Algae Culture

Feed an equal mix of Rhodomonas lens (CCMP 739) and Rhodomonas salina (CCMP 1319) to the copepods daily. Typical total feed concentrations are 30,000-50,000 cells/ml/day depending on population density and developmental stage.

The Provasoli-Guillard Center (Maine, USA) maintains alga cultures for sale, as does the University of Texas at Austin. Algae culture has its own set of protocols, be sure to review standard algal culture techniques prior to implementing the procedures described in this section.

The following algae species are utilized to boost egg production: Akashiwo sanguinea (strain GSBL), Prorocentrum micans, Pavlova lutheri, and Oxyrrhis marina. Algal cell densities for feeding copepods are based on reported picograms carbon per cell, relative cell volumes and experiential judgement. Calculate what you predict will be required, then adjust based on residual feed density after 24 h.

Culture Conditions: Rhodomonas lens and R. salina

19-23° C +/-1°
28-30 ppt
18 h light / 6 h dark
40 W Cool-White light bulbs
NaHCO₃ Buffer
Aerate moderately
Feed F/2 solution regularly
Place carboys 10-20 cm from lights

Expect densities approaching 2,000,000 cells/ml without CO₂ enrichment, and >5,000,000 cells/ml with CO₂ enrichment.

To accommodate cultures in 20L carboys we constructed a shelf and light system in an indoor room and maintained the room at specific lighting and temperature conditions (Figure 7).

1. Use sterile technique to maintain cultures effectively. Be fastidious.
2. Culture initial stocks in an incubator at 23° C, 18 h light/6 h dark, according to Provasolli-Guillard protocols, with progression from the 15 ml stock delivery vessel to covered 150 ml flasks to 1.5 L Fernbach flasks as culture densities allow.
3. Swirl flasks daily to maintain stocks in suspension.
4. Transfer to larger vessels as a function of cell density and culture age.
5. Transfer any cultures exceeding 500,000 cells/ml to the next larger vessel.
6. Transfer any cultures ten days old, irrespective of cell density, if its color remains healthy.
7. Fill all incubator vessels to working depth and microwave to 85° C for sanitation (Keller et al, 1988). Cool to 23° C, inoculate vessels with F/2 (0.15 ml/L each of F/2 stocks A and B) and algae stock under a laminar flow hood to minimize contamination.
8. Scrub all carboys (20L) inside and outside with a brush, and rinse thoroughly with hot water. Acid-wash all carboys with muriatic acid, outdoors.
9. Fill all carboys completely to the top with 1 µm filtered, UV-sterilized seawater.
10. Add 10% hypochlorite at 0.2 ml/L and treat vessels for 24 h
11. Dechlorinate vessels with thiosulfate stock solution at 0.2 ml/L, and allow to stand for 4-6 h.
12. Filter all air to 0.2 µm with Gelman Acro50 air filters, to minimize contamination during culture.
13. Sterilize your hands with 70% isopropyl alcohol. Wipe the mouth and neck of the carboy with alcohol prior to inoculation.
14. Decant carboys to 15 L working depth.
15. Inoculate carboys with F/2 stocks A&B at 0.15 ml/L each, 6 g sodium bicarbonate, NaHCO₃ (3 millimole, final concentration), and swirl to dissolve the bicarbonate.
16. Inoculate carboys with 750 ml algae culture from incubator Fernbach vessels and label for species and starting date.
17. Start moderate aeration immediately.
18. Swirl carboys twice daily to maintain algae in the water column.
19. After 5 days, add additional F/2 A&B stock and slightly increase aeration.
20. When the pH elevates above 9, bubble CO₂ to further increase cell density. If CO₂ is unavailable, add a little more NaHCO₃ to buffer the solution.
21. When cultures are 5-7 d old, cell densities should exceed 1,000,000 cells/ml.
22. Maintain the culture as long as cell density continues to increase. Nutritive values of algae are greatest during the log-phase of growth. Feed to copepods, or use as an inoculant for larger scale algae culture.

Discard cultures when cell density ceases to rise. The algae culture has reached senescence and its nutritive value plummets. Use as feed for copepods only in emergencies.


For each species of algae:

Feed concentration x liters of copepods cultured = Daily Production Demand

Algae concentration x liters of algae cultured = Daily Production Capacity

This demand specifies how many liters of a given species will be required daily (plus extra volume to maintain a production safety buffer).

Example:

Status: 200 L copepods @ 1 per ml, R. salina slightly higher cell count than R. lens

• 200 L of copepod culture requires:
• 15,000 cells/ml x 200 L x 1000 ml/L = 3 x 10⁹ cells R. lens per feeding
• R. lens culture at 1.5 million cells/ml requires:
• 3 x 10⁹ cells/ 1.5 x 10⁶ cells/ml = 2 L of R. lens per feeding
• R. salina culture at 2 million cells/ml requires:
• 3 x 10⁹ cells/ 2 x 10⁶ cells/ml = 1.5 L of R. salina per feeding
• Each week, 14 L of R. lens and 11 L of R. salina are required, at this culture density (more volume is required, if the density drops; less if it rises).