Decadal manure substitution reshapes microbial
communities to drive plant and microbial carbon
accumulation in soil carbon fractions

Zhengwu Wu, Yanyan Zhang, Yuchuan Fan, Changquan Wang, Bing Li, Zed

Rengel, Jianbo Shen, Edith Le Cadre, Joann Whalen, Xiaoyan Tang

► To cite this version:

Zhengwu Wu, Yanyan Zhang, Yuchuan Fan, Changquan Wang, Bing Li, et al.. Decadal manure substitution reshapes microbial communities to drive plant and microbial carbon accumulation in soil carbon fractions. Geoderma, 2025, 463 (117581), 10.1016/j.geoderma.2025.117581. hal-05365735

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# Decadal manure substitution reshapes microbial communities to drive plant and microbial carbon accumulation in soil carbon fractions

Zhengwu Wu<sup>a,b</sup>, Yanyan Zhang<sup>a</sup>, Yuchuan Fan<sup>c,d</sup>, Changquan Wang<sup>a</sup>, Bing Li<sup>a</sup>, Zed Rengel<sup>e,f,g</sup>, Jianbo Shen<sup>b</sup>, Edith le Cadre<sup>g</sup>, Joann Whalen<sup>d,h</sup>, Xiaoyan Tang<sup>a,* i,j</sup>

## ARTICLE INFO

Handling Editor: Dr Daniel Said-Pullicino

### Keywords:

- Flooded soil
- Composted pig manure
- Microbial community dynamics
- Organic residue
- Particulate organic carbon
- Mineral-associated organic carbon

## ABSTRACT

The transformation of organic residues into particulate organic carbon (POC) and mineral-associated organic carbon (MAOC) governs soil organic carbon (SOC) storage and stability. However, how long-term manure use steers plant- and microbe-derived SOC fractions in paddy soils remains unclear. The SOC in paddy soil was evaluated by measuring amino sugars, lignin and enzyme activities from a 12-yr field trial (rice-wheat rotation) with no fertilizer, 100% mineral N fertilizer, and pig manure (50% and 100% replacement of N fertilizer). Compared to mineral N, the 100% pig manure treatment increased SOC by 26%, mainly through enhanced lignin input and microbial necromass accumulation, leading to significantly higher POC and MAOC levels. Manure application, compared to mineral N, promoted rice growth, which increased total organic C input and nutrient availability, thereby selectively favoring *r*-strategist bacteria, as reflected by a lower vanillyl acid-to-aldehyde ratio, a higher ratio of cellulose- to lignin-degrading enzyme activity, and greater lignin and bacterial necromass production. In contrast, the combined NPK and pig manure treatment increased the proportion of MAOC in total SOC by 7% compared to the 100% manure treatment, yet this came with enhanced lignin degradation, a lower bacterial phospholipid fatty acid (PLFA)/fungal PLFA ratio, and greater abundance of *K*-strategist fungi, resulting in less POC accumulation. These findings demonstrate that substituting mineral N with pig manure reshapes microbial community, enhancing the microbial transformation of plant- and microbe-derived residue into stable and labile SOC pools, and more effectively promotes SOC sequestration and persistence than mineral fertilization alone in paddy systems.

## 1. Introduction

Maintaining and enhancing soil organic carbon (SOC) is a cornerstone of sustainable agriculture. SOC accrues primarily through the microbial transformation of plant-derived carbon (C) inputs, closely linked to crop productivity and residue management (Buckeridge et al., 2020; Ma et al., 2018). Fertilizer management regulates organic matter inputs and microbial activity, contributing an estimated 0.44 to 0.68 Gt C year⁻¹ globally (Lessmann et al., 2022). The type of fertilizer—mineral or organic—differentially affects both the quantity and

quality of SOC formed. SOC is commonly partitioned into particulate organic carbon (POC), enriched in plant-derived structural polymers with inherent biochemical recalcitrance, and mineral-associated organic carbon (MAOC), dominated by microbially processed compounds stabilized by minerals (Lavallee et al., 2019; Lugato et al., 2021; Witzgall et al., 2021). Because fertilization affects crop production and soil microbial communities, it is expected to increase the SOC content by augmenting the POC and MAOC pools in agricultural soil.

Fertilizer input, either mineral or organic fertilizer, changes the proportions of POC and MAOC in agricultural soils by affecting the

<footnote><sup>a</sup> College of Resource, Sichuan Agricultural university, Chengdu 611100, China</footnote>

<footnote><sup>b</sup> State Key Laboratory of Nutrient Use and Management, Department of Plant Nutrition, College of Resource and Environmental Science, China Agricultural University, Key Laboratory of Plant-Soil Interactions, Ministry of Education, Beijing 100193, China</footnote>

<footnote><sup>c</sup> High Performance Computing Collaboratory-Geosystems Research Institute, Mississippi State University, Starkville, MS 39759, USA</footnote>

<footnote><sup>d</sup> Department of Natural Resource Sciences, McGill University, Montreal, Quebec H9X 3V9, Canada</footnote>

<footnote><sup>e</sup> Soil Science and Plant Nutrition, UWA School of Agriculture and Environment, The University of Western Australia, Perth, WA 6009, Australia</footnote>

<footnote><sup>f</sup> Institute for Adriatic Crops and Karst Reclamation, Split 21000, Croatia</footnote>

<footnote><sup>g</sup> Institut Agro, INRAE, SAS, Rennes 35042, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: xytang@sicau.edu.cn (X. Tang).</footnote>
quantity of organic C available for microbial transformation. Mineral fertilizers and animal manure promote crop growth, increasing rhizodeposition and unharvested residues that serve as substrates for microbial utilization (Sokol et al., 2024). In addition, manure itself also contains partially decomposed plant residues that enter the POC pool in the short-term (Li et al., 2020; Zou et al., 2023). SOC accrual is jointly governed by *ex vivo* enzymatic depolymerization of plant-derived macromolecular C and *in vivo* microbial turnover generating necromass (Liang and Zhu, 2021; Romero-Olivares et al., 2017; Sinsabaugh et al., 2009; Tao et al., 2023). Both processes depend on the microbial life history strategies, according to soil nutrient availability and environmental conditions. In fertilized soils with abundant water-soluble nutrients, *r*-strategy microorganisms such as bacteria, metabolizing labile carbon substrates like cellulose for rapid growth and reproduction (Dijkstra et al., 2013; Fierer et al., 2007; Hagerty et al., 2014). In nutrient-limited soils, *K*-strategy microorganisms (such as fungi) dominate, producing lignin-degrading extracellular enzymes that decompose complex phenolic compounds in plants cell walls. Consequently, SOC storage increases in soils receiving N and P mineral fertilizers, which have fewer lignin-decomposing fungi, low lignin-decomposing enzyme activity, and increased lignin content (Carreiro et al., 2000; Luo et al., 2022; Whittinghill et al., 2012). Unlike mineral fertilizer, manure application not only enhances soil nutrient availability but also provides labile C substrates that support more *r*-strategy microorganisms, resulting in a 2.6-fold greater increase in bacterial necromass C (Xia et al., 2021; Zhou et al., 2023). Therefore, microbial life history strategies are crucial in predicting the transformations of plant-derived and microbe-derived C in agricultural soils under different fertilizer regimes.

