Gulosibacter molinativoxCK7 strain and application thereof in industrial wastewater decontamination
By using Gulosibacter molinativorax CK7 strain and its bacterial agent, the problem of phenol pollution in industrial wastewater in high concentration and low temperature environments is solved, and the efficient degradation effect is achieved. It is suitable for industrial wastewater treatment in high pollution load and low temperature environments.
Patent Information
- Application Number
- CN202510653802.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art is difficult to effectively deal with phenol pollution in industrial wastewater under high concentration and low temperature environments. The activity of traditional microbial bacterial agents is significantly reduced under this condition, making it difficult to achieve efficient degradation.
The Gulosibacter molinativorax CK7 strain and its bacterial agent were used to expand the culture in LB culture medium and inoculate it into industrial wastewater, and cultured at a temperature of 10-25°C and a shaker speed of 180 rpm to achieve the degradation of phenol.
The degradation rate of phenol in high concentrations reaches 91.03%, and the degradation rate of in low temperature environments reaches 65.09%, breaking through the activity limit of traditional bacterial agents and is suitable for wastewater treatment in high pollution loads and cold areas.
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Figure CN120442483A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbiology and particularly relates to a strain Gulosibacter molinativorax CK7 strain and its application in industrial wastewater decontamination. Background Art
[0002] China is a major industrial country, and the discharge of industrial wastewater is enormous. Compared with domestic and agricultural wastewater, industrial wastewater has complex composition, strong bioinhibition, and is difficult to degrade. If it is discharged without treatment, it will pose a serious threat to human health and the ecological environment.
[0003] Phenols are a typical and major pollutant in industrial wastewater, widely distributed in wastewater from industries such as petroleum, chemical, building materials, ceramics, papermaking, coal gas, textiles, plastics, synthetic fibers, and coking. Phenol is the primary phenolic compound among these phenols. Phenol is extremely harmful to organisms and the environment. It has a strong toxic effect on all organisms and can accumulate along the food chain. It is also highly mutagenic and carcinogenic, and is classified by the World Health Organization as a Class III carcinogen on its preliminary list.
[0004] As awareness of environmental protection grows, the treatment of industrial wastewater is gaining increasing attention. Biological methods are currently the ideal and leading method for industrial wastewater treatment, offering advantages such as cost-effectiveness, high efficiency, wide application, and no secondary pollution. However, they also suffer from limitations such as low tolerance to toxicants and poor temperature tolerance. Therefore, screening for microorganisms with tolerance to high concentrations of phenol and low temperatures could help improve the removal efficiency of pollutants such as phenol from industrial wastewater under low-temperature conditions. Summary of the Invention
[0005] The present invention aims to provide a Gulosibacter molinativorax CK7 strain and its bacterial agent for use in industrial wastewater decontamination, thereby achieving efficient degradation of phenol in industrial wastewater.
[0006] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides a Gulosibacter molinativorax strains, the Gulosibacter molinativorax The name of the strain is Gulosibacter molinativorax CK7 has been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 10, 2024, and the deposit number is CGMCC No. 33014.
[0007] Preferably, the Gulosibacter molinativoraxThe 16S rDNA sequence of CK7 is shown in SEQ ID NO.1.
[0008] Secondly, the present invention provides the use of the strain in preparing a phenol-degrading bacterial agent.
[0009] Preferably, the concentration of the strain in the phenol-degrading bacterial agent is ≥1×10 9 CFU / mL.
[0010] Preferably, the phenol-degrading bacterial agent is used for phenol degradation in industrial wastewater.
[0011] Preferably, the amount of the phenol-degrading bacterial agent used is 5-10% of the volume of the industrial wastewater; The initial phenol concentration of the industrial wastewater is ≤2000 mg / L.
[0012] Then, the present invention provides a degradation bacterial agent for degrading phenol pollution in industrial wastewater, the degradation bacterial agent contains a strain Gulosibacter molinativorax CK7, the strain Gulosibacter molinativorax The deposit number of CK7 is CGMCC No.33014.
[0013] Preferably, the concentration of the bacterial agent is ≥1×10 9 CFU / mL; the solvent of the bacterial agent is PBS.
