Global farming systems produce approximately 5 × 10⁹ tons of crop residues annually. Returning these residues to the soil is a common practice to maintain soil organic carbon (SOC) stock and productivity. The transformation of crop residues into SOC is complex, influenced by residue chemistry, microbial processes, and soil mineralogy. Research has increasingly focused on the role of microbial communities in this transformation, as their composition and activity are fundamental to residue-C turnover. The nitrogen (N) content of crop residues is a key factor affecting their degradability; N-rich residues decompose faster than those with high C/N ratios. This suggests that residue-N plays a significant role in soil C sequestration and SOC stability. The stability of residue-induced C is thought to be linked to how residue-N and C alter the SOC molecular composition and spatial distribution. Organic molecules vary in their resilience to microbial degradation; for example, plant-derived aliphatic compounds are more recalcitrant than O-alkyl compounds. However, a comprehensive understanding of how microbial community composition and functional activity link soil C and N dynamics in response to residue-N is lacking. This study aims to address this gap by using metagenomics to characterize microbial functional gene profiles and their contribution to residue-induced C accumulation in SOC pools and shifts in SOC molecular composition. The study was conducted in a severely degraded Mollisol, a typical fertile farming soil found globally. The findings could lead to mechanistic solutions for balancing SOC stock with crop residue return in agricultural systems.

Existing literature highlights the importance of crop residue amendment in enhancing soil organic carbon (SOC) stocks and soil productivity. Studies have shown that the decomposition of crop residues is influenced by various factors, including residue chemistry (e.g., C/N ratio), soil properties (e.g., mineralogy), and microbial community composition and activity. The role of nitrogen (N) in the decomposition process is particularly emphasized, with N-rich residues exhibiting faster degradation rates compared to those with high C/N ratios. This suggests a strong link between residue-N and soil C sequestration. However, the mechanisms through which microbial communities mediate this process remain unclear. While previous research has investigated the temporal shifts in microbial phylogenetic composition following residue amendment, integrative studies linking C and N metabolisms to SOC pool formation are limited. This study addresses this gap by employing metagenomic analysis to investigate the functional gene profiles of microbial communities and their role in residue-C transformation and SOC stabilization.

The study used a severely degraded Mollisol soil collected from a farming paddock under a soybean-maize rotation without fertilization. ¹⁵N-labeled soybean and maize residues were prepared by growing plants in soil amended with Ca(NO₃)₂ containing 20% ¹⁵N atom excess. An incubation experiment was conducted for 250 days with three treatments: maize residue amendment, soybean residue amendment, and a no-residue control. Soil samples were collected at 7, 30, 60, 100, and 250 days for various analyses. Soil respiration was measured by trapping CO₂ in NaOH solutions. Soil pH, SOC, total N, and available N were also determined. SOC was fractionated into coarse particulate organic carbon (POC), fine POC, and mineral-associated organic carbon (MOC). The chemical composition of SOC was analyzed using solid-state ¹³C NMR. DNA was extracted from soil samples for Illumina MiSeq sequencing of bacterial 16S rRNA genes and fungal ITS regions. Metagenome sequencing was also performed on samples from days 7 and 250. Bioinformatic analyses were conducted using QIIME, MEGAHIT, MetaGene, CD-HIT, SOAPaligner, and Diamond to identify and quantify microbial functional genes. Principal coordinate analysis (PCoA), redundancy analysis (RDA), ANOSIM, and Adonis were used to analyze microbial community composition. Co-occurrence networks were constructed using the R package "WGCNA" to identify keystone ecological clusters. Statistical analyses were performed using SPSS.

Residue amendment significantly altered the microbial community composition and functional gene profiles. Principal coordinate analysis (PCOA) revealed distinct shifts in bacterial and fungal communities in response to residue addition, with greater differences observed between maize and soybean residue treatments at the end of the incubation period. Redundancy analysis (RDA) showed a strong association between microbial community composition and SOC composition and C concentration in SOC pools. Residue amendment increased the abundance of N-ammonification genes (urease, glutaminase, leucyl aminopeptidase), particularly under soybean residue treatment. The abundance of C-degradation genes also increased after residue amendment, with higher abundances of genes involved in the mineralization of labile C compounds (starch, hemicellulose, pectin, cellulose) at day 7 and recalcitrant C compounds (aromatics, lignin) at day 250. Network analysis revealed a close connection between C-decomposition and N-mineralization genes with soil C and N status. Most C-decomposition and N-mineralization genes were positively correlated with the relative proportion of O-alkyl C but negatively correlated with the alkyl C/O-alkyl C ratio and aromatic C proportion. N-mineralization genes were positively associated with residue-N in fine-POC and MOC fractions. Ecological network analysis identified six major modules, with module 2 showing a strong correlation with C-decomposition and N-mineralization genes, suggesting it plays a keystone role. This module was enriched with genera such as *Massilia*, *Granulicella*, *Dyella*, *Sphingomonas*, *Luteimonas*, *Penicillium*, and *Ramophialophora*, known for their roles in degrading various organic compounds. The increase in aliphatic C in SOC, positively associated with N-mineralization genes (urease, glutamate synthase, glutaminase), contributed to SOC stabilization. Increased MOC with residue amendment indicated the physical binding of decomposed compounds to soil minerals, enhancing resistance to decomposition. The greater increase in C concentration in the POC fraction with soybean residue (compared to maize) suggests that residue-N promotes C accumulation and protection from decomposition through aggregation.

The findings demonstrate the pivotal role of residue-N in soil C sequestration. The observed increases in C and N concentrations in SOC fractions after residue amendment highlight the importance of nutrient stoichiometry within heterotrophic microorganisms. The C sequestration in SOC pools is largely N-limited due to the higher C/N ratio of residues compared to soil. This is supported by the significant decrease in mineral N in residue-amended soil, indicating microbial N mining from organic matter. The strong association between microbial community composition and C concentration in SOC pools, along with the altered functional gene profiles, indicates that microbial communities drive residue-C transformation into SOC. The integrated C and N metabolic profiles contribute significantly to C accumulation in SOC pools. Genes involved in N mineralization are closely linked to C-decomposition genes and C concentrations in SOC fractions. Keystone genera identified in the network analysis have known roles in degrading chitin, aromatic compounds, and lignocellulose, contributing to both C and N cycling. The increase in aliphatic C and MOC strengthens SOC stability. The study shows the mechanistic contribution of microbial N metabolism to crop residue turnover and SOC stabilization, particularly with N-enriched residues.

This study provides strong evidence for the crucial role of microbial C and N metabolisms in mediating the contribution of crop residues to soil organic carbon (SOC) accumulation and stability. The integrated approach, combining metagenomics and SOC characterization, reveals how residue-N stimulates microbial activity, leading to the transformation of labile and recalcitrant C compounds into persistent SOC pools. This knowledge is essential for developing strategies to optimize crop residue management practices for enhancing SOC sequestration and soil health. Future research could explore the interactions between specific microbial taxa and residue types, and the impact of varying environmental conditions on these processes.

The study was conducted in a single soil type (Mollisol) under controlled laboratory conditions. The results may not be directly generalizable to other soil types or field settings. Furthermore, the focus was primarily on bacterial and fungal communities, neglecting other potentially important microbial groups. The long-term stability of the observed effects needs further investigation through longer-term field studies.