The SOC accumulation also depends on soil environmental conditions, crop growth and cultivation practices. Soil water content strongly affects SOC accumulation by influencing net primary production, decomposition, and microbial activity (Schluter et al., 2022). Compared to dryland, the anaerobic conditions in paddy soils inhibited facultative aerobic microorganisms, including fungi, which limited lignin decomposition, leading to its accumulation in the POC pool, but also prevented the formation of microbial necromass C, which represented 28–36 % of the SOC in paddy soil versus 40–59 % SOC in dryland (Chen et al., 2021). Fertilization promoted microbial growth, increasing bacterial necromass C content by 26 % relative to the no fertilizer control in paddy soil (Bian et al., 2024; Chen et al., 2020), likely due to enhanced *ex vivo* modification of cellulose through hydrolytic enzymes and rapid diffusion of water-soluble nutrients to *r*-strategy microorganisms, including facultative and obligate anaerobic bacteria. These findings highlight the potential of manure to enhance SOC even under constrained oxygen conditions, though the underlying microbial pathways remain underexplored.

Overall, fertilization modifies SOC dynamics in paddy soil by regulating plant inputs and microbial processes, thereby shaping the POC and MAOC contents. To reveal the underlying microbial mechanisms, we conducted a 12-year field experiment in paddy soil to evaluate the differential impacts of manure, mineral N, and their combination on plant- and microbe-derived C accumulation in particulate organic carbon (POM) and mineral-associated organic matter (MAOM) fractions. Using lignin and amino sugars as molecular markers, we aimed to trace the fate of plant residues and microbial necromass under contrasting fertilization regimes. We hypothesized that: (1) Pig manure not only increases soil nutrient availability but also enhances carbon input from plant growth, which promotes *r*-strategy bacteria growth, reduces lignin degradation, and thereby enhances lignin accumulation in POM and bacterial necromass in MAOM; (2) The combination of pig manure and mineral N lowers soil pH while improving nutrient status, facilitating both bacterial and fungal activity and lignin-degrading enzyme production, resulting in greater microbial necromass C in MAOM and reduced lignin in POM.

# 2. Materials and methods

## 2.1. Experimental site and design

The long-term fertilizer experiment in a rice (*Oryza sativa* L.)-wheat (*Triticum aestivum* L.) cropping system was initiated in November 2011 at the Chengdu Plain in Sichuan Province, China (30°56′43″ N, 103°44′0″ E). The soil is classified as an Anthrosol-paddy soil, formed from gray alluvium in the Minjiang River basin. The region has a mid-subtropical monsoon climate with an average annual temperature of 15.2 °C, annual precipitation of 1200 mm, and frost-free period of 280 days. Soil contained 28.4 g soil organic matter kg⁻¹, 1.40 g total N kg⁻¹, 113 mg alkali-hydrolysable N kg⁻¹, 8.50 mg available P kg⁻¹ and 53.0 mg available potassium kg⁻¹, with pH~H₂O~ of 6.6 (1:2.5).

The field experiment was established in a randomized complete block design with four fertilizer treatments and three replicates. Each replicate field plot (5 m × 4 m) was separated by plastic film (0.2 mm thick), which was inserted vertically into the bunds down to the plow pan to prevent lateral movement of fertilizer and water between plots. The CK treatment had no nutrient additions. The fertilizer treatments were: (1) NPK (mineral fertilizers only; wheat and rice each received 180 kg N ha⁻¹, 32 kg P ha⁻¹ and 62 kg K ha⁻¹ during their respective growing seasons); (2) NPK + pig manure (50 % mineral N replaced with pig manure; wheat and rice each received 90 kg N ha⁻¹ and 10 kg K ha⁻¹ plus 5150 kg pig manure (dry weight) ha⁻¹); and (3) Pig manure (100 % mineral N replaced with pig manure; pig manure at 10300 kg dry weight ha⁻¹ to each wheat and rice).

## 2.2. Agronomic practices

Mineral fertilizers were applied as urea (46.4 % N), superphosphate (5.24 % P) and potassium chloride (49.8 % K). Composted pig manure was provided by the Dehong Agricultural Company, Dujiangyan City, China. It was stockpiled for approximately two months, naturally fermenting under ambient conditions, with temperatures reaching 50–60 °C. During this period, the manure was turned periodically to ensure proper aeration so it partially decomposed before being applied to the fields. Composted pig manure contained 56 % water, 25 % C, 1.75 % N, 1.27 % P and 1.0 % K (on dry weight basis).

Mineral fertilizer and pig manure were used as basal fertilizers and spread evenly on the soil surface 1 day before wheat sowing or rice transplanting. The farming system follows a winter wheat – summer rice rotation. Before seasonal planting, manual plowing, mixing, and leveling were carried out. Wheat was sown in November each year at a rate of 150 kg ha⁻¹ and harvested in May the next year after a growing period of about 180 days. Rice was sown in March for seedling preparation, transplanted in May with a plant spacing of 20 × 25 cm (3 seedlings per hole), and harvested in September after a growing period of approximately 130 days. For irrigation management, intermittent non-flood irrigation was conducted before wheat sowing, followed by dryland maintenance. During the rice season, irrigation started 3–5 days before rice transplanting. All plots were continuously flooded (with a water depth of 15 cm) until mid-season drainage, after which irrigation was resumed to keep soil moisture without waterlogging. The water was drained 14 days before rice harvest. After harvest, rice and wheat straws were cut into approximately 6 cm pieces and fully incorporated into the soil before sowing. The amounts of rice straw returned (dry matter basis) were 5366, 6400, 8635, and 11434 kg ha⁻¹ for CK, NPK, NPK + pig manure, and Pig manure treatments, respectively (Table 1), and the corresponding wheat straw returns were 3017, 3642, 4649, and 4526 kg ha⁻¹ (Table S1). Throughout the growing season, timely pest and disease control was implemented to ensure the normal growth of the crops.

## 2.3. Rice and soil sampling

Rice yield and biomass were determined in September 2022 at
<caption>**Table 1**</caption>

The organic carbon input from rice after 12 years of fertilization treatments in rice season in Chengdu Plain, Sichuan Province, China.