[0014] Finally, the present invention provides a method for treating phenol contamination in industrial wastewater, the method comprising the following steps: (1) Gulosibacter molinativorax The CK7 strain was inoculated into LB medium and cultured to a concentration of ≥ 1×10 9 CFU / mL of bacterial agent; (2) Inoculate the bacterial agent into the industrial wastewater containing phenol at a volume of 5% of the industrial wastewater; (3) Cultivate at a temperature of 10-25°C and a shaker speed of 180 rpm, with a degradation period of 24-72 hours.
[0015] Preferably, the deposit number of the Gulosibacter molinativorax CK7 strain is CGMCC No.33014.
[0016] The beneficial effects of the present invention are: (1) The strain provided by the present invention can quickly respond to medium concentrations of phenol: in phenol wastewater with an initial concentration of 1000 mg / L, the bacterial agent can degrade 90.88% of phenol within 24 hours and achieve complete degradation within 48 hours ( Figure 1 ), which was significantly better than the treatment efficiency of conventional strains.
[0017] (2) The strain provided by the present invention can adapt to high-concentration phenol environments: under high-concentration phenol (2000 mg / L) conditions, the strain gradually adapts to the environment and achieves a degradation rate of 91.03% within 72 hours ( Figure 3 ), which solves the problem of failure of traditional microorganisms due to toxic inhibition and is suitable for the treatment of industrial wastewater with high pollution load.
[0018] (3) The strain provided by the present invention has excellent cold resistance: in a low temperature environment of 15°C, the phenol degradation rate reaches 65.09% within 48 hours ( Figure 4 ); Under the harsh conditions of 10℃, the degradation rate still reached 49.06% after 48 hours ( Figure 5 ), which breaks through the limitation that the activity of traditional bacterial agents is significantly reduced at low temperatures, and provides a reliable solution for wastewater treatment in cold areas or in winter. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 is a morphological diagram of the Gulosibacter molinativorax CK7 strain of the present invention; Figure 2 This is a phylogenetic tree diagram of the Gulosibacter molinativorax CK7 strain of the present invention; Figure 3 is the difference in phenol concentration at different degradation times when the initial phenol concentration is 1000 mg / L; Figure 4 is the difference in phenol concentration at different degradation times when the initial phenol concentration is 1500 mg / L; Figure 5 is the difference in phenol concentration at different degradation times when the initial phenol concentration is 2000 mg / L; Figure 6 The phenol degradation efficiency of the strain at a low temperature of 15°C; Figure 7 The phenol degradation effect of the strain at a low temperature of 10℃. DETAILED DESCRIPTION
[0020] The following embodiments of the technical solution of the present invention are described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only provided as examples and are not intended to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.
[0021] The culture medium used in the embodiments of the present invention is as follows: LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1 L distilled water. Solid medium: add 15 g agar to the liquid medium.
[0022] Basal medium: 2.25 g potassium hydrogen phosphate (KHPO), 2.75 g potassium dihydrogen phosphate (KHPO), 1.00 g ammonium sulfate (AMSO), 0.20 g magnesium chloride hexahydrate (MgCl2), 0.10 g sodium chloride (NaCl), 0.02 g ferric chloride hexahydrate (FeCl3), 0.01 g calcium chloride (CaCl2), 1 L distilled water (pH 6.8-7.0). After sterilization, add varying volumes of phenol stock solution depending on the experimental purpose.
[0023] Example 1 Isolation and screening of strains The samples of the present invention were collected from the regulating tank wastewater and activated sludge (in winter) of a coking wastewater treatment plant in Changzhi, Shanxi Province. The phenol concentration was adjusted by adding phenol, and the samples were acclimated and enriched at 30°C and 180 rpm.
[0024] There were 6 cycles of acclimation, of which the first three cycles were 24 h each with a phenol concentration of about 2000 mg / L, and the last three cycles were 48 h each with a phenol concentration increased to about 2300 mg / L.
[0025] After each cycle, all samples were centrifuged and the supernatant was discarded. New wastewater from the regulating tank was added to continue the next cycle of enrichment culture. During this period, the removal of phenol in the culture medium was tested.
[0026] Take the mixed sample of wastewater and activated sludge after the last cycle, dilute it with sterile water, and spread 100 μL of the dilution liquid on LB solid culture medium for each gradient. Place it in a biochemical incubator at 30℃ and culture it for 48 hours. Observe the growth of the colonies, select single colonies with different morphologies, streak them on LB solid culture medium using the plate streak method, and culture them under the same conditions for 48 hours.
