Journal of

Marine Science
and Engineering

Article
Arthrospira platensis Variants: A Comparative Study Based on
C-phycocyanin Gene and Protein, Habitat, and Growth Conditions
Nawal Abd El-Baky * , Neama Mahmoud Fattouh Rezk and Amro A. Amara *

Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI),
City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City,
Alexandria P.O. Box 21934, Egypt
* Correspondence: [email protected] (N.A.E.-B.); [email protected] (A.A.A.)

Abstract: This study aimed to map the differences between Arthrospira sp. and Arthrospira platensis
strains and variants from the order Oscillatoriales at the gene and protein levels of C-phycocyanin
alpha chain via multiple alignment, phylogenetic trees of species, and analysis of the nucleotide and
amino acid composition of the studied sequences. The links between gene/protein and environmental
features of the habitat or source of isolation were also investigated. Phycocyanin was extracted from
three A. platensis strains: an Egyptian isolate cultivated in the laboratory under static conditions in
a highly saline medium and two commercial products. The French commercial strain showed the
highest extraction yield but the lowest C-phycocyanin purity, and the color intensity of the extracted
pigment from the Egyptian isolate was significantly weaker than those of the two commercial strains.
All the analyzed species and strains had GC content of more than 54.5% in C-phycocyanin alpha
chain gene and showed high abundance of alanine, an amino acid encoded exclusively by GC-biased
codons, in their protein. The frequencies of the acidic amino acids aspartic acid and glutamic acid
were 5.2% and 5.0% on average, respectively, which were slightly higher than those of the basic
residues (4.3% arginine, 0.6% histidine, and 5.0% lysine). Data relating to the isolation source of most
of the analyzed species revealed harsh conditions, such as high alkalinity, salinity, CO2 saturation,
and/or temperature. These findings may link the gene/protein of C-phycocyanin, which is one of
Citation: El-Baky, N.A.; Rezk, N.M.F.; the most important bioactive proteins of A. platensis, to the adaptation of this organism to harsh
Amara, A.A. Arthrospira platensis environmental conditions and associate the color of the pigment to cultivation conditions and/or
Variants: A Comparative Study isolation source.
Based on C-phycocyanin Gene and
Protein, Habitat, and Growth Keywords: Arthrospira platensis; C-phycocyanin; GC content; alignment; acidic and basic amino acids;
Conditions. J. Mar. Sci. Eng. 2023, 11,
static growth; alkalinity; salinity
663. https://doi.org/10.3390/
jmse11030663

Academic Editors: Francesco

Tiralongo and Azizur Rahman 1. Introduction
Received: 31 January 2023 From very early on, humans have benefited from their observation of birds utilizing
Revised: 8 March 2023 algae floating on the surface of water. From ancient times in Egypt up to now, farmers have
Accepted: 19 March 2023 collected floating algae to feed their birds. Among these floating algae, one species that has
Published: 21 March 2023 attracted considerable attention of scientists worldwide is the microalga or cyanobacterium
Arthrospira platensis [1,2].
Arthrospira platensis has been extensively studied since it was rediscovered as being
an edible microorganism (nontoxic nutritious food) that is consumed in large quantities in
Copyright: © 2023 by the authors.
Chad. People have observed that migratory birds and animals are interested in feeding on
Licensee MDPI, Basel, Switzerland.
Arthrospira platensis floating on the surface of different lakes in Africa and South America.
This article is an open access article
After a long migratory trip, the tired birds can recover their power through the consumption
distributed under the terms and
of this floating microalga. Even though the water is alkaline and salty, farm animals also
conditions of the Creative Commons
Attribution (CC BY) license (https://
drink this green water. In Ethiopia, farmers and herdsmen living in areas around soda lakes
creativecommons.org/licenses/by/
make their cattle drink water containing Arthrospira about once a month. They believe that
4.0/).
this microalga has therapeutic effects and supplement nutrients lacking in dietary food [3].

J. Mar. Sci. Eng. 2023, 11, 663. https://doi.org/10.3390/jmse11030663 https://www.mdpi.com/journal/jmse

J. Mar. Sci. Eng. 2023, 11, 663 2 of 19

In 1827, Turpin identified and described Arthrospira as spiral cyanobacteria using a light
microscope [4]. Rich (1931) reported that Arthrospira is a dominant phytoplankton in a number
of lakes in the Rift Valley of East Africa [5]. Dangeard (1940) reintroduced A. platensis to the
world from a sample collected by a pharmacist from a local market in Chad [1,6].
A. platensis occupies unique marine habitats, such as alkaline lakes [3]. A. platensis
producers mimic this natural phenomenon and increase the alkalinity to reduce the number
of other algal species [7]. In 2013, a study proved that in addition to alkalinity, salinity is an
essential factor that can suppress the growth of other algae and cyanobacteria, thus leading to
A. platensis dominating the environment and benefiting from the surrounding nutrients [2].
A. platensis can survive in both sunny and dark conditions. It can utilize sunlight
and CO2 to grow autotrophically. In the dark, it can utilize organic compounds and grow
auxotrophically. It can benefit from an appropriate day/night environment where organic
compounds exist and grow either autotrophically or auxotrophically or as a combination
of the two (autoauxotrophic growth) [8,9].
Amara and Steinbüchel (2013) suggested that salinity can play a significant role in
Arthrospira enrichment. They proposed that the rain/evaporation cycle on the lake and the
lake’s surrounding area would lead to salt accumulation [2]. Temperature can change the
amount of dissolved oxygen (pO2) and the pH value either by direct effect or by inducing
chemical or physical changes [2,10]. Salinity, alkalinity, temperature, organic compounds,
absence of other species due to these stresses, and other factors have been analyzed and
reported to affect the growth and cultivation of the genus Spirulina [10].
In addition to providing a good supply of bioactive ingredients in the diet, such
as essential amino acids and fatty acids, high protein synthesis capacity (60–70% of the
cell mass), minerals, and vitamins [11–13], A. platensis can synthetize phycobiliproteins
including C-phycocyanin (a blue natural pigment) [14]. In addition to its application
as natural colorants for food additives [15], C-phycocyanin also has anti-inflammatory,
antioxidant, and anticarcinogenic activities [16]. Unfortunately, this natural pigment has
been poorly explored by the food industry. The limitations for its application include the
extraction methods resulting in low-purity products and its low stability under storage and
during processing of foods. This natural pigment is preferably extracted at neutral pH of
5–8 and temperatures below 50 ◦ C [14].
Regions in the phycocyanin gene (e.g., intergenic spacer region) are highly conserved
and used in the molecular identification and characterization of the producing microor-
ganisms, including A. platensis [17]. It has previously been observed that the extracted
phycocyanin from strains that are phenotypically close to each other but were isolated
from different habitats (habitats with different light wavelengths and photoperiods) may
significantly vary in color [18,19]. Therefore, the extraction of phycocyanin from each strain
needs optimization [20,21].
This work aimed to map the differences between A. platensis strains and variants based
on gene and protein of the phycocyanin subunit alpha and determine any relationship
between the pigment structure, isolation source, cultivation conditions, and adaptation of
these microalgae to harsh environments. The frequencies of nucleotides in the phycocyanin
subunit alpha of nine cyanobacterial species and its amino acid composition in 25 cyanobac-
terial species, including Arthrospira platensis strains and variants, were calculated and linked
to their source of isolation. C-phycocyanin was also comparatively extracted from dried
biomass of three microalgae strains: an Egyptian A. platensis isolate cultivated in our lab
and French and Chinese commercial A. platensis products.

