Application of combination of nano molybdenum material and penicillium oxalicum in humification of traditional Chinese medicine residue compost

Abstract

The invention belongs to the technical field of organic waste resource utilization, and particularly relates to application of a nano molybdenum material combined with penicillium oxalicum in traditional Chinese medicine residue compost humification. Specifically, research finds that based on the synergistic effect of nano molybdenum disulfide (MoS2) and penicillium oxalicum, the growth and reproduction capability of the penicillium oxalicum and the activity of cellulase and xylanase can be remarkably improved, functional bacteria such as Thermobifida can be enriched, a compost microbial community structure can be optimized, and the composting yield can be improved. The conversion of dissolved organic matters (DOM) from easily degradable components to aromatic humic substances is accelerated, and the compost humification degree and the product fertility of the traditional Chinese medicine residues (such as cocklebur fruit residues) are improved. The method is simple to operate and controllable in cost, provides a new technical path and theoretical basis for efficient composting treatment of the traditional Chinese medicine residues and other agricultural organic wastes, and has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of waste resource utilization technology, specifically involving the application of nano-molybdenum materials combined with Penicillium oxalate in the composting and humification of traditional Chinese medicine residues. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] According to statistics, the annual output of Chinese medicinal herb residue (CMHRs) reaches as high as 70 million tons, of which Xanthium sibiricum (… Xanthii Fructus The annual production of medicinal herb residue is approximately 1 million tons. Xanthium sibiricum residue is characterized by its high carbon content, rich content of cellulose, hemicellulose, and highly active saponins. It also exhibits inhibitory effects on various plant pathogens, making it a potentially valuable biomass resource. However, Xanthium sibiricum residue has a high water content and is easily perishable; improper management can easily lead to soil, air, and water pollution, and currently, there is a lack of effective methods for its resource utilization.

[0004] Aerobic composting is an advanced technology for organic waste treatment, boasting advantages such as high decomposition efficiency and environmental cleanliness. However, traditional composting techniques suffer from drawbacks such as long cycles and low levels of humification. To address these issues, researchers have attempted to improve composting efficiency by adding microbial agents or nanomaterials. *Penicillium oxalate* possesses a strong cellulase secretion capacity, which can accelerate cellulose degradation and enhance composting efficiency. Nanomaterials such as ZnO NPs and Fe3O4 NPs can prolong the high-temperature period of composting and promote the conversion of fulvic acid to humic acid. However, the effects of adding microorganisms or nanomaterials alone are limited. Dissolved organic matter (DOM) is an important precursor to humic matter formation, and its molecular transformation process directly reflects the degree of humification in composting. The inventors discovered that existing technologies lack sufficient research on the molecular transformation mechanism of DOM under the synergistic effect of nanomaterials and microorganisms, limiting the optimization and upgrading of composting technology. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides the application of molybdenum nanomaterials combined with *Penicillium oxalate* in the composting and humification of traditional Chinese medicine residues. Specifically, this invention, through research, has found that based on the synergistic effect of nano-molybdenum disulfide (MoS2) and *Penicillium oxalate*, the growth and reproduction capacity of *Penicillium oxalate*, as well as the activities of cellulase and xylanase, can be significantly enhanced, enriching... Thermobifida This invention utilizes functional bacteria to optimize the microbial community structure of composting, accelerate the transformation of dissolved organic matter (DOM) from easily degradable components to aromatic humic substances, and improve the humification degree and fertility of composted medicinal herb residues (such as Xanthium sibiricum residue). Based on the above research results, this invention was completed.

[0006] To achieve the above-mentioned objectives, the present invention discloses the following technical solutions: The first aspect of the present invention provides the application of nano-molybdenum materials combined with Penicillium oxalate in the composting and humification of traditional Chinese medicine residues.

[0007] The nano-molybdenum material includes, but is not limited to, nano-Mo, MoO3, MoC and MoS2, with nano-MoS2 being preferred. Furthermore, experiments conducted in this invention have demonstrated that nano-MoS2 significantly promotes the growth, spore germination, and cellulase and xylanase activities of Penicillium oxalate.

[0008] The medicinal residue can be Xanthium sibiricum residue. Xanthium sibiricum residue is the waste residue after processing Xanthium sibiricum into a traditional Chinese medicine.

[0009] Furthermore, the application specifically includes: promoting the composting and humification process of cocklebur residue and enhancing the fertility of compost products.

[0010] A second aspect of the present invention provides a method for promoting the composting and humification of Chinese medicinal herb residue, the method comprising: applying nano-MoS2 and Penicillium oxalate to composting raw materials containing Xanthium sibiricum residue, and carrying out aerobic composting fermentation.

[0011] The composting raw material containing cocklebur residue includes cocklebur residue, cow dung and rice straw, with a mass ratio of 1-5:5-10:0.1-3, preferably 3:6:1; Furthermore, the initial C / N ratio of the composting raw materials is adjusted to 25-35:1 (preferably 30:1), and the moisture content is 50-75% (preferably 65%). Adjusting the C / N ratio and moisture content is beneficial for subsequent composting fermentation.

[0012] Furthermore, the amount of nano-MoS2 added accounts for 10-50 mg / kg of the compost raw material (preferably 20 mg / kg).

[0013] The *Penicillium oxalate* is added as a suspension of *Penicillium oxalate* conidia, and the amount of the *Penicillium oxalate* conidia suspension added is 1-5 wt% (preferably 2 wt%) of the compost raw material; furthermore, the concentration of *Penicillium oxalate* in the *Penicillium oxalate* conidia suspension is 0.1-10 × 10⁻⁶. 9 spores / mL, preferably 1×10 9 spores / mL.

