Phenol-degrading bacterial strain and application thereof in papermaking wastewater treatment

Abstract

The application discloses a strain for degrading phenol and application of the strain in papermaking wastewater treatment, and relates to the technical field of environmental treatment and microorganisms, wherein the strain takes phenol as the only carbon source and is a Bacillus halotolerans ACY; the strain is preserved in the China Center for Type Culture Collection of Wuhan University, has a preservation number of CCTCC M 20221689, and is preserved on November 7, 2022.The thermophilic and salt-tolerant strain ACY has a high degradation rate and degradation speed for phenol in papermaking wastewater, so that the strain can be used to realize the effect of efficient, clean and secondary pollution-free biological treatment; in addition, the thermophilic and salt-tolerant strain ACY has a wide adaptation range for temperature, pH and salt, can rapidly and effectively degrade high-concentration phenol in a high-temperature and high-salt environment, and provides high-quality strain resources for efficient biological remediation of papermaking wastewater.

Description

Technical Field

[0001] This invention relates to the fields of environmental treatment and microbial technology, specifically to a strain of bacteria capable of efficiently degrading phenol in papermaking wastewater, its screening method, and its application. Background Technology

[0002] Paper industry wastewater is a serious source of industrial pollution. Due to its difficulty in treatment, various countries regard paper industry wastewater as a key target for prevention and regulation of public hazards. According to estimates by the United Nations Environment Programme, the global paper industry discharges more than 27.4 billion tons of wastewater annually, accounting for approximately 10% of total industrial wastewater discharge. Its environmental pollution is second only to the chemical and metallurgical industries, posing a serious threat to people's lives and ecological safety. Currently, phenol, listed as a priority environmental pollutant, has been found in paper industry wastewater. This type of compound is a protoplasmic poison, toxic to almost all organisms. Irrigating farmland with phenol-contaminated water can lead to reduced crop yields or even crop death. Due to its ecotoxicological effects, long-term consumption of phenol-contaminated water can lead to nervous system poisoning and even death in animals. When phenol enters drinking water systems, it directly harms human cells, damaging the nervous system and mucous membranes, destroying the nervous system and liver, and may even induce cancer. Due to the high toxicity of phenol, if papermaking wastewater containing phenol is discharged directly into water bodies without treatment or effective treatment, it will lead to complex water composition, increased chemical oxygen demand (COD), large fluctuations in water quality, and easy blackening and odorization of water bodies, damaging the aquatic ecological environment. Therefore, it is imperative to treat phenol in papermaking wastewater.

[0003] Currently, the main methods for removing phenol from wastewater include physical, chemical, and biological methods. Physical methods primarily include adsorption, but this requires high-quality adsorbent materials, leading to increased costs, and the regeneration of these materials remains a pressing issue. Chemical methods mainly include oxidation, but this method consumes a lot of energy, has a narrow range of applications, and is prone to secondary pollution. With the increasing maturity of pollutant-degrading microbial treatment technologies, researchers believe that using microbial degradation methods to treat phenol in wastewater is a thorough, safe, and effective approach. Currently, scholars have isolated various phenol-degrading bacteria, such as Pseudomonas adaceae, Bacillus, Acinetobacter, Staphylococcus, and Rhodococcus. However, most of these degrading bacteria are only suitable for degrading low-concentration phenol-containing wastewater; at high concentrations, their degradation ability decreases due to inhibited microbial growth. In recent years, there have also been research reports on bacteria that degrade high-concentration phenol. For example, Zhang Ju isolated a Bacillus PD2 strain from methanol plant wastewater, which could degrade an initial phenol solution with a concentration of 1800 mg / L in 56 hours. Chen Xiaohua et al. screened a Bacillus anserinae CH10 strain with a maximum phenol tolerance of 1300 mg / L, which could degrade 82.2% of an initial phenol solution with a concentration of 1000 mg / L within 48 hours at a temperature of 30℃, pH 7.0, and an inoculum size of 5%. Xu Tiantian et al. isolated a phenol-degrading bacterium, Sphingobium sp., from activated sludge. With the addition of 0.2 g / L yeast extract as a co-metabolizing substrate, this bacterium could degrade 68% of an initial phenol concentration of 500 mg / L within 36 hours. Because high concentrations of phenol solutions have inhibitory and bactericidal effects on bacteria, and because papermaking wastewater has high environmental temperatures, pH levels, and salt concentrations, microorganisms find it difficult to adapt and grow in such extreme environments. Therefore, no literature reports studies on the degradation of phenol by bacteria under high temperature and high salt conditions. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a strain that degrades phenol. This strain is thermophilic and salt-tolerant, and can utilize phenol as a carbon source and energy source for degradation, showing great potential in the treatment of papermaking wastewater.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A strain, namely Bacillus halotolerans ACY, with accession number CCTCC M20221689.