<table><thead><tr><th>Treatment</th><th>Grain yield (kg ha<sup>-1</sup>)</th><th>Shoot biomass (kg ha<sup>-1</sup>)</th><th>Root biomass (kg ha<sup>-1</sup>)</th><th>Net rhizodeposition (kg ha<sup>-1</sup>)</th><th>Organic carbon input from rice (kg ha<sup>-1</sup>)</th></tr></thead><tbody><tr><td>CK</td><td>6562.9 ± 192.3b</td><td>5365.8 ± 149.1 d</td><td>1012.4 ± 28.1 d</td><td>506.2 ± 14.1 d</td><td>2753.8 ± 76.5 d</td></tr><tr><td>NPK</td><td>7933.9 ± 224.3b</td><td>6400.2 ± 252.2c</td><td>1280.0 ± 50.4c</td><td>640.0 ± 25.2c</td><td>3328.1 ± 131.2c</td></tr><tr><td>NPK + pig manure</td><td>9752.8 ± 192.3 a</td><td>8635.0 ± 393.1b</td><td>1798.9 ± 81.9b</td><td>899.5 ± 40.9b</td><td>4533.4 ± 139.3b</td></tr><tr><td>Pig manure</td><td>11037.7 ± 852.7 a</td><td>11434.0 ± 245.4 a</td><td>2432.7 ± 52.2 a</td><td>1216.4 ± 26.1 a</td><td>6033.2 ± 126.9 a</td></tr></tbody></table>

Note: Different letters in the same column indicated significant differences among treatments (p < 0.05). Means ± standard error (n = 3). CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.

maturity, with relatively uniform plant growth. The rice was harvested from three randomly selected quadrats (1.0 m²) within each plot (excluding the plot edges). Rice was divided into three parts: shoot, grains and root. After manual threshing and sun-drying to a constant weight, the rice was weighed.

Based on the root biomass, we calculated the net rhizodeposition of rice as follows:

$$ \text{Net rhizodeposition} = \text{Root biomass} * 0.5 \qquad (1) $$

0.5 is the ratio of net rhizodeposition/root biomass (Pausch and Kuzyakov, 2018; Villarino et al., 2021)

The total organic carbon input from rice is calculated as follows:

$$ \text{Total organic carbon input from rice} = (\text{Shoot biomass} + \text{Root biomass} + \text{Net rhizodeposition})*40\% \qquad (2) $$

Where 40 % is the carbon content of the rice tissue (Liu et al., 2019).

SOC is the largest component of the terrestrial organic carbon pool, and its dynamics depend on the balance between net primary productivity (inputs) and decomposition (outputs) (Wang et al., 2022). Because these processes are strongly influenced by human activities, vegetation, and environmental factors, SOC (particularly in the topsoil) is highly sensitive to such changes. Therefore, soil samples (0–20 cm) were collected after rice harvest in September 2022. Eight sampling points were randomly selected within each treatment plot and thoroughly mixed. Soil samples were transported to the lab in a refrigerated container and manually combed to remove visible roots and other debris, then divided into three parts. One part of fresh soil was air-dried for soil physicochemical analyses and SOM fractionation, the 2nd part was stored at 4 °C for soil microbial assessment, and the 3rd part was stored at –80 °C for DNA extraction and determination of microbial community structure.

## 2.4. SOM fractionation and physicochemical analysis

Soil was physically fractionated using sodium hexametaphosphate as a chemical dispersant and wet sieving to separate particulate organic matter (POM; > 53 µm) and mineral-associated organic matter (MAOM; < 53 µm) fractions (Sokol and Bradford, 2019). In brief, 10 g of air-dried soil was dispersed in 30 mL of 0.5 % (w/v) sodium hexametaphosphate solution, and shaken on a reciprocating shaker at 18°C for 18 h. The dispersed solution was sieved through a 53 µm mesh to separate POM and MAOM, which were then dried to constant weight at 70 °C. The POM and MAOM refer to soil fractions obtained through physically separation.

The organic carbon concentration in POM and MAOM was measured by the potassium dichromate oxidation-ferrous sulfate titrimetry method (Nelson and Sommers, 1996). Soil pH was measured in a soil-water suspension (the ratio of soil to water was 1:2.5, w/v) using a pH meter. Total N (TN) was quantified by a Kjeldahl analyzer (SKD-200, Shanghai, China), and alkali-hydrolysable (AN) was measured by the alkaline solution diffusion method (Chen et al., 2017). Total P (TP) and bicarbonate-extractable (available) P (AP) were quantified by the molybdenum-blue colorimetric method (Olsen et al., 1954). Microbial biomass carbon (MBC) was determined by the chloroform fumigation-incubation method (Vance et al., 1987). Dissolved organic carbon (DOC) was extracted with 0.5 M K₂SO₄ at a ratio of 1:5 (weight/volume) and then measured using a C/N analyzer (multi-N/C 3000, Analytik Jena, Jena, Germany).

## 2.5. Soil amino sugars and lignin analysis

Amino sugars, an indicator of microbe-derived carbon, were determined in POM and MAOM fractions according to Zhang and Amelung (1996) in four primary steps: hydrolysis, purification, derivation, and processing. Soil amino sugars are mainly divided into glucosamine (GluN), muramic acid (MurN), galactosamine (GalN), and mannosamine (ManN). Notably, GluN is derived mainly from fungi, whereas MurN originated exclusively from bacteria. Hence, the ratio of GluN/MurN is commonly utilized to indicate the contribution of fungal and bacterial necromass carbon to SOC accumulation (Liang et al., 2007). Assuming that MurN and GluN occur in bacterial cells in a molar ratio of 1:2, the fungal-derived GluN was determined by subtracting the bacterial-derived GluN from the total GluN (Li et al., 2015) as follows:

$$ \text{Fungal derived GluN} = \left( \frac{n}{179.17} - \frac{2m}{251.23} \right) * 179.17 \qquad (3) $$

On this basis, we calculated the fungal necromass carbon and bacterial necromass carbon as follows (Liang et al., 2019):

$$ \text{Fungal necromass carbon} = \left( \frac{n}{179.17} - \frac{2m}{251.23} \right) * 179.17 * 9 \qquad (4) $$

$$ \text{Bacterial necromass carbon} = m * 45 \qquad (5) $$

$$ \text{Microbial necromass carbon} = \left( \frac{n}{179.17} - \frac{2m}{251.23} \right) * 179.17 * 9 + m * 45 \qquad (6) $$

where *m* is the concentration of MurN, *n* is the concentration of GluN, 251.23 is the relative molecular mass of MurN, 179.17 is the relative molecular mass of GluN, 45 is the conversion coefficient of bacterial MurN to bacterial necromass carbon, and 9 is the conversion coefficient of fungal GluN to fungal necromass carbon.

Microbial necromass accumulation coefficient (NAC) indicates the amount of microbial necromass carbon produced per unit of microbial biomass carbon (Wang et al., 2021):

$$ \text{NAC} = \frac{\text{Microbial necromass carbon}}{\text{Microbial biomass carbon}} \qquad (7) $$

Lignin content, an indicator of plant-derived carbon, was analyzed in POM and MAOM according to Otto and Simpson (2006). The analysis included oxidation, acidification, extraction, nitrogen blowing, derivation, and processing. Lignin phenols are primarily categorized into vanillyl (V), syringyl (S), and cinnamyl (C) fractions (Feng et al., 2016). The sum (V + S + C) indicates the content and stability of plant-derived organic matter (Abiven et al., 2011). The acid-to-aldehyde ratio (Ad/Al) estimates the extent of lignin degradation (Thevenot et al., 2010).