[0014] Furthermore, the method also includes turning the compost pile every 2-5 days (preferably every 3 days) during aerobic composting fermentation until the composting is completed.

[0015] Preferably, the composting fermentation time is controlled to be 30-50 days (preferably 42 days).

[0016] A third aspect of the present invention provides the application of the above-described method or the compost material of Chinese herbal medicine residue obtained by the above-described method in crop cultivation.

[0017] In this invention, the crop can be a food crop or a cash crop, etc., and tobacco is preferred.

[0018] The application specifically promotes crop growth, specifically the growth of both the above-ground and underground parts of tobacco.

[0019] Compared with existing technologies, one or more of the above technical solutions have achieved the following beneficial effects: The above-mentioned technical solution significantly shortens the composting cycle of Xanthium sibiricum residue through the synergistic effect of nano-MoS2 and Penicillium oxalate, achieving high decomposition in just 42 days, thus solving the problem of long composting cycles in traditional methods. Simultaneously, this synergistic system significantly enhances the humification degree of the compost, resulting in a humification index (HI) of 46.47%, a humic acid (HA) content of 48.58%, and a seed germination index (GI) of 111.93%, demonstrating significantly superior fertility compared to traditional compost products. The synergistic effect of nano-MoS2 and Penicillium oxalate promotes the degradation of recalcitrant organic matter such as cellulose and hemicellulose, improving the resource utilization efficiency of Xanthium sibiricum residue and reducing environmental pollution. Furthermore, the compost product significantly promotes crop growth; when used in tobacco cultivation, the root fresh weight is 3.4 times that of traditional compost, while also exhibiting good disease prevention effects, with a control efficacy of 86.6%.

[0020] In summary, the above-mentioned technical solutions are simple to operate and cost-controllable, providing a new technical path and theoretical basis for the efficient composting treatment of Chinese medicinal herb residue and other agricultural organic wastes, and have good practical application value. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 Effects of different concentrations of nanomaterials on colony diameter growth and spore germination of Penicillium oxalate (a), colony growth diagram (b), and the effect of different concentrations of nano MoS2 on enzyme activity of Penicillium oxalate (c).

[0023] Figure 2 Different treatment temperatures (a), pH (b), EC (c), TN (d), and NH4 during composting. + -N(e), NO3- Changes in N(f), TC(g), OM(h), DOC(i), humic acid(j), fulvic acid(k), and HI(l) over time.

[0024] Figure 3 Microbial community dynamics in CK, CS, S, and SS treatment groups during composting and humification: Principal coordinate analysis (a), phylum-level microbial community (b), top 50 functional microorganisms at the genus level (c), and structural equation modeling (d, SEM) illustrate the direct and indirect effects of treatments on humic acid, fulvic acid, DOM, and bacterial community α-diversity (Shannon index). Continuous arrows and dashed arrows indicate significant and insignificant relationships, respectively. Adjacent numbers in the same direction as the arrow represent path coefficients, and the width of the arrow is proportional to the degree of the path coefficient. Green and red arrows indicate positive and negative relationships, respectively. 2 The value represents the proportion of variance explained by each variable. The significance level is expressed as... P<0.05 P<0.01 P < 0.001 indicates that the standardized total effect (direct plus indirect effect) calculated by SEM is shown below the SEM. The low chi-square (χ²) values ​​listed below the SEM are... 2 ), non-significant probability level (P>0.05) (d).

[0025] Figure 4 Venn diagrams of DOM molecules in different composting treatments (a); van Krevelen diagrams of DOM molecules common to different composting treatments (b) and unique DOM molecule variations in the SS treatment during composting (c); van Krevelen diagrams of DOM molecule variation in CK(l), CS(m), S(n), and SS(o) samples, respectively, according to different compound and formulation categories. The size of the spheres represents the relative abundance of a molecular formula.

[0026] Figure 5 Radar plots show the number of possible precursor-product pairs during different composting reactions in SS (a); quality difference network analysis plots show possible reaction pathways and possible structural formulas of CHO and CHON molecules (b); network correlation analysis of DOM composition with the top 50 microbial genera in CK (c), CS (d), S (e), and SS (f) (|r| ≥ 0.6, p<0.05).

[0027] Figure 6 . CK, CS, S processing quality network diagram.

[0028] Figure 7 Potential mechanisms by which compost products promote the composting and humification process. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] The present invention will be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Experimental conditions not specifically specified in the examples are generally performed under conventional conditions or as recommended by the selling company; unless otherwise specified in the present invention, these conditions are commercially available.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1. Materials and Methods 1.1 Experimental Materials and Procedures The tested *Penicillium oxalate* strain was obtained from the Tobacco Research Institute, Chinese Academy of Agricultural Sciences (accession number: CGMCC NO. 40499, this strain is disclosed in Chinese patent CN116606750A). Nano-molybdenum materials, including Mo, MoO3, MoS2, and MoC, were purchased from Zhongke Leiming (Beijing) Technology Co., Ltd. Nano-molybdenum concentrations of 1 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 50 mg / L were added to plate culture media, with a blank control included. Each treatment was repeated three times. Mycelial discs of uniform age and 5 mm in diameter were punched from the periphery of the purified *Penicillium oxalate* plates and inverted in the center of plates containing different nano-molybdenum concentrations. Each treatment was repeated three times. After incubation at 21 ℃ for 7 days, the colony diameter was measured using the cross-crossing method. A *Penicillium oxalate* conidial suspension was prepared, and the conidial suspension concentration was controlled to reach 4 × 10⁻⁶ using a hemocytometer. 3 / mL, take 0.1 mL and spread it evenly on plates containing different concentrations of nano-molybdenum. After growing for about 6, 12, and 18 hours, observe the spore germination under a microscope according to the spore germination standard (germ tube length greater than spore diameter) and count the spores. Activate *Penicillium oxalate* and inoculate it into liquid fermentation medium with the same concentration of nano-MoS2 as above. Incubate at 28 ℃ and 150 r / min for 96 h. Centrifuge the fermentation broth at 4 ℃ and 10,000 r / min for 10 min and collect the supernatant to complete the preparation of crude enzyme solution. The reducing sugar content in the crude enzyme solution is determined using the DNS method, and the enzyme activity, cellulase activity, and xylanase activity are also measured.

