Biochar and Straw Amendments over a Decade Divergently Alter Soil Organic Carbon Accumulation Pathways

Abstract

Exogenous organic carbon (C) inputs and their subsequent microbial and mineral transformation affect the accumulation process of soil organic C (SOC) pool. Nevertheless, knowledge gaps exist on how different long-term forms of crop straw incorporation (direct straw return or pyrolyzed to biochar) modifies SOC composition and stabilization. This study investigated, in a 13-year long-term field experiment, the functional fractions and composition of SOC and the protection of organic C by iron (Fe) oxide minerals in soils amended with straw or biochar. Under the equal C input, SOC accumulation was enhanced with both direct straw return (by 43%) and biochar incorporation (by 85%) compared to non-amended conventional fertilization, but by different pathways. Biochar had greater efficiency in increasing SOC through stable exogenous C inputs and inhibition of soil respiration. Moreover, biochar-amended soils contained 5.0-fold greater SOCs in particulate organic matter (POM) and 1.2-fold more in mineral-associated organic matter (MAOM) relative to conventionally fertilized soils. Comparatively, although the magnitude of the effect was smaller, straw-derived OC was preserved preferentially the most in the MAOM. Straw incorporation increased the soil nutrient content and stimulated the microbial activity, resulting in greater increases in microbial necromass C accumulation in POM and MAOM (by 117% and 43%, respectively) compared to biochar (by 72% and 18%). Moreover, straw incorporation promoted poorly crystalline (Feo) and organically complexed (Fep) Fe oxides accumulation, and both were significantly and positively correlated with MAOM and SOC. The results address the decadal-scale effects of biochar and straw application on the formation of the stable organic C pool in soil, and understanding the causal mechanisms can allow field practices to maximize SOC content. These results are of great implications for better predicting and accurately controlling the response of SOC pools in agroecosystems to future changes and disturbances and for maintaining regional C balance.

1. Introduction

Soil organic carbon (SOC) is well known as the core driver of global carbon (C) cycles and plays a pivotal role in climate regulation, maintaining soil health, and long-term ecosystem productivity [1,2]. Increasing SOC sequestration supports the food productivity and stability of agroecosystems, especially for low- and medium-yield fields that are in urgent demand for fertility enhancement [3]. Application of organic material is an effective way to maintain and enhance SOC storage by directly increasing the external C input to soils [4]. Simultaneously, SOC accumulation is highly dependent on C formation, stabilization, and preservation pathways in the soil. Theoretically greater quantities of organic material input are expected to be more favorable for maintaining or increasing SOC storage, while the efficiency of SOC sequestration is determined by the chemical properties of the organic material [5]. Therefore, for sustained advancement of SOC storage, it is necessary to gain a deeper understanding of the impact of different quality C management strategies on stabilizing SOC pool formation and stabilization pathways.

Biochar, a highly aromatic, C-rich material derived from plant residues and organic wastes, has proved to be a promising option for increasing SOC sequestration and C neutrality in soils [6,7]. The observed changes in biochar-induced SOC accrual have been associated with (i) direct introduction of stable, biochar-derived C into soils with enhanced negative priming effects [8]; (ii) direct or indirect alterations in microbial community structure and C metabolism through changes in physicochemical properties [9]; and (iii) enhancement of organo-mineral and organo-organic interactions to form more resistant OC [10]. By comparison, crop straw supply high levels of unstable compounds and nutrients that are more readily utilized by microbes and enhance microbial C metabolism [11]. According to the “microbial C pump” concept, straw amendment can contribute to SOC build-up by boosting the formation of microbial residues (metabolites and necromass) [12,13]. However, SOC accrual from straw may also be offset by accelerated mineralization of organic biomass and native soil organic matter via positive priming effect, thus reducing the efficiency of SOC sequestration [14]. This implies that the application of straw and biochar can affect the SOC sequestration efficiency by altering the formation and stabilization of SOC through different allocation pathways and decomposition. Increasing attention has been paid to the effects of these two amendments on SOC accumulation [15] and has greatly broadened the understanding regarding the role of organic amendments on SOC sequestration. However, there is still insufficient understanding to compare the distinct formation and stabilization pathways of SOC pools by straw and biochar amendments, especially on long time scales.

