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Article

Montmorillonite and Composite Amino Acid Overcome the Challenges of Straw Return in Cold-Region Soil: Synergistic Mechanisms of Rapid Straw Humification and Carbon Sequestration

by
Xingyan Chen
†,
Tchoumtchoua Foka Joseline Galliane
†,
Chongyang Zhao
,
Yanhui Feng
and
Mingtang Li
*
College of Resource and Environment, Jilin Agricultural University, Changchun 130118, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(8), 1979; https://doi.org/10.3390/agronomy15081979 (registering DOI)
Submission received: 8 July 2025 / Revised: 9 August 2025 / Accepted: 15 August 2025 / Published: 17 August 2025
(This article belongs to the Section Agroecology Innovation: Achieving System Resilience)
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Abstract

This study aimed to develop an effective method to overcome the challenge of straw return in cold-region soil. We systematically investigated the synergistic mechanism of montmorillonite (MMT) and composite amino acid (CAA) on straw humification and carbon sequestration through a low-temperature litterbag field experiment. The results indicate that the combined treatment (MMT-CAA) significantly increased the decomposition rate of straw by 42.1% compared to the control (CK), with MMT showing particular efficacy in lignin degradation (28.3% reduction), while the CAA preferentially decomposed cellulose (19.7% reduction). An FTIR analysis of the decomposition products confirmed these findings. Water-soluble organic carbon (WEOC) and its three-dimensional fluorescence spectra exhibited a 25.0% increase in MMT-CAA and enhanced aromaticity of humic acid-like substances. Humic substances and their 13C-NMR revealed that MMT-CAA enhanced humic acid formation and molecular stability by 31.4% (with a 47.8% increase in aromaticity). A further redundancy analysis and symbiotic network of microorganisms demonstrated that MMT-CAA increased the abundance of lignocellulose-degrading phyla (Actinomycetes and Stramenomycetes) and the formation of a complex co-degradation network. Field corn planting trials indicated that MMT-CAA increased plant height by 55.1%, stem thickness by 58.7%, leaf area by 70.2%, and the SPAD value by 41.1%. Additionally, MMT significantly reduced CO2 and N2O emission fluxes by 35.6% and 15.8%, respectively, while MMT-CAA increased CH4 uptake fluxes by 13.4%. This study presents an innovative strategy, providing mechanistic insights and practical solutions to synergistically address the challenges of slow straw decomposition and carbon loss in cold regions.

1. Introduction

Corn planting areas in Northeast China account for over 30% of the national total, yielding an annual corn output of approximately 120 million tons, which constitutes about 40% of China’s total corn production [1]. Corn straw is produced in large quantities as a byproduct of corn, and a large amount is used for straw return to the field. However, when corn straw is returned to the field, especially in autumn, the extended low-temperature period makes it difficult to degrade substances such as lignin and other compounds in corn straw, which continue to accumulate during the decomposition process [2,3]. Although the qualitative and quantitative traits of the corn straw returned to the field are fundamental for proper decomposition, climatic factors such as low temperatures and drought can strongly affect the entire process. In nutrient-poor tropical and temperate-zone soils and in frigid-zone soils, the efficient decomposition of organic matter and carbon sequestration are essential for crop nutrient access [4]. In addition, agricultural management and temperature directly influence carbon utilization by microorganisms, with temperature often correlating with carbon loss [5]. In tropical and temperate soils, organic matter is more extensively decomposed by microorganisms, allowing crops to fully utilize its nutrients and enhancing microbial carbon utilization efficiency [6]. Conversely, in cold regions, low temperatures frequently restrict microbial activity, resulting in slower decomposition of organic matter [7]. This significantly alters the microbial community structure and the efficiency of carbon conversion during the decomposition process, ultimately affecting carbon accumulation [8]. As global warming progresses, carbon loss is increasingly observed. This leads to pests and diseases, soil laxity, nutrient loss, and competition between microorganisms and crops for nitrogen, which is detrimental to crop growth and affects grain yield [9]. Therefore, accelerating the decomposition of organic matter and carbon sequestration at low temperatures in straw returning greatly helps improve the utilization rate of straw resources and address the challenges of straw return in cold regions.
A commonly used method to promote the decomposition of corn straw is adding nitrogen fertilizers to enhance microorganisms’ activity by adjusting the ratio of carbon to nitrogen, such as with urea [10]. Another method is microbial degradation using bacterial agents, biological enzymes, bacillus subtilis, and other microorganisms [11,12]. However, although these methods accelerate the decomposition of corn stover, their effectiveness is diminished at low temperatures [13,14,15]. Furthermore, they excessively promote the mineralization of organic carbon, resulting in increased CO2 emissions and a simplified humus structure, which undermines the long-term stability of the soil carbon pool [16,17]. Consequently, previous methods have predominantly focused on corn straw decomposition without adequately addressing concurrent crop growth and carbon sequestration. Furthermore, there is a deficiency of effective solutions to investigate the carbon conversion mechanisms and microbial regulation within the corn straw return system characterized in cold regions.
Montmorillonite is a 2:1 clay mineral composed of hydro-aluminosilicates that catalyzes the conversion of lignin to humus [18,19] and has the ability to inhibit organic carbon mineralization [20,21,22]. The microporous structure and large specific surface area of montmorillonite allow it to adsorb greenhouse gases such as carbon dioxide [23]. However, most studies on montmorillonite were conducted indoors, and its effect on organic carbon, water-soluble organic carbon (WEOC), and humus carbon, especially on the structure of humic acid (HA, which can affect carbon stabilization), needs further investigation. The effect of montmorillonite on corn straw decomposition in the actual field, on crop growth, and on microorganisms and their utilization of organic carbon also needs to be further studied and verified.
Microbes are also prone to competing with crops for nitrogen in straw return. Amino acids are substances that can provide nitrogen and be directly utilized by microorganisms and crops, and they have a variety of functions and roles [24,25]. For example, glutamic acid can assist microorganisms in secreting substances such as enzymes to promote the decomposition of corn straw, and lysine can improve the ability of microorganisms to resist low temperatures [26]. However, high-purity amino acids are costly and need new preparation methods. Therefore, in this study, low-cost composite amino acids were obtained by cooking livestock and poultry pancreas waste at high temperatures. However, it is not clear how composite amino acids affect the decomposition and carbon conversion of corn straw at low temperatures. Whether a stable corn straw degradation system will be formed when composite amino acids and montmorillonite are combined, and how they affect carbon, is also not known. Therefore, this study was carried out to investigate the single and synergistic effects of montmorillonite and composite amino acid under low-temperature conditions. This includes their effects on corn straw decomposition and humification, carbon sequestration and emission, microbial communities and co-degradation systems, and corn seedling growth. We hypothesized that montmorillonite and composite amino acids can not only promote the transformation of carbon in corn straw into structurally more stable humus and enhance carbon sequestration, but also reduce greenhouse gas emissions and promote seedling growth. We also reveal its possible underlying mechanisms.

