Brucella strain BF1, microbial agent and application of Brucella strain BF1 in degradation of phenol in water

Abstract

The invention belongs to the technical field of environmental microorganisms, and discloses a Brucella strain BF1, a microbial agent and application of the Brucella strain BF1 to degradation of phenol in water, the Brucella strain BF1 is Brucella thionovorans strain sp., the Brucella thionovorans strain sp. Is preserved in Guangdong Microbial Culture Collection Center on April 11, 2024, the preservation number is GDMCC No: 64516, and the length of a 16S rRNA gene sequence table is 1397. The invention further discloses a preparation method of the Brucella thionovorans strain BF1, the microbial agent and application of the Brucella thionovorans strain BF1, the microbial agent and the application of the Brucella thionovorans strain sp. Compared with an original strain before domestication and screening, the strain BF1 has the advantages that the body type is obviously increased, the strain BF1 has better temperature and acid-base resistance, after the strain BF1 is prepared into a microbial agent, phenol in various water bodies can be continuously subjected to biological enhancement degradation, the efficiency is high, and the requirements of low cost and wide-range popularization and application can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms and water pollution treatment, and specifically relates to a Brucella strain BF1, a microbial agent, and their application in degrading phenol in water bodies. Background Art

[0002] Phenol (C 6 H 5 OH) is an organic compound with an aromatic ring structure. It contains a hydroxyl group (-OH) directly connected to the benzene ring. Its characteristics include: Physical properties: white crystalline solid, slightly soluble in water (solubility at room temperature is about 8%), easily soluble in organic solvents such as ethanol and ether, and has a pungent "medicinal smell". Chemical properties: weak acidity (pKa≈10), can react with bases to form phenol salts; easily oxidized to form quinone substances; may polymerize under light or high temperature. Phenol is widely used in synthetic resins, plastics (such as phenolic resins), pharmaceuticals, dyes, and pesticides. Industrial wastewater (such as coking plants, chemical plants) is the main source of environmental phenol pollution.

[0003] Phenol causes great harm to the environment. Phenol is an important organic pollutant in the water environment and can accumulate in water bodies, soil, and sediments. The pollution of water bodies includes acute toxicity and chronic toxicity. In terms of acute toxicity, due to the high toxicity of phenol to aquatic organisms (fish, algae) (LC 50 as low as 1 - 10 mg / L), it can damage cell membranes and enzyme systems, leading to the death of organisms; in terms of chronic toxicity, long-term low-concentration exposure affects the reproduction and development of organisms and interferes with the endocrine system (estrogen-like effect); there is also sensory pollution of water bodies. Low concentrations (0.1 - 0.5 mg / L) can cause water bodies to produce odors, affecting the safety of drinking water. Soil pollution includes inhibiting the activity of soil microorganisms, disrupting the balance of the ecosystem, reducing soil fertility, and can also penetrate into groundwater through infiltration, expanding the pollution range, and then causing secondary pollution through bioaccumulation. Phenol metabolizes into more toxic products such as quinones and polycyclic aromatic hydrocarbons in organisms and may be transmitted to humans through the food chain, thus endangering human health. To protect water bodies and human health, it is necessary to closely monitor the pollution status of phenol and take corresponding prevention and control measures.