In recent years, a conceptual division of SOC into components of different properties, functions and residence times has been introduced in recent years to better recognize SOC dynamics and deepen the understanding of its persistence [16]. Furthermore, these two concepts of operationally definable fractions are gaining acceptance: particulate organic C (POC, persisting in soils by aggregates physical protection and inherent biochemical properties) and mineral-associated organic C (MAOC, binding with minerals and stabilizing as chemically protected C) [16]. POC is formed primarily of fragmentation and translocation of relatively undecomposed light organic matter and exhibit low stability [17]. MAOC, by contrast, is formed by low-molecular-weight compounds (e.g., microbial necromass) complexed to the surface of soil minerals or strongly chemisorbed, with longer turnover times [18]. Considering the fundamental differences in the formation, persistence, and functioning of these pools, it can therefore be assumed that POC and MAOC may respond divergently to different qualities of organic material amendments and potentially have different impacts on the SOC pool and its functioning and thus on the long-term SOC storage. Furthermore, given the prominent role of minerals, particularly of iron (Fe) phases, in stabilizing SOC [19], studies pairing soil organic matter (SOM) fractionation and Fe speciation can be particularly helpful to enhance our understanding on these effects.

Red soil (Ferric Acrisols) is an important soil resource for agricultural production in southern China [20,21]. The fine texture and highly reactive Fe (hydr) oxides coupled with low levels of organic matter suggest that biochar and straw amendments have a great capacity to sequester and stabilize C in this soil. Thus, based on a 13-year continuous field trial of equal C applications of straw and biochar, the present study aimed to investigate the effects of long-term organic amendments of varying quality on the formation and stabilization of SOC pools and the underlying mechanisms. This study specifically focused on (1) the responses of SOC accumulation and changes in SOC fractions (POC, MAOC) to long-term straw and biochar amendments and (2) how these changes would be interpreted as functional changes in the SOC pool through soil respiration, microbial necromass accumulation, and Fe oxide fractions. Exploring their distribution has been proposed to better predict SOC dynamics with agricultural management practices and to deepen our understanding of SOC persistence. We hypothesized that both long-term straw and biochar amendments would increase SOC accumulation and POC and MAOC content in soil, but the quality of C inputs led to inconsistent formation and stabilization mechanisms. Specifically, biochar amendment preferentially increases POC due to its poorly decomposable characteristics. Additionally, given the strong microbial involvement in the process of MAOC formation, straw amendments can be predicted to be more likely to increase MAOC due to their greater ability to influence microbial activity and quantity of products. This long-running field trial provides a chance to further our knowledge of the mechanisms of SOC accumulation and stabilization with different organic amendments in typical low-fertility soils.

