Sludge aerobic fermentation composite microbial inoculant, and preparation method and application thereof

Abstract

This invention proposes a composite microbial agent for aerobic fermentation of sludge, its preparation method, and its application, relating to the fields of sludge treatment and microbial technology. The composite microbial agent provided by this invention is a novel microbial agent prepared using three high-quality microorganisms—Z-1 Bacillus glycinoidis, DF-727 Bacillus subtilis, and J-64 Mucor spp.—as active ingredients. The effective viable count of the agent is 1.7 × 10⁻⁶. 8 CFU / g. Adding 3% (by mass) of microbial agent significantly optimized the aerobic fermentation process of sludge, with the high-temperature period (>50℃) lasting for 3 days and the highest temperature of the pile reaching 65.3℃. The fermentation and maturation cycle was shortened by 4 days, and the seed germination index reached 91%. Compared with the control group (without added microbial agent), the nitrate nitrogen content in the sludge fermentation products increased by 23%-35%, and the humic content increased to 285-298 g / kg.

Description

Technical Field

[0001] This invention relates to the fields of sludge treatment and microbial technology, specifically to aerobic fermentation compound bacterial agent for sludge, its preparation method, and its application. Background Technology

[0002] Current methods for treating sludge include landfilling, incineration, land application, and fermentation. However, landfilling has drawbacks such as consuming large amounts of land resources, leachate pollution, and greenhouse gas emissions (e.g., methane). Incineration suffers from high energy consumption, large equipment investment, and the potential for secondary pollution (e.g., dioxins, heavy metal fly ash). In contrast, aerobic fermentation of sludge, as a technology for sludge resource utilization, is gradually becoming an effective method for efficient sludge disposal due to its low cost, relatively simple operation, and the ability of its products to be used as soil conditioners or organic fertilizers. However, traditional aerobic fermentation technology suffers from problems in practical applications, including insufficient oxygen supply during fermentation, low fermentation efficiency, long fermentation cycle (usually 30-60 days), and the generation of large amounts of odorous gases (e.g., NH3-N, H2S).

[0003] Therefore, to solve the above problems, the fermentation efficiency and product quality can be improved by introducing aerobic fermentation compound microbial agents into the aerobic fermentation process of sludge. Summary of the Invention

[0004] The purpose of this invention is to provide a composite microbial agent for aerobic fermentation of sludge, its preparation method, and its application. This composite microbial agent is composed of self-selected functional strains of sludge fermentation: *Bacillus glyciniculatus*, *Bacillus subtilis*, and *Mucor*. Single microbial agents are prepared using liquid fermentation combined with solid-phase conversion technology using a microbial residue carrier, and then blended in equal proportions to form a solid composite microbial agent. Adding this solid composite microbial agent to the aerobic fermentation process of sludge can improve fermentation efficiency and enhance the quality of fermentation products. The final fermentation products can be used for ecological restoration in areas such as quarries.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A sludge aerobic fermentation compound microbial agent, the active ingredients of which include Z-1 Bacillus glycinifermentans, DF-727 Bacillus subtilis and J-64 Mucor circinelloides. Among them, Bacillus glycineus Z-1 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with the accession number CGMCC No. 33468; Bacillus subtilis DF-727 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC No. 33467; J-64 Mucor was deposited on April 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41140.

[0006] The aforementioned aerobic fermentation compound microbial agent for sludge is composed of solid microbial agents of Bacillus glycinus Z-1, Bacillus subtilis DF-727, and Mucor spp. J-64.

[0007] The aforementioned sludge aerobic fermentation compound microbial agent is composed of solid microbial agents of Z-1 glycine bacillus, DF-727 Bacillus subtilis and J-64 Mucor spp. in a ratio of 1-3:1-3:1-3.

[0008] The aforementioned sludge aerobic fermentation compound microbial agent is composed of solid microbial agents of Z-1 glycine bacillus, DF-727 Bacillus subtilis and J-64 Mucor spp. in a 1:1:1 ratio.

[0009] A method for preparing a sludge aerobic fermentation compound microbial agent, wherein the effective viable bacteria count of the sludge aerobic fermentation compound microbial agent is ≥1.7×10⁻⁶. 8 CFU / g.

