Extraction of mineralizable organic nitrogen from soils by a neutral phosphate buffer solution

The availability of soil nitrogen is often evaluated based on the amount of released inorganic N during incubation of a soil at 30°C under field moisture conditions (called the “incubation method”). In place of the incubation method, however, we have developed a phosphate buffer extraction method, as phosphate buffer-extractable organic nitrogen (PEON) appears to correlate strongly (r=0.92∗∗∗) with the inorganic N obtained by the incubation method, based on an analysis of 20 soil samples collected from various soil types. The substances in the soil solutions extracted with phosphate buffer were analyzed with size-exclusion HPLC equipped with a UV detector (280 nm) and sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS–PAGE). One major peak was detected in HPLC chromatograms irrespective of the soil types. The peak area in the HPLC showed a high correlation with both, the amount of PEON and the protein concentration in the soil solutions extracted with phosphate buffer. Further, the major peak detected in size-exclusion HPLC was identified as a uniform protein-like N compound as the SDS–PAGE results showed only a major band and the molecular weight of the band corresponded to that of the major peak. In order to determine the origin of this homogeneous N compound that appears as one major band in the SDS–PAGE, three kinds of organic materials (glucose and ammonium sulfate, a 4:1 mixture of rice bran and rice straw, and rapeseed cake) were added to soil and their soil solutions extracted with phosphate buffer were traced using SDS–PAGE. The original bands of the organic materials disappeared rapidly on the separation gel, and one major band that was present in the primary soil increased, even though different organic materials had been applied. However, when chloramphenicol and organic materials were added simultaneously, the appearance of this major band was retarded, but when cycloheximide and organic materials were added, the major band appeared more rapidly than in the case of organic materials without any antibiotic. Therefore, we were able to confirm that the source of mineralizable N may be a homogeneously-produced protein-like N compound derived from soil bacteria with a distinct molecular weight (about 8000–9000 Da).