[0004] In the prior art, the efficient degradation methods for phenol in water bodies include physical and chemical methods (adsorption method, advanced oxidation method, membrane separation method, etc.), all of which have problems such as high cost and easy generation of secondary pollution; the biodegradation method is to convert phenol into CO 2 and H 2O has the advantages of environmental protection and low cost. However, the degradation of phenol requires the selection of bacterial agents in combination with the pollution concentration and environmental conditions. Microorganisms capable of degrading phenol include Pseudomonas, Rhodococcus, and Bacillus. These strains decompose phenol into non-toxic products through metabolic enzymes (such as phenol hydroxylase). Conventional Brucella does not have the ability to degrade phenol. Conventional Brucella is a Gram-negative non-motile bacterium and an intracellular parasite, with the following characteristics: it is coccobacillary, without flagella, spores, or capsules (smooth strains have a microcapsule), catalase and oxidase positive, absolutely aerobic, and can reduce nitrates. It is divided into 6 species and 19 biotypes according to the host and biochemical characteristics. The main pathogenic species include Brucella melitensis (with the strongest pathogenicity), Brucella abortus, Brucella suis, and Brucella canis. It has strong survival ability in the natural environment and can survive in soil, water, fur, and dairy products for several weeks to several months; but it has poor tolerance to high temperatures and is sensitive to moist heat; it is also sensitive to chemical disinfectants such as ultraviolet light, chlorine-containing disinfectants (such as 3% bleach), and phenol. The metabolic characteristics of Brucella focus on intracellular parasitism and immune escape in the host, rather than the degradation of environmental pollutants. Its growth requires complex nutritional conditions (such as the host cell environment) and lacks known phenol metabolic pathways. The main applications of conventional Brucella focus on the medical and veterinary fields, rather than environmental remediation. The degradation of phenol requires the selection of specific degrading bacteria or engineered strains, such as Pseudomonas and Bacillus.

[0005] In the prior art, CN 119286699 A discloses a PAHs-degrading strain, which is Brucella anthorpi M1. This strain M1 is on the same branch as Brucella anthorpi DP5. Its morphological characteristics are as follows: the colony is light yellow, smooth, moist, flat on the surface, with a flat edge, and the diameter is about 3-4 mm; Gram staining is negative, facultative anaerobe, methyl red reaction is negative, and it has good degradation effect on BaP in the environment of 25°C - 30°C and pH 5.5 - 8.5. Its optimal growth environment has a pH of 7.5 and a temperature of 30°C; the prepared spherical-embedded Brucella M1 bacterial agent can grow, reproduce using benzo(a)pyrene (BaP), pyrene, or phenol as the sole carbon source and energy source, and can degrade PAHs. When cultured in an inorganic salt medium with an initial concentration of 5 mg / L of BaP, pyrene, and phenol for 5 days, the degradation efficiencies reach 81%, 37%, and 28% respectively. It can be seen that this strain M1 has poor high-temperature tolerance, low degradation efficiency for phenol, requires a long time, and only grows fastest in a slightly alkaline environment. Its method for preparing the bacterial agent is complex and difficult to meet the industrial needs (such as degrading phenol in thermally organic wastewater). Summary of the Invention

[0006] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a Brucella strain BF1 which has been specially collected, domesticated and screened and has significantly enhanced ecological characteristics and enhanced high temperature and pH tolerance, and to prepare it into three forms of microbial agents to accelerate its efficiency in degrading phenol in water, and further improve the strain's tolerance to temperature and pH to meet industrial needs.

[0007] The above-mentioned purpose of the present invention is achieved by adopting the following technical solutions: A Brucella strain BF1, characterized in that it belongs to Brucella Brucella thiophenivorans strain sp. , the taxonomic name is Brucella thiophenivorans It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCCNo: 64516, and the length of the 16S rRNA gene sequence list is 1397.

[0008] The Brucella strain BF1 is a preferred strain obtained after multiple high temperature and sunlight exposure acclimation and screening, which comprises the following steps: S1: Collect the surface soil of the coal chemical site with high phenol content and strain number as the strain inoculation soil, and obtain the inoculation soil powder after drying; S2: Continuously heating and turning the inoculated soil powder to raise its temperature from 25°C to 65°C, obtaining a small number of strains that continue to survive, have strong environmental resistance and are capable of degrading phenol as the original strains, and then cultivating, enriching, separating and screening to obtain the first generation of dominant bacterial flora; S3: Place the first generation dominant bacterial flora back into the soil of the coal chemical site with a high phenol content, mix it, and place it on the surface, expose it to sunlight, and after it grows in situ for no less than 5 days, collect the soil from that site and use it as the second generation strain inoculation soil, and dry it to obtain the second generation inoculation soil powder; S4: Repeat step S2, and after secondary domestication and screening, obtain the second generation of dominant bacterial flora; S5: Repeat steps S3 and S2 again, and obtain the third generation of dominant bacterial population after three acclimation and screening. A strain with the best growth shape (larger body size) is screened out from the third generation of dominant bacterial population. Its biological morphology is significantly larger than that of the original strain, and is club-shaped, without flagella, spores or capsules. The average length of the club is about 1-1.5 μm, and the average diameter is about 0.3-0.4 μm. It is named Brucella strain BF1 。