2. Materials and Methods

2.1. Experimental Design and Soil Sampling

Soil samples were collected after maize harvest at the end of July 2023 at the Yingtan National Agro-Ecosystem Observation and Research Station, operated by the Chinese Academy of Sciences, Jiangxi Province (116°55′ E, 28°15′ N). The sampling area is characterized by a warm subtropical climate with annual mean temperature of 17.6 °C and annual mean precipitation of 1795 mm. The soil is a Ferric Acrisols following the World Reference Base for Soil Resources [22] and an acidic loamy clay. The field trial was set up in 2010 in a randomized block design of 20 m × 5 m per plot with three replications. Four treatments were selected for this study: (1) unfertilized control (CK); (2) conventional chemical fertilizers (CF, nitrogen, phosphorus, and potassium fertilizers applied at 150 kg N ha⁻¹, 75 kg P₂O₅ ha⁻¹ and 60 kg K₂O ha⁻¹, respectively); (3) conventional chemical fertilizers plus maize straw (FS); (4) conventional chemical fertilizers plus straw biochar (FB). Maize straw and straw biochar were applied as equivalent organic C inputs at 1 Mg ha⁻¹ yr⁻¹. The total N amount applied to the CK, CF, FS, and FB treatments were 0, 150, 166, and 185.57 kg ha⁻¹, respectively. Maize straw was applied after being naturally air-dried and chopped into lengths of less than 5 cm. Biochar was produced by pyrolyzing maize straw as raw material at 450 °C for 48 h. The C and N contents of straw and biochar were listed in Table S1. Before seeding the crop, the straw or biochar were spread on the topsoil with chemical fertilizer and plowed to mix thoroughly. Maize was planted in April and harvested in July under the monoculture system.

Five intact soil cores (0–20 cm) were randomly selected within each plot and subsequently combined and mixed to form a composite sample. After removing stubble, debris, and stones, the samples were sieved through a 6 mm sieve, then homogenized and divided into three sub-samples. One sub-sample was stored in a refrigerator at 4 °C for incubation, and soil microbial biomass, available substrates analysis, and one sub-sample were stored at −20 °C for microbial community analysis. The other sub-sample was air-dried and used for physical and chemical analysis.

2.2. Soil Physicochemical Analyses

The SOC content was measured by the potassium dichromate oxidation method using concentrated K₂Cr₂O₇ and H₂SO₄ at 175–180 °C [23]. SOC storage (Mg C ha⁻¹) in 0−20 cm was estimated using SOC content (%) multiplied by soil depth (cm) and soil bulk density (g cm⁻³) (Table S1). Dissolved organic C (DOC) was extracted using boiled C dioxide-free water and measured using a TOC analyzer (Multi NC 3100, Analytic Jena, Jena, Germany). Microbial biomass C (MBC) was determined using a chloroform fumigation incubation method [24,25]. Soil pH was determined using a pH meter (PHS−3C, Shanghai, China) with a soil–water ratio of 1:5. Free iron oxide (Fed) is the Fe excluded from the layered silicate component of the soil and was extracted with dithionte–citrate–bicarbonate [26]. Soil amorphous Fe oxides (Feo) were extracted with ammonium oxalate [27]. Organically complexed Fe oxides (Fep) can form complexes with soil humus and were extracted by sodium–pyrophosphate solutions [28,29]. Crystalline Fe oxide is defined as the difference between Fed and Feo.

2.3. Physical Fractionation of Soil Organic Matter

SOM was separated into POM and MAOM following a modified procedure from Marriott and Wander [30]. Air-dried soils were placed into 200 mL plastic bottles with 5% sodium hexametaphosphate (soil:solution = 1:10) and then shaken at 180 rpm for 18 h to ensure adequate mixing. The dispersed soil was then rinsed through 53 μm sieve. The fractions retained on the sieve were collected as POM, and the fractions that passed through the sieve were collected as MAOM. Each fraction was dried in an oven at 50 °C to a constant mass and then analyzed for C concentration using the potassium dichromate oxidation method. The content of particulate organic C (POC, g kg⁻¹) and mineral-associated organic C (MAOC, g kg⁻¹) in soil were calculated C content in the POM or MAOM (g kg⁻¹) multiplied the weight of the POM or MAOM fraction (g), respectively.

2.4. Soil Incubation for Potential C Mineralization

Soil incubation experiment for the determination of potential C mineralization was performed according to Tang, et al. [31]. Briefly, 20 g dry weight equivalents of fresh soil were incubated in 250 mL flasks sealed with impermeable silicone rubber stoppers at 25 °C and 60% water-holding capacity in the dark. Soil moisture was maintained constant during incubation by weighing and regulating water every two days. Gas samples were obtained periodically from the headspace of the flask on days 0, 7, 15, 22, and 30 with a 20 mL syringe. Prior to gas sampling, the headspace air in the flask was mixed three times with a syringe. The rubber stopper was removed after gas sampling for 30 min to refresh the air in each flask, and the bottle was then resealed for 12 h. CO₂ concentrations were determined with a gas chromatograph (Agilent 7890, Santa Clara, CA, USA). Cumulative emissions were estimated according to the increasing concentration of headspace gas during the incubation period.

