Compost high-temperature degrading bacterium as well as method and application thereof for screening from vinasse compost

Abstract

The invention relates to the technical field of microbial screening and biodegradation, and discloses a compost high-temperature degrading bacterium as well as a screening method and application thereof. Microorganisms are enriched from vinasse compost, the bacillus licheniformis JZ-1 is obtained through TOC degradation rate screening, the bacillus licheniformis JZ-1 is preserved in Guangdong Microbial Culture Collection Center on September 22, 2025, and the preservation number is GDMCC NO: 67012. The strain efficiently degrades organic matters of the vinasse under the acidic conditions that the temperature is 50-55 DEG C and the pH value is smaller than or equal to 5, lime does not need to be added for neutralization, the problems of nutrient loss and mineral imbalance existing in a traditional method are solved, and an efficient microbial technical scheme is provided for vinasse resourceful treatment.

Description

Technical Field

[0001] This invention relates to the field of microbial screening and biodegradation technology, specifically to a composting high-temperature degrading bacterium and its screening method and application from distiller's grains compost. Background Technology

[0002] Distillery lees, a byproduct of the brewing industry, contains large amounts of recalcitrant organic matter such as cellulose, hemicellulose, and lignin, which can cause environmental pollution if directly discharged. Traditional distillery lees composting relies on natural microbial communities, but suffers from problems such as a short duration of the high-temperature phase, low efficiency in lignocellulose decomposition, long composting cycles, and high total organic carbon (TOC) residues. Existing microbial agents are mostly developed based on thermophilic bacteria, such as Bacillus licheniformis and Rhizopus microsporus, which maintain metabolic activity at 37–50 °C, while strains like Aspergillus niger and Aspergillus terreus are naturally adapted to acidic environments with pH < 5. However, thermophilic bacteria struggle to adapt to the high-temperature (above 50 °C) and acidic (pH < 5) environment of distillery lees composting, resulting in long composting cycles and high TOC residues. To improve composting efficiency, current technologies often add lime to neutralize the acidity, but the amount of lime used is difficult to control; excessive lime can inhibit microbial activity, lead to nitrogen loss, vitamin degradation, and mineral imbalances, affecting compost quality.

[0003] Recent studies have found that Bacillus licheniformis and Aspergillus strains are adaptable to high temperature and acidic environments, but research on the degradation of organic matter in distiller's grains by Bacillus licheniformis under high temperature environments above 50°C is still lacking. Summary of the Invention

[0004] This invention aims to provide a composting thermophilic degrading bacterium capable of efficiently degrading organic matter in high-temperature and acidic environments above 50°C; to provide a screening method for this strain, enabling its targeted enrichment and separation from distillers' grains compost; and to provide a method for applying this strain in distillers' grains compost to replace or reduce the use of lime, thereby solving problems such as inhibition of microbial activity, nutrient loss and mineral imbalance, low organic matter degradation efficiency, high TOC residue, and long composting cycle in existing lime neutralization methods.

[0005] To achieve the above objectives, the first aspect of this application provides a composting thermophilic degrading bacterium, wherein the strain is Bacillus licheniformis (B. licheniformis). Bacillus licheniformis JZ-1 was deposited at the Guangdong Microbial Culture Collection Center on September 22, 2025, with accession number GDMCC NO:67012.

[0006] Furthermore, the strain grows in a temperature range of 50-55°C and / or an acidic environment with pH ≤ 5, and degrades organic matter in the distiller's grains.

[0007] Secondly, this application also provides a screening method for the said strain, including the following steps: S1. Add the distiller's grains and compost products to the bacterial enrichment medium for enrichment culture. The enrichment culture is carried out for 5 to 7 days at a temperature of 45 to 50°C and a shaking speed of 150 to 170 r / min. S2. Screen for high-efficiency bacterial groups by TOC degradation rate, isolate and purify bacteria on bacterial solid culture medium by dilution plating method, and incubate at 45~50℃ for 2~3 days to screen single colonies; S3, single colonies were expanded and passaged on LB medium.

