Appendix - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36330/15/15_appendix.pdf · Appendix 241 Appendix I Description of collection sites of different macroalgae. Species

Appendix

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Appendix I

Description of collection sites of different macroalgae.

Species Collection site Location CHLOROPHYTA Ulvales Ulva lactuca Veraval N 20º 54.87'; E 70º 20.83' Ulva fasciata Veraval N 20º 54.87'; E 70º 20.83' Ulva taeniata Veraval N 20º 54.87'; E 70º 20.83' Ulva pertusa Kalubhar island N 22º 26.21'; E 69º 35.12’ Ulva reticulata Kalubhar island N 22º 26.21'; E 69º 35.12’ Ulva beytensis Kalubhar island N 22º 26.21'; E 69º 35.12’ Ulva compressa Kalubhar island N 22º 26.21'; E 69º 35.12’ Ulva rigida Veraval N 20º 54.87'; E 70º 20.83' Ulva linza Veraval N 20º 54.87'; E 70º 20.83' Ulva flexuosa Veraval N 20º 54.87'; E 70º 20.83' Ulva erecta Kalubhar island N 22º 26.21'; E 69º 35.12’ Ulva prolifera Kalubhar island N 22º 26.21'; E 69º 35.12’ Bryopsidales Caulerpa scalpelliformis Veraval N 20º 54.87'; E 70º 20.83' Caulerpa veravalensis Veraval N 20º 54.87'; E 70º 20.83' Caulerpa racemosa Veraval N 20º 54.87'; E 70º 20.83' Caulerpa racemosa v. corynephora Okha N 22º 27.06'; E 69º 4.02' Caulerpa racemosa v. occidentalis Okha N 22º 28.50'; E 69º 4.54' Caulerpa microphysa Veraval N 22º 28.43'; E 69º 4.17' Caulerpa verticillata Okha N 22º 28.39'; E 69º 4.50' Caulerpa sertularioides Okha N 22º 28.42'; E 69º 4.51' Codium dwarkense Veraval N 20º 54.87'; E 70º 20.83' Bryopsis pennata Okha N 22º 28.40'; E 69º 4.50' Bryopsis plumosa Okha N 22º 28.44'; E 69º 4.50' Trichosolen mucronatus Okha N 22º 28.41'; E 69º 3.58' Udotea indica Okha N 22º 28.43'; E 69º 4.10' Halimeda discoides Veraval N 20º 54.87'; E 70º 20.83' Halimeda tuna Veraval N 20º 54.87'; E 70º 20.83' Ulotrichales Monostroma oxyspermum Achara N 16º 11.59’; E 73º 26.38’ Siphonocladales Chamaedoris auriculata Veraval N 20º 54.87'; E 70º 20.83' Cladophoropsis javanica Okha N 22º 28.44'; E 69º 4.50' Valoniopsis pachynema Veraval N 20º 54.87'; E 70º 20.83' Cladophorales Chaetomorpha linum Kalubhar island N 22º 26.21'; E 69º 35.12’ Acrosiphonia orientalis Okha N 22º 28.46'; E 69º 04.34' PHAEOPHYTA Dictyotales Padina tetrastomatica Veraval N 20º 54.87'; E 70º 20.83' Padina gymnospora Kalubhar island N 22º 26.21'; E 69º 35.12’

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Dictyopteris deliculata Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota pinnatifida Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota bartayresiana Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota dichotoma Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota cervicornis Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota ciliolata Kalubhar island N 22º 26.21'; E 69º 35.12’ Dictyota haukiana Kalubhar island N 22º 26.21'; E 69º 35.12’ Stoechospermum marginatum Veraval N 20º 54.87'; E 70º 20.83' Lobophora variegata Veraval N 20º 54.87'; E 70º 20.83' Spatoglossum asperum Veraval N 20º 54.87'; E 70º 20.83' Fucales Sargassum tenerrimum Veraval N 20º 54.87'; E 70º 20.83' Sargassum johnstonii Okha N 22º 28.54'; E 69º 04.59' Sargassum sp. Kalubhar island N 22º 26.21'; E 69º 35.12’ Sargassum carpophyllum Kalubhar island N 22º 26.21'; E 69º 35.12’ Sargassum plagiophyllum Shivrajpur N 22º 19.87'; E 68º 56.95_ Sargassum cinereum Veraval N 20º 54.87'; E 70º 20.83' Sargassum cinctum Porbandar N 21º 38.24'; E 69º 35.81' Hormophysa cuneiformis Okha N 22º 28.43'; E 69º 04.10' Cystoseira indica Veraval N 20º 54.87'; E 70º 20.83' Cystoseira trinodis Dhani island N 22º 24.81’; E 69º 32.24’ Ectocarpales Hincksia mitchelliae Dhani island N 22º 24.81’; E 69º 32.24’ Scytosiphon lomentaria Dhani island N 22º 24.81’; E 69º 32.24’ RHODOPHYTA Gracilariales Gracilaria dura Adri N 20º 57.58'; E 70º 16.76' Gracilaria salicornia Veraval N 20º 54.87'; E 70º 20.83' Gracilaria textorii Veraval N 20º 54.87'; E 70º 20.83' Gracilaria corticata Veraval N 20º 54.87'; E 70º 20.83' Gracilaria corticata v. cylindrica Veraval N 20º 54.87'; E 70º 20.83' Gracilaria corticata v. folifera Veraval N 20º 54.87'; E 70º 20.83' Gracilaria debilis Okha N 22º 28.44'; E 69º 03.58' Gracilaria verrucosa Okha N22º 26.38'; E 69º 03.30' Gigartinales Sarconema scinaioides Veraval N 20º 54.87'; E 70º 20.83' Sarconema filiforme Veraval N 20º 54.87'; E 70º 20.83' Hypnea valentiae Okha N 22º 28.54'; E 69º 04.38' Hypnea musciformis Okha N 22º 28.51'; E 69º 04.48' Hypnea spinella Veraval N 20º 54.87'; E 70º 20.83' Solieria robusta Kalubhar island N 22º 26.21'; E 69º 35.12’ Bangiales Pyropia tenera Malvan N 16 º 03. 59'; E 73º 27. 31’ Pyropia yoezensis Malvan N 16 º 03. 59'; E 73º 27. 31’ Pyropia acanthophora Malvan N 16 º 03. 59'; E 73º 27. 31’ Pyropia acanthophora v. brasilensis Malvan N 16 º 03. 59'; E 73º 27. 31’ Pyropia sp. Malvan N 16 º 03. 59'; E 73º 27. 31’

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Rhodymeniales Rhodymenia sonderi Veraval N 20º 54.87'; E 70º 20.83' Coelarthrum muelleri Okha N 22º 28.47'; E 69º 04.55' Botryocladia leptopoda Okha N 22º 28.49'; E 69º 04.56' Botryocladia botryoides Boria reef N 22 º 24.49'; E 69º 13.33’ Gastroclonium iyengarii Dhani island N 22º 24.81’; E 69º 32.24’ Champia parvula Dhani island N 22º 24.81’; E 69º 32.24’ Gelidiopsis variabilis Okha N 22º 28.52'; E 69º 04.47' Gelidiales Gelidiella acerosa Veraval N 20º 54.87'; E 70º 20.83' Halymeniales Cryptonemia undulata Okha N 22º 28.48'; E 69º 04.56' Halymenia porphyraeformis Okha N 22º 28.51'; E 69º 04.50' Grateloupia indica Okha N 22º 28.50'; E 69º 04.55' Grateloupia filicina Okha N 22º 28.43'; E 69º 04.10' Ceramiales Odonthalia veravalensis Veraval N 20º 54.87'; E 70º 20.83' Acanthophora specifera Okha N 22º 28.39'; E 69º 03.56' Acanthophora nayadiformis Okha N 22º 28.48'; E 69º 04.37' Laurencia cruciata Veraval N 20º 54.87'; E 70º 20.83' Laurencia obstusa Veraval N 20º 54.87'; E 70º 20.83' Laurencia papillosa Veraval N 20º 54.87'; E 70º 20.83' Laurencia majusculus Veraval N 20º 54.87'; E 70º 20.83' Laurencia sp. Veraval N 20º 54.87'; E 70º 20.83' Polysiphonia ferulacea Veraval N 20º 54.87'; E 70º 20.83' Griffithsia corallinoides Okha N 22º 28.44'; E 69º 04.49' Corallinales Jania rubens Okha N 22º 28.50'; E 69º 04.33' Scinaia monoliformis Okha N 22º 28.48'; E 69º 04.55'

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Appendix II

Map showing the sampling locations along the Gujarat coast

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245

Appendix III

Fatty acid composition of different macroalgal species given in % of total fatty acid methyl esters (TFAs), expressed as means ± SD (n=3).

FAs 1 2 3 4 5 6 7 8 9 10 11 12 C12:0 1.1±0.1 0.2±0.1 0.7±0.2 0.5±0.2 0.6±0.1 1.1±0.9 0.5±0.2 0.6±0.4 0.6±0.3 1.7±0.6 0.8±0.1 0.8±0.4 C14:0 3.3±0.5 0.7±0.1 1.8±0.3 2.6±0.3 3.8±0.4 2.6±0.6 2.1±0.2 3.7±4.1 2.1±0.1 3.4±0.2 1.7±0.1 2.1±0.4 C15:0 1.0±0.1 0.3±0.2 0.5±0.1 0.5±0.1 0.2±0.01 0.8±0.1 0.6±0.2 0.4±0.1 0.7±0.3 1.0±0.1 1.1±0.2 0.7±0.2 C16:0 39.3±1.0 25.1±1.6 34.2±5.2 25.2±1.7 34±3.0 23.3±1.5 29.4±0.5 28.2±1.6 30.6±8.3 26.2±0.8 31±0.8 29.6±0.3 C17:0 0.5±0.1 0.2±0.1 0.3±0.1 0.2±0.01 0.2±0.02 0.5±0.2 0.3±0.1 0.2±0.1 0.1±0.1 0.5±0.1 0.2±0.1 0.4±0.3 C18:0 10.2±2.7 1.6±0.3 5.3±0.8 4.7±0.3 7.9±2.0 2.5±0.4 2.6±0.1 2.4±1.1 3.1±1.6 3.3±0.2 2.1±0.1 2.7±0.6 C20:0 0.6±0.1 0.2±0.1 2.4±2.4 0.3±0.01 0.7±0.2 0.3±0.1 0.6±0.1 0.5±0.2 0.8±0.6 0.3±0.4 1.0±0.5 1.0±0.3 C22:0 3.4±0.1 1.2±0.2 1.6±0.2 1.1±0.2 1.3±0.3 0.9±0.1 1.1±0.1 1.2±1.3 0.4±0.2 0.6±0.1 1.2±0.1 1.4±0.5 C24:0 0.5±0.1 n. d. n. d. 0.2±0.01 n. d. n. d. 0.3±0.1 0.5±0.3 n. d. 0.3±0.1 n. d. 0.6±0.5 C16:1(n-7) 2.5±0.4 1.9±0.3 1.8±0.3 5.2±0.1 2.9±0.3 1.3±0.3 1.4±0.2 3.4±2.7 n. d. 5.8±0.5 n. d. 1.5±0.3C16:1(n-9) 3.4±1.0 1.9±0.8 1.2±0.5 n. d. n. d. 2.1±0.3 3.6±0.1 1.9±1.8 1.5±0.4 0.0 3.1±2. 4.8±0.3 C17:1(n-7) 0.7±0.1 0.2±0.1 0.0 0.6±0.3 0.2±0.01 0.3±0.1 0.3±0.1 0.3±0.3 0.1±0.1 0.4±0.1 0.4±0.1 0.5±0.1 C18:1(n-9) 3.8±0.3 3.3±0.6 2.4±0.6 3.6± 4.0±0.3 6.4±1.1 2.6±0.1 2.4±0.6 2.4±0.4 1.3±0.1 2.4±0.4 2.7±0.3 C18:1(n-9) trans

n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.

C20:1(n-9) 0.4±0.1 0.3±0.1 n. d. n. d. n. d. 0.1±0.01 n. d. n. d. 0.1±0.1 n. d. n. d. n. d. C22:1(n-9 1.4±0.1 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. C18:2(n-6) 5.2±0.7 n. d. 8.1±0.3 7.6±0.6 7.3±2.4 n. d. 10.6±0.1 6.0±4.2 n. d. 10.8±0.3 13.4±0.2 14.3±0.5 C18:3(n-6) 0.5±0.2 1.0±0.1 2.9±0.8 0.5±0.1 1.0±0.3 0.4±0.1 1.3±0.3 0.5±0.3 0.7±0.2 1.0±0.1 1.0±0.2 1.2±0.3 C18:3(n-3) 14.3±0.2 38.9±1.2 22±2.4 22.8±1.0 17.3±1.7 35.5±2.0 27.5±0.2 23.2±3.5 28.5±5.0 36.8±0.6 29.2±0.6 24±2.4 C18:4(n-3) 2.7±0.4 9.5±0.6 7.5±1.5 19.9±0.8 14.7±1.2 11.2±1.7 8.4±0.7 20±11 15.5±2.5 3.6±0.2 4.8±0.2 5.4±1.7C20:3(n6) 0.4±0.1 0.4±0.1 1.2±0.6 0.3±0.1 n. d. 0.3±0.2 0.9±0.1 0.2±0.2 0.4±0.2 0.6±0.1 0.7±0.1 0.9±0.3 C20:3(n3) 0.6±0.1 0.3±0.3 0.8±0.2 1.0±0.1 n. d. 0.3±0.1 0.7±0.2 0.9±0.1 0.8±0.5 n. d. 0.5±0.2 0.8±0.5 C20:4(n-6) 0.9±0.1 1.3±0.2 2.0±1.4 0.5±0.1 0.9±0.3 1.7±1.0 1.8±0.1 1.0±0.8 1.4±0.5 n. d. 1.4±0.2 1.8±0.3 C20:5(n-3) 0.9±0.7 0.6±0.1 1.7±1.0 0.5±0.1 1.0±0.2 0.7±0.1 1.4±0.2 0.8±0.7 1.9±0.6 0.5±0.1 1.2±0.1 1.2±0.1 C22:6(n-3) 2.5±0.4 3.4±0.3 1.9±0.3 2.1±0.2 2.6±0.4 3.1±0.5 1.4±0.3 1.6±0.5 1.5±1.0 0.7±0.1 1.3±0.3 1.9±1.4

n.d.- not detected.

Table continued..

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FAs 13 14 15 16 17 18 19 20 21 22 23 24 C12:0 0.3±0.1 0.1±0.01 0.2±0.1 0.5±0.3 1.1±0.2 1.7±0.1 0.4±0.3 0.5±0.3 0.6±0.1 0.0 0.9±0.2 0.3±0.1 C14:0 2.4±0.2 0.8±0.1 1.2±0.2 4.4±3.0 4.4±1.0 3.6±0.8 7.0±3.5 2.8±0.3 11.1±1.1 3.4±2.8 5.6±3.1 7.0±1.9 C15:0 0.2±0.01 0.0 0.1±0.01 0.9±0.2 0.5±0.1 0.7±0.4 0.2±0.01 0.5±0.4 0.3±0.01 0.8±0.3 0.6±0.3 0.4±0.1 C16:0 30.1±0.6 25.2±0.9 30.7±3.5 44.6±1.7 39.4±3.5 27.2±1.3 29.5±2.9 34.4±0.6 43±4.4 32.3±3.5 36.6±5.3 36.9±8.1 C17:0 0.4±0.1 0.1±0.01 0.1±0.01 0.6±0.2 1.0±0.6 0.6±0.1 0.3±0.1 n. d. 0.7±0.1 n. d. n. d. 0.3±0.1 C18:0 2.0±0.2 0.8±0.3 1.1±0.4 3.1±0.3 3.2±0.5 3.0±0.4 2.3±0.4 n. d. 7.0±0.6 2.0±0.4 3.2±0.9 2.7±1.0C20:0 0.2±0.1 n.d. n. d. 0.5±0.3 n. d. 0.3±0.1 0.3±0.1 n. d. 0.7±0.1 0.8±0.1 0.7±0.6 0.3±0.2 C22:0 0.4±0.1 n.d. n. d. 0.3±0.2 n. d. 1.1±0.3 0.4±0.2 n. d. 1.2±0.1 1.1±0.3 1.0±0.2 0.5±0.2 C24:0 3.5±0.1 n. d. n. d. 3.3±3.2 3.6±1.0 2.2±1.1 3.6±1.6 1.3±0.8 0.9±0.1 1.5±0.5 2.9±1.1 0.9±0.6 C16:1(n-7) 1.9±0.1 1.4±0.2 2.9±0.5 2.1±0.3 3.5±3.8 3.2±0.5 3.3±3.5 1.9±0.6 1.8±0.1 1.8±0.7 2.2±0.6 1.9±0.5 C16:1(n-9) 2.5±0.1 0.1±0.01 n. d. n. d. 5.6±1.1 n. d. n. d. n. d. n. d. 3.4±1.7 4.6±0.7 0.5±0.8 C17:1(n-7) 0.3±0.1 0.3±0.02 0.1±0.01 0.4±0.1 0.0 n. d. 0.2±0.2 n. d. n. d. n. d. n. d. 0.2±0.2 C18:1(n-9) 1.8±0.1 0.3±0.01 2.6±0.8 0.4±0.2 1.0±0.1 0.5±0.2 0.7±0.4 0.3±0.1 4.8±0.1 1.3±0.3 1.4±0.5 4.1±2.8 C18:1(n-9) trans

n. d. n. d. n. d. n. d. n. d. n. d. 0.6±0.4 n. d. n. d. n. d. n. d. 1.3±1.2

C20:1(n-9) n. d. n. d. 0.2±0.1 n. d. n. d. n. d. 0.4±0.2 n. d. n. d. n. d. n. d. n. d. C22:1(n-9 n. d. n. d. n. d. n. d. n. d. 1.5±0.1 0.5±0.1 n. d. n. d. n. d. n. d. 0.2±0.2C18:2(n-6) 8.8±0.3 7.1±0.3 5.5±0.6 6.8±2.2 11.1±1.4 12.2±0.8 7.4±3.3 13.5±0.7 8.9±0.7 8.2±2.8 5.2±2.4 12.4±0.8 C18:3(n-6) 1.1±0.1 1.0±0.1 0.4±0.1 0.7±0.5 1.2±0.7 1.8±0.5 4.3±0.5 1.4±0.4 1.6±0.1 1.9±0.3 3.0±0.5 1.3±0.1 C18:3(n-3) 29.8±0.9 48±0.9 45.5±0.9 15.7±5.9 14.7±4.0 26.1±2.4 15.8±6.3 28.2±1.8 10.7±1.6 28.3±7.7 21.8±4.4 10.8±1 C18:4(n-3) 1.3±0.1 1.7±0.2 0.6±0.1 4.5±3.3 2.4±0.7 2.2±1.0 5.5±2.3 2.2±0.5 1.0±0.2 3.3±0.5 2.5±2.9 1.6±0.9 C20:3(n6) 0.8±0.1 0.5±0.1 0.4±0.1 1.0±0.5 n. d. 1.1±0.1 2.0±0.3 0.9±0.5 0.8±0.1 1.3±0.9 0.7±0.3 2.0±0.4 C20:3(n3) 0.3±0.1 0.2±0.1 0.1±0.01 n. d. n. d. n. d. 0.7±0.2 1.1±0.1 n. d. n. d. n. d. 0.8±0.7 C20:4(n-6) 4.6±0.1 7.8±0.1 2.2±0.4 3.0±1.3 3.2±0.1 5.0±0.6 9.8±4.1 2.0±0.8 3.0±0.1 6.6±1.8 4.4±0.9 7.5±0.4 C20:5(n-3) 5.4±0.1 3.6±0.1 5.1±0.8 3.6±0.2 3.9±0.5 4.7±1.5 3.6±0.7 4.7±0.9 0.7±0.2 1.8±0.4 2.8±0.9 5.7±0.4 C22:6(n-3) 0.8±0.1 0.6±0.1 0.4±0.3 1.1±0.3 n. d. 1.4±0.2 0.7±0.3 1.8±0.9 1.3±0.1 n. d. n. d. n. d. SFA 39.6±1.0 27.1±1.0 33.4±3.1 58.2±6.4 n. d. 40.3±2.0 44±2.7 41.4±0.9 65.4±2.6 42.1±6.2 51.5±4.4 49.3±10.8 MUFA 6.4±0.1 1.9±0.2 5.7±1.4 2.9±0.4 10.2±5 5.2±0.3 6.1±3.0 2.2±0.6 6.6±0.1 6.5±2.6 8.2±1.1 8.7±0.2 PUFA 54±1.0 71.1±0.9 60.8±3.0 39.1±6.0 36.5±3 54.6±1.9 50±5.3 56.8±1.1 28±2.6 51.5±7.9 40.4±3.4 42.3±10.5 n6/n3 0.4±0.1 0.3±0.01 0.2±0.01 0.6±0.1 0.8±0.3 0.6±0.01 1.0±0.3 0.5±0.1 1.1±0.1 0.6±0.2 0.5±0.1 1.9±1.6 U.I. 177.5±3 226±2.6 196.6±11 128.3±18 122.1±7 176.7±6.1 172.6±6 176.9±3 91±7.8 166±26 137±11.6 144.3±33 AI 0.5±0.02 0.4±0.02 0.5±0.08 1.2±0.3 1.0±0.13 0.5±0.05 0.7±0.05 0.6±0.02 1.6±0.22 0.6±0.17 0.9±0.12 0.9±0.41 TI 0.3±0.01 0.2±0.01 0.2±0.02 0.6±0.12 0.7±0.1 0.3±0.03 0.4±0.11 0.3±0.02 1.3±0.23 0.4±0.15 0.5±0.06 0.9±0.8

n.d.- not detected.

Table continued..

