DOI: 10.6084/m9.figshare.30464477
title: Original date

description: Prior to the autumn maize harvest in 2023, composite topsoil samples (0–20 cm depth) were obtained from each plot by mixing five 5-cm diameter cores collected in a S-shaped pattern, with three replicates per treatment. All samples were immediately taken back to the laboratory on dry ice. Visible plant/animal residues and gravel were carefully removed before each sample was divided into two parts: one for bulk soil analysis, and the other for aggregate fractionation. Aggregates were separated by the Savinov dry-sieving method to minimize disruption of non-water-stable aggregates and retain water-soluble substances . In brief, soil clods were gently broken along natural planes and passed through an 8-mm sieve, then air-dried under shaded. Subsequently, 60 g of air-dried soil was placed on the top sieve of a nested set (2 mm, 0.25 mm, and a collection pan) and shaken for 3 minutes using a mechanical shaker (amplitude: 2 mm, frequency: 50 Hz). Aggregates retained on each sieve and the pan were carefully collected and weighed to obtain large aggregates (LA, &gt; 2 mm), small aggregates (SA, 0.25–2 mm), and micro-aggregates (MA, &lt; 0.25 mm) . This procedure was repeated as necessary to obtain sufficient material for Q<sub>10</sub> assessment.Various forms of iron (Fe) and aluminum (Al) oxides were measured to assess their potential mediation of SOC persistence within aggregates. Free Fe and Al oxides (Fe<sub>d</sub> + Al<sub>d</sub>) were measured using a sodium dithionite, sodium citrate, and sodium bicarbonate extraction method. Amorphous Fe and Al oxides (Fe<sub>o</sub> + Al<sub>o</sub>) were extracted with acid ammonium oxalate buffer. Complex Fe and Al oxides (Fe<sub>p</sub> + Al<sub>p</sub>) were extracted using sodium pyrophosphate . Oxide contents were expressed as 0.5Fe + Al.The chemical stability (CS) of soil organic matter was evaluated by measuring aggregate-associated organic compounds via pyrolysis gas chromatography-mass spectrometry (Py-GC/MS). Briefly, 10 mg of aggregate sample was mixed with 1 ml of 25% tetramethylammonium hydroxide in a quartz boat and dried under N<sub>2</sub> flow. The dried residue was transferred to a Pyrex tube reactor (EGA/PY-303D) and pyrolyzed at 400°C for 5 min. The pyrolysis products were trapped in chloroform (50 ml, ice bath), concentrated by rotary evaporation, and re-dissolved in 0.2 ml chloroform for analysis on a GCMS-QP2010 Ultra (Shimadzu, Japan).Organic compounds were identified and quantified using the Automated Mass Spectral Deconvolution and Identification System and the NIST mass spectral library . The CS was calculated using the following equation: CS=OC<sub>R</sub>/OC<sub>L</sub>, where, OC<sub>R </sub>is the total concentration of recalcitrant organic compounds (phenols, aromatic hydrocarbons, carboxylic acids, and amides), and OC<sub>L</sub> is the total concentration of labile organic compounds (alcohols, lipids, ethers, and other aliphatics).Microbial activity in aggregates was assessed by measuring the activities of three extracellular enzymes involved in C cycling: α-glucosidase (AG), β-glucosidase (BG), and β-cellobiohydrolase (CBH). Briefly, 1 g of pre-incubated sample was mixed with 100 ml sodium acetate buffer to prepare a soil suspension. A 96-well microplate was set up with eight replicates of each of the following well types: 200 μl suspension + 50 μl buffer, 50 μl substrate (Table S1) + 200 μl buffer, 50 μl standard + 200 μl suspension, and 50 μl standard + 200 μl buffer. After incubation in the dark at 25°C for 4 h, the reaction was terminated by adding 10 µl of 1.0 mol L<sup>–</sup><sup>1</sup> NaOH to each well. Fluorescence was measured at 365 nm excitation and 450 nm emission using a microplate reader (Synergy H1M, USA).Bacterial and fungal community composition was assessed by high-throughput sequencing (Illumina MiSeq PE300, USA). The bacterial 16S rRNA gene was amplified using primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTT TRAGTTT-3′), and the fungal ITS region was amplified with primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA -3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). Microbial diversity indices (e.g., Shannon index) were calculated from OTU tables in Mothur (V1.30.2, https://www.mothur.org/ wiki/Download_mothur). Taxonomic classification of bacteria and fungi at class level was performed using the SILVA (release 138) and UNITE (release 8.0) databases, respectively.<br><br>