A Modified Molybdenum Blue Method for Orthophosphate Determination Suitable for Investigating Enzymatic Hydrolysis of Organic Phosphates

Abstract

In characterizing organic phosphorus (P o ) by phosphatase hydrolysis, the quantity of hydrolyzable P o is represented by the difference in orthophosphate [i.e., inorganic P (P i )] determined after and prior to enzymatic incubation. Therefore, precise determination of P i is of major importance for accurate application of the enzymatic hydrolysis approach. The strong acid conditions required for conventional molybdenum blue methods interferes with P i determination due to rapid hydrolysis of labile P o and precipitation of enzymes (proteins). The molybdenum blue method of Dick and Tabatabai in 1977 reduced errors pertaining to nonenzymatic hydrolysis of P o . This study revisited the method, finding that the absorption coefficient at 850 nm was 45 -49% higher than at 700 nm, and linear up to at least 80 nmol P i in 1-mL assay solution. Therefore, adaptation of the readings at 850 nm improved the sensitivities of P i determination by about 45%. Enzyme precipitation during P i determination was prevented by addition of 2% sodium dodecyl sulfate (SDS) before color-forming reagents were added. This method modification provides increased sensitivity for P i determination, thereby improving the accuracy of P o analysis by phosphatase hydrolysis.

Chemical Distribution of Phosphorus in Soils used during the Development of Sorption Isotherms

“…Soil residues retained in the tubes were then sequentially extracted with 20 mL each of ultrapure water and one strip of an anionic resin (2 cm 2 surface area per side), 0.5 M NaHCO 3 (pH 8.5), 0.1 M NaOH, and 1.0 M HCl for 16 h, with extraction conditions and supernatant collection as previously described. Phosphorus extracted in the resin fraction was recovered by allowing P desorption from the resin into 10 mL of a 0.5 M HCl solution by shaking the resin-HCl solution at 250 rpm for 2 h. Inorganic P in all extracting solutions was determined using the method of Murphy and Riley (1962), with the exception of the NaOH extract, which was determined according to the method of He and Honeycutt (2005).…”

“…where Q, c, and k are the same as previously described and b is the sorption maximum or the maximum amount of P that can adsorb to a given soil in units of mg P kg -1 soil (Olsen and Watanabe 1957;Sparks 2003;Havlin et al, 2005). Although Eq.…”

“…where Q and c are the same as previously described and k and b are coefficients that are unique to each soil (Sparks 2003;Havlin et al, 2005). The parameter k has been used as a measure of the sorption strength between soil particles and P (Sparks 2003).…”

“…Two primary equations have been used to described the adsorption behavior of soils: the Freunddlich and the Langmuir equations (Olsen and Watanabe 1957;Bache and Williams 1971;Leytem and Westermann 2003;Laboski and Lamb 2004;Guardini et al, 2012;Oliveira et al, 2014). The Freundlich equation has limitations and is preferred when the solution P concentrations used in the study are low (e.g., from 0 to 20 mg L -1 ), whereas the Langmuir equation is used at a wider range of P concentrations in solution (e.g., from 0 to 80 mg L -1 ) (Havlin et al, 2005). The Freundlich equation in its simplest form can be described as:…”

“…where Q is the amount of P adsorbed as a function of the P equilibrium concentration in the solution (c) (Havlin et al, 2005). Two primary equations have been used to described the adsorption behavior of soils: the Freunddlich and the Langmuir equations (Olsen and Watanabe 1957;Bache and Williams 1971;Leytem and Westermann 2003;Laboski and Lamb 2004;Guardini et al, 2012;Oliveira et al, 2014).…”