Using metabolic flux modeling to disentangle anabolic and catabolic contributions to soil heat dissipation

Metabolic flux analysis is an integrated experimental and computational approach for quantitative understanding of biochemical reaction networks with particular relevance in systems biology. Mass and energy flows through soil microbial metabolism are subject to the laws of thermodynamics. Carbon (C) allocation through central metabolic pathways (e.g. glycolysis, pentose phosphate, and Entner-Doudoroff) can be reconstructed by 13C-labelling coupled to metabolic flux analysis (13C-MFA) by tracing specific C atoms from within substrate molecules into metabolic products such as carbon dioxide (CO2) or fatty acids. However, mass flow calculated via 13C-MFA alone cannot fully characterise microbial carbon use. Here, we took the novel approach of coupling MFA with microcalorimetry, to also take bioenergetic constraints into account. We coupled energetics and mass flow on a metabolic level by selecting optimal sets of isotopomer tracers. Fifteen position-specific or uniformly 13C-labelled isotopomers - four alanine, seven glucose, and four glutamic acid ones – were added to a Luvisol (in total 4 folds of the microbial biomass C), and we analyzed substrate-derived 13CO2 fluxes as well as heat dissipation via isothermal microcalorimetry. Our results demonstrate that the temporal dynamics of catabolic CO2 release resembles that of the heat dissipation, i.e. peak respiration and peak heat dissipation were reached approximately 18 h after substrate addition, irrespective of whether the substance entered the central metabolic pathway at the monosaccharide level (glucose), at the pyruvate level (alanine) or in the citric acid cycle (glutamic acid). This indicates that heat dissipation in the initial growth period was strongly dominated by catabolic processes. However, whereas 13CO2 release leveled off during the 36 hours of incubation, the heat dissipation remained above its original level, suggesting that anabolic processes increasingly contribute to the heat dissipation in the later phases of incubation. Glucose isotopomer utilization indicated dominance of the pentose phosphate and Entner Douderoff pathways over glycolysis, suggesting a high activity of fast-growing organisms with considerable C allocation to anabolism. The dominance of this anabolic C use in the later stage of the incubation was confirmed by the isotopomer utilization of alanine and glutamic acid. This study shows that the heat dissipation of growing microbial communities under high C supply is closely linked to their catabolic CO2 release, whereas slow, potentially recycling-based growth after resource depletion releases energy more via anabolic reactions. We furthermore demonstrated that coupled MFA and calorespirometry provides a powerful tool to differentiate among metabolic contributions to the energy use of soil microbial communities in different growth phases.