2.6.1. Phosphate Solubilization Assay
Pikovskaya phosphate medium (PVK) with the following composition (g/L):
10.0 glucose, 1.0 yeast extract, 5.0 tri-calcium phosphate (TCP), 200.0
NaCl, 5.0 KCl, 31 MgCl2.6H2O, 35.0
MgSO4.7H2O, 0.5
CaCl2.2H2O and 0.5
KH2PO4 (pH 7.2) was used to test the
phosphate solubilizing ability of haloarchaea [34]. For inoculation
onto plates, the archaeal strains were grown in a liquid medium until
stationary phase at which time the cells were harvested by
centrifugation (8000xg, 10 min) and the pellets washed with sterile
water (SW) three times to remove any traces of the medium. The cell
pellets were then diluted to OD600 = 0.2 in SW. Ten μL droplets were
spotted onto the plates and allowed to air dry. then incubated upside
down at 37 ℃ for 5 days and the size of the clearing zone around the
colony was measured as an indication of positive activity.
Quantitative analysis of P-solubilization of archaeal strains was done
by molybdate blue color method [35]. Available phosphate was
calculated after 7 and 14 days. Cell-free supernatants were used for the
quantification of P-solubilization. After recording pH of cell-free
supernatants, they were filtered through 0.2 μm sterile filters (Orange
Scientific GyroDisc CA-PC, Belgium) to remove any residues. Solubilized
phosphates (primary and secondary orthophosphate) were measured by
spectrophotometer (Camspec M350-Double Beam UV-Visible
Spectrophotometer, UK) at 882 nm and values were calculated by using a
standard curve (KH2PO4 using 2, 4, 6, 8,
10, 12 ppm solutions).