[0010] The aforementioned method for preparing the aerobic fermentation compound microbial agent for sludge includes the following steps: Step 1, Preparation of seed solution: Z-1 glycine-bacterium, DF-727 Bacterium subtilis, and J-64 Mucor were inoculated into liquid culture medium and cultured until the logarithmic growth phase of the strains. After fermentation, seed liquids of Z-1 glycine-bacterium, DF-727 Bacterium subtilis, and J-64 Mucor were obtained. Step 2, Preparation of bacterial culture: Seed cultures of Bacillus Z-1 glycine, Bacillus subtilis DF-727, and Mucor truncatella J-64 were inoculated into liquid fermentation medium and cultured until the OD value of the culture was 1. After fermentation, the culture cultures of Bacillus Z-1 glycine, Bacillus subtilis DF-727, and Mucor truncatella J-64 were obtained. Step 3, Preparation of a single solid microbial agent: The sterilized substrate residue was mixed with the seed liquids of Bacillus glycinus Z-1, Bacillus subtilis DF-727, and Mucor truncatula at a ratio of 1:1. The mixture was then dried in a sterile incubator at 30°C (final moisture content was 40-50%) to produce solid inoculum of Bacillus glycinus Z-1, Bacillus subtilis DF-727, and Mucor truncatula. Step 4, Preparation of solid compound microbial agent: Solid inoculants of Z-1 glycine-containing Bacillus, DF-727 Bacillus subtilis, and J-64 Mucor were mixed in equal proportions to obtain a composite inoculant for aerobic fermentation of sludge.

[0011] Application of a sludge aerobic fermentation compound microbial agent: The sludge aerobic fermentation compound microbial agent is used in sludge aerobic fermentation operations.

[0012] In the aforementioned application of the aerobic fermentation compound microbial agent for sludge, the amount of the aerobic fermentation compound microbial agent added is 3% by mass ratio, based on the dry weight of the sludge.

[0013] The present invention has at least the following beneficial effects: The compound microbial agent provided by this invention is a novel microbial agent prepared using three high-quality microorganisms—Z-1 glycine-containing Bacillus, DF-727 Bacillus subtilis, and J-64 Mucor spp.—as active ingredients. The effective viable count of the agent is 1.7 × 10⁻⁶. 8 CFU / g. Adding 3% (by mass) of the microbial agent significantly optimized the aerobic fermentation process of sludge, maintaining a high-temperature period (>50℃) for 3 days with a maximum pile temperature of 65.3℃, shortening the fermentation and maturation cycle by 4 days, and achieving a seed germination index of 91%. Compared with the control group (without added microbial agent), the nitrate nitrogen content in the sludge fermentation products increased by 23%-35%, and the humic content increased to 285-298 g / kg. Furthermore, applying the sludge fermentation products obtained using this microbial agent in combination with phosphogypsum to the land helps accelerate plant growth, improve substrate quality, enhance plant absorption of heavy metals, reduce the risk of heavy metal pollution in the soil, and make the land more conducive to plant growth and ecological restoration, demonstrating enormous land use potential. Attached Figure Description

[0014] Figure 1 Image of the strain obtained after isolation and purification; Figure 2 This is a sieve diagram of the hydrolysis zone; Figure 3 Image showing the activity of amylase, protease, and lipase; Figure 4 Phylogenetic tree of bacteria Z-1; Figure 5 Phylogenetic tree of bacteria DF-727; Figure 6 Phylogenetic tree of fungus J-64; Figure 7 This is a graph showing the antagonism between bacterial strains. Figure 8 This is a graph showing the temperature changes during the aerobic fermentation of sludge. Figure 9 This is a graph showing the change in nitrogen levels during the aerobic fermentation of sludge. Figure 10 This is a graph showing the changes in ammonia nitrogen during the aerobic fermentation of sludge. Figure 11 This is a graph showing the changes in nitrate nitrogen during the aerobic fermentation of sludge. Figure 12 This is a graph showing the changes in the germination index during sludge fermentation. Figure 13 A graph showing the changes in humus at different fermentation stages; Figure 7 In the diagram, (a) shows the antagonistic state between bacteria Z-1 and DF-727; (b) shows the antagonistic state between bacteria Z-1 and fungus J-64; and (c) shows the antagonistic state between bacteria DF-727 and fungus J-64. Figures 8-13 In the study, CK: control group without added microbial agent; W1: group with 1% added microbial agent; W2: group with 3% added microbial agent; W3: group with 5% added microbial agent; SY: group with added commercial microbial agent. Detailed Implementation

[0015] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. For those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0016] Unless otherwise specified, the experimental materials used in the following embodiments of the present invention were purchased from conventional biochemical reagent stores. Unless otherwise specified, the methods in the embodiments are conventional methods in the art. The experimental materials used in the following embodiments are as follows: Casein medium: 10.0g casein, 3.0g beef extract, 5.0g sodium chloride, 2.0g dipotassium hydrogen phosphate, 15.0g agar, 0.05g bromothymol blue, 1L ultrapure water, sterilized at 121℃ for 20min.

