Phenol removing bacteria and phenol adsorption humus

Abstract

The invention relates to the technical field of microorganisms, in particular to phenol removing bacteria and phenol adsorption humus. Wherein the phenol removing bacteria comprise at least one of bacillus (bacillus luti) and bacillus (bacillus paranthracis), and the phenol removing bacteria comprise at least one of bacillus (bacillus luti) and bacillus (bacillus paranthracis); the Bacillus luti is preserved in the Guangdong Microbial Culture Collection Center on May 14, 2025, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 66330; the bacillus is preserved in the Guangdong Microbial Culture Collection Center on May 14, 2025, and the preservation number of the bacillus is GDMCC (China General Microbiological Culture Collection Center) NO: 66331. The phenol removing bacteria can grow and metabolize by taking phenol as a carbon source. In addition, the phenol prepared by adding the phenol removal bacterium liquid adsorbs humus, and the phenol removal rate of the phenol can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to phenol-removing bacteria and phenol-adsorbing humus. Background Art

[0002] Phenol is a highly toxic substance. When phenol enters the soil, it may have an adverse effect on soil quality, inhibit the activity of soil microorganisms, and thus affect soil fertility and plant growth. Phenol can also cause acute toxicity to aquatic organisms, affecting the balance and stability of aquatic ecosystems. If phenol can be removed by specific microorganisms, the pollution of water and soil resources by phenol can be reduced. Humus is an important component of soil. It can adsorb phenol in the soil, reduce its migration and diffusion in the soil, and reduce the pollution of the soil by phenol. At the same time, it is also beneficial to prevent phenol from percolating into groundwater through the soil, causing more extensive pollution. However, the current humus has a relatively low adsorption capacity for phenol.

[0003] It can be seen that the existing technology still needs to be improved. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide phenol-removing bacteria and phenol-adsorbing humus, aiming to remove phenol pollutants.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect of the present invention, phenol-removing bacteria are provided, which include at least one of Bacillus Bacillus luti and Bacillus Bacillus paranthracis ; the Bacillus Bacillus luti was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 14, 2025, with the deposit number GDMCC NO: 66330; the Bacillus Bacillus paranthracis was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 14, 2025, with the deposit number GDMCC NO: 66331.

[0006] In the second aspect of the present invention, the application of the above-mentioned phenol-removing bacteria in the preparation of microbial agents is provided.

[0007] In the third aspect of the present invention, a phenol-adsorbing humus is provided, which includes the above-mentioned phenol-removing bacteria.

[0008] For the said phenol-adsorbing humus, wherein, its preparation raw materials further include: tree branch debris pretreated with hydrochloric acid and a nitrogen source.

[0009] For the said phenol-adsorbing humus, wherein, its preparation method includes the following steps: Chop the tree branches into branch debris, soak them in a hydrochloric acid solution with a concentration of 0.4 - 0.6 mol / L for 12 - 14 hours. After acid treatment, rinse with water until the pH is 6 - 7, and air-dry until the moisture content is 18% - 22% to obtain the branch debris pretreated with hydrochloric acid; Add a nitrogen source, adjust the carbon-nitrogen ratio of the material to (24 - 26):1, add the phenol-removing bacteria solution, mix evenly, stack the material and carry out aerobic fermentation; Turn the pile once every 3 - 7 days and ferment for 85 - 95 days to obtain the phenol-adsorbing humus.

[0010] For the phenol-adsorbing humus, the volume ratio of the phenol-removing bacteria solution to the branch debris pretreated with hydrochloric acid is 1:(3200 - 3800); the bacterial content of the phenol-removing bacteria solution is 1×10 9 cfu / mL or more.

[0011] For the phenol-adsorbing humus, the length of the branch debris is 0.5 - 1.5 cm.

[0012] For the phenol-adsorbing humus, the nitrogen source includes at least one of urea, ammonium salts or nitrates.

[0013] Beneficial effects: The present invention provides phenol-removing bacteria, and the phenol-removing bacteria include Bacillus Bacillus luti and Bacillus Bacillus paranthracis at least one of them. The phenol-removing bacteria can use phenol as a carbon source for growth and metabolism, thereby removing phenol.

[0014] The present invention also provides phenol-adsorbing humus, which is obtained by aerobic fermentation using the above-mentioned phenol-removing bacteria. Compared with the humus obtained by fermentation without adding the bacteria solution, the phenol adsorption performance of the phenol-adsorbing humus of the present invention is significantly improved. Description of the Drawings

[0015] Figure 1 Is the culture solution at 0 h in the resazurin colorimetric method Figure 1 .

[0016] Figure 2 Is the culture solution at 0 h in the resazurin colorimetric method Figure 2 .

[0017] Figure 3 Is the culture solution at 0 h in the resazurin colorimetric method Figure 3 .

