Culture of microalgae in mollusc hatchery...Amino acid composition From Brown et al., 1989 • Essential amino acid profiles is similar between species and not effected by culture

1R. ROBERT Ifremer UMR PE2M,

Culture of Culture of microalgaemicroalgaein mollusc hatcheryin mollusc hatchery

Dr RENE ROBERTDr RENE ROBERTLaboratoireLaboratoire de de PhysiologiePhysiologie des des InvertébrésInvertébrés

IfremerIfremer--CentreCentre de Brest, Station de Brest, Station Expérimentale d’ArgentonExpérimentale d’Argenton


Relationship between species, egg size and larval size at formatRelationship between species, egg size and larval size at formationion

From Jones and Houde, 1981

SpeciesSpecies

Atlantic salmon (Atlantic salmon (Salmo salarSalmo salar))

Sole (Sole (Solea soleaSolea solea))

Grey mullet (Grey mullet (Mugil cephallusMugil cephallus))

European oyster (European oyster (Ostrea edulisOstrea edulis))

King scallop (King scallop (Pecten maximusPecten maximus))

Japanese Oyster (Japanese Oyster (CrassostreaCrassostrea gigasgigas))

Mature eggs Size (µm)Mature eggs Size (µm)

5 0005 000--6 0006 000

1 0001 000--1 4001 400

900900--1 0001 000

incubationincubation

6565--7070

5050--5555

D larvae size (µm)D larvae size (µm)

15 00015 000--25 00025 000

3 2003 200--3 7003 700

1 4001 400--2 4002 400

170170

100100

7070


Relationship between species, egg size and larval size at formatRelationship between species, egg size and larval size at formationion

From Jones and Houde, 1981

SpeciesSpecies

Atlantic salmon (Atlantic salmon (Salmo salarSalmo salar))

Sole (Sole (Solea soleaSolea solea))

Grey mullet (Grey mullet (Mugil cephallusMugil cephallus))

European oyster (European oyster (Ostrea edulisOstrea edulis))

King scallop (King scallop (Pecten maximusPecten maximus))

Japanese Oyster (Japanese Oyster (CrassostreaCrassostrea gigasgigas))

Mature eggs Size (µm)Mature eggs Size (µm)

5 0005 000--6 0006 000

1 0001 000--1 4001 400

900900--1 0001 000

incubationincubation

6565--7070

5050--5555

D larvae size (µm)D larvae size (µm)

15 00015 000--25 00025 000

3 2003 200--3 7003 700

1 4001 400--2 4002 400

170170

100100

7070


Combined effects of temperature and salinity Combined effects of temperature and salinity on the development of unfed larvaeon the development of unfed larvae

Mortality & growth

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Mortality rate (%)

Days since fertilization

Unfed 30 °C, 30 %O or 22 °C, 25 %O

From His et Seaman, 1992

Mean size (µm)

0 2 4 6 8 10 12 14 160

250

200

150

100

50

Fed control


Mortality & Growth

0 5 10 15 20 250 5 10 15 20 25

Days since fertilization

Mortality and growth ofMortality and growth of unfedunfed batches of larvae at batches of larvae at different periods of the yeardifferent periods of the year

0

20

40

60

80

100

Mortality rate (%)

Control: fed

Mean size (µm)

250

200

150

100

Starved MayDec

JanSept

ArgentonArgenton hatcheryhatchery

From Robert 2000, unpublished data


Today 3 groups of live diets are widely applied in industrial Today 3 groups of live diets are widely applied in industrial larviculturelarviculture of fish and shellfish:of fish and shellfish:•• Different species of 2 to 20 µm for bivalves, Different species of 2 to 20 µm for bivalves, penaeidpenaeid shrimps, rotifers, shrimps, rotifers, copépodscopépods and fishand fish•• The 50 to 200 µm rotifer The 50 to 200 µm rotifer Brachionus plicatilisBrachionus plicatilis for crustacean and marine fishfor crustacean and marine fish•• The 400 to 800 µm brine shrimps The 400 to 800 µm brine shrimps Artemia sppArtemia spp (meta(meta--) ) naupliinauplii for crustacean and fishfor crustacean and fish

From Brown et al., 1989


Feeding selection (Feeding selection (WiWi) patterns of ) patterns of Crasssotrea virginicaCrasssotrea virginica larvaelarvae

From Baldwin, 1995

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

1-2 2-4 4-6 6-8 8-10

Phytoplankton size fraction (µm)

Sele

ctiv

ity (W

i)

125 µm-larvae 260 µm-larvae A

Phytoplankton size (µm) and composition

0

0,2

0,4

0,6

0,8

1.0 (Syn) 4.5 (T-iso) 6.2 (Dun) 11.0 (Pro)

Sele

ctiv

ity (W

1)

106 µm-larvae 290 µm-larvae

BHorizontal line = neutral selectionHorizontal line = neutral selection

nnWWii = = CrCrii//ΣΣ CrCriii=1i=1


Size Size profilsprofils (volume and equivalent diameter)(volume and equivalent diameter)

C. «

min

us »

C. t

enui

ssim

us

C. p

umilu

mT

Iso

I. ga

lban

aP

. lut

heri

T. p

seud

onan

a

C. g

raci

lis/m

uelle

ri

C. c

alci

trans

T. s

triat

a

R. s

alin

a

T. s

ueci

ca

T. c

hui

050

100150200250300

350

Vo

lum

e (µ

m3)

Argenton strains CCAP strains

Chaetoceros sp « minus » 3.55 ± 0.46C. sp tenuissimus like 3.59 ± 0.44C. calcitrans forma pumilum 3.82 ± 0.52Pavlova lutheri CCAP 4.20 ± 0.23T Iso Argenton 4.29 ± 0.48T Iso CCAP 4.28 ± 0.18C.pumilum CCAP 4.37 ± 0.31I. galbana CCAP 1 4.51 ± 0.57I. galbana CCAP 2 4.55 ± 0.28P. lutheri 4.55 ± 0.47Thaliassosira pseudonana CCAP 4.72 ± 0.26T. pseudonana 5.13 ± 0.50C. muelleri CCAP 5.17 ± 0.19C. gracilis 5.30 ± 0.56C. calcitrans 5.58 ± 0.56Rhodomonas salina CCAP 7.19 ± 0.43Tetraselmis striata 7.35 ± 0.97R. salina 7.47 ± 1.13T. suecica CCAP 8.41 ± 0.52T. suecica 8.56 ± 0.98T. chui 8.64 ± 1.14

From Robert et al., 2004


Rates of ingestion and digestion by 2 day old Rates of ingestion and digestion by 2 day old C. C. gigasgigas larvaelarvae

From Robert, 1998

2 hours after feeding

S1


S1Y


S1Y

Nannochloris atomus

2 H

S1

4 H

S1

S2

24 H

S1

S2

S3

Stichoccocus bacillaris

2 H

S1

S2

4 H

S2

S3

24 H

S2

S3

S4

Isochrysis galbana

IngestionIngestion

IngestionIngestion+ Digestion+ Digestion

DigestionDigestion

End of digestionEnd of digestionor no ingestion or no ingestion


Rates of ingestion and digestion by 6 day old Rates of ingestion and digestion by 6 day old C. C. gigasgigas larvaelarvae

