Aerobic Composting of Auricularia auricula (L.) Residues: Investigating Nutrient Dynamics and Microbial Interactions with Different Substrate Compositions

Abstract

Auricularia auricula (L.) is a widely cultivated edible mushroom, and the resource utilization of its residues offers significant opportunities for sustainable waste management and nutrient recovery. This study investigated the effects of substrate composition on nutrient dynamics and microbial diversity during the aerobic composting of Auricularia auricula (L.) residues. Two treatments were established: composting of Auricularia auricula (L.) residues alone (CR) and composting supplemented with green grass (CRG) over a 49-day period. The results showed that both treatments achieved compost maturity, characterized by a slightly alkaline pH, a germination index (GI) above 80%, and an electrical conductivity below 4 mS/cm. Both composts were odorless, insect-free, and dark brown. Compared to CR, the CRG treatment exhibited higher total organic carbon (TOC) degradation, cumulative total phosphorus (TP) and potassium (TK) levels, as well as enhanced urease, cellulase, and β-glucosidase activities. In contrast, CR retained higher total nitrogen (TN), humic carbon (HEC), fulvic acid carbon (FAC), humic acid carbon (HAC), and a greater humic-to-fulvic acid (HA/FA) ratio. Microbial community analysis revealed diverse bacterial and fungal taxa, with certain species positively correlated with nutrient cycling. Notably, specific substrate compositions promoted beneficial microbial proliferation, essential for efficient composting and nutrient mineralization. These findings not only provide a scientific basis for optimizing composting strategies of mushroom residues but also offer a practical pathway to convert agricultural waste into high-quality organic fertilizers. By enhancing soil fertility, reducing reliance on synthetic fertilizers, and promoting circular bioeconomy practices, this study contributes directly to sustainable agricultural development. CR and CRG treatments, respectively, support either nutrient retention or release, allowing tailored application based on crop demand and soil condition. This study underscores the potential of Auricularia auricula (L.) residues in composting systems, contributing to waste reduction and soil fertility enhancement through tailored substrate management, and offers practical insights into optimizing composting strategies for Auricularia farming by-products.

1. Introduction

The global edible mushroom industry generates substantial organic waste in the form of cultivation residues, posing significant environmental and economic challenges to sustainable agriculture [1,2,3,4]. Auricularia auricula (L.) (hereafter referred to as A. auricula), commonly known as black fungus, is widely cultivated, with its production alone generating over 3.25 tons of lignocellulosic-rich residue per ton of harvested mushroom in China [5]. Current disposal practices, including incineration and landfilling, lead to nutrient wastage and environmental contamination, underscoring the urgent need for eco-friendly valorization strategies [6,7]. Composting has emerged as a scalable solution, converting organic waste into stable humic substances through microbial-mediated degradation while inactivating pathogens via thermophilic phases [8,9,10]. Compared to other organic waste management techniques, such as anaerobic digestion, pyrolysis, and vermicomposting, aerobic composting is particularly effective for lignocellulosic residues due to its capacity to degrade recalcitrant organic compounds via thermophilic microbial activity [11,12,13]. For example, anaerobic digestion primarily focuses on biogas recovery but struggles with lignin degradation, whereas vermicomposting is constrained by its reliance on earthworms and slower processing rates [14]. As a result, aerobic composting stands out as an efficient and scalable method for managing mushroom cultivation residues. However, the heterogeneous composition of fungal residues—characterized by recalcitrant lignin and variable C:N ratios—often limits composting efficiency, necessitating optimized substrate formulations to enhance biodegradation kinetics [15,16,17].

Substrate composition critically governs composting outcomes by modulating nutrient availability, microbial activity, and metabolic pathways [18,19,20]. While lignocellulosic materials like crop straws and sawdust dominate composting research [21,22], weed biomass represents an underutilized resource with high cellulose content, rapid degradability, and abundant availability [23]. Strategic blending of fungal residues with nitrogen-rich weed biomass may address key limitations in C: N balance, porosity, and microbial diversity [24,25]. Recent studies highlight that co-composting diverse substrates can synergize nutrient cycling; for instance, cellulose-rich materials accelerate lignin depolymerization by cross-feeding lignolytic and cellulolytic microbial consortia [26,27]. Nevertheless, systematic investigations into the composting of A. auricula residues—particularly when amended with weed biomass—remain scarce, leaving critical gaps in understanding nutrient dynamics and microbiome succession in such systems.

Study Aim and Importance

To address these gaps, this study aims to systematically evaluate the effects of substrate composition on nutrient dynamics and microbial diversity during the aerobic composting of A. auricula residues. Specifically, we hypothesize that

(1)

Supplementation of A. auricula residues with weed biomass would optimize the initial C:N ratio and porosity, thereby accelerating both organic matter mineralization and humification rates.

(2)

Co-composting would foster synergistic interactions between lignin-degrading fungi (e.g., Basidiomycota) and cellulose-degrading bacteria (e.g., Pseudomonas), enhancing functional diversity and metabolic efficiency.

(3)

This study uniquely emphasizes microbial diversity as a key factor in composting performance, hypothesizing that herbaceous materials may significantly stimulate microbial activity by augmenting carbon availability and structural heterogeneity, thereby advancing current knowledge of microbial succession in lignocellulosic waste composting.

Importantly, this study goes beyond mechanistic insight to address a critical agricultural need: the transformation of underutilized mushroom residues into value-added organic fertilizers. By optimizing substrate formulations, the composting process not only achieves efficient waste reduction but also produces nutrient-rich compost suitable for improving soil fertility, enhancing crop productivity, and reducing dependence on chemical fertilizers. This is particularly relevant for regions facing agricultural sustainability challenges, where resource recycling and soil health restoration are top priorities.

This study is distinguished from previous research by its focus on mushroom cultivation residues, particularly A. auricula, which has received limited attention in composting studies despite its significant contribution to agricultural waste. Moreover, while previous studies have extensively explored co-composting crop residues or animal manure, the use of weed biomass as a nitrogen-rich amendment remains underexplored. By investigating microbial diversity and nutrient dynamics, this work advances current knowledge on lignocellulosic waste management and co-composting strategies, offering insights into the sustainable integration of agricultural by-products into organic waste management.