[0008] Furthermore, the bacterial enrichment medium contains 5.0 g / L peptone, 5.0 g / L yeast extract, and 3.0 g / L NaCl, with the pH set to natural.

[0009] Furthermore, the bacterial solid culture medium contains 5.0 g / L peptone, 5.0 g / L yeast extract, 3.0 g / L NaCl, 15-20 g / L agar, and is at its natural pH.

[0010] Thirdly, this application also provides the application of the aforementioned composting thermophilic degrading bacteria in the degradation of organic matter in distiller's grains compost.

[0011] Fourthly, the method of using the aforementioned high-temperature degrading bacteria to degrade organic matter in distillers' grains involves adding the strain to the distillers' grains compost at an inoculation rate of 2%-3% to promote TOC degradation under conditions of 50-55℃.

[0012] Furthermore, in the method, an acidity correction experiment is simulated, in which the pH of the culture medium is periodically adjusted to the initial acidity value using 0.1 mol / L dilute hydrochloric acid during the culture process.

[0013] Furthermore, the culture temperature is 55°C.

[0014] Fifthly, this application also provides a composting agent containing Bacillus licheniformis JZ-1 as its active ingredient, which is suitable for promoting the organic matter degradation and composting of distiller's grains compost.

[0015] This invention enriches bacteria through high-temperature degradation of distiller's grains compost, obtaining strain JZ-1. The 16S rRNA sequence is shown in SEQ ID NO. 1. BLAST analysis was performed, and the gene sequence of the single-celled strain JZ-1 obtained in this invention was retrieved from NCBI. Its gene sequence is similar to that of Bacillus licheniformis (…). Bacillus licheniformis The similarity was over 98%, therefore the strain identified was Bacillus licheniformis.

[0016] The Bacillus licheniformis JZ-1 strain screened in this invention is adapted to the high-temperature (above 50°C) and acidic (pH≤5) environment of distiller's grains composting and can reproduce and grow. Furthermore, it can effectively degrade the organic matter in distiller's grains, significantly improve the TOC degradation rate, shorten the composting cycle, and avoid nutrient loss and mineral imbalance problems caused by lime addition. This provides an efficient and specific microbial solution for the high-value resource utilization of distiller's grains, offering both significant environmental and economic benefits. Attached Figure Description

[0017] Figure 1 This is a graph showing the change in TOC degradation rate of composting bacteria in bacterial enrichment solution. Figure 2 To assess the pH adaptability of Bacillus licheniformis to the primary screening medium in a high-temperature system at 50 °C; Figure 3 The growth curve of Bacillus licheniformis in the primary screening medium at a high temperature of 50 °C is shown. Figure 4 The graph shows the change of TOC content in the initial screening culture medium over time in a high-temperature system at 50 °C. Figure 5 The graph shows the pH changes of the initial culture medium for Bacillus licheniformis in a high-temperature system at 55 °C. Figure 6 The growth curve of Bacillus licheniformis in the primary screening medium at a high temperature of 55 °C is shown. Figure 7 The graph shows the change of TOC content of Bacillus licheniformis over time in a high-temperature system at 55 °C. Figure 8 The growth curve of Bacillus licheniformis in the primary screening medium under periodic acid regulation; Figure 9 The graph shows the change in TOC content of Bacillus licheniformis over time under periodic acid regulation. Figure 10 This graph shows the change in TOC degradation rate of Bacillus licheniformis in the primary screening medium. Figure 11 The TOC degradation rate of Bacillus licheniformis in the primary screening medium. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: Enrichment of functional microorganisms A mixture of distiller's grains and compost products is used to cultivate bacteria in a culture medium. The changes in the organic matter content of the culture medium are used to achieve the purpose of enriching functional microorganisms.

[0020] The culture medium formula used is as follows: Preparation method of bacterial enrichment medium: 5.0 g peptone, 5.0 g yeast powder, 3.0 g NaCl, 1000 mL deionized water, natural pH, sterilized at 121 ℃ for 20 min.

