Seasonal Changes in the Biochemical Composition of Dominant Macroalgal Species along the Egyptian Red Sea Shore

Abstract

Macroalgae are significant biological resources in coastal marine ecosystems. Seasonality influences macroalgae biochemical characteristics, which consequentially affect their ecological and economic values. Here, macroalgae were surveyed from summer 2017 to spring 2018 at three sites at 7 km (south) from El Qusier, 52 km (north) from Marsa Alam and 70 km (south) from Safaga along the Red Sea coast, Egypt. Across all the macroalgae collected, Caulerpa prolifera (green macroalgae), Acanthophora spicifera (red macroalgae) and Cystoseira myrica, Cystoseira trinodis and Turbinaria ornata (brown macroalgae) were the most dominant macroalgal species. These macroalgae were identified at morphological and molecular (18s rRNA) levels. Then, the seasonal variations in macroalgal minerals and biochemical composition were quantified to determine the apt period for harvesting based on the nutritional requirements for commercial utilizations. The chemical composition of macroalgae proved the species and seasonal variation. For instance, minerals were more accumulated in macroalgae C. prolifera, A. spicifera and T. ornata in the winter season, but they were accumulated in both C. myrica and C. trinodis in the summer season. Total sugars, amino acids, fatty acids and phenolic contents were higher in the summer season. Accordingly, macroalgae collected during the summer can be used as food and animal feed. Overall, we suggest the harvesting of macroalgae for different nutrients and metabolites in the respective seasons.

Full text

Citation: Kamal, M.; Abdel-Raouf,
N.; Alwutayd, K.; AbdElgawad, H.;
Abdelhameed, M.S.; Hammouda, O.;
Elsayed, K.N.M. Seasonal Changes in
the Biochemical Composition of
Dominant Macroalgal Species along
the Egyptian Red Sea Shore. Biology
2023,12, 411. https://doi.org/
10.3390/biology12030411
Academic Editor: John R. Turner
Received: 6 February 2023
Revised: 27 February 2023
Accepted: 1 March 2023
Published: 7 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biology
Article
Seasonal Changes in the Biochemical Composition of Dominant
Macroalgal Species along the Egyptian Red Sea Shore
Marwa Kamal 1, *, Neveen Abdel-Raouf 1,2, Khairiah Alwutayd 3, Hamada AbdElgawad 1,4 ,
Mohamed Sayed Abdelhameed 1, Ola Hammouda 1and Khaled N. M. Elsayed 1
1Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt
2Department of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz
University, Al-Kharj 11942, Saudi Arabia
3Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428,
Riyadh 11671, Saudi Arabia
4Integrated Molecular Plant Physiology Research, Department of Biology, University of Antwerp,
2020 Antwerp, Belgium
*Correspondence: marwakamal_211@yahoo.com
Simple Summary:
Macroalgae play a significant role as primary producers in marine ecosystems.
The most dominant species at the three collection sites studied along the Egyptian Red Sea were
seasonally harvested. Among several species collected, five dominant macroalgae (Caulerpa prolifera,
Acanthophora spicifera, Cystoseira myrica,Cystoseira trinodis and Turbinaria ornata) were selected for
further studies. These macroalgae were identified using morphological and molecular characteris-
tics. During summer and winter, the mineral content and biochemical composition of the selected
macroalgal species were evaluated. These analyses indicated that macroalgae are rich in minerals
as well as primary and secondary metabolites. Moreover, the findings reported that the macroalgae
studied possess high nutritional value in the summer more than in the winter season.
Abstract:
Macroalgae are significant biological resources in coastal marine ecosystems. Seasonality
influences macroalgae biochemical characteristics, which consequentially affect their ecological and
economic values. Here, macroalgae were surveyed from summer 2017 to spring 2018 at three sites at
7 km (south) from El Qusier, 52 km (north) from Marsa Alam and 70 km (south) from Safaga along
the Red Sea coast, Egypt. Across all the macroalgae collected, Caulerpa prolifera (green macroalgae),
Acanthophora spicifera (red macroalgae) and Cystoseira myrica,Cystoseira trinodis and Turbinaria ornata
(brown macroalgae) were the most dominant macroalgal species. These macroalgae were identified
at morphological and molecular (18s rRNA) levels. Then, the seasonal variations in macroalgal
minerals and biochemical composition were quantified to determine the apt period for harvesting
based on the nutritional requirements for commercial utilizations. The chemical composition of
macroalgae proved the species and seasonal variation. For instance, minerals were more accumulated
in macroalgae C. prolifera, A. spicifera and T. ornata in the winter season, but they were accumulated
in both C. myrica and C. trinodis in the summer season. Total sugars, amino acids, fatty acids and
phenolic contents were higher in the summer season. Accordingly, macroalgae collected during the
summer can be used as food and animal feed. Overall, we suggest the harvesting of macroalgae for
different nutrients and metabolites in the respective seasons.
Keywords: Red Sea; macroalgae; seasonality; molecular identification; biochemical composition
1. Introduction
The Red Sea is known to be the northernmost tropical sea in the world, possessing a
remarkable geography [
1
]. It is considered a landlocked and largely unperturbed marine
ecosystem, which is situated in one of the world’s hottest places along a small basin sepa-
rating the continents of Asia and Africa [
2
]. Its coastal areas of Egypt are very interesting
Biology 2023,12, 411. https://doi.org/10.3390/biology12030411 https://www.mdpi.com/journal/biology

Biology 2023,12, 411 2 of 21
to many researchers [
3
]. This is because the coastal areas of the Red Sea possess more
biotope and species diversity than the Mediterranean Sea and the world’s oceans [
4
]. The
Red Sea ecosystem comprises macroalgae, mangroves and coral reefs [
1
]. Macroalgae
diversity performs an important ecological role through the cycling of carbon, nitrogen and
phosphorus, which results in the regulation of marine water quality [5].
Based on their pigmentation, morphology, anatomy and biochemical composition [
3
],
macroalgae are classified into three categories: red (Rhodophyta), brown (Phaeophyta or
Ochrophyta) and green (Chlorophyta) [
6
]. Each class of macroalgae is characterized by
particular kinds of pigments, which give them their definite colors as well as distinctive
group names [
7
]. Globally, more than 4000 species of Rhodophyta, 1500 species of Phaeo-
phyta and 900 species of Chlorophyta have been recorded [
8
]. Approximately 500 species
of macroalgae were listed in the Red Sea [
9
]. Recently, the macroalgal biomass in the Red
Sea recorded an apparent increase, which may be attributed to nutrient enrichment from
urban and aquaculture outflow, as well as reduction in herbivores [
2
]. It is well known that
the surrounding environment can influence the biodiversity and abundance of macroalgal
flora, allowing some species to predominate over others [2,10].
Seaweeds are marine macroalgae that inhabit the littoral zone [
11
]. Seaweeds are
characterized as non-vascular plants, which represent the primary producers in oceans
and belong to the Protista not Planta kingdom [
3
]. They grow from intertidal to shallow
coastal waters, in addition to deep waters, up to 180 m in depth [
12
]. They can provide
oxygen, food resources and shelter substrates for many aquatic organisms. The floristic
composition of marine macroalgae [
13
], in addition to their distribution and periodicity
sequence, can be used for estimating several ecological changes [
14
]. For example, they
help in reducing ocean acidity and offer a solution to global warming [
15
,
16
]. Moreover,
they support the diversity and productivity of some communities because they provide
oxygen, food, as well as habitat for many kinds of aquatic biota [12].
Seaweeds attract attention as one of the most biologically active resources in nature
due to their great content of bioactive compounds. Macroalgae are known to be a wealthy
source of dietary fiber, essential amino acids, nutrients, vitamins, antioxidantsand lipids [
3
,
17
].
Thus, they are valuable natural sources for fertilizers and plant growth regulators, food
commodities, animal feeds and perform a crucial role in agriculture and horticulture [
18
,
19
].
Recently, seaweeds have also been used for a variety of purposes, including health benefits,
biofuel production, cosmetics, pharmaceuticals, textiles and bioplastic packaging [
20
,
21
].
In this context, seaweeds rich in bioactive components [
12
], including antioxidant, anti-
pigmentation, anticancer, anti-wrinkling and antimicrobial activities, have been of particular
interest [7,22,23].
The quality and concentration of bioactive compounds of seaweeds depend on various
factors, including the season, geographic location, harvesting period, in addition to biotic
factors, such as herbivory or direct competition with other organisms, and abiotic factors,
such as salinity, temperature, pH and nutrient composition of water [24,25]. These factors
could stimulate or inhibit the production of macroalgal bioactive constituents [
26
]. The
ability of macroalgae to produce distinctive secondary metabolites, such as polysaccharides,
proteins, lipids and phenolic compounds, enables them to quickly adapt to changes in the
marine environment, including temperature and solar radiation [
27
]. Moreover, the great
content of these metabolites in seaweeds may differ significantly according to the taxonomic
group, geographical, seasonal and physiological variations [
28
,
29
]. The formation of
marine macroalgal communities is regulated by a set of restrictions, such as light, depth,
temperature and nutritional content. As a result of macroalgal species’ diversity and
availability being affected, the marine environment ultimately changes [
30
]. In the Red
Sea, macroalgae are known to be one of the most significant biological resources in coastal
marine ecosystems, as well as supporting some communities’ diversity and productivity
because of their important role as primary producers in the marine environment [31].
Seaweed communities are considered significant as an indicator of environmental
stress, as their distribution and abundance are affected by disturbances, such as desiccation,

