Plankton Culture for Feeding Larval Fish
Introduction
• You’ve got larval fish!! Good job!!
• Now what??
• If you’ve researched then it shouldn’t be a big deal, because you’re
ready to feed those little critters!
• Mostly food for larval fish is size dependant. If they can get it down
and it doesn’t damage their gut lumen, then it might be a good food.
• Not all larval food is created equally. We’ll consider micro plants as
first feeding options, then progress toward larger and larger prey
items.
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.
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
Table 1
Species
Nannochloropsis oculata
Pavlora lutheir
Skeletonema costatum
Phaeodactylum tricornutum
Tetraselmus tetratheie
Isochrysis galbana
Isochrysis aff galbana
EPA % Total n-3 FA %
30.5
42.7
13.8
23.5
13.8
15.5
8.6
9.6
6.4
8.1
3.5
22.5
0.5
3.3
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)
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!!
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%
Phytoplankton Production
•
Feeding Larvae
– Cell Size 4-8 microns
– Species
• Isochrysis galbana
• Chaetoceros gracilis
• Nannochloris sp.
• Chlorella sp.
• Pavlova lutheri
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
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 - 28°C
•
Culture media
– Guillards f/2
•
Proximate Analysis
– 47% Protein
– 24% Carbs
– 17% Fat
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 - 30°C
•
Culture media
– Guillards f/2 with Si
•
Proximate Analysis
– 28% Protein
– 23% Carbs
– 9% Fat
Plankton for Larger Fry/Shellfish
• Broodstock and Spat
– Cell Size 10-24 microns
– Species
• Tetraselmis sp.
– Green
• Thalassiosra sp.
– Diatom
Tetraselmis sp.
•
Morphology
– Ovoid green cells
– 14 to 23 µm L X 8 µm W
– 4 flagella
•
Salinity
– 28-36 ppt
•
Temperature
– 22-26°C
•
Culture media
– Guillards f/2
•
Proximate Analysis
– 55% Protein
– 18% Carbs
– 14% Fat
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
Micro Algae Culture
•
•
•
•
•
•
•
•
•
General Conditions
Culture Phases
Culture Water
Sterilization
Nutrient Enrichment
Inoculation
Cell Counts
Harvest and Feeding
Stock Culture
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)
Photoperiod (light: dark,
hours)
pH
2,500-5,000
16:8
(minimum)
24:0
(maximum
)
7-9
8.2-8.7
Figure 2.3. Five growth phases of micro-algae cultures.
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.
Exponential Phase
•
Cell density increases as a function of time t according to a
logarithmic function:
C t = 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.
• 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.
Why Did My Culture Crash??
• A better question might be why did it not crash?
• Culture crashes causes:
Nutrient depletion
Overheating
All of the above
Oxygen deficiency
pH disturbance
(Those we didn’t 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.
Culture Water Bad?
• Sources
– Seawater
– Saltwater wells
– Prepared seawater
• Salinity
– 26-32 ppt
Nutrient Enrichment Not Right?
Nutrients
NaNO3
•
Conc.
(mg/l Seawater)
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
Guillard’s f/2
– Part A and B
– 0.5 ml/L each part
– Na2Si03 for diatoms
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
Figure 2.5. Aeration filter (Fox, 1983)
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.
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
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).
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
Figure 2.8.
Carboy culture apparatus
(Fox, 1983).
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.
Cell Counts
• Peak Algae Density
– I. Galbana
• 10-12 million cells/ml
• 10-14 days
• 2 wk stability
– T. pseudonana
• Hemacytometer
– Count total in
centermost 1 mm
– Multiply by 10,000
– Product = number/ml
• 4 million cells/ml
• 3 days
• 5 day stability
Motile cells should be killed
Harvest and Feeding to Fry
• Algae Density
• Larvae Density
– 5-10 larvae/ml
– 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)
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
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
Production
cost
(US$.kg-1
dry weight)
Remarks
Source
300
Tetraselmis suecica
200 l batch culture
calculated from Helm et al.
(1979)
167
various diatoms
continuous 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)