## 2.6. Soil enzymes

Four enzymes related to ex vivo degradation of plant polymers were
analyzed by spectrophotometry in fresh soil, including cellobiohy-
drolase (CBH), β-glucosidase (BG), peroxidase (POD), and polyphenol
oxidase (PPO) (Bach et al. 2013). The CBH catalyzes the formation of p-
nitrophenol from p-nitrobenzene celluloside, and BG catalyzes the for-
mation of p-nitrophenol from p-nitrobenzene-β-D-glucopyranoside, with
both measured as light absorption at 400 nm. The POD catalyzes the
oxidation of organic matter to quinone, and PPO catalyzes pyrogallol to
produce purple gallatin, with both reactions showing characteristic light
absorption at 430 nm. The ratio of Ln(cellulose-modifying enzymes)/Ln
(lignin-modifying enzymes) activity, where cellulose-modifying en-
zymes refer to CBH and BG, and lignin-modifying enzymes refer to PPO
and POD, was an indicator of microbial preference for carbon substrates:

$$
\frac{\text{Ln(cellulose - modifying enzymes)}}{\text{Ln(lignin - modifying enzymes)}} = \frac{\text{Ln(CBH + BG)}}{\text{Ln(PPO + POD)}} \quad (8)
$$

A higher Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes) ratio indicates that microorganisms used more labile carbon substrates (cellulose) than recalcitrant lignin (Mooshammer et al., 2022).

2.7. Phospholipid fatty acid analyses

Phospholipid fatty acid (PLFA) of fresh soil samples was analyzed
following Willers et al. (2015), including extraction, purification,
esterification, and processing with validation by the internal C10:0 and
C19:0 standards (Quideau et al., 2016). The Gram-positive bacteria (G⁺)
markers include i14:0, i15:0, i16:0, i17:0, a15:0, and a17:0. The Gram-
negative bacteria (G⁻) markers include 16:1ω5c, 16:1ω7c, 16:1ω9c,
17:1ω8c, 18:1ω5c, 18:1ω7c, cy17:0ω7c, and cy19:0ω7c. Other bacterial
markers include 14:0, 15:0, 16:0, 17:0, and 18:0. The fungal markers are
18:2ω6c and 18:1ω9c. The Actinobacteria markers are 10me16:0,
10me17:0, and 10me18:0 (Frostegård et al., 2011). The fungal/ bacte-
rial PLFA ratio (F/B), calculated as the nmol fungal PLFAs g⁻¹ divided by
the nmol g⁻¹ of all other biomarkers, including Actinobacteria, G⁺, G⁻
and other bacteria, indicate the relative composition of soil microbial
communities (Joergensen, 2021).

2.8. Soil microbial community analysis

The DNA extraction from soil samples was performed using an E.Z.N.
A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.). Concentration of
DNA was quantified using a NanoDrop®ND-2000 spectrophotometer
(Thermo Scientific, Wilmington, DE, USA), and its purity was assessed
by 2 % (w/v) agarose gel electrophoresis. The extracted DNA samples
were sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai,
China) for sequencing on a MiSeq PE300 platform. The V3–V4 hyper
variable regions of the bacterial 16S rRNA gene and the ITS region of the
fungal gene were amplified with the primers 338F/806R and ITS1F/
ITS2R, respectively (Adams et al., 2013; Xu et al., 2016). The raw
sequence data were stored in the National Center for Biotechnology
Information (NCBI) database with accession number: PRJNA1078611.
The raw amplification sequences were quality-controlled by fastp
version 0.19.6 (Chen et al., 2018), and then merged by FLASH version
1.2.7 (Magoč and Salzberg, 2011). Based on a threshold of ≥ 97 %
sequence similarity, the reads were grouped into operational taxonomic
units (OTUs). Each OTU was classified by Ribosomal Database Project
(RDP) (Van der Bom et al., 2018). The bacterial and fungal sequences
were standardized to 23,491 and 42,530 sequences per sample,
respectively, for subsequent data analysis.

Non-metric multidimensional scaling (NMDS) with the Bray-Curtis
distance metric was used to evaluate the differences in microbial com-
munity composition across different fertilization treatments. We applied
the r-/K- strategy framework to define microbial strategies based on
their response to nutrient availability. While the high diversity and
flexibility of microbial groups mean that even members of the same
group may exhibit different life strategies under varying environmental

conditions, the overall abundance patterns of soil microbial taxa still
tend to respond to changes in nutrient availability in a predictable
manner (Fierer et al., 2007; Zhao et al., 2023). Therefore, although our
analysis was conducted at phylum and class levels, this approach still
provides valuable insights for predicting microbial life-history strate-
gies. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and
Firmicutes were defined as r-strategy bacteria, whereas Actinobacteria,
Verrucomicrobia, Acidobacteria, Gemmatimonadetes, Chloroflexi, and
Planctomycetes were defined as K-strategy bacteria, Ascomycota were
defined as r-strategy fungi, and Basidiomycota as K-strategy fungi
(Fierer et al., 2007; Li et al., 2021).

2.9. Statistical analyses

The correlation between C derived from organic manure and crop
with SOC, POC and MAOC was analyzed to reveal how SOC fractions
respond to exogenous organic C. Data normality was assessed using the
Shapiro-Wilk test, and homogeneity of variance was tested using Lev-
ene's test. Then, a one-way ANOVA was conducted to test the effect of
fertilization treatments on SOC pools, microbial properties and soil en-
zymes activities, followed by Tukey's multiple range test at a signifi-
cance level of *p* < 0.05. In addition, differences between POM and
MAOM fractions were assessed using an independent two-sample *t*-test
at *p* < 0.05. Spearman's correlation was used to assess the relationships
between soil nutrient properties and microbial community structure and
activity. A general linear model was applied to investigate the re-
lationships between microbial community activity and organic carbon
content from various sources, under different fertilization management
practices. Based on the microbial model proposed by Tao et al. (2023),
partial least squares path modeling (PLS-PM) was applied to investigate
the microbial mechanisms for plant-derived and microbe-derived car-
bon turnover, as modulated by fertilizer effects, thereby influencing the
proportions of POC and MAOC within the SOC pool. The model assumes
that, in paddy soils under rice-wheat rotation, soil microorganisms can
contribute to SOC accumulation through necromass formation while
also promoting SOC loss through extracellular enzyme-mediated lignin
degradation.

3. Results

3.1. Soil organic C dynamics under long-term fertilization regimes

SOC concentration increased significantly by 16–28 % after 12-yr of
continuous application of pig manure compared to no fertilizer, reach-
ing the highest concentration of 23 g kg⁻¹ when applied alone (*p* < 0.05,
Table S2). Pig manure significantly increased POC and MAOC concen-
trations by 26 % and 34 %, respectively (*p* < 0.05, Fig. 1a). The com-
bined application of mineral fertilization and pig manure increased
MAOC by 32 % and the MAOC/SOC ratio by 10 % (*p* < 0.05, Figs. 1a, b).
In contrast, mineral fertilizer alone had no significant effect on SOC
concentration (Fig. 1a). Under pig manure substitution treatments, rice
yield, shoot biomass, root biomass, and net rhizodeposition were
significantly higher than those under mineral fertilizer and no fertilizer
treatment, leading to a significantly higher carbon input from rice, with
the highest value observed under 100 % pig manure substitution treat-
ment (*p* < 0.05, Table 1). SOC concentration increased linearly with
organic carbon input from pig manure, rice, and their combined sources,
accompanied by a decreasing accumulation rate of MAOC and an
increasing rate of POC (*p* < 0.05, Fig. 2 and S1). Fertilization signifi-
cantly increased soil nutrients (DOC, TN, AN, TP, and AP), while soil pH
decreased relative to the control (*p* < 0.05, Table S2). The DOC/AN ratio
was highest with NPK + pig manure, while the DOC/AP ratio was lowest
with sole pig manure application (*p* < 0.05, Table S2).