Biology 2023,12, 411 3 of 21
high temperatures and competition with coastal flora and fauna [
32
]. Therefore, it is very
important to study their variations and distribution at different times and places [
33
]. In
this study, green, red and brown macroalgae species were collected from the Red Sea shore,
Egypt, during four seasons. Out of several species collected, five species were selected
based on their dominance throughout the four seasons of the year in the geographical
locations under investigation. These five selected seaweeds were identified based on
morphological and molecular characterization. Then, the biochemical compositions of the
selected seaweeds, including primary metabolites (carbohydrates, amino acids (AAs), fatty
acids (FAs) and organic acids), secondary metabolites (phenolics) and mineral profiles,
were analyzed to evaluate the influence of seasons, i.e., summer and winter. To our
knowledge, the present study is the first to evaluate the seasonal impact on the biochemical
compositions of macroalgal species. This was also required in order to determine their
potential use in human food and other industries.
2. Materials and Methods
2.1. Collection Sites, Seasonal Climate Conditions and Identification of Macroalgae
Macroalgal specimens were collected from three sites at 7 km (south) from El Qu-
seir (26
◦
2
0
34.02
00
N; 34
◦
18
0
51.51
00
E), 52 km (north) from Marsa Alam (26
◦
11
0
30.75
00
N;
34
◦
13
0
43.92
00
E) and 70 km (south) from Safaga (25
◦
32
0
56.35
00
N; 34
◦
38
0
16.88
00
E) along the
Red Sea coast, Egypt, seasonally, from summer 2017 to spring 2018 during low tides when
seaweeds are exposed (Figure 1). These sites were selected because (1) they are fertile
seacoasts and they are markedly rich in flora and fauna, and (2) there is an absence of in-
dustrial activities, as well as (3) a significantly lower population of habitants. The quadrate
technique (steel quadrate 100 ×100 cm) was applied for the collection of macroalgal sam-
ples from the three collection sites [
34
]. Five quadrate samples were collected at each site.
Macroalgae were harvested at their maturation stage manually and washed thoroughly in
sea water to remove potential contaminants, such as adhering impurities, sand particles,
rock debris, epiphytes and animal castings. The fresh biomass was collected in polyethylene
bags containing sea water to prevent evaporation and washed with tap water followed by
distilled water to remove excess salts. The dried samples were fine-powdered using a food
mixer and stored in labeled plastic bags for further use [
35
]. Some of the collected seaweeds
were preserved for identification. The relative abundance of each macroalgal species was
determined according to the following equation: Abundance % = No of individuals of a
given species
×
100
÷
Total no. of all species [
36
]. The climate conditions of these sites were
as follows. Water temperature varied between 15.8 and 18.5
◦
C in the winter months and
31.7 and 32.7
◦
C in the summer at day time at the selected sites. During the summer, the
pH values were slightly alkaline; they fluctuated between 7.72 during the winter at Site 3
and 7.89 at Site 1 (Table 1). At first, the macroalgal samples collected were identified based
on their morphological characteristics with taxonomic references [37]. The morphological
identification was followed by molecular identification.
2.2. Molecular Identification
DNA was isolated using the Cetyl Trimethyl Ammonium Bromide (CTAB) method
from approximately 400 mg of macroalgal powder ground in liquid nitrogen [
38
]. The
purity and concentration of extracted DNA were determined using a spectrophotometer
at 260 nm and 280 nm. Purity was measured at the ratio of A 260: A 280 using agarose
gel electrophoresis. The purified DNA isolate was amplified through the polymerase
chain reaction (PCR) process using 18S rRNA primers (Table 2). Basic local alignment
search tool (BLAST) analysis was used to determine similarities in GenBank to confirm
the species of the macroalgal samples collected. The National Center for Biotechnology
NCBI (blast.ncbi.nih.nlm.gov) was used to carry out this analysis by entering the complete
sample sequences into the BLAST analysis. Phylogenetic trees were constructed using the
MEGA X program.

Biology 2023,12, 411 4 of 21
Biology 2023, 12, x FOR PEER REVIEW 4 of 21
Figure 1. Map illustrating the three collection sites along the Red Sea, Egypt.
Table 1. physico-chemical analysis of water samples collected from macroalgae collection sites.
Site 3
Site 2
Site 1
Water Analysis
Season
Winter
Summer
Winter
Summer
Winter
Summer
15.8 ± 2
32.2 ± 2
17.5 ± 2
32.7 ± 2
18.5 ± 2
31.5 ± 2
Temperature (°C)
7.72 ± 0.1
7.8 ± 0.2
7.76 ± 0.2
7.82 ± 0.2
7.78 ± 0.1
7.89 ± 0.2
PH
125.4 ± 4.2
116.6 ± 3.9
125.4 ± 5.2
126.6 ± 5.1
127.4 ± 6.3
113.3 ± 5.1
Total alkalinity
33.11 ± 2.2
21.70 ± 1.5
98.17 ± 4.1
42.85 ± 2.3
109.8 ± 4.5
43.44 ± 2.1
Nitrates (ppm)
20,486 ± 154
19,159 ± 124
19,409 ± 106
17,281 ± 118
1918 ± 55
18,345 ± 120
Chlorides (ppm)
3148 ± 62
2859 ± 51
2953 ± 54
2489 ± 38
2913 ± 48
2767 ± 53
Sulphate (ppm)
0.59 ±0.03
ND
ND
1.99 ±0.05
NR
0.55 ± 0.02
Phosphate (ppm)
12,519 ± 98
13,576 ± 102
13,029 ± 79
9932 ± 65
13,474 ± 83
11,261 ± 75
Sodium (ppm)
601 ± 21
571 ± 18
617 ± 29
479 ± 22
558 ± 25
506 ± 19
Potassium (ppm)
529 ± 23
433 ± 22
508 ± 19
438 ± 25
476 ± 22
454 ± 17
Calcium (ppm)
1438 ± 35
1384 ± 35
1432 ± 39
1214 ± 28
1645 ± 38
1327 ± 32
Magnesium (ppm)
ND
ND
ND
0.43 ± 0.02
ND
NR
Iron (ppm)
ND
ND
NR
NR
ND
NR
Copper (ppm)
0.026 ± 0.003
0.029 ± 0.002
0.024 ± 0.001
0.036 ± 0.002
0.032 ± 0.002
0.027 ± 0.001
Manganese (ppm)
0.195 ± 0.02
ND
ND
0.65 ± 0.01
NR
0.18 ± 0.02
Phosphorus (ppm)
40.4 ± 3
38.5 ± 2
41.2 ± 3
38.5 ± 4
40.8 ± 3
41.7 ± 4
Carbonate (ppm)
2.2. Molecular Identification
DNA was isolated using the Cetyl Trimethyl Ammonium Bromide (CTAB) method
from approximately 400 mg of macroalgal powder ground in liquid nitrogen [38]. The
Figure 1. Map illustrating the three collection sites along the Red Sea, Egypt.
Table 1. Physico-chemical analysis of water samples collected from macroalgae collection sites.
Water Analysis
Site 1 Site 2 Site 3
Season
Summer Winter Summer Winter Summer Winter
Temperature (◦C) 31.5 ±2 18.5 ±2 32.7 ±2 17.5 ±2 32.2 ±2 15.8 ±2
PH 7.89 ±0.2 7.78 ±0.1 7.82 ±0.2 7.76 ±0.2 7.8 ±0.2 7.72 ±0.1
Total alkalinity 113.3 ±5.1 127.4 ±6.3 126.6 ±5.1 125.4 ±5.2 116.6 ±3.9 125.4 ±4.2
Nitrates (ppm) 43.44 ±2.1 109.8 ±4.5 42.85 ±2.3 98.17 ±4.1 21.70 ±1.5 33.11 ±2.2
Chlorides (ppm) 18,345 ±120 1918 ±55 17,281 ±118 19,409 ±106 19,159 ±124 20,486 ±154
Sulphate (ppm) 2767 ±53 2913 ±48 2489 ±38 2953 ±54 2859 ±51 3148 ±62
Phosphate (ppm) 0.55 ±0.02 NR 1.99 ±0.05 ND ND 0.59 ±0.03
Sodium (ppm) 11,261 ±75 13,474 ±83 9932 ±65 13,029 ±79 13,576 ±102 12,519 ±98
Potassium (ppm) 506 ±19 558 ±25 479 ±22 617 ±29 571 ±18 601 ±21
Calcium (ppm) 454 ±17 476 ±22 438 ±25 508 ±19 433 ±22 529 ±23
Magnesium (ppm) 1327 ±32 1645 ±38 1214 ±28 1432 ±39 1384 ±35 1438 ±35
Iron (ppm) NR ND 0.43 ±0.02 ND ND ND
Copper (ppm) NR ND NR NR ND ND
Manganese (ppm) 0.027 ±0.001 0.032 ±0.002 0.036 ±0.002 0.024 ±0.001 0.029 ±0.002 0.026 ±0.003
Phosphorus (ppm) 0.18 ±0.02 NR 0.65 ±0.01 ND ND 0.195 ±0.02
Carbonate (ppm) 41.7 ±4 40.8 ±3 38.5 ±4 41.2 ±3 38.5 ±2 40.4 ±3