[0009] A microbial agent uses the Brucella strain BF1 as an active ingredient, specifically a suspended bacterial liquid, or a humus-fixed bacterial agent, or a humus-mineral composite fixed bacterial agent.

[0010] Application of Brucella strain BF1 or microbial inoculant in degrading phenol in water body. The conditions for the microbial inoculant to enhance the degradation of phenol in water body are as follows: under the natural sunlight environment in the wild, the water temperature is 25 - 40 °C, and the pH value is 5 - 9. The optimal working conditions are: the temperature is 30 - 35 °C, and the pH value is 6 - 9.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. A Brucella strain BF1 with significantly enlarged ecological characteristics, significantly enhanced high-temperature and pH tolerance obtained by specifically collecting, domestication and screening for multiple times. Through domestication, it has unique microbial genetic characteristics, and then it is prepared into an inoculant. After testing, the inoculant can tolerate high temperatures of 40 °C and above; the strains in the humus mineral composite fixed inoculant can survive at 40 °C, and its optimal temperature is 30 - 35 °C, which is more than 10 °C higher than that of conventional strains; the pH value that the inoculant can tolerate is 5 - 9, and the optimal working pH value is 6 - 9, that is, it can maintain a high degradation efficiency in acidic or alkaline environments.

[0012] 2. The Brucella strain BF1 provided by the present invention, after being prepared into two humus fixed inoculants and humus mineral composite fixed inoculants, can further accelerate the degradation efficiency of the strain for phenol in water body compared with the suspended bacterial liquid, and further improve the tolerance of the strain to temperature and pH, so as to meet various industrial needs such as low cost, high efficiency and easy promotion.

[0013] 3. The Brucella strain BF1 provided by the present invention, through actual testing, its inoculant has an enhanced degradation ability for phenol in water body. The environmental adaptability of the strain and the microbial inoculant is good, the degradation efficiency is high, it is environmentally friendly and has low cost, which can meet the needs of large-scale popularization and application, and has good economic and environmental benefits, and has good application prospects in the fields such as the treatment of hot organic sewage. Description of the Drawings

[0014] Figure 1 It is the scanning electron microscope SEM image of the bacterial morphology of BF1 strain in the embodiment of the present invention; Figure 2 It is the schematic diagram of the purified humic acid and fulvic acid powder in the embodiment of the present invention; Figure 3 It is the schematic diagram of the influence effect of different pH values on the growth curve of BF1 in the embodiment of the present invention; Figure 4 It is the schematic diagram of the influence effect of different temperatures on the growth curve of BF1 in the embodiment of the present invention; Figure 5 It is the schematic diagram of the degradation efficiency of BF1 strain for phenol under different pH conditions in the embodiment of the present invention; Figure 6 Schematic diagram of the degradation efficiency of phenol by the BF1 strain in the embodiments of the present invention under different temperature conditions; Figure 7 Schematic diagram of the degradation efficiency of phenol by the BF1 strain in the embodiments of the present invention under different inoculation amounts of the strain; Figure 8 Schematic diagram of the degradation efficiency of phenol by the HA humus - fixed microbial agent prepared in the embodiments of the present invention; Figure 9 Schematic diagram of the degradation efficiency of phenol by the FA humus - fixed microbial agent prepared in the embodiments of the present invention; Figure 10 Schematic diagram of the degradation efficiency of phenol by the HA humus - mineral composite fixed microbial agent prepared in the embodiments of the present invention. Detailed implementation manners

[0015] The following combines the attached Figures 1 - 10 and multiple specific embodiments to further elaborate on the present invention in detail, but the protection scope of the present invention is not limited thereto.

