A Brucella strain BF1, a microbial inoculum and their application in degrading phenol in water

Abstract

The present invention belongs to the field of environmental microbiological technology, and discloses a Brucella strain BF1, a microbial agent and its application in degrading phenol in water bodies. The strain is Brucella Brucella thiophenivorans strain sp. , which was deposited in the Guangdong Microbial Culture Collection Center on April 11, 2024, with the deposit number GDMCC No: 64516, and the length of the 16S rRNA gene sequence is 1397. The strain BF1 has a significantly larger body size than the original strain before domestication and screening, and has better temperature and acid-base tolerance. After being prepared into a microbial agent, it can continuously carry out bioaugmented degradation of phenol in various water bodies, with high efficiency, and can meet the needs of low-cost and large-scale popularization and application.

Description

Technical Field

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

[0002] Phenol (C6H5OH) 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: It is a 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: It has weak acidity (pKa≈10), can react with alkalis to form phenolates; is 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 has 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 make the water body produce an odor, affecting the safety of drinking water. Soil pollution includes inhibiting the activity of soil microorganisms, destroying the ecological balance, reducing soil fertility, and can also penetrate into groundwater through infiltration, expanding the pollution range, and then generating 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. In order 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 physicochemical 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 CO2 and H2O through microbial metabolism, with 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, and the microorganisms capable of degrading phenol include Pseudomonas, Rhodococcus, and Bacillus, etc. 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, belonging to intracellular parasites, with the following characteristics: it is coccobacillary, without flagella, spores or capsules (smooth strains have microcapsules), positive for catalase and oxidase, absolutely aerobic, and can reduce nitrates. It is divided into 6 species and 19 biotypes according to the host and biochemical characteristics, and 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 rays, chlorine-containing disinfectants (such as 3% bleaching powder), 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 it 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 engineering 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 a n tho rpi DP5, and its morphological characteristics are as follows: the colony is light yellow, with a smooth, moist, flat surface, a flat edge, and a diameter of about 3-4 mm; it is Gram-negative, a facultative anaerobe, with a negative methyl red reaction, and has a good degradation effect on BaP in an 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 bacterium agent can grow and 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, a low degradation efficiency for phenol, requires a long time, and only grows fastest in a slightly alkaline environment. Its bacterium agent preparation method is complex and difficult to meet the needs of the industry (such as degrading phenol in hot organic wastewater). Summary of the Invention

[0006] Aiming at the above problems existing in the prior art, the purpose of the present invention is to provide a Brucella strain BF1 that has been specially collected, domesticated, and screened, with significantly enlarged ecological characteristics and enhanced high-temperature and pH tolerance, and to prepare it into three forms of microbial bacterium agents to accelerate the efficiency of degrading phenol in water, and further improve the temperature and pH tolerance of this strain to meet the needs of the industry.

[0007] The above object of the present invention is achieved by adopting the following technical solutions:

[0008] A Brucella strain BF1, characterized in that it belongs to Brucella Brucella thiophenivorans strain sp. , and its taxonomic name is Brucella thiophenivorans , which was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 11, 2024. The deposit address is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64516. The length of the 16S rRNA gene sequence list is 1397.

[0009] The above-mentioned Brucella strain BF1 is an optimized strain obtained through multiple high-temperature and sun exposure domestication and screening, and it includes the following steps:

[0010] S1: Collect the surface soil of a coal chemical industry site with a relatively high phenol content and a large number of strains as the strain inoculation soil, and obtain the inoculation soil powder after drying.

