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Chapter 3
Fatty acid profiling of macroalgae: its nutritional and
chemotaxonomy perspectives
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3.1. Introduction
Benthic marine macroalgae, commonly known as seaweeds are multicellular
photosynthetic organisms with considerable potentials for using as a source of bioactive
compounds of immense pharmaceutical and nutraceutical importance. They are rich sources
of nutritionally beneficial components such as proteins, carbohydrates, polyunsaturated fatty
acids (PUFAs), antioxidants, minerals, dietary fibers and vitamins (Chandini et al. 2008;
Mohamed et al. 2011) and are thus consumed as functional foods. There are 250 macroalgal
species commercially utilized world-wide, of which 150 are consumed as human food
(Barrow et al. 2007). The macroalgal species, in general, are low in lipids and contain 1 to
5% on dry wt. basis. Nevertheless, the nutritionally important C18 and C20 PUFAs
including n-3 PUFAs are present in substantially high amounts with anti-inflammatory, anti-
thrombotic and anti-arrhythmic responses (Kumari et al. 2010; Gillies et al. 2011). The n-3
PUFAs are of particular importance as they cannot be synthesized by humans and are thus
obtained only through dietary sources.
Fatty acids (FAs) being metabolites of conserved acetyl-CoA pathway have been
extensively studied from the context of chemotaxonomic perspectives in higher plants
(Mongrand et al. 2001, 2005; Dussert et al. 2008), cyanobacteria (Shukla et al. 2011),
bacteria (Malviya et al. 2011; Núñez-Cardona, 2012), microalgae (Dunstan et al. 2005; Lang
et al. 2011) and fungi (Mishra et al. 2010). Further, Dunstan et al. (2005) deciphered the
evolutionary relationship between the FA composition of Rhodophyceaen and
Cryptophyceaen microalgae and the endosymbiotic theory. According to the endosymbiotic
theory, the micro- and macro-algae of both Chlorophyceae and Rhodophyceae have been
originated from primary endosymbiosis of photosynthetic cyanobacteria and eukaryotic host
while Phaeophyta diverged from red algae via secondary (or tertiary) endosymbiosis along
with other chlorophyll c bearing algae such as Cryptophytes, Haptophytes, diatoms,
dinoflagellates and non-photosynthetic Apicomplexans, ciliates and oomycetes, forming the
super-group Stramenopiles at the base of the tree of life (Baldauf et al. 2000; Baldauf, 2008;
Archibald, 2009, 2012; Dorrell and Smith 2011; Baurain et al. 2012; De Clerck et al. 2012;
Green et al. 2010; Woehle et al. 2012). But, there is no consensus among the researchers
regarding the number of secondary endosymbiosis and whether the endosymbiotic event
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involved different red algae and/or different heterotrophic hosts including the number of
plastid losses in algae containing chlorophyll c (Green et al. 2010 Archibald 2012; Baurain
et al. 2012; De Clerck et al. 2012). Further, these endosymbiont cyanobacteria that exhibited
the capability of synthesizing C18 n-3 and n-6 PUFAs, finally resulted into double
membrane chloroplasts in green and red algae, the main sites of FA biosynthesis in
photosynthetic eukaryotes (Sperling, 2003). Similarly, the red algal endosymbiont formed 3-
4 membrane bound plastids in Stramenopiles. Numerous studies and available EST
databases of algae have revealed that the evolution of green, red and brown algae has
undergone multiple endosymbiotic and horizontal gene transfer of plastidial and nuclear
genomes, gene duplication and losses, including those of FA metabolism (Domergue et al.
2003; Ryall et al. 2003; Bowler et al. 2008; John et al. 2008; Le Corguillé et al. 2009;
Michel et al. 2010; Coelho et al. 2012; Chan et al. 2012; Chen and Smith 2012; Cock et al.
2010; Dittami et al. 2012). Further, it is well known that de novo FA biosynthesis takes
place in chloroplast up to C16:0/C18:0/C18:1, after which these FAs are attached to
phosphatidylcholine (PC). They are then either desaturated and utilized for the synthesis of
glycerolipids of chloroplast membrane or transported to ER for further chain elongation and
desaturation and thereafter utilized for the synthesis of membrane lipids, storage lipids or
transported back to the chloroplast for membrane biosynthesis (Ohlrogge and Browse, 1995;
Uttaro et al. 2006; Joyard et al. 2010; Huerlimann and Heimann, 2012). As cyanobacteria
can produce FAs only up to C18 (Liu et al. 2010a; Iliev et al. 2011), Dunstan et al. (2005)
hypothesized that Rhodophytes inherited the capability of synthesizing C20 PUFAs from the
coupling of elongase and desaturase enzyme system of the eukaryotic host with the
photosynthetic n-3 C18 PUFA-producing prokaryote (cyanobacteria) during the primary
endosymbiosis. Similarly, the secondary endosymbiosis of the photosynthetic n-3 C20
PUFA-producing eukaryote and the eukaryotic host capable of further chain elongation and
desaturation resulted in the de novo production of C22 PUFAs along with C20 PUFAs in
Cryptophytes. However, a few studies are available where FA composition of macroalgae
has been investigated in correlation to the endosymbiotic history. Understanding the FA
profiles in the light of endosymbiotic evolution of different lineages of macroalgae could be
an important prerogative for gaining insights into the algal lipid metabolism.
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Furthermore, FA compositions of numerous macroalgae have been reported world-
wide for their nutritional potential but their chemotaxonomic implications gained
importance only in the last decade. Recently, Galloway et al. (2012) reported FA signatures
of 40 temperate macrophytes (both seaweeds and sea grasses) from San Juan Archipelago,
USA. But the diversity of macroalgal species investigated for FA composition is still low,
approximated to be < 200 (Gosch et al. 2012) which is minuscule against total estimated
species of 9255, to date (Guiry and Guiry, 2012)
In the present study, we analyzed total lipid (TL) and FA compositions of 100
macroalgae belonging to 46 different genera, 30 families and 18 orders encompassing
samples from northern and central west coast of India. An attempt was made to understand
their chemotaxonomic relationships at different taxonomic levels using multivariate
principal component and hierarchical cluster analysis and compared with the available
phylogenetic data. Furthermore, the results were also correlated with the endosymbiotic
origin of three extant macroalgal lineages besides mapping the algal species rich in
nutritionally essential n-3 PUFAs that could possibly be cultivated for utilizing in various
food and nutraceutical formulations.
3.2. Materials and Methods
3.2.1. Chemicals
For the identification and quantification of FAs, following analytical grade standards
were used: 37- component FAME Mix C4-C24 (Supelco, USA), 7-hexedecenoic acid
methyl ester (C16:1, n-7) and stearidonic acid methyl ester (C18:4, n-3) (Cayman chemicals,
USA). The internal standard nonadecanoic acid (C19:0) was purchased from Sigma. All the
solvents used (such as chloroform, methanol, dichloromethane and hexane) were of HPLC
grade and other reagents of analytical grade.
3.2.2. Algal samples
Macroalgal samples were collected in triplicates during low tide periods during
March-October 2011 from different sites along the Gujarat coast, India. The species of
Pyropia and Monostroma were collected during August, 2010 from Maharashtra coast,
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India. Fully grown thalli of each alga which were inundated below the small layer of water
(about 0.5-2.0 cm) were chosen for this investigation. A detailed list of macroalgal samples
along with the description of their habitat and the geographical co-ordinates are presented in
Appendix I. The samples were wrapped in wet tissue towels and transported to the
laboratory in cool conditions in an ice box. They were immediately cleaned thoroughly with
autoclaved seawater to remove the epiphytes and other undesired foreign matter from the
surface of algal fronds. The cleaned fronds were then blotted with tissue paper, weighed to
determine the fresh weight, thereafter, 0.5 g of each sample was immediately frozen in
liquid nitrogen in triplicate and stored at -40 °C, until the analysis commenced.
3.2.3. Lipid extraction, fatty acid methyl esters preparation and GC-MS analysis
Lipids were extracted by modified Bligh and Dyer method using chloroform-
methanol-phosphate buffer (pH-7.5) (1/2/0.8; v/v/v), as described in Chapter 2. The fatty
acids were converted to their fatty acid methyl esters (FAMEs) by transmethylation of lipid
samples with 1ml of 1% NaOH in methanol, followed by heating for 15 min at 55°C,
adding 2 ml of 5% methanolic HCl and again heated for 15 min at 55°C then adding 1ml
milli-Q water (Carreau and Dubacq, 1978). Nonadecanoic acid was used as internal standard
and FAMEs were extracted in hexane.