[0016] The "Viability Report" related to the strain in the present invention application records the following content: A biological material sample of a Brucella strain BF1 applied for preservation by the applicant belongs to Brucella thiophenivorans strain sp. , with the name Brucella thiophenivorans BF1, and the taxonomic name is Brucella thiophenivorans . This sample was preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 11, 2024. The preservation address is on the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou. The preservation number is GDMCC No. 64516, and the test result on April 11, 2024, is that it is viable.

[0017] Example 1 In this example, the collection, domestication, and cultivation of Brucella strains were carried out, and gene detection and naming were performed.

[0018] The Brucella strain BF1 provided in this example is an optimized strain obtained by domestication and screening through multiple high - temperature and sunlight exposures based on the original Brucella strain collected from contaminated soil in Ordos City, Inner Mongolia Autonomous Region, China. It includes the following steps: S1: In Ordos City, Inner Mongolia Autonomous Region, China, the surface soil of a coal chemical industry site with a relatively high phenol content and a large number of strains was collected as the strain inoculation soil. Multiple points were collected, with a total of 10 samples collected each time, 500 - 1000 grams for each sample. After mixing and drying, the inoculation soil powder was obtained; S2: Divide the inoculated soil powder evenly into 5 portions, continuously heat and turn each portion of the inoculated soil powder to increase its temperature from 25°C to 65°C within 5 minutes, then stop heating, take samples for cultivation respectively, and obtain a small number of strains (10 - 30 strains) in a colony that continue to survive, have strong environmental stress resistance, and have the ability to degrade phenol as the original strains. Then, through cultivation, enrichment, separation, and screening, obtain a dominant colony to get the first-generation dominant flora; S3: Put the first-generation dominant flora back into the coal chemical industry site soil with a relatively high phenol content, mix it, and place it on the surface layer. Expose it to sunlight under natural conditions. After it grows in situ for no less than 5 days (10 days in this example), the relatively resistant strains surviving in the soil re-form a flora. Repeat the sampling in step S1, then collect the soil at this place as the second-generation strain inoculated soil, and dry it to obtain the second-generation inoculated soil powder; S4: Repeat step S2, put the second-generation dominant flora back into the coal chemical industry site soil with a relatively high phenol content and expose it naturally for 10 days for secondary domestication. Then, after collection and screening, obtain the second-generation dominant flora; S5: Repeat steps S3 and S2 again. After three times of domestication and screening, obtain the third-generation dominant flora; Screen out a strain with the best growth shape (the largest size) from the third-generation dominant flora. Its biological morphology is significantly larger than that of the original strain. Please refer to the appendix Figure 1 , this strain is coccobacillus (with elliptical heads at both ends), without flagella, spores or capsules. The average length of the coccobacillus is about 1 - 1.5 μm, and the average diameter is about 0.3 - 0.4 μm; Conduct gene sequencing on the screened strain with relatively strong degradation ability. The length of its 16S rRNA gene sequence table is 1397. The specific gene DNA sequence is shown in the appendix. Name it Brucella strain BF1 and conduct microbial preservation 。

[0019] In this example, the three times of domestication, natural exposure, and screening can batch eliminate the strains with relatively poor resistance, and domesticate and retain the strains with relatively strong resistance, continuously strengthen their resistance characteristics, and finally screen them out. Utilize their strong resistance, fast growth rate, large size and other biological characteristics for the preparation of microbial agents and phenol degradation.

