Ochrobactrum terotinii with flocculation and growth promotion effects as well as culture method and application of ochrobactrum terotinii

Abstract

The invention provides ochrobactrum teretti with flocculation and growth promotion effects as well as a culture method and application of the ochrobactrum teretti, and provides ochrobactrum teretti JM8, and the preservation number of the ochrobactrum teretti JM8 is CCTCC (China Center For Type Culture Collection): M 20211666. The ochrobactrum terotinii JM8 strain provided by the invention is a new strain and has the effects of flocculation and plant growth promotion, and a microbial flocculant prepared from the JM8 strain and PFS have a synergistic effect, so that the ochrobactrum terotinii JM8 strain is suitable for reduction treatment of a cow dung separation liquid, and secondary pollution caused by use of a large amount of chemical flocculants is avoided. Meanwhile, the JM8 strain provides a potential application prospect for recycling the cow dung separation liquid.

Description

Technical Field

[0001] This invention belongs to the fields of environmental engineering and microbial engineering technology, and particularly relates to a type of *Territhione oryzae* with flocculation and growth-promoting effects, its culture method and application. Background Technology

[0002] Currently, flocculants can be broadly classified into three categories: inorganic flocculants, organic synthetic polymeric flocculants, and natural organic polymeric flocculants. Inorganic flocculants mainly include aluminum salts and iron salts, both of which easily leave metal ions behind. For example, iron salt flocculants such as polyferric sulfate (PFS) have good flocculation effects, but improper dosage can easily leave residues and cause secondary pollution. Artificially synthesized organic polymeric flocculants typically have long molecular chains and poor biodegradability, such as polyacrylamide and its derivatives. Natural organic polymeric flocculants, such as microbial flocculants, are considered the most promising next-generation green water treatment agents due to their non-toxicity, high efficiency, biodegradability, and lack of secondary pollution, representing an important direction for future flocculant development. Microbial flocculation is a process in which microorganisms or their metabolites (such as polysaccharides, proteins, and glycoproteins) flocculate suspended particles in water. Its principle mainly includes the adsorption of charges on the surface of microorganisms, the physical trapping of mycelial networks, and the bridging and charge neutralization effects of metabolites (such as extracellular polymers), causing suspended solids and colloidal particles in the liquid to coagulate and precipitate, achieving solid-liquid separation. Compared with traditional inorganic or organic synthetic flocculants, microbial flocculants have significant advantages such as biodegradability, no secondary pollution, and high safety. Currently, the main bottleneck limiting their large-scale application is the high production cost, specifically low strain yield, high fermentation costs, and complex extraction processes.

[0003] With the rise of sustainable and green agriculture, bio-fertilizers have gained widespread attention due to their advantages such as soil improvement, enhanced fertilizer efficiency, increased yield and quality, and no secondary pollution. In recent years, they have become an important component of my country's fertilizer industry. Bio-fertilizers are typically made by inoculating nutrient-rich organic waste with selected and domesticated microorganisms that promote growth, followed by fermentation. Existing bio-fertilizers come in both liquid and solid forms. The inoculated microorganisms include single or mixed strains, and trace elements and nutrients such as nitrogen, phosphorus, and potassium are often added to enhance their effects. Cow manure is one of the main sources of livestock and poultry waste, with a huge production volume; cow manure separation liquid accounts for 40% to 70% of the total raw cow manure. Cow manure separation liquid contains a large amount of suspended solids and colloidal particles, which contain a significant amount of macromolecular organic matter, nitrogen, phosphorus, and potassium. Utilizing these substances for bio-fertilizer fermentation is an important means of realizing the resource utilization of cow manure separation liquid. Flocculation and sedimentation is an effective method for collecting suspended solids and colloidal particles in the separation liquid. After flocculation, the organic matter and other nutrients in the cow manure separation liquid will enter the sediment along with the flocs. Using flocculation and sedimentation for bio-fertilizer fermentation has three advantages: first, the sediment can provide sufficient nutrients for microbial growth; second, the sediment after fermentation still contains nutrients such as N, P, and K, so there is no need to add more; and third, it can reduce the land area and transportation costs.

[0004] In the fields of agriculture and environmental ecology, research on plant rhizosphere growth-promoting bacteria (PGPR) has been quite extensive. These strains can directly promote plant growth and seed germination through nitrogen fixation, phosphorus solubilization, and the production of plant hormones, or indirectly exert their effects by antagonizing pathogens.