2.5. Measurement of Amino Sugar for Soil Microbial Residue C (MRC)

Amino sugars, used as biomarkers for microbial necromass, were hydrolyzed, purified, and derivatized by gas chromatography according to the method of Zhang and Amelung [32]. Briefly, soil samples containing approximately 0.4 mg of nitrogen were hydrolyzed in 6 M HCl at 105 °C for 8 h. After cooling, 100 μg of myoinositol was added as an agency standard. After filtration, the sample was evaporated to dryness under reduced pressure at 40 °C, redissolved, and pH-adjusted to 6.6–6.8. The supernatant was centrifuged at 3000× g and freeze-dried. Subsequently, methyl-glucosamine was added as a quantitative standard, which converted amino sugars to aldononitrile derivatives by heating the derivatization reagent in a pyridine and methanol mixture (4:1; v/v) containing hydroxylamine hydrochloride and 4-(dimethylamino) pyridine at 75–80 °C for 30 min. It was further acetylated with acetic anhydride, cooled, and mixed with dichloromethane. The methylene chloride phase containing amino sugar derivatives was dried under nitrogen after removal of excess derivatization reagent and prior to quantification.

The muramic acid (MurA) glucosamine (GluN) and galactosamine (GalN) were combined to determine the total amino sugars. Total microbial residual C (MRC) was determined by summing the fungal (FRC) and bacterial (BRC) residual C. According to the following equation, the estimation of FRC was derived by subtracting bacterial GluN from total GluN, with the assumption that MurN and GluN are present in bacterial cells in a 1:2 M ratio.

FRC = (GluN/179.2 − 2 × MurN/251.2) × 179.2 × 9

(1)

BRC = MurN × 45

(2)

where 179.2 and 251.2 (in g mol⁻¹) are the molecular weights of GluN and MurN, respectively.

2.6. Statistical Analyses

Data analyses were conducted by using R software (version 4.3.1) (R Core Team, 2014). The significant differences between fertilization treatments on all variables was tested using a one-way analysis of variance (ANOVA), followed by Tukey’s HSD test to determine the statistical significance at p < 0.05. The data were pre-checked for normality and homoscedasticity. The relationship between SOC content, physical C fractions (POC and MAOC contents), microbial necromass (MRC contents), and the soil response variables (i.e., soil physicochemical properties, MBC, soil respiration, and Fe oxides) was tested using regression analyses. A principal component analysis (PCA) was performed to evaluate the changes in SOC profiling between treatments and fractions.

3. Results

3.1. Soil Organic C Pools and Soil Properties

Both maize straw and biochar amendments significantly increased SOC content and storage compared with the CK and CF treatments (Figure 1a and Figure S1). Specifically, compared with the CF treatment, the FB treatment had the greatest increase in SOC content (by 85%), followed by the FS treatment (by 43%), although both had the equal annual exogenous organic C input. At 13 years after the setup of the experiment, the SOC storage in the topsoil (0–20 cm) was 7.18 ± 0.58 g kg⁻¹ for the CK treatment and 10.20 ± 0.31 g kg⁻¹ for CF treatment, whereas straw and biochar amendment stored 14.37 ± 0.53 and 17.14 ± 0.47 g kg⁻¹, respectively (Figure S1a). Compared to CF, the application of straw and biochar significantly increased SOC storage by 41% and 68%, respectively. Moreover, the SOC storage of the biochar treatment was significantly 19% higher than that of straw treatment.

