Fractionation of organic phosphorus
Organic P fractionation was carried out according to Bowman and Cole (1978) method modified by Ivanoff et al. (1998), and described in Zhang and Kovar (2009) wherein determination of microbial P was excluded from the fractionation scheme. Briefly, 1 g of soil was sequentially extracted with 0.5 M NaHCO3 at pH = 8.5, 1.0 M HCl, and 0.5 M NaOH. The 0.5 M NaHCO3 extract contained labile P fraction (POlab) and the 1.0 M HCl extract moderately labile P (POmod). The 0.5 M NaOH extract was acidified to pH = 1.0 to distinguish P contained in fulvic (Pfulv) and humic acids (Phum). The sample residue after sequential extraction was ashed at 550 °C and extracted with concentrated 1M H2SO4 to determine residual, non-labile organic P fraction (POres). The extracts were centrifuged at 4000 rpm for 10 min, filtered and measured for inorganic P (IP) and total P (TP) by the molybdate colorimetric method. Total P (TP) in the extracts was determined after digestion with 2.5 M H2SO4 and potassium persulfate. Organic P in the extracts was calculated as the difference between TP and IP.