2. Materials and Methods

2.1. Microalgae Strains
An extreme alkalophilic A. platensis previously isolated from the brackish Lake Mar-
iout southwest of Alexandria city in Egypt was used in this study. This Egyptian iso-
late was authenticated (Accession CBA13040, phycocyanin alpha subunit, partial from
Limnospira fusiformis LM) by Sharaf et al. [17]. The French commercial A. platensis product
J. Mar. Sci. Eng. 2023, 11, 663 3 of 19

(spirulina tablets, VITAMINEXT, France) was purchased from a pharmacy in Casablanca,

Morocco. The Chinese commercial A. platensis strain (spirulina powder organic, Starwest
Botanicals, China) was obtained from a local Egyptian pharmacy.

2.2. Cultivation Medium

Amara and Steinbuchel (A–St) medium at concentration of 1.5× (highly saline medium)
consisted of part A: 13.82 g/L NaHCO3 , 10.71 g/L NaCO3 , and 0.75 g/L K2 HPO4 ; part
B: 2.25 g/L NaNO3 , 0.85 g/L K2 SO4 , 1.5 g/L NaCl, 0.22 g/L MgSO4 ·7H2 O, 0.011 g/L
CaCl2 ·2H2 O, 0.012 g/L FeSO4 ·2H2 O, and 0.1 g/L EDTA-Na2 ·2H2 O; part C: 0.02 g/L ferric
citrate; and part D: 0.1 g/L peptone and 0.01 g/L yeast extract [2]. Parts A–C of this medium
were dissolved in a minimal amount of sterile distilled water and filter-sterilized through a
syringe filter (pore size of 0.22 µm) from TPP (St. Louis, MO, USA). Part D was dissolved in
distilled water and sterilized separately by autoclaving and then used as a broth. Finally, a
20 L glass bottle was used to grow Egyptian A. platensis under static conditions.

2.3. Cultivation Conditions

Egyptian A. platensis strain was cultivated in the laboratory under static cultivation
condition in a 20 L sterile glass bottle and kept at the regular day/night cycle at temper-
atures ranging from 24 ◦ C (day) to 18 ◦ C (night). The cultivation bottle was kept closed
during the cultivation process (for 27 days).

2.4. Cells Harvesting

The cells were harvested manually by passing the culture through a big funnel contain-
ing two layers: one of synthetic sponge and the other of multilayers of cotton tissue. After
that, the wet biomass of A. platensis was placed in a Petri dish and left to dry using a sterile
air
J. Mar. Sci. Eng. 2023, 11, x FOR PEER flow of microbial cabinet under sterile conditions for 2 days. The steps of cultivation
REVIEW 4 of 20 up
to the obtaining of air-dried biomass are illustrated in Figure 1.

Figure 1. Cultivation of Egyptian A. platensis strain in the laboratory under static conditions.
Figure 1. Cultivation of Egyptian A. platensis strain in the laboratory under static conditions. (A)
(A) Preparation of membrane filter 1.5× medium components from parts A–C and the autoclaved
Preparation of membrane filter 1.5× medium components from parts A–C and the autoclaved part
part D; mixing
D; (B) (B) mixing of medium
of the the medium components
components underunder aseptic
aseptic conditions;
conditions; (C) adding
(C) adding the culture
the seed seed culture
of of
A.
A.platensis
platensis and the
the medium
mediumcomponents
componentstoto a sterile
a sterile container;
container; (D,E)
(D,E) static
static cultivation
cultivation and collection
and collection
ofA.
of A. platensis;
platensis; (F)
(F) air
air drying
dryingofofthe
theharvested
harvested cells.
cells.

2.5. Extraction of C-phycocyanin from Dry Biomass of Three Microalgae Strains

The dried biomass (1 g) of Egyptian, French, and Chinese A. platensis was presoaked
in a 20 mL sodium phosphate buffer of 0.1 M and pH 7 for 2 h to prepare for cell disrup-
tion. Following the presoaking step, maceration using a mortar and pestle and repeated
J. Mar. Sci. Eng. 2023, 11, 663 4 of 19

2.5. Extraction of C-phycocyanin from Dry Biomass of Three Microalgae Strains

The dried biomass (1 g) of Egyptian, French, and Chinese A. platensis was presoaked
in a 20 mL sodium phosphate buffer of 0.1 M and pH 7 for 2 h to prepare for cell disruption.
Following the presoaking step, maceration using a mortar and pestle and repeated freezing
(at –40 ◦ C for 2 h) and thawing (at 4 ◦ C for 5 min) were carried out in the dark. Ultrasonica-
tion was performed in combination with conventional methods for primary extraction of
C-phycocyanin, as described previously by Tavanandi et al. [22]. The sonicator (Bandelin
Sonopuls Sonicator, Berlin, Germany) was programmed to provide 30 s pulses with 15 s
pauses for a total period of 5 min.

2.6. Protein Content and Concentration of the Extracted C-phycocyanin

Following ultrasonication, the mixture was centrifuged at 13,000× g and 4 ◦ C for
10 min to separate clear supernatants that contained extracted C-phycocyanin. The total
protein concentration of the extracted C-phycocyanin from dry biomass of the three microal-
gae strains was measured by Bradford assay [23]. The supernatant samples were run on
12% SDS-PAGE. Absorbance of the supernatant samples from the three microalgae strains
was evaluated at wavelengths of 600, 610, 615, 620, 630, 640, 650, and 652 nm (OPTIZEN
Scan UV/VIS spectrophotometer, KLab Co., Daejeon, Korea) for C-phycocyanin.
The concentration of C-phycocyanin extracted from the three microalgae strains was
estimated as previously reported by Siegelman and Kycia [24] and Sharmaa et al. [25]
as follows:
C-phycocyanin = (A615 − 0.474 × A652 )/5.34 (1)
where A615 is the absorbance measured at wavelength of 615 nm, and A652 is the absorbance
measured at wavelength of 652 nm.

2.7. The Template Nucleotide Sequence of A. platensis Used in This Study

NCBI database (www.ncbi.nlm.nih.gov, last accessed on 20 January 2023) was used to
obtain phycocyanin alpha subunit (cpcA) gene of Arthrospira platensis, and the obtained
nucleotide sequence was saved in FASTA format and used as template for collecting
sequences of nine Arthrospira sp. and Arthrospira platensis strains and variants from the
order Oscillatoriales. The following sequence was selected as a template.
>ABD64608.1:1-162 phycocyanin alpha chain (Arthrospira platensis)
ATGAAAACCCCGCTGACCGAAGCGGTGAGCATTGCGGATAGCCAGGGCCG
CTTTCTGAGCAGCACCGAAATTCAGGTGGCGTTTGGCCGCTTTCGCCAGGCGAAAG
CGGGCCTGGAAGCGGCGAAAGCGCTGACCAGCAAAGCGGATAGCCTGATTACCGG
CGCGGCGCAGGCGGTGTATAACAAATTTCCGTATACCACCCAGATGCAGGGCCCGA
ACTATGCGGCGAACCAGCGCGGCAAAGATAAATGCGCGCGCGATATTAGCTATTATC
TGCGCATGGTGACCTATTGCCTGATTGCGGGCGGCACCGGCCCGATGGATGAATATC
TGATTGCGGGCATTGATGAAATTAACCGCACCTTTGATCTGAGCCCGAGCTGGTATA
TTGAAGCGCTGAAATATATTAAAGCGAACCATGGCCTGAGCGGCGATGCGGCGGTG
GAAGCGAACAGCTATCTGGATTATGCGATTAACGCGCTGAGC
In the database BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on 14
January 2023), the template sequence was uploaded in the “Standard Nucleotide BLAST”
search (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastS
earch&LINK_LOC=blasthome, last accessed on 18 January 2023) [26]. Nine sequences were
selected based on equality of length and similarity to the template nucleotide sequence and
saved in FASTA format for further analysis.