There were also significant differences in other properties in the soil samples among the treatments (Figure 1). Compared with CF, straw and biochar amendment increased TN content and C/N, with the biochar amendment exhibiting the highest C/N (Figure 1b,d). In comparison, straw amendment had significantly higher DOC and MBC levels (Figure 1d,e). The cumulative CO₂ emission ranged from 2.88 ± 0.07 to 21.25 ± 0.56 mg CO₂ kg⁻¹ soil over 30 days of incubation (Figure 1f). Compared with CF, both straw and biochar amendment significantly promoted CO₂ emission, with a greater increase in total CO₂ emission in FS treatment (by 248%) than in FB treatment (by 33%), which was consistent with the changes in ratio between DOC and MBC content (Figure S1). Soil pH ranged from 4.4 to 4.8 across treatments with CF significantly reduced pH compared to CK, but none of the amendments had a major effect on soil pH compared to CF (Figure S1).

3.2. SOC Distribution in SOM Fractions

The MAOM fraction dominated the particle size distribution (>75% of the total recovered mass), with no significant differences in POM or MAOM mass among treatments (Figure 2a). Straw and biochar amendment altered the distribution of SOC stored in the POM and MAOM fractions (Figure 2b). There was a significant increase in POC and MAOC contents with the application of both straw and biochar compared with the CK and CF treatments. Specifically, compared to the CF soil, the FS and FB treatments significantly increased POC content by 65% and 415%, respectively, while significantly increased the MAOC content by 29 and 17%, respectively. FB was more efficient than FS in promoting POC (by 231%), and FS treatment tended to accumulate more MAOC (by 10%). The linear regression analyses indicated that the content of POC and MAOC increased linearly (R² = 0.89 and R² = 0.81, respectively; Figure 2c,d).

3.3. Amino Sugars and Microbial Necromass in the POM and MAOM Fractions

The fertilizer treatments had significant effects on soil amino sugars in different SOM fractions (Figure 3). The concentration of total amino sugars, including MurA, GalN, and GluN, was higher in the MAOM fraction (184.7–254.2 mg kg⁻¹) than in the POM fraction (56.6–238.6 mg kg⁻¹). Further, as compared to the levels in the CK and CF treatments, the total amino sugars contents in both POM and MAOM fractions were significantly increased under straw and biochar amendment, with the magnitude of changes being greater in the POM than in the MAOM (Figure 3a). Specifically, the straw and biochar amendments (cf. CF) significantly increased the total amino sugar concentration in the POM fraction by 106% and 63%, respectively. Whereas in the MAOM fraction, straw and biochar amendments increased by 35% and 25%, respectively, with biochar significantly lower than the straw amendment. In addition, CF increased the total amino acid sugar concentration in the POM fraction by 105% compared to CK, while there was no significant difference within the MAOAM fraction. Ratio of GluN–MurA was significantly highest in the straw treatment among all treatments in the POM fraction. However, in the MAOM fraction, there were no significant differences among treatments (Figure S2).

As expected, the amount of microbial residue C (MRC) was more abundant in the MAOM fraction than in the POM fraction across the treatments, with the levels ranging from 1.1 to 4.2 in the ratio of MAOM–POM (Figure 3e). Both straw and biochar amendments increased MRC in the MAOM fraction compared to CF, with higher proportions of increase in fungal MRC (47% and 33%, respectively) than in bacterial MRC (36% and 30%, respectively). By comparison, the increase in MRC content in the POM fraction was greater in the straw and biochar treatments compared to the CF treatment, with significant increases of 118% and 72%, respectively. In terms of fungal MRC, the straw and biochar amendments increased by 140% and 79%, respectively, which was higher than the proportion of bacterial MRC, which increased by 73% and 56%, respectively (Figure 3f,g). The contribution of microbially derived C to SOC in the POM fraction was the largest (34% of SOC) with straw amendment (Figure 3h), whereas the greatest contribution of microbially derived C to SOC in the MAOM fraction was observed in the CK treatment across all treatments. The ratio of fungal–bacterial MRC was generally higher in POM than MAOM fraction across the treatments and was significantly increased with straw amendment in the POM fraction rather than in the MAOM fraction (Figure S2).