2. Materials and Methods

2.1. Experimental Site

The present study was carried out in a corn-cultivated farmland in Changchun City, Jilin Province (125°21′ E, 43°52′ N). The terrain of the experimental area is flat, with an average winter temperature of −12 °C and an average annual temperature of 4.6 °C. Black soil was used for the test, and its basic physicochemical properties are shown in Table 1.

2.2. Experimental Materials

The corn straw used in this study was obtained from the experimental site in autumn of 2023, with the basic properties of 402 g/kg organic carbon, 5.46 g/kg total nitrogen, 0.43 g/kg total phosphorus, and 31.16 g/kg total potassium. The montmorillonite used in this study was a layered mineral containing hydrous aluminosilicates and was purchased from the Shanghai Sinopharm Chemical Reagent Group Co. Ltd., Shanghai, China. CAS No. 1318-93-0. Composite amino acids were obtained from livestock and poultry offal waste through high-temperature and high-pressure cooking. They contain 2.0% carbon and are rich in amino acid types, and their contents are shown in Table 2 [27].

2.3. Litterbag Experiments

Corn straws were chopped to a length of about 2 cm, and 10 g of montmorillonite (10% of the weight of corn straw) and a composite amino acid (2% of the weight of corn straw) were added with an adjustable carbon-to-nitrogen ratio of 40:1. After mixing with 100 g of corn straw, they were packed into nylon mesh litterbags (20 cm × 30 cm) and buried in field soil at a depth of 30 cm on 31 October 2022. Four treatments were set up: control (CK), 10% montmorillonite (MMT), 2% composite amino acid (CAA), and 10% montmorillonite + 2% composite amino acid (MMT-CAA). Each treatment involved 10 buried litterbags. On 9 May 2023, 5 litterbags were removed, and the remaining litterbags were used for the subsequent corn planting experiments. During the experiment, the lowest temperature was −28 °C. After removing the litterbags, the surface soil and other debris were cleaned, and the plant roots and stems were extracted. An amount of 3 g of decomposition products was weighed from each litterbag and placed in sterilized sealed bags, which were frozen and stored at −80 °C in a refrigerator. An amount of 10 g of decomposition products from each litterbag was taken for the determination of water-soluble organic carbon (WEOC). The remaining decomposition products were cleaned and dried to a constant weight, and the decomposition rate was calculated using the following formula [28]:
D = 100 × (m0 − m1)/m0,
where D is the decomposition rate (%); m0 is the weight of the straw before decomposition (g); and m1 is the remaining weight of straw after decomposition (g).
The washed and dried straw decomposition products were ground through a 150 µm sieve. An amount of 10 g of each treatment was taken to measure the contents of cellulose, hemicellulose, and lignin in the decomposition products; 1 g was used for the infrared spectroscopy determination of the decomposition products; 1 g was used to measure the remaining organic carbon content of the straw; 2 g was used to measure the content of humus; and 4 g was used to extract humic acid for 13C-NMR analysis.

2.4. Corn Planting Experiments

On 10 May 2023, the remaining litterbags were dug out, and the decomposition products were poured out. After covering with soil for 30 cm, corn was planted directly above the decomposition products. The variety of corn was “Liangyu 99”, planted in an area with a width of 11 m and a length of 10 m with a ridge spacing of 80 cm and a plant spacing of 30 cm. On 19 June 2023, the corn seedlings were uprooted and collected to measure their growth indicators. The height and stem diameter of the corn plants were measured using a steel tape measure and a vernier caliper, respectively. The relative chlorophyll content (SPAD value) of the leaves was determined using a handheld chlorophyll meter (SPAD-502, Konica Minolta, Toyko, Japan).
The static chamber method was utilized to collect greenhouse gas samples when the corn seedlings were planted. The static chamber was made of PVC, measuring 0.4 m long × 0.3 m wide × 0.5 m high. To avoid the sharp increase in air temperature outside the chamber during the sampling period under sunlight, each side of the top chamber was covered with a Styrofoam coating. The exterior of the static chamber was covered with a sponge and tin foil for heat preservation, and the base of the static chamber was made of steel that was 0.4 m long × 0.3 m wide × 0.2 m high. The static chamber base was stabilized horizontally, and water was placed in the sealing sink of the base. The fan power cord was connected to a 12 V DC power supply to ensure uniform gas mixing in the static chamber. The gas was sampled using a 100 mL syringe and then transferred to an evacuated vial once every 6 min for a total of 5 times. Greenhouse gas samples were continuously collected during the seedling stage of the corn, and the seedlings were all sealed in the static chamber. If it rained, collection was resumed after one day. Gas samples were collected from 9:00 to 11:00 AM on each sampling date. The analysis was completed within three days after collection. The gas samples were analyzed using an Agilent 7890B (Shanghai, China) gas chromatograph (GC) equipped with an electronic capture detector (ECD) and flame ionization detector (FID) operating at 300 °C. The fuel gas used for the chromatograph was H2, and the auxiliary gas was air. The carrier gas was high-purity dinitrogen (N2, 99.999%). The formula for the greenhouse gas emission flux is as follows [29]:
F = M P h R ( T + 273 ) × d c d t ,
where F is the gas emission flux, mg·(m2·h)−1; R is the gas constant (8.3144 Pa·m3)·(mol·K)−1; C is the gas fraction in the static chamber, 10−9; T is the time after the box is fastened; h is the height from the ground to the static chamber top, m; M is the molar mass of the gas (MN2O = 44 g·mol−1; MCO2 = 44 g·mol−1; MCH4 = 16 g·mol−1); T is the temperature inside the static chamber during measurement, °C; P is the standard atmospheric pressure, 101,325 Pa; and dc/dt is the gas emission rate, μL·(L·min)−1).
The formula for the cumulative greenhouse emissions is as follows [29]:
C E = ∑ ( F i + 1 + F i ) 2 × ( T i + 1 − T i ) ,
where Fi represents the greenhouse gas emission fluxes, i denotes the sampling count, and (Ti+1 − Ti) is the interval in days between greenhouse gas samplings.