In POM, total lignin phenols concentration was significantly higher,
whereas the (Ad/Al)ₗ ratio was significantly lower compared to MAOM
(*p* < 0.05, Fig. S2 and 1c). Under the NPK + pig manure treatment, total
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Response of organic carbon concentration (a) and proportion (b), lignin phenols concentration (c), and microbial necromass concentration (d) in particulate organic matter (POM) and mineral-associated organic matter (MAOM) fractions after 12-yr of different fertilization in rice season. Different lowercase letters indicate significant differences among fertilization treatments in particulate organic matter and uppercase letters indicate significant differences in mineral-associated organic matter (*p* < 0.05). \* indicate significant differences between POM and MAOM within the same fertilization treatment. \*\* *p* < 0.10, \*\*\* *p* < 0.01, \*\*\*\* *p* < 0.001. Means ± standard errors (n = 3). CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Relationship between the organic carbon input from pig manure (a) and rice (b) and the amount of soil organic carbon, particulate organic carbon, and mineral-associated organic carbon after 12-yr of different fertilization in rice season. CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure. The best-fit equations of the line, test statistics (R², *p* value) and 95 % confidence interval are shown for each line. The three data points for pig manure-derived carbon correspond to treatments with no pig manure (CK and NPK) and 50 % and 100 % pig manure substitution, which contribute 0, 2575 kg ha⁻¹, and 5150 kg ha⁻¹ of organic carbon, respectively. For rice-derived carbon input, multiple data points are shown because rice growth varied across different fertilization treatments and replicates, leading to differences in carbon input from rice.</figcaption></figure>

lignin phenols concentration in POM decreased by 14 % compared to CK, whereas the highest concentration, 18 % higher than CK, was observed under sole pig manure application (p < 0.05, Fig. 1c). In MAOM, the (Ad/Al)~v~ ratio decreased by 10 % and 17 % under the NPK and Pig manure treatments, respectively, but increased by 25 % under the NPK + pig manure treatment compared to CK (p < 0.05, Fig. S2d).

The microbial necromass carbon concentration in MAOM was significantly higher than in POM (*p* < 0.05, Fig. 1d and S3). The application of pig manure significantly increased the fungal necromass carbon concentration in both POM and MAOM, with the NPK + pig manure treatment showing a significantly higher fungal necromass carbon concentration in MAOM compared to the Pig manure treatment (*p* <

0.05, Fig. S4a). Fertilization significantly increased bacterial necromass carbon concentration by 38 %-81 % in MAOM compared to the CK treatment, with organic fertilization resulting in a greater increase than inorganic fertilization (*p* < 0.05, Fig. S4b). In POM, only the Pig manure treatment and NPK + pig manure treatment significantly increased bacterial necromass carbon concentration (*p* < 0.05, Fig. S4b). The glucosamine/muramic acid ratio in POM was significantly higher than in MAOM, and fertilization significantly decreased the glucosamine/muramic acid ratio compared to the CK treatment, with the lowest ratio observed in the Pig manure treatment (*p* < 0.05, Fig. S4c).
## 3.2. Microbial community structure and biological activity under long-term fertilization regimes

Fertilization significantly increased the microbial biomass carbon concentration (p < 0.05, Fig. 3a). Pig manure application led to a greater increase than mineral fertilization alone, with the highest concentration found under 100% pig manure substitution. The application of mineral fertilization and especially 100% pig manure significantly increased bacterial PLFA/fungal PLFA ratio, with the highest bacterial phospholipid fatty acid concentration observed in the 100% pig manure treatment (p < 0.05, Fig. 3b and S6). The NPK + pig manure treatment significantly decreased the alpha diversity of the bacterial community, as indicated by the Shannon index (p < 0.05, Fig. S7a). Fertilization significantly altered microbial community composition, as revealed by non-metric multidimensional scaling (NMDS) analysis (p < 0.05, Fig. S8 and S9). On the phylum level, the analysis of the top 10 most relative abundant bacterial taxa showed that compared to the CK treatment, the relative abundance of Chloroflexi (K-strategist) was significantly decreased under NPK and NPK + pig manure treatments, while Actinobacteria (K-strategist) was significantly increased. Gemmatimonadetes, K-strategist, was significantly increased under NPK and Pig manure treatments (p < 0.05, Table S3). At the fungal phylum level, fertilization significantly increased the relative abundance of

Basidiomycota (K-strategist), with the highest abundance observed under the NPK + pig manure treatment (p < 0.05, Table S4). Further analysis at the order level, analysis of the top 10 most relative abundant bacterial taxa showed that, compared to the CK treatment, the relative abundance of Rhizobiales (r-strategist) and Gaiellales (K-strategist) were significantly increased under the NPK + pig manure treatment, while Anaerolineales (K-strategist) was significantly decreased under both NPK + pig manure and Pig manure treatments (p < 0.05, Table S5). Bacillales, r-strategy bacteria, showed a significantly higher relative abundance under the Pig manure treatment compared to the NPK + pig manure treatment (p < 0.05, Table S5). At fungal order level, analysis of the top 10 most relatively abundant taxa showed that Pezizales (r-strategist) was significantly increased under the Pig manure treatment (p < 0.05, Table S6). Fertilization significantly increased the relative abundance of r-strategy bacteria, with the highest relative abundance observed in the Pig manure treatment, while K-strategy bacteria showed an opposite trend (p < 0.05, Fig. 3c, d). Fertilization significantly increased the relative abundance of K-strategy fungi, with the highest relative abundance observed when pig manure was applied with NPK fertilizer (p < 0.05, Fig. 3f). Compared to CK, fertilization significantly decreased lignin-modifying enzymes activity, except for the NPK + pig manure treatment, which showed no significant change in peroxidase activity (p < 0.05, Table 2). Fertilization significantly increased

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Response of microbial community structure after 12-yr of different fertilization in rice season. Different lowercase letters indicate significant differences among fertilization treatments (p < 0.05). Means ± standard errors (n=3). CK, no fertilizer; NPK, 100% mineral fertilizer; NPK+pig manure, 50% mineral N replaced with pig manure; Pig manure, 100% mineral N replaced with pig manure.</figcaption></figure>
<table><caption>**Table 2**