2.8. The Template Amino Acid Sequence of A. platensis Used in This Study
The following sequence was obtained from the NCBI protein database (www.ncbi.n
lm.nih.gov, last accessed on 20 January 2023) after searching for the phycocyanin alpha
subunit (cpcA) amino acid sequence of A. platensis and collected from the result of the
accession number ABD64608.1:1-162.
>ABD64608.1:1-162 phycocyanin alpha chain (Arthrospira platensis)
J. Mar. Sci. Eng. 2023, 11, 663 5 of 19

MKTPLTEAVSIADSQGRFLSSTEIQVAFGRFRQAKAGLEAAKALTSKADSLITGAAQ
AVYNKFPYTTQMQGPNYAANQRGKDKCARDISYYLRMVTYCLIAGGTGPMDEYLIAGI
DEINRTFDLSPSWYIEALKYIKANHGLSGDAAVEANSYLDYAINALS.
The amino acid sequence was saved in FASTA format to be used as a template. In
the database BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on 18 January
2023), the template sequence was uploaded in the Standard Protein BLAST (https://blast.nc
bi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=bla
sthome, last accessed on 20 January 2023) [26]. Additionally, Translated BLAST: blastx (http
s://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK
_LOC=blasthome, last accessed on 24 January 2023) was used. A total of 25 protein se-
quences of Arthrospira sp. and Arthrospira platensis strains and variants from the order
Oscillatoriales were selected based on the equality of length, presence of correct start codon,
and similarity to the template and then saved in FASTA format for further analysis.

2.9. Analysis of the Nucleotide and Protein Sequences

The selected 9 nucleotide sequences and 25 amino acid sequences were analyzed
using BioEdit version 7 for calculating the GC and AT content of the analyzed nucleotide
sequences, performing and editing multiple sequence alignment and creating consensus
sequence [27], using Molecular Evolutionary Genetics Analysis Version 11 (MEGA11)
for alignment, building phylogenetic trees of species, and analyzing the amino acid and
nucleotide composition of the studied sequences [28]. Phylogenetic tree visualization was
performed using the Tree Figure Drawing Tool Version 1.4.2 (FigTree 1.4.2), a graphical
viewer software, for each of the obtained phylogenetic trees (http://tree.bio.ed.ac.uk/soft
ware/figtree/, last accessed on 27 January 2023).

3. Results
3.1. Extraction of C-phycocyanin
The total protein content of the extracted C-phycocyanin from dried biomass of Egyp-
tian, French, and Chinese A. platensis as estimated by Bradford assay is presented in
Table 1. As shown in Table 1, the extracted C-phycocyanin of the Egyptian strain had
the highest total protein content, followed by the French strain and then the Chinese one.
Extracted C-phycocyanin from dry biomass of the three microalgae strains is shown in
Figure 2. As can be seen, there was a clear difference in color intensity between the ex-
tracted C-phycocyanin of the Egyptian strain (very faint blue extract) and the commercial
ones (deep blue extracts). SDS-PAGE analysis of the C-phycocyanin extract is presented
in Figure 3. The subunits of the extracted protein from the three microalgae strains were
observed at slightly different molecular weights on SDS-PAGE. Visible spectrum (at wave-
lengths of 600–650 nm) of the extracted C-phycocyanin from dry biomass of the three
microalgae strains is illustrated in Figure 4. The concentration of C-phycocyanin (mg/mL),
calculated by the equation of Siegelman and Kycia [24], is demonstrated in Table 2. Purity
of the extracted C-phycocyanin was calculated by dividing A615 (=2.186, 2.251, and 2.209)
by A280 (=2.61, 3.25, and 2.633) for the Egyptian, French, and Chinese strains, respectively,
as shown in Table 2. Regarding the concentration of C-phycocyanin, the French strain was
the highest in concentration but the lowest in purity.

Table 1. The total protein content of the extracted C-phycocyanin from dried biomass of the three
microalgae strains as measured by Bradford assay.

Strain Total Protein Concentration (mg/g Dried Biomass)

Egyptian A. platensis 1.233
French A. platensis 1.188
Chinese A. platensis 1.028
 Total Protein Co
Strain
(mg/g Dried B
Egyptian A. platensis 1.233
J. Mar. Sci. Eng. 2023, 11, 663
French A. platensis 6 of 19
1.188
Chinese A. platensis 1.028

J. Mar. Sci. Eng. 2023, 11, x FOR PEER REVIEW

Figure 2. Extracted C-phycocyanin from dry biomass of the three microalgae strains.
Figure 2. Extracted C-phycocyanin from dry biomass of the three microalgae

Figure 3. The 12% SDS-PAGE analysis of the extracted C-phycocyanin from dry biomass of the
Figure 3. The 12%
three microalgae SDS-PAGE
strains. M: Proteinanalysis
molecularof the extracted
weight C-phycocyanin
marker; 1, 2: fromfrom
C-phycocyanin extract drythe
biomass o
microalgae strains.
Chinese strain; M: Proteinextract
3, 4: C-phycocyanin molecular
from theweight marker;
French strain; 1, 2: C-phycocyanin
5: C-phycocyanin extract fromextract
the from
nese strain; 3, 4: C-phycocyanin extract from the French strain; 5: C-phycocyanin extrac
Egyptian strain. The arrow points to C-phycocyanin subunits.
Egyptian strain. The arrow points to C-phycocyanin subunits.
 Figure 3. The 12% SDS-PAGE analysis of the extracted C-phycocyanin from dry biomass of the three
J. Mar. Sci. Eng. 2023, 11, 663 microalgae strains. M: Protein molecular weight marker; 1, 2: C-phycocyanin extract from the Chi- 7 of 19
nese strain; 3, 4: C-phycocyanin extract from the French strain; 5: C-phycocyanin extract from the
Egyptian strain. The arrow points to C-phycocyanin subunits.

Visiblespectrum
Figure4.4.Visible
Figure spectrum(at(at wavelengths
wavelengths 600–650
600–650 nm)nm) of extracted
of the the extracted C-phycocyanin
C-phycocyanin from dry
from dry
biomass
biomassofofthe
thethree
threemicroalgae
microalgae strains.
strains.

