Phenol removal bacteria and phenol adsorbing humus

Abstract

The present invention relates to the field of microbial technology, in particular to phenol removal bacteria and phenol adsorption humus. The phenol removal bacteria include Bacillus sp. Clay bacillus and Bacillus Bacillus paranthracis At least one of; the Bacillus Clay bacillus It was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 14, 2025, with the deposit number GDMCC NO: 66330; the Bacillus Bacillus Paranthracian It was deposited with the Guangdong Provincial Microbial Culture Collection on May 14, 2025, with the deposit number GDMCC NO: 66331. The phenol-removing bacteria of the present invention can grow and metabolize using phenol as a carbon source. Furthermore, the phenol-adsorbing humus prepared by adding the phenol-removing bacteria solution of the present invention can significantly improve the phenol removal rate.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to phenol-removing bacteria and phenol-adsorbing humus. Background Art

[0002] Phenol is a highly toxic substance. Once it enters the soil, it can negatively impact soil quality, inhibiting the activity of soil microorganisms and, consequently, affecting soil fertility and plant growth. Phenol is also acutely toxic to aquatic organisms, impacting the balance and stability of aquatic ecosystems. If phenol could be removed by specific microorganisms, phenol's contamination of water and soil resources could be reduced. Humus, a key component of soil, can adsorb phenol, reducing its migration and diffusion within the soil, thereby reducing phenol contamination. It also helps prevent phenol from seeping through the soil into groundwater, causing wider contamination. However, current humus does not have a high enough adsorption capacity for phenol.

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

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

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a phenol removal bacterium comprising Bacillus sp. Bacillus luti and Bacillus Bacillus paranthracis At least one of; the Bacillus Bacillus luti It was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 14, 2025, with the deposit number GDMCC NO: 66330; the Bacillus Bacillus paranthracis It was deposited in Guangdong Provincial Microbiological Culture Collection on May 14, 2025, with the deposit number GDMCC NO:66331.

[0007] The second aspect of the present invention provides the use of the phenol-removing bacteria described above in the preparation of a bacterial agent.

[0008] A third aspect of the present invention provides a phenol-adsorbing humus, which comprises the phenol-removing bacteria described above.

[0009] The phenol-adsorbed humus, wherein its preparation raw materials also include: tree branch debris pretreated with hydrochloric acid and a nitrogen source.

[0010] The phenol adsorbed humus, wherein the preparation method thereof comprises the following steps:

[0011] chopping branches into branch chips, soaking them in a 0.4-0.6 mol / L hydrochloric acid solution for 12-14 hours, washing them with water to a pH of 6-7 after the acid treatment, and air-drying them to a moisture content of 18%-22% to obtain branch chips pretreated with hydrochloric acid;

[0012] Add nitrogen source, adjust the carbon-nitrogen ratio of the material to (24-26):1, add phenol removal bacteria liquid, mix evenly, pile the material and carry out aerobic fermentation;

[0013] The pile is turned over every 3 to 7 days, and the fermentation takes 85 to 95 days to obtain the phenol-adsorbed humus.

[0014] The phenol adsorption humus, wherein the volume ratio of the phenol removal bacteria solution to the branch debris pretreated with hydrochloric acid is 1: (3200-3800); the bacterial content of the phenol removal bacteria solution is 1×10 9 cfu / mL or above.

[0015] The phenol-adsorbed humus, wherein the length of the branch debris is 0.5 to 1.5 cm.

[0016] The phenol-adsorbed humus, wherein the nitrogen source includes at least one of urea, ammonium salt or nitrate.

[0017] Beneficial effect: The present invention provides a phenol removal bacterium, wherein the phenol removal bacterium includes Bacillus sp. Bacillus luti and Bacillus Bacillus paranthracis The phenol-removing bacteria can use phenol as a carbon source for growth and metabolism, thereby removing phenol.

[0018] The present invention also provides phenol-adsorbed humus, which is obtained by aerobic fermentation using the phenol-removing bacteria described above. Compared with humus obtained by fermentation without adding bacterial liquid, the phenol-adsorbed humus of the present invention has significantly improved phenol adsorption performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The culture medium at 0h in the resazurin colorimetric method Figure 1 .

[0020] Figure 2 The culture medium at 0h in the resazurin colorimetric method Figure 2 .

[0021] Figure 3 The culture medium at 0h in the resazurin colorimetric method Figure 3 .