[0020] Example 2 Based on Example 1, in this example, prepare Brucella strain BF1 into a suspension microbial agent and test the influence of different pH values on the growth characteristics of the microbial agent.

[0021] Refer to the appendix Figure 3 , after amplifying strain BF1 to OD600 of 0.45, inoculate it into the nutrient medium at an inoculation amount of 5%. Incubate it at a constant temperature under the conditions of 30°C, 160 r / min, and pH values of 5, 6, 7, 8, and 9 respectively; By Figure 3It can be seen that when the strain is cultured for 12 hours, its microbial concentration basically reaches the logarithmic growth phase, and it has good growth conditions at a pH of 6 - 8. It shows the highest growth rate and the highest microbial concentration at pH 7.

[0022] In Figures 3 - 4 each figure, the abscissa is time and the ordinate is OD600; OD600 among them represents the optical density (absorbance) of the sample at a wavelength of 600 nm, reflecting the amount of microorganisms.

[0023] Example 3 On the basis of Example 1, in this example, Brucella strain BF1 is prepared into a suspension bacterial agent, and the effects of different temperatures on the growth characteristics of the bacterial agent are tested.

[0024] See Appendix Figure 4 , after amplifying strain BF1 to an OD600 of 0.45, it is inoculated into the nutrient medium at an inoculation amount of 5%, and cultured at a constant temperature of 25°C, 30°C, and 35°C at a pH of 6 and 160 r / min. From Figure 4 it can be seen that the microbial concentration basically reaches the logarithmic growth phase at 12 hours. The growth of strain BF1 is similar at 30°C and 35°C, but choosing 30°C can reduce energy consumption while ensuring good growth. Therefore, 30°C is a more economical culture temperature.

[0025] Example 4 On the basis of Example 1, in this example, Brucella strain BF1 is prepared into a suspension bacterial agent, and the degradation test of phenol in water is carried out under the natural sunlight environment in the wild, and the change of the degradation efficiency of the bacterial agent under different environmental conditions is tested respectively. The specific test change conditions include pH (5, 6, 7, 8, 9), temperature (25°C, 30°C, 35°C, 40°C), and strain dosage (1%, 2%, 5%, 10%, 20%), and then the optimal degradation working conditions are selected.

[0026] First, in an inorganic salt medium with a phenol concentration of 50 mg / L, with a 5% strain addition amount, at a temperature of 30°C and a rotation speed of 130 r / min, degradation experiments are carried out under the conditions of pH being 5, 6, 7, 8, and 9 respectively, and the results are as Figure 5 shown. The test results show that when the pH is 6 or 7, the degradation rate has reached more than 99.8% at a degradation time of 36 hours, which is the most suitable degradation pH range. Especially when the pH is 7, the effect is more obvious. At this time, the degradation rates at pH 5 and 8 are 19.1% and 62.5% respectively, and all phenol degradation is completed in 48 hours; when the pH is 9, all phenol degradation can also be completed in 72 hours; it is proved that the strain has a wide pH adaptation range and has strong tolerance and high degradation efficiency for different pH values.

[0027] Secondly, in an inorganic salt medium with a phenol concentration of 50 mg / L, at a strain addition amount of 5%, at a pH of 7 and a rotation speed of 130 r / min, degradation experiments were carried out at temperatures of 25 °C, 30 °C, 35 °C, and 40 °C respectively. The results are shown in Figure 6 . The test results show that under the conditions of 25 - 30 °C, increasing the temperature is beneficial to phenol degradation. At 30 °C and 35 °C, phenol degradation was completed in 48 hours and 36 hours respectively; while at 25 °C, it took 120 hours to complete phenol degradation, and the degradation rate at 40 °C was 50.34% in 120 hours, which proves that it has strong high-temperature tolerance. In this test, it can be seen that the strain can also survive well at 40 °C, but the growth rate is significantly lower than that at 35 °C. From these test results and combined with common knowledge in this field, it can be known that the high-temperature tolerance of this strain is significantly enhanced and it can survive in a temperature range slightly higher than 40 °C, but the growth rate will further decrease as the temperature continues to rise.

