Extractable Phosphorus Determination Protocol

Determination of extractable phosphorus was modified from previous study [1 (link)]. Briefly, 5 g of each sample were mixed with 50 mL 0.2% pepsin solution, which was prepared with 0.2 g pepsin (Sigma, St. Louis, MO, USA) dissolved in 100 mL 0.1 M HCl solution. After 3 h of incubation at 37°C and 80 rpm, the mixtures were centrifuged at 3,000 rpm, followed by filtration through Advantec No.1 filter papers. The 0.3 mL extracts were added to 0.6 mL deionized water and 0.6 mL vanadium-ammonium molybdate solution, which was prepared with nitric acid, ammonium metavanadate (Sigma, USA) and ammonium molybdate tetrahydrate (Sigma, USA). Then, the mixtures were centrifuged again at 6,000 rpm for 10 min, and the absorbance of suspension was detected at 415 nm. Solutions of 0.3125 to 2.5 μmol/mL potassium dihydrogen phosphate were also detected to draw a standard curve. The 300 g WB treated with 750-unit Phy was also analyzed.