From Robert, 1998

Nannochloris atomus Isochrysis galbanaStichoccocus bacillaris


S1

S2


S1

S2

S3

S4

Y


S1

S2

S3

S4

Y

IngestionIngestion

IngestionIngestion+ Digestion+ Digestion

DigestionDigestion

End of digestionEnd of digestionor no ingestion or no ingestion

3 H

S1

S2

S3

6 H

S2

S3

S4

24 H

S3

S4

24 H

S2

S3

S4

6 H

S2

S3

S4

3 H

S1

S2

S3


Nutrient properties of Nutrient properties of microalgaemicroalgae

MicroalgaeMicroalgae contain between:contain between:

•• 20 to 45 % protein20 to 45 % protein•• 5 to 40 % carbohydrate 5 to 40 % carbohydrate •• 5 to 30 % lipid5 to 30 % lipid•• 5 to 25 % ash5 to 25 % ash

Can vary between species, and culture conditions (Can vary between species, and culture conditions (egeg. light, harvest stage). light, harvest stage)

Important nutrient fractions in Important nutrient fractions in microalgaemicroalgae::

Carbohydrate and lipid Carbohydrate and lipid -- energyenergyProtein Protein -- ‘balanced’ amino acid composition‘balanced’ amino acid composition

Polyunsaturated fatty acid (Polyunsaturated fatty acid (PUFAsPUFAs))VitaminsVitaminsPigmentsPigmentsOther trace nutrients (Other trace nutrients (egeg. sterols etc.). sterols etc.)

Variable between species; Variable between species; contribute to differences in contribute to differences in nutritional valuenutritional value



Amino acid compositionAmino acid composition


•• Essential amino acid profiles is Essential amino acid profiles is similar between species and similar between species and not effected by culture conditions.not effected by culture conditions.

•• Protein quality high with good match with protein from Protein quality high with good match with protein from aquaculturedaquacultured species.species.

0 4 8 12 16

% arginine

0 2 4

%histidine

0 5 10

% isoleucine

0 4 8 12 16

% leucine

0 4 8 12 16

%lysine

0 2 4 6

% methionine

0 4 8 12

% phenylalanine

0 4 8 12

% proline

0 4 8 12

% threonine

0 1 2 3

% tryptophan % valine

0 4 8 12

oyster larvae

oyster spat

C. calcitrans

P. lutheri

oyster larvae

oyster spat

C. calcitrans

P. lutheri

Iso sp (T. Iso)

Iso sp (T. Iso)


Long chain Long chain PUFAsPUFAs in in microalgaemicroalgae•• Essential for most larvae for membrane structure, Essential for most larvae for membrane structure, prostaglandinsprostaglandins

•• Algae generally a good source of these, but variableAlgae generally a good source of these, but variable between species andbetween species and culture conditionsculture conditions

% o

f to t

al fa

tty a

cids

Algal classes

AAEPADHA

20:5n-322:6n-3

20:4n-6Eicosapentaenoic acidDocosahexaenoic acid

Arachidonic acid



PUFAsPUFAs in algae and nutritional valuein algae and nutritional value

• Relationship not well understoodRelationship not well understood

•• Algae lacking PUFA Algae lacking PUFA -- poor nutritional value (unless in mixed poor nutritional value (unless in mixed

diet).diet).

•• Algae with 5 to 30% PUFA (i.e. 1 to 5% of DW): Algae with 5 to 30% PUFA (i.e. 1 to 5% of DW):

∗∗ Sufficient PUFA as direct feeds (oyster, shrimp larvae).Sufficient PUFA as direct feeds (oyster, shrimp larvae).

∗∗ May be insufficient for enriching rotifers, May be insufficient for enriching rotifers, ArtemiaArtemia (some (some marine fish larvae).marine fish larvae).



AscorbateAscorbate (C) (C) 1,000 1,000 -- 16,00016,000

ββ−−carotenecarotene 500 500 -- 1,0001,000

NiacinNiacin 100 100 -- 470470

αα−−tocopheroltocopherol (E) 70 (E) 70 -- 800800

Thiamin (B1)Thiamin (B1) 30 30 -- 110110

Riboflavin (B2)Riboflavin (B2) 25 25 -- 5050

PantothenatePantothenate 14 14 -- 3838

FolatesFolates 7 7 -- 2424

Pyridoxine (B6)Pyridoxine (B6) 4 4 -- 1717

Major vitamins in Major vitamins in microalgaemicroalgae

From Brown et al., 2000; Seguineau et al., 1996

Vitamin Conc. range (Vitamin Conc. range (µµg/g)g/g)

0 0.1 0.2 0.3 0.4

AlphaAlpha--tocopheroltocopherol (E)(E)

cod liver oil

Concentration (mg/g)

0 1 2 3 4 5

Pavlova lutheri

Skeletonema costatum

Isochrysis sp. (T.ISO)

Other foodstuff parsley

Ascorbic acid (C)Ascorbic acid (C)

Variable, but rich in vitaminsVariable, but rich in vitamins

16R. ROBERT Ifremer UMR PE2M, From Robert et al., 2004

Major Major sterolssterols in in microalgaemicroalgae in algaein algae

DIATOMEES (LN,STAT)

0

10

20

30

40

50

60

70

80

90

Choleste

rolBras

sicast

erol

Desmost

erol

Campe

sterol

24-M

ethylè

neStig

mastero

lMeth

ylPori

fera

B-Sito

sterol

Fucoster

olIso

fucoster

olMeth

ylPavl

ovol

EthylPavl

ovol

% d

es s

téro

ls to

taux

Chaetoceros calcitransChaetoceros gracilisChaetoceros pumilumChaetoceros T.LikeSkeletonema costatumThalassiosira pseudonana

FLAGELLES (LN,STAT)

0

10

20

30

40

50

60

70

80

90

100

Choleste

rol

Brassi

caste

rol

Desmos

terol

Campe

stero

l24

-Meth

ylène

Stigmas

terol

MethylP

orife

raB-S

itoste

rol

Fuco

stero

lIso

fuco

stero

lMeth

ylPav

lovol

EthylP

avlov

ol

% d

es s

téro

ls t

otau

x

Tetraselmis chuiTetraselmis striataTetraselmis suecicaPavlova lutheriRhodomonas salinaT. isochrysisIsochrysis galbana

Diatomées sont Diatomées sont riches en riches en cholestérolcholestérol, , fucosterol fucosterol

et en et en isofucosterolisofucosterol

Flagellés sont Flagellés sont riches en riches en brassicastérol brassicastérol et et campestérol campestérol


SummarySummaryMost algae are good source of protein (Most algae are good source of protein (well balanced in amino well balanced in amino acids)acids) and carbohydratesand carbohydrates

Most algae are good source of Most algae are good source of PUFAsPUFAs; sufficient as direct feeds for ; sufficient as direct feeds for most of most of molluscsmolluscs

Most algae are good source of vitaminsMost algae are good source of vitamins

Role of sterols is at present speculativeRole of sterols is at present speculative

Mixed algal diets Mixed algal diets -- excellent nutritional “package”excellent nutritional “package”