[0021] Culture medium preparation and culturing process: This procedure for enriching functional microorganisms involved two control groups and five parallel experimental groups. The containers used to store the bacterial culture medium were 250 mL Erlenmeyer flasks sterilized at 121 °C for 20 min. The control group Erlenmeyer flasks contained only 150 mL of bacterial enrichment medium, while the parallel experimental group Erlenmeyer flasks contained 150 mL of medium, plus 1 g of distiller's grains and 5 g of compost product in each flask. The prepared culture medium was placed in a 50 °C shaking incubator and cultured at 160 r / min. The expected culturing time was 5–7 days.

[0022] Determine the organic matter content in the culture medium: Take 5 mL of culture medium every 24 h, and stir at 5000 r / min. -1 Centrifuge at a speed of 10 min, determine the TOC concentration of the supernatant using a total organic carbon analyzer (Shimadzu, Japan, TOC-L CPH / CPN), and calculate the degradation rate for the day. Figure 1 The TOC degradation rate of bacteria decreased slightly in the early stage of enrichment, but the overall trend was upward. After 120 h (the fifth day of enrichment), the TOC degradation rate gradually stabilized, reaching a maximum of 81.60%. The second group of bacterial enrichment solution (final degradation rate of 79.44%) was taken at the end of the enrichment period of 144 h for bacterial separation and purification.

[0023] Example 2: Isolation, purification, and strain preservation of microorganisms Preparation of diluted bacterial culture: Take 1 mL of the bacterial culture with the highest organic matter degradation rate obtained in Example 1, add it to a 10 mL centrifuge tube sterilized at 121 °C for 20 min, and add 9 mL of deionized water sterilized in the same way. Shake well to obtain a dilution factor of 10. -1 The diluted bacterial solution was then added from 10... -1 Take 1 mL of the diluted bacterial culture and add it to a second centrifuge tube. Add 9 mL of deionized water and mix thoroughly to obtain 10 mL of the diluted bacterial culture. -2 The diluted bacterial suspensions were then diluted in this manner, resulting in a total dilution gradient of 10. -1 10 -2 , ... , 10 -9 Diluted bacterial solution.

[0024] Use a pipette to draw 200 μL of the diluted bacterial solution prepared above and spread it evenly on the surface of the bacterial solid culture medium. Invert the medium and incubate it in a 50 °C constant temperature incubator. After the strain has grown, select the culture medium with good strain growth for the next step of strain expansion culture.

[0025] The culture medium formulations involved are as follows: Preparation method of bacterial solid culture medium: 5.0 g peptone, 5.0 g yeast powder, 3.0 g NaCl, 15-20 g agar, 1000 mL deionized water, natural pH, sterilize at 121 ℃ for 20 min.

[0026] Ten vigorous bacterial strains were selected using an inoculation loop sterilized with an alcohol lamp and inoculated into 30 mL of bacterial LB medium. The medium was then shaken thoroughly and placed in a shaking incubator at 50 °C and 160 r / min for 2-3 days for expansion.

[0027] The formulation of the bacterial LB medium is as follows: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 1000 mL deionized water, natural pH, sterilized at 121 ℃ for 20 min.

[0028] Using a pipette, 200 μL of the expanded bacterial culture was transferred to the surface of the bacterial solid medium and spread evenly. The medium was then inverted and placed in a 50 °C constant temperature incubator for incubation. Finally, single bacteria were selected from 10 bacterial solid mediums and inoculated into 30 mL of bacterial LB medium. The inoculated medium was thoroughly shaken and then placed in a shaking medium at 50 °C and 160 r / min for subculture for 3-4 days.

[0029] At room temperature, the cultured bacterial solution after passage was incubated at 5000 r·min -1 Centrifuge for 10 min in a centrifuge, remove the supernatant to 1 mL with a pipette, mix the resuspended bacterial solution with a pipette, transfer 800 µL of the resuspended bacterial solution into a centrifuge tube, add 1 mL of 80% sterile glycerol, mix thoroughly, and store at -80 °C.