[0017] Rhodamine B medium: 15g agar, 0.02% (w / v) Rhodamine B, 6.6% (v / v) olive oil, 1L ultrapure water, sterilized at 121℃ for 20min.

[0018] LB solid medium: 5.0 g yeast extract, 10.0 g tryptone, 10.0 g sodium chloride, 20.0 g agar, 1.0 L ultrapure water, pH 7.0-7.5, sterilized at 121℃ for 20 min.

[0019] LB liquid medium: 5.0g yeast extract, 10.0g tryptone, 10.0g sodium chloride, 1.0L ultrapure water, pH 7.0-7.5, sterilized at 121℃ for 20min.

[0020] PDB liquid culture medium: 200.0g potato, 20.0g glucose, 1.0L ultrapure water, pH 7.0-7.5, sterilized at 121℃ for 20min.

[0021] Basic fermentation medium: 10.0g glucose, 10.0g tryptone, 10.0g sodium chloride, 1L ultrapure water, pH 7.0-7.5, sterilized at 121℃ for 20 min.

[0022] The functional bacteria used in the inoculum agent—Bacillus glycineosa (Z-1), Bacillus subtilis (DF-727), and Mucor spp. (J-64)—were all independently isolated in the laboratory previously, and all three bacteria have been deposited at the China General Microbiological Culture Collection Center (CGMCC). The deposit information is as follows: 1. Preservation Institution Code: CGMCC Survival status: Yes Deposit date: January 20, 2025 Collection Number: CGMCC No. 33468 Classification and nomenclature: Bacillus glycinifermentans 2. Preservation Institution Code: CGMCC Survival status: Yes Deposit date: January 20, 2025 Accession number: CGMCC No. 33467 Classification and nomenclature: Bacillus subtilis 3. Preservation Institution Code: CGMCC Survival status: Yes Deposit date: April 7, 2024 Accession number: CGMCC No. 41140 (Mucor circinelloide s) Example 1: Isolation, Screening and Identification of Strains Take a sterile 50mL centrifuge tube, add 10mL of NH3 selective medium, and add 10µL of ammonia water in a clean bench. After activating the test strain, inoculate it into the centrifuge tube at a 2% inoculum rate, seal it, and incubate it on a shaker at 55 ℃ and 160 r / min for 5 days. After incubation, if the bacterial solution is turbid, it indicates that the strain has the ability to assimilate ammonia; if the bacterial solution remains clear, it indicates that the strain cannot directly utilize ammonia. Select strains with higher turbidity as targets for subsequent rescreening. After isolation and purification, 9 bacterial strains and 1 fungal strain (e.g., ...) were obtained. Figure 1 ).

[0023] The purified 10 bacterial strains were then re-screened using the hydrolysis zone method on LBSP, casein, and Rhodamine B media. Bacteria were incubated at 55°C for 2 days, and fungi at 55°C for 4 days. The ratio of the hydrolysis zone diameter to the strain diameter (D / d) was used to identify the starch, protein, and fat degradation abilities of the strains. The results showed that J-64, Z-1, and DF-727 had stronger degradation abilities (e.g., ...). Figure 2 The D / d values ​​of J-64 in LBSP, casein, and Rhodamine B media were 1.12, 2.58, and 1.81, respectively; the D / d values ​​of Z-1 in LBSP, casein, and Rhodamine B media were 2.35, 1.82, and 1.36, respectively; and the D / d values ​​of DF-727 in LBSP, casein, and Rhodamine B media were 1.82, 2.21, and 2.33, respectively. The amylase, protease, and lipase activities of J-64, Z-1, and DF-727 were as follows: Figure 3 As shown.

[0024] Phylogenetic analysis based on 16S rDNA sequences revealed the taxonomic positions of strains Z-1 and DF-727. A neighbor-joining phylogenetic tree was constructed using ribosome database alignment and MEGA 11.0 software, and the phylogenetic analysis was completed with 1000 bootstrap tests.

[0025] The 16S rDNA sequence of strain Z-1 forms a monophyletic group with 100% support for the standard strain *Bacillus glycinifermentans*, further confirming its taxonomic position as *Bacillus glycinifermentans* (e.g., *Bacillus glycinifermentans*) within the phylum Firmicutes, class Bacillus, order Bacillusales, family Bacillusaceae, and genus *Bacillus*. Figure 4 ).