Variable in composition and can be manipulated by culture Variable in composition and can be manipulated by culture conditions attention has to be paid to maconditions attention has to be paid to maintain stable intain stable phytoplankton growing conditions (light, T°C, nutrients……)phytoplankton growing conditions (light, T°C, nutrients……)


Utilisation frequency ofUtilisation frequency of microalgalmicroalgal species in a mollusc hatchery :species in a mollusc hatchery :

From Robert and Trintignac, 1997

Utilisation Frequency (%)

Microalgal species Class Walne(1970)

Lucas(1980)

Coutteau &Sorgeloos

(1992)Chaetoceros calcitrans Bacillariophyceae 40 37.5 37

Chaetoceros gracilis ″ * * 53

Skeletonema costatum ″ 20 12.5 14

Phaeodactylum tricornutum ″ 50 12.5 5

Thalassiosira pseudonana, clone 3H ″ 40 62.5 33

Isochrysis galbana Prymnesiophyceae 80 75 19

Isochrysis affinis galbana (clone T-iso)

″ 20 0 72

Pavlova lutheri ″ 70 62.5 26

Pyramimonas virginica Prasinophyceae 0 37.5 *

Tetraselmis suecica ″ 60 25 35

Dunaliella sp Chlorophyceae 0 25 9

Nannochloropsis occulata Eustigmatophyceae 0 25 *

* no data


MainMain utilizationutilization ofof microalgaemicroalgae in hatcheriesin hatcheries

From Coutteau and Sorgellos, 1992

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8

Number of algal species

Num

ber o

f hat

cher

ies

CommercialfacilitiesAcademicfacilities

NoNo microalgaemicroalgae (0) = giant clams cultivation ((0) = giant clams cultivation (Tridacna gigasTridacna gigas andand Hippopus hippopusHippopus hippopus).).


Main parameters regulatingMain parameters regulating phytoplanctonphytoplancton growthgrowth

Photos from CSIRO, 2004

•• Culture medium/nutrientsCulture medium/nutrients•• LightLight•• pHpH•• Aeration/mixingAeration/mixing•• TemperatureTemperature•• SalinitySalinity

Nannochloropsis oculata

Chaetoceros sp.

Rhodomonas sp.

Skeletonema costatum

Pavlova lutheri


Main nutriments involved inMain nutriments involved in phytoplanctonphytoplancton growthgrowth

From Grobbelaar, 2004

Culture medium/nutrientsCulture medium/nutrients

CC C0C022, HCO, HCO3322--, C0, C033

--, organic molecules , organic molecules 175175--650 650 4 460 0004 460 000

OO 0022, H, H220, organic molecules 0, organic molecules 205205--330330 2 120 0002 120 000

HH HH220, organic molecules (H0, organic molecules (H22S inS in anoxieanoxie) ) 2929--100100 8 140 0008 140 000

NN NN22, NH, NH44++, NO, NO33

--, NO, NO22--, amino acids,, amino acids, purinespurines,, 1010--140140 487 000487 000

PyrimidinesPyrimidines, urea, …, urea, …

KK Several inorganic salts, i.e.,Several inorganic salts, i.e., KClKCl, K, K22SOSO44, K, K33POPO44 1.01.0--7575 55 00055 000

PP Several inorganic salts,Several inorganic salts, NaNa or K phosphateor K phosphate 0.50.5--3333 43 80043 800

NaNa Several inorganic salts, i.e.,Several inorganic salts, i.e., NaClNaCl, Na, Na22SOSO44, Na, Na33,, POPO4 4 0.40.4--4747 32 50032 500

Mg Mg Several inorganic salts, C0Several inorganic salts, C03322--, S0, S044

22-- oror ClCl -- saltssalts 0.50.5--7575 28 70028 700

CaCa Several inorganic salts, i.e., CaCOSeveral inorganic salts, i.e., CaCO33, Ca, Ca22-- (as chloride)(as chloride) 0.00.0--8080 27 50027 500

SS Several inorganic salts, MgSOSeveral inorganic salts, MgSO44--7H7H22O, amino acidsO, amino acids 1.51.5--1616 23 80023 800

Fe Fe FeClFeCl33, Fe(NH, Fe(NH44)) 22SOSO44, ferric citrate, ferric citrate 0.20.2--3434 13 80013 800

ZnZn S0S04422-- oror ClCl -- saltssalts 0.0050.005--11 540540

BB HH33BOBO33 0.0010.001--0.250.25 350350

CuCu S0S04422-- oror ClCl -- salts salts 0.0060.006--0.300.30 200200

CoCo Vitamins B12, S0Vitamins B12, S04422-- oror ClCl -- salts salts 0.00010.0001--0.20.2 125125

MnMn S0S04422-- oror ClCl -- saltssalts 0.020.02--0.240.24 138138

MoMo NaNa+ or NH4++ or NH4+ molybdatemolybdate saltssalts 0.00020.0002--0.0010.001 11

Element Compounds Cell composition range

µg/mg dry weightRelative no of atoms


Conway medium preparationConway medium preparation

From Walne, 1966

SolutionSolution principaleprincipale pourpour unun litrelitre ::··NaNa22 EDTAEDTA 5 g5 g··NaNONaNO33 100 g100 g··HH33BOBO33 33,6 g33,6 g··NaHNaH22POPO44 20 g20 g··MnClMnCl2 2 + 4H+ 4H22OO 0,36 g0,36 g··FeClFeCl3 3 + 6H+ 6H22OO 1,3 g1,3 g··HH22OO 1000 ml1000 mlSolution de traces deSolution de traces de métauxmétaux 1 ml1 mlDosageDosage :1 ml par litre:1 ml par litre d’eaud’eau dede mermer

Solution de traces deSolution de traces de métauxmétaux ::··ZnClZnCl22 2,1 g2,1 g··CoClCoCl22 + 6H+ 6H22OO 2,0 g2,0 g··(NH(NH44)6Mo)6Mo77OO2424 + 4H+ 4H22OO 0,9 g0,9 g··CuSOCuSO44 + H+ H22OO 2,0 g2,0 g··EauEau distilléedistillée 100 ml100 ml

Solution vitaminique :··ThiamineThiamine aneurineaneurine hydrochloride (B1)hydrochloride (B1) 200 mg200 mg··CyanocobalamineCyanocobalamine (B12)(B12) 10 mg10 mg··EauEau distilléedistillée 100 ml100 mlDosageDosage : 0,1 ml par litre: 0,1 ml par litre d’eaud’eau dede mermer

SolutionSolution silicatéesilicatée ((diatoméesdiatomées) :) :··NaNa22SiOSiO33 + 5H+ 5H22OO4 g4 g··EauEau distilléedistillée 100 ml100 ml

DosageDosage : 1 ml par litre: 1 ml par litre d’eaud’eau dede mermer



Soil extract:Soil extract: ErdschrieberErdschrieber mediummediumCulture medium/nutrientsCulture medium/nutrients

From Walne, 1966

IMPORTANT: soil must be collected from a natural uncultivated environment or a rich garden loam may be suitable.

No fungicides, insecticides or graden fertilizer should be present.