[0034] The composting materials mainly consist of cocklebur residue, cow dung, and rice straw. The cocklebur residue is the waste residue from the processing of cocklebur into a traditional Chinese medicine, sourced from Lanzhou Heren Pharmaceutical Co., Ltd. The cow dung and rice straw were purchased from farmers in Dezhou, Shandong Province, and Lianyungang, Jiangsu Province, respectively. The basic physicochemical properties of the composting materials are shown in Table 1. The cocklebur residue, cow dung, and straw were mixed in a dry weight ratio of 3:6:1, and the initial C / N ratio was adjusted to 30:1. This experiment included four treatment groups: CK (cocklebur residue + cow dung + rice straw), CS (cocklebur residue + cow dung + rice straw + 2 wt%)... Penicillium oxalicum at 1×10 9 (spores / mL), S (Xanthium sibiricum residue + cow dung + rice straw + 20 mg / kg nano-MoS2) and SS (Xanthium sibiricum residue + cow dung + rice straw + 2wt%) Penicillium oxalicum at 1×10 9 (Spores / mL + 20 mg / kg nano-MoS2). The moisture content of all raw materials was adjusted to 65% after mixing. The compost mixture was placed in a fermentation tank for aerobic composting, manually turning the pile every 3 days until the end of 42 days. Samples were collected from the compost pile using a five-point sampling method on days 1, 5, 10, 21, 35, and 42. The collected samples were evenly divided into three parts: the first part was used for subsequent air-dried sample analysis; the second part was stored at -4℃ for fresh sample analysis; and the third part was stored at -80℃ for microbial community analysis and other DOM analyses.

[0035] Table 1. Physicochemical properties of compost raw materials

[0036] 1.2 Determination of Physicochemical Properties of Compost Temperature at the center layer of the reactor core and ambient temperature were monitored twice daily (9:00 and 18:00), and the average temperature was recorded. Fresh samples were dissolved in water at a ratio of 1:10 (w / v) and shaken at 120 rpm for 60 minutes before measuring conductivity (EC) and pH. Organic matter (OM) was determined using the potassium dichromate titration method; dissolved organic carbon (DOC) was measured using a TOC analyzer (Shimadzu TOC-L CPH Total Organic Carbon Analyzer); total toxicity (TC) and total nitrogen (TN) were determined using an elemental analyzer (EA3100 elemental analyzer); and ammonium nitrogen (NH4) was measured. + -N) and nitrate nitrogen (NO3) - -N) were determined by indophenol blue colorimetry and ultraviolet spectrophotometry, respectively; the seed germination index (GI) of compost products was determined according to "Organic Fertilizer NY / T 525-2021"; humic acid and fulvic acid were determined by acid precipitation method.

[0037] 1.3 High-throughput sequencing and bioinformatics analysis Following the instructions, 0.25g of sample was weighed and DNA was extracted using the FastPure Bacteria DNAIsolation Mini Kit (Vazyme). DNA quality and concentration were assessed by 1.0% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified using universal primer pairs 338f (actcctacgggagcagcag) and 806R (GGACTACHVGGGTWTCTAAT). Because low-quality sequences and disproportionate N bases in the raw data could affect subsequent analysis, high-quality optimized sequence data was obtained through raw data quality control, sequence assembly and filtering, and chimera removal for subsequent information analysis. UPARSE was used to cluster these sequences into operational taxonomic units (OTUs) with 97% similarity. PCoA analysis was performed using R language with direct Euclidean distance plotting. Alpha diversity of the compost bacterial community (Chao and Shannon indices) was analyzed using Mothur version v.1.30.2 software. Relative species abundance was analyzed using Python 2.7 software, and bubble charts were generated using Origin 2021 software. The model was fitted using AMOS 17.0 software with maximum likelihood estimation. The model-data fit was comprehensively evaluated using indicators such as χ² test (P>0.05), goodness-of-fit index (GFI>0.90), and root mean square error of approximation (RMSEA<0.05).

