Heterotrophic cultivation of microalgae as a source of docosahexaenoic acid for aquaculture

203 Pages·2008·18.05 MB·English

Heterotrophic cultivation of
microalgae as a source of
docosahexaenoic acid for
aquaculture
ENEKO GANUZA TABERNA
Grupo de Investigacio´n en Acuicultura
Intituto Canario de Ciencias Marinas
Universidad de Las Palmas de Gran Canaria (IUSA)
Being a thesis submitted for the degree of
Doctor of Phylosophy
in the University of Las Palmas de Gran Canaria, 2008
Directors:
Prof. Marisol Izquierdo & Prof. Colin Ratledge
El haber nacido cerca del mar me gusta, me ha parecido siempre
como un augurio de libertad y cambio...
Pio Baroja
Contents
List of tables
III
List of figures
V
List of abbreviations
VII
Acknowledgements
IX
1. General introduction 1
1.1. Lipids and their role in living organisms . . . . . . . . . . . . 1
1.2. Lipid biochemistry . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3. Importance of HUFA for marine finfish . . . . . . . . . . . . . 6
1.4. Importance of HUFA for human health . . . . . . . . . . . . . 9
1.5. Fish oil as a source of HUFA . . . . . . . . . . . . . . . . . . . 10
1.6. Single cell oils as source of HUFA . . . . . . . . . . . . . . . . 12
1.7. DHA-producing microorganisms . . . . . . . . . . . . . . . . . 14
1.8. HUFA producing microorganisms in aquaculture . . . . . . . . 19
1.9. Gilthead seabream as a model to study the effect of hetero-
trophic organisms as a source of HUFA . . . . . . . . . . . . . 20
1.10.Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2. General materials and methods 39
2.1. Microbial cultures . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2. Rotifers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.3. Experimental microdiets . . . . . . . . . . . . . . . . . . . . . 45
2.4. Gilthead seabream larviculture . . . . . . . . . . . . . . . . . . 48
3. Lipid accumulation in Schizochytrium G13/2S produced in
continuous culture 53
4. The influence of air supply on fatty acid production by Schi-
zochytrium G13/2S and Crypthecodinium cohnii 67
5. Restriction in propionic acid precursors inhibits odd-chain
fatty acid synthesis in Schizochytrium G13/2S 79
i
Contents
6. High-cell-density cultivation of Schizochytrium G13/2S in
an ammonium/pH-auxostat fed-batch system 87
7. Docosahexaenoic acid production by Crypthecodinium coh-
nii grown in an ethanol fed-batch system 99
8. Crypthecodinium cohnii and Schizochytrium sp. as poten-
tial substitutes to fisheries-derived oils in seabream (Sparus
aurata) microdiets 109
9. General conclusions 127
10.Summary 129
10.1.Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
10.2.Resumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
10.3.Laburpena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
11.Resumen ampliado 135
11.1.Introduccio´n general . . . . . . . . . . . . . . . . . . . . . . . 135
11.2.Objetivos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
11.3.Materiales y m´etodos generales . . . . . . . . . . . . . . . . . 154
11.4.Discusi´on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
List of publications 187
Curriculum vitae 189
II
List of Tables
1.1. Fatty acid production by oleaginous microorganisms . . . . . . 13
3.1. Nitrogen source concentration in continuous culture . . . . . . 58
3.2. Carbon source concentration in continuous culture . . . . . . . 60
3.3. Schizochytrium: batch vs. continuous culture . . . . . . . . . . 63
4.1. Air supply in Schizochytrium G13/2S fatty acid profile . . . . 71
4.2. N -atmosphere in Schizochytrium G13/2S fatty acid profile . . 72
2
4.3. Air supply in Crypthecodinium cohnii fatty acid profile . . . . 73
4.4. N -atmosphere in Crypthecodinium cohnii fatty acid profile . . 74
