2.6.2. Indole Acetic Acid (IAA) Production Assay
For assessment of IAA, archaeal cultures were inoculated in 100 mL
sterile MGM broth containing 0.1% L-tryptophan and the flasks were kept
in shaking incubator at 120 rpm, 37 ℃ for 7 days. The culture medium was
transferred to sterile falcon tubes and centrifuged at 10,000 rpm for
15-20 minutes. The supernatant was transferred to a flask and pH was
adjusted at 2.8 using hydrochloric acid. Later, it was extracted twice
with equal volumes of ethyl acetate. The clear solution was transferred
to a beaker and anhydrous sodium sulfate was added to absorb left over
moisture. The clear solution was evaporated until dry using rotary
evaporator and was resuspended in 1 mL methanol. These samples were
analyzed by high-performance liquid chromatography (HPLC; Waters; e2995,
separations module) with 299h photodiode-array (PDA) detector using a
Nucleosil C18 column (4.6×250 mm, 3μM; Macherey-Nagel, Germany). The
mobile phase was a mixture of methanol/acetic acid/water (30:1:70,
v/v/v) and the flow rate was adjusted at 1.2 mL/min. Pure indole-3-
acetic acid (Sigma) was used to prepare standard solutions and IAA
production was quantified.