[0038] 1.4 Fourier Transform Example: Cyclotron Resonance Mass Spectrometry (FT-ICR MS) Analysis of the Molecular Composition of DOM The molecular composition of DOM was analyzed using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICRMS, SolariX 15T, Bruker) equipped with a negative ionization mode electrospray ionization source. Prior to analysis, DOM samples underwent solid-phase extraction (SPE) pretreatment using a Bond Elut-PPL column (500 mg, 6 mL, Abbott). The samples were then dried with ultrapure nitrogen and redissolved in 1 mL of a 1:1 methanol / water (v / v) solution for FT-ICR MS analysis. 200 μL of sample was directly injected for mass spectrometry analysis using an electrospray ionization (ESI) source in negative ion detection mode. The main detection parameters are as follows: continuous injection mode, injection rate of 120 μL / h, capillary inlet voltage of 4 kV, ion accumulation time of 0.03 s, mass range of 100-1600 Da, number of sampling points of 4 M 32-bit data, and time-domain signal superposition 300 times to improve signal-to-noise ratio. Before sample detection, the instrument was calibrated with 10 mmol / L sodium formate. After sample detection, soluble organic matter (known molecular formula) was used for internal standard calibration. After calibration, the mass error was less than 1 ppm. Mass spectrometry data were calibrated using known CHO compounds from the DOM as internal standards. After calibration, molecular formula matching was performed according to known compound parameters to derive mass spectrometry peaks with a signal-to-noise ratio (S / N) greater than 10 and a mass range of 150-1000, which were used for molecular formula allocation. 12 C 3-∞ , 1 H 1-∞ , 16 O 1-∞ , 14 N 0-3 , 32 S 0-3 P 0-2 The assigned quality tolerance was set to 1.0 ppm. Quality difference network analysis was performed using established R code to analyze reaction pathways based on quality difference networks. Joint network analysis involved calculating Spearman correlation coefficients using R and screening for significant correlations (r ≥ 0.5 and p < 0.05). Finally, visualization was performed using the open-source Gephi software (0.9.4). The microbial community-driven DOM molecular transformation and potential mechanism diagram were drawn using KingDraw software in conjunction with Adobe Illustrator 2020.

[0039] 1.5 Plant Pot Experiment Compost was mixed with potting soil at a ratio of 5% (by weight). Tobacco seedlings with uniform growth were selected and transplanted. The blank control used only potting soil. The seedlings were placed in an artificial climate chamber for cultivation. Each treatment consisted of 12 seedlings, and the results were repeated 3 times. About 14 days after transplanting, the leaf length, leaf width, root length, and root fresh weight of the tobacco plants were measured. The maximum photochemical efficiency (Fv / Fm) of the tobacco plants was determined using a chlorophyll fluorometer.

[0040] 1.6 Statistical Analysis and Graphing Data were analyzed using DPS software (Hangzhou Ruifeng Information Technology Co., Ltd., Hangzhou, China) in version 9.01. The mean difference was assessed at the p-significance level using the Tukey Hidden Significance Test (HSD), with p < 0.05.

[0041] 2 Results and Discussion 2.1 Effects of different molybdenum nanomaterials on Penicillium oxalate This study investigated the effects of four molybdenum nanomaterials (40 nm Mo, MoO3, MoC, and MoS2) at different concentrations on the colony growth, spore germination, and cellulase and xylanase activities of *Penicillium oxalate*. At concentrations of 0–10 mg / L, the effects of each molybdenum nanomaterial on colony diameter were not significant. Figure 1 (ab) indicates that *Penicillium oxalate* has a certain tolerance to low concentrations of nano-molybdenum. When the concentration increases to 20-50 mg / L, the effects of different nano-molybdenum materials on *Penicillium oxalate* show significant differentiation. In particular, at a concentration of 20 mg / L, the colony diameter of the nano-MoS2 treatment group was significantly larger than that of other groups (up to 5.0 cm), showing a significant growth-promoting effect. This may be due to the large specific surface area and slow-release capacity of molybdenum ions provided by the layered structure of MoS2, which is beneficial for hyphal absorption and utilization. Spore germination rate ( Figure 1 a) This further verified the superiority of MoS2 under the 20 mg / L condition, with a germination rate as high as 90.63%, which was significantly higher than that of other treatment groups. Figure 1Enzyme activity analysis showed that, under 20 mg / L conditions, the cellulase and xylanase activities in the MoS2-treated group reached 88.066 U / mL and 97.736 U / mL, respectively, significantly higher than those in other material treatment groups and the control group. This indicates that under these conditions, nano-MoS2 not only has no obvious toxicity but may also act as an inducer or cofactor, enhancing the enzyme synthesis ability of *Penicillium oxalate*. In contrast, the other three nano-molybdenum materials performed poorly at 20 mg / L concentrations. The MoC-treated group, in particular, exhibited the smallest colony diameter, significantly reduced spore germination rate, and low enzyme activity. The 40 nm Mo and MoO3-treated groups performed between MoS2 and MoC, but were significantly better than the MoC-treated group. When the concentration was further increased to 30-50 mg / L, all nano-molybdenum materials showed significant inhibitory effects, with significantly reduced colony diameter, a sharp decrease in spore germination rate, and a substantial reduction in enzyme activity. This may be related to their high surface activity and tendency to aggregate, leading to enhanced biotoxicity. Both MoC and Mo exhibited a sharp decline in spore germination rates at concentrations of 30-50 mg / L, with MoC showing a germination rate of only 2% at 30 mg / L, indicating its strong biotoxicity. This may be related to the small particle size, easy penetration of the spore wall, and oxidative stress. The good dispersibility and biocompatibility of MoS2 may have reduced the physical adsorption damage to the spore wall by nanoparticles. Simultaneously, molybdenum, as a trace element, participates in fungal metabolism, promoting germination. Furthermore, all nanomaterials resulted in a significant decrease in enzyme activity at 50 mg / L, particularly cellulase activity, which dropped to 6.323 U / mL. This suggests that high concentrations of nano-molybdenum generally inhibit metabolic activity, possibly related to cell membrane damage and ROS accumulation.