2.5. Measurement Method of Indicators

The contents of cellulose, hemicellulose, and lignin in the decomposed substances were determined using the agricultural industry standard NYT3494-2019 of the People’s Republic of China [30]. The infrared spectrum analysis of decomposition products was obtained by an FT-IR spectrometer (IRAffinidy-1S, Shimadzu, Toyko, Japan). With potassium bromide as the background, the wave number range is 4000 cm−1~400 cm−1. Furthermore, atmospheric correction and Savitzky–Golay filtering are used to reduce the interference in the infrared spectrum. The content of WEOC and its three-dimensional fluorescence spectrum were determined using the method of Li Yan [31]. The TOC instrument (Vario TOC, Elementer, Frankfurt Germany) was used for measurement, and absorbance at 254 nm was measured using a UV–visible spectrophotometer (UV 2401PC, Shimadzu, Toyko, Japan). The specific UV absorption value (SUV254) was calculated using the following formula [31]:
SUV254 = UV254/C × 100,
Here, SUV254 is the specific UV absorption value, L/(mg·m−1); UV254 is the absorbance of the extraction solution at 254 nm, cm−1; and C is the measured WEOC concentration, mg·L−1.
The determination and purification of humus were carried out using the IHSS (International Humus Society) method. The PQ value was the relative proportion of HA. A 150 mg sample of HA extracted from the decomposition products was weighed and dissolved in 1.5 mL of 0.5 mol/L NaOD. After shaking and dissolving, it was filtered through a 0.45 µm filter membrane and subjected to liquid 13C-NMR using a nuclear magnetic resonance instrument (Advance Neo 500M, Bruker, Karlsruhe, Germany).
The method of microbial DNA extraction and high-throughput sequencing in the decomposed products is as follows: The genomic DNA of the sample was extracted using the CTAB method, and agarose gel electrophoresis was used to detect the purity and concentration of DNA. An appropriate amount of sample DNA was placed in a centrifuge tube, and the sample was diluted to 1 ng/μL with sterile water. Using the diluted genomic DNA as a template and based on the selection of sequencing regions, specific primers with a Barcode were used. The Phusion High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs (Beijing, China) was used to perform PCR with efficient and high-fidelity enzymes to ensure amplification efficiency and accuracy. Notably, 16S V4 primers 515F and 806R were used to identify bacterial diversity; ITS1 primers (ITS5-1737F and ITS2-2043R) were used to identify fungal diversity. PCR products were detected using 2% agarose gel electrophoresis. Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, California, USA) following the manufacturer’s recommendations, and index codes were added. Sequences were clustered into operational taxonomic units at the 97% threshold.

2.6. Data Statistics and Analysis

The experimental data was organized using Excel 2021 and plotted using Origin 2021. The results are presented as mean values. SPSS 26.0 (IBM, Chicago, IL, USA) statistical software was used for relevant statistical analysis and difference testing (p < 0.05). A redundancy analysis was performed using Canoco 5.0 software to evaluate the impact of environmental factors on changes in the straw bacterial community composition. A relevant network analysis was conducted using Gephi (version 0.10.1). Rstudio (v4.2.0) was used to draw NMDS diagrams and chord diagrams. SPSSAU was used for structural equation modeling (SEM).

