Simple method to enable the high resolution determination of total free amino acids in soil solutions and soil extracts

Amino acids, either in a free or polymeric state, represent a significant addition of N to most ecosystems and therefore comprise a key pool in the soil nitrogen cycle. The ability of some plants to take up amino acids directly from soil may confer a competitive advantage particularly in N-limited environments. Whilst measurements of inorganic N concentrations (NH4(+) and NO3(-) are routinely undertaken on either soil solutions or soil water/KCl extracts, there is a need for a supplementary procedure to determine free amino acids and enable determination of all plant-available pools. The aim of this study is to develop and test a procedure for the rapid and sensitive determination of total free amino acids in soil solutions and soil extracts (water or 2 M KCl) and to compare this with the standard ninhydrin method of Moore and Stein (1954). The spectrofluorometric procedure relies on the reaction of free amino acids with o-phthaldialdehyde and beta-mercaptoethanol. The fluorometric method is much more sensitive (working range 0.1-50 micromolar) than typical spectrophotometric analysis procedures for free amino acids which employs the ninhydrin reagent (working range 10-500 micromolar). In addition, the method only requires small sample volumes (1-50 microliter), is rapid (1 min sample-1), simple to perform, and linear over a concentration range of three orders of magnitude (0.1-100 micromolar). The method was used to determine free amino acid concentrations across a range of ecosystem types (upland and lowland grasslands, forest, heathland and coastal saltmarsh). Generally, the concentration of free amino acids in soil solution was low and relatively independent of soil type. The average soluble N concentrations across all sites (n=7) were 24 +/- 8 micromolar for amino acids, 39 +/- 14 micromolar for NH4(+) and 67 +/- 42 micromolar for NO3(-). Free amino acids typically constituted 10-40% of the total soluble N in the soil solution and therefore comprise a significant soluble N and plant available pool in soil.