Analysis of super(15)N incorporation into D-alanine: a new method for tracing nitrogen uptake by bacteria

The quantitative contribution of bacteria to total microbial uptake of nitrogenous substrates is an aspect of the aquatic nitrogen cycle that is still largely unclear, mainly because existing methods are generally inadequate. We investigated the feasibility of measuring super(15)N incorporation into bacterial D-amino acids by gas chromatography- combustion-isotope ratio mass spectrometry (GC-c-IRMS) and the potential of this method as a new tool for quantification of super(15)N uptake by bacteria. The presented method allowed analysis of super(15)N incorporation into various hydrolysable amino acids (HAAs), including the bacterial biomarker D-alanine (D-Ala), at trace levels. The potential of the method was tested in a super(15)N labeling experiment in which sediment slurries were incubated with super(15)NH sub(4) super(+) and a super(15)N-labeled amino acid mixture. super(15)N incorporation into D-Ala was used to calculate total bacterial super(15)N uptake while comparison of super(15)N incorporation into D-Ala versus L-Ala provided a direct measure for the relative contributions of bacteria versus algae to the total microbial super(15)N uptake. Subsequently, it was also possible to calculate super(15)N uptake by algae. Results for the test experiment showed that bacteria accounted for 38% of total super(15)NH sub(4) super(+) uptake and dominated uptake of the super(15)N-amino acid mixture (90%). Analysis of super(15)N incorporation into other (non-biomarker) HAAs yielded useful additional information on the transformation of these HAAs during organic matter degradation. In conclusion, GC-c-IRMS analysis of D-Ala combined with super(15)N labeling is a unique approach in aquatic sciences that provides a powerful new method for quantification of nitrogen flows through bacteria in natural microbial communities.