[0007] The thermophilic and halophilic Bacillus ACY of this invention was deposited on October 31, 2022, at the China Center for Type Culture Collection (CCTCC, address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO: M 20221689, and classified as Bacillus halotolerans ACY. It was found to be viable.

[0008] The phenol-degrading bacterium of the present invention, Bacillus halotolerans ACY, was isolated from activated sludge in a paper mill.

[0009] Furthermore, the 16S rRNA sequence of the Bacillus halotolerans ACY strain is shown in SEQ ID NO: 1.

[0010] This invention also provides a method for screening a bacterial strain, wherein the strain is Bacillus halotolerans ACYCCTCC M 20221689, and the screening method includes the following steps:

[0011] (1) Take sludge samples from the wastewater treatment pond of the paper mill and culture them in a shaking culture medium containing phenol. Then, take the culture and add it to a new liquid inorganic salt culture medium and add phenol for subculture.

[0012] (2) Dilute the culture obtained after 5-6 subcultures to obtain diluted bacterial solution. Spread the diluted bacterial solution on an inorganic salt solid culture medium plate, add phenol and culture. Pick a single colony that grows well on the medium, isolate and purify it to obtain the screening strain.

[0013] (3) The selected strains were inoculated into a new liquid inorganic salt medium, phenol was added and cultured in a shaker, and the phenol degradation rate was measured. Finally, the strain ACY with the strongest phenol degradation ability was screened out (100% degradation within 48h), which is the efficient phenol degradation strain.

[0014] (4) The strain obtained in step (3) is identified by 16S rRNA and constructed by BLAST alignment in NCBI.

[0015] The screening method for highly efficient phenol-degrading strains provided by this invention can obtain strains with phenol as the sole carbon source in a simple and efficient manner. These strains have high degradation efficiency for phenol, a pollutant in papermaking wastewater, and can adapt to the high temperature and high salinity environment of papermaking wastewater.

[0016] Further, in step (1), the concentration of phenol in the liquid culture medium is 1.0 g / L, and the culture conditions during shaking culture are: culture temperature of 50℃, shaking frequency of 170 rpm, and shaking culture time of 48 h.

[0017] Further, the 1L formulation of the liquid inorganic salt culture medium containing phenol is as follows: phenol 1.0g, (NH4)2SO4 1.6g, MgSO4·7H2O 0.7g, K2HPO4 2.5g, NaCl 0.5g, CaCl2 0.5g, FeSO4·7H2O 0.05g, MnSO4 0.001g, ZnCl2 0.001g and CoCl2 0.001g; 1000mL of distilled water, and the pH value of the liquid inorganic salt culture medium is 7.0-7.5.

[0018] Further, the specific operation of step (2) is as follows: dilute the culture that has been passaged 5-6 times into a diluted bacterial solution, spread the diluted bacterial solution onto an inorganic salt solid culture medium plate, add phenol and incubate at 50°C for 5-6 days.

[0019] Furthermore, the universal primers used in step (5) for the identification of the 16S rRNA include 7F: 5'-CAGAGTTTGATCCTGGCT-3' and 1540R: 5'-AGGAGGTGATCCAGCCGCA-3'.

[0020] This invention also provides Bacillus halotolerans ACY CCTCC NO: M 20221689

[0021] Application of strains in the field of environmental remediation.

[0022] This invention also provides Bacillus halotolerans ACY CCTCC NO: M 20221689

[0023] The strain was used to treat phenol-containing papermaking wastewater.

[0024] Preferably, the optimal growth and degradation conditions for the strain include: a temperature of 50°C, a pH of 10.0, an inoculum size of 3%, a shaking speed of 170 rpm, a culture time of 48 h, and a substrate concentration of 1000 mg / L, wherein the upper limit of the substrate concentration tolerable is 4500 mg / L.

[0025] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0026] (1) This invention provides a novel strain of Bacillus halotolerans ACY CCTCC NO: M20221689;

[0027] (2) The strain Bacillus halotolerans ACY can grow using phenol as the sole carbon source and energy source, and can rapidly and effectively degrade phenol in a high-temperature and high-salt environment.