3.4. Relationships between Fe Oxides, Necromass, and SOC Fractions

The concentration of different forms of Fe oxides in the soil was influenced by long-term different exogenous organic C input (Figure 4). Feo content ranged from 0.98 to 1.65 g kg⁻¹ and was highest in the CF treatment, while Fed ranged from 24.43 to 31.61 g kg⁻¹, with the highest in the FS treatment, followed by FB (Figure 4). Similarly, crystalline Fe and Fep content was significantly increased with straw and biochar amendments compared to CF and CK treatments, with straw amendment significantly higher than that of biochar (Figure 4). In addition, Fed, crystalline Fe, and Fep contents were positively correlated with SOC contents (Figure 4f–h), suggesting that straw and biochar application could efficiently activate the function of Fe oxides and thus preserved the SOC. Furthermore, the correlation analyses show that MAOC were highly positively correlated with total amino sugars, MRC, Fed, and Fep contents, but in contrast, such relationships were not observed for POC (Figure 5).

Using the OC content and composition of the SOM fractions together with related soil indicators analyzed above, changes in the SOM status of the POM and MAOM fractions with different fertilizer applications were assessed using principal component analysis (Figure 6). The result showed that the first two principal components explained 67.47% and 23.83% of the overall variance, respectively. These parameters clearly differentiated the different treatments, where PC1 separated the CK and CF samples from the organically amended samples and PC2 separated the two different types of amendments. Based on the sample locations of the arrows, it was clearly evident that soil respiration, Fe oxides, and MRC content in the MAOM and POC fraction were the key factors in differentiating the straw amendment. In contrast, the biochar amendment was shaped by the SOC and POC content and the ratio of C/N and POC/MAOC.

4. Discussion

4.1. Long-Term Straw and Biochar Management Raises Soil Carbon Storage

Thirteen years of continuous straw and biochar amendment significantly increased SOC content and storage in the topsoil, representing an important C sequestration. This finding is consistent with previous studies showing that long-term straw and biochar incorporation increased SOC levels [33,34]. The SOC stock is governed by exogenous C inputs such as organic amendments, plant primary productivity, or rhizodeposits, as well as by the mineralization of these inputs. Enhancing the biomass of aboveground plants and root systems could introduce additional OC into soils, thus becoming an optimum for potential soil C sequestration [35]. Significant increased plant productivity (straw biomass and grain yield) with both straw and biochar amendments were recorded in a previous study at the same experimental site, with CF also significantly higher than CK [36]. This suggests that the increases in SOC were partly driven by increased plant productivity, which enhanced belowground C inputs [37]. Furthermore, the present study found that biochar sequestered C more efficiently than straw at equal exogenous C inputs. Straw and biochar treatments were expected to yield the same amount of soil C stocks (13 g kg⁻¹ soil), which was 47.5% higher and 134.8% lower than the actual measured gains in soil C stocks (6.1 and 5.8 g kg⁻¹ soil), respectively (Figure 1, Table S1). This suggests that although both organic amendments increased SOC storage, they have different accumulation mechanisms, resulting in different increments of organic C content.

The relatively lesser increase in SOC storage with straw amendment compared to biochar may be due to the fact that straw-derived C is more readily utilized by microbial decomposition, resulting in greater SOC loss from mineralization [33]. The 30-day soil respiration experiment in this study provided preliminary evidence that the straw treatment promoted SOC mineralization (Figure 1). MBC also increased substantially with straw amendment compared to the non-amended control, which was explained by the increase in available C for microbial growth as indicated by the high DOC content in the straw treatment. On the contrary, most of biochar-derived C is able to in the soil for decades to hundreds of years due to its aromatic structure and chemical stability that prevents it from being decomposed [38]. Long-term biochar amendment reduced soil C availability to microbes, leading to decreased accumulation of SOC mineralization, which suggests a reduction in native SOC decomposition, known as negative priming effect [39,40]. This was confirmed by earlier observations that straw incorporation significantly promoted native SOC mineralization with a positive priming effect, whereas biochar reduced SOC mineralization with a negative priming effect [41].