[0005] *Treptesiae*, belonging to the genus *Treptesiae*, is a widely distributed Gram-negative bacterium. This species is commonly found in soil, water, and plant rhizospheres, exhibiting strong environmental adaptability. Existing research indicates that some *Treptesiae* strains show potential in environmental remediation and as rhizosphere growth-promoting bacteria. Therefore, the screening and development of strains has ongoing research value. Developing a microorganism with flocculation and growth-promoting effects has significant application value. Summary of the Invention

[0006] This invention proposes a *Territtia leucocephala* bacterium with flocculation and growth-promoting effects, its culture method and application, which have good flocculation and growth-promoting effects.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention proposes a Territoryl aristocetis with flocculation and growth-promoting effects, wherein the Territoryl aristocetis is named Territoryl aristocetis JM8 and its accession number is CCTCCNO: M 20211666.

[0008] The second aspect of this invention provides a method for culturing *Territtasidium* bacillus with flocculation and growth-promoting effects, wherein the *Territtasidium* bacillus JM8 strain is inoculated onto a solid culture medium and cultured, and then the resulting single colonies are inoculated into a liquid culture medium and fermented until OD (Organic Degree) is reached. 600 The value is 1, the C / P mass ratio of the liquid culture medium is 1-10, the C / N mass ratio is 5-40, and the pH is 5-11. After overnight incubation, the microbial flocculant is obtained.

[0009] Preferably, the liquid culture medium is glucose at 18–22 g / L. -1 K2HPO4 4~6g·L -1 KH2PO4 2~3g·L -1 NaCl 0.09~0.11g·L -1 (NH4)2SO4 0.18~0.22 g·L -1 Urea 0.45–0.55 g·L -1 Yeast extract 0.45–0.55 g / L -1 MgSO4 0.18~0.22g·L -1 The solvent is deionized water.

[0010] The third aspect of this invention proposes the use of *Trietacia leucocephala*, which has flocculation and growth-promoting effects, as a plant growth regulator for promoting seed germination, either as a whole or in the preparation of such a regulator.

[0011] The fourth aspect of this invention proposes the use of *Teretoxigenicus tarda*, which has flocculation and growth-promoting effects, as a microbial flocculant or in the preparation of such a flocculant.

[0012] The fifth aspect of this invention proposes the application of *Trietacia leucocytogenes*, which has flocculation and growth-promoting effects, as a microbial flocculant in the reduction of cow manure separation liquid.

[0013] The sixth aspect of this invention proposes the application of *Trichoderma pallida*, which has flocculation and growth-promoting effects, as a microbial flocculant in a mixed solution with PFS (polyferric sulfate) in flocculants. Through research, this invention has found that the microbial flocculant prepared by this strain has a synergistic effect with PFS, producing a significantly better flocculation effect at the same dosage than using only the microbial flocculant or only PFS. Furthermore, compared to using the microbial flocculant alone, its production and usage costs are lower, and it avoids the secondary pollution caused by the large-scale use of chemical flocculants.

[0014] Furthermore, the amount of the microbial flocculant added accounts for 3% of the volume ratio of the mixed solution, and the OD of the microbial flocculant is... 600 Value = 1, the final concentration of PFS in the mixed solution is 1 g·L⁻¹ -1 .

[0015] The seventh aspect of this invention proposes the application of *Trichoderma pallida*, which has flocculation and growth-promoting effects, as a microbial flocculant in combination with PFS in the preparation of cow manure separation liquid bio-fertilizer.

[0016] Beneficial effects: The present invention proposes Territory Painful Bacterium ( Ochrobactrum haematophilum The JM8 strain is a new strain with the preservation number CCTCCNO: M20211666. The JM8 strain has flocculation and plant growth promotion effects. The microbial flocculant prepared from it can produce flocculation. At the same time, this strain also has the effect of promoting seed germination.

[0017] This invention also discovered through research that the microbial flocculant prepared by strain JM8 has a synergistic effect with PFS (polyferric sulfate). The flocculation composition compounded with PFS can achieve a flocculation rate of up to 50% in cow manure separation liquid, making it suitable for volume reduction treatment of cow manure separation liquid. The flocculation effect produced at the same dosage is significantly better than using only the microbial flocculant or only PFS, and its production and usage costs are lower than using the microbial flocculant alone, while avoiding secondary pollution caused by the large-scale use of chemical flocculants.