Scope and Contributions

This study contributes to advancing sustainable agricultural practices by demonstrating the potential of converting mushroom cultivation residues into high-quality organic fertilizers through optimized aerobic composting strategies. By systematically evaluating changes in total organic carbon, nitrogen, phosphorus, potassium, humic substances, key enzyme activities, and microbial diversity, this work provides a comprehensive understanding of how substrate composition influences composting efficiency and product quality.

The findings not only offer practical guidance for mushroom growers and waste managers seeking environmentally sound disposal solutions, but also inform broader efforts to improve soil health, reduce chemical fertilizer inputs, and promote circular resource use in agriculture. The differentiated composting outcomes between CR and CRG treatments—favoring either nutrient retention or rapid release—enable flexible application scenarios tailored to specific crop needs or soil conditions.

By integrating underutilized agricultural by-products such as Auricularia auricula residues and weed biomass, this study promotes a scalable, low-cost approach to organic waste valorization. Furthermore, by highlighting the role of microbial communities in compost performance, the study encourages future microbiome-informed composting strategies to enhance nutrient mineralization and agroecosystem sustainability.

2. Materials and Methods

2.1. Composting Feedstock and Experimental Design

The composting experiment was conducted on the west campus of Changchun University (43°49′42″ N, 125°17′32″ E) in July 2023 and lasted for 49 days. Two composting treatments were established: (1) independent composting of Auricularia auricula (L.) residues (CR), and (2) co-composting of residues supplemented with green grass at a volumetric ratio of 3:1 (CRG). The basic physical and chemical properties of the raw materials are presented in

Table 1. Basic physical and chemical properties of test materials (dry base).

Treatment	TOC (g/kg)	TN (g/kg)		TP (g/kg)		TK (g/kg)		Urea (g/kg)	Moisture Content (%)	C/N
CR	310.29 ± 5.29 a	8.60 ± 0.20 a		7.33 ± 0.21 a		25.52 ± 0.34 a		3.74 ± 0.04	65–70%	30:1
CRG	381.43 ± 6.43 b	12.45 ± 0.15 b		12.31 ± 0.23 b		15.74 ± 0.23 b		/	65–70%	30:1
Basic physical and chemical properties of test materials (dry base)
Treatment	pH		EC (mS/cm)		Lignin (g/kg)		Cellulose (g/kg)		Hemicellulose (g/kg)
CR	6.52 ± 0.03 a		4.22 ± 0.12 a		55.56 ± 5.52 a		100.29 ± 10.01 a		65.18 ± 14.82 a
CRG	6.54 ± 0.02 a		3.80 ± 0.13 a		40.22 ± 12.08 b		137.50 ± 12.59 b		90.24 ± 9.72 b

Note: Different letters represent different treatments with significant differences. (p < 0.05).

. The A. auricula residues are from Huangsongdian black fungus planting base in China, while the grass materials are mainly Setaria viridis (L.) Beauv, Eleusine indica (L.) Gaertn, and Digitaria sanguinalis (L.) Scope. The initial moisture content of the composting mixtures was maintained at approximately 60%, and the materials were piled into fermentation units measuring 2 m × 3 m × 1.5 m (length × width × height), as illustrated in Figure 1. In this experiment, the 3:1 volumetric ratio of A. auricula residue and grass was selected based on considerations of carbon-to-nitrogen (C:N) balance and pore structure optimization. The A. auricula residue served as the primary carbon source, while the grass adjusted the overall C:N ratio to approximately 30:1 and enhanced porosity to improve aeration. The pile size was determined using thermal mass calculations to ensure that the composting system maintained a high temperature (>55 °C) for at least five consecutive days, meeting sanitation and pathogen inactivation requirements. This composting approach not only minimizes energy consumption but also aligns with the characteristics of local waste materials, thereby achieving low-cost and sustainable resource utilization.

Temperatures were recorded daily at 9:00 AM and 2:00 PM, and the compost piles were regularly turned to ensure aeration. Samples were collected on days 0, 2, 4, 6, 8, 10, 12, 14, 21, 28, 35, 42, and 49 of the composting process. A five-point sampling method was employed, with each sample weighing approximately 0.5 kg. The sampling schedule for each treatment is detailed in

Table 2. Sampling time during the composting process.

Sample Collection	CR	CRG
Initial Stage	0 d (7.2)	0 d (7.2)
Peak Thermophilic Stage	2, 4, 6, 8 d (7.4, 7.6, 7.8, 7.10)	2, 4, 6, 9 d (7.4, 7.6, 7.8, 7.11)
Cooling Stage	10, 12, 14, 21, 28, 35, 42 d (7.12, 7.14, 7.16, 7.23, 7.30, 8.6, 8.13)	10, 12, 14, 21, 28, 35, 42 d (7.12, 7.14, 7.16, 7.23, 7.30, 8.6, 8.13)
Decay Stage	49 d (8.20)	49 d (8.20)

. A portion of the fresh samples was used to measure pH and electrical conductivity (EC), while the remaining samples were air-dried, ground, and analyzed for organic constituents, including total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), total potassium (TK), and humic substances (humic substance carbon [HSc], humic acid carbon [HAc], fulvic acid carbon [FAc]), supplemented by infrared spectroscopic analysis. These samples were labeled as CR, CR1, CR2, CR3, and CRG, CRG1, CRG2, CRG3, respectively, corresponding to samples collected during the initial, thermophilic, and maturation stages of composting (days 2, 8, and 49, respectively). Stage-specific samples were stored at −80 °C in an ultra-low-temperature freezer for high-throughput microbial sequencing. The experimental workflow is illustrated in Figure 2.

2.2. Measurement of Experimental Parameters During Composting

2.2.1. Basic Physiochemical Properties

The pH value was determined by desktop pH meter (PHS-3C), electrical conductivity (EC) by conductivity meter (DDS-307), and total organic carbon (TOC) [28] was measured by potassium dichromate external heating, total nitrogen (TN) was determined using a Kayan ometer [29], total phosphorus (TP) by spectrophotometer [30], and total potassium (TK) by flame photometer [31]. The seed germination index (GI) was verified with Chinese cabbage seeds, and the seed germination index was calculated using Equation (1).

$$I_{GI}={\displaystyle \frac{M1\times R1}{M2\times R2}} \times 100\%$$

(1)

In the equation, I_GI is the seed germination index, M1 and M2 are the mean germination rate for compost treatment and blank control, R1 and R2 are the mean root length in cm for compost treatment and blank control, respectively.