4.2. Straw and Biochar Differently Affect Soil Organic Carbon Pool Composition and Functional Groups

Beyond affecting the total SOC content, straw and biochar altered the distribution of SOC and MRC in soil fractions to varying degrees. This study found that 13 years of straw and biochar amendments both significantly improved the SOC stored in the POM and MAOM, although the yield (proportional distribution) of these fractions was not affected (Figure 2). Furthermore, the ratio of POC–MAOC ranged from 0.20 in the CK soil to 1.18 in the biochar-amended soils, with POM-related C significantly greater in biochar-amended soils than in straw-amended soils. This supported our first hypothesis and the findings of Giannetta, et al. [42] who also found that biochar amendments significantly increased C occurred in the particulate fraction. It could be reasonably explained by the fact that POC is a sensitive C pool that can respond rapidly to exogenous C additions, whereas MAOC is considered to be a resistant C pool and typically has a larger pool sizes and slower turnover [43]. The increased C content in the POM from biochar amendment could be partly due to the coarser organic particles are inherently contained in the biochar used. Thus, biochar particles were distributed to these soil fractions with relatively large or similar size [42]. In addition, reactive functional groups on the surface of biochar, such as COOH, C–OH, C–O, and CN, can act as persistent bridges for interactions with mineral surfaces, facilitating the binding of coarse organic fragments with mineral particles [44], thus promoting the formation of C in the POM. According to the amendments, the increases in POC under straw amendments also occurred. This is due to the direct formation of POM from partially decomposed or undecomposed residue debris through physical migration [45]. As fresh crop residue enters the POM fraction, the unstable components break down rapidly, promoting microbial turnover [46,47]. This was evidenced by the higher (2.2 times) concentration of total amino sugars in the POM fraction under straw treatment relative to CF in this study (Figure 3).

Interestingly, the ratio of POC to MAOC under straw amendment was less than 1, and C storage was higher in the MAOM compared to the biochar amendments (3.9 vs. 3.5 g kg⁻¹ bulk soil), indicating that straw-derived C inputs resulted in higher increases in MAOC than POC. This challenges some common perceptions that inputs of high C/N materials, like straw, should contribute more effectively to POC [17,48]. A reasonable explanation for this phenomenon is that MAOC is not yet saturated under the low soil fertility in this study area. Generally, under limited SOC or energy, microbes maintain high metabolism and turnover time with high activities to absorb exogenous organic substrates [49,50]. According to previous studies [51,52], microbes synthesize biomass through assimilation, followed by continuous generation and accumulation of microbial metabolites and dead necromass in the soil. This iterative processes promoted high formation efficiency of MAOC formation via in vivo turnover pathway under exogenous C input [53]. Consistent with our second hypothesis, the soils under the straw treatment exhibited higher MRC concentrations in MAOM compared to the other treatments. Corroborated by the fact that MRC was strongly and positively correlated with MAOC, suggesting that microbial-derived products are essential for MAOC accumulation. These indicate that straw amendment may act as a soil microbial community “pump” by promoting microbial turnover, which facilitates the formation of a range of organic matter including microbial residues. Furthermore, the increased microbial-derived low-molecular OM and root exudation could potentially boost MAOC formation with clay minerals through direct adsorption and ex vivo modification pathways [54].