[0028] (3) The phenol-degrading strain Bacillus halotolerans ACY provided by this invention has extremely high removal efficiency of phenol in papermaking wastewater (100% degradation within 48h), thereby solving the pollution problem of phenol in various media in the environment and can be used for environmental remediation projects.

[0029] (4) The phenol-degrading strain Bacillus halotolerans ACY provided by the present invention can grow well in an environment with a temperature of 50-60℃, a pH value of 10-12, and a salt concentration of 0-30g / L. It has strong environmental adaptability and can effectively solve the problem that existing microorganisms are difficult to use directly to treat phenol in papermaking wastewater.

[0030] (5) The phenol-degrading strain Bacillus halotolerans ACY provided by this invention has the advantages of high efficiency, no secondary pollution and low cost in biodegrading phenol in papermaking wastewater, and has good application prospects. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0032] Figure 1 A scanning electron microscope image of the cell morphology of Bacillus halotolerans ACY strain according to an embodiment of the present invention;

[0033] Figure 2 A schematic diagram of a phylogenetic tree constructed after 16S rRNA sequence analysis and alignment of the strain Bacillus halotolerans ACY, which is an embodiment of the present invention.

[0034] Figure 3 The graph shows the cell growth density and phenol degradation rate of a strain of the present invention in an inorganic salt liquid culture medium under different temperature conditions.

[0035] Figure 4 The graph shows the cell growth density and phenol degradation rate of a strain of the present invention in inorganic salt liquid culture medium under different pH conditions.

[0036] Figure 5 This is a graph showing the cell growth density and phenol degradation rate of a strain of the present invention in an inorganic salt liquid culture medium under different inoculation conditions according to an embodiment of the present invention.

[0037] Figure 6 The graph shows the cell growth density and phenol degradation rate of a strain of the present invention in an inorganic salt liquid culture medium under different shaking speeds.

[0038] Figure 7 The graph shows the cell growth density and phenol degradation rate of a strain of the present invention in an inorganic salt liquid culture medium under different culture time conditions.

[0039] Figure 8 This is a graph showing the cell growth density and phenol degradation rate of a strain of the present invention in inorganic salt liquid culture medium under different substrate concentrations.

[0040] Figure 9 This is a graph showing the cell growth density and phenol degradation rate of a strain of the present invention in inorganic salt liquid culture medium at different salt concentrations, according to an embodiment of the present invention.

[0041] Figure 10 This is a graph showing the cell growth density and phenol concentration data of a strain in one embodiment of the present invention when the substrate concentration was measured to the maximum tolerance limit;

[0042] Figure 11 This is a graph showing the removal data of phenol, color, COD, and BOD from papermaking wastewater treated by strain Bacillus halotolerans ACY in one embodiment of the present invention. Detailed Implementation

[0043] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the data are the average of the three replicates or the average ± standard deviation.

[0045] Example 1: Isolation, screening and identification of Bacillus halotolerans ACY 1. Samples

[0046] Sludge sample: Activated sludge was taken from the wastewater treatment pond of a paper mill in Harbin, Heilongjiang Province, China. The sludge was placed in sterilized triangular flasks, sealed, and stored in a refrigerator at 4°C.

[0047] 2. Culture medium:

[0048] The enrichment medium was LB liquid medium (g / L): 10g tryptone, 5g yeast extract, 10g NaCl, pH 7.0, autoclaved at 121℃ for 20min.

[0049] Inorganic salt culture medium: (NH4)2SO4 1.6g, MgSO4·7H2O 0.7g, K2HPO4 2.5g, NaCl 0.5g, CaCl2 0.5g, FeSO4·7H2O 0.05g, MnSO4 0.001g, ZnCl2 0.001g and CoCl2 0.001g, 1000mL distilled water, and pH adjusted to 7.0-7.5 with NaOH.

[0050] The phenol-containing culture medium is prepared by adding 1.0 g of phenol to the above liquid culture medium.

[0051] Solid culture medium is made by adding 1.5% agar powder to liquid culture medium.

