A screening method of a lysing bacterial strain, the lysing bacterial strain and application thereof

Abstract

The present application relates to a kind of screening methods of lysing bacterial strain, lysing bacterial strain and its application, the lysing bacterial strain is soil Bacillus (Bacillus terrae) Geobacillus sp. ) DX11, preserved in China typical culture preservation center, and the preservation number is CCTCC M 2023509;Single colony isolation process used plate solid culture medium includes: D-calcium pantothenate, nitrilotriacetic acid, NaCl, MgSO4·7H2O, Na2HPO4·H2O, NH4Cl, proteose peptone, yeast extract, K2HPO4, D-glucose, agar and trace elements.The lysing bacterial strain of the present application can grow and multiply using organic matter in sludge, and the enzymes produced in its metabolic process can accelerate the hydrolysis of extracellular polymeric substance and the lysis of sludge cells, achieve the purpose of sludge lysis, greatly improve the degradation rate of VSS in sludge reduction process, reduce the amount of residual sludge.

Description

Technical Field

[0001] This invention belongs to the field of biological sludge treatment, specifically relating to a method for screening lysing strains, lysing strains and their applications. Background Technology

[0002] Biological wastewater treatment generates a large amount of residual sludge. With the increasing prevalence of wastewater treatment, the treatment and disposal of this residual sludge has become a significant new challenge in the field of environmental engineering. Currently, methods for reducing residual sludge during wastewater treatment are mainly divided into physical methods, chemical methods, and biological pretreatment methods. Among these, physical and chemical methods lead to additional energy consumption, high costs, secondary pollution, and other economic expenses. In contrast, biological pretreatment shows a clear advantage in accelerating the sludge hydrolysis process.

[0003] Biological pretreatment methods for sludge reduction can be divided into enzymatic methods and microbial strain methods. Enzymatic methods require the addition of hydrolytic enzymes to the sludge, which presents challenges due to high costs and stringent reaction conditions, making large-scale application difficult. Microbial strain methods, on the other hand, primarily utilize anaerobic or cryogenic strains, requiring anaerobic or cryogenic conditions for sludge reduction, which are difficult to control and generally result in poor lysing effects. While our laboratory has previously screened several aerobic thermophilic strains for sludge reduction, achieving some improvement in sludge reduction compared to traditional anaerobic or cryogenic strains, the desired sludge reduction effect is still not achieved. Therefore, further screening of lysing strains is needed to obtain superior sludge reduction strains. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the prior art and provide a method for screening lysogenic strains, lysogenic strains and their applications. The lysogenic strains can grow and reproduce using organic matter in sludge. The enzymes produced during the metabolism of the strains can accelerate the hydrolysis of extracellular polymers and the lysis of sludge cells, thereby achieving the purpose of sludge lysation. This greatly improves the degradation rate of VSS in the sludge reduction process and reduces the amount of residual sludge in wastewater treatment.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Technical Solution 1:

[0007] A lysed strain, Geobacillus sp. DX11, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 10, 2023, with accession number CCTCC M2023509.

[0008] As a further technical solution, the 16S rDNA sequence of the *Geobacillus sp.* DX11 is shown in SEQ ID: 1.

[0009] Technical Solution Two:

[0010] A method for screening the above-mentioned lysed bacterial strains includes the following steps:

[0011] Step 1, Sludge Acclimation: Acclimate the sewage sludge under aeration conditions of 0.1-1.0 vvm and at a temperature of 55-80℃ for 5-60 days.

[0012] Step 2: Isolation of single colonies: After serial dilution of the acclimatized sludge, the strains were isolated and purified using the dilution pour method and the three-zone streak method to obtain single colonies.

[0013] Step 3, Initial screening: The isolated single colony strains are initially screened by the hydrolysis zone test, and strains with larger hydrolysis zones are retained;

[0014] Step 4: Further screen strains with larger hydrolysis zones using sludge lysis tests and extracellular enzyme activity tests. The lysing strains with higher sludge VSS degradation rates and higher extracellular enzyme activity are retained as the dominant strains.