SOIL extract: (solution 1)SOIL extract: (solution 1)

• Sift 1 kg dry soil through 1mm mesh sieve and mix with 2L of dSift 1 kg dry soil through 1mm mesh sieve and mix with 2L of distilled wateristilled water

•• Autoclave for 60Autoclave for 60 mnmn at 121°C and cool for 1 h duringat 121°C and cool for 1 h during wich decantion occureswich decantion occures

•• Filter through coarse filter paper (Joseph paper)Filter through coarse filter paper (Joseph paper)

•• Filter again through fineFilter again through fine WhatmanWhatman paper (N°1) and glass fibre paper (GF/C) paper (N°1) and glass fibre paper (GF/C)

•• Autoclave for 20Autoclave for 20 mnmn at 121°C, cool overnight and store at 4°C (cold room or refrigeat 121°C, cool overnight and store at 4°C (cold room or refrigerator) until required.rator) until required.

Nitrate/Phosphate stock solution (solution 2)Nitrate/Phosphate stock solution (solution 2)

Dissolve 40 g NaNODissolve 40 g NaNO3 3 and 4 g Naand 4 g Na22HPOHPO4 4 in 200 ml distilled water.in 200 ml distilled water.

Silicate stock solution (solution 3)Silicate stock solution (solution 3)

Dissolve 8 g NaDissolve 8 g Na22SiOSiO33, 5 H, 5 H22OO in 200 ml distilled water.in 200 ml distilled water.

ErdschrieberErdschrieber mediummediumDissolve 50 ml of solution 1, 1Dissolve 50 ml of solution 1, 1 mLmL of solution 2 and 1of solution 2 and 1 mLmL of solution 3 in 1L of seawaterof solution 3 in 1L of seawater

Autoclave for 20Autoclave for 20 mnmn at 121°C, cool overnight and store 3 days atat 121°C, cool overnight and store 3 days at ambiantambiant temperature before use.temperature before use.


Outside productionOutside production

From Pelanisamy et al., 1991

A B C D E FAmmonium sulfate 150 100 300 100 - -

Urea 7,5 5 - 10-15 - 12-15Calcium superphosphate 25 15 50 - - -

Clewat 32 - 5 - - - -N:P 16/20 fertilizer - - - 10-15 - -N:P:K: 16-20-20 - - - - 12-15 -N:P:K: 14-14-14 - - - - - 30

Concentration (mg.l-1)Fertilizers


25R. ROBERT Ifremer UMR PE2M, From Sverdrup et al., 1942

2HCO2HCO--3 3 ⇔⇔ COCO33

22-- + H+ H220 + CO0 + CO22

HCOHCO--3 3 ⇔⇔ COCO2 2 + OH+ OH--

COCO3322--+ H+ H220 0 ⇔⇔ COCO2 2 + 2OH+ 2OH--

pHpH

0

1

2

3

4

5

6 7 8 9 10

pH

Car

bone

mm

ol.l-

1COCO33

22--

HCOHCO--33

COCO22 libre dissouslibre dissous + H+ H2 2 COCO33

pH des eaux de mer de surface

26R. ROBERT Ifremer UMR PE2M, From Trintignac, 1998

General equation: CO2 + 2H20---light----> (CH20) + 02 + H20LightLight

Carbon dioxideCarbon dioxide

2H2H22O (water)O (water)

light

CCOO

OO2H2H

2H2H

HH22OO

CHCH22OOGlucids

OO

OOOO22

1. Water1. Water photolysephotolyse

2. Carbon dioxide 2. Carbon dioxide reductionreduction


The nature of lightThe nature of light

From Masojidek et al., 2004

1010--1414 1010--1212 1010--1010 1010--88 1010--66 1010--44 1010--22 101000

Cosmic raysCosmic raysGamma raysGamma rays

X raysX rays

Ultra violetUltra violetInfraInfra--redred

Radio WavesRadio Waves

Visi

ble

Visi

ble

400

450

550

500

600

650

700

Colour spectrum

of white light

Viol

et

750

Blu

e

Gre

en

Yello

w

Ora

nge

Red

Wavelength (nm)

Light: Usefull range for microalgae = 20-200 µEm-2s-1

Direct sunlight (midday in tropics) is approximatively

1 700 µEm-2s-1

LightLight


Negative effects of aerationNegative effects of aeration


Aeration/mixingAeration/mixing

Chaetoceros calcitransChaetoceros calcitrans

Chaetoceros calcitransChaetoceros calcitransdetail ofdetail of frustulefrustule

Thalassiosira pseudonanaThalassiosira pseudonana

Thalassiosira pseudonanaThalassiosira pseudonana::detail ofdetail of processusprocessus

Observations of young Observations of young intermediate scale cultures intermediate scale cultures (D4/D6) on SEM showed (D4/D6) on SEM showed damaged cells particularly in damaged cells particularly in diatoms. diatoms.

⇓⇓Observations were then made Observations were then made on non aerated stock cultures on non aerated stock cultures and particular attention was and particular attention was paid to diatoms spines.paid to diatoms spines.


Effects of temperature onEffects of temperature on phytoplanktonphytoplankton growthgrowth

From Jeffrey et al., 1994

TemperatureTemperature

30R. ROBERT Ifremer UMR PE2M, From Jeffrey et al., 1994

SalinitySalinityEffects of salinity onEffects of salinity on phytoplanktonphytoplankton growthgrowth


Five growth phases of microFive growth phases of micro--algae culturesalgae cultures

0 4 8 12 16

Age of culture

Con

cent

ratio

n (lo

g of

cel

l num

besr

)

P1

P2

P3

P4

P5

P1: lag or induction phase

P2: exponential phase

P3: Declining relative

growth phase or end of

exponential phase

P4: stationary phase

P5: death phase


Gro

wth

in d

iato

ms

Gro

wth

in d

iato

ms


0

10

20

30

40

0 2 4 6 8Age de la c ulture de puis ino c ulatio n (jo ur)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

C. calcitrans A

C. calcitrans B

0

20

40

60

80


Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

C.sp tenuis s imus A

C.sp tenuis s imus B

0

20

40

60

80


Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

C. pumilum A

C. pumilum B

0

10

20

30

40

0 2 4 6 8Age de la c ulture de puis ino c ula tio n (jo ur)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

T. pseudonana A

T. pseudonana B

0

10

20

30

40


Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

C. gracilis A

C. gracilis B

0

10

20

30

40

0 2 4 6 8Age de la c ulture de puis ino c ula tio n (jo ur)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

S. cos tatum A

S. cos tatum B

0

10

20

30

40

50

0 2 4 6 8 10Age de la culture depuis inoculation (jour)

Con

cent

ratio

n ce

llula

ire

(mill

ion)

C. pumilum A

C. pumilum B

0

10

20

30

40


Con

cent

ratio

n ce

llula

ire

(mill

ion)

T. pseudonana A

T. pseudonana B

0

10

20

30

40


Con

cent

ratio

n ce

llula

ire

(mill

ion)

C. muelleri A

C. muelleri B

0

10

20

30

40


Con

cent

ratio

n ce

llula

ire

(mill

ion)