[0052] 3. Separation, screening, and domestication procedures:

[0053] Sludge was collected from the wastewater treatment pond of a paper mill. A 20 mL sample was placed in an Erlenmeyer flask containing 250 mL of enrichment medium (phenol concentration 0.5 g / L) and incubated at 170 rpm and 50 °C. The sludge was then further incubated with sterile water to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6The following methods were used to prepare a phenol-degrading culture medium. Six different dilutions (100 μL each) were then spread evenly on enrichment plates and incubated at 30°C for 3 days. Single colonies were then picked and cultured. A 3% inoculum was added to an inorganic salt medium with an initial phenol concentration of 500 mg / L. The phenol concentration was increased in 500 mg / L increments until a maximum concentration of 5500 mg / L was reached. Strains that could not utilize phenol were eliminated, thus initially screening for strains capable of degrading phenol and exhibiting strong tolerance. After 5-6 subcultures, the culture medium from the last subculture was prepared into different dilutions and spread on inorganic salt solid medium containing 1000 mg / L phenol. The medium was incubated at 50°C until single colonies appeared, and colony characteristics were observed. Well-grown colonies with different morphologies were streaked, and after 3-5 days, single colonies were re-streaked and cultured. This process was repeated 3-5 times to obtain a purified strain. Further, the screened pure strains were cultured in inorganic salt medium containing phenol, and their phenol degradation rate was measured to determine their phenol degradation characteristics. Finally, the strain with the strongest phenol degradation ability, ACY, was selected and named the degrading bacterium ACY. This strain could degrade 100% of phenol at an initial concentration of 1000 mg / L within 48 hours under pure culture conditions. For the pure strains confirmed to have phenol degradation characteristics through secondary screening, one copy was inoculated into LB slant medium and stored at 4°C; simultaneously, it was stored in 30% glycerol at -80°C for later use.

[0054] HPLC determination conditions: The culture medium was extracted with an equal volume of ethyl acetate, and the extract was detected by high-performance liquid chromatography (HPLC). HPLC determination conditions: Waters 600E HPLC system (Waters, USA); Detector: Waters 2487 UV detector (Waters, USA); Column: C18 reversed-phase column (SunFire). TM The column was 150 nm x 4.60 mm; the mobile phase was water / methanol (50 / 50, V / V); the flow rate was 0.5 mL / min; the column temperature was 25 °C; the detection wavelength was 270 nm; and the injection volume was 10 μL. Phenol standards with different concentration gradients were prepared, and the peak areas at the corresponding concentrations were measured to plot a standard curve. The phenol concentration was calculated from the phenol peak area in the sample using the standard curve.

[0055] Degradation rate = (initial concentration - final concentration) / initial concentration.

[0056] 4. Identification of degrading bacteria

[0057] (1) Morphological identification of the degrading bacterium ACY

[0058] The degrading bacteria ACY obtained from the isolation and purification in step one above, which are in the logarithmic growth phase and have stable colony size, are described as single colony states. The morphology of the degrading bacteria ACY in the logarithmic growth phase is observed by optical microscopy after smear staining.

[0059] The results showed that the degrading bacteria ACY, isolated and purified in step one, grew rapidly on LB medium. The colonies were milky white, opaque, rough, wrinkled, and round or nearly round. Scanning electron microscopy results are shown below. Figure 1 The bacterial cells are rod-shaped, and under an electron microscope, the long diameter of the rod-shaped cells is >1μm.

[0060] (2) Physiological and biochemical characteristics analysis

[0061] The physiological and biochemical characteristics of the degrading bacterium ACY were determined with reference to the methods described in "Microbiology Experiments" (Shen Ping, Fan Xiurong, and Li Guangwu. Microbiology Experiments (3rd Edition). Beijing: Higher Education Press, 1999) and "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press, 2011).

[0062] Gram staining was performed on the bacterial cells cultured to the logarithmic growth phase. Under an optical microscope, the strain stained purple after Gram staining, indicating a positive result. Physiological and biochemical assays showed that the strain was positive for catalase, catalase, and VP test, as detailed in Table 1.

[0063] Table 1. Physiological and biochemical characteristics of strain Bacillus halotolerans ACY

[0064] glucose + sucrose + lactose + maltose + fructose + catalase + VP test + Nitrate reductase test + Hydrogen sulfide production + citrate + Mannitol + Sorbitol + Starch hydrolysis +

[0065] + Positive; - Negative

[0066] (3) Homology analysis of 16S rDNA of degrading bacteria

[0067] Genomic DNA was extracted from the bacterial strain according to the instructions of the bacterial genomic DNA extraction kit. 16S rDNA was amplified using universal bacterial primers 7F: 5'-CAGAGTTTGATCCTGGCT-3' and 1540R: 5'-AGGAGGTGATCCAGCCGCA-3'.

