# J412: Fe(III)-REDUCER FRESHWATER ENRICHMENT MEDIUM ## Main sol. J412 100 mM **Poorly crystalline iron(III) oxide** 0.33 g MgCl2 x 6 H2O 0.33 g CaCl2 x 2 H2O 0.33 g KCl 0.33 g KH2PO4 0.25 g NH4Cl 2.5 g NaHCO3 18.9 mg Na2SeO4 10 ml **Trace vitamins** 10 ml **Trace mineral solution** 0.044 g L-Cysteine HCl x H2O 0.26 g FeCl2 x 4 H2O 1. Add components except L-cysteine x HCl x H2O and FeCl2 x 4H2O to distilled water, bring volume to 1.0 L and adjust pH to 7.0. Distribute the medium into appropriate culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a H2-CO2 (4:1, v/v) gas mixture, sparge with the same gas mixture for 10 - 15 min, seal with butyl rubber stoppers and autoclave. Separately autoclave concentrated (100- or 200-fold) L-cysteine x HCl x H2O and FeCl2 x 4H2O solutions under a N2 gas atmosphere. Prior to inoculation, add the sterile L-cysteine x HCl x H2O and FeCl2 x 4H2O solutions. Pressurize the inoculated vessels to 100 kPa H2-CO2 (4:1, v/v) and cultivate without shaking. 2. Comment: For strain JCM 12379, inoculate with a 10% inoculum. ## Poorly crystalline iron(III) oxide 1. Dissolve 108 g FeCl3 in 1.0 L distilled water. Adjust pH to 7.0 with 10 N NaOH. Allow the suspension to sit for 30 min, then readjust pH to 7.0. Centrifuge the suspension at 5,000 x g for 20 min and pour off supernatant. Wash the precipitate with distilled water by resuspending in distilled water followed by the centrifugation. Repeat the washing at least 5 times, then, resuspend in 1.0 L distilled water. Use the Fe(III) suspension as 0.67 M solution. More precisely, check the concentration of Cl- to be less than 1 mM and determine the concentration of Fe(III) by using hydroxylamine as described by Lovley and Phillips (Appl. Environ. Microbiol. 54, 751-757, 1986). ## Trace vitamins 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg Lipoic acid 1000 ml Distilled water ## Trace mineral solution 1. Use Trace minerals of Medium No. 151 supplemented with 0.025 g (final) Na2MoO4 x 2H2O, 0.024 g NiCl2 x 6H2O and 0.025 g Na2WO4 x 2H2O (per liter). 2. Comment: For strain JCM 12378, inoculate with a 10% inoculum.