Appendix

247

FAs 25 26 27 28 29 30 31 32 33 34 35 36 C12:0 0.4±0.2 1.0±0.4 0.5±0.1 1.7±0.3 0.3±0.1 1.5±0.9 1.1±0.5 0.6±0.3 1.3±0.4 0.9±0.2 0.4±0.1 0.3±0.2 C14:0 2.8±0.7 6.8±0.5 6.2±0.1 5.7±0.4 7.7±0.7 7.9±0.7 8.5±2.9 6.8±1.0 5.8±1.3 7.0±0.3 7.5±0.5 11.2±1.3 C15:0 0.5±0.3 0.9±0.3 0.5±0.1 0.8±0.1 0.4±0.1 1.4±0.7 1.0±0.7 0.7±0.5 0.6±0.3 0.7±0.1 0.6±0.1 1.4±1.7 C16:0 35±2.2 30±3.8 29.6±0.8 26.7±3.9 33.5±3.3 29.3±4.5 31.6±1.4 24.9±2.2 30.6±3.7 32.9±0.6 20.2±0.5 32.2±2.7 C17:0 0.3±0.1 0.5±0.1 0.4±0.1 0.6±0.1 0.3±0.1 1.4±1.8 0.3±0.2 0.9±0.4 0.7±0.4 0.3±0.1 0.4±0.1 0.5±0.4 C18:0 1.2±0.2 2.8±08 2.0±0.4 17.4±7.0 1.3±0.9 2.4±0.8 2.1±0.5 2.2±0.7 1.2±0.2 3.7±0.3 2.5±0.1 4.0±2.2C20:0 0.3±0.1 n. d. 0.1±0.01 1.1±0.3 0.2±0.1 0.4±0.1 0.2±0.1 n. d. n. d. 0.3±0.1 1.8±1.0 0.4±0.1 C22:0 0.6±0.4 n. d. 0.2±0.01 1.1±0.1 0.2±0.1 0.5±0.1 0.5±0.3 0.4±0.2 0.6±0.2 0.4±0.1 0.3±0.1 0.2±0.2 C24:0 2.5±0.6 3.6±1.6 2.5±0.9 0.0 0.0 0.9±0.6 1.0±0.4 0.9±0.5 0.9±0.4 0.3±0.1 0.1±0.1 0.2±0.2 C16:1(n-7) 2.3±2.0 2.3±0.6 2.3±0.2 1.0±0.7 5.8±1.5 1.7±0.4 9.2±6.5 1.7±0.7 1.9±0.2 2.7±0.2 4.8±0.3 1.0±0.5 C16:1(n-9) n. d. n. d. n. d. n. d. 2.9±0.6 3.7±2.0 1.8±0.2 2.0±0.4 3.7±0.5 1.8±0.2 5.3±0.4 1.6±0.4 C17:1(n-7) n. d. n. d. n. d. n. d. 0.2±0.1 n. d. n. d. n. d. 0.2±0.1 0.5±0.1 n. d. n. d. C18:1(n-9) 1.3±0.3 2.0±1.5 1.0±0.1 1.9±1.6 4.1±0.6 3.4±0.6 3.7±0.8 16.7±0.1 2.9±0.5 6.0±0.4 5.3±0.3 6.9±0.6 C18:1(n-9) trans

n. d. n. d. 1.7±0.7 n. d. n. d. n. d. n. d. 1.5±0.6 n. d. 3.5±0.1 1.5±1.1 1.6±0.6

C20:1(n-9) 1.3±0.7 n. d. 1.4±0.4 0.5±0.2 0.6±0.4 0.0 0.8±0.4 n. d. n. d. n. d. n. d. n. d. C22:1(n-9 0.7±0.4 n. d. 1.1±0.4 n. d. n. d. 0.3±0.1 0.0 n. d. n. d. 0.4±0.2 n. d. n. d.C18:2(n-6) 7.1±1.8 12.8±5.0 11.4±0.3 4.9±1.9 4.6±0.4 12.3±0.6 8.6±0.7 8.0±2.0 17.2±3.8 8.3±1.0 7.6±0.6 9.2±1.2 C18:3(n-6) 0.8±0.2 1.1±0.4 0.9±0.1 1.4±0.4 3.0±0.4 8.9±1.7 9.3±4.1 6.1±3.5 2.5±0.4 2.2±0.2 2.8±0.2 1.1±0.4 C18:3(n-3) 19.8±8.9 11.6±3.2 6.8±1.5 25.3±2.4 16.1±2 9.0±3.8 6.5±0.7 6.4±3.5 16.1±7.6 n. d. n. d. n. d. C18:4(n-3) 2.9±0.6 1.8±0.8 1.6±0.3 3.6±0.3 0.7±0.4 1.8±1.3 1.4±0.3 6.8±3.4 0.9±0.5 n. d. 23.5±0.6 9.4±0.4 C20:3(n6) 0.9±0.4 0.7±0.3 0.7±0.1 n. d. 0.3±0.1 1.0±0.7 1.2±0.7 1.5±0.5 0.6±0.1 1.2±0.1 1.7±0.1 1.5±1.3 C20:3(n3) 1.0±0.5 0.8±0.3 0.9±0.3 n. d. 0.2±0.1 0.7±0.7 0.4±0.2 1.0±0.4 n. d. 1.2±0.1 1.9±0.3 0.5±0.5 C20:4(n-6) 1.1±0.3 3.7±1.1 7.9±1.2 0.8±0.5 10.7±0.8 5.8±1.4 4.1±0.5 5.0±0.2 n. d. 9.2±0.8 10.6±0.6 15.4±1.3 C20:5(n-3) 11±4 9.3±3.2 11.4±1.0 1.3±0.8 6.0±0.4 2.9±0.5 2.7±0.5 2.1±1.2 3.4±1.4 1.2±0.1 1.2±0.2 1.5±0.3 C22:6(n-3) 5.1±2.1 5.9±1.9 7.1±0.9 1.2±0.8 0.9±0.4 2.3±0.9 3.5±1.7 3.6±0.8 1.9±0.6 n. d. n. d. n. d. n.d.- not detected.

Table continued..

Appendix

248

FAs 37 38 39 40 41 42 43 44 45 46 47 48 C12:0 1.4±0.9 0.6±0.3 0.6±0.1 n. d. 0.3±0.1 0.4±0.2 0.4±0.2 0.6±0.2 0.2±0.1 0.5±0.1 1.1±0.4 0.6±0.1 C14:0 4.6±0.3 5.1±1.0 8.2±1.0 7.4±1.0 14.9±0.5 10.7±1.2 24.7±4.0 14.1±1.2 12.3±0.2 7.5±0.7 5.4±0.2 5.5±0.7 C15:0 0.8±0.1 0.7±0.5 0.9±0.1 2.1±0.6 0.7±0.1 0.4±0.1 0.9±0.2 0.8±0.1 0.7±0.2 0.7±0.1 1.9±2.2 0.7±0.1 C16:0 41.5±4.9 18.8±1.3 24.5±9.8 18.4±2.0 20.1±0.4 19±1.2 27.7±1.2 28.9±2.1 35±3.0 36.9±1.0 31.7±4.1 33.7±0.4 C17:0 0.6±0.2 0.5±0.1 0.6±0.4 0.5±0.1 0.3±0.1 0.1±0.01 0.5±0.2 0.3±0.1 0.1±0.1 0.3±0.1 0.4±0.1 0.4±0.1 C18:0 4.5±0.8 1.8±0.7 2.6±0.4 4.8±0.1 2.7±1.1 2.1±0.4 3.5±3.1 1.5±0.2 2.1±0.2 2.2±1.7 4.0±0.2 6.6±0.8 C20:0 0.4±0.1 0.6±0.3 0.8±0.5 1.4±0.3 0.3±0.1 1.0±0.2 0.7±0.6 0.8±0.1 0.4±0.1 0.4±0.01 0.4±0.2 0.7±0.2 C22:0 1.0±0.2 0.3±0.1 0.4±0.1 1.3±0.3 0.4±0.1 0.3±0.1 0.5±0.1 0.5±0.2 0.1±0.1 0.6±0.01 0.8±0.3 0.7±0.1 C24:0 1.0±0.6 0.5±0.2 0.4±0.1 0.8±0.1 0.2±0.1 0.2±0.01 0.5±0.1 0.7±0.2 0.2±0.01 0.4±0.01 0.6±0.2 0.6±0.1 C16:1(n-7) 0.7±0.2 2.8±0.5 2.1±0.5 2.5±0.5 0.7±0.2 10.1±0.5 1.7±0.5 1.0±0.3 8.3±0.1 5.3±0.4 3.2±0.5 7.4±0.6 C16:1(n-9) 2.8±0.7 2.0±0.4 4.2±3.6 2.0±0.2 1.7±0.1 1.8±0.3 1.8±1.2 1.5±0.4 4.1±6.5 0.0 0.0 0.0 C17:1(n-7) 0.8±0.3 0.4±0.5 0.6±0.4 0.0 0.2±0.1 0.1±0.1 0.6±0.3 0.3±0.1 0.4±0.2 0.3±0.2 0.4±0.1 0.3±0.2 C18:1(n-9) 3.5±0.2 5.6±1.4 7.5±3.3 5.7±0.4 6.4±0.4 6.4±0.3 11.4±0.3 9.2±1.1 12.6±0.3 7.1±0.1 7.1±0.8 6.8±0.3 C18:1(n-9) trans

3.5±0.5 2.3±0.9 2.0±0.7 1.8±0.1 2.8±0.1 3.3±0.8 n. d. n. d. n. d. 1.4±0.4 1.4±1.1 1.6±0.2

C20:1(n-9) n. d. n. d. n. d. n. d. n. d. n. d. 0.8±0.1 0.3±0.2 n. d. 1.9±0.2 1.9±0.1 1.0±0.2 C22:1(n-9 n. d. n. d. n. d. n. d. 0.1±0.1 0.3±0.5 0.7±0.3 0.3±0.1 n. d. 1.7±0.1 1.1±0.1 1.2±0.1 C18:2(n-6) 4.3±0.1 3.0±0.3 3.8±1.5 6.3±0.4 5.3±0.4 0.7±0.4 3.2±1.7 2.4±0.7 0.6±0.3 4.0±0.3 3.3±0.3 5.0±0.4 C18:3(n-6) 0.7±0.5 1.7±0.8 1.6±0.4 3.6±0.5 1.3±0.1 2.0±0.1 3.7±3.0 4.0±0.2 0.4±0.2 n. d. 0.5±0.1 n. d. C18:3(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.C18:4(n-3) 3.3±1.3 18.7±2.0 13.4±6.0 15.9±1.5 17.4±0.4 22.4±0.6 0.0 3.6±0.7 4.8±0.5 4.5±0.5 8.2±2.3 5.4±0.5 C20:3(n6) 1.5±0.3 2.8±0.7 2.8±0.4 1.8±0.3 2.8±0.1 0.3±0.1 3.6±1.4 0.9±0.1 1.3±0.3 0.6±0.1 0.7±0.1 0.9±0.1 C20:3(n3) 1.7±0.6 5.2±1.1 3.4±2.5 2.3±0.2 8.0±0.3 1.7±1.2 3.9±3.0 1.4±0.2 2.5±0.4 1.0±0.1 1.2±0.5 0.7±0.1 C20:4(n-6) 19.0±1.0 14.5±1.1 10.9±2.1 12.2±0.8 9.7±0.2 9.0±0.2 6.2±3.3 10.2±1.4 11.2±1.3 18.1±0.6 18.7±0.7 15.6±0.3 C20:5(n-3) 2.4±0.9 12.2±0.2 8.4±3.7 9.4±0.2 2.8±0.3 7.2±0.1 2.6±0.2 16.7±1.2 2.9±0.5 3.5±0.4 5.9±1.5 3.8±0.3 C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.n.d.- not detected.

Table continued..

Appendix

249

FAs 49 50 51 52 53 54 55 56 57 58 59 60 C12:0 0.8±0.1 0.7±0.1 0.7±0.2 0.3±0.1 0.3±0.1 0.8±0.1 0.7±0.2 0.2±0.1 0.0 0.1±0.01 1.3±0.1 1.0±0.5 C14:0 3.7±0.5 6.6±0.3 4.6±1.7 5.4±1.3 5.0±1.2 7.1±0.3 6.3±0.2 7.7±0.8 8.7±0.3 1.9±0.1 4.2±0.1 5.2±0.7 C15:0 0.8±0.6 0.7±0.1 0.6±0.1 0.4±0.1 0.4±0.1 0.5±0.1 0.8±0.1 0.4±0.01 2.1±2.2 0.4±0.2 2.1±0.1 0.9±0.3 C16:0 37.7±3.9 28.9±1.7 35.4±4.3 26±17.3 26.4±0.5 34.5±6.0 26.8±1.8 23.5±1.3 41.5±3.1 27.8±1.5 58.1±1.5 44.8±2.7 C17:0 0.3±0.2 0.3±0.1 0.3±0.1 0.2±0.1 0.4±0.2 0.6±0.8 0.4±0.3 0.2±0.01 0.4±0.2 0.2±0.01 1.9±0.1 1.1±0.6 C18:0 7.0±5.1 4.3±0.3 0.7±0.5 2.3±0.6 6.6±2.1 1.8±0.1 2.6±0.5 1.5±0.4 3.6±0.5 2.6±0.1 5.6±0.2 4.8±3.3 C20:0 0.6±0.3 0.4±0.1 0.2±0.1 0.2±0.1 0.4±0.01 0.4±02 0.3±0.1 0.6±0.1 1.4±0.7 0.2±0.2 1.1±0.1 0.7±0.4 C22:0 1.0±0.6 0.4±0.1 0.5±0.1 0.6±0.2 0.7±0.01 0.9±0.4 0.6±0.2 1.4±0.6 0.8±0.2 n. d. 1.0±0.8 1.0±0.5 C24:0 0.5±0.4 0.6±0.1 0.3±0.1 0.4±0.1 0.4±0.01 0.4±0.1 0.6±0.3 0.2±0.1 1.1±0.4 n. d. 2.9±0.3 0.5±0.2 C16:1(n-7) 2.6±0.3 3.0±0.2 5.4±1.2 7.6±1.6 1.8±0.2 4.4±0.2 4.7±0.3 1.6±0.6 1.4±0.4 1.4±0.3 2.5±0.2 3.2±0.8 C16:1(n-9) n. d. n. d. n. d. n. d. 2.3±0.1 n. d. 1.3±0.2 2.6±1.1 1.4±0.2 n. d. n. d. n. d.C17:1(n-7) 0.3±0.1 n. d. 0.2±0.1 0.4±0.1 n. d. 0.4±0.3 n. d. 0.1±0.01 n. d. n. d. n. d. 0.5±0.2 C18:1(n-9) 6.8±1.0 7.4±0.3 24.2±9.8 7.6±1.9 9.4±0.8 6.6±1.4 6.2±0.4 7.6±0.5 6.6±1.2 1.4±0.1 3.9±0.2 4.4±0.6 C18:1(n-9) trans

n. d. n. d. 1.3±0.3 n. d. n. d. n. d. n. d. n. d. 1.1±0.1 1.4±0.2 4.3±0.1 2.7±0.1

C20:1(n-9) 1.2±0.3 1.9±0.1 1.1±0.1 1.8±0.4 0.2±0.01 1.7±0.7 n. d. n. d. n. d. n. d. n. d. n. d.C22:1(n-9 0.4±0.3 0.8±0.1 0.7±0.1 1.2±0. 2.2±0.5 0.6±0.1 n. d. 0.2±0.1 n. d. n. d. n. d. n. d.C18:2(n-6) 5.1±1.0 5.2±0.4 0.2±0.01 7.7±1.5 15.4±0.3 4.8±1.0 11±0.6 2.0±0.9 5.4±1.1 2.2±0.5 1.8±0.8 9.2±0.9 C18:3(n-6) 0.8±0.1 1.0±0.1 0.4±0.01 0.7±0.1 0.9±0.1 0.6±0.3 2.3±0.2 1.7±0.1 1.4±0.3 n. d. n. d. 0.6±0.3 C18:3(n-3) n. d. n. d. n. d. n. d. n. d. 2.9±2.0 n. d. n. d. n. d. n. d. n. d. n. d.C18:4(n-3) 11.5±2.8 12.3±0.9 0.9±1.1 8.8±1.8 4.3±0.3 6.9±3.9 11.6±1.4 23.5±0.4 6.5±1.6 n. d. n. d. n. d.C20:3(n6) 0.9±0.3 0.9±0.1 4.3±0.1 0.9±0.2 4.6±0.3 0.8±0.2 0.9±0.2 0.5±0.01 1.2±0.2 1.9±0.3 2.3±0.1 1.2±0.3 C20:3(n3) 0.9±0.4 0.7±0.1 0.6±0.1 1.3±0.2 0.5±0.01 1.0±0.2 1.2±0.4 1.3±0.2 n. d. n. d. n. d. n. d.C20:4(n-6) 14.1±4.5 18.0±0.3 2.3±1.2 20.9±5.1 16.9±0.9 17.6±4.8 15.4±0.1 13.6±0.6 9.4±1.3 58.3±0.5 7.3±0.1 17.6±4.2 C20:5(n-3) 3.1±0.7 6.4±0.7 14.9±1.0 4.7±1.4 0.4±0.3 4.2±1.6 5.2±0.4 9.3±0.1 5.9±1.5 0.3±0.1 n. d. n. d.C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.n.d.- not detected.

Table continued..

Appendix

250

FAs 61 62 63 64 65 66 67 68 69 70 71 C12:0 0.2±0.01 0.5±0.2 0.4±0.2 n. d. 0.4±0.01 n. d. 0.3±0.1 0.4±0.1 n. d. n. d. 0.9±0.3 C14:0 1.1±0.1 1.4±0.2 2.4±0.2 5.2±2.1 2.1±1.6 4.1±0.2 3.2±1.3 9.8±0.7 10±0.1 3.4±0. 8.6±1.6 C15:0 0.6±0.01 0.6±0.1 0.6±0.1 0.3±0.6 0.5±0.1 1.4±0.3 0.3±0.1 0.9±0.2 1.5±0.2 1.0±0.1 1.3±0.2 C16:0 25.1±2.3 49.5±3.8 54.9±1.9 31.5±3.3 39.6±2.9 46.8±3.4 21.6±2.7 44.5±4.0 50.3±1.5 53.1±2.6 38.1±3.4 C17:0 0.2±0.01 0.4±0.1 0.4±0.1 0.0 0.2±0.1 6.6±1.1 0.2±0.01 0.2±0.1 0.0 1.9±3.2 0.2±0.1 C18:0 2.3±0.2 4.8±2.0 2.3±1.7 3.1±1.0 2.2±0.4 3.9±0.8 12±8.1 2.1±0.9 2.1±0.2 4.0±2.7 10.9±0.2 C20:0 0.2±0.01 0.7±0.4 0.4±0.3 0.0 0.5±0.3 0.8±0.3 0.9±0.1 0.5±0.2 0.5±0.1 0.8±0.2 0.5±0.1 C22:0 0.4±0.01 1.3±0.7 0.4±0.2 0.0 0.5±0.3 0.5±0.1 0.7±0.4 0.5±0.3 0.7±0.2 1.1±0.5 0.6±0.1 C24:0 0.8±0.1 1.6±0.7 0.7±0.4 0.0 0.6±0.4 0.6±0.1 0.7±0.01 0.0 0.0 0.5±0.4 0.3±0.1 C16:1(n-7) 0.7±0.01 1.1±0.4 0.7±0.3 2.3±1.8 1.0±1.0 7.0±2.9 9.3±1.7 7.9±0.5 2.1±0.8 1.6±1.3 0.9±0.3 C16:1(n-9) n. d. n. d. n. d. 2.0±0.1 1.6±0.9 5.1±1.1 n. d. n. d. n. d. n. d. 4.4±1.0 C17:1(n-7) n. d. 0.3±0.1 0.3±0.1 n. d. 0.2±0.1 0.7±0.2 n. d. 0.1±0.1 n. d. n. d. n. d. C18:1(n-9) 1.1±0.01 2.3±1.5 0.4±0.1 2.5±1.3 2.6±0.7 3.0±0.6 3.1±2.7 3.6±0.2 5.0±0.4 1.0±0.4 6.1±0.1 C18:1(n-9) trans

1.4±0.1 4.8±0.6 3.4±0.6 4.9±3.0 3.0±0.8 2.0±0.7 1.8±0.2 6.6±0.6 7.9±1.5 3.6±0.7 4.3±0.3

C20:1(n-9) 0.3±0.01 0.6±0.3 0.2±0.1 n. d. 0.5±0.3 n. d. n. d. n. d. n. d. n. d. n. d. C22:1(n-9 0.0 0.1±0.1 0.2±0.1 n. d. 0.2±0.1 n. d. 5.4±0.4 n. d. n. d. n. d. 1.6±0.3 C18:2(n-6) 1.9±0.2 2.1±0.9 1.5±0.8 5.1±1.5 1.6±0.6 n. d. 23.2±4.2 2.2±0.3 2.3±0.8 11.4±2.4 n. d.C18:3(n-6) n. d. 0.8±0.4 n. d. n. d. 0.4±0.2 1.0±0.5 n. d. 0.3±0.1 n. d. n. d. n. d.C18:3(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.C18:4(n-3) n. d. n. d. n. d. n. d. 2.4±0.1 n. d. 11.8±1.8 n. d. n. d. n. d. n. d.C20:3(n6) 4.9±0.1 5.4±0.5 3.6±0.6 1.3±1.1 1.9±0.6 0.9±0.4 0.4±0.1 0.7±0.2 2.7±0.9 n. d. 0.6±0.3 C20:3(n3) n. d. 0.2±0.1 2.9±2.4 n. d. n. d. n. d. n. d. n. d. 2.8±0.8 n. d. n. d. C20:4(n-6) 58.8±2.5 20.8±4.0 23.9±1.6 41.1±3.1 12.9±2.5 10.7±3.8 5.0±0.8 7.3±2.6 5.6±0.8 15.1±1.2 8.7±1.0 C20:5(n-3) 0.0 0.5±0.2 0.4±0.3 0.8±0.3 25±3.7 5.0±3.0 n. d. 12.7±2.7 6.7±2.8 1.7±0.4 12.0±1.3 C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.n.d.- not detected.