Response of carbon cycling-related extracellular enzymes activity after 12-yr of different fertilization in rice season.</caption><thead><tr><th>Treatment</th><th>Polyphenol oxidase activity (nmol g<sup>-1</sup>h<sup>-1</sup>)</th><th>Peroxidase activity (nmol g<sup>-1</sup>h<sup>-1</sup>)</th><th>Cellobiohydrolase activity (nmol g<sup>-1</sup>h<sup>-1</sup>)</th><th>β-glucosidase activity (nmol g<sup>-1</sup>h<sup>-1</sup>)</th><th>Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes)</th></tr></thead><tbody><tr><td>CK</td><td>943.2 ± 36.0 a</td><td>12527.9 ± 314.2 a</td><td>352.3 ± 17.7c</td><td>433.6 ± 28.1c</td><td>0.70 ± 0.01 d</td></tr><tr><td>NPK</td><td>490.4 ± 31.8b</td><td>7222.0 ± 361.4b</td><td>416.8 ± 16.6 bc</td><td>571.9 ± 7.8b</td><td>0.77 ± 0.01b</td></tr><tr><td>NPK + pig manure</td><td>345.6 ± 33.2c</td><td>13293.8 ± 255.5 a</td><td>480.6 ± 32.6 ab</td><td>600.8 ± 21.5 ab</td><td>0.73 ± 0.01c</td></tr><tr><td>Pig manure</td><td>518.3 ± 32.6b</td><td>6247.5 ± 139.5c</td><td>495.1 ± 19.8 a</td><td>658.8 ± 11.5 a</td><td>0.80 ± 0.01 a</td></tr></tbody></table>

Note: Different letters in the same column indicated significant differences among treatments (*p* < 0.05). Means ± standard errors (n = 3). CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.

cellulose-modifying enzymes activity, except there was no significant difference in cellobiohydrolase activity between the control and NPK treatment (*p* < 0.05, Table 2). Notably, fertilizer input significantly increased the Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes) ratio, with the Pig manure treatment showing the highest ratio (*p* < 0.05, Table 2).

Microbial biomass C, bacterial PLFA/fungal PLFA ratio, the relative abundance of *r*-strategy bacteria, cellobiohydrolase, β-glucosidase and Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes) ratio increased with enhanced total and available nutrient content, while the relative abundance of *K*-strategy bacteria showed the opposite trend (*p* < 0.05, Fig. S10). In contrast, polyphenol oxidase activity decreased with increasing dissolved C, total nitrogen and available nitrogen (*p* < 0.05, Fig. S10). Peroxidase activity decreased with higher available nitrogen, total phosphorus and available phosphorus (*p* < 0.05, Fig. S10). The relative abundance of *K*-strategy fungi increased with the dissolved C/available nitrogen ratio, but declined as pH increased (*p* < 0.05, Fig. S10).

### 3.3. The correlation between microbial community composition and carbon origin

The concentration of microbial necromass carbon increased with MBC (*p* < 0.05, Fig. S11a, b). Specifically, the concentration of bacterial necromass carbon increased with the relative abundance of *r*-strategy bacteria, but decreased with the relative abundance of *K*-strategy bacteria in both POM and MAOM (*p* < 0.05, Fig. 4a-d). The fungal necromass carbon concentration increased with the relative abundance of *K*-strategy fungi in both fractions (*p* < 0.05, Figs. 4g, h). The GluN/MurN ratio decreased with increasing bacterial PLFA/fungal PLFA ratio in POM and MAOM (*p* < 0.05, Fig. S11c, d). The concentration of lignin phenols increased with the increase of bacterial PLFA/fungal PLFA ratio and Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes) ratio, while (Ad/Al)~v~ decreased with the increase of these ratios in both fractions (*p* < 0.05, Fig. 5).

### 3.4. Mechanism of SOC change under different fertilization regimes

The PLS-PM approach was used to determine the effect of fertilization (and thus changing soil nutrient availability) on microbial community that influenced the SOC balance (Fig. 6). In the accumulation pathway (Fig. 6a), fertilization significantly increased availability of C, N, P and the total N, P concentration, which increased microbial biomass and alter microbial composition, promoting microbial necromass carbon production. Microbial necromass carbon primarily contributed to SOC through MAOC, improving the SOC persistence (Fig. 6a). In the decomposition pathway, fertilization increased the ratio of Ln(cellulose-modifying enzyme)/Ln(lignin-modifying enzyme), which influenced the degradation of plant-derived C, such as lignin, significantly promoting POC formation, without a significant contribution to MAOC (Fig. 6b).

Under the NPK treatment, bacterial necromass carbon content and lignin content in MAOC significantly increased by 37 % and 10 %,

respectively (Fig. 7b). In the NPK + pig manure treatment, microbial necromass carbon content increased significantly in POM and MAOC fractions, while lignin content decreased significantly in POM, which increased the proportion of MAOC to SOC, resulting in the greatest improvement in SOC persistence (Fig. 7c). Pig manure treatment significantly increased the content of microbial necromass carbon and lignin phenols in both fractions, leading to the most significant increase (by 28 %) in SOC content (Fig. 7d).

## 4. Discussion

### 4.1. Long-term pig manure substitution increased SOC storage and persistence

Our results showed that pig manure substitution significantly increased SOC content compared to the CK and NPK treatments, with the increase mainly driven by MAOC (Fig. 1a and Table S2). This contrasted with previous studies where SOC accumulation in paddy soils was primarily linked to increase in POC (Lan et al., 2022). One possible explanation was that pig manure input provides a range of organic compounds of various availability, accelerates microbial anabolism, and continuously forms microbial necromass and MAOC through entombment effects (Chen et al., 2020; Hu et al., 2023). Given MAOC's greater stability and slower turnover, this may have led to sustained SOC accumulation (Lavallee et al., 2019; Lugato et al., 2021). Alternatively, increased microbial activity may have enhanced extracellular enzyme production, thereby accelerating the decomposition of organic matter in the POC fraction (Tao et al., 2023). Additionally, we observed that the rate of MAOC increase decreased with higher external carbon input from pig manure or rice, while POC showed the opposite trend (Fig. 2). This could be due to the 100 % replacement of mineral N with pig manure, which introduced high levels of P that competed with organic carbon for adsorption onto mineral surface, thus limiting MAOC accumulation driven by microbial biomass proliferation (Spohn et al., 2022). Additionally, when the soil carbon approached or exceeded the MAOC saturation threshold, POC continued to increase (Bian et al., 2024). Therefore, excessive P input may have hindered the persistence of SOC. Due to the lack of measurement of lignin and non-lignin compounds in the rice and pig manure added to the soil, we were unable to compare the difference in carbon quality, which influenced the allocation of carbon across different SOC fractions (Cotrufo et al., 2013). Future research could use nuclear magnetic resonance (NMR) technology to explore the relationship between the quality of carbon input and SOC fractions, thereby providing deeper insights into the impact of pig manure substitution on the dynamics and stability of SOC.