[0030] Example 3: Identification of Functional Microorganisms The colony characteristics of strain JZ-1 are as follows: After isolation and purification, it grows well on the surface of bacterial solid culture medium at 50 ℃. The colonies are nearly round, dull, raised in the center, with neat edges, and grayish-white.

[0031] Molecular identification based on the 16S sequence: its 16S rDNA sequence is shown in SEQ ID NO. 1. Upon comparison, its gene sequence is similar to that of Bacillus licheniformis (…). Bacillus licheniformisThe similarity was over 98%, therefore the strain identified was Bacillus licheniformis.

[0032] Example 4: Screening strains' adaptability and degradation effect on organic matter in distiller's grains Add 0.1 mL of bacterial culture medium with organic matter degradation function to 10 mL of bacterial LB medium, shake well, and incubate at 50 ℃ and 160 r / min for 2-3 days. Then incubate at 5000 r·min. -1 Centrifuge at a speed of 10 min, discard the supernatant, dilute with sterile deionized water to prepare a bacterial suspension, so that the bacterial suspension for testing is accurate. OD 600 The value was approximately 1.0. The bacterial suspension was added to 100 mL of primary screening medium at an inoculation rate of 3%, with three replicates for each bacterial strain. The primary screening medium without added bacterial suspension served as a control group. All samples were incubated at 50 °C and 160 r / min. -1 They are cultured in a vibrating incubator.

[0033] The initial screening culture medium was formulated as follows: 25 g of distiller's grains sample, 5.0 g of NaCl, 1000 mL of deionized water, natural pH, sterilized at 121 ℃ for 30 min.

[0034] Every 8 hours, 5 mL of culture medium was collected and measured using a UV-Vis spectrophotometer (Shimadzu UVmini-1240, Japan). OD 600 The pH of the culture medium was measured using a portable multi-parameter analyzer, and then the solution was analyzed at 5000 r·min. -1 Centrifuge at a speed of 10 min, collect the supernatant and place it in a total organic carbon analyzer to determine the TOC, and calculate the TOC degradation rate and degradation rate of the strain on the initial screening medium. This is used to evaluate the degradation effect of the screened strain on organic matter.

[0035] The initial pH of the primary screening medium was 3.6. Figure 2 The pH of the control group remained almost unchanged, while *Bacillus licheniformis* rapidly adapted to the acidic environment within the first 40 hours and efficiently regulated the pH. Within a time range of 40-56 hours, the pH of the primary screening medium remained stable at around 7.7, indicating that *Bacillus licheniformis* of this invention can improve the acidic environment of the primary screening medium to a neutral environment. This bacterium rapidly multiplied and grew within the first 16 hours (…). Figure 3 The colony count initially increased, then grew slowly over the next 16-48 hours, reaching its maximum OD600 value at 48 hours, after which the colony count began to decline. *Bacillus licheniformis* significantly reduced the TOC content of the initial screening medium in the first 32 hours. Figure 4 and Figure 10Among them, the TOC degradation rate increased rapidly in the first 8 hours. Figure 11 The degradation rate gradually decreased after 32 hours, with TOC degradation slowing down and stabilizing after 48 hours. The results indicate that *Bacillus licheniformis* can effectively degrade organic matter in distiller's grains at 50 °C, achieving a degradation rate as high as 77.24%.

[0036] Example 5: Degradation effect of Bacillus licheniformis on organic matter in distiller's grains under a constant temperature system of 55 ℃ The bacterial suspension was prepared using the same method as in Example 4. The Bacillus licheniformis suspension was added to the primary screening medium, and a control group without the bacterial suspension was set up. Each group had three replicates. The culture was incubated at 55 °C and 160 rpm. -1 The culture medium was incubated in a shaking incubator, and samples were taken every 8 hours. Simultaneously, the pH of the culture medium was measured according to the method described in Example 4. OD 600 The study investigated whether the strain of this invention could effectively reduce the organic matter content in distiller's grains and adapt to the high-temperature acidic environment under a culture system at 55°C (above 50°C).