Table
Table 2.
2. The
Theconcentration
concentrationand purity
and of the
purity extracted
of the C-phycocyanin
extracted from from
C-phycocyanin drieddried
biomass of the of the
biomass
three microalgae strains.
three microalgae strains.
C-phycocyanin Concentration C-phycocyanin Purity
Strain Strain
C-phycocyanin Concentration (mg/mL)
(mg/mL) C-phycocyanin (Dividing
Purity (Dividing A615 by A280 )
A615 by A280)
Egyptian A. platensis Egyptian A. platensis0.323 0.323 0.84 0.84
French A. platensis French A. platensis 0.333 0.333 0.69 0.69
Chinese A. platensis 0.327 0.84
Chinese A. platensis 0.327 0.84

3.2. Bioinformatics Analysis of the Nucleotide Sequences of C-phycocyanin Subunit Alpha

After searching for many sequences in the NCBI database and carrying out BLAST
analysis, only nine nucleotide sequences were selected to prove that phycocyanin subunit
alpha sequences of Arthrospira species and Arthrospira platensis strains and variants are
close to each other and different from other unrelated species of cyanobacteria. BioEdit and
MEGA11 were used to run the alignment and build a phylogenetic tree. The multiple align-
ment of the nine selected nucleotide sequences revealed the presence of minor differences
(only 31 from 486 nucleotides) among the aligned species and strains, as shown in Figure 5.
This proved the conservation (455 from 486 nucleotides) of the sequence of phycocyanin
subunit alpha among the nine aligned sequences. When the sequences of Arthrospira sp.
CCALA 030 (Accession MG777151), Arthrospira erdosensis ez (Accession AEV40868), and
Arthrospira sp. SH-MAG29 (Accession MBS0014833) were excluded from the alignment,
the conservation of the phycocyanin subunit alpha sequence was even higher (461 from
486 nucleotides). This can be explained by the positions of the species Arthrospira sp.
SH-MAG29 and the strain Arthrospira erdosensis ez in the upper end of the phylogenetic tree
(Figure 6). The species Arthrospira sp. CCALA 030 (Accession MG777151) and the variant
strain of Arthrospira platensis (Accession CAA70296) were found in the lower end of the
tree. MEGA11 was used to analyze the nucleotide content of the investigated sequences.
The nucleotide frequencies in the phycocyanin subunit alpha genes of the nine Arthrospira
species and Arthrospira platensis strains and variants are presented in Table 3. As can be seen,
the G content of 31.5% was found in Arthrospira sp. CCALA 030 (Accession MG777151)
and three Arthrospira platensis variants (the species Limnoraphis sp. WC205 (Accession
MCG5058249), Arthrospira platensis (Accession CAA70296), and Oscillatoriales (Accession
WP 006620876)). The T content of 19.3% was found in only one strain of Arthrospira platensis
(Accession ABD64608) and three other Arthrospira strains and species (Accessions WP
006620876, AEV40868, and MBS0014833). The C content of 25.1% was found in two strains
of Arthrospira platensis (Accessions ABD64608 and P72509) in addition to Arthrospira sp.
 J. Mar. Sci. Eng. 2023, 11, 663 8 of 19

CCALA 030 (Accession MG777151), while that of 24.7% was more common in the analyzed
species and strains (Accessions WP 006620876, 1GH0 A, AEV40868, and MBS0014833). The
A content of 24.7% was found in two strains of Arthrospira platensis (Accessions ABD64608
and P72509) and Arthrospira erdosensis ez (Accession AEV40868), while that of 24.5% was
observed in Arthrospira sp. CCALA 030 (Accession MG777151), Oscillatoriales (Accession
WP 006620876), and Arthrospira platensis (Accession CAA70296). T-1 of 11.7%, C-1 of 20.4%,
and A-3 of 10.5% were common in eight of the nine analyzed sequences, while A-2 of 31.5%
was constant in all of them. Table 4 demonstrates the GC and AT content in phycocyanin
J. Mar. Sci. Eng. 2023, 11, x FOR PEER REVIEW
subunit alpha nucleotide sequences of the analyzed A. platensis strains and variants. As can 9 of 20
be seen, all the analyzed sequences had GC content of more than 54.5%.

Figure 5. Multiple
Figure 5. Multiplealignment ofnucleotide
alignment of nucleotide sequences
sequences of phycocyanin
of phycocyanin subunitsubunit
alpha of alpha of nine cyano-
nine cyanobac-
bacterial species,including
terial species, includingArthrospira
Arthrospira platensis
platensis strains
strains and and variants,
variants, usingusing
BioEdit.BioEdit. The A,
The letters letters
T, A, T,
C, and G symbolize the four nucleotides adenine, thymine, cytosine, and guanine,
C, and G symbolize the four nucleotides adenine, thymine, cytosine, and guanine, respectively; respectively; *
represents conserved nucleotides.
* represents conserved nucleotides.
J. Mar. Sci. Eng. 2023, 11, 663 9 of 19

Table 3. The frequencies of the nucleotides in phycocyanin subunit alpha nucleotide sequences of nine cyanobacterial species, including A. platensis strains and
variants. All nucleotide frequencies were calculated by MEGA11 and are given as percentages.

Accession and Species T(U) C A G Total T-1 C-1 A-1 G-1 Pos #1 T-2 C-2 A-2 G-2 Pos #2 T-3 C-3 A-3 G-3 Pos #3
MG777151, Arthrospira sp. CCALA 030 18.9 25.1 24.5 31.5 486.0 11.7 20.4 31.5 36.4 162.0 22.2 23.5 31.5 22.8 162.0 22.8 31.5 10.5 35.2 162.0
P72509, Arthrospira platensis 19.1 25.1 24.7 31.1 486.0 11.7 20.4 32.1 35.8 162.0 22.8 24.1 31.5 21.6 162.0 22.8 30.9 10.5 35.8 162.0
WP 006620876, Oscillatoriales 19.3 24.7 24.5 31.5 486.0 11.7 20.4 31.5 36.4 162.0 23.5 23.5 31.5 21.6 162.0 22.8 30.2 10.5 36.4 162.0
1GH0 A, Arthrospira platensis 19.1 24.7 24.3 31.9 486.0 11.7 20.4 30.9 37.0 162.0 23.5 23.5 31.5 21.6 162.0 22.2 30.2 10.5 37.0 162.0
AEV40868, Arthrospira erdosensis ez 19.3 24.7 24.7 31.3 486.0 11.7 20.4 32.1 35.8 162.0 23.5 22.8 31.5 22.2 162.0 22.8 30.9 10.5 35.8 162.0
MBS0014833, Arthrospira sp. SH-MAG29 19.3 24.7 24.9 31.1 486.0 11.7 20.4 32.7 35.2 162.0 23.5 22.2 31.5 22.8 162.0 22.8 31.5 10.5 35.2 162.0
ABD64608, Arthrospira platensis 19.3 25.1 24.7 30.9 486.0 11.7 20.4 32.7 35.2 162.0 23.5 24.1 31.5 21.0 162.0 22.8 30.9 9.9 36.4 162.0
MCG5058249, Limnoraphis sp. WC205 18.9 25.3 24.3 31.5 486.0 11.7 21.0 30.9 36.4 162.0 22.8 24.1 31.5 21.6 162.0 22.2 30.9 10.5 36.4 162.0
CAA70296, Arthrospira platensis 20.6 23.5 24.5 31.5 486.0 12 20.4 31.5 36.4 162.0 22 23.5 31.5 22.8 162.0 28 26.5 10.5 35.2 162.0
Average % 19.3 24.8 24.6 31.3 486.0 12 20.4 31.8 36.1 162.0 23 23.5 31.5 22.0 162.0 23 30.4 10.4 35.9 162.0
J. Mar. Sci. Eng. 2023, 11, 663 10 of 19
J. Mar. Sci. Eng. 2023, 11, x FOR PEER REVIEW 10 of 20