Table continued..

Appendix

251

FAs 72 73 74 75 76 77 78 79 80 81 82 C12:0 0.3±0.1 0.3±0.1 0.9±0.1 0.3±0.1 0.5±0.3 0.5±0.3 n. d. 1.0±0.2 0.4±0.2 3.1±0.8 2.0±0.1 C14:0 0.9±0.1 1.0±0.1 1.8±0.1 1.2±0.3 1.1±0.2 4.5±0.7 4.6±0.8 7.0±0.7 6.4±1.3 9.3±4.3 11.3±0.7 C15:0 0.4±0.1 0.7±.1 0.7±0.1 0.9±0.1 0.8±0.1 0.5±0.3 1.7±0.2 0.9±0.3 0.8±0.1 2.7±0.9 0.9±0.1 C16:0 30.1±1.1 31±3.9 26.5±1.1 34.6±3.0 29.9±0.7 39.6±3.0 34.3±5.4 41.2±1.7 34.8±0.9 31±2.8 39.7±0.8 C17:0 0.2±0.01 0.5±0.1 0.4±0.1 0.4±0.2 0.6±0.3 0.4±0.2 1.0±0.1 0.4±0.1 0.3±1.2 1.1±0.8 0.4±0.1 C18:0 1.5±0.2 3.1±0.4 5.3±0.1 3.2±0.8 3.5±0.3 2.8±0.4 6.6±0.5 4.6±1.0 3.5±0.1 6.8±0.5 4.5±0.6 C20:0 0.2±0.01 0.2±0.2 0.2±0.1 0.2±0.1 0.4±0.2 0.2±0.01 0.7±0.1 0.3±0.01 0.2±0.3 0.3±0.3 0.2±0.01 C22:0 0.2±0.02 0.5±0.3 0.2±0.1 0.5±0.4 0.7±0.5 0.4±0.1 0.8±.1 0.5±0.2 0.8±0.2 1.8±0.3 0.5±0.1 C24:0 0.6±0.1 n. d. n. d. n. d. 0.00.8 0.4±.1 0.7±.1 0.7±0.3 0.4±0.7 0.8±0.3 0.3±0.1 C16:1(n-7) 0.7±0.1 0.3±0.1 0.5±0.1 1.3±0.9 1.1±0.1 1.5±.1 1.0±.1 3.1±0.6 4.1±0.1 3.6±3.0 3.0±0.2 C16:1(n-9) 0.7±0.1 0.8±0.1 1.6±0.3 0.6±0.1 0.8±0.1 n. d. 7.9±0.8 n. d. 3.2±0.3 n. d. 4.3±0.1 C17:1(n-7) 0.1±0.01 0.1±0.1 0.2±0.1 0.3±0.3 0.3±0.1 0.2±0.1 4.7±.1 n. d. 0.7±0.1 n. d. 0.2±0.1 C18:1(n-9) 1.2±0.1 2.9±0.4 5.7±1.0 1.8±1.3 2.3±0.8 2.4±0.2 1.1±0.2 4.5±0.8 4.4±1.0 3.8±1.8 3.5±0.4 C18:1(n-9) trans

1.0±0.1 3.2±0.4 2.1±0.1 2.2±1.5 2.1±0.1 3.7±0.2 1.8±0.1 6.3±0.7 13±1.3 2.5±0.3 4.3±0.3

C20:1(n-9) 0.7±0.2 1.4±0.3 1.6±0.1 1.7±0.2 1.4±0.1 n. d. n. d. n. d. n. d. n. d. 0.4±0.2 C22:1(n-9 0.8±0.1 1.4±0.3 1.3±0.2 1.3±0.3 1.4±0.5 n. d. n. d. 2.0±1.2 0.8±0.2 n. d. 1.3±0.6 C18:2(n-6) 2.7±1.2 4.9±0.5 4.3±0.5 3.7±0.1 4.3±1.0 4.5±1.3 1.3±0.2 7.9±1.7 6.1±1.6 24.8±0.8 6.7±0.5 C18:3(n-6) 0.3±0.1 0.7±0.3 0.7±0.1 0.8±0.2 1.6±0.3 0.6±0.1 n. d. n. d. 0.6±0.2 1.7±0.7 0.7±0.1 C18:3(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.C18:4(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. 3.2±0.4 1.4±0.2 C20:3(n6) 2.4±0.6 2.6±0.2 3.7±0.2 2.7±0.2 3.7±0.4 2.3±0.1 2.2±1.0 0.5±0.1 0.6±0.2 0.0 0.4±0.1 C20:3(n3) 0.2±0.1 0.3±0.2 0.1±0.01 0.5±0.01 0.5±0.1 n. d. n. d. n. d. n. d. n. d. n. d.C20:4(n-6) 16.7±1.2 21±0.5 17.4±1.1 19.3±3.0 19.5±1.6 35±3.2 25.1±3.8 9.0±1.8 12.9±0.9 1.3±0.2 3.8±1.0 C20:5(n-3) 35.9±2.1 23.6±3.1 23.7±1.2 20.8±5.8 21.3±2.3 0.5±0.1 5.0±0.8 10.3±1.5 5.6±0.9 2.2±1.3 10±0.5 C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.n.d.- not detected.

Table continued..

Appendix

252

FAs 83 84 85 86 87 88 89 90 91 92 93 C12:0 0.3±0.2 0.5±0.1 0.3±0.2 1.7±0.1 0.8±0.2 0.5±0.3 0.5±0.1 1.0±0.6 0.3±0.1 3.0±0.9 3.1±0.8 C14:0 2.8±0.7 2.7±0.5 3.2±0.4 3.1±0.5 1.9±0.4 2.2±1.0 8.6±0.1 5.8±0.5 2.5±2.3 6.8±5.1 13.4±3.0 C15:0 0.7±0.1 0.5±0.3 0.8±0.1 1.1±0.4 0.8±0.5 0.5±0.2 0.5±0.1 0.9±0.1 0.3±0.01 0.7±0.1 0.7±0.2 C16:0 37.7±1.3 37±1.4 26.4±2.0 29.6±0.04 41.2±0.8 40±2.8 34.3±1.2 38.8±3.2 45.1±4.4 37±2.9 38.5±2.7 C17:0 0.2±0.1 0.4±0.3 0.2±0.1 0.1±0.01 0.7±0.4 0.2±0.1 0.2±0.1 0.3±0.1 n. d. n. d. 0.5±0.2 C18:0 3.3±0.8 2.1±0.7 1.7±0.2 3.5±0.1 3.2±0.5 2.6±0.4 1.9±0.3 3.5±0.5 1.0±0.1 2.6±0.8 3.0±0.9 C20:0 0.4±0.3 0.3±0.02 0.1±0.1 n. d. 0.4±0.1 0.4±0.2 0.2±0.1 0.2±0.1 0.2±0.1 n. d. 0.4±0.2 C22:0 0.6±0.1 0.6±0.1 0.2±0.1 0.2±0.1 0.5±0.1 0.3±0.01 0.2±0.1 0.5±0.3 0.1±0.1 n. d. 0.5±0.2 C24:0 1.5±1.7 0.6±0.4 0.1±0.01 0.0 0.4±0.3 0.5±0.4 0.0 0.2±0.1 0.2±0.1 n. d. 0.5±0.1 C16:1(n-7) 4.9±0.2 5.4±0.5 4.6±0.7 0.6±0.1 1.2±0.4 0.9±0.1 6.1±3.4 2.5±0.7 0.7±0.3 1.7±0.6 1.7±0.3 C16:1(n-9) n. d. n. d. n. d. n. d. n. d. 2.0±0.2 n. d. 1.5±0.9 n. d. 1.5±0.15 3.1±1.1 C17:1(n-7) n. d. 0.0 0.4±0.2 n. d. n. d. n. d. n. d. 0.6±0.1 n. d. n. d. n. d.C18:1(n-9) 2.4±0.1 2.1±0.2 3.1±0.5 3.4±0.5 3.5±0.3 2.6±0.7 2.6±0.8 3.5±0.4 2.9±1.2 3.3±0.6 3.4±1.0 C18:1(n-9) trans

6.0±1.8 4.2±1.2 16.9±2.1 4.1±1.7 8.3±1.0 4.8±1.0 4.5±1.4 3.7±0.4 0.9±0.2 4.9±1.0 3.5±1.0

C20:1(n-9) n. d. n. d. 0.3±0.2 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.C22:1(n-9 n. d. n. d. n. d. n. d. n. d. n. d. 0.4±0.3 n. d. n. d. n. d. n. d.C18:2(n-6) 1.9±1.5 2.8±0.3 2.2±0.2 5.8±1.0 3.8±0.5 3.9±1.0 2.9±0.2 3.7±0.4 0.3±0.1 3.1±0.4 n. d.C18:3(n-6) 1.1±0.6 1.1±0.3 1.4±1 1.3±0.8 0.0 1.6±0.9 0.7±0.3 0.4±0.2 n. d. n. d. n. d.C18:3(n-3) n. d. n. d. 0.9±0.3 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.C18:4(n-3) 1.6±.07 0.8±0.4 0.6±0.6 n. d. n. d. 2.1± 1.0±0.1 0.8±0.1 n. d. n. d. n. d.C20:3(n6) 3.7±0.5 1.4±1.0 0.8±0.2 1.0±0.3 0.7±0.2 0.5±0.3 0.3±0.1 1.1±0.2 0.3±0.1 3.6±2.3 1.0±0.5 C20:3(n3) n. d. n. d. 0.2±0.1 n. d. n. d. n. d. n. d. 0.3±0.1 n. d. n. d. n. d.C20:4(n-6) 10.8±5.4 11.1±2.7 35.4±1.9 25.2±6.3 19.8±0.5 8.2±2.2 15.5±1.1 9.2±1.5 20.6±4.4 18.2±2.2 14.3±2.1 C20:5(n-3) 20.2±6.8 26.4±3.6 0.4±0.3 19.4±4.1 12.9±1.1 25.8±6.1 19.6±4.3 21.2±2.7 24.8±1.5 14.1±1.4 12.5±1.9 C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.n.d.- not detected.

Table continued..

Appendix

253

FAs 94 95 96 97 98 99 100 LSD (0.1%)A C12:0 0.5±0.5 1.2±0.3 0.6±0.1 1.0±0.1 0.8±0.4 0.7±0.8 2.3±0.8 0.9 C14:0 8.0±2.5 7.5±5.6 11.9±0.9 4.2±0.4 4.3±0.8 5.0±0.7 5.3±0.6 4.2 C15:0 0.4±0.3 1.0±0.1 1.3±0.1 1.0±0.4 1.5±1.0 1.3±0.2 1.4±0.4 1.2 C16:0 34.3±1.8 45.8±15.4 56.6±2.7 37.5±1.1 36.9±2.9 54.3±3.7 44.2±4.8 10.7 C17:0 0.2±0.01 0.3±0.1 0.3±0.01 0.6±0.1 0.9±0.6 0.4±0.1 2.6±2.6 1.1 C18:0 3.9±2.9 4.5±1.6 2.7±0.8 3.6±1.3 4.3±0.4 3.3±1.8 7.5±2.1 4.4 C20:0 0.2±0.01 0.4±0.2 0.3±0.02 0.6±0.3 1.2±0.3 0.2±0.1 0.4±0.1 0.9 C22:0 0.3±0.1 0.7±0.4 0.5±0.1 1.2±0.6 1.6±0.4 0.3±0.01 1.0±0.01 0.8 C24:0 0.5±0.2 0.6±0.1 0.3±0.1 1.8±0.1 1.1±0.4 0.3±0.01 0.8±0.2 1.5 C16:1(n-7) 4.7±0.8 2.5±1.3 3.3±0.4 1.8±0.5 2.5±0.3 2.0±0.1 1.1±0.1 4.6 C16:1(n-9) 1.7±0.7 3.5±1.3 1.7±1.7 3.3±0.2 4.5±1.8 2.9±0.4 6.7±2.4 3.9 C17:1(n-7) n. d. 0.1±0.01 0.5±0.2 n. d. n. d. n. d. 0.7±0.1 0.4 C18:1(n-9) 3.7±0.9 3.8±1.2 4.1±0.5 2.8±0.3 3.4±1.4 2.8±1.0 4.0±0.4 4.0 C18:1(n-9) trans

4.4±0.8 3.7±0.8 4.9±0.3 6.3±1.6 7.2±1.7 3.4±1.1 3.9±1.9 2.1

C20:1(n-9) n. d. n. d. n. d. n. d. 2.0±0.5 0.6±0.1 0.9±0.1 0.5 C22:1(n-9 0.1±0.01 n. d. 0.4±0.1 n. d. n. d. n. d. 0.9±0.1 0.7 C18:2(n-6) 4.4±0.7 2.4±0.7 1.8±0.1 6.7±1.1 8.1±0.7 6.1±2.1 4.9±1.2 3.8 C18:3(n-6) 0.1±0.01 0.0 0.3±0.1 1.3±0.4 0.8±0.6 n. d. 1.3±0.3 2.3 C18:3(n-3) 0.0 n. d. n. d. n. d. n. d. n. d. n. d. 6.4 C18:4(n-3) 1.4±0.1 n. d. n. d. n. d. 1.9±0.8 n. d. 1.8±0.2 5.6 C20:3(n6) 0.2±0.1 0.5±0.3 0.2±0.1 1.4±0.4 1.4±0.4 0.5±0.1 0.5±0.1 2.0 C20:3(n3) n. d. n. d. n. d. n. d. n. d. n. d. 0.3±0.1 1.5 C20:4(n-6) 14.6±0.9 12.1±5.0 4.2±0.3 2.8±0.6 4.4±0.9 9.2±3.1 1.2±0.8 5.3 C20:5(n-3) 16.2±2.2 9.4±2.0 3.9±0.1 22.2±1.5 10.9±2.1 5.4±1.3 5.1±1.7 5.2 C22:6(n-3) n. d. n. d. n. d. n. d. n. d. n. d. n. d. 1.2 n.d.- not detected; A: LSD values obtained from ANOVA, The values in a row are significantly different at p≤0.001.

Appendix

254

Appendix IV

Percent relative abundance of polar lipid molecular species present in different macroalgae as determined by ESI-MS scans.

Mass Compound name UL UF UT UB UTb GC GCY GFO GD GS GTx GFU PT ST CI SA

766.1 MGDG (34:5) 28 98 58 28 98 10 8 10 8 8 18 5 10 8 32 12 768.1 MGDG (34:4) 70 50 45 22 28 15 10 12 10 5 8 8 12 10 24 15 770.5 MGDG (34:3) 27 8 20 20 18 20 8 10 18 5 12 5 15 6 22 24 772 MGDG (34:2) 20 15 22 10 8 10 5 8 15 5 8 5 28 15 18 20 794 MGDG (36:8) 8 5 8 21 5 15 10 8 12 5 18 5 95 10 15 18 796 MGDG (36:6) 6 5 5 12 5 12 8 8 10 5 8 5 98 80 12 24 823 MGDG (38:5) 5 5 8 14 5 18 10 15 8 5 12 12 5 6 16 98 843.7 MGDG (40:9) 8 6 5 98 5 72 88 9 88 30 5 5 8 15 12 12 845 MGDG (40:8) 5 5 15 15 5 22 12 80 10 8 18 5 5 10 18 6 908.6 DGDG (32:3) 8 5 5 10 5 8 10 30 12 8 12 5 5 8 5 5 910 DGDG (32:2) 5 10 15 22 5 70 60 48 10 8 15 5 8 12 15 24 931 DGDG (34:6) 10 5 15 28 5 18 15 12 12 15 12 5 8 50 32 5 941.3 DGDG (34:1) 22 12 25 28 5 48 68 60 22 12 24 8 8 10 12 6 958 DGDG (36:8) 12 5 5 22 5 10 15 18 15 5 22 5 8 6 22 6 960 DGDG (36:6) 18 5 10 22 5 8 10 12 18 5 8 5 5 10 18 6 962 DGDG (36:5) 11 5 8 18 5 18 12 15 18 5 5 5 15 28 15 6 968 DGDG (36:4) 68 5 32 60 5 98 50 45 68 78 8 5 10 10 12 8 1004.6 DGDG (40:9) 5 5 8 10 5 12 12 12 12 8 70 5 5 6 8 5 1006.6 DGDG (40:8) 5 6 8 10 5 52 55 52 52 18 10 8 6 8 10 6 761.34 SQDG (30:2) 22 18 12 5 5 20 5 10 30 10 8 12 5 12 18 12 765.77 SQDG (30:0) 18 12 11 5 5 18 5 8 32 8 5 8 40 51 40 38 785.86 SQDG (32:4) 30 70 15 10 30 15 5 5 18 8 8 5 30 10 20 32 791.5 SQDG (32:1) 15 10 10 5 8 22 5 8 52 10 8 18 80 98 85 98 793.84 SQDG (32:0) 12 12 60 5 8 12 5 8 50 18 22 60 35 78 80 56 815.37 SQDG (34:3) 40 98 35 12 28 25 5 5 22 8 10 40 15 30 20 28 819 SQDG (38:9) 12 92 98 10 8 22 5 5 18 5 18 15 28 98 97 85 821 SQDG (38:8) 45 30 35 10 5 18 5 5 30 5 8 10 15 48 25 42 704.5 DGTS (32:4) 6 4 6 8 5 0 0 0 0 0 0 0 0 0 0 0 706.5 DGTS (32:3) 8 6 8 8 5 0 0 0 0 0 0 0 0 0 0 0 710.5 DGTS (32:1) 10 8 8 18 5 0 0 0 0 0 0 0 0 0 0 0 732 DGTS (34:4) 6 10 10 15 5 0 0 0 0 0 0 0 0 0 0 0 734 DGTS (34:3) 6 12 11 18 5 0 0 0 0 0 0 0 0 0 0 0 736.5 DGTS (34:2) 8 12 12 22 5 0 0 0 0 0 0 0 0 0 0 0 738.5 DGTS (34:1) 18 20 18 60 5 0 0 0 0 0 0 0 0 0 0 0 762 DGTS (36:3) 20 18 15 95 10 0 0 0 0 0 0 0 0 0 0 0 764 DGTS (36:2) 30 25 20 25 20 0 0 0 0 0 0 0 0 0 0 0 676.49 PC(28:1) 0 0 0 0 10 18 28 40 36 8 5 5 0 0 0 0 678.5 PC(28:0) 0 0 0 0 5 8 16 12 4 4 12 18 0 0 0 0 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-

Gracilaria salicornia, GTx-Gracilaria textorii, GFU- Gracilaria fergusonii, PT- Padina tetrastomatica, ST- Sargassum tenerrimum, CI- Cystoseira indica, SA- Spatoglossum asperum.

Table continued..

