Biochar-based composite reinforced microbial agent and application thereof

Abstract

The invention relates to a reinforced microbial inoculum, in particular to a charcoal-based composite reinforced microbial inoculum with a grease degradation function and application of the charcoal-based composite reinforced microbial inoculum. According to the invention, bacillus coagulans and Aspergillus niger are found as grease degradation functional bacteria for the first time, and can be loaded on a charcoal base to form a composite reinforced microbial inoculum for improving the quality of a compost product. According to the invention, the research and development experiment of the biochar-based composite reinforced microbial inoculum is carried out, the optimal preparation method and composting process parameters are compared and selected, the grease degradation efficiency of the biochar-based composite reinforced microbial inoculum is experimentally investigated, the action mechanism of the biochar-based composite reinforced microbial inoculum for improving the quality of compost products is clarified, and a feasible technical path is provided for improving the efficiency of household kitchen garbage compost.

Description

Technical Field

[0001] This invention relates to microbial inoculants, specifically to a biochar-based composite microbial inoculant and its application. Background Technology

[0002] Kitchen waste refers to easily perishable food waste generated by residents in their daily lives. It typically includes food scraps from cooking, leftover food from the table, and waste portions of fruits and vegetables. Using miniaturized kitchen waste treatment equipment for source reduction and resource utilization allows for on-site processing of kitchen waste, avoiding the additional costs and secondary pollution associated with centralized transportation, reducing operating energy consumption and costs, and improving the overall efficiency of waste resource utilization. The main problems with household kitchen waste composting technology in actual production are: insufficient compost stability, low composting efficiency, and low quality and application potential of compost products.

[0003] Existing technology reports mainly focus on improving composting efficiency by adding auxiliary materials such as biochar. While related inventions indicate that biochar can improve composting efficiency, further improvements in oil degradation efficiency are needed to address the material adhesion problem within the compost pile. Immobilizing functional microorganisms is a feasible approach to enhance composting efficiency. Due to its porous structure and strong adsorption capacity, biochar can effectively reduce nitrogen volatilization and odor emissions during composting, promote the humification process of kitchen waste, and improve the quality of compost products. Biochar-based composite inoculants consist of microbial cells attached to the porous structure of biochar, forming an immobilized system. The addition of these inoculants can significantly increase the diversity and abundance of dominant microbial communities in the composting system, promote the growth and reproduction of dominant microorganisms during composting, optimize the microbial community structure, enhance the stability of the composting system, significantly improve the organic matter degradation efficiency during aerobic composting of kitchen waste, accelerate pile heating, and shorten the maturation period. Biochar can also serve as an excellent attachment carrier for microorganisms, enhancing the activity of the microbial community. Therefore, biochar is actually a functional biological carrier. Its rich pore structure, large specific surface area and stable chemical properties can provide a large number of attachment sites for the growth and reproduction of microorganisms, promote the extracellular mass transfer of their metabolic substrates and products, and improve the extracellular electrochemical environment.

[0004] However, different bacteria and fungi have different metabolic pathways and functions, resulting in differences in their adsorption methods and effects on biochar. The selection of functional bacteria and preparation methods of biochar-based composite enhanced microbial agents have a significant impact on the composting effect of kitchen waste. Therefore, finding suitable biochar-based composite enhanced microbial agents is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] Purpose of the invention

[0006] This invention is the first to discover that Bacillus coagulans and Aspergillus niger are functional bacteria for lipid degradation, and that they can be immobilized with biochar to form a biochar-based composite enhanced bacterial agent.

[0007] Technical solution

[0008] A biochar-based bacterial agent, characterized in that it is prepared from Bacillus coagulans.

[0009] The biochar-based bacterial agent is characterized in that it is prepared by the following steps:

[0010] (1) Pretreated biochar

[0011] Sterilize the biochar and set aside for later use;

[0012] (2) Preparation of Bacillus coagulans bacterial culture

[0013] Bacillus coagulans was inoculated into liquid culture medium and activated at 45°C and 150 rpm; then it was transferred into fresh sterile culture medium at a volume ratio of 5% and amplified under the same culture conditions.

[0014] (3) Preparation of biochar-based bacterial inoculants

[0015] Weigh out biochar at a weight-to-volume ratio of 2%, add it to the liquid culture medium and mix well. Inoculate with Bacillus coagulans bacterial solution at a volume ratio of 5%, and culture at 45℃ and 150rpm for 24h. After the culture is completed, filter to separate the biochar loaded with bacteria, wash with water, and dry to obtain biochar-based bacterial inoculum.

