A TECHNIQUE TO RECOVER TRACER AS CARBOXYL-CARBON AND ALPHA-NITROGEN FROM AMINO ACIDS IN SOIL HYDROLYSATES

Authors: MARSH, K - UNIV OF ILLINOIS; MULVANEY, RICHARD - UNIV OF ILLINOIS; Sims, Gerald

Submitted to: Biology and Fertility of Soils
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2003
Publication Date: 9/15/2003
Citation: MARSH, K., MULVANEY, R.L., SIMS, G.K. 2002. A technique to recover tracer as carboxyl-carbon and alpha-nitregen from amino acids in soil hydrolysates. Biology and Fertility of Soils. 02175.

Interpretive Summary: Research on the activities of microorganisms in soil is often limited by lack of dependable methods. In order to examine use of various agricultural chemicals for microbial growth, we developed a procedure that allowed measurement of the assimilation of nitrogen and carbon derived from agricultural chemicals into microbial amino acids. Such assimilation can be used as evidence that the organisms used the chemical under investigation for growth. The method is based on a previously published assay for measurement of amino acid nitrogen, and uses a very specific reaction (ninhydrin) to release labeled carbon and nitrogen atoms from amino acids isolated from soil. The method employs commonly available materials and reagents in order to minimize costs associated with adopting the method. Recoveries of the target carbon and nitrogen atoms ranged from 98 to 101% in evaluations with standard solutions of amino acids, whereas these recoveries from soil samples amended with labeled amino acids exceeded 90% for labeled carbon and 94% for labeled nitrogen. The method developed by this research will facilitate further studies on the microbial degradation of a wide variety of chemicals in the soil environment. The greatest impact of this work will be realized as research using this method is applied to reduce negative environmental effects of agriculture, such as off-site migration of pesticides.

Technical Abstract: Isotope analysis of biochemical compounds provides an unequivocal means for detecting assimilation of tracer C and N into microbial biomass. A diffusion method recently developed to determine amino acid-N by ninhydrin oxidation of soil hydrolysates was modified to permit simultaneous collection of the CO2 liberated during this oxidation. In the technique described herein, this is accomplished following removal of (NH4 +amino sugar)-N, by performing ninhydrin oxidation at 90ºC for 7 h in a 1.9-L Mason jar sealed with a lid equipped to support a petri dish containing 5 mL of 0.2 M NaOH. Recoveries of carboxyl-C and -N ranged from 98 to 101% in evaluations with standard solutions of amino acids, whereas these recoveries from soil hydrolysates amended with labeled amino acids exceeded 90% for 14C and 94% for 15N.