3. Results and Discussion

3.1. Direct Evidence of Montmorillonite and Composite Amino Acid Promoting Straw Decomposition

Our results demonstrate that montmorillonite and composite amino acids can promote the decomposition of corn straw in cold-region soil. The addition of montmorillonite (MMT), composite amino acid (CAA), and their combination (MMT-CAA) significantly increased the straw decomposition rate by 18.2%, 19.5%, and 42.1%, respectively, compared to the control (CK) (Figure 1a). The FTIR spectrograms of the decomposition products show similar profiles across all treatments, and MMT-CAA has the weakest absorption peaks (Figure 1b). The main absorption peaks were identified based on previous reports on straw decomposition [32,33]. MMT and CAA weakened the stretching vibration of C-O, O-H, and C-H, likely due to the breakdown of cellulose, hemicellulose, proteins, lipids, and soluble sugars under the catalytic influence of montmorillonite and composite amino acid. Additionally, the weakening of the C=O and C=C stretching vibration suggested the degradation of an aromatic ring containing substances such as lignin [33]. MMT, CAA, and MMT-CAA reduced the contents of cellulose, hemicellulose, and lignin in the decomposition products by 8.9%, 19.2%, and 39.6%; 19.7%, 22.6%, and 43.4%; and 28.3%, 15.9%, and 37.4%, respectively (Figure 1c). These enhancements exceeded the previous degradation enhancement rates achieved through the use of either a microbial agent or clay minerals alone during low-temperature periods [20,34]. This shows that composite amino acid primarily enhances the decomposition of cellulose and hemicellulose. This may be because amino acids supply the nutrients needed by microorganisms, promoting their growth and proliferation, which aids in decomposing corn straw [24]. MMT was effective in decomposing lignin, probably due to its ability to aggregate lignin molecules on the montmorillonite surface, and it also facilitated lignin decomposition through hydrogen bonding and ligand exchange [35,36,37]. Conversely, MMT was less effective than CAA in decomposing cellulose and hemicellulose, possibly due to the low abundance of nutrients required by microorganisms in montmorillonite, which resulted in lower activity of microorganisms capable of breaking down cellulose and hemicellulose. When combined, montmorillonite and composite amino acid leverage their respective strengths to enhance corn straw decomposition.
After the decomposition of corn straw, montmorillonite and composite amino acids facilitated the release of organic carbon (Table 3). Compared to the CK, the total organic carbon (TOC) content in corn straw decreased by 10.7%, 6.8%, and 12.8%, respectively. Our findings indicate that montmorillonite significantly promoted the decomposition of total organic carbon in corn straw, likely because montmorillonite provides a habitat for microbial communities, and it has a catalytic effect on microbial metabolism [38]. Then, TOC was converted into WEOC (WEOC). WEOC is an important active component, easily utilized by microorganisms as a carbon and energy source [39,40]. The WEOC content of decomposed products increased by 10.5%, 19.7%, and 25.0%, respectively. Additionally, the SUV254 value of the CAA decreased by 11.0% compared to the CK, while the SUV254 values for MMT and their combination increased significantly by 14.3% and 9.6%, respectively. An analysis of the WEOC content using three-dimensional fluorescence (Figure 1d) showed that CAA decreased the fluorescence intensity of WEOC, whereas MMT increased it. This suggests that composite amino acids form humic acid-like substances with lower aromaticity, which may be due to the nitrogen in the composite amino acid stimulating the formation of WEOC or the amino acid promoting microbial growth, leading to increased WEOC production [41]. In addition, the rapid and strong decomposition of cellulose by composite amino acids resulted in a small accumulation of primary and intermediate metabolites with aromatic hydrocarbon structures, affecting water-soluble organic aromaticity [31]. Montmorillonite enhances the aromaticity of humic acid-like substances due to its large specific surface area, which provides sites for microbial activity [36]. It allows lignin, proteins, and other insoluble polymers to be decomposed into small water-soluble molecules like peptides. Montmorillonite facilitated the decomposition of these small molecules into more stable and complex organic compounds [42]. When combined with composite amino acids, montmorillonite neutralized the low aromaticity of humic acid-like substances generated by the amino acids. The combination maintains a high WEOC content while increasing its degree of aromatization, indicating a synergistic effect in enhancing WEOC aromaticity. This is a new model for collaboratively promoting the “quality” and “quantity” of WEOC.

3.2. Montmorillonite and Composite Amino Acid Promote Humification of Corn Straw and Make Humic Acid Structure More Stable

Humus, a primary component of organic matter, consists of complex and stable organic compounds synthesized through microbial metabolism and degradation [42]. It mainly includes lipids, Alkyl phenyl esters, and carbohydrates. Compared to the CK, CAA and MMT-CAA increased the total humic content in the decomposed material by 6.3% and 13.9%, respectively. MMT, CAA, and MMT-CAA increased the HA content in the humus by 4.7%, 11.9%, and 31.4%, respectively; raised the ratio of HA to fulvic acid (FA) by 12.0%, 9.9%, and 31.5% (Figure 2a); and increased the PQ value by 6.3%, 5.2%, and 15.4%, respectively (Figure 2b). Our results indicate that CAA and MMT-CAA significantly enhanced the conversion of organic carbon into humus in corn straw, and MMT can promote the accumulation of HA. They increased the conversion of FA into HA while reducing FA formation to varying degrees (Figure 2a). This observation aligns with the “polyphenol theory”, which suggests that FA forms first and then polymerizes into HA [43]. Initially, cellulose and hemicellulose decompose into small-molecular-weight, low-aromaticity FA. This FA then combines with other small molecules and undecomposed lignin to recondense into HA, eventually forming larger HA molecules from an HA monomer [44]. Montmorillonite and composite amino acids therefore accelerated the humification of corn straw by promoting the polymerization of FA into HA. It is noteworthy that montmorillonite, due to its layered structure and large specific surface area, possesses a strong water retention capacity. The accumulated moisture may maintain a persistently moist state in the decomposition environment, protecting the activity of microbial communities involved in humification and indirectly promoting this transformation process [45]. Research teams led by Wang and Qiu confirmed that clay minerals can decompose cellulose and lignin, catalyzing the formation of HA polymers involved in the synthesis of humus [20,46]. Filip’s research also demonstrated that montmorillonite can shorten the formation time of humus [18]. The reason that the total humus content did not significantly increase in MMT might be the lack of nitrogen sources necessary for microorganisms, slowing the humification process of corn straw and resulting in a smaller amount being converted into humus. However, MMT can enhance the conversion of organic carbon from corn straw to HA in humus, improve humus quality, and increase the degree of humification of corn straw. This effect is likely due to montmorillonite’s adsorption capacity for HA, which continuously accumulates HA, increasing the ratio of HA to FA and the PQ value. Adding composite amino acids with montmorillonite can address nitrogen deficiency in microorganisms, enhancing the benefits of montmorillonite and resulting in a combined effect greater than their individual effects. In summary, both the individual and combined applications of montmorillonite and composite amino acids significantly influence humus formation in decomposition products, promoting the conversion of organic carbon from corn straw to humus, increasing humus quantity, and improving the degree and quality of humus.
We further compared the 13C-NMR of humic acids under various treatments to explore their different properties (Figure 2c). The relative proportions of different carbon types in HA were obtained by integrating the spectrograms (Table 4). In 13C-NMR liquid nuclear magnetic resonance, the Aliphatic C/Aromatic C ratio reflects the complexity of the molecular structure of humic substances. A lower ratio indicates a more complex molecular structure in humic substances [47]. MMT, CAA, and MMT-CAA decreased the Aliphatic C/Aromatic C ratios by 29.9%, 25.5%, and 44.1%, respectively. The ratio of Alkyl C to Alkoxy C, with Alkyl C being more difficult to decompose, reflects the degree of alkylation of humic substances and is often used as an indicator of the degree of organic carbon decomposition [48]. Aromaticity indicates the degree of aromatization of an HA structure, with a higher value indicating a more stable structure [49]. MMT, CAA, and MMT-CAA decreased the Alkyl C/Alkoxy C ratio by 15.5%, 19.7%, and 22.7%, respectively, while aromaticity increased the ratio by 28.1%, 23.0%, and 47.8%, respectively, compared to the CK. This suggests that these substances can increase the aromatic structure of humic substances, reduce Aliphatic side chains, and enhance the degree of condensation, making humic substances more complex and stable [50,51]. The increased aromaticity of the HA structure due to montmorillonite and its combined effect may be because montmorillonite promotes the decomposition of lignin, a natural polymer and highly aromatic cross-linked aromatic substance [52]. This is consistent with the mechanism identified by previous researchers, which demonstrates that clay minerals facilitate the conversion of lignin to humus [18,36]. This promoted the binding of humus and increased the aromaticity and stability of HA.