[0018] The JM8 strain proposed in this invention has a promising application prospect for the resource utilization of cow manure separation liquid and the promotion of plant growth. Attached Figure Description

[0019] Figure 1 The present invention is based on Territory Painful Bacillus (Territory Painful Bacillus). Ochrobactrum haematophilum Scanning electron microscope image of JM8 bacterial cell morphology; Figure 2 The present invention is based on Territory Painful Bacillus (Territory Painful Bacillus). Ochrobactrum haematophilum Phylogenetic tree of the 16S rRNA gene sequence of strain JM8; Figure 3 Comparative figures for experiments optimizing the amount of microbial flocculation applied: (a) flocculation experiment with kaolin, (b) flocculation experiment with cow dung separation liquid; Figure 4 The following is a comparison of experiments on the optimization of PFS flocculation application amount: (a) is the flocculation experiment of kaolin, and (b) is the flocculation experiment of cow manure separation liquid. Note: a, b and c represent the results of the significance difference analysis, and the value marked a > the value marked b > the value marked c. The same letter indicates that the difference is not significant, and different letters indicate that the difference is significant. Figure 5 A diagram illustrating the combined effect of JM8 microbial flocculation and PFS chemical flocculation; Figure 6The figures show a comparison of the growth-promoting effects of bio-fertilizers; (a) compares plant height, (b) compares root length, (c) compares fresh weight, and (d) compares dry weight. Note: a, b, c, d, and e represent the results of the significance analysis, and the value marked a > the value marked b > the value marked c > the value marked d > the value marked e. The same letter indicates no significant difference, and different letters indicate significant difference. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] The culture medium used in this invention is as follows: The beef extract peptone solid medium is prepared as follows: 3 g / L beef extract -1 10 g / L of peptone -1 NaCl 5 g·L -1 Add 15-20 g / L of agar powder. -1 Prepare 1000mL of deionized water, adjust the pH to 7.0-7.2, and pour it into sterilized petri dishes to form plates for later use.

[0022] Flocculation liquid culture medium: glucose 18–22 g·L -1 K2HPO4 4~6 g·L -1 KH2PO4 2~3 g·L -1 NaCl 0.09~0.11g·L -1 (NH4)2SO4 0.18~0.22 g·L -1 Urea 0.45–0.55 g·L -1 Yeast powder 0.45–0.55 g·L -1 MgSO4 0.18~0.22 g·L -1 The pH was adjusted to 7.0–8.0, and the solvent was deionized water.

[0023] Preferably, glucose 20 g·L -1 , K2HPO4 5g·L -1 , KH2PO4 2g·L -1 NaCl 0.1 g·L -1 0.2 g·L⁻¹ of (NH₄)₂SO₄ -1 urea 1.5 g·L -10.5g / L yeast powder -1 MgSO4·7H2O 0.2g·L -1 Adjust the pH to 7.0–8.0.

[0024] LB liquid medium: 10 g / L tryptone -1 5 g·L yeast powder -1 NaCl 10 g·L -1 Adjust the pH to 7.0.

[0025] All of the above culture media must be sterilized before use, specifically by sterilizing at 121℃ for 30 minutes.

[0026] Example 1: Isolation and Culture of Strains Screening and isolation of bacterial strains: 1g of rhizosphere soil collected from farmland vegetable fields in Shenbei New District, Liaoning Province, was added to a centrifuge tube containing 9mL of sterile water and incubated at 30℃ and 180rpm for 30min with shaking. The strains were isolated using the dilution plating method, with a dilution concentration of 10... -3 10 -4 10 -5 Using a pipette, 150 µL of each of the three concentration dilutions was spread onto beef extract peptone solid medium and incubated at 25–30 °C, preferably 30 °C, for 24–36 h. Single colonies with vigorous growth and a viscous surface were picked using a sterile inoculation loop and isolated and purified on beef extract peptone solid medium using the streak plate method to obtain the target strain.