[0015] As a further technical solution, the solid plate culture medium used in the single colony isolation process is as follows: D-calcium pantothenate 50mg, hyponitrotriacetic acid 100mg, NaCl 8mg, MgSO4·7H2O 100mg, Na2HPO4·H2O 980mg, NH4Cl 0.49g, peptone 1g, yeast extract 1g, K2HPO4 1.57g, D-glucose 2.94g, agar 20g; trace elements: Na2MoO4·2H2O 98mg, FeSO4·7H2O 28mg, CuSO4·5H2O 16mg, MnSO4·H2O 2.2mg, H3BO3 2.5mg, ZnSO4·7H2O 1.2mg, CoSO4·7H2O 0.86mg, CaSO4 60mg, KAl(SO4)2·12H2O 1.96 mg; add 1 L of deionized water, adjust the pH to 7-7.2, place in an autoclave, and sterilize at 121℃ for 20 min.

[0016] Technical Solution 3:

[0017] A microbial agent comprising the above-mentioned lysing strains or comprising lysing strains obtained by screening using the above-mentioned screening method.

[0018] Technical Solution Four:

[0019] The application of a lysogenic strain comprising the above-described lysogenic strain or a lysogenic strain obtained by screening using the above-described screening method in sludge reduction treatment.

[0020] Technical Solution 5:

[0021] The application of the above-mentioned microbial agent in sludge reduction treatment.

[0022] Technical Solution Six:

[0023] A method for sludge reduction treatment using the above-mentioned lysing strains or lysing strains screened using the above-mentioned screening method includes the following steps:

[0024] Step 1: Activation culture of the dominant strain:

[0025] Inoculate the dominant strain DX11 into liquid culture medium at an inoculation rate of 1%-5%, and activate it by shaking culture at 50℃-85℃ for 2-3 cycles to obtain OD. 600 Seed culture at 1.0-1.2, then centrifuged to collect bacterial cells; each shaking culture lasted 12-15 hours at a shaking rate of 100-120 rpm.

[0026] Step 2: The collected bacterial cells are agitated with sterile water to obtain a bacterial solution, which is then set aside. The OD value of the bacterial solution is... 600 It is 1.0-1.2;

[0027] Step 3: Sludge lysing treatment

[0028] Add bacterial solution to the organic sludge to be treated at a volume of 10-15%, adjust the pH to 7.0±0.2, and then carry out the lysis reaction at 60-70℃ with stirring and aeration for 24-36 hours; the aeration rate is 0.2-0.5 vvm, and the stirring rate is 120-150 rpm.

[0029] As a further technical solution, in step 1, the liquid culture medium comprises: macroelements: D-calcium pantothenate 50mg, hyponitrotriacetic acid 100mg, NaCl 8mg, MgSO4·7H2O 100mg, Na2HPO4·H2O 980mg, NH4Cl 0.49g, peptone 1g, yeast extract 1g, K2HPO4 1.57g, D-glucose 2.94g; microelements: Na2MoO4·2H2O 98mg, FeSO4·7H2O 28mg, CuSO4·5H2O 16mg, MnSO4·H2O 2.2mg, H3BO3 2.5mg, ZnSO4·7H2O 1.2mg, CoSO4·7H2O 0.86mg, CaSO4 60mg, KAl(SO4)2·12H2O 1.96 mg; add 1 L of deionized water, adjust the pH to 7-7.2, place in an autoclave, and sterilize at 121°C for 20 min;

[0030] The liquid culture was based on inoculation with 5% sterile sludge immediately before use.

[0031] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0032] 1. In the process of strain screening, this invention adjusts the plate solid culture medium used for single colony isolation to make it more suitable for the growth of dominant strains, thereby screening out the lysogenic strain *Geobacillus sp.* DX11. This strain grows occultly in the sludge to be treated, secretes extracellular enzymes, promotes the hydrolysis of macromolecular organic matter in the sludge, and releases organic matter from the solid phase of the sludge into the liquid phase for subsequent use. Compared with the lysogenic strain *Geobacillus sp.* THE-14 in the prior art, its lysogenic effect is improved by 5.9%, and compared with the blank control, its lysogenic effect is improved by 27.1%.

[0033] 2. In the process of sludge reduction treatment of the strain, the present invention optimizes the sludge reduction treatment conditions and obtains the optimal treatment conditions for Geobacillus sp. DX11, which improves the sludge reduction treatment effect of strain DX11 to a certain extent. Attached Figure Description

[0034] Figure 1 It is a phylogenetic tree of lysing strains;

[0035] Figure 2 This is a graph showing the changes in the activity of the DX11 protease in *Geobacillus sp.* in Example 2.