Figure 6. Phylogenetic tree of nucleotide

Figure 6. Phylogenetic sequences
tree of nucleotide of phycocyanin
sequences subunit
of phycocyanin subunit alpha
alpha of nineof nine cyanobac-
cyanobac-
terial species, including Arthrospira platensis strains and variants. The branching order and score
terial species, including Arthrospira platensis strains and variants. The branching order and score were
were calculated by MEGA11 and visualized by FigTree 1.4.2.
calculated by MEGA11 and visualized by FigTree 1.4.2.
Table 3. The frequencies of the nucleotides in phycocyanin subunit alpha nucleotide sequences of
nine cyanobacterial species, including A. platensis strains and variants. All nucleotide frequencies
Table 4. The GCwere
andcalculated
AT content of theand
by MEGA11 analyzed
are given nucleotide
as percentages.sequences of nine cyanobacterial species,
Accession and Species A. platensis
including T(U) strains
C A and
G variants.
Total T-1 C-1 A-1 G-1 Pos #1 T-2 C-2 A-2 G-2 Pos #2 T-3 C-3 A-3 G-3 Pos #3
MG777151, Arthrospira sp.
18.9 25.1 24.5 31.5 486.0 11.7 20.4 31.5 36.4 162.0 22.2 23.5 31.5 22.8 162.0 22.8 31.5 10.5 35.2 162.0
CCALA 030
Accession GC Content (%) AT Content (%)
P72509, Arthrospira platensis 19.1 25.1 24.7 31.1 486.0 11.7 20.4 32.1 35.8 162.0 22.8 24.1 31.5 21.6 162.0 22.8 30.9 10.5 35.8 162.0
WP 006620876, Oscillatoriales MG777151
19.3 24.7 24.5 31.5 486.0 11.7 20.4 31.556.58
36.4 162.0 23.5 23.5 31.5 21.6 162.0 22.8 30.2 10.5 36.4 162.0
43.42
1GH0 A, Arthrospira platensis 19.1 24.7 24.3 31.9 486.0 11.7 20.4 30.9 37.0 162.0 23.5 23.5 31.5 21.6 162.0 22.2 30.2 10.5 37.0 162.0
AEV40868, Arthrospira erdosensis P72509
19.3 24.7 24.7 31.3 486.0 11.7 20.4 32.156.17 43.83
35.8 162.0 23.5 22.8 31.5 22.2 162.0 22.8 30.9 10.5 35.8 162.0
ez
MBS0014833, Arthrospira sp.WP SH- 006620876 56.17 43.83
19.3 24.7 24.9 31.1 486.0 11.7 20.4 32.7 35.2 162.0 23.5 22.2 31.5 22.8 162.0 22.8 31.5 10.5 35.2 162.0
MAG29
ABD64608, Arthrospira platensis 1GH019.3 A 25.1 24.7 30.9 486.0 11.7 20.4 32.756.58 43.42
35.2 162.0 23.5 24.1 31.5 21.0 162.0 22.8 30.9 9.9 36.4 162.0
MCG5058249, Limnoraphis sp.
18.9
AEV40868 25.3 24.3 31.5 486.0 11.7 21.0 30.955.97
36.4 162.0 22.8 24.1 31.5 21.6 162.0 22.2 30.9 10.5 36.4 162.0
44.03
WC205
CAA70296, Arthrospira platensis 20.6 23.5 24.5 31.5 486.0 12 20.4 31.5 36.4 162.0 22 23.5 31.5 22.8 162.0 28 26.5 10.5 35.2 162.0
Average % MBS0014833
19.3 24.8 24.6 31.3 486.0 12 20.4 31.855.76
36.1 162.0 23 23.5 31.5 22.0 162.0 44.24
23 30.4 10.4 35.9 162.0

ABD64608 55.97 44.03

Table 4. The GC and AT content of the analyzed nucleotide sequences of nine cyanobacterial spe-
cies, including A. platensis strains and variants.
MCG5058249 56.79 43.21
CAA70296 GC Content
54.94 AT Content
45.06
Accession
(%) (%)
MG777151 56.58 43.42
3.3. Bioinformatics Analysis of the Amino Acid Sequences of C-phycocyanin Subunit Alpha
From the NCBI database, 25 protein sequences of phycocyanin subunit alpha of
Arthrospira sp. and Arthrospira platensis strains and variants from the order Oscillatori-
ales were selected to compare their amino acid composition. The alignment of these
25 sequences proved the presence of 97 conserved amino acids (about 60% sequence sim-
ilarity) among the analyzed sequences, as shown in Figure 7. Phylogenetic tree of the
amino acid sequences of phycocyanin subunit alpha of the 25 cyanobacterial species, in-
cluding Arthrospira platensis strains and variants, is shown in Figure 8. The frequencies of
amino acids (%) in phycocyanin subunit alpha protein sequences of the 25 cyanobacterial
species are demonstrated in Table 5. As can be seen from Table 5, all essential amino acids
(F, H, I, K, L, M, R, T, V, and W) were found in all the analyzed phycocyanin subunit
alpha protein sequences. Alanine (A) was the most abundant amino acid (average 15.1%)
J. Mar. Sci. Eng. 2023, 11, 663 11 of 19

among all the analyzed sequences, followed by glycine (G) and leucine (L) with average
of 8.1% and then serine (S), tyrosine (Y), and threonine (T) with average of 7.7, 6.7, and
6.4%, respectively. The basic amino acid histidine (H) with content of 0.6% was nearly
constant (24 of the 25 cyanobacterial species and strains), and the tryptophan (W) content
of 0.6% was observed in all species and strains. Regarding acidic amino acids, aspartic
acid (D) content of 5.6% and glutamic acid (E) content of 4.9% were found in 12 and
9 cyanobacterial species and strains, respectively, including Arthrospira species and strains
of Arthrospira platensis. The frequencies of basic amino acids of 4.3% for arginine (R) and
5.6% for lysine (K) were present in 11 and 10 cyanobacterial species and strains, respectively,
including Arthrospira sp., Arthrospira platensis, and their related strains. The contents of
3.1% phenylalanine (F) and 2.5% methionine (M) were found in 21 and 16 cyanobacterial
J. Mar. Sci. Eng. 2023, 11, x FOR PEER REVIEW
species 12 of 20
and strains, respectively. Table 6 demonstrates the unique and harsh environments
from which the investigated cyanobacterial species and strains were isolated.

Figure7.7.Multiple
Figure Multiplealignment
alignmentofofamino
aminoacidacidsequences
sequencesofofphycocyanin
phycocyanin subunit
subunit alpha
alpha of of
25 25 cyano-
cyanobac-
bacterial species, including A. platensis strains and variants, using BioEdit. Amino
terial species, including A. platensis strains and variants, using BioEdit. Amino acids are shown acids are shown in
in one-letter coded form. A: alanine, C: cysteine, D: aspartic acid, E: glutamic acid, F: phenylalanine,
one-letter coded form. A: alanine, C: cysteine, D: aspartic acid, E: glutamic acid, F: phenylalanine,
G: glycine, H: histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N: asparagine, P: proline,
G:
Q:glycine, H: histidine,
glutamine, I: isoleucine,
R: arginine, K:threonine,
S: serine, T: lysine, L: leucine, M:W:
V: valine, methionine,
tryptophan, N:Y:asparagine, P: proline,
tyrosine. Consensus
Q: glutamine, R: arginine, S: serine, T: threonine, V: valine, W: tryptophan, Y: tyrosine.
key: * (asterisk): positions that have a single, fully conserved residue; : (colon): conservation betweenConsensus
key: * (asterisk):
groups that havepositions
strongly that have aproperties;
similar single, fully conservedconservation
. (period): residue; : (colon):
between conservation
groups thatbetween
have
groups
weaklythat have
similar strongly blank
properties; similarspaces
properties;
mean no . (period):
consensus.conservation between groups that have
weakly similar properties; blank spaces mean no consensus.
J. Mar. Sci. Eng. 2023, 11, 663 12 of 19
J. Mar. Sci. Eng. 2023, 11, x FOR PEER REVIEW 13 of 20

Figure 8. Phylogenetic tree

Figure of amino acid
8. Phylogenetic sequences
tree of phycocyanin
of amino acid subunit alpha
sequences of phycocyanin of 25alpha
subunit cyanobacterial
of 25 cyanobac-
species, including A. platensis strains and variants. The branching order and score were calculated
terial species, including A. platensis strains and variants. The branching order and scorebywere
calculated
MEGA11 and visualized byby MEGA11
FigTree and visualized by FigTree 1.4.2.
1.4.2.
Table 5. The frequencies of amino acids (%) in phycocyanin subunit alpha protein sequences of 25
cyanobacterial species, including A. platensis strains and variants, as calculated using MEGA11.