2.2.2. Enzyme Activities

Urease activity was measured by phenol sodium–sodium hypochlorite, cellulase activity by 3,5-dinitrosalicylic acid, β-glucosidase activity by nitrophenol colorimeteran assay [32].

2.2.3. Humic Substance Characterization

Humus carbon fractions were analyzed following International Humic Substances Society protocols [33]. Sequential extraction techniques isolate humic acid and fulvic acid components, with organic carbon content determined through dichromate oxidation. Structural evolution of humic fractions was investigated using lyophilized specimens analyzed by FT-IR spectroscopy (4000–500 cm⁻¹ spectral range), focusing on functional treatment transitions [34].

2.2.4. High-Throughput Sequencing

Microbial diversity was assessed using the Illumina NovaSeq sequencing platform with a paired-end (PE) sequencing strategy, generating small fragment libraries for sequencing. Bacterial diversity was analyzed based on the 16S rRNA gene (V3–V4 regions), yielding a total of 1,049,870 paired-end reads, from which 445,786 high-quality clean reads were obtained after quality control. Each sample retained a minimum of 39,469 clean reads, with an average of 74,298 clean reads per sample.

To ensure data accuracy, Trimmomatic v0.33 was used for raw read filtering, followed by Cutadapt v1.9.1, which removed primer sequences to obtain high-quality reads. The DADA2 algorithm in QIIME2 v2020.6 was applied for denoising, chimera removal, and generation of final non-chimeric reads.

Fungal diversity was assessed based on internal transcribed spacer (ITS) region sequencing, specifically targeting the ITS1 region. This analysis yielded 814,996 paired end reads, from which 396,480 high-quality clean reads were obtained. Each sample retained at least 25,670 clean reads, with an average of 66,080 clean reads per sample. The same quality control and denoising procedures were applied as described for bacterial data.

All sequencing and bioinformatics analyses were conducted by Biomarker Technologies Co., Ltd. (Beijing, China). The raw sequence data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject ID PRJNA1230376 (Study Accession Number: SRP567228).

2.3. Data Processing and Statistical Analysis

The data were analyzed and drawn using Origin Ro 2024, IBM SPSS Statistics 26.0, and RStudio 4.4.2. Differences in bacterial and fungal community composition were assessed using principal component analysis (PCA) based on Bray–Curtis distance. T-test of species abundance data between treatments using Meta stats software (version 1.2, http://metastats.cbcb.umd.edu/, accessed on 10 June 2024). Functional prediction of the bacterial community was performed using PICRUSt2 and FAPROTAX, and the closed OTU Table obtained by QIIME was compared with the KEGG database to obtain functional prediction information [35]. Classification of fungal nutrient types and their functional treatments was performed with the FUNGuild tool (https://www.funguild.org/).

3. Results and Discussion

3.1. Compost Safety Index

The heavy metal content and seed germination index (GI) under compost treatment are presented in

Table 3. Heavy metal content and seed germination index under compost treatment.

Samples	GI (%)	Pb (mg/kg)	Cd (mg/kg)	Cr (mg/kg)	As (mg/kg)	Hg (mg/kg)
CR	132 ± 5.2 a	13.2 ± 0.7 a	0.7 ± 0.1 a	34.2 ± 1.2 a	1.8 ± 0.2 a	0.2 ± 0.05 a
CRG	118 ± 4.9 b	17.2 ± 0.6 b	1.3 ± 0.2 b	44.2 ± 3.3 b	3.3 ± 0.2 b	0.6 ± 0.1 b

Note: Different letters represent different treatments with significant differences. (p < 0.05).

. GI is the key indicator for assessing compost maturity. Generally, when the GI value is greater than 80%, composting can be considered to completely reach [36]. In this study, the GI values of both treatments exceeded 80%, with the GI of the CR treatment being 14% higher than that of the CRG treatment. This suggests that the CR treatment is more suitable for plant cultivation. Thus, both composting treatments successfully achieved maturation. The limits of heavy metals in the international European Union organic fertilizer standard are lead (Pb) 50 mg/kg, cadmium (Cd) 3 mg/kg, chromium (Cr) 100 mg/kg, arsenic (As) 10 mg/kg, and mercury (Hg) 2.0 mg/kg [37]. In this study, the heavy metal content in both treatments was significantly lower than the international limit values, indicating their safety for agricultural use. The GI of the CR treatment was significantly higher than that of the CRG treatment, while the heavy metal content in the CR treatment was significantly lower, suggesting better maturation and more stable heavy metal conditions. In contrast, the CRG treatment exhibited higher heavy metal content, which may be attributed to the high lignin content of the paper lignin (PL). The slow degradation of PL during composting reduces the microbial efficiency in heavy metal conversion [38,39].

3.2. Basic Physical and Chemical Indexes in Composting Process

3.2.1. Temperature, pH, and EC Values

Compost temperature serves as a key indicator of the composting process and reflects its sanitization efficiency [40]. In the CR treatment, the peak temperature of 57 °C was recorded on day 8 and maintained above 50 °C for 7 days. In the CRG treatment, the maximum temperature of 63 °C was observed on day 9 and sustained above 50 °C for 12 days (Figure 3). Temperature during aerobic composting not only influences microbial metabolism but also affects the degradation of organic matter, ultimately impacting its decomposition and the maturation process of the compost pile [41]. Under the condition of regulating the environmental temperature, the two treatments of reactors can reach the temperature rise under simple composting conditions. Compared to CR, CRG exhibited a higher peak temperature and a longer thermophilic phase, with both treatments meeting the temperature requirements specified in composting standards. The CRG reactors heat up quickly and the high temperature period lasts long, which is more conducive to composting and decay.

The pH trends in both treatments were similar, starting at approximately 6.5. The pH increased progressively with composting duration, reaching peak values by day 49. In CR, the pH ranged from 6.52 to 8.17, while in CRG, it ranged from 6.54 to 8.27 (Figure 4). The pH value of the two stacks is maintained between 6.52 and 8.27, which meets the requirements of composting fermentation environment and is beneficial to maintain the normal activity of microorganisms [42].