[0042] 2.2 Changes in matter during composting This study systematically analyzed the transformation patterns of compost materials by monitoring the changes in various physicochemical indicators during the composting process under different treatments (CK, CS, S, SS). Compost temperature is an important indicator reflecting microbial activity and the composting process. During the 11-day thermophilic period, the temperature of the S group rapidly reached a peak of 64.5℃ on day 2. The SS and CS groups reached their peak temperatures of 63.8℃ and 61.6℃ respectively on day 4. Meanwhile, the temperature of the CK group continued to rise, reaching its peak of 60.9℃ on day 5. Figure 2 a). Electrical conductivity reflects the content of soluble salts in compost. Overall, the EC value increased slightly in the middle stage of composting and tended to stabilize in the later stage. The EC values ​​of the SS and S treatments were higher in the later stage (reaching 1.59 and 1.65 mS / cm, respectively), which may be related to the fact that the addition of MoS2 promoted the release of mineral salts. The EC value of the CS treatment was moderate, indicating that Penicillium oxalate has a certain regulatory effect on salt accumulation. Figure 2b). During composting, the pH value initially rose and then fell, remaining generally alkaline. All treatments reached their peak values ​​(8.64–8.76) around day 14, subsequently gradually decreasing to 7.6–7.9; the pH values ​​of the SS and S treatments decreased more rapidly in the later stages. Figure 2 c). The total nitrogen (TN) content generally increased during composting, indicating that organic nitrogen mineralization and fixation occurred simultaneously. The highest TN content (26.30 g / kg) was observed in the SS treatment at day 21, suggesting that combined inoculation was beneficial for nitrogen retention. Figure 2 d). NH4 + -N is higher in the early stages of composting ( Figure 2 e), and then gradually decreased. At 42 days, the CK and CS treatments dropped to 30–40 g / kg, while the S and SS treatments remained at 250–290 g / kg, indicating that MoS2 delayed the conversion of ammonium nitrogen and NO3. - -N gradually accumulated in the later stages, with the highest SS concentration (0.965 g / kg) at 42 days, indicating strong nitrification. TC and OM generally decreased during composting. Figure 2 The concentrations of organic matter (OM) were high at 42 days, indicating that organic matter was continuously degraded. OM levels remained high in the CK and CS treatments (>200 g / kg), while they were low in the S and SS treatments (180–186 g / kg), indicating that MoS2 promoted the decomposition of organic matter. DOC is an important indicator of readily degradable organic components in compost, and its changes directly reflect the dynamics of organic matter decomposition and transformation. The DOC content in all treatments gradually decreased during composting. Figure 2 i) indicates that readily degradable organic matter is continuously utilized by microorganisms. The SS treatment had a higher DOC content in the early stage (9.814 g / kg), indicating that the combined inoculation promoted the dissolution and initial degradation of organic matter. In the later stages, the DOC content in all treatments tended to stabilize, ranging from 3.7 to 4.1 mg / g, indicating that the readily degradable components had been largely decomposed and the compost had stabilized. The decrease in DOC was consistent with the decreasing trend of TC and OM, further confirming the mineralization and transformation process of organic matter. HA and FA are important components of humus. The HA content gradually increased during composting ( Figure 2 j), the SS treatment showed the highest humification level at 42 days (48.58%), indicating the highest degree of humification. FA content, however, showed a decreasing trend. Figure 2 The HI value decreased significantly, especially in the SS and S treatments, indicating that MoS2 promoted the conversion of FA to HA. The CS treatment also showed higher HA accumulation, suggesting that Penicillium oxalate is beneficial for humus formation. The HI value gradually increased with the composting process (k). Figure 2The HI (hygroscopic index) reflects the increased degree of humification. The SS treatment had the highest HI (hygroscopic index) at 42 days (46.47), significantly higher than other treatments, indicating that the combined inoculation significantly promoted the synthesis and stabilization of humus. The S treatment also had a high HI (42.84%), further confirming the positive role of MoS2 in the humification process. The GI (hygroscopic index) is an important indicator for evaluating compost maturity and toxicity. At 42 days, the SS treatment had the highest GI (111.93%), indicating the best maturity and lowest toxicity. The CK treatment had a GI of 101.25%, also meeting the maturity standard. The CS treatment had a lower GI (74.93%), possibly related to the incomplete degradation of some metabolites.

[0043] 2.3 Dynamic succession of microbial communities during composting and humification To reveal the role of composting microorganisms in the humification process, the microbial communities of different treatments were analyzed. PCoA analysis showed that the bacterial communities of the CS, S, and SS treatments at composting days 5, 10, and 35 were different from the CK sample. Figure 3 a) The sampling time in composting determines the differences in the compost microbial community; different sampling times have a greater impact on the microbial community than different composting treatments. Dynamic monitoring of bacterial community α-diversity during composting revealed that the effects of exogenous addition of nano-MoS2 and Penicillium oxalate were significantly stage-specific. Differences were observed among the treatment groups at the start of composting; the CS treatment showed potential to promote microbial reproduction from the initial stage of composting, and its Chao index remained at the highest level throughout the entire maturation stage. Figure 3 (b) In contrast, the S treatment generally exhibited an inhibitory effect on bacterial community richness. The SS treatment, however, showed a continuous increase in bacterial community richness after the compost entered the maturation stage, effectively mitigating the inhibitory effect of nano-MoS2 on the microbial community. It reached its peak at the end of composting (42 days), significantly higher than other treatments, and had a positive promoting effect on the deep degradation of organic matter and the formation of humus.