Biology 2023,12, 411 5 of 21
Table 2. Polymerase chain reaction primers used in the present study.
Sample ID Barcode Sequence Linker Primer Sequence
A1 “GCCACATA,
GGTGCGAA” “GCGGTAATTCCAGCTCCAA,AATCCRAGAATTTCACCTCT”
A2 “GCCACATA,
GTCGTAGA” “GCGGTAATTCCAGCTCCAA,AATCCRAGAATTTCACCTCT”
A3 “GCCACATA,
TCTTCACA” “GCGGTAATTCCAGCTCCAA,AATCCRAGAATTTCACCTCT”
A4 “GCCACATA,
TTCACGCA” “GCGGTAATTCCAGCTCCAA,AATCCRAGAATTTCACCTCT”
A5 “GCCACATA,
AATCCGTC” “GCGGTAATTCCAGCTCCAA,AATCCRAGAATTTCACCTCT”
2.3. Physico-Chemical Analysis of Water Samples
Samples of water (approx. 2 L) were collected from the study sites in clean, plastic
bottles and transferred to the laboratory in cold condition. Water temperature and pH were
measured in situ using Hydrolab, Model (Multi Set 430i WTW). For the other chemical
analysis, water samples were collected and transferred to the laboratory to measure the
chemical parameters. Calcium (Ca
++
), magnesium (Mg
++
), salinity, total hardness as CaCO
3
,
chloride (Cl
−
), Sulfate (SO
4−
), bicarbonate (HCO
3−
), nitrate (NO
3−
), total phosphate (TP),
copper, zinc and lead were measured following the protocol of the American Public Health
Association standard methods (APHA) [39].
2.4. Primary Metabolites’ Analysis
The sugars, amino acids (AAs) and fatty acids (FAs) and contents of macroalgal
biomass were evaluated and recorded in both seasons (summer and winter). Sugars were
measured in an acetonitrile/water (2 mL, 1:1, v/v) extract and determined using high-
performance liquid chromatography (HPLC) according to Alasalvar et al. [
40
]. Individual
sugars were measured using standard curves built using definite concentrations of standard
sugar solutions from 1 to 10 mg/100 mL of acetonitrile/water (1:1, v/v).
The AAs of macroalgal samples were measured according to Sinha et al. [
41
] using
1 mL of 80% (v/v) aqueous ethanolic extract. Seaweed extracts were centrifuged, and
then, the supernatant was evaporated under vacuum. Pellets were dissolved in 1 mL of
chloroform, and the suspension was re-extracted using 1 mL of HPLC-grade water. Then,
the aqueous phase was gathered after centrifugation and filtered using 0.2
µ
M Millipore
microfilters. AAs were analyzed using a Waters Acquity UPLC-tqd system (Milford,
Worcester County, MA, USA) equipped with BEH amide 2.1 ×50 columns.
The FAs of macroalgal samples were estimated using GC/MS using aqueous methano-
lic extract (1:1 w/v) until discoloration occurred according to Torras-Claveria et al. [
42
].
The FAs of macroalgal extracts were identified with GC/MS using a Hewlett Packard 6890,
MSD 5975 mass spectrometer (Hewlett Packard, Palo Alto, CA, USA). Different FAs were
quantified with the NIST 05 database and plant-specific databases.
The organic acids of macroalgal samples were measured according to De Sousa et al. [
43
].
Samples of macroalgae powder were milled and extracted using 0.1% phosphoric acid contain-
ing butylated hydroxyanisole. The internal standard (ribitol) was added during the extraction
steps. After centrifugation for 30 min at 14,000 rpm, the supernatant was transferred to new
tubes for HPLC evaluation (LaChrom L-7455 diode array, LaChrom, Tokyo, Japan). Methanol
was used for samples’ elution as mobile phase A and 5% potassium dihydrogen phosphate
(pH 2.5) as mobile phase B at 0.5 mL/min and 40 µL injection volume.
2.5. Minerals’ Analysis
Macroalgal samples were digested using HNO3/H2O (5:1 ratio) in an oven. Various
minerals were measured using mass spectrometry (ICP—MS Finnigan Element XR; Scien-

Biology 2023,12, 411 6 of 21
tific, Bremen, Germany) according to Ref. [
44
]. Standard mixtures were prepared in 1%
nitric acid.
2.6. Phenolic Compounds
Total polyphenols and flavonoids were assessed in macroalgal biomass extracted in
80% ethanol. The phenolic content was quantified by the Folin–Ciocalteu method [
45
], while
flavonoids were determined by the modified aluminum chloride colorimetric method [
46
]. Toco-
pherols were determined using hexane extract quantified by HPLC according to Siebert et al. [
47
].
2.7. Statistical Analysis
The results were expressed as mean ±SD (standard deviation) and analyzed by one-
way ANOVA using IBM SPSS Statistical software package (SPSS
®
Inc., Chicago, IL, USA).
In cases of significant interactions between the factors, one-way ANOVA was performed for
each factor, and Tukey’s multiple range tests were used to determine significant differences
among means between the two seasons of the same species (p< 0.05). A significance level
of p< 0.05 was used for rejection of the null hypothesis. All experiments were carried out
in three replicates (n= 3).
3. Results and Discussion
3.1. Macroalgal Species Collection
The dominance of macroalgal species along the Red Sea coast was determined accord-
ing to the relative abundance of species from all collection sites throughout the year [
36
].
The green macroalgae (C. prolifera), the red macroalgae (A. spicifera) and the brown macroal-
gae (C. myrica, C. trinodis and T. ornata) (Figure 2) were the most prominent macroalgal
species among all collected macroalgae.
Biology 2023, 12, x FOR PEER REVIEW 7 of 21
Figure 2. The dominant macroalgal species collected from three sites along the Red Sea shore,
Egypt. (A) Caulerpa prolifera, (B) A. spicifera, (C) Cystoseira myrica, (D) Cystoseira trinodis and (E) T.
ornata.
For years, the biodiversity of these seaweeds has been largely classified based on
their morphological features [48]. Recent developments have inspired scientists to use
molecular approaches to investigate the biodiversity of marine macroalgae [49]. Molec-
ular studies were used by algal taxonomists for species’ discovery and identification, in
addition to many routine taxonomic studies [50]. Therefore, the five dominant seaweeds
collected were first morphologically identified, followed by molecular identification us-
ing 18S rRNA sequencing. The sequences of 18S rRNA were analyzed on NCBI using the
BLAST tool to determine the sequences’ percentage of similarity with the sequences in
GenBank. All of the obtained sequences corresponded to known macroalgal species with
significant sequence similarity. Based on the results of phylogenetic tree analysis, the
harvested macroalgal species were closely related to Caulerpa prolifera, red seaweed Ac-
anthophora spicifera, brown seaweeds Cystoseira myrica, C. trinodis and Turbinaria ornata,
respectively (Figure 3).
Figure 2.
The dominant macroalgal species collected from three sites along the Red Sea shore, Egypt.
(A)Caulerpa prolifera, (B)A. spicifera, (C)Cystoseira myrica, (D)Cystoseira trinodis and (E)T. ornata.

