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Strain improvement of long-chain fatty acids producing Micractinium sp.

by flow cytometry

Deepi Deka, Riwandahun Marwein, Channakeshavaiah

Chikkaputtaiah, Shiva Shanker Kaki, Tirupathi Azmeera, Hari
Prasanna Deka Boruah, Natarajan Velmurugan

PII: S1359-5113(20)30306-8
DOI: https://doi.org/10.1016/j.procbio.2020.06.004
Reference: PRBI 12055

To appear in: Process Biochemistry

Received Date: 20 February 2020

Revised Date: 30 May 2020
Accepted Date: 2 June 2020

Please cite this article as: Deka D, Marwein R, Chikkaputtaiah C, Kaki SS, Azmeera T, Boruah
HPD, Velmurugan N, Strain improvement of long-chain fatty acids producing Micractinium sp.
by flow cytometry, Process Biochemistry (2020),
doi: https://doi.org/10.1016/j.procbio.2020.06.004

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Strain improvement of long-chain fatty acids producing
Micractinium sp. by flow cytometry

Deepi Dekaa,b • Riwandahun Marweinb,c • Channakeshavaiah Chikkaputtaiahb,c • Shiva

Shanker Kakid • Tirupathi Azmeerad • Hari Prasanna Deka Boruahb,c • Natarajan
Velmurugana,b,*

aBiological Sciences Division, CSIR-North East Institute of Science and Technology

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(CSIR-NEIST), Branch Laboratory-Itanagar, Naharlagun 791 110, Arunachal Pradesh,
Republic of India

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bAcademy of Scientific and Innovative Research (AcSIR), CSIR-NEIST, Jorhat, Assam,
Republic of India

cBiological
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Sciences and Technology Division, CSIR-North East Institute of Science and
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Technology (CSIR-NEIST), Jorhat 785 006, Assam, Republic of India

dCentre for Lipid Science and Technology, CSIR-Indian Institute of Chemical

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Technology (CSIR-IICT), Hyderabad 500 007, Telangana, Republic of India

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*Correspondence:
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Dr. Natarajan Velmurugan – Biological Sciences Division, CSIR-North East Institute of

Science and Technology, Branch Laboratory-Itanagar, Naharlagun 791 110, Arunachal
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Pradesh, Republic of India.

Ph.: +91-360-2244220

Fax: +91-360-2244220

E-mail: [email protected], [email protected]

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Graphical abstract

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Highlights
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 ► Bioprocess of long-chain fatty acids accumulating new isolate Micractinium sp.

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 ► Optimized FACS conditions in combination with fluorescent dyes for cell sorting

 ► Established of FACS-based adaptive evolution strategy for strain improvement

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 ► Achieved improved phenotypes with high biomass and desirable fatty acids
composition
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ABSTRACT

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A new green microalga isolate Micractinium sp. GA001 was found to accumulate long-chain
fatty acids, and the strain was subjected to flow cytometry-based adaptive evolution approach
to produce improved phenotypes. At first, original phenotype of new isolate GA001 was well
characterized followed by establishment of flow cytometry conditions in combination with
fluorescent dyes BODIPY and Nile Red, to screen intracellular long-chain fatty acids in
GA001. Fluorescent dyes staining and flow cytometry analysis revealed the progressive
accumulation of desirable lipid components in GA001. Further, a flow cytometry-based
strategy was used to selectively isolate and enrich particular GA001 phenotypes with higher
accumulation of long-chain fatty acids, under nitrogen-depletion and –repletion conditions.
This strategy yielded an improved population with 198% high lipid content than original

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population. Micractinium sp. GA001 was proved to be a promising strain with improved
phenotypes for the production of large-scale target-specific long-chain fatty acids.

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Keywords: Long-chain fatty acids; Micractinium sp.; Strain improvement; Fluorescent
activated cell sorter; Adaptive evolution
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1. Introduction:

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 Microalgae are unicellular photo- or heterotrophic microorganisms contributing to the
global primary production in marine ecosystem [1, 2]. In recent times, they are gaining wide
importance as viable, eco-friendly, renewable and alternative source for green energy
production. Microalgae are promising sources of raw materials for different industries
including nutraceuticals, aquaculture, pharmaceuticals, and cosmetics because of their
capabilities to produce long-chain polyunsaturated fatty acids (PUFA), carbohydrates,
carotenoids, vitamins, chlorophyll and other pigments [3, 4, 5]. These sources are also
considered as the third-generation feedstock contributing to the global green energy
requirement [6]. They are believed to replace the non-renewable fossil fuels due to the
presence of some unique characteristics such as high photosynthetic efficiency, thereby

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achieving increased biomass production as well as high-level yield of lipids [7]. In general,
the growth rate of microalgae was quite high with doubling time of 24 hours which was

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significantly faster in comparison to other oil-yielding higher plants [8]. Additional salient
feature was their ability to tolerate wide ranges of environmental stress conditions [9].

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Intracellular lipid bodies of microalgae composed of a mixture of fatty acids methyl esters
(FAMEs), and those were synthesized in the chloroplast using a single set of enzymes with
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acetyl CoA carboxylase (ACCase) as the key factor [10, 11]. A study by Lorenzen et al.
(2017) reported that microalgae can produce up to 70% w/w triglycerides (TAG) with respect
to the cell dry weight [12]. Microalgae are believed to be alternative production host for
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commercially/biologically important PUFA’s especially arachidonic acid (AA),

docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) which are originally derived
from fish oil at the moment [13]. Omega-3 (PUFA ω3) and omega-6 (PUFA ω6) were well
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known derivative products of PUFA used in aquaculture industry and disease control [14,
15]. Traditionally, development of screening pipelines for robustness in production of high
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biomass as well as fatty acid methyl esters (FAME) content (with specific target on ω-3 fatty
acids, EPA and DHA) was in practice based on rational sampling, isolation, and culturing in
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both nutrient-repletion and -depletion conditions [16]. Of the different screening techniques
for selection of particular phenotypes with desirable characteristics, high-throughput
screening methodologies (HTMs) have gained substantial importance for better ability to
screen a large number of cells within a few seconds followed by interrogation and selection
(or isolation) of particular phenotype at single cell level with desired properties [17]. In
recent times, high throughput screening techniques such as fluorescent activated cell sorter

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(FACS) coupled with lipophilic fluorescent dyes (especially BODIPY and Nile Red)were
drawing the interests of researchers for selection of phenotype at single cell level with
desirable characteristics [18, 19, 20]. Importantly, flow cytometry based HTMs were more
effective to distinguish neutral and polar lipids within a phenotype. By combining flow
cytometry and chromatography techniques, Jara et al. (2003) distinguished neutral and polar
lipids in marine dinoflagellate Crypthecodinium cohnii [21].

Strain improvement in microalgae is becoming a promising strategy in algal

biotechnology industries focusing on turning of low-value bioproducts and chemicals into an
economically feasible venture [22]. In general, in industries, spontaneous mutations and
random mutagenesis followed by intelligent (HTMs) screening processes were used to

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construct improved strains with desirable properties [22]. At the beginning, increased lipid
content in microalgal strains Chlorella vulgaris UTEX 1803, Micractinium pusillum

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GU732425, and Micractinium inermum NLP-F014 were achieved using traditional media
manipulation techniques [23, 24, 25]. Further, microalgal strains were improved using
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random mutagenesis either by physical (ultraviolet (UV) or chemical (ethyl methane
sulfonate (EMS), quizalofob) mutagens [26]. Interestingly, improved strains achieved using
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random mutagenesis was directly used as feedstock material in aquaculture industry [26]. In
addition, rational metabolic engineering approaches have also been successful in improving
strain performance in several cases [27]. However, success in metabolic engineering attempts
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to improve microalgae strains were limited [28]. Recent advances in HTMs supported by
biological manipulations have resulted in rapid accumulation of a wide range of bio products
at various levels [29]. Further, combinations of HTMs with “omics” techniques were
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providing a foundation for in-depth understanding of biological processes at single cell level
[30]. Innovative FACS strategy along with “omics” opens up a new door to develop
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microalgal strain with desired properties. The FACS-based adaptive evolution strategy under
various abiotic stress conditions, including, nutrient, temperature, light, pH, salinity, is a
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rapid, precise, non-labour-intensive, cost-effective and ready-to-go process for scaling-up

without any genetic manipulations. It involves repetitive selection rounds of sorting of
particular phenotypes in order to obtain the best evolved population in terms of the desired
phenotype with high-level accumulation of target compound [22]. At first, FACS-based
adaptive evolution strategy was successfully established in the model microalgal strain
Chlamydomonas reinhardtii CC124 and their mutants [30]. It was further followed by

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improving Chlorococcum littorale NBRC 10276, and Tetraselmis sp. CTP4 strains with high-
level lipid content [31, 32]. In addition with enhancing intracellular lipid content in
microalgae, FACS-based strategies were also helpful for improving other
commercially/biologically important compounds in microalgae. For example, FACS-based
screening and sorting of wild type Dunaliella salina species yielded improved D. salina with
high level carotene producing phenotype [33]. The combination of random mutagenesis and
flow cytometry were altered the FAME contents in haptophyte Isochrysis sp., and green
microalgae Nannochloropsis maritime [13, 34]. EMS-based random mutagenesis and FACS
sorting in N. maritime yielded a mutant strain with a 30% increase in fatty acids palmitoleic
acid (16:1) and a 45% decrease in eicosapentaenoic acid (EPA) (20:5n3) than wild type [13].

