A kind of acid and alkali resistant and efficient phenol-degrading bacteria and its application

Abstract

The present invention discloses an acid-resistant and alkali-resistant high-efficiency phenol-degrading bacterium and its application, belonging to the field of environmental microbiology technology. The screened strain G0-1 ​​was identified as Acinetobacter berezzii ( Acinetobacter bereziniae ), the strain has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20251392 and the deposit date is June 16, 2025. Strain G0‑1 can use phenol as the sole carbon source and energy source for metabolic activities, and can completely degrade 500 mg / L of phenol within 12 h, and its maximum tolerance concentration can reach 1400 mg / L. It is worth noting that the strain can maintain stable phenol degradation activity over a wide range of pH 4‑10. The G0‑1 strain obtained by the present invention shows significant advantages in treating phenol-containing wastewater, including high treatment efficiency, low cost, no secondary pollution and strong environmental tolerance, and has important theoretical and application value in the field of environmental pollution control.

Description

Technical Field

[0001] The present invention relates to the fields of microbial technology and coking wastewater treatment, and more particularly to an acid-resistant and alkali-resistant high-efficiency phenol-degrading bacterium and application thereof. Background Art

[0002] Approximately 14% of phenolic compounds discharged in industrial wastewater migrate through soil infiltration or adsorption onto airborne particles. Phenol pollution poses serious health risks: long-term exposure can cause neurological symptoms such as dizziness and vomiting, and in severe cases, can even lead to poisoning. Concerning the ecological environment, phenol concentrations of 0.1-0.2 mg / L in water can affect the reproduction of aquatic organisms. When phenol levels exceed 100 mg / L in irrigation water, soil structure damage and crop mortality can result. Due to the severity of phenol pollution, countries around the world have established strict control standards. The US Environmental Protection Agency (EPA) has designated phenol as a priority pollutant, setting a discharge limit of 0.01 mg / L or less in surface water and 0.001 mg / L or less in drinking water. To address this environmental challenge, developing efficient phenol treatment technologies has become a focus of research.

[0003] There are many methods to remove phenol, among which biological methods have great development potential and are currently the best choice for removing phenol because they have the characteristics of lower cost, better remediation effect and no secondary pollution compared with other physical and chemical methods. Studies have shown that microorganisms with phenol degradation ability are widely distributed in the bacterial kingdom, mainly including Pseudomonas spp. Pseudomonas ), Stenotrophomonas maltophilia ( Stenotrophomonas ), Acinetobacter spp. ( Acinetobacter ) and Candida tropicalis ( Candida tropicalis ) etc. High concentrations of phenol are toxic to microorganisms, hindering their growth and metabolic activity. Therefore, isolating strains with strong tolerance to high concentrations of phenol and outstanding degradation capabilities will significantly optimize the biodegradation efficiency of phenolic wastewater. Furthermore, coking wastewater is typically acidic or alkaline due to differences in treatment processes at different stages of the production process. Selected strains can maintain stable degradation performance across a wide pH range, which not only helps enrich microbial strain resources but also provides more efficient solutions for the biotreatment of acidic and alkaline phenolic wastewater. Summary of the Invention

[0004] The primary purpose of the present invention is to address the technical issues of low degradation efficiency and narrow acid-base tolerance of phenol-degrading bacteria in the prior art, providing a highly tolerant and efficient phenol-degrading bacterial strain. This strain maintains stable activity and rapidly degrades phenol over a wide pH range, expanding the resource of efficient phenol-degrading microbial strains. It also provides a biological solution with practical application potential for environmental pollution control, with significant economic benefits and ecological significance.

[0005] According to the first aspect of the present invention, a phenol-degrading bacterium with acid and alkali resistance is provided, wherein the phenol-degrading bacterium is Acinetobacter berezzii with a deposit number of CCTCC NO: M 20251392. Acinetobacter bereziniae .

[0006] According to another aspect of the present invention, there is provided the use of the phenol-degrading bacteria having acid and alkali resistance for degrading phenol.

