T+ (Modified from: Chen Y. B., Zehr J. P., Mellon M., 1996. Growth and nitrogen fixation of the diazotrophic filamentous non‐heterocystous cyanobacterium Trichodesmium sp. IMS 101 in defined media: evidence for a circadian rhythm . J Phycol. 32: 916‐923.) Minerals Stock Solutions (g/L) Quantity (mL Stock/L Media) NaCl MgCl2 .6 H2O KCl MgSO4. 7 H2O CaCl2 .2 H2O 245.45 100 10 10 10 10 406.6 74.60 603.8 147 Final Concentration (mM) 420 20 10 25 10 Adjust to 900mL with mQ water and autoclave. After cooling, add the following filter sterilized (0.2 µm) components to complete the medium: Minerals Stock Solutions (g/L) Quantity (mL Stock/L Media) NaNO3 NaHCO3 K2HPO4 . 3H2O Na2CO3 Fe‐NH4‐citrate KBr NaF Trace Metal Mix 4 Trace Metal Mix Vitamins3 Mix 150 21 6.8 26.5 6 115.7 2.9 See recipe below See recipe below See recipe below 10 10 1 0.6 0.25 1 1 1 1 1 Final Concentration (mM) 16 2.5 0.03 0.15 ‐ 0.97 0.07 ‐ ‐ ‐ Check the pH, has to be between 8.12 and 8.2. For solid medium use 7g/L of agarose. Sterilize the agarose separately in 550 ml of milliQ water. In this case the mineral solution is filled up to 400 ml. Trace Metal Mix 4: Trace metals H3BO3 SrCl2. 6H2O LiCl Na2SeO3. 5H2O Quantity g/L 35.9 17.3 1.1 0.5mL of a stock of 32mg/L Concentration in the final media (mM) 0.0006 0.00006 0.00003 0.00000006 Trace Metal Mix: Trace metals Stock 1 (g/100mL) EDTA FeCl3 . 6H2O MnCl2 . 4H2O ZnSO4 . 7H2O CoCl2 . 6H2O Na2MoO4 . 2H2O CuSO4 . 5H2O ‐ ‐ 0.4 0.12 0.06 0.27 0.025 Trace metal mix (Stock1 mL/L) 0.74 g 0.11 g 1 1 1 1 1 Vitamins 3 Mix: Vitamins Thiamine‐HCl d‐biotin* VItamin B12 Vitamins 3 mix (quantity/100mL) 10 mg 100 µL (from a stock of 5 mg in 10mL) 100 µL (from a stock of 5 mg in 10mL) *Dissolve first in 0.1 mL 2M NaOH. Then add 9.9 mL of mQ water. Concentration in the final media (mM) 2.5 0.0004 0.00002 0.000004 0.000002 0.00001 0.000001