The GC-MS analysis of FAME samples was carried out on a QP2010 gas chromatography
mass spectrometer (GC-2010 coupled with GC-MS QP-2010) equipped with an autosampler
(AOC-5000) from Shimadzu (Japan) using a RTX-5 fused silica capillary column, 30 m x
0.25 mm x 0.25 µm (Rastek). Helium (99.9% purity) was used as the carrier gas with the
column flow rate of 1 ml min-1 and the pre-column pressure of 49.7 KPa. The column
temperature regime was 40 °C for 3 min, followed by a 5 °C min-1 ramp up to 230 °C
followed by 40 min at 230 °C. The injection volume and temperature was 0.2 µl and 240 °C
and the split ratio was 1/30. The mass spectrometer was operated in electron compact mode
with electron energy of 70eV. Both the ion source temperature and the interface temperature
were set at 200 °C. FAME peaks were identified by comparison of their retention times with
authentic standards by GC-MS Post run analysis and quantified by area normalization.
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3.2.4. Indices
The unsaturation index (U.I.) was calculated by multiplying the percentage of each
fatty acid by the number of double bonds followed by summing up their contributions
(Poerschmann et al. 2004). Atherogenic and thrombogenic indices (AI and TI) were
calculated according to De Lorenzo et al. (2001), where:
AI = (C12:0 + C14:0 + C16:0)/(n-3 PUFAs + n-6 PUFAs + MUFAs), and
TI = (C14:0 + C16:0 + C18:0)/(0.5n-6 PUFAs + 3n-3PUFAs + n3/n-6 PUFAs)
3.2.5. Statistical and Multivariate analysis
All analytical determinations were performed in triplicate and the mean values were
recorded. The percentages of fatty acids were compared by analysis of variance (ANOVA)
with values significant at p ≤ 0.001. The multivariate analyses were performed using
software Unscrambler X (Camo, USA) to establish the relationship between and within
different orders and families belonging to the three phyla and to evaluate the distribution of
essential FAs in macroalgal samples. Three principal component analyses (PCA) were
performed separately on FA, FA groups and PUFA + nutritional indices data matrices of 100
macroalgal samples. FA data matrix included all the FAs reported in Appendix III with the
exception of dodecanoic acid (C12:0), pentadecanoic acid (C15:0), heptadecanoic acid
(C17:0), 10-heptadecenoic acid (C17:0, n-7), eicosanoic acid (C20:0), 11-eicosenoic acid
(C20:1, n-9), docosanoic acid (C22:0), 13-docosenoic acid (C22:1, n-9) and tetradocosanoic
acid (C24:0) due to their insignificant amounts and lack of correlation with the data matrix
which otherwise might led to misclassification of species. Such variables could also increase
the number of outliers owing to their higher contribution to the score/loading values.
Further, FA groups’ data matrix included seven variables; SFA, MUFA, PUFA, C18 PUFA,
C20 PUFA, n-3 PUFAs and n-6 PUFAs and PUFA + nutritional indices data matrix
included LA, ALA, GLA, STA, AA, EPA, DHA, PUFA/SFA, UI, AI and TI. In addition, a
dendrogram was obtained by Ward hierarchical clustering, using squared Euclidean distance
(Ward, 1963) described by the first 8 principal components with eigen-values >1. Ward
linkage is an agglomerative clustering algorithm which starts with n singleton clusters (each
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consisting of one element of the data set) and merges two clusters on the basis of a similarity
measure.
3.3. Result
3.3.1. Lipids
The total lipid (TL) contents investigated in this study varied significantly (p ≤ 0.001)
among different algal species (Table 3.1). The brown algae showed the highest TL contents
(5.7-20.1 mg g-1 FW), followed by green algae (3.1-20 mg g-1 FW) and red algae (2.3-12.2
mg g-1 FW). The species belonging to Ulvales (Ulva erecta), Bryopsidales (except Caulerpa
microphysa, C. racemosa v. occidentalis), Ulotrichales, Dictyotales and Sargassum
cinereum showed high contents of TL (10-20 mg g-1 FW). The variations observed between
different species of the same genus was more likely to be due to the inter-specific/intra-
generic variations rather than the geographical and environmental conditions as apparent
from the minor variations found in the environmental parameters for the studied collection
sites. Also, there was no trend observed between TL contents of macroalgal species
belonging to the same genus and collection sites.
3.3.2. Fatty acid composition
Chlorophyta
The FA compositions of 33 species belonging to the orders of Ulvales, Ulotrichales,
Bryopsidales, Siphonocladales and Cladophorales are presented in Table 3.2 and Appendix
III. The green algal samples showed higher contents of unsaturated fatty acids (UFAs) with
the exception of Ulva lactuca, C. racemosa v. occidentalis, C. racemosa v. corneyphora,
Codium dwarkense, Bryopsis plumosa and Monostroma oxyspermum which had 1.1-1.4-fold
higher contents of saturated fatty acids (SFAs) (p ≤ 0.001). The PUFA contents in them
ranged between 28% (U. lactuca) and 71% (C. veravalensis) of total FAs (TFAs). The major
FAs detected in Chlorophyta members were myristic (C14:0), palmitic (C16:0), stearic
(C18:0), palmitoleic (C16:1, n-9), oleic (C18:1, n-9), linoleic (C18:2, n-6; LA), α-linolenic
(18:3, n-3; ALA) and stearidonic acid (C18:4, n-3; STA) that collectively contributed to
58.7%-88.9% of TFAs pool. Interestingly, no trans FAs were encountered in any green
algae.
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Table 3.1 Total lipid content of macroalgal samples (means ± S.D.; n=3). The lipid values for different algae
were significant at p ≤ 0.001.
S.No. Species TL (mg g-1 FW) S.No. Species TL (mg g-1 FW)CHLOROPHYTA 52 Sargassum cinctum 9.8 ± 1.3 Ulvales 53 Hormophysa cuneiformis 11 ± 3.0
