Acclimatized high-temperature-resistant aerobacillus pallidus Cb and application thereof in aerobic composting

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to domesticated aerobacillus pallidus Cb resistant to high temperature and application of the domesticated aerobacillus pallidus Cb in aerobic composting. The Aerobacter pallidum Cb which is at least resistant to the high temperature of 70 DEG C and has cellulose degradation capacity is provided for the first time, the application effect of the Aerobacter pallidum Cb is verified in aerobic composting of chicken manure and pig manure, and the Aerobacter pallidum Cb can accelerate composting temperature rise, improve the composting temperature, the seed germination rate and the cellulase activity of a compost body, promote accumulation of nitrate nitrogen and total nitrogen in the compost and improve the yield of the compost. The compost maturity is accelerated, the emission of ammonia gas and carbon dioxide in the composting process is reduced, and a high-quality seed strain is provided for harmless treatment of livestock and poultry manure compost.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a heat-resistant Aerobacillus pallidum Cb after acclimation and application thereof in aerobic composting. Background Art

[0002] Aerobic composting is an effective method for the harmless treatment and resource utilization of livestock and poultry manure. However, the aerobic composting process still suffers from significant challenges, including slow temperature rise, low bioconversion efficiency, and high carbon and nitrogen gas emissions. Exogenous microbial inoculants can accelerate the composting process, but currently, there is a lack of microbial inoculants that can withstand the high temperatures encountered in composting and exhibit efficient degradation. This study screened thermostable cellulose-degrading functional bacteria through high-temperature directed continuous subculture acclimation and investigated their application to achieve efficient bioconversion of livestock and poultry manure.

[0003] Studies have reported that Bacillus pallidus has the ability to withstand high temperatures of 50°C, efficiently degrade polyethylene plastics, produce bioemulsifiers, and remove ammonia. However, there are currently no reports on Bacillus pallidus with the ability to withstand high-temperature cellulose degradation and its application in livestock and poultry manure composting.

[0004] To address the above problems, the present application isolated a strain of Bacillus pallidus Cb that can withstand temperatures of at least 70°C from compost samples in the high-temperature period, and verified its application effect in aerobic composting of chicken manure and pig manure, respectively. The bacteria can accelerate the temperature rise of compost, increase compost temperature, seed germination rate and cellulase activity, promote the accumulation of nitrate nitrogen and total nitrogen in compost, accelerate compost maturity, and reduce ammonia and carbon dioxide emissions during the composting process. Summary of the Invention

[0005] The invention aims to provide a high-temperature-resistant Bacillus pallidum Cb after acclimation. The deposit number of the Bacillus pallidum Cb is CCTCC NO: M20251395.

[0006] Another object of the present invention is to provide the use of the above-mentioned Bacillus pallidum Cb in aerobic composting.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A functional strain with the ability to decompose cellulose and produce a transparent zone was screened from high-temperature samples of biogas residue compost by the dilution spread plate method and cellulose identification medium. After high-temperature acclimation, the strain can withstand a high temperature of at least 70°C. The strain was deposited in the China Center for Type Culture Collection on June 16, 2025, with the classification name: Aeribacillusspallidus Cb, the preservation number: CCTCC NO: M20251395, and the address: Wuhan University, Wuhan, China.

[0009] The single colony characteristics of the pale air Bacillus are: 4 mm in diameter, round in shape, moist in texture on the surface of the colony, smooth and rounded in edge, milky white and translucent in color, and suitable for growth at a temperature of 37° C. to 60° C.

[0010] The protection scope of the present invention also includes:

[0011] A fermentation broth of Aerobacillus pallidus Cb, containing live Aerobacillus pallidus Cb bacteria and / or spores thereof, and application of Aerobacillus pallidus Cb or its fermentation broth in aerobic composting.

[0012] Application of Bacillus pallidum Cb or its fermentation liquid in the preparation of aerobic composting microbial inoculants.

[0013] In the above application, preferably, the aerobic composting is high-temperature aerobic composting.

[0014] In the above application, preferably, the high temperature is 50-70°C.

[0015] Application of Bacillus pallidum Cb or its fermentation broth in cellulose degradation.

[0016] Application of Bacillus pallidum Cb or its fermentation broth in the preparation of cellulose-degrading microbial agents.

[0017] An aerobic composting microbial agent, comprising Aerobacillus pallidum Cb and / or the fermentation broth according to claim 2.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention provides for the first time a strain of Bacillus pallidus Cb that is resistant to high temperatures of at least 70°C and has the ability to degrade cellulose. The application effect of the strain was verified in aerobic composting of chicken manure and pig manure, respectively. The strain can accelerate the temperature rise of the compost, increase the compost temperature, seed germination rate and cellulase activity in the compost, promote the accumulation of nitrate nitrogen and total nitrogen in the compost, accelerate compost maturity, and reduce ammonia and carbon dioxide emissions during the composting process, providing a high-quality seed strain for the harmless treatment of livestock and poultry manure compost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To determine the cellulase activity of different strains.