[0011] S2: Continuously heat and turn the inoculated soil powder so that its temperature increases from 25°C to 65°C, obtaining a small number of strains that continue to survive, have strong environmental stress resistance, and the ability to degrade phenol as the original strains. Then, through cultivation, enrichment, separation, and screening, a first-generation dominant bacterial community is obtained;

[0012] S3: Put the first-generation dominant bacterial community back into the soil of the coal chemical industry site with a relatively high phenol content, mix it, and place it on the surface. After exposing it to sunlight for at least 5 days for in-situ growth, then collect the soil at this place as the second-generation strain inoculated soil, and obtain the second-generation inoculated soil powder after drying;

[0013] S4: Repeat step S2. After secondary domestication and screening, a second-generation dominant bacterial community is obtained;

[0014] S5: Then repeat steps S3 and S2. After three times of domestication and screening, a third-generation dominant bacterial community is obtained. Select a strain with the best growth shape (larger in size) from the third-generation dominant bacterial community. Its biological morphology is significantly larger than that of the original strain, showing a coccobacillus shape, 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, named Brucella strain BF1 。

[0015] A microbial inoculant uses the above-mentioned Brucella strain BF1 as the active ingredient, specifically a suspension bacterial liquid, or a humus-fixed inoculant, or a humus-mineral composite fixed inoculant.

[0016] The application of the above-mentioned Brucella strain BF1 or the microbial inoculant in degrading phenol in water. The conditions for the microbial inoculant to enhance the degradation of phenol in water are: in the field natural sunlight environment, 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.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The present invention obtains a Brucella strain BF1 with significantly enlarged ecological characteristics, significantly enhanced high-temperature and pH tolerance through special collection, multiple domestication, and screening. 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.

[0019] 2. The Brucella strain BF1 provided by the present invention, when prepared into two humus - fixing agents and humus - mineral composite fixing agents, can further accelerate the efficiency of the strain in degrading phenol in water compared with the suspended bacterial liquid, and further improve the tolerance of the strain to temperature and pH, enabling it to meet various industrial requirements such as low cost, high efficiency, and easy promotion.

[0020] 3. The Brucella strain BF1 provided by the present invention, through actual tests, has an enhanced ability to degrade phenol in water. The strain and the microbial agent have 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 treating heat - containing organic sewage and other fields. Brief Description of the Drawings

[0021] Figure 1 It is a scanning electron microscope SEM image of the bacterial morphology of the BF1 strain in the embodiment of the present invention;

[0022] Figure 2 It is a schematic diagram of the purified humic acid and fulvic acid powders in the embodiment of the present invention;

[0023] Figure 3 It is a schematic diagram of the effect of different pH values on the growth curve of BF1 in the embodiment of the present invention;

[0024] Figure 4 It is a schematic diagram of the effect of different temperatures on the growth curve of BF1 in the embodiment of the present invention;

[0025] Figure 5 It is a schematic diagram of the degradation efficiency of the BF1 strain on phenol under different pH conditions in the embodiment of the present invention;

[0026] Figure 6 It is a schematic diagram of the degradation efficiency of the BF1 strain on phenol under different temperature conditions in the embodiment of the present invention;

[0027] Figure 7 It is a schematic diagram of the degradation efficiency of the BF1 strain on phenol under different strain dosage conditions in the embodiment of the present invention;

[0028] Figure 8 It is a schematic diagram of the degradation efficiency of the HA humus - fixing agent prepared in the embodiment of the present invention on phenol;

[0029] Fig. 9 It is a schematic diagram of the degradation efficiency of the FA humus - fixing agent prepared in the embodiment of the present invention on phenol;

[0030] Fig.10 It is a schematic diagram of the degradation efficiency of the HA humus - mineral composite fixing agent prepared in the embodiment of the present invention on phenol. Detailed Embodiments

[0031] The following is combined with Figure 1-Figure 10 The present invention is further described in detail with reference to a plurality of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0032] The "Survival Report" of the strain involved in the present application records the following content: The Brucella strain BF1 biological material sample applied for preservation by the applicant belongs to Brucella thiophenivorans strain sp. , named Brucella thiophenivorans BF1, taxonomic name Brucella thiophenivorans The sample was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) 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. The test result on April 11, 2024 was alive.

[0033] Example 1

[0034] This example involves the collection, domestication and cultivation of Brucella strains, as well as gene detection and naming.