[0027] Then, single colonies were selected and purified by multiple partitioning and streaking until a pure strain was obtained. The purified strain was placed in 20% glycerol and stored in a -80℃ refrigerator for future use. The morphology is shown in the figure below. Figure 1 shown.
[0028] Example 2 Identification of strains After the separation and purification in Example 1, the present invention obtained a bacterium with phenol degradation function. The strain obtained by the present invention was subjected to molecular biological identification, and sequence alignment was performed after PCR amplification of the 16S rRNA gene sequence (SEQ ID NO.1).
[0029] The amplification primer sequences are: 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2), 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID NO. 3); The reaction system was as follows: 10× Buffer 2 μL, 2.5 mM dNTP 1.5 μL, Primer1 1 μL, Primer2 1 μL, template 1 μL, enzyme 0.3 μL, water 13.2 μL, total volume 20 μL; The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, extension at 72°C for 10 min, and insulation at 4°C forever.
[0030] The PCR products were detected by agarose gel electrophoresis and then sequenced.
[0031] The 16S rRNA gene sequence of the bacterium (SEQ ID NO.1) was obtained after forward and reverse sequencing and splicing. The sequence was compared with the NCBI database by Blast. Gulosibactermolinativorax ON4 has the highest homology, reaching 99.14% (system diagram as shown in Figure 2 The strain was named Gulosibactermolinativorax CK7, hereinafter referred to as strain CK7 in this application.
[0032] Gulosibacter molinativorax CK7 was deposited on December 10, 2024 in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, with its address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33014.
[0033] Example 3 Strain expansion culture Strain CK7 was inoculated into 50 mL of LB medium and cultured at 30°C with a shaker at 150 rpm until the bacterial seed solution reached the logarithmic growth phase, with an OD600 of 0.6-0.8. The seed solution was inoculated into fresh LB medium at a volume ratio of 1:10 and cultured at 30°C with a shaker speed of 150 rpm until OD600 was ≈ 1.2-1.5; The expanded cultured strain was centrifuged at 5000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the bacterial precipitate was collected; The cells were washed twice with sterile saline (0.9% NaCl solution), and then resuspended in PBS to adjust the concentration of the bacterial suspension to 10 9CFU / mL, and the phenol-degrading bacterial agent was obtained.
[0034] Example 4 Detection of strain CK7's tolerance and degradation ability to high concentrations of phenol Phenol was added to the basal medium to adjust the final concentrations of phenol to 1000 mg / L, 1500 mg / L, and 2000 mg / L, respectively; The obtained phenol-degrading bacterial agent was inoculated into a basal culture medium containing different concentrations of phenol at an inoculum size of 5%; The culture was carried out in a shaker at 25°C and 180 rpm for 72 h, and the phenol concentration was measured at 6 h, 12 h, 24 h, 48 h and 72 h. The results were as follows: Figure 3-5 shown.
[0035] from Figure 3 As can be seen, the phenol-degrading bacterial agent of the present invention exhibited excellent degradation capabilities at an initial phenol concentration of 1000 mg / L. Within 24 hours of inoculation, the agent efficiently degraded 90.88% of the phenol in the basal medium, demonstrating its rapid response and efficient metabolic activity. By 48 hours, the phenol in the basal medium had been virtually completely degraded. This result not only demonstrates the strain's strong adaptability to moderate phenol concentrations but also provides strong support for its application in practical wastewater treatment.
[0036] from Figure 4 As can be seen, at an initial phenol concentration of 1500 mg / L, while phenol degradation was relatively modest in the initial stages (e.g., the first 6 or 12 hours), likely due to the toxicity of high phenol concentrations, degradation efficiency increased significantly over time as the strain gradually adapted to the environment and activated relevant metabolic pathways. At 48 hours, the phenol degradation rate reached 86.49%, and by 72 hours, phenol in the basal medium had been virtually completely degraded. This demonstrates that even at higher concentrations, the bacterial agent of the present invention can achieve complete phenol removal through gradual adaptation and efficient metabolism, demonstrating excellent environmental adaptability.