3.3. Microbial Communities and Co-Degradation Systems Revealed the Important Roles of Montmorillonite and Composite Amino Acid in Straw Degradation

Significant differences in microbial community characteristics were observed among different treatments due to the influence of montmorillonite and composite amino acids on decomposition products (Figure 3). MMT and CAA increased the unique bacterial OTU and fungal ASV numbers, with the combined effect being the most significant. Composite amino acids significantly increased the Shannon and Simpson diversity indices of bacteria (Table 5). MMT increased these indices for fungi, and the combined effect significantly increased the Chao1, Shannon, and Simpson indices of fungi. This indicates that montmorillonite and composite amino acids significantly increase bacterial diversity, and fungal richness and diversity during corn straw decomposition. Montmorillonite had a more prominent effect, increasing fungal diversity, whereas composite amino acids had a more significant impact on bacterial diversity and a lesser effect on fungi. When combined, montmorillonite and composite amino acids significantly increased both bacterial and fungal diversity. This may be due to the rich carbon, nitrogen, and trace elements in composite amino acids, which provide essential nutrition and favorable conditions for microorganisms [10,24], enhancing their ability to resist low temperatures. Montmorillonite further enhanced microbial metabolic activity and provided an adhesive interface for them to thrive [18,36]. Thus, montmorillonite and composite amino acids can influence the number, species, and activity of microorganisms. Differences in microbial phyla across treatments were compared using chordal plots (Figure 3c), with effects on the relative abundance of microbial species composition being shown in Table 6. The species abundance of Bacteroidetes in bacteria and Ascomycetes and Zygomycetes in fungi significantly increased with CAA. MMT and MMT-CAA significantly increased the species abundance of basidiomycetes in fungi (Figure 3c and Table 6). Additionally, MMT-CAA significantly increased the species abundance of the bacterial phyla Mycobacterium and Actinobacterium. The phyla Ascomycetes, Actinobacteria, Bacteroidetes, and Basidiomycetes are key for degrading cellulose, hemicellulose, and lignin. An increase in their relative abundance promotes the degradation of lignocellulose and provides more precursors for humus synthesis [43,53]. This finding aligns with the research conducted by Bao and Meng, which demonstrates that Actinobacteria and Ascomycetes possess the capability to effectively degrade lignocellulose at low temperatures [52,54,55,56].
We evaluated the relationship between environmental factors and bacterial and fungal communities through a redundancy analysis (Figure 4a). Most environmental factors significantly explained the variation in community structure. Among them, cellulose (explained 86.9%, p = 0.001), HA (88.1%, p = 0.001), and decomposition rate (78.8%, p = 0.002) made the highest contributions. Second, hemicellulose (77.4%, p = 0.003), WEOC (75.9%, p = 0.004), and lignin (63.6%, p = 0.015) also showed significant effects. The decomposition rate, WEOC, humus, and PQ values were negatively correlated with straw cellulose, hemicellulose, and lignin, demonstrating that straw decomposition influences the production of humus and WEOC. Actinobacteria and Bacteroidetes were positively correlated with cellulose and hemicellulose degradation, while Proteobacteria were positively correlated with FA formation. Firmicutes and verrucomicrobiota of bacteria, as well as Basidiomycota and Ascomycota of fungi, are significantly positively correlated with the decomposition rate, WEOC, humus components, and PQ, indicating their role in promoting the microbial decomposition of organic matter into humus. Jiao also discovered the important role of Firmicutes in humification [57]. There was a negative correlation between cellulose, hemicellulose, lignin, HE, and the PQ value.
Exploring the differences in the degree of microbial interaction between different treatments through a co-occurrence network analysis (Figure 4b) revealed the network complexity and interaction of bacterial and fungal communities using topological indices (Table 7). The bacterial co-occurrence network for the MMT-CAA was the densest. It had the highest number of nodes (71) and edges (348) with a positive correlation ratio of 61.8%, and it had the highest average connectivity (AD = 9.1622) and the shortest average path length (APL = 1.8938). Among the fungal co-occurrence networks, MMT-CAA had the highest number of edges (95), the highest network density (GD = 0.1218), and the highest average connectivity (AD = 4.7500), with the shortest average path length (APL = 2.1462). The results show that the MMT-CAA led to tighter connections, higher modularity, and more complex network relationships, enhancing microbial interaction in straw degradation systems. This suggests that the combination of montmorillonite and CAA enhances the interrelationships between various bacterial phyla capable of undergoing the decomposition process, strengthening the interactions within the microbial straw degradation system [58,59]; the combination therefore formed a more complex co-degradation system.