[0044] Figure 3 c shows the succession of the top 50 most abundant bacterial genera in different composting treatments. Although the microbial composition of each treatment group was dominated by aerobic heterotrophic bacteria at the beginning of composting, differences existed due to the different additives. The dominant bacterial genera were the same in the CK and CS treatments, mainly... Bacillus (44.51-47.57%) and norank_f__Bacillaceae (75.39-84.73%), the S treatment showed different genera distributions. Sinibacillus (57.02%) and Bacillus (55.07%) were the main bacterial genera, while the dominant bacteria in the SS treatment were... Enterobacter (98.63%) and Bacillus(39.26%). As composting enters the high-temperature stage, the microbial community structure undergoes dramatic succession, with each treatment exhibiting different adaptation strategies. The dominant bacteria in the early stage of the CK treatment... Bacillus and norank_f__Bacillaceae Abundance decreased sharply, and was replaced by Tepidimicrobium (33.18%) and Thermobacillus A significant increase in thermophilic bacteria, such as (22.98%), was reported. Tepidmicrobium Widely present in composting systems, it can degrade cellulose components during the thermophilic phase. CS treatment also exhibits a similar trend. Neobacillus (22.23%) and Thermobacillus (42.82%) became the core microbial community at this stage. It is noteworthy that the S treatment at this stage... Symbiobacterium (68.11%) and norank_c__Bacilli (66.40%) was the dominant strain. The changes were most significant in the SS treatment, with the initial absolute dominant bacteria... Enterobacter They almost disappeared (reduced to 0.002%), and the community structure became more diversified. Neobacillus (28.24%) and Tepidimicrobium (26.91%) became the new dominant bacteria, indicating that microbial succession was more rapid and thorough under the combined treatment. Compared with the S treatment, the SS treatment... Neobacillus The higher abundance of certain bacteria (such as *I. spp.*) may be attributed to the improved porosity and nutrient balance of the heap after SS treatment, which avoided overly extreme microenvironments, allowing more thermophilic bacteria to work synergistically, potentially resulting in higher and more stable high-temperature decomposition efficiency. It is worth noting that... Thermobifida As a thermophilic actinomycete, its relative abundance showed explosive growth in all treatments, but significant differences existed between treatments. In the control (CK) treatment, its abundance rose to 12.6-21.4%, while treatments with added exogenous inoculants exhibited even higher increases, with CS, S, and SS treatments reaching 34.0-36.3%, 29.8-37.7%, and 35.4-37.0%, respectively. Importantly, the SS treatment successfully maintained its abundance throughout the high-temperature period. Thermobifida The highest and most stable abundance level (35-37%). ThermobifidaThis is a core functional genus of bacteria that degrades stubborn polysaccharides (such as cellulose) during the high-temperature period of composting. It secretes abundant carbohydrate-active enzymes, especially glycoside hydrolases and polysaccharide monooxygenases (AAs, such as AA10), which are tightly adsorbed onto the surface of cellulose crystals through their carbohydrate-binding modules (CBMs, such as CBM2). The unique function of AA10 (Lytic Polysaccharide Monooxygenase, LPMO) lies in its ability to oxidatively cleave cellulose chains in the crystalline region, exposing more hydrolysis sites and thus greatly improving the degradation efficiency of subsequent cellulases. In this study, the SS treatment was applied during the high-temperature period... Thermobifida The sustained high abundance of this substance indicates its robust cellulose oxidative degradation capacity, providing crucial microbiological evidence to explain the efficient organic matter degradation and humification processes observed in SS treatment. Furthermore, Thermobifida The function of this bacterium is not limited to direct enzymatic degradation. It can also indirectly enhance the metabolic activity and stability of the entire system by sharing metabolites such as vitamin B12 (cobalamin) and promoting the growth of other bacteria in the compost microbial community. Therefore, SS treatment, through a combined addition strategy, may create a system centered on… Thermobifida The dominant reciprocal functional microbial community is the fundamental reason why it can drive the degradation of lignocellulose and the formation of humic precursors more effectively during high temperatures compared to single addition (CS or S) and control group (CK).

[0045] DOC is a core component of DOM, and its concentration changes are widely considered the most direct and fundamental indicator for measuring DOM quantity dynamics. This study uses structural equation modeling to further understand the impact of various post-treatment factors on compost DOM changes. Figure 3 d). Standardized total effects analysis showed that the treatment groups had a direct effect on the levels of humic acid (HA) and fulvic acid (FA). Furthermore, the treatment groups also had a significant indirect impact on changes in dissolved organic matter (DOM), primarily by regulating the production of HA and FA. Meanwhile, bacterial community structure not only directly drove changes in DOM but also played a crucial mediating role in the dynamic changes of DOM by significantly influencing the transformation of HA and FA.

