Bioprospecting for high PUFA content in Northern marine microalgae Pia Steinrücken University of Bergen Norway 1
Focus new microalgae with industrial potential University of Bergen, Norway University of Las Palmas de Gran Canaria University of Antofagasta, Chile 1. Bioprospecting 2. Characterization of selected strains 3. utdoor mass cultivation 2
Focus new microalgae with industrial potential University of Bergen, Norway 1. Bioprospecting Polyunsaturated fatty acids H 3
Fatty acids SAFA saturated fatty acids H MUFA monounsaturated fatty acids H PUFA polyunsaturated fatty acids H Polar lipids Algae cell P Neutral lipids Triacylglycerol (TAG) Chloroplast Nucleus mitochondria 4
Fatty acids SAFA saturated fatty acids H MUFA monounsaturated fatty acids H PUFA polyunsaturated fatty acids H Polar lipids Algae cell P Neutral lipids Triacylglycerol (TAG) Chloroplast Nucleus mitochondria 5
Fatty acids SAFA saturated fatty acids H MUFA monounsaturated fatty acids H PUFA polyunsaturated fatty acids H Polar lipids Algae cell P Neutral lipids Triacylglycerol (TAG) Chloroplast TAG Nucleus mitochondria TAG TAG 6
PUFAs EPA Eicosapentaenoic acid 20:5 DHA Provide health benefits Cardiovascular health Neural system Immune system Essential in human nutrition Docosahexaenoic acid 22:6 Major source Fish oil EPA Algae EPA DHA DHA EPA DHA Health products EPA DHA aquaculture Food Health products EPA DHA aquaculture Food 7
PUFAs EPA Eicosapentaenoic acid 20:5 DHA Provide health benefits Cardiovascular health Neural system Immune system Essential in human nutrition Docosahexaenoic acid 22:6 Major source Fish oil EPA Algae EPA DHA DHA EPA DHA EPA DHA Health products aquaculture Food 8
Concept 1. Bioprospecting 9
Concept 1. Bioprospecting Sampling Isolation Cultivation Screening Selection 10
Sampling 11
Sampling 12
Sampling Arctic Cruises May 2014 July 2014 Nov2014 August 2016 13
Sampling Bergen 14
Sampling Bergen 15
Sampling Bergen 16
Sampling Bergen 17
Isolation 18
Isolation 19
Isolation 20
Cultivation 15 C PAR: 50 µmol photons m 2 s 1 10 C PAR: 50 µmol photons m 2 s 1 Conditions Medium: 80% Sea Water Nutrients (Walne`s) Light : 50 µmol photons m 2 s 1 16:8 light/dark Temp.: 15 C Fjord summer 10 C Arctic, Fjord winter 21
Cultivation 15 C PAR: 50 µmol photons m 2 s 1 10 C PAR: 50 µmol photons m 2 s 1 22
Cultivation 15 C PAR: 50 µmol photons m 2 s 1 10 C PAR: 50 µmol photons m 2 s 1 23
Cultivation 15 C PAR: 50 µmol photons m 2 s 1 25 strains Diatoms Green Algae 149 clonal strains 10 C PAR: 50 µmol photons m 2 s 1 124 strains Diatoms 24
Screening D 0.4 0.2 0.0 0 1 2 3 4 5 6 7 8 9 10 Day Conditions Medium: 80% Sea Water Nutrients (Walne`s) Light : 100 120 µmol photons m 2 s 1 continuous Temp.: 15 C Fjord summer 10 C Arctic, Fjord winter 25
Screening D 0.4 + Nutrients Nutrients 0.2 0.0 0 1 2 3 4 5 6 7 8 9 10 Day Conditions Medium: 80% Sea Water Nutrients (Walne`s) Light : 100 120 µmol photons m 2 s 1 continuous Temp.: 15 C Fjord summer 10 C Arctic, Fjord winter 26
Screening D 0.4 + Nutrients Nutrients 0.2 TAG 0.0 0 1 2 3 4 5 6 7 8 9 10 Day Conditions Medium: 80% Sea Water Nutrients (Walne`s) Light : 100 120 µmol photons m 2 s 1 continuous Temp.: 15 C Fjord summer 10 C Arctic, Fjord winter 27
Screening D 0.4 + Nutrients Nutrients 0.2 0.0 0 1 2 3 4 5 6 7 8 9 10 Day Analysis Daily: D 750 Exp & stat: 3x Dry weight 3x Fatty acids End: Molecular Identification partial LSU sequences 28
Screening Fatty acid analysis ne step extraction / methylation Internal standard 23:0 FAME Gas chromatography Analysis Daily: D 750 Exp & stat: 3x Dry weight 3x Fatty acids End: Molecular Identification partial LSU sequences 29
Screening 15 C 10 Strains Fjord 8 Diatoms 2 Green algae 1 2 3 4 5 6 7 8 9 10 10 C 10 Strains 8 Arctic 2 Fjord winter Diatoms 11 12 13 14 15 16 17 18 19 20 30
Screening No ID 10 C 15 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 31
Screening No ID 10 C 15 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 32