[0007] Preferably, the application is specifically: inoculating the phenol-degrading bacteria into a liquid containing phenol, and then placing the liquid in a shaking incubator for cultivation.

[0008] Preferably, the pH of the liquid is 4-10.

[0009] Preferably, the concentration of phenol in the liquid is less than or equal to 1400 mg / L.

[0010] Preferably, the shaking culture temperature is 25°C-30°C.

[0011] Preferably, the rotation speed of the shaking culture is 100 rpm-180 rpm.

[0012] The strain G0-1 ​​of the present invention is Acinetobacter berezzii ( Acinetobacter bereziniae ), the strain has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO:M 20251392, and its Latin name is: Acinetobacter bereziniae G0-1. This strain is deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and was deposited on June 16, 2025. Strain G0-1 ​​can metabolize phenol using phenol as its sole carbon and energy source, completely degrading 500 mg / L of phenol within 12 hours, with a maximum tolerance of 1400 mg / L. Notably, this strain maintains stable phenol-degrading activity over a wide pH range of 4-10.

[0013] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0014] (1) Phenol-degrading bacteria provided by the present invention Acinetobacter bereziniaeG0-1 can grow and reproduce using phenol as the sole carbon source within a phenol concentration of 1400 mg / L, and has a high removal rate and efficiency of phenol.

[0015] (2) Phenol-degrading bacteria provided by the present invention Acinetobacter bereziniae G0-1 has the characteristics of high efficiency, low cost and strong environmental tolerance in treating phenol-containing wastewater, and has good application prospects.

[0016] (3) Phenol-degrading bacteria provided by the present invention Acinetobacter bereziniae G0-1 can grow stably and efficiently degrade phenol in the pH range of 4-10, which helps solve the problem of low treatment efficiency of acidic and alkaline phenol-containing wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The strain of the present invention Acinetobacter bereziniae Colony morphology of G0-1 ​​on LB solid medium.

[0018] Figure 2 The strain of the present invention Acinetobacter bereziniae Scanning electron microscopy image of the bacterial morphology of G0-1 ​​in LB medium.

[0019] Figure 3 The strain of the present invention Acinetobacter bereziniae Schematic diagram of the phylogenetic tree constructed after 16S rRNA gene sequence analysis and alignment of G0-1.

[0020] Figure 4 The strain of the present invention Acinetobacter bereziniae Growth curve of G0-1 ​​in LB liquid medium.

[0021] Figure 5 The strain of the present invention Acinetobacter bereziniae The OD value of G0-1 ​​in the inorganic salt medium containing 500 mg / L phenol was 600 The curve of the change of phenol concentration with time.

[0022] Figure 6 The strain of the present invention Acinetobacter bereziniae Phenol degradation of G0-1 ​​in inorganic salt culture medium containing different phenol concentrations.

[0023] Figure 7 The strain of the present invention Acinetobacter bereziniae Phenol degradation of G0-1 ​​at different pH values.

[0024] Figure 8 The strain of the present invention Acinetobacter bereziniae Phenol degradation of G0-1 ​​at different inoculation rates. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0026] The screening method for phenol-degrading bacteria of the present invention comprises the following steps:

[0027] (1) Activated sludge obtained from the primary aerobic tank of a coking wastewater plant in Wuhan was pretreated and inoculated into a laboratory-scale sequencing batch reactor (SBR). During the cultivation process, the concentration of the influent was continuously increased to acclimate and enrich the phenol-degrading functional bacteria in the activated sludge.

[0028] (2) Take a sample from the activated sludge after acclimation and spread it on the surface of LB solid culture medium using the dilution coating method. Culture it until a single colony is formed. Repeated streaking culture is then performed to obtain a single strain.

[0029] (3) Inoculate a single strain into an inorganic salt medium containing phenol, measure the phenol degradation rate, and screen out strains with stronger phenol degradation ability. Store them in cryopreservative solution and place them in a -80℃ ultra-low temperature freezer.

