Fibrolytic bacterium j6 and use thereof
By using the fiber microbacterium J6, the problems of long composting time and poor quality were solved. It achieved efficient degradation of cellulose, protein and starch in harsh environments, promoted the rapid maturation of cow manure aerobic compost, and improved compost quality.
Patent Information
- Application Number
- CN202510496918.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2026-03-03
- Estimated Expiration
- 2045-04-21
AI Technical Summary
Traditional composting processes suffer from problems such as long composting time, significant nutrient loss, and poor quality. Furthermore, environmental factors in aerobic composting have a substantial impact on microbial growth and biological activity.
The fiber microbacterium J6 is used. This strain has the ability to degrade cellulose, protein and starch, and is resistant to acid, alkali, salt and heavy metals. It is adapted to environments with pH 5~10, temperature 10℃~50℃ and salt concentration of 0.5%~9.5%, and is used in aerobic composting of cow manure.
Accelerating the composting process, shortening the maturation cycle, increasing the degree of maturation, promoting the compost pile to enter the high-temperature stage more quickly, and improving the quality of compost.
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Figure CN120399939B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a fibrous microbacterium J6 and its applications. Background Technology
[0002] Composting is a key measure to achieve the harmless, resource-based, and fertilizer-based utilization of agricultural waste, and to promote the development of circular agriculture. Traditional composting relies solely on the action of microorganisms, which results in problems such as long composting time, significant nutrient loss, and poor quality.
[0003] Inoculating exogenous microorganisms during aerobic composting can effectively address these issues. However, differences in substrate composition, salinity, pH, and temperature in aerobic composting significantly impact the growth and bioactivity of microorganisms. Therefore, exploring the effects of microbial agents on composting and screening new strains for research is crucial for the harmless, resource-based, and fertilizer-based treatment of agricultural waste, holding immense potential value in environmental protection. Summary of the Invention
[0004] The purpose of this invention is to provide a cellulose microbacterium J6 that is degradable to cellulose, protein, and starch, and is resistant to acid, alkali, salt, and heavy metals, as well as its applications.
[0005] The present invention adopts the following technical solution:
[0006] A type of fibrous microbacteria ( Cellulosimicrobium cellulans J6 was deposited on January 13, 2025 at the China Center for Type Culture Collection, Wuhan, Hubei Province, China, with accession number CCTCC NO:M2025107.
[0007] Furthermore, the fibrous microbacterium J6 has the ability to degrade cellulose, protein, and starch.
[0008] Furthermore, the optimal temperature range for the growth of the fibrous microbacteria J6 is 10℃~50℃.
[0009] Furthermore, the fibrous microbacteria J6 can grow at a pH of 5-10.
[0010] Furthermore, the fibrous microbacteria J6 can grow in a salt concentration range of 0.5% to 9.5%.
[0011] Furthermore, the fibrous microbacteria J6 are tolerant to Cr. 2+ Ions and Zn 2+ ion.
[0012] Application of the aforementioned fibrous microbacterium J6 in aerobic composting.
[0013] Application of the aforementioned fibrous microbacterium J6 in aerobic composting of cow manure.
[0014] A composting fermentation agent comprising the aforementioned fibrous microbacteria J6.
[0015] The beneficial effects of this invention are as follows: the fiber microbacterium J6 of this invention can degrade cellulose, protein, and starch. Furthermore, this strain can grow at pH 5-10, salt concentration 0.5%-9.5%, and temperature 10℃-50℃, and it can also tolerate Cr. 2+ Ions and Zn 2+ Ions can be used for harsh substrates and aerobic composting environments. Experiments using this strain in aerobic composting of cow manure demonstrated that it effectively promotes faster entry of the compost pile into the high-temperature stage, accelerates the composting process, shortens the maturation period, and increases the degree of maturation. Attached Figure Description
[0016] Figure 1 The results show the cellulose degradation ability of strain J6.
[0017] Figure 2 The results show the protein degradation ability of strain J6.
[0018] Figure 3 The results show the starch degradation ability of strain J6.
[0019] Figure 4 The growth curve of strain J6 is shown.
[0020] Figure 5 The effect of temperature on the growth of strain J6.
[0021] Figure 6 The effect of pH on the growth of strain J6.