[0046] 2.4 Molecular composition of DOM during composting To further investigate the molecular composition and transformation patterns of DOM (morphological components) during the composting of cocklebur residue, this study systematically analyzed the DOM molecular composition of four treatments (CK, CS, S, SS) at different composting time points based on FT-ICR MS data. Figure 4 As shown in figure a, the DOM molecules in the initial stage of composting in different treatments (CK, CS, S, SS) showed similar composition, mainly including protein / aliphatic, lipid, and lignin / carboxyl-rich alicyclic molecules. Figure 4 b), which originates from cocklebur residue, cow dung, and rice straw. Furthermore, only a few DOM molecules with aromatic structures (H / C < 1.5) were found in the SS treatment, which were completely removed after the composting process. Figure 4 c). For example Figure 4 As shown in dg, in the early stages of composting, the proportions of protein / aliphatic and lipid compounds were relatively high in all treatments. However, by day 35 of composting, both protein and lipid content significantly decreased in all treatments, indicating that these readily degradable components were preferentially utilized by microorganisms. The relative abundance of lignin / CRAMs further increased, suggesting that these recalcitrant aromatic structures gradually enriched and became a major component of the DOM during composting. Furthermore, tannins also accumulated in the later stages of composting (days 21 and 35), possibly related to polyphenolic intermediates produced by lignin degradation. From an elemental composition perspective ( Figure 4 CHO and CHON molecules are the main components of DOM. During composting, the relative abundance of CHO compounds decreases, while the abundance of CHON compounds increases significantly, indicating that nitrogen-containing organic matter (such as proteins and amino acids) accumulates relatively during degradation or is enriched through microbial synthesis. The intensity-weighted average molecular weight (MW) of all treatments... wa The intensity-weighted aromaticity index (AI_mod) increased with composting time (Table 2), indicating that small molecules gradually aggregated to form larger molecular weight humic substances. wa ) and double bond equivalence (DBE) wa The increase in H / C indicates enhanced aromaticity and unsaturation of the DOM, further confirming the advancement of the humification process. wa The decrease and O / C wa A slight increase or stabilization indicates a decrease in aliphatic components and an increase in oxidation, which is consistent with the degradation and oxidation processes of organic matter. N / C wa The general increase in CK (from 0.04 to 0.07) also corresponds to the accumulation of CHON-like molecules, indicating that nitrogen-containing structures are gradually occupying a more important position in DOM. SS-treated DBE wa (10.15) and AI_mod waThe values ​​(0.32) were the highest, indicating that their DOM contained more aromatic structures and fused-ring compounds, and had the highest degree of humification. Meanwhile, although the number of molecules in the SS group (3331) was lower than other treatments, its MWwa (387.07 Da) was the lowest. Combined with higher DBE and AI_mod values, this indicates that the DOM molecules in the SS group underwent more significant condensation and aromatization reactions, generating more complex and stable humic substances. Although the CK group had the highest number of molecules (5400), its DBEwa and AI_modwa were lower, indicating that its DOM was still mainly composed of aliphatic and easily degradable components, with a lower degree of humification. The molecular structure parameters of the CS and S groups were between those of CK and SS, indicating that the addition of Penicillium oxalate or nano-MoS2 alone could promote humification, but the effect was not as good as the synergistic treatment. To further reveal the transformation pathway of DOM molecules, this study compared and analyzed the DOM molecules decomposed, generated, and retained during composting. Figure 4 The decomposed DOM molecules are mainly lipids, proteins / aliphatic compounds, and carbohydrates. These easily degradable components are rapidly utilized by microorganisms during the high-temperature phase, generating humic precursors such as small-molecule organic acids, amines, and polyphenols. Newly generated DOM molecules are mainly concentrated in lignin / CRAM compounds, tannins, and polycyclic aromatic hydrocarbon regions, exhibiting higher aromaticity and oxygen content, which helps improve the humification degree and stability of the compost product. The retained DOM molecules are mainly structurally stable CRAM compounds and lignin compounds. These substances are difficult to further degrade and are ultimately retained in the mature compost product, improving the compost's stability.

[0047] Table 2. Intensity-weighted parameters of DOM molecular composition in the samples

[0048] 2.5 Microbial Community-Driven DOM Molecular Transformation To better understand DOM transformation in SS-treated compost, quality difference network analysis was employed, a practical method for associating precursor and product pairs. Stage A is dominated by oxidation (+O) and preliminary hydrolysis (+H2O); Stage B is dominated by deep oxidation (+2O-2H) and sulfur metabolism (-SH2). Dealkylation reactions (such as C2H4, C3H6) are prominent in the intermediate stage (Stage B), potentially accelerating small molecule degradation and providing precursors for humic substances. Figure 5 a). Deamination is active at this stage; in the Streck deamination reaction (–NH3 + O), the terminal amino group readily converts to a carbonyl group. This reaction shifts the molecule towards a higher O / C ratio and a lower H / C ratio, and many nitrogen-containing compounds react via this pathway. Figure 5b); The dominant reactions in stage C are reducing condensation (-2H) and humification (-CO2). Phenolic acids and aliphatic acids (such as acetic acid) form ester or ether bonds through dehydrogenation to generate humic acid (HA) and fulvic acid (FA). The decarboxylation reaction reaches its highest value in the later stage (stage C), significantly better than other groups, indicating a significant synergistic effect. Figure 6 Amino groups (-NH2) are integrated into the humic skeleton via Maillard reactions (CHON ratio 48.15%-44.05%). The intermediates of these reactions, as important precursors, readily undergo aromatization and polymerization, exhibiting amino acid-like structures to produce unsaturated and aromatic compounds that resemble humic substances in some physicochemical properties. In terms of relative abundance by molecular category, groups S and SS promote the formation of nitrogen- and sulfur-containing humic substances by accelerating the decomposition of CHO compounds and increasing the ratio of nitrogen- and sulfur-containing organic matter (CHON / CHONS), consistent with the enhanced dealkylation and dealkylation reactions in the reaction data. In summary, the cascade reaction, arranged chronologically, can be summarized as follows: starting with DOM hydrolysis and oxidation, followed by dealkylation, desulfurization, and decarboxylation cascades, finally condensing into humic substances.

[0049] like Figure 5 As shown in the diagram, a two-factor network analysis was used to explore the potential relationships between DOM composition and microbial genera in different composting treatments. The CK group had 40 nodes and 213 edges, CS had 45 nodes and 219 edges, S had 44 nodes and 204 edges, and SS had 54 nodes and 226 edges, reflecting a more complex and closer interaction between SS microorganisms inoculated with MoS2 and Penicillium oxalate and organic matter. Protein / aliphatic, lipid, fused aromatic structures, CHON, and CHOS had the highest centrality in the three network graphs, indicating that proteins and lipids are the main active organic substrates interacting with microorganisms in compost. At the microbial genus level, the core microorganisms also differed among the treatment groups, with the CK and CS treatments showing the most significant differences. Sporosarcina Anaeromyxobacter,Phaselicystis,Vulgatibacter The main focus is on S and SS processing. Thermobifida Vulgatibacter, Paenibacillus, Bacillus It is the core genus. Most of these microorganisms belong to... Firmicutes Actinobacteria and Myxococcus The bacteria exhibit strong organic matter degradation capabilities and environmental adaptability, especially in the S and SS groups, suggesting that nano-molybdenum sulfide may promote DOM transformation by enhancing the activity of these functional bacterial communities.