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A recent study by Gnouma et al. (2018) reported significant alterations in FAME contents in
haptophyte Isochrysis sp. after UV-based random mutagenesis and FACS sorting [34].

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Herein, a complete characterization of newly isolated long-chain fatty acids synthesizing
Micractinium sp. GA001 is presented. Firstly, this study established a relationship between
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cellular growth and accumulation of intracellular long-chain fatty acids in GA001.
Specifically, the use of FACS with different fluorescent dyes was tested for the screening,
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isolation and culturing of Micractinium sp. GA001. Furthermore, the primary focus in this
study was to establish a flow cytometry-based strategy to improve long-chain fatty acids
accumulating phenotypes without compromising intracellular FAME composition. To this
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effect, a FACS-based adaptive evolution procedure was established that is suitable for
improving long-chain fatty acids accumulating Micractinium sp.
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2. Materials and Methods

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2.1. Strains, culture conditions and molecular characterization

Marine samples were collected from Bay of Bengal near Pulicat, Chennai. All
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experiments were carried out in artificial sea water (ASW)containing (per L) 23.5 g NaCl, 5 g
MgCl2, 3.9 g Na2SO4, 1.1 g CaCl2, 0.66 g KCl, 0.20 g NaHCO3, 0.10 g KBr, 0.026 g H3BO3,
0.024 g SrCl2 and 0.003 g NaF, supplemented media. To isolate microalgae strains, 1 mL of
water sample was inoculated with 9 mL of f/2 medium containing (per L)8 mg NaNO3, 5 mg
NaH2PO4.2H2O, and 1 mL of trace metal stock solution (TMSS) (containing (per L) 21 mg
ZnCl2, 15 mg Cobalt (II) chloride, 15 mg Na2MoO4.2H2O, 180 mg MnCl2.4H2O,7 mg
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(NH4)6Mo7O24.4H2O, 10 mg CuSO4.5H2O, 3.15 g FeCl3.6H2O , 4.36 g Na2EDTA.2H2O and
22 mg ZnSO4.7H2O), and 100 µL of vitamin stock solution (VSS) (containing (per mL) 5 mg
thiamine HCl, 0.1 mg biotin and 0.1 mg cyanocobalamin) and incubated at 25°C for 14 days
under a light intensity of 125 µmol/m2s. After incubation period, culture medium was
centrifuged at 10000 rpm for 5 mins (5424R, Eppendorf, Hamburg, Germany) and entire
pellet was inoculated in f/2 agar plates at 25°C for 14 days under a light intensity of 125
µmol/m2s. Photo period was maintained as 16 h light and 8 h dark conditions. Control plates
inoculated with sterilized double distilled water were incubated under the same conditions.
After the incubation period, microalgal species grown on the f/2 plates were transferred to
fresh f/2 plates as pure microalgal species. Axenic algal cultures were maintained both on
agar plates and in broths under a light intensity of 125 µmol/m2s. Cell size was determined by

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particle size analyser (Nano ZS, Malvern, Worcestershire, UK). Haptophyte strain Isochrysis

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galbana NIES 2590 was purchased from National Institute for Environmental Studies
(NIES), Ibaraki, Japan, and maintained in a modified ESM medium.

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All the marine-derived microalgal isolates were initially tested for their growth.
Micractinium sp. GA001 was found to be fast growing microalgal strain. GA001 was
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maintained in f/2 broth for microalgal genomic DNA extraction. Genomic DNA was isolated
from this actively growth microalgal strain after 10 days’ incubation. Total microalgal
genomic DNA extraction was carried out using Nucleospin Plant II mini kit (Macherey-
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Nagel, Duren, Germany). The LSUD region of the isolated microalgal genomic DNA was
amplified using primers LSUD1F (5-AGCGGAGGAAAAGAAACTA-3) and LSUD1R (5-
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TACTAGAAGGTTCGATTAGTC-3).Microalgal LSUD region amplification was carried

out as initial denaturation step at 94°C for 5 mins, followed by 30 cycles each at 94°C for 30
sec, 55 °C for 30 sec, 72°C for 2 mins, further followed by one final extension at 72°C for 10
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mins(C1000, BioRad, Hercules,CA). Amplified fragments were visualized on 1.5% agarose

gel (XR+, BioRad, Hercules,CA).The amplified region was sequenced by the AgriGenome
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sequencing Company (Cochin, Kerala, India). Sequences were analysed using GENETYX
software (Version 6.1). BLAST was used to confirm the sequence identity in NCBI. A
phylogenetic tree (neighbour joining method) was constructed by Molecular Evolutionary
Genetic Analysis 2.1 Software (MEGA, Version4, Mega software, Phoenix, AZ). The
microalgal sequence obtained from this study was submitted to GenBank and an accession
number was obtained (MN990350).

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2.2. Phenotypic characterizations
GA001 was tested in modified 6 different media namely, f/2 (composition as mentioned
above), walnes containing (per L) 500 µl from 1.82 mM stock of MnCl2.4H2O, 100 mg
NaNO3, 34 mg H3BO3, 45 mg Na2EDTA.2H2O, 20 mg NaH2PO4.2H2O, ESM containing (per
L) 120 mg NaNO3, 1 g TRIS base, 5 mg K2HPO4, 20 mg Fe-Na-EDTA.3H20, 10 nM from
10 mM Na2SeO3 stock, chry containing (per L) 7.5 mg NaNO3, 1 mg NaH2PO4.2H2O, 3 mg
NH4Cl, 6 mg Urea, 10 nM from 10 mM Na2SeO3 stock, MNK containing (per L) 120 mg
NaNO3, 280 mg Na2HPO4.12H2O, 1 g K2HPO4, and TMRL containing (per L) 100 mg
KNO3, 10 mg Na2HPO4.12H2O, 3 mg FeCl3.6H2O, for optimization of better growth

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medium. All the media were prepared in ASW supplemented with (per L) 1 mL of TMSS and
100 µL of VSS. Cultures were grown at 25°C under a light intensity of 125 µmol/m2s.Photo

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period was maintained as 16 h light and 8 h dark conditions. Samples for cell density
measurement were taken at different intervals including 0 h (samples were taken immediately
after re-suspension). Optical density was measured using a UV spectrophotometer (Lambda
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25, PerkinElmer, Singapore). Experimental triplicates were maintained for all experiments.
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For total cell dry weight determination of GA001 in all tested media, a seed culture was
prepared in 1 L flask containing 300 mL of respective media. Further 10% (v/v) axenic
cultures from mid exponential phase of seed cultures were inoculated in 1 L of 6 different
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media, separately, and allowed to grown at 25°C under a light intensity of 125
µmol/m2s.Photo period was maintained as 16 h light and 8 h dark conditions. The cells were
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harvested at late stationary phase, lyophilized (Heto FD3, Heto-Holten, Allerod, Denmark)
followed by determination of total cell dry weight. Experimental triplicates were maintained
for all experiments.
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Elemental composition of GA001 in 6 different media was determined. The lyophilized

microalgal biomasses were used for elemental analysis. Carbon (C), hydrogen (H), and
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nitrogen (N) contents were analysed using CHN analyzer (PE2400, PerkinElmer, Singapore).
Experimental triplicates were maintained for all experiments.