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255

Mass Compound name UL UF UT UB UZ GC GD GS GTx GFU GFO GCY PT ST CI SA

724.49 PC(32:5) 0 0 0 0 0 2 12 14 16 5 2 2 0 0 0 0

726.5 PC(32:4) 0 0 0 0 0 2 9 12 8 2 5 5 0 0 0 0

728.52 PC(32:3) 0 0 0 0 0 2 2 2 5 2 4 5 0 0 0 0

730.53 PC(32:2) 0 0 0 0 0 2 2 2 4 2 4 5 0 0 0 0

732.55 PC(32:1) 0 0 0 0 35 2 2 2 4 2 4 5 0 0 0 0

734.56 PC(32:0) 0 0 0 0 98 2 2 2 4 2 4 5 0 0 0 0

746.56 PC(33:1) 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0

748.58 PC(33:0) 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0

750.5 PC(34:6) 0 0 0 0 2 2 2 2 2 2 2 2 0 0 0 0

752.25 PC(34:5) 0 0 0 0 2 2 2 2 2 2 2 3 0 0 0 0

754.53 PC(34:4) 0 0 0 0 20 8 6 2 2 2 2 5 0 0 0 0

756.55 PC(34:3) 0 0 0 0 80 6 6 2 2 2 2 4 0 0 0 0

758.56 PC(34:2) 0 0 0 0 48 4 6 2 2 2 2 4 0 0 0 0

760.58 PC(34:1) 0 0 0 0 22 2 6 2 2 2 2 4 0 0 0 0

762.6 PC(34:0) 0 0 0 0 12 2 4 2 2 2 2 5 0 0 0 0

774.5 PC(36:8) 0 0 0 0 4 2 10 2 2 2 2 2 0 0 0 0

776.5 PC(36:7) 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0

778.5 PC(36:6) 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0

780.5 PC(36:5) 0 0 0 0 4 2 4 80 20 8 2 2 0 0 0 0

782.56 PC(36:4) 0 0 0 0 12 2 4 6 4 4 3 5 0 0 0 0

784.5 PC(36:3) 0 0 0 0 10 2 4 4 4 2 3 4 0 0 0 0

786.6 PC(36:2) 0 0 0 0 8 2 4 5 4 2 3 4 0 0 0 0

788.6 PC(36:1) 0 0 0 0 6 3 4 4 4 2 3 4 0 0 0 0

790.6 PC(36:0) 0 0 0 0 12 6 4 6 4 2 3 4 0 0 0 0

800.52 PC(38:9) 0 0 0 0 8 5 4 6 18 17 3 4 0 0 0 0

802.5 PC(38:8) 0 0 0 0 0 8 98 36 12 8 4 3 0 0 0 0

804.55 PC(38:7) 0 0 0 0 0 10 27 24 4 6 2 2 0 0 0 0

806.56 PC(38:6) 0 0 0 0 0 5 16 12 2 5 2 2 0 0 0 0 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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808.5 PC(38:5) 0 0 0 0 0 5 8 5 2 5 2 2 0 0 0 0

810.6 PC(38:4) 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0

812.6 PC(38:3) 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0

814.6 PC(38:2) 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0

816.64 PC(38:1) 0 0 0 0 2 2 2 15 2 2 2 2 0 0 0 0

818.6 PC(38:0) 0 0 0 0 4 2 2 8 2 2 2 2 0 0 0 0

826.53 PC(40:10) 0 0 0 0 0 2 2 6 4 2 4 2 0 0 0 0

828.5 PC(40:9) 0 0 0 0 0 2 2 4 4 2 4 3 0 0 0 0

830.5 PC(40:8) 0 0 0 0 0 2 2 4 4 2 2 3 0 0 0 0

832.5 PC(40:7) 0 0 0 0 0 2 2 4 4 2 2 4 0 0 0 0

834.6 PC(40:6) 0 0 0 0 0 2 2 4 4 2 4 2 0 0 0 0

836.6 PC(40:5) 0 0 0 0 0 2 2 4 4 2 5 4 0 0 0 0

838.63 PC(40:4) 0 0 0 0 0 2 2 4 4 2 5 8 0 0 0 0

840.64 PC(40:3) 0 0 0 0 4 2 2 8 5 2 5 5 0 0 0 0

842.6 PC(40:2) 0 0 0 0 0 2 2 6 5 2 4 0 0 0 0 0

844.6 PC(40:1) 0 0 0 0 0 2 2 6 5 2 4 0 0 0 0 0

846.6 PC(40:0) 0 0 0 0 0 2 2 4 5 2 4 0 0 0 0 0

852.5 PC(42:11) 0 0 0 0 0 2 2 20 10 8 2 2 0 0 0 0

854.56 PC(42:10) 0 0 0 0 0 2 2 8 6 6 2 2 0 0 0 0

856.5 PC(42:9) 0 0 0 0 0 2 2 6 5 4 2 2 0 0 0 0

858.6 PC(42:8) 0 0 0 0 0 2 2 4 4 3 2 2 0 0 0 0

860.6 PC(42:7) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

862.63 PC(42:6) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

864.6 PC(42:5) 0 0 0 0 4 2 2 25 2 3 2 2 0 0 0 0

866.6 PC(42:4) 0 0 0 0 4 2 2 12 2 3 2 2 0 0 0 0

874.72 PC(42:0) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

892.67 PC(44:5) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

440.0 LPC(12:0) 0 0 0 0 0 8 12 20 12 6 12 15 0 0 0 0 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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257


468.0 LPE(14:0) 0 0 0 0 0 2 8 8 5 2 6 5 0 0 0 0

494.32 LPC(16:1) 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0

496.34 LPC(16:0) 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0

508.33 LPC(17:1) 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0

542.32 LPC(20:5) 0 0 0 0 0 2 2 12 15 2 2 2 0 0 0 0

544.33 LPC(20:4) 0 0 0 0 0 2 2 6 8 4 4 2 0 0 0 0

546.35 LPC(20:3) 0 0 0 0 0 2 2 5 6 5 4 2 0 0 0 0

580.39 PE(24:0) 4 4 5 4 0 2 2 2 4 2 5 5 4 4 5 4

660.45 PE(30:2) 0 0 0 0 0 0 0 0 0 0 0 0 5 5 6 6

676.49 PE(31:1) 6 5 6 98 12 17 26 2 4 8 12 6 7 5 6 4

678.5 PE(31:0) 5 4 4 28 7 12 22 2 5 4 5 15 13 12 15 11

682.44 PE(32:5) 4 2 2 4 2 6 8 2 5 4 4 4 5 4 4 3

684.45 PE(32:4) 4 2 2 2 2 2 6 2 5 4 28 4 5 4 4 3

686.47 PE(32:3) 0 0 0 0 0 2 5 2 4 4 15 4 12 12 14 10

688.49 PE(32:2) 0 0 0 0 0 2 5 8 21 4 8 2 11 12 10 8

690.5 PE(32:1) 0 0 0 0 0 2 5 5 10 4 4 2 5 6 4 4

692.52 PE(32:0) 0 0 0 0 0 2 5 5 8 3 4 2 15 15 14 12

704.5 PE(33:1) 0 0 0 0 0 0 0 0 0 0 0 0 4 5 4 2

706.53 PE(33:0) 0 0 0 0 0 0 0 0 0 0 0 0 3 5 5 2

708.45 PE(34:6) 0 0 0 0 0 0 0 0 0 0 0 0 3 4 5 3

710.47 PE(34:5) 4 4 2 2 10 0 0 0 0 0 0 0 3 4 5 4

712.49 PE(34:4) 4 4 2 2 4 2 4 4 4 3 4 4 3 6 5 2

714.5 PE(34:3) 4 4 2 2 4 2 4 10 5 3 4 4 6 7 8 6

716.52 PE(34:2) 0 0 0 0 0 2 4 8 4 2 10 4 4 5 6 4

718.54 PE(34:1) 0 0 0 0 0 2 4 6 4 2 4 5 4 5 6 5

720.55 PE(34:0) 0 0 0 0 0 2 6 4 2 2 2 2 4 5 6 5

732.45 PE(36:8) 5 5 6 2 30 2 6 2 2 2 2 2 4 5 7 6

734.47 PE(36:7) 5 4 7 2 98 2 5 2 8 2 4 4 4 5 6 6 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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736.49 PE(36:6) 5 4 7 2 55 2 5 2 7 2 4 4 4 5 6 4

738.5 PE(36:5) 5 3 6 2 20 2 5 2 4 2 6 5 4 5 7 4

740.52 PE(36:4) 4 3 5 2 10 2 5 2 4 2 5 4 4 5 4 4

742.5 PE(36:3) 2 2 2 3 4 2 5 2 5 2 6 4 2 5 4 4

744.45 PE(36:2) 2 2 2 10 5 2 5 2 4 2 6 4 2 4 4 3

746.56 PE(36:1) 2 2 2 8 2 0 0 0 0 0 0 0 2 4 2 2

748.58 PE(36:0) 2 2 2 2 2 0 0 0 0 0 0 0 2 2 2 2

758.47 PE(38:9) 6 4 16 12 40 0 0 0 0 0 0 0 6 10 8 6

760.49 PE(38:8) 5 5 12 4 20 0 0 0 0 0 0 0 0 0 0 0

762.5 PE(38:7) 0 0 0 5 10 0 0 0 0 0 0 0 0 0 0 0

764.52 PE(38:6) 2 2 2 14 6 0 0 0 0 0 0 0 0 0 6 5

766.5 PE(38:5) 2 2 2 8 6 0 0 0 0 0 0 0 5 4 5 4

768.5 PE(38:4) 2 2 2 5 18 0 0 0 0 0 0 0 7 8 6 4

770.56 PE(38:3) 2 2 2 5 6 0 0 0 0 0 0 0 4 5 4 4

772.58 PE(38:2) 0 0 0 0 0 0 0 0 0 0 0 0 3 5 3 3

774.6 PE(38:1) 0 0 0 0 0 0 0 0 0 0 0 0 2 4 4 2

776.6 PE(38:0) 2 2 4 4 5 2 8 2 4 2 3 3 4 4 4 2

784.49 PE(40:10) 0 0 0 0 0 2 6 2 4 2 4 2 15 16 20 10

786.5 PE(40:9) 0 0 0 0 0 2 6 2 3 2 3 5 6 8 8 4

788.5 PE(40:8) 0 0 0 0 0 2 5 2 3 2 4 5 6 6 6 4

790.5 PE(40:7) 0 0 0 0 0 2 5 18 22 2 3 3 5 4 4 5

792.5 PE(40:6) 0 0 0 0 0 2 5 98 98 2 4 4 4 2 2 4

794.56 PE(40:5) 0 0 0 0 0 2 5 35 36 2 2 2 2 2 2 4

796.58 PE(40:4) 0 0 0 0 0 2 5 12 24 2 2 2 2 2 2 3

798.6 PE(40:3) 0 0 0 0 0 2 5 8 6 2 2 2 2 2 2 3

800.6 PE(40:2) 0 0 0 0 0 8 5 4 2 14 4 4 2 2 2 3

802.6 PE(40:1) 0 0 0 0 0 4 90 4 3 8 3 3 2 2 4 3

804.6 PE(40:0) 4 4 4 4 4 10 30 3 3 4 4 5 2 2 4 3 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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259


810.5 PE(42:11) 0 0 0 0 0 6 12 3 3 3 3 3 2 2 4 3

812.5 PE(42:10) 0 0 0 0 0 5 12 10 27 2 2 2 2 2 4 2

814.5 PE(42:9) 0 0 0 0 0 4 10 6 24 2 2 2 2 2 4 2

816.5 PE(42:8) 0 0 0 0 0 4 8 8 10 2 2 2 2 2 4 2

818.5 PE(42:7) 4 4 2 2 2 2 8 5 4 2 2 2 2 2 4 2

820.58 PE(42:6) 4 2 2 2 2 3 4 5 5 2 2 2 2 2 4 2

822.6 PE(42:5) 4 2 3 4 2 3 75 2 2 20 2 2 2 2 4 4

824.6 PE(42:4) 4 2 3 4 2 2 36 2 2 10 2 2 2 2 4 4

832.67 PE(42:0) 0 0 0 0 0 0 0 0 0 0 0 0 10 15 16 12

850.63 PE(44:5) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

852.6 PE(44:4) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

426.2 LPE(14:0) 0 0 0 0 0 0 0 0 0 0 0 0 8 8 12 8

440 LPE(15:0) 0 0 0 0 0 0 0 0 0 0 0 0 7 8 10 6

452 LPE(16:1) 0 0 0 0 0 0 0 0 0 0 0 0 4 6 5 3

454 LPE(16:0) 0 0 0 0 0 0 0 0 0 0 0 0 4 4 5 3

426.2 LPE(14:0) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

440 LPE(15:0) 0 0 0 0 0 4 12 4 5 6 10 15 0 0 0 0

452 LPE(16:1) 0 0 0 0 0 4 9 2 22 2 6 10 0 0 0 0

454 LPE(16:0) 0 0 0 0 0 4 8 2 8 2 6 5 0 0 0 0

508.3 LPE(20:1) 6 5 22 12 20 0 0 0 0 0 0 0 0 0 0 0

609.37 PG(24:0) 4 8 4 12 2 2 2 2 4 2 4 4 4 2 2 2

635.39 PG(26:1) 2 4 2 4 2 0 0 0 0 0 0 0 0 0 0 0

665.4 PG(28:0) 0 0 0 0 0 2 2 2 4 2 4 4 2 2 2 2

689.43 PG(30:2) 8 6 5 14 4 4 5 8 16 4 5 10 4 2 2 4

691.4 PG(30:1) 6 5 3 48 15 0 4 4 10 5 22 5 4 2 2 5

693.4 PG(30:0) 6 2 2 14 8 2 2 2 6 4 12 5 5 2 2 3 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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260


705.47 PG(31:2) 5 2 2 2 2 0 0 0 0 0 0 0 4 2 2 2

707.47 PG(31:1) 5 2 2 2 2 0 0 0 0 0 0 0 5 2 2 2

709.4 PG(31:0) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 2 2

711.42 PG(32:5) 0 0 0 0 0 0 0 0 0 0 0 0 6 2 2 2

713.4 PG(32:4) 0 0 0 0 0 0 0 0 0 0 0 0 5 2 2 2

715.4 PG(32:3) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 2 2

717.4 PG(32:2) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 2 2

719.4 PG(32:1) 0 0 0 0 0 2 4 5 5 2 50 6 4 2 2 2

721.4 PG(32:0) 0 0 0 0 0 2 2 2 5 2 30 5 4 2 2 2

733.5 PG(33:1) 4 4 2 2 2 2 2 2 5 3 15 4 5 2 2 4

735.5 PG(33:0) 4 4 2 2 2 2 2 2 5 4 12 5 5 2 2 2

737.4 PG(34:6) 4 12 2 2 6 2 2 2 6 3 10 4 5 2 4 3

739.4 PG(34:5) 8 10 5 4 6 2 2 2 5 3 8 4 5 2 3 4

741.4 PG(34:4) 7 8 20 5 25 2 2 2 4 2 4 3 5 4 4 4

743.4 PG(34:3) 8 6 16 4 18 2 2 2 4 2 5 3 5 5 2 4

745.4 PG(34:2) 6 6 6 3 15 2 2 2 4 2 16 3 5 3 2 4

747.4 PG(34:1) 5 7 5 3 8 2 2 2 4 2 11 0 5 2 2 5

749.4 PG(34:0) 4 8 4 3 5 0 2 2 4 4 9 0 4 4 4 4

761.4 PG(36:8) 5 2 2 2 2 0 0 0 0 0 0 0 4 5 6 6

763.4 PG(36:7) 5 2 2 2 2 0 0 0 0 0 0 0 10 6 15 7

765.4 PG(36:6) 5 6 2 8 2 18 14 20 20 16 14 25 33 18 45 25

767.4 PG(36:5) 5 7 22 4 12 8 8 8 6 8 8 12 18 19 16 11

769.4 PG(36:4) 5 8 2 4 2 7 6 6 5 6 7 8 14 8 8 8

771.4 PG(36:3) 5 15 2 4 6 6 5 4 5 4 4 4 8 4 6 2

773.4 PG(36:2) 5 12 2 4 6 5 4 4 4 4 4 4 0 0 0 0

775.4 PG(36:1) 5 10 2 4 6 0 0 0 0 0 0 0 0 0 0 0

791.4 PG(38:7) 5 8 2 3 6 2 2 2 23 11 8 4 40 25 50 44

793.4 PG(38:6) 78 98 98 48 42 98 98 98 90 75 6 98 27 30 74 36 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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261


795.4 PG(38:5) 25 30 35 15 12 32 41 30 40 36 98 26 16 15 33 24

797.4 PG(38:4) 12 16 80 12 8 17 12 12 20 24 50 10 11 12 16 12

799.4 PG(38:3) 6 7 5 8 5 12 8 8 10 17 28 5 7 4 8 8

801.4 PG(38:2) 4 4 2 4 4 8 6 4 2 2 8 4 5 4 4 6

803.4 PG(38:1) 4 4 2 2 2 6 4 2 2 2 6 3 4 4 5 4

805.5 PG(38:0) 0 0 0 0 0 4 4 2 2 2 5 4 4 4 4 6

813.47 PG(40:10) 24 6 12 12 12 4 39 40 17 10 10 4 22 12 15 6

815.47 PG(40:9) 16 12 14 10 98 8 21 18 15 8 20 5 16 15 16 11

817.5 PG(40:8) 12 18 10 6 33 4 22 12 14 6 11 4 12 16 18 14

819.5 PG(40:7) 98 97 80 18 30 4 10 8 6 5 10 4 28 52 98 98

821.5 PG(40:6) 34 34 25 6 16 4 8 6 6 4 8 5 16 22 30 38

823.5 PG(40:5) 12 14 8 5 8 2 5 4 4 3 7 5 14 14 11 16

825.5 PG(40:4) 8 10 8 18 8 2 5 4 4 3 6 5 8 8 8 8

827.5 PG(40:3) 3 6 6 12 9 2 5 4 4 3 5 4 7 6 5 7

829.5 PG(40:2) 3 5 4 6 2 2 5 4 4 3 4 0 5 4 4 4

831.5 PG(40:1) 4 5 4 4 2 2 5 4 2 3 4 0 5 8 20 5

833.5 PG(40:0) 4 5 4 4 2 2 5 4 2 3 4 0 5 6 16 5

839.48 PG(42:11) 4 4 2 4 2 12 5 4 2 4 4 0 4 8 8 5

841.48 PG(42:10) 4 4 2 2 2 10 36 42 30 31 14 12 4 7 6 6

843.48 PG(42:9) 4 4 2 2 2 8 15 26 15 22 10 10 4 6 7 4

845.48 PG(42:8) 4 4 2 2 2 5 8 12 8 16 9 8 4 5 8 3

847.48 PG(42:7) 4 4 2 2 2 4 9 8 6 10 6 6 0 0 0 0

849.48 PG(42:6) 4 4 2 2 2 4 6 8 6 4 6 6 0 0 0 0

851.48 PG(42:5) 4 4 2 5 2 2 6 6 4 3 5 5 0 0 0 0

853.48 PG(42:4) 4 4 2 8 2 2 4 6 4 2 5 5 0 0 0 0

861.65 PG(42:0) 0 0 0 0 0 2 28 34 25 2 4 4 0 0 0 0

889.6 PG(44:0) 0 0 0 0 0 2 2 2 12 5 13 10 0 0 0 0

455.24 LPG(14:0) 0 0 0 0 0 4 2 2 2 2 3 4 0 0 0 0 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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262


481.25 LPG(16:1) 4 8 4 2 4 0 0 0 0 0 0 0 0 0 0 0

483.27 LPG(16:0) 4 12 4 4 2 0 0 0 0 0 0 0 0 0 0 0

511.3 LPG(18:0) 0 0 0 0 0 0 0 0 0 0 0 0 8 7 6 5

529.25 LPG(20:5) 8 12 5 4 4 6 4 5 8 18 8 8 20 10 12 7

531.27 LPG(20:4) 0 0 0 0 0 4 4 6 7 7 10 4 0 0 0 0

622.37 PS(24:0) 2 4 2 2 4 2 2 2 4 2 4 4 4 3 2 2

648.38 PS(26:1) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 3 2

650.4 PS(26:0) 0 0 0 0 0 4 2 2 4 2 4 5 4 2 5 4

674.4 PS(28:2) 0 0 0 0 0 4 3 2 4 2 4 4 6 2 2 4

676.41 PS(28:1) 0 0 0 0 0 0 0 0 0 0 0 0 8 2 2 2

678.4 PS(28:0) 0 0 0 0 0 2 5 2 4 2 4 4 4 2 2 2

690.4 PS(29:1) 0 0 0 0 0 2 5 6 17 4 18 8 5 3 2 2

692.4 PS(29:0) 0 0 0 0 0 2 4 4 10 5 12 5 5 3 2 2

702.43 PS(30:2) 4 6 2 8 4 2 2 2 5 2 4 5 4 3 2 3

704.4 PS(30:1) 0 0 0 0 0 2 2 2 6 2 4 4 4 3 2 3

706.4 PS(30:0) 0 0 0 0 0 2 2 2 5 2 4 5 6 2 2 3

716.45 PS(31:2) 0 0 0 0 0 2 8 5 4 5 10 4 6 3 2 3

718.45 PS(31:1) 0 0 0 0 0 2 6 3 5 4 51 8 5 2 2 2

720.48 PS(31:0) 0 0 0 0 0 2 8 2 4 2 22 5 5 4 2 3

724.4 PS(32:5) 0 0 0 0 0 2 2 2 5 2 27 4 4 4 4 3

726.4 PS(32:4) 0 0 0 0 0 2 2 2 6 2 5 4 4 4 4 4

728.4 PS(32:3) 0 0 0 0 0 2 2 2 4 2 5 4 4 4 4 4

730.4 PS(32:2) 0 0 0 0 0 2 4 2 4 6 75 22 4 2 3 2

732.4 PS(32:1) 0 0 0 0 0 2 4 2 4 4 38 11 5 2 3 2

734.4 PS(32:0) 0 0 0 0 0 2 4 2 4 2 20 10 4 2 3 2

746.5 PS(33:1) 7 4 5 4 5 2 3 2 4 2 17 8 4 2 3 4

748.5 PS(33:0) 8 5 2 2 4 2 3 2 4 2 14 6 4 2 3 5

750.4 PS(34:6) 8 5 2 2 2 2 3 2 4 2 12 2 5 2 2 3 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


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263


752.4 PS(34:5) 4 5 2 4 2 2 3 2 4 2 10 3 5 2 3 3

754.4 PS(34:4) 5 5 2 4 2 2 3 2 4 2 5 2 5 2 3 2

756.4 PS(34:3) 4 5 2 2 2 2 4 2 4 2 4 2 5 2 3 2

758.4 PS(34:2) 4 5 2 2 2 2 4 2 4 2 4 2 5 2 2 2

760.4 PS(34:1) 7 4 2 2 2 2 4 2 4 2 4 2 5 2 5 2

762.4 PS(34:0) 8 4 2 2 2 2 4 2 4 2 4 2 5 2 4 8

774.4 PS(36:8) 4 12 2 2 4 0 0 0 0 0 0 0 8 2 4 2

776.4 PS(36:7) 4 11 2 2 5 0 0 0 0 0 0 0 8 2 5 2

778.4 PS(36:6) 4 8 4 2 16 0 0 0 0 0 0 0 8 2 4 2

780.4 PS(36:5) 4 6 5 2 8 0 0 0 0 0 0 0 8 2 5 6

782.4 PS(36:4) 2 4 5 2 6 0 0 0 0 0 0 0 8 4 8 7

784.4 PS(36:3) 2 4 2 2 4 0 0 0 0 0 0 0 7 4 10 6

786.4 PS(36:2) 2 4 2 2 4 0 0 0 0 0 0 0 7 2 7 6

788.4 PS(36:1) 2 4 2 2 4 0 0 0 0 0 0 0 8 4 8 6

790.5 PS(36:0) 2 4 2 4 2 5 2 8 22 2 4 2 5 5 50 45

800.45 PS(38:9) 2 4 3 6 8 4 2 2 2 2 4 2 4 6 12 33

802.4 PS(38:8) 2 4 2 4 4 4 2 2 2 2 5 2 4 2 8 5

804.4 PS(38:7) 2 4 2 5 5 3 2 2 2 2 5 2 4 2 6 6

806.4 PS(38:6) 2 5 2 2 2 3 2 2 2 2 4 2 4 2 5 8

808.4 PS(38:5) 2 5 2 4 2 3 2 2 2 2 6 2 16 2 5 6

810.4 PS(38:4) 8 4 4 4 2 2 2 2 2 2 6 4 12 2 5 8

812.4 PS(38:3) 10 4 4 4 8 2 12 40 16 12 6 5 10 2 4 9

814.4 PS(38:2) 12 12 12 4 98 8 10 15 10 8 15 5 14 2 4 11

816.4 PS(38:1) 24 18 56 4 30 5 8 18 15 7 7 5 8 2 4 12

818.5 PS(38:0) 25 12 72 16 18 4 6 10 8 5 8 4 8 6 98 10

826.46 PS(40:10) 6 8 2 20 8 2 2 5 2 2 4 4 4 2 3 5

828.4 PS(40:9) 4 6 2 12 5 2 2 2 2 2 5 4 5 2 3 3

830.5 PS(40:8) 4 4 2 5 3 2 2 2 2 2 5 4 4 2 3 3 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


Table continued..