[0028] Finally, in an inorganic salt medium with a phenol concentration of 50 mg / L, at a pH of 7, a temperature of 30 °C, and a rotation speed of 130 r / min, degradation experiments were carried out at strain addition amounts of 1%, 2%, 5%, 10%, and 20% respectively. The results are shown in Figure 7 . The test results show that when degradation experiments were carried out with strain addition amounts of 10% and 20%, obvious degradation occurred when the degradation time reached 12 hours. When the degradation experiment was carried out with a strain addition amount of 5%, obvious degradation occurred when the degradation time reached 18 hours, and at this time, there was still no obvious change in the degradation rate under the conditions of 1% and 2% strain addition amounts. Considering the economic cost, the cost-effectiveness ratio is the highest when the strain addition amount is 5%.

[0029] Figures 5 - 10 They are all schematic diagrams of the strain or bacterial agent BF1 for C / C0 of phenol. The degradation efficiency, that is, the removal rate (%) = (1 - C / C0) × 100%, and the mapping basis is that the degradation efficiency (%) = (initial mass - remaining mass) / (initial mass) × 100%.

[0030] Example 5 On the basis of Example 4, in this example, the Brucella strain BF1 was further prepared into a humus-fixed bacterial agent, and its degradation efficiency for phenol in water was tested, including the following content: (I) Preparation of humus materials 1. Pretreatment of cherry wood At 110 °C, cherry wood was mixed with water and 0.1 mol / L Ca(OH) 2Mix in a ratio of 1:10 and conduct a high-temperature pretreatment for 2 hours; prepare and alkali-treated cherry wood (AlHTYT); place the pretreated cherry wood in a shady place to dry until the water content is about 20%, and use it as a conditioner for subsequent composting.

[0031] 2. Composting method Use a conventional composting device for composting. Mix cow dung and the conditioner in a dry weight ratio of 2:1. The total amount of composting materials is about 45 kg (dry weight), 30 kg (dry weight) of cow dung and 15 kg (dry mass) of conditioner. Stop composting after the temperature drops and remains basically unchanged. The entire composting process lasts for 40 days.

[0032] 3. Preparation of humus materials Use the product after 40 days of composting to prepare humus. Extraction: Weigh 2 g of the frozen compost product and add it to a 50 mL centrifuge tube, and add 35 mL of alkaline extraction solution (0.1 mol / L sodium hydroxide: 0.1 mol / L sodium pyrophosphate = 1:20). Oscillate in a water bath constant temperature oscillator at 240 r / min for 10 min, mix well, heat at 60 °C for 2 h. Centrifuge at 4000 r / min in a centrifuge for ten minutes and then filter. Add 1 mol / L hydrochloric acid to the supernatant to acidify the pH to below 2, keep it in a constant temperature water bath at 60 °C for 1 h, then centrifuge at 4000 r / min for 10 min, and filter the supernatant. The filtrate is the fulvic acid (FA) solution, and the filter residue is the humic acid (HA) solution.

[0033] Purification of humic acid and fulvic acid: Wash the humic acid with water multiple times to obtain purified humic acid. After adsorbing fulvic acid with a MAD-8 column, elute it with 0.1 mol / L NaOH and water; adjust the pH of the effluent to 1 with 6 mol / L HCl, and the solution continues to pass through H + Saturated ion exchange resin to obtain purified fulvic acid. The purified humic acid (HA) and fulvic acid (FA) powders are as Figure 2 shown.

[0034] (II) Preparation of humus-fixing microbial agents 1. Preparation of two humus-fixing microbial agents Respectively add humic acid (HA) or fulvic acid (FA) to the Brucella strain BF1 bacterial solution in mass ratios of 1%, 2%, 5%, 10%, and 20%, and let it stand for 2 hours to allow the strain BF1 to spontaneously attach and fix on the surface of the humic acid (HA) or fulvic acid (FA) powder, respectively obtaining HA humus-fixing microbial agents and FA humus-fixing microbial agents with different addition mass ratios.