Biology 2023,12, 411 7 of 21
For years, the biodiversity of these seaweeds has been largely classified based on their
morphological features [
48
]. Recent developments have inspired scientists to use molecular
approaches to investigate the biodiversity of marine macroalgae [
49
]. Molecular studies
were used by algal taxonomists for species’ discovery and identification, in addition to
many routine taxonomic studies [
50
]. Therefore, the five dominant seaweeds collected
were first morphologically identified, followed by molecular identification using 18S rRNA
sequencing. The sequences of 18S rRNA were analyzed on NCBI using the BLAST tool to
determine the sequences’ percentage of similarity with the sequences in GenBank. All of the
obtained sequences corresponded to known macroalgal species with significant sequence
similarity. Based on the results of phylogenetic tree analysis, the harvested macroalgal
species were closely related to Caulerpa prolifera, red seaweed Acanthophora spicifera, brown
seaweeds Cystoseira myrica,C. trinodis and Turbinaria ornata, respectively (Figure 3).
Biology 2023, 12, x FOR PEER REVIEW 8 of 21
Figure 3. Phylogenetic tree of 18s rRNA sequences of macroalgae. (A) Caulerpa prolifera, (B) A.
spicifera, (C) Cystoseira myrica, (D) Cystoseira trinodis and (E) T. ornata. constructed by NCBI/BLAST
3.2. Minerals’ Level Change with Season and Species
Macroalgae accumulate minerals, which are necessary for seaweeds survival, as well
as improve their nutritional value and as a medicinal source [32,51]. Fifty-two essential
minerals, including macrominerals, such as Na, K, Ca, Mg and P, and trace elements,
such as Cd, Fe, Zn, Cu and Mn, were identified. The minerals’ profiles of the five
macroalgae investigated in this study exhibited various amounts of essential metals. P, K,
Na and Mg were the most abundant elements among the different species. Their con-
centrations were as the the following ranges: 1.65–5.62 mg/g dry weight (DW), 0.64–2.54
mg/g DW, 0.29–0.91 mg/g DW, and 0.23–0.82 mg/g DW, 0.64–2.54 mg, 0.29–0.91 mg, and
0.23–0.82 mg, respectively. Significant difference at p < 0.05 was observed between the
content of minerals in the summer and winter. C. prolifera and T. ornata had a high content
of K in the winter season, but high content of K was recorded in A. spicifera and C. myrica
in the summer . C. trinodis. C. prolifera, A. spicifera and T. ornata had a high content of P in
the winter, but C. myrica and C. trinodis had a high P content in the summer. Na and Mg
rendered the same results with different tested macroalgae. Significant increase was ob-
served in the winter season for C. prolifera, A. spicifera and T. ornata. In contrast, significant
increase was observed in the summer season for both C. myrica and C. trinodis (Table 3).
Figure 3.
Phylogenetic tree of 18s rRNA sequences of macroalgae. (
A
)Caulerpa prolifera, (
B
)A.
spicifera, (C)Cystoseira myrica, (D)Cystoseira trinodis and (E)T. ornata. constructed by NCBI/BLAST.
3.2. Minerals’ Level Change with Season and Species
Macroalgae accumulate minerals, which are necessary for seaweeds survival, as well
as improve their nutritional value and as a medicinal source [
32
,
51
]. Fifty-two essential
minerals, including macrominerals, such as Na, K, Ca, Mg and P, and trace elements, such
as Cd, Fe, Zn, Cu and Mn, were identified. The minerals’ profiles of the five macroalgae
investigated in this study exhibited various amounts of essential metals. P, K, Na and
Mg were the most abundant elements among the different species. Their concentrations
were as the the following ranges: 1.65–5.62 mg/g dry weight (DW), 0.64–2.54 mg/g DW,

Biology 2023,12, 411 8 of 21
0.29–0.91 mg/g DW, and 0.23–0.82 mg/g DW, 0.64–2.54 mg, 0.29–0.91 mg, and 0.23–0.82 mg,
respectively. Significant difference at p< 0.05 was observed between the content of minerals
in the summer and winter. C. prolifera and T. ornata had a high content of K in the winter
season, but high content of K was recorded in A. spicifera and C. myrica in the summer.
C. trinodis,C. prolifera,A. spicifera and T. ornata had a high content of P in the winter, but
C. myrica and C. trinodis had a high P content in the summer. Na and Mg rendered the
same results with different tested macroalgae. Significant increase was observed in the
winter season for C. prolifera,A. spicifera and T. ornata. In contrast, significant increase was
observed in the summer season for both C. myrica and C. trinodis (Table 3).
Macroalgae do not biosynthesize minerals, but they absorb them from the surrounding
environment based on many factors, such as temperature, pH, salinity and light [
52
]. Thus,
both internal and external factors have an impact on minerals’ accumulation in macroalgae.
The former involve sulfhydryl ester, amino, carboxyl, hydroxyl, proteins and/or lipids,
while the latter include sea water, temperature, salinity, pH and disruptions [
53
]. Regarding
their biological and nutritional value, Ca is a crucial element in the body skeleton, in
heart strength and smooth muscle contraction, in addition to the nervous and muscular
equilibria [
54
], while Mg is a very important cofactor of several enzymes, including those
involved in respiration. Other minerals, such as Fe, Mg, Cu, Zn and Co, are involved in
several metabolic processes, as well as working as enzyme cofactors [
32
]. According to
metal analysis of the four seaweeds Laminaria digitata,L.hyperborea,Saccharina latissima and
Alaria esculenta, the concentrations of K and Na in the winter were more than the doubleof
their concentrations in the summer [
55
]. The current result is in line with previous findings
for Laminaria digitata [
56
]. Saldarriaga-Hernandez et al. [
57
] reported a high concentration
of P in Sargassum and concluded that Sargassum is recommended as an alternative source of
P. A similar result was described by Gaillande et al. [
58
] who indicated that high quantities
of Na, K, Ca and Mg were also reported in Caulerpa species.
3.3. Species and Seasonal Variation in Primary and Secondary Metabolites
In this study, seasonal variations in macroalgal biochemical composition were ob-
served, which affect the apt period for harvesting based on the nutritional requirements for
commercial utilizations. Seasonal characterization is a prerequisite for future valorization
of macroalgal biomass as a component of feed additives or fertilizers [
51
]. Thus, it is
important to understand these variations in the production of biologically active com-
pounds in order to determine the ideal time for harvesting macroalgal biomass based on
its proposed applications in food, animal feed, biofuel production, pharmaceutical and
various industries.
Several studies proved the effect of seasonality on the biochemical constituent of
different species of seaweeds. Kumar et al. [
59
] observed significant individual differences
in the biochemical composition of all investigated marine macroalgae. Ajayan et al. [
60
]
studied the fatty acid contents, metals and other elemental compositions of 25 macroalgal
species and proved that the lipids, proteins and carbohydrate levels varied significantly
among the species studied. For instance, Samanta et al. [
61
] illustrated that the chemical
composition of Agarophyton vermiculophyllum was changed by variable climatic conditions,
such as temperature, pH and nutrient availability. Pérez et al. [
62
] proved that seaweed
harvested in the summer showed superior physiological activities as a result of the presence
of active metabolites, such as fatty acids, pigments, phlorotannins, lectins, terpenoids,
alkaloids and halogenated compounds, as a pattern of adaptation. Overall, more studies
are required to evaluate the use of macroalgae as a healthy and sustainable alternative in
the nutraceutical, cosmetics, as well as well-being industries because seaweed exploitation
in Egypt is still in its early stages [63].

Biology 2023,12, 411 9 of 21
Table 3.
Seasonal variations in the concentrations (mg/g DW) of minerals in macroalgae C. prolifera, A. spicifera, C. myrica, C. trinodis and T. ornata. Values are shown
as means ±S.E. (n= 3). Different letters show significance between the two seasons of the same species (p< 0.05).
Minerals C. prolifera A. spicifera C. myrica C. trinodis T. ornata
Summer Winter Summer Winter Summer Winter Summer Winter Summer Winter
P 1.6 ±0.03 b 2.67 ±0.15 a 3.53 ±0.21 a 3.76 ±0.36 a 5.62 ±0.18 b 3.44 ±0.16 a 4.25 ±0.29 b 2.61 ±0.31 a 3.1 ±0.18 b 4.2 ±0.34 a
K 0.64 ±0.03 b 1.42 ±0.07 a 1.96 ±0.02 a 1.27 ±0.20 a 1.89 ±0.06 b 0.94 ±0.04 a 2.18 ±0.04 a 1.95 ±0.31 a 1.29 ±0.13 b 2.54 ±0 a
Na 0.29 ±0 b 0.37 ±0.01 a 0.57 ±0.05 b 0.8 ±0.08 a 0.81 ±0.02 b 0.63 ±0.03 a 0.91 ±0.06 b 0.6 ±0.03 a 0.4 ±0.09 b 0.84 ±0.06 a
Mg 0.23 ±0.02 b 0.41 ±0.01 a 0.53 ±0.04 a 0.57 ±0.09 a 0.82 ±0.02 b 0.49 ±0.02 a 0.8 ±0.06 b 0.54 ±0.06 a 0.52 ±0.10 b 0.74 ±0.08 a
Ca 0.02 ±0 a 0.03 ±0 a 0.04 ±0 a 0.03 ±0.01 a 0.04 ±0 a 0.03 ±0 a 0.05 ±0 a 0.04 ±0 a 0.04 ±0.01 b 0.06 ±0 a
Cd 0.21 ±0.01 b 0.03 ±0.01 a 0.21 ±0.01 a 0.17 ±0 a 0.16 ±0.01 b 0.13 ±0 a 0.2 ±0.01 b 0.08 ±0.01 a 0.23 ±0.01 b 0.06 ±0.01 a
Fe 0.04 ±0 b 0.06 ±0.01 a 0.07 ±0.01 a 0.07 ±0.01 a 0.1 ±0 a 0.07 ±0 a 0.1 ±0.01 b 0.06 ±0 a 0.07 ±0.01 a 0.1 ±0.01 a
Zn 0.03 ±0 b 0.06 ±0.01 a 0.08 ±0.01 a 0.08 ±0.01 a 0.11 ±0 b 0.08 ±0 a 0.1 ±0.02 b 0.08 ±0 a 0.1 ±0.01 a 0.09 ±0 a
Mn 0.02 ±0 a 0.03 ±0 a 0.04 ±0 a 0.05 ±0.01 a 0.07 ±0 b 0.05 ±0 a 0.05 ±0 a 0.04 ±0 a 0.04 ±0.01 a 0.05 ±0 a
Cu 0.01 ±0 a 0.01 ±0 a 0.01 ±0.01 b 0.02 ±0 a 0.02 ±0 a 0.02 ±0 a 0.02 ±0.001 a 0.01 ±0 a 0.02 ±0 a 0.02 ±0 a