[0016] The biochar-based bacterial agent is characterized in that the liquid culture medium in step (2) or (3) is: 5g peptone, 3g beef extract powder, 5g sodium chloride (NaCl), 5mg manganese sulfate (MnSO4·H2O), and 1000mL distilled water.

[0017] The biochar-based bacterial agent is characterized in that the OD600 of the Bacillus coagulans bacterial solution inoculated in step (3) is 0.8.

[0018] A biochar-based fungal agent, characterized in that it is prepared from Aspergillus niger.

[0019] The biochar-based fungal agent is characterized in that it is prepared by the following steps:

[0020] (1) Pretreated biochar

[0021] Sterilize the biochar and set aside for later use.

[0022] (2) Preparation of Aspergillus niger spore suspension

[0023] The Aspergillus niger strain was inoculated onto potato solid medium and cultured. After the hyphae covered the plate and formed a spore layer, the spores were scraped off to prepare a suspension. The resulting suspension was fully dispersed, and the hyphae and solid medium residue were removed to obtain the Aspergillus niger spore suspension.

[0024] (3) Preparation of biochar-based fungal inoculants

[0025] Weigh out biochar at a weight-to-volume ratio of 3%, add it to potato solid culture medium and mix well. Inoculate with Aspergillus niger suspension at a volume ratio of 5%, and culture at 37℃ and 150 rpm for 48 h. Filter to separate the biochar loaded with mycelia, wash with water, and dry to obtain biochar-based fungal inoculant.

[0026] The biochar-based fungal agent is characterized in that the potato solid culture medium in step (2) or (3) consists of 200g peeled potatoes, 20g glucose, 1000mL distilled water, and 15g agar.

[0027] The biochar-based fungal agent is characterized in that the OD620 of the inoculated Aspergillus niger strain suspension is 1.26.

[0028] A biochar-based fungal enhancement agent, characterized in that the biochar-based fungal enhancement agent comprises a biochar-based bacterial agent and a biochar-based fungal agent, wherein the biochar-based bacterial agent and the biochar-based fungal agent are mixed in an equal mass ratio.

[0029] Application of the biochar-based bacterial agent, the biochar-based fungal agent, or the biochar-based fungal enhancement agent in composting.

[0030] As an optimization method:

[0031] A biochar-based bacterial agent, characterized in that it is prepared from Bacillus coagulans.

[0032] The biochar-based bacterial agent is characterized in that it is prepared by the following steps:

[0033] (1) Pretreated biochar

[0034] The biochar was placed in a sterile bottle and sterilized under high pressure steam at 121°C for 15 minutes. After being removed and allowed to cool naturally, it was stored under sterile conditions for later use.

[0035] (2) Preparation of Bacillus coagulans bacterial culture

[0036] The bacterial powder was inoculated into liquid culture medium and activated at 45°C and 150 rpm. Subsequently, it was transferred to fresh sterile culture medium at a 5% inoculation ratio and amplified under the same conditions. The OD600 value of the bacterial culture was monitored using a spectrophotometer during the culture process.

[0037] (3) Preparation of biochar-based bacterial inoculants

[0038] Weigh out biochar at a dosage ratio of 2%, add it to the liquid culture medium and mix thoroughly. Inoculate with standardized bacterial suspension (OD600 = 0.8) at a volume ratio of 5%, and incubate at 45℃ and 150 rpm for 24 h. After incubation, filter to separate the bacteria-loaded biochar, wash three times with sterile physiological saline, and air dry at room temperature for 24 h to obtain biochar-based bacterial inoculum.

[0039] A biochar-based fungal agent, characterized in that it is prepared from Aspergillus niger.

[0040] The biochar-based fungal agent is characterized in that it is prepared by the following steps:

[0041] (1) Pretreated biochar

[0042] The biochar was placed in a sterile bottle and sterilized under high pressure steam at 121°C for 15 minutes. After being removed and allowed to cool naturally, it was stored under sterile conditions for later use.

[0043] (2) Preparation of Aspergillus niger spore suspension

[0044] Aspergillus niger strains were inoculated onto sterilized potato broth and cultured at 37°C for 7 days. After the mycelium covered the plate and formed a spore layer, the colony surface was rinsed with sterile physiological saline, and the spores were gently scraped off using a sterile spreader to prepare a suspension. The resulting suspension was treated in a vortex mixer to fully disperse the spores and reduce aggregation. Subsequently, the mycelium and solid culture medium residue were removed by filtration through sterile gauze to obtain the Aspergillus niger spore suspension. After preparation, the OD620 value of the spore suspension was monitored using a spectrophotometer.