[0027] Bacterial culture: The isolated strain was inoculated onto beef extract peptone solid medium and cultured at 25–30°C; then, the resulting single colonies were inoculated into liquid medium and fermented until OD reached. 600 Value = 1, culture temperature is 25-30℃, the C / P mass ratio of the liquid culture medium is 1-10, the C / N mass ratio is 5-40, and the pH is 5-11.

[0028] Example 2: Identification of the flocculation ability of strains Single colonies obtained after repeated streak purification on beef extract peptone solid plates were inoculated into 30 mL of beef extract peptone liquid medium and cultured at 30°C with shaking at 180 rpm until the fermentation broth reached OD. 600 With a value of 1, the inoculum was transferred to the flocculation liquid culture medium at a volume fraction of 1%. After culturing at 30°C and 180 rpm for 24 hours, the microbial flocculant was obtained and used for kaolin flocculation experiments and cow dung separation liquid flocculation experiments.

[0029] Kaolin flocculation experiment: Add 0.5g of kaolin and 100mL of deionized water to a 200mL Erlenmeyer flask to prepare a 5g·L⁻¹ solution.-1 Add 2 mL of microbial flocculant to the kaolin suspension, stir for 2 min, let stand for 30 min, and then measure the flocculation rate of the supernatant.

[0030] Flocculation experiment of cow dung separation liquid: The cow manure used in this invention was taken from a beef cattle farm in Tieling, Liaoning Province. After being filtered through a nylon filter screen (100 mesh), cow manure separation liquid was obtained. The separation liquid contained a large amount of suspended solids and colloidal particles. 1 mL of microbial flocculant was added to 50 mL of cow manure separation liquid and stirred for 2 min. After standing at room temperature for 12 h, the flocculation rate of the supernatant was measured to determine the microbial flocculation activity.

[0031] Flocculation effect determination: The absorbance (DB) of the supernatant was measured at 550 nm, with the absorbance of the supernatant without flocculant as the control (DA). Flocculation activity was expressed as the flocculation rate, and the formula for the flocculation rate is as follows: ; Three strains with flocculation activity, JM3, JM4 and JM8, were isolated from the soil. The comparison of the flocculation activities of the three strains is shown in Table 1.

[0032] Table 1 Comparison of flocculation activities of strains

[0033] As shown in Table 1, strain JM8 had a significantly higher flocculation effect in kaolin and cow manure liquid than strains JM3 and JM4. The flocculation rate of strain JM8 reached 17.02% in the kaolin experiment and 13.03% in the cow manure liquid experiment.

[0034] Example 3: Identification of the growth-promoting ability of the strain The isolated strains were cultured in LB liquid medium until the logarithmic growth phase, and the fermentation broth was adjusted to OD200. 600 A value of 0.5 was used to obtain the seed soaking bacterial solution. The effect of the bacterial solution on the germination potential and germination rate of cucumber seeds, as well as the average root length and shoot length, was used to determine its growth-promoting effect.

[0035] Cucumber seeds were soaked in 75% alcohol for 5 minutes, rinsed 5 times with sterile water, and then blotted dry with filter paper. 5 mL of different bacterial solutions (strains JM3, JM4, and JM8) were added, and the seeds were soaked for 2 hours, after which the surface bacterial solution was blotted dry. The seeds were then cultured at 30°C under constant light for 7 days in sterile Petri dishes with two layers of qualitative filter paper at the bottom, with water added periodically. Each treatment was prepared in triplicate, with 25 seeds per replicate. Seeds soaked in water served as a control (CK).

[0036] Seed germination numbers were observed and recorded on days 3 and 7, with germination defined as the radicle breaking through 1 mm from the seed. Germination potential and germination rate were calculated. On day 7, root and shoot lengths of germinated seeds were measured using calipers, and the average root and shoot lengths for each treatment were calculated. The effects of each functional strain's inoculum solution on seed germination potential, germination rate, average root length, and average shoot length were compared to evaluate the growth-promoting effects of each strain (Table 2).

[0037] Table 2 Comparison of growth-promoting effects of strains

[0038] As shown in Table 2, cucumber seeds soaked in bacterial solution had higher germination potential, germination rate, average shoot length, and average root length than the control (CK) treated with water. Among them, strain JM8 had the best growth-promoting effect, which was significantly higher than that of strains JM3 and JM4.

[0039] In summary, strain JM8 possesses both the functions of a microbial flocculant and a seed germination promoter, and can be used as or in the preparation of a microbial flocculant, or as or in the preparation of a plant growth regulator that promotes seed germination.