[0036] Figure 3This is a diagram showing the cell lysis effect of the strain in Example 3; Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments.

[0038] The wastewater sludge used in the separation and screening of this invention comes from: Nanjing Qiaobei Treatment Plant A 2 Sludge from the / O process; Sludge parameters: TSS: 10545±200mg / L; VSS: 6214±180mg / L; TCOD: 5236±160mg / L; SCOD: 72±10mg / L; pH: 6.8±0.2;

[0039] The wastewater sludge used in the cytolysis test of this invention is derived from: A 2 The wastewater sludge from the return pump station of the wastewater treatment plant operating under the / O process has the following properties: Total suspended solids (TSS) 12585±140 mg / L, volatile suspended solids (VSS) 5821±20 mg / L, total chemical oxygen demand (TCOD) 4275±65 mg / L, dissolved chemical oxygen demand (DOD) 63±5 mg / L, and pH 6.8±0.2. After sieving to remove particles larger than 1 mm, the sludge is stored at 4°C for no more than one week.

[0040] The control strain THE14 belongs to the genus Geobacillus sp. and is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: May 8, 2021; accession number: CCTCC M2021514; control strain THE14 is a strain in the prior art and is publicly available.

[0041] Liquid culture medium: Macroelements: D-calcium pantothenate 50mg, hyponitrotriacetic acid 100mg, NaCl 8mg, MgSO4·7H2O 100mg, Na2HPO4·H2O 980mg, NH4Cl 0.49g, peptone 1g, yeast extract 1g, K2HPO4 1.57g, D-glucose 2.94g; Microelements: Na2MoO4·2H2O 98mg, FeSO4·7H2O 28mg, CuSO4·5H2O 16mg, MnSO4·H2O 2.2mg, H3BO3 2.5mg, ZnSO4·7H2O 1.2mg, CoSO4·7H2O 0.86mg, CaSO4 60mg, KAl(SO4)2·12H2O 1.96mg. Add 1L of deionized water, adjust the pH to 7-7.2, place in an autoclave, and sterilize at 121℃ for 20 minutes.

[0042] Example 1: Isolation, screening, identification and application of dominant lysinic bacteria

[0043] This invention mainly includes the following parts: 1. Isolating and screening dominant strains from acclimatized sludge; 2. Identifying the dominant strains and determining their species relationships; 3. Optimizing the process to obtain the best process for sludge reduction using the screened dominant strains.

[0044] I. Isolating and screening dominant bacterial strains from acclimatized sludge, specifically including the following steps:

[0045] Step 1, Sludge Acclimation: Acclimate the sewage sludge under aeration conditions of 0.1-1.0 vvm and at a temperature of 55-80℃ for 5-60 days.

[0046] Step 2: After gradient dilution of the acclimated sludge, the strains were separated and purified using the dilution pouring method and the three-zone streak method to obtain single-colony strains, and the strains were named and numbered respectively.

[0047] Step 2.1, Pour the flat plate:

[0048] The solid agar plates used for single colony isolation consisted of: 50 mg D-calcium pantothenate, 100 mg hypozinotriacetic acid, 8 mg NaCl, 100 mg MgSO4·7H2O, 980 mg Na2HPO4·H2O, 0.49 g NH4Cl, 1 g peptone, 1 g yeast extract, 1.57 g K2HPO4, 2.94 g D-glucose, and 20 g agar. Trace elements were also included: 98 mg Na2MoO4·2H2O, 28 mg FeSO4·7H2O, 16 mg CuSO4·5H2O, 2.2 mg MnSO4·H2O, 2.5 mg H3BO3, 1.2 mg ZnSO4·7H2O, 0.86 mg CoSO4·7H2O, 60 mg CaSO4, and 1.96 mg KAl(SO4)2·12H2O.

[0049] Preparation method: Weigh all raw materials according to the above solid culture medium formula, add 1L of deionized water, adjust the pH to 7-7.2, and place in an autoclave for sterilization at 121℃ for 20 minutes. After cooling to 50℃-55℃, pour the mixture into sterile petri dishes, ensuring that it covers about 2 / 3 of the bottom of the dish. Once the agar has solidified, invert the plates onto a sterile table.