[0045] (3) Preparation of biochar-based fungal inoculants

[0046] Weigh out biochar at a ratio of 3%, add it to the liquid culture medium and mix thoroughly. Inoculate with a standardized spore suspension (OD620 = 1.26) at a volume ratio of 5%, and incubate at 37℃ and 150 rpm for 48 h. Filter to separate the biochar loaded with mycelia, wash three times with sterile physiological saline, and air dry at room temperature for 24 h to obtain a biochar-based fungal inoculum.

[0047] A composite bio-enhanced inoculant is obtained by mixing biochar-based fungal inoculant and biochar-based bacterial inoculant in equal proportions.

[0048] The application of the aforementioned biochar-based composite enhanced microbial agent in composting. The main invention is as follows:

[0049] A biofortification scheme is proposed to construct a composite microbial agent by loading functional bacteria and fungi with biochar. Based on the identification of oil-degrading functional bacteria in the composting system, a biochar-based composite fortification microbial agent is developed and its preparation process is optimized. Its effect on improving oil degradation efficiency, composting efficiency and compost product quality in the composting process is evaluated.

[0050] Beneficial effects

[0051] This invention, through systematic experiments, revealed that the addition of biochar significantly altered the microbial community in aerobic composting of household kitchen waste compared to composting without biochar. Further species difference analysis identified, for the first time, *Bacillus coagulans* and *Aspergillus niger* exhibiting the highest LDA values, indicating their ability to improve the oil degradation efficiency during kitchen waste composting. According to existing reports, *Bacillus coagulans*, used for fermentation and probiotic preparations, produces lipases; *Aspergillus niger* secretes various hydrolases and oxidases, participating in the degradation of complex organic matter. However, no literature reports have documented the application of these two strains in the treatment of household kitchen waste.

[0052] The present invention prepares biochar-based bacterial agents, fungal agents, and biochar-based composite enhanced bacterial agents, respectively. Among them, the biochar-based composite enhanced bacterial agent has a synergistic enhancing effect on degrading oils and improving composting efficiency, which has not been reported in the literature.

[0053] Specifically as follows:

[0054] (1) Identification of functional bacteria related to oil degradation in biochar-mediated household kitchen waste composting systems. The effects of biochar addition on the physicochemical properties and microbial community structure of aerobic household kitchen waste compost were investigated. Results showed that biochar addition significantly improved the pH, electrical conductivity, oil content, C / N ratio, and seed germination index of the compost pile, but the pile remained compacted. Microbial community analysis indicated that the microbial community structure was relatively stable in the control group during composting, while the community structure in the biochar-added pile changed significantly during the composting process. Species difference analysis identified the bacteria *Bacillus coagulans* and the fungus *Aspergillus niger*, both possessing oil degradation functions.

[0055] (2) A method for preparing biochar-based bacterial agents was established. The effects of immobilization time and biochar addition ratio on the loading of bacteria and fungi on biochar were investigated. The results showed that when immobilized for 24 h and with a biochar addition ratio of 2%, the bacterial loading of Bacillus coagulans was the highest and the attachment state was stable; when immobilized for 48 h and with a biochar addition ratio of 3%, the fungal immobilization effect of Aspergillus niger was the best, with hyphae covering the biochar surface, a uniform and stable structure, and good suspension properties.

[0056] (3) Using biochar as a control group, the effects of three biochar-based microbial agents on oil degradation efficiency, composting efficiency, and compost product quality were investigated. The results showed that, compared with the control group, the addition of biochar-based microbial agents significantly improved all three compost performance indicators: with biochar-based bacterial agents, oil content decreased by 8.73%, and the compost weight loss rate and seed germination index increased by 2.54% and 8.92%, respectively; with biochar-based fungal agents, oil content decreased by 18.12%, and the compost weight loss rate and seed germination index increased by 3.82% and 13.91%, respectively; with biochar-based composite strengthening microbial agents, oil content decreased by 24.72%, and the compost weight loss rate and seed germination index increased by 5.42% and 14.80%, respectively. The biochar-based composite strengthening microbial agent showed the best improvement effect, and the material was loose and non-sticky.

[0057] The invention provides a feasible technical approach to improve the efficiency of household kitchen waste composting. Attached Figure Description

[0058] Figure 1 Community difference analysis between biochar-enhanced compost and control group; including A) LEfSe multi-level species hierarchy tree; B) LDA discrimination of key bacterial species; C) LDA discrimination of key fungal species;

[0059] Figure 2 Kitchen waste composting reactor;

[0060] Figure 3 Bacillus coagulans streak plating culture;

[0061] Figure 4 Experiment on the preparation of biochar-based bacterial inoculants; including A) inoculation of bacterial culture; B) shaker culture;

[0062] Figure 5 Aspergillus niger streak inoculation culture;

[0063] Figure 6 Experiment on the preparation of biochar-based fungal inoculants, including (a) inoculation with fungal spore suspension; and (b) shaking culture. Detailed Implementation

[0064] Overall Design

[0065] (1) Analyze the effects of adding biochar on the changes in the physicochemical properties and microbial community structure of the compost pile during the composting of household kitchen waste, and identify and select oil-degrading functional bacteria;

[0066] (2) A method for preparing biochar-based bacterial agents of two lipid-degrading bacteria, Aspergillus niger and Bacillus coagulans supported on biochar, was established, and then a biochar-based composite enhanced bacterial agent was prepared.