[0035] The Brucella strain BF1 provided in this embodiment is based on the original Brucella strain collected from the contaminated soil in Ordos City, Inner Mongolia Autonomous Region, China, and is a preferred strain obtained after multiple high temperature and sun exposure domestication and screening, which comprises the following steps:

[0036] S1: In Ordos City, Inner Mongolia Autonomous Region, China, surface soil from a coal chemical site with high phenol content and high number of strains was collected as strain inoculation soil. The soil was collected at multiple points, with a total of 10 samples collected at a time, each weighing 500-1000 grams. The soil was mixed and dried to obtain inoculation soil powder;

[0037] S2: Divide the inoculated soil powder into 5 equal parts, heat and turn each inoculated soil powder continuously to raise its temperature from 25°C to 65°C within 5 minutes, then stop heating, sample and culture them respectively, and obtain a small number of strains (10-30 strains) from a colony that continues to survive, has strong environmental resistance, and has the ability to degrade phenol as the original strains, and then cultivate, enrich, separate and screen a dominant colony to obtain the first generation of dominant bacterial flora;

[0038] S3: Place the first generation dominant bacterial community 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 under natural conditions, and after it grows in situ for no less than 5 days (10 days in this embodiment), the bacterial community is re-formed by the strains with strong resistance that survive in the soil, repeat step S1 for sampling, and then collect the soil from this place as the second generation strain inoculation soil, and obtain the second generation inoculation soil powder after drying;

[0039] S4: Repeat step S2, put the second-generation dominant flora back to the soil of the coal chemical industry site with a relatively high phenol content for natural exposure for 10 days for secondary domestication, and then collect and screen to obtain the second-generation dominant flora;

[0040] S5: Repeat steps S3 and S2 again. After three rounds of domestication and screening, obtain the third-generation dominant flora; Screen out a strain with the optimal 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-shaped (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 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 。

[0041] The three rounds of domestication, natural exposure and screening in this example can batch-eliminate strains with relatively poor resistance, and enable strains with relatively strong resistance to be domesticated and retained, so that their resistance characteristics are continuously strengthened and finally screened out. Utilize its strong resistance, fast growth rate, large size and other biological characteristics for the preparation of microbial agents and phenol degradation.

[0042] Example 2

[0043] Based on Example 1, this example prepares the Brucella strain BF1 into a suspension microbial agent and tests the influence of different pH values on the growth characteristics of the microbial agent.

[0044] Refer to the appendix Figure 3 , after amplifying the strain BF1 to OD600 of 0.45, inoculate it into the nutrient medium at an inoculation amount of 5%, and 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; It can be seen from Figure 3 that when the strain is cultured for 12 hours, its microbial concentration basically reaches the logarithmic growth phase. The growth conditions are good when the pH is 6-8, and it shows the highest growth rate and the highest microbial concentration at pH 7.

[0045] In Figure 3-Figure 4 , the abscissa of each graph is time, and the ordinate is OD600; Among them, OD600 represents the optical density (absorbance) of the sample at a wavelength of 600 nm, reflecting the amount of microorganisms.

[0046] Example 3

[0047] Based on Example 1, this example prepares the Brucella strain BF1 into a suspension microbial agent and tests the influence of different temperatures on the growth characteristics of the microbial agent.

[0048] See the appendix Figure 4 , after amplifying the strain BF1 to an OD600 of 0.45, inoculate it into the nutrient medium at an inoculation amount of 5%, and incubate it at a constant temperature of 25 °C, 30 °C, and 35 °C with a pH of 6 and a rotation speed of 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 conditions of strain BF1 at 30 °C and 35 °C are similar, but choosing 30 °C can reduce energy consumption while ensuring good growth. Therefore, 30 °C is a more economical culture temperature.

[0049] Example 4

[0050] On the basis of Example 1, in this example, the Brucella strain BF1 was prepared into a suspension bacterial agent, and the degradation test of phenol in water was carried out under the natural sunlight environment in the wild, and the changes in the degradation efficiency of the bacterial agent under different environmental conditions were tested respectively. The specific test change conditions include pH (5, 6, 7, 8, 9), temperature (25 °C, 30 °C, 35 °C, 40 °C), and the strain addition amount (1%, 2%, 5%, 10%, 20%), and then the optimal degradation working conditions were selected.