EC value can reflect whether composting has a toxic effect on crop growth. In the two piles, the EC value is between 2.6 and 4.3 ms/cm, and when the EC value is <9.0 ms/cm, it will not inhibit seed germination [43]. In the CR treatment, EC decreased by 27.3%, from 4.2 mS/cm to 3.3 mS/cm. In the CRG treatment, EC dropped by 31.6%, from 3.8 mS/cm to 2.6 mS/cm. EC is directly proportional to the soluble salt content in organic fertilizers and serves as an indicator of salinity levels [44]. Electrical conductivity (EC) values decreased over the composting period, declining as composting progressed. According to USDA standards, the EC value after composting should be below 4.3 mS/cm to avoid classification as high-salt compost, which could adversely affect plant growth. By the end of the composting period (day 49), both CR and CRG treatments had EC values below 4 mS/cm, complying with these standards (Figure 5).

3.2.2. Total Organic Carbon (TOC)

TOC content decreased over time in both the CR and CRG treatments (Figure 6). In the CR treatment, TOC decreased from 310.29 g/kg to 281.54 g/kg, a reduction of 10.2%. In the CRG treatment, TOC declined more significantly, from 381.43 g/kg to 281.98 g/kg, representing a 35.3% decrease. This higher TOC degradation rate in the CRG treatment suggests that microbial activity was more intense, leading to more efficient decomposition. The greater reduction in organic carbon in the CRG treatment could also be attributed to the higher initial nutrient content of the organic materials, which may have facilitated more rapid microbial activity and carbon mineralization [45]. This finding aligns with previous research indicating that organic matter decomposition is more efficient in materials with higher microbial activity, such as those composted with biogas residue and kitchen waste [46].

3.2.3. Total Nitrogen (TN)

TN content increased in both treatments (Figure 7). In the CR treatment, TN rose from 8.6 g/kg to 18.49 g/kg, an increase of 115%. The peak TN content (23.7 g/kg) occurred on day 14, after which it gradually decreased and then increased again by day 49. In the CRG treatment, TN content increased from 12.45 g/kg to 16.42 g/kg, representing a 31.9% increase. The highest TN content (20.52 g/kg) was recorded on day 12, followed by a gradual decline and a slight increase by day 49. The cumulative TN content was higher in the CR treatment compared to the CRG treatment. This suggests that the CR treatment facilitated greater nitrogen accumulation, likely due to enhanced nitrogen retention and microbial conversion during composting [47]. The increase in TN content in both treatments can be attributed to the composting process promoting the adsorption of NH₄⁺ and NH₃, accelerating nitrification, and enhancing the cumulative nitrogen content [48].

3.2.4. Total Phosphorus (TP)

In both treatments, TP content increased over time, albeit with relatively small changes (Figure 8). In the CR treatment, TP increased from 7.3 g/kg to 7.7 g/kg, a 5.5% increase. In the CRG treatment, TP increased from 12.3 g/kg to 13.2 g/kg, representing a 7.3% increase. Although the increase in TP was modest in both treatments, the cumulative TP increase was more pronounced in the CRG treatment. This may be attributed to the higher initial phosphorus content of the materials used in the CRG treatment, which facilitated more release and accumulation of phosphorus during composting [49].

3.2.5. Total Potassium (TK)

Figure 9 shows the changes in TK content. TK content increased in both treatments over time. In the CR treatment, TK content rose by 26.3%, from 25.5 g/kg to 32.2 g/kg. Similarly, the CRG treatment exhibited a 66.2% increase, from 15.7 g/kg to 26.1 g/kg. The larger increase in the CRG treatment could be due to the higher initial potassium content in the organic materials, which resulted in a greater release of potassium during composting [50]. The higher percentage increase in TK in the CRG treatment may reflect the higher efficiency of potassium mineralization and microbial uptake in the more nutrient-rich materials [51].

3.3. Changes in Enzyme Activity During the Composting Process

3.3.1. Urease Activity

As shown in Figure 10, urease activity in both the CR and CRG treatments followed a similar trend: an initial increase followed by a decrease over time. In the CR treatment, urease activity gradually increased from the start, peaking at 361.3 μg/g/d on day 10. Afterward, it decreased to 249.2 μg/g/d by day 49, reflecting a reduction of 9.2%. Similarly, the CRG treatment reached its highest urease activity of 401.7 μg/g/d on day 10, which then declined to 319.2 μg/g/d by day 49, a 6.9% reduction. Throughout the composting process, urease activity in the CR treatment remained consistently higher than in the CRG treatment, with a difference of 2.3%.

The decrease in urease activity over time is likely due to the shift in microbial processes as the composting progressed [52]. As the temperature in the pile dropped and nitrifying bacteria became more active, a significant portion of ammonium nitrogen was converted to nitrate nitrogen, reducing the need for urease in nitrogen transformation [53]. This aligns with the increase in total nitrogen content observed in both treatments, highlighting the ongoing nitrogen cycle during composting. The reduction in urease activity suggests that the composting process gradually transitions from nitrogen fixation to nitrification, a common feature in microbial-driven composting systems [54].

3.3.2. Cellulase Activity

As shown in Figure 11, cellulase activity showed a general decline over time in both treatments. In the CR treatment, cellulase activity decreased sharply in the first 4 days, reaching its lowest value of 119.5 μg/g/d. From day 21 to day 49, activity increased slightly from 178.3 μg/g/d to 150.2 μg/g/d, a 15.8% decrease. The CRG treatment also showed a similar trend, with a sharp initial decline followed by a brief increase from day 6 to day 8. Between days 8 and 21, activity gradually decreased, reaching a minimum of 172.4 μg/g/d on day 21, and by day 49, it decreased by 24.4%, from 257.7 μg/g/d to 194.9 μg/g/d.

The reduction in cellulase activity is indicative of the breakdown of cellulose and lignin, two of the most recalcitrant organic compounds in composting [55]. As the compost pile matured, microbial activity shifted toward decomposing more complex and resistant substances [53]. The decrease in cellulase activity correlates with the color deepening and particle size reduction in the pile, suggesting that cellulose and lignin were gradually disintegrated, aiding in the compost’s maturation. Additionally, the lower cellulase activity in the CR treatment compared to the CRG treatment may be due to the higher lignin content in the CRG material, which is more resistant to microbial degradation, requiring more time for breakdown [56].