[0067] (3) Through composting experiments, the effects of adding biochar-based bacterial agents, fungal agents and biochar-based compound enhanced agents on improving the efficiency of household kitchen waste composting were systematically evaluated.

[0068] Example 1

[0069] I. Identification of Key Microorganisms in Biochar-Mediated Household Kitchen Waste Composting Systems

[0070] (1) Experimental Design

[0071] This invention references the research group's findings in supplementary aerobic composting experiments of household kitchen waste, employing an initial addition strategy of 8:4:5 ratio of green waste, biochar, and daily supplementary kitchen waste [Reference: Sun Haoran. The Influence of Added Biochar and Green Waste on Aerobic Composting of Kitchen Waste [D]: [Master's Thesis]. Southeast University, 2024.]. Specifically, when 400g of biochar with a particle size of 1-3mm and 800g of green waste are added at the beginning of composting, followed by daily addition of 500g of kitchen waste, the compost pile exhibits good stability and maturation effects.

[0072] Based on this, to further explore the impact of biochar on the microbial community during household kitchen waste composting and to identify key functional bacteria in the household kitchen waste composting system under biochar addition conditions, this invention conducted a control experiment with biochar addition as the variable, with two parallel replicates in each group. Specific groupings and material addition amounts are shown in Table 1.

[0073] Table 1 Operation Scheme of Feed-Type Reactor

[0074]

[0075] (2) Experimental Procedure

[0076] During the composting process, the apparent properties of the compost pile were observed daily, and changes in temperature and mass were recorded. Every 3 days, approximately 20g samples were taken from each reactor to measure indicators such as moisture content, pH, conductivity, total nitrogen, organic matter content, oil content, and seed germination index. Additionally, starting from the 3rd day of composting, samples were collected every 6 days for microbial community analysis.

[0077] DNA extraction was performed using the FastPure Soil DNA Isolation Kit (Magnetic Bead) (MJYH, Shanghai, China). The integrity of the extracted products was detected by 1% agarose gel electrophoresis, and their concentration and purity were determined using NanoDrop 2000 (Thermo Scientific, USA).

[0078] Specific primers with barcodes were synthesized according to the specified sequencing region (see Table 2), using ABI. Amplification was performed using a 9700 PCR instrument.

[0079] To ensure the accuracy and reliability of subsequent analyses, the number of amplification cycles was kept as low as possible, and all samples were kept consistent. All samples were performed in triplicate. Amplification products were mixed and analyzed by 2% agarose gel electrophoresis. The gel was then excised using the AxyPrep DNA Gel Extraction Kit (AXYGEN), eluted with Tris-HCl, and analyzed again by 2% agarose gel electrophoresis.

[0080] The sequencer used was a Nextseq 2000 (Illumina, San Diego, California, USA), and sequencing was performed on an Illumina PE300 platform. Alpha diversity knowledge, such as the Chao1 and Shannon indices, was calculated using Mothur software. Principal Coordinate Analysis (PCoA) based on the Bray-Curtis distance algorithm was used to examine the similarity of microbial community structure among samples. Linear Discriminant Analysis Effect Size (LDA>2, P<0.05) was used to identify microbial taxa with significant differences in abundance among different groups.

[0081] Table 2 Primers used for PCR amplification

[0082]

[0083] (3) Experimental Results

[0084] Bacterial community analysis of the compost pile showed that: in the early stage of composting, the dominant bacteria in the control group were *Thermoactinomyces* and *Lactobacillus*. After the addition of biochar, *Bacillus* and *Streptomyces* became significantly dominant, and α-diversity was high. In the middle stage of composting, *Lactobacillus* remained the dominant bacteria in the control group. After the addition of biochar, *Pediococcus* became significantly dominant, and α-diversity did not increase significantly. In the later stage of composting, *Lactobacillus* remained the dominant bacteria in the control group. After the addition of biochar, *Lactobacillus* and *Aeriscardovia* became significantly dominant, and α-diversity increased significantly. PCoA analysis showed that the bacterial community structure in the control group was relatively stable during composting, while the bacterial community structure in the biochar-added compost changed significantly during the composting process. LEfSe analysis showed that, compared with the control group, the addition of biochar significantly enriched Bacillus, Aneurinibacillus, Brevibacillus, Ureibacillus, Saccharomonospora, and Aeribacillus.

