1, Method 1
Pyrococcus furiosus cells can be grown at 90 oC in a 100 l enamel-coated fermenter (Braun-Biotech) in a slightly modified medium used for cultivation of Thermococcales containing 1.0 g/l yeast extract (Difco), 5.0 g/l peptone (Difco), 5.0 g/l soluble starch (Merck), 18.4 g/l NaCl, 12.6 g/l MgCl 2 ·6H 2 O,0.16 g/l NaHCO 3, 3.24 g/l Na 2SO4 , 2.38 g/l CaCl 2 ·2H2O, 0.56 g/lKCl, 0.5 g/l L -cysteine and 0.0001% resazurine. Trace minerals (80 mg/l KBr, 57 mg/l SrCl2 ·6H 2 O, 22 mg/l H 3 BO 4, 10 mg/l Na 2 HPO 4 , 4 mg/l sodium metasilicate·5H 2 O, 2.4 mg/l NaF and 1.6 mg/l KNO 3 ) were added from a stock solution. The pH was adjusted to 6.0 with sulphuric acid. The fermenter was gassed with N 2 /CO 2 at a ratio of 80:20 (2 l/min) to remove hydrogen formed during growth of Pyrococcus . The agitation rate was 200 r.p.m. Cells were rapidly cooled before harvesting, collected by centrifugation and stored at -70 C.
2, Method 2
The following describes how the hyperthermophilic archaebacterium, P. furiosus, is routinely grown in a 500 liter fermentor for the purpose of obtaining cell mass in sufficient quantities for large scale protein purification. It is a modified version (Bryant et al., J. Biol. Chem., 264:5070-5079 (1989)) of the original protocol of Fiala et al., Arch. Microbiol., 145:56-61 (1986).
For culture maintenance, P. furiosus (DSM 3638) is routinely grown at 85°-88° C. as a closed static culture in 100 ml of the medium described in Table 2.
TABLE 2
______________________________________
Maltose 5 g/l
NH4 Cl 1.25 g/l
Elemental Sulfur 5 g/l
Na2 S 0.5 g/l
Synthetic Sea Water1
Vitamin mixture2
1 ml/l
FeCl3 25 µM
Na2 WO4 10 µM
Yeast Extract 0.01%
______________________________________
1 Synthetic Sea Water:
NaCl, 13.8 g/l
MgSO4, 3.5 g/l
MgCl2, 2.7 g/l
KCl, 0.3 g/l
CaCl2, 0.75 g/l
KH2 PO4, 0.5 g/l
NaBr, 0.0-5 g/l
KI, 0.05 g/l
H3 BO3, 0.015 g/l
Sodium citrate, 0.005 g/l
2. Vitamin mixture (Balch et al., Microbiol. Rev., 43:260-296 (1979)):
Biotin, 2 mg/l
Folic acid, 2 mg/l
Pyridoxine hydrochloride, 10 mg/l
Thiamine hydrochloride, 5 mg/l
Riboflavin, 5 mg/l
Nicotinic acid, 5 mg/l
DLCalcium pantothenate, 5 mg/l
Vitamin B12, 0.1 mg/l
pAminobenzoic acid, 5 mg/l
Lipoic acid, 5 mg/l
Loading ...
