Soils are the largest terrestrial carbon (C) pool, with the potential to sequester C but also to act as source for CO2 to the atmosphere. Microorganisms are key drivers of soil organic matter (SOM) transformation and mineralization. Since soil processes are often very slow and/or in steady state, 13C or 14C enriched tracer molecules have been used to gain insights into SOM transformations. Uniformly labeled tracers (i.e. all C atoms within the molecule are replaced with heavy isotopes) only allow for quantitative conclusions on the C fate. Thus, the application of position-specifically 13/14C labeled tracers combined with compound-specific analysis of the 13C signature in soil pools such as microbial biomarkers (e.g. phospholipid fatty acids ?PLFAs) is increasingly used to reconstruct metabolic processes. Using position-specific labeling, this project aimed to a) unravel metabolic transformations of glucose under field conditions, and b) identify the impacts of sorption of organic substances to soil minerals on microbial processes in soil. As Glucose does not possess functional groups allowing for sorption, Alanine was used in this study.
Medium-term transformations (3 and 10 days after tracer application) of position specifically labeled Glucose and Ribose are analyzed in soil under field conditions. 13C incorporation is quantified in bulk soil, microbial biomass and in cell membranes of microbial groups classified by 13C-PLFA. A laboratory experiment, studying the effect of sorption on microbial metabolism is conducted in soil sampling rings installed in glass microcosms. Tracer 13C and 14C incorporation into CO2 was measured in increasing time steps starting 15 min after application of tracer. Tracer incorporation into bulk soil, microbial biomass and PLFAs was quantified after 1, 3 and 10 days.
Through the position-specific pattern in microbial biomass it is possible to observe the basic metabolism of Glucose: in the field experiment, the incorporations of position C-1 und C-4 into microbial biomass is lower than that of C-2 and C-6, revealing that both glycolysis (preferentially mineralizing C-4) and pentose phosphate pathway (ppp, preferentially mineralizing C-1), exist in soil in parallel. The 13C pattern in PLFA reveals an even more complex Glucose metabolism. Position C-2 is incorporated the most into PLFAs, which can only be explained by a succession of Glucose degrading and rebuilding pathways: First, Glucose is transformed within the glycolysis into two C3 molecules. The enzyme of triose isomerase mirrored the two C3 molecules, so that during the rebuilding of Glucose via the reversible reactions of the gluconeogenesis, the newly formed Glucose has a 50:50 chance to either retain its positions in their original or the reverse order. If this newly formed Glucose molecule is transformed via ppp, 50% of C-6 and 50% of C-1 are mineralized and consequently both positions are incorporated into PLFA lower than C-2. Thus, we could show the possibility to trace gluconeogenesis via PLFA incorporation ? the prerequisite for incorporating this pathway in metabolic flux models in soils (see the respective DAAD project).
Comparing the mineralization and incorporation of Alanine in dissolved organic carbon (DOC) and sorbed to soil reveals that the stabilization effect of sorption does not only rely on inaccessibility: Sorption decreased the initial mineralization peak of Alanine by ~80% while increasing the time until the respiration peak was reached for 4 times. However, almost all C-1 is mineralized after one day independent of sorption. This means that after one day, microorganisms were able to take almost all sorbed Alanine, deaminate it to Pyruvate and decarboxylate it to Acetyl-CoA. Sorption increases the carbon use efficiency (CUE) by 60% compared to Alanine in solution and increased C incorporation in microbial biomass up to four times. Incorporation into PLFA shows that this shift is mostly driven by the group of fungi, which incorporate more C from sorbed than free Alanine into their PLFA, while all other microbial groups take up free and sorbed Alanine indifferently. The fungis metabolization is also much more complex, including gluconeogenesis, followed by ppp. Only by applying position-specific tracer it is possible to show how sorption promotes stabilization of SOM not only by reducing accessibility, but by inducing a shift in the microbial metabolism.
Whereas previous studies established the basis of tracing metabolic transformations by position-specific labelling, this project provides a range of experiments applying this technique to unravel distinct reactions and metabolic behaviour of microorganisms under environmental stress such as restricted access ot C at mineral surfaces. It could be shown that the ability to identify metabolic pathways in situ is crucial to understanding C allocation and stabilization in soils and how these will be influenced by changing environmental conditions and land-use practices.