[0068] PCR amplification system (25 μL): template DNA 1-2 μL, primer F 2 μL, primer R 2 μL, dNTP (mix) 2 μL, ddH2O 9.5 μL, Taq Buffer (with MgCl2) 5 μL, Taq enzyme 0.5 μL.

[0069] PCR amplification program: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s; 57℃ annealing for 30 s; 72℃ extension for 90 s, 32 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0070] The PCR amplification products were purified and sequenced by Beijing BGI Genomics Co., Ltd. The 16S rDNA sequence of ACY is shown in SEQ ID NO: 1.

[0071] The obtained sequences were submitted to NCBI (www.ncbi.nlm.nih.gov) for Megablast analysis to obtain highly homologous related sequences. ClustalX software was used for alignment, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 5.0 software. The phylogenetic tree based on the 16S rDNA sequence is shown below. Figure 2 As shown. Strain ACY has been submitted to GenBank, accession number MW559481. The 16S rDNA sequence of strain ACY was submitted to the GenBank database. A homologous sequence search was performed in the GenBank nucleic acid sequence database. ACY showed 100% similarity to Bacillus halotolerans F41-3 (CP041357.1). Based on the physiological and biochemical characteristics of the strain, ACY was identified as a halophilic Bacillus (Bacillus halotolerans ACY).

[0072] Example 2: Growth of halophilic Bacillus ACY and optimization of its phenol degradation ability under different culture conditions

[0073] ①Optimal culture temperature for strain growth and degradation:

[0074] 3% of activated ACY bacterial strain suspension was inoculated into sterilized inorganic salt medium containing 1000 mg / L phenol and cultured for 3 days at 20℃, 30℃, 40℃, 50℃, and 60℃, respectively, with environmental parameters of 170 rpm and pH 8.0. Cell growth was detected by measuring OD600 nm using a spectrophotometer, and phenol concentration was detected by high-performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown, the phenol degradation rate is optimal at 50℃.

[0075] ②Optimal pH for bacterial growth and degradation:

[0076] Inorganic salt media containing 1000 mg / L phenol were prepared with pH values ​​of 6, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. Cell suspensions of ACY strains in PBS were inoculated with 3% ACY and cultured in a shaker at 50℃ and 170 rpm for 48 h. Cell growth was detected by measuring OD 600 nm using a spectrophotometer, and phenol concentration was detected by high-performance liquid chromatography (HPLC). Results are as follows: Figure 4 As shown, the degradation rate of phenol is better at a pH of 10.0.

[0077] ③ Effect of inoculum size on the degradation rate of degrading bacteria AXJ-M:

[0078] 1%, 2%, 3%, 4%, 5%, and 6% of the PBS suspension of the degrading bacterium ACY was inoculated into sterilized inorganic salt medium containing 1000 mg / L phenol. The cultures were then incubated for 48 h on a constant-temperature shaker at 50 °C, pH 10.0, and 150 rpm. The degradation effect of each treatment was then measured. The results are as follows: Figure 5 As shown, the phenol degradation rate was optimal in a PBS suspension containing 3% ACY degrading bacteria.

[0079] ④ Effect of shaker speed on the degradation rate of ACY bacteria:

[0080] A 3% suspension of activated degrading bacteria ACY was inoculated into a sterilized inorganic salt medium containing 1000 mg / L phenol. The culture was then incubated at 130 rpm, 150 rpm, 170 rpm, 190 rpm, and 210 rpm on a shaker at 50 °C and pH 10.0 for 48 h. The degradation effect of each treatment was then measured. Results are as follows: Figure 6 As shown, the phenol degradation rate is optimal at 170 rpm.

[0081] ⑤ Effect of culture time on the degradation effect of ACY by degrading bacteria:

[0082] A 3% suspension of the degrading bacterium ACY was inoculated into a sterilized inorganic salt medium containing 1000 mg / L phenol. The medium was then incubated on a constant temperature shaker at 50°C, pH 10.0, and 170 rpm for 48 hours. Samples were taken every 8 hours during incubation to determine the degradation effect of each treatment. Results are as follows: Figure 7 As shown.