1 Ulva lactuca 15.7 ± 0.8 54 Cystoseira indica 6.1 ± 0.4 2 Ulva fasciata 13.7 ± 0.8 55 Cystoseira trinodis 6.7 ± 2.5 3 Ulva taeniata 16 ± 1.0 Ectocarpales 4 Ulva pertusa 14 ± 4.0 56 Hincksia mitchelliae 12.4 ± 1.1 5 Ulva reticulata 10 ±2.0 57 Scytosiphon lomentaria 6.3 ±0.2 6 Ulva beytensis 20 ±2.0 RHODOPHYTA 7 Ulva compressa 9.2 ±1.0 Gracilariales 8 Ulva rigida 16.3±1.4 58 Gracilaria dura 6.3 ± 0.3 9 Ulva linza 16 ± 0.8 59 Gracilaria salicornia 7.6± 0.5 10 Ulva flexuosa 13.8±1.3 60 Gracilaria textorii 7.3 ± 1.2 11 Ulva erecta 7.0 ± 0.2 61 Gracilaria corticata 8 ± 2.0 12 Ulva prolifera 14.7 ± 0.2 62 Gracilaria corticata v. cylindrica 5.2 ± 1.1
Bryopsidales 63 Gracilaria corticata v. folifera 5.7 ± 0.6 13 Caulerpa scalpelliformis 13.3 ± 1.5 64 Gracilaria debilis 2.9 ± 0.2 14 Caulerpa veravalensis 16 ± 0.3 65 Gracilaria verrucosa 9.7 ± 0.6 15 Caulerpa racemosa 15 ± 0.8 Gigartinales 16 Caulerpa racemosa v. corynephora 10.3 ± 2.1 66 Sarconema scinaioides 4.3 ± 0.8 17 Caulerpa racemosa v. occidentalis 5.5 ± 1.1 67 Sarconema filiforme 9.8 ± 0.7 18 Caulerpa microphysa 4.7 ± 1.3 68 Hypnea valentiae 5.8 ± 0.8 19 Caulerpa verticillata 15.3 ± 1.3 69 Hypnea musciformis 7 ± 0.12 20 Caulerpa sertularioides 9.1 ± 2.2 70 Hypnea spinella 7.8 ± 0.7 21 Codium dwarkense 14 ± 2.2 71 Solieria robusta 9 ± 0.6 22 Bryopsis pennata 9.6 ± 1.7 Bangiales 23 Bryopsis plumosa 11.3 ± 1.5 72 Pyropia tenera 7.7 ± 0.5 24 Trichosolen mucronatus 5.4 ± 1.1 73 Pyropia yoezensis 7.0 ± 0.2 25 Udotea indica 12 ± 4.7 74 Pyropia acanthophora 7.3 ± 0.3 26 Halimeda discoides 3.1 ± 0.4 75 Pyropia acanthophora v. brasilensis 7.3 ± 0.5 27 Halimeda tuna 10 ± 1.7 76 Pyropia sp. 7.3 ± 0.4
Ulotrichales Rhodymeniales 28 Monostroma oxyspermum 17.3 ± 1.2 77 Rhodymenia sonderi 7.1 ± 1.2
Siphonocladales 78 Coelarthrum muelleri 7.7 ± 0.6 29 Chamaedoris auriculata 8 ± 2.0 79 Botryocladia leptopoda 5.2 ± 2.0 30 Cladophoropsis javanica 10.6 ± 1.3 80 Botryocladia botryoides 2.3 ± 0.4 31 Valoniopsis pachynema 6.1 ± 0.4 81 Gastroclonium iyengarii 4.3 ± 0.6
Cladophorales 82 Champia parvula 9.3 ± 2.7 32 Chaetomorpha linum 4.8 ± 0.2 83 Gelidiopsis variabilis 5.5 ± 0.6 33 Acrosiphonia orientalis 10.8 ± 2.2 Gelidiales
PHAEOPHYTA 84 Gelidiella acerosa 6.7 ± 1.0 Dictyotales Halymeniales
34 Padina tetrastomatica 13.3 ± 2.1 85 Cryptonemia undulata 11.3 ± 1.6 35 Padina gymnospora 17.7 ± 0.6 86 Halymenia porphyraeformis 10 ± 1.7 36 Dictyopteris deliculata 9.3 ± 0.6 87 Grateloupia indica 6.4 ± 0.1 37 Dictyota pinnatifida 13.3 ± 1.2 88 Grateloupia filicina 5 ± 0.9 38 Dictyota bartayresiana 10 ± 2.2 Ceramiales 39 Dictyota dichotoma 10.3 ± 1.0 89 Odonthalia veravalensis 11.4 ± 4.4 40 Dictyota cervicornis 7.3 ± 0.1 90 Acanthophora specifera 6.8 ± 0.5 41 Dictyota ciliolata 13.4 ± 0.9 91 Acanthophora nayadiformis 10.4 ± 1.1 42 Dictyota haukiana 8.4 ± 2.1 92 Laurencia cruciata 8.2 ± 1.2 43 Stoechospermum marginatum 11.5 ± 1.1 93 Laurencia obstusa 5.4 ± 0.4 44 Lobophora variegata 15 ± 1.6 94 Laurencia papillosa 9.3 ± 0.5 45 Spatoglossum asperum 13.8 ± 0.7 95 Laurencia majusculus 8 ± 1.5
Fucales 96 Laurencia sp. 6.6 ± 1.8 46 Sargassum tenerrimum 7.2 ±1.1 97 Polysiphonia ferulacea 9.6 ± 2.8 47 Sargassum johnstonii 7.3 ±1.2 98 Griffithsia corallinoides 4.2 ± 1.3 48 Sargassum sp. 5.7 ± 1.5 Corallinales 49 Sargassum carpophyllum 6.3 ± 0.6 99 Jania rubens 12.2 ± 1.4 50 Sargassum plagiophyllum 7.6 ± 2.6 100 Scinaia monoliformis 5.2 ± 0.4 51 Sargassum cinereum 20 ± 1.0
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Table 3.2 Fatty acid groups (% of total fatty acid methyl esters; TFAs) and nutritional indices in different
macroalgal species expressed as means ± SD (n=3).
Macroalgae SFAs MUFAs PUFAs PUFA/SFA n6/n3 U.I. AI TI Ulva lactuca 59.9±2.3 12.2±1.8 28.0±0.7 0.47±0.03 0.3±0.01 104.1±2.8 1.1±0.06 0.8±0.03 Ulva fasciata 29.6±2.1 7.5±0.9 63.1±1.9 2.14±0.2 0.2±0.01 211.4±5.2 0.4±0.03 0.2±0.02 Ulva taeniata 46.8±7.1 5.4±1.0 48±7.8 1.06±0.3 0.4±0.1 159.7±27 0.7±0.2 0.4±0.11 Ulva pertusa 35.5±2.3 9.4±0.4 55.2±2.1 1.56±0.15 0.2±0.01 195±6.4 0.4±0.05 0.2±0.02 Ulva reticulata 48.8±5.9 7.1±0.7 44.8±5.9 0.94±0.22 0.3±0.2 159.4±28 0.8±0.15 0.4±0.17 Ulva beytensis 32.3±0.8 10.9±0.8 56.9±0.2 1.76±0.05 0.1±0.1 201.6±6.1 0.4±0.01 0.2±0.01 Ulva compressa 37.6±0.6 8.3±0.2 54.2±0.8 1.44±0.04 0.4±0.1 173.3±3.2 0.5±0.01 0.3±0.01 Ulva rigida 37.8±5.0 8.1±4.0 54.2±8.9 1.47±0.44 0.2±0.2 191.8±37 0.5±0.09 0.3±0.11 Ulva linza 38.4±9.9 6.0±0.8 55.6±9.1 1.54±0.56 0.2±0.01 193±35 0.6±0.25 0.3±0.12 Ulva flexuosa 37.3±0.5 7.5±0.4 55.2±0.6 1.48±0.03 0.3±0.01 170±1.9 0.5±0.02 0.3±0.01 Ulva erecta 39.2±1.5 7.1±2.4 53.7±0.9 1.37±0.03 0.5±0.1 167±6.9 0.6±0.03 0.3±0.01 Ulva prolifera 39.1±0.9 9.5±0.8 51.4±0.9 1.31±0.05 0.6±0.1 164.3±6.9 0.5±0.02 0.3±0.01 Caulerpa scalpelliformis
39.6±1.0 6.4±0.1 54±1.0 1.36±0.06 0.4±0.1 177.5±3 0.5±0.02 0.3±0.01
Caulerpa veravalensis 27.1±1.0 1.9±0.2 71.1±0.9 2.63±0.13 0.3±0.01 226±2.6 0.4±0.02 0.2±0.01 Caulerpa racemosa 33.4±3.1 5.7±1.4 60.8±3.0 1.84±0.24 0.2±0.01 196.6±11 0.5±0.08 0.2±0.02 Caulerpa racemosa v. corynephora
58.2±6.4 2.9±0.4 39.1±6.0 0.68±0.17 0.6±0.1 128.3±18 1.2±0.3 0.6±0.12
Caulerpa racemosa v. occidentalis
n. d. 10.2±5 36.5±3 0.68±0.05 0.8±0.3 122.1±7 1.0±0.13 0.7±0.1
Caulerpa microphysa 40.3±2.0 5.2±0.3 54.6±1.9 1.36±0.12 0.6±0.01 176.7±6.1 0.5±0.05 0.3±0.03 Caulerpa verticillata 44±2.7 6.1±3.0 50±5.3 1.14±0.18 1.0±0.3 172.6±6 0.7±0.05 0.4±0.11
Caulerpa sertularioides 41.4±0.9 2.2±0.6 56.8±1.1 1.38±0.06 0.5±0.1 176.9±3 0.6±0.02 0.3±0.02 Codium dwarkense 65.4±2.6 6.6±0.1 28±2.6 0.43±0.06 1.1±0.1 91±7.8 1.6±0.22 1.3±0.23 Bryopsis pennata 42.1±6.2 6.5±2.6 51.5±7.9 1.26±0.36 0.6±0.2 166±26 0.6±0.17 0.4±0.15 Bryopsis plumosa 51.5±4.4 8.2±1.1 40.4±3.4 0.79±0.13 0.5±0.1 137±11.6 0.9±0.12 0.5±0.06 Trichosolen mucronatus 49.3±10.8 8.7±0.2 42.3±10.5 0.91±0.37 1.9±1.6 144.3±33 0.9±0.41 0.9±0.8 Udotea indica 43.5±3.3 5.7±2.1 50.9±5.4 1.18±0.21 0.3±0.01 191.3±8.8 0.7±0.09 0.3±0.05 Halimeda discoides 46.1±2.2 6.1±2.3 47.9±0.7 1.04±0.05 0.6±0.2 178±19.3 0.7±0.12 0.4±0.07 Halimeda tuna 41.9±0.9 7.9±1.3 50.2±1. 