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Plankton Culture for Feeding Larval Fish

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Title: Plankton Culture for Feeding Larval Fish


1
Plankton Culture for Feeding Larval Fish
2
Introduction
  • Youve got larval fish!! Good job!!
  • Now what??
  • If youve researched then it shouldnt be a big
    deal, because youre ready to feed those little
    critters!
  • Mostly food for larval fish is size dependant.
    If they can get it down and it doesnt damage
    their gut lumen, then it might be a good food.
  • Not all larval food is created equally. Well
    consider micro plants as first feeding options,
    then progress toward larger and larger prey items.

3
Microalgae (phytoplankton)
  • Nutritionally, microalgae are a good source of
    macro and micronutrients for some larval fish.
  • Fatty acids and pigments gained from ingestion of
    microalgae are especially important for larval
    fish health.
  • Table 1 and 2 highlights some of these features.

4
Table 1. Approximate percent nutritional
composition of several microalgae fed to larval
fish.
  • Species Protein Fat Carb Ash
  • Chaetoceros muelleri 35 30 20 15
  • Pavlora virdis 60 16 16 8
  • Tetraselmus tetratheie 30 5 27 38
  • Isochrysis galbana 46 22 22 10

5
Table 1
  • Species EPA Total n-3 FA
  • Nannochloropsis oculata 30.5 42.7
  • Pavlora lutheir 13.8 23.5
  • Skeletonema costatum 13.8 15.5
  • Phaeodactylum tricornutum 8.6 9.6
  • Tetraselmus tetratheie 6.4 8.1
  • Isochrysis galbana 3.5 22.5
  • Isochrysis aff galbana 0.5 3.3

6
Spirulina The Ultimate Food?
  • Cultured for over 600 years.
  • 65-68 protein
  • (similar to herring)
  • One acre of this stuff
  • produces 10 tons of protein
  • (wheat only gets you 0.16 tons)

7
Other Goodies
  • Chlorella and Scenedesmus are also excellent
    sources of protein.
  • Could yield 40 tons/acre/yr
  • That would be feeding 1000 cows for a year with a
    one acre pond of this stuff 3 ft deep!!

8
  • Spirulina is a single-celled, spiral-shaped
    blue-green microalgae. Highly digestible food,
    60 vegetable protein, which is predigested by
    the algae. It is higher in protein than any other
    food. 1 tsp of Spirulina contains 280 DV Beta
    Carotene, 110 B12, 15 Iron, 2 Calcium and no
    fat. Its outstanding nutritional profile also
    includes the essential fatty acids, GLA fatty
    acid, lipids, the nucleic acids (RNA and DNA), B
    complex, vitamin C and E and phytochemicals, such
    as carotenoids, chlorophyll (blood purifier), and
    phycocyanin (a blue pigment), which is a protein
    that is known to inhibit cancer. The carotenoids
    and chlorophyll may also contribute to
    Spirulina's anticancer and apparent immunogenic
    effects. Spirulina is two to six times richer in
    B12 than its nearest rival, raw beef liver.
    Spirulina is 58 times richer than raw spinach in
    iron. Spirulina is nature's richest whole-food
    source of Vitamin E. It's 3 times richer than raw
    wheat germ and its biological activity is 49
    greater than synthetic vitamin E. Spirulina is
    nature's richest whole-food source of
    Beta-Carotene (Pro Vitamin A). It's 25 times
    richer than raw carrots. Unlike the preformed
    vitamin A of synthetics and fish liver oils,
    beta-carotene is completely nontoxic even in mega
    doses. Spirulina is nature's richest whole-food
    source of Antioxidants. It contains a spectrum of
    every natural antioxidant known, including the
    antioxidant vitamins B-1 and B-6 the minerals
    zinc, manganese and copper the amino acid
    methionine and the superantioxidants
    beta-carotene, vitamin E and trace element
    selenium. Spirulina is nature's richest
    whole-food source of Gamma Linolenic Acid (GLA).
    Its oils are 3 times richer in GLA than evening
    primrose oil. Studies have indicated that GLA
    helps lower blood cholesterol and high blood
    pressure and eases such conditions as arthritis,
    premenstrual pain, eczema and other skin
    conditions. Spirulina is nature's richest
    whole-food source of Chlorophyll - many times
    richer than alfalfa or wheat grass! Spirulina is
    nature's richest whole-food source of Complete
    High-Biological Value Protein Spirulina - 60-70
    Soybeans - 30-35 Beef - 18-22 Eggs - 12-16
    Tofu - 8 Milk - 3