[0021] Figure 2 Schematic diagram of the morphological characteristics of strain Cb with cellulase activity.

[0022] Figure 3 is the survival rate of strains (Cb) of different generations after high temperature acclimation.

[0023] Figure 4Schematic diagram of temperature changes during composting at different stages.

[0024] Figure 5 Schematic diagram of changes in cellulase activity at different periods;

[0025] Among them: *P<0.05.

[0026] Figure 6 Schematic diagram of the changes in ammonium nitrogen in different periods.

[0027] Figure 7 Schematic diagram of the changes in nitrate nitrogen in different periods.

[0028] Figure 8 Schematic diagram of the changes in the C / N ratio of compost at different periods.

[0029] Figure 9 Schematic diagram of ammonia concentration in the tank during composting at different stages.

[0030] Figure 10 Schematic diagram of CO2 concentration in the tank during the composting process at different stages.

[0031] Figure 11 Schematic diagram of the changes in pile temperature at different periods.

[0032] Figure 12 Schematic diagram of changes in cellulase activity at different periods;

[0033] Among them: *P<0.05, **P<0.01, ***P<0.001.

[0034] Figure 13 NH4 in the pile at different periods + -Schematic diagram of the changes in N.

[0035] Figure 14 NO3 in the pile at different periods - -N change diagram.

[0036] Figure 15 Schematic diagram of the changes in the C / N ratio in the pile at different periods. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field; the reagents or materials described are all from commercial channels unless otherwise specified.

[0038] Example 1:

[0039] Screening and identification of Bacillus pallidum Cb:

[0040] (1) Screening of cellulose-degrading strains

[0041] Eight strains with the ability to decompose cellulose and produce transparent zones were screened from biogas residue compost samples during the high-temperature period by using the dilution plate method and cellulose identification medium. The strains were added to the CMC cellulose enzyme production medium at an inoculum of 2%, shaken, and cultured at 37°C and 150 rpm for 72 h, 120 h, and 148 h, respectively, and their CMCase activity was measured. Figure 1 The CMCase activity of strain Cb was 9.86 U / mL.

[0042] (2) Strain identification

[0043] The strain Cb was milky white in color and smooth and round in appearance on the LB plate ( Figure 2 ); Physiological and biochemical identification is shown in Table 1. Strain Cb has the ability to secrete amylase, can use glucose as a growth material, and has a certain ability to degrade gelatin; the phylogenetic tree of the strain was established, indicating that strain Cb is Bacillus pallidum.

[0044] Table 1 Physiological and biochemical identification of strain Cb

[0045]

[0046] High temperature acclimation culture of strains:

[0047] The above-screened Bacillus pallidum strain was repeatedly subcultured in a 45°C environment. When the bacterial survival rate exceeded 80%, the next subculture was carried out, and the temperature was increased by 5°C each time. After culturing for 14-16 hours, the survival rate was calculated again. Based on this, a relationship between the number of acclimation times and the survival rate of Bacillus pallidum Cb at different temperatures was drawn as shown below. Figure 3 As shown, the finally acclimated Bacillus pallidum Cb can tolerate a high temperature of at least 70°C, and the survival rate after being cultured in an environment of 70°C for 14-16 hours is ≥80%.

[0048] The domesticated strain was deposited in the China Center for Type Culture Collection on June 16, 2025, with the classification name: Aeribacillus pallidus Cb, the deposit number: CCTCC NO: M20251395, address: Wuhan University, Wuhan, China, and was used in the following examples.

[0049] Example 2:

[0050] Growth curve of Bacillus pallidum Cb:

[0051] LB liquid medium was used, 37°C, 180r / min, and the seed liquid of Bacillus pallidum Cb was inoculated at a volume fraction of 1%, and cultured for 12-16h to obtain the fermentation liquid of Bacillus pallidum Cb. The OD of the fermentation liquid was600 The value is 0.85-0.95. The fermentation broth contains Bacillus pallidum Cb and its spores and is used in the following examples.

[0052] Example 3:

[0053] Application of Aerobacillus pallidum Cb in aerobic composting of chicken manure

[0054] This composting experiment was conducted at the experimental base of Huazhong Agricultural University. The test materials included chicken manure from the university's experimental chicken farm and sawdust from the university's veterinary hospital. The manure and sawdust were mixed in a ratio based on their carbon and nitrogen content. After mixing, the compost had a carbon to nitrogen ratio of approximately 25:1 and a moisture content of 65%.