3.4. Montmorillonite and Composite Amino Acid Promote the Growth of Corn Seedlings and Reduce Greenhouse Gas Emissions

Corn was planted on top of the decomposition products after winter, with a low temperature of incubation. The plant height and stem diameter are crucial indicators of corn growth and development, significantly influencing corn yield [60]. The size of corn leaves affects their ability to capture and utilize light energy, while chlorophyll is an essential substance for photosynthesis, impacting overall plant growth [61]. The study results demonstrate that the single and combined effects of montmorillonite and composite amino acids increase the plant height, stem diameter, leaf area, and SPAD value of corn seedlings, with the combined effect being greater than the effect of single applications (Table 8). Specifically, the plant height increased by 24.9%, 38.3%, and 55.1%; the stem diameter increased by 14.7%, 40.4%, and 58.7%; and the leaf area increased by 25.2%, 62.5%, and 70.2%. Additionally, the leaf SPAD value increased by 28.1%, 29.9%, and 41.2%. This may be attributed to the nitrogen in humus and compound amino acids being absorbed and utilized by corn seedlings, with nitrogen primarily being distributed in the leaves, thereby promoting growth and photosynthesis [62]. In addition, the powerful water retention capacity of montmorillonite may help maintain the stability of rhizosphere moisture, providing a more suitable water-fertilizer synergistic environment for corn, thereby promoting the growth of corn seedlings [63].
Greenhouse emissions were also measured during the corn seedling stage (Figure 5 and Table 9). MMT significantly reduced CO2 and N2O average emission fluxes (35.6% and 15.8%, respectively) and cumulative emissions (34.5% and 16.3%, respectively). However, CAA significantly increased CO2 and N2O average emission fluxes (33.5% and 18.6%, respectively) and cumulative emissions (29.1% and 12.8%, respectively). The combined effect of both had no significant impact on CO2 and N2O emissions. Compared to the CK, CAA and MMT-CAA significantly increased CH4 average uptake fluxes (30.8% and 13.4%, respectively) and cumulative uptake (28.2% and 11.0%, respectively).
Regarding carbon dioxide and nitrous oxide emissions, MMT significantly reduced emissions compared to the CK, while CAA significantly increased them. This could be due to the high carbon and nitrogen contents in composite amino acid and corn straw, providing energy and nutrients for microbial respiration [16,64,65]. Moreover, CAA significantly increased the content of WEOC, which is easily decomposable and readily utilized by microorganisms, thus promoting microbial respiration and increasing carbon dioxide emissions [10,66]. CAA significantly increased N2O emissions, which might be due to the abundant nitrogen and carbon sources provided by it and corn straw during deep burial, stimulating denitrification reactions among denitrifying microorganisms [67,68,69]. Montmorillonite may reduce CO2 emissions by humifying more of the organic matter in straw rather than mineralizing it. Additionally, other decomposition products may accumulate on the montmorillonite surface, including the microbial biomass that is safeguarded by the minerals, clay–organic complexes that are stabilized in mineral-bound organic carbon, and some small-molecule organic intermediates [23,37,45]. This indicates that montmorillonite may play a role in adsorbing or inhibiting carbon dioxide and nitrous oxide emissions due to its pore structure and ion exchange effect.
Following snowfall in cold regions, a portion of the snowmelt infiltrates the soil. When corn straw is buried deeply, microorganisms consume oxygen as they proliferate, leading to the formation of anaerobic zones [70,71]. During the decomposition of corn straw by these microorganisms, CH4 was produced. Regarding CH4 uptakes, MMT showed no significant difference compared to the CK, while the single application of composite amino acid and its combination with montmorillonite significantly increased CH4 uptake. This indicates that methane in black soil during the corn seedling stage is mainly an absorption sink. The addition of composite amino acid and corn straw likely provided methane-oxidizing bacteria with ample easily decomposable carbon, creating favorable conditions for their growth or inhibiting the activity of methanogenic bacteria, thereby facilitating methane oxidation and increasing CH4 uptakes [68,72,73].
In summary, montmorillonite has a more significant role in inhibiting greenhouse gas emissions, thereby mitigating the main negative effect of composite amino acid and reducing carbon emissions when combined.

3.5. Possible Mechanisms of Humification and Carbon Sequestration Promoted by Montmorillonite and Composite Amino Acid

A structural equation model was developed to reveal how montmorillonite and composite amino acids promote the carbon conversion of corn straw and the growth of corn seedlings (Figure 6). Montmorillonite and composite amino acids accelerate the further decomposition of recalcitrant substances such as cellulose, hemicellulose, and lignin in straw, increasing the decomposition rate of deeply buried straw (−1.000) and accelerating the humification process. This decomposition significantly influences the conversion of carbon elements into WEOC (0.974) and humic substances (1.000). The content of humic substances determines the degree of straw humification (0.989), while WEOC, as an easily utilized carbon source for microorganisms, also promotes microbial activity. The decomposition rate of straw and humus significantly impacts the growth of corn seedlings (−0.229). The composite amino acid and nutrients released from the straw are fully utilized by crops, increasing the stem diameter, plant height, leaf area, and SPAD value (1.214), which affect carbon dioxide emissions. Carbon dioxide emissions and the growth of corn seedlings are mutually influential (0.571). The decomposition of straw fibers releases carbon dioxide, and the carbon dioxide concentration affects straw fibers’ decomposition (−1.834). Montmorillonite and composite amino acids accelerate straw humification and carbon sequestration, reducing carbon dioxide emissions.

4. Conclusions

Montmorillonite and composite amino acids can form a stable and complex co-degradation microbial system in cold-region soil to rapidly decompose and humify corn straw and convert more organic carbon into structurally stable WEOC and humus carbon. This helps to reduce greenhouse gas emissions, thereby enhancing carbon sequestration and promoting the growth of corn seedlings. However, this study primarily employs litterbag experiments in the field under controlled conditions. Future studies must combine production practices to overcome limitations and explore the effects of different soil types and straw return methods on crop growth and yield, and the carbon and nitrogen transformation mechanisms.

Author Contributions

X.C.: Writing—Original Draft, Data Curation, Software, Methodology, and Investigation. T.F.J.G.: Validation, and Writing—Review and Editing. C.Z.: Conceptualization, Investigation, Methodology, and Validation. Y.F.: Methodology, Supervision, and Validation. M.L.: Formal Analysis, Funding Acquisition, Project Administration, Supervision, and Writing—Review and Editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (42077137) and the Jilin Scientific and Technological Development Program (20220101186JC).