9
Phytoplankton Production
  • Feeding Larvae
  • Cell Size 4-8 microns
  • Species
  • Isochrysis galbana
  • Chaetoceros gracilis
  • Nannochloris sp.
  • Chlorella sp.
  • Pavlova lutheri

10
Pavlova lutheri
  • Morphology
  • Golden brown
  • Spherical with 2 flagella
  • 3-6 µm
  • Salinity
  • 8-32 ppt
  • Temperature
  • 11-26 C
  • Culture media
  • Guillards f/2
  • Proximate Analysis
  • 52 Protein
  • 24 Carbs
  • 29 Fat

11
Isochrysis galbana
  • Morphology
  • Tahiti (T-Iso strain)
  • Golden brown
  • Cells spherical with 2 flagella
  • 5-6 µm length, 2-4 µm wide
  • Salinity
  • 8-32 ppt
  • Temperature
  • 23 - 28C
  • Culture media
  • Guillards f/2
  • Proximate Analysis
  • 47 Protein
  • 24 Carbs
  • 17 Fat

12
Chaetoceros gracilis
  • Morphology
  • Golden brown diatom
  • Medium-size 12 µm wide, 10.5 µm long
  • Cells united in chains
  • Salinity
  • 26 - 32 ppt
  • Temperature
  • 28 - 30C
  • Culture media
  • Guillards f/2 with Si
  • Proximate Analysis
  • 28 Protein
  • 23 Carbs
  • 9 Fat

13
Plankton for Larger Fry/Shellfish
  • Broodstock and Spat
  • Cell Size 10-24 microns
  • Species
  • Tetraselmis sp.
  • Green
  • Thalassiosra sp.
  • Diatom

14
Tetraselmis sp.
  • Morphology
  • Ovoid green cells
  • 14 to 23 µm L X 8 µm W
  • 4 flagella
  • Salinity
  • 28-36 ppt
  • Temperature
  • 22-26C
  • Culture media
  • Guillards f/2
  • Proximate Analysis
  • 55 Protein
  • 18 Carbs
  • 14 Fat

15
Thalassiosra sp.
  • Morphology
  • Golden brown diatom
  • Cells united in chains
  • Barrel-shaped
  • Non-motile
  • 4 µm
  • Salinity
  • 26 32 ppt
  • Temperature
  • 22-29 C
  • Culture media
  • Guillards f/2 with Si
  • Other characteristics

16
Micro Algae Culture
  • General Conditions
  • Culture Phases
  • Culture Water
  • Sterilization
  • Nutrient Enrichment
  • Inoculation
  • Cell Counts
  • Harvest and Feeding
  • Stock Culture

17
Table 2.2. A generalized set of conditions for
culturing micro-algae (modified from Anonymous,
1991).
Parameters Range Optima
Temperature (C) 16-27 18-24
Salinity (g.l-1) 12-40 20-24
Light intensity (lux) 1,000-10,000(depends on volume and density) 2,500-5,000
Photoperiod (light dark, hours) 168 (minimum)240 (maximum)
pH 7-9 8.2-8.7
18
Figure 2.3. Five growth phases of micro-algae
cultures.
19
Lag/Induction Phase
  • This phase, during which little increase in cell
    density occurs, is relatively long when an algal
    culture is transferred from a plate to liquid
    culture.
  • Cultures inoculated with exponentially growing
    algae have short lag phases, which can seriously
    reduce the time required for upscaling.
  • The lag in growth is attributed to the
    physiological adaptation of the cell metabolism
    to growth, such as the increase of the levels of
    enzymes and metabolites involved in cell division
    and carbon fixation.