[0030] The following are specific embodiments

[0031] Example 1: Isolation and identification of strains

[0032] (1) Sample collection and pretreatment

[0033] In order to activate the microbial community in the sludge and deplete other residual nutrients in the sludge, the activated sludge obtained from the primary aerobic tank of a coking wastewater treatment plant in Wuhan was pretreated by aeration in a closed aeration device for 48 hours.

[0034] (2) Domestication and isolation of strains

[0035] Treated sludge was inoculated into a laboratory-scale sequencing batch reactor (SBR) with a 1-liter effective volume. The reactor used synthetic wastewater to simulate a real-world wastewater treatment environment. Influent and effluent were controlled by a peristaltic pump and flow valves. An air pump provided continuous aeration, and a mechanical stirrer ensured uniform mixing of the reactor fluids. The initial mixed liquor suspended solids concentration (MLSS) was set at 4000 mg / L. The SBR had a daily liquid exchange volume of 500 mL, and the operating cycle consisted of 5 minutes of water inlet, 23 hours of agitation and aeration, 50 minutes of sedimentation, and 5 minutes of drainage.

[0036] During the acclimation process, the concentration of phenol was gradually increased from 300 mg / L and the phenol degradation rate was tested each time. After continuous acclimation, sludge that could degrade higher concentrations of phenol (1300-1400 mg / L) was obtained, and a microbial community with high phenol degradation ability was obtained.

[0037] Samples were taken from the acclimated active sludge and spread onto the surface of LB solid culture medium using the dilution spreading method. Cultures were then cultured until single colonies formed. Repeated streaking cultures were then performed to obtain a single strain. The single strain was then inoculated into an inorganic salt medium containing phenol. The phenol degradation rate was measured, and a strain with strong phenol degradation ability was selected and designated G0-1. This strain was stored in cryopreservative solution in a -80°C ultra-low temperature freezer.

[0038] Inorganic salt medium composition: 0.12 g / L MgSO4, 0.4 g / L KH2PO4, 0.5 g / L NaCl, 0.2 g / L NaHCO3, 0.02 g / L FeSO4·7H2O, 0.5 g / L NH4Cl, 500 mg / L phenol, and 1 mL / 1000 mL of trace element solution (1.5 g / L FeCl2·4H2O, 190 mg / L CoCl2·6H2O, 100 mg / L MnCl2·6H2O, 70 mg / L ZnCl2, 62 mg / L H3BO3, 36 mg / L Na2MoO4·2H2O, 24 mg / L NiCl2·6H2O, and 17 mg / L CuCl2·2H2O). The final pH was controlled at 7.

[0039] LB medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7 with 1 mol / L NaOH solution. To prepare a solid medium, add 15 g / L (1.5%) agar powder to the LB liquid medium.

[0040] (3) Morphological characteristics of the strain

[0041] The isolated and purified strains with good growth were characterized by single colony morphology. The results showed that the phenol-degrading bacteria G0-1 ​​had a Gram-negative stain and grew well on LB medium. The colonies were yellow and opaque, with neat, round edges and a raised center. They were easy to pick up and separate, and grew healthily. Figure 1 At the same time, the strains grown to the logarithmic phase in LB liquid medium were isolated and scanned by SEM electron microscope. The results are as follows: Figure 2 , it can be seen that the strain is in good growth condition and the bacterial cells are rod-shaped.

[0042] (4) Physiological and biochemical characteristics

[0043] The physiological and biochemical characteristics of the degrading bacteria were determined with reference to the Manual of Identification of Common Bacteria (Dong Xiuzhu, Cai Miaoying. Manual of Identification of Common Bacteria. Beijing: Science Press, 2011). The results of the physiological and biochemical tests are shown in the table below.

[0044] Table 1 Physiological and biochemical characteristics of phenol-degrading bacteria Acinetobacter bereziniae G0-1.