[0051] 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 were carried out under the conditions of pH 5, 6, 7, 8, and 9 respectively. 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 at 48 hours; when the pH is 9, all phenol degradation can also be completed at 72 hours; it is proved that the strain has a wide pH adaptation range and strong tolerance and high degradation efficiency for different pH values.

[0052] Secondly, in an inorganic salt medium with a phenol concentration of 50 mg / L, with a 5% strain addition amount, at a pH of 7 and a rotation speed of 130 r / min, degradation experiments were carried out under the conditions of 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. The phenol degradation is completed at 30 °C and 35 °C in 48 hours and 36 hours respectively; while it takes 120 hours to complete phenol degradation at 25 °C, and the degradation rate at 40 °C is 50.34% in 120 hours, which proves that it has strong high - temperature tolerance ability. In this test, it can be seen that the strain can also survive well at 40 °C, only the growth rate decreases significantly compared with 35 °C. From these test results and combined with the common knowledge in this field, it can be known that the high - temperature tolerance of this strain is significantly enhanced, and it can also survive within a temperature range slightly higher than 40 °C, but the growth rate will further decrease as the temperature continues to rise.

[0053] Finally, in an inorganic salt medium with a phenol concentration of 50 mg / L, at pH 7, temperature 30 °C, and rotation speed 130 r / min, degradation experiments were carried out under the conditions of strain addition amounts of 1%, 2%, 5%, 10%, and 20% respectively, and 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, the degradation rates under the conditions of strain addition amounts of 1% and 2% still did not change significantly. Considering the economic cost, the cost - effectiveness ratio is the highest when the strain addition amount is 5% for degradation.

[0054] Figure 5-Figure 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%.

[0055] Example 5

[0056] On the basis of Example 4, in this example, Brucella strain BF1 was further prepared into a humus - fixing bacterial agent, and its degradation efficiency for phenol in water was tested, including the following content:

[0057] (I) Preparation of humus materials

[0058] 1. Pretreatment of cherry wood

[0059] At 110 °C, cherry wood was mixed with water and 0.1 mol / L Ca(OH)2 in a ratio of 1:10, and subjected to high - temperature pretreatment for 2 hours; prepared and alkali - treated cherry wood (AlHTYT); the pretreated cherry wood was placed in a cool place to dry until the water content was about 20%, and it was used as a conditioner for subsequent composting.

[0060] 2. Composting method

[0061] Composting was carried out using a conventional composting device. Cow dung and conditioner were mixed at a dry weight ratio of 2:1. The total amount of composting materials was about 45 kg (dry weight), including 30 kg (dry weight) of cow dung and 15 kg (dry mass) of conditioner. Composting was stopped after the temperature dropped and remained basically unchanged. The entire composting process lasted for 40 days.

[0062] 3. Preparation of humus materials

[0063] Humus was prepared using the product after 40 days of composting. Extraction: Weigh 2 g of the frozen compost product and add it to a 50 mL centrifuge tube. Then add 35 mL of alkaline extraction solution (0.1 mol / L sodium hydroxide: 0.1 mol / L sodium pyrophosphate = 1:20). Mix it evenly by shaking at 240 r / min in a water bath thermostatic shaker for 10 min, and then heat it at 60 °C for 2 h. Centrifuge at 4000 r / min for ten minutes in a centrifuge 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.

[0064] Purification of humic acid and fulvic acid: Humic acid was rinsed with water multiple times to obtain purified humic acid. After adsorbing fulvic acid using a MAD-8 column, it was eluted with 0.1 mol / L NaOH and water; the effluent was adjusted to pH 1 using 6 mol / L HCl, and the solution was passed through a + saturated ion exchange resin to obtain purified fulvic acid. The purified humic acid (HA) and fulvic acid (FA) powders are as Figure 2 shown.

[0065] (II) Preparation of humus-fixing microbial agents

[0066] 1. Preparation of two kinds of humus-fixing microbial agents

[0067] Humic acid (HA) or fulvic acid (FA) was added to the liquid of Brucella strain BF1 at mass ratios of 1%, 2%, 5%, 10%, and 20% respectively, and left standing for 2 hours to allow strain BF1 to spontaneously attach and fix on the surface of the humic acid (HA) or fulvic acid (FA) powder, thus preparing HA humus-fixing microbial agents and FA humus-fixing microbial agents with different added mass ratios respectively.