[0055] Bacillus pallidus Cb group: The bacterial weight after centrifugation (8000r / min) of the fermentation broth prepared in Example 2 was used as the standard, and the bacterial agent was added at a ratio of 1% of the weight of the compost. After centrifugation of the fermentation broth, the bacterial cells were diluted with purified water and added to the compost. The effective concentration of Bacillus pallidus after dilution was 10 8 CFU / ml; the CK control group was added with an equal volume of purified water, and all other procedures were the same as the experimental group except that no bacterial agent was added.

[0056] The above composting adopts aerobic composting reactor for aerobic composting. There are vents at the bottom of the tank for aeration, and sampling ports on the front for temperature measurement and sample collection. The ventilation device ventilates at regular intervals to ensure uniform ventilation volume.

[0057] (1) Compost temperature changes

[0058] like Figure 4 As shown, the temperature in each group showed an initial upward trend. Once the maximum temperature was reached, it began to decrease with increasing composting time, ultimately remaining constant. On the first day, temperatures in all experimental groups, except the control group, exceeded 55°C. By the second day, the control group also reached the high-temperature phase of 55°C. The group containing the Bacillus pallidus Cb bacteria reached the high-temperature phase 12 hours earlier than the control group. The addition of the inoculum increased the compost temperature and accelerated the temperature onset. The maximum temperature in the blank control group reached 58.1°C, while that in the Bacillus pallidus Cb group reached 62.2°C, 4.1°C higher than the control group. These results indicate that the addition of Bacillus pallidus Cb bacteria shortened the time to the high-temperature phase, increased the compost temperature, and accelerated the degradation of organic matter.

[0059] (2) Changes in compost seed germination index (GI)

[0060] The GI (Growth Index) is an important indicator for determining the toxicity of compost products and whether they meet standards. It reflects the number of seed germinations and root length. Table 2 shows the effects of compost products on the growth of bok choy seeds. The Aerobacterium pallidus Cb group significantly improved the seed germination index, reaching 74.15% on day 14, meeting the technical requirements for livestock and poultry manure composting (seed germination index ≥ 70.00%) and significantly higher than the control group (Growth Index 58.11%).

[0061] Table 2 Changes in seed germination index in each group

[0062]

[0063] (3) Changes in cellulase activity in compost

[0064] Cellulase activity is an important indicator of cellulose decomposition. Higher cellulase activity is conducive to the reduction of total carbon in the compost at different stages. Cellulase activity determination of samples at different stages in the compost can reflect the degree of composting maturity. Figure 5 The cellulase activity of the two compost groups fluctuated, but the cellulase activity in the group inoculated with Aerobacillus pallidus Cb increased significantly in the later stages of composting, reaching 3.83 U / g, a significant difference from the control group (P < 0.05). This was a 28.63% increase in cellulase activity compared to the control group. This suggests that inoculating compost with Aerobacillus pallidus Cb can increase cellulase activity and promote compost maturity.

[0065] (4) Changes in ammonium nitrogen and nitrate nitrogen content

[0066] Ammonium nitrogen and nitrate nitrogen are important parameters that reflect the maturity of compost, and the changes in nitrogen can be seen intuitively. Figure 6 、 Figure 7 As shown, the ammonium nitrogen content of the group inoculated with Bacillus pallidus Cb was 3.83g / kg on the 15th day, which was significantly lower than that of the control group (5.6g / kg), while the nitrate nitrogen content was significantly higher than that of the control group (0.338g / kg in the Cb group and 0.222g / kg in the control group) on the 15th day. These results show that inoculation with Bacillus pallidus Cb can effectively promote the accumulation of nitrate nitrogen and improve the quality of compost products.

[0067] (5) Changes in total C, total N content and C / N ratio in compost

[0068] The carbon-nitrogen ratio in compost is an important indicator for measuring the composting process and can reflect the degree of maturity of the compost. Figure 8As can be seen from the figure, at the end of the 15th day of composting, the carbon-nitrogen ratio of the group containing Bacillus pallidus Cb was the lowest, at 19.96, and was significantly different from the control group (20.8) (P < 0.05). This indicates that adding Bacillus pallidus Cb can reduce the carbon-nitrogen ratio of compost and improve compost maturity.

[0069] (6) Changes in compost ammonia concentration

[0070] Ammonia concentration is an important parameter indicator of compost odor generation. Ammonia concentration is closely related to nitrogen conversion and the generation of compost odor gas. Figure 9 As shown in the figure, the ammonia concentration in the compost generally showed a trend of first increasing and then decreasing. On the third day, the ammonia concentration in the tank of the control group and the pale aerobic Bacillus Cb group reached the highest value of 1516 mg / m 3 , 1396mg / m 3 , adding Bacillus pallidum Cb significantly reduced the ammonia concentration in compost.