Appendix

264


832.5 PS(40:7) 4 4 2 2 2 2 2 2 2 2 4 0 4 4 17 3

834.5 PS(40:6) 4 4 2 2 2 2 2 2 2 0 0 0 4 5 12 5

836.5 PS(40:5) 4 4 2 3 2 2 2 2 22 0 0 0 4 5 10 6

838.5 PS(40:4) 4 4 2 3 2 2 2 2 2 0 0 0 4 2 8 6

840.5 PS(40:3) 4 4 2 3 2 2 34 37 31 32 12 12 6 2 8 4

842.5 PS(40:2) 4 4 2 2 2 12 18 16 21 22 10 8 4 6 4 6

844.5 PS(40:1) 4 4 2 2 2 8 8 8 12 18 6 6 4 5 6 4

846.5 PS(40:0) 4 4 2 2 2 4 6 6 4 4 5 4 6 5 7 4

852.48 PS(42:11) 0 0 0 0 0 2 5 5 5 2 3 5 0 0 0 0

854.48 PS(42:10) 0 0 0 0 0 2 4 4 4 2 3 4 0 0 0 0

856.48 PS(42:9) 0 0 0 0 0 2 2 4 4 2 3 4 0 0 0 0

858.48 PS(42:8) 0 0 0 0 0 2 2 4 5 2 3 3 0 0 0 0

860.48 PS(42:7) 0 0 0 0 0 2 15 28 16 22 3 4 0 0 0 0

862.48 PS(42:6) 0 0 0 0 0 2 8 18 14 14 3 3 0 0 0 0

864.48 PS(42:5) 0 0 0 0 0 2 8 15 6 8 3 2 0 0 0 0

866.48 PS(42:4) 0 0 0 0 0 2 6 12 4 6 2 2 0 0 0 0

874.65 PS(42:0) 0 0 0 0 0 2 2 8 2 7 2 2 0 0 0 0

892.6 PS(44:5) 0 0 0 0 0 2 2 5 2 3 8 11 0 0 0 0

765.45 PI(29:1) 4 10 4 8 4 14 15 10 18 14 16 24 10 18 42 20

767.4 PI(29:0) 4 11 5 6 2 2 8 4 8 6 6 10 35 8 21 11

777.4 PI(30:2) 4 12 6 4 17 2 8 4 2 5 4 8 16 0 0 0

791.4 PI(31:2) 0 0 0 0 0 98 90 98 90 81 96 94 8 6 50 51

793.48 PI(31:1) 76 98 98 48 33 30 32 25 40 38 40 20 48 24 68 45

795.5 PI(31:0) 25 36 27 18 16 16 16 12 18 18 20 6 32 34 42 12

799.4 PI(32:5) 6 16 6 6 2 0 0 0 0 0 0 0 8 6 24 8

821.5 PI(33:1) 32 35 22 8 12 0 0 0 0 0 0 0 24 27 30 12

823.5 PI(33:0) 16 18 16 4 8 0 0 0 0 0 0 0 12 15 14 8

825.4 PI(34:6) 6 8 11 22 5 0 0 0 0 0 0 0 8 6 8 7 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


Table continued..

Appendix

265


827.5 PI(34:5) 3 4 2 26 5 0 0 0 0 0 0 0 7 7 7 2

829.4 PI(34:4) 3 4 2 6 2 0 0 0 0 0 0 0 6 7 5 3

831.4 PI(34:3) 2 4 2 4 2 0 0 0 0 0 0 0 6 10 18 8

833.4 PI(34:2) 2 4 2 4 2 0 0 0 0 0 0 0 6 8 16 6

835.4 PI(34:1) 2 4 2 4 2 0 0 0 0 0 0 0 6 6 14 5

837.4 PI(34:0) 2 4 2 5 2 0 0 0 0 0 0 0 5 6 12 4

849.4 PI(36:8) 0 0 0 0 0 0 0 0 0 0 0 0 5 7 10 4

851.4 PI(36:7) 0 0 0 0 0 0 0 0 0 0 0 0 5 5 9 2

853.4 PI(36:6) 2 4 2 5 2 0 0 0 0 0 0 0 4 4 13 2

855.4 PI(36:5) 2 2 4 4 2 0 0 0 0 0 0 0 4 4 14 2

857.4 PI(36:4) 2 4 2 2 2 0 0 0 0 0 0 0 4 4 8 2

859.4 PI(36:3) 2 3 4 2 2 0 0 0 0 0 0 0 4 2 7 2

861.4 PI(36:2) 2 2 2 2 2 4 28 18 24 29 4 4 2 2 4 2

863.8 PI(36:1) 3 3 3 2 2 4 8 6 18 19 4 4 2 2 4 2

865.8 PI(36:0) 2 2 2 2 2 3 6 4 7 8 3 3 2 2 4 2

875.4 PI(38:9) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 12 2

877.4 PI(38:8) 0 0 0 0 0 0 0 0 0 0 0 0 5 10 16 4

879.4 PI(38:7) 0 0 0 0 0 0 0 0 0 0 0 0 4 8 11 4

889.4 PI(38:2) 0 0 0 0 0 3 3 2 10 3 12 12 0 0 0 0

891.4 PI(38:1) 0 0 0 0 0 2 3 2 8 4 8 8 0 0 0 0

893.5 PI(38:0) 0 0 0 0 0 4 3 2 7 3 6 7 0 0 0 0

975.6 PI(44:1) 0 0 0 0 0 2 8 10 20 12 2 2 0 0 0 0

977.6 PI(44:0) 0 0 0 0 0 2 6 8 14 7 2 2 0 0 0 0

535.34 PA(24:0) 5 5 2 4 2 0 0 0 0 0 0 0 0 0 0 0

603.4 PA(29:1) 0 0 0 0 0 2 4 2 8 5 4 4 0 0 0 0

645.4 PA(32:1) 0 0 0 0 0 4 2 2 4 4 4 8 5 5 5 4

687.4 PA(36:8) 2 2 4 2 2 2 2 2 2 4 5 5 4 2 5 4

689.4 PA(36:7) 6 8 3 2 2 4 2 2 18 5 6 9 4 2 3 5 UL-Ulva lactuca, UF-Ulva fasciata, UT-Ulva taeniata, UB- Ulva beytensis, UTb-Ulva tubulosa, GC- Gracilaria corticata, GCY- Gracilaria corticata v. cylindrica, GFO- Gracilaria corticata v. folifera, GD-Gracilaria dura, GS-


Table continued..

Appendix

266


691.4 PA(36:6) 4 6 4 8 2 2 2 2 11 4 12 6 4 2 4 4

693.4 PA(36:5) 5 5 3 6 2 2 2 2 4 4 16 5 3 2 3 3

695.4 PA(36:4) 2 3 3 4 2 2 2 2 3 4 10 4 3 2 4 2

697.4 PA(36:3) 0 0 0 0 0 2 2 2 3 4 6 4 5 2 3 2

699.4 PA(36:2) 0 0 0 0 0 2 2 2 3 3 7 4 5 2 4 2

739.43 PA(40:10) 0 0 0 0 0 0 0 0 0 0 0 0 4 2 5 2

741.4 PA(40:9) 0 0 0 0 0 0 0 0 0 0 0 0 4 4 5 3

713.4 PA(38:9) 0 0 0 0 0 2 2 2 4 2 8 4 0 0 0 0

715.4 PA(38:8) 0 0 0 0 0 2 4 8 5 2 12 5 0 0 0 0

717.4 PA(38:7) 0 0 0 0 0 2 5 6 4 2 40 5 0 0 0 0

719.4 PA(38:6) 0 0 0 0 0 2 4 4 4 2 26 8 0 0 0 0

721.4 PA(38:5) 0 0 0 0 0 2 2 2 4 2 12 7 0 0 0 0

723.4 PA(38:4) 0 0 0 0 0 2 2 2 4 2 11 5 0 0 0 0

725.4 PA(38:3) 0 0 0 0 0 2 2 2 4 2 6 5 0 0 0 0

727.4 PA(38:2) 0 0 0 0 0 2 2 2 4 2 5 5 0 0 0 0

729.4 PA(38:1) 0 0 0 0 0 2 2 2 4 5 60 5 0 0 0 0

731.5 PA(38:0) 2 6 2 2 2 2 3 2 4 3 30 15 0 0 0 0

739.43 PA(40:10) 3 12 18 4 4 2 4 3 4 3 11 12 0 0 0 0

741.4 PA(40:9) 6 12 11 3 18 0 0 0 0 0 0 0 0 0 0 0

743.4 PA(40:8) 6 10 8 2 12 0 0 0 0 0 0 0 4 4 4 4

745.4 PA(40:7) 7 8 4 2 8 0 0 0 0 0 0 0 4 4 4 5

747.4 PA(40:6) 6 7 4 2 4 0 0 0 0 0 0 0 4 4 4 6

749.4 PA(40:5) 0 0 0 0 0 0 0 0 0 0 0 0 4 4 3 4

751.4 PA(40:4) 0 0 0 0 0 0 0 0 0 0 0 0 5 3 3 2

753.4 PA(40:3) 0 0 0 0 0 0 0 0 0 0 0 0 5 3 3 2

755.4 PA(40:2) 0 0 0 0 0 0 0 0 0 0 0 0 5 3 2 2

765.45 PA(42:11) 0 0 0 0 0 15 12 8 22 21 12 20 10 20 28 18



Table continued..

Appendix

267


769.45 PA(42:9) 0 0 0 0 0 6 4 2 7 6 6 7 20 10 8 7

775.45 PA(42:6) 2 7 6 2 2 0 0 0 0 0 0 0 0 0 0 0

777.45 PA(42:5) 2 7 7 2 2 0 0 0 0 0 0 0 0 0 0 0

779.45 PA(42:4) 2 6 6 2 16 0 0 0 0 0 0 0 0 0 0 0

787.6 PA(42:0) 2 4 4 2 2 0 0 0 0 0 0 0 0 0 0 0

813.5 PA(44:1) 17 10 11 4 98 6 14 12 16 16 4 4 24 12 11 7



Publications

1. Puja Kumari, A. J. Bijo, Vaibhav A. Mantri, C. R. K Reddy, Bhavanath Jha (2013)

Fatty acid profiling of tropical marine macroalgae: An analysis from chemotaxonomic

and nutritional perspectives. Phytochemistry 86: 44-56. (IF: 3.35, Citations: 1)

2. Puja Kumari, Ravindra Pal Singh, A. J. Bijo, C. R. K. Reddy, Bhavanath Jha (2012)

Estimation of lipid hydroperoxide levels in tropical marine macroalgae. Journal of

Phycology 48: 1362-1373. (IF: 2.07, Citations: 0)

3. Puja Kumari, C. R. K. Reddy, Bhavanath Jha (2011) Comparative evaluation and

selection of a method for lipid and fatty acid extraction from macroalgae. Analytical

Biochemistry 415: 134-144. (IF: 2.99, Citations: 10)

4. Puja Kumari, Manoj Kumar, Vishal Gupta, C. R. K. Reddy, Bhavanath Jha (2010)

Tropical marine macroalgae as potential sources of nutritionally important PUFAs. Food

Chemistry 120: 749-757. (IF: 3.45, Citations: 36)

5. Manoj Kumar, Puja Kumari, Nitin Trivedi, Mahendra Kumar Shukla, Vishal Gupta, C.

R. K. Reddy, Bhavanath Jha (2011). Minerals, PUFAs and antioxidant properties of some

tropical seaweeds from Saurashtra coast of India. Journal of Applied Phycology 23: 797-

810. (IF: 2.4, Citations: 11)

6. Vishal Gupta, Manoj Kumar, Puja Kumari, C. R. K. Reddy, Bhavanath Jha (2011)

Optimization of protoplast yields from the red algae Gracilaria dura (C. Agardh) J.

Agardh and G. verrucosa (Huds.) Papenfuss. Journal of Applied Phycology 23: 209-218.

(IF: 2.4, Citations: 5)

7. Vaibhav A. Mantri, Ravindra Pal Singh, A. J. Bijo, Puja Kumari, C. R. K. Reddy,

Bhavanath Jha (2011) Differential response of varying salinity and temperature on

zoospore induction, regeneration and daily growth rate in Ulva fasciata (Chlorophyta,

Ulvales). Journal of Applied Phycology 23: 243-250. (IF: 2.4, Citations: 3)

8. Manoj Kumar, Vishal Gupta, Nitin Trivedi, Puja Kumari, A. J. Bijo, C. R. K. Reddy,

Bhavanath Jha (2011) Desiccation induced oxidative stress and its biochemical responses

in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta). Environmental

and Experimental Botany 72: 194-201. (IF: 2.98, Citations: 4)

9. Ravindra Pal Singh, Mahendra Kumar Shukla, Avinash Mishra, Puja Kumari, C. R. K.

Reddy, Bhavanath Jha (2011) Isolation and characterization of exopolysaccharides from

seaweed associated bacteria Bacillus licheniformis. Carbohydrate Polymers. 84(3): 1019-

1026. (IF: 3.62, Citations: 11)

10. Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C. R. K. Reddy, Bhavanath

Jha. (2011) An alkali-halotolerant cellulase from Bacillus flexus isolated from green

seaweed Ulva lactuca. Carbohydrate Polymers. 83(2): 891-897. (IF: 3.62, Citations: 9)

11. Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C. R. K. Reddy, Bhavanath

Jha (2011) Solvent tolerant marine bacterium Bacillus aquimaris secreting organic

solvent stable alkaline cellulose. Chemosphere 83: 706-712. (IF: 3.20; Citations: 3)

12. Vishal Gupta, Ravi S. Baghel, Manoj Kumar, Puja Kumari, Vaibhav A. Mantri, C. R. K.

Reddy, Bhavnath Jha (2011) Growth and agarose characteristics of isomorphic

gametophyte (male and female) and sporophyte of Gracilaria dura and their marker

assisted selection. Aquaculture 318 (3-4): 389-396. (IF: 2.04; Citations: 0)

13. Ravi S. Baghel, Puja Kumari, A. J. Bijo, Vishal Gupta, C. R. K. Reddy, Bhavanath Jha

(2011) Genetic analysis and marker assisted identification of life phases of red alga

Gracilaria corticata (J. Agardh). Molecular Biology Reports 38: 4211-4218. (IF: 2.99;

Citations: 0)

14. Manoj Kumar, Vishal Gupta, Puja Kumari, C. R. K. Reddy, Bhavanath Jha (2010).

Assessment of Caulerpa species for nutritional, fatty acids and antioxidant potentials.

Journal of Food Composition and Analysis. 24: 270-278. (IF: 1.94, Citations: 4)

15. Manoj Kumar, Puja Kumari, Vishal Gupta, P. A. Anisha, C. R. K. Reddy and

Bhavanath Jha (2010). Differential responses to cadmium induced oxidative stress in

marine macroalga Ulva lactuca (Ulvales, Chlorophyta). Biometals 23: 315-325. (IF: 2.3,

Citations: 15)

16. Manoj Kumar, Puja Kumari, Vishal Gupta, C. R. K. Reddy, Bhavanath Jha (2010)

Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to

salinity induced oxidative stress. Journal of Experimental Marine Biology and Ecology

391: 27-34. (IF: 1.91, Citations: 10)

17. Ravindra Pal Singh, Vishal Gupta, Puja Kumari, Manoj Kumar, C. R. K. Reddy,

Kamalesh Prasad, Bhavanath Jha. (2010) Purification and partial characterization of an

extracellular alginate lyase from Aspergillus oryzae isolated from brown seaweed.

Journal of Applied Phycology. 23: 755-762. (IF: 1.79, Citations: 3)

18. Puja Kumari, C. R. K. Reddy, Bhavanath Jha (2012) Quantification of select

endogenous hydroxy-oxylipins from tropical marine macroalgae. Marine Biotechnology

(Submitted).

Book Chapter

1. Puja Kumari, Manoj Kumar, C. R. K. Reddy, Bhavanath Jha (2013) Algal lipids, fatty

acids and sterols. In Herminia Domínguez ed. Functional ingredients from algae for food

and nutraceuticals. Woodhead Publishing Ltd. UK. In press.

Author's personal copy

Fatty acid profiling of tropical marine macroalgae: An analysisfrom chemotaxonomic and nutritional perspectives

Puja Kumari, A.J. Bijo, Vaibhav A. Mantri, C.R.K. Reddy ⇑, Bhavanath Jha

Discipline of Marine Biotechnology and Ecology, CSIR – Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat 364002, India

a r t i c l e i n f o

Article history:

Received 4 May 2012

Received in revised form 18 October 2012

Available online 17 November 2012

Keywords:

Macroalgae

Lipids

Fatty acids

PUFAs

n6/n3 Ratio

Chemotaxonomy

Endosymbiosis

a b s t r a c t

The lipid and fatty acid (FA) compositions for 100 marine macroalgae were determined and discussed

from the context of chemotaxonomic and nutritional perspectives. In general, the lipid contents in mac-

roalgae were low (2.3–20 mg/g fr. wt.) but with substantially high amounts of nutritionally important

polyunsaturated fatty acids (PUFAs) such as LA, ALA, STA, AA, EPA and DHA, that ranged from 10% to

70% of TFAs. More than 90% of the species showed nutritionally beneficial n6/n3 ratio (0.1:1–3.6:1)

(p 6 0.001). A closer look at the FA data revealed characteristic chemotaxonomic features with C18 PUFAs

(LA, ALA and STA) being higher in Chlorophyta, C20 PUFAs (AA and EPA) in Rhodophyta while Phaeophyta

depicted evenly distribution of C18 and C20 PUFAs. The ability of macroalgae to produce long-chain

PUFAs could be attributed to the coupling of chloroplastic FA desaturase enzyme system from a photo-

synthetic endosymbiont to the FA desaturase/elongase enzyme system of a non-photosynthetic eukary-

otic protist host. Further, the principal component analysis segregated the three macroalgal groups with a

marked distinction of different genera, families and orders, Hierarchical cluster analyses substantiated

the phylogenetic relationships of all orders investigated except for those red algal taxa belonging to Gig-

artinales, Ceramiales, Halymeniales and Rhodymeniales for which increased sampling effort is required

to infer a conclusion. Also, the groups deduced from FA compositions were congruent with the clades

inferred from nuclear and plastid genome sequences. This study further indicates that FA signatures

could be employed as a valid chemotaxonomic tool to differentiate macroalgae at higher taxonomic lev-

els such as family and orders.

Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Benthic marine macroalgae, commonly known as seaweeds are

multicellular photosynthetic organisms with considerable poten-

tials 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, 2007).

The macroalgal species, in general, are low in lipids and contain

1–5% on dry wt. basis. Nevertheless, the nutritionally important

C18 and C20 PUFAs including n3 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 che-

motaxonomic 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), mic-

roalgae (Dunstan et al., 2005; Lang et al., 2011) and fungi (Mishra

et al., 2010). Further, Dunstan et al. (2005) deciphered the evolu-

tionary relationship between the FA composition of Rhodo-

phyceaen and Cryptophyceaen microalgae and the endosymbiotic

theory. According to the endosymbiotic theory, the micro- and

macroalgae of both Chlorophyceae and Rhodophyceae have been

originated from primary endosymbiosis of photosynthetic cyano-

bacteria 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, 2010; Woehle et al., 2012).

0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.phytochem.2012.10.015

⇑ Corresponding author. Tel.: +91 278 256 5801x614; fax: +91 278 2567562/

2566970.

E-mail address: [email protected] (C.R.K. Reddy).

Phytochemistry 86 (2013) 44–56

Contents lists available at SciVerse ScienceDirect

Phytochemistry

journal homepage: www.elsevier .com/locate /phytochem

ESTIMATION OF LIPID HYDROPEROXIDE LEVELS IN TROPICALMARINE MACROALGAE1

Puja Kumari, Ravindra Pal Singh, A. J. Bijo, C. R. K. Reddy,2 and Bhavanath Jha

Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, 364002,

Gujarat, India

The incipient levels of lipid hydroperoxides(LHPOs) were determined in selected green, brown,and red macroalgae by the FOX assay usinghydroperoxy HPLC mix. The LHPOs contents variedbetween the investigated species and showedrelatively low values in this study. Among the greens,it varied from 12 ± 6.2 lg g!1 (Codium sursum) to31.5 ± 2.8 lg g!1 (Ulva lactuca), whereas in reds,from 5.7 ± 1.6 lg g!1 (Gracilaria corticata) to46.2 ± 6 lg g!1 (Sarconema filiforme), and in browns,from 4.6 ± 4.4 lg g!1 (Dictyota bartayresiana) to79 ± 5.0 lg g!1 (Sargassum tenerrimum), on freshweight basis. These hydroperoxides represented aminor fraction of total lipids and ranged from 0.04%(S. swartzii) to 1.1% (S. tenerrimum) despite being arich source of highly unsaturated fatty acids. Thesusceptibility of peroxidation was assessed by specificlipid peroxidazibility (SLP) values for macroalgaltissues. The LHPO values were found to beindependent of both the PUFAs contents and theirdegree of unsaturation (DBI), as evident from thePCA analysis. SLP values were positively correlatedwith the LHPOs and negatively with DBI. The FOXassay gave " 20-fold higher values for LHPOs ascompared to the TBARS method for all the samplesinvestigated in this study. Furthermore, U. lactucacultured in artificial seawater (ASW) enriched withnutrients (N, P, and NP) showed a sharp decline inLHPOs contents relative to those cultured in ASWalone P # 0.05. It is inferred from this study that theFOX assay is an efficient, rapid, sensitive, andinexpensive technique for detecting the incipientlipid peroxidation in macroalgal tissues.