3.3.3. β-Glucosidase Activity

As shown in Figure 12, β-glucosidase activity exhibited a general increase over time in both treatments. In the CR treatment, β-glucosidase activity surged from day 0 to day 2 and again from day 4 to day 6, followed by a brief dip on days 10 to 12, reaching a low of 2.2 μg/g/d. Afterward, the enzyme activity steadily increased from 2.3 μg/g/d to 3 μg/g/d by day 49, representing a 23.3% increase. In the CRG treatment, β-glucosidase activity increased from day 0 to day 6, then slightly decreased before rising again from day 14 to day 49, reaching a final value of 3.1 μg/g/d, which is a 47.6% increase.

The greater increase in β-glucosidase activity in the CRG treatment compared to the CR treatment suggests that the CRG material provided more available carbon sources, promoting the hydrolysis of cellulose into glucose [55]. β-glucosidase plays a crucial role in breaking down cellulose, and its increased activity indicates that composting is progressing by facilitating the breakdown of complex organic materials into simpler compounds [57]. The increase in β-glucosidase activity was accompanied by a decrease in cellulase activity, confirming that as the compost matures, the decomposition process transitions from cellulose breakdown to more advanced biochemical reactions, where hydrolytic enzymes like β-glucosidase become more involved in facilitating microbial processes [58].

The contrasting trends observed in the changes in urease, cellulase, and β-glucosidase activity highlight distinct microbial processes occurring throughout the composting process. The decrease in urease and cellulase activities indicates the microbial shift from nitrogen transformation and cellulose degradation to more stable, complex biochemical breakdowns. In contrast, the steady increase in β-glucosidase activity suggests an ongoing transformation of organic matter, providing carbon sources to promote microbial activity [59]. These changes reflect the complex interactions between microorganisms and organic matter, as they drive the composting process toward maturity.

3.4. Changes in Humic Substance Components During the Composting Process

3.4.1. Humic Substance Carbon (HSc)

As shown in Figure 13, the accumulation of humic substance carbon (HSc) increased in both the CR and CRG treatments over the composting period. In the CR treatment, HSc increased from 42.1 g/kg to 62.5 g/kg, reflecting a 48.5% increase. In the CRG treatment, HSc increased from 47.6 g/kg to 57.9 g/kg, a 21.6% increase. The CR treatment exhibited a higher accumulation of HSc compared to the CRG treatment, and this trend remained consistent throughout the composting process. The observed increase in HSc in both treatments can be attributed to the humification process during composting. Humification is the transformation of organic matter into the humus, and it generally involves the stabilization of organic carbon [60]. The higher increase in HSc in the CR treatment indicates that the organic material in this treatment was more effectively transformed into stable humic substances. This could be due to the higher temperature in the CR treatment, which promotes the breakdown of complex organic compounds into simpler precursors that are more easily converted into humic substances [61].

3.4.2. Humic Acid Carbon (HAc)

As shown in Figure 14, humic acid carbon (HAc) exhibited an overall increase overtime in both treatments, with the CR treatment displaying a more substantial rise. In the CR treatment, HAc increased from 9.2 g/kg to 27.1 g/kg, a cumulative increase of 194.6%. In the CRG treatment, HAc increased from 11.3 g/kg to 19.7 g/kg, a 74.3% increase. The CR treatment accumulated more HAc than the CRG treatment, and this trend persisted throughout the composting process. The increase in HAc content reflects the ongoing humification process, as HAc is a key component of humic substances [62]. The more pronounced increase in HAc in the CR treatment suggests that the material in this treatment was more conducive to the formation of humic acids, likely due to a higher rate of organic matter decomposition and more favorable conditions for humification. The higher temperatures and microbial activity in the CR treatment likely accelerated the transformation of organic matter into HAc [63].

3.4.3. Fulvic Acid Carbon (FAc)

As shown in Figure 15, fulvic acid carbon (FAc) exhibited a general decrease over time in both treatments during the composting period. In the CR treatment, FAc decreased from 32.5 g/kg to 27.7 g/kg, a reduction of 14.8%. In the CRG treatment, FAc decreased from 35.1 g/kg to 26.0 g/kg, a 25.9% reduction. The CRG treatment exhibited a more significant decrease in FAc compared to the CR treatment, and after day 21, the FAc content in the CRG treatment remained lower than in the CR treatment. The decrease in FAc content is consistent with the typical decomposition process in composting, where more unstable and easily decomposable substances like FAc are broken down by microorganisms [63]. FAc, being more readily available to microbes, is generally the first to degrade and can be further transformed into more stable forms such as humic acids [64]. This shift from FAc to HAc indicates the stabilization of organic matter and the ongoing humification process, which is crucial for enhancing the stability of the compost.

3.4.4. Humification Index (HA/FA)

As shown in Figure 16, the humification index (HA/FA ratio) increased over time in both treatments, reflecting the ongoing humification process. In the CR treatment, the HA/FA ratio increased from 0.283 to 0.978, a 245.6% increase. In the CRG treatment, the HA/FA ratio increased from 0.322 to 0.758, a 135.4% increase. Throughout the composting process, the HA/FA ratio was higher in CR treatment compared to the CRG treatment. The increase in the HA/FA ratio in both treatments indicates that the organic matter is undergoing humification, with a shift from the more labile fulvic acids (FA) to the more stable humic acids (HA). The higher HA/FA ratio in the CR treatment suggests a greater degree of humification, likely due to more favorable conditions for humus formation, such as higher temperatures and more intensive microbial activity [65]. The increase in this ratio reflects the stabilization and maturation of the compost, which is essential for improving the soil fertility of the final product [66].

3.5. HA and FA Infrared Spectral Changes

3.5.1. Humic Acid (HA)

Figure 17a,b illustrate the FTIR spectra of humic acid (HA) in the CR and CRG treatments. The observed spectra indicate comparable functional groups in both treatments, suggesting similar compositional transformations during composting. Notably, the absorption peaks at 3435 cm⁻¹ (CRG) and 3420 cm⁻¹ (CR) correspond to the -OH stretching vibration in alcohols, phenols, and organic acids. The gradual attenuation of these peaks suggests microbial degradation of small organic molecules, including amino acids [67].