S. costatum A

S. costatum B


S1

0

10

20

30

40

0 2 4 6 8 10 12

Age de la culture depuis inoculation (jour)C

once

ntra

tion

cellu

laire

(m

illio

n)

T. Iso AT. Iso B

S2

0

10

20

30

40

0 2 4 6 8 10 12

Age de la culture depuis inoculation (jour)

Con

cent

ratio

n ce

llula

ire

(mill

ion)

T. Iso AT. Iso B

S2

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12Age de la culture depuis inoculum (jour)

Con

cent

ratio

n ce

llula

ire

(mill

ion)

I. galbana AI. galbana B

S1

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12


Con

cent

ratio

n ce

llula

ire

(mill

ion)

I. galbana AI. galbana B

S1

0

10

20

30

40

0 2 4 6 8 10 12


Con

cent

ratio

n ce

llula

ire

(mill

ion)

P. lutheri AP. lutheri B

S2

0

10

20

30

40

0 2 4 6 8 10 12

Age de la culture depuis inoculation (jour)C

once

ntra

tion

cellu

laire

(m

illio

n)

P. lutheri AP. lutheri B

S1

0

1

2

3

4

0 2 4 6 8 10 12


Con

cent

ratio

n ce

llula

ire

(mill

ion)

T. suecica AT. suecica B

S2

0

1

2

3

4

0 2 4 6 8 10 12


Con

cent

ratio

n ce

llula

ire

(mill

ion)

T. suecica AT. suecica B

0

1

2

3

4

5

0 2 4 6 8 1 0A g e d e l a c u l t u r e d e p u i s i n o c u l a t i o n ( jo u r )

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

T e t ra s e lm is s u e c ic a A

T e t ra s e lm is s u e c ic a B

0

1

2

3

4

5

0 2 4 6 8 1 0A g e d e l a c u l t u re d e p u i s i n o c u l a t i o n ( jo u r)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

T e t ra s e lm is c h u i A

T e t ra s e lm is c h u i B

0

1

2

3

4

5

0 2 4 6 8 10A g e d e l a c u l t u re d e p u is in o c u la t io n ( jo u r)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

T e t ra s e lm is s u e c ic a A

T e t ra s e lm is s u e c ic a B

0

10

20

30

40

0 2 4 6 8 10A g e d e l a c u l t u re d e p u is in o c u la t io n ( jo u r)

Con

cen

trat

ion

cel

lula

ire

(mil

lion

)

T . Is o A

T . Is o B

Gro

wth

in fl

agel

late

sG

row

th in

flag

ella

tes


Competition betweenCompetition between microalgaemicroalgae and bacteriaand bacteria

From Prieur, 1980

0 4 8 12 16

Age of culture

Conc

entr

atio

n (lo

g of

cel

l num

besr

)


Stock cultures : 20Stock cultures : 20 mLmL, 500, 500 mLmL

AlgalAlgal collection of CSIRO (Australia): different cabinetscollection of CSIRO (Australia): different cabinets AlgalAlgal collection of CSIRO (Australia): inside a cabinetcollection of CSIRO (Australia): inside a cabinet


36R. ROBERT Ifremer UMR PE2M, From Robert 2005, unpublished data

Stock cultures : 50Stock cultures : 50 mLmL, 500, 500 mLmL

Stock cultures maintenance on a table with light Stock cultures maintenance on a table with light

above : experimental hatchery of above : experimental hatchery of ArgentonArgentonStock cultures maintenance in integrate illuminated Stock cultures maintenance in integrate illuminated

drawers: experimental hatchery of drawers: experimental hatchery of ArgentonArgenton


Stock cultures : 50 Stock cultures : 50 mLmL, 500 , 500 mLmL

JM JM LeroiLeroi from CSIRO Hobart (Australia)from CSIRO Hobart (Australia)

transferring stock culturestransferring stock cultures


JP JP ConnanConnan fromfrom IfremerIfremer ArgentonArgenton (France)(France)

transferring stock culturestransferring stock cultures


Stock cultures : 50 Stock cultures : 50 mLmL, 500 , 500 mLmLSpecies OriginHaptophyceae:Isochrysis galbana (Parke 1949) CCAP 927/1Isochrysis affinis galbana : T. Iso CCAP 927/14Pavlova lutheri (Green 1975) CCAP 931/1Pavlova pinguis Marseille (Algobank)Pavlova sp Baie des Veys (Algobank)Pavlova sp Thaïlande (Algobank)Pavlova sp Saint Honora (Algobank)Pavlova sp Menton (Algobank)Pavlova sp Saint Martin (Algobank)Pseudoisochrysis paradoxa (Ott nom nud.) CCAP 949/1Diacronema vlkianum (Prauser emend. Green & Hibberd 1Algobank HAP 67Imantonia rotunda Algobank HAP 23Bacilariophyceae:Chaetoceros calcitrans Argenton hatchery (origin ? ?)Chaetoceros simplex (Ostenfeld) CS 251Chaetoceros simplex Ostenfeld (Paulsen) var. calcitrans CCAP 1085/3Chaetoceros gracilis (Schütt) Utex LB 2375 (2658)Chaetoceros muelleri (Lemmermann, 1898) CCAP 1010/3Chaetoceros muelleri CS 176Chaetoceros calcitrans forma pumilus Argenton hatchery (origin ? ?)Chaetoceros calcitrans forma pumilus (Takano 1968) CCAP 1010/11Chaetoceros sp tenuissimus like La Tremblade (recent field isolation)Chaetoceros sp minus Tahiti (recent field isolation) Skeletonema costatum Argenton hatchery (origin ? ?)Skeletonema costatum ((Greville)Cleeve 1873) CCAP 1077/5Thalassiosira pseudonana ((Hustedt) Hasle & Heimdal 1970) CCAP 1085/12Thalassiosira pseudonana CS 173Thalassiosira weissflogii (Fryxell & Hasle 1977) CCAP 1085/1Cyclotella meneghiniana (Kützing) CCMP334Cryptophyceae:Rhodomonas salina ((Wilslouch) Hill & Wetherbee 1869) CCAP 978/24Prasinophyceae:Tetraselmis suecica ((Kylin) Butcher 1959) CCAP 66/4Tetraselmis chui Milford laboratoryTetraselmis striata Milford laboratory


Inside batch production : 2L, 6L and 10LInside batch production : 2L, 6L and 10L


2 to 6Lcarboys in the experimental hatchery of 2 to 6Lcarboys in the experimental hatchery of ArgentonArgenton 10L flasks in an industrial hatchery (France)10L flasks in an industrial hatchery (France)


Inside batch production : 300Inside batch production : 300--500L500L

270 L 270 L cylindrocylindro--conical tanks in theconical tanks in the

experimental hatchery of experimental hatchery of ArgentonArgenton500 L plastic bags in an industrial hatchery (France)500 L plastic bags in an industrial hatchery (France)


Inside batch production : 300LInside batch production : 300L

0

2

4

6

8

10

0 2 4 6 8

Time since inoculation (day)

Con

cent

ratio

n (n

b of

cel

ls x

106 )

C. calcitrans 01-02

0

1

2

3

4

5

0 2 4 6 8


Con

cent

ratio

n (n

b of

cel

ls

x 10

6 )

C.gracilis 01-04

0

1

2

3

4

5

0 2 4 6 8


Con

cent

ratio

n (n

b of

cel

ls

x 10

6 )

Skeleto 02-04

Mean diatoms concentration in 300 L

cylinders from 2001 to 2004 in Argenton

hatchery under standard conditions with

10<n<250. Note the low heterogeneity

except on day 7.