[0035] 2. Test for removing phenol by humus-fixing microbial agents In an inorganic salt medium with a phenol concentration of 50 mg / L, with a 5% strain addition amount, at a pH of 7, a temperature of 30 °C, and a rotation speed of 130 r / min, a phenol degradation experiment of the humus-fixing bacterium agent was carried out. The test results of the HA humus-fixing bacterium agent are shown in Figure 8 , and the test results of the FA humus-fixing bacterium agent are shown in Figure 9 .

[0036] The test results show that under the conditions of 1%, 2%, and 5% addition amounts of HA or FA, as the addition amount increases, the humus in the humus-fixing bacterium agent can significantly promote the strain to degrade phenol. Although 10% and 20% of HA and FA can promote the growth of the strain, they will also compete for substrates too much, which will instead slow down the degradation of phenol. Therefore, HA and FA can complete the degradation of all phenol in the water body in 18 hours at an addition amount of 2%. At the same time, considering the economic effect, when the addition amount of HA or FA is 2%, the prepared humus-fixing bacterium agent has the best effect of promoting the strain to degrade phenol and the best economic benefit.

[0037] Example 6 On the basis of Example 5, in this example, the Brucella strain BF1 was further prepared into a humus-mineral composite fixing bacterium agent, and its degradation efficiency for phenol in water was tested, including the following contents: 1. Preparation of two humus-mineral composite fixing bacterium agents Two clay mineral montmorillonite (Mon) and kaolinite (Kln) powder materials were prepared and added to the HA humus-fixing bacterium agent at three ratios of solid-liquid ratios of 1:1000, 1:5000, and 1:10000, and left standing for 2 hours to allow the strain and the mineral powder to spontaneously attach naturally, and they were all attached to the surface of HA to obtain the montmorillonite humus-mineral composite fixing bacterium agent and the kaolinite humus-mineral composite fixing bacterium agent.

[0038] 2. Test of the humus-mineral composite fixing bacterium agent for removing phenol In an inorganic salt medium with a phenol concentration of 50 mg / L, with a 5 wt% strain addition amount and a 2 wt% HA addition amount, a phenol degradation experiment was carried out at a pH of 7, a temperature of 30 °C, and a rotation speed of 130 r / min. The results are as Figure 10As shown. The test results show that the addition of montmorillonite and kaolinite with a low solid-liquid ratio significantly promotes the removal efficiency of the bacterial agent in the range of 0-6 hours. The degradation efficiency of the low proportion of clay minerals with a solid-liquid ratio of 1:1000 in the degradation system is significantly higher than that of the solid-liquid ratios of 1:5000 and 1:10000 between the clay minerals and the degradation system, but it will hinder the final degradation effect of the strain, and the degradation of all phenol is still not completed after 12 hours. Under the condition of a solid-liquid ratio of 1:1000 between montmorillonite (Mon) and kaolinite (Kln) and the degradation system, the degradation rates after 12 hours are 92.36% and 87.75% respectively; when montmorillonite (Mon) and kaolinite (Kln) are added at the solid-liquid ratios of 1:5000 and 1:10000 between the clay minerals and the degradation system in the 2% HA humic acid-fixed bacterial agent, the removal rates of the two humic acid-mineral composite fixed bacterial agents can reach 91.94-96.16% at 9 hours and can be completely removed at 12 hours, and the improvement effect of the degradation efficiency is the most significant.