Data Availability Statement

The data presented in this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 01979 g001
Figure 1. Montmorillonite and composite amino acids promoted straw decomposition. The decomposition rate of corn straw (a); the FTIR spectrograms of the decomposition products (b); the contents of cellulose, hemicellulose, and lignin in the decomposition products (c); and WEOC three-dimensional fluorescence spectra of the decomposition products in soil from litterbags buried at a depth of 30 cm during winter with low-temperature incubation (d). The data are expressed as the mean ± standard deviation. Different letters represent significantly different means (p < 0.05) among treatments. Note: Region V (250–420 nm/380–520 nm) is a humic acid-like substance.
Figure 1. Montmorillonite and composite amino acids promoted straw decomposition. The decomposition rate of corn straw (a); the FTIR spectrograms of the decomposition products (b); the contents of cellulose, hemicellulose, and lignin in the decomposition products (c); and WEOC three-dimensional fluorescence spectra of the decomposition products in soil from litterbags buried at a depth of 30 cm during winter with low-temperature incubation (d). The data are expressed as the mean ± standard deviation. Different letters represent significantly different means (p < 0.05) among treatments. Note: Region V (250–420 nm/380–520 nm) is a humic acid-like substance.
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Figure 2. Variations in humification parameters. Content of humus fractions in decomposition products (a), degree of humification (b), and 13C-NMR liquid nuclear magnetic resonance of humic acid (HA) (c). Different letters represent significantly different means (p < 0.05) among treatments.
Figure 2. Variations in humification parameters. Content of humus fractions in decomposition products (a), degree of humification (b), and 13C-NMR liquid nuclear magnetic resonance of humic acid (HA) (c). Different letters represent significantly different means (p < 0.05) among treatments.
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Figure 3. Bacteria and fungi microbial beta diversity (a), bacteria and fungi Venn diagram (b) and bacterial and fungal communities at phylum level (c) of decomposition products in soil from litterbags buried at depth of 30 cm during winter with low-temperature incubation.
Figure 3. Bacteria and fungi microbial beta diversity (a), bacteria and fungi Venn diagram (b) and bacterial and fungal communities at phylum level (c) of decomposition products in soil from litterbags buried at depth of 30 cm during winter with low-temperature incubation.
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Figure 4. A redundancy analysis (RDA) was used to evaluate the relationship between microbial community structure and various environmental factors (a). Note: In the RDA, arrows represent different environmental factors, and the longer the arrow, the greater the impact of that environmental factor. Different colored dots represent samples in different groups. An acute angle between environmental factors indicates a positive correlation between the two environmental factors, and an obtuse angle indicates a negative correlation. Differences in the microbial co-occurrence network in each group (b). Note: The red line represents a positive correlation, while the blue line represents a negative correlation.
Figure 4. A redundancy analysis (RDA) was used to evaluate the relationship between microbial community structure and various environmental factors (a). Note: In the RDA, arrows represent different environmental factors, and the longer the arrow, the greater the impact of that environmental factor. Different colored dots represent samples in different groups. An acute angle between environmental factors indicates a positive correlation between the two environmental factors, and an obtuse angle indicates a negative correlation. Differences in the microbial co-occurrence network in each group (b). Note: The red line represents a positive correlation, while the blue line represents a negative correlation.
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Figure 5. The greenhouse gas emission fluxes during the stage of corn seedling above the decomposition products.
Figure 5. The greenhouse gas emission fluxes during the stage of corn seedling above the decomposition products.
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Figure 6. A structural equation model (SEM) revealing the potential mechanisms by which montmorillonite and composite amino acids affect the degree of straw humification, promote the growth of corn seedlings, and reduce carbon dioxide emissions. Note: The number on the arrow represents the path strength. The significance level is represented by * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 6. A structural equation model (SEM) revealing the potential mechanisms by which montmorillonite and composite amino acids affect the degree of straw humification, promote the growth of corn seedlings, and reduce carbon dioxide emissions. Note: The number on the arrow represents the path strength. The significance level is represented by * p < 0.05, ** p < 0.01, and *** p < 0.001.
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Table 1. The basic physicochemical properties of the primary soil.
Table 1. The basic physicochemical properties of the primary soil.
pHOrganic Matter
(g·kg−1)		Total N
(g·kg−1)		Alkaline N
(mg·kg−1)		Available P
(mg·kg−1)		Available K
(mg·kg−1)
Primary soil6.1312.871.27103.7423.90180.79
Table 2. Composite amino acid components.
Table 2. Composite amino acid components.
NameAspThrSerGluGlyCysValMetIleLeuPheHisLysArg
Content (mg/mL)0.4390.9681.1212.111.2920.6923.1442.6441.7473.7162.820.3954.1733.575
Table 3. Total organic carbon content (TOC) and UV absorption characteristics of water-soluble organic carbon in decomposition products.
Table 3. Total organic carbon content (TOC) and UV absorption characteristics of water-soluble organic carbon in decomposition products.
TreatmentTOC
(g·kg−1)		WEOC
(g·kg−1)		UV254
(cm−1)		SUV254
(L·mg−1·m−1)		f 450/f 500
CK488.393 ± 10.400 a19.503 ± 0.438 d0.3660.938 c1.21278
MMT436.243 ± 14.984 bc21.549 ± 0.436 c0.4621.072 a1.39466
CAA455.157 ± 9.989 b23.343 ± 0.319 b0.3900.835 d1.17967
MMT-CAA425.747 ± 10.137 c24.375 ± 0.477 a0.5011.028 b1.20813
CK, Control; MMT, Montmorillonite; CAA, Composite Amino Acid; MMT-CAA, Montmorillonite + Composite Amino Acid. Different lowercase letters indicate significant differences (p < 0.05).
Table 4. Relative contents of various types of carbon in 13C-NMR liquid nuclear magnetic resonance of humic acids.
Table 4. Relative contents of various types of carbon in 13C-NMR liquid nuclear magnetic resonance of humic acids.
Alkyl C
(0–50 ppm)		O-Alkyl C
(50–110 ppm)		Aromatic C
(110–160 ppm)		Carbonyl C
(160–200 ppm)		Aliphatic C
(0–110 ppm)		Aliphatic C
/Aromatic C		Alkyl C
/O-Alkyl C		Aromaticity
CK0.2260.4770.2560.0410.7032.7420.47226.726
MMT0.2210.4060.3260.0470.6271.9210.54534.237
CAA0.2240.3970.3040.0750.6212.0430.56532.860
MMT-CAA0.2100.3630.3740.0540.5731.5320.57939.487
Table 5. Microbial abundance and alpha diversity.
Table 5. Microbial abundance and alpha diversity.
TreatmentChao1ShannonSimpson
BacteriaCK246.31 ± 6.16 a2.68 ± 0.06 b0.86 b
MMT255.42 ± 47.01 a3.10 ± 0.10 a0.90 a
CAA290.38 ± 12.77 a3.23 ± 0.25 a0.91 a
MMT-CAA259.87 ± 33.58 a2.96 ± 0.086 a0.91 a
FungiCK149.81 ± 4.99 ab2.14 ± 0.174 b0.77 b
MMT158.26 ± 4.52 ab2.67 ± 0.34 a0.86 a
CAA134.77 ± 12.01 b2.02 ± 0.10 b0.73 b
MMT-CAA187.30 ± 4.84 a2.81 ± 0.06 a0.88 a
Different lowercase letters indicate significant differences (p < 0.05).
Table 6. The relative abundance of microbial species.
Table 6. The relative abundance of microbial species.
Species AbundanceTreatment
CKMMTCAAMMT-CAA
BacteriaProteobacteria0.819 a0.784 ab0.755 b0.663 c
Bacteroidetes0.173 c0.204 bc0.227 b0.307 a
Actinobacteria0.006 b0.012 ab0.012 ab0.024 a
FungiAscomycota0.796 a0.652 b0.857 a0.800 a
Basidiomycota0.057 b0.108 a0.028 b0.126 a
Different lowercase letters indicate significant differences (p < 0.05).
Table 7. Topological indices of bacterial and fungal co-occurrence networks.
Table 7. Topological indices of bacterial and fungal co-occurrence networks.
TreatmentNodesEdgesPositive/%MDGDADAPL
BacteriaCK5319956.780.23920.14447.50941.9289
MMT6527663.320.26200.13278.49231.9519
CAA6721364.790.30530.09636.35822.1746
MMT-CAA7134861.780.22260.14009.80281.8938
FungiCK427950.530.33880.12184.75002.1462
MMT436855.880.47530.07533.16282.8671
CAA355771.930.34760.09583.25712.2505
MMT-CAA409555.700.40300.09183.76192.2285
CK, Control; MMT, Montmorillonite; CAA, Composite Amino Acid; MMT-CAA, Montmorillonite + Composite Amino Acid. MD (modularity), GD (Graph Density), AD (Average Degree), APL (average path length).
Table 8. Corn seedling growth above decomposition products.
Table 8. Corn seedling growth above decomposition products.
TreatmentPlant Height (cm)Stem Diameter (cm)Leaf Area (cm2)Leaf SPAD
CK44.28 ± 3.21 d1.09 ± 0.15 d90.64 ± 15.91 d31.86 ± 0.53 c
MMT55.31 ± 2.98 c1.25 ± 0.16 c113.45 ± 18.64 c40.80 ± 0.93 b
CAA61.22 ± 3.02 b1.53 ± 0.11 b147.32 ± 10.93 b41.38 ± 2.39 b
MMT-CAA68.67 ± 1.98 a1.73 ± 0.08 a154.30 ± 14.79 a44.96 ± 0.78 a
Different lowercase letters indicate significant differences (p < 0.05).
Table 9. Greenhouse gas emissions.
Table 9. Greenhouse gas emissions.
TreatmentAverage Greenhouse Gas Emission FluxesCumulative Greenhouse Gas Emissions
CO2
(mg m−2 h−1)		N2O
(μg m−2 h−1)		CH4
(μg m−2h−1)		CO2
(kg ha−1)		N2O
(kg ha−1)		CH4
(kg ha−1)
CK215.24 ± 7.14 b68.48 ± 2.62 b−36.59 ± 1.71 a518.195 ± 16.69 b0.164 ± 0.013 b−0.090 ± 0.005 a
MMT138.69 ± 6.08 c57.66 ± 1.49 c−34.84 ± 1.91 a319.064 ± 14.35 c0.137 ± 0.006 c−0.086 ± 0.002 a
CAA287.43 ± 6.75 a81.23 ± 2.73 a−47.85 ± 1.5 c695.057 ± 26.41 a0.185 ± 0.017 a−0.117 ± 0.004 c
MMT-CAA223.759 ± 5.77 b65.41 ± 1.72 b−41.48 ± 1.64 b542.533 ± 29.02 b0.152 ± 0.009 b−0.101 ± 0.003 b
Different lowercase letters indicate significant differences (p < 0.05)
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MDPI and ACS Style