2.3. Fluorescent staining and visualization of intracellular lipid bodies of Micractinium

sp.GA001

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 Nile Red and BODIPY 505/515 staining were performed as reported previously [35]. Nile
Red and BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-31, 41-diaza-s-indacen)
were purchased from Himedia (Mumbai, India) and Sigma Aldrich (St. Louis, Missouri,
USA), respectively. A laser scanning confocal microscope (TCS SP8 SMD, Leica,
Mannheim, Germany) with the excitation set at 358 nm and the emission set at 500-579 nm
was used for visualization of fluorescent stained intracellular lipid bodies of Micractinium sp.
GA001. Chloroplast autofluorescence was selectively detected with the excitation set at 358
nm and the emission set at 676-696 nm. Quantitative analysis of digitized images was carried
out using the Leica Applied System X (LASX) software according to the protocols provided
by the manufacturer.

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2.4. Total lipid and FAME content analysis

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The total lipids were extracted from 25 mg of lyophilized microalgal biomass with a
chloroform-methanol (2:1 v/v) solvent mixture using a procedure similar to Folch’s method
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[36]. Fatty acids methyl esters (FAMEs) were produced from the extracted lipid by a
transesterification reaction. Briefly, methanol was added to the extracted lipids with sulfuric
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acid as a catalyst and the transesterification reaction was allowed to occur at 100°C for 10
min. After the reaction, 1 ml of deionized water was added and the organic phase was
separated from the water phase by centrifugation on at 4000 rpm for 10 min. The fatty acid
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composition of the extracted lipid samples was determined by gas chromatography (GC). GC
was performed on an Agilent 6890 gas chromatography equipped with a flame ionization
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detector (FID). The column used as a DB-225 having a length of 30 m, 0.25 nm i.d. and 25
µm film thickness. The carrier gas was nitrogen at a flow rate of 1 mL/min. The oven
programming was as follows: 160°C for 2 min, which rose to 230°C at a rate of 5°C/min and
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held at 230°C for 20 min. The injector and detector temperatures were maintained at 220°C
and 250°C, respectively. The fatty acids were identified by comparing the retention times
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with a mixture of standard FAME’s, C4-C24 (Supelco, St. Louis, MO). Each FAME sample
was analysed in duplicate and average values are reported.

2.5. Flow cytometry analysis

A high speed CytoFlex S flow cytometry (Beckmann Coulter, Fullerton, California,

United States) was used for the screening of intracellular lipid bodies in Micractinium sp.
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GA001. The fluorescence reading was obtained using an excitation of 358 nm with blue laser.
The emission signal was measured using three channels upon excitation, FITC (525/40 nm)
for BODIPY 505/515 stained cells, ECD (610/20 nm) for Nile Red stained cells, andAPC-
A700 (712/25 nm) and APC-A750 A750 (780/60 nm) for chloroplast autofluorescence. The
samples mean fluorescence intensity values and images were analysed using CytExpert
Software Version 2.0 (Beckmann Coulter, Fullerton, California, United States).The Flow
cytometry settings of all channels were same for all the samples. Micractinium sp. GA001
samples for analysis were taken at different growth phases. Experimental triplicates were
maintained for all experiments.

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2.6. Adaptive evolution and FACS-based cell sorting

BODIPY 505/515 stained cells of Micractinium sp. GA001 were used for FACS-based

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adaptive evolution for strain improvement. A high speed FACS instrument MoFlo XDP
(Beckmann Coulter, Fullerton, California, United States) was used for the analysis of
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BODIPY 505/515 stained intracellular lipid bodies in GA001. The fluorescence reading was
obtained using an excitation of 358 nm with an argon laser representing the blue laser. The
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emission signal was measured in detectors upon excitation (FL1 channel centred at 530/40
nm and FL5 channel centred at 740 LP). The samples mean fluorescence intensity values and
images were analysed using SUMMIT Software Version 5.4.0 (Beckmann Coulter, Fullerton,
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California, United States). The FACS settings of all channels were the same for all sorting
procedures. Puraflow 8X Sheath Fluid (Beckmann Coulter, Fullerton, California, United
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States) was used in all experiments as the FACS sheath fluid. Cell sorting was carried out
using cell sort precision mode, with a 100 μm nozzle. The 0 hour seed cell population was
divided into three groups (as shown in Figure 3: blue, green and red colour indicating low,
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average, and high lipid content cells, respectively) in both 2-dimensional dot plot and flow
cytogram respectively (FL1 vs FL5). FL1 represented compensated fluorescence signal for
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neutral lipid and FL5 represented compensated fluorescence signal for chloroplast
autofluorescence. A total of 10,000 cells of the top 2% of the total population from red
regions representing the high lipid content cells were sorted directly into the sterilized tubes
containing1 mL of nitrogen-repleted ESM broth and re-suspended in nitrogen-repleted and
nitrogen-depleted ESM media, separately (see SI Table 1 for detailed compositions for
nitrogen-repleted and nitrogen-depleted ESM media). Samples were taken for cell density

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analysis at different intervals and cells were maintained until they reached high cell density
(or late-stationary phase) at 25°C under light intensity of 125 µmol/m2s. Photo period was
maintained as 16 h light and 8 h dark conditions. A portion of high cell density (or late-
stationary phase) cells were further screened using FACS and 10,000 cells from the top 2%
cells were sorted into the sterilized tubes containing 1 mL of nitrogen-repleted ESM broth.
Experimental triplicates were maintained for all experiments. The above mentioned same
protocols were followed for visualization of intracellular lipid bodies, elemental analysis,
total lipid contents and FAME compositions in evolved cells.

2.7. Statistical analysis

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Experimental triplicates were analysed throughout the experiments. Statistical analyses
were performed using SigmaPlot 10.0 software (Systat Software Inc., Chicago, IL). The

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results were expressed as means ± SD (standard deviation) of triplicate experiments.
Differences were considered significant at p<0.05.

3. Results and Discussion

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3.1. Phenotypic characterization of long-chain fatty acids producing Micractinium sp.
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GA001

The strain GA001was isolated from the marine samples collected from the Bay of Bengal
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near Pulicat, Chennai. A pure culture of GA001 was maintained in artificial sea water
supplemented with f/2 medium. The isolate GA001 was unicellular strain with average size
of 5.5 µM at stationary phase (SI. F.1). On the basis of molecular analysis using the LSUD
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region, the sequence of Micractinium sp. GA001 was found to be 99.87% identical to
Micractinium reisseri (LC153789). Members of Micractinium species especially M. reisseri
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originally derived from Chlorella species [37]. Micractinium sp. GA001 falls within the class
of Trebouxiophyceae and within the division of Chlorophyta. Phylogenetic tree constructed
with previously reported sequences of rRNA showing that Micractinium sp. GA001 falls
within the Chlorella clade, and exhibited close similarities with M. reisseri RAIW01
(JN169781) and Chlorella variabilis (AB437257) (SI. F. 2). Interestingly, members of
Micractinium species have shown highly unique characteristics than other microalgae [38,

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39]. Micractinium conductrix SAG 241.80 an endosymbiont of ciliate Paramecium bursaria
secreted significant amounts of maltose and glucose when grown at specific pH range 5.7
[38]. Most importantly, cryotolerant Micractinium sp. isolated from freshwater samples of
Antarctica region reported to accumulate unique combination of FAME content with
nutritionally important linoleic (C18:2) and α-linolenic (C18:3 ω3) fatty acids [39].