[0040] Example 4: Strain Identification and Preservation The strain JM8 obtained from Examples 2 and 3 exhibits both flocculation activity and growth-promoting effect, therefore, further morphological and molecular biological identification of this strain was conducted.

[0041] Bacterial species identification: Territory albendazole ( Ochrobactrum haematophilum The biological characteristics of strain JM8 are Gram-negative bacteria; colonies are pale yellow, round, with regular edges, smooth, moist, opaque, and slightly raised; Figure 1 As shown, the bacteria are short rod-shaped, single, with a relatively thick and blunt end, do not produce spores, and are obligate aerobic.

[0042] Strain preservation: The *Territtsiclos* strain proposed in this invention (… Ochrobactrum haematophilum The JM8 strain, with accession number CCTCCNO: M20211666, was deposited on December 23, 2021, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The confirmed name of the deposited organism is... Ochrobactrum haematophilum JM8.

[0043] Gene fragments were amplified from the 16S rDNA of the strain using PCR. These fragments were identified by Sangon Biotech (Shanghai) Co., Ltd. as *Territis aureus*. Ochrobactrum haematophilum JM8. The phylogenetic tree of the 16S rRNA amplification sequencing system of this strain is as follows: Figure 2 As shown, the amplification and sequencing results are as shown in SEQ ID NO: 1.

[0044]

[0045] Example 5: Optimization of Microbial Flocculation Conditions The methods for the kaolin experiment and the cow dung separation liquid flocculation experiment are the same as in Example 2.

[0046] The experiment used strains JM3, JM4, and a mixed bacterial strain as controls. The mixed bacterial strain included *Tretinoinus tarda* (…). Ochrobactrum haematophilum JM8, JM3, and JM4 strains were prepared by mixing suspensions of the three strains in equal volume ratios. *Territhione albendazole* (JM8, JM3, and JM4) was then used to prepare the strain. Ochrobactrum haematophilum The preparation method of the bacterial suspension flocculants of strains JM8, JM3, and JM4 is as described in Example 2. Flocculation experiments were conducted on the kaolin and cow dung liquid separation liquid using strains JM8, JM3, JM4, and the mixed strains at application rates of 1%, 3%, and 5% by volume. The flocculation rate was measured, and the experimental results are shown in [Figure 2]. Figure 3 The results show that a 3% microbial flocculant dosage is optimal for both kaolin flocculation and cow dung separation liquid flocculation experiments. Strain JM8 exhibits better flocculation performance than strains JM3 and JM4, while the mixed strain shows slightly better performance than the single strain JM8. Therefore, a 3% dosage of microbial flocculant is the best choice. To further improve flocculation, mixed-strain flocculants can be prepared. Thus, mixed-strain flocculants prepared from strains JM3, JM4, and JM8 can also be used as or in the preparation of microbial flocculants.

[0047] Example 6: Optimization of Flocculation Conditions for Polyferric Sulfate (PFS) Dissolve 5g of PFS in 50mL of deionized water to obtain 100g·L⁻¹ -1 PFS flocculant stock solution was prepared. The PFS stock solution was added to the kaolin suspension and cow dung separation liquid at a certain ratio, with a final PFS concentration of 0.5 g / L in the solution. -1 1.0 g·L -1 1.5g·L -1 2.0 g·L -1 2.5g·L -1 3.0 g·L -1 Chemical flocculation experiments were conducted on kaolin and cow dung liquid to determine the flocculation rate of each treatment and to evaluate the flocculation effect of different PFS application amounts. Figure 4 a). By Figure 4 It can be seen that when the final concentration of PFS is greater than 1.0 g·L⁻¹ -1 At that time, the flocculation rates of both kaolin and cow dung liquid were significantly higher than 0.5 g·L⁻¹. -1 However, the final concentration of PFS was 1.0 g·L⁻¹. -1 1.5g·L -1 2.0 g·L -1 2.5g·L -1 and 3.0 g·L -1There were no significant differences between the treatments.