1.20±0.06 0.8±0.1 198.6±7.0 0.6±0.02 0.4±0.02 Monostroma oxyspermum 55.1±10 3.5±2.6 41.6±7.4 0.79±0.27 0.2±0.1 133.8±27 0.8±0.26 0.5±0.16 Chamaedoris auriculata 43.9±3.2 13.7±1.0 42.6±3.4 0.98±0.15 0.8±0.01 163±10 0.7±0.11 0.5±0.08 Cladophoropsis javanica 45.7±1.4 9.4±1.2 45.1±1.3 0.99±0.05 1.8±0.7 152±4.9 0.7±0.09 0.6±0.17 Valoniopsis pachynema 46.4±1.4 15.7±5.1 38±6.4 0.82±0.16 1.6±0.4 141.2±18 0.8±0.08 0.8±0.15 Chaetomorpha linum 37.5±3.5 21.9±5.5 40.7±3.3 1.09±0.11 1.3±0.9 162.9±12 0.5±0.05 0.5±0.17 Acrosiphonia orientalis 41.7±4.8 8.7±0.7 49.8±5.6 1.21±0.28 1.4±0.5 161.3±18 0.7±0.14 0.5±0.17 Padina tetrastomatica 46.5±0.4 14.9±0.6 38.7±1.0 0.83±0.03 1.2±0.3 149.8±5.9 0.8±0.12 0.7±0.09 Padina gymnospora 33.8±0.2 16.9±0.7 49.3±0.9 1.46±0.04 0.9±0.01 193.7±2.0 0.4±0.01 0.3±0.01 Dictyopteris deliculata 50.6±2.8 11±1.2 38.7±3.1 0.77±0.1 2.4±0.2 145.7±9.4 0.9±0.08 1.0±0.05 Dictyota pinnatifida 55.8±3.4 11.3±1.3 33.0±2.3 0.60±0.07 3.9±1.8 133.3±9.9 1.1±0.2 1.5±0.51 Dictyota bartayresiana 28.9±1.0 13.1±0.6 58.0±4.0 2.01±0.08 0.6±0.01 241.8±5.6 0.3±0.02 0.2±0.01 Dictyota dichotoma 39.0±9.1 16.6±5.8 44.4±14.9 1.23±0.59 0.9±0.4 187.2±53 0.6±0.25 0.5±0.46 A: LSD values obtained from ANOVA. The values in a column are significantly different at p ≤ 0.001; Note: SFAs: Saturated fatty acids, MUFAs: Monounsaturated fatty
acids, PUFAs: Polyunsaturated fatty acids, U.I.: Unsaturation index, AI: Atherogenic index, TI: Thrombogenic index
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Macroalgae SFAs MUFAs PUFAs PUFA/SFA n6/n3 U.I. AI TI Dictyota cervicornis 36.5±1.9 12.1±0.7 51.4±2.7 1.41±0.15 0.9±0.01 207±9.5 0.4±0.06 0.3±0.04 Dictyota ciliolata 39.9±0.9 12.0±0.4 48.2±0.6 1.21±0.04 0.7±0.01 184.6±3.5 0.6±0.01 0.4±0.02 Dictyota haukiana 34.2±1.7 22.2±0.7 43.7±1.9 1.28±0.11 0.4±0.01 197.4±5.8 0.5±0.03 0.3±0.03 Stoechospermum marginatum
59.3±8.7 17.2±1.9 23.6±8.6 0.42±0.2 2.8±0.7 95.2±2.7 1.4±0.42 2.4±1.6
Lobophora variegata 48.3±1.2 12.9±0.9 39.2±1.9 0.81±0.06 0.8±0.1 175.3±7.1 0.8±0.05 0.6±0.06 Spatoglossum asperum 51.1±3.4 25.4±2.1 23.7±1.5 0.47±0.06 1.3±0.1 117.6±8.1 1.0±0.13 1.3±0.14 Sargassum tenerrimum 49.5±1.0 17.6±0.6 33±1.4 0.67±0.04 2.7±0.2 141±5.0 0.9±0.01 1.2±0.1 Sargassum johnstonii 46.3±4.4 15.2±0.3 38.5±4.2 0.84±0.16 1.6±0.3 166±18.3 0.7±0.15 0.7±0.22 Sargassum sp. 49.6±0.6 18.2±0.5 32.4±0.2 0.65±0.01 2.3±0.1 137.8±0.9 0.8±0.03 1.1±0.03 Sargassum carpophyllum 52.4±10.4 11.3±1.3 36.4±9.1 0.73±0.29 1.3±0.1 147±36.2 0.9±0.31 0.9±0.4 Sargassum plagiophyllum 42.8±1.9 13.0±0.4 44.7±2.2 1.05±0.1 1.3±0.1 185±8.4 0.6±0.05 0.6±0.06 Sargassum cinereum 43.2±6.4 32.9±8.7 24.1±2.5 0.56±0.02 0.5±0.1 139±3.0 0.7±0.2 0.7±0.11 Sargassum cinctum 35.9±14.7 18.5±4.4 45.7±10.5 1.61±1.25 2.1±0.1 186.4±44 0.5±0.33 0.6±0.35 Hormophysa cuneiformis 40.7±1.4 15.9±1.5 43.5±0.7 1.07±0.04 7.4±0.4 152.3±3.0 0.5±0.02 1.1±0.07 Cystoseira indica 47.0±7.4 14.0±1.4 38.9±8.6 0.86±0.29 1.6±0.2 159±35.1 0.8±0.26 0.8±0.34 Cystoseira trinodis 39.1±1.8 12.6±0.6 48.4±1.7 1.24±0.1 1.7±0.2 183.7±7.9 0.6±0.04 0.5±0.06 Hincksia mitchelliae 35.7±1.5 12.4±1.6 51.9±3.0 1.46±0.06 0.5±0.01 221±1.0 0.5±0.02 0.3±0.01 Scytosiphon lomentaria 59.7±1.8 10.5±1.6 29.8±0.2 0.50±0.02 1.4±0.3 122.4±6.4 0.2±0.01 1.2±0.12 Gracilaria dura 33.2±1.1 4.1±0.5 62.8±1.2 1.89±0.1 194.7±41 249±3.6 0.4±0.03 1.0±0.07 Gracilaria salicornia 78.2±0.5 10.6±0.2 11.5±0.8 0.15±0.01 ∞ 50.6±1.3 2.9±0.14 11.9±1.0 Gracilaria textorii 60.0±4.4 11.5±1.6 28.6±2.9 0.48±0.08 ∞ 105.8±15.1 1.3±0.21 3.9±0.61 Gracilaria corticata 31±2.5 3.5±0.1 65.6±2.5 2.13±0.25 ∞ 257±10 0.4±0.05 0.9±0.11 Gracilaria corticata v. cylindrica
61±2.5 9.3±1.1 29.8±2.8 0.49±0.06 42±17.4 118.4±12.4 1.3±0.14 3.4±.11
Gracilaria corticata v. folifera
62.7±3.0 5.1±0.6 32.3±3.4 0.52±0.08 8.7±2.7 125.3±9.8 1.5±0.15 2.6±1.03
Gracilaria debilis 40.1±3.6 11.7±4.5 48.4±3.4 1.21±0.15 59.2±29 194.5±11.3 0.6±0.1 1.5±0.16 Gracilaria verrucosa 46.6±3.2 9.2±1.6 44.3±4.1 0.96±0.15 0.6±0.01 205.3±21 0.8±0.1 0.5±0.08 Sarconema scinaioides 64.7±3.1 17.9±3.8 17.6±0.8 0.27±0.01 2.5±2.0 91.4±1.7 1.4±0.2 2.7±0.8 Sarconema filiforme 39.9±8.5 19.7±9.3 40.4±2.0 1.04±0.23 2.4±0.6 134.5±12.6 0.4±0.07 0.7±0.22 Hypnea valentiae 59.0±6.0 18.2±1.3 23.2±5.2 0.40±0.12 0.8±0.01 118.2±25.0 1.4±0.35 1.3±0.5 Hypnea musciformis 65.1±1.7 15±1.8 20±2.7 0.31±0.05 1.2±0.5 91.6±12.4 1.7±0.12 1.9±0.56 Hypnea spinella 65.7±1.5 6.2±1.1 28.2±2.1 0.43±0.04 16±4.6 98.1±3.9 1.6±0.15 3.3±0.27 Solieria robusta 61.4±1.3 17.4±0.7 21.3±2.1 0.35±0.04 0.8±0.01 113.9±9.0 1.2±0.08 1.4±0.2 Pyropia tenera 34.3±1.0 5.4±1.1 60.4±2.3 1.76±0.08 0.7±0.1 272±5.2 0.4±0.03 0.4±0.05 Pyropia yoezensis 37.2±2.7 10.1±1.4 52.9±2.8 1.43±0.18 1.2±0.2 232±13.7 0.4±0.05 0.3±0.01 Pyropia acanthophora 35.9±2.1 12.9±1.2 51.3±3.1 1.36±0.14 1.2±0.1 226±5.8 0.5±0.03 0.4±0.03 Pyropia acanthophora v. brasilensis
41.2±5.0 9.2±3.8 49.6±8.2 1.23±0.32 1.3±0.2 213.4±35.6 0.6±0.12 0.5±0.18
Pyropia sp. 37.7±1.1 9.4±2.1 53±3.0 1.46±0.12 1.4±0.1 224±13.0 0.5±0.18 0.4±0.03 Rhodymenia sonderi 49.1±2.2 8.0±0.8 42.9±1.6 0.87±0.07 88.2±9.4 168±25.7 0.5±0.04 0.01±0.01Coelarthrum muelleri 50.4±6.2 16.6±8.3 33.6±2.0 0.67±0.05 5.7±0.8 151±3.3 0.6±0.07 0.2±0.03 Botryocladia leptopoda 56.6±2.3 15.9±0.9 27.7±2.4 0.49±0.06 1.7±0.2 121±12.3 1.1±0.12 0.6±0.06 Botryocladia botryoides 47.7±0.5 26.6±0.7 25.8±0.3 0.54±0.01 3.6±0.7 122±3.9 0.9±0.06 0.3±0.06 A: LSD values obtained from ANOVA. The values in a column are significantly different at p ≤ 0.001; Note: SFAs: Saturated fatty acids, MUFAs: Monounsaturated fatty
acids, PUFAs: Polyunsaturated fatty acids, U.I.: Unsaturation index, AI: Atherogenic index, TI: Thrombogenic index
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67