Six different modified media supplemented with ASW, TMSS and VSS were tested to
select best suitable medium for efficient growth of new isolate Micractinium sp. GA001.
Previous reports have provided insights into media composition for effective heterotrophic
cultivation of microalgae [40]. However, there is limited information available on role and
relationship between carbon and nitrogen on cellular metabolism in microalgae. Therefore,

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we conducted a comparative characterization of 6 different modified media. It is well known
that higher carbon to nitrogen (C:N) ratio reported to be major trigger for high-level

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intracellular lipid accumulation. However, more nitrogen limitation condition may negatively
impact the microalgae growth [30]. Therefore, a more balanced recipe is essential for cell
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growth as well as lipid accumulation. The growth rates of Micractinium sp. GA001 in 6
different modified media were measured and are shown in Fig. 1. Modified ESM, TMRL,
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and walnes were found to be the first best 3 media for growth of Micractinium sp. GA001. It
was observed that f/2, chry, and MNK were found to be less effective to support the growth
of GA001. Therefore, it was further evaluated in ESM, TMRL, and walnes media.
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Micractinium sp. GA001 showed better growth pattern in modified ESM medium while
modified walnes and TMRL media were shared similar growth pattern. GA001 was found to
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have a longer lag phase that lasted for 4 days. In these best 3 media, GA001 showed
exponential growth from 5th day to 20th day with reaching the stationary phase after this time
frame. GA001 has reached early stationary phase by 20 days, followed by mid and late
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stationary phases by 25 days (Fig. 1). In comparison, our modified ESM recipe contains
sodium selenite and micronutrients manganese/iron EDTA while walnes and TMRL recipes
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contained various forms of sodium sources, and potassium nitrate, respectively. Selenium
was considered as an important ingredient for the growth of biologically important
haptophyte Emiliania huxleyi [41]. Selenium was known to be an inducer of glutathione
peroxidise and act as modified soil [41]. It is interesting to note that addition of manganese
and iron micronutrients were positively induced microalgae cell growth while supply of
additional sodium sources have not shown remarkable changes in microalgal growth pattern.

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Comparatively, in this study, manganese, iron and sodium sources were replaced with the
addition of potassium nitrate in modified TMRL medium. Growth pattern difference of
Micractinium sp. GA001 in modified ESM and TMRL was moderate (Fig. 1). The nitrate
sources available in the above mentioned modified media can induce accumulation of best
PUFA level as previously reported [40, 42]. The overall goal of this optimization was to find
a best suitable medium for efficient growth of new isolate GA001. Further modifications in
nitrogen content in a selective medium was achieved and discussed in strain improvement
section.

Elemental analysis of Micractinium sp. GA001 grown in modified ESM, walnes and
TMRL showed no significant differences between the values of the cellular carbon to

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nitrogen ratio (C:N). The C:N ratio was found to be 7.5:1, 8.0:1, and 8.0:1 for ESM, walnes
and TMRL, respectively. The C:N values of GA001 in different media were similar to those

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of earlier reports on other microalgae [43]. The C:N ratio plays major role in intracellular
fatty acids accumulation, and the values can be directly correlated with fatty acids
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3.2. Fatty acids composition of Micractinium sp. GA001

To confirm the accumulation of long-chain fatty acids in Micractinium sp. GA001, the
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fatty acids methyl esters (FAME) contents of GA001 cells grown under different media were
quantified by gas chromatography. In general, long-chain fatty acids are considered to
contain ≥20 carbon atoms in length, however, 18 carbon fatty acids also classified under
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commercially important ω3 and ω6 fatty acids. Long-chain fatty acids profiles of the
Micractinium sp. GA001 in the different media are summarized in Table 1. Micractinium sp.
GA001 accumulated various forms of long-chain fatty acids in all tested media. Except for
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walnes and MNK media, predominant fatty acids components observed in other 4 modified
media were C20:0, C20:1, and C20:5 (Table 1). It is interesting to note that GA001
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accumulated significant levels of C16 and C18 fatty acids only when grown in walnes and
MNK media in which presence of C20 and other long-chain fatty acids were considered to be
minor (Table 1). Meanwhile, presence of C16 and C18 fatty acids in GA001 when grown in
f/2, ESM, chry and TMRL were considered to be minor. As growth pattern of Micractinium
sp. GA001 in f/2 and chry was not significant, the fatty acids composition of GA001 grown
in ESM and TMRL were further evaluated. Comparatively, long-chain fatty acids
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accumulation of GA001 in ESM was higher than in TMRL. Micractinium sp. GA001 in ESM
medium accumulated 8%, 25% and 2% of C20:0, C20:1, and C20:5 fatty acids, respectively
(Table 1). Among microalgae fatty acids, various forms of C18 and C20 long-chain fatty
acids have been considered as high-value and commercially important components especially
in food and health industries [44]. However, majority of green microalgal strains were not
able to replace fish-derived ω3 and ω6 fatty acids because of their limited ability to synthesis
long-chain fatty acids. As this study aimed at characterization of long-chain fatty acids
producing microalgae, for comparison, we have characterized fatty acids profiling of the long
and very long-chain producing haptophyte strain I. galbana NIES 2590 (Table 1).
Haptophytes are well-known for their capability to accumulate long and very long-chain fatty

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acids [45]. Long-chain fatty acids profiles of Micractinium sp. GA001 and I. galbana NIES
2590 have exhibited similarities. Though haptophytes are considered to be promising sources

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for biosynthesis of long-chain fatty acids, bioprocessing of haptophytes perceived as difficult
because of the complexity associated with growth and maintenance of haptophyte strains
[45]. Therefore, it is necessary to find alternative strains in other microalgal family with long-
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chain fatty acids synthesising characteristics. In this context, Micractinium sp. GA001 can
emerge as promising strain because it is easy to grow and easy to set-up bioprocess
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techniques in the laboratory as well in race way ponds.
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3.3. Flow cytometry analysis of intracellular lipid bodies in Micractinium sp. GA001

As major objective of the present work was to establish flow cytometry-based strategy for
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strain improvement of long-chain fatty acids accumulating microalgal phenotypes, efficient

procedures were optimized for staining of intracellular lipid bodies in Micractinium sp.
GA001 using fluorescent dyes. For this purpose, the effectiveness of Nile Red and BODIPY
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505/515 dyes were compared to stain lipid bodies in Micractinium sp. GA001 for flow
cytometry-based screening and cell sorting (Fig. 2). Though the fluorescent dyes-based
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measurement of lipid contents in microalgae has been well established, the evaluation of lipid
bodies of Micractinium sp. by lipophilic dyes and subsequent processing using flow
cytometry are scarce [46, 47, 48]. The pre-treatment conditions were optimized for better
penetration of fluorescent dyes and proper binding with intracellular lipid bodies. The cell
viability is an important point to be considered because one of the major aims of this study
was to sort potential phenotypes of GA001 with high lipid content and recover those cells for

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further evaluation. Therefore, as reported in our earlier studies, minimum concentration of
DMSO and glycerol were used as carrier solvents for BODIPY 505/515 and Nile Red,
respectively [30]. Confocal microscopy images of Nile Red stained Micractinium sp. GA001
shown in Fig. 2. From the confocal images, we can clearly conclude that Nile Red assisted
with glycerol efficiently transported into the cytoplasm of Micractinium sp. GA001 and binds
to lipid bodies emitting strong yellow fluorescence (Fig. 2). These results were consistent
with the earlier reports in green microalgae and cyanobacteria [48]. While BODIPY 505/515
assisted with significantly lower concentration of DMSO was able to efficiently penetrate the
cell wall of Micractinium sp. GA001 and binds with intracellular lipid bodies emitting green
fluorescence Fig. 2. Confocal images of both Nile Red and BODIPY 505/515 stained

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Micractinium sp. GA001 were clearly distinguished from chloroplasts and other intracellular
organelles by their fluorescence (Fig. 2).

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Further, the suitability of Nile Red and BODIPY 505/515 have been evaluated for flow
cytometry-based analysis. Channels ECD and FITC in CytoFlex S were used for detection of
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fluorescent signals of Nile Red and BODIY 505/515, respectively (Fig. 3). Micractinium sp.
GA001 grown in ESM, walnes, and TMRL were subjected to flow cytometry analysis. Fig. 3
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presents a sequence of fluorescence histograms related to different cell growth phases of
Micractinium sp. GA001 in above mentioned 3 media. The results clearly exhibiting higher
relative fluorescence intensity for BODIPY 505/515 stained cells than Nile red stained cells
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in all growth phases in all tested media. The mean fluorescent intensity of both fluorescent
dyes stained GA001 cells gradually increased from the exponential to the stationary growth
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phase. In addition, we can also clearly distinguish the peaks between different cell growth
phases in BODIPY 505/515 stained cells than Nile Red stained cells. These results regarding
flow cytometry analysis of BODIPY 505/515 and Nile Red stained GA001 cells were
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consistent with our previous reports [30, 35]. BODIPY fluorescent dye based screening of
lipid bodies in Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and
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Nannochloropsis atomus has been well established [49]. Overall, fluorescent staining and
flow cytometry analysis results clearly suggesting that BODIPY 505/515 is more suitable
than Nile Red for analysis and cell sorting of high-lipid producing phenotypes of
Micractinium sp. GA001 (Fig. 3).