[0083] ⑥ Optimal substrate concentration for strain growth and degradation:

[0084] The strain was inoculated into 50 mL of inorganic salt medium with different phenol concentrations of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, and 5500 mg / L, at an optimal pH of 10.0. The medium was then incubated at the optimal temperature of 50°C and a shaking speed of 170 rpm for 48 h. Cell growth was detected by measuring OD600 nm using a spectrophotometer, and phenol concentration was determined by high-performance liquid chromatography (HPLC). Figure 8 As shown, the strains were able to degrade phenol relatively quickly at various phenol concentrations. The optimal phenol concentration for strain growth and phenol degradation was 1000 mg / L.

[0085] according to Figures 3 to 8 The results showed that the optimal degradation conditions for the phenol-degrading bacterium ACY were: 50℃, pH 10.0, inoculum size of 3%, and shaking speed of 170 rpm. Under these optimal conditions, the strain could completely degrade 1000 mg / L phenol after 48 hours of cultivation. Notably, even at an ambient temperature of 60℃, the ACY strain could still degrade more than 80% of the phenol.

[0086] Example 4: Verification of the maximum tolerance of halophilic Bacillus ACY to phenol

[0087] The strain was inoculated into 100 mL of MSM medium with different phenol concentrations of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, and 5500 mg / L, and cultured in a shaker at an optimal temperature of 50°C, pH 10.0, and a rotation speed of 170 rpm. Cell density was measured at OD 600 nm using a spectrophotometer at regular intervals, and phenol concentration was determined by high-performance liquid chromatography (HPLC). Figure 9 As shown in a and 9b, the growth and degradation of the strains worsened with increasing concentration. At phenol concentrations above 5000 mg / L, cells showed almost no growth or degradation, possibly because higher phenol concentrations are more toxic to cells. The ACY strain can tolerate phenol at 4500 mg / L.

[0088] Example 5 Salt tolerance test of salt-tolerant Bacillus cylindrica (ACY)

[0089] Different amounts of NaCl were added to MSM liquid medium to achieve NaCl concentrations of 0 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, and 70 g / L. 3% of the bacterial strain was added to this medium, with a control group not containing NaCl. The medium was incubated at 170 rpm and 50 °C for 48 h using a shaker. Samples were then taken to measure the absorbance (OD) at 600 nm. Figure 10As shown, strain ACY can tolerate a certain concentration of salt. It grows well under NaCl concentrations of 0-30 g / L, but its growth is significantly affected when the NaCl concentration exceeds 40 g / L. These results indicate that strain ACY is a salt-tolerant bacterium, capable of rapidly adapting to and accumulating biomass under high NaCl concentrations, demonstrating strong adaptability to high-salt environments.

[0090] Example 6: Treatment effect of degrading bacteria ACY on papermaking wastewater

[0091] The papermaking wastewater was taken from a paper mill in Jiangxi Province. Table 2 shows the values ​​of various parameters in the actual wastewater.

[0092] Table 2 Results of physicochemical parameter determination of papermaking wastewater

[0093]

[0094] 10 mL of papermaking wastewater was placed in a 250 mL Erlenmeyer flask. After adjusting the pH to 8.95, the degrading bacteria ACY were inoculated at a rate of 3%. Uninoculated papermaking wastewater was used as a control. Both were fermented at 170 rpm and 50 °C. COD, BOD, color, and phenol content were measured periodically. The results are as follows: Figure 11 As shown in the figure. The results showed that after 7 days of treatment, the COD removal rate, BOD removal rate, color removal rate, and phenol removal rate of the wastewater reached 87%, 83%, 98%, and 83%, respectively. Therefore, the phenol-reducing microorganism of this invention can efficiently remove phenol, the main pollutant in papermaking wastewater, and achieves ideal removal rates for other target parameters. It is suitable for papermaking wastewater with high pollution loads and complex environments, and can achieve good treatment results in a short time.

[0095] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A strain, characterized in that, The strain is Bacillus halotolerans ACY, with accession number CCTCC NO: M2020273.

2. The application of the strain described in claim 1 in the field of environmental remediation, wherein the environmental remediation field is the treatment of phenol-containing papermaking wastewater.

3. The application of the strain described in claim 1 in the degradation of phenol in papermaking wastewater.

4. The application according to claim 3, characterized in that, The growth and degradation conditions of the strain include: a temperature of 50 °C, a pH of 10.0, an inoculum size of 3%, a shaking speed of 170 rpm, a culture time of 24 h, and a substrate concentration of 1000 mg / L, wherein the upper limit of the substrate concentration tolerable is 4500 mg / L.

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