Macroalgae SFAs MUFAs PUFAs PUFA/SFA n6/n3 U.I. AI TI Gastroclonium iyengarii 57.1±4.7 9.9±1.7 33.3±3.0 0.59±0.1 5.1±1.4 94±12.3 0.7±0.05 0.2±0.02 Champia parvula 59.7±1.5 17.4±1.6 23±1.0 0.39±0.01 1.0±0.1 105±3.6 1.5±0.06 1.2±0.07 Gelidiopsis variabilis 47.4±4.7 13.3±1.7 39.4±6.4 0.84±0.21 0.8±0.5 182±38 0.8±0.11 0.6±0.27 Gelidiella acerosa 44.6±1.8 11.7±2.1 43.8±3.2 0.98±0.06 0.6±0.1 204±9.2 0.7±0.03 0.5±0.07 Cryptonemia undulata 32.9±2.4 25.3±1.3 41.9±1.4 1.28±0.13 18.8±6.2 185.3±5.7 0.4±0.05 1.2±0.06 Halymenia porphyraeformis 39.2±5.0 8.1±2.5 52.7±4.2 1.37±0.27 1.7±0.7 225±26.2 0.6±0.12 0.5±0.18 Grateloupia indica 49.9±0.7 13.1±1.0 37.2±1.2 0.74±0.03 1.9±0.2 166.3±5.9 0.9±0.02 0.9±0.07 Grateloupia filicina 47.2±3.0 10.8±0.8 42.2±3.2 0.90±0.13 0.5±0.2 195.4±21.3 0.8±0.08 0.5±0.1 Cryptonemia undulata 46.3±0.9 13.7±4.1 40.1±3.5 0.86±0.07 0.9±0.3 187±14.1 0.8±0.04 0.6±0.1 Odonthalia veravalensis 51.2±3.0 12.1±1.3 36.7±3.2 0.72±0.1 0.6±0.1 171±14.6 0.9±0.1 0.6±0.1 Acanthophora specifera 49.7±4.9 4.5±0.8 46.1±4.1 0.94±0.18 0.9±0.2 212.6±17.0 1.0±0.18 0.6±0.07 Acanthophora nayadiformis 50±3.8 11.7±2.1 38.9±5.4 0.79±0.16 1.8±0.1 171.6±19.5 0.8±0.08 0.9±0.0 Laurencia cruciata 60.7±1.4 11.7±0.3 27.8±1.7 0.46±0.04 1.2±0.3 134±7.7 1.6±0.73 1.2±0.16 Laurencia obstusa 48.4±3.4 14.7±2,1 37.0±15 0.77±0.08 1.1±0.2 169.6±9.2 0.8±0.08 0.7±0.1 Laurencia papillosa 62±9.9 13.7±3.9 24.4±7.2 0.41±0.18 1.6±0.3 115.3±32.1 1.6±0.73 1.7±0.71 Laurencia majusculus 74.5±7.7 15.1±1.7 10.6±0.4 0.14±0.01 1.7±0.1 56.6±2.2 2.7±0.27 4.6±0.2 Laurencia sp. 51.5±1.4 14.2±1.3 34.4±1.5 0.67±0.04 0.5±0.1 157.8±4.9 0.9±0.04 0.6±0.03 Polysiphonia ferulacea 52.5±2.8 20.0±2.3 27.6±2.2 0.53±0.06 1.1±0.2 123±10.5 0.9±0.08 1.0±0.15 Jania rubens 65.8±5.8 13.1±2.8 21.2±2.4 0.32±0.03 2.9±0.5 90.7±12.1 1.7±0.09 2.6±0.4 Scinia monoliformis 65.6±5.4 19.3±6.9 15.2±1.6 0.23±0.01 1.1±0.01 73.3±2.1 1.5±0.4 2.1±0.13 LSDA 11.9 7.1 11.8 0.45 17.5 46.1 0.44 0.84 A: LSD values obtained from ANOVA. The values in a column are significantly different at p ≤ 0.001; Abbreviations: SFAs: Saturated fatty acids, MUFAs:
Monounsaturated fatty acids, PUFAs: Polyunsaturated fatty acids, U.I.: Unsaturation index, AI: Atherogenic index, TI: Thrombogenic index
This study revealed that all the species exhibited the characteristic profile of C18>C20
PUFAs, except H. tuna. The ratio of different C18 PUFAs (LA, ALA and STA) further
revealed that the levels of ALA exceeded the contents of LA and STA taken together in all
the species except those of Trichosolen, Halimeda, Cladophoropsis, Valoniopsis,
Chaetomorpha and Acrosiphonia. Docosahexaenoic acid (C22:6, n-3; DHA), was present in
almost all the samples (0.4%-7.1% of TFAs) except Bryopsis spp., Trichosolen mucronatus
and C. racemosa v. occidentalis. Moreover, members of the same genus exhibited similar
FA patterns but differed significantly in their individual FA contents (p ≤ 0.001). All the
members of Ulvales had characteristic FA profiles of high palmitic, oleic, C18 PUFAs with
ALA>LA (2-9.8 times) and low C20 PUFAs. STA was found to be another characteristic of
Ulva spp. varying from 3.6%-20% of TFAs. In contrast, the members of Bryopsidales,
Siphonocladales and Cladophorales showed 2.0-6.4-fold higher C20 PUFAs than those of
Ulvales and Ulotrichales.
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Rhodophyta
The FA compositions of 43 taxa belonging to the orders of Gracilariales,
Gigratinales, Bangiales, Rhodymeniales, Gelidiales, Halymeniales, Ceramiales and
Corallinales are presented in Table 3.2 and Appendix III. Of the 43 species, 24 species had
higher UFAs while 19 had higher SFAs with UFA/SFA ratio varying from 1.0 (Rhodymenia
sonderii, Coelarthrum muelleri, Grateloupia indica, Acanthophora spp. and Laurencia
papillosa) to 2.2 (Gracilaria corticata). Only 13 species showed higher PUFAs with
65.6±2.5 % of TFAs being the highest in G. corticata followed by 60.4±2.3% in Pyropia
tenera, due to higher AA and EPA contents respectively. All the species showed
characteristic red algal profiles of higher C20 PUFAs with the exception of Sarconema
filiforme, Gastroclonium iyengarii and Scinaia monoliformis. In addition, red algae showed
large variations in MUFAs and ranged from 3.5%-26.6% of TFAs. Myristic, palmitic,
stearic, palmitoleic, oleic, elaidic, dihomogammalinolenic acid (C20:3, n-6; DGLA), AA
and EPA were among the predominant FAs encountered and collectively accounted for
77.6%-99.2% of TFAs.
Phaeophyta
The FA compositions of 24 different brown algal species belonging to the three
orders, Dictyotales, Fucales and Ectocarpales are presented in Table 3.2 and Appendix III.
The major FAs encountered were myristic, palmitic, stearic, palmitoleic, oleic, LA, STA,
AA and EPA that collectively contributed to 80.1%-92.2% of TFAs. In contrast to green
and red algae, both the polyenoic C18 and C20 FAs were predominant in these species and
varied from 23.6 ± 8.6% to 58 ± 4.0% of TFAs. This trend was a typical character of
Phaeophyta group that distinguishes them from rest of the Chlorophyta and Rhodophyta
members. The comparative analysis of C18 and C20 PUFAs distribution revealed that the
members of Phaeophyta could be divided into three groups. The first group comprised of
Padina spp., Dictyota hauckiana, Cystoseira trinodis and Hincksia mitchelliae that exhibited
higher C18 PUFAs (1.1-2.2-fold) owing to higher STA contents. The second group included
Dictyota spp. (D. pinnatifida, D. bartayresiana, D. dichotoma), Sargassum spp.,
Hormophysa cuneiformis, Cystoseira indica and Scytosiphon lomentaria that exhibited
higher C20 PUFAs than C18 PUFAs (due to higher AA and EPA contents) while the third
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69
group consisted of Dictyopteris deliculata, Dictyota crevicornis and D. ciliolata that
recorded equal amounts of C18 and C20 PUFAs. The Phaeophyta members also reported
higher MUFAs than both the Chlorophyta and Rhodophyta and accounted for 19%-51% of
unsaturation. Among the six Dictyota species studied, D. pinnatifida, D. bartayresiana and
D. dichotoma recorded higher C20 PUFAs (AA and EPA), D. crevicornis and D. ciliolata
recorded equal amounts while D. hauckiana recorded 1.4-fold higher C18 PUFAs chiefly
due to STA (22.4 ± 0.6% of TFAs). Fucales members had marginal variations and relatively
lower myristic acid contents (5.0%-7.5% of TFAs) as compared with Dictyotales (4.6%-
24.7% of TFAs). They further showed lower STA contents (0.9%-8.8% of TFAs) except S.
carpophyllum and S. plagiophyllum recording 11.5% and 12.3 % of TFAs respectively.