[0037] Depend on Figure 5 It can be seen that under high-temperature cultivation at 55 ℃, Bacillus licheniformis can rapidly adjust the pH of the initial screening culture medium from pH=3.6 to pH=7.4 within the first 32 hours, then gradually slow down, finally stabilizing at around 7.7. The strain can rapidly multiply within the first 16 hours and enter a stable growth phase, after which its numbers begin to decrease. Figure 6 This strain can significantly reduce TOC content, with a degradation rate of up to 76.6%. Figure 7 and Figure 10 It undergoes rapid degradation in the first 8 hours, after which the degradation gradually slows down. Figure 11 TOC content tended to stabilize after 48 h. Figure 7 ).

[0038] Example 6: Periodic pH Adjustment Investigates the Adaptability of Bacillus licheniformis to Acidic Environments and its Ability to Degrade Organic Matter in Distillers' Grains The bacterial suspension was prepared using the same method as in Example 4. The bacterial suspension was added to the initial screening medium and used as a control group, with three replicates for each group. The culture medium was incubated at 55 °C and 160 r / min. -1 The culture medium was incubated in a shaking incubator, and samples were taken every 8 hours. Simultaneously, the concentration of the culture medium was determined using the method described in Example 4. OD 600TOC. After sampling, the pH of the culture medium system was adjusted to approximately 3.6 using 0.1 mol / L dilute hydrochloric acid to investigate the adaptability of Bacillus licheniformis to acidic environments and its TOC degradation ability, as well as the technical limitations of using lime to neutralize the acidic environment of composting.

[0039] Within the first 24 hours, Bacillus licheniformis can rapidly multiply ( Figure 8 After that, the increase slows down, and then begins to decrease after 48 hours. Bacillus licheniformis can efficiently reduce TOC content under periodic acidic conditions, also rapidly degrading it within the first 8 hours, then the degradation rate decreases, and the TOC content tends to stabilize after 48 hours. Figure 9 and Figure 11 Furthermore, the degradation rate of TOC can reach a relatively high level, with a maximum of 71.21%. Figure 10 ).

[0040] Therefore, the present invention (Bacillus licheniformis) Bacillus licheniformis JZ-1 can adapt to the high-temperature, acidic environment of organic matter in distiller's grains compost, and can adjust the acidity to neutral, and can reproduce and grow in this environment. In addition, this Bacillus licheniformis (… Bacillus licheniformis JZ-1 can rapidly and effectively degrade the organic matter content of distiller's grains compost, achieving a degradation rate of up to 77.24% at 50℃, and maintaining a similar rate of 76.6% at 55℃ (above 50℃). In addition, Bacillus licheniformis (… Bacillus licheniformis JZ-1 has a strong ability to adapt to acidity, and its degradation rate is as high as 71.21% under the condition of periodic pH adjustment, which provides scientific support for the high-temperature composting of distillers' grains above 50 ℃ and the limitations of current lime acid neutralization composting.

[0041] Application Experiment: To clarify the advantages of the microbial agent of this invention in actual fertilizer production, the applicant cooperated with a local organic fertilizer manufacturer to conduct a comparative experiment. With "simulated factory composting conditions" as the core, the actual application effects of the two groups of microbial agents were compared by controlling variables. The method is highly operable and meets production needs.

[0042] I. Basic Information for Experimental Design

[0043] II. Experimental Procedure 1. Inoculation with microbial agent: The control group was evenly spread and mixed according to the recommended dosage of commercially available microbial agent (e.g., 1‰, based on the dry weight of the pile); the experimental group was sprayed after diluting the inoculation amount (approximately 20 L JZ-1 microbial agent) at 2% of the total volume of the pile according to the method described in this article. Both groups were mixed 3 times by a pile turner to ensure uniformity.