20
Exponential Phase
  • Cell density increases as a function of time t
    according to a logarithmic function
  • Ct C0 x emt
  • Ct and C0 being the cell concentrations at time t
    and 0, respectively.
  • m specific growth rate. The specific growth
    rate is mainly dependent on algal species, light
    intensity and temperature.

21
  • Phase of declining growth rate
  • Cell division slows down when nutrients, light,
    pH, carbon dioxide or other physical and chemical
    factors begin to limit growth.
  • Stationary phase
  • In the fourth stage the limiting factor and the
    growth rate are balanced, which results in a
    relatively constant cell density.
  • Death or crash phase
  • During the final stage, water quality
    deteriorates and nutrients are depleted to a
    level incapable of sustaining growth. Cell
    density decreases rapidly and the culture
    eventually collapses.

22
Why Did My Culture Crash??
  • A better question might be why did it not crash?
  • Culture crashes causes
  • Nutrient depletion Oxygen deficiency
  • Overheating pH disturbance
  • All of the above (Those we didnt mention.)
  • The key to the success of algal production is
    maintaining all cultures in the exponential phase
    of growth.
  • Moreoever, the nutritional value of the produced
    algae is inferior once the culture is beyond
    phase 3 due to reduced digestibility, deficient
    composition, and possible production of toxic
    metabolites.

23
Culture Water Bad?
  • Sources
  • Seawater
  • Saltwater wells
  • Prepared seawater
  • Salinity
  • 26-32 ppt

24
Nutrient Enrichment Not Right?
  • Guillards f/2
  • Part A and B
  • 0.5 ml/L each part
  • Na2Si03 for diatoms

Nutrients Conc.(mg/l Seawater)
NaNO3 75
NaH2PO4.H2O 5
Na2SiO3.9H2O 30
Na2C10H14O8N2.H2O (Na2EDTA) 4.36
CoCl2.6H2O 0.01
CuSO4.5H2O 0.01
FeCl3.6H2O 3.15
MnCl2.4H2O 0.18
Na2MoO4.2H2O 0.006
ZnSO4.7H2O 0.022
Thiamin HCl 0.1
Biotin 0.0005
B12 0.0005
25
Sterilization Techniques Poor?
  • Methods
  • Heat Pasteurization
  • 80 C and cool naturally
  • Autoclave
  • Sodium Hypochlorite (bleach)
  • 0.5 ml/L (10 drops)
  • Neutralize 10-15 ml sodium thiosulfate (248 g/L)
    per liter
  • Hydrochloric acid (muriatic)
  • 0.2 ml/L (4 drops)
  • Neutralize Na2CO3 0.4-0.9 g/L

26
Figure 2.5. Aeration filter (Fox, 1983)
27
Culture Types
  • Indoor/Outdoor. Indoor culture allows control
    over illumination, temperature, nutrient level,
    contamination with predators and competing algae,
    whereas outdoor algal systems make it very
    difficult to grow specific algal cultures for
    extended periods.
  • Open/Closed. Open cultures such as uncovered
    ponds and tanks (indoors or outdoors) are more
    readily contaminated than closed culture vessels
    such as tubes, flasks, carboys, bags, etc.
  • Axenic (sterile)/Xenic. Axenic cultures are free
    of any foreign organisms such as bacteria and
    require a strict sterilization of all glassware,
    culture media and vessels to avoid contamination.
    The latter makes it impractical for commercial
    operations.