[0050] Step 2.2, Separation:

[0051] The bacterial solution was spread using the dilution pouring method, as follows: Take several sterilized EP tubes, number them sequentially, and add 0.9 ml of sterile water to each tube. Take 0.1 ml of sludge that has been acclimated at 65℃ and add it to the sterile water in tube #1. Mix thoroughly. Transfer 0.1 ml of the diluted solution from tube #1 to the next sterilized EP tube, mix well, and repeat this process until the fourth and fifth tubes are reached. The dilution ratios from tube #1 to tube #5 are 10:10. -1 10 -2 10 -3 10 -4 10 -5 ; Add 10 drops -4 and 10 -5 Two tubes of diluent, each containing 0.1 ml, were inoculated into the corresponding plates and spread evenly using a sterile spreader. The spread plates were placed on a sterile table for 20-25 minutes to allow the bacterial suspension to penetrate the culture medium. The plates were then inverted and placed in an incubator for 12 hours at 50-85°C. Single colonies were selected using a sterile inoculation loop and isolated by streaking the plates using the three-zone streak method. This process was repeated 3-4 times to obtain purified colonies. In this example, a total of 11 bacterial strains were isolated.

[0052] Step 3: The isolated single-colony strains were initially screened using the hydrolysis zone test. Strains with larger hydrolysis zones were retained for further screening using the sludge lysing test and extracellular enzyme activity test. The lysing strains with higher sludge VSS degradation rate and higher extracellular enzyme activity were retained as the dominant strains. In this example, the dominant strain obtained by screening was DX11.

[0053] Hydrolysis zone test: The lysinic strains isolated from domesticated and mature sludge were inoculated into skim milk powder solid culture medium, placed in a constant temperature incubator, and the changes in the hydrolysis zone of each strain were observed after 12 hours.

[0054] The components of the skim milk powder solid culture medium used for strain screening were as follows: Macroelements: D-calcium pantothenate 50mg, hyponitrotriacetic acid 100mg, NaCl 8mg, MgSO4·7H2O 100mg, Na2HPO4·H2O 980mg, NH4Cl 0.49g, peptone 1g, yeast extract 1g, K2HPO4 1.57g, D-glucose 2.94g, agar 20g, skim milk powder 20g, distilled water 1L; Microelements: Na2MoO4·2H2O 98mg, FeSO4·7H2O 28mg, CuSO4·5H2O 16mg, MnSO4·H2O 2.2mg, H3BO3 2.5mg, ZnSO4·7H2O 1.2mg, CoSO4·7H2O 0.86mg, CaSO4 60mg, KAl(SO4)2·12H2O 1.96mg; adjust pH to 7.0-7.2.

[0055] Preliminary sludge dissolution test: Take several conical flasks and add sludge with a solid content of 1.2-1.5% to each flask; after culturing the strains after initial screening for 12-15 hours, mix them evenly with sterile water, and then inoculate them into the corresponding conical flasks. Incubate them in a constant temperature shaker at a temperature of 50-85℃ and a shaking rate of 120 rpm. At the same time, use ultrapure water as a blank control. Measure the volatile suspended solids (VSS) content of the sludge in each conical flask after 24 hours and calculate the VSS reduction rate of the sludge.

[0056] Extracellular enzyme activity assay: The strains after initial screening were inoculated into liquid culture medium at an inoculation rate of 1%, and the activity of the protease was determined by the azo casein method.

[0057] II. Identify the dominant strains and determine their species relationships; this includes physiological and biochemical identification and 16S rDNA identification:

[0058] 1) Gram staining microscopy: Gram staining microscopy was performed on the dominant strain DX11. The strain was stained with oxalic acid crystal violet solution for 1 min, iodine solution for 1 min, 95% ethanol for 30 s for decolorization, and safranin solution for 2 min-3 min. The strain was covered with a coverslip and observed under a microscope. The results showed that the cells of DX11 were pale yellow, rod-shaped, and Gram negative.

[0059] 2) 16S rDNA identification: The 16S rDNA base sequence of the dominant strain THE-4 is shown in SEQ ID:1.

[0060] The dominant strain DX11 was identified as belonging to the genus *Bacillus*. A phylogenetic tree constructed using the neighbor-joining method is shown below. Figure 1 .

[0061] Third, through process optimization, the optimal process for sludge reduction using the selected dominant strains was obtained.