Chen, X.; Galliane, T.F.J.; Zhao, C.; Feng, Y.; Li, M. Montmorillonite and Composite Amino Acid Overcome the Challenges of Straw Return in Cold-Region Soil: Synergistic Mechanisms of Rapid Straw Humification and Carbon Sequestration. Agronomy 2025, 15, 1979. https://doi.org/10.3390/agronomy15081979

AMA Style

Chen X, Galliane TFJ, Zhao C, Feng Y, Li M. Montmorillonite and Composite Amino Acid Overcome the Challenges of Straw Return in Cold-Region Soil: Synergistic Mechanisms of Rapid Straw Humification and Carbon Sequestration. Agronomy. 2025; 15(8):1979. https://doi.org/10.3390/agronomy15081979

Chicago/Turabian Style

Chen, Xingyan, Tchoumtchoua Foka Joseline Galliane, Chongyang Zhao, Yanhui Feng, and Mingtang Li. 2025. "Montmorillonite and Composite Amino Acid Overcome the Challenges of Straw Return in Cold-Region Soil: Synergistic Mechanisms of Rapid Straw Humification and Carbon Sequestration" Agronomy 15, no. 8: 1979. https://doi.org/10.3390/agronomy15081979

APA Style

Chen, X., Galliane, T. F. J., Zhao, C., Feng, Y., & Li, M. (2025). Montmorillonite and Composite Amino Acid Overcome the Challenges of Straw Return in Cold-Region Soil: Synergistic Mechanisms of Rapid Straw Humification and Carbon Sequestration. Agronomy, 15(8), 1979. https://doi.org/10.3390/agronomy15081979

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