15
3.4. Selective enrichment of long-chain fatty acids producing phenotypes of
Micractinium sp. GA001

3.4.1. Selective isolation of high-level lipids accumulating phenotypes using FACS

As overall goals of this study were to selectively isolate and enrich high-level long-
chain fatty acids producing phenotypes of Micractinium sp. GA001, cell sorting protocols
have been established in fluorescent stained Micractinium sp. GA001 using FACS. Based on
the results obtained from above experiments, ESM and BODIPY 505/515 were selected as
the best medium for enrichment and best fluorescent dye for staining of intracellular lipid
bodies, respectively. In this adaptive evolution approach, modified ESM medium was used

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with nitrogen-repletion and –depletion conditions, separately. The use of nutrient stress
phenomena for enhancing intracellular lipid content were previously reported in diatom

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Phaeodactylum tricornutum, green microalgae Schizochytrium sp., C. littorale, and C.
reinhardtii, as microalgal strains respond in a highly dynamic way under nutrient stress
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condition [31, 50, 51]. Highly healthy seed population of Micractinium sp. GA001 was
obtained from late-log phase in nitrogen-supplemented ESM medium with initial
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concentration of approximately 0.05 at OD750 nm. BODIPY 505/515 was selected as
lipophilic fluorescent dye and FACS sorting conditions were used as described previously
with minor modifications [30]. In detail, FL1 (530/40 nm) and FL5 (740 LP) were used for
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detection of BODIPY 505/515 fluorescence and autofluorescence, respectively. A two

dimensional plots of autofluorescence (FL5) vs. BODIPY 505/515 (FL1) channel obtained
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from BODIPY 505/515 stained cells of Micractinium sp. GA001 (Fig. 4). A plot between
chloroplast autofluorescence and green fluorescence of BODIPY stained lipid bodies
exhibited best variables with better correlation values. A plot between chloroplast
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autofluorescence and lipid fluorescence can help us to retrieve the cells with low levels of
chlorophyll degradation upon exposure to nitrogen-depletion conditions [31]. Therefore, the
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cells could efficiently grow in freshly supplied medium, and achieve high-level of biomass
during subsequent rounds of sorting. As this study aims to selectively enrich high-lipid
content phenotypes of GA001, 10,000 cells from the high-lipid content population of
BODIPY 505/515 stained seed cells of GA001 was sorted directly into ESM medium and
was re-suspended in nitrogen-depleted or –replete ESM medium separately. Experimental
triplicates were maintained for both conditions.

16
3.4.2. Change in biomass and intracellular lipid accumulation in Micractinium sp.
GA001 during adaptive evolution period

After sorting of 10,000 cells, the cells were initially allowed to grow in 10 mL of
ESM medium for 7 days further the cells were transferred into fresh ESM medium and
allowed to growth until they reached late-stationary phase (or high cell density). Nitrogen-
depletion and –repletion conditions were maintained separately. Fig. 5 shows the growth
curves for all generations in both nitrogen-depletion and –repletion conditions. At the initial
stage of evolution period, it was found that Micractinium sp. GA001 showed slow growth in
nitrogen-depletion condition (by 28 days) than in nitrogen-repletion condition (Fig. 5b). Cells
from day 21 and day 28 cultures of GA001 in nitrogen-repletion and –depletion conditions,

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respectively, were considered as first generation cultures and prepared for next consecutive
rounds of sorting (Fig. 5). After sorting 10,000 cells at indicated intervals, the remaining

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portions of the cells were used for fatty acids analysis using confocal microscopy and GC-
FID and elemental analysis. During second generation culturing, GA001 was found to have
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better growth rate compared to first generation growth. The second generation phenotypes of
GA001 were reached high cell density in Day 43 and Day 50 in nitrogen-repletion and –
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depletion conditions respectively (Fig. 5). The adaptation period for sorted phenotypes of
GA001 was higher in nitrogen-depletion condition than in –repletion condition (Fig. 5). This
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may be the possible reason for slow growth of sorted phenotypes of GA001 at the beginning
of evolution period in nitrogen-depletion condition. However, the sorted phenotypes of
GA001 showed healthy growth in subsequent generations. The zero hour seed and first
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generation cultures were considered as positive controls in our strain improvement

experiments. Therefore, the microalgal growth and intracellular lipid growth patterns of
subsequent generations were compared with positive controls. Though Micractinium sp.
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GA001 had attained relatively high cell growth in nitrogen-repletion condition than in -
depletion condition at some points, overall, no significant changes were found in cell growth
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pattern between nitrogen-repletion and –depletion conditions (Fig. 5). In addition, we also
observed no significant variations in the cell size of GA001 between nitrogen-depletion (5.35
µm) and nitrogen-repletion (5.36 µm) conditions. This growth pattern is in line with our
earlier observation in wild type strain of model microalgae C. reinhardtii CC124. Wild type
CC124 attained similar growth pattern in both nitrogen-repletion and -depletion conditions
during adaptive evolution period [30]. Therefore, it was concluded that the cell division and

17
regeneration of GA001 in both nitrogen-repletion and –depletion conditions are similar
regardless of the presence of nitrogen.

As a final goal of this study was to obtain improved biomass without alternations in
FAME contents in GA001, this study highlight that the evolved population had attained 3-
fold more biomass (1.98 g/L) than original population (0.7 g/L) at the end of evolution period
(Table 2). To analyse the changes in intracellular lipid content during adaptive evolution
period, GA001 phenotypes periodically collected during adaptive evolution period were
stained with BODIPY 505/515, and this indicates relative measurement of intracellular lipids
[31]. A gradual increase in BODIPY 505/515 fluorescence intensities was observed during

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adaptive evolution period, and that indicates a highly progressive increase in accumulation of
total lipids in evolved phenotypes of GA001. Fig. 6 shows progressive increase in
fluorescence signals of BODIPY 505/515-stained GA001 in nitrogen-depletion and –

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repletion conditions. A well-defined shift in fluorescent intensities of BODIPY 505/515
signals was indicating increased accumulation of intracellular lipid bodies in GA001 (Fig. 6).
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A gradual increase and a clear shift in BODIPY 505/515 fluorescence signals of GA001 cells
was found in nitrogen-repletion conditions, from first generation to the end of evolution
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period. The GA001 had attained the highest mean fluorescent intensities in third generation
population under nitrogen-depletion conditions (Fig. 6b). The highest mean fluorescent
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intensities of GA001 phenotypes achieved in nitrogen-repletion conditions were significantly

lower than that obtained in nitrogen-depletion conditions. The gradual increase in lipid
accumulation in GA001 cells during adaptive evolution period was analysed using confocal
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microscopy. Fig. 7 and 8 represents the confocal images of BODIPY 505/515 stained cells of
GA001 under nitrogen-repletion and –depletion conditions. As we mentioned in the earlier
section, green fluorescence of BODIPY 505/515 stained intracellular lipid bodies can be
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easily distinguished from the strong red autofluorescence emitted from the chlorophyll. The
confocal microscopy images clearly showed an increase in accumulation of intracellular lipid
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bodies in GA001 under nitrogen-repletion and -depletion conditions (Fig. 7 and 8). Very
interestingly, the confocal images showed that size of intracellular lipid bodies significantly
increased at the end of adaptive evolution period in nitrogen-depletion conditions (Fig. 8g-i).
This observation is in line with earlier reports that an increase in intracellular lipid bodies can
be achieved in microalgae under nitrogen stress conditions [52]. As particular phenotypes
were selected based on gate values between chlorophyll and lipid signals, the chlorophyll
18
signals were analysed. Interestingly, no significant change in chlorophyll signals was found
during adaptive evolution period. This indicating the fact that no degradation in chlorophyll
content in GA001 under nitrogen-stress conditions thus the cells can efficiently function.
However, further sorting and regeneration of GA001 have not produced more lipids
accumulating populations (data not shown). This could be due to one of the facts that GA001
phenotypes achieved their highest lipid accumulation in nitrogen stress conditions. And
another fact is that since this strategy aims to increase intracellular lipid contents without
addition of any mutagens, and purely based on nutrient stress conditions, the phenotypes may
revert back to original characteristics.