In this study, microbial necromass carbon contributed primarily to MAOC, whereas lignin contributed mainly to POC (Figs. 1 and 6). Changes in the POC and MAOC amounts reflect the differential accumulation of plant-derived and microbe-derived carbon. These findings are consistent with previous studies (Bian et al., 2024; Li et al., 2023). This is because microbial necromass, with its high surface activity and strong affinity for minerals, contrasts with lignin that needs to be
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Linear correlations between microbe-derived necromass carbon and relative abundance of *r*-and *K*-strategy microbe in particulate organic matter (POM) and mineral-associated organic matter (MAOM) fractions after 12-yr of different fertilization in rice season. The best-fit equations of the line, test statistics (R², *p* value) and 95 % confidence interval are shown for each line. CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.</figcaption></figure>

depolymerized before interacting with mineral surfaces (Kleber et al., 2007; Thevenot et al., 2010). In the NPK treatment, bacterial necromass carbon and lignin content in the MAOM fraction significantly increased, though still lower than under organic fertilizer treatments, resulting in no significant changes in POC and MAOC compared to the CK treatment (Fig. 1 and S4). The NPK + pig manure treatment significantly increased microbial necromass carbon in both fractions, while significantly decreased lignin content in POM, resulting in significant increase in MAOC content and its proportion of total SOC (Fig. 1). Under the Pig manure treatment, microbial necromass carbon and lignin content significantly increased in both fractions, resulting in the largest increase in POC, MAOC and SOC (Fig. 1). These results were consistent with our

hypothesis, indicating that fertilization regulates the accumulation characteristic of plant-derived and microbe-derived carbon in POM and MAOM, influencing the storage and persistence of SOC (Liang et al., 2020; Liu et al., 2023).

## 4.2. Microbial traits mediate the retention of microbial necromass and lignin

We observed that fertilization significantly increased the microbial necromass carbon content in MAOM, with higher content observed in the NPK + pig manure and Pig manure treatments compared to NPK treatments (Fig. 1d). This may be attributed to fertilization improving
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Linear correlations between plant-derived carbon and extracellular enzymes activity and Bacterial PLFA/Fungal PLFA in particulate organic matter (POM) and mineral-associated organic matter (MAOM) fractions after 12-yr of different fertilization in rice season. The best-fit equations of the line, test statistics (R², *p* value) and 95 % confidence interval are shown for each line. CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.</figcaption></figure>

soil nutrient availability, which enhanced microbial biomass and promoted microbial necromass carbon accumulation. This was supported by the significantly positive relationships between available nutrient concentration and microbial biomass carbon, and between microbial biomass and necromass carbon (Fig. S10 and S11), consistent with previous studies highlighting microbial biomass as a key precursor in microbial necromass formation (Liang et al., 2016). The higher microbial necromass carbon content under pig manure application may be due to the provision of labile carbon, which further increased microbial biomass (Gul and Whalen, 2022; Tian et al., 2022; Zhou et al., 2023). During the composting process of pig manure, microbial growth and the resulting microbial necromass carbon directly contribute to the increase

in soil microbial necromass carbon content (Jost et al., 2011). Additionally, compared to other fertilization treatments, 100 % pig manure substitution significantly increased the carbon input from root and net rhizodeposition to the rhizosphere (Table 1), where microbial community exhibits higher SOC formation efficiency compared to bulk soil (Sokol and Bradford, 2019).

Fertilization increased the bacterial PLFA/fungal PLFA ratio and decreased the GluN/MurN ratio in both POM and MAOM fractions, enhancing the contribution of bacterial necromass carbon to SOC (Fig. 3b and S4c). This is because higher soil nutrient availability favors bacterial proliferation over fungal growth (Xia et al., 2021). Among the treatments, the lowest GluN/MurN ratio was observed in the Pig manure
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Two contrasting pathways, microbial accumulation pathway favouring SOC accumulation (a) and extracellular enzyme decomposition pathway stimulating SOC loss (b) determine the relationship between microbial community and SOC storage based on the concept model proposed by Tao *et al.* (2023). The black solid arrows indicate the pathways with significant positive results, and grey solid arrows indicate non-significant correlation. Gof denotes the proportion of variance explained by the model. \*p < 0.10, \*\*p < 0.01, \*\*\*p < 0.001. DOC, dissolved organic C; TN, total nitrogen; AN, available nitrogen; TP, total phosphorus; AP, available phosphorus; DOC/AN, dissolved organic C/available nitrogen; DOC/AP, dissolved organic C/available phosphorus; rB, the relative abundance of r-strategy bacteria; KB, the relative abundance of K-strategy bacteria; rF, the relative abundance of r-strategy fungi; KF, the relative abundance of K-strategy fungi; B/F, bacterial PLFA/fungal PLFA; MBC, microbial biomass C; Enzyme ratio, Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes); FNCp, fungal necromass carbon in POM; FNCm, fungal necromass carbon in MAOM; BNCp, bacterial necromass carbon in POM; BNCm, bacterial necromass carbon in MAOM; VSCp, the concentration of total lignin phenols in POM; VSCm, the concentration of total lignin phenols in MAOM.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Framework shows the influence of soil microorganisms on SOC content and persistence through changes in plant-derived carbon and microbe-derived carbon content. rB, the relative abundance of *r*-strategy bacteria; KB, the relative abundance of *K*-strategy bacteria; rF, the relative abundance of *r*-strategy fungi; KF, the relative abundance of *K*-strategy fungi; B/F, bacterial PLFA/fungal PLFA; MBC, microbial biomass C; FNCp, fungal necromass carbon in POM; FNCm, fungal necromass carbon in MAOM; BNCp, bacterial necromass carbon in POM; BNCm, bacterial necromass carbon in MAOM; VSCp, the concentration of total lignin phenols in POM; VSCm, the concentration of total lignin phenols in MAOM. CK, no fertilizer; NPK, 100 % mineral fertilizer; NPK + pig manure, 50 % mineral N replaced with pig manure; Pig manure, 100 % mineral N replaced with pig manure.</figcaption></figure>

treatment (Fig. S4c), likely because the 100 % replacement of mineral N with pig manure not only contributed substantial amounts of available N and P, but also provided a high concentration of easily degradable carbon substrates (Table S2). These inputs alleviated carbon limitations under high nutrient conditions and exerted selective pressure on bacterial communities, favoring the proliferation of *r*-strategy bacteria while suppressing *K*-strategy bacteria (Fontaine et al., 2003; Zhang et al., 2024). This was further supported by the highest relative abundance of *r*-strategy bacteria and their significant positive correlation with bacterial necromass carbon, whereas *K*-strategy bacteria exhibited the lowest relative abundance and a significant negative correlation (Fig. 3 and 4), supporting our first hypothesis. By contrast, the NPK + pig manure treatment exhibited the highest GluN/MurN ratio among all fertilization regimes (hypothesis 2, Fig. S4c), indicating microbial necromass carbon was stored primarily as fungal necromass carbon. This could be attributed to: (i) nutrient limitation caused by high carbon input from pig manure, which intensified microbial competition and favored *K*-strategy fungi with higher nutrient acquisition efficiency (Cheng and Kuzyakov, 2005; Dijkstra et al., 2013). This was supported by the highest relative abundance of *K*-strategy fungi observed in this

treatment (Fig. 3f), along with their positive correlations with the DOC/AN ratio and fungal necromass carbon (Fig. 4 and S10); and (ii) reduced soil pH (Table S2), which favored fungal growth (Grosso et al., 2016), as indicated by the negative correlation between pH and the relative abundance of *K*-strategy fungi (Fig. S10). The NPK + pig manure treatment also exhibited the highest microbial necromass accumulation coefficient (Fig. S5), highlighting its stronger potential for necromass carbon accumulation compared to other treatments. This finding was consistent with previous studies that fungal-dominated microbial communities possess greater carbon storage potential (Six et al., 2006).

