Food for microorganisms: Position-specific 13 C labeling and 13 C-PLFA analysis reveals preferences for sorbed or necromass C

Sorption to mineral surfaces is one of the most important processes contributing to soil organic matter (SOM) stabilization. While this stabilization is commonly attributed to inaccessibility of the sorbed SOM to microorganisms, recent findings have revealed increased metabolic efficiency from sorbed substrates. The application of position-specifically 13C labeled sorbed tracer, combined with compound-specific 13C analysis of phospholipid fatty acids (PLFA), enables determination of whether this higher efficiency is due to a metabolic shift in a) the whole microbial community or b) microbial specialists adapted to sorbed C sources. To assess metabolic changes induced by sorption in individual microbial groups, we added uniformly and position-specifically 13C labeled alanine to a loamy Luvisol and incubated the soil samples for 10 days. Two different amendments were compared: i) alanine sorbed to sterilized soil, and ii) free alanine in solution. Incorporation of C from individual alanine positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Most of the microbial groups took up sorbed and free alanine equally. The metabolic pattern was also similar in most microbial groups: incorporation of C from carboxylic C-1 into PLFA was negligible, whereas C from the amino-bound C-2 and the methyl C-3 were preferentially incorporated into PLFA. This pattern reflects the basic microbial metabolism of C3 molecules - fast mineralization of C-1 via pyruvate decarboxylation. Only fungi incorporated more C from sorbed than free alanine into their PLFA. Their metabolic pattern also revealed a more complex metabolization via gluconeogenesis, followed by pentose-phosphate pathway. While the incorporation into most microbial groups’ PLFA remained stable or decreased over 10 days, the 13C in Actinobacteria PLFA increased up to 7 times from day 1 to day 10. This strong 13C increase in Actinobacteria was explained by their uptake of secondary substrates derived from microbial necromass or secretion products. This experiment showed that the more efficient metabolization of sorbed substrates is the work of microbial specialists: While uniform labeling revealed the higher incorporation of sorbed than free substrate into fungus PLFAs, position-specific labeling allowed the reconstruction of metabolic changes triggered by sorption of the substrate. To assess changes in the transformation pathways of individual microbial groups caused by sorption, we added uniformly and position-specifically 13C and 14C labeled alanine—a common amino acid in soil—in two ways: i) sorbed to sterilized soil (loamy Luvisol) or ii) free in solution, and incubated for 10 days. Microbial incorporation of alanine’s individual C positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Most of the microbial groups were able to take up sorbed and free alanine equally. The metabolic pattern also remained similar in most microbial groups: the C from carboxylic C-1 was nearly not incorporated into PLFA, whereas the C from the amino-bound (C-2) and the methylic C-3 were preferentially incorporated into PLFA of microorganisms. This reflects the basic microbial metabolism of C3 molecules via glycolysis. Only fungi incorporated more C from sorbed than free alanine into their PLFA. Their metabolic pattern also revealed a much more complex metabolization via gluconeogenesis, followed by pentose-phosphate pathway. While the incorporation into all microbial groups’ PLFA remained stable or decreased over 10 days, the 13C in Actinomycetes PLFA increased up to 7 times from day 1 to day 10. This strong 13C increase in Actinomycetes was explained by their uptake of secondary substrates derived from microbial necromass or secretion products. While uniform labeling was able to reveal the preference of fungi for sorbed versus free substrates, position-specific labeling allowed the reconstruction of metabolic changes triggered by sorption of the substrate and reflected a metabolic preference in fungi for sorbed, charged monomers.