[0085] Fungal community analysis of the compost pile showed that in the early stage of composting, the dominant microorganisms in both the control group and after biochar addition were Kazachstania, Pichia, Aspergillus, and Candida, with slightly higher α-diversity after biochar addition. In the middle stage of composting, the dominant microorganisms in both the control group and after biochar addition were still Kazachstania, Pichia, and Candida, with no significant increase in α-diversity. In the later stage of composting, the dominant microorganisms in the control group remained Pichia, Kazachstania, and Candida, but after biochar addition, Aspergillus, Kazachstania, Pichia, and Candida became significantly dominant, and α-diversity increased significantly. PCoA analysis showed that the community structure in the control group was relatively stable during composting, while the fungal community structure in the biochar-added compost changed significantly during the composting process. LEfSe analysis showed that, compared with the control group, the addition of biochar significantly enriched Aspergillus, Lodderomyces, Paecilomyces, Thermomyces, and Flammulina.

[0086] It would be best to add data for the control group to Tables 3 and 4 below, so that comparisons can be made more convenient.

[0087] Table 3. LDA values ​​and functional characteristics of differentially expressed bacteria

[0088]

[0089] Table 4. Differentially derived fungal LDA values ​​and functional characteristics

[0090]

[0091] Conclusion: Analysis of fungi and bacteria whose abundance significantly increased after biochar addition revealed that the fungus Aspergillus niger and the bacterium Bacillus coagulans had the highest LDA scores, making them the functional bacteria for oil degradation in biochar-mediated household kitchen waste composting systems.

[0092] II. Preparation of Biochar-Based Inoculants

[0093] (1) Preparation of biochar-based bacterial inoculants

[0094] 1) Pretreatment of biochar

[0095] To reduce interference from other microorganisms, the biochar was pretreated before loading. The specific procedure was as follows: the biochar was placed in a sterile bottle and sterilized under high pressure steam at 121°C for 15 minutes. After removal, it was allowed to cool naturally and stored under sterile conditions for later use.

[0096] 2) Experimental bacterial strains

[0097] The bacteria used in this invention are Bacillus coagulans CICC 20138, purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0098] 3) Culture medium

[0099] Liquid culture medium: 5g peptone, 3g beef extract, 5g sodium chloride (NaCl), 5mg manganese sulfate (MnSO4·H2O), 1000mL distilled water. For solid culture medium, add 15g agar. The prepared culture media were autoclaved at 121℃ for 15min.

[0100] The results of Bacillus coagulans streak inoculation culture are as follows: Figure 2 As shown.

[0101] 4) Determination of bacterial load on biochar

[0102] To estimate the number of bacteria loaded on biochar, a standard curve was established between the concentration of Bacillus coagulans bacterial suspension and optical density before the immobilization experiment. Specifically, bacterial suspension samples of different concentrations were prepared, and their optical density (OD) was measured. 600 The bacterial concentration (cfu / mL) was determined using the plate count method, and the optical density was measured using a spectrophotometer at a wavelength of 600 nm. To ensure the accuracy of the results, the OD values ​​of each sample were... 600 The concentration was controlled between 0.1 and 0.8, and multiple concentration points were set for repeated measurements.

[0103] Weigh 1g of biochar-based bacterial agent and place it in a 50mL sterile Erlenmeyer flask. Add 30mL of sterile distilled water and 4 to 6 sterilized glass beads. Incubate with shaking at 200rpm for 60min to obtain a bacterial suspension. Measure the OD of the bacterial suspension. 600 The measured absorbance is substituted into the established standard curve to calculate the bacterial concentration and convert it into the bacterial load per unit mass of biochar.

[0104] 5) Standardized bacterial culture preparation

[0105] Before the immobilization experiment, the bacterial powder was inoculated into liquid culture medium and activated at 45℃ and 150 rpm. Subsequently, it was transferred to fresh sterile culture medium at a 5% inoculation ratio and amplified under the same conditions. During the culture process, the OD of the bacterial culture was monitored using a spectrophotometer. 600 To ensure consistent inoculation conditions across experimental treatments, optical density was used as a control index to determine the initial inoculum size. All bacterial suspensions were diluted to an OD value. 600 =0.8. The standardized bacterial solution was used as the inoculum for subsequent loading experiments.

[0106] 6) Immobilization process optimization experiments and preparation of biochar-based bacterial inoculants

[0107] To optimize the bacterial loading effect on biochar, single-factor experiments were conducted to investigate the influence of immobilization time and biochar dosage ratio on the loading effect. The preparation experiment of the biochar-based bacterial inoculant is as follows: Figure 3 As shown.

