochi T, Honda D, Higashihara T, Nakahara T (1998) Optimization
of docosahexaenoic acid production by Schizochytrium limacinum
SR21. ApplMicrobiolBiotechnol 49(1):72–76