Clostridium alkalicellum Medium (DSMZ Medium 1036)

Composition per liter:

NaCl .......................................................................10.0g

NaHCO3  .....................................................................7.6g

Na2CO3  .....................................................................1.0g

NH4Cl .......................................................................0.5g

KH2PO4 ......................................................................0.2g

KCl .........................................................................0.2g

Yeast extract ...............................................................0.2

MgSO4 ·7H2O.................................................................0.1g

Cellosbiose solution.....................................................100.0mL

Na2S·9H2O solution.......................................................10.0mL

Trace element solution SL-10...............................................1.0mL

Selenite/tungstate solution ...............................................1.0mL

pH 8.9 ± 0.2 at 25°C

Cellobiose Solution :

Composition per 100.0mL:

Cellobiose ................................................................3.0g

Preparation of Cellobiose Solution: Add cellobiose to distilled/

deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge

with 100% N2 . Filter sterilize.

Na2S·9H2O Solution :

Composition per 10.0mL:

Na2S·9H2O..................................................................0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool

to room temperature.

Selenite/Tungstate Solution:

Composition per liter:

NaOH.........................................................................0.5g

Na2WO4 ·2H2O...............................................................4.0mg

Na2SeO3 ·5H2O..............................................................3.0mg

Preparation of Selenite/Tungstate Solution:  Add components

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin Solution:

Composition per liter:

Pyridoxine-HCl.............................................................10.0mg

Thiamine-HCl·2H2O..........................................................5.0mg

Riboflavin .................................................................5.0mg

Nicotinic acid..............................................................5.0mg

D -Ca-pantothenate..........................................................5.0mg

p-Aminobenzoic acid.........................................................5.0mg

Lipoic acid ................................................................5.0mg

Biotin .....................................................................2.0mg

Folic acid..................................................................2.0mg

Vitamin B 12 ...............................................................0.1mg

Preparation of Vitamin Solution:  Add components to distilled/

deionized water and bring volume to 1.0L. Mix thoroughly. Sparge

with 80% H2 + 20% CO2 . Filter sterilize.

Trace Elements Solution SL-10:

Composition per liter:

FeCl2 ·4H2O..................................................................1.5g

CoCl2 ·6H2O ..............................................................190.0mg

MnCl2 ·4H2O...............................................................100.0mg

ZnCl2 ......................................................................70.0mg

Na2MoO4 ·2H2O .............................................................36.0mg

NiCl2 ·6H2O................................................................24.0mg

H3BO3 .......................................................................6.0mg

CuCl2 ·2H2O ................................................................2.0mg

HCl (25% solution)..........................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2 ·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized

water and bring volume to 1.0L. Add remaining components. Mix thor-

oughly. Sparge with 100% N2 . Autoclave for 15 min at 15 psi pressure–

121°C.

Preparation of Medium: Add components, except cellobiose and

sulfide solutions, to distilled/deionized water and bring volume to

890.0mL. Mix thoroughly. Sparge with 100% N2 for 30–60 min. Dis-

pense  under  100% N2  gas atmosphere.  Autoclave under 100% N2 for

15 min at 15 psi pressure–121°C. Aseptically and anoxically add cel-

lobiose and sulfide solutions. Adjust final pH of the medium to pH 8.8–

9.0.

Use:  For the cultivation of Clostridium alkalicellum.