[0062] In this embodiment, the optimal process for sludge reduction using the dominant strain Geobacillus sp. DX11 is as follows:

[0063] Step 1: Activation culture of the dominant strain:

[0064] Inoculate the dominant strain DX11 into liquid culture medium at an inoculation rate of 1%-5% (the liquid culture is based on inoculation with 5% sterile sludge immediately before use), and activate it by shaking culture at 50℃-85℃ 2-3 times to obtain OD. 600 Seed culture at 1.0-1.2, then centrifuged to collect bacterial cells; each shaking culture lasted 12-15 hours at a shaking rate of 100-120 rpm.

[0065] Step 2: The collected bacterial cells are agitated with sterile water to obtain a bacterial solution, which is then set aside. The OD value of the bacterial solution is... 600 It is 1.0-1.2;

[0066] Step 3: Sludge lysing treatment

[0067] Add bacterial solution to the organic sludge to be treated at a volume of 10-15%, adjust the pH to 7.0±0.2, and then carry out the cell lysis reaction at 50-85℃ with stirring and aeration for 1-36 hours; the aeration rate is 0.1-1.0 vvm, and the stirring rate is 40-200 rpm.

[0068] Example 2: Extracellular enzyme activity analysis of the dominant strain Geobacillus sp. DX11

[0069] Wastewater sludge is an organic wastewater containing a high concentration of suspended solids. Its main components include small-molecule soluble organic matter (monosaccharides, amino acids, etc.) and recalcitrant macromolecules (mainly proteins, polysaccharides, and lipids), with carbohydrates accounting for up to 20% of the total organic matter. Studies have found that glucosidase, protease, and α-amylase are closely related to the degradation of extracellular polymers in wastewater sludge during dissolution, with protein hydrolysis being a key factor. Therefore, the activity of extracellular proteases secreted by bacteria is crucial.

[0070] The dominant strain of this invention, *Geobacillus sp.* DX11, was inoculated into corresponding liquid culture media at an inoculum size of 1%. The protease activity was determined using the azocasein method. Bacterial cultures at different time points were centrifuged in a benchtop centrifuge (4℃, 10 min, 14,000 × g). The supernatant was reacted with the substrate azocasein at 60-70℃ for 30 min. Immediately afterward, 5% trichloroacetic acid (TCA) was added, and the mixture was vortexed for 5 seconds. The mixture was then equilibrated on ice for 10 minutes. After centrifugation, the supernatant was added dropwise with NaOH to a concentration of 0.4 M. The absorbance at 440 nm was measured using a UV / VIS spectrophotometer. One protease activity unit is defined as the amount of enzyme required per unit time, under specific experimental conditions, to hydrolyze soluble casein into 1 micromolar tyrosine. Results are shown below. Figure 2 The study concluded that the enzyme activity of the DX11 strain of the present invention reached its peak of 1354 U / L after 8 hours of culture; between 8 and 36 hours, due to the rapid consumption of nutrients, the extracellular enzyme activity of the strain began to decline.

[0071] Example 3: Investigation on the sludge-dissolving performance of the dominant strain Geobacillus sp. DX11

[0072] Step 1: Strain Activation: The dominant strain of this invention, *Geobacillus sp.* DX11, and the control strain, THE14 (accession number CCTCC M 2021514), were inoculated into their respective solid culture media and activated at 65°C for 12 hours. Using a sterile pipette tip, the activated strains were inoculated into 250 ml Erlenmeyer flasks containing 50 ml of liquid culture medium (inoculated with 5% sterile sludge) and incubated at 65°C for 12 hours with a shaking rate of 120 rpm. This activation process was repeated three times. After activation and maturation, the bacterial cells were centrifuged (6000 rpm, 10 min) using a benchtop centrifuge. The cells were collected, and sterile water was used to agitate the cells evenly. The OD value of the bacterial solution was adjusted. 600 Up to 1, for use in subsequent experiments.

[0073] Step 2: Sludge reduction treatment:

[0074] Geobacillus sp. DX11 and control strain THE14 were subjected to sludge reduction treatment under their respective optimal lysing conditions, with a blank control group also included.

[0075] The lysing treatment conditions for Geobacillus sp. DX11 were as follows: 100 mL of dissolved sludge was taken, pH 7.0, and 20 mL of DX11 bacterial solution was added, i.e., the inoculum size was 4 x 10⁻⁶. 10CFU was added to bring the liquid level to a total volume of 125 ml with sterile water and reacted at 65 °C with aeration for 36 h with stirring. The aeration rate was 0.2 vvm and the stirring rate was 60 ± 10 rpm.