[0022] Figure 4 6h culture medium in the resazurin colorimetric method Figure 1 .

[0023] Figure 5 6h culture medium in the resazurin colorimetric method Figure 2 .

[0024] Figure 6 The results show the removal rate of phenol by humus in each group. DETAILED DESCRIPTION

[0025] The present invention provides phenol-removing bacteria and phenol-adsorbing humic substances. To make the objectives, technical solutions, and effects of the present invention more clear and explicit, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0026] Bacillus of the present invention Bacillus luti (Self-numbered: MS-3-85-1) was deposited on May 14, 2025, at Guangdong Provincial Microbiological Culture Collection, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO: 66330;

[0027] Bacillus Bacillus paranthracis (Self-numbered: MS-3-85-3) was deposited on May 14, 2025 in the Guangdong Provincial Microbiological Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO: 66331.

[0028] Example 1 Verification of strain activity by resazurin colorimetry

[0029] Resazurin color development mechanism: Resazurin is a redox indicator. When oxidized, it displays a blue color (inactive). During bacterial metabolism, resazurin is progressively reduced by metabolites to pink intermediates, ultimately becoming colorless (complete reduction occurs in anaerobic conditions). Significance of color development: The color change reflects the metabolic activity of the strain, indirectly demonstrating its ability to utilize the target carbon source (phenol).

[0030] Here are the steps:

[0031] 1. Preparation of standby bacterial solution: Take 5 mL of bacterial solution in the logarithmic growth phase, centrifuge at 4000 rpm for 10 minutes, discard the supernatant; add 5 mL of sterile MSM medium to resuspend, repeat centrifugation and wash twice; resuspend the bacteria in 5 mL of sterile MSM, and prepare Bacillus luti Reserve bacterial solution and Bacillus paranthracis Reserve bacterial solution.

[0032] 2. The experimental groups are as follows:

[0033] Experimental group A: 0.5 mL of 0.1 g / L phenol solution and 50 μL of 0.01 g / mL resazurin solution were added to 49.5 mL of inorganic salt medium (MSM), and 100 μL of Bacillus luti Reserve bacterial solution;

[0034] Experimental group B: 0.5 mL of 0.1 g / L phenol solution and 50 μL of 0.01 g / mL resazurin solution were added to 49.5 mL of inorganic salt medium (MSM), and 100 μL of Bacillus paranthracis Reserve bacterial solution;

[0035] Positive control group A: Add 0.5 mL of glucose stock solution and 50 μL of 0.01 g / mL resazurin solution to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus luti Reserve bacterial solution;

[0036] Positive control group B: Add 0.5 mL of glucose stock solution and 50 μL of 0.01 g / mL resazurin solution to 49.5 mL of inorganic salt medium (MSM), and inoculate 100 μL Bacillus paranthracis Reserve bacterial solution;

[0037] Negative control group: 0.5 mL of 0.1 g / L phenol solution and 50 μL of 0.01 g / mL resazurin solution were added to 49.5 mL of inorganic salt medium (MSM);

[0038] Blank control group: add 50 μL of 0.01 g / mL resazurin solution into 50.0 mL of inorganic salt medium (MSM);

[0039] Each group was replicated three times and the samples were divided into sterile test tubes for culture.

[0040] 3. Culture conditions: Culture in a 37°C shaker (180 rpm) in the dark. Record the color of the culture medium at 0, 6, and 48 hours.

[0041] The results are shown in Table 1. Figures 1 to 5 shown.

[0042] Table 1

[0043]

[0044] Figures 1 to 3 The color of each group at 0h, Figure 4 and Figure 5 The color of each group at 6 h is shown in Table 1 and the figures. Experimental group A, experimental group B, positive control group A and positive control group B all showed pink at the same time, indicating that the strain can use phenol for growth metabolism.

[0045] Example 2 Preparation of phenol-adsorbed humus

[0046] Here are the steps:

[0047] 1. Stacking: Chop the collected discarded branches into fragments of about 0.5cm to 1.5cm, soak them in 0.5mol / L hydrochloric acid solution for 12 hours, then rinse them to a pH of 6-7, and finally air-dry them to a moisture content of 18% to 22%; evenly spray a 5% urea solution on the material and stir it to adjust the carbon-nitrogen ratio of the material to 25:1; divide the material into 3 piles and put them into foam boxes respectively. The weight of each pile is the same, and they are stacked into a conical pile with a bottom width of 30cm and a height of 30cm.