In addition, the proportion of fungal MRC in the total variation of SOC was greater than that of bacterial MRC (Figure 3). Changes in fungal to bacterial MRC ratios could reflect the accumulation and turnover of fungal and bacterial MRC. The fungal to bacterial MRC ratio was much higher under the straw treatment across the treatments, implying that the decomposition process were dominated by fungi, their residues were more likely to accumulate in the soil than bacterial ones. This is possibly attributed to greater utilization of plant residue C by fungi, whereas the majority of bacteria prefer microbial biomass [55,56]. Moreover, soil fungal cell walls originated from decomposition-resistant components (e.g., chitin and melanin), which directly influences the persistence of fungal MRC in the soil and protects it from decomposition [57,58]. The above together highlights that MAOC accumulation can be enhanced by manipulating the structure of the soil microbiome and increasing the surface area of minerals.

4.3. Soil Organic Carbon Stabilization Pathways under Straw and Biochar Amendments

The mechanism of SOC stabilization occurs through organic–mineral interactions, the evaluation of whether long-term agricultural practices affect the reaction sites of minerals in the soil matrix has been considered interesting [42,59]. The Fe (hydr)oxides provide physical and chemical protection against microbial decomposition through the formation of organic C–Fe complexes that enable clay minerals to stabilize SOM in soils, especially in acidic soils with high clay mineral content in Ferric Acrisols soils [60]. In this study, the concentration of total free Fe oxides (Fed) and organically bound Fe oxides (Fep) concentrations was found to be significantly increased following straw and biochar incorporation, with straw being higher than that of biochar (Figure 4). The possible explanation is that low-molecular-weight acids produced by microbial decomposition of straw, which induced minerals to transform from orderly crystalline phases to short-range order (SRO) phases, forming organic–mineral compounds to prevent recrystallization [28]. Organic fertilization has been reported to enhance the capacity of SOC preservation by promoting the formation of Fe oxides in the soil, which in turn aided binding with SOC to form soil macro-aggregates and SOC–Fe associations [61]. Interaction with Fe oxides in soil clays are considered to be one of the most important mechanisms among the reaction sites [19]. Moreover, the concentration of Fed, Fep, and crystalline Fe were found positively correlated with MRC in MAOM instead of POM (Figure 4), suggesting that OC application facilitated the binding of MAOC to Fe and ultimately increased the SOC content, in line with the results for SOC. This is consistent with previous reports that organic matter leads to the transformation of Fe oxide minerals, enhancing the capacity of their surface reaction and consequently elevating the sequestration potential of SOC inherent in the soil [62]. This further implies that Fe oxides preferential bind to microbial-derived OC, and that C storage in the MAOM fraction is mainly associated with the presence of this Fe oxide form. By emphasizing the important role of soil straw amendments in enhancing soil Fe–OC sequestration in our study, further research is needed to elucidate the factors affecting the stability of Fe–C association and hence soil C balance.

5. Conclusions

In conclusion, this study demonstrated that long-term straw and biochar incorporation favored SOC accumulation, but sequestration efficiency and the formation mechanisms of stable SOC pools varied between the two amendments (Figure 7). Straw and biochar amendments differently altered the distribution and chemical composition of functional fractions of SOC pools. Biochar sequestered C primarily by increasing the resistance of SOC to decomposition to reduce the degradability of the resource, thus reducing SOM mineralization, with the raised OC concentrated largely in the POM. Conversely, straw incorporation increased nutrient availability and promoted microbial activity, and accelerated mineralization. Nevertheless, this simultaneously enhanced MRC production, promoting OC enriched mainly in MAOM. Furthermore, the SOC stabilization under straw amendment was linked to chemical stability of Fe oxides, protected SOM from further decomposition. Altogether these provide new understanding of continued application of straw and biochar on the long-term effects of SOC accumulation and stabilization in different soil functional fractions to assist in maximizing SOC content in the field. Future efforts are proposed for explicitly linking mineral–microbial interactions to SOC stabilization with advanced characterization techniques and metabolomics, focusing on how metabolic processes under redox conditions interact with Fe–SOC and the probability of these products contributing to SOC persistence.