1630c . modified Nitrosospaera medium

Equipment needed: Autoclave Filter

Final pH: 7.5

Description: based on M. 1630, but modified after Melcher&Schleper during deposition in 2021-2022

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Main sol. 1630c #

	Compound	Amount	Unit
	NaCl	1.00	g
	MgCl₂ x 6 H₂O	0.40	g
	CaCl₂ x 2 H₂O	0.10	g
	KH₂PO₄	0.20	g
	KCl	0.50	g
	Na₂SO₄	0.05	g
	Trace element solution (Nitrososphaera)	1.00	ml
	FeNaEDTA solution (7.5 mM)	1.00	ml
	NaHCO3 buffer (1M)	2.00	ml
	HEPES buffer solution	10.00	ml
	NH4Cl (1 M)	2.00	ml
	Sodiumpyruvate (1 M)	1.00	ml
	Double distilled water	1000.00	ml
1 Incubate all used glass ware and magnetic stirring rods in 0.1 M HCl and rinse 3x with double distilled water to remove residual detergents (always check visual for soap bubbles).
2 Autoclave the Basic-FWM and cool to room temperature. This medium should be free of any precipitations!
3 Aseptically, add per L of BASIC-FWM the following solutions from sterile stocks (do not store complemented FWM): - Trace element solution (Nitrososphaera) - FeNaEDTA solution - NaHCO₃ solution - HEPES buffer solution - NH₄Cl solution - Sodiumpyruvate solution
4 Adjust pH to 7.5.

Trace element solution (Nitrososphaera) #

1000

	Compound	Amount	Unit
	HCl	8	mL
	H₃BO₃	30	mg
	MnCl₂ x 4 H₂O	100	mg
	CoCl₂ x 6 H₂O	190	mg
	NiCl₂ x 6 H₂O	24	mg
	CuCl₂ x 2 H₂O	2	mg
	ZnSO₄ x 7 H₂O	144	mg
	Na₂MoO₄ x 2 H₂O	36	mg
	Double distilled water	1000	ml
1 This solution contains no iron! Sterilise by autoclaving or filtration (0.2 µm) and store in the dark at 4 °C.

HEPES buffer solution #

1000

	Compound	Amount	Unit
	NaOH (pellets)	24.0	g
	HEPES (free acid)	238.4	g
	Distilled water	1000.0	ml
1 Fist dissolve NaOH in ca 600 ml water, then add HEPES, adjust pH to 7.5 and fill up to 1000 ml volume.

FeNaEDTA solution (7.5 mM) #

1000

	Compound	Amount	Unit
	FeNa-EDTA	2753	mg
	Double distilled water	1000	ml
1 Sterilise by filtration (0.2 µm) and store in the dark at 4 °C.

NaHCO3 buffer (1M) #

	Compound	Amount	Unit
	NaHCO₃	4.2	g
	Double distilled water	50.0	ml
1 Dissolve 4.2 g of NaHCO₃ in 50 mL Double distilled water (use a small beaker with stirring rod to dissolve) and sterilize by filtration (0.2 µm) in the laminar flow hood. Store in a sterile 50 mL falcon tube at 4 °C. Avoid high headspace to liquid ratios to ensure stable concentration.

NH4Cl (1 M) #

	Compound	Amount	Unit
	NH₄Cl	2.67	g
	Double distilled water	50.00	ml
1 Dissolve 2.67 g ammonium chloride in 50 mL Double distilled water.
2 Filtrate (0.2 µm).

Sodiumpyruvate (1 M) #

	Compound	Amount	Unit
	Sodium pyruvate	5.5	g
	Double distilled water	50.0	ml
1 Dissolve 5.5 g in 50 mL and filter it through 0.2 µm sterile filter in a sterile falcon tube. Store at 4 °C

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1630c . modified Nitrosospaera medium

Main sol. 1630c #

Trace element solution (Nitrososphaera) #

HEPES buffer solution #

FeNaEDTA solution (7.5 mM) #

NaHCO3 buffer (1M) #

NH4Cl (1 M) #

Sodiumpyruvate (1 M) #

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