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Table 2 shows the elemental analysis of Micractinium sp. GA001 phenotypes during
adaptive evolution period. The carbon to nitrogen ratio (C:N) of GA001 increased at the end
of adaptive evolution period, in both nitrogen-repletion and –depletion conditions. Increase in

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C:N ratio of GA001 was gradual but not significant in nitrogen-repletion condition (Table 2).
This is in agreement with lipid accumulation pattern that increase in lipid accumulation was
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gradual but not very significant in nitrogen-repletion condition (Table 2). However, we have
observed a significant increase in C:N ratio in GA001 phenotypes in nitrogen-depletion
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condition (Table 2). The C:N ratio results of GA001 correlates well with lipid accumulation
pattern in both nitrogen-repletion and –depletion conditions (Table 2). A study by Zhen et al.
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(2016) reported significant alterations in total lipid contents in Chlorella sp. in nitrogen-
depletion conditions under different C:N ratios (1.0, 3.0, and 4.0) [54]. The study reported
gradual increase in total lipid content and attained highest lipid content of 58.33% when C:N
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ratios were changed from 1.0 to 3.0 to 5.0. Increase in lipid content was likely because high
C:N (5.0 compared with 3.0) ratio caused salt osmotic and toxic ionic stress for microalgal
growth and biomass production. In addition, it was also reported that any change in C:N ratio
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can influence the intracellular fatty acids composition in microalgae [43, 54]. It is because of
the fact that C:N ratios influence the competitive metabolic pathways between the lipid
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biosynthesis and photosynthesis [43, 54]. Previous studies suggest that change in intracellular
C:N ratios in algal systems attributing to the enhancement in biomass and lipid accumulation.
In this regard, in the present study, under nitrogen-depletion conditions, increase in C:N ratio
(from 11.1 to 13.0) enhanced the total lipid yield (9.6% to 16.6%) when compared with
nitrogen-repletion conditions (Table 2). This finding also supports earlier finding in diatom P.
tricornutum and green algae C. reinhardtii that change in C:N ratio in context with change in
19
intracellular lipids accumulation in microalgae [43].

One of the most important aims in this study was to specifically increase lipid contents
without alterations in long-chain fatty acids composition. For this purpose, FAME contents of
evolved phenotypes of Micractinium sp. GA001 were quantified using gas chromatography.
At the end of evolution period, GA001 exhibited one fold increase in total lipid content (8.4%
to 16.6%, compared than mother population) in nitrogen-depletion conditions, and a
moderate increase (8.4% to 12.2%, compared than mother population) was observed in
nitrogen-repletion conditions. Most importantly, no change in fatty acids composition was
observed in both nitrogen-depletion and –repletion conditions. Evolved phenotypes of
GA001 accumulated commercially important ω3 and ω6 fatty acids. Especially, evolved

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Micractinium sp. GA001 accumulated C20:0, C20:1, and C20:5 fatty acids. Overall, by

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combining all our results related to strain improvement proved that the present strategy
achieved in yielding evolved phenotypes of Micractinium sp. GA001 with target specific
increased accumulation of long-chain fatty acids. FACS-based microalgae strain
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improvement yielded high-level lipid accumulating population in different strains including
N. maritime, C. reinhardtii, and C. littorale [13, 31]. However, till date, no successful studies
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were reported for targeting long-chain fatty acids accumulating microalgal strains.
Haptophytes are well recognized microalgae for the synthesis of long-chain fatty acids. A
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traditional random mutagenesis using UV irradiation followed by flow cytometry selection in

haptophyta Isochrysis affinis galbana have not shown significant increase in the total fatty
liacids content [53]. To be a successful microalgal phenotype at commercial scale, any
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phenotype should achieve a biomass of 4g/L culture medium with accumulation of

intracellular lipid content of about 20%. However, for target specific goals, for example, to
achieve improved strains specifically with long-chain fatty acids, achieving 4g/L may not be
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feasible. As Micractinium sp. GA001 phenotypes specifically accumulating long-chain fatty

acids, the biomass and target specific lipid content achieved in this study were highly
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reasonable. In addition, considering the limitations in long-chain fatty acids producing

haptophytes, Micractinium sp. GA001 can be a better candidate for the establishment of
further bioprocessing and scaling-up with emphasis on long-chain fatty acids production.

4. Conclusions

20
 FACS-based bioprocessing technique in combination with nitrogen stress condition
yielded improved phenotypes of Micractinium sp. GA001 with high biomass and desirable
FAME composition. The results of media optimization, FACS-sorting, confocal microscopic
analysis, lipid composition and elemental analysis reported here highlight the characterization
of significantly improved phenotypes of GA001. By considering limitations on bioprocessing
and scaling-up of long-chain fatty acids producing haptophytes, the improved phenotypes
reported here can be promising materials for the biosynthesis of long-chain fatty acids in
higher scale. Further understanding on metabolic pathways in GA001 can improve our
knowledge on biosynthesis of long-chain fatty acids in green microalgae.

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Author Agreement
I, Dr. Natarajan Velmurugan, the undersigned, on behalf of all authors, declare that this manuscript is

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original, has not been published before and is not currently being considered for publication
elsewhere.

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I confirm that the manuscript has been read and approved by all named authors and that there are no
other persons who satisfied the criteria for authorship but are not listed. I further confirm that the
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order of authors listed in the manuscript has been approved by all of us.
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I understand that the Corresponding Author is the sole contact for the Editorial process. He/she is
responsible for communicating with the other authors about progress, submissions of revisions and
final approval of proofs.
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Authors’ contributions
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N.V. conceived the idea, and designed and supervised the experiments. D.D., R.M.,
T.M., and N.V. performed the experiments. D.D., R.M., S.S.K., T.M., C.C., H.P.D.B., and
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N.V. analysed data and wrote the article.

Acknowledgment

The authors wish to thank the Director, CSIR-NEIST, Jorhat, Assam for his kind
encouragement in carrying out this work. This work was supported by research funds of

21
DST-INSPIRE Faculty program (IFA 15-LSPA 31) funded by the Department of Science and
Technology (DST), Government of India.

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals

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performed by any of the authors.

References

ro
[1] H.C. Greenwell, L.M.L. Laurens, R.J. Shields, Placing microalgae on the biofuels
priority list : a review of the technological challenges, J. R. Soc. Interface. 7 (2010)
-p
703–726.
[2] M. Jaubert, J. Bouly, M. Ribera, Light sensing and responses in marine microalgae,
re
Curr. Opin. Plant Biol. 37 (2017) 70–77.
[3] T. Ishika, N.R. Moheimani, P.A. Bahri, Sustainable saline microalgae co-cultivation
for biofuel production : a critical review, Renew. Sustain. Energy Rev. 78 (2017) 356–
lP

368.
[4] E.C. Odjadjare, T. Mutanda, A.O. Olaniran, E.C. Odjadjare, T. Mutanda, A.O.
Olaniran, Critical reviews in biotechnology potential biotechnological application of
na

microalgae : a critical review, Crit. Rev. Biotechnol. 37 (2017) 37–52.

[5] A. Zonouzi, M. Auli, M.J. Dakheli, M.A. A.M. Hejazi, Oil extraction from microalgae
Dunalliela sp . by polar and non-polar solvents, Int. J. Agri. Biosys. Eng. 10 (2016)
642–645.
ur

[6] B.K. Shurtz, B. Wood, J.C. Quinn, Nutrient resource requirements for large-scale
microalgae biofuel production : multi-pathway evaluation, Sustain. Energy Technol.
Jo

Assessments. 19 (2017) 51–58.

[7] J. Milano, H. Chyuan, H.H. Masjuki, W.T. Chong, M. Kee, Microalgae biofuels as an
alternative to fossil fuel for power generation, Renew. Sustain. Energy Rev. 58 (2016)
180–197.
[8] O.K. Dalrymple, T. Halfhide, I. Udom, B. Gilles, J. Wolan, Q. Zhang, S. Ergas,
Wastewater use in algae production for generation of renewable resources : a review
and preliminary results, Aquat. Biosyst. 9 (2013) 1–11.

22
[9] X. Sun, L. Ren, Z. Bi, X. Ji, Q. Zhao, H. Huang, Adaptive evolution of microalgae
Schizochytrium sp . under high salinity stress to alleviate oxidative damage and
improve lipid biosynthesis, Bioresour. Technol. 267 (2018) 438–444.
[10] H. Taher, S. Al-zuhair, A. Al-marzouqi, Y. Haik, M. Farid, Growth of microalgae
using CO2 enriched air for biodiesel production in supercritical CO2, Renew. Energ. 82
(2015) 61–70.
[11] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, A. Darzins,
Microalgal triacylglycerols as feedstocks for biofuel production : perspectives and
advances, Plant J. 54 (2008) 621–639.