Biology 2023,12, 411 10 of 21
3.3.1. Carbohydrates
Carbohydrates are considered the primary source of energy in the majority of human
diets in addition to their importance in respiration and other metabolic processes [
64
,
65
].
Furthermore, they can be used for biofuel production [
66
]. In the present study, total
sugars recorded a high content in the summer season for all macroalgal species tested.
Red macroalgae A. spicifera had the greatest concentration (564.7 mg/g DW), followed by
green macroalgae C. prolifera (557.9 mg/g DW), while brown macroalgae showed minimum
contents (Figure 4). Monosaccharide glucose exhibited a different pattern, whereby a
significant increase in glucose quantity was observed in the winter season in all macroalgae
tested. The highest content of fructose was recorded in the winter for brown macroalgae in
contrast to red macroalgae A. spicifera and green macroalgae C. prolifera.
Biology 2023, 12, x FOR PEER REVIEW 10 of 21
on its proposed applications in food, animal feed, biofuel production, pharmaceutical
and various industries.
Several studies proved the effect of seasonality on the biochemical constituent of
different species of seaweeds. Kumar et al. [59] observed significant individual differ-
ences in the biochemical composition of all investigated marine macroalgae. Ajayan et al.
[60] studied the fatty acid contents, metals and other elemental compositions of 25
macroalgal species and proved that the lipids, proteins and carbohydrate levels varied
significantly among the species studied. For instance, Samanta et al. [61] illustrated that
the chemical composition of Agarophyton vermiculophyllum was changed by variable cli-
matic conditions, such as temperature, pH and nutrient availability. Pérez et al. [62]
proved that seaweed harvested in the summer showed superior physiological activities
as a result of the presence of active metabolites, such as fatty acids, pigments, phloro-
tannins, lectins, terpenoids, alkaloids and halogenated compounds, as a pattern of ad-
aptation. Overall, more studies are required to evaluate the use of macroalgae as a
healthy and sustainable alternative in the nutraceutical, cosmetics, as well as well-being
industries because seaweed exploitation in Egypt is still in its early stages [63].
3.3.1. Carbohydrates
Carbohydrates are considered the primary source of energy in the majority of hu-
man diets in addition to their importance in respiration and other metabolic processes
[64,65]. Furthermore, they can be used for biofuel production [66]. In the present study,
total sugars recorded a high content in the summer season for all macroalgal species
tested. Red macroalgae A. spicifera had the greatest concentration (564.7 mg/g DW), fol-
lowed by green macroalgae C. prolifera (557.9 mg/g DW), while brown macroalgae
showed minimum contents (Figure 4). Monosaccharide glucose exhibited a different
pattern, whereby a significant increase in glucose quantity was observed in the winter
season in all macroalgae tested. The highest content of fructose was recorded in the
winter for brown macroalgae in contrast to red macroalgae A. spicifera and green
macroalgae C. prolifera.
Figure 4. Seasonal variations in the concentrations (mg/g DW) of (A) Glucose, (B) Fructose, (C)
Sucrose, (D) Soluble sugars, (E) Insoluble sugars and (F) Total sugars in macroalgae Caulerpa pro-
Figure 4.
Seasonal variations in the concentrations (mg/g DW) of (
A
) Glucose, (
B
) Fructose, (
C
) Su-
crose, (
D
) Soluble sugars, (
E
) Insoluble sugars and (
F
) Total sugars in macroalgae Caulerpa prolifera, A.
spicifera, Cystoseira myrica, Cystoseira trinodis, and T. ornata. Values are shown as means
±
S.E. (n= 3).
Different letters show significance between the two seasons of the same species (p< 0.05).
The result obtained revealed that the greatest content of total sugars was found in the
summer season in all macroalgae investigated. A similar pattern was noticed by Khairy and
El-Shafay [
64
] who reported that the highest amount of carbohydrates in U. lactuca and P.
capillacea was produced during the summer season. According to García-Sanchez et al. [
67
],
Sargassum exhibited rapid growth during the summer season owing to greater sunlight
exposure, storing carbohydrates for the rainy season, which is characterized by reduced
photosynthesis. Variations in carbohydrates’ production among macroalgal species may be
attributed to their various life cycles and abiotic oscillations [65].
3.3.2. Proteins and Amino Acids
Proteins are macromolecules and serve a variety of functions in all living organisms,
including repair and maintenance, mechanical support and energy [
32
]. A total of 19 AAs
were evaluated in the five macroalgae, including essential (EAAs) (which must be obtained
from food) and non-essential amino acids (NEAAs). Lysine, histidine, phenylalanine and
valine were the most prominent EAA. Lysine concentration was somewhat higher in the
summer season in C. prolifera, A. spicifera and T. ornata, unlike the concentrations in C. myrica

Biology 2023,12, 411 11 of 21
and C. trinodis, which were higher in the winter season. The content of phenylalanine
in the summer was double that in the winter (significant difference at p< 0.05). There
was a non-significant difference at p< 0.05 in the histidine level between both seasons.
Glycine, alanine, asparagine and glutamic acid were the most abundant NEAA. There was
a significant difference at p< 0.05 in their levels between both seasons. Glycine content was
the highestamong both EAAs and NEAAs. Glycine concentration was increased by 20–30%
in the summer season for all seaweeds studied, except C.trinodis. The greatest levels of
alanine were observed during the summer in C. prolifera and T. ornata, while they were
higher in A.spicifera, C. myrica and C. trinodis during the winter season. A. spicifera and C.
trinodis had a high content of asparagine (4.49 mg/g DW) in the summer, while T. ornata
had a high content of asparagine (5.04 mg/g DW) in the winter (Table 4).
The quality of the protein is just as important as its quantity. The protein quality of
foods is frequently assessed by the amount and composition of its essential amino acids [
26
].
Macroalgae are an important source of proteins because their protein content is rich in
essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan and valine) [
68
]. In this regard, macroalgae proteins are also signifi-
cant as a source of peptides and amino acid extracts, principally after enzymatic digestion,
which increases their solubility in water, making them acceptable to be employed in a
variety of industries [69].
Red macroalgae Palmaria palmata exhibited a similar pattern, displaying variations in
macroalgal protein content, with the winter–spring season showing greater protein content
than the summer–early autumn season [
70
]. Protein content differs greatly with seasons; the
highest concentration was recorded during the beginning of spring and winter, while the
lowest concentration was recorded in the early autumn and summer season [
71
]. Afonso
et al. [
72
] proposed that a gradual decrease in protein levels from March to August may be
due to the lower availability of nitrogenous compounds. However, other seasonal factors
may also influence the protein content, namely the high temperature of water, salinity and
eutrophication [
71
,
72
]. Balboa et al. [
51
] indicated that protein content exhibited a negative
relationship with temperature and salinity. Overall, the macroalgal protein is considered
an excellent source of EAAs and represents almost half of the total AAs they produce [
73
].
3.3.3. Lipids and Fatty Acids
Lipids play a basic role not only in energy supply, but they are also necessary for the
production of hormones and for maintaining the integrity of cell membranes [
74
]. Lipids are
also required for the transportation and absorption of fat-soluble vitamins, including A, D,
E and K [
75
]. Therefore, a total of 16 individual FAs were identified and quantified in the
five macroalgal species studied, including 8 saturated fatty acids (SFAs), 6 monounsaturated
fatty acids (MUFAs) and 2 polyunsaturated fatty acids (PUFAs). Palmitic (C16:0) and stearic
(C18:0) acids were the most abundant SFA in all macroalgal species, with high concentrations
observed in the summer season (significant difference at p< 0.05). The greatest content
of MUFAs was recorded for oleic acid (C18:1) in the summer season for all macroalgae
studied, except T. ornata in the winter season. Oleic acid (C18:1) was followed by eicosenoic
(C20:1), heptadecenoic (C17:1), palmitoleic (C16:1), and finally, tetracosenoic (C24:1) acid, at
a lower concentration. Two PUFA were reported, namely linoleic (C18:2
ω
-6) and linolenic
(C18:3
ω
-3) acid, with slightly higher concentrations in the summer than in the winter season
in C. prolifera and T. ornata, while there was no difference in their amounts in both seasons in
C. myrica, C. trinodis and A. spicifera (Table 5).