The peak at 1650 cm⁻¹, indicative of C=C stretching vibration in aromatic rings, became more pronounced over time, reflecting enhanced aromaticity and stabilization of HA [68]. Additionally, the peak near 1450 cm⁻¹, attributed to asymmetric C-O stretching and O-H deformation in carboxyl groups, intensified, signifying ongoing organic matter decomposition and mineralization [69].

Further structural modifications were evident in the peak at 1143 cm⁻¹, associated with C-O stretching in polysaccharides, which diminished as composting progressed. This decline indicates microbial decomposition of carbohydrate fractions [70]. The peak at 871 cm⁻¹, linked to alkanes, olefins, and alcohols, increased in the CR treatment, suggesting the accumulation of these compounds during composting [69]. Similarly, the peaks at 625 cm⁻¹ (CR) and 623 cm⁻¹ (CRG), related to simple carbohydrates like arabinose and xylose, weakened, highlighting carbohydrate breakdown [71].

3.5.2. Fulvic Acid (FA)

Figure 17c,d present the FTIR spectra of fulvic acid (FA) in the CR and CRG treatments. The spectrum of FA exhibited similar trends to HA, with key absorption peaks indicative of humification and organic matter transformation. The peak at 2910 cm⁻¹, corresponding to aliphatic C-H₃ and C-H₂ stretching, intensified in the CRG treatment, reflecting an increase in aliphatic compounds [72]. Aromatic structural changes were observed at 1650 cm⁻¹ and 1638 cm⁻¹, which is characteristic of humic substances. These peaks became more pronounced in both treatments, indicating the development of stable aromatic components [73]. The additional peak around 2350 cm⁻¹ suggested the formation of C=C bonds, further supporting the transformation of organic matter into humic substances [74].

Carbohydrate decomposition was evident through the peaks at 1186 cm⁻¹, 1146 cm⁻¹, and 1050 cm⁻¹, all associated with C-O stretching vibrations in polysaccharides. These peaks increased over the composting period, reflecting the progressive breakdown of carbohydrate fractions [75]. Additionally, the peak at 870 cm⁻¹, linked to simple carbohydrates like arabinose and xylose, narrowed as composting proceeded, reflecting a reduction in carbohydrate content [71]. The peak near 587 cm⁻¹, attributed to the decomposition of aliphatic compounds, grew more distinct, suggesting microbial decomposition [73].

FTIR analysis revealed consistent functional groups in both HA and FA, including -OH, C=C, and C-O in HA, and C-H3, C-H2, C=C, and C-O in FA. These findings align with previous composting studies involving waste mushroom substrates and livestock manure [76]. The attenuation of peaks at 3435 cm⁻¹, 1143 cm⁻¹, and 625 cm⁻¹, along with the narrowing of the 870 cm⁻¹ peak, underscores the progressive breakdown of carbohydrates. This transformation corresponds to a decline in total organic carbon (TOC) and a shift from simple sugars to humified, stable organic matter [77]. The enhanced 1650 cm⁻¹ peak in HA and the increased 1650 cm⁻¹ and 1638 cm⁻¹ peaks in FA further support the formation of humic substances, reinforcing compost stabilization.

3.6. Microbial Community Dynamics and Functional Implications

3.6.1. Microbial Dynamics

Based on temperature variations, the composting process was divided into three phases: the initial stage (CR1, CRG1), high temperature period (CR2, CRG2), and decay stage (CR3, CRG3). Distinct microbial succession patterns emerged in both treatments, primarily driven by substrate composition and thermophilic conditions (Figure 18 and Figure 19).

Throughout the composting phases, bacterial communities were primarily dominated by Proteobacteria and Bacteroidota, albeit with contrasting trends (Figure 18a). Proteobacteria abundance declined more steeply in CR (24%) compared to CRG (17%), likely due to the higher lignin content in CR, which restricted labile carbon availability and suppressed copiotrophic taxa. In contrast, Bacteroidota exhibited an 18% increase in CR and a 16% increase in CRG, benefiting from their ability to degrade recalcitrant polymers (e.g., chitin) and solubilize phosphorus, which is consistent with the higher TP accumulation observed in CR (

Table 4. Statistics of the bacterial Alpha diversity index.

Sample ID	ACE Index	Chao1 Index	Simpson Index	Shannon Index	Coverage
CR1	657.81 ± 65.78 Aa	661.00 ± 66.10 Aa	0.92 ± 0.09 Aa	5.57 ± 0.56 Aa	0.9999
CR2	797.53 ± 79.75 Ba	798.50 ± 79.85 Ba	0.96 ± 0.10 Aa	6.36 ± 0.64 Ba	0.9999
CR3	951.90 ± 95.19 Ca	954.00 ± 95.40 Ca	0.97 ± 0.10 Aa	7.45 ± 0.75 Ca	0.9996
CRG1	524.89 ± 52.49 Ab	524.60 ± 52.46 Ab	0.97 ± 0.10 Aa	6.06 ± 0.61 Ab	0.9999
CRG2	886.15 ± 88.62 Bb	887.46 ± 88.75 Bb	0.99 ± 0.10 Aa	7.71 ± 0.77 Bb	0.9997
CRG3	1092.06 ± 109.21 Cb	1093.21 ± 109.32 Cb	0.97 ± 0.10 Aa	7.51 ± 0.75 Ba	0.9997

Note: Different capital letters represent the same pile in different composting periods have significant differences; different lowercase letters represent significant difference between different piles in the same composting period (p < 0.05), the same below.

) [78,79].

At the genus level, both Pseudomonas (cellulolytic) and Pseudoxanthomonas (ligninolytic) declined markedly in both treatments (CR: 26.3% and 21%; CRG: 24% and 15%), reflecting substrate depletion as composting progressed (Figure 18b). Notably, CRG exhibited higher bacterial diversity (Chao1: 1093 vs. 954 in CR; Shannon: 7.71 vs. 7.45), suggesting that weed-derived cellulose promoted niche diversification, enhancing microbial resilience in the later composting stages [80].

Fungal community dynamics displayed contrasting trends across treatments (Figure 19a). Ascomycota abundance increased by 55% in CR, while Basidiomycota dominance rose by 39% in CRG. The enhanced presence of Ascomycota in CR correlated with its ability to produce lignocellulolytic enzymes (e.g., cellulases), thereby promoting nitrogen retention via increased urease activity (Figure 19b) [81]. Conversely, Basidiomycota enrichment in CRG aligned with its preference for degrading herbaceous cellulose, accelerating total organic carbon (TOC) mineralization [82]. At the genus level, Auricularia and Aspergillus significant declines in both treatments (CR: 42% and 6%; CRG: 11% and 6%), indicating substrate depletion during maturation.

