2.6.3. Acetylene Reduction Assay
Nitrogenase activity was assessed by using acetylene reduction assay.
Single archaeal colonies of each strain were inoculated in vials
containing modified NFM (nitrogen free malate) semi-solid medium (5
mL/vial) and incubated at 37 ℃. NFM semi-solid medium contained (g L-1)
malic acid, 5.0; K2HPO4, 0.6; KH2PO4, 1.8; MgSO4.7H2O, 0.2; NaCl, 0.1;
CaCl2.2H2O, 0.02; micronutrient solution (above), 2 mL; bromothymol blue
(5 g L-1 in 0.2 N KOH), 2 mL; FeEDTA (16.4 g L-1), 4 mL; vitamin
solution, 1mL and NaCl, 200 g L-1; adjusted pH to 6.8 with KOH and 2%
agar [36]. After 48 h of growth, acetylene (10% v/v) was injected,
and vials were re-incubated for 24 h. Ethylene production was measured
by GC-2014 System (Shimadzu Corporation, Japan) fitted with a Porapak
column using flame ionization detector. Helium was used as a carrier
gas. Nitrogenase activity was described in terms of nanomoles of
ethylene per hour per milligram archaeal protein [37].