[0022] Figure 7 The effect of salt concentration on the growth of strain J6.
[0023] Figure 8 Analysis of the tolerance of strain J6 to metallic chromium.
[0024] Figure 9 Analysis of the tolerance of strain J6 to metallic zinc.
[0025] Figure 10 The effect of strain J6 on temperature changes during the aerobic composting of cow manure.
[0026] Figure 11 The effect of strain J6 on the seed germination index (GI) during aerobic composting of cow manure. Detailed Implementation
[0027] The specific technical solutions of the present invention will be described in detail below with reference to embodiments and accompanying drawings.
[0028] Example 1: Isolation and purification of strains
[0029] (1) Sample collection: The compost was collected from the composting experimental field of Hebei University of Science and Technology in Shijiazhuang, Hebei Province, China, during the high-temperature period.
[0030] (2) Sample pretreatment: Take 5g of sample and add it to a 100mL sterile conical flask. Add 50mL of sterile water and shake with a shaker. Take 1mL of the supernatant and put it into LB liquid medium and incubate at 35℃ for 2~3 days.
[0031] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, bring to a final volume of 1000mL, pH 7.0, and autoclave at 121℃.
[0032] (3) Serial dilution: Take 100 μL of bacterial culture and dilute it stepwise to 10 μL. -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 Take 100 μL from the conical flask and inoculate it into LB solid medium (LB liquid medium with 18 g agar powder added). Spread three copies of each dilution simultaneously. Incubate at 35°C for 2-3 days.
[0033] (4) Purification: Observe the colony morphology, pick out single colonies of different morphologies with an inoculation needle, and streak them onto LB solid medium for culture. After several streak purifications, pure single colonies are obtained.
[0034] Example 2 Screening of strains
[0035] (1) Cellulose degradation test
[0036] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it onto sodium carboxymethyl cellulose Congo red medium. Inoculate 4 replicates on each plate and incubate at 30°C for 48 h. Observe the growth of colonies. After incubation, stain with Congo red reagent and observe whether a clear zone appears around the colonies on the medium. Measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value (HC=D / d).
[0037] Carboxymethyl cellulose sodium Congo red medium: NaNO3 2.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4 0.01g, CMC-Na 10.0g, agar 20.0g, pH 9.5, distilled water 1000mL, sterilized at 121℃ for 20min.
[0038] (2) Protein degradation experiment
[0039] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it onto casein agar medium, inoculate 4 replicates on each plate, incubate at 30°C for 48 h, and observe the growth of colonies. After incubation, observe whether a clear zone appears around the colonies on the medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value.
[0040] Casein agar medium: 10.0g casein, 3.0g beef extract powder, 2.0g disodium hydrogen phosphate Na2HPO4, 5.0g NaCl, 0.05g bromothymol blue, 15.0g agar, pH 7.4±0.2, sterilized at 121℃ for 20min.
[0041] (3) Starch degradation experiment
[0042] Pure bacterial colonies were picked and activated in LB liquid medium. The activated bacterial solution was then centrifuged to obtain bacterial cells, which were diluted with sterile water to an OD value of 1. 600 =1, take 10 μL and spot it onto starch medium, inoculate 4 replicates on each plate, incubate at 30°C for 48 h, and observe the growth of colonies. After incubation, add iodine solution and observe whether a clear zone appears around the colonies on the medium. Measure the diameter of the clear zone (D) and the diameter of the colony (d), and calculate the HC value.
[0043] Starch culture medium: 10.0g soluble starch, 1.0g K2HPO4, 1.0g MgSO4, 1.0g NaCl, 2.0g (NH4)2SO4, 2.0g CaCO3, 0.001g FeSO4, 0.001g MnCl2, 0.001g ZnSO4, pH 7.2±0.2, sterilized at 121℃ for 20min.
[0044] The results showed that strain J6 had the ability to degrade cellulose, protein, and starch, as indicated by the assay results. Figures 1-3 And as shown in Table 1.
[0045] Table 1. HC values from degradation experiments
[0046] .
[0047] Example 3 Identification and Preservation of Strains
[0048] Strain J6 was identified as a fibrobacterium (Fibriophyte) by 16S rRNA gene sequencing (SEQ ID No. 1) and NT comparison. Cellulosimicrobium cellulans The pure culture of strain J6 was deposited at the China Center for Type Culture Collection on January 13, 2025, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCCNO:M2025107.