[0108] Effect of immobilization time: Weigh out biochar at a ratio of 3%, add it to the liquid culture medium and mix thoroughly. Inoculate with standardized bacterial suspension (OD) at a volume ratio of 5%. 600 =0.8), and cultured at 45℃ and 150rpm. Immobilization times were set at 6h, 12h, 24h, 36h, and 48h. After culture, the biochar was collected by filtration, washed three times with sterile physiological saline, and then dried at 45℃. Following the method in "4) Determination of bacterial load on biochar", sterile distilled water was added and shaken for culture. The absorbance of the bacterial suspension was measured, the bacterial concentration was calculated, and the bacterial load per unit mass of biochar was obtained. The loading effect under different immobilization times was compared.

[0109] Effect of Biochar Dosage Ratio: With the immobilization time already determined, the effect of different biochar dosage ratios on the immobilization effect was further evaluated. Biochar dosage ratios of 1%, 2%, 3%, 4%, and 5% were set. In each treatment, biochar was weighed according to the ratio and mixed with liquid culture medium, and a standardized bacterial suspension (OD) was inoculated at a volume ratio of 5%. 600 =0.8), and cultured at 45℃ and 150rpm. After culture, the biochar was collected by filtration, washed three times with sterile physiological saline, and then dried at 45℃. Following the method in "4) Determination of bacterial load on biochar" in this section, sterile distilled water was added and shaken for culture. The absorbance of the bacterial suspension was measured, the bacterial concentration was calculated, and the bacterial load per unit mass of biochar was obtained. The loading effect under different biochar addition ratios was compared.

[0110] Based on the determined optimal immobilization time and biochar dosage ratio, the same operating procedure was used for bacterial biochar immobilization. After immobilization, the biochar loaded with bacteria was filtered and separated, washed three times with sterile physiological saline, and air-dried at room temperature for 24 hours to obtain a biochar-based bacterial inoculum, which was then sealed and stored in a refrigerator at 4°C for later use.

[0111] (2) Preparation of biochar-based fungal fortification agents

[0112] 1) Pretreatment of biochar carrier

[0113] To reduce interference from other microorganisms and improve the surface adhesion environment of biochar, the biochar was pretreated before loading. The specific procedure was as follows: the weighed biochar was placed into a sterile bottle, sterilized in a high-pressure steam environment at 121℃ for 15 minutes, removed and allowed to cool naturally, and then stored under aseptic conditions for later use.

[0114] 2) Experimental bacterial strains

[0115] The bacteria used in this invention is Aspergillus niger (CICC 41796), which was purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0116] 3) Preparation of culture medium and spore suspension

[0117] Liquid culture medium: 200g peeled potatoes, 20g glucose, 1000mL distilled water. For solid culture medium, add 15g agar. The prepared culture medium is then autoclaved at 121℃ for 15min.

[0118] Aspergillus niger strains were inoculated onto sterilized potato solid medium and cultured at 37°C for 7 days. After the mycelium covered the plate and a spore layer formed, the colony surface was rinsed with sterile physiological saline, and the spores were gently scraped off using a sterile spreader to prepare a suspension. Aspergillus niger streak plating culture was performed as follows. Figure 4 As shown.

[0119] The resulting suspension was treated in a vortex mixer to fully disperse the spores and reduce aggregation. Subsequently, it was filtered through sterile gauze to remove mycelia and solid culture medium residue, yielding a *Aspergillus niger* spore suspension. To ensure consistent inoculation conditions across all experimental treatments, optical density was used as a control index to determine the initial spore inoculum size. The absorbance of the spore suspension was measured at 620 nm and diluted to a standardized level; this standardized spore suspension was used as the inoculum for subsequent loading experiments.

[0120] 4) Immobilization process optimization experiments and preparation of biochar-based fungal inoculants

[0121] To optimize the fungal loading effect on biochar, single-factor experiments were conducted to investigate the influence of immobilization time and biochar dosage ratio on the loading effect. The preparation experiments of the biochar-based fungal inoculant are as follows: Figure 5 As shown.

[0122] Effect of immobilization time: Biochar was weighed at a ratio of 3%, added to the liquid culture medium and mixed thoroughly. Standardized spore suspension (OD) was then inoculated at a volume ratio of 5%. 620 =1.26), and cultured at 37℃ and 150 rpm. Immobilization times were set at 12h, 24h, 48h, 72h, and 96h. After cultivation, the samples were filtered and washed three times with sterile water to remove as many unattached free hyphae as possible, and then dried at 37℃ to constant weight. The dry weight of hyphae per unit mass of biochar was calculated for each treatment, and the loading effect under different immobilization times was compared.

