Straw return is a widespread agricultural practice for improving cropland nitrogen (N) stocks. However, the contribution of microbial N to the soil aggregate N pool and the underlying microbial metabolic regulation mechanisms remain uncertain. This study was based on a 13-year field experiment with rice (Oryza sativa L.) and wheat (Triticum aestivum L.) rotation, using only a chemical fertilizer alone (CF) as the control. We analyzed the effects of the chemical fertilizer combined with (CS, 9500 kg ha−1 y−1) and wheat (4000 kg ha−1 y−1) straw on microbial derived-N, microbial carbon (C) and N limitations. We also assessed microbial N use efficiency (NUE) in various aggregates of ferric lixisols (0–20 cm). Rotary tillage reached a depth of 20 cm. The CS significantly increased microbial-derived N concentrations in soil aggregates and enhanced the contribution of fungal residual N to the N pool in aggregates < 0.25 mm, but did not affect those > 0.25 mm. Conversely, the bacterial contribution to the N pool was not affected by CS. Meanwhile, CS significantly increased the soil organic C and microbial biomass in the aggregates. The results of our eco-enzymatic stoichiometric model revealed that the CS significantly alleviated microbial C limitations and increased microbial NUE in soil aggregates. Structural equation modeling further revealed that the microbial biomass and soil organic C contents are key drivers of the microbial C limitation. The increased contribution of fungal residual N to the N pools in < aggregates 0.25 mm was attributed to improved microbial NUE resulting from the straw, without altering net N mineralization rates or β-1,4-N-acetylglucosidase activity. Our findings suggest that straw return promotes microbial-derived N production and sequestration by alleviating microbial C limitation. The strategies governing these microbial-derived N responses in aggregates to straw return might vary. This might be valuable for designing cropland management practices to improve N storage.
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