[0048] 2. Experimental groups:

[0049] Blank group: no bacterial agent was added;

[0050] Experimental group A: Add 200 mL Bacillus luti The bacterial content of the bacterial solution is 1×10 9 cfu / mL;

[0051] Experimental group B: add 200mL Bacillus paranthracis The bacterial content of the bacterial solution is 1×10 9 cfu / mL.

[0052] 3. Pile fermentation: fully mix the bacterial liquid and the pile material, and turn the pile regularly by manual turning to ensure that the pile obtains sufficient oxygen for aerobic fermentation. Turn the pile every 3 days from 0 to 30 days, every 5 days from 30 to 60 days, and every 7 days from 60 to 90 days; the humidity of the pile is maintained at 50% RH to 55% RH; the ambient temperature is maintained at 20℃ to 32℃.

[0053] 4. Adsorption experiment: The branch humus samples obtained after 90 days of compost fermentation were collected. Samples were collected from three different locations of each group of compost (bottom, middle, and surface), and a total of 9 humus samples were obtained. The adsorption test was carried out under laboratory ambient temperature of 26°C: 4g of humus was added to a 50mL test tube for each sample, 20mL of phenol standard solution (0.1g / L) was added to the test tube, sealed with a cover, and placed in a low-temperature constant temperature oscillator. The low-temperature oscillator temperature was set to 15°C (to avoid volatilization of the phenol solution at high temperature), the oscillation speed was 180r / min, and the oscillation was continued for 72h. The phenol solution and humus in the test tube were centrifuged (3200r / min, 30min), and the supernatant after centrifugation was taken and the pollutant content was analyzed by gas chromatography-mass spectrometry. The results are shown in Table 2 and Figure 6 shown.

[0054] Table 2

[0055]

[0056] From the results in Table 2, it can be seen that the removal rate of phenol by the humus samples in the blank group is the highest, which is 87.24%, while the removal rate of phenol by the humus samples in the experimental group A is the highest, which is 96.6%, and the removal rate of phenol by the humus samples in the experimental group B is the highest, which is 98.73%.

[0057] Figure 6 In the table, “*” indicates significant difference, P less than 0.05; “**” indicates highly significant difference, P less than 0.01. Figure 6 The histogram analysis shows that the phenol removal rates of experimental group A and experimental group B are significantly higher than those of the blank group, indicating that the addition of Bacillus luti Bacterial solution or Bacillus paranthracis Bacterial liquid can effectively improve the removal rate of phenol by humus. The removal of phenol by humus is due to its own adsorption on one hand, and on the other hand it may be due to the action of Bacillus Bacillus luti or Bacillus paranthracis The growth and metabolism of bacteria removed some phenol. In addition, during the composting process, the bacteria used the acid-treated branch debris as a carbon source, which promoted the formation of more pores in the branch debris and improved the adsorption of humus.

[0058] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached 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; the Bacillus Bacillus luti It was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 14, 2025, with the deposit number GDMCC NO: 66330; the Bacillus Bacillus paranthracis It was deposited in Guangdong Provincial Microbiological Culture Collection on May 14, 2025, with the deposit number GDMCC NO:66331.

2. Use of the phenol-removing bacteria as claimed in claim 1 in preparing a bacterial agent.

3. A phenol adsorption humus, characterized in that, The invention comprises the phenol-removing bacteria according to claim 1.

4. The phenol-adsorbed humus according to claim 3, characterized in that The preparation raw materials also include: tree branch debris pretreated with hydrochloric acid and a nitrogen source.

5. The phenol-adsorbed humus according to claim 3, characterized in that: The preparation method comprises the following steps: chopping branches into branch chips, soaking them in a 0.4-0.6 mol / L hydrochloric acid solution for 12-14 hours, washing them with water to a pH of 6-7 after the acid treatment, and air-drying them to a moisture content of 18%-22% to obtain branch chips pretreated with hydrochloric acid; Add nitrogen source, adjust the carbon-nitrogen ratio of the material to (24-26):1, add phenol removal bacteria liquid, mix evenly, pile the material and carry out aerobic fermentation; The pile is turned over every 3 to 7 days, and the fermentation takes 85 to 95 days to obtain the phenol-adsorbed humus.

6. The phenol-adsorbed humus according to claim 5, characterized in that 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 above.

7. The phenol-adsorbed humus according to claim 5, characterized in that: The length of the branch debris is 0.5 to 1.5 cm.

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

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