Key index words: fatty acid; FOX; lipid hydroperoxides;macroalgae; TBARS

Abbreviations: 12(S)-HpETE, 12(S)-Hydroperoxyeico-satetraenoic acid; 15(S)-HpETE, 15(S)-Hydroperoxy-eicosatetraenoic acid; 5(S)-HpETE, 5(S)-Hydroperoxyeicosatetraenoic acid; 13(S)-HpODE, 13(S)-Hydro-peroxyoctadecadienoic acid; 9(S)-HpODE, 9(S)-Hy-droperoxyoctadecadienoic acid; 13-HpOTE, 13-Hydroperoxyoctadecatrienoic acid; ANOVA, analysis ofvariance; ASW, artificial seawater; BHT, butylatedhydroxytoluene; CAT, catalase; DBI, double bond

index; FA, fatty acid; FAME, fatty acid methyl ester;FOX, ferrous oxidation-xylenol orange; FOX1, fer-rous oxidation-xylenol orange version 1; FOX2, fer-rous oxidation-xylenol orange version 2; H2O2,hydrogen peroxide; HPO, hydroperoxy HPLC mix;GC, gas chromatography; GC–MS, gas chromatographymass spectrometry; LC–MS, liquid chromatographymass spectrometry; LHPOs, lipid hydrop-eroxides; MDA, malondialdehyde; PC1, principalcomponent 1; PC2, principal component 2; PCA,principal component analysis; POD, peroxidase;PUFA, polyunsaturated fatty acid; ROS, reactiveoxygen species; SLP, specific lipid peroxidazibility;SOD, superoxide dismutase; TBARS, thiobarbituricacid-reactive substances; TFA, total fatty acid; TL,total lipid; TPP, triphenylphosphine

Lipid peroxidation is a well recognized indicatorof oxidative stress in all types of organisms, includ-ing plants, bacteria, fungi, algae, and mammaliansystems. In addition, their products exhibit a widevariety of biological and cell signaling functions(Spickett et al. 2010). Lipid peroxidation can beeither enzymatic mediated by lipoxygenases andcyclooxygenases, or nonenzymatic free radicalmediated, where several chain reactions are medi-ated by reactive oxygen species (ROS), whichthemselves are unavoidable consequences of aerobicmetabolism.Although lipid peroxidation is a well-investigated

phenomenon, lipid hydroperoxides (LHPOs) havereceived comparatively less attention than the otherlipid oxidized products because of their either insta-bility or lengthy procedures involving of expensiveand sophisticated equipments for detection. TheLHPOs have been estimated by several analyticalapproaches, such as high-performance liquidchromatography (HPLC; Nakamura and Maeda1991, Hui et al. 2005), gas chromatography (GC; Tur-nipseed et al. 1993), electrospray mass spectrometry(Spickett et al. 1998), iodine oxidation (Jessup et al.1994), heme degradation of peroxides (Frei et al.1998), cyclooxygenase activation (Pendleton andLands 1987, Calzada et al. 1997), chemiluminescencedetection (Yamamoto 1994), immunological assays(Paradis et al. 1997), conjugated diene measurement(Moore and Roberts 1998), and thiobarbituric

1Received 29 February 2012. Accepted 5 June 2012.2Author for correspondence: e-mail: [email protected].

J. Phycol. 48, 1362–1373 (2012)

© 2012 Phycological Society of America

DOI: 10.1111/j.1529-8817.2012.01208.x

1362


Comparative evaluation and selection of a method for lipid and fatty acidextraction from macroalgae

Puja Kumari, C.R.K. Reddy ⇑, Bhavanath Jha

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021,

Gujarat, India


Article history:

Received 27 January 2011

Received in revised form 16 March 2011

Accepted 6 April 2011

Available online 16 April 2011

Keywords:

Lipids

Fatty acids

Macroalgae

Sonication

Buffer

Extraction method

a b s t r a c t

A comparative evaluation of Bligh and Dyer, Folch, and Cequier-Sánchez methods for quantitative deter-

mination of total lipids (TLs) and fatty acids (FAs) was accomplished in selective green (Ulva fasciata), red

(Gracilaria corticata), and brown algae (Sargassum tenerrimum) using a full factorial categorical design.

Applications of sonication and buffer individually on lipid extraction solvent systems were also evalu-

ated. The FA recoveries obtained from the aforementioned methods were compared with those of direct

transesterification (DT) methods to identify the best extraction methods. The experimental design

showed that macroalgal matrix, extraction method, and buffer were key determinants for TL and FA

recoveries (P 6 0.05), exhibiting significant interactions. But sonication gave erratic results with no inter-

action with any of the factors investigated. The buffered solvent system of Folch rendered the highest TL

yield in U. fasciata and G. corticatawhile the buffered system of Bligh and Dyer gave the highest yield in S.

tenerrimum. DT methods were more convenient and accurate for FA quantification and rendered 1.5–2

times higher yields when compared with the best conventional method, minimizing the use of chlori-

nated solvents, their cost of analysis, and disposal. The buffered solvent system was found to be the most

appropriate for lipid research in macroalgae.

Ó 2011 Elsevier Inc. All rights reserved.

Macroalgae have been reported to contain more than 2400 nat-

ural products of commercial importance in pharmaceutical, bio-

medical, and nutraceutical industries [1]. They have also been

extensively utilized as ingredients in human and animal food prep-

arations owing to their high contents of polyunsaturated fatty

acids (PUFAs),1 carbohydrates, vitamins, minerals, and dietary fibers

[2,3]. Nowadays, algal resources have been studied with renewed

interest across the world as an alternative source of renewable en-

ergy feedstock that circumvents the controversy of ‘‘fuel versus

food’’. The attributes for such choice are their relatively higher pro-

duction turnover and amenability for depolymerization of substrate

in addition to greater carbon sequestration potentials than terrestrial

feedstock [4]. Most recently, Petcavich succeeded in producing

hydrocarbon biofuels from transformed kelp Macrocystis pyrifera

with high hydrocarbon producing genes of microalgae, Botryococcus

braunii [5].

Fatty acid (FA) analysis has been increasingly gaining impor-

tance due to the realization of their beneficial applications in nutri-

tional and health products. Further, they have also been used for

addressing various fundamental and pragmatic research problems

in experimental biochemical, physiological, and clinical studies

[6,7]. Further, in biodiesel production, clean burn properties of

the fuel are influenced by FA structural features including chain

length and degree of unsaturation [8]. Thus, a precise quantifica-

tion of FA can also be used to predict the quality of biodiesel, which

is reduced considerably with the increase in the amount of satu-

rated FAs.

Traditionally, the fatty acid composition of lipid samples is

determined by assessing the corresponding methyl esters via gas

chromatography (GC). A large number of analytical approaches

based on initial lipid extraction by solvents, followed by their

transmethylation (i.e., conventional methods), are employed and

where FAs are sought, they are extracted and methylated with

one-step procedures wherein methylation reagent is added di-

rectly to the samples without previous extraction (direct transeste-

rification methods). However, both types of methods have their

own advantages and disadvantages that are well illustrated in

the literature [9–16]. Conventional methods are time consuming

0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2011.04.010

⇑ Corresponding author. Fax: +91 278 2567562/2566970.

E-mail address: [email protected] (C.R.K. Reddy).1 Abbreviations used: BD, Bligh and Dyer; BDB, Bligh and Dyer with buffer; BDS,

Bligh and Dyer with sonication; CM, conventional methods; CS, Cequier-Sánchez

method; CSB, Cequier-Sánchez method with buffer; CSS, Cequier-Sánchez method

with sonication; DT, direct transesterification; FA, fatty acid; FAMEs, fatty acid methyl

esters; FM, Folch method; FMB, Folch method with buffer; FMS, Folch method with

sonication; GM, Garcia method; LRC, Lepage and Roy modified by Cohen method;

PUFAs, polyunsaturated fatty acids; TFA, total fatty acid; MUFA, monounsaturated

fatty acids; SFA, saturated fatty acid.

Analytical Biochemistry 415 (2011) 134–144

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio


Tropical marine macroalgae as potential sources of nutritionally important PUFAs

Puja Kumari, Manoj Kumar, Vishal Gupta, C.R.K. Reddy *, Bhavanath Jha *

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India


Article history:

Received 27 January 2009

Received in revised form 25 September

2009

Accepted 4 November 2009

Keywords:

Chlorophyta

Fatty acids

Lipids

n6/n3 ratio

Phaeophyta

PUFAs

Rhodophyta

Tropical macroalgae

a b s t r a c t

The lipid and fatty acid compositions of 27 tropical macroalgae belonging to the three phyla, Chlorophyta,

Phaeophyta and Rhodophyta, were studied from a nutritional and chemotaxonomic perspective. The lipid

content varied widely among the species and ranged from 0.57% to 3.5% on a dry weight basis (p 6 0.01).

Chlorophyta members showed higher C18PUFAs contents than did C20 PUFAs while for Rhodophyta the

trend was opposite. The Phaeophyta members displayed a profile of C18PUFAs similar to that of Chloro-

phyta and of C20PUFAs to that of Rhodophyta. Both Phaeophyta and Rhodophyta species were rich in

arachadonic acid (AA) and eicosopentaenoic acid (EPA) and Ulvales in docosahexaenoic acid (DHA) con-

tent. Most of the species studied had a nutritionally beneficial n6/n3 ratio (0.61–5.15:1). Further, the

principal component analysis clearly segregated the three phyla by their FA composition and hierarchical

cluster analysis altogether classified them into six distinct groups, suggesting that FAs can be used as a

tool for chemotaxonomic studies.


1. Introduction

Benthic marine macroalgae, commonly known as seaweeds,

are increasingly viewed as potential sources of bioactive com-

pounds with immense pharmaceutical, biomedical and nutraceu-

tical importance (Veena, Josephine, Preetha, & Varalakshmi,

2006). Many macroalgal species have been used as ingredients

in both medicinal and food preparations, traditionally, in different

regions across the world (Chandini, Ganesan, Suresh, & Bhaskar,

2008). In addition, some are common sources of phycocolloids

(gelling agents) of commercial value (Cardozo et al., 2007). There

are 250 macroalgal species which have been listed as commer-

cially utilised worldwide, among which 150 are consumed as

human food (Barrow, 2007). They are also considered as low cal-

orie foods with high contents of minerals, vitamins, proteins and

carbohydrates (Chandini et al., 2008). Although macroalgae have

been reported to have low lipid contents, their polyunsaturated

fatty acid (PUFA) contents are superior to those of the terrestrial

vegetables (Darcy-Vrillon, 1993). They are rich in C18 and C20

PUFAs with nutritional implications and are thus, studied

extensively for biotechnological, food, feed, cosmetic and pharma-

ceutical applications (Chandini et al., 2008). Long-chain n 3 PU-

FAs, such as EPA and DHA, have various beneficial clinical and

nutraceutical applications (Ginzberg et al., 2000; Lombardo, Hein,

& Chicco, 2007). Their role in growth, development and reproduc-

tion of both marine invertebrates and fish has also been well

documented (Rodriguez et al., 2004). The n 3 PUFAs cannot be

synthesized by humans and are thus obtained through diet. In

view of their promising medical and nutritional applications, they

have been extensively investigated, However, the studies on effi-

cient exploitation of natural sources for these compounds are lim-

ited (Colombo et al., 2006). At present, marine fishes and fish oils

are the main commercial sources of PUFAs but their suitability for

human consumption has been questioned from a biosafety

perspective, raising the need to search for alternative sources of

high quality PUFAs (Bhosale, Velankar, & Chaugule, 2008). Conse-

quently, marine macroalgae have been studied as alternative

potential sources, as many of them could easily be cultivated in

the sea on a large scale (Critchely, Ohno, & Largo, 2006). Also,

the PUFAs present in the fishes enter the food chain from differ-

ent trophic levels as a result of consuming primary producers,

such as phytoplankton and seaweeds, which synthesize and store

them in good quantities (Visentainer et al., 2007).

Further, FAs have been extensively used as a chemotaxonomic

tool to classify the species in higher plants (Mongrand et al.,

2001) and cyanobacteria (Temina, Rezankovab, Rezankac, &

Dembitsky, 2007). Recently, Marsham, Scott, and Tobin (2007)

grouped seaweeds by their nutritional composition using multi-

variate analysis. To the best of our knowledge, classification of

macroalgae by FAs remains largely unexplored to date.


doi:10.1016/j.foodchem.2009.11.006

* Corresponding authors. Tel.: +91 278 256 7352; fax: +91 278 257 0885/256

6970/256 7562.

E-mail address: [email protected] (B. Jha).

Food Chemistry 120 (2010) 749–757


Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Minerals, PUFAs and antioxidant properties of some

tropical seaweeds from Saurashtra coast of India

Manoj Kumar & Puja Kumari & Nitin Trivedi &

Mahendra K. Shukla & Vishal Gupta & C. R. K. Reddy &

Bhavanath Jha

Received: 16 April 2010 /Revised and accepted: 17 August 2010 /Published online: 7 September 2010# Springer Science+Business Media B.V. 2010

Abstract Twenty-two tropical seaweeds from the Rhodo-

phyta, Phaeophyta and Chlorophyta were examined for

their possible use as nutritional supplements. All seaweeds

contained balanced Na/K and C/N ratio and high amounts

of macroelements (Na, K, Ca, and Mg) as compared to the

terrestrial vegetables. Among the microelements, Fe was

the highest followed by Zn, Mn, Cu and other trace

elements. Fatty acid distribution showed high level of n-6

and n-3 polyunsaturated fatty acids (PUFAs), and their

ratios were within the WHO prescribed limits. The higher

ratios of PUFA/SFA (>0.4) are in agreement with the

recommendations of nutritional guidelines. Most of the

species, especially the Chlorophyta and Phaeophyta, had

permissible intake values of unsaturation, atherogenic and

thrombogenic indexes comparable to milk-based products.

Principal component analysis demonstrated a correlation

between total phenolic content, total antioxidant activity,

DPPH, and O2•− radical scavenging activity, suggesting

polyphenols as the chief contributor to the antioxidant

activity in seaweeds. These results indicate that these

seaweeds could be a potential source of natural antiox-

idants, minerals and high-quality PUFAs and may be

efficiently used as ingredients in functional foods.

Keywords Antioxidant potential . Minerals . PUFAs .

Tropical seaweeds

Introduction

Increasing awareness among consumers about health-

promoting foods has aroused interest in food supplement

research worldwide. In addition to food supplements,

consumption of exotic foods with proven nutritional

values has also been gaining prominence in several

developed countries (Herrero et al. 2006). Many of these

foods are presently promoted and marketed as function-

al foods with premium price. The beneficial actions of

these foods are reported to be mainly due to their function-

al components such as minerals, antioxidants and n-3 fatty

acids, which are either absent in the analogous traditional

foods or present only in trace concentrations. Consequent-

ly, there has been a quest to explore and utilize foods from

nonconventional sources of both terrestrial and marine

origin to enhance the nutritional quality of human foods

which in turn also reduces the dependability on traditional

foods.

Seaweeds with their diverse bioactive compounds (Lee

et al. 2008; Zubia et al. 2009) have opened up potential

opportunities in pharmaceutical and agri-food processing

industries. The consumption of seaweeds as part of diet has

been shown to be one of the prime reasons for low

incidence of breast and prostate cancer in Japan and China

compared to North America and Europe (Pisani et al.

2002). Seaweeds also contain sufficient amounts of protein,

polysaccharides (e.g., alginates, fucans and laminarans),

and amino acids of considerable nutritional importance.

Algal lipids (1–3% dry matter) contain a high proportion of

essential fatty acids particularly n-3 polyunsaturated fatty

acids (PUFAs). At present, marine fish and their oil are the

major commercial sources of PUFAs, but their suitability

for human consumption has been questioned from the

biosafety perspective.

M. Kumar : P. Kumari :N. Trivedi :M. K. Shukla :V. Gupta :

C. R. K. Reddy (*) : B. Jha

Discipline of Marine Biotechnology and Ecology,

Central Salt and Marine Chemicals Research Institute,

Council of Scientific and Industrial Research (CSIR),

G.B. Marg,

Bhavnagar 364021, India

e-mail: [email protected]

J Appl Phycol (2011) 23:797–810

DOI 10.1007/s10811-010-9578-7

Optimization of protoplast yields from the red algae

Gracilaria dura (C. Agardh) J. Agardh and G. verrucosa

(Huds.) Papenfuss

Vishal Gupta & Manoj Kumar & Puja Kumari &

C. R. K. Reddy & Bhavanath Jha

Received: 4 February 2010 /Revised and accepted: 17 August 2010# Springer Science+Business Media B.V. 2010

Abstract This study reports on the optimization of

protoplast yield from two important tropical agarophytes

Gracilaria dura and Gracilaria verrucosa using different

cell-wall-degrading enzymes obtained from commercial

sources. The conditions for achieving the highest protoplast

yield was investigated by optimizing key parameters such

as enzyme combinations and their concentrations, duration

of enzyme treatment, enzyme pH, mannitol concentration,

and temperature. The significance of each key parameter

was also further validated using the statistical central

composite design. The enzyme composition with 4%

cellulase Onozuka R-10, 2% macerozyme R-10, 0.5%

pectolyase, and 100 U agarase, 0.4 M mannitol in seawater

(30‰) adjusted to pH 7.5 produced the highest protoplast

yields of 3.7±0.7×106 cells g−1 fresh wt for G. dura and

1.2±0.78×106 cells g−1 fresh wt for G. verrucosa when

incubated at 25°C for 4–6 h duration. The young growing

tips maximally released the protoplasts having a size of 7–

15 μm in G. dura and 15–25 μm in G. verrucosa, mostly

from epidermal and upper cortical regions. A few large-size

protoplasts of 25–35 μm, presumably from cortical region,

were also observed in G. verrucosa.

Keywords Agarophytes . Commercial enzymes .

Gracilaria . Protoplast production . Rhodophyta

Introduction

Among the red algae, the genus Gracilaria has considerable

industrial importance as an agarophyte and is the principal

source of raw material to the agar industry worldwide

(Zemke-White and Ohno 1999; Smit 2004). These agar

industries consume 72,300 dry tons of agarophytes annually

and produce approximately 9,600 tons agar with a value

of US$173 million. Of this, Gracilaria alone accounts for

about 80% of the world’s agar market with a value of

US$ 138 million (Bixler and Porse 2010). The increasing

demand for agar worldwide, coupled with short supplies of

agarophytes from wild stocks, has led to the development

of viable field cultivation methods for their large-scale

cultivation in the sea (Critchley 1993). Following the success

in large-scale cultivation, cellular biotechnology techniques

are also being applied to improve the cultivated germplasm

of this important resource (see reviews of Reddy et al.

2008a, b, 2010). However, these techniques have largely

remained underdeveloped and are thus in their nascent stage.

The high structural complexity and diversity in cell wall

composition (Duckworth and Yaphe 1971; Rochas and

Lahaye 1989) have rendered the agarophytes in particular

recalcitrant to enzymatic digestion of the cell walls and have

thus become an impediment for realizing the potentials

offered by the application of biotechnology tools and

techniques for seaweeds in general. The skeletal and matrix

polysaccharides of cell walls of red seaweeds mainly consist

of cellulose and agar. Therefore, the mixture of enzymes

having cellulase and agarase are invariably required to digest

the cell wall components of agarophytic red algal species for

preparing protoplasts.

To date, protoplast isolation has been accomplished for

48 red algal species, including agarophytes, belonging to 13

genera (Reddy et al. 2010). In the genus Gracilaria, with

This paper was presented at the 7th Asia Pacific Congress on Algal

Biotechnology, New Delhi, 2009.

V. Gupta :M. Kumar : P. Kumari :C. R. K. Reddy (*) :B. Jha






J Appl Phycol

DOI 10.1007/s10811-010-9579-6

Differential response of varying salinity and temperature

on zoospore induction, regeneration and daily growth rate

in Ulva fasciata (Chlorophyta, Ulvales)

Vaibhav A. Mantri & Ravindra Pal Singh & A. J. Bijo &

Puja Kumari & C. R. K. Reddy & Bhavanath Jha

Received: 17 January 2010 /Revised and accepted: 11 June 2010# Springer Science+Business Media B.V. 2010

Abstract Seaweed cultivation is imperative to augment

increasing industrial demand. Ulva fasciata Delile is a

potential seaweed for cultivation with applications in food

industries. There is a renewed interest in large-scale

aquaculture of this species in India due to its envisaged

demand in snack food products. In the present study, we

have successfully demonstrated the possibility of inducing

zoospores in vegetative tissue, effective regeneration and

improved growth in this seaweed by manipulating salinity

(from 15 to 30 psu) and temperature (from 15 to 35°C). The

optimum salinity and temperature requirement for zoo-

spores induction were found to be 15 psu and 25°C,

respectively. The quadriflagellate zoospores showed nega-

tive phototaxis and the settlement and germination pattern

similar to several other green seaweeds. The optimum

regeneration (78.53±10.05%) was recorded at 25°C and

30 psu salinity. The maximum daily growth rate (16.1±

0.28%) was at 25°C and 30 psu salinity which corre-

sponded to the field conditions. This method could be

further refined at nursery culture to achieve artificial

seeding essential for the success of commercial cultivation

of this seaweed.

Keywords Cultivation . Edible seaweed . Salinity .

