Soils are crucial carbon (C) and nitrogen (N) sinks, exceeding atmospheric and vegetation stores. Grasslands, a major component of agricultural land, contain substantial soil organic carbon (SOC). Microbial necromass, composed of microbial products and residues, contributes significantly to persistent SOM, particularly in grasslands. Its formation is influenced by factors such as spatial location, microbial density and taxa, abiotic conditions, and soil mineralogy, often beginning with adsorption to mineral surfaces. Microbial metabolism influences both formation and loss of mineral-associated necromass C, but the roles of necromass recycling and the persistence of mineral associations are unclear. This study addresses key uncertainties: 1) the impact of microbial substrate type (necromass vs. plant litter) on persistent soil C formation; 2) the proportion of necromass C and N recycled versus mineral-associated; and 3) the rate of loss of mineral-associated necromass C and N. Understanding these processes is crucial for predicting soil C sequestration under various management practices and improving ecosystem models.

Existing research highlights the importance of microbial necromass in soil organic matter persistence, particularly in grasslands. Studies have shown that the formation of stable necromass C is influenced by factors affecting live and dead microbes (spatial location, microbial density, taxa, abiotic conditions) and soil mineralogy. Mineral association is often a crucial first step in stabilization. However, the relative importance of microbial necromass consumption by live microbes (necromass recycling) and the persistence of mineral-associated necromass remain less clear. The influence of grassland management intensity on necromass formation, persistence, and loss is also an area requiring further investigation. Previous work suggests that management affects plant and microbial community structure, microbial processing of C and N, and ultimately C persistence. The higher N content of necromass compared to plant inputs may lead to a stronger affinity for mineral surfaces. Decoupling of C and N in necromass dynamics has not been comprehensively explored and understanding this decoupling is key for improving ecosystem models.

This study employed both laboratory and field experiments to investigate microbial substrate mineralization and the sorption/desorption of necromass from soil minerals. In the laboratory incubation, soils from low- and high-intensity grasslands were amended with dual-isotope-labeled (¹³C¹⁵N) Escherichia coli (bacterial necromass), Lolium perenne (ryegrass leaf or root litter), or no addition (controls). Microbial decomposition was assessed through substrate-derived CO₂ and N₂O efflux, and the remaining C and N in bulk soil and the fine mineral fraction (<53 µm). The field experiment traced isotopically labeled E. coli necromass in the same grasslands over approximately eight months. Necromass was injected into soil collars, and samples were collected at multiple time points to measure pool sizes and isotopic signals of C and N in CO₂, N₂O, microbial biomass, bulk SOM, and mineral-associated SOM. Physical fractionation was used to separate the fine mineral fraction. Isotope mixing models were used to quantify the proportion of substrate-derived C and N in various pools. Statistical analyses, including ANOVA and linear mixed effects models, were used to analyze the data.

The laboratory incubation revealed higher microbial substrate mineralization rates for leaf litter compared to root litter and necromass. However, there was no significant difference in C persistence after one year. This contradicts the hypothesis that substrate quality (C:N ratio) solely dictates decomposition rates. The field experiment showed that a substantial portion (64%) of the added necromass C was mineral-associated within three days. However, over eight months, mineral-associated C declined significantly faster than N. Low-intensity grasslands exhibited greater loss of mineral-associated C in the upper soil layers compared to high-intensity grasslands. High-intensity management led to enhanced persistence of both mineral-associated C and N at 5-10 cm depth. Microbial biomass incorporated only a small percentage of the added necromass C and N. Mean residence times of necromass C and N in the mineral-associated fraction varied, with N exhibiting much longer residence times than C, suggesting a decoupling of C and N persistence.

The findings challenge the conventional understanding that readily decomposable substrates lead to more stable soil organic matter. The decoupling of C and N persistence in necromass has significant implications for C and N cycling in grasslands. The faster loss of C than N, particularly under low-intensity management, contributes to N accumulation in agricultural soils. The effects of management intensity on mineral-associated C and N persistence highlight the role of plant communities and rhizosphere processes in influencing SOM dynamics. The relatively low incorporation of necromass into microbial biomass suggests selective utilization by a specialized microbial community (the necrobiome). The results emphasize the need for improved ecosystem models that explicitly represent the complexities of organo-mineral interactions and C and N decoupling.

This study reveals a previously underappreciated decoupling of carbon and nitrogen persistence in microbial necromass, demonstrating that carbon is less persistent than nitrogen in agricultural grasslands. Management intensity significantly influences this decoupling, highlighting the importance of considering both carbon and nitrogen dynamics for effective soil carbon sequestration strategies. Future research should focus on the necrobiome and the mechanisms driving C and N decoupling to improve predictions of soil organic matter dynamics and inform sustainable grassland management practices.

The use of a single species of necromass and plant litter may not fully capture the chemical complexity of natural microbial and plant communities. The experimental design, while robust, might not fully represent the spatial heterogeneity of necromass production and turnover in real soil systems. Further studies are needed to assess the role of other factors, such as soil moisture variations, in affecting necromass dynamics.