[0018] Figure 4 Is the culture solution at 6 h in the resazurin colorimetric method Figure 1 .

[0019] Figure 5 The culture medium for 6 hours in the resazurin colorimetric method Figure 2 。

[0020] Figure 6 The results of the removal rate of phenol by humus in each group Specific implementation mode

[0021] The present invention provides phenol-removing bacteria and phenol-adsorbing humus. To make the purpose, technical solution and effect of the present invention clearer and more definite, the following further elaborates the present invention with reference to the attached drawings and by way of examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The Bacillus of the present invention Bacillus luti (self-numbered: MS-3-85-1) was deposited on May 14, 2025 at the Guangdong Provincial Microbial Culture Collection Center, located at the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO: 66330; Bacillus Bacillus paranthracis (self-numbered: MS-3-85-3) was deposited on May 14, 2025 at the Guangdong Provincial Microbial Culture Collection Center, located at the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO: 66331.

[0023] Example 1 Verification of strain activity by resazurin colorimetric method Resazurin color development mechanism: Resazurin is an oxidation-reduction indicator. When in the oxidized state, it shows blue (no microbial activity). During the metabolism of live bacteria, resazurin is gradually reduced by metabolites to pink intermediate products and can finally become colorless (completely reduced in an anaerobic environment). Significance of color development: The color change reflects the metabolic activity of the strain and indirectly proves its ability to utilize the target carbon source (phenol).

[0024] The steps are as follows: 1. Preparation of spare bacterial liquid: Take 5 mL of bacterial liquid in the logarithmic growth phase, centrifuge at 4000 rpm for 10 minutes, and discard the supernatant; add 5 mL of sterile MSM medium to resuspend, and repeat the centrifugation and washing 2 times; resuspend the bacteria in 5 mL of sterile MSM, and prepare Bacillus luti spare bacterial liquid and Bacillus paranthracis spare bacterial liquid respectively according to the above method.

[0025] 2. The experimental groups are as follows: Experimental group A: Add 0.5 mL of phenol solution with a concentration of 0.1 g / L and 50 μL of resazurin solution with a concentration of 0.01 g / mL to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus luti spare bacterial liquid; Experimental Group B: Add 0.5 mL of phenol solution with a concentration of 0.1 g / L and 50 μL of resazurin solution with a concentration of 0.01 g / mL to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus paranthracis of the standby bacterial solution; Positive Control Group A: Add 0.5 mL of glucose stock solution and 50 μL of resazurin solution with a concentration of 0.01 g / mL to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus luti of the standby bacterial solution; Positive Control Group B: Add 0.5 mL of glucose stock solution and 50 μL of resazurin solution with a concentration of 0.01 g / mL to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus paranthracis of the standby bacterial solution; Negative Control Group: Add 0.5 mL of phenol solution with a concentration of 0.1 g / L and 50 μL of resazurin solution with a concentration of 0.01 g / mL to 49.5 mL of inorganic salt medium (MSM); Blank Control Group: Add 50 μL of resazurin solution with a concentration of 0.01 g / mL to 50.0 mL of inorganic salt medium (MSM); Set 3 replicates for each group, and aliquot them into sterile test tubes for culture.

[0026] 3. Culture conditions: Culture in the dark in a shaker at 37 °C (180 rpm), and record the color of the culture solution at 0, 6, and 48 hours.

[0027] The results are shown in Table 1, Figures 1 to 5 as follows.

[0028] Table 1

[0029] Figures 1 to 3 shows the color conditions of each group at 0 h, Figure 4 and Figure 5 shows the color conditions of each group at 6 h. It can be seen from Table 1 and the figures that Experimental Group A, Experimental Group B, Positive Control Group A, and Positive Control Group B all showed a pink color synchronously, indicating that the strain can utilize phenol for growth and metabolism.

[0030] Example 2 Preparation of phenol-adsorbing humus The steps are as follows: 1. Stockpiling: The collected waste branches are shredded into debris about 0.5 cm to 1.5 cm in size, soaked in a 0.5 mol / L hydrochloric acid solution for 12 hours, then rinsed until the pH is 6 - 7, and finally air-dried to a moisture content of 18% - 22%; a 5% urea solution is evenly sprayed on the material and stirred evenly to adjust the carbon-nitrogen ratio of the material to 25:1; the material is divided into 3 piles, which are respectively put into foam boxes, and each pile has the same weight, and piled into a conical pile with a bottom width of 30 cm and a height of 30 cm.

[0031] 2. Experimental grouping: Blank group: No microbial agent is added; Experimental group A: 200 mL Bacillus luti of microbial agent solution is added, and the microbial content of the microbial agent solution is 1×10 9 cfu / mL; Experimental group B: 200 mL Bacillus paranthracis of microbial agent solution is added, and the microbial content of the microbial agent solution is 1×10 9 cfu / mL.