[0045] project result maltose - glucose - sucrose - Nitrate (reduced) - hydrogen sulfide - Xylose - Glucophosphodiesterone water -

[0046] +: positive result; -: negative result

[0047] (5) 16S rRNA identification

[0048] Amplification was performed using 16S rRNA bacterial universal primers (27F 5'-AGAGTTTGATCCTGGCTCA-3' and 1492R 5'AGAGTTTGATCCTGGCTCA-3'), and the amplified results were sent to a sequencing company for sequencing. After sequencing, a gene sequence of approximately 1400 bp was obtained. The sequence was submitted to NCBI (National Center for Biotechnology Information (nih.gov)) for BLAST comparison to obtain related sequences with high homology. A phylogenetic tree was constructed using the neighbor-joining method using MEGA11 software ( Figure 3 ).

[0049] Based on the bacterial morphology, physiological and biochemical characteristics and 16S rRNA gene sequence results, strain G0-1 ​​was identified as Acinetobacter bereziniae.

[0050] Example 2: Growth and degradation curves of strains

[0051] Optical density (OD 600 ) Measure the absorbance change of the bacterial solution to characterize the bacterial concentration. 600 The determination value is 0.1 inoculum inoculated into 100 mL of sterilized LB medium and inorganic salt medium containing 500 mg / L phenol, and cultured in a shaking incubator at 30°C and 150 rpm. Samples were taken regularly to determine the phenol concentration and OD in the culture medium. 600 The growth curve and phenol degradation curve of the strain were drawn according to the changes in the values ​​to see the growth characteristics of the strain in different culture media and its phenol degradation ability.

[0052] The growth curve of strain G0-1 ​​in LB liquid medium is shown in Figure 2. Figure 4As shown in the figure, the strain is in the lag phase from 0 to 4 hours, with a relatively slow growth rate. It enters the logarithmic phase at around 4 hours, with very rapid growth, and enters the stable phase at around 20 hours, with the bacterial count reaching its maximum.

[0053] The growth curve and phenol degradation of strain G0-1 ​​in inorganic salt medium are as follows Figure 5 As shown. In MSM medium, at the beginning, because phenol is biologically toxic, 0-4 h OD 600 The value decreased, part of the strain died, and the phenol utilization rate was slow; after 4 h, the strain G0-1 ​​adapted to the environment and began to grow rapidly, and the OD 600 The OD value increased rapidly, and the degradation rate of phenol was fast. At 12 h, phenol was almost completely degraded. After 12 h, phenol was completely degraded. 600 The value began to decrease, and some strains died due to lack of nutrients. As can be seen from the figure, the strain OD 600 As the value increased, the phenol concentration continued to decrease, and the two trends showed an obvious negative correlation. After the phenol was basically degraded, the strain began to die. These results clearly showed that the strain could use phenol as the only carbon source and energy source for its own growth and reproduction to synthesize nutrients.

[0054] Example 3: Phenol degradation performance experiment of strains

[0055] The growth and dephenolization characteristics of phenol-degrading bacteria G0-1 ​​under different phenol concentrations, pH values ​​and inoculation amounts were studied through experiments.

[0056] Degradation characteristics of phenol-degrading bacteria G0-1 ​​at different phenol concentrations

[0057] The strain G0-1 ​​was isolated at OD 600 The determination value was 0.1. The inoculum was inoculated into 100 mL of inorganic salt liquid culture medium at pH 7, where the phenol concentrations were 1200, 1300, 1400, 1500, and 1600 mg / L, respectively. The culture was shaken at 30°C and 150 rpm. An appropriate amount of culture medium was regularly taken and the residual phenol concentration was measured using the 4-aminoantipyrine spectrophotometric method to evaluate the degradation effect of strain G0-1. The results are as follows Figure 6 As shown,

[0058] Strain G0-1 ​​can degrade phenol at concentrations up to 1400 mg / L, achieving a phenol removal rate of approximately 60% in 96 hours. Complete degradation of 1200 and 1300 mg / L phenol was achieved in approximately 84 hours, with degradation increasing at lower phenol concentrations. However, when the phenol concentration increased to 1500 mg / L, strain G0-1 ​​became unable to grow and degrade the phenol. This is because at 1500 mg / L, the high concentration of phenol is toxic to strain G0-1, leading to its death. It is unable to utilize phenol as its sole carbon source for growth and degradation. The degradation rate slows with higher phenol concentrations because higher phenol concentrations increase substrate inhibition, resulting in a longer retardation period for cell growth and thus affecting its degradation performance.