[0044] 2. Composting Management: Both groups are placed in the same workshop, with the ambient temperature controlled at 15-25℃. The same turning frequency is used (turning the pile once every 2 days for the first 7 days, and once every 3 days thereafter). The moisture content of the pile is maintained at 50%-60% by spraying water, and no lime is added for neutralization.

[0045] 3. Sampling and testing: Sampling was carried out on day 0 (initial), day 3, day 7, day 14 and day 21 (end of composting). For each group, a mixed sample was taken from 3 different locations (depth 30-50 cm) and key indicators were tested.

[0046] III. Core Testing Indicators and Judgment Criteria

[0047] IV. Results and Data Analysis Direction 1. Temperature comparison: The experimental group was expected to reach a high temperature of 55℃ on the 3rd day, and the high temperature (55-60℃) lasted for ≥7 days; the control group may need more than 5 days to reach 50℃, and the high temperature lasted for ≤5 days. This result reflects the high temperature adaptability advantage of JZ-1 bacterial agent.

[0048] 2. Efficiency comparison: When composting for 21 days, the experimental group had a TOC degradation rate ≥75% and a composting period ≤21 days; the control group had a TOC degradation rate ≤60% and a composting period ≥28 days, indicating that JZ-1 microbial agent significantly improves production efficiency.

[0049] 3. Nutrient comparison: The total nitrogen, total phosphorus and total potassium content of the experimental product was expected to be ≥6%, which was higher than that of the control group (expected ≤5%), indicating that JZ-1 inoculant can reduce nutrient loss and improve the quality of the finished product.

Claims

1. A composting thermophilic degrading bacterium, characterized in that: The strain is Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis JZ-1 was deposited at the Guangdong Microbial Culture Collection Center on September 22, 2025, with accession number GDMCC NO:67012.

2. The composting thermophilic degrading bacteria according to claim 1, characterized in that: The strain grows in a temperature range of 50-55°C and / or an acidic environment with pH ≤ 5, and degrades organic matter in the distiller's grains.

3. The method for screening strains as described in claim 1, characterized in that: Includes the following steps: S1. Add the distiller's grains and compost products to the bacterial enrichment medium for enrichment culture. The enrichment culture is carried out for 5 to 7 days at a temperature of 45 to 50°C and a shaking speed of 150 to 170 r / min. S2. Screen for high-efficiency bacterial groups by TOC degradation rate, isolate and purify bacteria on bacterial solid culture medium by dilution plating method, and incubate at 45~50℃ for 2~3 days to screen single colonies; S3, single colonies were expanded and passaged on LB medium.

4. The method according to claim 3, characterized in that, The bacterial enrichment medium contains 5.0 g / L peptone, 5.0 g / L yeast extract, and 3.0 g / L NaCl, with the pH set to natural.

5. The method according to claim 3, characterized in that, The bacterial solid culture medium contains 5.0 g / L peptone, 5.0 g / L yeast extract, 3.0 g / L NaCl, and 15-20 g / L agar, with the pH set to natural.

6. The application of the composting thermophilic degrading bacteria as described in claim 1 in the degradation of organic matter in distiller's grains compost.

7. A method for degrading organic matter from distiller's grains using the thermophilic composting bacteria as described in claim 1, characterized in that: The strain was inoculated at 2% to 3% of the total volume of the composting system, and TOC degradation was promoted at 50 to 55°C.

8. The method according to claim 7, characterized in that: A simulated acidity correction experiment was conducted, in which the pH of the culture medium was periodically adjusted to the initial acidity value using 0.1 mol / L dilute hydrochloric acid during the culture process.

9. The method according to claim 8, characterized in that: The culture temperature is 55℃.

10. A composting agent comprising the strain of claim 1, characterized in that: Its active ingredient includes Bacillus licheniformis JZ-1, which is suitable for the degradation and maturation promotion of organic matter in distiller's grains compost.

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