[0071] (7) Changes in CO2 concentration in compost

[0072] like Figure 10 As shown, the CO2 concentration in the tanks of the two groups initially decreased and then increased. Adding Bacillus pallidum Cb significantly reduced the CO2 concentration in the compost. The CO2 concentrations in the control and Cb-added groups were 14.6% and 10.6% on day 1, 9.6% and 4.5% on day 3, and 33.4% and 25.7% on day 15, respectively.

[0073] Example 4:

[0074] Application of Aerobacillus pallidum Cb in aerobic composting of pig manure

[0075] This composting experiment was conducted at the experimental base of Huazhong Agricultural University. The test materials included pig manure from the university's experimental pig farm and sawdust from the university's veterinary hospital. The manure and sawdust were mixed in a ratio based on their carbon and nitrogen content. After mixing, the compost had a carbon to nitrogen ratio of approximately 25:1 and a moisture content of 65%.

[0076] Bacillus pallidus Cb group: The bacterial weight of the fermentation broth prepared in Example 2 after centrifugation was used as the standard, and the bacterial agent was added at a ratio of 1% of the weight of the compost. After the fermentation broth was centrifuged, the bacterial cells were diluted with purified water and added to the compost. The effective concentration of Bacillus pallidus Cb after dilution was 10 8 CFU / ml; the CK control group was added with an equal volume of purified water, and all other procedures were the same as the experimental group except that no bacterial agent was added.

[0077] The above composting adopts aerobic composting reactor for aerobic composting. There are vents at the bottom of the tank for aeration, and sampling ports on the front for temperature measurement and sample collection. The ventilation device ventilates at regular intervals to ensure uniform ventilation volume.

[0078] (1) Temperature changes

[0079] like Figure 11 As shown in the figure, the temperature of the control group and the inoculated group reached above 50℃ on the second day of pig manure composting, and the highest temperature reached 55.3℃ and 57.2℃ respectively on the third day. The addition of Bacillus aeruginosa Cb increased the temperature by 1.9℃ compared with the control group (the control group was 55.3℃), indicating that the addition of Bacillus aeruginosa Cb to pig manure can significantly increase the temperature of the pile.

[0080] (2) Changes in seed germination index GI

[0081] As can be seen from Table 3, inoculating Bacillus pallidum Cb into pig manure compost can significantly increase the GI value. On the 14th day, the GI of the Bacillus pallidum Cb group (GI was 81%) was higher than that of the CK group (GI was 77.6%).

[0082] Table 3 Seed germination index of each group

[0083]

[0084] (3) Changes in cellulase activity

[0085] Similar to the chicken manure compost, on the 7th day of composting, the cellulase activity in the compost of the pale aerobic Bacillus Cb group increased significantly, reaching 2.83U / g, which was significantly higher than that of the control group (P<0.001) (the control group was 2.32U / g), and the cellulase activity increased by 21.9% ( Figure 12 ).

[0086] (4) Changes in ammonium nitrogen, nitrate nitrogen, and C / N ratio in compost

[0087] The ammonium nitrogen content in the compost initially increased and then decreased, while the nitrate nitrogen content showed an overall upward trend. At the end of composting, the ammonium nitrogen content in the control group and the Aerobacillus pallidus Cb group was 6.05 g / kg and 5.93 g / kg, respectively; the nitrate nitrogen content was 0.2 g / kg and 0.25 g / kg, respectively. These results indicate that inoculation with Aerobacillus pallidus Cb can effectively promote nitrate nitrogen accumulation and improve compost quality during pig manure composting. Furthermore, the carbon-nitrogen ratio of the compost in the Aerobacillus pallidus Cb group was significantly lower than that in the control group (19.86 in the Aerobacillus pallidus Cb group and 20.76 in the control group), indicating improved compost maturity.

Claims

1. An isolated strain of Bacillus pallidum ( Aeribacillus pallidus ) Cb, the deposit number of the strain is CCTCC NO: M20251395.

2. The fermentation liquid of Bacillus aeruginosa Cb according to claim 1, wherein the fermentation liquid contains live bacteria of Bacillus aeruginosa Cb and / or spores thereof.

3. Use of the pale aerobic Bacillus Cb according to claim 1 or the fermentation liquid according to claim 2 in aerobic composting.

4. Use of the Bacillus pallidum Cb according to claim 1 or the fermentation liquid according to claim 2 in preparing an aerobic composting microbial agent.

5. The use according to claim 3 or 4, wherein the aerobic composting is high-temperature aerobic composting.

6. The use according to claim 5, wherein the high temperature is 50-70°C.

7. Use of the Aerobacillus pallidum Cb according to claim 1 or the fermentation liquid according to claim 2 in cellulose degradation.

8. Use of the Bacillus pallidum Cb according to claim 1 or the fermentation broth according to claim 2 in the preparation of a cellulose-degrading microbial agent.

9. An aerobic composting microbial agent, comprising Aerobacillus pallidum Cb and / or the fermentation broth according to claim 2.

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