[0049] Example 4: Growth characteristics of strain J6
[0050] The optical density (OD) value is used to measure and reflect the growth status of bacteria, generally ranging from 0 to 4. The higher the OD value, the higher the concentration of microorganisms and the better their growth.
[0051] Strain activation and culture process: After partially thawing the strain frozen at -80℃ in an ice bath, use a sterile inoculation loop to pick up the unmelted ice crystals on the surface of the cryopreservation solution, streak the strains on the surface of LB solid medium, and incubate them upside down at 35℃ for 24 h to obtain single colonies; to eliminate potential contamination or genetic drift during cryopreservation, pick single colonies and transfer them to LB liquid medium, pre-culture at 35℃ and 150 rpm for 24 h to prepare the logarithmic growth phase seed culture.
[0052] (1) Growth curve of strain J6
[0053] The logarithmic growth phase seed culture of strain J6 was inoculated into LB liquid medium (pH 7.0, NaCl 10 g·L⁻¹) at a rate of 1% (v / v). -1 The culture was placed in a shaker at 20℃ and 150 rpm, using fresh LB liquid medium as a reference. A wavelength of 600 nm was selected, and 2 mL of bacterial solution was sampled every 2 hours to measure the optical density (OD) value. A growth curve for the strain was then plotted. Figure 4 As shown in the growth curve of strain J6, J6 enters the logarithmic growth phase after 4 hours and the stationary growth phase after 16 hours. During this process, the OD... 600 The value is approximately between 0.5 and 2.6, indicating that strain J6 is growing well.
[0054] (2) Effect of temperature on the growth of strain J6
[0055] Experiment and detection: The logarithmic growth phase seed culture of strain J6 was inoculated into LB liquid medium at an inoculum of 1% and placed at different incubation temperatures, at 150 r·min. -1 The sample was cultured at a rotating speed for 24 hours, and the absorbance at a wavelength of 600 nm was measured. The results are as follows: Figure 5As shown, within the temperature range of 10~40℃, the OD of strain J6... 600 It increases with increasing temperature; when the temperature is above 40℃, OD 600 Rapid decline; when the temperature exceeds 60℃, OD 600 The concentration dropped below 0.5. The J6 strain is adapted to a temperature range of 10~50℃, with an optimal growth temperature of 30~40℃.
[0056] (3) Effect of pH on the growth of strain J6
[0057] The pH gradient of LB liquid medium was prepared as follows: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0. The logarithmic growth phase seed culture of strain J6 was inoculated at a rate of 1% into Erlenmeyer flasks containing LB liquid medium at different pH values and incubated at 40℃ for 24 hours. The results are as follows... Figure 6 As shown, the suitable pH range for the growth of strain J6 is 5-10, with the optimal pH being 8.
[0058] (4) Effect of salt concentration on the growth of strain J6
[0059] The logarithmic growth phase seed culture of strain J6 was inoculated into LB liquid medium at a 1% inoculation rate, and the NaCl content in the medium was adjusted to 5 g·L⁻¹. -1 10g·L -1 15g·L -1 25g·L -1 35g·L -1 50g·L -1 65g·L -1 80g·L -1 95g·L -1 The pH of the culture medium was adjusted to 8, and the medium was incubated at 30°C and 150 rpm. -1 Incubate with shaking for 24 hours. Results are as follows: Figure 7 As shown, the OD of strain J6 within the measured salt concentration range... 600 All values exceeded 0.5, indicating good tolerance to salinity; the optimal salt concentration for growth was 10 g·L⁻¹. -1 .
[0060] Example 5 Heavy Metal Tolerance Analysis
[0061] (1) Resistance analysis to metallic chromium
[0062] The logarithmic growth phase seed culture of strain J6 was inoculated into LB liquid medium at an inoculum volume of 1%, with Cr in the medium... 2+The concentrations were adjusted to 0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The pH of the culture medium was adjusted to 7, and the medium was incubated at 30℃ and 150 rpm. -1 After 24 hours of shaking culture, the results are as follows: Figure 8 As shown. In the measured Cr... 2+ Within the concentration range, the growth curves of strain J6 all entered the logarithmic growth phase after 4 hours, and the growth curves were good, indicating that strain J6 has good tolerance to metallic chromium.