According to alpha diversity indices (Chao1, Simpson, Shannon), microbial diversity was initially higher in CR during the thermophilic phase, but CRG exhibited greater diversity in the maturation stage (

Table 5. Statistics of the fungal Alpha diversity index.

Sample ID	ACE Index	Chao1 Index	Simpson Index	Shannon Index	Coverage
CR1	314.91 ± 31.49 Aa	320.33 ± 32.03 Aa	0.89 ± 0.09 Aa	4.63 ± 0.46 Aa	0.9996
CR2	276.75 ± 27.68 Ba	276.25 ± 27.63 Ba	0.87 ± 0.09 Aa	3.92 ± 0.39 Ba	0.9998
CR3	187.95 ± 18.80 Ca	188.50 ± 18.85 Ca	0.28 ± 0.03 Ba	1.36 ± 0.14 Ca	0.9999
CRG1	273.58 ± 27.36 Ab	276.71 ± 27.67 Aa	0.88 ± 0.09 Aa	4.27 ± 0.43 Aa	0.9997
CRG2	183.87 ± 18.39 Bb	189.83 ± 18.98 Bb	0.68 ± 0.07 Ba	2.73 ± 0.27 Ba	0.9998
CRG3	233.87 ± 23.39 Ba	235.00 ± 23.50 Ba	0.74 ± 0.07 Bb	3.72 ± 0.37 Ba	0.9992

Note: Different capital letters represent the same pile in different composting periods have significant differences; different lowercase letters represent significant difference between different piles in the same composting period (p < 0.05).

). These findings underscore the role of substrate heterogeneity in shaping microbial succession and influencing composting efficiency and nutrient dynamics.

While bacterial diversity increased throughout composting, fungal diversity decreased, a trend with significant functional implications. The initial dominance of fungi, particularly Ascomycota, facilitated early-stage lignocellulose degradation, enhancing organic matter breakdown and nitrogen retention. However, as composting progressed, fungal activity declined, likely due to substrate depletion and increasing competition from bacterial communities specializing in secondary metabolite utilization and organic matter mineralization. The gradual shift toward higher bacterial diversity in CRG suggests greater microbial functional redundancy, which may enhance compost stability and accelerate the formation of humified organic matter [83].

3.6.2. Functional Implications

Bacterial Functions: PICRUSt2 analysis revealed elevated methanol oxidation (CR: +32%; CRG: +28%) and aerobic chemoheterotrophy (CR: +25%; CRG: +21%) during thermophilic phases, coinciding with Proteobacteria activity (Figure 20a). Fermentation pathways declined post-thermophilic phase (CR: −18%; CRG: −15%), marking maturation. CRG treatment sustained Firmicutes abundance (high-temperature phase: 22% vs. CR’s 15%) facilitated TN accumulation through amino acid metabolism, corroborating its higher TN retention [82].

Fungal Functions: FUN Guild annotation highlighted divergent saprotrophic strategies (Figure 20b). CR’s Ascomycota-driven “undefined saprotrophs” decreased (−40%), reflecting lignin depletion, while CRG’s “wood saprotrophs” increased (+25%), which is consistent with weed cellulose degradation. Pathogenic fungi (e.g., Cercospora) were suppressed in both treatments (−90%), validating compost sanitization [84].

3.7. Composting Correlation Between Microbial Community Structure and Nutrient Indicators in Compost

This study analyzed the complex relationships between microbial community structures and nutrient dynamics during the composting of CR and CRG. While statistical correlations were identified (Figure 21), interpreting these patterns provided insights into biological mechanisms and their connection to composting processes.

In the CR treatment, nitrogen-related microbial interactions revealed distinct metabolic trade-offs among taxa. TN exhibited a negative correlation with Proteobacteria, emphasizing their role as fast-growing copiotrophs in the early composting stages. These bacteria rapidly utilize labile carbon sources, potentially leading to nitrogen immobilization, which in turn reduces TN availability. Conversely, TN showed a positive correlation with Bacteroidota and Basidiomycota. The enrichment of Bacteroidota was associated with their ability to degrade nitrogen-rich polymers, such as chitin, thereby releasing ammonium through protease activity. Similarly, Basidiomycota contributed to lignin decomposition, indirectly facilitating the release of bound nitrogen [85]. TOC negatively correlated with Basidiomycota, reflecting their enzymatic preference for lignin degradation over labile carbon utilization. In contrast, TOC positively correlated with Pseudomonas, Firmicutes, and Auricularia, microbial taxa known for their roles in cellulose and hemicellulose hydrolysis, thereby promoting carbon mineralization [86]. TP positively correlated with Bacteroidota, highlighting their role in phosphatase secretion, which enhances phosphorus solubilization. However, the negative correlation between TP and Proteobacteria (including Pseudomonas) suggests competition for phosphorus uptake, potentially limiting TP availability in the composting matrix [87]. Additionally, TK exhibited a negative correlation with Aspergillus, indicating a possible immobilization of potassium within fungal biomass or organic complexes during the composting process [88]. This suggests that fungal-driven potassium sequestration could play a key role in nutrient retention and stabilization within the composting system.

In the CRG treatment, the incorporation of green grass residues significantly influenced microbial-nutrient interactions, leading to distinct patterns in nitrogen, carbon, and phosphorus cycling. TN showed a positive correlation with Aspergillus and Pseudoxanthomonas. These taxa played complementary roles in nitrogen retention, with Aspergillus enhancing urease activity and Pseudoxanthomonas facilitating nitrogen fixation, collectively improving compost nitrogen availability [89]. TOC exhibited a strong positive correlation with Firmicutes, which thrived on cellulose-rich substrates derived from grass biomass. Their production of β-glucosidase enzymes promoted carbon turnover and accelerated TOC degradation [90]. TP and TK both positively correlated with Bacteroidota, a microbial group known for organic acid excretion, which enhances inorganic phosphate solubilization and potassium release [91]. However, TK displayed a negative correlation with Firmicutes in CRG, suggesting a competitive exclusion mechanism—where the dominance of Firmicutes in carbon metabolism may suppress potassium-releasing microbial taxa. Urease activity was strongly correlated with Ascomycota, reflecting their functional specialization in nitrogen cycling. Additionally, pH negatively correlated with Firmicutes, reinforcing their role in organic acid production during cellulose fermentation, which may contribute to localized acidification [92].