Biology 2023,12, 411 12 of 21
Table 4.
Seasonal variations in the concentrations (mg/g DW) of amino acids—essential (EAAs) and non-essential amino acids (NEAAs)—in macroalgae C. prolifera,
A. spicifera, C. myrica, C. trinodis and T. ornata. Values are shown as means
±
S.E. (n= 3). Different letters show significance between the two seasons of the same
species (p< 0.05).
Amino Acids C. prolifera A. spicifera C. myrica C. trinodis T. ornata
Summer Winter Summer Winter Summer Winter Summer Winter Summer Winter
EAAs
Lysine 3.6 ±0.30 a 3.2 ±0.10 a 2.8 ±0.20 a 2.6 ±0.10 a 3.1 ±0.20 b 6.2 ±0.30 a 3.7 ±0.30 a 3.9 ±0.10 a 3.1 ±0.20 a 2.1 ±0.10 a
Histidine 1.1 ±0.10 a 1.2 ±0 a 1.1 ±0.10 a 1 ±0.20 a 1.1 ±0.10 a 1.1 ±0.20 a 1.5 ±0.20 a 1.4 ±0.10 a 0.9 ±0.10 b 1.4 ±0.10 a
Phenylalanine 1.01 ±0.10 b 0.53 ±0.01 a 1.19 ±0.08 b 0.34 ±0.08 a 0.52 ±0.09 b 0.27 ±0.09 a 1.31 ±0.11 b 0.91 ±0.13 a 1.14 ±0.08 b 0.67 ±0.13 a
Valine 0.91 ±0.03 b 0.34 ±0.01 a 0.73 ±0.04 a 0.72 ±0.02 a 0.95 ±0.03 b 2.22 ±0.03 a 0.71 ±0.05 b 0.63 ±0.02 a 0.62 ±0.04 b 0.38 ±0.01 a
Threonine 0.29 ±0.12 b 0.15 ±0.04 a 0.63 ±0.08 b 0.31 ±0.10 a 0.35 ±0.12 a 0.38 ±0.33 a 0.55 ±0.08 b 0.21 ±0.08 a 0.29 ±0.07 a 0.57 ±0.04 a
Isoleucine 0.11 ±0 a 0.09 ±0 a 0.19 ±0.03 a 0.16 ±0 a 0.13 ±0.01 a 0.16 ±0 a 0.15 ±0.02 a 0.17 ±0.01 a 0.12 ±0.01 b 1.77 ±0.02 a
Methionine 0.03 ±0.01 a 0.03 ±0.01 a 0.3 ±0.06 b 0.05 ±0.02 a 0.13 ±0.01 b 0.03 ±0 a 0.24 ±0.05 b 0.07 ±0.01 a 0.11 ±0.02 b 0.31 ±0.06 a
Leucine 0.02 ±0 a 0.02 ±0 a 0.26 ±0.02 a 0.02 ±0 a 0.15 ±0.01 b 0.03 ±0 a 0.19 ±0.02 b 0.08 ±0.01 a 0.1 ±0.01 b 0.24 ±0.02 a
ΣEAAs 7.07 5.56 7.20 5.20 6.43 10.39 8.35 7.37 6.38 7.44
NEAAs
Glycine 79.5 ±8 b 57.1 ±3 a 49.5 ±4.9 b 37.7 ±1.9 a 57.8 ±5.7 a 41.6 ±1.8 a 40.1 ±3.8 b 70.9 ±3.8 a 49.8 ±4.9 b 34 ±1.8 a
Alanine 19.6 ±2 b 4.2 ±0.2 a 4.5 ±0.4 b 11.2 ±0.5 a 8.5 ±0.8 a 9.1 ±0.4 a 7.6 ±0.7 b 12.4 ±0.6 a 11.8 ±1.2 b 5.8 ±0.2 a
Asparagine 0.83 ±0.07 b 0.63 ±0.03 a 4.49 ±0.4 b 1.17 ±0.05 a 1.2 ±0.1 b 0.31 ±0.02 a 3.9 ±0.4 b 0.58 ±0.02 a 1.37 ±0.1 b 5.04 ±0.21 a
Glutamic acid 0.78 ±0.01 b 0.09 ±0.01 a 0.7 ±0.01 b 0.55 ±0.01 a 0.65 ±0.01 a 0.65 ±0.01 a 0.93 ±0.02 a 0.98 ±0.01 a 0.67 ±0.01 b 0.92 ±0.01 a
Arginine 0.7 ±0.08 b 0.5 ±0.03 a 0.8 ±0.06 a 0.8 ±0.18 a 0.8 ±0.07 b 1.4 ±0.06 a 1.4 ±0.12 b 1 ±0.04 a 1.3 ±0.39 a 1.1 ±0.04 a
Glutamine 0.63 ±0.1 a 0.63 ±0.08 a 1.79 ±0.62 a 1.95 ±0.13 a 1.16 ±0.14 a 1.13 ±0.04 a 2.77 ±0.4 b 0.71 ±0.07 a 1.39 ±0.1 b 2.05 ±0.69 a
Serine 0.54 ±0.1 b 0.28 ±0.08 a 0.65 ±0.14 b 0.24 ±0.02 a 0.42 ±0.04 b 0.21 ±0.01 a 0.76 ±0.14 b 0.38 ±0.08 a 0.63 ±0.13 b 0.22 ±0.05 a
Tyrosine 0.47 ±0.05 a 0.7 ±0.04 a 0.59 ±0.06 b 0.37 ±0.02 a 0.36 ±0.04 b 0.76 ±0.05 a 0.46 ±0.05 b 0.89 ±0.05 a 0.07 ±0.01 b 0.7 ±0.04 a
Ornithine 0.28 ±0.02 b 0.13 ±0.05 a 0.34 ±0.03 b 0.21 ±0.02 a 0.32 ±0.04 b 0.24 ±0.02 a 0.59 ±0.11 b 0.34 ±0.02 a 0.67 ±0.1 a 0.55 ±0.29 a
Aspartate 0.15 ±0 b 0.21 ±0.08 a 0.16 ±0 b 0.08 ±0 a 0.09 ±0 a 0.07 ±0 a 0.19 ±0 a 0.15 ±0 a 0.15 ±0 a 0.19 ±0.02 a
Cysteine 0.11 ±0.06 b 0.73 ±0.07 a 0.12 ±0.08 a 0.12 ±0.05 a 0.08 ±0.05 a 0.05 ±0.11 a 0.15 ±0.06 b 0.07 ±0.13 a 0.02 ±0.01 b 0.39 ±0.1 a
ΣNEAAs 103.59 65.20 63.64 54.39 71.38 55.52 58.85 88.40 67.87 50.96

