Biochemical pathways of amino acids in soil: Assessment by position-specific labeling and 13C-PLFA analysis

Microbial utilization is a key transformation process of soil organic matter (SOM). For the first time, position-specific 13C labeling was combined with compound-specific 13C-PLFA analysis to trace metabolites of two amino acids in microbial groups and to reconstruct detailed biochemical pathways. Short-term transformation was assessed by applying position-specifically 13C labeled alanine and glutamic acid to soil in a field experiment. Microbial utilization of the amino acids' functional groups was quantified by 13C incorporation in total microbial biomass and in distinct microbial groups classified by 13C-PLFA. Loss from PLFAs was fastest for the highly oxidized carboxyl group of both amino acids, whereas the reduced C positions, e.g. C3–5, were preferentially incorporated into microorganisms and their PLFAs. The incorporation of C from alanines' C2 position into the cell membrane of gram negative bacteria was higher by more than one order of magnitude than into all other microbial groups. Whereas C2 of alanine was still bound to C3 at day 3, the C2 and C3 positions were partially split at day 10. In contrast, the C2 of glutamic acid was lost faster from PLFAs of all microbial groups. The divergence index, which reflects relative incorporation of one position to the incorporation of C from all positions in a molecule, revealed that discrimination between positions is highest in the initial reactions and decreases with time. Reconstruction of microbial transformation pathways showed that the C2 position of alanine is lost faster than its C3 position regardless of whether the molecule is used ana- or catabolically. Glutamic acid C2 is incorporated into PLFAs only by two out of eight microbial groups (fungi and part of gram positive prokaryotes). Its incorporation in PLFA can only be explained by either the utilization of the glyoxolate bypass or the transformation of glutamic acid into aspartate prior to being fed into the citric acid cycle. During these pathways, no C is lost as CO2 but neither is energy produced, making them typical C deficiency pathways. Glutamic acid is therefore a promising metabolic tracer in regard to ecophysiology of cells and therefore changing environmental conditions. Analyzing the fate of individual C atoms by position-specific labeling allows insight into the mechanisms and kinetics of microbial utilization by various microbial groups. This approach will strongly improve our understanding of soil C fluxes.