Microbial succession patterns provided further insight into nutrient transformations during composting. Bacterial diversity increased throughout the process, driven by the progressive decomposition of macromolecules and the subsequent release of available nutrients. In contrast, fungal diversity declined, likely due to the depletion of simple sugars and lignin derivatives, which serve as primary fungal substrates [93]. The key microbial groups identified in this study exhibited distinct functional roles during composting. Proteobacteria, prevalent in both treatments, were central to organic matter decomposition and nutrient cycling, particularly through their urease activity, which facilitated nitrogen transformations [94]. Bacteroidota, significantly enriched as composting progressed, contributed to carbon degradation and phosphorus mobilization, with their increase being more pronounced in the CR treatment, corresponding to enhanced organic matter decomposition and TOC reduction [95]. Firmicutes, known for their acid-producing capability, were particularly active in the CRG treatment, where they accelerated cellulose hydrolysis and promoted nitrogen mineralization, resulting in higher TN accumulation [96]. Patescibacteria, which peaked during the decay stage, thrived in conditions of elevated bacterial diversity, likely contributing to nutrient recycling and metabolic exchanges. Ascomycota, the dominant fungal phylum in the CR treatment, played a pivotal role in nitrogen cycling by driving urease activity, which led to higher TN retention in CR compared to CRG. These findings highlight the substrate-specific metabolic activities of microbial groups and their critical contributions to nutrient dynamics.

The lignin-rich substrate characteristics of CR dictated microbial functional trade-offs: Basidiomycota dominated lignin metabolism, driving humification and nitrogen retention. In contrast, the cellulose-rich grass in CRG fostered Firmicutes and Bacteroidota communities, promoting rapid carbon mineralization and nutrient (phosphorus and potassium) release. The role of Auricularia, a lignocellulolytic fungus, is hypothesized to bridge these microbial interactions by initiating lignin-cellulose decomposition in the early stages, which facilitates subsequent bacterial activity. Future studies should explore Auricularia’s enzymatic synergy with cellulose-degrading Firmicutes and nitrogen-metabolizing Proteobacteria.

3.8. Partial Least Squares Path Model Analysis of Compost Maturity

These well-fitted models revealed intricate associations governing humus content, demonstrating variable impacts (positive, neutral, or negative) on compost maturation.

To quantify the complex interactions among microbial communities, enzyme activities, and physicochemical properties during composting, structural equation modeling (SEM) was employed using the Partial Least Squares Path Modeling (PLS-PM) approach [97]. The models for CR (Figure 22a) and CRG (Figure 22b) stacks visually represent dynamic inter-variable relationships, with goodness-of-fit (GOF) values of 0.62 and 0.72, respectively, indicating robust explanatory power for data variability [98].

The results indicate that microbial community plays a crucial role in regulating enzymatic activity and chemical properties in both treatments, with a stronger influence observed in CRG (1.326 **) compared to CR (1.219 **). Enzymatic activities, including β-glucosidase, cellulase, and urease, are positively correlated with microbial dynamics, showing a slightly higher impact on humus content in CRG (1.082 *) than in CR (1.009 *). Furthermore, chemical properties such as EC, pH, TK, TP, TN, and TOC exhibit a more pronounced effect on humus content in CRG (0.892 **) than in CR (0.21), highlighting improved organic matter stabilization with grass addition. The humus fraction analysis reveals that humic acid (HAc), fulvic acid (FAc), and the HA/FA ratio exhibit stronger positive effects in the mixed compost system, indicating enhanced humification processes [99].

In CR treatment, microbial communities primarily influence humus content indirectly by improving chemical properties and enhancing enzymatic activity [100]; however, the synergy between these factors remains relatively weak. In contrast, the mixed compost (A. auricula + grass) significantly strengthens the synergy among microbial functions, enzymatic activity, and chemical properties, ultimately promoting the formation of high-quality humus. These findings suggest that integrating grass into fungal residue composting optimizes microbial diversity, nutrient cycling, and soil organic matter stabilization, making it a more effective strategy for improving soil health and agricultural sustainability.

4. Conclusions

Both CR and CRG treatments achieved full compost maturity and met established safety criteria, including neutral-to-alkaline pH, acceptable electrical conductivity, and absence of phytotoxicity. However, the two treatments exhibited distinct nutrient transformation patterns and compost characteristics, highlighting their differentiated applications in agricultural systems.

The CR treatment, consisting of Auricularia auricula residues alone, demonstrated superior nitrogen retention and humic substance formation, with significantly higher total nitrogen (+115%), humic substance carbon (+48.5%), humic acid carbon (+194.6%), and a greater humic-to-fulvic acid (HAc/FAc) ratio (0.978). These attributes indicate greater organic matter stabilization, making CR compost particularly suitable as a long-term soil amendment for enhancing soil organic matter pools and improving nitrogen conservation.

In contrast, the CRG treatment, which incorporated green grass as a nitrogen-rich amendment, significantly enhanced microbial activity and enzymatic function, leading to accelerated organic matter degradation. This was reflected by a greater reduction in total organic carbon (−35.3%) and increased accumulation of total phosphorus (+7.3%) and total potassium (+66.2%), alongside elevated activities of urease, cellulase, and β-glucosidase. These results suggest CRG compost is more appropriate for short-term agricultural use, especially where rapid nutrient release and immediate fertilization effects are desired.

Collectively, these findings underscore the critical role of feedstock composition in determining compost quality, nutrient availability, and application scenarios. Tailored composting strategies based on specific substrate blends can support more targeted and sustainable soil fertility management in diverse agroecosystems.

Study Limitations and Future Directions

While this study provides valuable insights into composting performance under controlled conditions, it did not assess the long-term agronomic effectiveness of CR and CRG composts under field application. Future research should focus on evaluating their impact on crop productivity, soil microbial ecology, nutrient cycling, and environmental safety over extended periods to further optimize their integration into sustainable agricultural practices.