[0026] Strain DF-727 is classified as *Bacillus subtilis*, belonging to the phylum Firmicutes, class Bacillus, order Bacillusales, family Bacillusaceae, and genus *Bacillus*. This result is highly consistent with phenotypic characteristics (Gram positivity, spore formation, etc.) (p<0.001). Figure 5 ).

[0027] The ITS sequence of fungus J-64 was blast-aligned on NCBI and a phylogenetic tree was constructed using MEGA (Version 11.0) software.

[0028] The ITS sequence of fungus J-64 has 100% similarity to the gene sequence of *Aspergillus fumigatus*, therefore J-64 is identified as *Aspergillus fumigatus*, belonging to the phylum Fungi, class Oomycetes, order Mucorales, family Mucoraceae, and genus *Aspergillus*. Figure 6 ).

[0029] Example 2: Determination of antagonism between strains The interaction between bacterial strains was assessed using a plate confrontation assay. Bacteria Z-1 and DF-727 were inoculated onto LB solid medium using the streak method and incubated upside down at 30°C for 24 h. After incubation, no boundary line formed at the intersection of the strains, and both strains grew well, indicating that there was no antagonistic effect between them (e.g., no cross-linking). Figure 7 a).

[0030] A 5 mm diameter fungal J-64 mycelial disc was inoculated at the center of a PDA plate, and 10 μL of bacterial Z-1 suspension (OD600=0.6) was spot-inoculated 2 cm from the edge of the mycelial disc. The plates were incubated upside down at 30 ℃ for 3-5 days. After mixed culture, no boundary line was observed at the point where the strains were tangent, and their growth was not affected by each other, indicating that there is no antagonism between them (e.g., ...). Figure 7 b). Furthermore, using the same method, no antagonism was observed between bacteria DF-727 and fungus J-64 (e.g., ...). Figure 7 c).

[0031] Example 3: Preparation of solid compound microbial agent (1) Preparation of seed liquid Z-1 glycine Bacillus was subjected to liquid fermentation until the strain reached the logarithmic phase. The liquid culture medium was LB liquid medium. The fermentation broth obtained after the fermentation was completed was the glycine Bacillus seed culture.

[0032] DF-727 Bacillus subtilis was subjected to liquid fermentation until the strain reached the logarithmic phase. The liquid culture medium was LB liquid medium. The fermentation broth obtained after the fermentation was completed was the Bacillus subtilis seed culture.

[0033] J-64 Mucor was subjected to liquid fermentation until the strain reached the logarithmic phase. The liquid culture medium was PDB liquid culture medium. The fermentation liquid obtained after the fermentation was completed was the Mucor seed liquid.

[0034] (2) Preparation of microbial agents The seed culture of strain Z-1 was inoculated into a basal fermentation medium with soluble starch as the carbon source and ammonium chloride as the nitrogen source. The culture temperature was 50℃, the pH of the basal fermentation medium was 7.0, the rotation speed was 150 r / min, and the seed culture volume was 11%. The culture was carried out in a constant temperature shaker for 12 hours. After fermentation, the fermentation broth obtained was the Z-1 inoculum.

[0035] The seed culture of DF-727 strain was inoculated into a basal fermentation medium with sucrose as the carbon source and ammonium chloride as the nitrogen source. The culture temperature was 50℃, the pH of the basal fermentation medium was 7.0, the rotation speed was 150 r / min, and the seed culture volume was 9% in a constant temperature shaker for 12 hours. After fermentation, the fermentation broth obtained was the DF-727 inoculum.

[0036] The seed culture of strain J-64 was inoculated into a basal fermentation medium with glucose as the carbon source and ammonium chloride as the nitrogen source. The culture temperature was 45℃, the pH of the basal fermentation medium was 6.0, the rotation speed was 150 r / min, and the seed culture volume was 7% in a constant temperature shaker for 6 days. After fermentation, the fermentation broth obtained was the inoculum of J-64.

[0037] Sterilized edible mushroom residue was mixed 1:1 with fermentation broth from three different bacterial strains, and then dried in a 55℃ aseptic drying oven (final moisture content 40-50%) to obtain a single solid compound microbial agent. These single solid compound microbial agents were then mixed in equal proportions to obtain a solid compound microbial agent. Testing showed that the effective viable count of the obtained agent was 1.7 × 10⁻⁶. 8 CFU / g.