Biology 2023,12, 411 13 of 21
Table 5.
Seasonal variations in the concentrations (mg/g DW) of various fatty acids in macroalgae C. prolifera, A. spicifera, C. myrica, C. trinodis and T. ornata. Saturated
fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs). Values are shown as means
±
S.E. (n= 3). Different letters show
significance between the two seasons of the same species (p< 0.05).
Fatty Acids C. prolifera A. spicifera C. myrica C. trinodis T. ornata
Summer Winter Summer Winter Summer Winter Summer Winter Summer Winter
SFAs
Palmitic (C16:0) 34.7 ±17 a 25.1 ±10 a 33.7 ±8.5 b 21.1 ±5.8 a 35.4 ±10 b 23.5 ±6 a 27.3 ±6 a 26.3 ±13 a 29 ±9.4 b 22.7 ±4 a
Myristic (C14:0) 3.7 ±20.9 b 2.4 ±13 a 3.1 ±12.9 b 1.9 ±8.5 a 3.6 ±15.1 a 2.7 ±9.1 a 3.4 ±10.3 a 2.8 ±16.1 a 2.9 ±13 b 1.9 ±7.7 a
Stearic (C18:0) 1.7 ±0.2 a 1.7 ±0.1 a 2.6 ±0.1 b 1.6 ±0.1 a 1.5 ±0.1 a 1.2 ±0.1 a 2.6 ±0.1 b 1.8 ±0.1 a 1.7 ±0.1 a 1.3 ±0.1 a
Arachidic (C20:0) 1.1 ±0.11 a 1.3 ±0.02 a 1.6 ±0.15 a 1.1 ±0.02 a 0.8 ±0.02 a 1 ±0.03 a 1.8 ±0.05 a 1.1 ±0.04 a 1.1 ±0.02 a 1.5 ±0.15 a
Docosanoic (C22:0) 0.7 ±0.04 a 0.6 ±0.03 a 0.5 ±0.06 a 0.5 ±0.02 a 0.6 ±0.02 b 0.4 ±0.02 a 0.6 ±0.09 a 0.5 ±0.03 a 0.4 ±0.02 b 0.7 ±0.09 a
Pentacosanoic (C25:0) 0.16 ±21 b 0.08 ±13.1 a 0.08 ±13 a 0.07 ±8.6 a 0.1 ±15.2 a 0.07 ±9.2 a 0.11 ±10.4 a 0.11 ±16.2 a 0.13 ±13.1 b 0.09 ±7.8 a
Heptadecanoic (C17:0) 0.07 ±0.01 b 0.03 ±0 a 0.05 ±0 b 0.03 ±0 a 0.04 ±0 a 0.03 ±0 a 0.03 ±0 a 0.04 ±0 a 0.07 ±0 b 0.05 ±0 a
Tricosanoic (C23:0) 0.04 ±0.01 b 0.02 ±0 a 0.042 ±0.02 b 0.02 ±0.02 a 0.043 ±0.04 b 0.02 ±0.01 a 0.039 ±0.01 b 0.02 ±0.01 a 0.065 ±0.02 b 0.03 ±0.01 a
ΣSFAs 42.17 31.23 41.67 26.32 42.08 28.92 35.88 32.67 35.37 28.27
MUFAs
Oleic (C18:1) 47.7 ±1.8 b 35.9 ±0.9 a 37.1 ±0.4 a 37.8 ±1.3 a 42.3 ±0.7 b 30.1 ±1.5 a 42.6 ±0.6 b 35.3 ±1 a 43.4 ±0.7 a 47.3 ±5 a
Eicosenoic (C20:1) 4.8 ±0.8 b 3.3 ±12.9 a 4.2 ±2.8 a 3.9 ±8.4 a 4.3 ±0.5 a 4 ±9 a 3.3 ±0.3 a 3.7 ±16.1 a 4.4 ±0.8 b 3.3 ±7.6 a
Heptadecenoic (C17:1) 1.5 ±0.18 a 1 ±0.11 a 1.5 ±0.11 a 1.2 ±0.07 a 1.5 ±0.13 a 1.2 ±0.08 a 1.1 ±0.09 a 1.1 ±0.14 a 1.3 ±0.11 a 1 ±0.07 a
Palmitoleic (C16:1) 0.11 ±0 aa 0.07 ±0.01 a 0.08 ±0.01 aa 0.08 ±0.01 a 0.09 ±0 a 0.06 ±0.01 a 0.07 ±0.01 a 0.07 ±0.01 a 0.08 ±0.01 a 0.07 ±0 a
Eicosenoic (C20:1) 0.072 ±0 b 0.04 ±0 a 0.087 ±0.01 b 0.063 ±0 a 0.191 ±0.19 b 0.062 ±0 a 0.075 ±0.01 b 0.043 ±0 a 0.071 ±0 b 0.056 ±0 a
Tetracosenoic (C24:1) 0.01 ±0.001 b 0.006 ±0 a 0.009 ±0 b 0.008 ±0 a 0.009 ±0 b 0.007 ±0 a 0.011 ±0 b 0.006 ±0 a 0.001 ±0 a 0.009 ±0 a
ΣMUFAs 54.69 40.32 42.98 33.05 48.39 35.43 47.10 40.22 49.25 51.60
PUFAs
Linoleic (C18:2 ω-6) 0.3 ±0.2 b 0.2 ±0.1 a 0.3 ±0.1 a 0.3 ±0.6 a 0.3 ±0.2 a 0.3 ±0.2 a 0.2 ±0.4 a 0.2 ±0.1 a 0.3 ±0.2 a 0.2 ±0.08 a
Linolenic (C18:3 ω-3) 0.3 ±21 a 0.2 ±13.1 a 0.3 ±0.01 a 0.2 ±8.6 a 0.3 ±0.2 a 0.3 ±9.2 a 0.2 ±0.01 a 0.2 ±16.2 a 0.3 ±0.1 a 0.2 ±7.8 a
ΣPUFAs 0.6 0.4 0.6 0.5 0.6 0.6 0.4 0.4 0.6 0.4

Biology 2023,12, 411 14 of 21
Oleic acid (C18:1) followed by palmitic acid (C16:0) were the most abundant FA, in
agreement with Morales et al. [
76
]. PUFA help macroalgae survive by acting as precursors
for the biosynthesis of a variety of secondary metabolites with crucial ecological roles [
77
].
Khairy and El shafey [
64
] reported that palmitic acid (C16:0) is the most abundant saturated
fatty acid in seaweeds, accounting for 74.3%. The essential C18 fatty acids, linoleic acid
(18:2,
ω
6) and linolenic acid (18:3,
ω
3), were recorded in the same amounts, with the
highest contents in March and April (5.7–7.2%) [
51
]. Macroalgal lipid contents are directly
affected by many variables, such as macroalgal species, location, sampling period and
environmental conditions, in addition to the extraction method and solvent polarity [78].
Although many studies proved that macroalgae possess relatively low lipid contents,
their PUFAs contents are equal to or may be greater than those of terrestrial plants [
59
].
Macroalgae accumulate high concentrations of PUFAs, which have beneficial impacts on
human health, such as reducing cardiovascular risk and improving both the brain function
and immune response [
32
,
79
]. It was also described that the PUFAs content of Caulerpa is
greater than those in coconut and palm oils [
80
]. In addition, Ajayan et al. [
60
] stated that
linolenic acid and oleic acid comprised the majority of the total fatty acids of macroalgae.
Francavilla et al. [
81
] described the increase in PUFAs and decrease in SFAs in macroal-
gae G. gracilis during the winter season. They attributed this result to the increased tightness
of cell membranes due to lower temperatures. Due to the mild winters recorded along the
Egyptian coast, the lowest temperatures recorded do not seem to significantly change the
PUFAs content. Balboa et al. [
51
] concluded that the unsaturation degree of FAs depends
primarily on the water temperature; macroalgae harvested from cold water have a greater
content of PUFAs and unsaturation degree than those collected from tropical water. Some
seaweed fatty acids are distinctive and play crucial roles in nutrition and cell membrane
construction, such as the essential
α
-linolenic fatty acid, which cannot be synthesized by
mammals, while it can only be synthesized in limited amounts by terrestrial plants [
82
].
Both FAs content and profile differ based on the variation of geographical location, biotic
(temperature, salinity, pH, light, nutrient) and abiotic parameters (herbivory), in addition
to the genetic characteristics of each macroalgal species [
51
,
75
]. Based on the results, high
FAs content can best be obtained during the summer season. SFAs, C14:0 and C16:0, are
essential for the cholesterol synthesis and thus important for human health [72].
3.3.4. Organic Acids
Six organic acids were identified and measured in the macroalgae tested. Malic acid,
isobutyric acid, citric acid and oxalic acid were the most abundant in the two seasons. The
malic acid quantity showed a comparative increase in the summer season in C. prolifera and
A. spicifera, but the three brown macroalgae recorded a high content in the winter. Succinic
acid was found in high concentration in A. spicifera during the summer season, while
fumaric acid was observed in minimum quantity in all the macroalgae studied (Figure 5).
Carpena et al. [
83
] also recorded the presence of several organic acids, including malic,
oxalic and citric acids, in the three seaweeds Chondrus crispus,Mastocarpus stellatus and
Gigartina pistillata. Tanna et al. [
82
] also reported that lactic and oxalic acids were found in
the green macroalgae Caulerpa scalpelliformis.
These detected organic acids are known for their high biological and medical values.
Succinic acid has high potential in many biological production processes, including food,
pharmaceutical, cosmetics, detergents and lubricants [
84
]. Fumaric acid is an intermediate
of the TCA cycle, and it is generally used in the food industries, such as a beverage
constituent and food acidulant [
85
]. Malic acid, as a low-calorie food additive, is used
in a variety of industries, including food, beverage, metal cleaning, pharmaceuticals and
plastics [
86
]. Malic and citric acids have antioxidant properties and are frequently used in
the food, agriculture, pharmaceutical and chemical industries [
83
]. Butyric acid is used to
produce butyric acid esters, cellulose butyrate, food and medicine, as well as serving as an
emulsifier, varnish and cosmetic [87].