[0076] The lysing treatment conditions for the control strain THE14 were as follows: 100 mL of dissolved sludge, pH 7.2, and 25 mL of DX11 bacterial solution were added, resulting in an inoculum size of 5 x 10⁻⁶ cells / mL. 10 CFU, with a total volume of 125 mL, was reacted at 65 °C with aeration for 36 h with stirring; the aeration rate was 0.2 vvm and the stirring rate was 60 ± 10 rpm.

[0077] The treatment conditions for the blank control group were as follows: 100 mL of dissolved sludge with a pH of 7.0 was taken and 25 mL of sterile water was added, with a total volume of 125 mL. The mixture was stirred and reacted at 65 °C under aeration conditions for 36 h. The aeration rate was 0.2 vvm and the stirring rate was 60 ± 10 rpm.

[0078] Step 3: The VSS concentration in each sample was measured using the national standard method, and the VSS degradation rate of the sludge was calculated. The results are shown in Table 1. The study concluded that under the optimal lysing treatment conditions for each strain, the VSS degradation rate of *Geobacillus sp.* DX11 of this invention was 27.1% higher than that of the blank control group and 5.9% higher than that of the control strain THE14. The lysing effect of DX11 was significantly higher than that of the blank control group and the control strain, which may be due to the optimal conditions of 65℃, pH 7, and inoculum size of 4 x 10⁻⁶. 10 During CFU (cell lysis) treatment, DX11 can maximize the use of nutrients in the sludge to complete its own growth and reproduction, quickly becoming the dominant strain and exerting its cell lysis performance.

[0079] Table 1

[0080]

[0081] Example 4: Effect of temperature on cell lysis

[0082] The dominant strain of *Geobacillus sp.* DX11 was cultured at 60℃, 65℃, and 70℃ in liquid culture medium inoculated with sterilized sludge (5% sterilized sludge inoculum) for 15-18 hours, and the activation was repeated three times to achieve its OD value. 600The bacterial cells were collected by centrifugation (6000 rpm, 10 min) between pH 1.0 and 1.2 for subsequent cell lysis experiments. The temperatures of each reactor were 60℃, 65℃, and 70℃, with a pH of 7.0 ± 0.2. The volume of dissolved sludge was 360 ml, and the volume of bacterial solution added was 40 ml, representing 10% of the total reaction system. The degradation rate of VSS by *Geobacillus sp.* DX11 at different temperatures is shown in Table 2. The best sludge dissolution rate (31.7%) was observed at 65℃.

[0083] Table 2 Effect of temperature on VSS degradation rate

[0084]

[0085] Example 5: Effect of pH on cell lysis

[0086] The dominant strain of *Geobacillus sp.* DX11 was cultured at 65°C in liquid medium inoculated with sterilized sludge (5% sterilized sludge inoculum) for 15-18 hours, and the culture was repeatedly activated three times to achieve its OD value. 600 The bacterial cells were collected by centrifugation (6000 rpm, 10 min) between pH 1.0 and 1.2 for subsequent cell lysis experiments. The temperature of each reactor was 65℃, and the pH was set to 5, 6, 7, 8, and 9, respectively. The volume of dissolved sludge was 360 ml, and the volume of bacterial solution added was 40 ml, accounting for 10% of the total reaction system. The degradation rate of VSS by *Geobacillus sp.* DX11 under different pH conditions is shown in Table 3. The best sludge dissolution effect of *Geobacillus sp.* DX11 was observed at pH 7, reaching 31.7%.

