1. Plankton Culture for Feeding Larval Fish

2. 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.

3. Microalgae (phytoplankton) Nutritionally, microalgae are a good source of macro and micronutrients for some larval fish.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.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 and 2 highlights some of these features.

4. Table 1. Approximate percent nutritional composition of several microalgae fed to larval fish. Species ProteinFatCarbAsh Chaetoceros muelleri 35302015 Pavlora virdis 601616 8 Tetraselmus tetratheie 30 52738 Isochrysis galbana 46222210

5. Table 1 SpeciesEPA % Total n-3 FA % Nannochloropsis oculata30.542.7 Pavlora lutheir13.823.5 Skeletonema costatum13.815.5 Phaeodactylum tricornutum 8.6 9.6 Tetraselmus tetratheie 6.4 8.1 Isochrysis galbana 3.522.5 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% --BOTTOM LINE, IT’S GOOD STUFF!!

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

10. 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 - 28°C 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 - 30°C 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 –22-26°C 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). ParametersRangeOptima Temperature (°C)16-2718-24 Salinity (g.l -1 )12-4020-24 Light intensity (lux)1,000-10,000 (depends on volume and density) 2,500-5,000 Photoperiod (light: dark, hours) 16:8 (minimum) 24:0 (maximum ) pH7-98.2-8.7

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

19. 1. 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. 2. Exponential Phase Cell density increases as a function of time t according to a logarithmic function: C t = C 0 x e mt Ct and C 0 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. 3. 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. 4. Stationary phase In the fourth stage the limiting factor and the growth rate are balanced, which results in a relatively constant cell density. 5. 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 depletionOxygen deficiency OverheatingpH disturbance All of the above exponential phase of growthThe key to the success of algal production is maintaining all cultures in the exponential phase of growth. Moreover, 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? Guillard’s f/2 –Part A and B –0.5 ml/L each part –Na 2 Si0 3 for diatoms NutrientsConc. (mg/l Seawater) NaNO 3 75 NaH 2 PO 4.H 2 O5 Na 2 SiO 3.9H 2 O30 Na 2 C 10 H 14 O 8 N 2.H 2 O (Na 2 EDTA)4.36 CoCl 2.6H 2 O0.01 CuSO 4.5H 2 O0.01 FeCl 3.6H 2 O3.15 MnCl 2.4H 2 O0.18 Na 2 MoO 4.2H 2 O0.006 ZnSO 4.7H 2 O0.022 Thiamin HCl0.1 Biotin0.0005 B 12 0.0005

25. Sterilization Techniques Poor? Methods –Heat Pasteurization 80 ◦ C and cool naturally –Autoclave (heat and pressure) –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: Na 2 CO 3 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. Culture typeAdvantagesDisadvantages IndoorsA high degree of control (predictable) Expensive OutdoorsCheaperLittle control (less predictable) ClosedContamination less likelyExpensive OpenCheaperContamination more likely Axenic (sterile)Predictable, less prone to crashes Expensive, difficult Non-axenicCheaper, less difficultMore prone to crashes ContinuousEfficient, 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-continuousEasier, somewhat efficientSporadic quality, less reliable BatchEasiest, most reliableLeast efficient, quality may be inconsistent Table 2.6. Advantages and disadvantages of various algal culture techniques.

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).

31. 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

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

34. 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.

35. 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

36. Harvest and Feeding to Fry Algae Density –Wk 1 = 50,000 cells/ml –Wk 2+ = 100,000 cells/ml –Onset of spatting = 200,000/ml Tank cleared in 24hrs Larvae Density –5-10 larvae/ml Liters to feed = (TD x V)/CD TD = Target Density (1,000s/ml) TD = Target Density (1,000s/ml) V = Volume of larval tank (thousands of L) V = Volume of larval tank (thousands of L) CD = Cell Density (millions/ml) CD = Cell Density (millions/ml)

37. 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

38. 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

39. Production cost (US$.kg -1 dry weight) RemarksSource 300Tetraselmis suecica 200 l batch culture calculated from Helm et al. (1979) 167various diatoms continuous flow cultures (240 m 3 ) a calculated from Walsh et al. (1987) 4-20outdoor cultureDe Pauw and Persoone (1988) 160-200indoor culture 23-115summer-winter production continuous flow cultures in bags (8 m 3 ) and tanks (150 m 3 ) a Dravers (pers. comm. 1990) 50tank culture (450 m 3 ) a Donaldson (1991) 50 - 400international survey among bivalve hatchery operators in 1991 Coutteau and Sorgeloos (1992)