Temperature .Ulva fasciata

Introduction

The green alga Ulva fasciata Delile (Chlorophyta, Ulvales)

is a potential edible seaweed distributed widely along the

Northern west coast of India. This seaweed is a rich source

of proteins, essential amino acids, fatty acids, vitamins,

dietary fibers and minerals vital for human nutrition (Sitaka

Rao and Tipnis 1964; Lewis 1966; McDermid and Stuercke

2003; Carvalho et al. 2009; Kumari et al. 2010). The iodine

content has been estimated in a range of 29–37 mg per

100 g dry wt. (Kesava Rao and Indusekhar 1989). A

substantial amount of beta-carotene and alpha tocopherol

has also been reported (de Sousa et al. 2008). The naturally

harvested biomass from the West coast of India has been

reported to possess antiviral properties against Japanese

encephalitis virus (Sharma et al. 1996). Recently, local food

industries have developed interest in incorporating this

species in popular snack items. Its dark green color, rich

flavor, and sweet aroma along with wide range of

nutritionally beneficial substances including omega-3-fatty

acids can improve dietetic composition of the food items.

This suggests a potential high demand for this species by

the food processing sector. However, inadequate natural

resources may hamper its prospects for utilization by food

industry. The development of reliable cultivation technique

based on the artificial seed production can ensure sustain-

able supply of quality biomass thereby negating the

consequences of over-harvesting.

Species of Monostroma, Enteromorpha, and Ulva are

popular in Japanese cuisine and are being cultivated

commercially in Japan and other Southeast Asian countries

(Ohno 2006; Hiraoka and Oka 2008). The seedling

production required for large-scale cultivation of these

seaweeds has traditionally been achieved through entrap-

ment of the zoospores on cultivation nets kept under the

This paper was presented at the 7th Asia Pacific Congress on Algal

Biotechnology, New Delhi 2009.

V. A. Mantri (*) : R. P. Singh : A. J. Bijo : P. Kumari :

C. R. K. Reddy : B. Jha

Marine Biotechnology and Ecology Discipline,



Gijubhai Badheka Marg,

Bhavnagar 364 021, India


J Appl Phycol

DOI 10.1007/s10811-010-9544-4

Environmental and Experimental Botany 72 (2011) 194–201


Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

Desiccation induced oxidative stress and its biochemical responses in intertidal

red alga Gracilaria corticata (Gracilariales, Rhodophyta)

Manoj Kumar, Vishal Gupta, Nitin Trivedi, Puja Kumari, A.J. Bijo, C.R.K. Reddy ∗, Bhavanath Jha



Article history:

Received 9 July 2010

Received in revised form 4 March 2011

Accepted 11 March 2011

Keywords:

Antioxidative enzymes

Desiccation

Gracilaria corticata

Polyamines

PUFAs

Reactive oxygen species

a b s t r a c t

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stresses

during the low tides. The aim of this study was to determine whether desiccation exposure would lead

to oxidative stress and its effect varies with exposure periods. This study gives an account of various

biochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1,

2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic loss

of water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species

(ROS) and increased lipid peroxidation observed during the exposure of 3–4 h were chiefly contributed

by higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX2, ∼85 kDa;

LOX3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin)

were increased during initial 2 h exposure compared to control and thereafter declined in the succeeding

exposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX),

glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate

(AsA) and glutathione (GSH) increased during desiccation up to 2–3 h. Further, the isoforms of antiox

idant enzymes MnSOD (∼150 kDa), APX4 (∼110 kDa), APX5 (∼45 kDa), GPX1 (∼80 kDa) and GPX2

(∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher con

tent of both free and bound insoluble putrescine and spermine together with enhanced n6 PUFAs namely

C20:4(n6) and C20:3(n6) fatty acids found during 2 h exposure reveals their involvement in defence

reactions against the desiccation induced oxidative stress.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The red alga Gracilaria corticata (J. Agardh) J. Agardh occurs

extensively in intertidal zone of the Indian coast and regularly

experiences the desiccation during low tide periods. The organ

isms living in the intertidal zone of tropical shores are subjected

to various types of abiotic stresses due to periodic exposure to

a wide range of fluctuating environmental factors such as desic

cation, salinity, radiation, temperature and pollutants (Apel and

Hirt, 2004; Liu and Pang, 2010; Kumar et al., 2010a). The environ

mental exposure during low tide condition, demands the intertidal

macroalgae to prepare early for the desiccation followed by rehy

dration and associated cellular damage (Burritt et al., 2002). This

constant state of readiness requires a great deal of energy budget

and could be a contributing factor to the slow growth rates of algae

dwelling at the upper littoral zone as compared to those at lower

littoral zone (Stengal and Dring, 1997). The possible explanation

∗ Corresponding author. Tel.: +91 278 256 5801/3805x614;

fax: +91 278 256 6970/7562.

Email address: [email protected] (C.R.K. Reddy).

for the success of an alga exposed to drought could be either being

physiologically more tolerant or better at resisting the water loss

(Ji and Tanaka, 2002).

During desiccation many of the intertidal seaweeds experience

extreme drying rates, reaching air dryness within hours (Schonbeck

and Norton, 1979; Nelson et al., 2010), generally depends on the cli

matic conditions as well as the evaporating surfacetovolume ratio

of the thallus (Lobban et al., 1985). Also, desiccation causes cellu

lar dehydration, which increases the concentration of electrolyte

within the cell, causing changes to membranebound structures

including the thylakoid (Kim and Garbary, 2007). It has been sug

gested that instant responses of marine plants to adverse milieu

involve excess production of reactive oxygen species (ROS) such

as hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide

(O2•−) and hydroxyl radical (OH−) (Burritt et al., 2002). The abil

ity to withstand the oxidative assault imposed by ROS depends

on the enzymatic and non enzymatic oxidants of the cell. This

antioxidant system functions in a coordinated manner to alle

viate the cellular hypo/hyper osmolarity, ion disequilibrium and

detoxification of ROS which otherwise cause oxidative destruc

tion to cell (Wu and Lee, 2008; Liu and Pang, 2010; Kumar et al.,

2010a,b).

00988472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.envexpbot.2011.03.007

Carbohydrate Polymers 84 (2011) 1019–1026


Carbohydrate Polymers

journa l homepage: www.e lsev ier .com/ locate /carbpol

Isolation and characterization of exopolysaccharides from seaweed associated

bacteria Bacillus licheniformis

Ravindra Pal Singh, Mahendra K. Shukla, Avinash Mishra, Puja Kumari, C.R.K. Reddy ∗, Bhavanath Jha



Article history:

Received 2 December 2010

Received in revised form

17 December 2010

Accepted 19 December 2010

Available online 25 December 2010

Keywords:

Exopolysaccharide

MALDITOFTOF MS

Bacillus licheniformis

Seaweed

Atomic force microscopy

Xray diffraction

a b s t r a c t

In the present study, EPS secreted by the endophytic bacterium Bacillus licheniformis was isolated and

characterized. The molecular masses of the EPS were 1540 and 44,565 kDa and 1H NMR, FTIR and UV–Vis

spectral analyses revealed prevalence of characteristic primary aminegroup, aromaticcompound, halide

and aliphatic alkylgroup in addition to Na, P, Ca, C, O, Cl and S as inferred from EDX analysis. XRD and DSC

analysis confirmed the amorphous nature of EPS, showing an average particle size of 24.977 mm (d 0.5)

with 191 nm average roughness. The positive ion reflector mode of MALDI TOFTOF MS exhibited a series

of low and high mass peaks corresponding to various oligosaccharides and polysaccharides respectively.

Further, GC–MS analysis revealed its four monosaccharide constituents glucose, galactose, mannose and

arabinose. The potential heterogeneous properties of EPS as revealed in the present study may be explored

in various biotechnological and industrial applications.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Microbial exopolysaccharides (EPSs) are biosynthetic polymers

mainly consisting of carbohydrates secreted by bacteria (Freitas

et al., 2009) and cyanobacteria (Chi, Su, & Lu, 2007; Decho,

1990; Parikh & Madamwar, 2006), however, it is also reported

from Cryptophyta (Bermudez, Rosales, Loreto, Briceno, & Morales,

2004), mushroom (Zou, Sun, & Guo, 2006), yeast (Duan, Chi,

Wang, & Wang, 2008) and basidiomycetes (Chi & Zhao, 2003;

Manzoni & Rollini, 2001). EPSs constitute different classes of

organic macromolecules such as polysaccharides, proteins, nucleic

acids, phospholipids and other polymeric compounds thereby car

rying different organic functional groups such as acetyl, succinyl

or pyruvyl and some inorganic constituent like sulfate (Nielsen &

Jahn, 1999). Depending on their location, EPSs occur in two forms

either in capsular (polymers being closely associated with the cell

surface) or slimy polysaccharides (loosely associated with the cell

surface) (Costerton, 1999).

In recent years, interest in the exploitation of valuable EPSs

has been considerably increased towards polysaccharide produc

ing bacteria and cyanobacteria for various industrial applications

(Mishra & Jha, 2009). The EPSs produced by marine bacteria have

∗ Corresponding author. Tel.: +91 278 256 5801/256 3805x614;

fax: +91 278 256 6970/256 7562.

Email address: [email protected] (C.R.K. Reddy).

been utilized as ingredients in food products, pharmacy, petroleum

industry and emulsification of crude oil, hydrocarbons, vegetable,

mineral oils and bioremediation agents in environment manage

ment system (Costerton, 1999; Jia, Yu, Lin, & Dai, 2007). Apart

from these, they have also been implicated in biofilm formation

in marine ecosystem thereby enhancing the survival of microbes

under abiotic and biotic stress by influencing their physicochemical

environment (Bhaskar & Bhosle, 2005; Duan et al., 2008).

The bacteria are ubiquitous colonizers on the surface of seaweed

and are reported to play important roles in their growth, develop

ment and reproduction (Marshall, Joint, Callow, & Callow, 2006).

Similar findings have been demonstrated with Bacillus licheniformis

which influence the life history and developmental morphology of

Ulva fasciata (Singh, Mantri, Reddy, & Jha, 2011) in corresponding

to increase the zoospore production and in attaining the normal

foliose morphology. Further, seaweed–bacteria interactions are

partly attributed to the extracellular substances secreted by associ

ated marine bacteria. The previous reports reveal that the addition

of supernatants of morphogenesisinducing bacterial isolates were

capable of giving the same effect (Marshall et al., 2006; Tatewaki,

Provasoli, & Pintner, 1983).

In this study, an attempt was made to extract and character

ize the chemical and physical properties of EPS produced by an

endophytic bacterium B. licheniformis associated with Gracilaria

dura with the help of advanced analytical techniques. Despite the

biotechnological potential of these biopolymers from marine and

estuarine environments, the role of EPS in seaweed–bacteria inter

01448617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.carbpol.2010.12.061


Carbohydrate Polymers 83 (2011) 891–897


Carbohydrate Polymers

journa l homepage: www.e lsev ier .com/ locate /carbpol

An alkalihalotolerant cellulase from Bacillus flexus isolated from green seaweed

Ulva lactuca

Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C.R.K.Reddy ∗, B. Jha

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR),



Article history:

Received 10 May 2010

Received in revised form 25 August 2010

Accepted 31 August 2010

Available online 6 September 2010

Keywords:

Cellulose

Cellulase

Haloalkali tolerance

Marine habitat

a b s t r a c t

An extracellular alkalihalotolerant cellulase from the strain Bacillus flexus NT isolated from Ulva lactuca

was purified to homogeneity with a recovery of 25.03% and purity fold of 22.31. The molecular weight of

the enzyme was about 97 kDa and the Vmax and Km was 370.17 U/ml/min and 6.18 mg/ml respectively.

The optimum pH and temperature for enzyme activity was 10 and 45 ◦C respectively. The enzymatic

hydrolysis of the CMC was confirmed with GPC and GCMS analysis. The stabilized activity of the enzyme

even at high pH of 9.0–12.0 and residual activity of about 70% at salt concentration (NaCl 15%) revealed for

its alkalihalotolerance nature. The metal ions Cd2+ and Li1+ were found as inducers while Cr2+, Co2+, Zn2+

and metal chelator EDTA have significantly inhibited the enzyme activity. Enzyme activity was insensitive

to ethanol and isopropanol while partially inhibited by acetone, cyclohexane and benzene.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The industrial and agriculture wastes contain considerable

amounts of cellulose that can effectively be utilized either as a

major source of energy feedstock or as a raw material for production

of high value chemicals (Cherry & Fidantsef, 2003; Kim, Yoo, Oh, &

Kim, 2003). Cellulose, the most abundant carbohydrate in nature,

is a linear polysaccharide of repeating units of glucose linked with

1,4 bacetal bond and mainly forms the primary structural cell wall

component in both the lower and higher plants (Saha, Roy, Sen,

& Ray, 2006). The herbaceous and woody plants are the primary

sources of cellulose intact with complex hemicelluloses, lignin and

pectin. Conversely, seaweeds contain mainly acellulose without

much complex lignin thus differentiating them those of terrestrial

plants and make them preferential source of cellulose (Siddhanta

et al., 2009). The efficient hydrolytic conversion of cellulose into

its monomers, i.e. glucose as source for highenergy molecule will

facilitate to meet the future energy need and also will be an alter

nate to starch. Chemical hydrolysis (acid hydrolysis) is one of the

viable methods currently being employed as a promising means of

producing sugar from cellulose. The combination of high tempera

tures and strong acids in acid hydrolysis leads to the degradation of

products, accumulation of nonsugar byproducts (such as inhibitors

∗ Corresponding author. Tel.: +91 278 256 5801/256 3805x614; fax: +91 278 256

6970/256 7562.

Email address: [email protected] ( C.R.K.Reddy).

to subsequent chemical and biological conversion), and also pose

problem of recovery of reaction agents and resulting saccharides

(Sasaki et al., 1998).

The microorganisms with potential cellulolytic activities could

provide unique opportunity towards the biodegradation of cel

lulosic matter through efficient enzymatic conversion into high

energetic molecules (Wen, Liao, & Chen, 2005). Cellulases are

inducible enzymes synthesized by microorganisms during their

growth on the media containing cellulose as a sole source for

carbon (Lee & Koo, 2001). At commercial scale, cellulases have

been obtained mainly from fungal species of Aspergillus and Tri

choderma due to their high activity but at moderate temperature

(Nandakumar, Thankur, Raghavarao, & Ghildyal, 1994). Several bac

terial genera reported for cellulolytic activities include Bacillus,

Clostridium, Cellomonas, Rumminococcus, Alteromonas, Acetivibrio,

Bacteriodes (Roboson & Chambliss, 1989). Industrial applications

of cellulases have been potentially utilized in leather, textile, agri

culture, food, paper and pulp industries (Bhat, 2000; Kim, Hur, &

Hong, 2005). The industrial utility of cellulase enzymes can fur

ther be improved by investigating the functional efficiency of these

enzymes under extreme conditions of temperature and pH. In

comparison to terrestrial environment, marine habitat with hyper

variable conditions could represent the novel functional abilities of

the microbes that can be further elucidated for their potential as

source of extracellular enzymes.

This study describes the potential of marine bacteria B. flexus

as a source of extracellular cellulase with promising applications in

various industries. The strain was studied for salt tolerance with the

01448617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.carbpol.2010.08.069

Author's Personal Copy

Technical Note

Solvent tolerant marine bacterium Bacillus aquimaris secreting organic solventstable alkaline cellulase

Nitin Trivedi, Vishal Gupta, Manoj Kumar, Puja Kumari, C.R.K. Reddy ⇑, Bhavanath Jha

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364 021, India


Article history:


Received in revised form 1 February 2011

Accepted 1 February 2011

Keywords:

Solvent tolerance

Bacillus aquimaris

Fatty acid

Cellulase

Ionic liquids

a b s t r a c t

The organic solvent tolerant bacteria with their physiological abilities to decontaminate the organic pol-

lutants have potentials to secrete extracellular enzymes of commercial importance. Of the 19 marine bac-

terial isolates examined for their solvent tolerance at 10 vol.% concentration, one had the significant

tolerance and showed a relative growth yield of 86% for acetone, 71% for methanol, 52% for benzene,

35% for heptane, 24% for toluene and 19% for ethylacetate. The phylogenetic analysis of this strain using

16S rDNA sequence revealed 99% homology with Bacillus aquimaris. The cellulase enzyme secreted by this

strain under normal conditions showed an optimum activity at pH 11 and 45 °C. The enzyme did show

functional stability even at higher pH (12) and temperature (75 °C) with residual activity of 85% and

95% respectively. The enzyme activity in the presence of different additives were in the following order:

Co+2 > Fe+2 > NaOCl2 > CuSO4 > KCl > NaCl. The enzyme stability in the presence of solvents at 20 vol.%

concentration was highest in benzene with 122% followed by methanol (85%), acetone (75%), toluene

(73%) and heptane (42%). The pre-incubation of enzyme in ionic liquids such as 1-ethyl-3-methylimida-

zolium methanesulfonate and 1-ethyl-3-methylimidazolium bromide increased its activity to 150% and

155% respectively. The change in fatty acid profile with different solvents further elucidated the physio-

logical adaptations of the strain to tolerate such extreme conditions.


1. Introduction

The environment nowadays is increasingly contaminated with

highly toxic monocyclic aromatic hydrocarbons (benzene, toluene,

ethyl benzene and xylenes) as a consequence of rapidly developing

petrochemical and power generating industries worldwide (Wang

et al., 2008). These hydrocarbons are collectively grouped under

BTEX and reported to be carcinogens and pose serious concern to

human health and other kinds of life (Fang et al., 2004). The marine

environment receives about 700 m3 of oil and a variety of hydro-

carbons annually from shipping activities, accidental fuel spills,

and petrochemical based industries (Islam and Tanaka, 2004).

Organic solvents are extremely toxic to cells by virtue of their

ability to disrupt the normal functioning of biological membranes.

The toxicity of solvents is classified on the basis of the measured

values of their partition coefficient in n-octanol and water and

estimated in terms of log Pow value. Solvents with a low log Pow(1.5–4.0) are considered extremely toxic, while others with a high-

er log Pow are less toxic (Inoue et al., 1991). The toxicity level of

organic solvents leads to the cell death by dissolving and disorga-

nizing the cell membrane and finally causes the loss of lipids and

proteins. The microbes dwelling in such toxic organic solvent

habitats can effectively be utilized for bioremediation and various

biotransformation processes of industrial effluents (Inoue et al.,

1991; Ramos et al., 1995; Na et al., 2005; Fang et al., 2006; Chen

et al., 2009). Micro-organisms develop versatile mechanisms such

as rigidification and alteration of cell membrane, or by forming

vesicles to resist the entry of organic solvents (Heipieper et al.,

2007; Segura et al., 2008). The cis–trans isomerization of fatty acids

(FAs) has also been reported as adaptive mechanism in Pseudomo-

nas and Vibrio (Ramos et al., 1997; Heipieper et al., 2003). The other

adaptive mechanisms include modification of lipopolysaccharide

or the porines, active export of the solvent and transformation of

the solvent (Isken and de Bont, 1998). The organic solvent tolerat-

ing microbes particularly the BTEX degraders have been investi-

gated from terrestrial environments that includes largely from

the soil, sewage and also from anaerobic habitat (Botton and

Parsons, 2006). However, only a few micro-organisms from marine

habitat have been investigated for solvent tolerance. Marine

microbes are often exposed to diverse conditions ranging from

extreme temperature, pressure, salinity, pH, photo-radiations, hea-

vy metals, organic solvents and hydrocarbons. In addition to phys-

iological adaptive characteristics, synthesis of exozymes is one of

the notable adaptive catabolic mechanisms of micro-organisms


doi:10.1016/j.chemosphere.2011.02.006

⇑ Corresponding author. Tel.: +91 278 256 5801/3805x614; fax: +91 278 256

6970/7562.


Chemosphere 83 (2011) 706–712


Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere


Growth and agarose characteristics of isomorphic gametophyte (male and female)

and sporophyte of Gracilaria dura and their marker assisted selection

Vishal Gupta, Ravi S. Baghel, Manoj Kumar, Puja Kumari, Vaibhav A. Mantri, C.R.K. Reddy ⁎, Bhavnath Jha ⁎


a b s t r a c ta r t i c l e i n f o

Article history:

Received 7 February 2011

Received in revised form 4 June 2011

Accepted 7 June 2011

Available online 15 June 2011

Keywords:

Agarose

Chromosome

Gracilaria dura

Marker assisted selection

Life cycle stage

The characteristics of agarose and growth for three isomorphic life phases of G. durawith their bio-molecular

marker assisted selection have been described in this study. The tetrasporophyte showed superior quality of

agarose over gametophytes and recorded growth rate was highest for females. The genetic relatedness

studied with ISSR markers showed quadratic line of correlation between these phases (R2=1). Their genetic

diversity determinants as percentage of polymorphic loci (PPL), average heterozygosity (He) and Shannon's

Weaver index (I) were 55.55%, 0.5±0.07 and 0.33 respectively. The cytological analysis for chromosome

count revealed 8 chromosomes in haploid gametophytic thallus (N) and 16 for diploid tetrasporophyte (2N)

together with genetic structure analysis confirmed to their sexual mating behaviour. Their marker assisted

selection based on ISSR generated characteristic band of 430 bp specific to male, 860 bp for female and two

bands of 800 and 1600 bp for tetrasporophytic thallus from primer ‘A’. Similarly ISSR primer ‘E’ also generated

bands specific to male, female and tetrasporophytes while others gave bands specific to either of life phase.

Interestingly, endogenic ABA content was significantly higher for haploid gametophytes (female more than

male) than diploid tetrasporophyte while no significant difference was observed in IBA content. Thus the

study described not only the features of three life phases of G. dura but also reliable biomarkers for

differentiating such isomorphic life phases which could be beneficial for the selection of cultivar and in

breeding programmes.


1. Introduction

Among the red algae, genus Gracilaria constitutes important

agarophytes with more than 150 species reported across the world

(Byrne et al., 2002). The development of improved processing

technologies for agar extraction has increased the annual harvest

rate of this genus. The agar industries throughout the world consume

over 72,300 dry tonnes of agarophytes annually to produce ca.

9600 tonnes of agar worth of US$173 million (Bixler and Porse,

2011). Of this, Gracilaria alone accounts for about 80% of the world's

agar market with a value of US$ 138 million (Bixler and Porse, 2011).