[0063] (2) Resistance to zinc
[0064] The logarithmic growth phase seed culture of strain J6 was inoculated into LB liquid medium at a 1% inoculum, with Zn in the medium. 2+ The concentrations were adjusted to 0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The pH of the culture medium was adjusted to 7, and the medium was incubated at 30℃ and 150 rpm. -1 Incubate under shaking for 24 hours.
[0065] The results are as follows Figure 9 As shown, the growth performance of strain J6 increased with Zn 2+ The concentration increases and then decreases. When Zn 2+ At a concentration of 100 mg / L, the growth of strain J6 was somewhat inhibited, but after 4 hours of culture, its growth curve reached the logarithmic growth phase; when Zn 2+ When the concentration reaches 200 mg / L or above, Zn 2+ It showed a significant inhibitory effect on the growth of strain J6, and its OD... 600 The value remained below 0.25 throughout the 24-hour incubation period. This indicates that strain J6 has some tolerance to zinc, but this tolerance is significantly affected by concentration. Strain J6's tolerance to Zn... 2+ The tolerable concentration is no more than 200 mg / L.
[0066] Example 6: Aerobic composting of cow manure
[0067] The J6 strain was inoculated into a compost pile containing cow dung as a substrate and wheat straw as an auxiliary material for aerobic composting. The J4 bacterial suspension (effective viable count ≥3×10⁻⁶) was then used for composting. 8 CFU / mL ~4×10 8 The dosage of the microbial agent (CFU / mL) was 0.5% of the wet weight of the compost material. A control group (labeled A) was set up without the addition of microbial agent. Before composting, the heavy metal content in the compost pile was tested, and the results are shown in Table 2.
[0068] Table 2 Heavy metal content of the compost pile before composting
[0069] .
[0070] According to the "Technical Specification for Harmless Treatment of Livestock and Poultry Manure" (GB / T 36195-2018), changes in the temperature of the manure pile can reflect changes in the activity of microorganisms within the pile, and also indicate the progress of aerobic fermentation. For example... Figure 10 As shown, the temperature of the J6 strain (J6 group) reached above 50℃ on day 3 (maximum 52.85℃), while the control group A reached above 50℃ on day 4. The high-temperature period of the J6 group lasted for 6 days, while that of the A group lasted for 5 days. During the high-temperature period, the temperature of the J6 group was about 2-6℃ higher than that of the A group. This indicates that adding the J6 strain during composting can effectively promote the compost pile to enter the high-temperature stage more quickly, which is more conducive to increasing the high temperature during composting and accelerating the composting process.
[0071] The germination index (GI) is a biological indicator reflecting the toxicity of compost to plant seeds and the degree of compost maturity. For example... Figure 11 As shown, after 18 days of composting, the GI value of group J6 reached over 130%, exceeding the 70% requirement of the "Organic Fertilizer Standard" (GB NY 525-2021); while group A was far from meeting the requirement.
[0072] Combination Figure 10 , Figure 11 As can be seen from Table 2, strain J6 still showed good applicability in the aerobic composting process of cow manure in the presence of heavy metals.
Claims
1. A type of fibrous microbacterium ( Cellulosimicrobium cellulans J6, characterized in that, Its accession number is CCTCC NO:M2025107.
2. The fibrous microbacterium J6 according to claim 1, characterized in that, It has the ability to degrade cellulose, protein and starch.
3. The fibrous microbacterium J6 according to claim 1, characterized in that, Its suitable temperature range for growth is 10℃~50℃.
4. The fibrous microbacterium J6 according to claim 1, characterized in that, It can grow at a pH of 5-10.
5. The fibrous microbacterium J6 according to claim 1, characterized in that, It can grow in salt concentrations ranging from 0.5% to 9.5%.
6. The fibrous microbacterium J6 according to claim 1, characterized in that, It can tolerate Cr 2+ Ions and Zn 2+ ion.
7. The application of the fibrous microbacterium J6 as described in claim 1 in aerobic composting.
8. The application of the fibrous microbacterium J6 as described in claim 1 in aerobic composting of cow manure.
9. A composting fermentation agent, characterized in that, It contains the fibrous microbacteria J6 as described in claim 1.
Citation Information
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