[0039] In the above embodiments of the present invention, the optimal Brucella strain BF1 is collected, domesticated, cultivated and screened. It has biological characteristics such as a significantly increased body size and strong resistance, and has strong survival and reproduction abilities under the conditions of natural exposure and a water environment with a relatively high temperature; various fixed bacterial agents prepared from it can significantly improve the enhanced degradation ability of phenol in water compared with the bacterial liquid, greatly improving the degradation efficiency and at the same time improving the resistance (environmental adaptability) of the strain. Therefore, the Brucella strain BF1 and its microbial bacterial agents have outstanding biological characteristics, good environmental adaptability, high degradation efficiency, environmental friendliness and low cost, can meet the needs of large-scale popularization and application, have good economic and environmental benefits, and have good application prospects in the field of organic sewage treatment and other fields.

[0040] Finally, it should be noted that although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A Brucella strain BF1, characterized in that Brucella Brucella thiophenivorans strain sp. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 64516, and the length of the 16S rRNA gene sequence list is 1397.

2. Brucella strain BF1 according to claim 1, characterized in that, The Brucella strain BF1 is a preferred strain obtained after multiple high temperature and sunlight exposure acclimation and screening, which comprises the following steps: S1: Collect the surface soil of the coal chemical site with high phenol content and strain number as the strain inoculation soil, and obtain the inoculation soil powder after drying; S2: Continuously heating and turning the inoculated soil powder to raise its temperature from 25°C to 65°C, obtaining a small number of strains that continue to survive, have strong environmental resistance and are capable of degrading phenol as the original strains, and then cultivating, enriching, separating and screening to obtain the first generation of dominant bacterial flora; S3: Place the first generation dominant bacterial flora back into the soil of the coal chemical site with a high phenol content, mix it, and place it on the surface, expose it to sunlight, and after it grows in situ for no less than 5 days, collect the soil from that site and use it as the second generation strain inoculation soil, and dry it to obtain the second generation inoculation soil powder; S4: Repeat step S2, and after secondary domestication and screening, obtain the second generation of dominant bacterial flora; S5: Repeat steps S3 and S2 again, and obtain three generations of dominant bacterial colonies after three acclimation and screening. A strain with the best growth shape is screened out from the three generations of dominant bacterial colonies. Its biological morphology is significantly larger than that of the original strain, and is club-shaped, without flagella, spores or capsules. The average length of the club is about 1-1.5 μm, and the average diameter is about 0.3-0.4 μm. It is named Brucella strain BF1 。 3. A microbial agent, characterized in that: The Brucella strain BF1 according to claim 1 or 2 is used as an active ingredient.

4. The microbial agent according to claim 3, characterized in that: It is a suspended bacterial solution, or a humus-fixed bacterial agent, or a humus-mineral composite fixed bacterial agent.

5. The microbial agent according to claim 4, characterized in that: The humus-fixing bacterial agent is obtained by mixing Brucella strain BF1 with humus and allowing Brucella strain BF1 to naturally adhere to the humus; the humus is humic acid HA or fulvic acid FA.

6. The microbial agent according to claim 4, characterized in that: The humus-mineral composite fixative bacterial agent is obtained by mixing Brucella strain BF1 with humus and clay minerals, and allowing the BF1 strain and the clay mineral to adhere to the composite humus at the same time.

7. The microbial agent according to claim 6, characterized in that: The humus mineral composite fixative bacterial agent is prepared by adding clay mineral powder to a mixed solution of 2wt% humus and Brucella strain BF1 at a solid-liquid ratio of 1:5000 and 1:10000, respectively, so that the clay mineral powder and the strain are naturally attached to the humus to form the humus mineral composite fixative bacterial agent; the clay mineral is one of montmorillonite and kaolinite.

8. Use of the Brucella strain BF1 described in claim 1 or 2 or the microbial agent described in any one of claims 3 to 7 in degrading phenol in water.

9. The use according to claim 8, characterized in that: The conditions for the microbial agent to enhance the degradation of phenol in water are: under the natural sunlight environment in the wild, the water temperature is 25~40℃, the pH value is 5~9, and the optimal working conditions are: temperature 30~35℃, pH value 6~9.

Images