Media and Culture condition

○Preparation of Enriched Sea Water
1. Collect offshore water and remove particulate matter with filters.
2. Sterilize filterated sea water by autoclaving at 121 ℃ for 20 min.
3. Dissolve 20 ml of PESI or PES medium into 1000 ml of sterilized sea water.

○Preparation of PESI medium
1. Dissolve appropriate quantities of weighted Tris, NaNO3, C3H7Na2O6P・5H2O, Fe solution, PII metal solution and KI solution into about 300 ml of distilled water in order.
2. Check the pH and adjust to 7.8 with 1N HCl or NaOH, and then dilute to 1000 ml of final volume with distilled water.
3. Dispense 20 ml of the medium into each of seeled tube and sterilize the medium by autoclaving at 121 ℃ for 20 min, and store at 4 ℃.


○Preparation of PES medium
1. Dissolve appropriate quantities of weighted Tris, NaNO3, C3H7Na2O6P・5H2O, Fe solution, PII metal solution, VitamineB12, Tyamine and Biotine into about 300 ml of distilled water in order.
2. Check the pH and adjust to 7.8 with 1N HCl or NaOH, and then dilute to 1000 ml of final volume with distilled water.
3. Dispense 20 ml of the medium into each of seeled tube and sterilize the medium by autoclaving at 121 ℃ for 20 min, and store at 4 ℃.



Preparation of Fe solution
1. Dissolve appropriate quantities of weighted Na2-EDTA・2H2O and Fe(NH4)2(SO4)2・6H2O into about 450 ml of diltilled water in order.
2. Dilute to 500 ml of final volume with distilled water, and store at 4 ℃


Preparation of P-2 metal solution
1. Disolve appropriate quantities of weighted Na2-EDTA・2H2O, H3BO3, FeCl3・6H2O, MnSO4・4H2O, CoSO4・7H2O stock solution, ZnSO4・7H2O into about 450 ml of distilled water in order.
2. Dilute to 500 ml of final volume with distilled water, and store at 4 ℃

PESI medium

Tris hydroxymethyl aminomethane ---- 5.0 g
NaNO3 -------- 3.5 g
Na2-glycerophosphate -------- 500 mg
Fe stock solution * --------- 250 ml
P-2 metal mix ** ---------- 250 ml
KI stock solution (0.1 mg / ml) ------ 10 ml
DW

Total ------------------------------------ 1000 ml
pH 7.8

PES medium

Tris hydroxymethyl aminomethane -------- 5.0 g
NaNO3 -------- 3.5 g
Na2-glycerophosphate -------- 500 mg
Fe stock solution * --------- 250 ml
P-2 metal mix ** ---------- 250 ml
Vitamine B12 stock solution (0.1 mg / ml) ---- 1.0 ml
Thiamine-HCl stock solution (1.0 mg / ml) ---- 5.0 ml
Biotine stock solution (0.1 mg / ml) ---- 0.5 ml
DW

Total ----------------------------------- 1000 ml
pH 7.8

*Fe stock solution

Na2-EDTA・2H2O ----------------- 330 mg
Fe(NH4)2(SO4)2・6H2O ------------ 351 mg
DW

Total ----------------------------- 500 ml

**P-2 metal mix

Na2-EDTA・2H2O ----------------- 500 mg
H3BO3 ---------------- 570 mg
FeCl3・6H2O ------------ 24.5 mg
MnSO4・4H2O ------------ 82.0 mg
CoSO4・7H2O stock solution (4.8 mg / ml) - 0.5 ml
ZnSO4・7H2O -------------- 11.0 mg
DW

Total ----------------------------------- 500 ml

Reference:
○Robert A. Andersen. 2005. Algal Culturing TEQUNIQUES. Elsevier Academic Press, San Diego, California. In: H. Kawai, T. Motomura, K. Okuda, Chapter9, pp. 133-143.
○Provasoli L. 1966. Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A (end) Cultures and collections of algae. Proceedings of the US-Japan conference held at Hakone, 12-15 Sept 1966.
Jpn. Soc. Plant Physiol., pp. 63-75