Biology 2023,12, 411 15 of 21
Biology 2023, 12, x FOR PEER REVIEW 15 of 21
season, while fumaric acid was observed in minimum quantity in all the macroalgae
studied (Figure 5). Carpena et al. [83] also recorded the presence of several organic acids,
including malic, oxalic and citric acids, in the three seaweeds Chondrus crispus, Masto-
carpus stellatus and Gigartina pistillata. Tanna et al. [82] also reported that lactic and oxalic
acids were found in the green macroalgae Caulerpa scalpelliformis.
Figure 5. Seasonal variations in the concentrations (mg/g DW) of (A) Oxalic acid, (B) Malic acid, (C)
Succinic acid, (D) Citric acid, (E) Isobutyric acid and (F) Fumaric acid in macroalgae Caulerpa pro-
lifera, A. spicifera, Cystoseira myrica, Cystoseira trinodis and T. ornata. Values are shown as means ± S.E.
(n = 3). Different letters show significance between the two seasons of the same species (p < 0.05).
These detected organic acids are known for their high biological and medical values.
Succinic acid has high potential in many biological production processes, including food,
pharmaceutical, cosmetics, detergents and lubricants [84]. Fumaric acid is an intermedi-
ate of the TCA cycle, and it is generally used in the food industries, such as a beverage
constituent and food acidulant [85]. Malic acid, as a low-calorie food additive, is used in a
variety of industries, including food, beverage, metal cleaning, pharmaceuticals and
plastics [86]. Malic and citric acids have antioxidant properties and are frequently used in
the food, agriculture, pharmaceutical and chemical industries [83]. Butyric acid is used to
produce butyric acid esters, cellulose butyrate, food and medicine, as well as serving as
an emulsifier, varnish and cosmetic [87].
3.4. Secondary Metabolites
Phenolic Compounds
Phenolic compounds are a group of metabolites with the most structural variety and
the greatest concentration in macroalgae [88]. Phenolic compounds produced by sea-
weeds in the present study were assessed and quantified in the five macroalgal species
tested during the two seasons (summer and winter). The greatest levels of phenolic
compounds, such as polyphenols and flavonoids, were recorded in the summer season
for all macroalgal species studied. In contrast, tocopherols recorded a slight increase in
the winter (31.1 mg/g DW) compared to the summer season (29.4 mg/g DW) (Figure 6).
Figure 5.
Seasonal variations in the concentrations (mg/g DW) of (
A
) Oxalic acid, (
B
) Malic acid,
(
C
) Succinic acid, (
D
) Citric acid, (
E
) Isobutyric acid and (
F
) Fumaric acid in macroalgae Caulerpa pro-
lifera, A. spicifera, Cystoseira myrica, Cystoseira trinodis and T. ornata. Values are shown as means
±
S.E.
(n= 3). Different letters show significance between the two seasons of the same species (p< 0.05).
3.4. Secondary Metabolites
Phenolic Compounds
Phenolic compounds are a group of metabolites with the most structural variety and the
greatest concentration in macroalgae [
88
]. Phenolic compounds produced by seaweeds in
the present study were assessed and quantified in the five macroalgal species tested during
the two seasons (summer and winter). The greatest levels of phenolic compounds, such as
polyphenols and flavonoids, were recorded in the summer season for all macroalgal species
studied. In contrast, tocopherols recorded a slight increase in the winter (31.1 mg/g DW)
compared to the summer season (29.4 mg/g DW) (Figure 6).
The total phenolic content of macroalgae changes with seasonal variations in temper-
ature, salinity, light intensity, geographical region and water depth, in addition to other
biological factors, such as age, size, the stage of the seaweed’s life cycle and herbivores’
presence [
89
]. The greatest levels of phenolic compounds were found in the summer season.
Schiener et al. [
55
] concluded that the highest polyphenol quantity was observed between
May and July in all seaweeds tested, while the lowest quantity was found in October for
the Laminaria spp. and March for the Alaria esculenta and Saccharina latissima. Mancuso
et al. [
90
] proved an increase in the total phenolic content in brown seaweed Cystoseira
compressa as the water temperature rose. This may be attributed to greater light irradiance
during the spring season; the exposure of seaweeds to UV radiation promotes the forma-
tion of phenolic compounds to provide protection from oxidative stress [
91
]. Polyphenolic
compounds extracted from macroalgae exhibited antioxidant [92], anti-inflammatory and
antidiabetic [
93
], anticarcinogenic [
94
] and antimicrobial properties [
7
]. Moreover, these
compounds could be used in several industries and applications, generating innovative
products, such as natural food stabilizer, skin care and anti-aging cosmetic products [
32
,
88
].

Biology 2023,12, 411 16 of 21
Biology 2023, 12, x FOR PEER REVIEW 16 of 21
Figure 6. Seasonal variations in the concentrations (mg/g DW) of (A) TAC, (B) Polyphenol, (C)
Flavonoids and (D) Tocopherols in macroalgae Caulerpa prolifera, A. spicifera, Cystoseira myrica,
Cystoseira trinodis and T. ornata. Values are shown as means ± S.E. (n = 3). Different letters show
significance between the two seasons of the same species (p < 0.05).
The total phenolic content of macroalgae changes with seasonal variations in tem-
perature, salinity, light intensity, geographical region and water depth, in addition to
other biological factors, such as age, size, the stage of the seaweed’s life cycle and herbi-
vores’ presence [89]. The greatest levels of phenolic compounds were found in the
summer season. Schiener et al. [55] concluded that the highest polyphenol quantity was
observed between May and July in all seaweeds tested, while the lowest quantity was
found in October for the Laminaria spp. and March for the Alaria esculenta and Saccharina
latissima. Mancuso et al. [90] proved an increase in the total phenolic content in brown
seaweed Cystoseira compressa as the water temperature rose. This may be attributed to
greater light irradiance during the spring season; the exposure of seaweeds to UV radia-
tion promotes the formation of phenolic compounds to provide protection from oxida-
tive stress [91]. Polyphenolic compounds extracted from macroalgae exhibited antioxi-
dant [92], anti-inflammatory and antidiabetic [93], anticarcinogenic [94] and antimicro-
bial properties [7]. Moreover, these compounds could be used in several industries and
applications, generating innovative products, such as natural food stabilizer, skin care
and anti-aging cosmetic products [32,88].
There is variability in seaweeds’ phenolic content throughout the year, which rep-
resents the cellular defensive response, as well as prevents the attack of bacteria, micro-
algae, fungi, invertebrates, and enables survival in these difficult conditions [95]. The
strong antioxidant potential of macroalgae was attributed to the higher levels of antiox-
idant molecules, such as flavonoids, ascorbate, phenols and glutathione. Wang et al. [96]
reported that seaweed phenols have scavenging potential because of the presence of a
hydroxyl group, which is a remarkable constituent of seaweed.
In general, flavonoids are found in epidermal cells to absorb UV light; therefore,
their concentration is higher in the summer season, with high light intensity and duration
[97]. Water salinity decreases during the rainy season, lower salinity effects the bio-
Figure 6.
Seasonal variations in the concentrations (mg/g DW) of (
A
) TAC, (
B
) Polyphenol,
(
C
) Flavonoids and (
D
) Tocopherols in macroalgae Caulerpa prolifera, A. spicifera, Cystoseira myrica,
Cystoseira trinodis and T. ornata. Values are shown as means
±
S.E. (n= 3). Different letters show
significance between the two seasons of the same species (p< 0.05).
There is variability in seaweeds’ phenolic content throughout the year, which repre-
sents the cellular defensive response, as well as prevents the attack of bacteria, microalgae,
fungi, invertebrates, and enables survival in these difficult conditions [
95
]. The strong
antioxidant potential of macroalgae was attributed to the higher levels of antioxidant
molecules, such as flavonoids, ascorbate, phenols and glutathione. Wang et al. [
96
] reported
that seaweed phenols have scavenging potential because of the presence of a hydroxyl
group, which is a remarkable constituent of seaweed.
In general, flavonoids are found in epidermal cells to absorb UV light; therefore, their
concentration is higher in the summer season, with high light intensity and duration [
97
].
Water salinity decreases during the rainy season, lower salinity effects the biochemical
composition of macroalgae by reducing their phenolic content [
98
]. Marinho et al. [
99
]
recorded the same seasonal fluctuation pattern of flavonoids’ concentration in Saccharina
latissima. Seasonal environmental factors may result in a considerable difference in an-
tioxidant activity [
97
]. The total antioxidant activity (TAC) was enhanced in the summer
compared to the winter season in all studied macroalgal species.
4. Conclusions
Out of several macroalgae, Caulerpa prolifera (green macroalgae), Acanthophora spicifera
(red macroalgae) and Cystoseira myrica,Cystoseira trinodis and Turbinaria ornata (brown
macroalgae) were the most dominant species