[0087] Table 3 Effect of pH on VSS degradation rate

[0088]

[0089] Example 6: Effect of different inoculum sizes on cell lysis

[0090] Inoculate 5% sterilized sludge into the liquid culture medium used in this invention, and co-culture the frozen bacterial strain at 65°C for 15-18 hours, repeating the activation process three times. Take OD... 600 1 ml of bacterial culture with a concentration of 1 was added to 9 ml of sterile water to obtain a bacterial culture diluted 10 times. Then, 1 ml of this diluted bacterial culture was added to 9 ml of sterile water. This dilution process was repeated 6 times to obtain a bacterial culture with a concentration of 10 times the original concentration. -6Dilute the bacterial suspension by 10 μl. Spread 10 μl of the diluted bacterial suspension onto a solid culture medium and incubate at 65°C. Three parallel groups were set up, and the colony count was checked after 24 hours. The result showed 20 single bacteria per solid culture medium, i.e., OD0.05. 600 When the concentration of viable bacteria in the bacterial culture is 1, the concentration of viable bacteria is approximately 2 x 10� 9 cfu / ml;

[0091] The temperature of each reactor was adjusted to 65℃, and the pH was adjusted to the optimal reaction pH, i.e., pH 7. The volume of dissolved sludge was 100 mL, and 5, 10, 15, 20, and 25 mL of bacterial solution were added respectively, representing inoculum sizes of 1 x 10⁻⁶ bacteria. 10 CFU, 2x10 10 CFU, 3x10 10 CFU, 4x10 10 CFU, 5x10 10 CFU was added to bring the liquid level up with sterile water, and an equal volume of sterile water was added to the control group to ensure that the total volume of each group was 125 ml. The degradation rate of VSS by *Geobacillus sp.* DX11 at different inoculum sizes is shown in Table 4, with the degradation rate at an inoculum size of 4 x 10⁻⁶ being the highest. 10 Under CFU conditions, Geobacillus sp. DX11 showed the best sludge dissolution effect, reaching 32.7%.

[0092] Table 4. Effect of inoculum size on VSS degradation rate

[0093]

[0094] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A lysinic bacterial strain, characterized in that, Bacillus aureus ( Geobacillus sp. DX11, deposited at the China Center for Type Culture Collection, accession number CCTCC M 2023509.

2. A method comprising Bacillus aeruginosa as described in claim 1 ( Geobacillus sp. DX11 microbial inoculant.

3. A type of Bacillus aeruginosa as described in claim 1 ( Geobacillus sp. Application of DX11 in VSS degradation of sludge.

4. The application of the microbial agent as described in claim 2 in the degradation of VSS in sludge.

5. A method utilizing the Bacillus aeruginosa described in claim 1 ( Geobacillus sp. The method for degrading VSS in sludge using DX11 is characterized by, Includes the following steps: Step 1: Activation culture of the dominant strain: Inoculate with the dominant strain Bacillus aeruginosa at a rate of 1%-5%. Geobacillus sp. DX11 was inoculated into liquid culture medium and activated by shaking culture at 60-70℃ for 2-3 cycles to obtain OD. 600 Seed culture at 1.0-1.2, then centrifuged to collect bacterial cells; each shaking culture lasted 12-15 hours at a shaking rate of 120-150 rpm. Step 2: The collected bacterial cells are agitated with sterile water to obtain a bacterial solution, which is then set aside. The OD value of the bacterial solution is... 600 It is 1.0-1.2; Step 3: Sludge lysing treatment Add bacterial solution to the organic sludge to be treated at 10% of its volume, and then adjust the pH to 7.

0. Then, the cell lysis reaction was carried out at 60-70℃ under stirring and aeration conditions for 24-36 h; the aeration rate was 0.2-0.5 vvm and the stirring rate was 120-150 rpm.

6. The method of utilizing Bacillus terrestris (as described in claim 5) Geobacillus sp. The method for degrading VSS in sludge using DX11 is characterized by, In step 1, the liquid culture medium consists of: macroelements: D-calcium pantothenate 50 mg, hyponitrotriacetic acid 100 mg, NaCl 8 mg, MgSO4•7H2O 100 mg, Na2HPO4•H2O 980 mg, NH4Cl 0.49 g, peptone 1 g, yeast extract 1 g, K2HPO4 1.57 g, D-glucose 2.94 g; microelements: Na2MoO4•2H2O 98 mg, FeSO4•7H2O 28 mg, CuSO4•5H2O 16 mg, MnSO4•H2O 2.2 mg, H3BO3 2.5 mg, ZnSO4•7H2O 1.2 mg, CoSO4•7H2O 0.86 mg, CaSO4 60 mg, KAl(SO4)2•12H2O 1.96 mg. mg; add 1L of deionized water, adjust the pH to 7-7.2, place in an autoclave, and sterilize at 121℃ for 20 min; The liquid culture is based on pre-use inoculation with sterilized sludge.

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