[0123] Effect of Biochar Dosage Ratio: With the immobilization time already determined, the effect of different biochar dosage ratios on the immobilization effect was further evaluated. Biochar dosage ratios of 1%, 2%, 3%, 4%, and 5% were set. In each treatment, biochar was weighed according to the ratio and mixed with liquid culture medium. Standardized spore suspension (OD200) was added at 5% of the inoculum size. 620 =1.26), and cultured at 37℃ and 150 rpm. After culture, the mixture was filtered and washed three times with sterile water to remove as many unattached free hyphae as possible, and then dried at 37℃ to constant weight. The dry weight of hyphae per unit mass of biochar was calculated for each treatment, and the loading effect under different biochar addition ratios was compared.

[0124] Based on the determined optimal immobilization time and biochar dosage ratio, the same operating procedure was used for cultivation. After cultivation, the biochar loaded with mycelia was filtered and separated. It was washed three times with sterile physiological saline and air-dried at room temperature for 24 hours to obtain a biochar-based fungal inoculum, which was then sealed and stored in a refrigerator at 4°C for later use.

[0125] (3) Experimental Results

[0126] By comparing the number of bacteria loaded on biochar, the optimal immobilization time for Bacillus coagulans was determined to be 24 hours. Based on this, five biochar dosages of 1%, 2%, 3%, 4%, and 5% were set. Similarly, by comparing the number of bacteria loaded on the biochar, the optimal biochar dosage of 2% was determined to be 2%.

[0127] By comparing the dry weight of mycelia loaded with biochar, the optimal immobilization time for Aspergillus niger was determined to be 48 h. Based on this, five biochar dosages of 1%, 2%, 3%, 4%, and 5% were set. Similarly, by comparing the dry weight of mycelia loaded with biochar, the optimal biochar dosage of 3% was determined to be 3%.

[0128] (3) Preparation of biochar-based composite fortified bacterial agent

[0129] A biochar-based composite enhanced microbial agent is obtained by mixing biochar-based fungal agents and biochar-based bacterial agents in an equal mass ratio (1:1).

[0130] III. Application of Compound Bio-enhancing Microbial Agents

[0131] (1) Experimental Design

[0132] To further investigate the application effect of the prepared biochar-based microbial agent in household kitchen waste composting, four groups of supplementary household kitchen waste composting experiments were set up. The specific grouping and material addition amount are shown in Table 5.

[0133] Table 5 Operation Scheme of Feed-Type Reactor

[0134]

[0135]

[0136] (2) Experimental Procedure

[0137] During the composting process, the apparent properties of the compost pile were observed daily, and temperature and mass changes were recorded. Every 3 days, approximately 20g samples were taken from each reactor to measure indicators such as moisture content, pH, conductivity, total nitrogen, organic matter content, oil content, and seed germination index.

[0138] (3) Experimental Results

[0139] Using biochar as a control group, this experiment investigated the effects of three biochar-based inoculants on improving oil degradation efficiency, composting efficiency, and compost product quality. The main results are as follows:

[0140] 1) Compared to adding biochar, adding biochar-based bacterial inoculants resulted in a looser compost structure, slightly improved odor, faster initial temperature rise, higher average temperature, and more stable nitrogen accumulation. At the end of composting, the weight loss rate increased by 2.54%, oil content decreased by 8.73%, organic matter content decreased by 5.56%, nitrogen accumulation remained stable, the C / N ratio decreased more significantly, the seed germination index increased by 8.92%, and the compost pile entered the maturation stage earlier. However, after adding biochar-based bacterial inoculants, the compost pile's moisture retention capacity was weaker in the early stages, and the pH fluctuation range was larger.

[0141] 2) Compared with the addition of biochar, the addition of biochar-based fungal inoculants improved the structural stability of the compost pile, resulted in better odor control, faster heating rate, higher average temperature, and more stable nitrogen accumulation. The final weight loss rate increased by 3.82%, oil content decreased by 18.12%, organic matter content decreased by 4.61%, nitrogen accumulation remained stable, the C / N ratio decreased significantly, the seed germination index increased by 13.91%, and the compost pile matured faster.

[0142] 3) Compared with the addition of biochar, the addition of a biochar-based composite inoculant in a 1:1 ratio with the same total amount resulted in a continuously loose pile structure, the best odor control, rapid temperature rise, and a higher average temperature. At the end of composting, the weight loss rate increased by 5.42%, the oil content decreased by 24.72%, the organic matter content decreased by 7.76%, nitrogen accumulation remained stable, the C / N ratio decreased the most, and the seed germination index increased by 14.80%. All indicators were superior to the single inoculant group, the pile maturation process was accelerated, and the system operation stability was stronger.

