Zarrouk medium

Standard medium for culturing spirulina

Application: Arthrospira platensis, A. maxima and other alkaliphilic and haloalkaliphilic cyanobacteria
pH: 8.5–11.0 (working optimum 9.5–10.0)
Source: Zarrouk, 1966

Composition

Component	Amount per 1 L
NaHCO₃	16.8 g
K₂HPO₄	0.5 g
NaNO₃	2.5 g
K₂SO₄	1.0 g
NaCl	1.0 g
MgSO₄·7H₂O	0.2 g
CaCl₂ (anhydrous)	0.04 g (or CaCl₂·2H₂O 0.053 g)
FeSO₄·7H₂O	0.01 g
Na₂EDTA	0.08 g
A5 trace element solution	1.0 mL
B6 rare metals solution	1.0 mL
Distilled water	to 1000 mL

A5 + Co trace element solution

per 100 mL of solution · usage rate 1 mL per 1 L of medium (sufficient for 100 L) · storage at 4 °C in dark glass, 12 months

Component	Weighed amount / volume
H₃BO₃	286 mg
MnCl₂·4H₂O	181 mg
ZnSO₄·7H₂O	22.2 mg
Na₂MoO₄·2H₂O	39.0 mg
CuSO₄·5H₂O	7.9 mg
Co(NO₃)₂·6H₂O	4.94 mg (or 1 mL of a primary stock solution at 4.94 g/L)
Distilled water	to 100 mL

B6 rare metals solution (Zarrouk's)

per 100 mL of solution · usage rate 1 mL per 1 L of medium · storage at 4 °C, 12 months

Component	Weighed amount / volume
K₂Cr₂(SO₄)₄·24H₂O	9.6 mg
NiSO₄·7H₂O	4.78 mg (1 mL of a primary stock solution at 4.78 g/L)
Co(NO₃)₂·6H₂O	4.40 mg (1 mL of a primary stock solution at 4.40 g/L)
Ti₂(SO₄)₃	4.00 mg (1 mL of a primary stock solution at 4.00 g/L)
NH₄VO₃	2.29 mg (1 mL of a primary stock solution at 2.29 g/L)
Na₂WO₄·2H₂O	1.79 mg (1 mL of a primary stock solution at 1.79 g/L)
Distilled water	to 100 mL

Preparation

TWO solutions are prepared. Solution "A" (500 mL): NaHCO₃ 16.8 g + K₂HPO₄ 0.5 g. Solution "B" (500 mL): NaNO₃ → K₂SO₄ → NaCl → MgSO₄·7H₂O → CaCl₂ → the FeSO₄ + EDTA complex → 1 mL of A5 → 1 mL of B6. The two solutions are sterilised separately and combined only after they have cooled completely to room temperature.

Notes

CRITICAL POINT: NaHCO₃ and CaCl₂ must not be mixed in concentrated form — CaCO₃ precipitates instantly. Hence the mandatory separation into solutions "A" and "B".
NaHCO₃ decomposes during autoclaving, releasing CO₂ and raising the pH to 10.5–11. Solution "A" is preferably sterilised by filtration through a 0.22 µm membrane; if it is autoclaved, set the target pH 0.5–1.0 units lower.
The iron complex is prepared in advance: FeSO₄·7H₂O 0.01 g and Na₂EDTA 0.08 g (a ratio of ≈ 1:8 by mass) are dissolved in 50 mL of water and stirred for 30 min until a stable straw-yellow colour develops with no precipitate. Without EDTA the iron precipitates completely as Fe(OH)₃ at pH 10.
Molar equivalents: NaHCO₃ — 200 mM (the principal source of carbon and of alkalinity); NaNO₃ — 29.4 mM.
The high pH is itself a protection against contamination: above pH 9.5 the overwhelming majority of green algae and bacteria fail to develop — this is precisely the basis of industrial spirulina cultivation in open ponds.
Dissolve the salts one at a time, in the order given; H₃BO₃ dissolves slowly — warming to 40 °C is permissible. Cobalt is added from a primary stock solution: a weighed amount of 4.94 mg cannot be measured on an ordinary balance. Primary Co stock solution: 494 mg Co(NO₃)₂·6H₂O in 100 mL of water, take 1 mL. Without cobalt the solution is simply called "A5". The appearance of a precipitate or turbidity indicates that the solution is no longer usable.
The primary stock solutions are prepared in 100 mL: the concentration given in g/L corresponds to a weighed amount ten times smaller per 100 mL (for example, 229 mg per 100 mL for NH₄VO₃). Ti₂(SO₄)₃ hydrolyses — the finished solution is acidified with 0.1 mL of conc. H₂SO₄. Solution B6 is not obligatory: when cultures are grown on technical-grade water these elements are already present as impurities.