Although, all the seven Sargassum species differed significantly in their FA contents, they
showed similar FA profiles with higher MUFAs, PUFAs (C20>C18 PUFAs) and low SFAs
with the exception of S. carpophyllum.
To the best of our knowledge, the FA profiles for Trichosloen mucronatus,
Chamaedoris auriculata and Cladophoropsis javanica belonging to Chlorophyta,
Coelarthrum muelleri, Gastroclonium iyengarii and Griffithsia corallinoides belonging to
Rhodophyta are reported for the first time in the present study (Table 3.2 and Appendix III).
3.3.3. Multivariate analysis and Fatty acid phylogeny
Principal component analysis (PCA) of FA data matrix provided a global statistical
distinction between the three groups, Chlorophyta, Rhodophyta and Phaeophyta although it
explained only 36% of the variations (PC1-21% and PC2-15%) revealing many correlations
of taxonomic implications. Axis I separated the Chlorophyta members (group I) from
Rhodophyta (group II) while Phaeophyta members (group III) were positioned intermediate
between these two, as evident from the PCA bi-plot (Fig. 3.1). The most discriminant
variables along axis I were STA, GLA, LA, ALA, DHA, AA, EPA and elaidic acid while
along axis II were oleic, myristic, palmitic, 11,14,17-eicosatrienoic acid (ETA) and DGLA.
All the species belonging to the same genus were clustered together with a few exceptions of
U. lactuca (due to higher loadings of stearic acid), S. cinereum, H. musciformis (due to
DGLA) and C. trinodis (due to LA, STA and GLA).
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70
Fig. 3.1 Bi-plot representation of macroalgal samples obtained from PCA analysis of FA data matrix, with first
two principal components. The macroalgal species are indexed from 1 to 100 according to Table 3.1.
Fig. 3.2 Bi-plot of macroalgal samples, obtained by PCA analysis of FA groups namely, SFA, MUFA, PUFA,
C18 PUFA, C20 PUFA, n-3 PUFA and n-6 PUFA, with first two principal components. The
macroalgal species are indexed from 1 to 100 according to Table 3.1.
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Fig. 3.3 Dendrogram obtained from hierarchical cluster analysis of 100 macroalgal samples indexed according
to Table 3.1.
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A large number of outliers were detected in this model, U. flexuosa, C. racemosa and C.
veravalensis owing to the higher loadings of ALA, P. gymnospora, H. mitchalliae and S.
filiforme due to STA, D. ciliolata, D. bartayresiana due to ETA, S. marginatum, S. asperum,
G. corticata, P. tenera, G. iyengarii and G. salicornia due to oleic, myristic, AA, EPA, LA
and palmitic acid respectively. Further, bi-plot of FA groups’ data matrix successfully
explained 80% of the variances (PC1-48% and PC2-32%), (Fig. 3.2). This indicated the vast
diversity present in FA data matrix in macroalgae; when PCA was applied on individual
FAs, the model could explain only 36% of the variations satisfactorily, but when they were
clustered together in groups, FA trends were observed to be conserved within the respective
phyla. Moreover, the dendrogram generated from Ward hierarchical clustering to assess the
phylogenetic relationship between different macroalgal species grouped them into nine
clusters (Fig. 3.3).
The groups deduced from FA compositions were congruent with the clades inferred
from their nuclear and plastid genomes for the genus Ulva, Bryopsis, Halimeda, Sargassum,
Dictyota and Gracilaria. Ulva spp. and Monostroma sp. were closely related to each other
forming Ulvales-Ulotrichales clade. Similarly, Caulerpa, Bryopsis, Halimeda, Codium and
Trichosolen belonging to the same order Bryopsidales, Padina, Dictyota, Dictyopteris,
Stoechospermum and Spatoglossum belonging to Dictyotales (except P. tetrastomatica) and
Sargassum (except S. cinereum), Hormophysa and Cystoseira belonging to Fucales were
grouped together. Similar trends were observed for Rhodophyta where FA trends remained
conserved up to orders except for Gigartinales, Halymeniales, Ceramiales and
Rhodymeniales. The algal species belonging to the families of Cystocloniaceae,
Solieriaceae, Rhodymeniaceae, Champiaceae, Lomentariaceae, Halymeniaceae and
Rhodomelaceae were grouped separately. This deviation could be due to inadequate taxon
sampling and further efforts including extensive sampling may give a better resolution.
Further, it was found that the fatty acid compositions of macroalgae could be
correlated with their evolutionary history. The results revealed that the production of C18
and C20 PUFAs in green and red macroalgae could be attributed to their primary
endosymbiotic origin of engulfment of C18 PUFA producing photosynthetic
cyanobacterium by an eukaryotic host capable of further elongation and desaturation de
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73
novo. Phaeophyta members that originated from secondary endosymbiosis of red algae and
an eukaryotic host further capable of elongation and desaturation of FAs de novo produce
C18 and C20 PUFAs along with minor amounts of C22 PUFAs (C22:4, n-6) such as in
Dictyota spp. (<0.4% of TFAs, not included in present analysis).
3.3.4. Nutritional assessment
The present study revealed that the investigated macroalgae were rich in nutritionally
important PUFAs with their unsaturation index (U.I.) varying from 104 ± 2.8 (U. lactuca) to
226 ± 2.6 (C. veravalensis) among green; 95.2 ± 2.7 (S. marginatum) to 241.8 ± 5.6 (D.
bartayresiana) among brown and 50.6 ± 1.3 (G. salicornia) to 272 ± 5.2 (P. tenera). PCA
analysis of PUFA and nutritional indices data matrix (LA, ALA, GLA, STA, AA, EPA,
DHA, PUFA/SFA, UI, AI and TI) to evaluate the distribution of essential FAs in
macroalgae, explained 49% of the variations (PC1-31% and PC2-18%), (Fig. 3.4). The
discriminant variables along axis I were AA, EPA and UI while along axis II were ALA,
DHA, LA, GLA, STA, PUFA/SFA, AI and TI. The analysis revealed higher amounts of LA
and ALA in all the Chlorophyta members while STA was higher in Ulva spp. along with the
species of Padina, Dictyota, Cystoseira and S. filiforme. DHA which is essential for visual
and neurological development in infants was higher in the species of Halimeda, Udotea,
Ulva, Cladophoropsis, Valoniopsis, Acrosiphonia, and Chaetomorpha. The species of
Gracilaria, Cryptonemia, Rhodymenia and Coelarthrum had higher loadings of AA while S.
cinereum, L. variegata, D. bartayresiana, G. filicina, as well as species of Pyropia,
Acanthophora, Gelidiella, Gelidiopsis and Polysiphonia had higher loadings of EPA.
Further, all the species had nutritionally beneficial n6/n3 ratio ranging from 0.1 ± 0.1:1 (U.
beytensis) to 1.7 ± 0.7:1 (C. javanica) in green, from 0.4 ± 0.01:1 (D. hauckiana) to 3.9 ±
1.8 (D. pinnatifida) in brown (except H. cuneiformis) and from 0.5 ± 0.1:1 (P. ferulacea) to
3.6 ± 0.7:1 (B. botryoides) in red with the exception of Gracilaria spp., H. spinella, R.
sonderii and C. undulata (p ≤ 0.001, Table 3.2). In addition, the PUFA/SFA ratio was ≥ 0.4
in all the macroalgal samples except seven red algae, J. rubens, S. monoliformis, Laurencia
sp., S. robusta, H. musciformis, S. scinaioides and G. salicornia. The artherogenic (AI) and
thrombogenic (TI) indices were < 3 in all the macroalgal species studied except Laurencia
sp., G. textorii, G. salicornia and G. corticata v. cylindrica.
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Fig. 3.4 Bi-plot of macroalgal species, indexed from 1 to 100 according to Table 3.1, obtained by PCA analysis
of PUFA and nutritional indices data matrix (LA, ALA, GLA, STA, AA, EPA, DHA, PUFA/SFA,
U.I., AI and TI).
3.4. Discussion
The lipid content in the investigated macroalgae varied significantly among the three
phyla with the highest lipid content shown by browns, followed by greens and red
macroalgae (Table 3.1). The lipid contents in the present study were lower than those
reported for the same or related species (such as Acrosiphonia sp., Cystoseira sp., Laurencia
sp., Polysiphonia sp. and Scytosiphon sp.) from Caspian Sea (Dembitsky et al. 1993), Sea of
Japan (Khotimchenko 1998), Bohai Sea (Li et al. 2002) and Seribu island, Indonesia
(Santoso et al. 2006). However, the lipid contents of Caulerpa racemosa, C. sertularioides,
Cladophoropsis javanica, Sargassum cinereum, Padina spp. and Ulva spp. reported either
similar or 1.2-2.0-fold higher values with an exception of U. erecta that showed equal
content to the sibling species of U. intestinalis studied from Bohai sea (Li et al. 2002). These
variations could be attributed to the interplay of inter- or intra-specific variations along with
the spatiotemporal variations in environmental parameters across the world (Chandini et al.