[0038] Example 4: Verification of the ability of compound microbial agents to promote aerobic fermentation of sludge The sludge used in the fermentation experiment came from a wastewater treatment plant in Zunyi City, Guizhou Province, China. The experiment was designed with 5 groups, with 1%, 3% and 5% of the prepared compound bacterial agent added respectively as experimental groups, and no bacterial agent added and commercially available bacterial agent added as control groups. The fermentation raw materials were mixed and then carried out in an 80L polyethylene tank for aerobic reaction.

[0039] Experimental results showed that adding 3% compound microbial agent significantly optimized the fermentation process. The high-temperature phase in the experimental group was advanced by 1-2 days (e.g., Figure 8 The highest temperature reached 65.3℃, and the composting period was shortened to 21 days; the total nitrogen retention rate was improved (e.g., Figure 9 Ammonia nitrogen volatilization decreased (e.g.) Figure 10 ), nitrate nitrogen content increased by 23%-35% (e.g. Figure 11 ); Germination index (GI) reached 91% (e.g. Figure 12 The humus content increases to 285-298 g / kg (e.g. Figure 13Inoculation with the compound microbial agent altered the microbial community structure during composting, inhibiting the growth of harmful strains such as Brucella. This verified the synergistic effect of the compound microbial agent in the aerobic fermentation of sludge.

Claims

1. A sludge aerobic fermentation composite microbial agent, characterized in that: The active ingredients in the microbial agent include Bacillus glycinifermentans (Z-1), Bacillus subtilis (DF-727), and Mucor circinelloides (J-64). Among them, Bacillus glycineus Z-1 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with the accession number CGMCC No. 33468; Bacillus subtilis DF-727 was deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC No. 33467; J-64 Mucor was deposited on April 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41140.

2. The sludge aerobic fermentation composite microbial agent according to claim 1, characterized in that: It consists of a solid inoculum composed of Z-1 glycine bacillus, DF-727 Bacillus subtilis and J-64 Mucor.

3. The sludge aerobic fermentation composite microbial agent according to claim 2, characterized in that: The solid inoculum is composed of Z-1 glycine bacillus, DF-727 Bacillus subtilis and J-64 Mucor spp. in a ratio of 1-3:1-3:1-3.

4. The sludge aerobic fermentation composite microbial agent according to claim 3, characterized in that: The solid inoculum consists of Z-1 glycine bacillus, DF-727 Bacillus subtilis and J-64 Mucor in a 1:1:1 ratio.

5. The sludge aerobic fermentation composite microbial agent according to claim 3, characterized in that: The effective viable cell number of the sludge aerobic fermentation composite microbial agent is ≥1.7×10 8 CFU / g.

6. The method for preparing the aerobic fermentation compound microbial agent for sludge according to any one of claims 1-5, characterized in that: Includes the following steps: Step 1, Preparation of seed solution: Z-1 glycine-bacterium, DF-727 Bacterium subtilis, and J-64 Mucor were inoculated into liquid culture medium and cultured until the logarithmic growth phase of the strains. After fermentation, seed liquids of Z-1 glycine-bacterium, DF-727 Bacterium subtilis, and J-64 Mucor were obtained. Step 2, Preparation of bacterial culture: Seed cultures of Bacillus Z-1 glycine, Bacillus subtilis DF-727, and Mucor truncatella J-64 were inoculated into liquid fermentation medium and cultured until the OD value of the culture was 1. After fermentation, the culture cultures of Bacillus Z-1 glycine, Bacillus subtilis DF-727, and Mucor truncatella J-64 were obtained. Step 3, Preparation of a single solid microbial agent: The sterilized mushroom residue was mixed with the seed liquids of Bacillus glycinus Z-1, Bacillus subtilis DF-727, and Mucor truncatula at a ratio of 1:

1. The mixture was then dried in a sterile incubator at 30°C to produce solid inoculum of Bacillus glycinus Z-1, Bacillus subtilis DF-727, and Mucor truncatula. Step 4, Preparation of solid compound microbial agent: Solid inoculants of Z-1 glycine-containing Bacillus, DF-727 Bacillus subtilis, and J-64 Mucor were mixed in equal proportions to obtain a composite inoculant for aerobic fermentation of sludge.

7. The application of the aerobic fermentation compound microbial agent for sludge according to any one of claims 1-5, characterized in that: The aerobic fermentation compound microbial agent for sludge is used in the aerobic fermentation operation of sludge.

8. The application of the aerobic fermentation compound microbial agent for sludge according to claim 7, characterized in that: The aerobic fermentation compound microbial agent for sludge is added at a rate of 3% by mass, based on the dry weight of the sludge.

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