[12] J. Lorenzen, N. Igl, M. Tippelt, A. Stege, F. Qoura, U. Sohling, T. Brück, Extraction of

microalgae derived lipids with supercritical carbon dioxide in an industrial relevant
pilot plant, Bioprocess Biosyst. Eng. 40 (2017) 911–918.

of
[13] T.T.Y. Doan, J.P. Obbard, Enhanced intracellular lipid in Nannochloropsis sp . via
random mutagenesis and flow cytometric cell sorting, Algal Res. 1 (2012), 17–21.
[14] P. Charoonnart, S. Purton, Applications of microalgal biotechnology for disease

ro
control in aquaculture, Biology (Basel). 7 (2018), 1–14.
[15] C.M. Beal, L.N. Gerber, T. Supis, W. Phromkunth, V. Kiron, J. Granados, I.
-p
Archibald, C.H. Greene, M.E. Huntley, Marine microalgae commercial production
improves sustainability of global fisheries and aquaculture, Sci. Rep. 8 (2018) 1–8.
[16] P. Steinrücken, S. Rune, S. Are, H. Kleivdal, S. Kristin, Bioprospecting North Atlantic
re
microalgae with fast growth and high polyunsaturated fatty acid (PUFA) content for
microalgae-based technologies, Algal Res. 26 (2017) 392–401.
lP

[17] S. Sonowal, C. Chikkaputtaiah, N., Velmurugan, Role of flow cytometry for the
improvement of bioprocessing of oleaginous microorganisms, J. Chem. Technol.
Biotechnol. 94 (2019) 1712–1726.
[18] T. Katayama, M. Kishi, K. Takahashi, K. Furuya, Isolation of lipid-rich marine
na

microalgae by flow cytometric screening with nile red staining, Aquac. Int. 27 (2019)
509–518.
[19] H.S. Kim, S.H.S. Han, H.R. Thapa, A.R. Guzman, D.R. Browne, M. Tatli, T.P.
ur

Devarenne, D.B. Stern, A. Han, High-throughput droplet microfluidics screening

platform for selecting fast-growing and high lipid-producing microalgae from a mutant
library, Plant Direct. 1 (2017) 1–13.
Jo

[20] Z. Yi, Y. Su, M. Xu, A.B. Id, S. Ingthorsson, Chemical mutagenesis and fluorescence-
based high-throughput screening for enhanced accumulation of carotenoids in a model
marine diatom Phaeodactylum tricornutum, Mar. Drugs. 16 (2018) 1–16.
[21] A. De Jara, A. Martel, C. Molina, L. Nordströn, V. De Rosa, D. Ricardo, Flow
cytometric determination of lipid content in a marine dinoflagellate , Crypthecodinium
cohnii, J. Appl. Phycol. 15 (2003) 433–438.
[22] H. Pereira, P.S.C. Schulze, L. Maylin, T. Santos, L. Barreira, Fluorescence activated
23
 cell-sorting principles and applications in microalgal biotechnology, Algal Res. 30
(2018) 113–120.
[23] R.A.I. Abou-shanab, S. V Raghavulu, N.M.A. Hassanin, S. Kim, Y.J. Kim, S.U. Oh,
Y. Oh, B. Jeon, Manipulating nutrient composition of microalgal growth media to
improve biomass yield and lipid content of Micractinium pusillum, Afr. J. Biotechnol.
11 (2012) 16270–16276.
[24] L.L. Estévez-Landazábal, A.F. Barajas-Solano, C. Barajas-Ferreira, V. Kafarov,
Improvement of lipid productivity on Chlorella vulgaris using waste glycerol and
sodium acetate, Cienc. Tecnol. Futuro. 5 (2013) 113–126.

[25] S. Park, J. Kim, Y. Park, S. Kim, S. Cho, J. Yu, C. Kang, Comparison of trophic
modes to maximize biomass and lipid productivity of Micractinium inermum NLP-
F014, Biotechnol. Bioproc. E. 245 (2018) 238–245.

of
[26] J.D. Moha-león, I.A. Pérez-legaspi, I. Rubio-franchini, E. Ríos-leal, I.A. Pérez-legaspi,
Improving the lipid content of Nannochloropsis oculata by a mutation-selection
program using UV radiation and quizalofop, J. Appl. Phycol. 31 (2018) 191–199.

ro
[27] G. De Bhowmick, L. Koduru, R. Sen, Metabolic pathway engineering towards
enhancing microalgal lipid biosynthesis for biofuel application — a review, Renew.
Sustain. Energy Rev. 50 (2015) 1239–1253.
-p
[28] J.L. Blatti, J. Michaud, M.D. Burkart, Engineering fatty acid biosynthesis in
microalgae for sustainable biodiesel, Curr. Opin. Chem. Biol. 17 (2013) 496–505.
re
[29] S.Y. Lee, D. Lee, T.Y. Kim, Systems biotechnology for strain improvement, Trends
Biotechnol. 23 (2005) 349–358.
lP

[30] N. Velmurugan, M. Sung, S.S. Yim, M.S. Park, J.W. Yang, K.J. Jeong, Systematically
programmed adaptive evolution reveals potential role of carbon and nitrogen pathways
during lipid accumulation in Chlamydomonas reinhardtii, Biotechnol. Biofuels. 7
(2014) 1–15.
na

[31] I.T.D. Cabanelas, M. Van Der Zwart, D.M.M. Kleinegris, R.H. Wijffels, M.J. Barbosa,
Sorting cells of the microalga Chlorococcum littorale with increased triacylglycerol
productivity, Biotechnol. Biofuels. 9 (2016) 1–12.
ur

[32] H. Pereira, K.N. Gangadhar, P.S.C. Schulze, T. Santos, C.B. De Sousa, L.M. Schueler,
L. Custódio, F.X. Malcata, L. Gouveia, J.C.S. Varela, L. Barreira, Isolation of a
euryhaline microalgal a robust feedstock for biodiesel production, Sci. Rep. 6 (2016)
Jo

1–11.
[33] A.M. Nonomura, D.M. Coder, Improved phycocatalysis of carotene production by
flow cytometry and cell sorting, Biocatalysis. 1 (1988) 333–338.
[34] A. Gnouma, E. Sehli, W. Medhioub, R. Ben, D. Mahmoud, M. Norbert, Strain
selection of microalgae isolated from Tunisian coast : characterization of the lipid
profile for potential biodiesel production, Bioprocess Biosyst. Eng. 41 (2018) 1449–
1459.

24
[35] N. Velmurugan, M. Sung, S.S. Yim, M.S. Park, J.W. Yang, K.J. Jeong, Evaluation of
intracellular lipid bodies in Chlamydomonas reinhardtii strains by flow cytometry,
Bioresour. Technol. 138 (2013) 30–37.

[36] J. Folch, M. Lees, G.H.S. Stanley, A simple method for the isolation and purification of
total lipids from animal tissues. J. Biol. Chem. 226 (1957) 497-509.
[37] R. Hoshina, M. Iwataki, N. Imamura, (Chlorellaceae, Trebouxiophyceae): redescription
of the endosymbiotic green algae of Paramecium bursaria (Peniculia ,
Oligohymenophorea) in the 120th year, Phycological Res. 58 (2010) 188–201.
[38] Arriola, M.B., Velmurugan, N., Zhang, Y., Plunkett, M.H., Hondzo, H., Barne, B.M.,
Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium
conductrix SAG 241.80: implications to maltose excretion by a green alga, Plant J. 93
(2018) 566–586.

of
[39] J.W. Hong, S. Jo, H. Cho, S.W. Nam, W. Shin, K.M. Park, K.I. Lee, Phylogeny,
morphology, and physiology of Micractinium strains isolated from shallow ephemeral
freshwater in Antarctica strains isolate, Phycological Res. 63 (2015) 212–218.

ro
[40] V.C.A. Ward, L. Rehmann, Fast media optimization for mixotrophic cultivation of
Chlorella vulgaris, Sci. Rep. 9 (2019) 19262.
-p
[41] A. Danbara, Y. Shiraiwa, The requirement of selenium for the growth of marine
coccolithophorids , Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp.
(Prymnesiophyceae), Plant Cell Physiol. 40 (1999) 762–766.
re
[42] J. Dörner, P. Carbonell, S. Pino, A. Farías, Fisheries and aquaculture cultured under
different nitrate availabilities, Fish. Aquac. J. 5 (2014) 3–5.
lP