Screening No ID 10 C 15 C 1 2 3 100% Nitzshia laevis 4 5 96% Acrocellulus cornucervis 6 99% Nanofrustulum shiloi 7 99% Phaeodactylum tricornutum 8 9 99% Phaeodactylum tricornutum 10 99% Phaeodactylum tricornutum 11 12 13 100% Thalassiosira hispida 100% Thalassiosira hispida 99% Attheya septentrionalis 14 15 16 17 18 100% Thalassiosira hispida 100% Thalassiosira hispida 100% Thalassiosira hispida 100% Thalassiosira hispida 19 20 100% Thalassiosira hispida 100% Thalassiosira hispida 33
Screening 10 C 15 C No ID 1 88% Micractinium sp. 2 3 100% Nitzshia laevis 4 5 96% Acrocellulus cornucervis 6 99% Nanofrustulum shiloi 7 99% Phaeodactylum tricornutum 8 9 99% Phaeodactylum tricornutum 10 99% Phaeodactylum tricornutum 11 12 13 100% Thalassiosira hispida 100% Thalassiosira hispida 99% Attheya septentrionalis 14 15 16 17 18 100% Thalassiosira hispida 100% Thalassiosira hispida 100% Thalassiosira hispida 100% Thalassiosira hispida 19 20 100% Thalassiosira hispida 100% Thalassiosira hispida 34
Screening Biomass 10 C 15 C No ID Growth EPA DHA 1 Industrial benchmark levels: 2 3 4 EPA > 18% of total fatty acids DHA > 12% of total fatty acids 5 6 7 8 9 µ av > 0,7 10 11 12 13 14 15 16 17 Time 18 19 20 35
Screening Biomass 10 C 15 C No ID Growth EPA DHA 1 Industrial benchmark levels: 2 3 4 5 6 7 8 9 10 EPA > 18% of total fatty acids DHA > 12% of total fatty acids µ av > 0,7 11 12 13 14 15 16 17 Time 18 19 20 36
Screening 10 C 15 C Biomass No ID Growth EPA DHA 1 Industrial benchmark levels: 2 3 4 5 6 7 8 9 10 EPA > 18% of total fatty acids DHA > 12% of total fatty acids µ av > 0,7 11 12 13 14 15 16 17 Time 18 19 20 37
Screening 10 C 15 C Biomass No ID Growth EPA DHA 1 Industrial benchmark levels: 2 3 4 5 6 7 8 9 10 EPA > 18% of total fatty acids DHA > 12% of total fatty acids µ av > 0,7 11 12 13 14 15 16 17 Time 18 19 20 38
Screening 10 C 15 C Biomass No ID Growth EPA DHA 1 Industrial benchmark levels: 2 3 4 5 6 7 8 9 10 EPA > 18% of total fatty acids DHA > 12% of total fatty acids µ av > 0,7 11 12 13 14 15 16 17 Time 18 19 20 39
1 2 3 4 5 6 7 8 9 10 No 11 12 13 14 15 16 17 18 19 20 Growth EPA DHA ID 7 9 10 3 13 13 Screening 40 10 C 15 C P. tricornutum P. tricornutum P. tricornutum Attheya septentrionalis
Selection g l 1 D 750 13 1 0.1 exp stat Attheya septentrionalis 0.01 0 5 10 15 Day 0.3 0.2 total DW total FA 0.1 0.0 12 % exp 19 % stat 41
Selection D 750 13 1 stat 0.1 exp Attheya septentrionalis 0.01 0 5 10 15 Day m g g 1 D W 100 80 60 40 exp stat 25 % total FA 24 % total FA g l 1 0.3 0.2 0.1 total DW total FA 20 0 0.0 12 % 19 % exp stat FA 42
m g g 1 D W D 750 Selection 9 200 100 80 60 40 stat exp P. tricornutum 20 % total FA 7 % total FA 1 0.1 exp stat 20 0.01 0 5 10 15 0 Day 100 80 exp stat FA 25 % total FA 24 % total FA 0.3 total DW total FA m g g 1 D W 60 40 g l 1 0.2 0.1 20 0 0.0 12 % 19 % exp stat FA 43
Selection D 750 13 1 stat 0.1 exp Attheya septentrionalis 0.01 0 5 10 15 Day m g g 1 D W 100 80 60 40 exp stat 25 % total FA 24 % total FA g l 1 0.3 0.2 0.1 total DW total FA 20 0 0.0 12 % 19 % exp stat FA 44
Selection D 750 13 1 stat 0.1 exp Attheya septentrionalis 0.01 0 5 10 15 Day m g g 1 D W 100 80 60 40 20 0 exp stat 25 % total FA 24 % total FA 25 % total FA g l 1 0.3 0.2 0.1 0.0 total DW total FA 12 % 19 % 26 % exp stat stat FA Increase in EPA content in stationary phase from 3% to 4,6 % to 6,4% of DW 45
Perspective 13 Attheya septentrionalis 2. Characterization of selected strains 46
Perspective 1. Bioprospecting 2. Characterization of selected strains 47
MiraclesBergenTeam Svein Rune Erga Pia Steinrücken Siv Kristin Prestegard Hans Kleivdal Fatty Acids Svein Mjos 48
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Selection m g g 1 D W D 750 9 200 100 80 60 exp P. tricornutum 20 % total FA 1 0.1 exp stat 40 20 0.01 0 5 10 15 0 Day m g g 1 D W 100 80 60 40 exp stat FA 25 % total FA 24 % total FA g l 1 0.3 0.2 0.1 total DW total FA 20 0 0.0 12 % 19 % exp stat FA 50