Plankton Culture for Feeding Larval Fish

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Plankton Culture for Feeding Larval Fish
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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.

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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.

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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

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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

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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)

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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!!

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• 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

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Phytoplankton Production

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

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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

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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

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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

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Plankton for Larger Fry/Shellfish

• Broodstock and Spat
• Cell Size 10-24 microns
• Species
• Tetraselmis sp.
• Green
• Thalassiosra sp.
• Diatom

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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

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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

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Micro Algae Culture

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

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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
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Figure 2.3. Five growth phases of micro-algae
cultures.
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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.

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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.

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• 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.

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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.

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Culture Water Bad?

• Sources
• Seawater
• Saltwater wells
• Prepared seawater
• Salinity
• 26-32 ppt

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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
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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

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Figure 2.5. Aeration filter (Fox, 1983)
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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.

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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
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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).

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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

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Figure 2.8. Carboy culture apparatus (Fox,
1983).
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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.

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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
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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)
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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

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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

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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)