Phycocyanin Subunit Alpha A C D E F G H I K L M N P Q R S T V W Y Total

ABD64608.1:1-162 Arthrospira
14.8 1.2 5.6 4.3 3.1 7.4 0.6 6.8 5.6 8.0 2.5 4.3 3.1 4.3 4.3 7.4 6.2 3.1 0.6 6.8 162
platensis
1GH0 A:1-162 Arthrospira
14.8 1.2 5.6 4.9 3.1 8.0 0.6 6.2 5.6 8.0 2.5 3.7 3.1 4.3 4.3 7.4 5.6 3.7 0.6 6.8 162
platensis
AEV40868.1:1-162 Arthrospira
14.2 1.2 5.6 4.9 3.1 8.0 0.6 6.8 5.6 8.0 2.5 3.7 3.1 4.3 4.3 8.0 5.6 3.1 0.6 6.8 162
erdosensis ez
MBS0014833.1:1-162 Arthrospira
13.6 1.2 5.6 4.9 3.1 8.0 0.6 6.8 5.6 8.0 2.5 3.7 3.1 4.3 4.3 8.6 5.6 3.1 0.6 6.8 162
sp. SH-MAG29
CAA70296.1:1-162 Arthrospira
14.8 1.2 5.6 4.9 3.1 8.6 0.6 6.8 5.6 7.4 2.5 3.7 3.1 4.3 4.9 7.4 5.6 2.5 0.6 6.8 162
platensis
NJO70792.1:1-162
Oscillatoriales cyanobacterium 15.4 1.2 5.6 4.9 3.1 8.0 0.6 5.6 5.6 8.0 3.1 3.7 3.1 4.3 4.3 6.2 7.4 3.1 0.6 6.2 162
RM1 1 9
OIP70521.1:1-162 Oscillatoriales
14.8 1.2 4.3 5.6 3.1 8.0 0.6 5.6 5.6 7.4 1.9 3.7 3.1 4.3 4.3 6.8 8.0 4.3 0.6 6.8 162
cyanobacterium CG2 30 40 61
WP 026793960.1:1-162
14.2 1.2 4.3 5.6 3.1 8.0 0.6 4.9 5.6 8.0 1.9 4.3 3.1 3.7 4.3 8.6 6.8 4.3 0.6 6.8 162
Planktothrix mougeotii
WP 083621658.1:1-162
14.8 1.2 4.3 5.6 3.1 8.6 0.6 4.9 5.6 8.6 1.2 3.7 3.1 4.3 4.3 6.8 7.4 4.3 0.6 6.8 162
Planktothrix paucivesiculata
J. Mar. Sci. Eng. 2023, 11, 663 13 of 19

Table 5. The frequencies of amino acids (%) in phycocyanin subunit alpha protein sequences of 25 cyanobacterial species, including A. platensis strains and variants,
as calculated using MEGA11.

Phycocyanin Subunit Alpha A C D E F G H I K L M N P Q R S T V W Y Total

ABD64608.1:1-162 Arthrospira platensis 14.8 1.2 5.6 4.3 3.1 7.4 0.6 6.8 5.6 8.0 2.5 4.3 3.1 4.3 4.3 7.4 6.2 3.1 0.6 6.8 162
1GH0 A:1-162 Arthrospira platensis 14.8 1.2 5.6 4.9 3.1 8.0 0.6 6.2 5.6 8.0 2.5 3.7 3.1 4.3 4.3 7.4 5.6 3.7 0.6 6.8 162
AEV40868.1:1-162 Arthrospira erdosensis ez 14.2 1.2 5.6 4.9 3.1 8.0 0.6 6.8 5.6 8.0 2.5 3.7 3.1 4.3 4.3 8.0 5.6 3.1 0.6 6.8 162
MBS0014833.1:1-162 Arthrospira sp. SH-MAG29 13.6 1.2 5.6 4.9 3.1 8.0 0.6 6.8 5.6 8.0 2.5 3.7 3.1 4.3 4.3 8.6 5.6 3.1 0.6 6.8 162
CAA70296.1:1-162 Arthrospira platensis 14.8 1.2 5.6 4.9 3.1 8.6 0.6 6.8 5.6 7.4 2.5 3.7 3.1 4.3 4.9 7.4 5.6 2.5 0.6 6.8 162
NJO70792.1:1-162 Oscillatoriales cyanobacterium RM1 1 9 15.4 1.2 5.6 4.9 3.1 8.0 0.6 5.6 5.6 8.0 3.1 3.7 3.1 4.3 4.3 6.2 7.4 3.1 0.6 6.2 162
OIP70521.1:1-162 Oscillatoriales cyanobacterium CG2 30 40 61 14.8 1.2 4.3 5.6 3.1 8.0 0.6 5.6 5.6 7.4 1.9 3.7 3.1 4.3 4.3 6.8 8.0 4.3 0.6 6.8 162
WP 026793960.1:1-162 Planktothrix mougeotii 14.2 1.2 4.3 5.6 3.1 8.0 0.6 4.9 5.6 8.0 1.9 4.3 3.1 3.7 4.3 8.6 6.8 4.3 0.6 6.8 162
WP 083621658.1:1-162 Planktothrix paucivesiculata 14.8 1.2 4.3 5.6 3.1 8.6 0.6 4.9 5.6 8.6 1.2 3.7 3.1 4.3 4.3 6.8 7.4 4.3 0.6 6.8 162
PSN18344.1:1-162 filamentous cyanobacterium CCP5 14.8 1.2 4.9 5.6 3.1 8.6 0.6 3.7 4.9 8.6 3.1 3.7 3.1 4.9 3.7 8.0 6.2 3.7 0.6 6.8 162
WP 194023545.1:1-162 Nodosilinea sp. LEGE 07298 14.8 1.2 4.9 5.6 3.1 8.6 0.6 3.1 4.9 8.0 3.1 3.7 3.1 4.3 3.7 8.0 6.8 4.9 0.6 6.8 162
MCA2686410.1:1-162 Microcystis sp. M046S2 15.4 1.2 5.6 4.3 3.1 7.4 0.6 6.2 4.3 7.4 2.5 4.9 3.1 4.3 4.9 7.4 6.2 3.7 0.6 6.8 162
WP 015143844.1:1-162 MULTISPECIES: Pleurocapsales 15.4 1.9 4.9 4.9 2.5 8.0 0.6 6.2 4.9 8.0 3.1 5.6 3.1 4.9 3.7 6.8 5.6 2.5 0.6 6.8 162
WP 147071652.1:1-162 Microcystis aeruginosa 15.4 1.2 5.6 4.3 3.1 8.0 0.6 6.2 4.3 7.4 2.5 3.7 3.1 4.3 4.9 8.0 6.2 3.7 0.6 6.8 162
RPH86579.1:1-162 Chroococcales cyanobacterium metabat2.561 16.0 1.2 4.9 4.9 3.1 7.4 0.6 6.2 4.3 7.4 2.5 4.3 3.1 4.3 4.9 7.4 6.2 3.7 0.6 6.8 162
WP 002771746.1:1-162 Microcystis aeruginosa 16.0 1.2 5.6 4.3 3.1 6.8 0.6 6.2 4.9 7.4 2.5 3.7 3.1 4.3 4.3 8.6 6.2 3.7 0.6 6.8 162
WP 079207951.1:1-162 Microcystis aeruginosa 15.4 1.2 5.6 4.3 3.1 8.6 0.6 6.2 4.3 7.4 2.5 3.7 3.1 4.3 4.9 7.4 6.2 3.7 0.6 6.8 162
WP 035990438.1:1-162 Leptolyngbya sp. KIOST-1 14.8 1.2 4.9 5.6 3.1 8.6 0.6 3.7 4.9 8.0 3.1 3.7 2.5 4.3 3.7 9.3 6.2 4.3 0.6 6.8 162
MBF2078792.1:1-162 Synechococcales cyanobacterium T60 A2020 003 14.8 1.2 5.6 4.9 3.1 7.4 0.6 6.2 4.9 7.4 2.5 3.7 3.1 6.2 4.3 7.4 5.6 3.7 0.6 6.8 162
NJN32399.1:1-162 Synechococcales cyanobacterium RM1 1 8 14.8 1.2 5.6 4.3 3.1 8.0 0.6 4.3 5.6 8.6 2.5 3.7 3.7 4.3 3.1 8.6 6.8 3.7 0.6 6.8 162
WP 190519399.1:1-162 MULTISPECIES: unclassified Nodosilinea 16.7 1.9 4.9 5.6 2.5 8.6 0.6 3.1 4.9 8.6 2.5 4.3 2.5 3.7 3.7 7.4 6.2 4.9 0.6 6.8 162
WP 010871860.1:1-162 MULTISPECIES: Synechocystis 14.2 1.2 6.8 3.7 3.1 8.0 0.6 4.9 4.3 9.9 0.6 6.2 2.5 5.6 4.9 6.2 6.8 3.7 0.6 6.2 162
WP 194029021.1:1-162 Lusitaniella coriacea 14.8 1.2 4.9 6.2 2.5 9.3 1.2 4.3 4.9 8.6 2.5 3.7 2.5 3.1 3.7 9.9 5.6 4.3 0.6 6.2 162
TAG95764.1:1-162 Oscillatoriales cyanobacterium 16.0 1.2 4.3 4.9 3.1 8.0 0.6 4.3 4.3 8.6 2.5 3.7 3.1 4.9 4.3 6.2 8.6 3.7 0.6 6.8 162
HIK44559.1:1-162 Leptolyngbyaceae cyanobacterium M65 K2018 010 16.7 1.9 4.9 5.6 2.5 8.0 0.6 4.3 3.7 8.6 2.5 4.3 2.5 3.7 4.9 8.0 6.2 3.7 0.6 6.8 162
Average % 15.1 1.3 5.2 5.0 3.0 8.1 0.6 5.3 5.0 8.1 2.4 4.0 3.0 4.4 4.3 7.7 6.4 3.7 0.6 6.7 162
J. Mar. Sci. Eng. 2023, 11, 663 14 of 19