Period of utilisation as

food for molluscs


Inside batch production : 300LInside batch production : 300L

0

2

4

6

8

10

12

14

0 2 4 6 8 10


Con

cent

ratio

n (n

b of

cel

ls x

106 ) T iso 01-02

T iso CCAP 02-04

0.0

0.5

1.0

1.5

2.0

0 2 4 6 8 10 12

Time since inoculation (day)C

once

ntra

tion

(nb

of c

ells

x 1

06 )

Tetra chui 02-03

Mean flagellate concentration in 300 L cylinders from 2001 to 2004

in Argenton hatchery under standard conditions with 10<n<250.

Note the change in T. iso cultures performance due to a change

of strain since 2002 and a brightness decrease in the alga room.

Note the heterogeneity with Tetraselmis chui .

Period of utilisation as food for molluscs


Schedule for batch production at Schedule for batch production at ArgentonArgentonMaster stockMaster stock

5-10 ml phyto+ 300 ml ESS


Day 0Day 0

Day 7Day 7


Phyto rest +1. 7L ESS

Day 14Day 14

Conc. =20/40 M

500 ml flask

2L flask

0.8-1.7 L phyto+ 4 L ESS

6L flaskDay 19Day 19

6 L phyto+ 294 L ESS

Concentration =10 M for T. iso;5 M for C. gracilis

Utilisation from

day 24-26 (5-7d)

for T. iso and

day 22-24 (3-5d)

for Chaeto and

Skeleto.

BroodstockBroodstock/spat feeding/spat feeding


Continuous cultures: biotechnology conceptionContinuous cultures: biotechnology conception

250 m2 tubular continuous production system

(France)

5L flat panel continuous production system

(Germany)


After technical adjustments, the first T. After technical adjustments, the first T. isoiso continuous cultures, performed in continuous cultures, performed in 2L 2L photobioreactorsphotobioreactors, led to stable productions of 60 to 80 millions of cell .ml, led to stable productions of 60 to 80 millions of cell .ml--11

(two fold higher than 2L bath cultures) for a daily 33% enriche(two fold higher than 2L bath cultures) for a daily 33% enriched seawater daily d seawater daily renewal (Miner and Robert, 1999).renewal (Miner and Robert, 1999).

2L continuous cultures 2L continuous cultures

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1,0E+09

0 10 20 30 40 50 60

Time since inoculation days

Num

ber

of c

ells

per

ml (

Log)

Culture 1

Culture 2

From Miner and Robert, 1999


Crassostrea gigasCrassostrea gigas larval development was similar when larval development was similar when veligersveligers were fed either were fed either T.T.isoiso, maintained in continuous culture since 2 months, or with a 5, maintained in continuous culture since 2 months, or with a 5--7 day old 7 day old batch culture. In both experiments the addition of batch culture. In both experiments the addition of C. C. calcitranscalcitrans forma forma pumilumpumilum, , produced in batch, was performed as complementary diet.produced in batch, was performed as complementary diet.

T Is o batch AT Is o batch B

T Is o cont AT Is o cont B

Day 7

Day 140

20

40

60

80

100

120

140

160

180

Lar

val l

engt

h (µ

m)

Die ts

T Is o batchA

T Is o batchB

T Is o batchC T Is o cont

AT Is o cont

B T Is o contC

Day 9

Day 180

50

100

150

200

250

300

Lar

val l

eng

th (

µm)

Die ts

From Miner and Robert, 1999



The 2L to 300LThe 2L to 300L scale up required the development of a bulk seawater treatment scale up required the development of a bulk seawater treatment which had to be robust, compact and with low running cost which had to be robust, compact and with low running cost ⇒⇒ continuous continuous pasteurisation apparatus. The demand was planed to be 5 mpasteurisation apparatus. The demand was planed to be 5 m33.d.d--11, with a total , with a total residence time in heating chambers of 10 min at 90residence time in heating chambers of 10 min at 90°°C. C.

0

0.5

1

1.5

2

2.5

0 20 40 60 80

Time (min.)

fluor

esce

nce

Flow (80L/h)

Flow (260L/h)

Frequent culture contaminations with an Frequent culture contaminations with an heterotrophicheterotrophic flagellate flagellate Paraphysomonas Paraphysomonas vestitavestita (7(7--9 µm) 9 µm) with sudden culture with sudden culture crash.crash.

Pasteurisation was inefficient accordingly Pasteurisation was inefficient accordingly ⇒⇒ by use of by use of fluoresceine fluoresceine colourful test colourful test residence time in heating chambers was residence time in heating chambers was shown to be 1 to 5 min shown to be 1 to 5 min ⇒⇒ ModificationsModifications

From Fichez, 2000



Actual apparatus= Actual apparatus= pasteurisator pasteurisator with “PPP” with “PPP” chambers (chambers (vsvs inoxinox 316L)316L)seawater income: bottom chamber/outcome at seawater income: bottom chamber/outcome at the opposite side of the top chamber, both the opposite side of the top chamber, both chambers are chambers are coupledcoupled (in series)in series). Seawater Seawater heat= 95°C, but seawater temperature = 25°C heat= 95°C, but seawater temperature = 25°C after heat exchanger outflow.after heat exchanger outflow.


From Felix 2001-2002


A daily enriched seawater renewal of 20 to 25 % is adequate for A daily enriched seawater renewal of 20 to 25 % is adequate for T. T. isoiso and allows mean cells and allows mean cells concentration of 6 to 8 millions.mlconcentration of 6 to 8 millions.ml--11 in our current conditions (in our current conditions (vsvs 10 millions.ml10 millions.ml--11 in 300L in 300L batch culture). This renewal has to be specify for batch culture). This renewal has to be specify for C. C. gracilisgracilis but a 30% daily renewal is but a 30% daily renewal is expected with concentrations of 4 millions. mlexpected with concentrations of 4 millions. ml--11 ((vsvs 6 millions.ml6 millions.ml--11 in 300L batch culture).in 300L batch culture).

0

2

4

6

8

10

0 10 20 30 40 50 60Age of cultures since inoculation (day)

Cel

l con

cent

ratio

n pe

r ml (

mill

ion)

C. gracilis 30%

T. Iso 25%


From Robert et al., 2003, unpublished data


Continuous cultures: Continuous cultures: attempsattemps to industrial aquaculture productionto industrial aquaculture production

AAPS

Biofence


Continuous cultures: successful applicationContinuous cultures: successful applicationfor mollusc hatchery: for mollusc hatchery: BayesBayes systemssystems

500 L vertical plastic bags in an industrial 500 L vertical plastic bags in an industrial

hatchery (UK).hatchery (UK).