2008).
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The FA profiling of 100 macroalgal samples revealed that all the Chlorophyta members
(except H. tuna) were characterized by higher content of C18 PUFAs (LA, ALA and STA)
while Rhodophyta members had higher C20 PUFAs, AA and EPA (except S. filiforme, G.
iyengarii and S. monoliformis) while Phaeophyta members contained both C18 and C20
PUFAs in abundant amounts in accordance with the earlier studies (Khotimchenko et al.
2002; Li et al. 2002; Kumari et al. 2010). About 70% of the species had higher UFAs (Table
3.2). Phaeophyta members had the highest MUFA contents (10.5%-32.9% of TFAs)
followed by Rhodophyta members (3.6%-26.6% of TFAs) and Chlorophyta (3.5%-21.9% of
TFAs) similar to earlier reports (Khotimchenko et al. 2002; Li et al. 2002; Kumari et al.
2010). Further, members of the same genus exhibited similar FA patterns but differed
significantly in their individual FA contents (p ≤ 0.001). Ulva spp. had characteristic FA
profiles of high palmitic, oleic, C18 PUFAs with ALA>LA (2.0- to 9.8-times), STA and low
C20 PUFAs as illustrated earlier for different Ulva spp. (Vaskovsky, 1996; Khotimchenko et
al. 2002; Li et al. 2002; Colombo et al. 2006; Yazici et al. 2007; Chakraborty and Santra,
2008; Pereira et al. 2012). Moreover, DHA in Ulva spp. has earlier only been reported from
Yellow Sea (Vaskovsky, 1996), Black Sea and Dardenelles (Yazici et al. 2007) in low
amounts (0.2%-0.6% of TFAs). Monostroma oxyspermum belonging to Ulotrichales
recorded higher SFAs due to higher stearic acid (C18:0) (17.4% of TFAs) but with lower
PUFAs in contrast with those values reported for M. nitidum collected from Yamaguchi,
Japan (Kaneniwa et al. 1998). Moreover, higher C20 PUFAs contents found in the taxa of
Bryopsidales, Siphonocladales and Cladophorales have also been reported in previous
studies (Khotimchenko, 1993; Carbelleira et al. 1999; Khotimchenko, 2003a; Goecke et al.
2010). Codium dwarkense showed relatively higher SFAs due to high palmitic (43% of
TFAs) and myristic acid (11.1% of TFAs) with low MUFAs and PUFAs with minor DHA
(1.3% of TFAs). These values were comparatively higher than those reported for C.
elongatum (0.2 % of TFAs) and C. tomentosum (1.1% of TFAs) (Khotimchenko, 2003a; Xu
et al. 1998; Goecke et al. 2010). The taxa belonging to the genus of Bryopsis and Halimeda
showed FA patterns similar to those recorded earlier for the same or related species
(Vaskovsky, 1996; Carbelleira et al. 1999). A few deviations were observed in the FA
composition of Chaetomorpha spp. where LA was reported as the predominant PUFA while
ALA and STA together accounted to less than 2% of TFAs (Khotimchenko, 1993;
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Khotimchenko et al. 2002). However, in the present investigation, though LA content was
1.2-fold higher than ALA and STA, they together contributed to 13.2% of TFAs (p ≤ 0.001).
Similar variations in FA profiles of Chaetomorpha spp. were reported by Yazici et al.
(2007) from Black Sea and Dardenelles. Similarly, FA profiles obtained for red and brown
algal species of Gracilaria, Sarconema, Hypnea, Solieria, Pyropia, Rhodymenia,
Cryptonemia, Botryocladia, Champia, Gelidiopsis, Grateloupia, Halymenia, Odonthalia,
Acanthophora, Polysiphonia, Laurencia, Jania, Scinaia, Padina, S. marginatum, S. asperum
Dictyota, Sargassum, Hormophysa and Cystoseira were consistent with those reported in
previous studies from other regions (Aknin, 1990; Levy et al. 1992; Khotimchenko, 1995;
Vaskovsky, 1996; Khotimchenko et al. 2002; Li et al. 2002; Bhaskar and Miyashita, 2005;
Colombo et al. 2006; Chakraborty & Santra, 2008; Ortiz et al. 2009; Kumari et al. 2010;
Kendel et al. 2012; Pereira et al. 2012).
PCA proved to be an important tool to unravel chemotaxonomic relationships among
different macroalgae. PCA of both the FA and FA groups’ data matrix (Fig. 3.1 and 3.2)
segregated green, red and brown macroalgal samples into three groups. The comparative
assessment of the PCA of two data matrices revealed that PCA of FA data matrix could
explain only 36% of the variations satisfactorily as compared to FA groups’ data matrix that
explained greater variations (80% of the variances). This indicated the vast diversity present
in FA data matrix in macroalgae; when PCA was applied on individual FAs the model could
explain only 36% of the variations satisfactorily, but when they were clustered together in
groups, FA trends were observed to be conserved within the respective phyla in congruence
to the earlier findings of Galloway et al. (2012).
Moreover, dendrogram obtained from Ward hierarchical clustering demonstrated that
the groups deduced from FA compositions were congruent with the clades inferred from
their nuclear and plastid genomes for the genus Ulva, Bryopsis, Halimeda, Sargassum,
Dictyota and Gracilaria, (Verbruggen et al. 2009; Pareek et al. 2010; Silberfeld et al. 2010;
Tronholm et al. 2010; Caisová et al. 2012; Duan et al. 2012). Further, Ulva spp. and
Monostroma sp. were closely related to each other forming Ulvales-Ulotrichales clade
identical to the one obtained from molecular data (Leliaert et al. 2012). Caulerpa, Bryopsis,
Halimeda, Codium and Trichosolen belonging to the same order Bryopsidales were grouped
together and their inter-relationship were in agreement with the multi-locus-time calibrated
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phylogeny of siphonaceous green algae based on rbcL, tufA, atpB, 18S rRNA gene
sequences (Lam and Zechman, 2006; Vebruggen et al. 2009). Similarly, Padina, Dictyota,
Dictyopteris, Stoechospermum, Spatoglossum belonging to Dictyotales (except P.
tetrastomatica) and Sargassum (except S. cinereum), Hormophysa and Cystoseira belonging
to Fucales were grouped together in agreement with the multi-marker phylogeny deduced by
Silberfeld et al. (2010). Similar trends were observed for Rhodophyta where FA trends
remained conserved up to orders except for Gigartinales, Halymeniales, Ceramiales and
Rhodymeniales. Our study corroborates with the findings of Lang et al. (2011), Galloway et
al. (2012) and Gosch et al. (2012) that FA distribution reflects phylogenetic relationships
among phyla, classes and orders as seen in the genomic and molecular phylogenies at higher
ranks in algae. However, higher variations in FA contents at the levels of genus and species
pose difficulty in discriminating species as observed in the present study especially in the
case of red macroalgae.
The production of C18 and C20 PUFAs in green and red macroalgae could be attributed
to their primary endosymbiotic origin of engulfment of C18 PUFA producing photosynthetic
cyanobacteria by eukaryotic host capable of further elongation and desaturation de novo
similar to the green and red microalgae as explained by Dunstan et al. (2005) since it is
noteworthy to note that cyanobacteria contain FAs only up to the chain length of 18-carbon
(Liu et al. 2010a; Iliev et al. 2011). Phaeophyta members that originated from secondary
endosymbiosis of red algae and an eukaryotic host further capable of elongation and
desaturation of FAs de novo produce C18 and C20 PUFAs along with minor amounts of
C22 PUFAs (C22:4, n-6) such as in Dictyota spp. (<0.4% of TFAs). Similarly,
Cryptophytes, Haptophytes, dinoflagellates, other Heterokont algae and Apicomplexans
belonging to the red algal lineage that diverged along with brown algae during secondary
endosymbiosis also produce C22 PUFAs (Dunstan et al. 2005; Leblond et al. 2005; Lang et
al. 2012). Some of the species belonging to these groups such as Phaeodactylum tricornutm,
Skeletonema sp., Crypthecodinium cohnii, Schizochytrium sp., Rhodomonas sp., Pavlova sp.
and Isochrysis sp. are commercially explored for their EPA and DHA contents in
aquaculture and pharmaceuticals (Patil et al. 2007; Mendes et al. 2008; Lang et al. 2012).
Similarly, Chlororachinophytic algae such as Bigelowiella sp., Gymnochlora sp. and
Lotharella sp. that diverged from the secondary endosymbiosis of green algae and the
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eukaryotic host also produce C22 PUFAs, C22:5 (n-3) and C22:6 (n-3) (Leblond et al.