2
5.1. Nitrogen source induces odd-chain fatty acid production . . . 82
6.1. Optimum ammonium concentration . . . . . . . . . . . . . . . 91
6.2. Fatty acid profile in a NH /pH-auxostat fermentation . . . . . 94
4
7.1. Fatty acid profile in an ethanol fed-batch fermentation . . . . 105
7.2. C. cohnii: acetic acid vs. ethanol . . . . . . . . . . . . . . . . 106
8.1. Dietary formulation and proximate composition . . . . . . . . 112
8.2. Dietary fatty acid composition . . . . . . . . . . . . . . . . . . 113
8.3. Larval fatty acid composition . . . . . . . . . . . . . . . . . . 119
11.1.Fuentes microbianas de ´acidos grasos . . . . . . . . . . . . . . 147
11.2.Schizochytrium: cultivo tipo “batch” vs. continuo . . . . . . . 156
11.3.C. cohnii: ´acido ac´etico vs. etanol . . . . . . . . . . . . . . . . 163
iii
List of Tables
IV
List of Figures
1.1. Docosahexaenoic acid (DHA; 22:6 n-3) . . . . . . . . . . . . . 2
1.2. DHA biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3. Fish oil price . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4. Biochemistry of lipid accumulation . . . . . . . . . . . . . . . 15
1.5. Fermentation facilities . . . . . . . . . . . . . . . . . . . . . . 16
1.6. Microphotographs of DHA producing microorganisms . . . . . 18
1.7. Gilthead seabream global production . . . . . . . . . . . . . . 21
3.1. Growth-associated lipid accumulation in batch culture . . . . 57
3.2. Carbon source limited batch culture . . . . . . . . . . . . . . . 59
3.3. Dilution rate in continuous culture . . . . . . . . . . . . . . . 61
4.1. Anaerobic culture . . . . . . . . . . . . . . . . . . . . . . . . . 72
5.1. Fatty acid GC chromatograms . . . . . . . . . . . . . . . . . . 83
6.1. Optimum glucose concentration . . . . . . . . . . . . . . . . . 91
6.2. NH /pH-auxostat fermentation . . . . . . . . . . . . . . . . . 93
4
7.1. Ethanol fed-batch fermentation . . . . . . . . . . . . . . . . . 103
7.2. O enrichment in an ethanol fed-batch fermentation . . . . . . 104
2
8.1. Overall survival and survival to air-exposure of larvae . . . . . 117
8.2. Larval growth . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
v
List of Figures
VI
List of abbreviations
ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
AMP Adenosine monophosphate
ARA Arachidonic acid (20:4 n-6)
ATP Adenosine triphosphate
ATTC American type culture collection
CCMP Culture collection marine phytoplankton
CDW Cell dry weight
D Dilution rate
DGLA Dihomogammalinolenic acid (20:3 n-6)
DHA Docosahexaenoic acid (22:6 n-3)
DPA-3 Docosapentaenoic acid (22:5 n-3)
DPA-6 Docosapentaenoic acid (22:5 n-6)
EDTA Ethylenediamine tetraacetic acid
EFA Essential fatty acids
EPA Eicosapentaenoic acid (20:5 n-3)
FA Fatty acid
FAME Fatty acid methyl esters
FAS Fatty acid synthase
GC Gas cromatography
GLA γ-linolenic acid (18:3 n-6)
GRAS Generally recognized as safe
HUFA Highly unsaturated fatty acids
LA Linoleic acid
LNA α-linolenic acid (18:3 n-3)
MOPS 3-(N-morpholino)propanesulfonic acid
NADH Nicotinamide adenine dinucleotide
NADPH Nicotinamide adenine dinucleotide phosphate
OA Oleic acid (19:1 n-9)
OCFA Odd chain fatty acids
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PI Phosphatidylinositol
PKS Polyketide synthase
PUFA Polyunsaturated fatty acids
List of abbreviations
r Biomass volumetric productivity (g l−1 h−1)
CDW
r DHA volumetric productivity (mg DHA l−1 h−1)
DHA
r Fatty acid volumetric productivity (mg FA l−1 h−1)
FA
rpm Revolutions per minute
SCO Single cell oils
SEM Standard error of the mean
TFA Total fatty acids
TRIS Trishydroxymethylaminomethane
µ Specific rate of DHA formation (mg DHA g CDW−1 h−1)
DHA
µ Maximum specific growth rate
max
VIII