500500--1000 L horizontal plastic bags in an industrial 1000 L horizontal plastic bags in an industrial

hatchery (Spain)hatchery (Spain)


PERSPECTIVESPERSPECTIVESExperimental larval demand with Experimental larval demand with partner ship LPBA IFREMER Nantes.partner ship LPBA IFREMER Nantes.

••2004:2004: Installation of two Installation of two photobioreactorsphotobioreactors of 17 L: feasibility of of 17 L: feasibility of continuous cultures with T. continuous cultures with T. IsoIso and and Chaetoceros calcitransChaetoceros calcitrans//gracilisgracilis..

•• 2005:2005: Comparative feeding bioassays Comparative feeding bioassays (batch and continuous) on larvae.(batch and continuous) on larvae.

••20042004--2007:2007: Development and scale up of Development and scale up of a a photobioreactorphotobioreactor suitable for suitable for commercial hatcheries (partnership with commercial hatcheries (partnership with industry). industry).



Inside batch production : 1mInside batch production : 1m33--5m5m33

1m1m33 batch production in a commercial hatchery (France)batch production in a commercial hatchery (France) 5 m5 m33 batch production in a commercial hatchery (France)batch production in a commercial hatchery (France)

(background)(background)


Inside batch production : 5mInside batch production : 5m33--20m20m33

10 m10 m33 batch production in a commercial hatchery (batch production in a commercial hatchery (ChiliChili))


Inside batch production in greenhouse:Inside batch production in greenhouse: 22--5m5m33

22--5 m5 m33 batch production in a commercial hatchery: (Francebatch production in a commercial hatchery: (France) ) greenhousegreenhouse


Inside batch production in greenhouse:Inside batch production in greenhouse: 10m10m33--20m20m33

55--20 m20 m33 batch production in commercial hatcheries: (Francebatch production in commercial hatcheries: (France) ) greenhousegreenhouse


OusideOuside batch production : > 20mbatch production : > 20m33

> 20 m> 20 m33 batch outside production in a commercial nursery: (Francebatch outside production in a commercial nursery: (France) )



> 20 m> 20 m33 batch outside production in La batch outside production in La TrembladeTremblade IfremerIfremer hatchery (France):hatchery (France):

concrete basins on left and fibreglass circular tanks on rightconcrete basins on left and fibreglass circular tanks on right



> 20 m> 20 m33 batch outside production in a commercial nursery: (Francebatch outside production in a commercial nursery: (France) )

60R. ROBERT Ifremer UMR PE2M, From Trintignac, 1998



> 20 m> 20 m33 natural bloom production in a commercial nursery: (Francenatural bloom production in a commercial nursery: (France) )



> 20 m> 20 m33 natural bloom production in a commercial nursery: (Francenatural bloom production in a commercial nursery: (France) )


Substitutes for live Substitutes for live microalgaemicroalgae in in mariculturemariculture



Criteria selection for larval food sources from two viewpoints:Criteria selection for larval food sources from two viewpoints:

AvailabilityAvailability

Cost Cost effectivityeffectivity

SimplicitySimplicity

VersatilityVersatility

PHYSICAL CONSIDERATIONS PHYSICAL CONSIDERATIONS

PurityPurityAvailabilityAvailability

AcceptabilityAcceptability

NUTRITIONAL ASPECTSNUTRITIONAL ASPECTS

DigestibilityDigestibilityEnergetic requirementsEnergetic requirementsNutrient requirementsNutrient requirements

Culturist PredatorPredator

From Léger et al., 1987

65R. ROBERT Ifremer UMR PE2M, From Southgate et al., 1992

ProteinProtein--walled microcapsuleswalled microcapsules

MicrocapsulesMicrocapsules

Proteinaceous Proteinaceous capsule wallcapsule wall

Diatery Diatery materialmaterial

LipidLipid--walled microcapsuleswalled microcapsules

Hardened lipidHardened lipid

GelatinGelatin--acacia microcapsulesacacia microcapsules

GelatinGelatin--acacia complexacacia complex

MicrogelMicrogel microcapsulesmicrocapsules

Aqueous and particulateAqueous and particulate

nutriments trappednutriments trapped

Alginate matrixAlginate matrixLipid/oilLipid/oil

Aqueous coreAqueous core

66R. ROBERT Ifremer UMR PE2M, From chu et al., 1987; Numagushi and Nell, 1991


Crassostrea virginica

0

50

100

150

200

250

300

0 5 10 15 20 25

Time from fertilization (days)

Larv

al le

ngth

(µm

)

UnfedMicrocapsulesMicrocapsules + algal extractControl (live mixed algae)

Saccostrea commercialis

0

50

100

150

200

250

300

0 5 10 15 20 25

Time from fertilization (days)

Larv

al le

ngth

(µm

)

UnfedMicrocapsules + microalgaeControl (live Isochrysis)

Effects of microcapsules on larval growth Effects of microcapsules on larval growth increment (length) on a 3 week period.increment (length) on a 3 week period.

67R. ROBERT Ifremer UMR PE2M, From Laing and Verdugo, 1991

Crassostrea gigas

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 20 25 30 35Time from beginning of experimentation (days)

Dry

wei

ght (

mg)

Ruditapes philippinarum

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 5 10 15 20 25 30 35

Time from beginning of experimentation (days)

Dry

wei

ght (

mg)

Live TetraselmisDry TetraselmisControl (live mixed algae)


Effects of Effects of TetraselmisTetraselmis suecicasuecica (fresh and dry) on spat growth increment (dry weight) on a 35 d(fresh and dry) on spat growth increment (dry weight) on a 35 day perioday period


Heterotrophic - “algal-like”-fungi related to red and brown algae

Rapid growth, high cell densities – cheap production cost

Rich in polyunsaturated fatty acids, especially 22:6(n-3):> 15 % of total fatty acids

A spray-dried thraustochytrid (Schizochytrium sp.) is commercially available; i.e. AlgaMac 2000

– effective as an enrichment diet for rotifers

Aquaculture potential have not been broadly assessed


ThraustochytridsThraustochytrids


-25

0

25

50

75

100

125

150

175

200

-25

0

25

50

75

100

125

150

175

200

Dry weight Organic weight

Unfedcontrol

T. suecica- 10 mg

T. suecica- 2 mg

Thr. # 2- 10 mg

Thr. # 2- 8 mg

Thr. # 3- 8 mg

AlgaMac 2000- 8 mg

plus T. suecica - 2 mg

Growth(% increase)

Pacific oysters (1 Pacific oysters (1 mLmL of 1500 of 1500 μμm) were fed the following daily diet rations for 3 weeks at 19º m) were fed the following daily diet rations for 3 weeks at 19º C.C.


