2.2. Determination of nitrogen fixing capacity
Pure diazotrophic bacterial isolates were grown in 10 ml Ashby broth in 20 ml test tube on a rotary shaker at 125 rpm under continuous airflow at 30 1C for 72 h in triplicate. The uninoculated media served as control. Afterward, the concentration of nitrogen in each liquid culture was measured by digestion and subsequent measurement by the Kjeldahl method (Bremner, 1965).
2.3. Quantitative estimation of phosphate solubilization and pH changes
Bacterial cultures were inoculated in 100ml Pikovskaya's broth medium in 250ml of Erlenmeyer flasks and incubated at 28721C for 2 and 5 days in rotary shaker at 120rpm. Triplicates were maintained foreachtreatment.Afterincubationthebacterialcultureswere filtered through Whatman no.1 filter paper and were clarified by centrifuga- tion at 10,000rpm for 15min. Uninoculated broth served as control. After that pH of the filtrate was recorded with a digital pH meter and amount of soluble phosphate was measured by a Molybdenum blue method (Strickland and Parsons, 1972). Potassium di-hydrogen phos- phate was used as standard. The intensity of blue color was measured by an UV–vis spectrophotometer at 690nm.