Table 6. Characteristics of the investigated cyanobacterial organisms and source of isolation.

Strain Characteristics of Isolate Accession Reference

- Phycocyanin alpha chain
Arthrospira platensis - Country: India ABD64608 Unpublished
- Submitted by Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, India
- Crystal structure of C-phycocyanin
Arthrospira platensis (Gomont) Geitler 1925 1GH0 A [29]
- Isolation source: urban reservoir in Poland, Central Europe.
- Phycocyanin alpha chain
Arthrospira erdosensis ez - Submitted by College of Life Science and Technology, Inner Mongolia Normal University, P.R. China AEV40868 Unpublished
- Isolation source: alkaline lake 1200–1600 m above sea level
- Phycocyanin subunit alpha
- Isolation source: shallow sediments of the arsenic-rich Salar de Huasco
Arthrospira sp. SH-MAG29 MBS0014833 [30]
lagoon
- Submitted by Departamento de Ingenieria Quimica, Universidad Catolica del Norte, Chile
- Phycocyanin alpha subunit
Arthrospira platensis - Submitted by W. Jeamton, School of Bioresources and CAA70296 Unpublished
Technology, King Mongkut’s Institute of Technology Thonburi (KMITT), Thailand
- Phycocyanin subunit alpha
Oscillatoriales cyanobacterium RM1_1_9 - Environmental sample NJO70792 Unpublished
- Country: South Africa, Cape Recife
- Phycocyanin subunit alpha
Oscillatoriales cyanobacterium CG2_30_40_61 - Isolation source: Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are OIP70521 [31]
erupted at the surface
Planktothrix mougeotii Phycocyanin subunit alpha WP_026793960 Unpublished
Planktothrix paucivesiculata Phycocyanin subunit alpha WP_083621658 Unpublished
- Phycocyanin subunit alpha- Submitted by Earth, Atmospheric and Planetary Sciences,
Filamentous cyanobacterium CCP5 Massachusetts Institute of Technology, USA PSN18344 Unpublished
- Isolation source: salt marsh
Nodosilinea sp. LEGE 07298 Phycocyanin subunit alpha WP_194023545 Unpublished
- Phycocyanin subunit alpha
Microcystis sp. M046S2 MCA2686410 [32]
- Isolation source: fresh water surface (50 cm depth)
Pleurocapsales Phycocyanin subunit alpha WP_015143844 Unpublished
Microcystis aeruginosa Phycocyanin subunit alpha WP_147071652 Unpublished
- Phycocyanin subunit alpha
Chroococcales cyanobacterium metabat2.561 RPH86579 [33]
- Isolation source: Prairie Pothole Region wetland sediments
Microcystis aeruginosa Phycocyanin subunit alpha WP_002771746 Unpublished
J. Mar. Sci. Eng. 2023, 11, 663 15 of 19

Table 6. Cont.

Strain Characteristics of Isolate Accession Reference

Microcystis aeruginosa Phycocyanin subunit alpha WP_079207951 Unpublished
Leptolyngbya sp. KIOST-1 Phycocyanin subunit alpha WP_035990438 Unpublished
- Phycocyanin subunit alpha
- Isolation source: nonacidic hot spring microbial mat
Synechococcales cyanobacterium T60_A2020_003 MBF2078792 Unpublished
- Submitted by Departamento de Genetica Molecular y Microbiologia, Pontificia Universidad Catolica
de Chile, Chile
-Phycocyanin subunit alpha
Synechococcales cyanobacterium RM1_1_8 -Isolation source: stromatolite NJN32399 Unpublished
- Submitted by Pharmaceutical Sciences, University of Wisconsin, USA
Unclassified Nodosilinea Phycocyanin subunit alpha WP_190519399 Unpublished
Synechocystis Phycocyanin subunit alpha WP_010871860 Unpublished
Lusitaniella coriacea Phycocyanin subunit alpha WP_194029021 Unpublished
- Phycocyanin subunit alpha
- Isolation source: microbial mat material
Oscillatoriales cyanobacterium TAG95764 [34]
- Environmental sample
- Country: USA, California, Eel River, Elder Creek
-Phycocyanin subunit alpha
Leptolyngbyaceae cyanobacterium M65_K2018_010 HIK44559 [35]
-Isolation source: Hot spring_65deg.
J. Mar. Sci. Eng. 2023, 11, 663 16 of 19