Live Live thraustochytridsthraustochytrids as feed for juvenile Pacific oystersas feed for juvenile Pacific oysters

70R. ROBERT Ifremer UMR PE2M, From Heasman et al., 2000

MicroalgalMicroalgal paste as feed for paste as feed for S. glomeraS. glomera larvaelarvae

Effects on growth increment and survival (up numbers)Effects on growth increment and survival (up numbers)

0

5

10

15

20

25

30

35U

nfed

con

trol

Live

fed

cont

rol

Pav

C. c

al

Skel

T. Is

o

Thal

C. m

uell

T.ch

ui

Pav

& C

. cal

Pav

& S

kel

Pav

& C

. mue

ll

Pav

& T

hal

Pav

& T

. chu

i

Pav

& T

. Iso

Skel

& C

. mue

ll

Diet

Incr

ease

in sh

ell h

eigh

t (µm

)

95%

10%

35%

32%25%

17%15%20%

8%

95%75%

70%

70%65% 55%

85%


High concentrated biomass produced in High concentrated biomass produced in flat flat pannel photobioreactorspannel photobioreactors: cooperation : cooperation

with Florence University (with Florence University (ItalieItalie))

Concentration by Concentration by floculationfloculation method using method using traditionnaltraditionnal algal algal batch production (low biomass): cooperation with CSIRO batch production (low biomass): cooperation with CSIRO

(Australia)(Australia)

MicroalgalMicroalgal paste and slurry as feed for paste and slurry as feed for C. gigasC. gigas larvae and larvae and postlarvaepostlarvaeTwo strategies developed:Two strategies developed:

From Ponis, 2002


a

0

1

2

3

NoFood

Slurry Te tra T. Is o +Slurry

T. Is o Cha e to+ Slurry

T. Is o +Cha e to+ Slurry

Cha e to T. Is o +Cha e to

Spa

t len

gth

(mm

)

Effects of Effects of TetraselmisTetraselmis suecicasuecica on spat growth increment (length and weight) on day 21on spat growth increment (length and weight) on day 21

MicroalgalMicroalgal paste as feed for paste as feed for C. gigasC. gigas postlarvaepostlarvae

b

0

0.5

1

1.5

2

No Food

Slurry

Te traT. Is

o + Slurry

T. Iso

Chae to + S

lurry

T. Iso + C

ha eto +

Slur

ryCha

e toT. Is

o + Cha eto

DietsS

pat

dry

wei

gh

t (m

g)

73R. ROBERT Ifremer UMR PE2M, From Ponis et al., 2003a

0

1

2

3

4

5

6

7

8

9

Pfp

Pfb

Pcp4

Pcp1

Pfp+

Cp

Pfb+

Cp

Pcp4

+Cp

Pcp1

+Cp Cp

Unf

ed

Gro

wth

rat

e ( μ

m d

ay-1

)Trial 1 Trial 2

Effects of Effects of Pavlova Pavlova lutherilutheri on growth increment (length) on day 14on growth increment (length) on day 14

MicroalgalMicroalgal paste as feed for paste as feed for C. gigasC. gigas larvaelarvae


20% T.ISO + 80 % of20% T.ISO + 80 % of

SKELSKEL..--conc.conc.

20% T.ISO fresh20% T.ISO fresh

C.TENC.TEN-- freshfresh

C.TENC.TEN-- conc.conc.

C.PUMC.PUM--conc.conc.

C.PUMC.PUM-- freshfresh

0 1 2 3 4 5

Postlarval length (mm)

MicroalgalMicroalgal flocculated paste as feed for flocculated paste as feed for C. gigasC. gigas larvaelarvae

From Brown and Robert, 2002

Initial size ≈ 2mm

Effects of diatoms on growth increment (length) on days 21 ( Effects of diatoms on growth increment (length) on days 21 ( ) and 28 ( )) and 28 ( )


0.00 2.00 4.00 6.00 8.00 10.00

Initial

Control

C.cal fresh

C.cal conc.

C.muel fresh

C.muel conc.

Diets

Individual dry weight (mg)

MicroalgalMicroalgal flocculated paste as feed for flocculated paste as feed for C. gigasC. gigas postlarvaepostlarvaeEffects of diatoms on growth increment (length) on day 28Effects of diatoms on growth increment (length) on day 28

From Robert and Brown, unpublished data


FloculationFloculation of of TT--IsoIso, , Pavlova lutheriPavlova lutheri and and Chaetoceros calcitrans f. pumilumChaetoceros calcitrans f. pumilum

Harvested in late Log , Harvested in late Log , microalgaemicroalgae are concentrated by use of a chemical treatment based on are concentrated by use of a chemical treatment based on a progressive increase of culture pH to 10.2a progressive increase of culture pH to 10.2--10.6 by addition of 10.6 by addition of NaOHNaOH (1M, 15 (1M, 15 mnsmns) and ) and aggregation of aggregation of microalgalmicroalgal cells by addition of a cells by addition of a polyacrylamidpolyacrylamid compound (compound (MagnaflocMagnafloc LTLT--25: 25: 300 ml of a solution at 0,05 %, 15 300 ml of a solution at 0,05 %, 15 mnsmns) and ) and swithswith off air/Co2 bubbling for 10 off air/Co2 bubbling for 10 mnsmns..

Elimination of the supernatant and adjustment of pH at 8,5Elimination of the supernatant and adjustment of pH at 8,5--8,9 by addition of 8,9 by addition of Hcl Hcl 1M.1M.

From Brown and Robert, 2002


P. lutheri

0

0.1

0.2

0.3

0.4

0.5

0 7 14 21 28jours

Phé

opigm

ent /

chlo

ro a

T-Iso

0

0.1

0.2

0.3

0.4

0.5

0 7 14 21 28jours

Phéo

pigm

ents/

chlro

a

C. calcitrans f. pumilum

0

0.1

0.2

0.3

0.4

0.5

0 7 14 21 28jours


Preservation of concentrates: photosynthesis abilityPreservation of concentrates: photosynthesis ability

From Ponis et al., 2003b


0

5

10

15

20

25

30

bf af 7 14 21 28 bf af 7 14 21 28 bf af 7 14 21 28day

T-Iso

pg c

el-1

LipidsProteinsCarbohydrates

P. lutheri C. calcitrans f. pumilum

bf : before flocculationbf : before flocculation

af : af : after after floculationfloculation

Harvest Harvest recoveryrecovery

100 %100 % 55 %55 % 100 %100 %



Preservation of concentrates: proteinPreservation of concentrates: protein--lipidslipids--carbohydrates evolutioncarbohydrates evolution


0 40 80 120 160

control

TC

PC

PT

PTC

freshp reserved

shell length (μm )0 20 40 60 80 100

survival (%)

freshpreserved

Effects of mixed fresh or preserved diets on spat development (lEffects of mixed fresh or preserved diets on spat development (length and survival) on day 9ength and survival) on day 9

MicroalgalMicroalgal flocculated paste as feed for flocculated paste as feed for C. gigasC. gigas larvaelarvae



0 0.5 1 1.5 2 2.5 3 3.5 4

control

TC

PC

PT

PTC

shell length (mm)

freshpreserved

*

*

*

*

MicroalgalMicroalgal flocculated paste as feed for flocculated paste as feed for C. gigasC. gigas postlarvaepostlarvae

Effects of mixed fresh or preserved diets on spat development (lEffects of mixed fresh or preserved diets on spat development (length) on day 28ength) on day 28



AND NOW IT IS TIME TO SAY GOOD BYAND NOW IT IS TIME TO SAY GOOD BY

Documents

Culture of microalgae in mollusc hatchery...Amino acid composition From Brown et al., 1989 • Essential amino acid profiles is similar between species and not effected by culture