The lignin content increased with both Ln(cellulose-modifying enzymes)/Ln(lignin-modifying enzymes) ratio and bacterial PLFA/fungal PLFA ratio, but, conversely, (Ad/Al)v ratio showed opposite trends with these ratios (Fig. 5), suggesting that the degradation and preservation of lignin were affected by the composition and activity of microbial community (Li et al., 2023), consistent with our hypothesis. The highest lignin content in POM and the lowest (Ad/Al)v in MAOM were recorded in the Pig manure treatment (Fig. 1c and S2d). One explanation is that pig manure input introduced a substantial amount of C, and microorganisms preferentially degraded more easily available carbon sources,
thus reducing lignin degradation, leading it to accumulate in the soil (Bahri et al., 2008; Dijkstra et al., 2013). This was supported by the significant negative relationship between the activity of lignin-modifying enzymes and the soil available nutrient concentrations (Fig. S10). Additionally, pig manure not only contained considerable amounts of lignin itself (Li et al., 2020), but also significantly increased the rice litter, such as shoot and root (Table 1), which were rich in chemically recalcitrant substance like lignin (Villarino et al., 2021). The NPK + pig manure treatment exhibited the lowest lignin content in POM and the highest (Ad/Al)v in MAOM (Fig. 1c and S2d), because the relative abundance of K-strategy fungi was significantly higher than in other treatments (Fig. 3f), which play a key role in the decomposition of recalcitrant organics such as lignin (Deng and Liang, 2022).

### 4.3. Implications for using fertilizer management to enhance C storage and persistence

We conducted a comprehensive assessment of the effect of long-term fertilization on SOC storage and persistence in response to changes in microbial community structure and activity under field conditions. Even though current research often emphasizes MAOC, it should be borne in mind POC (as major SOC reservoir) (Cotrufo et al., 2019) is equally important for effective carbon management (Angst et al., 2023). The POC also acts as a precursor for MAOC that features organo-mineral association (Witzgall et al., 2021) and contributes to soil structure through fungal hyphae and microbial extracellular polymeric substances on its surface (Tian et al., 2021). Therefore, effective carbon management must consider both POC and MAOC dynamics. When assessing the impact of fertilization on SOC, it is essential to also consider the net primary production of crops. Crops act as carbon sinks by assimilating atmospheric CO₂ through photosynthesis and incorporating it into their biomass, which is a major contributor to SOC accumulation (Prince et al., 2001). Therefore, by optimizing fertilization to promoted crop growth, more effective carbon management strategies can be developed to enhance SOC sequestration. Microbial life strategies significantly influence the dynamics of SOC (Yang et al., 2024). For example, an increase in the relative abundance of K-strategy fungi enhances the utilization of recalcitrant carbon, promotes nutrient release, and stimulates microbial necromass carbon formation, which contribute to improving the stability of SOC. Thus, regulating soil microbial life strategies by mediating the DOC: available nutrient ratio is an important measure for the continuous and stable increase of SOC. Although the r-/K-strategy framework based on environmental resource availability provided a valuable theoretical basis for understanding how microbial life-history strategies regulate SOC turnover, it cannot fully explain microbial adaptation strategies under extreme environmental stress, which affect allocation within microbes. Therefore, future research may benefit from incorporating additional dimensions, such as the Y-A-S framework, which classifies microbial life-history strategies into high yield (Y), resource acquisition (A), and stress tolerance (S) strategies (Malik et al., 2020). The tradeoff between these strategies may also be related to genome size (Saifuddin et al., 2019). In addition, other conceptual frameworks such as the copiotroph-oligotroph classification, which has been implemented in some biogeochemical soil models, may also provide complementary perspectives for describing microbial responses to nutrient availability. These factors will help enhance our understanding of microbial life-history strategies and improve our comprehension of the microbial mechanisms driving soil organic carbon turnover. Nutrient balance is fundamentally important for crop growth, but in fertilization strategies whereby organic fertilizers are used to substitute a part or the whole amount of mineral fertilizer N to be applied, the excessive application of P is a prevalent concern (the rates of organic fertilizers needed to supply the optimal N amounts may result in over-application of P), which leads to nutrient imbalance. Phosphorus over-application not only hinders the biological potential of plant roots, leading to decreased root-derived C input and P-use efficiency, but also

exacerbates environmental risks such as water eutrophication. Moreover, soil P dynamics are increasingly recognized as an important but often overlooked factor in regulating the formation of mineral-associated organic carbon (MAOC). P enrichment not only mediates organo-mineral associations but also influences microbial life-history strategies, thereby affecting microbial necromass and by-product contributions to MAOC (Lieberman et al., 2025; Luo et al., 2025). So, P-based substitution strategies may better balance yield goals and carbon sequestration. Importantly, integrating microbial community structure and carbon dynamics into fertilization practices is essential for enhancing SOC storage (and even sequestration) and advancing sustainable agriculture, contributing to global carbon management goals to support the “4 per 1000” initiative (Rumpel et al., 2018).

### 5. Conclusions

Long-term pig manure substitution regulated the accumulation of plant- and microbe-derived carbon in soil fractions by influencing microbial community composition and activity and rice growth, ultimately affecting SOC storage. Pig manure application proved significantly more effective than inorganic fertilizers in enhancing SOC content and persistence, primarily due to the increased accumulation of microbially derived MAOC. Notably, pig manure applications provide 100 % of the crop N needs promoted rice growth, significantly increased the total organic carbon input, resulting in the largest increase in SOC content. However, 50 % N substitution with pig manure promoted the degradation of lignin in POM, notably improving SOC persistence. These findings provided vital insights into the microbial mechanisms driving the transformation of microbe- and plant-derived carbon during SOC accumulation, offering a scientific basis for refining fertilization strategies to enhance SOC storage and persistence in paddy soils.

#### CRediT authorship contribution statement

Zhengwu Wu: Writing - review & editing, Writing - original draft, Visualization, Investigation, Formal analysis, Data curation, Conceptualization. Yanyan Zhang: Writing - review & editing. Yuchuan Fan: Writing - review & editing. Changquan Wang: Supervision. Bing Li: Supervision, Resources. Zed Rengel: Writing - review & editing. Jianbo Shen: Writing - review & editing. Edith le Cadre: Joann Whalen: Writing - review & editing, Visualization. Xiaoyan Tang: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

#### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

#### Acknowledgments

This work was supported by the National Key R&D Program of China (2022YFD1901400); the Sichuan Science and Technology Program (2024YFHZ0226, 2025YFHZ0256); the National Natural Science Foundation of China (31972505), the Chengdu Science and Technology Program (2024-YF05-01653-SN) and the International Science and Technology Cooperation Base for Efficient Utilization of Nutrient Resources and Fertilizer Innovation.

#### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.geoderma.2025.117581.
# Data availability

Data will be made available on request.

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