4. Discussion
Phycobiliproteins are large protein aggregates produced by cyanobacterial cells at a
concentration of 40–60% of their total soluble protein. They are involved in harvesting of
light for these cells during photosynthesis [36]. They can be categorized into phycoerythrin
(maximum wavelength of 565 nm), phycocyanin (maximum wavelength of 620 nm), and
allophycocyanin (maximum wavelength of 650 nm) based on their spectral properties [37].
Phycobiliproteins have antimicrobial, anti-inflammatory, antioxidant, and hepatoprotective
effects [38,39]. They are commonly used as natural pigments and fluorescent proteins in
several applications, such as food and cosmetic industries [40,41]. The phycocyanin gene is
commonly used in the molecular identification and characterization of A. platensis strains [17].
This work investigated the variation between A. platensis strains and variants from
the order Oscillatoriales based on the nucleotide and amino acid composition of the gene
and protein sequences of C-phycocyanin alpha chain. The obtained data were linked to
their habitat or source of isolation. C-phycocyanin was comparatively extracted from three
different A. platensis strains: one isolated from the brackish Lake Mariout at southwest of
Alexandria city in Egypt and two commercial French and Chinese strains. The Egyptian
strain was cultivated in the laboratory using highly saline medium under static conditions
at a temperature of 24 ◦ C (day) and 18 ◦ C (night). The extracted pigment was expected
to vary based on the difference in isolation source of the three A. platensis strains and
cultivation conditions used with the Egyptian isolate.
In 2013, salinity was verified to be an essential factor in addition to alkalinity for
suppressing the growth of algae and cyanobacteria except for A. platensis, thus leading
to this microalga dominating the environment [2]. In this study, an Egyptian isolate
was cultivated in 1.5× (highly saline) Amara and Steinbuchel medium derived from a
combination of George and Zarrouk media under the regular cycle of day and night and
static cultivation condition, but the work was conducted in a photobioreactor [2]. The
pigment extracted from the cultivated Egyptian strain was expected to be affected by these
cultivation conditions, which was the case as the pigment was very faint in color compared
to that extracted from the commercial strains. Interestingly, when the same strain was
previously grown in Zarrouk medium at room temperature and exposed to fluorescent
lamp/day–light, the extracted pigment using potassium phosphate buffer of 0.1 M and pH
7 was green in color, not even blue [17]. Therefore, further investigation should be carried
out to control the quality of the pigment obtained from this microalga for biotechnological
applications either by mimicking conditions of its natural environment or optimizing
conditions in a bioreactor.
Genomes rich in GC are anticipated to be more adapted to high growth temperatures
than those rich in AT as GC pairs are usually more stable than the AT ones [42]. In this study,
Arthrospira platensis (Accession 1GH0 A) had GC content of 56.58% in the phycocyanin
subunit alpha coding gene and was isolated from urban reservoir in Poland, which is
characterized by high temperature and reduced air humidity [29,43]. A. platensis (Accession
ABD64608) with GC content of 55.97% was isolated from India, while Arthrospira erdosensis
ez (Accession AEV40868) with the same GC content was isolated from an alkaline lake in
China. Arthrospira sp. SH-MAG29 (Accession MBS0014833) with GC content of 55.76% was
isolated from shallow sediments of the arsenic-rich Salar de Huasco Lagoon in Chile, which
is an extreme environment with high daily variations in temperature, high UV radiation,
arsenic and salinity, and low pressure [30]. In addition, Arthrospira platensis (Accession
CAA70296) with GC content of 54.94% was isolated from Thailand. These results prove
that the GC content of the gene of phycocyanin subunit alpha of A. platensis strains and
variants is linked with the extreme conditions in their habitat, such as high temperatures
and alkalinity.
Amino acids play a vital role in metabolism and growth of microorganisms [44].
Amino acids are categorized as essential and nonessential; acidic, basic, and neutral;
hydrophilic and hydrophobic; and polar and nonpolar amino acids based on their source
and nature of their side chain [45]. Alanine is encoded exclusively or primarily by GC-
J. Mar. Sci. Eng. 2023, 11, 663 17 of 19

biased codons, so it is closely related to the GC content [46]. In this work, all essential amino
acids (F, H, I, K, L, M, R, T, V, and W) were found in all the analyzed protein sequences
of phycocyanin subunit alpha of cyanobacterial species, including A. platensis strains and
variants. Alanine was the most abundant amino acid (average of 15.1%) among all the
analyzed sequences. Histidine was encoded equally by both GC-and AT-biased codons, but
only one residue was found in nearly all the analyzed sequences. The increase in the acidic
nature of proteins as an adaptation to hypersalinity has been previously reported [47].
Accordingly, the acidic amino acids aspartic acid and glutamic acid were more abundant
than basic residues, especially histidine, in all the analyzed sequences. Overall, these
findings confirm that the amino acid composition of the protein sequences of phycocyanin
subunit alpha of A. platensis strains and variants are closely related to both GC content and
the extreme conditions in their habitat, such as salinity.

5. Conclusions
Arthrospira platensis is an edible cyanobacterium that has great significance for different
applications, including environmental, food, feed, biotechnological, and pharmaceutical.
A. platensis shows dense growth under conditions of high salinity and alkalinity and
dominates lakes in certain periods of the year. It can survive alone under many harsh
environmental conditions, and its ability to adapt to environmental stresses exceeds that of
other competitors. Phycobiliproteins (natural pigments), especially C-phycocyanin, are one
of the most important bioactive products produced by Arthrospira platensis. C-phycocyanin
is commonly used in the molecular identification and characterization of A. platensis due
to the high conservation of its gene among cyanobacterial species. In this study, an Egyp-
tian A. platensis strain was cultivated in our lab under static conditions in a highly saline
medium, and then its C-phycocyanin was extracted and compared to two commercial
strains. Additionally, the link between the amino acids and nucleotide composition of the
alpha chain of this pigment and the adaptation of Arthrospira platensis to harsh environmen-
tal conditions was investigated. Overall, the extracted pigment from the Egyptian strain
had a very faint color compared to the pigment of the commercial strains, which may be
due to the difference in cultivation conditions and/or source of isolation. The analyzed
species and strains had GC content of more than 54.5% in the C-phycocyanin alpha chain
gene, which may be linked to adaptation of these cyanobacteria to high temperatures.
Moreover, high frequencies of the acidic amino acids aspartic acid and glutamic acid in
the C-phycocyanin alpha chain protein can be attributed to adaptation to hypersalinity.
Understanding the differences between the strains and variants of Arthrospira platensis at the
gene and protein levels of C-phycocyanin alpha chain and determining if these differences
are environmentally based and affected by cultivation conditions on the pigment may help
in optimizing biotechnological applications of this microalga and its pigment by mimicking
the best-suited growth conditions for each habitat.

Author Contributions: N.A.E.-B. and A.A.A. conceived the research topic and designed the research;
N.A.E.-B. and N.M.F.R. conducted the experimental work; N.A.E.-B. and A.A.A. collected and
analyzed the data; N.A.E.-B. and A.A.A. wrote the manuscript; A.A.A. proofread and revised
the manuscript; and N.A.E.-B. finalized the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
J. Mar. Sci. Eng. 2023, 11, 663 18 of 19

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