[0037] from Figure 5 As can be seen, phenol degradation was initially poor at an initial concentration of 2000 mg / L. This was likely due to the strain needing time to adapt to the high phenol concentration of 2000 mg / L. Subsequently, the phenol content gradually decreased over time. At 72 hours, the degradation rate reached 91.03%, demonstrating excellent phenol degradation.
[0038] In summary, the phenol-degrading bacterial agent of the present invention exhibited excellent degradation performance at various initial concentrations (1000 mg / L, 1500 mg / L, and 2000 mg / L). Whether rapidly responding to moderate phenol concentrations or gradually adapting to high-concentration environments, the agent achieved efficient phenol removal within a reasonable timeframe, demonstrating its broad application prospects in industrial wastewater treatment.
[0039] Example 5 Detection of differences in phenol degradation ability of strain CK7 under low temperature environment Phenol was added to the basal medium to adjust the final concentration of phenol to 1000 mg / L; The obtained phenol-degrading bacterial agent was inoculated into a basic culture medium containing phenol at an inoculum size of 5%; The basal culture medium of the inoculated strain was cultured in a low-temperature culture shaker at 10°C and 15°C, respectively, at 180 rpm for 48 hours. The concentration of phenol was measured at 6 hours, 12 hours, 24 hours and 48 hours. The results were as follows: Figure 6-Figure 7 shown.
[0040] from Figure 6 As can be seen, strain CK7's degrading agent exhibited excellent phenol degradation at a low temperature of 15°C. Although degradation efficiency was relatively low in the initial stages (e.g., the first 6 or 12 hours) due to the inhibitory effect of low temperatures on microbial metabolic activity, this efficiency increased significantly with increased incubation time as the strain gradually adapted to the environment and activated relevant metabolic pathways. By 48 hours, the phenol degradation rate reached 65.09%, demonstrating strain CK7's strong adaptability and efficient degradation capabilities in moderately low-temperature environments. This characteristic makes it particularly valuable for industrial wastewater treatment in spring and autumn.
[0041] from Figure 7 As can be seen, under the more stringent conditions of 10°C, although the degradation capacity of strain CK7 was reduced, it still showed good tolerance. At 48 hours, the phenol degradation rate reached 49.06%, demonstrating the strain's excellent performance in low-temperature environments.
Claims
1. One plant Gulosibacter molinativorax strain, characterized in that described Gulosibacter molinativorax The strain is named Gulosibacter molinativorax CK7 has been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 10, 2024, and the deposit number is CGMCC No. 33014.
2. The strain according to claim 1, characterized in that described Gulosibacter molinativorax The 16S rDNA sequence of CK7 is shown in SEQ ID NO.
1.
3. Use of the strain as claimed in claim 1 in preparing a phenol-degrading bacterial agent.
4. The use according to claim 3, characterized in that In the phenol-degrading bacterial agent, the concentration of the strain is ≥1×10 9 CFU / mL.
5. The use according to claim 4, characterized in that The phenol-degrading bacterial agent is used for degrading phenol in industrial wastewater.
6. The use according to claim 5, characterized in that The dosage of the phenol-degrading bacterial agent is 5-10% of the volume of the industrial wastewater; The initial phenol concentration of the industrial wastewater is ≤2000 mg / L.
7. A bacterial agent for degrading phenol pollution in industrial wastewater, characterized in that: The degrading bacterial agent contains a strain Gulosibacter molinativorax CK7, the strain Gulosibacter molinativorax The deposit number of CK7 is CGMCC No.33014.
8. The degrading bacterial agent according to claim 7, characterized in that In the bacterial agent, the concentration of the bacterial agent is ≥1×10 9 CFU / mL; the solvent of the bacterial agent is PBS.
9. A method for treating phenol pollution in industrial wastewater, characterized in that: The processing method comprises the following steps: (1) Gulosibacter molinativorax The CK7 strain was inoculated into LB medium and cultured to a concentration of ≥ 1×10 9 CFU / mL of bacterial agent; (2) Inoculate the bacterial agent into the industrial wastewater containing phenol at a volume of 5% of the industrial wastewater; (3) Cultivate at a temperature of 10-25°C and a shaker speed of 180 rpm, with a degradation period of 24-72 hours.
10. The processing method according to claim 9, characterized in that: The deposit number of the Gulosibacter molinativorax CK7 strain is CGMCC No.33014.