[0048] Prepare PFS to a final concentration of 0 g·L -1 1.0 g·L -1 2.0 g·L -1 and 3.0 g·L -1 The beef extract peptone solid medium was used to dilute overnight cultured JM3, JM4, and JM8 bacterial suspensions using a 10-fold dilution method. 150 μL of each suspension was then taken. -4 10 -5 10 -6 Serial dilutions were plated on beef extract peptone solid medium and incubated at 30°C for 3 days. Dilutions with colony counts ranging from 30 to 300 were selected to evaluate the effect of PFS concentration on microbial growth. Figure 4 b). As can be seen from the figure, when the PFS concentration is 1 g·L⁻¹ -1 and 0g·L -1 At that time, the number of single bacteria JM3, JM4, JM8 and mixed bacteria did not differ significantly, while when the PFS concentration was 2 g·L⁻¹ -1 and 3g·L -1 At that time, the number of microorganisms was significantly lower than that at a PFS concentration of 1 g·L⁻¹. -1 and 0g·L -1 Therefore, when the PFS concentration is 1.0 g·L⁻¹, it is possible to achieve the desired effect. -1 It did not affect the growth of microorganisms.

[0049] Based on the above results and considering economic costs, the optimal PFS flocculant concentration was selected as 1.0 g·L⁻¹. -1 .

[0050] Example 7: Verification of the combined effect of JM8 microbial flocculation and PFS chemical flocculation Based on Examples 2, 5, and 6, strain JM8 was selected to prepare a microbial flocculant, and its combined effect with PFS flocculation was verified. The amount of microbial flocculant added accounted for 3% of the volume of the mixed solution. The OD of the microbial flocculant was... 600 Value = 1, the final concentration of PFS in the mixed solution is 1 g·L⁻¹ -1 Three treatments were set up, including JM8 flocculation (BIO), PFS flocculation (CHE), and combined JM8 and PFS flocculation (BIO-CHE), and their flocculation effects on kaolin and cow dung separation liquid were measured. Figure 5As shown in the figure, in the flocculation experiments of kaolin and cow dung separation liquid, the combined flocculation effect of JM8 and PFS (BIO-CHE) was higher than that of JM8 (BIO) alone and PFS (CHE) alone. In the kaolin flocculation experiment, the flocculation rate of the BIO-CHE group reached 80.49%, which was 10.15% and 57.65% higher than the CHE and BIO treatment groups, respectively. The flocculation experiment of cow dung separation liquid showed the same trend as the kaolin flocculation experiment, with the BIO-CHE group reaching 53.55%, which was 14.13% and 47.26% higher than the CHE and BIO treatment groups, respectively.

[0051] Example 8: Preparation and Growth-Promoting Effect of Cow Manure Separation Liquid Flocculation and Sedimentation Bio-fertilizer Cow manure is one of the main sources of livestock and poultry waste. After solid-liquid separation, the liquid phase component is cow manure separation liquid, which accounts for 40% to 70% of the original cow manure. Since cow manure separation liquid contains a large amount of suspended solids and colloidal particles, it can be reduced in volume through flocculation separation. The precipitate after separation is converted into organic fertilizer or prepared into bio-fertilizer through anaerobic or aerobic fermentation, while the purified clear liquid can be used for flushing cattle sheds or irrigating farmland.

[0052] Using the flocculated precipitate produced by combined flocculation of JM8 and PFS (BIO-CHE) in Example 7 as a substrate, bio-fertilizer was prepared. Two treatment methods were employed: one involved adding 0.5% (v / v) OD to the flocculated precipitate. 600 Value = 1 JM8 bacterial suspension; secondly, add 0.5% volume percentage OD to the flocculation sediment. 600 After suspending JM8 bacteria with a concentration of 1, the culture was aerobic fermented at 30℃ for 10 days. The number of microorganisms under the two treatments was determined by real-time quantitative PCR. At the same time, the number of microorganisms in the initial cow dung separation liquid and the initial flocculated sediment was also determined. The results are shown in Table 3.

[0053] Table 3 Comparison of microbial numbers under different treatment methods

[0054] As shown in Table 3, when preparing bio-fertilizer using flocculation sedimentation (B1), flocculation sedimentation + JM8 (B2), and flocculation sedimentation + JM8 + fermentation (B3), the number of microorganisms was significantly higher than that of the initial cow manure separation liquid. Furthermore, the number of microorganisms in the flocculated sedimentation liquid after inoculation with JM8 and subsequent fermentation treatment increased by nearly one order of magnitude compared to the unfermented treatment. This indicates that flocculation sedimentation can provide sufficient nutrients for microbial growth, promoting microbial growth and serving as a good substrate for bio-fertilizer preparation. Therefore, the preferred method for preparing cow manure separation liquid bio-fertilizer is inoculation with *Trichoderma repens* strain JM8 followed by aerobic fermentation.