[0050] 2.6 Potential mechanisms of DOM molecular transformation As shown in Table 3, after adding compost CS, S, or SS treatments to the substrate soil, all measured indicators were significantly promoted (p<0.05), with effects significantly superior to the substrate soil and CK treatments. The root fresh weight of CS, S, and SS treatments was 3.1 times, 3.6 times, and 3.4 times that of CK treatment, respectively. This indicates that the addition of Penicillium oxalate and / or nano-molybdenum sulfide can significantly promote the biomass accumulation of tobacco roots by promoting compost humification.

[0051] Therefore, based on the above analysis, a potential mechanism by which nano-MoS2 and Penicillium oxalate promote rapid humification during composting is proposed. Figure 7 The addition of nano-MoS2 and Penicillium oxalate enhances the degradation capacity of functional bacteria by regulating the microbial community structure, promoting the conversion of fatty acids (FA) to hyaluronic acid (HA). Simultaneously, the bacterial community and humic acid work together to transform domino acids (DOM). In terms of reaction pathways, all treatments primarily involved oxidation reactions (+O, +H2O) in the early stages of composting, while dealkylation and deamination reactions dominated in the middle stages. The SS treatment showed a significant enhancement in decarboxylation in the later stages of composting, promoting the efficient conversion of small-molecule organic acids into humic acid, ultimately significantly improving the degree of humification and product quality of the compost. This strategy not only accelerates the resource utilization of Xanthium sibiricum medicinal residue but also provides a theoretical basis and technical pathway for the efficient composting of other agricultural organic wastes.

[0052] Table 3. Effects of different compost products on tobacco growth

[0053] In summary, nano-MoS2 significantly promoted the growth of Penicillium oxalate and its ability to produce key hydrolytic enzymes, laying a microbiological and enzymatic foundation for efficient degradation in the early stages of composting. The SS group treatment enriched [the enzymes] by regulating the microbial community structure. Thermobifida Functional microorganisms with highly efficient cellulose-degrading capabilities accelerated the decomposition and transformation of organic matter, significantly promoted the conversion of fulvic acid to humic acid, and greatly enhanced the humification degree of composting. The combination of nanomaterials and functional microorganisms produced a synergistic effect. FT-ICR MS analysis showed that oxidation reactions (+O, +H2O) dominated in the early stage of composting in all treatments, while dealkylation and deamination reactions dominated in the middle stage. The SS group enhanced the aromaticity and condensation degree of DOM components and promoted the efficient conversion of small molecule organic acids to humic acid by strengthening the decarboxylation reaction in the later stage of composting. The main active organic substrates in DOM, proteins and lipids, are mainly related to… Thermobifida, Vulgatibacter, Paenibacillus, Bacillus Interactions. Therefore, the combined treatment of nano-MoS2 and Penicillium oxalate is an effective strategy to improve the efficiency and quality of Xanthium sibiricum medicinal residue composting. This study is the first to apply a nanomaterial-microorganism synergistic system to medicinal residue composting and reveals its mechanism of action at the DOM molecular level, providing a new approach for the resource utilization of organic solid waste.

[0054] Matters not covered in this invention are common knowledge.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of nano-molybdenum materials combined with Penicillium oxalate in the composting and humification of traditional Chinese medicine residues.

2. The application as described in claim 1, characterized in that, The nano-molybdenum material includes nano-Mo, MoO3, MoC, and MoS2.

3. The application as described in claim 2, characterized in that, The molybdenum nanomaterial is nano-MoS2.

4. The application as described in claim 1, characterized in that, The medicinal residue mentioned is Xanthium sibiricum residue.

5. The application as described in any one of claims 1-4, characterized in that, The specific applications are: promoting the composting and humification process of cocklebur residue and enhancing the fertility of compost products.

6. A method for promoting the composting and humification of traditional Chinese medicine residue, characterized in that, The method includes: applying nano-MoS2 and Penicillium oxalate to compost raw materials containing cocklebur residue to carry out aerobic composting fermentation.

7. The method as described in claim 6, characterized in that, The composting raw materials containing cocklebur residue include cocklebur residue, cow dung, and rice straw, with a mass ratio of 1-5:5-10:0.1-3. Furthermore, the initial C / N ratio of the composting raw materials is adjusted to 25-35:1, and the moisture content is 50-75%.

8. The method as described in claim 6, characterized in that, The amount of nano-MoS2 added accounts for 10-50 mg / kg of the compost raw material; The *Penicillium oxalate* was added as a suspension of *Penicillium oxalate* conidia, and the amount of the *Penicillium oxalate* conidia suspension added was 1-5 wt% of the compost raw material; furthermore, the concentration of *Penicillium oxalate* in the *Penicillium oxalate* conidia suspension was 0.1-10 × 10⁻⁶. 9 spores / mL.

9. The method as described in claim 6, characterized in that, The method also includes turning the compost pile every 2-5 days during aerobic composting fermentation until the composting is completed. Preferably, the composting fermentation time is controlled to be 30-50 days.

10. The application of the method according to any one of claims 6-9 or the compost material of traditional Chinese medicine residue obtained by the method according to any one of claims 6-9 in crop cultivation; Furthermore, the crop is a food crop or a cash crop; preferably tobacco; Furthermore, the application specifically manifests as promoting crop growth.

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