[0143] 4) Compared to the group treated with biochar-based composite inoculants, the group treated with biochar-based bacterial inoculants experienced faster initial temperature rise and rapid reaction initiation, but lower weight loss and organic matter removal rates. Simultaneously, the system exhibited greater pH fluctuations, poor moisture retention, delayed maturation, and limited improvement in seed germination index. Furthermore, this group showed insufficient oil degradation in the later stages, resulting in noticeable adhesion and off-odors, indicating poor system stability. Compared to the group treated with biochar-based composite inoculants, the group treated with biochar-based fungal inoculants showed slightly lower weight loss, organic matter removal, and total nitrogen accumulation, a slower maturation pace, lower seed germination rates in the later stages, and still higher oil accumulation.

[0144] In comparison, the addition of biochar-based composite enhanced microbial agent resulted in the best performance across multiple key indicators: high system heating efficiency and the longest duration of high temperature; a consistently loose pile structure, good odor control, the lowest oil content, and the highest final weight loss rate and organic matter removal efficiency. Total nitrogen content steadily increased, the C / N ratio decreased more significantly, the seed germination index further improved, and the pile entered the decomposition stage earlier, demonstrating the most outstanding overall control effect.

Claims

1. A biochar-based bacterial inoculant, characterized in that, A biochar-based bacterial inoculum was prepared from Bacillus coagulans.

2. The biochar-based bacterial agent according to claim 1, characterized in that, The bacterial agent is prepared by the following steps: (1) Pretreated biochar Sterilize the biochar and set aside for later use; (2) Preparation of Bacillus coagulans bacterial culture Bacillus coagulans was inoculated into liquid culture medium and activated at 45°C and 150 rpm; then it was transferred into the culture medium at a volume ratio of 5% and amplified under the same culture conditions. (3) Preparation of biochar-based bacterial inoculants Weigh out biochar at a weight-to-volume ratio of 2%, add it to the liquid culture medium and mix well. Inoculate with Bacillus coagulans bacterial solution at a volume ratio of 5%, and culture at 45℃ and 150rpm for 24h. After the culture is completed, filter to separate the biochar loaded with bacteria, wash with water, and dry to obtain biochar-based bacterial inoculum.

3. The biochar-based bacterial agent according to claim 2, characterized in that, The liquid culture medium mentioned in step (2) or (3) is: 5g peptone, 3g beef extract powder, 5g sodium chloride, 5mg manganese sulfate, and 1000mL distilled water.

4. The biochar-based bacterial agent according to claim 2, characterized in that, The OD600 of the Bacillus coagulans bacterial culture in step (3) was 0.

8.

5. A biochar-based fungal inoculant, characterized in that, Inoculum preparations were made from Aspergillus niger.

6. The biochar-based fungal agent according to claim 5, characterized in that, The bacterial agent is prepared by the following steps: (1) Pretreated biochar Sterilize the biochar and set aside for later use; (2) Preparation of Aspergillus niger spore suspension The Aspergillus niger strain was inoculated onto potato solid medium and cultured. After the hyphae covered the plate and formed a spore layer, the spores were scraped off to prepare a suspension. The resulting suspension was fully dispersed, and the hyphae and solid medium residue were removed to obtain the Aspergillus niger spore suspension. (3) Preparation of biochar-based fungal inoculants Weigh out biochar at a weight-to-volume ratio of 3%, add it to potato solid culture medium and mix well. Inoculate with Aspergillus niger suspension at a volume ratio of 5%, and culture at 37℃ and 150 rpm for 48 h. Filter to separate the biochar loaded with mycelia, wash with water, and dry to obtain biochar-based fungal inoculant.

7. The biochar-based fungal agent according to claim 6, characterized in that, The steps described in (2) or (3) Potato solid culture medium: 200g peeled potatoes, 20g glucose, 1000mL distilled water, 15g agar.

8. The biochar-based fungal agent according to claim 6, characterized in that, The OD620 of the suspension of Aspergillus niger was 1.

26.

9. A biochar-based fungal strengthening agent, characterized in that, The biochar-based fungal strengthening agent includes the biochar-based bacterial agent according to any one of claims 1-4 and the biochar-based fungal agent according to any one of claims 5-8, wherein the biochar-based bacterial agent and the biochar-based fungal agent are mixed in equal mass ratios.

10. The application of the biochar-based bacterial agent according to any one of claims 1-4, the biochar-based fungal agent according to any one of claims 5-8, or the biochar-based fungal strengthening agent according to claim 9 in composting.

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