[43] J. Valenzuela, A. Mazurie, R.P. Carlson, R. Gerlach, K.E. Cooksey, B.M. Peyton,
M.W. Fields, Potential role of multiple carbon fixation pathways during lipid
accumulation in Phaeodactylum tricornutum, Biotechnol. Biofuels. 5 (2012) 1–17.
[44] B.R. Kumar, G. Deviram, T. Mathimani, P.A. Duc, A. Pugazhendhi, A.,
na

Microalgae as rich source of polyunsaturated fatty acids, Biocat. Agri. Biotechnol. 17

(2019) 583–588.
[45] N. Velmurugan, D. Deka, Transformation techniques for metabolic engineering of
ur

diatoms and haptophytes : current state and prospects, Appl. Microbiol. Biotechnol.
102 (2018) 204–210.
[46] M.S. Cooper, W.R. Hardin, T.W. Petersen, R.A. Cattolico, Visualizing green oil in live
Jo

algal cells, J. Biosci. Bioeng. 109 (2010) 198–201.

[47] T.T.Y. Doan, J.P. Obbard, Improved nile red staining of Nannochloropsis sp ., Algal
Res. 1 (2011) 895–901.
[48] T. Govender, L. Ramanna, I. Rawat, F. Bux, BODIPY staining , an alternative to the
nile red fluorescence method for the evaluation of intracellular lipids in microalgae,
Bioresour. Technol. 114 (2012) 507–511.
[49] L. Brennan, A. Blanco, A.S. Mostaert, P. Owende, Enhancement of BODIPY 505/515
25
 lipid fl uorescence method for applications in biofuel-directed microalgae production,
J. Microbiol. Methods. 90 (2012) 137–143.
[50] X.M. Sun, L.J. Ren, Q.Y. Zhao, X.J. Ji, H. Huang, Microalgae for the production of
lipid and carotenoids : a review with focus on stress regulation and adaptation,
Biotechnol. Biofuels, 11 (2018) 1–16.
[51] X. Wang, S. Luo, W. Luo, W. Yang, J. Liu, H. Li, Adaptive evolution of microalgal
strains empowered by fulvic acid for enhanced polyunsaturated fatty acid production,
Bioresour. Technol. 277 (2019) 204–210.
[52] C. Goodson, R. Roth, Z.T. Wang, U. Goodenough, Structural correlates of cytoplasmic
and chloroplast lipid body synthesis in Chlamydomonas reinhardtii and stimulation of
lipid body production with structural correlates of cytoplasmic and stimulation of lipid
body production with acetate boost, Eukaryot. Cell. 10 (2011) 1592–1606.
[53] G. Bougaran, C. Rouxel, N. Dubois, R. Kaas, S. Grouas, E. Lukomska, J.R. Le Coz,

of
J.P. Cadoret, Enhancement of neutral lipid productivity in the microalga Isochrysis
affinis galbana (T-Iso) by a mutation-selection procedure, Biotechnol. Bioeng. 109
(2012) 2737-2745.

ro
[54] J. Zhan, Y. Hong, H. Hu, Effects of nitrogen sources and C/N ratios on the lipid
producing potential of Chlorella sp. HQ, J. Microbiol. Biotechnol. 26 (2016) 1290-1302.
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Figure Captions
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Fig. 1. Growth curve for Micractinium sp. GA001 in 6 different media. Each data point
represents average of triplicates. Symbols representing the following media (●) walnes, (▼)
ESM, (∆) Chry, (□) TMRL, (○) f/2, (■) MNK.
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Fig. 2. Confocal images of BODIPY 505/515 (a-c) and Nile Red (d-f) strained strains of
Micractinium sp. GA001 at early stationary phase, arrows indicate lipid droplets in a and d,
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arrows indicate chloroplast signals in b and e.

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Fig. 3. Flow cytograms of BODIPY 505/515 (1), and Nile Red (2) stained Micractinium sp.
GA001 in walnes (a), ESM (b), and TMRL (c) media. The growth phase representations of
numerical numbers in the flow cytograms are as follows: 1: exponential phase, 2: early-stationary
phase, and 3: late-stationary phase.

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Fig. 4. Two-dimensional dot plots (a) and flow cytograms (b) of BODIPY 505/515 stained 0
hour seed cells of Micractinium sp. GA001. (a) two-dimension plot representing signal
pattern between BODIPY 505/515 stained lipid bodies and autofluorescence of chlorophyll
of seed cells of Micractinium sp. GA001. (b) region R1 (blue), region R2 (green), and region
R3 (red) in flow cygrams (as well in two-dimensional plot) represent low, medium, and high
lipid content phenotypes of Micractinium sp. GA001, respectively. A total of 10,000 cells
from the top 2% of R3 region were sorted and regenerated in nitrogen-depletion –repletion
ESM medium, separately.

Fig. 5. Growth curve of Micractinium sp. GA001 in nitrogen-repletion (a) and nitrogen-

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depletion (b) ESM medium.

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Fig. 6. Flow cytograms of adaptive evolved phenotypes of Micractinium sp. GA001 in
nitrogen-repletion (a) and nitrogen-depletion (b) ESM medium. Graphs show the
fluorescence signals of BODIPY 505/515-stained GA001 between first and third generation
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populations. A well-defined progressive shift in fluorescent intensities of probe signals
between first and third generation populations was indicating increased accumulation of
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intracellular lipid bodies in GA001. At the end of evolution period, the third-generation
populations shown higher values of fluorescent intensities (ie. produced high lipids) than the
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first-generation populations.

Fig. 7. Confocal images of BODIPY 505/515-stained adaptive evolved Micractinium sp.

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GA001 strains in nitrogen-repletion conditions, arrows indicate lipid bodies. (a-c) first
generation population and (d-f) third generation population, arrows indicate lipid droplets in
a, c, d and f, arrows indicate chloroplast signals in b, and e.
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Fig. 8. Confocal images of BODIPY 505/515-stained adaptive evolved Micractinium sp.

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GA001 strains in nitrogen-depletion conditions, arrows indicate lipid bodies. (a-c) first
generation population, (d-f) second generation population, (g-i) third generation population,
arrows indicate lipid droplets in a, c, d, f, g, and i, arrows indicate chloroplast signals in b, e,
and h.

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Table 1 GC quantification of long-chain fatty acids in Micractinium sp. GA001 grown in

different media
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Media Total lipid Fatty acid composition

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(%)a C20:0 C20:1 C20:5 (EPA)

(ARA) Eicosenoic acid

f/2 4.0±0.03 8.31 31.9 2.1

ESM 8.4±0.1 7.9 24.96 2.0

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 Chry 4.8±0.03 6.7 23.9 1.9

MNK 4.4±0.02 17.0 5.34 12.60

TMRL 7.2±0.2 5.20 15.84 6.1

walnes 6.9±0.1 4.0 10.8 (C18:0) 12.7 (C18:2)

Isochrysis galbana NIES 2590 8.0±0.1 7.17 25.40 2.93

a
All data are expressed as average and SD of triplicate experiments

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Table 2 Elemental analysis of adaptive evolved strains of Micractinium sp. GA001 at

different stages
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Conditions Generations Biochemical elementsa C/N Total lipid Biomass

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C H N ratio (%)a (g/L) a

first 23.59±0.2 5.84±0.02 3.09±0.01 7.63:1 8.9±0.3 1.87±0.4

Nitrogen- second 22.27±0.11 5.64±0.001 2.88±0.03 7.73:1 11.4±0.3 1.92±0.21

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repletion third 29.46±0.23 6.19±0.01 3.65±0.01 8.80:1 12.2±0.2 1.90±0.2

first 18.32±0.13 4.99±0.001 1.64±0.02 11.1:1 9.6±0.2 1.66±0.5

Nitrogen- second 33.24±0.1 6.97±0.03 2.98±0.04 11.15:1 11.8±0.4 1.78±0.8

depletion
third 26.43±0.1 6.07±0.02 2.02±0.01 13.10:1 16.6±0.22 1.98±0.3

a
All data are expressed as average and SD of triplicate experiments

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