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).