The increasing demand for agar worldwide coupled with short

supplies of raw material from wild stocks has led to the development

of viablemethods for their large scale cultivation in the sea (Peng et al.,

2009). The cultivation of Gracilaria for commercial purposes is being

carried out in several countries including Chile, China and Taiwan and

on pilot scale in Namibia, Venezuela, Mexico and India.

Gracilaria dura (C. Agardh) J. Agardh from the Indian waters has

been reported to produce superior quality agarose (1%

gelN1900 g cm−2) suitable for biotechnological applications (Meena

et al., 2007). Like other tropical seaweeds the limited distribution

coupled with short lifespan prevented its commercial utilisation thus

underlie the need for their cultivation. Further, selection of cultivars

or life phases with superior traits of growth and phycocolloid will

ascertain the economical viability of resources.

The genus Gracilaria has a characteristic Polysiphonia type life cycle

with an alternation of isomorphic gametopyhtic (male and female)

and tetrasporophytic generations (Engel et al., 2004). Moreover,

isomorphic life phases with high resemblances in their morphological

features have frequently misled the identification. Thus, segregation

of life phases at early stages of their development is desired for

cultivar selection, breeding and other biotechnological interventions

aimed at genetic improvement.

In higher plants, several physiological processes are controlled

with synchronised hormonal signals (Stirk et al., 2009). The reported

implication of plant growth regulators (PGRs) seems to be very

promising since some of these compounds can be used as potential

markers for segregating the isomorphic life phases of species such as

G. dura. Along with the PGRs, the adoption of molecular sex-linked

markers offers additional tools for differentiation of life phases of

species even at their juvenile stages dispensing the long wait till the

development of reproductive bodies (Sim et al., 2007).

The RAPD based molecular markers have been extensively

employed for determination of sex in higher plants (Chaves-Bedoya

Aquaculture 318 (2011) 389–396

⁎ Corresponding authors. Tel.: +91 278 256 5801/256 3805x614; fax: +91 278 256

6970/256 7562.


0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2011.06.009


Aquaculture

j ourna l homepage: www.e lsev ie r.com/ locate /aqua-on l ine

Genetic analysis and marker assisted identification of life phases

of red alga Gracilaria corticata (J. Agardh)

Ravi S. Baghel • Puja Kumari • A. J. Bijo •

Vishal Gupta • C. R. K. Reddy • B. Jha

Received: 5 June 2010 / Accepted: 16 November 2010 / Published online: 30 November 2010

Ó Springer Science+Business Media B.V. 2010

Abstract The present study firstly reports the cytological

and molecular marker assisted differentiation of isomor-

phic population of Gracilaria corticata (J. Agardh) with

inter and intra-phasic genetic diversity analysis using ISSR

markers. The genetic diversity of inbreeding population of

G. corticata as determined in terms of percentage of

polymorphic loci (PPL), average heterozygosity (He) and

Shannon’s Weaver index (I) were 59.80, 0.59 and 1.21,

respectively. The inter-phasic pair-wise average polymor-

phism were found to be 31.6% between male and female,

24.0% in male and tetrasporophyte and 25.3% in female

and tetrasporophyte. The intra-phasic average polymor-

phisms were calculated as a maximum of 5.5% between

females, 4.2% between males and the lowest 2.4% between

tetrasporophytes. The primer 10 generated a marker of

800 bp specific to male and 650 bp to female gametophyte,

while the primer 17 generated a marker of 2,500 bp spe-

cific to tetrasporophyte. Both the UPGMA based dendro-

gram and PCA analysis clustered all the three life phases

differentially as distinct identity. Cytological analysis by

chromosome count revealed 24 chromosomes in both

haploid male and female gametophytes (N) and 48 for

diploid (2 N) tetrasporophyte further confirming their

genetic distinctness. The life phase specific markers

reported in this study could be of help in breeding pro-

grammes where differentiation of life phases at the early

developmental stages is crucial.

Keywords Gracilaria corticata Life phase

Inter-Simple Sequence Repeat (ISSR) markers

Genetic structure Chromosome

Introduction

Among the seaweeds, Gracilaria is the third largest genus

with more than 150 species reported across the world and

consists of many commercially important agarophytes [2].

At present, it contributes to more than half of the global

agar industry requirement [18]. Farming of Gracilaria is

being carried out in several countries including the Phil-

ippines, Chile, China, Korea, Indonesia, Namibia, Vietnam

and Argentina at a commercial scale [14] to complement

the growing raw material demand by world agar industry.

Of the Gracilaria species, G. corticata which commonly

inhabit in the intertidal pools and lower intertidal zone has

been reported as a potential source of raw material for food

grade agar [1, 8]. G. corticata from the west coast of India

has been reported to yield food grade agar of about 16%

with the gel strength of 100 g/cm2 [15].

The G. corticata has an isomorphic triphasic life cycle

wherein an alternation of morphologically inseparable yet

genetically distinct generations of haploid and diploid

phases occurs. Of the three life phases only the haploid

female gametophytes can easily be identified by the pres-

ence of cystocarps on the surface of thallus after fertiliza-

tion. The other two life phases i.e. male gametophyte and

tetrasporophyte can be identified microscopically only after

reproductive maturity. At present, there are no reliable

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-010-0543-y) contains supplementarymaterial, which is available to authorized users.

R. S. Baghel P. Kumari A. J. Bijo V. Gupta

C. R. K. Reddy (&) B. Jha






123

Mol Biol Rep (2011) 38:4211–4218

DOI 10.1007/s11033-010-0543-y

Original Article

Assessment of nutrient composition and antioxidant potential of Caulerpaceae

seaweeds

Manoj Kumar, Vishal Gupta, Puja Kumari, C.R.K. Reddy *, B. Jha


1. Introduction

An increasing human population, global climate change and the

diversificationof terrestrial food resources for energyneeds in recent

times have raised serious global food security concerns (Rosegrant

and Cline, 2003). Further, the globalization of markets has also

brought about an increasing globalization of foods, diminishing the

boundaries of human races andgeographical regions of the countries

throughout the world. There has also been a quest to explore and

utilize foods from non-conventional sources, of both terrestrial and

marine origin, to enhance and supplement the nutritional quality of

human foods. Also, this in turn eases off the growing burden on

traditional foods. Marine macroalgae, commonly known as sea-

weeds, are one of the living renewable resources of the oceans with

potential food applications. Consumption of seaweeds as sea

vegetables in human diets has been the common practice in several

Asian countries (Nisizawa, 2002). Presently, interest in supplement-

ing the human foods with antioxidants particularly from natural

sources has been on the raise as synthetic antioxidants have been

suspected to be a possible cause for liver damage and carcinogenesis

(Farag et al., 2003; Tang et al., 2001). Therefore, there is a need for

isolationandcharacterizationofantioxidantshavingleastsideeffects

from natural sources as an alternative to synthetic antioxidants.

The previous studies have demonstrated the potential of

enzymatic superoxide dismutase (SOD), catalase (CAT), ascorbate

peroxidase (APX) and glutathione reductase (GR) and non-

enzymatic (polyphenols, glutathione, ascorbic acid and carotenoids)

antioxidants scavenging the reactive oxygen species (ROS) thus

relieving from the oxidative stresses and other associated health

risks such as cancer, coronary heart diseases, neurodegenerative

diseases and inflammation (Duan et al., 2006; Kuda and Ikemori,

2009; Nagai and Yukimoto, 2003). The recent findings have also

revealedavailability ofusefulmetaboliteswithmedicinal properties

from some marine biota (Blunt et al., 2005; Mayer et al., 2009).

Recently, the genus Caulerpa has attracted the attention of

researchers due to its important secondary metabolite caulerpe-

nyne (CYN) that is reported to exhibit the antineoplastic,

antibacterial and antiproliferative activities (Barbier et al., 2001;

Cavas et al., 2006). Further, it has also been shown to inhibit the cell

division of sea urchin eggs as well as cancer cell lines (Fischel et al.,

1995; Lemee et al., 1993). Three species of Caulerpa namely C.

racemosa, C. scalpelliformis and C. veravelensis have been found

growing luxuriantly in the intertidal region during October–

February along theVeraval coast of Gujarat (north-western coast of

India). Among these three species, C. racemosa with a wide

Journal of Food Composition and Analysis 24 (2011) 270–278

A R T I C L E I N F O

Article history:


Received in revised form 5 July 2010

Accepted 31 July 2010

Available online 8 December 2010

Keywords:

Antioxidants

Biochemical constituents

Caulerpa

Minerals

Nutritional supplement

Pigments

Polyunsaturated fatty acids

Seafood

Food analysis

Food composition

A B S T R A C T

The proximate nutrient composition, mineral contents, enzymatic and non-enzymatic antioxidant

potential of three Caulerpa species were investigated. All three species were high in ash (24.20–33.70%)

and carbohydrate content (37.23–48.95%) on dry weight basis (DW). The lipid content ranged between

2.64 and 3.06% DW. The mineral contents varied marginally among the species but were in the order of

Na > K > Ca >Mg. The Na/K ratio among the species varied from 1.80 to 2.55 and was lowest in C.

scalpelliformis. A 10 g DW of Caulerpa powder contains 11–21% Fe, 52–60% Ca and 35–43% Mg, which is

higher than the recommended daily allowance (RDA), compared with non-seafood. The percentage sum

of PUFAs (C18:2, C18:3, C20:4 and C20:5) in total fatty acids was highest in both C. scalpelliformis

(39.25%) and C. veravelensis (36.73%) while it was the lowest in C. racemosa (24.50%). The n 6/n 3 ratio

among the species varied from 1.44 to 7.72 and remained within the prescribed WHO standards (<10).

Further, the higher enzymatic dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and

glutathione reductase (GR) and non-enzymatic antioxidant potential of Caulerpa species found in the

present study confirm their usefulness in terms of nutrients and antioxidants.

ß 2010 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +91 278 256 5801/256 3805x614;

fax: +91 278 256 6970/256 7562.



Journal of Food Composition and Analysis

journa l homepage: www.e lsev ier .com/ locate / j fca

0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2010.07.007

Differential responses to cadmium induced oxidative stress

in marine macroalga Ulva lactuca (Ulvales, Chlorophyta)

Manoj Kumar • Puja Kumari • Vishal Gupta •

P. A. Anisha • C. R. K. Reddy • Bhavanath Jha

Received: 7 January 2010 / Accepted: 12 January 2010 / Published online: 30 January 2010

Ó Springer Science+Business Media, LLC. 2010

Abstract This study describes various biochemical

processes involved in the mitigation of cadmium

toxicity in green alga Ulva lactuca. The plants when

exposed to 0.4 mM CdCl2 for 4 days showed twofold

increase in lipoperoxides and H2O2 content that

collectively decreased the growth and photosynthetic

pigments by almost 30% over the control. The

activities of antioxidant enzymes such as superoxide

dismutase (SOD), ascorbate peroxidase (APX), glu-

tathione reductase (GR) and glutathione peroxidase

(GPX) enhanced by twofold to threefold and that of

catalase (CAT) diminished. Further, the isoforms of

these enzymes, namely, Mn-SOD (*85 kDa), GR

(*180 kDa) and GPX (*50 kDa) responded specif-

ically to Cd2? exposure. Moreover, the contents of

reduced glutathione (3.01 fold) and ascorbate

(1.85 fold) also increased substantially. Lipoxyge-

nase (LOX) activity increased by two fold coupled

with the induction of two new isoforms upon Cd2?

exposure. Among the polyunsaturated fatty acids,

although n - 3 PUFAs and n - 6 PUFAs (18:3n - 6

and C18:2n - 6) showed relatively higher contents

than control, the latter ones showed threefold increase

indicating their prominence in controlling the cad-

mium stress. Both free and bound soluble putrescine

increased noticeably without any change in spermi-

dine. In contrast, spermine content reduced to half over

control. Among the macronutrients analysed in

exposed thalli, the decreased K content was accom-

panied by higher Na and Mn with no appreciable

change in Ca, Mg, Fe and Zn. Induction of antioxidant

enzymes and LOX isoforms together with storage of

putrescine and n - 6 PUFAs in cadmium exposed

thallus in the present study reveal their potential role in

Cd2? induced oxidative stress in U. lactuca.

Keywords Antioxidant enzymes

Cadmium LOX Minerals Oxidative stress

PUFAs Ulva lactuca

Introduction

Of the toxic substances contaminating the aquatic

environment, heavy metals particularly cadmium,

lead and mercury are of great concern for humans as

well as for the environment because of their acute

toxicity and high mobility in food chain (Sokolova

et al. 2005). Cadmium (Cd2?), with no reported

biological function except one occasion as a cofactor

for carbonic anhydrase in marine diatom (Lane and

M. Kumar P. Kumari V. Gupta

C. R. K. Reddy (&) B. Jha

Discipline of Marine Biotechnology and Ecology, Central

Salt and Marine Chemicals Research Institute, Council

of Scientific and Industrial Research (CSIR), Bhavnagar

364021, India


P. A. Anisha

School of Environmental Studies, Cochin University

of Science and Technology, Cochin, India

123

Biometals (2010) 23:315–325

DOI 10.1007/s10534-010-9290-8


Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to

salinity induced oxidative stress

Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy ⁎, Bhavanath Jha


a b s t r a c ta r t i c l e i n f o

Article history:

Received 26 February 2010

Received in revised form 31 May 2010

Accepted 1 June 2010

Keywords:

Antioxidant enzymes

Gracilaria corticata

Minerals

Oxidative stress

Phycobiliproteins

PUFAs

Salinity stress

The biochemical responses of Gracilaria corticata (J. Agardh) J. Agardh to salinity induced oxidative stress were

studied following the exposure to different salinities ranging from 15, 25, 35 (control), 45 to 55 in laboratory

conditions. The growth was highest under 25 (3.14±0.69% DGR) and 35 (3.58±0.32% DGR) and decreased

significantly in both extreme lower (15) and hyper (55) salinities. Both phycoerythrin (PE) and

allophycocyanin (APC) were significantly higher in hyper-salinity (45) with an increase of almost 70% and

52% from their initial contents. Conversely, the level of increase of the same in hypo-salinitieswas considerably

lower as compared with that of hyper-salinity. Both hypo- and hyper-salinity treatments induced almost two

fold increase in the contents of polyphenols, proline and the activities of antioxidative enzymes such as

superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) especially for 6 d

exposure. The Na+ ions readily displaced the K+ and Ca2+ from their uptake sites at extreme hyper-salinity

(55) and accounted for substantial increase in the ratio of Na+/K+ and Na+/Ca2+ that impeded the

growth under long term exposure (N6 d). The survivability at salinity 45 evenwith considerably higher ratio of

Na+/K+ and Na+/Ca2+ suggests the compartmentalization of Na+ into the vacuoles. Further, the micro

nutrients such as Zn, Fe and Mn were decreased at both high and low end salinities with highest at extreme

hyper-salinity. The C18:1(n−9) cis, C18:2(n−6), C18:3(n−3) and C20:4(n−6) were found in significant

amounts in hyper-salinities. The C18:1(n−9) cis in particular increased by 60.25% and 70.51% for salinities 45

and 55, respectively from their initial amounts. The ratio of total unsaturated to saturated fatty acids (UFA/SFA)

also increased linearly with increasing salinity. These results collectively suggest the potential role of

antioxidative enzymes, phycobiliproteins, PUFAs and mineral nutrients to combat the salinity induced

oxidative stress in G. corticata.


1. Introduction

The red alga Gracilaria corticata (J. Agardh) J. Agardh is one of the

common algae of the Indian coast and occurs predominantly in the

lower littoral zone. It also inhabits occasionally in the intertidal rock

pools as submerged population. The intertidal algae often get exposed

to the atmosphere periodically during low tide regimes and experi-

ence an oxidative stress on regular basis with the turning tides. In

marine waters, salinity around 35 is the most common, but it could

also vary from 10 to 70 as a result of evaporation or precipitation/

freshwater influxes (Graham and Wilcox, 2000). Osmotic stress most

often resulting from fluctuating salinities exerts considerable oxida-

tive stress on seaweeds in the intertidal zone. The previous studies

have investigated the responses of estuarine macroalgae for either

individual or combined abiotic factors (light, pH, temperature,

nutrient load and salinity) in the context of growth andphotosynthetic

performance (Macler, 1988; Dawes et al., 1999; Israel et al., 1999; Choi

et al., 2006; Phoopronget al., 2007). Subsequent studies have also dealt

with the possible effects of environmental stresses on floristic

variations of intertidal benthic macro algal communities (Helmuth

et al., 2005).

It has been suggested that instant responses of marine plants to

adverse environmental conditions involve excess production of

reactive oxygen species (ROS) such as hydrogen peroxide (H2O2),

singlet oxygen (1O2), superoxide radical (O2•−) and hydroxyl radical

(OH−) (Dring, 2006). Increased physiological stress conditions lead to

the rapid formation of ROS that reacts with most cellular components

and thus they need to be neutralized instantly once formed.

Acclimation to altered osmotic conditions particularly to salinity

induced stress involves changes in physiological processes including

antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT),

ascorbate peroxidase (APX) and glutathione reductase (GR)] and non-

enzymatic antioxidants (ascorbate, glutathione and carotenoids).

All these processes function in coordinated manner in order to

alleviate the cellular hypo/hyper osmolarity, ion disequilibrium and

Journal of Experimental Marine Biology and Ecology 391 (2010) 27–34

⁎ Corresponding author. Tel.: +91 278 256 5801/256 3805x614; fax: +91 278 256

6970 / 256 7562.


0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.jembe.2010.06.001


Journal of Experimental Marine Biology and Ecology

j ourna l homepage: www.e lsev ie r.com/ locate / jembe

Purification and partial characterization of an extracellular

alginate lyase from Aspergillus oryzae isolated

from brown seaweed

Ravindra Pal Singh & Vishal Gupta & Puja Kumari &

Manoj Kumar & C. R. K. Reddy & Kamalesh Prasad &

Bhavanath Jha

Received: 15 April 2010 /Revised and accepted: 9 August 2010 /Published online: 2 September 2010# Springer Science+Business Media B.V. 2010

Abstract The extracellular enzyme alginate lyase produced

from marine fungus Aspergillus oryzae isolated from brown

algaDictyota dichotoma was purified, partially characterized,

and evaluated for its sodium alginate depolymerization

abilities. The enzyme characterization studies have revealed

that alginate lyase consisted of two polypeptides with about

45 and 50 kDa each on 10% sodium dodecyl sulfate

polyacrylamide gel electrophoresis and showed 140-fold

higher activity than crude enzyme under optimized pH (6.5)

and temperature (35°C) conditions. Zn2+, Mn2+, Cu2+, Mg2+,

Co2+ and NaCl were found to enhance the enzyme activity

while (Ca2+, Cd2+, Fe2+, Hg2+, Sr2+, Ni2+), glutathione, and

metal chelators (ethylenediaminetetraacetic acid and eth-

ylene glycol tetraacetic acid) suppressed the activity.

Fourier transform infrared and thin-layer chromatography

analysis of depolymerized sodium alginate indicated the

enzyme specificity for cleaving at the β-1,4 glycosidic

bond between polyM and polyG blocks of sodium alginate

and therefore resulted in estimation of relatively higher

polyM content than polyG. Comparison of chemical shifts

in 13C nuclear magnetic resonance spectra of both polyM

and polyG from that of sodium alginate also showed

further evidence for enzymatic depolymerization of sodi-

um alginate.

Keywords Alginate lyase . Aspergillus oryzae . Fungus .

PolyM . PolyG . Sodium alginate

Introduction

Alginate occurs as a structural cell wall polysaccharide in a

wide variety of brown seaweeds. It can also be obtained

from bacteria such as Azotobacter vinelandii (Gorin and

Spencer 1966) and Pseudomonas aeruginosa (Evans and

Linker 1973) but with poor gelling characteristics. Alginates

are linear unbranched polymers consisting of 1,4-linked β-D-

mannuronic acid (M) and α-L-guluronic acid (G) blocks,

arranged as either homopolymeric (M–M or G–G blocks) or

heteropolymeric (M–G and G–M blocks) random sequences

(Gacesa 1992). Alginates are commercially important cell

wall polysaccharides and widely used as stabilizers, visco-

sifiers, and gelling agents in diverse products such as food,

beverages, and pharmaceuticals industries (Wong et al.

2000). The polyM has been well studied and reported as a

potent inducer of cytokines under acute inflammatory

responses (Jahr et al. 1997). In contrast, polyG inhibits the

secretion of cytokines, resulting to the alleviation of the

immunostimulation during tissue grafting and other autoim-

mune disorders (Otterlei et al. 1992). The alginate derivatives

with sulfate groups have been reported to have high tumor

inhibition activity against solid sarcoma 180 in vivo (Hu et

al. 2004) in addition to tissue engineering applications

(Kataoka et al. 2004). Further, the depolymerized products

of alginate have also been stated to promote germination,

growth, and development in crop plants (Cao et al. 2007).

Alginate lyases, characterized as either mannuronate (EC

4.2.2.3) or guluronate lyases (EC 4.2.2.11), catalyze the

degradation of alginate. Alginates can be depolymerized

into respective oligosaccharide fragments using either

enzymatic lyases or acid hydrolysis. Alginate lyase uses a

β-elimination in which a non-reducing unsaturated bond is

produced during cleavage of the uronic acid, giving rise to

R. P. Singh : V. Gupta : P. Kumari : M. Kumar :

C. R. K. Reddy (*) : K. Prasad : B. Jha

Discipline of Marine Biotechnology and Ecology, Central Salt and

Marine Chemicals Research Institute, Council of Scientific and

Industrial Research (CSIR),



J Appl Phycol (2011) 23:755–762

DOI 10.1007/s10811-010-9576-9

Documents

Appendix - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36330/15/15_appendix.pdf · Appendix 241 Appendix I Description of collection sites of different macroalgae. Species