[0068] 2. Test for removing phenol by humus-fixing microbial agents

[0069] In an inorganic salt medium with a phenol concentration of 50 mg / L, a phenol degradation experiment of humus-fixing microbial agents was carried out at a strain addition amount of 5%, pH 7, temperature 30 °C, and rotation speed 130 r / min. The test results of HA humus-fixing microbial agents are shown in Figure 8 , the test results of the FA humus-fixing bactericide are shown in Fig. 9 .

[0070] 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 bactericide can significantly promote the degradation of phenol by the strain. Although 10% and 20% of HA and FA can promote the growth of the strain, they will also compete for too many substrates, which instead slows 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 bactericide has the best effect on promoting the degradation of phenol by the strain and the best economic benefit.

[0071] Example 6

[0072] On the basis of Example 5, in this example, Brucella strain BF1 was further prepared into a humus-mineral composite fixing bactericide, and its degradation efficiency for phenol in water was tested, including the following contents:

[0073] 1. Preparation of two humus-mineral composite fixing bactericides

[0074] Two powder materials of clay minerals montmorillonite (Mon) and kaolinite (Kln) were prepared and added to the HA humus-fixing bactericide at three ratios of solid-liquid ratio 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 montmorillonite humus-mineral composite fixing bactericide and kaolinite humus-mineral composite fixing bactericide.

[0075] 2. Test for the removal of phenol by the humus-mineral composite fixing bactericide

[0076] 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 pH 7, temperature 30 °C, and rotation speed 130 r / min. The results are as Fig.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 within 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 all phenol degradation is not completed even 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 at 12 hours are 92.36% and 87.75% respectively; when montmorillonite (Mon) and kaolinite (Kln) are added at 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.

[0077] 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 natural exposure and water environment conditions 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 agent 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.

[0078] Finally, it should be noted that although the content of the present invention has been introduced 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 to 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, It belongs to Brucella Brucella thiophenivorans strain sp. , and was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 11, 2024. The deposit address is the 5th floor, Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64516.

2. A microbial inoculant, characterized in that, It uses the Brucella strain BF1 described in claim 1 as the active ingredient.

3. The microbial inoculant according to claim 2, wherein, It is a suspension bacterial liquid, or a humus-fixed bacterial agent, a humus-mineral composite fixed bacterial agent.

4. The microbial inoculum according to claim 3, wherein The described humus-fixed bacterial agent is obtained by mixing the Brucella strain BF1 with humus, allowing the Brucella strain BF1 to naturally adhere to the humus; the humus is humic acid HA or fulvic acid FA.

5. The microbial inoculant according to claim 3, characterized in that, The described humus-mineral composite fixed bacterial agent is obtained by mixing the Brucella strain BF1 with humus and clay minerals, allowing the BF1 strain and the clay minerals to simultaneously adhere to the composite humus.

6. The microbial inoculant according to claim 5, wherein The described humus-mineral composite fixed bacterial agent is obtained by adding powder of clay minerals to a 2wt% mixture of humus and the Brucella strain BF1 at a solid-liquid ratio of 1:5000 and 1:10000 respectively, so that the powder of clay minerals and the strain both naturally adhere to the humus to form a humus-mineral composite fixed bacterial agent; the clay mineral is one of montmorillonite or kaolinite.

7. Application of the Brucella strain BF1 described in claim 1 or the microbial bacterial agent described in any one of claims 2 to 6 in degrading phenol in water.

8. The application according to claim 7, characterized in that, The conditions for the microbial bacterial agent to enhance the degradation of phenol in water are: in the natural sunlight environment in the wild, the water temperature is 25 - 40 °C, and the pH value is 5 - 9.

9. The application according to claim 8, wherein The optimal working conditions for the microbial bacterial agent to enhance the degradation of phenol in water are: the temperature is 30 - 35 °C, and the pH value is 6 - 9.

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