The standard Zarrouk medium
The Zarrouk medium (1966) remains the reference for spirulina cultivation. Per litre of culture:
- NaHCO3: 16.8 g (carbon source, pH buffer)
- KNO3: 2.5 g (nitrogen source)
- K2HPO4: 0.5 g (phosphorus, potassium)
- NaCl: 1.0 g (osmotic balance)
- MgSO4·7H2O: 0.2 g (magnesium, sulfur)
- CaCl2·2H2O: 0.04 g (calcium)
- FeSO4·7H2O: 0.01 g (iron)
- EDTA (Na): 0.08 g (iron chelator — keeps Fe in solution at high pH)
- Micronutrient solution: 1 ml/L (containing MnCl2, ZnSO4, CuSO4, Na2MoO4, H3BO3, CoCl2)
Nitrogen: the primary growth driver
- Source:KNO3 (potassium nitrate) at 2.5 g/L provides nitrate as the primary nitrogen source. Spirulina can also use urea and ammonium, but nitrate is standard for food-grade production (ammonia can accumulate to toxic levels).
- Deficiency signs:Nitrogen depletion causes chlorosis — cells turn yellow/orange as phycocyanin and chlorophyll synthesis both require nitrogen. This is the most common cause of yellow culture in home growing. Measure biomass colour change with a spectrophotometer or visually — vivid teal-blue-green = nitrogen replete; yellow-orange = deficient.
- Top-up protocol:Add KNO3at 1–2 g per litre of total culture volume weekly during active growth. Monitor colour recovery over 2–3 days.
Carbon: the most commonly depleted
- Source:NaHCO3 provides both the carbon for photosynthetic fixation (CO2from bicarbonate dissociation) and the alkaline pH buffer (pH 9–10.5). Spirulina consumes bicarbonate rapidly in active growth.
- Deficiency sign:pH rises above 10.8 as all CO2is consumed and no more is buffered. Culture stops growing. This is the daily management challenge in outdoor systems without CO2injection.
- Correction:Inject CO2gas to bring pH back to 9.5–10.2, or add 2–4 g/L NaHCO3. pH-triggered CO2solenoids automate this in photobioreactor systems.
Iron: phycocyanin synthesis cofactor
- Why iron matters:Phycocyanin and phycocyanobilin synthesis both require iron as a cofactor. Iron deficiency in the medium produces pale-blue spirulina with reduced phycocyanin content — the biomass is nutritionally impaired even if cell count is normal.
- The solubility problem:At pH 9–10, free Fe³+ precipitates as Fe(OH)3. Without EDTA (ethylene diamine tetraacetic acid) chelation, iron falls out of solution and becomes unavailable. Add FeSO4always complexed with EDTA: the EDTA ratio should be 8:1 EDTA:Fe by weight to ensure chelation at high pH.
- Deficiency sign:Pale blue-green instead of deep teal. OD&sub6;&sub5;&sub0;/OD&sub6;&sub2;&sub0; ratio shifts (chlorophyll A relatively preserved, phycocyanin reduced).
- Excess iron:Excess unchelated iron causes Fenton chemistry (superoxide → hydroxyl radical → oxidative damage to spirulina cells). Use food-grade FeSO4 at exactly 0.01 g/L with EDTA chelation.
Phosphorus and potassium
- K2HPO4provides both potassium and phosphorus. Phosphorus is essential for ATP, DNA, and membrane synthesis. Deficiency causes impaired cell division and reduced growth rate (not colour change). Standard 0.5 g/L is sufficient for most growth rates.
- In very fast-growing outdoor cultures (doubling time <2 days), phosphorus can become limiting. Add 0.1–0.2 g/L K2HPO4weekly as maintenance in dense outdoor cultures.
Magnesium and sulfur
- MgSO4provides magnesium (chlorophyll synthesis cofactor — the Mg at the centre of the chlorophyll porphyrin ring) and sulfur (cysteine, methionine synthesis). Standard 0.2 g/L is rarely limiting. Deficiency is uncommon but causes chlorosis similar to nitrogen deficiency.
Micronutrients: trace elements
- Manganese (Mn):Photosystem II cofactor; essential for oxygen evolution in photosynthesis. Deficiency causes impaired photosynthetic efficiency.
- Zinc (Zn):Carbonic anhydrase cofactor (CO2 hydration to bicarbonate). Also RNA polymerase. Deficiency reduces growth rate at high pH where CO2is rate-limiting.
- Copper (Cu):Plastocyanin cofactor in photosynthetic electron transport. Very low requirement; excess copper is toxic to cyanobacteria (used as algicide in other contexts). Keep copper strictly at trace levels.
- Molybdenum (Mo):Nitrate reductase cofactor — essential for converting nitrate (NO3–) to ammonium for amino acid synthesis. Without Mo, nitrogen from nitrate cannot be assimilated.
Medium refreshment vs top-up protocol
- Small-scale batch (1–10 L):Prepare fresh Zarrouk medium fully every 4–6 weeks (complete medium exchange during harvest). Top up NaHCO3and KNO3weekly between harvests.
- Semi-continuous growing:After each 20–30% harvest, replace harvested volume with fresh Zarrouk medium. Nutrients are proportionally refreshed with each harvest.
- Conductivity monitoring:Medium conductivity (measured with an aquarium EC meter) drops as nutrients are consumed. Target EC 6–8 mS/cm for Zarrouk medium. If EC falls below 4 mS/cm: refresh with full Zarrouk medium or add concentrated nutrient stock.