[0032] 3. Stockpiling fermentation: The microbial agent solution is fully stirred and mixed evenly with the stockpiling material, and manual turning of the pile is adopted for regular turning to ensure that the stockpiling material obtains sufficient oxygen for aerobic fermentation. It is turned every 3 days within the first 0 - 30 days, every 5 days within the 30 - 60 days, and every 7 days within the 60 - 90 days; the humidity of the stockpiling material is maintained at 50%RH - 55%RH; the environmental temperature is kept at 20℃ - 32℃.

[0033] 4. Adsorption experiment: Collect the branch humus samples obtained after 90 days of stockpiling fermentation. Samples are collected at 3 different positions (bottom, middle, surface) of each compost pile, and a total of 9 humus samples are obtained. The adsorption experiment is carried out at a laboratory environmental temperature of 26℃: 4 g of humus is taken from each sample and added to a 50 mL test tube, 20 mL of phenol standard solution (0.1 g / L) is added to the test tube, sealed with a lid, placed in a low-temperature constant-temperature shaker, the temperature of the low-temperature shaker is set at 15℃ (to avoid the volatilization of the phenol solution at high temperatures), the oscillation speed is 180 r / min, and the oscillation is carried out for 72 h. The phenol solution and humus in the test tube are centrifuged (3200 r / min, 30 min), the supernatant after centrifugation is taken, and the pollutant content is analyzed by gas chromatography - mass spectrometry. The results are shown in Table 2 and Figure 6 as follows.

[0034] Table 2

[0035] From the results in Table 2, it can be seen that the highest removal rate of phenol by the humus sample in the blank group was 87.24%, while the highest removal rate of phenol by the humus sample in experimental group A was 96.6%, and the highest removal rate of phenol by the humus sample in experimental group B was 98.73%.

[0036] Figure 6 In it, "*" represents a significant difference, with P less than 0.05; "**" represents a highly significant difference, with P less than 0.01. From Figure 6 the analysis of the bar graph, it can be seen that the phenol removal rates in experimental group A and experimental group B were both significantly higher than those in the blank group, indicating that adding Bacillus luti bacterial solution or Bacillus paranthracis bacterial solution during the preparation of humus can effectively improve the removal rate of phenol by humus. The removal of phenol by humus is on the one hand due to its own adsorption effect, and on the other hand, it may be that the growth and metabolism of Bacillus Bacillus luti or Bacillus paranthracis removed part of the phenol. In addition, during the composting process, the acid-treated branch debris was used as a carbon source by bacteria, which promoted the formation of more pores on the branch debris and improved the adsorption effect of humus.

[0037] It can be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and its inventive concept, and all such changes or substitutions should fall within the protection scope of the claims appended to the present invention.

Claims

1. A phenol-removing bacterium, characterized in that, including Bacillus Bacillus luti and Bacillus Bacillus paranthracis at least one of them; the Bacillus Bacillus luti was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on May 14, 2025, with the deposit number GDMCC NO: 66330; the Bacillus Bacillus paranthracis was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on May 14, 2025, with the deposit number GDMCC NO: 66331.

2. Use of the phenol-removing bacterium according to claim 1 in the preparation of a microbial agent.

3. A phenol-adsorbing humus, characterized in that, It includes the phenol-removing bacterium according to claim 1.

4. The phenol-adsorbing humus according to claim 3, wherein Its preparation raw materials further include: tree branch debris pretreated with hydrochloric acid and a nitrogen source.

5. The phenol-adsorbing humus according to claim 3, wherein Its preparation method includes the following steps: Cut the tree branches into tree branch debris, soak them in a 0.4 - 0.6 mol / L hydrochloric acid solution for 12 - 14 hours, after acid treatment, wash them with water until the pH is 6 - 7, and air-dry until the moisture content is 18% - 22% to obtain the tree branch debris pretreated with hydrochloric acid; Add a nitrogen source, adjust the carbon-nitrogen ratio of the material to (24 - 26):1, add the phenol-removing bacterium bacterial liquid, mix evenly, stack the materials and carry out aerobic fermentation; Turn the pile once every 3 - 7 days, ferment for 85 - 95 days to obtain the phenol-adsorbing humus.

6. The phenol-adsorbing humus according to claim 5, wherein, The volume ratio of the phenol-removing bacterial liquid to the tree branch debris pretreated with hydrochloric acid is 1:(3200 - 3800); the bacterial content of the phenol-removing bacterial liquid is 1×10 9 cfu / mL or more.

7. The phenol-adsorbing humus according to claim 5, wherein The length of the tree branch debris is 0.5 - 1.5 cm.

8. The phenol-adsorbing humus according to claim 5, characterized in that, The nitrogen source includes at least one of urea, ammonium salts or nitrates.

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