The transformation of plant inputs into stable soil organic carbon (SOC) is a crucial yet poorly understood process with significant implications for carbon storage, nutrient availability, primary production, and ecosystem responses to global change. The most substantial and slowly cycling SOC pool consists primarily of microbial products stabilized by interactions with soil minerals, suggesting a mediating role of microbial production in transferring plant inputs to this mineral-associated SOC pool. Microbial necromass (dead microbial cells and their byproducts) contributes significantly to SOC in many ecosystems and its production is governed by microbial growth rate (MGR), carbon use efficiency (CUE), and biomass turnover rate (MTR). Despite their potential importance in SOC formation and decomposition, the environmental controls on these microbial traits and their influence on SOC across environmental gradients remain unclear. Current SOC theory posits that these microbial physiological traits mediate SOC stabilization, proposing a positive relationship between microbial growth, efficiency, or turnover and the amount of mineral-associated SOC (necromass stabilization hypothesis). Several isotope tracing studies support the idea that high-quality, rapidly decomposing plant inputs are more efficiently transferred into mineral-associated SOC. This is often attributed to the necromass stabilization hypothesis. However, this hypothesis lacks substantial empirical support beyond small-scale studies and overlooks other potential mechanisms. These alternative mechanisms include direct interactions between plant compounds and mineral surfaces, microbial extracellular production, and litter-induced priming effects (stimulation of SOC decay by fresh inputs). The current research aims to rigorously test the necromass stabilization hypothesis using a combination of experimental microcosms and a multi-site field study across a range of eastern U.S. temperate forests. The study specifically investigates the relationship between microbial physiological traits and mineral-associated SOC, and explores competing hypotheses that other factors contribute significantly to SOC dynamics.

The literature review extensively cites previous research supporting the necromass stabilization hypothesis, highlighting studies using isotope tracing to demonstrate that high-quality, rapidly decomposing plant inputs lead to faster and more efficient mineral-associated SOC formation. However, the review also points out the limited empirical support for this hypothesis, particularly in natural field settings. The review then introduces alternative hypotheses that could explain the observed relationships between plant litter quality and SOC. These include: direct sorption of plant-derived compounds to mineral surfaces, independent of microbial involvement; the contribution of microbial extracellular products to SOC formation; and litter-induced priming effects that increase the decomposition of SOC, potentially offsetting the positive impact of microbial necromass. The review underscores the lack of studies that directly compare the relative importance of these alternative mechanisms alongside the traditional necromass stabilization pathway, framing the need for a comprehensive investigation that simultaneously considers these competing hypotheses.

The study employed a two-pronged approach: a controlled microcosm experiment and a multi-site field study. The microcosm experiment involved incubating 16 different temperate tree leaf litters with isotopically distinct soil. The researchers monitored litter decomposition by measuring respiration rates and isotopic signatures over 185 days. Microbial physiological traits (MGR, CUE, MTR) were quantified using a substrate nonspecific technique (¹⁸O incorporation into DNA) at early and intermediate stages of decomposition. The flow of litter-derived carbon into the mineral-associated SOC pool was also tracked. In the field study, the researchers sampled six eastern US temperate forests with varying tree mycorrhizal associations, using these associations as a proxy for differences in litter quality and other potential drivers of microbial physiological traits. They quantified SOC pools (mineral-associated and necromass-derived), microbial physiological traits, and other biotic and abiotic factors across wide environmental gradients within each forest. Both the microcosm experiment and the field study used stable isotopes (¹³C and ¹⁸O) to trace carbon flow and microbial processes. Statistical analyses, including path analysis, were used to determine the relationships between litter quality, microbial physiological traits, and SOC formation and decomposition. Specific methods included size fractionation to separate particulate and mineral-associated SOC, chloroform fumigation extraction to quantify microbial biomass, and amino sugar analysis to estimate microbial necromass. The researchers calculated several indices (litter quality index, microbial physiological trait index) using principal component analysis (PCA) to reduce the dimensionality of their data.

The key findings consistently demonstrated a decoupling between microbial physiological traits and mineral-associated SOC formation. In the microcosm experiment, high-quality litters stimulated microbial growth, CUE, and turnover, but these traits were negatively correlated with the amount of litter carbon recovered in the mineral-associated SOC pool. Path analysis revealed that the positive effect of litter quality on mineral-associated SOC was direct, not mediated through microbial physiological traits; in fact, the indirect effect of microbial traits counteracted the positive effect of litter quality. This suggests that the increased microbial activity associated with high-quality litter led to increased SOC decomposition (priming effect), outweighing the positive contribution of microbial necromass. The field study showed similar results. Litter quality was positively associated with mineral-associated SOC, but this relationship was not explained by microbial physiological traits, which were unrelated to total mineral-associated SOC. Furthermore, a negative relationship was observed between microbial physiological traits and the proportion of soil carbon stored in mineral-associated SOC. Analysis of microbial necromass revealed no significant relationship between microbial physiological traits and the size of the soil microbial necromass pool. The study also found that soil abiotic properties, such as clay content and oxalate-extractable Fe (Feox), were strong predictors of mineral-associated SOC. Additionally, fine root biomass was negatively associated with mineral-associated SOC, potentially indicating root-driven SOC priming.

The findings strongly challenge the widely held necromass stabilization hypothesis, indicating that microbial physiological traits are not sufficient to explain the link between plant input quality and mineral-associated SOC formation in temperate forests. The negative relationships observed between microbial traits and SOC suggest that increased microbial activity, stimulated by high-quality litter, primarily leads to SOC decomposition rather than accumulation. The study highlights four critical factors that decouple microbial necromass production from mineral-associated SOC: variations in the stabilization potential of necromass from different microbial communities; alternative pathways for SOC formation (direct stabilization of plant inputs or stabilization of microbial extracellular products); priming effects leading to both new and old SOC decomposition; and the overriding influence of soil abiotic properties. The balance of these factors likely varies across different ecosystems, emphasizing the need for context-specific models of SOC dynamics. The study's results have important implications for modeling soil carbon and predicting future soil carbon dynamics under changing environmental conditions.

This research demonstrates that the necromass stabilization hypothesis is an insufficient explanation for the relationship between plant litter quality and mineral-associated SOC in temperate forests. The negative association between microbial activity and SOC highlights the importance of considering alternative SOC formation pathways, priming effects, and soil abiotic properties. Future research should focus on further elucidating the relative contributions of these various factors and developing more mechanistic models that incorporate this complexity. Specifically, studies investigating the role of microbial extracellular products, the differential stabilization of necromass from various microbial sources, and the interactions between roots, microbes, and SOC are needed.

The study focused on surface soils and aboveground inputs, potentially overlooking the role of roots and belowground inputs in deeper soil layers. The use of mycorrhizal association as a proxy for variation in litter quality and other factors may not fully capture the complexity of environmental influences on microbial processes. The relatively short duration of the microcosm experiment may limit the ability to fully assess long-term SOC dynamics. Although the researchers addressed potential confounding factors, there may be other unmeasured variables influencing the observed relationships.