28
Table 2.6. Advantages and disadvantages of
various algal culture techniques.
Culture type Advantages Disadvantages
Indoors A high degree of control (predictable) Expensive
Outdoors Cheaper Little control (less predictable)
Closed Contamination less likely Expensive
Open Cheaper Contamination more likely
Axenic Predictable, less prone to crashes Expensive, difficult
Non-axenic Cheaper, less difficult More prone to crashes
Continuous Efficient, provides a consistent supply of high-quality cells, automation, highest rate of production over extended periods Difficult, usually only possible to culture small quantities, complex, equipment expenses may be high
Semi-continuous Easier, somewhat efficient Sporadic quality, less reliable
Batch Easiest, most reliable Least efficient, quality may be inconsistent
29
Batch Culture
  • The batch culture consists of a single
    inoculation of cells into a container of
    fertilized seawater followed by a growing period
    of several days and finally harvesting when the
    algal population reaches its maximum or
    near-maximum density.
  • In practice, algae are transferred to larger
    culture volumes prior to reaching the stationary
    phase and the larger culture volumes are then
    brought to a maximum density and harvested.
  • Your handout depicts an example of how
    consecutive stages might be utilized test tubes,
    2 l flasks, 5 and 20 l carboys, 160 l cylinders,
    500 l indoor tanks, 5,000 l to 25,000 l outdoor
    tanks (Figs. 2.6., 2.7).

30
Inoculation
  • Culture vessels
  • 1,000 ml flask
  • 18.7 L (5 gal.) Carboy (glass)
  • 178 L (47 gal) Transparent Tank
  • Add enough algae to give a strong tint to the
    water
  • 100,000-200,000/ml
  • Lighting
  • Types
  • Sunlight
  • Fluorescent
  • VHO fluorescent
  • Metal halide
  • Highest Densities 24/7

31
(No Transcript)
32
Figure 2.8. Carboy culture apparatus (Fox,
1983).
33
Continuous Culture
  • The continuous culture method (supplied with
    fertilized seawater continuously, the excess
    culture is simultaneously washed out)
  • Permits the maintenance of cultures very close to
    the maximum growth rate! Very desireable.
  • Turbidostat culture Algal concentration (cell
    density) is kept at a preset level by diluting
    the culture with fresh medium by means of an
    automatic system.
  • Chemostat culture Fresh medium is introduced
  • into the culture at a steady, predetermined
    rate.
  • Addition of a limiting vital nutrient (e.g.
    nitrate) at
  • a fixed rate is also required. This way the
    growth
  • rate and not the cell density is kept constant.

34
Cell Counts
  • Peak Algae Density
  • I. Galbana
  • 10-12 million cells/ml
  • 10-14 days
  • 2 wk stability
  • T. pseudonana
  • 4 million cells/ml
  • 3 days
  • 5 day stability
  • Hemacytometer
  • Count total in centermost 1 mm
  • Multiply by 10,000
  • Product number/ml

Motile cells should be killed
35
Harvest and Feeding to Fry
  • Larvae Density
  • 5-10 larvae/ml
  • Algae Density
  • Wk 1 50,000 cells/ml
  • Wk 2 100,000 cells/ml
  • Onset of spatting 200,000/ml
  • Tank cleared in 24hrs

Liters to feed (TD x V)/CD TD Target
Density (1,000s/ml) V Volume of larval tank
(thousands of L) CD Cell Density
(millions/ml)
36
Harvesting and Feeding
  • Batch
  • Total harvest occurs once or over several days
  • Semi-Continuous
  • Works well with diatoms
  • Part of the algae remains in the vessel
  • New media is added to replenish the algae removed

37
Stock Culture
  • Purchase pure strain
  • Avoid contamination
  • No aeration
  • Half filed container
  • Redundancy
  • Holding
  • Test tubes
  • Conical flasks
  • Transfer
  • 1 drop/wk for T. pseudonana
  • 1 drop/2 wk for I. galbana

38
Production cost(US.kg-1 dry weight) Remarks Source
300 Tetraselmis suecica200 l batch culture calculated from Helm et al. (1979)
167 various diatomscontinuous flow cultures (240 m3)a calculated from Walsh et al. (1987)
4-20 outdoor culture De Pauw and Persoone (1988)
160-200 indoor culture
23-115 summer-winter production continuous flow cultures in bags (8 m3) and tanks (150 m3)a Dravers (pers. comm. 1990)
50 tank culture (450 m3)a Donaldson (1991)
50 - 400 international survey among bivalve hatchery operators in 1991 Coutteau and Sorgeloos (1992)
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