[0055] The growth-promoting effect of bio-fertilizer was verified using a pot experiment. The test plant was Chinese cabbage, variety 'Jinjian Kuaicai F1'. Five treatments were set up: water treatment CK1, cow manure liquid treatment CK2, B1 treatment XN1, B2 treatment XN2, and B3 treatment XN3. The experimental setup is shown in Table 4. Each treatment was replicated in triplicate. Each pot contained 500g of soil. Initially, the bio-fertilizer was added at 10% by mass. Afterward, 20mL of water was added daily. After 60 days, the above-ground and below-ground parts of the Chinese cabbage were collected, and plant height, root length, dry weight, and wet weight were measured. (See Table 4 for details.) Figure 6 .

[0056] Table 4 Experimental Setup

[0057] Depend on Figure 6 It is evident that the application of bio-fertilizers XN1, XN2, and XN3 all significantly promoted the growth of pak choi. The plant height, root length, dry weight, and fresh weight of pak choi in the bio-fertilizer treatment groups all increased to a certain extent. Among them, the XN3 treatment had the most significant effect on the growth indicators of pak choi. The plant height, root length, dry weight, and fresh weight were 1.50, 2.02, 9.41, and 10.95 times that of the CK1 treatment, respectively, and 1.7, 1.72, 2.90, and 2.75 times that of the CK2 treatment, respectively.

[0058] In summary, the microbial flocculant with flocculation and growth-promoting effects provided by this invention and its application have greater potential for the resource utilization of cow manure separation liquid and the promotion of plant growth.

[0059] Although the present invention has been described in detail in this specification with general description and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A type of *Territis* bacterium with flocculation and growth-promoting effects, characterized in that, The *Territtasis* bacterium was named *Territtasis* JM8, and its accession number is CCTCCNO: M 20211666.

2. A method for culturing *Terexacum mongolicum* with flocculation and growth-promoting effects as described in claim 1, characterized in that, The *Treptesia leucocephala* JM8 strain was inoculated onto a solid culture medium and cultured. Then, the resulting single colonies were inoculated into a liquid culture medium and fermented until OD (Organic Degree) was reached. 600 Value = 1, the C / P mass ratio of the liquid culture medium is 1 to 10, the C / N mass ratio is 5 to 40, and the pH is 5 to 11.

3. The method for culturing *Terexamic Aristolochic acid* with flocculation and growth-promoting effects according to claim 2, characterized in that, The liquid culture medium is glucose at 18–22 g·L⁻¹. -1 K2HPO4 4~6g·L -1 KH2PO4 2~3g·L -1 NaCl 0.09~0.11g·L -1 (NH4)2SO4 0.18~0.22g·L -1 Urea 0.45–0.55 g·L -1 Yeast extract 0.45–0.55 g / L -1 MgSO4 0.18~0.22g·L -1 The solvent is deionized water.

4. The use of *Trietacia leucocephala* as described in claim 1, which has flocculation and growth-promoting effects, as a plant growth regulator for promoting seed germination, in the preparation of such a regulator.

5. The use of *Terexacum mongolicum* as described in claim 1, which has flocculation and growth-promoting effects, in the preparation of a microbial flocculant.

6. The application of *Tripterygium wilfordii*, which has flocculation and growth-promoting effects as described in claim 1, as a microbial flocculant in the reduction of cow manure separation liquid.

7. The application of a mixed solution of *Tripterygium wilfordii* as described in claim 1, which has flocculation and growth-promoting effects, as a microbial flocculant and PFS, in flocculants.

8. The application according to claim 7, characterized in that, The amount of the microbial flocculant added is 3% of the volume ratio of the mixed solution, and the OD of the microbial flocculant is... 600 Value = 1, the final concentration of PFS in the mixed solution is 1 g·L⁻¹ -1 .

9. The application of *Trichoderma pallida*, which has flocculation and growth-promoting effects as described in claim 1, as a microbial flocculant in combination with PFS in the preparation of cow manure separation liquid bio-fertilizer.

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