An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions

Characterizing labile P forms in animal manure is a challenge due to their susceptibility to hydrolysis. In this study, we enzymatically characterized P forms in dairy manure (no bedding), collected from a representative dairy farm in New York, by separating into soluble and residual components under mild assay conditions using water and sodium acetate buffer (pH 5.0). About 75% of total manure P in the fresh manure was characterized, with the remainder (25%) regarded as recalcitrant or biochemically unidentified P. The hydrolyzable organic P in soluble and residual fractions was then characterized by using phosphatase enzymes to simple monoester P, polynucleotide P, phytate-like P, and non-hydrolyzable P. Of the total P in water extracts, 77% was inorganic P, 11% hydrolyzable organic P and 12% non-hydrolyzable P. In the residual resuspension, the distribution of characterized P was 25% spontaneous labile P, 32% simple monoester P, 7% polynucleotide P, 9% phytate-like P, and 26% non-hydrolyzable P. Ultrasonication increased the Pi release from the manure residues, but the deviation in Pi concentrations due to the sampling variance was greater that the increase in Pi due to sonication. Autoclaving sped up the release of both spontaneously labile P and enzymatically hydrolyzable P trapped in the manure residual matrix. Quantifying labile P forms by this approach may advance our ability to predict amount of manure P that will be hydrolyzed and eventually become bioavailable. The information obtained by our modified method would be complementary to that obtained by other methods (such as P-31 NMR and sequential fractionation) for a full spectrum of P species in animal manure.