2005). Such endosymbiotic relationship with the FA profiles of algae is well supported by
the fact that green and red microalgae and macroalgae inherited their type II fatty acid
synthase (FASII) genes from their cyanobacterial endosymbiont and brown algae from their
red algal endosymbiont (Chan et al. 2012; Ryall et al. 2003). Similarly, two distinct types of
ACCase that catalyze the key step of FA biosynthesis has been found in algae, heteromeric
form in green and red algae of primary endosymbiotic origin and homomeric ACCase in
brown and other stramenopiles algae of secondary endosymbiotic origin, (Huerlimann and
Heimann 2012). Chan et al. (2012) proposed the co-regulation of FA biosynthesis and
intracellular lipid trafficking in red algae on the basis of their analysis of 482 EST contigs
encoding putative membrane transporters in Pyropia spp. They also deciphered the
evolution of these transporters and showed that among the three proteins of transporter
complex of chloroplast inner membrane which is involved in lipid trafficking (TGD1, TGD2
and TGD3), TGD1 and TGD2 are located in the plastid genome in red algae. Moreover
TGD2 is located in the same operon as accA that encodes the α-subunit of ACCase while
TGD3 is nuclear encoded and showed strong phylogenetic association with the TGD3 of
cyanobacteria and higher plants. No homolog for TGD4 involved in FA import in
chloroplast in Arabidopsis was found in EST sequences or plastid genomes of red alga
Pyropia sp. Further, Chan et al. (2012) also did not find any candidate gene for any of these
TGD-genes in secondary plastids of stramenopiles. In contrast, all the three TGD orthologs
are located in the nucleus in green algae (Xu et al. 2008).
Furthermore, the low amounts of C20 PUFAs in Chlorophyta members as compared to
Rhodophyta could be due to the low rate of conversion of C18 to C20 PUFAs. However, the
question arises, if C22 PUFA production in algae is attributed to secondary endosymbiosis
then, how DHA (C22 PUFA) is synthesized in Chlorophyta species. Whether the enzymes
that elongate and desaturate C18 PUFAs also exhibit the specificity for C20 PUFAs as
substrates or they inherited some type I polyketide synthase (PKS) genes through lateral
gene transfer from bacteria like lower unicellular chlorophytes, Ostrerococcus tauri, O.
lucimarinus and Chlamydomonas reinhardtii (John et al. 2008). Such type I PKS genes have
not been found in any other Plantae lineages neither in higher plants nor in red algae.
However, type III PKS gene has recently been reported in brown alga Sargassum binderi
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similar to the higher plants but its role in lipid biosynthesis has not yet been established
(Baharum 2011). Also, Dittami et al. (2012) found three desaturases similar to ∆4-
desaturases of haptophytes along with three putative polyketide synthases in brown
microalgae Pseudochattonella farcimen, some of these genes have been acquired via
horizontal gene transfer from the common ancestor of brown algae and dictyochophyceae.
However, the presence of C22 PUFAs in green macroalgae is quite debatable as no ∆4-
desaturase responsible for DHA production in microalgae has been reported in green
macroalgae and thus requires further investigation.
The nutritional assessment of investigated macroalgal samples revealed that all the
species had nutritionally beneficial n6/n3 ratio (0.1:1 to 3.9:1) with the exception of a few
Gracilaria spp., H. spinella, R. sonderii and C. undulata (p ≤ 0.001, Table 3.2), that reported
higher n6/n3 ratios exceeding the ratio of n6/n3<5:1 recommended by World Health
Organization (WHO) (WHO and FAO joint consultation, 1995; Simopoulos, 2002).
According to the recent FAO report 2010, if a diet contains the recommended amount of
essential FAs then, whether the n6/n3 ratio of 4/1 or1/1 or < 1 is meaningless. It should be
ensured that n3 PUFAs are in substantial amount in the food product. Thus, macroalgae
exhibiting such low n6/n3 FAs could help in decreasing low density lipoproteins, cholesterol
and prevent inflammatory, cardiovascular and nervous system disorders. In addition, the
PUFA/SFA ratio, which is one of the important parameter to assess the nutritional quality of
the lipid fraction of food, was in accordance to the nutritional guidelines, ≥ 0.4 in all the
seaweeds except seven red algae, J. rubens, S. monoliformis, Laurencia sp., S. robusta, H.
musciformis, S. scinaioides and G. salicornia (Department of Health, 1994). The
artherogenic (AI) and thrombogenic (TI) indices were <3 in all the macroalgal species
studied except Laurencia sp., G. textorii, G. salicornia and G. corticata v. cylindrica.
Similar AI and TI values were observed for different macroalgae in our previous work
(Kumar et al. 2011c). Recently, López-López et al. (2009) demonstrated that the addition of
macroalgae such as Undaria sp., Himanthalia sp. and Pyropia sp. to meat products
improved their thrombogenic and atherogenic indices thereby illustrating their potential in
the development of healthier lipid formulations.
Despite the health-benefitting n6/n3 ratio displayed by most of the investigated
macroalgae, microalgae have been considered better promising sustainable sources of
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essential FAs (especially, EPA and DHA) for commercial use till date as they are capable of
producing much more lipids than any conventional crops. However, many of the
investigated macroalgal species contained comparable ALA, AA and EPA contents. Most of
the green macroalgal species such as Ulva, Caulerpa, Monostroma, Bryopsis, Udotea and
Acrosiphonia had ALA contents in the range of 14%-48% of TFAs comparable to those of
Dunaliela and Chlorella commercially utilized for ALA (Zhukova and Aizdacher, 1995;
Lang et al. 2012). Further, C. auriculata, C. verticillata, all the brown algae except P.
tetrastomatica, D. ciliolata and S. cinereum and red algae had AA contents 10-25% of
TFAs. C. undulata and R. sonderi had AA content >30% of TFAs while G. corticata
showed higher content >50% of TFAs that is higher than commercial sources of microlagal
AA such as Porphyridium sp. (34.7% of TFA) and Parietochloris sp. (46% of TFA) (Guil-
Guerrero et al. 2001; Lang et al. 2011). Similarly, EPA contents of the species Pyropia,
Halymenia, Polysiphonia, Acanthophora, Gelidium and Gelidiella (20%-35% of TFAs)
were comparable to those of Phaeodactylum tricornutum (12-14% of TFAs), Pavlova
lutheri (10%-28% of TFAs), Nannochloropsis spp., Thalassiosira sp., Chaetoceros spp.
(12%-18% of TFAs), Nitzschia sp., Skeletonema sp., Chattonella spp. and Navicula sp.
(20%-26% of TFAs) (Marshall et al. 2002; Pratoomyot et al. 2005; Lang et al. 2012) But,
the DHA content in macroalgae was very low (<7% of TFAs found in Chlorophyta
members) as compared to the species of Schizochytrium, Crypthecodinium, Scripsiella and
Peridinium (30%-50% of TFAs) (Leblond et al. 2006; Fan et al. 2007; Mendes et al. 2008).
The only bottleneck in the utilization of macroalgae for pure oil-based products such as
PUFA-oils is the low lipid content of macroalgae as compared to microalgae. Conversely,
this low lipid content rich in PUFAs is a boon for their utilization as whole macroalgae in
both fresh and dried form in human nutrition and aquaculture as it helps in improving
cardiac, mental health and combating inflammatory diseases.
In conclusion, the macroalgae investigated in this study revealed low lipid contents but
exhibited nutritionally high amounts of essential n-6 and n-3 PUFAs (LA, ALA, STA, AA,
EPA and DHA). The health benefiting n6/n3 ratio in macroalgae offers their utilization in
the formulation of functional foods and nutraceuticals. The species of green macroalgae
investigated could be utilized as potential sources of LA and ALA while the species of Ulva,
Padina, Dictyota, Cystoseira and S. filiforme for STA. The red algal species of Gracilaria,
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Cryptonemia, Rhodymenia and Coelarthrum could be utilized for AA while Pyropia,
Polysiphonia, Acanthophora, Gelidiella, Gelidiopsis, G. filicina, S. cinereum L. variegata
and D. bartayresiana for EPA. The contents of ALA in green, AA and EPA in most of the
red macroalgae were comparable with those of commercially utilized microalgae. Thus, an
appropriate choice of macroalgae can be utilized as ingredients in the formulation of low fat
foods and PUFA-rich nutraceuticals that would improve the quality of human diet and also
reduce the dependency on traditional terrestrial sources. Further, the statistical analysis
demonstrated that FA based chemotaxonomic relationships were conserved at different
taxonomic ranks within the phyla and corroborated well with their molecular phylogenies.
This signifies the use of FA signatures as an additional chemotaxonomic tool for closer
understanding the relationships among different species at higher ordinal levels such as
families, orders and phyla. Moreover, the green, red and brown macroalgae acquired their
ability of producing long-chain PUFAs from the coupling of chloroplastic desaturase
enzymatic systems of their respective endosymbionts and FA desaturase and elongase
system of the eukaryotic host. Although the inheritance of DHA production by Chlorophyta
members is not yet understood, further phylogenetic investigations coupled with increased
availability of EST sequences may decode this enigma.
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