[0059] (2) Degradation characteristics of phenol-degrading bacteria G0-1 ​​at different pH

[0060] The strain G0-1 ​​was isolated at OD 600 The determination value is 0.1 inoculum inoculated into 100 mL of inorganic salt liquid culture medium with a phenol concentration of 500 mg / L, wherein the pH is 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively, and cultured in a shaking incubator at 30°C and 150 rpm. An appropriate amount of culture medium was taken regularly and the residual phenol concentration was measured by 4-aminoantipyrine spectrophotometry to evaluate the degradation effect of strain G0-1. The results are as follows Figure 7 As shown, strain G0-1 ​​was able to grow and completely degrade phenol at an initial concentration of 500 mg / L within a pH range of 4-10. Its optimal pH was neutral. At pH 5-9, the microorganism degraded phenol extremely rapidly, with a short lag phase. Within 12 hours of inoculation, the strain degraded over 50% of 500 mg / L phenol, and completely degraded the 500 mg / L phenol within 24 hours. At more acidic and alkaline pHs of 4 and 10, the strain showed little phenol degradation for the first 36 hours, likely due to the toxic effects of these environments on the cells. However, after an acclimatization period, strain G0-1 ​​began degrading 500 mg / L phenol at pH 4 and 10 by 36 hours, and degradation was very rapid within 12 hours between 36 and 48 hours. The 500 mg / L phenol was essentially completely degraded by 48 hours, and completely degraded by 60 hours.

[0061] (3) Degradation characteristics of phenol-degrading bacteria G0-1 ​​at different inoculation rates

[0062] The strain was inoculated into an inorganic salt medium with a pH of 7 at different inoculum sizes (0.10, 0.15, and 0.20) as determined by optical density. The phenol concentration was set at 500 mg / L and the culture temperature was maintained at 30°C. Appropriate amounts of culture medium were regularly sampled and the residual phenol concentration was measured using 4-aminoantipyrine spectrophotometry to evaluate the degradation effect of strain G0-1. Figure 8 As shown, it can be seen that the inoculum size OD 600 When the inoculum OD was 0.10 and 0.15, the degradation rate of phenol by the strain was not much different. The strain could achieve basic degradation of 500 mg / L phenol in 12 h and completely degrade it in 16 h. 600 When the inoculum size was 0.20, the degradation rate was faster than when the inoculum size was 0.10 and 0.15, and complete degradation of 500 mg / L phenol was achieved in 12 hours. This shows that with increasing inoculum size, the strain growth rate accelerated, the lag phase was shortened, and the utilization rate of phenol increased.

[0063] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A phenol-degrading bacterium having acid and alkali resistance, characterized in that: The phenol-degrading bacteria is Acinetobacter berezzii with a deposit number of CCTCC NO: M 20251392. Acinetobacter bereziniae .

2. Use of the phenol-degrading bacteria having acid and alkali resistance as claimed in claim 1 for degrading phenol.

3. The use according to claim 2, characterized in that The application specifically includes: inoculating the phenol-degrading bacteria into a liquid containing phenol, and then placing the liquid in a shaking table for cultivation.

4. The use according to claim 3, characterized in that The pH of the liquid is 4-10.

5. The use according to claim 3, characterized in that The concentration of phenol in the liquid is less than or equal to 1400 mg / L.

6. The use according to claim 3, characterized in that The temperature of the shaking culture is 25°C-30°C.

7. The use according to claim 3, characterized in that The rotation speed of the shaking culture is 100 rpm-180 rpm.

Images